Inhibition of phosphatidylinositol 3-kinase by PX-866 suppresses temozolomide-induced autophagy and promotes apoptosis in glioblastoma cells
Identifieur interne : 000135 ( Pmc/Corpus ); précédent : 000134; suivant : 000136Inhibition of phosphatidylinositol 3-kinase by PX-866 suppresses temozolomide-induced autophagy and promotes apoptosis in glioblastoma cells
Auteurs : Bryan G. Harder ; Sen Peng ; Christopher P. Sereduk ; Andrej M. Sodoma ; Gaspar J. Kitange ; Joseph C. Loftus ; Jann N. Sarkaria ; Nhan L. TranSource :
- Molecular Medicine [ 1076-1551 ] ; 2019.
Abstract
Temozolomide (TMZ) is the most commonly used chemotherapeutic agent used to treat glioblastoma (GBM), which causes significant DNA damage to highly proliferative cells. Our observations have added to accumulating evidence that TMZ induces stress-responsive cellular programs known to promote cell survival, including autophagy. As such, targeting these survival pathways may represent new vulnerabilities of GBM after treatment with TMZ.
Using the T98G human glioma cell line, we assessed the molecular signaling associated with TMZ treatment, the cellular consequences of using the pan-PI3K inhibitor PX-866, and performed clonogenic assays to determine the effect sequential treatment of TMZ and PX-866 had on colony formation. Additionally, we also use subcutaneous GBM patient derived xenograft (PDX) tumors to show relative LC3 protein expression and correlations between survival pathways and molecular markers which dictate clinical responsiveness to TMZ.
Here, we report that TMZ can induce autophagic flux in T98G glioma cells. GBM patient-derived xenograft (PDX) tumors treated with TMZ also display an increase in the autophagosome marker LC3 II. Additionally, O6-methylguanine-DNA-methyltransferase (MGMT) expression correlates with PI3K/AKT activity, suggesting that patients with inherent resistance to TMZ (MGMT-high) would benefit from PI3K/AKT inhibitors in addition to TMZ. Accordingly, we have identified that the blood-brain barrier (BBB) penetrant pan-PI3K inhibitor, PX-866, is an early-stage inhibitor of autophagic flux, while maintaining its ability to inhibit PI3K/AKT signaling in glioma cells. Lastly, due to the induction of autophagic flux by TMZ, we provide evidence for sequential treatment of TMZ followed by PX-866, rather than combined co-treatment, as a means to shut down autophagy-induced survival in GBM cells and to enhance apoptosis.
The understanding of how TMZ induces survival pathways, such as autophagy, may offer new therapeutic vulnerabilities and opportunities to use sequential inhibition of alternate pro-survival pathways that regulate autophagy. As such, identification of additional ways to inhibit TMZ-induced autophagy could enhance the efficacy of TMZ.
Url:
DOI: 10.1186/s10020-019-0116-z
PubMed: 31726966
PubMed Central: 6854621
Links to Exploration step
PMC:6854621Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Inhibition of phosphatidylinositol 3-kinase by PX-866 suppresses temozolomide-induced autophagy and promotes apoptosis in glioblastoma cells</title><author><name sortKey="Harder, Bryan G" sort="Harder, Bryan G" uniqKey="Harder B" first="Bryan G." last="Harder">Bryan G. Harder</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0000 8875 6339</institution-id><institution-id institution-id-type="GRID">grid.417468.8</institution-id><institution>Departments of Cancer Biology and Neurosurgery,</institution><institution>Mayo Clinic Arizona,</institution></institution-wrap>13400 E. Shea Blvd, MCCRB 03-055, Scottsdale, AZ 85259 USA</nlm:aff></affiliation></author><author><name sortKey="Peng, Sen" sort="Peng, Sen" uniqKey="Peng S" first="Sen" last="Peng">Sen Peng</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0507 3225</institution-id><institution-id institution-id-type="GRID">grid.250942.8</institution-id><institution>Cancer and Cell Biology Division,</institution><institution>Translational Genomics Research Institute,</institution></institution-wrap>Phoenix, AZ USA</nlm:aff></affiliation></author><author><name sortKey="Sereduk, Christopher P" sort="Sereduk, Christopher P" uniqKey="Sereduk C" first="Christopher P." last="Sereduk">Christopher P. Sereduk</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0000 8875 6339</institution-id><institution-id institution-id-type="GRID">grid.417468.8</institution-id><institution>Departments of Cancer Biology and Neurosurgery,</institution><institution>Mayo Clinic Arizona,</institution></institution-wrap>13400 E. Shea Blvd, MCCRB 03-055, Scottsdale, AZ 85259 USA</nlm:aff></affiliation></author><author><name sortKey="Sodoma, Andrej M" sort="Sodoma, Andrej M" uniqKey="Sodoma A" first="Andrej M." last="Sodoma">Andrej M. Sodoma</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0000 8875 6339</institution-id><institution-id institution-id-type="GRID">grid.417468.8</institution-id><institution>Departments of Cancer Biology and Neurosurgery,</institution><institution>Mayo Clinic Arizona,</institution></institution-wrap>13400 E. Shea Blvd, MCCRB 03-055, Scottsdale, AZ 85259 USA</nlm:aff></affiliation></author><author><name sortKey="Kitange, Gaspar J" sort="Kitange, Gaspar J" uniqKey="Kitange G" first="Gaspar J." last="Kitange">Gaspar J. Kitange</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0459 167X</institution-id><institution-id institution-id-type="GRID">grid.66875.3a</institution-id><institution>Department of Radiation Oncology,</institution><institution>Mayo Clinic,</institution></institution-wrap>Rochester, MN USA</nlm:aff></affiliation></author><author><name sortKey="Loftus, Joseph C" sort="Loftus, Joseph C" uniqKey="Loftus J" first="Joseph C." last="Loftus">Joseph C. Loftus</name><affiliation><nlm:aff id="Aff4"><institution-wrap><institution-id institution-id-type="ISNI">0000 0000 8875 6339</institution-id><institution-id institution-id-type="GRID">grid.417468.8</institution-id><institution>Department of Biochemistry and Molecular Biology,</institution><institution>Mayo Clinic Arizona,</institution></institution-wrap>Scottsdale, AZ USA</nlm:aff></affiliation></author><author><name sortKey="Sarkaria, Jann N" sort="Sarkaria, Jann N" uniqKey="Sarkaria J" first="Jann N." last="Sarkaria">Jann N. Sarkaria</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0459 167X</institution-id><institution-id institution-id-type="GRID">grid.66875.3a</institution-id><institution>Department of Radiation Oncology,</institution><institution>Mayo Clinic,</institution></institution-wrap>Rochester, MN USA</nlm:aff></affiliation></author><author><name sortKey="Tran, Nhan L" sort="Tran, Nhan L" uniqKey="Tran N" first="Nhan L." last="Tran">Nhan L. Tran</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0000 8875 6339</institution-id><institution-id institution-id-type="GRID">grid.417468.8</institution-id><institution>Departments of Cancer Biology and Neurosurgery,</institution><institution>Mayo Clinic Arizona,</institution></institution-wrap>13400 E. Shea Blvd, MCCRB 03-055, Scottsdale, AZ 85259 USA</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31726966</idno><idno type="pmc">6854621</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854621</idno><idno type="RBID">PMC:6854621</idno><idno type="doi">10.1186/s10020-019-0116-z</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000135</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000135</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Inhibition of phosphatidylinositol 3-kinase by PX-866 suppresses temozolomide-induced autophagy and promotes apoptosis in glioblastoma cells</title><author><name sortKey="Harder, Bryan G" sort="Harder, Bryan G" uniqKey="Harder B" first="Bryan G." last="Harder">Bryan G. Harder</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0000 8875 6339</institution-id><institution-id institution-id-type="GRID">grid.417468.8</institution-id><institution>Departments of Cancer Biology and Neurosurgery,</institution><institution>Mayo Clinic Arizona,</institution></institution-wrap>13400 E. Shea Blvd, MCCRB 03-055, Scottsdale, AZ 85259 USA</nlm:aff></affiliation></author><author><name sortKey="Peng, Sen" sort="Peng, Sen" uniqKey="Peng S" first="Sen" last="Peng">Sen Peng</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0507 3225</institution-id><institution-id institution-id-type="GRID">grid.250942.8</institution-id><institution>Cancer and Cell Biology Division,</institution><institution>Translational Genomics Research Institute,</institution></institution-wrap>Phoenix, AZ USA</nlm:aff></affiliation></author><author><name sortKey="Sereduk, Christopher P" sort="Sereduk, Christopher P" uniqKey="Sereduk C" first="Christopher P." last="Sereduk">Christopher P. Sereduk</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0000 8875 6339</institution-id><institution-id institution-id-type="GRID">grid.417468.8</institution-id><institution>Departments of Cancer Biology and Neurosurgery,</institution><institution>Mayo Clinic Arizona,</institution></institution-wrap>13400 E. Shea Blvd, MCCRB 03-055, Scottsdale, AZ 85259 USA</nlm:aff></affiliation></author><author><name sortKey="Sodoma, Andrej M" sort="Sodoma, Andrej M" uniqKey="Sodoma A" first="Andrej M." last="Sodoma">Andrej M. Sodoma</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0000 8875 6339</institution-id><institution-id institution-id-type="GRID">grid.417468.8</institution-id><institution>Departments of Cancer Biology and Neurosurgery,</institution><institution>Mayo Clinic Arizona,</institution></institution-wrap>13400 E. Shea Blvd, MCCRB 03-055, Scottsdale, AZ 85259 USA</nlm:aff></affiliation></author><author><name sortKey="Kitange, Gaspar J" sort="Kitange, Gaspar J" uniqKey="Kitange G" first="Gaspar J." last="Kitange">Gaspar J. Kitange</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0459 167X</institution-id><institution-id institution-id-type="GRID">grid.66875.3a</institution-id><institution>Department of Radiation Oncology,</institution><institution>Mayo Clinic,</institution></institution-wrap>Rochester, MN USA</nlm:aff></affiliation></author><author><name sortKey="Loftus, Joseph C" sort="Loftus, Joseph C" uniqKey="Loftus J" first="Joseph C." last="Loftus">Joseph C. Loftus</name><affiliation><nlm:aff id="Aff4"><institution-wrap><institution-id institution-id-type="ISNI">0000 0000 8875 6339</institution-id><institution-id institution-id-type="GRID">grid.417468.8</institution-id><institution>Department of Biochemistry and Molecular Biology,</institution><institution>Mayo Clinic Arizona,</institution></institution-wrap>Scottsdale, AZ USA</nlm:aff></affiliation></author><author><name sortKey="Sarkaria, Jann N" sort="Sarkaria, Jann N" uniqKey="Sarkaria J" first="Jann N." last="Sarkaria">Jann N. Sarkaria</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0459 167X</institution-id><institution-id institution-id-type="GRID">grid.66875.3a</institution-id><institution>Department of Radiation Oncology,</institution><institution>Mayo Clinic,</institution></institution-wrap>Rochester, MN USA</nlm:aff></affiliation></author><author><name sortKey="Tran, Nhan L" sort="Tran, Nhan L" uniqKey="Tran N" first="Nhan L." last="Tran">Nhan L. Tran</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0000 8875 6339</institution-id><institution-id institution-id-type="GRID">grid.417468.8</institution-id><institution>Departments of Cancer Biology and Neurosurgery,</institution><institution>Mayo Clinic Arizona,</institution></institution-wrap>13400 E. Shea Blvd, MCCRB 03-055, Scottsdale, AZ 85259 USA</nlm:aff></affiliation></author></analytic><series><title level="j">Molecular Medicine</title><idno type="ISSN">1076-1551</idno><idno type="eISSN">1528-3658</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p id="Par1">Temozolomide (TMZ) is the most commonly used chemotherapeutic agent used to treat glioblastoma (GBM), which causes significant DNA damage to highly proliferative cells. Our observations have added to accumulating evidence that TMZ induces stress-responsive cellular programs known to promote cell survival, including autophagy. As such, targeting these survival pathways may represent new vulnerabilities of GBM after treatment with TMZ.</p></sec><sec><title>Methods</title><p id="Par2">Using the T98G human glioma cell line, we assessed the molecular signaling associated with TMZ treatment, the cellular consequences of using the pan-PI3K inhibitor PX-866, and performed clonogenic assays to determine the effect sequential treatment of TMZ and PX-866 had on colony formation. Additionally, we also use subcutaneous GBM patient derived xenograft (PDX) tumors to show relative LC3 protein expression and correlations between survival pathways and molecular markers which dictate clinical responsiveness to TMZ.</p></sec><sec><title>Results</title><p id="Par3">Here, we report that TMZ can induce autophagic flux in T98G glioma cells. GBM patient-derived xenograft (PDX) tumors treated with TMZ also display an increase in the autophagosome marker LC3 II. Additionally, O<sup>6</sup>-methylguanine-DNA-methyltransferase (MGMT) expression correlates with PI3K/AKT activity, suggesting that patients with inherent resistance to TMZ (MGMT-high) would benefit from PI3K/AKT inhibitors in addition to TMZ. Accordingly, we have identified that the blood-brain barrier (BBB) penetrant pan-PI3K inhibitor, PX-866, is an early-stage inhibitor of autophagic flux, while maintaining its ability to inhibit PI3K/AKT signaling in glioma cells. Lastly, due to the induction of autophagic flux by TMZ, we provide evidence for sequential treatment of TMZ followed by PX-866, rather than combined co-treatment, as a means to shut down autophagy-induced survival in GBM cells and to enhance apoptosis.</p></sec><sec><title>Conclusions</title><p id="Par4">The understanding of how TMZ induces survival pathways, such as autophagy, may offer new therapeutic vulnerabilities and opportunities to use sequential inhibition of alternate pro-survival pathways that regulate autophagy. As such, identification of additional ways to inhibit TMZ-induced autophagy could enhance the efficacy of TMZ.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Apel, A" uniqKey="Apel A">A Apel</name></author><author><name sortKey="Herr, I" uniqKey="Herr I">I Herr</name></author><author><name sortKey="Schwarz, H" uniqKey="Schwarz H">H Schwarz</name></author><author><name sortKey="Rodemann, Hp" uniqKey="Rodemann H">HP Rodemann</name></author><author><name sortKey="Mayer, A" uniqKey="Mayer A">A Mayer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brennan, Cw" uniqKey="Brennan C">CW Brennan</name></author><author><name sortKey="Verhaak, Rg" uniqKey="Verhaak R">RG Verhaak</name></author><author><name sortKey="Mckenna, A" uniqKey="Mckenna A">A McKenna</name></author><author><name sortKey="Campos, B" uniqKey="Campos B">B Campos</name></author><author><name sortKey="Noushmehr, H" uniqKey="Noushmehr H">H Noushmehr</name></author><author><name sortKey="Salama, Sr" uniqKey="Salama S">SR Salama</name></author><author><name sortKey="Zheng, S" uniqKey="Zheng S">S Zheng</name></author><author><name sortKey="Chakravarty, D" uniqKey="Chakravarty D">D Chakravarty</name></author><author><name sortKey="Sanborn, Jz" uniqKey="Sanborn J">JZ Sanborn</name></author><author><name sortKey="Berman, Sh" uniqKey="Berman S">SH Berman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Briceno, E" uniqKey="Briceno E">E Briceno</name></author><author><name sortKey="Calderon, A" uniqKey="Calderon A">A Calderon</name></author><author><name sortKey="Sotelo, J" uniqKey="Sotelo J">J Sotelo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Buccarelli, M" uniqKey="Buccarelli M">M Buccarelli</name></author><author><name sortKey="Marconi, M" uniqKey="Marconi M">M Marconi</name></author><author><name sortKey="Pacioni, S" uniqKey="Pacioni S">S Pacioni</name></author><author><name sortKey="De Pascalis, I" uniqKey="De Pascalis I">I De Pascalis</name></author><author><name sortKey="D Alessandris, Qg" uniqKey="D Alessandris Q">QG D'Alessandris</name></author><author><name sortKey="Martini, M" uniqKey="Martini M">M Martini</name></author><author><name sortKey="Ascione, B" uniqKey="Ascione B">B Ascione</name></author><author><name sortKey="Malorni, W" uniqKey="Malorni W">W Malorni</name></author><author><name sortKey="Larocca, Lm" uniqKey="Larocca L">LM Larocca</name></author><author><name sortKey="Pallini, R" uniqKey="Pallini R">R Pallini</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Button, Rw" uniqKey="Button R">RW Button</name></author><author><name sortKey="Vincent, Jh" uniqKey="Vincent J">JH Vincent</name></author><author><name sortKey="Strang, Cj" uniqKey="Strang C">CJ Strang</name></author><author><name sortKey="Luo, S" uniqKey="Luo S">S Luo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Catasus, L" uniqKey="Catasus L">L Catasus</name></author><author><name sortKey="D Angelo, E" uniqKey="D Angelo E">E D'Angelo</name></author><author><name