Fig. 5. Genome-wide drop-out screens identify new therapeutic targets.

a Summary of the experimental approach. Parental and ME-Transformed cells were obtained as described in Fig. 1a from Cas9-expressing Hoxb8-FL cells. Then, Parental cells (cultured in self-renewal conditions) and ME-Transformed cells (IL-3 dependent) were infected with a lentiviral pool bearing a genome-wide gRNA library. Forty-eight hours post transduction, puromycin was added in order to select for efficiently infected cells. Cell aliquots were then sampled at days 6, 10 and 12 post infection. DNA was extracted, gRNAs were PCR amplified and subsequent libraries were sequenced. Enriched or depleted gRNAs were determined by comparison to the library used for infection. Depleted guides represent genes whose expression is required for cell survival. b Intersection of drop-outs selected with FDR < 0.25 of Parental and ME-Transformed samples. c Table showing top significant pathways enriched in each cell subgroup identified in (b). The GSEA database was used and an FDR < 0.05 was applied to define a pathway as statistically significant. Individual FDR values are shown. d Venn diagram showing intersection between unique ME-Transformed drop-outs and upregulated genes obtained from comparing the transcriptomic profile of MLL-ENL1 and Parental cells (refers to Fig. 4). e GSEA analysis of pathways using the 127 overlapping genes identified. An FDR < 0.05 was applied to define a pathway as statistically significant. f MA plot showing expression levels of each of the 127 overlapping genes in ME-Transformed cells (expressed as log10) against its difference of expression in the comparison MLL-ENL1 vs. Parental (expressed as log2 of the fold-change). Genes defined as druggable are highlighted in red; genes selected for validation (Supplementary Fig. 5) and further analysis are in bold.