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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">FLT3-ITDs Instruct a Myeloid Differentiation and Transformation Bias in Lymphomyeloid Multipotent Progenitors</title><author><name sortKey="Mead, Adam J" sort="Mead, Adam J" uniqKey="Mead A" first="Adam J." last="Mead">Adam J. Mead</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Kharazi, Shabnam" sort="Kharazi, Shabnam" uniqKey="Kharazi S" first="Shabnam" last="Kharazi">Shabnam Kharazi</name><affiliation><nlm:aff id="aff3">Hematopoietic Stem Cell Laboratory, Lund Stem Cell Center, Lund University, Lund 22184, Sweden</nlm:aff></affiliation></author><author><name sortKey="Atkinson, Deborah" sort="Atkinson, Deborah" uniqKey="Atkinson D" first="Deborah" last="Atkinson">Deborah Atkinson</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Macaulay, Iain" sort="Macaulay, Iain" uniqKey="Macaulay I" first="Iain" last="Macaulay">Iain Macaulay</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Pecquet, Christian" sort="Pecquet, Christian" uniqKey="Pecquet C" first="Christian" last="Pecquet">Christian Pecquet</name><affiliation><nlm:aff id="aff4">Ludwig Institute for Cancer Research, Brussels B1200, Belgium</nlm:aff></affiliation><affiliation><nlm:aff id="aff5">de Duve Institute, Université Catholique de Louvain, Brussels B1200, Belgium</nlm:aff></affiliation></author><author><name sortKey="Loughran, Stephen" sort="Loughran, Stephen" uniqKey="Loughran S" first="Stephen" last="Loughran">Stephen Loughran</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Lutteropp, Michael" sort="Lutteropp, Michael" uniqKey="Lutteropp M" first="Michael" last="Lutteropp">Michael Lutteropp</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Woll, Petter" sort="Woll, Petter" uniqKey="Woll P" first="Petter" last="Woll">Petter Woll</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Chowdhury, Onima" sort="Chowdhury, Onima" uniqKey="Chowdhury O" first="Onima" last="Chowdhury">Onima Chowdhury</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Luc, Sidinh" sort="Luc, Sidinh" uniqKey="Luc S" first="Sidinh" last="Luc">Sidinh Luc</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Buza Vidas, Natalija" sort="Buza Vidas, Natalija" uniqKey="Buza Vidas N" first="Natalija" last="Buza-Vidas">Natalija Buza-Vidas</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Ferry, Helen" sort="Ferry, Helen" uniqKey="Ferry H" first="Helen" last="Ferry">Helen Ferry</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Clark, Sally Ann" sort="Clark, Sally Ann" uniqKey="Clark S" first="Sally-Ann" last="Clark">Sally-Ann Clark</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Goardon, Nicolas" sort="Goardon, Nicolas" uniqKey="Goardon N" first="Nicolas" last="Goardon">Nicolas Goardon</name><affiliation><nlm:aff id="aff2">MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Vyas, Paresh" sort="Vyas, Paresh" uniqKey="Vyas P" first="Paresh" last="Vyas">Paresh Vyas</name><affiliation><nlm:aff id="aff2">MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Constantinescu, Stefan N" sort="Constantinescu, Stefan N" uniqKey="Constantinescu S" first="Stefan N." last="Constantinescu">Stefan N. Constantinescu</name><affiliation><nlm:aff id="aff4">Ludwig Institute for Cancer Research, Brussels B1200, Belgium</nlm:aff></affiliation><affiliation><nlm:aff id="aff5">de Duve Institute, Université Catholique de Louvain, Brussels B1200, Belgium</nlm:aff></affiliation></author><author><name sortKey="Sitnicka, Ewa" sort="Sitnicka, Ewa" uniqKey="Sitnicka E" first="Ewa" last="Sitnicka">Ewa Sitnicka</name><affiliation><nlm:aff id="aff3">Hematopoietic Stem Cell Laboratory, Lund Stem Cell Center, Lund University, Lund 22184, Sweden</nlm:aff></affiliation></author><author><name sortKey="Nerlov, Claus" sort="Nerlov, Claus" uniqKey="Nerlov C" first="Claus" last="Nerlov">Claus Nerlov</name><affiliation><nlm:aff id="aff2">MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation><affiliation><nlm:aff id="aff6">Institute for Stem Cell Research, MRC Centre for Regenerative Medicine, University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh EH93JQ, UK</nlm:aff></affiliation></author><author><name sortKey="Jacobsen, Sten Eirik W" sort="Jacobsen, Sten Eirik W" uniqKey="Jacobsen S" first="Sten Eirik W." last="Jacobsen">Sten Eirik W. Jacobsen</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">23727242</idno><idno type="pmc">3701326</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701326</idno><idno type="RBID">PMC:3701326</idno><idno type="doi">10.1016/j.celrep.2013.04.031</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">000255</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">FLT3-ITDs Instruct a Myeloid Differentiation and Transformation Bias in Lymphomyeloid Multipotent Progenitors</title><author><name sortKey="Mead, Adam J" sort="Mead, Adam J" uniqKey="Mead A" first="Adam J." last="Mead">Adam J. Mead</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Kharazi, Shabnam" sort="Kharazi, Shabnam" uniqKey="Kharazi S" first="Shabnam" last="Kharazi">Shabnam Kharazi</name><affiliation><nlm:aff id="aff3">Hematopoietic Stem Cell Laboratory, Lund Stem Cell Center, Lund University, Lund 22184, Sweden</nlm:aff></affiliation></author><author><name sortKey="Atkinson, Deborah" sort="Atkinson, Deborah" uniqKey="Atkinson D" first="Deborah" last="Atkinson">Deborah Atkinson</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Macaulay, Iain" sort="Macaulay, Iain" uniqKey="Macaulay I" first="Iain" last="Macaulay">Iain Macaulay</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Pecquet, Christian" sort="Pecquet, Christian" uniqKey="Pecquet C" first="Christian" last="Pecquet">Christian Pecquet</name><affiliation><nlm:aff id="aff4">Ludwig Institute for Cancer Research, Brussels B1200, Belgium</nlm:aff></affiliation><affiliation><nlm:aff id="aff5">de Duve Institute, Université Catholique de Louvain, Brussels B1200, Belgium</nlm:aff></affiliation></author><author><name sortKey="Loughran, Stephen" sort="Loughran, Stephen" uniqKey="Loughran S" first="Stephen" last="Loughran">Stephen Loughran</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Lutteropp, Michael" sort="Lutteropp, Michael" uniqKey="Lutteropp M" first="Michael" last="Lutteropp">Michael Lutteropp</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Woll, Petter" sort="Woll, Petter" uniqKey="Woll P" first="Petter" last="Woll">Petter Woll</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Chowdhury, Onima" sort="Chowdhury, Onima" uniqKey="Chowdhury O" first="Onima" last="Chowdhury">Onima Chowdhury</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Luc, Sidinh" sort="Luc, Sidinh" uniqKey="Luc S" first="Sidinh" last="Luc">Sidinh Luc</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Buza Vidas, Natalija" sort="Buza Vidas, Natalija" uniqKey="Buza Vidas N" first="Natalija" last="Buza-Vidas">Natalija Buza-Vidas</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Ferry, Helen" sort="Ferry, Helen" uniqKey="Ferry H" first="Helen" last="Ferry">Helen Ferry</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Clark, Sally Ann" sort="Clark, Sally Ann" uniqKey="Clark S" first="Sally-Ann" last="Clark">Sally-Ann Clark</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Goardon, Nicolas" sort="Goardon, Nicolas" uniqKey="Goardon N" first="Nicolas" last="Goardon">Nicolas Goardon</name><affiliation><nlm:aff id="aff2">MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Vyas, Paresh" sort="Vyas, Paresh" uniqKey="Vyas P" first="Paresh" last="Vyas">Paresh Vyas</name><affiliation><nlm:aff id="aff2">MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author><author><name sortKey="Constantinescu, Stefan N" sort="Constantinescu, Stefan N" uniqKey="Constantinescu S" first="Stefan N." last="Constantinescu">Stefan N. Constantinescu</name><affiliation><nlm:aff id="aff4">Ludwig Institute for Cancer Research, Brussels B1200, Belgium</nlm:aff></affiliation><affiliation><nlm:aff id="aff5">de Duve Institute, Université Catholique de Louvain, Brussels B1200, Belgium</nlm:aff></affiliation></author><author><name sortKey="Sitnicka, Ewa" sort="Sitnicka, Ewa" uniqKey="Sitnicka E" first="Ewa" last="Sitnicka">Ewa Sitnicka</name><affiliation><nlm:aff id="aff3">Hematopoietic Stem Cell Laboratory, Lund Stem Cell Center, Lund University, Lund 22184, Sweden</nlm:aff></affiliation></author><author><name sortKey="Nerlov, Claus" sort="Nerlov, Claus" uniqKey="Nerlov C" first="Claus" last="Nerlov">Claus Nerlov</name><affiliation><nlm:aff id="aff2">MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation><affiliation><nlm:aff id="aff6">Institute for Stem Cell Research, MRC Centre for Regenerative Medicine, University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh EH93JQ, UK</nlm:aff></affiliation></author><author><name sortKey="Jacobsen, Sten Eirik W" sort="Jacobsen, Sten Eirik W" uniqKey="Jacobsen S" first="Sten Eirik W." last="Jacobsen">Sten Eirik W. Jacobsen</name><affiliation><nlm:aff id="aff1">Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</nlm:aff></affiliation></author></analytic><series><title level="j">Cell Reports</title><idno type="eISSN">2211-1247</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Summary</title><p>Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function <italic>Flt3-ITD</italic> mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with <italic>Runx1</italic> mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor <italic>Pu.1</italic>, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Cell Rep</journal-id><journal-id journal-id-type="iso-abbrev">Cell Rep</journal-id><journal-title-group><journal-title>Cell Reports</journal-title></journal-title-group><issn pub-type="epub">2211-1247</issn><publisher><publisher-name>Cell Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">23727242</article-id><article-id pub-id-type="pmc">3701326</article-id><article-id pub-id-type="publisher-id">S2211-1247(13)00213-1</article-id><article-id pub-id-type="doi">10.1016/j.celrep.2013.04.031</article-id><article-categories><subj-group subj-group-type="heading"><subject>Report</subject></subj-group></article-categories><title-group><article-title>FLT3-ITDs Instruct a Myeloid Differentiation and Transformation Bias in Lymphomyeloid Multipotent Progenitors</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Mead</surname><given-names>Adam J.