sortKey="Pons, C" uniqKey="Pons C">C Pons</name></author><author><name sortKey="Espinosa, I" uniqKey="Espinosa I">I Espinosa</name></author><author><name sortKey="Prat, J" uniqKey="Prat J">J Prat</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cheng, Ck" uniqKey="Cheng C">CK Cheng</name></author><author><name sortKey="Fan, Qw" uniqKey="Fan Q">QW Fan</name></author><author><name sortKey="Weiss, Wa" uniqKey="Weiss W">WA Weiss</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Degenhardt, K" uniqKey="Degenhardt K">K Degenhardt</name></author><author><name sortKey="Mathew, R" uniqKey="Mathew R">R Mathew</name></author><author><name sortKey="Beaudoin, B" uniqKey="Beaudoin B">B Beaudoin</name></author><author><name sortKey="Bray, K" uniqKey="Bray K">K Bray</name></author><author><name sortKey="Anderson, D" uniqKey="Anderson D">D Anderson</name></author><author><name sortKey="Chen, G" uniqKey="Chen G">G Chen</name></author><author><name sortKey="Mukherjee, C" uniqKey="Mukherjee C">C Mukherjee</name></author><author><name sortKey="Shi, Y" uniqKey="Shi Y">Y Shi</name></author><author><name sortKey="Gelinas, C" uniqKey="Gelinas C">C Gelinas</name></author><author><name sortKey="Fan, Y" uniqKey="Fan Y">Y Fan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Degtyarev, M" uniqKey="Degtyarev M">M Degtyarev</name></author><author><name sortKey="De Maziere, A" uniqKey="De Maziere A">A De Maziere</name></author><author><name sortKey="Orr, C" uniqKey="Orr C">C Orr</name></author><author><name sortKey="Lin, J" uniqKey="Lin J">J Lin</name></author><author><name sortKey="Lee, Bb" uniqKey="Lee B">BB Lee</name></author><author><name sortKey="Tien, Jy" uniqKey="Tien J">JY Tien</name></author><author><name sortKey="Prior, Ww" uniqKey="Prior W">WW Prior</name></author><author><name sortKey="Van Dijk, S" uniqKey="Van Dijk S">S van Dijk</name></author><author><name sortKey="Wu, H" uniqKey="Wu H">H Wu</name></author><author><name sortKey="Gray, Dc" uniqKey="Gray D">DC Gray</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ding, Zb" uniqKey="Ding Z">ZB Ding</name></author><author><name sortKey="Hui, B" uniqKey="Hui B">B Hui</name></author><author><name sortKey="Shi, Yh" uniqKey="Shi Y">YH Shi</name></author><author><name sortKey="Zhou, J" uniqKey="Zhou J">J Zhou</name></author><author><name sortKey="Peng, Yf" uniqKey="Peng Y">YF Peng</name></author><author><name sortKey="Gu, Cy" uniqKey="Gu C">CY Gu</name></author><author><name sortKey="Yang, H" uniqKey="Yang H">H Yang</name></author><author><name sortKey="Shi, Gm" uniqKey="Shi G">GM Shi</name></author><author><name sortKey="Ke, Aw" uniqKey="Ke A">AW Ke</name></author><author><name sortKey="Wang, Xy" uniqKey="Wang X">XY Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Eliopoulos, Ag" uniqKey="Eliopoulos A">AG Eliopoulos</name></author><author><name sortKey="Havaki, S" uniqKey="Havaki S">S Havaki</name></author><author><name sortKey="Gorgoulis, Vg" uniqKey="Gorgoulis V">VG Gorgoulis</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fan, Qw" uniqKey="Fan Q">QW Fan</name></author><author><name sortKey="Cheng, C" uniqKey="Cheng C">C Cheng</name></author><author><name sortKey="Hackett, C" uniqKey="Hackett C">C Hackett</name></author><author><name sortKey="Feldman, M" uniqKey="Feldman M">M Feldman</name></author><author><name sortKey="Houseman, Bt" uniqKey="Houseman B">BT Houseman</name></author><author><name sortKey="Nicolaides, T" uniqKey="Nicolaides T">T Nicolaides</name></author><author><name sortKey="Haas Kogan, D" uniqKey="Haas Kogan D">D Haas-Kogan</name></author><author><name sortKey="James, Cd" uniqKey="James C">CD James</name></author><author><name sortKey="Oakes, Sa" uniqKey="Oakes S">SA Oakes</name></author><author><name sortKey="Debnath, J" uniqKey="Debnath J">J Debnath</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fan, Qw" uniqKey="Fan Q">QW Fan</name></author><author><name sortKey="Weiss, Wa" uniqKey="Weiss W">WA Weiss</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Franken, Na" uniqKey="Franken N">NA Franken</name></author><author><name sortKey="Rodermond, Hm" uniqKey="Rodermond H">HM Rodermond</name></author><author><name sortKey="Stap, J" uniqKey="Stap J">J Stap</name></author><author><name sortKey="Haveman, J" uniqKey="Haveman J">J Haveman</name></author><author><name sortKey="Van Bree, C" uniqKey="Van Bree C">C van Bree</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Golden, Eb" uniqKey="Golden E">EB Golden</name></author><author><name sortKey="Cho, Hy" uniqKey="Cho H">HY Cho</name></author><author><name sortKey="Jahanian, A" uniqKey="Jahanian A">A Jahanian</name></author><author><name sortKey="Hofman, Fm" uniqKey="Hofman F">FM Hofman</name></author><author><name sortKey="Louie, Sg" uniqKey="Louie S">SG Louie</name></author><author><name sortKey="Schonthal, Ah" uniqKey="Schonthal A">AH Schonthal</name></author><author><name sortKey="Chen, Tc" uniqKey="Chen T">TC Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Golding, Se" uniqKey="Golding S">SE Golding</name></author><author><name sortKey="Morgan, Rn" uniqKey="Morgan R">RN Morgan</name></author><author><name sortKey="Adams, Br" uniqKey="Adams B">BR Adams</name></author><author><name sortKey="Hawkins, Aj" uniqKey="Hawkins A">AJ Hawkins</name></author><author><name sortKey="Povirk, Lf" uniqKey="Povirk L">LF Povirk</name></author><author><name sortKey="Valerie, K" uniqKey="Valerie K">K Valerie</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Han, W" uniqKey="Han W">W Han</name></author><author><name sortKey="Pan, H" uniqKey="Pan H">H Pan</name></author><author><name sortKey="Chen, Y" uniqKey="Chen Y">Y Chen</name></author><author><name sortKey="Sun, J" uniqKey="Sun J">J Sun</name></author><author><name sortKey="Wang, Y" uniqKey="Wang Y">Y Wang</name></author><author><name sortKey="Li, J" uniqKey="Li J">J Li</name></author><author><name sortKey="Ge, W" uniqKey="Ge W">W Ge</name></author><author><name sortKey="Feng, L" uniqKey="Feng L">L Feng</name></author><author><name sortKey="Lin, X" uniqKey="Lin X">X Lin</name></author><author><name sortKey="Wang, X" uniqKey="Wang X">X Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hanzelmann, S" uniqKey="Hanzelmann S">S Hanzelmann</name></author><author><name sortKey="Castelo, R" uniqKey="Castelo R">R Castelo</name></author><author><name sortKey="Guinney, J" uniqKey="Guinney J">J Guinney</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hegi, Me" uniqKey="Hegi M">ME Hegi</name></author><author><name sortKey="Diserens, Ac" uniqKey="Diserens A">AC Diserens</name></author><author><name sortKey="Gorlia, T" uniqKey="Gorlia T">T Gorlia</name></author><author><name sortKey="Hamou, Mf" uniqKey="Hamou M">MF Hamou</name></author><author><name sortKey="De Tribolet, N" uniqKey="De Tribolet N">N de Tribolet</name></author><author><name sortKey="Weller, M" uniqKey="Weller M">M Weller</name></author><author><name sortKey="Kros, Jm" uniqKey="Kros J">JM Kros</name></author><author><name sortKey="Hainfellner, Ja" uniqKey="Hainfellner J">JA Hainfellner</name></author><author><name sortKey="Mason, W" uniqKey="Mason W">W Mason</name></author><author><name sortKey="Mariani, L" uniqKey="Mariani L">L Mariani</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hombach Klonisch, S" uniqKey="Hombach Klonisch S">S Hombach-Klonisch</name></author><author><name sortKey="Mehrpour, M" uniqKey="Mehrpour M">M Mehrpour</name></author><author><name sortKey="Shojaei, S" uniqKey="Shojaei S">S Shojaei</name></author><author><name sortKey="Harlos, C" uniqKey="Harlos C">C Harlos</name></author><author><name sortKey="Pitz, M" uniqKey="Pitz M">M Pitz</name></author><author><name sortKey="Hamai, A" uniqKey="Hamai A">A Hamai</name></author><author><name sortKey="Siemianowicz, K" uniqKey="Siemianowicz K">K Siemianowicz</name></author><author><name sortKey="Likus, W" uniqKey="Likus W">W Likus</name></author><author><name sortKey="Wiechec, E" uniqKey="Wiechec E">E Wiechec</name></author><author><name sortKey="Toyota, Bd" uniqKey="Toyota B">BD Toyota</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hori, Ys" uniqKey="Hori Y">YS Hori</name></author><author><name sortKey="Hosoda, R" uniqKey="Hosoda R">R Hosoda</name></author><author><name sortKey="Akiyama, Y" uniqKey="Akiyama Y">Y Akiyama</name></author><author><name sortKey="Sebori, R" uniqKey="Sebori R">R Sebori</name></author><author><name sortKey="Wanibuchi, M" uniqKey="Wanibuchi M">M Wanibuchi</name></author><author><name sortKey="Mikami, T" uniqKey="Mikami T">T Mikami</name></author><author><name sortKey="Sugino, T" uniqKey="Sugino T">T Sugino</name></author><author><name sortKey="Suzuki, K" uniqKey="Suzuki K">K Suzuki</name></author><author><name sortKey="Maruyama, M" uniqKey="Maruyama M">M Maruyama</name></author><author><name sortKey="Tsukamoto, M" uniqKey="Tsukamoto M">M Tsukamoto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hu, Yl" uniqKey="Hu Y">YL Hu</name></author><author><name sortKey="Delay, M" uniqKey="Delay M">M DeLay</name></author><author><name sortKey="Jahangiri, A" uniqKey="Jahangiri A">A Jahangiri</name></author><author><name sortKey="Molinaro, Am" uniqKey="Molinaro A">AM Molinaro</name></author><author><name sortKey="Rose, Sd" uniqKey="Rose S">SD Rose</name></author><author><name sortKey="Carbonell, Ws" uniqKey="Carbonell W">WS Carbonell</name></author><author><name sortKey="Aghi, Mk" uniqKey="Aghi M">MK Aghi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jutten, B" uniqKey="Jutten B">B Jutten</name></author><author><name sortKey="Keulers, Tg" uniqKey="Keulers T">TG Keulers</name></author><author><name sortKey="Peeters, Hjm" uniqKey="Peeters H">HJM Peeters</name></author><author><name sortKey="Schaaf, Mbe" uniqKey="Schaaf M">MBE Schaaf</name></author><author><name sortKey="Savelkouls, Kgm" uniqKey="Savelkouls K">KGM Savelkouls</name></author><author><name sortKey="Compter, I" uniqKey="Compter I">I Compter</name></author><author><name sortKey="Clarijs, R" uniqKey="Clarijs R">R Clarijs</name></author><author><name sortKey="Schijns, O" uniqKey="Schijns O">O Schijns</name></author><author><name sortKey="Ackermans, L" uniqKey="Ackermans L">L Ackermans</name></author><author><name sortKey="Teernstra, Opm" uniqKey="Teernstra O">OPM Teernstra</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kanzawa, T" uniqKey="Kanzawa T">T Kanzawa</name></author><author><name sortKey="Germano, Im" uniqKey="Germano I">IM Germano</name></author><author><name sortKey="Komata, T" uniqKey="Komata T">T Komata</name></author><author><name sortKey="Ito, H" uniqKey="Ito H">H Ito</name></author><author><name sortKey="Kondo, Y" uniqKey="Kondo Y">Y Kondo</name></author><author><name sortKey="Kondo, S" uniqKey="Kondo S">S Kondo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kitange, Gj" uniqKey="Kitange G">GJ Kitange</name></author><author><name sortKey="Carlson, Bl" uniqKey="Carlson B">BL Carlson</name></author><author><name sortKey="Schroeder, Ma" uniqKey="Schroeder M">MA Schroeder</name></author><author><name sortKey="Grogan, Pt" uniqKey="Grogan P">PT Grogan</name></author><author><name sortKey="Lamont, Jd" uniqKey="Lamont J">JD Lamont</name></author><author><name sortKey="Decker, Pa" uniqKey="Decker P">PA Decker</name></author><author><name sortKey="Wu, W" uniqKey="Wu W">W Wu</name></author><author><name sortKey="James, Cd" uniqKey="James C">CD James</name></author><author><name sortKey="Sarkaria, Jn" uniqKey="Sarkaria J">JN Sarkaria</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kitange, Gj" uniqKey="Kitange G">GJ Kitange</name></author><author><name sortKey="Mladek, Ac" uniqKey="Mladek A">AC Mladek</name></author><author><name sortKey="Carlson, Bl" uniqKey="Carlson B">BL Carlson</name></author><author><name sortKey="Schroeder, Ma" uniqKey="Schroeder M">MA Schroeder</name></author><author><name sortKey="Pokorny, Jl" uniqKey="Pokorny J">JL Pokorny</name></author><author><name sortKey="Cen, L" uniqKey="Cen L">L Cen</name></author><author><name sortKey="Decker, Pa" uniqKey="Decker P">PA Decker</name></author><author><name sortKey="Wu, W" uniqKey="Wu W">W Wu</name></author><author><name sortKey="Lomberk, Ga" uniqKey="Lomberk G">GA Lomberk</name></author><author><name sortKey="Gupta, Sk" uniqKey="Gupta S">SK Gupta</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Koul, D" uniqKey="Koul D">D Koul</name></author><author><name sortKey="Shen, R" uniqKey="Shen R">R Shen</name></author><author><name sortKey="Kim, Yw" uniqKey="Kim Y">YW Kim</name></author><author><name sortKey="Kondo, Y" uniqKey="Kondo Y">Y Kondo</name></author><author><name sortKey="Lu, Y" uniqKey="Lu Y">Y Lu</name></author><author><name sortKey="Bankson, J" uniqKey="Bankson J">J Bankson</name></author><author><name sortKey="Ronen, Sm" uniqKey="Ronen S">SM Ronen</name></author><author><name sortKey="Kirkpatrick, Dl" uniqKey="Kirkpatrick D">DL Kirkpatrick</name></author><author><name sortKey="Powis, G" uniqKey="Powis G">G Powis</name></author><author><name sortKey="Yung, Wk" uniqKey="Yung W">WK Yung</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kuger, S" uniqKey="Kuger S">S Kuger</name></author><author><name sortKey="Corek, E" uniqKey="Corek E">E Corek</name></author><author><name sortKey="Polat, B" uniqKey="Polat B">B Polat</name></author><author><name sortKey="Kammerer, U" uniqKey="Kammerer U">U Kammerer</name></author><author><name sortKey="Flentje, M" uniqKey="Flentje M">M Flentje</name></author><author><name sortKey="Djuzenova, Cs" uniqKey="Djuzenova C">CS Djuzenova</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Learn, Ca" uniqKey="Learn C">CA Learn</name></author><author><name sortKey="Hartzell, Tl" uniqKey="Hartzell T">TL Hartzell</name></author><author><name sortKey="Wikstrand, Cj" uniqKey="Wikstrand C">CJ Wikstrand</name></author><author><name sortKey="Archer, Ge" uniqKey="Archer G">GE Archer</name></author><author><name sortKey="Rich, Jn" uniqKey="Rich J">JN Rich</name></author><author><name sortKey="Friedman, Ah" uniqKey="Friedman A">AH Friedman</name></author><author><name sortKey="Friedman, Hs" uniqKey="Friedman H">HS Friedman</name></author><author><name sortKey="Bigner, Dd" uniqKey="Bigner D">DD Bigner</name></author><author><name sortKey="Sampson, Jh" uniqKey="Sampson J">JH Sampson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lee, Sw" uniqKey="Lee S">SW Lee</name></author><author><name sortKey="Kim, Hk" uniqKey="Kim H">HK Kim</name></author><author><name sortKey="Lee, Nh" uniqKey="Lee N">NH Lee</name></author><author><name sortKey="Yi, Hy" uniqKey="Yi H">HY Yi</name></author><author><name sortKey="Kim, Hs" uniqKey="Kim H">HS Kim</name></author><author><name sortKey="Hong, Sh" uniqKey="Hong S">SH Hong</name></author><author><name sortKey="Hong, Yk" uniqKey="Hong Y">YK Hong</name></author><author><name sortKey="Joe, Ya" uniqKey="Joe Y">YA Joe</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Levine, B" uniqKey="Levine B">B Levine</name></author><author><name sortKey="Kroemer, G" uniqKey="Kroemer G">G Kroemer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Liberzon, A" uniqKey="Liberzon A">A Liberzon</name></author><author><name sortKey="Birger, C" uniqKey="Birger C">C Birger</name></author><author><name sortKey="Thorvaldsdottir, H" uniqKey="Thorvaldsdottir H">H Thorvaldsdottir</name></author><author><name sortKey="Ghandi, M" uniqKey="Ghandi M">M Ghandi</name></author><author><name sortKey="Mesirov, Jp" uniqKey="Mesirov J">JP Mesirov</name></author><author><name sortKey="Tamayo, P" uniqKey="Tamayo P">P Tamayo</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Mizushima, N" uniqKey="Mizushima N">N Mizushima</name></author><author><name sortKey="Yoshimori, T" uniqKey="Yoshimori T">T Yoshimori</name></author><author><name sortKey="Levine, B" uniqKey="Levine B">B Levine</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nazio, F" uniqKey="Nazio F">F Nazio</name></author><author><name sortKey="Strappazzon, F" uniqKey="Strappazzon F">F Strappazzon</name></author><author><name sortKey="Antonioli, M" uniqKey="Antonioli M">M Antonioli</name></author><author><name sortKey="Bielli, P" uniqKey="Bielli P">P Bielli</name></author><author><name sortKey="Cianfanelli, V" uniqKey="Cianfanelli V">V Cianfanelli</name></author><author><name sortKey="Bordi, M" uniqKey="Bordi M">M Bordi</name></author><author><name sortKey="Gretzmeier, C" uniqKey="Gretzmeier C">C Gretzmeier</name></author><author><name sortKey="Dengjel, J" uniqKey="Dengjel J">J Dengjel</name></author><author><name sortKey="Piacentini, M" uniqKey="Piacentini M">M Piacentini</name></author><author><name sortKey="Fimia, Gm" uniqKey="Fimia G">GM Fimia</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ogata, M" uniqKey="Ogata M">M Ogata</name></author><author><name sortKey="Hino, S" uniqKey="Hino S">S Hino</name></author><author><name sortKey="Saito, A" uniqKey="Saito A">A Saito</name></author><author><name sortKey="Morikawa, K" uniqKey="Morikawa K">K Morikawa</name></author><author><name sortKey="Kondo, S" uniqKey="Kondo S">S Kondo</name></author><author><name sortKey="Kanemoto, S" uniqKey="Kanemoto