Sphingosine-1-phosphate receptor modulators resensitize FLT3-ITD acute myeloid leukemia cells with NRAS mutations to FLT3 inhibitors

Abstract FLT3 inhibitor efficacy in AML with FLT3-ITD is short-lived, frequently due to new mutations, most commonly in NRAS. Sphingosine kinase 1 (SPHK1), which phosphorylates sphingosine to generate sphingosine-1-phosphate (S1P), is upregulated and localized to the plasma membrane in RAS-mutated cells. We studied S1P and FLT3 co-targeting to overcome FLT3 inhibitor resistance in NRAS-mutated FLT3-ITD AML cells. NRAS-mutated FLT3-ITD AML cell lines and patient blasts were treated with FLT3 inhibitors and/or S1P receptor (S1PR) modulators. FLT3 inhibitor sensitivity was assessed by immunoblotting, cytotoxicity and apoptosis assays. Co-treatment was also assessed in vivo in an orthotopic mouse model. Downstream RAS and SPHK1 effectors were measured by immunoblotting and qRT-PCR. The S1PR modulators fingolimod (FTY720) and mocravimod (KRP-203) resensitized FLT3-ITD-expressing MOLM-14 and MV4-11 human AML cells with G12D, G12S, Q61K or Q61H, but not G12C, and patient blasts with G13D or G13V NRAS mutations to FLT3 inhibitors. Moreover, FTY720 co-treatment resensitized G12D NRAS-mutated M14(R)701 cells to gilteritinib in vivo. Co-treatment inactivated ERK, transcriptionally downregulated SPHK1, and inactivated downstream AKT, p70S6K and BAD, with inactivation abrogated by constitutive SPHK1 expression. The clinically applicable S1PR modulators fingolimod and mocravimod resensitize NRAS-mutated FLT3-ITD AML cells to FLT3 inhibitors, supporting potential clinical efficacy of these combinations. Competing Interest Statement C.C.S. reports research funding from Revolution Medicines, ERASCA, Abbvie; honoraria from Daiichi Sankyo; funding for clinical trials from Zentalis and Biomea; has served on advisory boards for Abbvie, Genentech, Servier and Biomea; and has served as a consultant for Astellas. E.T. reports research funding from AstraZeneca, Genentech, Prelude, Rigel, Schrodinger; funding for clinical trials from Incyte; and has served as a consultant for Abbvie, Astellas, Daiichi-Sankyo, Incyte, Rigel, Servier and Syndax. Footnotes Grant support; Supported by Merit Review Award BX005120 from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service (M.R.B.), University of Maryland Greenebaum Comprehensive Cancer Center American Cancer Society Institutional Research Grant IRG-24-1290479-19 (G.S.), Maryland’s Cancer Moonshot Initiative in Pediatric Cancer Research (Y.W.), National Cancer Institute (NCI) Cancer Center Support Grant (CCSG) P30CA134274, the State of Maryland Department of Health’s Cigarette Restitution Fund Program, the Valanda Wilson Leukemia Research Fund and Mary Ellen’s Angelic Fund for Leukemia Research, NCI CCSG P30CA006973 (D.S.), Giant Food Pediatric Cancer Fund (D.S.), NCI R01CA277031 (C.C.S.), NCI grants U01CA271412 (E.T.), U54CA224019 (E.T.), American Cancer Society MRSG-17-040-01-LIB (E.T.) and a Hildegard Lamfrom Physician Scientist Award (E.T.). C.C.S. is a Scholar in Clinical Research of Blood Cancer United and a Damon Runyon-Richard Lumsden Foundation Clinical Investigator supported (in part) by the Damon Runyon Cancer Research Foundation (CI-99-18).