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, apoptosis and colony formation. 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, G13V or G12D 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, p70 S6K 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.