S">S Kanemoto</name></author><author><name sortKey="Murakami, T" uniqKey="Murakami T">T Murakami</name></author><author><name sortKey="Taniguchi, M" uniqKey="Taniguchi M">M Taniguchi</name></author><author><name sortKey="Tanii, I" uniqKey="Tanii I">I Tanii</name></author><author><name sortKey="Yoshinaga, K" uniqKey="Yoshinaga K">K Yoshinaga</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Paillas, S" uniqKey="Paillas S">S Paillas</name></author><author><name sortKey="Causse, A" uniqKey="Causse A">A Causse</name></author><author><name sortKey="Marzi, L" uniqKey="Marzi L">L Marzi</name></author><author><name sortKey="De Medina, P" uniqKey="De Medina P">P de Medina</name></author><author><name sortKey="Poirot, M" uniqKey="Poirot M">M Poirot</name></author><author><name sortKey="Denis, V" uniqKey="Denis V">V Denis</name></author><author><name sortKey="Vezzio Vie, N" uniqKey="Vezzio Vie N">N Vezzio-Vie</name></author><author><name sortKey="Espert, L" uniqKey="Espert L">L Espert</name></author><author><name sortKey="Arzouk, H" uniqKey="Arzouk H">H Arzouk</name></author><author><name sortKey="Coquelle, A" uniqKey="Coquelle A">A Coquelle</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Parsons, Dw" uniqKey="Parsons D">DW Parsons</name></author><author><name sortKey="Jones, S" uniqKey="Jones S">S Jones</name></author><author><name sortKey="Zhang, X" uniqKey="Zhang X">X Zhang</name></author><author><name sortKey="Lin, Jc" uniqKey="Lin J">JC Lin</name></author><author><name sortKey="Leary, Rj" uniqKey="Leary R">RJ Leary</name></author><author><name sortKey="Angenendt, P" uniqKey="Angenendt P">P Angenendt</name></author><author><name sortKey="Mankoo, P" uniqKey="Mankoo P">P Mankoo</name></author><author><name sortKey="Carter, H" uniqKey="Carter H">H Carter</name></author><author><name sortKey="Siu, Im" uniqKey="Siu I">IM Siu</name></author><author><name sortKey="Gallia, Gl" uniqKey="Gallia G">GL Gallia</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Patel, Ap" uniqKey="Patel A">AP Patel</name></author><author><name sortKey="Tirosh, I" uniqKey="Tirosh I">I Tirosh</name></author><author><name sortKey="Trombetta, Jj" uniqKey="Trombetta J">JJ Trombetta</name></author><author><name sortKey="Shalek, Ak" uniqKey="Shalek A">AK Shalek</name></author><author><name sortKey="Gillespie, Sm" uniqKey="Gillespie S">SM Gillespie</name></author><author><name sortKey="Wakimoto, H" uniqKey="Wakimoto H">H Wakimoto</name></author><author><name sortKey="Cahill, Dp" uniqKey="Cahill D">DP Cahill</name></author><author><name sortKey="Nahed, Bv" uniqKey="Nahed B">BV Nahed</name></author><author><name sortKey="Curry, Wt" uniqKey="Curry W">WT Curry</name></author><author><name sortKey="Martuza, Rl" uniqKey="Martuza R">RL Martuza</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pitz, Mw" uniqKey="Pitz M">MW Pitz</name></author><author><name sortKey="Eisenhauer, Ea" uniqKey="Eisenhauer E">EA Eisenhauer</name></author><author><name sortKey="Macneil, Mv" uniqKey="Macneil M">MV MacNeil</name></author><author><name sortKey="Thiessen, B" uniqKey="Thiessen B">B Thiessen</name></author><author><name sortKey="Easaw, Jc" uniqKey="Easaw J">JC Easaw</name></author><author><name sortKey="Macdonald, Dr" uniqKey="Macdonald D">DR Macdonald</name></author><author><name sortKey="Eisenstat, Dd" uniqKey="Eisenstat D">DD Eisenstat</name></author><author><name sortKey="Kakumanu, As" uniqKey="Kakumanu A">AS Kakumanu</name></author><author><name sortKey="Salim, M" uniqKey="Salim M">M Salim</name></author><author><name sortKey="Chalchal, H" uniqKey="Chalchal H">H Chalchal</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Prados, Md" uniqKey="Prados M">MD Prados</name></author><author><name sortKey="Byron, Sa" uniqKey="Byron S">SA Byron</name></author><author><name sortKey="Tran, Nl" uniqKey="Tran N">NL Tran</name></author><author><name sortKey="Phillips, Jj" uniqKey="Phillips J">JJ Phillips</name></author><author><name sortKey="Molinaro, Am" uniqKey="Molinaro A">AM Molinaro</name></author><author><name sortKey="Ligon, Kl" uniqKey="Ligon K">KL Ligon</name></author><author><name sortKey="Wen, Py" uniqKey="Wen P">PY Wen</name></author><author><name sortKey="Kuhn, Jg" uniqKey="Kuhn J">JG Kuhn</name></author><author><name sortKey="Mellinghoff, Ik" uniqKey="Mellinghoff I">IK Mellinghoff</name></author><author><name sortKey="De Groot, Jf" uniqKey="De Groot J">JF de Groot</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Roos, A" uniqKey="Roos A">A Roos</name></author><author><name sortKey="Dhruv, Hd" uniqKey="Dhruv H">HD Dhruv</name></author><author><name sortKey="Peng, S" uniqKey="Peng S">S Peng</name></author><author><name sortKey="Inge, Lj" uniqKey="Inge L">LJ Inge</name></author><author><name sortKey="Tuncali, S" uniqKey="Tuncali S">S Tuncali</name></author><author><name sortKey="Pineda, M" uniqKey="Pineda M">M Pineda</name></author><author><name sortKey="Millard, N" uniqKey="Millard N">N Millard</name></author><author><name sortKey="Mayo, Z" uniqKey="Mayo Z">Z Mayo</name></author><author><name sortKey="Eschbacher, Jm" uniqKey="Eschbacher J">JM Eschbacher</name></author><author><name sortKey="Loftus, Jc" uniqKey="Loftus J">JC Loftus</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rosenfeld, Mr" uniqKey="Rosenfeld M">MR Rosenfeld</name></author><author><name sortKey="Ye, X" uniqKey="Ye X">X Ye</name></author><author><name sortKey="Supko, Jg" uniqKey="Supko J">JG Supko</name></author><author><name sortKey="Desideri, S" uniqKey="Desideri S">S Desideri</name></author><author><name sortKey="Grossman, Sa" uniqKey="Grossman S">SA Grossman</name></author><author><name sortKey="Brem, S" uniqKey="Brem S">S Brem</name></author><author><name sortKey="Mikkelson, T" uniqKey="Mikkelson T">T Mikkelson</name></author><author><name sortKey="Wang, D" uniqKey="Wang D">D Wang</name></author><author><name sortKey="Chang, Yc" uniqKey="Chang Y">YC Chang</name></author><author><name sortKey="Hu, J" uniqKey="Hu J">J Hu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Roy, S" uniqKey="Roy S">S Roy</name></author><author><name sortKey="Lahiri, D" uniqKey="Lahiri D">D Lahiri</name></author><author><name sortKey="Maji, T" uniqKey="Maji T">T Maji</name></author><author><name sortKey="Biswas, J" uniqKey="Biswas J">J Biswas</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Russell, Rc" uniqKey="Russell R">RC Russell</name></author><author><name sortKey="Tian, Y" uniqKey="Tian Y">Y Tian</name></author><author><name sortKey="Yuan, H" uniqKey="Yuan H">H Yuan</name></author><author><name sortKey="Park, Hw" uniqKey="Park H">HW Park</name></author><author><name sortKey="Chang, Yy" uniqKey="Chang Y">YY Chang</name></author><author><name sortKey="Kim, J" uniqKey="Kim J">J Kim</name></author><author><name sortKey="Kim, H" uniqKey="Kim H">H Kim</name></author><author><name sortKey="Neufeld, Tp" uniqKey="Neufeld T">TP Neufeld</name></author><author><name sortKey="Dillin, A" uniqKey="Dillin A">A Dillin</name></author><author><name sortKey="Guan, Kl" uniqKey="Guan K">KL Guan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Siegel, Rl" uniqKey="Siegel R">RL Siegel</name></author><author><name sortKey="Miller, Kd" uniqKey="Miller K">KD Miller</name></author><author><name sortKey="Jemal, A" uniqKey="Jemal A">A Jemal</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sottoriva, A" uniqKey="Sottoriva A">A Sottoriva</name></author><author><name sortKey="Spiteri, I" uniqKey="Spiteri I">I Spiteri</name></author><author><name sortKey="Piccirillo, Sg" uniqKey="Piccirillo S">SG Piccirillo</name></author><author><name sortKey="Touloumis, A" uniqKey="Touloumis A">A Touloumis</name></author><author><name sortKey="Collins, Vp" uniqKey="Collins V">VP Collins</name></author><author><name sortKey="Marioni, Jc" uniqKey="Marioni J">JC Marioni</name></author><author><name sortKey="Curtis, C" uniqKey="Curtis C">C Curtis</name></author><author><name sortKey="Watts, C" uniqKey="Watts C">C Watts</name></author><author><name sortKey="Tavare, S" uniqKey="Tavare S">S Tavare</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Stjepanovic, G" uniqKey="Stjepanovic G">G Stjepanovic</name></author><author><name sortKey="Baskaran, S" uniqKey="Baskaran S">S Baskaran</name></author><author><name sortKey="Lin, Mg" uniqKey="Lin M">MG Lin</name></author><author><name sortKey="Hurley, Jh" uniqKey="Hurley J">JH Hurley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Stupp, R" uniqKey="Stupp R">R Stupp</name></author><author><name sortKey="Mason, Wp" uniqKey="Mason W">WP Mason</name></author><author><name sortKey="Van Den Bent, Mj" uniqKey="Van Den Bent M">MJ van den Bent</name></author><author><name sortKey="Weller, M" uniqKey="Weller M">M Weller</name></author><author><name sortKey="Fisher, B" uniqKey="Fisher B">B Fisher</name></author><author><name sortKey="Taphoorn, Mj" uniqKey="Taphoorn M">MJ Taphoorn</name></author><author><name sortKey="Belanger, K" uniqKey="Belanger K">K Belanger</name></author><author><name sortKey="Brandes, Aa" uniqKey="Brandes A">AA Brandes</name></author><author><name sortKey="Marosi, C" uniqKey="Marosi C">C Marosi</name></author><author><name sortKey="Bogdahn, U" uniqKey="Bogdahn U">U Bogdahn</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Thomas, Aa" uniqKey="Thomas A">AA Thomas</name></author><author><name sortKey="Brennan, Cw" uniqKey="Brennan C">CW Brennan</name></author><author><name sortKey="Deangelis, Lm" uniqKey="Deangelis L">LM DeAngelis</name></author><author><name sortKey="Omuro, Am" uniqKey="Omuro A">AM Omuro</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Tellingen, O" uniqKey="Van Tellingen O">O van Tellingen</name></author><author><name sortKey="Yetkin Arik, B" uniqKey="Yetkin Arik B">B Yetkin-Arik</name></author><author><name sortKey="De Gooijer, Mc" uniqKey="De Gooijer M">MC de Gooijer</name></author><author><name sortKey="Wesseling, P" uniqKey="Wesseling P">P Wesseling</name></author><author><name sortKey="Wurdinger, T" uniqKey="Wurdinger T">T Wurdinger</name></author><author><name sortKey="De Vries, He" uniqKey="De Vries H">HE de Vries</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="White, E" uniqKey="White E">E White</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wurstle, S" uniqKey="Wurstle S">S Wurstle</name></author><author><name sortKey="Schneider, F" uniqKey="Schneider F">F Schneider</name></author><author><name sortKey="Ringel, F" uniqKey="Ringel F">F Ringel</name></author><author><name sortKey="Gempt, J" uniqKey="Gempt J">J Gempt</name></author><author><name sortKey="Lammer, F" uniqKey="Lammer F">F Lammer</name></author><author><name sortKey="Delbridge, C" uniqKey="Delbridge C">C Delbridge</name></author><author><name sortKey="Wu, W" uniqKey="Wu W">W Wu</name></author><author><name sortKey="Schlegel, J" uniqKey="Schlegel J">J Schlegel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Xie, Q" uniqKey="Xie Q">Q Xie</name></author><author><name sortKey="Mittal, S" uniqKey="Mittal S">S Mittal</name></author><author><name sortKey="Berens, Me" uniqKey="Berens M">ME Berens</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yin, Z" uniqKey="Yin Z">Z Yin</name></author><author><name sortKey="Pascual, C" uniqKey="Pascual C">C Pascual</name></author><author><name sortKey="Klionsky, Dj" uniqKey="Klionsky D">DJ Klionsky</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yu, X" uniqKey="Yu X">X Yu</name></author><author><name sortKey="Long, Yc" uniqKey="Long Y">YC Long</name></author><author><name sortKey="Shen, Hm" uniqKey="Shen H">HM Shen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhao, Hf" uniqKey="Zhao H">HF Zhao</name></author><author><name sortKey="Wang, J" uniqKey="Wang J">J Wang</name></author><author><name sortKey="Shao, W" uniqKey="Shao W">W Shao</name></author><author><name sortKey="Wu, Cp" uniqKey="Wu C">CP Wu</name></author><author><name sortKey="Chen, Zp" uniqKey="Chen Z">ZP Chen</name></author><author><name sortKey="To, St" uniqKey="To S">ST To</name></author><author><name sortKey="Li, Wp" uniqKey="Li W">WP Li</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zou, Z" uniqKey="Zou Z">Z Zou</name></author><author><name sortKey="Yuan, Z" uniqKey="Yuan Z">Z Yuan</name></author><author><name sortKey="Zhang, Q" uniqKey="Zhang Q">Q Zhang</name></author><author><name sortKey="Long, Z" uniqKey="Long Z">Z Long</name></author><author><name sortKey="Chen, J" uniqKey="Chen J">J Chen</name></author><author><name sortKey="Tang, Z" uniqKey="Tang Z">Z Tang</name></author><author><name sortKey="Zhu, Y" uniqKey="Zhu Y">Y Zhu</name></author><author><name sortKey="Chen, S" uniqKey="Chen S">S Chen</name></author><author><name sortKey="Xu, J" uniqKey="Xu J">J Xu</name></author><author><name sortKey="Yan, M" uniqKey="Yan M">M Yan</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Mol Med</journal-id><journal-id journal-id-type="iso-abbrev">Mol. Med</journal-id><journal-title-group><journal-title>Molecular Medicine</journal-title></journal-title-group><issn pub-type="ppub">1076-1551</issn><issn pub-type="epub">1528-3658</issn><publisher><publisher-name>BioMed Central</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31726966</article-id><article-id pub-id-type="pmc">6854621</article-id><article-id pub-id-type="publisher-id">116</article-id><article-id pub-id-type="doi">10.1186/s10020-019-0116-z</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Inhibition of phosphatidylinositol 3-kinase by PX-866 suppresses temozolomide-induced autophagy and promotes apoptosis in glioblastoma cells</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Harder</surname><given-names>Bryan G.</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Peng</surname><given-names>Sen</given-names></name><xref ref-type="aff" rid="Aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Sereduk</surname><given-names>Christopher P.</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Sodoma</surname><given-names>Andrej M.</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kitange</surname><given-names>Gaspar J.</given-names></name><xref ref-type="aff" rid="Aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Loftus</surname><given-names>Joseph C.</given-names></name><xref ref-type="aff" rid="Aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Sarkaria</surname><given-names>Jann N.</given-names></name><xref ref-type="aff" rid="Aff3">3</xref></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0003-0908-9964</contrib-id><name><surname>Tran</surname><given-names>Nhan L.</given-names></name><address><phone>480-301-4462</phone><email>Tran.Nhan@mayo.edu</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><aff id="Aff1"><label>1</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0000 8875 6339</institution-id><institution-id institution-id-type="GRID">grid.417468.8</institution-id><institution>Departments of Cancer Biology and Neurosurgery,</institution><institution>Mayo Clinic Arizona,</institution></institution-wrap>13400 E. Shea Blvd, MCCRB 03-055, Scottsdale, AZ 85259 USA</aff><aff id="Aff2"><label>2</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0507 3225</institution-id><institution-id institution-id-type="GRID">grid.250942.8</institution-id><institution>Cancer and Cell Biology Division,</institution><institution>Translational Genomics Research Institute,</institution></institution-wrap>Phoenix, AZ USA</aff><aff id="Aff3"><label>3</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0459 167X</institution-id><institution-id institution-id-type="GRID">grid.66875.3a</institution-id><institution>Department of Radiation Oncology,</institution><institution>Mayo Clinic,</institution></institution-wrap>Rochester, MN USA</aff><aff id="Aff4"><label>4</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0000 8875 6339</institution-id><institution-id institution-id-type="GRID">grid.417468.8</institution-id><institution>Department of Biochemistry and Molecular Biology,</institution><institution>Mayo Clinic Arizona,</institution></institution-wrap>Scottsdale, AZ USA</aff></contrib-group><pub-date pub-type="epub"><day>14</day><month>11</month><year>2019</year></pub-date><pub-date pub-type="pmc-release"><day>14</day><month>11</month><year>2019</year></pub-date><pub-date pub-type="collection"><year>2019</year></pub-date><volume>25</volume><elocation-id>49</elocation-id><history><date date-type="received"><day>21</day><month>8</month><year>2019</year></date><date date-type="accepted"><day>18</day><month>10</month><year>2019</year></date></history><permissions><copyright-statement>© The Author(s) 2019</copyright-statement><license license-type="OpenAccess"><license-p><bold>Open Access</bold>This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/publicdomain/zero/1.0/">http://creativecommons.org/publicdomain/zero/1.0/</ext-link>) applies to the data made available in this article, unless otherwise stated.