</given-names></name><email>adam.mead@imm.ox.ac.uk</email><xref rid="aff1" ref-type="aff">1</xref><xref rid="cor1" ref-type="corresp">∗</xref></contrib><contrib contrib-type="author"><name><surname>Kharazi</surname><given-names>Shabnam</given-names></name><xref rid="aff3" ref-type="aff">3</xref></contrib><contrib contrib-type="author"><name><surname>Atkinson</surname><given-names>Deborah</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Macaulay</surname><given-names>Iain</given-names></name><xref rid="aff1" ref-type="aff">1</xref><xref rid="aff2" ref-type="aff">2</xref></contrib><contrib contrib-type="author"><name><surname>Pecquet</surname><given-names>Christian</given-names></name><xref rid="aff4" ref-type="aff">4</xref><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author"><name><surname>Loughran</surname><given-names>Stephen</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Lutteropp</surname><given-names>Michael</given-names></name><xref rid="aff1" ref-type="aff">1</xref><xref rid="aff2" ref-type="aff">2</xref></contrib><contrib contrib-type="author"><name><surname>Woll</surname><given-names>Petter</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Chowdhury</surname><given-names>Onima</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Luc</surname><given-names>Sidinh</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Buza-Vidas</surname><given-names>Natalija</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Ferry</surname><given-names>Helen</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Clark</surname><given-names>Sally-Ann</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Goardon</surname><given-names>Nicolas</given-names></name><xref rid="aff2" ref-type="aff">2</xref></contrib><contrib contrib-type="author"><name><surname>Vyas</surname><given-names>Paresh</given-names></name><xref rid="aff2" ref-type="aff">2</xref></contrib><contrib contrib-type="author"><name><surname>Constantinescu</surname><given-names>Stefan N.</given-names></name><xref rid="aff4" ref-type="aff">4</xref><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author"><name><surname>Sitnicka</surname><given-names>Ewa</given-names></name><xref rid="aff3" ref-type="aff">3</xref></contrib><contrib contrib-type="author"><name><surname>Nerlov</surname><given-names>Claus</given-names></name><xref rid="aff2" ref-type="aff">2</xref><xref rid="aff6" ref-type="aff">6</xref></contrib><contrib contrib-type="author"><name><surname>Jacobsen</surname><given-names>Sten Eirik W.</given-names></name><email>sten.jacobsen@imm.ox.ac.uk</email><xref rid="aff1" ref-type="aff">1</xref><xref rid="aff2" ref-type="aff">2</xref><xref rid="cor2" ref-type="corresp">∗∗</xref></contrib></contrib-group><aff id="aff1"><label>1</label>Haematopoietic Stem Cell Biology Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</aff><aff id="aff2"><label>2</label>MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK</aff><aff id="aff3"><label>3</label>Hematopoietic Stem Cell Laboratory, Lund Stem Cell Center, Lund University, Lund 22184, Sweden</aff><aff id="aff4"><label>4</label>Ludwig Institute for Cancer Research, Brussels B1200, Belgium</aff><aff id="aff5"><label>5</label>de Duve Institute, Université Catholique de Louvain, Brussels B1200, Belgium</aff><aff id="aff6"><label>6</label>Institute for Stem Cell Research, MRC Centre for Regenerative Medicine, University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh EH93JQ, UK</aff><author-notes><corresp id="cor1"><label>∗</label>Corresponding author <email>adam.mead@imm.ox.ac.uk</email></corresp><corresp id="cor2"><label>∗∗</label>Corresponding author <email>sten.jacobsen@imm.ox.ac.uk</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>27</day><month>6</month><year>2013</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><day>27</day><month>6</month><year>2013</year></pub-date><volume>3</volume><issue>6</issue><fpage>1766</fpage><lpage>1776</lpage><history><date date-type="received"><day>12</day><month>1</month><year>2012</year></date><date date-type="rev-recd"><day>12</day><month>3</month><year>2013</year></date><date date-type="accepted"><day>29</day><month>4</month><year>2013</year></date></history><permissions><copyright-statement>© 2013 The Authors</copyright-statement><copyright-year>2013</copyright-year><license xlink:href="https://creativecommons.org/licenses/by-nc-nd/3.0/"><license-p>Open Access under <ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by-nc-nd/3.0/">CC BY-NC-ND 3.0</ext-link> license</license-p></license></permissions><abstract><title>Summary</title><p>Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function <italic>Flt3-ITD</italic> mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with <italic>Runx1</italic> mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor <italic>Pu.1</italic>, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies.</p></abstract><abstract abstract-type="graphical"><title>Graphical Abstract</title><fig id="undfig1" position="anchor"><graphic xlink:href="fx1"></graphic></fig></abstract><abstract abstract-type="author-highlights"><title>Highlights</title><p><list list-type="simple"><list-item id="u0010"><label>•</label><p>Flt3-ITDs collaborate with <italic>Runx1</italic> mutation to cause acute myeloid leukemia exclusively</p></list-item><list-item id="u0015"><label>•</label><p>Flt3-ITDs instruct myeloid lineage bias in lymphoid-primed multipotent precursors</p></list-item><list-item id="u0020"><label>•</label><p>Flt3-ITDs inhibit thymic seeding by bone marrow progenitors</p></list-item><list-item id="u0025"><label>•</label><p>Flt3-ITD-induced myeloid bias and progenitor phenotype involve upregulation of Pu.1</p></list-item></list></p></abstract><abstract abstract-type="teaser"><p>In this study, Mead, Jacobsen, and colleagues demonstrate that constitutive growth factor receptor (GFR) signaling through an <italic>Flt3-ITD</italic> mutation instructs a myeloid-lineage differentiation bias to multipotent hematopoietic progenitor cells. <italic>Runx1</italic> mutation collaborated with Flt3-ITD to induce aggressive, universally myeloid-lineage leukemias, indicating that Flt3-ITD GFR signaling acts to dictate the phenotype of resulting malignancies. The Flt3-ITD-induced myeloid lineage bias involves upregulation of the transcription factor Pu.1, thus establishing how GFR signaling might elicit lineage-instructive signaling in vivo.</p></abstract></article-meta><notes><p id="misc0010">Published: May 30, 2013</p></notes></front><body><sec sec-type="intro" id="sec1"><title>Introduction</title><p>Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in normal multipotent progenitors (MPPs) and stem cells remains unclear and disputed. New insights into this process have recently been gained through studies in vitro (<xref rid="bib25" ref-type="bibr">Rieger et al., 2009</xref>); however, whether GFR signaling instructs lineage specification in vivo remains a key unresolved issue (<xref rid="bib6" ref-type="bibr">Enver and Jacobsen, 2009</xref>). Studying the impact of gain-of-function mutations is an alternative approach to determine whether and how GFRs might instruct lineage fate in vivo. Indeed, activating mutations of GFRs and downstream signaling pathways are common events in cancer, particularly in hematopoietic malignancies such as acute myeloid leukemia (AML), and often show striking associations with distinct clinical and cell-lineage phenotypes (<xref rid="bib3" ref-type="bibr">Croce, 2008</xref>). In some cases, this might simply reflect the fact that such mutations primarily target a specific cell lineage. However, more intriguingly, if a mutation targets a primitive multipotent cell, it might instruct lineage-fate decisions. As recent investigations of human AML have suggested that the propagating cell might frequently represent the counterpart of normal MPPs (<xref rid="bib8" ref-type="bibr">Goardon et al., 2011</xref>), it is possible but still unclear to what degree GFR signaling mutations might also influence lineage specification in multipotent cells.</p><p>A good example of aberrant GFR signaling associated with specific leukemia phenotypes relates to the FMS-like tyrosine kinase 3 receptor (FLT3), which is expressed in the majority of cases of AML and acute lymphoblastic leukemia (ALL) (<xref rid="bib33" ref-type="bibr">Stirewalt and Radich, 2003</xref>). Constitutively activating internal tandem duplications (ITDs) within the juxtamembrane domain of FLT3 occur in ∼25% of cases of AML, conferring an adverse prognosis (<xref rid="bib33" ref-type="bibr">Stirewalt and Radich, 2003</xref>). However, despite the high frequency of FLT3 expression in ALL and the key role of Flt3 in early lymphoid development (<xref rid="bib30" ref-type="bibr">Sitnicka et al., 2002</xref>), including high-level Flt3 expression in lymphoid-primed MPPs (LMPPs) with combined lymphoid and myeloid potential (<xref rid="bib1" ref-type="bibr">Adolfsson et al., 2005</xref>), FLT3-ITDs are rare in cases of ALL, occurring in <1% in larger series (<xref rid="bib18" ref-type="bibr">Leow et al., 2011</xref>).</p><p>As is the case for most leukemic mutations, it is unclear how often FLT3-ITDs are a true initiating event in myeloid malignancies. Indeed, a number of lines of evidence support the notion that FLT3-ITDs are often acquired secondarily to an initiating clonogenic event (<xref rid="bib7" ref-type="bibr">Gale et al., 2008</xref>; <xref rid="bib13" ref-type="bibr">Jan et al., 2012</xref>), although when involved in chronic myelomonocytic leukemia (CMML), they may indeed be the initiating mutation (<xref rid="bib17" ref-type="bibr">Lee et al., 2007</xref>). Nevertheless, in line with the normal expression pattern of Flt3 (<xref rid="bib1" ref-type="bibr">Adolfsson et al., 2005</xref>), results from studies in patients are compatible with the notion that FLT3-ITDs occur within the human MPP compartment (<xref rid="bib19" ref-type="bibr">Levis et al., 2005</xref>) and are an essential driver mutation within the founding leukemic clone (<xref rid="bib5" ref-type="bibr">Ding et al., 2012</xref>; <xref rid="bib31" ref-type="bibr">Smith et al., 2012</xref>). Thus, even though FLT3-ITDs might occur secondarily to an initiating clonogenic event in many cases, these mutations occur in multipotent cells (<xref rid="bib19" ref-type="bibr">Levis et al., 2005</xref>) and FLT3-ITD GFR signaling appears to be an essential requirement for leukemia propagation (<xref rid="bib31" ref-type="bibr">Smith et al., 2012</xref>). It is possible, therefore, that FLT3-ITDs may act to dictate the lineage fate and phenotype of the resulting leukemia. Compatible with a role of Flt3-ITD signaling in lineage determination, two different knockin mouse models of <italic>Flt3-ITD</italic> have been reported to develop a myeloproliferative phenotype exclusively (<xref rid="bib17" ref-type="bibr">Lee et al., 2007</xref>; <xref rid="bib20" ref-type="bibr">Li et al., 2008</xref>). However, although it was recently suggested that Flt3-ITDs deplete hematopoietic stem cells (HSCs) (<xref rid="bib2" ref-type="bibr">Chu et al., 2012</xref>), the key progenitor population that propagates FLT3-ITD-induced myeloid disease, as well as the cellular and molecular bases of their myeloid lineage bias, remains unclear.