Genome-wide CRISPR-Cas9 screen identifies rationally designed combination therapies for CRLF2-rearranged Ph-like ALL

Acute lymphoblastic leukemia (ALL) harboring the IgH-CRLF2 rearrangement (IgH-CRLF2-r) exhibits poor clinical outcomes and is the most common subtype of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). While multiple chemotherapeutic regimens, including ruxolitinib monotherapy and/or its combination with chemotherapy, are being tested, their efficacy is reportedly limited. To identify molecules/pathways relevant for IgH-CRLF2-r ALL pathogenesis, we performed genome-wide CRISPR-Cas9 dropout screens in the presence or absence of ruxolitinib using 2 IgH-CRLF2-r ALL lines that differ in RAS mutational status. To do so, we employed a baboon envelope pseudotyped lentiviral vector system, which enabled, for the first time, highly efficient transduction of human B cells. While single-guide RNAs (sgRNAs) targeting CRLF2, IL7RA, or JAK1/2 significantly affected cell fitness in both lines, those targeting STAT5A, STAT5B, or STAT3 did not, suggesting that STAT signaling is largely dispensable for IgH-CRLF2-r ALL cell survival. We show that regulators of RAS signaling are critical for cell fitness and ruxolitinib sensitivity and that CRKL depletion enhances ruxolitinib sensitivity in RAS wild-type (WT) cells. Gilteritinib, a pan-tyrosine kinase inhibitor that blocks CRKL phosphorylation, effectively killed RAS WT IgH-CRLF2-r ALL cells in vitro and in vivo, either alone or combined with ruxolitinib. We further show that combining gilteritinib with trametinib, a MEK1/2 inhibitor, is an effective means to target IgH-CRLF2-r ALL cells regardless of RAS mutational status. Our study delineates molecules/pathways relevant for CRLF2-r ALL pathogenesis and could suggest rationally designed combination therapies appropriate for disease subtypes.