</license-p></license></permissions><abstract id="Abs1"><sec><title>Background</title><p id="Par1">Temozolomide (TMZ) is the most commonly used chemotherapeutic agent used to treat glioblastoma (GBM), which causes significant DNA damage to highly proliferative cells. Our observations have added to accumulating evidence that TMZ induces stress-responsive cellular programs known to promote cell survival, including autophagy. As such, targeting these survival pathways may represent new vulnerabilities of GBM after treatment with TMZ.</p></sec><sec><title>Methods</title><p id="Par2">Using the T98G human glioma cell line, we assessed the molecular signaling associated with TMZ treatment, the cellular consequences of using the pan-PI3K inhibitor PX-866, and performed clonogenic assays to determine the effect sequential treatment of TMZ and PX-866 had on colony formation. Additionally, we also use subcutaneous GBM patient derived xenograft (PDX) tumors to show relative LC3 protein expression and correlations between survival pathways and molecular markers which dictate clinical responsiveness to TMZ.</p></sec><sec><title>Results</title><p id="Par3">Here, we report that TMZ can induce autophagic flux in T98G glioma cells. GBM patient-derived xenograft (PDX) tumors treated with TMZ also display an increase in the autophagosome marker LC3 II. Additionally, O<sup>6</sup>-methylguanine-DNA-methyltransferase (MGMT) expression correlates with PI3K/AKT activity, suggesting that patients with inherent resistance to TMZ (MGMT-high) would benefit from PI3K/AKT inhibitors in addition to TMZ. Accordingly, we have identified that the blood-brain barrier (BBB) penetrant pan-PI3K inhibitor, PX-866, is an early-stage inhibitor of autophagic flux, while maintaining its ability to inhibit PI3K/AKT signaling in glioma cells. Lastly, due to the induction of autophagic flux by TMZ, we provide evidence for sequential treatment of TMZ followed by PX-866, rather than combined co-treatment, as a means to shut down autophagy-induced survival in GBM cells and to enhance apoptosis.</p></sec><sec><title>Conclusions</title><p id="Par4">The understanding of how TMZ induces survival pathways, such as autophagy, may offer new therapeutic vulnerabilities and opportunities to use sequential inhibition of alternate pro-survival pathways that regulate autophagy. As such, identification of additional ways to inhibit TMZ-induced autophagy could enhance the efficacy of TMZ.</p></sec></abstract><kwd-group xml:lang="en"><title>Keywords</title><kwd>Glioblastoma (GBM)</kwd><kwd>Autophagy</kwd><kwd>Temozolomide (TMZ)</kwd><kwd>PX-866</kwd><kwd>PI3K</kwd><kwd>Bafilomycin</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>R01 NS086853</award-id><award-id>U01 CA220378-01</award-id><award-id>U54 CA210180</award-id></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2019</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1"><title>Introduction</title><p id="Par21">Glioblastoma (GBM) is the most common and deadly primary adult brain tumor in adults with a median survival time of about 15 months from diagnosis (Thomas et al. <xref ref-type="bibr" rid="CR50">2014</xref>; Siegel et al. <xref ref-type="bibr" rid="CR46">2018</xref>). Numerous factors such tumor heterogeneity, invasiveness, and the blood-brain barrier (BBB) make treating this disease extremely challenging without compromising healthy brain tissue (Sottoriva et al. <xref ref-type="bibr" rid="CR47">2013</xref>; Patel et al. <xref ref-type="bibr" rid="CR39">2014</xref>; Xie et al. <xref ref-type="bibr" rid="CR54">2014</xref>; van Tellingen et al. <xref ref-type="bibr" rid="CR51">2015</xref>). Currently, investigations are underway to identify new therapeutics or novel ways to deliver drugs to the tumor site, but there have been minimal advancements since the implementation of the Stupp protocol in 2005, which only provides a moderate benefit on overall survival (Stupp et al. <xref ref-type="bibr" rid="CR49">2005</xref>). Therefore, an emphasis has been placed on identifying mechanisms to enhance the efficacy of temozolomide (TMZ), an orally administered alkylating agent, or radiation therapy (RT) to prolong patient survival.</p><p id="Par22">Cells at the rim of a GBM are highly invasive and migratory which prevents complete surgical resection of tumors prior to standard therapy (Xie et al. <xref ref-type="bibr" rid="CR54">2014</xref>). The remaining cells are then targeted with RT and concomitant TMZ regimens. Both RT and TMZ are highly effective DNA-damaging agents, but recent work has shown that the remaining migratory cells display a certain degree of resistance to these therapies. Ultimately, the cells that survive this treatment regimen form a recurrent tumor. Currently, there are no effective treatment options for recurrent GBM that prolong overall survival and patients typically succumb to the disease after approximately 8 months (Roy et al. <xref ref-type="bibr" rid="CR44">2015</xref>).</p><p id="Par23">Emerging evidence has begun to shed light on additional consequences of TMZ treatment. Notably, TMZ-induced methylation of DNA induces the DNA-damage response (DDR), which has recently been implicated to be associated with the induction of cell stress pathways such as macroautophagy (henceforth ‘autophagy’) (Hombach-Klonisch et al. <xref ref-type="bibr" rid="CR20">2018</xref>). Autophagy is a major catabolic process that is responsible for degrading cellular cargo too large or abundant for the proteasome (Levine and Kroemer <xref ref-type="bibr" rid="CR31">2008</xref>; Yin et al. <xref ref-type="bibr" rid="CR55">2016</xref>). Cargo targeted for degradation, such as misfolded proteins, protein aggregates, and damaged organelles are sequestered into forming autophagosome structures. These double-membrane autophagosomes containing cargo then fuse with lysosomes to create autolysosomes, where degradation takes place. After degradation of the internal components and completion of autophagic flux, the autolysosome breaks down and the resulting cellular constituents are released into the cytosol to be recycled by anabolic processes.</p><p id="Par24">Cancer cells from multiple tumor types have been shown to have high basal autophagy, which is used as a cell-survival mechanism (White <xref ref-type="bibr" rid="CR52">2015</xref>; Degenhardt et al. <xref ref-type="bibr" rid="CR8">2006</xref>; Hu et al. <xref ref-type="bibr" rid="CR22">2012</xref>). When cells are deprived of nutrients, growth factor-induced signaling from receptor tyrosine kinases in the membrane is shut off. The PI3K/AKT/mTOR axis is a central signaling pathway that is activated as a result of growth factor stimulation and activation of class I PI3K functions as a negative regulator of autophagy (Nazio et al. <xref ref-type="bibr" rid="CR35">2013</xref>). When the PI3K/AKT/mTOR axis is not active, there is release of the inhibition on autophagic formation and autophagosome biogenesis begins through a signaling cascade that begins with the ULK1 complex (Russell et al. <xref ref-type="bibr" rid="CR45">2013</xref>). As a result of limited nutrients, autophagy is initiated, thus providing a growth advantage to tumor cells. However, there are some reports that showed that the class III PI3K Vps34(PIK3C3) forms a protein complex with BECN1 and PIK3R4 to generate phosphatidylinositol 3-phosphate, which is required for the initiation and progression of autophagy (Yu et al. <xref ref-type="bibr" rid="CR56">2015</xref>). In general, dual inhibitors of PI3K/mTOR are considered autophagy activators as both class I PI3K and mTOR (both of which suppresses autophagy) are simultaneously inhibited (Kuger et al. <xref ref-type="bibr" rid="CR28">2014</xref>). However, there is evidence that some of these dual inhibitors such as dactolisib and PI-103 can suppress autophagic flux in cancer cells (Button et al. <xref ref-type="bibr" rid="CR5">2016</xref>) via inhibition of class III PI3K. Moreover, autophagy is also a stress-responsive pathway, and cellular insult from chemotherapy also has shown to activate this process (Han et al. <xref ref-type="bibr" rid="CR17">2011</xref>; Ding et al. <xref ref-type="bibr" rid="CR10">2011</xref>; Paillas et al. <xref ref-type="bibr" rid="CR37">2012</xref>; Zou et al. <xref ref-type="bibr" rid="CR58">2012</xref>).</p><p id="Par25">Previous studies have identified that the PI3K/AKT/mTOR axis is overactive in GBM and has been linked with enhanced proliferation, chemoresistance, and suppressed apoptosis (Parsons et al. <xref ref-type="bibr" rid="CR38">2008</xref>; Cheng et al. <xref ref-type="bibr" rid="CR7">2009</xref>). As a result, PI3K inhibitors have shown promising pre-clinical potential against GBM, however, none have achieved matriculation into the clinic (Zhao et al. <xref ref-type="bibr" rid="CR57">2017</xref>). Moreover, identification of compounds that can sensitize glioma cells to the effects of TMZ would be of major interest in the clinic. Here, we provide evidence that TMZ induces autophagic flux, and identify PX-866, an analog of wortmannin, as an early-stage blocker of autophagosome formation. Given these findings, we argue for the combined use of PX-866 to block TMZ-induced autophagy and augment GBM cell death.</p></sec><sec id="Sec2"><title>Materials and methods</title><sec id="Sec3"><title>Cell culture</title><p id="Par26">Human T98G glioma cells were purchased from the American Type Culture Collection (ATCC) and were maintained in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). U373vIII cells were maintained in DMEM supplemented with 10% tetracycline-free FBS and have been described previously (Roos et al. <xref ref-type="bibr" rid="CR42">2018</xref>). All cells were grown at 37 °C with 5% CO<sub>2</sub>.</p></sec><sec id="Sec4"><title>Generation of TMZ-resistant GBM PDX tumors</title><p id="Par27">Parental GBM PDX lines were derived from either primary or recurrent tumor resections and have been described previously (Kitange et al. <xref ref-type="bibr" rid="CR26">2012</xref>). A comprehensive description of these parental GBM PDX models can be found at the (Mayo Clinic Brain PDX National Resource Portal <xref ref-type="bibr" rid="CR33">n.d.</xref>). The TMZ-resistant lines were generated by treating subcutaneous parental GBM xenografts with escalating doses of TMZ (20 mg/kg/day X 3 days and then 66 mg/kg/day X 3 days after initial tumor re-growth). The resulting TMZ-resistant lines were completely resistant to a challenged 120 mg/kg/day X 5 days as described previously (Kitange et al. <xref ref-type="bibr" rid="CR26">2012</xref>).</p></sec><sec id="Sec5"><title>Western blot analysis</title><p id="Par28">For experiments involving western blot analysis, cells were seeded in 6-well plates and left to adhere overnight. Cells were then treated with the indicated drugs and corresponding vehicle as a control for the appropriate time points. At the time of lysis, cells were washed once with ice-cold PBS containing protease and phosphatase inhibitor (Thermo scientific) cocktails. Cells were then lysed with 1x radioimmunoprecipitation assay (RIPA) buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were sonicated, and the total protein concentration was determined using the BCA assay kit, using bovine serum albumin as a standard. Equal protein amounts were loaded per well and resolved using SDS-PAGE. Protein was transferred to a nitrocellulose membrane using the Biorad fast transfer system, followed by blocking with Odyssey Blocking Buffer (LI-COR Biosciences) for 1 hour. The Odyssey CLx Near-Infrared (NIR) Western Blot Detection System (LI-COR Biosciences) was used to detect protein bands. Densitometry analysis was performed using the Image Studio Lite Version 5.2 software.</p></sec><sec id="Sec6"><title>Antibodies and reagents</title><p id="Par29">The following primary antibodies purchased from Cell Signaling Technology (CST) include: PARP (9542), MGMT (2739), phospho-AKT (ser473) (4060), AKT (2920), and GAPDH (5174). The antibody against LC3 was purchased from Sigma (L7543), the antibody against p62 was purchased from Abnova [(M01), clone 2C11], and the antibody against α-Tubulin was purchased from Millipore. Primary antibodies were diluted in Odyssey Blocking Buffer with 0.2% Tween 20 and incubated overnight with gentle shaking at 4 °C. IRDye 800CW secondary antibodies (LI-COR Biosciences) were also diluted in Odyssey Blocking Buffer with 0.2% Tween 20 and incubated for 1 h at room temp with gentle shaking.</p></sec><sec id="Sec7"><title>Analysis of correlation between MGMT methylation status and PI3K-AKT activity</title><p id="Par30">Patients included in this analysis were limited to the provisional TCGA microarray dataset with MGMT clinical status availability. Primary GBM were selected by filtering the “Sample Type” column in the patient clinical information table. Among the GBM microarray dataset, 190 MGMT unmethylated and 156 MGMT methylated samples were identified. Gene expression data is presented as normalized z-scores, which reflect the number of standard deviations away from the mean expression in the reference population. Gene set variation analysis (GSVA) was then performed to determine gene set enrichment scores of PI3K-AKT signaling pathway (Hanzelmann et al. <xref ref-type="bibr" rid="CR18">2013</xref>). The PI3K-AKT gene set was downloaded from Molecular Signatures Database (MSigDB) and modified to include additional canonical targets (Liberzon et al. <xref ref-type="bibr" rid="CR32">2015</xref>; Catasus et al. <xref ref-type="bibr" rid="CR6">2010</xref>). Log transformation was applied and default parameter settings were followed for gene expression data in GSVA. Student’s t-test was used to test the statistical significance of pathway activity difference between MGMT unmethylated and methylated groups. All figures were generated and statistical tests were performed using R 3.5.0 software. <italic>P</italic> values < 0.05 were considered significant.</p></sec><sec id="Sec8"><title>Assessment of cell viability</title><p id="Par31">In order to determine the apoptotic state of cells after treatments involving TMZ, cleaved PARP was used as a marker. After completed treatment times, floating cells were collected from the media and adherent cells were harvested as mentioned above. Lysates from each treatment were combined and prepared for western analysis. Total cell lysate was resolved via SDS-PAGE and membranes were probed with a PARP antibody (CST) that recognizes both total and cleaved PARP.</p></sec><sec id="Sec9"><title>Colony formation assay</title><p id="Par32">A colony formation (clonogenic) assay was used in order to determine cell survival after TMZ treatment either alone, or in combination with other treatments (Franken et al. <xref ref-type="bibr" rid="CR14">2006</xref>). Briefly, T98G or U373vIII cells were treated for 24 h with the indicated treatments. Remaining cells were trypsinized and plated in-triplicate into 6-well plates at a density of 1500 cells per well. Colonies were allowed to grow (approximately 9–10 days) before fixation with a 6% (v/v) glutaraldehyde (Sigma-Aldrich) solution. Fixed cells were then stained with a 0.5% (v/v) crystal violet (Sigma-Aldrich) solution and washed 3 times with de-ionized water. Only colonies consisting of ≥50 cells were counted. Statistical significance was determined using the Student’s <italic>t</italic> test and <italic>P</italic> values of < 0.05 were considered to be statistically significant.</p></sec></sec><sec id="Sec10"><title>Results</title><sec id="Sec11"><title>Temozolomide induces autophagic flux in glioma cells</title><p id="Par33">Previous research has shown that treatment with TMZ has an effect on autophagy in various GBM cell lines. Our study used the T98G cell line to confirm the effect of TMZ on autophagy. Forty-eight hours treatment with TMZ induced a dose-dependent increase in LC3 II protein expression indicating that autophagy is being activated. To verify autophagy flux is induced by TMZ, we also measured the protein level of p62, a receptor for cargo destined to be degraded by autophagy, and itself is degraded upon autophagy induction. Upon TMZ treatment, p62 protein levels also degraded accordingly (Fig. <xref rid="Fig1" ref-type="fig">1</xref>a). To further confirm that the effect on autophagy is an induction rather than an inhibition of basal autophagy, the LC3 flux (turnover) assay was performed as previously reported (Mizushima et al. <xref ref-type="bibr" rid="CR34">2010</xref>). T98G cells were treated with vehicle or TMZ (500 μM) for 48 h, with or without the late-stage autophagy inhibitor Bafilomycin (Baf, 10 nM). Cells treated with TMZ and Baf showed a higher expression of LC3 II compared to the LC3 II expression of Baf treatment alone, confirming the production of autophagosomes by TMZ over the course of 48 h (Fig. <xref rid="Fig1" ref-type="fig">1</xref>b). Moreover, p62 levels decrease when treated with TMZ alone, but accumulate when treated with Baf or Baf plus TMZ, confirming that p62 is not being degraded because of an inhibition of autophagy completion.