</p><p>Using a mouse knockin model of the <italic>Flt3-ITD</italic> mutation, we investigated the cellular and molecular mechanisms by which constitutive GFR signaling might subvert lineage specification in MPPs and alter the cell fate of early lymphoid progenitors, in order to explain the myeloid bias of the resulting leukemias.</p></sec><sec sec-type="results" id="sec2"><title>Results</title><sec id="sec2.1"><title><italic>Flt3-ITD</italic> Collaborates with <italic>Runx1</italic> Mutation to Induce Aggressive AML</title><p>To definitively determine whether physiologically expressed FLT3-ITD impacts the establishment of myeloid versus lymphoid leukemia development, we crossed <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> with <italic>Runx1</italic><sup>fl/fl</sup> (<xref rid="bib10" ref-type="bibr">Growney et al., 2005</xref>) and <italic>Mx1-Cre</italic> mice to induce <italic>Runx1</italic> deletion in MPPs. Importantly, <italic>Runx1</italic> loss-of-function mutation is associated with both lymphoid and myeloid leukemia (<xref rid="bib9" ref-type="bibr">Grossmann et al., 2011</xref>; <xref rid="bib28" ref-type="bibr">Schnittger et al., 2011</xref>). Unexpectedly, even without poly I:C induction, <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1-Cre</italic><sup>+</sup> mice developed a high-penetrance, short-latency acute leukemia (<xref rid="fig1" ref-type="fig">Figure 1</xref>A) characterized by marked leukocytosis, anemia, and thrombocytopenia (<xref rid="figs1" ref-type="fig">Figures S1</xref>A–S1C) and hepatosplenomegaly. Peripheral blood (PB) and bone marrow (BM) morphology resembled AML in all diseased mice (<xref rid="fig1" ref-type="fig">Figures 1</xref>B–1D), and myeloid lineage was confirmed by flow cytometry (<xref rid="fig1" ref-type="fig">Figures 1</xref>E and <xref rid="figs1" ref-type="fig">S1</xref>D). Development of ALL was never observed. Leukemias showed deletion of the Runx1 DNA-binding domain (<xref rid="figs1" ref-type="fig">Figure S1</xref>E). The uniform myeloid-lineage leukemias in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1-Cre</italic><sup>+</sup> mice demonstrate a key role for Flt3-ITD signaling in introducing a myeloid-lineage bias during leukemogenesis.</p></sec><sec id="sec2.2"><title>Flt3-ITDs Expand Myeloid-Biased LMPPs</title><p>We next investigated the cellular and molecular bases for FLT3-ITD-induced myeloid bias. As shown previously (<xref rid="bib17" ref-type="bibr">Lee et al., 2007</xref>), the multipotent Lin<sup>−</sup>Sca1<sup>+</sup>c-Kit<sup>+</sup> (LSK) compartment was expanded in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (<xref rid="fig2" ref-type="fig">Figure 2</xref>A). We applied CD150 and CD48 to determine the nature of the expanded cells within the LSK compartment (<xref rid="bib15" ref-type="bibr">Kiel et al., 2005</xref>), as Flt3-ITD is not detectable at the cell surface (<xref rid="figs2" ref-type="fig">Figure S2</xref>A). Notably, the expansion of LSKs was wholly attributable to a marked expansion of LSKCD150<sup>−</sup>48<sup>+</sup> MPPs in 8- to 10-week-old <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (<xref rid="fig2" ref-type="fig">Figures 2</xref>B and <xref rid="fig2" ref-type="fig">2</xref>C), in line with the recent observation that Flt3-ITD may suppress the HSC compartment (<xref rid="bib2" ref-type="bibr">Chu et al., 2012</xref>). Heterozygous (<italic>Flt3</italic><sup><italic>ITD/+</italic></sup>) mice had an intermediate phenotype between <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (<xref rid="figs2" ref-type="fig">Figures S2</xref>B and S2C). High-level Flt3 expression in the normal LSK compartment defines LMPPs that lack self-renewal and megakaryocytic (Mk) and erythroid (E) potential but sustain lymphomyeloid capability (<xref rid="bib1" ref-type="bibr">Adolfsson et al., 2005</xref>), a progenitor that is also implicated in human AML (<xref rid="bib8" ref-type="bibr">Goardon et al., 2011</xref>). Because Flt3<sup>high</sup> LMPPs reside almost exclusively in the LSKCD150<sup>−</sup>48<sup>+</sup> compartment (<xref rid="figs2" ref-type="fig">Figure S2</xref>D), we explored whether the expanded LSKCD150<sup>−</sup>48<sup>+</sup> cells in <italic>Flt3-ITD</italic> mice had LMPP-like characteristics. As expected for LMPPs (<xref rid="bib22" ref-type="bibr">Månsson et al., 2007</xref>), HSC- and MkE-affiliated gene expression was downregulated in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> LSKCD150<sup>−</sup>48<sup>+</sup> MPPs (<xref rid="fig2" ref-type="fig">Figures 2</xref>D, 2E, and <xref rid="figs2" ref-type="fig">S2</xref>E), and in line with the molecular data they possessed little or no Mk potential in vitro (<xref rid="fig2" ref-type="fig">Figure 2</xref>F). In contrast, myeloid-affiliated gene expression was upregulated in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> LSKCD150<sup>−</sup>48<sup>+</sup> cells (<xref rid="fig2" ref-type="fig">Figure 2</xref>G), paralleled by high granulocyte-macrophage (GM) potential in vitro (<xref rid="fig2" ref-type="fig">Figure 2</xref>H). Early lymphoid transcriptional programs were downregulated in both <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/+</italic></sup> LSKCD150<sup>−</sup>48<sup>+</sup> MPPs (<xref rid="fig2" ref-type="fig">Figures 2</xref>I and 2J), paralleled by severely reduced B cell potential in vitro (<xref rid="fig2" ref-type="fig">Figure 2</xref>K). Importantly, the lymphoid transcriptional program was already suppressed in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> LSKCD150<sup>−</sup>48<sup>+</sup> MPPs in embryonic day 15 (E15) fetal liver (FL) (<xref rid="figs2" ref-type="fig">Figure S2</xref>F), at which time the phenotype and number of LSKCD150<sup>−</sup>48<sup>+</sup> MPPs were unaffected (<xref rid="figs2" ref-type="fig">Figure S2</xref>G). In keeping with this suppression of early lymphoid programs in fetal MPPs, the number of B220<sup>+</sup>CD19<sup>+</sup> B cells was suppressed in <italic>Flt3-ITD</italic> E15 FL (<xref rid="figs2" ref-type="fig">Figure S2</xref>H), preceding the emergence of a myeloproliferative phenotype at this early stage of ontogeny (<xref rid="figs2" ref-type="fig">Figure S2</xref>I). This supports the notion that lymphoid suppression and myeloid bias in MPPs occurs as a cell-intrinsic and direct consequence of Flt3-ITD signaling.</p><p>Because FLT3-ITDs in humans often occur secondarily to an initiating clonogenic event (<xref rid="bib13" ref-type="bibr">Jan et al., 2012</xref>), it is possible that in such cases it is the other genetic events and not the FLT3-ITD that influence the leukemic phenotype. In order to address this issue, we used <italic>Vav-Cre</italic> mediated recombination to examine the impact of <italic>Runx1</italic> loss of function on lineage priming in MPPs in the absence of Flt3-ITD. The data obtained demonstrate a significant upregulation of <italic>Il7r</italic> and <italic>Rag1</italic> expression in MPPs with no significant impact on <italic>sIgH</italic> expression (<xref rid="figs2" ref-type="fig">Figure S2</xref>J). This finding is in keeping with the high incidence of lymphoid malignancies in mouse models of <italic>Runx1</italic> mutation (<xref rid="bib12" ref-type="bibr">Jacob et al., 2010</xref>; <xref rid="bib16" ref-type="bibr">Kundu et al., 2005</xref>; <xref rid="bib24" ref-type="bibr">Putz et al., 2006</xref>) and contrasts markedly with the suppression of lymphoid programs caused by Flt3-ITDs in MPPs. This supports the notion that it is GFR signaling through Flt3-ITD that instructs the uniform myeloid phenotype of leukemias resulting from the collaboration between Runx1 deletion and FLT3-ITD in LMPPs.</p></sec><sec id="sec2.3"><title><italic>Flt3-ITD</italic> Suppresses Early T- and B-Lymphoid Progenitors</title><p>Because <italic>Flt3-ITD</italic> compromised lymphoid-transcriptional priming in LMPPs, which have been implicated as critical thymus-seeding progenitors (<xref rid="bib21" ref-type="bibr">Luc et al., 2012</xref>), we next investigated whether T lymphopoiesis might be suppressed in <italic>Flt3-ITD</italic> mice. Thymic cellularity was found to be progressively reduced, and the earliest thymic progenitors (double-negative 1 Kit+ [DN1Kit<sup>+</sup>]) were almost completely lost in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (<xref rid="fig3" ref-type="fig">Figures 3</xref>A–3C). Also, subsequent stages of DN2Kit<sup>+</sup> and DN3 thymocyte progenitors were severely reduced (<xref rid="figs3" ref-type="fig">Figures S3</xref>A and S3B). Of relevance, gene and protein expression for the chemokine receptor Ccr9, which is critical for migration of thymus-seeding progenitors from the BM to the thymus (<xref rid="bib29" ref-type="bibr">Schwarz et al., 2007</xref>), was suppressed in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> LSKCD150<sup>−</sup>48<sup>+</sup> MPPs (<xref rid="fig3" ref-type="fig">Figures 3</xref>D, <xref rid="figs3" ref-type="fig">S3</xref>C, and S3D). Moreover, DN1 cells showed large reductions in expression of <italic>Notch1</italic> and its targets (<italic>Hes1</italic> and <italic>Hes5</italic>; <xref rid="figs3" ref-type="fig">Figure S3</xref>E), which are critical for early T cell development. Although expression of some early lymphoid genes (<italic>Il2ra</italic>, <italic>Il7r</italic>, and <italic>Gata3</italic>) was maintained, myeloid genes (<italic>Cebpa</italic>, <italic>Sfpi1</italic>, <italic>Csf1r</italic>, <italic>Csf2r</italic>, and <italic>Csf3r</italic>) were highly upregulated in DN1 thymic progenitors (<xref rid="figs3" ref-type="fig">Figure S3</xref>E). In keeping with this myeloid bias, <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> DN1 progenitors showed low T cell potential in vitro (<xref rid="fig3" ref-type="fig">Figure 3</xref>E).