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Acute lymphoblastic leukemia (ALL) harboring the IgH-CRLF2 rearrangement (IgH-CRLF2-r) exhibits poor clinical outcomes and is the most common subtype of 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 two 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 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.

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Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL, also known as BCR-ABL1-like ALL) is a disease entity of B-cell ALL that exhibits a gene expression profile, similar to that of Philadelphia chromosome-positive ALL. Ph-like ALL is categorized into two disease subtypes: "ABL-class"- and "CRLF2/JAK pathway"-types, both of which harbor gene alterations that constitutively activate cytokine/growth factor-related signals. Ph-like ALL with the CRLF2 rearrangement exhibits poor clinical outcomes and is the most common subtype of Ph-like ALL. Tyrosine kinase inhibitor (TKI)-based treatment regimens are effective in treating ABL-class type Ph-like ALL; however, no standard regimen has been established for the CRLF2/JAK pathway type. While multiple chemotherapeutic regimens, including Ruxolitinib monotherapy and/or its combination with chemotherapy, are being tested, their efficacy is reportedly limited. Thus, novel approaches are needed to treat CRLF2/JAK pathway type Ph-like ALL, in particular for CRLF2-rearranged (CRLF2-r) ALL. To identify molecules/pathways relevant for CRLF2-r ALL pathogenesis, we performed genome-wide CRISPR-Cas9 dropout screens in the presence or absence of Ruxolitinib using two IgH-CRLF2-r ALL lines (MUTZ5 and KOPN49) that differ in RAS mutational status. To do so, we employed a baboon envelope pseudotyped lentiviral vector system, which, for the first time, enabled highly efficient transduction of human B cell amenable for genome-wide CRSPR/Cas9 screens. While sgRNAs targeting CRLF2, IL7RA or JAK1/2 significantly affected cell fitness in both lines, those targeting STAT5A, STAT5B or STAT3 did not, suggesting that the JAK-STAT axis 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 and FLT3 depletion enhances Ruxolitinib sensitivity in RAS wild-type (WT) cells. Gilteritinib, a pan-tyrosine kinase inhibitor that reduces CRKL activity, 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. Disclosures No relevant conflicts of interest to declare.