Identifying Targeted Therapies for CBFA2T3::GLIS2 Acute Myeloid Leukemia.

fusion positive pediatric acute myeloid leukemia (AML) remains one of the worst prognostic AML subgroups. To uncover innovative targeted therapeutic approaches in this disease subtype we performed genome-scale CRISPR-Cas9 screening that highlighted a strong, selective dependency on compared to other types of cancer. Using a doxycycline-inducible knockout (KO) system, we validated dependency in cell lines, observing impaired proliferation in vitro and in vivo and induced apoptosis with KO. Both type I (ruxolitinib) and type II (CHZ868) JAK2 inhibitors showed selective in vitro activity in positive AML models. To identify resistance and sensitizer mechanisms to JAK2 inhibitors, we used CRISPR-Cas9 ruxolitinib anchor screening in AML. sgRNAs targeting negative regulators of the MAPK pathway were enriched in the ruxolitinib-treated cells. Similarly, AML sublines grown to resistance under chronic ruxolitinib treatment expressed pathogenic mutations. Both approaches converged on MAPK pathway activation as a resistance mechanism to ruxolitinib treatment. Combining ruxolitinib with MEK inhibitors showed a synergistic effect in cell lines and patient-derived xenograft (PDX) cells expressing the fusion and in vivo activity in a AML PDX, suggesting a potential approach to target this signaling circuitry in this poor outcome AML subtype.