</p><p>Previous studies demonstrated a reduction of mature B cells in <italic>Flt3-ITD</italic> mice (<xref rid="bib17" ref-type="bibr">Lee et al., 2007</xref>; <xref rid="bib20" ref-type="bibr">Li et al., 2008</xref>). We investigated whether this might reflect <italic>Flt3-ITD</italic>-induced perturbation at the earliest B cell commitment stages, and in support of this found a notable 11-fold expansion of Lin<sup>−</sup>CD19<sup>−</sup>CD24<sup>−</sup>AA4.1<sup>+</sup>CD43<sup>mid</sup>B220<sup>+</sup> pre-pro-B cells in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (<xref rid="fig3" ref-type="fig">Figures 3</xref>F and 3G), whereas pro-B cells, the next stage in B cell development, were severely reduced (<xref rid="fig3" ref-type="fig">Figures 3</xref>H and <xref rid="figs3" ref-type="fig">S3</xref>F). Because pre-pro-B cells are known to sustain low-level myeloid potential (<xref rid="bib26" ref-type="bibr">Rumfelt et al., 2006</xref>), we next investigated lymphoid and myeloid gene expression in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> pre-pro-B cells. Key early lymphoid genes (<italic>Cd79a</italic>, <italic>Il7r</italic>, <italic>sIgh</italic>, <italic>Ebf1</italic>, <italic>Pax5</italic>, <italic>Rag1</italic>, and <italic>Rag2</italic>) were all suppressed (<xref rid="fig3" ref-type="fig">Figure 3</xref>I), whereas myeloid-affiliated genes (<italic>Cebpa</italic>, <italic>Cebpb</italic>, <italic>Sfpi1</italic>, <italic>Csf1r</italic>, and <italic>Csf2r</italic>) were upregulated in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> pre-pro-B cells (<xref rid="fig3" ref-type="fig">Figure 3</xref>J). In agreement with this, <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> pre-pro-B cells had severely reduced B cell potential (<xref rid="fig3" ref-type="fig">Figure 3</xref>K). These findings demonstrate that Flt3-ITDs markedly impair the earliest stage of both T and B lymphopoiesis and upregulate myeloid gene expression in the earliest B and T lymphoid progenitors, in agreement with the notion that myeloid bias initiates in multipotent LMPPs.</p></sec><sec id="sec2.4"><title>Flt3-ITD-Induced Myeloid Bias Is Dependent on Upregulation of Pu1</title><p>To further explore the mechanistic basis of Flt3-ITD-induced myeloid bias of early lymphoid progenitors, we examined Pu1 protein expression using a Pu1-YFP reporter line. Whereas in wild-type (WT) mice Lin<sup>−</sup> cells showed a bimodal distribution between Pu1<sup>low/−</sup> and Pu1<sup>high</sup> cells, almost all Lin<sup>−</sup> cells in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice were Pu1<sup>high</sup> (<xref rid="fig4" ref-type="fig">Figure 4</xref>A). <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice showed a modest increase in Pu1 expression in LSKCD150<sup>−</sup>48<sup>+</sup> MPPs (<xref rid="fig4" ref-type="fig">Figure 4</xref>B) and markedly enhanced levels of Pu1-YFP in pre-pro-B cells (<xref rid="fig4" ref-type="fig">Figure 4</xref>C). In keeping with the observed increased expression of Pu1, gene set enrichment analysis (GSEA) demonstrated upregulation of Pu1 target genes (<xref rid="bib32" ref-type="bibr">Steidl et al., 2006</xref>) in <italic>Flt3-ITD</italic> LSKCD150<sup>−</sup>48<sup>+</sup> MPPs (<xref rid="fig4" ref-type="fig">Figure 4</xref>D). Furthermore, GSEA also demonstrated a similar upregulation of Pu1 target genes in <italic>FLT3-ITD</italic> mutated “LMPP-like” leukemia stem cells in human AML (<xref rid="bib8" ref-type="bibr">Goardon et al., 2011</xref>) in comparison with FLT3-WT counterparts (<xref rid="figs4" ref-type="fig">Figure S4</xref>A).</p><p>Because transition from pre-pro-B cells to subsequent stages of B cell development is associated with downregulation of Pu1 expression (<xref rid="figs4" ref-type="fig">Figure S4</xref>B), we attempted to rescue the suppressed B cell phenotype in Flt3-ITD mice through generation of <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> Pu1<sup>+/−</sup> mice. Notably, whereas Pu1 haploinsufficiency did not increase the number of B cells or their progenitors on a WT background, it resulted in a 10.3-fold increase in B cells in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (<xref rid="fig4" ref-type="fig">Figure 4</xref>E). Furthermore, Pu1 haploinsufficiency led to a 2-fold reduction in the expanded pre-pro-B cell population in Flt3-ITD mice (<xref rid="fig4" ref-type="fig">Figure 4</xref>F) together with a 13.5-fold rescue of the suppression of pro-B cells (<xref rid="fig4" ref-type="fig">Figure 4</xref>G). Strikingly, in the presence of Pu1 haploinsufficiency, the expansion of MPPs was also restored to WT levels (<xref rid="fig4" ref-type="fig">Figure 4</xref>H) together with a significant amelioration of the myeloproliferative phenotype in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (<xref rid="fig4" ref-type="fig">Figure 4</xref>I).</p><p>Stat3 is aberrantly activated by Flt3-ITD signaling (<xref rid="bib27" ref-type="bibr">Schmidt-Arras et al., 2009</xref>) and Pu1 is a direct target gene of Stat3 (<xref rid="bib11" ref-type="bibr">Hegde et al., 2009</xref>). In line with this, <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> BM showed aberrant activation of STAT3 (<xref rid="figs4" ref-type="fig">Figure S4</xref>C), and MPPs and pre-pro-B cells showed significantly increased expression of Stat3 target genes (<xref rid="fig4" ref-type="fig">Figures 4</xref>J–4L). Collectively, these data suggest that Pu1 upregulation is a key mediator of the Flt3-ITD-induced myeloid bias and progenitor phenotype.</p></sec></sec><sec sec-type="discussion" id="sec3"><title>Discussion</title><p>In the studies presented here, we explored whether and how GFR signaling might influence lineage specification in vivo. To that end, we used a knockin model of <italic>Flt3-ITD</italic> to investigate whether oncogenic mutations that result in constitutive GFR signaling influence the lineage fate of MPPs in vivo.</p><p>In order to first establish the physiological relevance of Flt3-ITD as an AML-inducing mutation, we developed a mouse model in which <italic>Flt3-ITD</italic> was coexpressed with an inducible deletion of the DNA-binding domain of <italic>Runx1</italic> (<xref rid="bib10" ref-type="bibr">Growney et al., 2005</xref>) in MPPs. Importantly, although Runx1 loss-of-function mutations are found in patients with both ALL and AML, only the latter is often found in association with FLT3-ITDs (<xref rid="bib9" ref-type="bibr">Grossmann et al., 2011</xref>; <xref rid="bib28" ref-type="bibr">Schnittger et al., 2011</xref>). Deletion of the DNA-binding domain of Runx1 during adult murine hematopoiesis results in mild myeloproliferation (<xref rid="bib10" ref-type="bibr">Growney et al., 2005</xref>) or lymphoblastic leukemias/lymphomas (<xref rid="bib12" ref-type="bibr">Jacob et al., 2010</xref>; <xref rid="bib16" ref-type="bibr">Kundu et al., 2005</xref>; <xref rid="bib24" ref-type="bibr">Putz et al., 2006</xref>). Here, we demonstrate that the combination of <italic>Runx1</italic> mutation with Flt3-ITDs results in aggressive leukemia with 100% penetrance and a uniform myeloid phenotype. In addition to demonstrating the functional relevance for FLT3-ITD-induced AML, this AML model has a number of important features. First, in contrast to many other mutation collaboration models, both mutations are targeted to the physiologically relevant endogenous genetic loci. Second, in isolation each mutation results in only a modest phenotype, but in combination a minor clone of deleted cells rapidly expands and becomes clonally dominant, paralleling somatic mutations during leukemogenesis. Third, the evolution of leukemia is rapid, supporting the notion that it occurs without the requirement for additional genetic events. Finally, the model recapitulates a particularly poor prognostic form of AML with a major unmet need for novel therapeutic approaches and thus provides a powerful model for future studies of the cellular and molecular mechanistic bases for collaboration of these mutations in AML.</p><p>The most primitive progenitor population that was expanded by Flt3-ITDs consisted of LMPP-like cells, demonstrating upregulation of the myeloid program, whereas the transcriptional expression of lymphoid-affiliated genes was markedly reduced in both heterozygous and homozygous Flt3-ITD mice. This myeloid transcriptional bias and suppression of lymphoid transcripts was also present during early B and T cell development and, importantly, was already present in Flt3-ITD FL MPPs, preceding the expansion of MPPs and development of a myeloproliferative phenotype, supporting the notion that it is a direct consequence of Flt3-ITD signaling in LMPPs. The severe suppression of the earliest thymic progenitors in <italic>Flt3-ITD</italic> mice and concomitant lack of Ccr9 upregulation in LMPPs, which have been implicated as key thymus-seeding progenitors (<xref rid="bib21" ref-type="bibr">Luc et al., 2012</xref>), further suggests that thymic seeding might also be impaired by Flt3-ITDs. These findings demonstrate that the myeloid propensity of FLT3-ITDs results from FLT3-ITD introducing a myeloid bias in multipotent lymphomyeloid progenitors as well as in the earliest B and T lymphoid progenitors.