3149 – DNA REPAIR AND CELL CYCLE ARE SYNTHETIC LETHAL PATHWAYS IN SRSF2P95H MUTATED CELLS

Current strategies to target cells with mutations in the RNA splicing machinery have focused on the disruption of normal splicing activities. However, splicing inhibitors are also toxic to wild-type cells as has been demonstrated in the clinical trial of spliceosome inhibitors. To understand splicing changes when there is a mutation in the RNA splicing factors, we analyzed splicing across a collated collection of SRSF2P95H mutant samples from both human and murine, incorporating multiple myeloid cancer types (AML, MDS, CMML). Using the gene lists of transcripts that were mis-spliced across multiple datasets we found that DNA repair and cell cycle are represented amongst the top mis-spliced pathways. To determine which cellular pathway/s are essential for SRSF2P95H cell survival, we performed a genome-wide CRISPR negative selection screen using Hoxb8 immortalized Srsf2P95H/+ and Srsf2+/+ myeloid progenitor cell lines (n=3 biologically independent lines per genotype). These cells were stably engineered to express Cas9. Mutant and wild-type cells were transfected with murine sgRNA Brie library, which targets 19,674 genes. CRISPR-Cas9 mediated gene deletion was allowed to occur and then the Srsf2P95H/+ mutation was activated. DNA were collected at three time points: day 4 (immediate after Srsf2P95H/+ activation), day 11 (one-week post Srsf2P95H/+ activation) and day 18 (two-week post Srsf2P95H/+ activation) to determine gRNA dropouts. All genotypes had divided >10 times by day 18. Overall, the screen achieved good coverage in both wild-type and Srsf2P95H/+ cell lines (wild-type: 371x, Srsf2P95H/+: 344x). There were 762 genes dropped out in Srsf2P95H/+ cells, compared to 235 in wild-type controls (FDR