<fig id="Fig1"><label>Fig. 1</label><caption><p>Temozolomide induces autophagic flux in glioma cells. <bold>a</bold> T98G cells were treated with increasing doses of TMZ for 48 h and only living (attached) cells were collected for analysis. Western blotting shows an increase in LC3 II protein levels and a decrease in p62 protein levels. GAPDH was used as a loading control. <bold>b</bold> The LC3 flux assay was performed on T98G cells. T98G cells were treated with vehicle or TMZ (500 μM) for 48 h containing either DMSO or Baf (10 nM) and only living (adherent) cells were collected. Western blotting for LC3 indicates an increase in LC3 II for the TMZ + Baf lane compared to Baf alone, proving TMZ-induced autophagosome formation. The data (LC3 II/GAPDH) was quantified and graphed to mark this increase. Data represents three independent experiments</p></caption><graphic xlink:href="10020_2019_116_Fig1_HTML" id="MO1"></graphic></fig></p></sec><sec id="Sec12"><title>TMZ-resistant GBM-PDX tumors display increased LC3 II expression</title><p id="Par34">Next, we sought to assess the effect of TMZ on autophagy in vivo by utilizing subcutaneous GBM-PDX tumor samples from mice that received either vehicle or a dose-escalating regimen of TMZ. Lysate was created from tumors harvested from animals and western analysis was performed. Relative autophagosome number was determined by LC3 II expression between the parental TMZ-sensitive (P) and TMZ-resistant (T) groups for both primary (Fig. <xref rid="Fig2" ref-type="fig">2</xref>a) and recurrent (Fig. <xref rid="Fig2" ref-type="fig">2</xref>b) GBM-PDX tumor samples. In multiple GBM-PDX models, the TMZ-treated groups showed to have higher LC3-II expression compared to the parental groups, potentially indicating increased autophagosome number. Densitometry analysis revealed 6 out of 9 TMZ-treated tumors displayed a steady-state of 1.5-fold or higher increase in LC3 II expression (Fig. <xref rid="Fig2" ref-type="fig">2</xref>c).
<fig id="Fig2"><label>Fig. 2</label><caption><p>TMZ-resistant GBM-PDX tumors display increased LC3 II expression. Parental TMZ-sensitive (P) and TMZ-resistant (T) GBM-PDX subcutaneous tumors that were derived from either (<bold>a</bold>) primary tumor resections or (<bold>b</bold>) recurrent tumor resections were harvested and lysed for western blot analysis. LC3 protein levels were examined by western blotting and α-tubulin was used as a loading control. <bold>c</bold> Densitometry was used to quantify the western blot results</p></caption><graphic xlink:href="10020_2019_116_Fig2_HTML" id="MO2"></graphic></fig></p></sec><sec id="Sec13"><title>MGMT expression correlates with PI3K-AKT pathway activation</title><p id="Par35">O<sup>6</sup>-methylguanine-methyltransferase (MGMT) is an important prognostic marker for patients with GBM (Hegi et al. <xref ref-type="bibr" rid="CR19">2005</xref>; Kitange et al. <xref ref-type="bibr" rid="CR25">2009</xref>). Because patients with high expression (low promoter methylation) of MGMT show resistance to TMZ, we examined if there was a correlation between MGMT expression and other pro-survival pathways in GBM clinical samples. Western blotting of GBM-PDX tumor lysate reveals differential expression of MGMT between tumor samples, which correlated with the activation of the PI3K-AKT axis across all primary-derived GBM-PDX tumor specimens, but not for recurrent GBM-PDX (GBM10) (Fig. <xref rid="Fig3" ref-type="fig">3</xref>a). Relative AKT activation and MGMT expression was correlated based on the GBM5 sample, which shows low p-AKT and low MGMT expression (Fig. <xref rid="Fig3" ref-type="fig">3</xref>b). We next characterized this correlation further by exploring the TCGA GBM cohort of large-scale patient samples with genomic and clinical profiles. Patient samples with available <italic>MGMT</italic> promoter methylation status and gene expression data were used to query PI3K-AKT pathway activity. Primary GBM patients with unmethylated <italic>MGMT</italic> have statistically significant higher PI3K-AKT activity compared to <italic>MGMT</italic> methylated cases (Fig. <xref rid="Fig3" ref-type="fig">3</xref>c).
<fig id="Fig3"><label>Fig. 3</label><caption><p>MGMT expression correlates with PI3K-AKT pathway activation. <bold>a</bold> Total protein from parental GBM-PDX subcutaneous tumor lysates was used to assess the differential expression of MGMT and relative activation of p-AKT between the samples. <bold>b</bold> Densitometry was used to quantify the western blot results and all PDX tumors were normalized to GBM5. <bold>c</bold> Gene set variation analysis (GSVA) was used to determine gene set enrichment scores of the PI3K-AKT signaling pathway according to the methylation status of the <italic>MGMT</italic> promoter in clinical samples</p></caption><graphic xlink:href="10020_2019_116_Fig3_HTML" id="MO3"></graphic></fig></p></sec><sec id="Sec14"><title>PX-866 blocks autophagy in glioma cells</title><p id="Par36">Given that over-activation of the PI3K pathway is frequently reported in primary GBM, we decided to explore the signaling consequences of the pan-PI3K inhibitor PX-866, due to its previous use in the clinic for recurrent GBM. T98G cells treated with increasing doses of PX-866 dose-dependently inhibited the phosphorylation of AKT (ser473), but also increased the expression of both LC3 I and LC3 II, which is indicative of a late-stage autophagy blocker (Fig. <xref rid="Fig4" ref-type="fig">4</xref>a). In addition, p62 protein was increased with the treatment of PX-866 (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Figure S1). We then used 3-MA (PI3K inhibitor/early autophagy blocker) to determine if there was any functional similarity between PX-866 and Baf. Co-treatment of 3-MA with PX-866 was able to inhibit the accumulation of both LC3 I and II compared to PX-866 alone, while co-treatment of 3-MA with Baf showed no alteration in LC3 protein levels compared to Baf alone, indicating that PX-866 is not a late stage inhibitor like Baf (Fig. <xref rid="Fig4" ref-type="fig">4</xref>b). To further explore this mechanism, an LC3 flux assay was performed using early (4 h) and late (24 h) time points. At a 4 h time point, PX-866 alone and in combination with Baf was able to cause an accumulation of LC3 I and a reduction in LC3 II. At 24 h, PX-866 in combination with Baf showed a reduction of LC3 II levels compared to Baf alone (Fig. <xref rid="Fig4" ref-type="fig">4</xref>c). These data provide evidence that PX-866 is an early-stage inhibitor of autophagosome formation, thereby preventing the conversion of LC3 I to LC3 II.
<fig id="Fig4"><label>Fig. 4</label><caption><p>PX-866 blocks autophagy in glioma cells. <bold>a</bold> T98G cells were treated with increasing doses of PX-866 for 24 h and then total protein was isolated. <bold>b</bold> T98G cells were subjected to single treatment with 3-MA (2.5 mM) or co-treatment with either PX-866 (1 μM) or Baf (10 nM) for 24 h, according to the treatment legend. <bold>c</bold> The LC3 flux assay was performed on T98G cells at 4 h and 24 h using Baf (10 nM), PX-866 (1 μM) or a combination of the two. Data represents three independent experiments</p></caption><graphic xlink:href="10020_2019_116_Fig4_HTML" id="MO4"></graphic></fig></p></sec><sec id="Sec15"><title>TMZ and PX-866 cooperate to diminish survival</title><p id="Par37">TMZ-induced autophagy could potentially facilitate GBM cell survival, but it may also represent a therapeutic vulnerability. It is well established that inhibition of autophagy with lysosomotropic compounds enhances the efficacy of conventional chemotherapy (Degtyarev et al. <xref ref-type="bibr" rid="CR9">2008</xref>; Apel et al. <xref ref-type="bibr" rid="CR1">2008</xref>; Buccarelli et al. <xref ref-type="bibr" rid="CR4">2018</xref>). Given that PX-866 inhibits autophagy, we set out to determine if the addition of PX-866 to TMZ could augment apoptosis in T98G cells. The addition of increasing doses of PX-866 to TMZ for 48 h had no effect on the cleavage of PARP, a traditional marker of apoptosis, compared to TMZ alone, but did show an induction of both LC3 I and LC3 II (Fig. <xref rid="Fig5" ref-type="fig">5</xref>a) and p62 (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Figure S1) protein levels, indicating a blockage of autophagy. This observation was confirmed in U373vIII cells, which have high activation of the PI3K pathway due to the presence of the EGFRvIII receptor which is constitutively active (Learn et al. <xref ref-type="bibr" rid="CR29">2004</xref>; Golding et al. <xref ref-type="bibr" rid="CR16">2009</xref>). Interestingly, treatment with TMZ first for 24 h, followed by the addition of PX-866 for another 24 h, induced the cleavage of PARP in a dose-dependent manner in both T98G and U373vIII cells, while consistently causing an induction of both LC3 I and LC3 II protein levels (Fig. <xref rid="Fig5" ref-type="fig">5</xref>b). Accordingly, both T98G and U373vIII cells exhibited a decrease in the ability to form colonies after co-treatment with TMZ (500 μM for T98G, and 15 μM for U373vIII) and PX-866 (both 1 μM) compared to TMZ alone (Fig. <xref rid="Fig5" ref-type="fig">5</xref>c and d). The addition of Baf to TMZ had a more pronounced effect on colony formation compared to TMZ and PX-866, potentially indicating that lysosomotropic inhibitors may be more effective due to their ability to inhibit the late stage of autophagy compared to inhibition of initiation.
<fig id="Fig5"><label>Fig. 5</label><caption><p>TMZ and PX-866 cooperate to diminish survival. <bold>a</bold> T98G (EGFR WT) and U373vIII (EGFRvIII) were treated with TMZ (500 μM) and increasing doses of PX-866 for 48 h (co-treatment). Both floating and remaining adherent cells were collected for analysis and western blotting for PARP and LC3 was used to assess apoptosis and autophagy, respectively. The triangle representing the dose escalation for PX-866 indicates: 0.125, 0.25, 0.5, and 1 μM. <bold>b</bold> T98G and U373vIII cells were treated with TMZ (500 μM) for 24 h, followed by the addition of PX-866 for another 24 h (sequential treatment). The triangle representing the dose escalation for PX-866 indicates: 0.125, 0.25, 0.5, and 1 μM. T98G cells (<bold>c</bold>) and U373vIII cells (<bold>d</bold>) were treated with the indicated treatments for 24 h. Cells were trypsinized 24 h after drug treatment and re-plated in 35 mm dishes in triplicate and allowed to form colonies. After about 9–10 days, colonies were fixed (6% glutaraldehyde) and stained (0.5% crystal violet). The number of colonies were counted and presented as a bar graph. Values are the mean ± the standard deviation of three separate measurements, *, <italic>P</italic> < 0.05 and, **, <italic>P</italic> < 0.01. Data represents the mean and S.D. from three independent experiments with each experiment conducted in triplicates</p></caption><graphic xlink:href="10020_2019_116_Fig5_HTML" id="MO5"></graphic></fig></p></sec></sec><sec id="Sec16"><title>Discussion</title><p id="Par38">In this report we have provided mechanistic evidence for the use of a new treatment strategy for GBM. TMZ was shown to induce autophagic flux and inhibition of TMZ-mediated autophagy with PX-866 increased apoptosis and significantly diminished the capacity of GBM cells to form colonies compared to TMZ alone.</p><p id="Par39">TMZ has been the mainstay chemotherapy for GBM since 2005 and is effective against highly proliferative cells through alkylation of DNA. TMZ has been shown to activate the autophagy pathway, but there is little evidence to indicate how this occurs (Kanzawa et al. <xref ref-type="bibr" rid="CR24">2004</xref>; Wurstle et al. <xref ref-type="bibr" rid="CR53">2017</xref>). Induction of the DNA damage response (DDR) has recently been associated with the activation of autophagy and other cellular programs to mitigate stress and promote survival (Eliopoulos et al. <xref ref-type="bibr" rid="CR11">2016</xref>). Stress-responsive programs, such as the unfolded protein response (UPR), could play a role in the induction of autophagy, potentially through transcriptional activation of key autophagy genes (Ogata et al. <xref ref-type="bibr" rid="CR36">2006</xref>).</p><p id="Par40">The autophagy inhibitor chloroquine (CQ) has shown pre-clinical promise as a chemosensitizing agent which can enhance the effects of TMZ (Lee et al. <xref ref-type="bibr" rid="CR30">2015</xref>; Hori et al. <xref ref-type="bibr" rid="CR21">2015</xref>; Golden et al. <xref ref-type="bibr" rid="CR15">2014</xref>). The addition of CQ to radiation and TMZ was shown to be clinically effective for newly diagnosed GBM compared to standard care alone, but the efficacy of this combination was restricted by dose-limiting toxicity associated with CQ (Briceno et al. <xref ref-type="bibr" rid="CR3">2007</xref>; Rosenfeld et al. <xref ref-type="bibr" rid="CR43">2014</xref>). In this study, we have provided evidence that PX-866 blocks autophagosome maturation in T98G cells, identifying it as an inhibitor of autophagy. PX-866, an irreversible pan-PI3K inhibitor, is able to penetrate the BBB and has previously been shown to minimize tumor growth in a flank xenograft model of GBM in mice (Koul et al. <xref ref-type="bibr" rid="CR27">2010</xref>). It has also been reported that PX-866 does not induce apoptosis in GBM cells, but significantly reduces the expression of VEGF and the invasion of GBM cells in vitro (Koul et al. <xref ref-type="bibr" rid="CR27">2010</xref>).</p><p id="Par41">Multiple genomic alterations confer overactive PI3K-AKT-mTOR signaling in GBM. Amplification of <italic>EGFR</italic>, the expression of EGFRvIII, loss of <italic>PTEN</italic>, and mutations in <italic>PIK3CA</italic> represents a large portion of driver mutations in GBM which converge on the AKT-mTOR signaling axis (Prados et al. <xref ref-type="bibr" rid="CR41">2015</xref>; Brennan et al. <xref ref-type="bibr" rid="CR2">2013</xref>). Despite a plethora of signaling effectors for potential therapeutic intervention, no compounds targeting this pathway have made it into the clinic for the treatment of GBM. A phase II clinical trial was performed with PX-866 in patients with recurrent GBM (Pitz et al. <xref ref-type="bibr" rid="CR40">2015</xref>). Used as a monotherapy, PX-866 did not offer an advantage in overall survival. Other molecules, such as BEZ235, a dual PI3K and mTOR inhibitor, has shown promising pre-clinical evidence against GBM, however, BEZ235 has poor BBB-penetrance and is a strong autophagic inducer, potentially allowing cells to achieve autophagy-dependent survival.</p><p id="Par42">There is evidence to support the fact that autophagic induction promotes GBM cell survival and may be a critical mechanism of adaptive chemoresistance (Fan et al. <xref ref-type="bibr" rid="CR12">2010</xref>; Fan and Weiss <xref ref-type="bibr" rid="CR13">2011</xref>). The majority of drugs targeting the PI3K/AKT/mTOR axis inhibit class 1 PI3K, which promotes autophagy due to the inhibitory effect on mTOR. Interestingly, our findings using PX-866 showed a blocking effect on autophagy, potentially through inhibition of class III PI3K (VPS34) which helps to control autophagosome biogenesis (Russell et al. <xref ref-type="bibr" rid="CR45">2013</xref>; Stjepanovic et al. <xref ref-type="bibr" rid="CR48">2017</xref>). Understanding the expression and activity of the different types of PI3K enzymes present in certain GBM cell lines or PDX models may help to ultimately determine the overall effect certain inhibitors may have on autophagy. As such, this potentially may lead to the identification of therapeutic vulnerabilities in GBM and provide a translatable approach to create rational drug combinations to treat GBM (Jutten et al. <xref ref-type="bibr" rid="CR23">2018</xref>).</p><p id="Par43">Patients with newly diagnosed GBM will invariably receive the standard care of radiation therapy and TMZ after surgical removal of the tumor regardless of their expression status of MGMT. In cases where MGMT expression is high, these patients will respond poorly to TMZ, however, if we begin to understand additional features of tumors with inherent resistance to TMZ, this also may offer new therapeutic combinations to drugs that are readily available for repurposing efforts. The correlation between PI3K pathway activation and MGMT promoter methylation status may be an early insight into treating patients with inherent TMZ resistance with PI3K inhibitors that can inhibit autophagy or late-stage autophagy blockers such as CQ. Thus, these data provide mechanistic evidence that patients with high expression of MGMT and a high degree of PI3K activity may respond well to the use of TMZ followed by a pan-PI3K inhibitor/autophagy blocker added in a sequential manner.</p></sec><sec id="Sec17"><title>Conclusion</title><p id="Par44">We have identified that treating with TMZ followed by the addition of the pan-PI3K inhibitor PX-866 promotes glioma cell death. We provide evidence that TMZ-induced autophagy is a delayed response, thus providing an opportunity to sequentially target this cell survival mechanism with PX-866 (Fig. <xref rid="Fig6" ref-type="fig">6</xref>). Understanding the correlation between MGMT status and relative activation of key survival pathways in GBM may provide therapeutically-relevant discernments for a more personalized approach towards treating newly diagnosed GBM in the future.