</p><p>Pu1 is a dosage-sensitive regulator of myeloid-lymphoid cell-fate decisions that promotes myeloid differentiation when overexpressed (<xref rid="bib4" ref-type="bibr">DeKoter and Singh, 2000</xref>). Using a Pu1-YFP reporter, we demonstrated that Flt3-ITDs upregulate Pu1 expression in MPPs and the earliest B lymphoid progenitors, paralleled by increased expression of Pu1 target genes in MPPs. Furthermore, we confirmed the relevance of these findings for human AML by demonstrating upregulation of Pu1 target genes in FLT3-ITD mutated LMPP-like human AML stem cells (<xref rid="bib8" ref-type="bibr">Goardon et al., 2011</xref>). In agreement with a key mechanistic role for Pu1 overexpression, Flt3-ITD-induced suppression of B cell development, as well as the Flt3-ITD-associated progenitor phenotype, was partially rescued by Pu1 haploinsufficiency.</p><p>Constitutive Flt3-ITD signaling is distinct from WT Flt3 signaling due to abnormal anchoring of FLT3-ITD in the endoplasmic reticulum, with markedly reduced surface Flt3 expression resulting in aberrant Stat3 activation, as confirmed in our study (<xref rid="bib27" ref-type="bibr">Schmidt-Arras et al., 2009</xref>). Because Pu1 is a direct target gene of Stat3 (<xref rid="bib11" ref-type="bibr">Hegde et al., 2009</xref>) and Stat3 target genes were upregulated in <italic>Flt3-ITD</italic> LSKCD150<sup>−</sup>48<sup>+</sup> MPPs and pre-pro-B cells, this provides a putative direct link between aberrant constitutive Flt3-ITD signaling and Pu1 overexpression as a cause of aberrant myeloid bias.</p><p>Several lines of evidence indicate that FLT3-ITDs frequently arise early in the transformation process in MPPs (<xref rid="bib19" ref-type="bibr">Levis et al., 2005</xref>) and as a driver mutation in the founding leukemic clone (<xref rid="bib5" ref-type="bibr">Ding et al., 2012</xref>; <xref rid="bib31" ref-type="bibr">Smith et al., 2012</xref>), and thus may influence the lineage of resulting leukemias. Together with our findings, this is consistent with a model in which LMPPs, which normally express high levels of FLT3 (<xref rid="bib1" ref-type="bibr">Adolfsson et al., 2005</xref>), acquire FLT3-ITD mutations that confer both a strong clonal advantage and a marked myeloid bias. This results in myeloid expansion and suppression of early lymphoid development, strongly supporting a fundamental role for FLT3-ITDs in promoting myeloid lineage leukemia development at the MPP level. Our study also further highlights LMPPs as a key target population in AML, as also supported by recent findings in human AML (<xref rid="bib8" ref-type="bibr">Goardon et al., 2011</xref>). Transformation to an aggressive leukemia exclusively of a myeloid phenotype by introduction of Runx1 mutation demonstrates the functional relevance of this FLT3-ITD-induced myeloid bias and clonal dominance, providing insights into the process by which oncogenic mutations might determine the lineage fate of the resulting leukemias at the precommitment stage. Our findings are also of considerable relevance for normal hematopoiesis, as it remains disputed whether key cytokine receptor signaling pathways mediate critical in vivo functions in blood lineage development through purely permissive rather than instructive actions (<xref rid="bib6" ref-type="bibr">Enver and Jacobsen, 2009</xref>). Thus, although Flt3-ITD elicits aberrant signaling, our findings clearly provide support for the notion that cytokine receptors are also capable of eliciting lineage-instructive signaling in MPPs in vivo.</p></sec><sec sec-type="methods" id="sec4"><title>Experimental Procedures</title><sec id="sec4.1"><title>Animals</title><p>All animals used were bred and maintained in accordance with regulations of the UK Home Office. Details regarding the mouse lines are provided in the <xref rid="dtbox1" ref-type="boxed-text">Extended Experimental Procedures</xref>.</p></sec><sec id="sec4.2"><title>Patient Samples</title><p>BM samples from AML patients were obtained with informed consent and the approval of Oxford Ethics Committee B (protocols 06/Q1606/110 and 05/MRE07/74).</p></sec><sec id="sec4.3"><title>Fluorescence-Activated Cell Sorting</title><p>Details of the antibody staining panels and protocols are provided in the <xref rid="dtbox1" ref-type="boxed-text">Extended Experimental Procedures</xref>. Antigens and antibodies used for identification of specific cell populations are shown in <xref rid="mmc1" ref-type="supplementary-material">Tables S1</xref> and <xref rid="mmc1" ref-type="supplementary-material">S2</xref>.</p></sec><sec id="sec4.4"><title>Western Blotting</title><p>Cells were lysed in 1% NP40 buffer and analyzed by western blot as previously described (<xref rid="bib23" ref-type="bibr">Pecquet et al., 2010</xref>) with antibodies against pStat3 (Cell Signaling) and beta-actin (Sigma).</p></sec><sec id="sec4.5"><title>In Vitro Evaluation of Lineage Potentials</title><p>Evaluation of megakaryocytic, GM, and lymphoid potentials was carried out as previously described (<xref rid="bib22" ref-type="bibr">Månsson et al., 2007</xref>) and detailed in the <xref rid="dtbox1" ref-type="boxed-text">Extended Experimental Procedures</xref>.</p></sec><sec id="sec4.6"><title>Gene Expression by Dynamic Arrays</title><p>Multiplex quantitative PCR was performed as previously described (<xref rid="bib14" ref-type="bibr">Kharazi et al., 2011</xref>) and detailed in the <xref rid="dtbox1" ref-type="boxed-text">Extended Experimental Procedures</xref>. Assays used for dynamic arrays are shown in <xref rid="mmc1" ref-type="supplementary-material">Table S3</xref>.</p></sec><sec id="sec4.7"><title>Microarray Analysis</title><p>Analysis by Affymetrix Mouse Genome 430 2.0 Arrays was carried out at the Stanford Protein and Nucleic Acid Facility as described previously (<xref rid="bib22" ref-type="bibr">Månsson et al., 2007</xref>) and in the <xref rid="dtbox1" ref-type="boxed-text">Extended Experimental Procedures</xref>.</p><p><boxed-text id="dtbox1"><label>Extended Experimental Procedures</label><sec id="dtbox1sec1"><title>Quantification of Hematologic Indices</title><p>Hemoglobin, WBC, and platelet count per milliliter of blood were quantified on a Sysmex<sup>®</sup> KX-21N 3-part differential hematology analyzer.</p></sec><sec id="dtbox1sec2"><title>Animals</title><p><italic>Flt3-ITD</italic> knockin mice on C57BL/6 background have been previously described (<xref rid="bib14" ref-type="bibr">Kharazi et al., 2011</xref>; <xref rid="bib17" ref-type="bibr">Lee et al., 2007</xref>). Homozygous (<italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup>) and heterozygous (<italic>Flt3</italic><sup><italic>ITD/+</italic></sup>) <italic>Flt3-ITD</italic> mice were studied as specified. <italic>Runx1</italic><sup><italic>fl/fl</italic></sup> mice have been previously described (<xref rid="bib37" ref-type="bibr">Grundler et al., 2005</xref>) and were on a 129S1 x CBA background. <italic>VavCre</italic> mice have been previously described (<xref rid="bib36" ref-type="bibr">de Boer et al., 2003</xref>) and were on a C57BL/6 background. <italic>Mx1-Cre</italic> mice were on C57BL/6 background and have been previously described (<xref rid="bib42" ref-type="bibr">Kühn et al., 1995</xref>). Mice carrying a germline deletion of <italic>Pu1</italic> (<italic>Pu1</italic><sup><italic>+/−</italic></sup>) were derived by crossing a previously reported <italic>Pu1</italic><sup><italic>fl/+</italic></sup> line (<xref rid="bib40" ref-type="bibr">Iwasaki et al., 2005</xref>) on a C57BL/6 × 129S1 x CBA background with a constitutive CMV-Cre recombinase, on a C57BL/6 background. Pu1-YFP mice on a C57BL/6 background have been previously described (<xref rid="bib41" ref-type="bibr">Kirstetter et al., 2006</xref>). Mice were analyzed with littermate controls of the appropriate genotype when available.</p></sec><sec id="dtbox1sec3"><title>Flow Cytometry Analysis and FACS Purification</title><p>Antibodies and viability dyes used for flow cytometry analysis are shown in <xref rid="mmc1" ref-type="supplementary-material">Table S1</xref>. Combinations of antibodies used for identification and purification of indicated populations are shown in <xref rid="mmc1" ref-type="supplementary-material">Table S2</xref>.</p><p>All antibodies were used at predetermined (titrated) optimized concentrations. Cell acquisition and analysis were performed on a 4-laser LSRII (Becton Dickinson, San Jose, CA) using FlowJo software (TreeStar, Ashland, OR). Cell sorting was done on a BD FACSAriaII cell sorter (BD Biosciences). Cells used in cell sorting experiments were either un-enriched or enriched for CD117 with MACS cell separation (Miltenyi Biotec) followed by Fc-block incubation and staining with anti-mouse antibodies. Fluorescence minus-one controls as well as negative populations were used as gate-setting controls. Gates were set using a combination of fluorescence minus one controls and also populations that are known to be negative for the antigen. For example, LSKCD150<sup>+</sup>CD48<sup>−</sup> cells, which are known to not express Flt3, were used to set the gate for surface Flt3 expression on LSKCD150<sup>−</sup>CD48<sup>+</sup> cells.</p></sec><sec id="dtbox1sec4"><title>Microarray Analysis</title><p>Global gene expression analysis was performed on LSKCD150<sup>−</sup>CD48<sup>+</sup> cells from <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> (4 mice of each genotype) as previously reported (<xref rid="bib21" ref-type="bibr">Luc et al., 2012</xref>). Two thousand cells were sorted directly into Trizol (Invitrogen) and the RNA extraction carried out as per manufacturer’s instructions and previously as described (<xref rid="bib21" ref-type="bibr">Luc et al., 2012</xref>). Using the same total amount of input RNA, samples were amplified using the NuGEN kit WT-Ovation Pico RNA Amplifications System followed by the WT Ovation cDNA Biotin Module V2 for cDNA labeling (NuGEN) and fragmentation and finally hybridized to Affymetrix Mouse Genome 430 2.0 Arrays using standard protocols (Affymetrix) at the Stanford Protein and Nucleic Acid facility. These data will be available through GEO accession number GSE35805 (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=dfcvjocouaoccds&acc=GSE35805" id="intref0010">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=dfcvjocouaoccds&acc=GSE35805</ext-link>).