Genome-Wide CRISPR-Cas9 Screen Identifies Rationally Designed Combination Therapies Relevant for CRLF2-Rearranged Ph-like ALL

Abstract

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.

BCL6 inhibition ameliorates resistance to ruxolitinib in <i>CRLF2</i>-rearranged acute lymphoblastic leukemia

“…Cells used in this study are listed in Online Supplementary Tables S1 and S2. YCUB5 (30) and KOPN49 (22,31) were provided by Hiroaki Goto (Kanagawa Children's Medical Center) and Takeshi Inukai (University of Yamanashi), respectively. Cell authentication was made using the GenePrint 10 System (Promega) with Expasy Cellosaurus short tandem repeat (STR) reference, except for YCUB5 and KOPN49, which lacked such reference.…”

“…Others include combinatorial inhibition targeting JAK2 together with Bcl2 and BclxL (18) or phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (19,20). Additionally, recent studies reported that GSPT1 (21), CRKL, MEK1/2 (22), HMGN1 (23), and B cell receptor-like signaling (24) are important targets for better treatment. Combinations of ruxolitinib with chemotherapeutic drugs provide additional options (25).…”