Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming "boosters" also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

The MLL-AF4 (MA4) fusion gene is the genetic hallmark of an aggressive infant pro-B-acute lymphoblastic leukemia (B-ALL). Our understanding of MA4-mediated transformation is very limited. Whole-genome sequencing studies revealed a silent mutational landscape, which contradicts the aggressive clinical outcome of this hematological malignancy. Only RAS mutations were recurrently detected in patients and found to be associated with poorer outcome. The absence of MA4-driven B-ALL models further questions whether MA4 acts as a single oncogenic driver or requires cooperating mutations to manifest a malignant phenotype. We explored whether KRAS activation cooperates with MA4 to initiate leukemia in cord blood (CB)-derived CD34+ hematopoietic stem/progenitor cells (HSPCs). Clonogenic and differentiation/proliferation assays demonstrated that KRAS activation does not cooperate with MA4 to immortalize CD34+ HSPCs. Intra-bone marrow transplantation into immunodeficient mice further showed that MA4 and KRASG12V alone or in combination enhanced hematopoietic repopulation without impairing myeloid-lymphoid differentiation, and that mutated KRAS did not cooperate with MA4 to initiate leukemia. However, KRAS activation enhanced extramedullary hematopoiesis of MA4-expressing cell lines and CD34+ HSPCs that was associated with leukocytosis and central nervous system infiltration, both hallmarks of infant t(4;11)+B-ALL. Transcriptional profiling of MA4-expressing patients supported a cell migration gene signature underlying the mutant KRAS-mediated phenotype. Collectively, our findings demonstrate that KRAS impacts the homeostasis of MA4-expressing HSPCs, suggesting that KRAS activation in MA4+ B-ALL is important for tumor maintenance rather than initiation.

B-cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B-cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B-cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus (SeV). Co-expression of c/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B-cells. We also demonstrate that myeloid cells are much easier to reprogram than B- and T-lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B-cells since biased reprogramming was avoided by using freshly-isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B-cells offers an unprecedented opportunity for studying developmental B-lymphopoiesis and modeling B-cell malignances.Leukemia accepted article preview online, 26 October 2015. doi:10.1038/leu.2015.294.

Infant B-cell acute lymphoblastic leukemia (B-ALL) accounts for 10% of childhood ALL. The genetic hallmark of most infant B-ALL are chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene. Despite improvement in the clinical management and survival (~85-90%) of childhood B-ALL, the outcome of infants with MLL-rearranged (MLL-r) B-ALL remains dismal, with overall survival <35%. Among MLL-r infant B-ALL, t(4;11)+ patients harboring the fusion MLL-AF4 (MA4), display a particularly poor prognosis and a pro-B/mixed phenotype. Studies in monozygotic twins and archived blood spots have provided compelling evidence of a single cell of prenatal origin as target for MA4 fusion, explaining the brief leukemia latency. Despite its aggressiveness and short latency, current progress about its etiology, pathogenesis and cellular origin is limited as evidenced by the lack of mouse/human models recapitulating the disease phenotype/latency. We propose this is because infant cancer is from an etiological and pathogenesis standpoint distinct to adult cancer and should be seen as a developmental disease. This is supported by whole-genome sequencing studies suggesting that opposite to the view of cancer as "multiple-and-sequential-hit" model, t(4;11) alone might be sufficient to spawn leukemia. The stable genome of these patients suggests that in infant developmental cancer one "big-hit" might be sufficient for overt disease, and supports a key contribution of epigenetics and a prenatal cell-of-origin during a critical developmental window of stem cell vulnerability in the leukemia pathogenesis. Here, we revisit the biology of t(4;11)+ infant B-ALL with emphasis on its origin, genetics and disease models.

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.