FLT3 activation cooperates with MLL-AF4 fusion protein to abrogate the hematopoietic specification of human ESCs Bueno, Clara Ayllon, Veronica Montes Lorenzo, Rosa María Navarro-Montero, Oscar Ramos Mejía, Verónica Real Luna, Pedro José Romero-Moya, Damia Araúzo-Bravo, Marcos J Menendez, Pablo This work was funded by FIS/FEDER (PI10/00449 to P.M. and PI11/00119 to C.B.) and by The Spanish Association Against Cancer to P.M. D.R.-M. is supported by PFIS scholarship (FI11/ 00511). C.B., P.J.R., and V.R.-M. are supported by “Miguel Servet” Fellowships (CP07/0059, CP09/0063, and CP12/03175). R.M. is supported by the ISCIII (CA10/01332). P.M. is an ICREA investigator supported by the ISCIII Red de Terapia Celular (Tercel, RD12/0019/0006). Mixed-lineage leukemia (MLL)-AF4 fusion arises prenatally in high-risk infant acute pro-B-lymphoblastic leukemia (pro-B-ALL). In human embryonic stem cells (hESCs), MLL-AF4 skewed hematoendothelial specification but was insufficient for transformation, suggesting that additional oncogenic insults seem required for MLL-AF4-mediated transformation. MLL-AF4+ pro-B-ALL expresses enormous levels of FLT3, occasionally because of activating mutations, thus representing a candidate cooperating event in MLL-AF4+ pro-B-ALL. Here, we explored the developmental impact of FLT3 activation alone, or together with MLL-AF4, in the hematopoietic fate of hESCs. FLT3 activation does not affect specification of hemogenic precursors but significantly enhances the formation of CD45(+) blood cells, and CD45(+)CD34(+) blood progenitors with clonogenic potential. However, overexpression of FLT3 mutations or wild-type FLT3 (FLT3-WT) completely abrogates hematopoietic differentiation from MLL-AF4-expressing hESCs, indicating that FLT3 activation cooperates with MLL-AF4 to inhibit human embryonic hematopoiesis. Cell cycle/apoptosis analyses suggest that FLT3 activation directly affects hESC specification rather than proliferation or survival of hESC-emerging hematopoietic derivatives. Transcriptional profiling of hESC-derived CD45(+) cells supports the FLT3-mediated inhibition of hematopoiesis in MLL-AF4-expressing hESCs, which is associated with large transcriptional changes and downregulation of genes involved in hematopoietic system development and function. Importantly, FLT3 activation does not cooperate with MLL-AF4 to immortalize/transform hESC-derived hematopoietic cells, suggesting the need of alternative (epi)-genetic cooperating hits 2026-02-26T12:09:40Z 2026-02-26T12:09:40Z 2013-05 journal article Bueno C, Ayllón V, Montes R, Navarro-Montero O, Ramos-Mejia V, Real PJ, Romero-Moya D, Araúzo-Bravo MJ, Menendez P. (2013). FLT3 activation cooperates with MLL-AF4 fusion protein to abrogate the hematopoietic specification of human ESCs. Blood; 121 (19): 3867-78, S1-3. doi: 10.1182/blood-2012-11-470146 0006-4971 https://hdl.handle.net/10481/111588 10.1182/blood-2012-11-470146 eng open access American Society of Hematology