<fig id="Fig6"><label>Fig. 6</label><caption><p>Schematic of the proposed mechanism of how PX-866 cooperates with TMZ to enhance apoptosis through inhibition of TMZ-induced autophagy</p></caption><graphic xlink:href="10020_2019_116_Fig6_HTML" id="MO6"></graphic></fig></p></sec><sec sec-type="supplementary-material"><title>Supplementary information</title><sec id="Sec18"><p><supplementary-material content-type="local-data" id="MOESM1"><media xlink:href="10020_2019_116_MOESM1_ESM.pptx"><caption><p><bold>Additional file 1: Figure S1.</bold> T98G cells were treated with PX-866 or TMZ alone or in combination for 24 h and then total protein was isolated. p62 protein levels were examined by western blotting, and GAPDH was used as a loading control.</p></caption></media></supplementary-material></p></sec></sec></body><back><glossary><title>Abbreviations</title><def-list><def-item><term>(3-MA</term><def><p id="Par5">3-Methyladenine</p></def></def-item><def-item><term>Baf</term><def><p id="Par6">Bafilomycin</p></def></def-item><def-item><term>BBB</term><def><p id="Par7">Blood-brain barrier</p></def></def-item><def-item><term>CQ</term><def><p id="Par8">Chloroquine</p></def></def-item><def-item><term>DDR</term><def><p id="Par9">DNA-damage response</p></def></def-item><def-item><term>EGFR</term><def><p id="Par10">Epidermal growth factor receptor</p></def></def-item><def-item><term>GBM</term><def><p id="Par11">Glioblastoma</p></def></def-item><def-item><term>GSVA</term><def><p id="Par12">Gene set variation analysis</p></def></def-item><def-item><term>MGMT</term><def><p id="Par13">O<sup>6</sup>-methylguanine-DNA- methyltransferase</p></def></def-item><def-item><term>MSigDB</term><def><p id="Par14">Molecular Signatures Database</p></def></def-item><def-item><term>PARP</term><def><p id="Par15">Poly (ADP-ribose) polymerase</p></def></def-item><def-item><term>PDX</term><def><p id="Par16">Patient-derived xenograft</p></def></def-item><def-item><term>PI3K</term><def><p id="Par17">Phosphatidylinositol 3-kinase</p></def></def-item><def-item><term>PTEN</term><def><p id="Par18">Phosphatase and tensin homolog</p></def></def-item><def-item><term>RT</term><def><p id="Par19">Radiation therapy</p></def></def-item><def-item><term>TMZ</term><def><p id="Par20">Temozolomide</p></def></def-item></def-list></glossary><fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> accompanies this paper at 10.1186/s10020-019-0116-z.</p></sec><ack><title>Acknowledgements</title><p>We thank Serdar Tuncali for technical assistance.</p></ack><notes notes-type="author-contribution"><title>Authors’ contributions</title><p>Concept and design- all authors, Data acquisition- BGH, SP, AMS, CPS, GJK, Drafting of the manuscript- BGH, GJK, JCL, JNS, NLT, Interpretation of analysis and revising the manuscript critically for important intellectual content- all authors. All authors present read and approved the final manuscript.</p></notes><notes notes-type="funding-information"><title>Funding</title><p>Supported in part by NIH grants R01 NS086853 (JCL and NLT), U01 CA220378–01 (JCL and NLT), and U54 CA210180 (NLT, JNS).</p></notes><notes notes-type="data-availability"><title>Availability of data and materials</title><p>Readily available upon request.</p></notes><notes><title>Ethics approval and consent to participate</title><p id="Par45">Not applicable.</p></notes><notes><title>Consent for publication</title><p id="Par46">Not applicable.</p></notes><notes notes-type="COI-statement"><title>Competing interests</title><p id="Par47">The authors declare that they have no competing interests.</p></notes><ref-list id="Bib1"><title>References</title><ref id="CR1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Apel</surname><given-names>A</given-names></name><name><surname>Herr</surname><given-names>I</given-names></name><name><surname>Schwarz</surname><given-names>H</given-names></name><name><surname>Rodemann</surname><given-names>HP</given-names></name><name><surname>Mayer</surname><given-names>A</given-names></name></person-group><article-title>Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy</article-title><source>Cancer Res</source><year>2008</year><volume>68</volume><issue>5</issue><fpage>1485</fpage><lpage>1494</lpage><pub-id pub-id-type="doi">10.1158/0008-5472.CAN-07-0562</pub-id><pub-id pub-id-type="pmid">18316613</pub-id></element-citation></ref><ref id="CR2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brennan</surname><given-names>CW</given-names></name><name><surname>Verhaak</surname><given-names>RG</given-names></name><name><surname>McKenna</surname><given-names>A</given-names></name><name><surname>Campos</surname><given-names>B</given-names></name><name><surname>Noushmehr</surname><given-names>H</given-names></name><name><surname>Salama</surname><given-names>SR</given-names></name><name><surname>Zheng</surname><given-names>S</given-names></name><name><surname>Chakravarty</surname><given-names>D</given-names></name><name><surname>Sanborn</surname><given-names>JZ</given-names></name><name><surname>Berman</surname><given-names>SH</given-names></name><etal></etal></person-group><article-title>The somatic genomic landscape of glioblastoma</article-title><source>Cell</source><year>2013</year><volume>155</volume><issue>2</issue><fpage>462</fpage><lpage>477</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2013.09.034</pub-id><pub-id pub-id-type="pmid">24120142</pub-id></element-citation></ref><ref id="CR3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Briceno</surname><given-names>E</given-names></name><name><surname>Calderon</surname><given-names>A</given-names></name><name><surname>Sotelo</surname><given-names>J</given-names></name></person-group><article-title>Institutional experience with chloroquine as an adjuvant to the therapy for glioblastoma multiforme</article-title><source>Surg Neurol</source><year>2007</year><volume>67</volume><issue>4</issue><fpage>388</fpage><lpage>391</lpage><pub-id pub-id-type="doi">10.1016/j.surneu.2006.08.080</pub-id><pub-id pub-id-type="pmid">17350410</pub-id></element-citation></ref><ref id="CR4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Buccarelli</surname><given-names>M</given-names></name><name><surname>Marconi</surname><given-names>M</given-names></name><name><surname>Pacioni</surname><given-names>S</given-names></name><name><surname>De Pascalis</surname><given-names>I</given-names></name><name><surname>D'Alessandris</surname><given-names>QG</given-names></name><name><surname>Martini</surname><given-names>M</given-names></name><name><surname>Ascione</surname><given-names>B</given-names></name><name><surname>Malorni</surname><given-names>W</given-names></name><name><surname>Larocca</surname><given-names>LM</given-names></name><name><surname>Pallini</surname><given-names>R</given-names></name><etal></etal></person-group><article-title>Inhibition of autophagy increases susceptibility of glioblastoma stem cells to temozolomide by igniting ferroptosis</article-title><source>Cell Death Dis</source><year>2018</year><volume>9</volume><issue>8</issue><fpage>841</fpage><pub-id pub-id-type="doi">10.1038/s41419-018-0864-7</pub-id><pub-id pub-id-type="pmid">30082680</pub-id></element-citation></ref><ref id="CR5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Button</surname><given-names>RW</given-names></name><name><surname>Vincent</surname><given-names>JH</given-names></name><name><surname>Strang</surname><given-names>CJ</given-names></name><name><surname>Luo</surname><given-names>S</given-names></name></person-group><article-title>Dual PI-3 kinase/mTOR inhibition impairs autophagy flux and induces cell death independent of apoptosis and necroptosis</article-title><source>Oncotarget</source><year>2016</year><volume>7</volume><issue>5</issue><fpage>5157</fpage><lpage>5175</lpage><pub-id pub-id-type="doi">10.18632/oncotarget.6986</pub-id><pub-id pub-id-type="pmid">26814436</pub-id></element-citation></ref><ref id="CR6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Catasus</surname><given-names>L</given-names></name><name><surname>D'Angelo</surname><given-names>E</given-names></name><name><surname>Pons</surname><given-names>C</given-names></name><name><surname>Espinosa</surname><given-names>I</given-names></name><name><surname>Prat</surname><given-names>J</given-names></name></person-group><article-title>Expression profiling of 22 genes involved in the PI3K-AKT pathway identifies two subgroups of high-grade endometrial carcinomas with different molecular alterations</article-title><source>Mod Pathol</source><year>2010</year><volume>23</volume><issue>5</issue><fpage>694</fpage><lpage>702</lpage><pub-id pub-id-type="doi">10.1038/modpathol.2010.44</pub-id><pub-id pub-id-type="pmid">20173732</pub-id></element-citation></ref><ref id="CR7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cheng</surname><given-names>CK</given-names></name><name><surname>Fan</surname><given-names>QW</given-names></name><name><surname>Weiss</surname><given-names>WA</given-names></name></person-group><article-title>PI3K signaling in glioma--animal models and therapeutic challenges</article-title><source>Brain Pathol</source><year>2009</year><volume>19</volume><issue>1</issue><fpage>112</fpage><lpage>120</lpage><pub-id pub-id-type="doi">10.1111/j.1750-3639.2008.00233.x</pub-id><pub-id pub-id-type="pmid">19076776</pub-id></element-citation></ref><ref id="CR8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Degenhardt</surname><given-names>K</given-names></name><name><surname>Mathew</surname><given-names>R</given-names></name><name><surname>Beaudoin</surname><given-names>B</given-names></name><name><surname>Bray</surname><given-names>K</given-names></name><name><surname>Anderson</surname><given-names>D</given-names></name><name><surname>Chen</surname><given-names>G</given-names></name><name><surname>Mukherjee</surname><given-names>C</given-names></name><name><surname>Shi</surname><given-names>Y</given-names></name><name><surname>Gelinas</surname><given-names>C</given-names></name><name><surname>Fan</surname><given-names>Y</given-names></name><etal></etal></person-group><article-title>Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis</article-title><source>Cancer Cell</source><year>2006</year><volume>10</volume><issue>1</issue><fpage>51</fpage><lpage>64</lpage><pub-id pub-id-type="doi">10.1016/j.ccr.2006.06.001</pub-id><pub-id pub-id-type="pmid">16843265</pub-id></element-citation></ref><ref id="CR9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Degtyarev</surname><given-names>M</given-names></name><name><surname>De Maziere</surname><given-names>A</given-names></name><name><surname>Orr</surname><given-names>C</given-names></name><name><surname>Lin</surname><given-names>J</given-names></name><name><surname>Lee</surname><given-names>BB</given-names></name><name><surname>Tien</surname><given-names>JY</given-names></name><name><surname>Prior</surname><given-names>WW</given-names></name><name><surname>van Dijk</surname><given-names>S</given-names></name><name><surname>Wu</surname><given-names>H</given-names></name><name><surname>Gray</surname><given-names>DC</given-names></name><etal></etal></person-group><article-title>Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents</article-title><source>J Cell Biol</source><year>2008</year><volume>183</volume><issue>1</issue><fpage>101</fpage><lpage>116</lpage><pub-id pub-id-type="doi">10.1083/jcb.200801099</pub-id><pub-id pub-id-type="pmid">18838554</pub-id></element-citation></ref><ref id="CR10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ding</surname><given-names>ZB</given-names></name><name><surname>Hui</surname><given-names>B</given-names></name><name><surname>Shi</surname><given-names>YH</given-names></name><name><surname>Zhou</surname><given-names>J</given-names></name><name><surname>Peng</surname><given-names>YF</given-names></name><name><surname>Gu</surname><given-names>CY</given-names></name><name><surname>Yang</surname><given-names>H</given-names></name><name><surname>Shi</surname><given-names>GM</given-names></name><name><surname>Ke</surname><given-names>AW</given-names></name><name><surname>Wang</surname><given-names>XY</given-names></name><etal></etal></person-group><article-title>Autophagy activation in hepatocellular carcinoma contributes to the tolerance of oxaliplatin via reactive oxygen species modulation</article-title><source>Clin Cancer Res</source><year>2011</year><volume>17</volume><issue>19</issue><fpage>6229</fpage><lpage>6238</lpage><pub-id pub-id-type="doi">10.1158/1078-0432.CCR-11-0816</pub-id><pub-id pub-id-type="pmid">21825039</pub-id></element-citation></ref><ref id="CR11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Eliopoulos</surname><given-names>AG</given-names></name><name><surname>Havaki</surname><given-names>S</given-names></name><name><surname>Gorgoulis</surname><given-names>VG</given-names></name></person-group><article-title>DNA damage response and autophagy: a meaningful partnership</article-title><source>Front Genet</source><year>2016</year><volume>7</volume><fpage>204</fpage><pub-id pub-id-type="doi">10.3389/fgene.2016.00204</pub-id><pub-id pub-id-type="pmid">27917193</pub-id></element-citation></ref><ref id="CR12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fan</surname><given-names>QW</given-names></name><name><surname>Cheng</surname><given-names>C</given-names></name><name><surname>Hackett</surname><given-names>C</given-names></name><name><surname>Feldman</surname><given-names>M</given-names></name><name><surname>Houseman</surname><given-names>BT</given-names></name><name><surname>Nicolaides</surname><given-names>T</given-names></name><name><surname>Haas-Kogan</surname><given-names>D</given-names></name><name><surname>James</surname><given-names>CD</given-names></name><name><surname>Oakes</surname><given-names>SA</given-names></name><name><surname>Debnath</surname><given-names>J</given-names></name><etal></etal></person-group><article-title>Akt and autophagy cooperate to promote survival of drug-resistant glioma</article-title><source>Sci Signal</source><year>2010</year><volume>3</volume><issue>147</issue><fpage>ra81</fpage><pub-id pub-id-type="doi">10.1126/scisignal.2001017</pub-id><pub-id pub-id-type="pmid">21062993</pub-id></element-citation></ref><ref id="CR13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fan</surname><given-names>QW</given-names></name><name><surname>Weiss</surname><given-names>WA</given-names></name></person-group><article-title>Autophagy and Akt promote survival in glioma</article-title><source>Autophagy</source><year>2011</year><volume>7</volume><issue>5</issue><fpage>536</fpage><lpage>538</lpage><pub-id pub-id-type="doi">10.4161/auto.7.5.14779</pub-id><pub-id pub-id-type="pmid">21266843</pub-id></element-citation></ref><ref id="CR14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Franken</surname><given-names>NA</given-names></name><name><surname>Rodermond</surname><given-names>HM</given-names></name><name><surname>Stap</surname><given-names>J</given-names></name><name><surname>Haveman</surname><given-names>J</given-names></name><name><surname>van Bree</surname><given-names>C</given-names></name></person-group><article-title>Clonogenic assay of cells in vitro</article-title><source>Nat Protoc</source><year>2006</year><volume>1</volume><issue>5</issue><fpage>2315</fpage><lpage>2319</lpage><pub-id pub-id-type="doi">10.1038/nprot.2006.339</pub-id><pub-id pub-id-type="pmid">17406473</pub-id></element-citation></ref><ref id="CR15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Golden</surname><given-names>EB</given-names></name><name><surname>Cho</surname><given-names>HY</given-names></name><name><surname>Jahanian</surname><given-names>A</given-names></name><name><surname>Hofman</surname><given-names>FM</given-names></name><name><surname>Louie</surname><given-names>SG</given-names></name><name><surname>Schonthal</surname><given-names>AH</given-names></name><name><surname>Chen</surname><given-names>TC</given-names></name></person-group><article-title>Chloroquine enhances temozolomide cytotoxicity in malignant gliomas by blocking autophagy</article-title><source>Neurosurg Focus</source><year>2014</year><volume>37</volume><issue>6</issue><fpage>E12</fpage><pub-id pub-id-type="doi">10.3171/2014.9.FOCUS14504</pub-id><pub-id pub-id-type="pmid">25434381</pub-id></element-citation></ref><ref id="CR16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Golding</surname><given-names>SE</given-names></name><name><surname>Morgan</surname><given-names>RN</given-names></name><name><surname>Adams</surname><given-names>BR</given-names></name><name><surname>Hawkins</surname><given-names>AJ</given-names></name><name><surname>Povirk</surname><given-names>LF</given-names></name><name><surname>Valerie</surname><given-names>K</given-names></name></person-group><article-title>Pro-survival AKT and ERK signaling from EGFR and mutant EGFRvIII enhances DNA double-strand break repair in human glioma cells</article-title><source>Cancer Biol Ther</source><year>2009</year><volume>8</volume><issue>8</issue><fpage>730</fpage><lpage>738</lpage><pub-id pub-id-type="doi">10.4161/cbt.8.8.7927</pub-id><pub-id pub-id-type="pmid">19252415</pub-id></element-citation></ref><ref id="CR17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Han</surname><given-names>W</given-names></name><name><surname>Pan</surname><given-names>H</given-names></name><name><surname>Chen</surname><given-names>Y</given-names></name><name><surname>Sun</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Li</surname><given-names>J</given-names></name><name><surname>Ge</surname><given-names>W</given-names></name><name><surname>Feng</surname><given-names>L</given-names></name><name><surname>Lin</surname><given-names>X</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name><etal></etal></person-group><article-title>EGFR tyrosine kinase inhibitors activate autophagy as a cytoprotective response in human lung cancer cells</article-title><source>PLoS One</source><year>2011</year><volume>6</volume><issue>6</issue><fpage>e18691</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0018691</pub-id><pub-id pub-id-type="pmid">21655094</pub-id></element-citation></ref><ref id="CR18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hanzelmann</surname><given-names>S</given-names></name><name><surname>Castelo</surname><given-names>R</given-names></name><name><surname>Guinney</surname><given-names>J</given-names></name></person-group><article-title>GSVA: