arn-509 Here we attempted to reprogram

Here, we attempted to reprogram highly fluorescence-activated cell sorting (FACS)-purified (100% purity) leukemia blasts from three subtypes of B-ALL, t(4;11)/MLL-AF4+, t(1;11)+MLL-EPS15+, and t(12;21)/ETV6-RUNX1 B-ALL, to establish novel iPSC-based disease models to address the developmental impact of these leukemia-specific fusion genes on human stem cell fate. Our data demonstrate that despite multiple technical and biological reprogramming strategies, neither primary blasts nor B-ALL cell lines could be reprogrammed to pluripotency. Functional assays coupled with global transcriptome and DNA methylome profiling suggest a developmental/differentiation refractoriness of MLL-rearranged human B-ALL to reprogramming to pluripotency.
Results
Discussion iPSCs reprogrammed from cancer cells have the potential to illuminate molecular mechanisms underlying the pathogenesis of cancer (Barrett et al., 2014; Curry et al., 2015; Ramos-Mejia et al., 2012c; Yilmazer et al., 2015). However, reprogramming human primary cancer cells remains challenging, and only a few reports have demonstrated successful reprogramming of malignant cells. Moreover, iPSCs from primary leukemic cells have exclusively been generated from chronic hematological malignances, including Philadelphia+ CML, PMF, JAK2-V617F+ PV, and JMML (Bedel et al., 2013; Carette et al., 2010; Gandre-Babbe et al., 2013; Hu, 2014; Kumano et al., 2012; Yamamoto et al., 2015; Ye et al., 2009) (Table S1). No iPSCs reprogrammed from acute leukemias have been reported, but iPSCs have been successfully generated from normal myeloid, T cells, and arn-509 using non-integrative tetracistronic OKSM-expressing SeV (Bueno et al., 2016; Munoz-Lopez et al., 2016). Here, we attempted to establish an iPSC-based disease model to address the developmental impact of leukemia-specific fusion genes on human stem cell fate. We provide insights into the difficulty of reprogramming primary leukemia blasts from cytogenetically different subtypes of B-ALL, including t(4;11)/MLL-AF4+, t(1;11)+MLL-EPS15+, and t(12;21)/ETV6-RUNX1 B-ALL. Our data demonstrate that neither primary blasts nor B-ALL cell lines could be reprogrammed to pluripotency. The few iPSC clones that were generated consistently lacked the leukemic fusion gene, indicating that only residual/contaminating non-leukemic myeloid cells, which are less demanding to reprogram than lymphoid cells, were reprogrammed (Bueno et al., 2016). The question remains open as to whether biological or technical reprogramming barriers underlie the inability of B-ALL leukemic blasts to be reprogrammed. Technically, multiple reprogramming strategies were attempted, but neither transient nor stable expression of reprogramming factors using a variety of monocistronic and polycistronic vectors rendered leukemic iPSCs. The choice of reprogramming factors (OKSM, OKSL, or OKSML) also had little impact on the reprogramming outcome, suggesting that the methods used for transgene delivery and c-myc dependency are not causal mechanisms responsible for the lack of success. From a biological standpoint, cell identity is a reflection of cell-type-specific gene expression and epigenetic signatures. A variety of transcription factors, tumor suppressors, microRNAs, and chromatin-remodeling enzymes, as well as chemical regulators of histone and DNA modifications, have been extensively reported to provide a permissive environment for cell-fate change during cellular reprogramming (Nashun et al., 2015). We combined different reprogramming strategies with several reprogramming “boosters” acting on transcription factor expression and chromatin structure; yet, iPSCs could not be generated. Recently, the differentiation blockage of BCR-ABL1+ B cell ALL cells was overcome by forcing cells to reprogram to the myeloid lineage through exposure to myeloid differentiation-promoting cytokines in vitro or by transient expression of the myeloid transcription factor C/EBPα (McClellan et al., 2015). Similarly, mouse and human healthy B cells were efficiently reprogrammed upon C/EBPα-mediated myeloid priming (Bueno et al., 2016; Di Stefano et al., 2014). Unfortunately, neither C/EBPα expression (Tables 1 and 2) nor exposure to myeloid differentiation-promoting cytokines (interleukin-3 [IL-3], IL-6, FLT3, granulocyte macrophage colony-stimulating factor, and macrophage colony-stimulating factor; data not shown) sensitized MLL-AF4+ blasts to undergo myeloid priming and subsequent reprogramming.