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

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.