gene set variation analysis for microarray and RNA-seq data</article-title><source>BMC Bioinformatics</source><year>2013</year><volume>14</volume><fpage>7</fpage><pub-id pub-id-type="doi">10.1186/1471-2105-14-7</pub-id><pub-id pub-id-type="pmid">23323831</pub-id></element-citation></ref><ref id="CR19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hegi</surname><given-names>ME</given-names></name><name><surname>Diserens</surname><given-names>AC</given-names></name><name><surname>Gorlia</surname><given-names>T</given-names></name><name><surname>Hamou</surname><given-names>MF</given-names></name><name><surname>de Tribolet</surname><given-names>N</given-names></name><name><surname>Weller</surname><given-names>M</given-names></name><name><surname>Kros</surname><given-names>JM</given-names></name><name><surname>Hainfellner</surname><given-names>JA</given-names></name><name><surname>Mason</surname><given-names>W</given-names></name><name><surname>Mariani</surname><given-names>L</given-names></name><etal></etal></person-group><article-title>MGMT gene silencing and benefit from temozolomide in glioblastoma</article-title><source>N Engl J Med</source><year>2005</year><volume>352</volume><issue>10</issue><fpage>997</fpage><lpage>1003</lpage><pub-id pub-id-type="doi">10.1056/NEJMoa043331</pub-id><pub-id pub-id-type="pmid">15758010</pub-id></element-citation></ref><ref id="CR20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hombach-Klonisch</surname><given-names>S</given-names></name><name><surname>Mehrpour</surname><given-names>M</given-names></name><name><surname>Shojaei</surname><given-names>S</given-names></name><name><surname>Harlos</surname><given-names>C</given-names></name><name><surname>Pitz</surname><given-names>M</given-names></name><name><surname>Hamai</surname><given-names>A</given-names></name><name><surname>Siemianowicz</surname><given-names>K</given-names></name><name><surname>Likus</surname><given-names>W</given-names></name><name><surname>Wiechec</surname><given-names>E</given-names></name><name><surname>Toyota</surname><given-names>BD</given-names></name><etal></etal></person-group><article-title>Glioblastoma and chemoresistance to alkylating agents: involvement of apoptosis, autophagy, and unfolded protein response</article-title><source>Pharmacol Ther</source><year>2018</year><volume>184</volume><fpage>13</fpage><lpage>41</lpage><pub-id pub-id-type="doi">10.1016/j.pharmthera.2017.10.017</pub-id><pub-id pub-id-type="pmid">29080702</pub-id></element-citation></ref><ref id="CR21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hori</surname><given-names>YS</given-names></name><name><surname>Hosoda</surname><given-names>R</given-names></name><name><surname>Akiyama</surname><given-names>Y</given-names></name><name><surname>Sebori</surname><given-names>R</given-names></name><name><surname>Wanibuchi</surname><given-names>M</given-names></name><name><surname>Mikami</surname><given-names>T</given-names></name><name><surname>Sugino</surname><given-names>T</given-names></name><name><surname>Suzuki</surname><given-names>K</given-names></name><name><surname>Maruyama</surname><given-names>M</given-names></name><name><surname>Tsukamoto</surname><given-names>M</given-names></name><etal></etal></person-group><article-title>Chloroquine potentiates temozolomide cytotoxicity by inhibiting mitochondrial autophagy in glioma cells</article-title><source>J Neurooncol</source><year>2015</year><volume>122</volume><issue>1</issue><fpage>11</fpage><lpage>20</lpage><pub-id pub-id-type="doi">10.1007/s11060-014-1686-9</pub-id><pub-id pub-id-type="pmid">25528635</pub-id></element-citation></ref><ref id="CR22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hu</surname><given-names>YL</given-names></name><name><surname>DeLay</surname><given-names>M</given-names></name><name><surname>Jahangiri</surname><given-names>A</given-names></name><name><surname>Molinaro</surname><given-names>AM</given-names></name><name><surname>Rose</surname><given-names>SD</given-names></name><name><surname>Carbonell</surname><given-names>WS</given-names></name><name><surname>Aghi</surname><given-names>MK</given-names></name></person-group><article-title>Hypoxia-induced autophagy promotes tumor cell survival and adaptation to antiangiogenic treatment in glioblastoma</article-title><source>Cancer Res</source><year>2012</year><volume>72</volume><issue>7</issue><fpage>1773</fpage><lpage>1783</lpage><pub-id pub-id-type="doi">10.1158/0008-5472.CAN-11-3831</pub-id><pub-id pub-id-type="pmid">22447568</pub-id></element-citation></ref><ref id="CR23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jutten</surname><given-names>B</given-names></name><name><surname>Keulers</surname><given-names>TG</given-names></name><name><surname>Peeters</surname><given-names>HJM</given-names></name><name><surname>Schaaf</surname><given-names>MBE</given-names></name><name><surname>Savelkouls</surname><given-names>KGM</given-names></name><name><surname>Compter</surname><given-names>I</given-names></name><name><surname>Clarijs</surname><given-names>R</given-names></name><name><surname>Schijns</surname><given-names>O</given-names></name><name><surname>Ackermans</surname><given-names>L</given-names></name><name><surname>Teernstra</surname><given-names>OPM</given-names></name><etal></etal></person-group><article-title>EGFRvIII expression triggers a metabolic dependency and therapeutic vulnerability sensitive to autophagy inhibition</article-title><source>Autophagy</source><year>2018</year><volume>14</volume><issue>2</issue><fpage>283</fpage><lpage>295</lpage><pub-id pub-id-type="doi">10.1080/15548627.2017.1409926</pub-id><pub-id pub-id-type="pmid">29377763</pub-id></element-citation></ref><ref id="CR24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kanzawa</surname><given-names>T</given-names></name><name><surname>Germano</surname><given-names>IM</given-names></name><name><surname>Komata</surname><given-names>T</given-names></name><name><surname>Ito</surname><given-names>H</given-names></name><name><surname>Kondo</surname><given-names>Y</given-names></name><name><surname>Kondo</surname><given-names>S</given-names></name></person-group><article-title>Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells</article-title><source>Cell Death Differ</source><year>2004</year><volume>11</volume><issue>4</issue><fpage>448</fpage><lpage>457</lpage><pub-id pub-id-type="doi">10.1038/sj.cdd.4401359</pub-id><pub-id pub-id-type="pmid">14713959</pub-id></element-citation></ref><ref id="CR25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kitange</surname><given-names>GJ</given-names></name><name><surname>Carlson</surname><given-names>BL</given-names></name><name><surname>Schroeder</surname><given-names>MA</given-names></name><name><surname>Grogan</surname><given-names>PT</given-names></name><name><surname>Lamont</surname><given-names>JD</given-names></name><name><surname>Decker</surname><given-names>PA</given-names></name><name><surname>Wu</surname><given-names>W</given-names></name><name><surname>James</surname><given-names>CD</given-names></name><name><surname>Sarkaria</surname><given-names>JN</given-names></name></person-group><article-title>Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts</article-title><source>Neuro-Oncology</source><year>2009</year><volume>11</volume><issue>3</issue><fpage>281</fpage><lpage>291</lpage><pub-id pub-id-type="doi">10.1215/15228517-2008-090</pub-id><pub-id pub-id-type="pmid">18952979</pub-id></element-citation></ref><ref id="CR26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kitange</surname><given-names>GJ</given-names></name><name><surname>Mladek</surname><given-names>AC</given-names></name><name><surname>Carlson</surname><given-names>BL</given-names></name><name><surname>Schroeder</surname><given-names>MA</given-names></name><name><surname>Pokorny</surname><given-names>JL</given-names></name><name><surname>Cen</surname><given-names>L</given-names></name><name><surname>Decker</surname><given-names>PA</given-names></name><name><surname>Wu</surname><given-names>W</given-names></name><name><surname>Lomberk</surname><given-names>GA</given-names></name><name><surname>Gupta</surname><given-names>SK</given-names></name><etal></etal></person-group><article-title>Inhibition of histone deacetylation potentiates the evolution of acquired temozolomide resistance linked to MGMT upregulation in glioblastoma xenografts</article-title><source>Clin Cancer Res</source><year>2012</year><volume>18</volume><issue>15</issue><fpage>4070</fpage><lpage>4079</lpage><pub-id pub-id-type="doi">10.1158/1078-0432.CCR-12-0560</pub-id><pub-id pub-id-type="pmid">22675172</pub-id></element-citation></ref><ref id="CR27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Koul</surname><given-names>D</given-names></name><name><surname>Shen</surname><given-names>R</given-names></name><name><surname>Kim</surname><given-names>YW</given-names></name><name><surname>Kondo</surname><given-names>Y</given-names></name><name><surname>Lu</surname><given-names>Y</given-names></name><name><surname>Bankson</surname><given-names>J</given-names></name><name><surname>Ronen</surname><given-names>SM</given-names></name><name><surname>Kirkpatrick</surname><given-names>DL</given-names></name><name><surname>Powis</surname><given-names>G</given-names></name><name><surname>Yung</surname><given-names>WK</given-names></name></person-group><article-title>Cellular and in vivo activity of a novel PI3K inhibitor, PX-866, against human glioblastoma</article-title><source>Neuro-Oncology</source><year>2010</year><volume>12</volume><issue>6</issue><fpage>559</fpage><lpage>569</lpage><pub-id pub-id-type="doi">10.1093/neuonc/nop058</pub-id><pub-id pub-id-type="pmid">20156803</pub-id></element-citation></ref><ref id="CR28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kuger</surname><given-names>S</given-names></name><name><surname>Corek</surname><given-names>E</given-names></name><name><surname>Polat</surname><given-names>B</given-names></name><name><surname>Kammerer</surname><given-names>U</given-names></name><name><surname>Flentje</surname><given-names>M</given-names></name><name><surname>Djuzenova</surname><given-names>CS</given-names></name></person-group><article-title>Novel PI3K and mTOR inhibitor NVP-BEZ235 Radiosensitizes breast Cancer cell lines under normoxic and hypoxic conditions</article-title><source>Breast Cancer (Auckl)</source><year>2014</year><volume>8</volume><fpage>39</fpage><lpage>49</lpage><pub-id pub-id-type="pmid">24678241</pub-id></element-citation></ref><ref id="CR29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Learn</surname><given-names>CA</given-names></name><name><surname>Hartzell</surname><given-names>TL</given-names></name><name><surname>Wikstrand</surname><given-names>CJ</given-names></name><name><surname>Archer</surname><given-names>GE</given-names></name><name><surname>Rich</surname><given-names>JN</given-names></name><name><surname>Friedman</surname><given-names>AH</given-names></name><name><surname>Friedman</surname><given-names>HS</given-names></name><name><surname>Bigner</surname><given-names>DD</given-names></name><name><surname>Sampson</surname><given-names>JH</given-names></name></person-group><article-title>Resistance to tyrosine kinase inhibition by mutant epidermal growth factor receptor variant III contributes to the neoplastic phenotype of glioblastoma multiforme</article-title><source>Clin Cancer Res</source><year>2004</year><volume>10</volume><issue>9</issue><fpage>3216</fpage><lpage>3224</lpage><pub-id pub-id-type="doi">10.1158/1078-0432.CCR-03-0521</pub-id><pub-id pub-id-type="pmid">15131063</pub-id></element-citation></ref><ref id="CR30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>SW</given-names></name><name><surname>Kim</surname><given-names>HK</given-names></name><name><surname>Lee</surname><given-names>NH</given-names></name><name><surname>Yi</surname><given-names>HY</given-names></name><name><surname>Kim</surname><given-names>HS</given-names></name><name><surname>Hong</surname><given-names>SH</given-names></name><name><surname>Hong</surname><given-names>YK</given-names></name><name><surname>Joe</surname><given-names>YA</given-names></name></person-group><article-title>The synergistic effect of combination temozolomide and chloroquine treatment is dependent on autophagy formation and p53 status in glioma cells</article-title><source>Cancer Lett</source><year>2015</year><volume>360</volume><issue>2</issue><fpage>195</fpage><lpage>204</lpage><pub-id pub-id-type="doi">10.1016/j.canlet.2015.02.012</pub-id><pub-id pub-id-type="pmid">25681668</pub-id></element-citation></ref><ref id="CR31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Levine</surname><given-names>B</given-names></name><name><surname>Kroemer</surname><given-names>G</given-names></name></person-group><article-title>Autophagy in the pathogenesis of disease</article-title><source>Cell</source><year>2008</year><volume>132</volume><issue>1</issue><fpage>27</fpage><lpage>42</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2007.12.018</pub-id><pub-id pub-id-type="pmid">18191218</pub-id></element-citation></ref><ref id="CR32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liberzon</surname><given-names>A</given-names></name><name><surname>Birger</surname><given-names>C</given-names></name><name><surname>Thorvaldsdottir</surname><given-names>H</given-names></name><name><surname>Ghandi</surname><given-names>M</given-names></name><name><surname>Mesirov</surname><given-names>JP</given-names></name><name><surname>Tamayo</surname><given-names>P</given-names></name></person-group><article-title>The molecular signatures database (MSigDB) hallmark gene set collection</article-title><source>Cell Syst</source><year>2015</year><volume>1</volume><issue>6</issue><fpage>417</fpage><lpage>425</lpage><pub-id pub-id-type="doi">10.1016/j.cels.2015.12.004</pub-id><pub-id pub-id-type="pmid">26771021</pub-id></element-citation></ref><ref id="CR33"><mixed-citation publication-type="other">Mayo Clinic Brain PDX National Resource Portal [<ext-link ext-link-type="uri" xlink:href="http://www.mayo.edu/research/labs/translational-neuro-oncology/mayo-clinic-brain-tumor-patient-derived-xenograft-national-resource">http://www.mayo.edu/research/labs/translational-neuro-oncology/mayo-clinic-brain-tumor-patient-derived-xenograft-national-resource</ext-link>]. 2018.</mixed-citation></ref><ref id="CR34"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mizushima</surname><given-names>N</given-names></name><name><surname>Yoshimori</surname><given-names>T</given-names></name><name><surname>Levine</surname><given-names>B</given-names></name></person-group><article-title>Methods in mammalian autophagy research</article-title><source>Cell</source><year>2010</year><volume>140</volume><issue>3</issue><fpage>313</fpage><lpage>326</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2010.01.028</pub-id><pub-id pub-id-type="pmid">20144757</pub-id></element-citation></ref><ref id="CR35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nazio</surname><given-names>F</given-names></name><name><surname>Strappazzon</surname><given-names>F</given-names></name><name><surname>Antonioli</surname><given-names>M</given-names></name><name><surname>Bielli</surname><given-names>P</given-names></name><name><surname>Cianfanelli</surname><given-names>V</given-names></name><name><surname>Bordi</surname><given-names>M</given-names></name><name><surname>Gretzmeier</surname><given-names>C</given-names></name><name><surname>Dengjel</surname><given-names>J</given-names></name><name><surname>Piacentini</surname><given-names>M</given-names></name><name><surname>Fimia</surname><given-names>GM</given-names></name><etal></etal></person-group><article-title>mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6</article-title><source>Nat Cell Biol</source><year>2013</year><volume>15</volume><issue>4</issue><fpage>406</fpage><lpage>416</lpage><pub-id pub-id-type="doi">10.1038/ncb2708</pub-id><pub-id pub-id-type="pmid">23524951</pub-id></element-citation></ref><ref id="CR36"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ogata</surname><given-names>M</given-names></name><name><surname>Hino</surname><given-names>S</given-names></name><name><surname>Saito</surname><given-names>A</given-names></name><name><surname>Morikawa</surname><given-names>K</given-names></name><name><surname>Kondo</surname><given-names>S</given-names></name><name><surname>Kanemoto</surname><given-names>S</given-names></name><name><surname>Murakami</surname><given-names>T</given-names></name><name><surname>Taniguchi</surname><given-names>M</given-names></name><name><surname>Tanii</surname><given-names>I</given-names></name><name><surname>Yoshinaga</surname><given-names>K</given-names></name><etal></etal></person-group><article-title>Autophagy is activated for cell survival after endoplasmic reticulum stress</article-title><source>Mol Cell Biol</source><year>2006</year><volume>26</volume><issue>24</issue><fpage>9220</fpage><lpage>9231</lpage><pub-id pub-id-type="doi">10.1128/MCB.01453-06</pub-id><pub-id pub-id-type="pmid">17030611</pub-id></element-citation></ref><ref id="CR37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Paillas</surname><given-names>S</given-names></name><name><surname>Causse</surname><given-names>A</given-names></name><name><surname>Marzi</surname><given-names>L</given-names></name><name><surname>de Medina</surname><given-names>P</given-names></name><name><surname>Poirot</surname><given-names>M</given-names></name><name><surname>Denis</surname><given-names>V</given-names></name><name><surname>Vezzio-Vie</surname><given-names>N</given-names></name><name><surname>Espert</surname><given-names>L</given-names></name><name><surname>Arzouk</surname><given-names>H</given-names></name><name><surname>Coquelle</surname><given-names>A</given-names></name><etal></etal></person-group><article-title>MAPK14/p38alpha confers irinotecan resistance to TP53-defective cells by inducing survival autophagy</article-title><source>Autophagy</source><year>2012</year><volume>8</volume><issue>7</issue><fpage>1098</fpage><lpage>1112</lpage><pub-id pub-id-type="doi">10.4161/auto.20268</pub-id><pub-id pub-id-type="pmid">22647487</pub-id></element-citation></ref><ref