</p><p>Microarray data were normalized using the Robust Multi-array Averages (RMA)(<xref rid="bib39" ref-type="bibr">Irizarry et al., 2003</xref>) method in Biocondcutor/R and analyzed using gene set enrichment analysis (GSEA) (<xref rid="bib45" ref-type="bibr">Subramanian et al., 2005</xref>). Erythroid, myeloid (<xref rid="bib35" ref-type="bibr">Chambers et al., 2007</xref>), Pu1 (<xref rid="bib32" ref-type="bibr">Steidl et al., 2006</xref>), and STAT3 (<xref rid="bib34" ref-type="bibr">Alvarez et al., 2005</xref>) target gene lists have been previously described. A signature for early B cell commitment was generated using data from the Immunological Genome Project (<xref rid="bib38" ref-type="bibr">Heng et al., 2008</xref>) (<ext-link ext-link-type="uri" xlink:href="http://www.immgen.org" id="intref0015">http://www.immgen.org</ext-link>) by comparing the gene expression profile of long term reconstituting stem cells with that of pre-pro-B cells using the limma package (<xref rid="bib44" ref-type="bibr">Smyth, 2004</xref>) in R; genes which were greater than 2-fold upregulated (limma B statistic > 3) were used for the B cell commitment signature. GSEA analysis of LMPP-like human AML stem cells, stratified according to the presence of <italic>FLT3-ITD</italic> mutation, was carried out using previously described data set (<xref rid="bib8" ref-type="bibr">Goardon et al., 2011</xref>), using human equivalents of the above murine Pu1 target genes (<xref rid="bib32" ref-type="bibr">Steidl et al., 2006</xref>).</p></sec><sec id="dtbox1sec5"><title>Multiplex Quantitative Real-Time PCR Analysis</title><p>Multiplex quantitative PCR was performed as previously described (<xref rid="bib14" ref-type="bibr">Kharazi et al., 2011</xref>). For each cell population, two to four biological replicates (different mice) were prepared, and two to three separately sorted cell samples from each mouse were cell sorted and analyzed. For LSKCD150<sup>−</sup>CD48<sup>+</sup> cells, LSKFlt3<sup>−</sup> cells, and pre-pro-B cells, 100 cells per replicate were analyzed. For DN1 cells, 20 cells per replicate were analyzed as cell numbers of this population were highly limited in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice. Details of the TaqMan assays used are shown in <xref rid="mmc1" ref-type="supplementary-material">Table S3</xref>.</p><p>For cDNA synthesis and preamplification of target genes CellsDirect One-Step qRT-PCR kit (Invitrogen) was used. Cells were sorted directly into PCR plates or 0.2 ml PCR tubes containing 2.5 μl gene specific 0.2x TaqMan gene expression assays (Applied Biosystems), 5 μl of CellsDirect 2x Reaction mix (Invitrogen), 1.2 μl CellsDirect RT/Taq mix, 1.2 μl TE buffer and 0.1 μl SUPERase-In RNase Inhibitor (Ambion). Conditions for reverse transcription and target gene amplification were: 15 min at 50°C; 2 min at 95°C; 22 cycles of 95°C for 15 s and 60°C for 4 min. Preamplified products were diluted 1:5 in TE buffer and analyzed on Dynamic Array (Fluidigm) using the following PCR cycling condition: 95°C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 60 s. Data was analyzed using the ΔCt method; results were normalized to <italic>Hprt</italic> expression and expressed as mean expression level relative to <italic>Hprt</italic>.</p></sec><sec id="dtbox1sec6"><title>In Vitro Assays for Megakaryocyte, GM, and Lymphoid Potentials</title><p>Evaluation of megakaryocytic and GM potentials was carried out as previously described (<xref rid="bib43" ref-type="bibr">Luc et al., 2008</xref>). For evaluation of megakaryocytic potential, 150 cells were sorted into 3 ml of X-vivo 15 (Lonza) supplemented with 0.5% detoxified Bovine Serum Albumin (BSA; StemCell Technologies Inc), 10% fetal calf serum (FCS), 2-mercaptoethanol (2-ME; 10<sup>−4</sup> M; Sigma-Aldrich Co), 1% penicillin/streptomycin (PAA) and 50 ng/mL murine stem cell factor (mSCF; PeproTech, Rocky Hill, NJ), 50 ng/mL human fms-like tyrosine kinase 3 ligand (hFL; Immunex, Seattle, WA), 50 ng/mL human thrombopoietin (hTHPO; PeptroTech), 5 U/mL human erythropoietin (hEPO; Boehringer Mannheim, Mannheim, Germany) and 20 ng/mL murine interleukin 3 (mIL-3; PeproTech). For evaluation of GM potential, cells were sorted into media as above but with the following cytokines: mSCF, hFL, hTHPO, hG-CSF (Neopogen, Amgen, Thousand Oaks, CA) (all 25 ng/mL), mGM-CSF (Immunex, Seattle, WA) and mIL-3 (both 20 ng/mL). The cell suspension was thoroughly mixed and 20 μl was pipetted into each well of two 60-well plates per replicate (Nunc Minitrays catalog number: 163118, Nunc A/S, Roskilde, Denmark). Wells were scored for clonal growth and/or frequency of wells with megakaryocytes with an inverted light microscope after 8 days of culture. The reliability of this approach to score cultures for megakaryocytic potential has been validated by morphological validation of Giemsa stained cytospins of the cultures (<xref rid="bib22" ref-type="bibr">Månsson et al., 2007</xref>). Percentage of cloning efficiency was calculated according to the Poisson distribution which predicts that 63% of wells should contain 1 or more cells following manual plating (76 of 120 wells).</p><p>B cell and T cell potential was evaluated by sorting 10 MPPs, DN1 or pre-pro-B cells onto approximately 80% confluent monolayers of OP9 (B cell potential) and OP9DL1 (T cell potential) stromal cells, as previously described (<xref rid="bib1" ref-type="bibr">Adolfsson et al., 2005</xref>; <xref rid="bib21" ref-type="bibr">Luc et al., 2012</xref>). Clones were identified and picked at 3 to 4 weeks (depending on clonal size), and subsequently analyzed by FACS for the presence of B cell (defined as B220<sup>+</sup>CD19<sup>+</sup>) or T cell (CD4/8 double positive) committed progeny.</p></sec><sec id="dtbox1sec7"><title>Statistical Analysis</title><p>The statistical significance of differences between samples was determined using the 2-tailed t test.</p></sec></boxed-text></p></sec></sec></body><back><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal" id="sref1"><person-group person-group-type="author"><name><surname>Adolfsson</surname><given-names>J.</given-names></name><name><surname>Månsson</surname><given-names>R.</given-names></name><name><surname>Buza-Vidas</surname><given-names>N.</given-names></name><name><surname>Hultquist</surname><given-names>A.</given-names></name><name><surname>Liuba</surname><given-names>K.</given-names></name><name><surname>Jensen</surname><given-names>C.T.</given-names></name><name><surname>Bryder</surname><given-names>D.</given-names></name><name><surname>Yang</surname><given-names>L.</given-names></name><name><surname>Borge</surname><given-names>O.J.</given-names></name><name><surname>Thoren</surname><given-names>L.A.</given-names></name></person-group><article-title>Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment</article-title><source>Cell</source><volume>121</volume><year>2005</year><fpage>295</fpage><lpage>306</lpage><pub-id pub-id-type="pmid">15851035</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal" id="sref2"><person-group person-group-type="author"><name><surname>Chu</surname><given-names>S.H.</given-names></name><name><surname>Heiser</surname><given-names>D.</given-names></name><name><surname>Li</surname><given-names>L.</given-names></name><name><surname>Kaplan</surname><given-names>I.</given-names></name><name><surname>Collector</surname><given-names>M.</given-names></name><name><surname>Huso</surname><given-names>D.</given-names></name><name><surname>Sharkis</surname><given-names>S.J.</given-names></name><name><surname>Civin</surname><given-names>C.</given-names></name><name><surname>Small</surname><given-names>D.</given-names></name></person-group><article-title>FLT3-ITD knockin impairs hematopoietic stem cell quiescence/homeostasis, leading to myeloproliferative neoplasm</article-title><source>Cell Stem Cell</source><volume>11</volume><year>2012</year><fpage>346</fpage><lpage>358</lpage><pub-id pub-id-type="pmid">22958930</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal" id="sref3"><person-group person-group-type="author"><name><surname>Croce</surname><given-names>C.M.</given-names></name></person-group><article-title>Oncogenes and cancer</article-title><source>N. 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Article plus Supplemental Information</title></caption><media xlink:href="mmc2.pdf"></media></supplementary-material></p></sec><ack id="ack0010"><title>Acknowledgments</title><p>We thank Gary Gilliland for kindly providing Flt3-ITD knockin mice. A.M. and D.A. were funded by a Leukaemia and Lymphoma Research Senior Bennett Fellowship, S.K. was funded by the Pasteur Institute of Iran, and S.E.W.J. was funded through a Strategic Appointment and Programme grant from the Medical Research Council (UK) and a Hemato-Linné grant (Swedish Research Council Linnaeus). A.M., P.V., and S.E.J. were supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre based at Oxford University Hospitals NHS Trust, and by the NCRN MDSBio sample collection study. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health. A.M. designed, performed, and analyzed experiments and wrote the manuscript. Support to S.N.C. from Fondation contre le cancer, Salus Sanguinis, Action de Recherche Concertée and Interuniversity Attraction Poles Programs and Fonds de la Recherche Scientifique, Belgium and Ludwig Institute for Cancer Research is acknowledged. S.K. performed and analyzed experiments. D.A. provided technical assistance. I.M. and N.G. analyzed microarray data. S. Loughran, M.L., P.W., O.C., S. Luc, N.B.V., H.F., and S.A.C. were involved in FACS analysis/sorting. C.P. performed western blotting. E.S., P.V., S.C., and C.N. provided input on experimental design and analysis. S.E.W.J. conceived and supervised the project, designed and analyzed experiments, and wrote the manuscript.</p></ack><fn-group><fn id="app2" fn-type="supplementary-material"><p>Supplemental Information includes Extended Experimental Procedures, four figures, and three tables and can be found with this article online at <ext-link ext-link-type="doi" xlink:href="10.1016/j.celrep.2013.04.031" id="intref0025">http://dx.doi.org/10.1016/j.celrep.2013.04.031</ext-link>.</p></fn><fn id="app4" fn-type="supplementary-material"><p>This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.</p></fn></fn-group></back><floats-group><fig id="fig1"><label>Figure 1</label><caption><p>Mutation of <italic>Runx1</italic> in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> Mice Results in High-Penetrance Aggressive Myeloid Leukemia</p><p>(A) Leukemia-free survival curves of <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup> mice stratified according to <italic>Mx1Cre</italic> genotype (n = 50–68 mice of each genotype).