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.

Background: Indications for allogeneic hematopoietic stem cell transplantation (allo-HCT) with reduced-intensity conditioning (RIC) have expanded widely for elderly patients and those with comorbidities. However, the optimal RIC regimen remains uncertain. A combination of fludarabine phosphate (Flu) and melphalan (Mel) is widely used in RIC regimen with comparable results to that of fludarabine phosphate and busulfan. In our hospital, Flu/Mel and low-dose total body irradiation (Flu/Mel/TBI) is mainly used in RIC regimen. We analyzed safety and efficacy in allo-HCT patients with Flu/Mel/TBI. Methods: This study was a retrospective analysis of patients with hematopoietic malignancies who received their first allo-HCT with Flu/Mel/TBI from between January 2005 to December 2015. Patients were included for Flu/Mel/TBI treatment for elderly, those with a recent fungal infection or organ dysfunction. Flu/Mel/TBI consists of intravenous Flu (125-150 mg/m2), Mel (140 mg/m2), and TBI (2-4 Gy). GVHD prophylaxis consists of tacrolimus and short-term methotrexate with or without low-dose anti-thymocyte globulin (ATG). The primary end point was non-relapse mortality (NRM) on day 100. As the secondary end point, we verified the effects about Flu/Mel/TBI for patients with non-remission. Results: From January 2005 to December 2015, 168 patients received conditioning regimens with Flu/Mel/TBI. The median age was 59 years (16-71 years). Hematopoietic malignancies consisted of acute myeloid leukemia (AML) (n=74), myelodysplastic syndrome (MDS) (n=63), acute lymphoblastic leukemia (ALL) (n=12), malignant lymphoma (ML) (n=10), chronic myeloid leukemia (CML) (n=5), adult T-cell leukemia (ATL) (n=2), acute myeloid/NK cell leukemia (n=1), and chronic active Epstein-Barr virus infection (CAEBV) (n=1). Twenty-eight donors were HLA matched siblings, 6 were mismatched siblings, and 2 were haplo-identical donors. Of these, 55 had matched unrelated bone marrow donors, and 47 had 1-allele mismatched unrelated donors and 30 cord blood donors. With regard to disease status at allo-HCT, 44 patients (26.0%) were in remission and 124 patients (74.0%) were non-remission. Forty-nine patients were administered low-dose ATG. The median observation period of survivors was 2021 days (758-4660 days). Neutrophil engraftment was achieved in 89.3%. Median neutrophil recovery to over 500/µl was obtained on day 19 (11-87). Cumulative incidence of NRM at day 100 was 17.4%. Early death, defined as death before day 100, from allo-HCT occurred in 32 patients. Three patients had early relapse. Causes for NRM were infection in 15 patients (8 pneumonia, 7 sepsis), acute cardiac failure in 3 patients, cerebral hemorrhage in 2 patients, thrombotic microangiopathy in 2 patients, acute GVHD in 1 patient, hemophagocytic syndrome in 1 patient, and 5 other causes. NRM rates at day 100 by disease status at allo-HCT showed no significant differences (remission: 15.9% vs non-remission: 17.9%, p=0.15). Overall survival (OS) and disease-free survival (DFS) at 2 years were 50.6% and 43.5%, respectively. Survival rates with regard to disease status showed significant differences. Patients in remission had a 2-year OS of 65.9%, whereas patients in non-remission had a 2-year OS of 45.2% (p=0.031). Patients in remission had a 2-year DFS of 59.1%, whereas patients in non-remission had a 2-year DFS of 37.9% (p=0.016). Cumulative incidences of grade 2-4 and 3-4 acute GVHD were 30.7% and 17.1%, respectively. Cumulative incidence of relapse at 2 years was 29.8%. Conclusion: The current study proved that disease status at allo-HCT were mainly in non-remission. In such a situation, RIC using Flu/Mel/TBI was well tolerated with relatively low NRM, and was sufficient to allow engraftment for elderly or frail patients with hematopoietic malignancies. Disclosures No relevant conflicts of interest to declare.

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