id="CR38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Parsons</surname><given-names>DW</given-names></name><name><surname>Jones</surname><given-names>S</given-names></name><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Lin</surname><given-names>JC</given-names></name><name><surname>Leary</surname><given-names>RJ</given-names></name><name><surname>Angenendt</surname><given-names>P</given-names></name><name><surname>Mankoo</surname><given-names>P</given-names></name><name><surname>Carter</surname><given-names>H</given-names></name><name><surname>Siu</surname><given-names>IM</given-names></name><name><surname>Gallia</surname><given-names>GL</given-names></name><etal></etal></person-group><article-title>An integrated genomic analysis of human glioblastoma multiforme</article-title><source>Science</source><year>2008</year><volume>321</volume><issue>5897</issue><fpage>1807</fpage><lpage>1812</lpage><pub-id pub-id-type="doi">10.1126/science.1164382</pub-id><pub-id pub-id-type="pmid">18772396</pub-id></element-citation></ref><ref id="CR39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Patel</surname><given-names>AP</given-names></name><name><surname>Tirosh</surname><given-names>I</given-names></name><name><surname>Trombetta</surname><given-names>JJ</given-names></name><name><surname>Shalek</surname><given-names>AK</given-names></name><name><surname>Gillespie</surname><given-names>SM</given-names></name><name><surname>Wakimoto</surname><given-names>H</given-names></name><name><surname>Cahill</surname><given-names>DP</given-names></name><name><surname>Nahed</surname><given-names>BV</given-names></name><name><surname>Curry</surname><given-names>WT</given-names></name><name><surname>Martuza</surname><given-names>RL</given-names></name><etal></etal></person-group><article-title>Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma</article-title><source>Science</source><year>2014</year><volume>344</volume><issue>6190</issue><fpage>1396</fpage><lpage>1401</lpage><pub-id pub-id-type="doi">10.1126/science.1254257</pub-id><pub-id pub-id-type="pmid">24925914</pub-id></element-citation></ref><ref id="CR40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pitz</surname><given-names>MW</given-names></name><name><surname>Eisenhauer</surname><given-names>EA</given-names></name><name><surname>MacNeil</surname><given-names>MV</given-names></name><name><surname>Thiessen</surname><given-names>B</given-names></name><name><surname>Easaw</surname><given-names>JC</given-names></name><name><surname>Macdonald</surname><given-names>DR</given-names></name><name><surname>Eisenstat</surname><given-names>DD</given-names></name><name><surname>Kakumanu</surname><given-names>AS</given-names></name><name><surname>Salim</surname><given-names>M</given-names></name><name><surname>Chalchal</surname><given-names>H</given-names></name><etal></etal></person-group><article-title>Phase II study of PX-866 in recurrent glioblastoma</article-title><source>Neuro-Oncology</source><year>2015</year><volume>17</volume><issue>9</issue><fpage>1270</fpage><lpage>1274</lpage><pub-id pub-id-type="pmid">25605819</pub-id></element-citation></ref><ref id="CR41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Prados</surname><given-names>MD</given-names></name><name><surname>Byron</surname><given-names>SA</given-names></name><name><surname>Tran</surname><given-names>NL</given-names></name><name><surname>Phillips</surname><given-names>JJ</given-names></name><name><surname>Molinaro</surname><given-names>AM</given-names></name><name><surname>Ligon</surname><given-names>KL</given-names></name><name><surname>Wen</surname><given-names>PY</given-names></name><name><surname>Kuhn</surname><given-names>JG</given-names></name><name><surname>Mellinghoff</surname><given-names>IK</given-names></name><name><surname>de Groot</surname><given-names>JF</given-names></name><etal></etal></person-group><article-title>Toward precision medicine in glioblastoma: the promise and the challenges</article-title><source>Neuro-Oncology</source><year>2015</year><volume>17</volume><issue>8</issue><fpage>1051</fpage><lpage>1063</lpage><pub-id pub-id-type="doi">10.1093/neuonc/nov031</pub-id><pub-id pub-id-type="pmid">25934816</pub-id></element-citation></ref><ref id="CR42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Roos</surname><given-names>A</given-names></name><name><surname>Dhruv</surname><given-names>HD</given-names></name><name><surname>Peng</surname><given-names>S</given-names></name><name><surname>Inge</surname><given-names>LJ</given-names></name><name><surname>Tuncali</surname><given-names>S</given-names></name><name><surname>Pineda</surname><given-names>M</given-names></name><name><surname>Millard</surname><given-names>N</given-names></name><name><surname>Mayo</surname><given-names>Z</given-names></name><name><surname>Eschbacher</surname><given-names>JM</given-names></name><name><surname>Loftus</surname><given-names>JC</given-names></name><etal></etal></person-group><article-title>EGFRvIII-Stat5 signaling enhances Glioblastoma cell migration and survival</article-title><source>Mol Cancer Res</source><year>2018</year><volume>16</volume><issue>7</issue><fpage>1185</fpage><lpage>1195</lpage><pub-id pub-id-type="doi">10.1158/1541-7786.MCR-18-0125</pub-id><pub-id pub-id-type="pmid">29724813</pub-id></element-citation></ref><ref id="CR43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rosenfeld</surname><given-names>MR</given-names></name><name><surname>Ye</surname><given-names>X</given-names></name><name><surname>Supko</surname><given-names>JG</given-names></name><name><surname>Desideri</surname><given-names>S</given-names></name><name><surname>Grossman</surname><given-names>SA</given-names></name><name><surname>Brem</surname><given-names>S</given-names></name><name><surname>Mikkelson</surname><given-names>T</given-names></name><name><surname>Wang</surname><given-names>D</given-names></name><name><surname>Chang</surname><given-names>YC</given-names></name><name><surname>Hu</surname><given-names>J</given-names></name><etal></etal></person-group><article-title>A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme</article-title><source>Autophagy</source><year>2014</year><volume>10</volume><issue>8</issue><fpage>1359</fpage><lpage>1368</lpage><pub-id pub-id-type="doi">10.4161/auto.28984</pub-id><pub-id pub-id-type="pmid">24991840</pub-id></element-citation></ref><ref id="CR44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Roy</surname><given-names>S</given-names></name><name><surname>Lahiri</surname><given-names>D</given-names></name><name><surname>Maji</surname><given-names>T</given-names></name><name><surname>Biswas</surname><given-names>J</given-names></name></person-group><article-title>Recurrent Glioblastoma: where we stand</article-title><source>South Asian J Cancer</source><year>2015</year><volume>4</volume><issue>4</issue><fpage>163</fpage><lpage>173</lpage><pub-id pub-id-type="doi">10.4103/2278-330X.175953</pub-id><pub-id pub-id-type="pmid">26981507</pub-id></element-citation></ref><ref id="CR45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Russell</surname><given-names>RC</given-names></name><name><surname>Tian</surname><given-names>Y</given-names></name><name><surname>Yuan</surname><given-names>H</given-names></name><name><surname>Park</surname><given-names>HW</given-names></name><name><surname>Chang</surname><given-names>YY</given-names></name><name><surname>Kim</surname><given-names>J</given-names></name><name><surname>Kim</surname><given-names>H</given-names></name><name><surname>Neufeld</surname><given-names>TP</given-names></name><name><surname>Dillin</surname><given-names>A</given-names></name><name><surname>Guan</surname><given-names>KL</given-names></name></person-group><article-title>ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase</article-title><source>Nat Cell Biol</source><year>2013</year><volume>15</volume><issue>7</issue><fpage>741</fpage><lpage>750</lpage><pub-id pub-id-type="doi">10.1038/ncb2757</pub-id><pub-id pub-id-type="pmid">23685627</pub-id></element-citation></ref><ref id="CR46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Siegel</surname><given-names>RL</given-names></name><name><surname>Miller</surname><given-names>KD</given-names></name><name><surname>Jemal</surname><given-names>A</given-names></name></person-group><article-title>Cancer statistics, 2018</article-title><source>CA Cancer J Clin</source><year>2018</year><volume>68</volume><issue>1</issue><fpage>7</fpage><lpage>30</lpage><pub-id pub-id-type="doi">10.3322/caac.21442</pub-id><pub-id pub-id-type="pmid">29313949</pub-id></element-citation></ref><ref id="CR47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sottoriva</surname><given-names>A</given-names></name><name><surname>Spiteri</surname><given-names>I</given-names></name><name><surname>Piccirillo</surname><given-names>SG</given-names></name><name><surname>Touloumis</surname><given-names>A</given-names></name><name><surname>Collins</surname><given-names>VP</given-names></name><name><surname>Marioni</surname><given-names>JC</given-names></name><name><surname>Curtis</surname><given-names>C</given-names></name><name><surname>Watts</surname><given-names>C</given-names></name><name><surname>Tavare</surname><given-names>S</given-names></name></person-group><article-title>Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics</article-title><source>Proc Natl Acad Sci U S A</source><year>2013</year><volume>110</volume><issue>10</issue><fpage>4009</fpage><lpage>4014</lpage><pub-id pub-id-type="doi">10.1073/pnas.1219747110</pub-id><pub-id pub-id-type="pmid">23412337</pub-id></element-citation></ref><ref id="CR48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stjepanovic</surname><given-names>G</given-names></name><name><surname>Baskaran</surname><given-names>S</given-names></name><name><surname>Lin</surname><given-names>MG</given-names></name><name><surname>Hurley</surname><given-names>JH</given-names></name></person-group><article-title>Vps34 kinase domain dynamics regulate the Autophagic PI 3-kinase complex</article-title><source>Mol Cell</source><year>2017</year><volume>67</volume><issue>3</issue><fpage>528</fpage><lpage>534</lpage><pub-id pub-id-type="doi">10.1016/j.molcel.2017.07.003</pub-id><pub-id pub-id-type="pmid">28757208</pub-id></element-citation></ref><ref id="CR49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stupp</surname><given-names>R</given-names></name><name><surname>Mason</surname><given-names>WP</given-names></name><name><surname>van den Bent</surname><given-names>MJ</given-names></name><name><surname>Weller</surname><given-names>M</given-names></name><name><surname>Fisher</surname><given-names>B</given-names></name><name><surname>Taphoorn</surname><given-names>MJ</given-names></name><name><surname>Belanger</surname><given-names>K</given-names></name><name><surname>Brandes</surname><given-names>AA</given-names></name><name><surname>Marosi</surname><given-names>C</given-names></name><name><surname>Bogdahn</surname><given-names>U</given-names></name><etal></etal></person-group><article-title>Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma</article-title><source>N Engl J Med</source><year>2005</year><volume>352</volume><issue>10</issue><fpage>987</fpage><lpage>996</lpage><pub-id pub-id-type="doi">10.1056/NEJMoa043330</pub-id><pub-id pub-id-type="pmid">15758009</pub-id></element-citation></ref><ref id="CR50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thomas</surname><given-names>AA</given-names></name><name><surname>Brennan</surname><given-names>CW</given-names></name><name><surname>DeAngelis</surname><given-names>LM</given-names></name><name><surname>Omuro</surname><given-names>AM</given-names></name></person-group><article-title>Emerging therapies for glioblastoma</article-title><source>JAMA Neurol</source><year>2014</year><volume>71</volume><issue>11</issue><fpage>1437</fpage><lpage>1444</lpage><pub-id pub-id-type="doi">10.1001/jamaneurol.2014.1701</pub-id><pub-id pub-id-type="pmid">25244650</pub-id></element-citation></ref><ref id="CR51"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Tellingen</surname><given-names>O</given-names></name><name><surname>Yetkin-Arik</surname><given-names>B</given-names></name><name><surname>de Gooijer</surname><given-names>MC</given-names></name><name><surname>Wesseling</surname><given-names>P</given-names></name><name><surname>Wurdinger</surname><given-names>T</given-names></name><name><surname>de Vries</surname><given-names>HE</given-names></name></person-group><article-title>Overcoming the blood-brain tumor barrier for effective glioblastoma treatment</article-title><source>Drug Resist Updat</source><year>2015</year><volume>19</volume><fpage>1</fpage><lpage>12</lpage><pub-id pub-id-type="doi">10.1016/j.drup.2015.02.002</pub-id><pub-id pub-id-type="pmid">25791797</pub-id></element-citation></ref><ref id="CR52"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>White</surname><given-names>E</given-names></name></person-group><article-title>The role for autophagy in cancer</article-title><source>J Clin Invest</source><year>2015</year><volume>125</volume><issue>1</issue><fpage>42</fpage><lpage>46</lpage><pub-id pub-id-type="doi">10.1172/JCI73941</pub-id><pub-id pub-id-type="pmid">25654549</pub-id></element-citation></ref><ref id="CR53"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wurstle</surname><given-names>S</given-names></name><name><surname>Schneider</surname><given-names>F</given-names></name><name><surname>Ringel</surname><given-names>F</given-names></name><name><surname>Gempt</surname><given-names>J</given-names></name><name><surname>Lammer</surname><given-names>F</given-names></name><name><surname>Delbridge</surname><given-names>C</given-names></name><name><surname>Wu</surname><given-names>W</given-names></name><name><surname>Schlegel</surname><given-names>J</given-names></name></person-group><article-title>Temozolomide induces autophagy in primary and established glioblastoma cells in an EGFR independent manner</article-title><source>Oncol Lett</source><year>2017</year><volume>14</volume><issue>1</issue><fpage>322</fpage><lpage>328</lpage><pub-id pub-id-type="doi">10.3892/ol.2017.6107</pub-id><pub-id pub-id-type="pmid">28693171</pub-id></element-citation></ref><ref id="CR54"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Xie</surname><given-names>Q</given-names></name><name><surname>Mittal</surname><given-names>S</given-names></name><name><surname>Berens</surname><given-names>ME</given-names></name></person-group><article-title>Targeting adaptive glioblastoma: an overview of proliferation and invasion</article-title><source>Neuro-Oncology</source><year>2014</year><volume>16</volume><issue>12</issue><fpage>1575</fpage><lpage>1584</lpage><pub-id pub-id-type="doi">10.1093/neuonc/nou147</pub-id><pub-id pub-id-type="pmid">25082799</pub-id></element-citation></ref><ref id="CR55"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yin</surname><given-names>Z</given-names></name><name><surname>Pascual</surname><given-names>C</given-names></name><name><surname>Klionsky</surname><given-names>DJ</given-names></name></person-group><article-title>Autophagy: machinery and regulation</article-title><source>Microb Cell</source><year>2016</year><volume>3</volume><issue>12</issue><fpage>588</fpage><lpage>596</lpage><pub-id pub-id-type="doi">10.15698/mic2016.12.546</pub-id><pub-id pub-id-type="pmid">28357331</pub-id></element-citation></ref><ref id="CR56"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yu</surname><given-names>X</given-names></name><name><surname>Long</surname><given-names>YC</given-names></name><name><surname>Shen</surname><given-names>HM</given-names></name></person-group><article-title>Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy</article-title><source>Autophagy</source><year>2015</year><volume>11</volume><issue>10</issue><fpage>1711</fpage><lpage>1728</lpage><pub-id pub-id-type="doi">10.1080/15548627.2015.1043076</pub-id><pub-id pub-id-type="pmid">26018563</pub-id></element-citation></ref><ref id="CR57"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhao</surname><given-names>HF</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Shao</surname><given-names>W</given-names></name><name><surname>Wu</surname><given-names>CP</given-names></name><name><surname>Chen</surname><given-names>ZP</given-names></name><name><surname>To</surname><given-names>ST</given-names></name><name><surname>Li</surname><given-names>WP</given-names></name></person-group><article-title>Recent advances in the use of PI3K inhibitors for glioblastoma multiforme: current preclinical and clinical development</article-title><source>Mol Cancer</source><year>2017</year><volume>16</volume><issue>1</issue><fpage>100</fpage><pub-id pub-id-type="doi">10.1186/s12943-017-0670-3</pub-id><pub-id pub-id-type="pmid">28592260</pub-id></element-citation></ref><ref id="CR58"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zou</surname><given-names>Z</given-names></name><name><surname>Yuan</surname><given-names>Z</given-names></name><name><surname>Zhang</surname><given-names>Q</given-names></name><name><surname>Long</surname><given-names>Z</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name><name><surname>Tang</surname><given-names>Z</given-names></name><name><surname>Zhu</surname><given-names>Y</given-names></name><name><surname>Chen</surname><given-names>S</given-names></name><name><surname>Xu</surname><given-names>J</given-names></name><name><surname>Yan</surname><given-names>M</given-names></name><etal></etal></person-group><article-title>Aurora kinase a inhibition-induced autophagy triggers drug resistance in breast cancer cells</article-title><source>Autophagy</source><year>2012</year><volume>8</volume><issue>12</issue><fpage>1798</fpage><lpage>1810</lpage><pub-id pub-id-type="doi">10.4161/auto.22110</pub-id><pub-id pub-id-type="pmid">23026799</pub-id></element-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/ChloroquineV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000135 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000135 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= ChloroquineV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:6854621
|texte= Inhibition of phosphatidylinositol 3-kinase by PX-866 suppresses temozolomide-induced autophagy and promotes apoptosis in glioblastoma cells
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:31726966" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a ChloroquineV1
| This area was generated with Dilib version V0.6.33. |  |