</p><p>(B and C) Low-power (20×; B) and high-power (100×; C) morphology of typical leukemic cells in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1Cre</italic><sup><italic>+</italic></sup> mice.</p><p>(D) Differential morphology results from BM of WT (+/+), <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1Cre</italic><sup><italic>−</italic></sup>, and leukemic <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1Cre</italic><sup><italic>+</italic></sup> mice; percentage of total BM cells. Mean (SEM) values for 4–11 mice of each genotype.</p><p>(E) Fluorescence-activated cell sorting (FACS) analysis of PB and BM from an <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1Cre</italic><sup><italic>+</italic></sup> mouse with myeloid leukemia (representative of 19 further analyzed cases).</p><p>Error bars represent SEM. See also <xref rid="figs1" ref-type="fig">Figure S1</xref>.</p></caption><graphic xlink:href="gr1"></graphic></fig><fig id="fig2"><label>Figure 2</label><caption><p>Flt3-ITDs Expand Myeloid-Biased MPPs</p><p>(A) Expansion of LSK cells in 8- to 10-week-old <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (+/+: n = 27; ITD/ITD: n = 23).</p><p>(B) Expression of CD48 and CD150 on BM LSK cells from <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice.</p><p>(C) Expansion of LSKCD48<sup>+</sup>CD150<sup>−</sup> MPPs in 8- to 10-week-old <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (+/+: n = 27; ITD/ITD: n = 23).</p><p>(D) GSEA demonstrating downregulation of E-affiliated genes in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> versus <italic>Flt3</italic><sup><italic>+/+</italic></sup>LSKCD48<sup>+</sup>150<sup>−</sup> MPPs (n = 4 of each genotype). FDR, false discovery rate; NES, normalized enrichment score.</p><p>(E) Reduced MkE-affiliated gene expression in LSKCD48<sup>+</sup>150<sup>−</sup> cells from <italic>Flt3</italic><sup><italic>+/+</italic></sup>, <italic>Flt3</italic><sup><italic>ITD/+</italic></sup>, and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice relative to <italic>Flt3</italic><sup><italic>+/+</italic></sup> LSK FLT3<sup>−</sup> cells; p values represent a comparison of <italic>Flt3</italic><sup><italic>+/+</italic></sup>LSK FLT3<sup>−</sup> cells versus LSKCD48<sup>+</sup>150<sup>−</sup><italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> cells by unpaired t test (n = 6–8 replicates from 2–3 mice per genotype). Differences among <italic>Flt3</italic><sup><italic>+/+</italic></sup>, <italic>Flt3</italic><sup><italic>ITD/+</italic></sup>, and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> MPPs were not significant.</p><p>(F) Single-cell Mk potential of <italic>Flt3</italic><sup><italic>+/+</italic></sup>and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> LSKCD48<sup>+</sup>150<sup>−</sup> MPPs in comparison with <italic>Flt3</italic><sup><italic>+/+</italic></sup>LSKCD48<sup>−</sup>150<sup>+</sup> HSCs (n = 3 independent experiments).</p><p>(G) GSEA demonstrating upregulation of myeloid affiliated genes in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> versus <italic>Flt3</italic><sup><italic>+/+</italic></sup>LSKCD48<sup>+</sup>150<sup>−</sup> MPPs (n = 4 of each genotype).</p><p>(H) Single-cell GM potential of <italic>Flt3</italic><sup><italic>+/+</italic></sup>and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> LSKCD48<sup>+</sup>150<sup>−</sup> MPPs. Open bars show the frequency of clones formed, and closed bars show the frequency of highly proliferative clones (covering >50% of the well; n = 3 independent experiments).</p><p>(I) GSEA demonstrating downregulation of lymphoid transcriptional program in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> versus <italic>Flt3</italic><sup><italic>+/+</italic></sup> LSKCD48<sup>+</sup>150<sup>−</sup> MPPs (n = 4 of each genotype).</p><p>(J) Expression of lymphoid-affiliated genes in LSKCD48<sup>+</sup>150<sup>−</sup> MPPs from <italic>Flt3</italic><sup><italic>+/+</italic></sup>, <italic>Flt3</italic><sup><italic>ITD/+</italic></sup>, and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (n = 6–8 replicates from 2–3 mice per genotype); p values indicate comparisons of <italic>Flt3</italic><sup><italic>+/+</italic></sup> versus <italic>Flt3</italic><sup><italic>ITD/+</italic></sup> MPPs, and <italic>Flt3</italic><sup><italic>+/+</italic></sup> versus <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> MPPs by unpaired t test (p < 0.001 for all analyses).</p><p>(K) B cell potential (B220<sup>+</sup>CD19<sup>+</sup>) of <italic>Flt3</italic><sup><italic>+/+</italic></sup> versus <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> BM LSKCD48<sup>+</sup>150<sup>−</sup> MPPs (10 cells/well). Data are mean (SEM) values of 2–3 experiments, with 9–12 replicate wells in each experiment.</p><p>Error bars represent SEM. See also <xref rid="figs2" ref-type="fig">Figure S2</xref>.</p></caption><graphic xlink:href="gr2"></graphic></fig><fig id="fig3"><label>Figure 3</label><caption><p>Early T and B Cell Progenitors Are Suppressed and Myeloid Biased in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> Mice</p><p>(A) Thymic cellularity of <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice at 2–3 and 8–10 weeks (n = 7–10 mice of each genotype at each age).</p><p>(B) Representative DN staging of <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> thymocytes from 2-week-old mice; percentages are relative to total thymocytes.</p><p>(C) Progressive reduction of DN1Kit<sup>+</sup> cells in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> thymus.</p><p>(D) Expression (SEM) of <italic>Ccr9</italic> in LSKCD48<sup>+</sup>150<sup>−</sup> BM cells from 8- to 10-week-old <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (n = 3–4 mice per genotype, 2–3 separately sorted replicates per mouse).</p><p>(E) T cell potential on OP9DL1 cells of DN1 cells (10/cells per well) from 2- to 3-week-old <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (n = 2 mice per genotype, 12 replicate wells per mouse). ND, not detected.</p><p>(F) Representative staging of early B cell progenitors in <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> 2- to 3-week-old mice. pre-pro-B cells are identified as Lin<sup>−</sup>B220<sup>+</sup>CD19<sup>−</sup>CD24<sup>−</sup>AA4.1<sup>+</sup>CD43<sup>mid</sup>; percentage of total BM cells.</p><p>(G) Increased numbers of pre-pro-B cells in 2- to 3-week-old <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (mean [SEM] values for 2- to 3-week-old mice, n = 8–10 of each genotype).</p><p>(H) Reduced numbers of pro-B cells (Lin<sup>−</sup>B220<sup>+</sup>CD43<sup>+</sup>CD19<sup>+</sup>CD24<sup>int</sup>AA4.1<sup>+</sup>) in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice.</p><p>(I) Mean (SEM) expression of lymphoid-affiliated genes in pre-pro-B cells from 2- to 3-week-old mice (n = 2 mice per genotype, 3 separately sorted replicates per mouse).</p><p>(J) Mean (SEM) expression of myeloid-affiliated genes in pre-pro-B cells from <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice.</p><p>(K) In vitro B cell potential (B220<sup>+</sup>CD19<sup>+</sup> cells) of pre-pro-B cells from 2- to 3-week-old mice (n = 10 cells/well, 2 mice per genotype, 10 replicate wells per mouse).</p><p>Error bars represent SEM. See also <xref rid="figs3" ref-type="fig">Figure S3</xref>.</p></caption><graphic xlink:href="gr3"></graphic></fig><fig id="fig4"><label>Figure 4</label><caption><p>Involvement of PU1 Upregulation in the Myeloid Bias of <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> Mice</p><p>(A–C) Representative FACS analysis of PU1-YFP expression in lineage-negative (A), LSKCD48<sup>+</sup>150<sup>−</sup> MPPs (B), and Lin<sup>−</sup>CD19<sup>−</sup>CD24<sup>−</sup>AA4.1<sup>+</sup>CD43<sup>mid</sup>B220<sup>+</sup> pre-pro-B (C) cells in BM of 2- to 3-month-old <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice. Also shown is the mean fold difference in PU1-YFP expression for each population from six mice of each genotype.</p><p>(D) GSEA demonstrating upregulation of Pu1 target genes in LSKCD48<sup>+</sup>150<sup>−</sup> MPPs from <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> versus <italic>Flt3</italic><sup>+/+</sup> mice (n = 4 of each genotype).</p><p>(E) Percentage of B220<sup>+</sup>CD19<sup>+</sup> in the BM of 6- to 8-week-old <italic>Flt3</italic><sup><italic>+/+</italic></sup><italic>Pu1</italic><sup><italic>+/+</italic></sup>, <italic>Flt3</italic><sup><italic>+/+</italic></sup><italic>Pu1</italic><sup><italic>+/−</italic></sup>, <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Pu1</italic><sup><italic>+/+</italic></sup>, and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Pu1</italic><sup><italic>+/−</italic></sup> mice (n = 8–11 mice of each genotype; fold difference and p value are for the difference between <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Pu1</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Pu1</italic><sup><italic>+/−</italic></sup> mice).</p><p>(F) Percentage of pre-pro-B cells in the BM of 6- to 8-week-old <italic>Flt3</italic><sup><italic>+/+</italic></sup><italic>Pu1</italic><sup><italic>+/+</italic></sup>, <italic>Flt3</italic><sup><italic>+/+</italic></sup><italic>Pu1</italic><sup><italic>+/−</italic></sup>, <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Pu1</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Pu1</italic><sup><italic>+/−</italic></sup> mice (n = 3 mice of each genotype).</p><p>(G) Percentage of pro-B cells in the BM of 6- to 8-week-old <italic>Flt3</italic><sup><italic>+/+</italic></sup><italic>Pu1</italic><sup><italic>+/+</italic></sup>, <italic>Flt3</italic><sup><italic>+/+</italic></sup><italic>Pu1</italic><sup><italic>+/−</italic></sup>, <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Pu1</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Pu1</italic><sup><italic>+/−</italic></sup> mice (n = 3 mice of each genotype).