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Inhibition of DOT1L and PRMT5 promote synergistic anti-tumor activity in a human MLL leukemia model induced by CRISPR/Cas9

ABSTRACT: Previous studies have identified frequent MLL-AF4 chromosomal translocation breakpoints in patients. Using CRISPRscan, we designed different sgRNAs recognizing patient-specific sequences in intron 11 of the MLL and exon 3 of the AF4 genes, respectively. Using plasmid- and virus-free delivery of sgRNAs with Cas9 protein, we nucleofected CD34+ target cells derived from human umbilical cord blood. Following nucleofection, the cells were maintained in liquid culture supplemented with cytokines and chemokines optimized for growth of MLLr cells. On day 30 (MLL-AF4) of liquid culture, MLL translocations were detcted in 100% of cells by FISH analysis allowing for further analysis of pure MLLr cells. MLL-AF4 translocated cells and the respective control cells from the two different donors were maintained in similar culture conditions. After reaching 100% purity of the MLLr cells, they were subjected to RNA-seq.

ORGANISM(S): Homo sapiens

PROVIDER: GSE128342 | GEO | 2019/03/16

REPOSITORIES: GEO

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Project description:MLL-AF4 is a hallmark genomic aberration which arises prenatally in high-risk infant acute lymphoblastic leukemia (ALL). In human embryonic stem cells (hESCs), MLL-AF4 skewed hemato-endothelial specification but was not sufficient for transformation. Additional cooperating genetic insults seem required for MLL-AF4-mediated leukemogenesis. FLT3 is highly expressed in MLL-AF4+ ALL through activating mutations (FLT3-TKD or FLT3-ITD) or increased transcriptional expression, being therefore considered a potential cooperating event in MLL-AF4+ ALL. Here, we explored the developmental impact of FLT3 activation on its own or in cooperation with MLL-AF4 in the hematopoietic fate of hESCs. FLT3 activation did not impact specification of CD45-CD31+ hemogenic precursors but significantly enhanced the formation of CD45+CD34+ and CD45+ blood cells and blood progenitors with clonogenic potential. Importantly, FLT3 activation through FLT3 mutations or FLT3-WT overexpression completely abrogated 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 impacts hESC specification rather than selective proliferation/survival of hESC-emerging hematopoietic derivatives. Transcriptional profiling supported the limited impact of FLT3 activation on hESC specification towards CD45-hemogenic precursors and the enhanced hematopoiesis upon FLT3 activation, and inhibited hematopoiesis upon MLL-AF4 expression in FLT3-activated hESCs which was associated to large transcriptional changes and regulation of master early hematopoietic genes. Also, although FLT3 activation and MLL-AF4 cooperate to inhibit embryonic hematopoiesis the underlying molecular/genetic mechanisms differ depending on how FLT3 activation is achieved. Finally, FLT3 activation did not cooperate with MLL-AF4 to immortalize/transform hESC-derived hematopoietic cells. 18 samples were analyzed. CD45- hemogenic precursors EV, 2 biological rep CD45- hemogenic precursors FLT3-TKD, 2 biological rep CD45- hemogenic precursors FLT3-WT, 2 biological rep CD45- hemogenic precursors FLT3-TKD/MLLAF4, 2 biological rep CD45- hemogenic precursors FLT3-WT/MLLAF4, 2 biological rep CD45+ blood cells EV, 1 biological rep CD45+ blood cells FLT3-TKD, 2 biological rep CD45+ blood cells FLT3-WT, 2 biological rep CD45+ blood cells FLT3-TKD/MLLAF4, 2 biological rep CD45+ blood cells FLT3-WT/MLLAF4, 1 biological rep

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Inhibition of DOT1L and PRMT5 promote synergistic anti-tumor activity in a human MLL leukemia model induced by CRISPR/Cas9

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