</p><p>(H) Percentage of LSKCD48<sup>+</sup>150<sup>−</sup> MPPs in the BM of 6- to 8-week-old <italic>Flt3</italic><sup><italic>+/+</italic></sup><italic>Pu1</italic><sup><italic>+/+</italic></sup>, <italic>Flt3</italic><sup><italic>+/+</italic></sup><italic>Pu1</italic><sup><italic>+/−</italic></sup>, <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Pu1</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Pu1</italic><sup><italic>+/−</italic></sup> mice (n = 7–9 mice of each genotype).</p><p>(I) Percentage of Mac1<sup>low</sup>cKit<sup>low</sup> myeloid precursor cells in the BM of 6- to 8-week-old <italic>Flt3</italic><sup><italic>+/+</italic></sup><italic>Pu1</italic><sup><italic>+/+</italic></sup>, <italic>Flt3</italic><sup><italic>+/+</italic></sup><italic>Pu1</italic><sup><italic>+/−</italic></sup>, <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Pu1</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Pu1</italic><sup><italic>+/−</italic></sup> mice (n = 7–9 mice of each genotype).</p><p>(J) GSEA demonstrating upregulation of STAT3 target genes in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> versus <italic>Flt3</italic><sup><italic>+/+</italic></sup> LSKCD48<sup>+</sup>150<sup>−</sup> MPPs (n = 4 mice of each genotype).</p><p>(K and L) Expression of Stat3 target genes in <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> LSKCD48<sup>+</sup>150<sup>−</sup> MPPs (K) and pre-pro-B cells (L) (n = 2–4 mice of each genotype, 2–3 replicates per mouse). ND, not detected.</p><p>Error bars represent SEM. See also <xref rid="figs4" ref-type="fig">Figure S4</xref>.</p></caption><graphic xlink:href="gr4"></graphic></fig><fig id="figs1"><label>Figure S1</label><caption><p>Mutation of <italic>Runx1</italic> in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> Mice Results in High-Penetrance Aggressive Myeloid Leukemia, Related to <xref rid="fig1" ref-type="fig">Figure 1</xref></p><p>(A–C) Hematologic indices in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1Cre</italic><sup><italic>+</italic></sup> leukemic mice demonstrating a marked increase in white blood cell count (WBC) (A), anemia (B), and thrombocytopenia (C) compared with <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1Cre</italic><sup><italic>−</italic></sup> controls. Mean (SEM) values from 6-14 mice from 5-11 independent experiments. Hematologic indices were measured using a Sysmex<sup>®</sup> automated analyzer.</p><p>(D) Representative FACS analysis of infiltrated livers and spleens from <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1Cre</italic><sup><italic>+</italic></sup> leukemic mice showing lineage surface markers typical of myeloid leukemic cells (results representative of 3 [liver] and 11 [spleen] further analyzed cases).</p><p>(E) Recombination at the <italic>Runx1</italic> locus without poly I:C induction in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1Cre</italic><sup><italic>+</italic></sup> leukemic BM. Representative PCR analysis of <italic>Runx1</italic><sup><italic>fl/fl</italic></sup> recombination in paired BM and earclip (EC) DNA from <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1Cre</italic><sup><italic>+</italic></sup> leukemic and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1Cre</italic><sup><italic>−</italic></sup> and <italic>Flt3</italic><sup><italic>+/+</italic></sup><italic>Runx1</italic><sup><italic>fl/fl</italic></sup><italic>Mx1Cre</italic><sup><italic>+</italic></sup> controls, all without polyI:C induction. The upper band represents non-deleted <italic>Runx1</italic> allele and the lower band indicates deleted <italic>Runx1</italic> alleles. Results are representative of 3 further analyzed leukemic samples. Error bars represent SEM.</p></caption><graphic xlink:href="figs1"></graphic></fig><fig id="figs2"><label>Figure S2</label><caption><p>Flt3-ITDs Expand Myeloid-Biased LMPPs, Related to <xref rid="fig2" ref-type="fig">Figure 2</xref></p><p>(A) Lack of cell surface Flt3 expression in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice; representative FACS analysis of surface Flt3 expression in LSK cells from <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice. Percentages in the graph represent mean frequency of Flt3-positive cells in the LSK compartment of <italic>Flt3</italic><sup><italic>+/+</italic></sup> (n = 27) and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> (n = 23) mice.</p><p>(B) <italic>Flt3</italic><sup><italic>ITD/+</italic></sup> mice show an intermediate expansion of LSK cells relative to <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (<italic>Flt3</italic><sup><italic>+/+</italic></sup> n = 27; <italic>Flt3</italic><sup><italic>ITD/+</italic></sup> n = 13; and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> n = 23).</p><p>(C) <italic>Flt3</italic><sup><italic>ITD/+</italic></sup> mice show an expansion of LSKCD48<sup>+</sup>CD150<sup>−</sup> MPPs intermediate between <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (<italic>Flt3</italic><sup><italic>+/+</italic></sup> n = 27; <italic>Flt3</italic><sup><italic>ITD/+</italic></sup> n = 13; and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> n = 23).</p><p>(D) LMPPs reside in the LSKCD150<sup>−</sup>48<sup>+</sup> compartment; representative FACS analysis of CD48 and CD150 expression in relation to high-level Flt3 expressing LMPPs within the LSK compartment. High-level Flt3 expression was defined as the highest 20% of Flt3 staining cells in <italic>Flt3</italic><sup><italic>+/+</italic></sup> LSK cells. Percentages in the graph represent mean frequency of indicated cells relative to parental gate from <italic>Flt3</italic><sup><italic>+/+</italic></sup> (n = 17) mice.</p><p>(E) HSC associated gene expression is not upregulated in the LSKCD150<sup>−</sup>48<sup>+</sup> compartment in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice. Mean (SEM) HSC affiliated gene expression in indicated cell populations from <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice. P-values are shown if < 0.05 (not reached for any comparison of <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> MPPs); ND indicates not detected.</p><p>(F) Expression of lymphoid affiliated genes in E15 FL LSKCD150<sup>+</sup>CD48<sup>−</sup> HSCs and LSKCD150<sup>−</sup>CD48<sup>+</sup> MPPs from <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (n = 9 replicates from 3 mice per genotype). P values less than 0.05 are shown for comparison of <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> MPPs.</p><p>(G) LSKCD150<sup>−</sup>48<sup>+</sup> MPPs are not expanded in the fetal liver (FL) of <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice; representative FACS analysis from E15 FL from <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice. Numbers in the graph represent mean frequencies of total FL cells from 7-9 mice of each genotype from 3 independent experiments. There were no significant differences in the frequency of MPPs between <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice.</p><p>(H) Percentage of B220<sup>+</sup>CD19<sup>+</sup> cells in E15 <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> FL (8-9 FL of each genotype).</p><p>(I) Percentage of Mac1<sup>low</sup>cKit<sup>low</sup> cells in E15 <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> FL (8-9 FL of each genotype). NS indicates not significant.</p><p>(J) Expression of lymphoid affiliated genes in LSKFlt3<sup>+</sup>CD48<sup>+</sup>150<sup>−</sup> MPPs from Runx1<sup>fl/fl</sup>VavCre<sup>−</sup> and Runx1<sup>flfl</sup>VavCre<sup>+</sup> mice (n = 4 mice 4-6 weeks of age per genotype). Error bars represent SEM.</p></caption><graphic xlink:href="figs2"></graphic></fig><fig id="figs3"><label>Figure S3</label><caption><p>Early T and B Cell Progenitors Are Suppressed and Myeloid Biased in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> Mice, Related to <xref rid="fig3" ref-type="fig">Figure 3</xref></p><p>(A and B) Progressive loss of DN2Kit<sup>+</sup> (A) and DN3 (B) thymocyte progenitors in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> thymus (mean (SEM) values from 7-8 mice of each genotype at indicated ages in 3 independent experiments).</p><p>(C) Representative FACS profile demonstrating loss of Ccr9 expression on the cell surface of LSKCD150<sup>−</sup> cells in <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice.</p><p>(D) Quantification of Ccr9 positive LSKCD150<sup>−</sup>CD48<sup>+</sup> cells in <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> BM (mean (SEM) values from 5-6 mice of each genotype analyzed at 6-8 weeks of age).</p><p>(E) Lineage-affiliated gene expression analysis of DN1 thymocytes from <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (mean (SEM) expression, 2 mice per genotype; 3 separately sorted replicates per mouse).</p><p>(F) Representative FACS analysis showing gating strategy for identification of pro-B (Lin<sup>−</sup>B220<sup>+</sup>CD19<sup>+</sup>CD24<sup>mid</sup>AA4.1<sup>+</sup>) cells in <italic>Flt3</italic><sup><italic>+/+</italic></sup> and <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice (percentages are relative to total bone marrow cells). Error bars represent SEM.</p></caption><graphic xlink:href="figs3"></graphic></fig><fig id="figs4"><label>Figure S4</label><caption><p>Involvement of Pu1 Upregulation in the Myeloid Bias of <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> Progenitors, Related to <xref rid="fig4" ref-type="fig">Figure 4</xref></p><p>(A) Gene set enrichment analysis demonstrating upregulation of Pu1 target genes in Lin<sup>−</sup>CD34<sup>+</sup>CD38<sup>−</sup>CD90<sup>−</sup>CD45RA<sup>+</sup> cells from <italic>FLT3-ITD</italic> mutated human AML (n = 8) relative to <italic>FLT3-WT</italic> counterparts (n = 18).</p><p>(B) Representative FACS analysis of Pu1-YFP expression levels during early B cell commitment demonstrating marked downregulation of Pu1 expression at the pre-pro-B to pro-B transition. Representative of 2 experiments.</p><p>(C) Western blot showing increased pStat3 activation in the BM of <italic>Flt3</italic><sup><italic>ITD/ITD</italic></sup> mice in comparison with <italic>Flt3</italic><sup><italic>+/+</italic></sup> BM. The blot shown is representative of two independent experiments.</p></caption><graphic xlink:href="figs4"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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