Background

FLT3-ITD is present in acute myeloid leukemia (AML) in 25% of patients and is associated with poor treatment outcomes. FLT3 inhibitors effectively target FLT3-ITD, but resistance develops during FLT3 inhibitor treatment, frequently due to new mutations that activate the Ras-Raf-MEK-ERK signaling pathway. NRAS mutations altering RAS protein amino acids at codons G12, G13 or Q61 constitutively activate RAS proteins by impairing GTPase activity, with consequent constitutive activation of the Ras-Raf-MEK-ERK pathway, are most commonly associated with development of resistance to FLT3 inhibitors during treatment. Sphingosine kinase 1 (SphK1) is upregulated and localized to the plasma membrane in RAS-mutated cells. SphK1 phosphorylates sphingosine to form the pro-survival lipid sphingosine-1-phosphate (S1P), which promotes cell survival and proliferation and inhibits apoptosis. SphK1 is linked to FLT3 inhibitor resistance, as prolonged sorafenib exposure was shown to activate the Sphk1/S1P axis. Here we studied the efficacy of targeting Sphk1 with sphingosine-1-phosphate receptor (S1PR) modulators in conjunction with FLT3 inhibitors to overcome FLT3 inhibitor resistance mediated by NRAS mutations in AML cells with FLT3-ITD.

Methods MOLM-14 and MV4-11 human FLT3-ITD AML cell lines with NRAS mutations including G12D, G12S, G12C, Q61K and Q61H and FLT3-ITD AML patient blasts with G13V and G13D mutations were cultured with the FLT3 inhibitors gilteritinib (10 nM) or quizartinib (1 nM) and/or the S1PR modulators fingolimod (FTY720; 2.5 μM) or mocravimod (KRP203; 5 μM). Apoptosis was measured by Annexin V and propidium iodide labeling, detected by flow cytometry. p-FLT3 (Y591), FLT3, p-STAT5 (Y694), STAT5, Sphk1, p-AKT (S473), AKT, p-p70 S6K (T389), p70 S6K, p-BAD (S136) and BAD protein expression were measured by immunoblotting, and Sphk1 mRNA by qPCR. MOLM-14 cells with an NRAS G12D mutation infected with lentiviral pLenti-Sphk1 plasmid overexpressing Sphk1 and pLenti-empty vector control were treated with gilteritinib and FTY720 combination to confirm the key role of Sphk1 in FLT3 inhibitor resensitization and inactivation of Sphk1 downstream proteins. To test efficacy in vivo, NSG mice engrafted with NRAS G12D-mutated MOLM-14 cells following intravenous injection were treated with the FLT3 inhibitor gilteritinib (7.5 mg/kg) and/or the S1PR modulator FTY720 (10 mg/kg), or vehicle control. Leukemia burden was assessed weekly by non-invasive luciferin imaging and compared by 2-Way ANOVA with Sidak's multiple comparison test. Survival was compared by Kaplan-Meier analysis.

Results The S1PR modulators fingolimod (FTY720) and mocravimod (KRP203) resensitized FLT3-ITD-expressing MOLM-14 and MV4-11 cells with NRAS G12D, G12S, Q61K or Q61H, but not G12C, mutations to the FLT3 inhibitors gilteritinib and quizartinib, evidenced by decreased FLT3 and STAT5 phosphorylation and increase in apoptotic cells from 5% up to 90%. Concurrent treatment of G12D, G12S, Q61K and Q61H, but not G12C, NRAS-mutated MOLM-14 and MV4-11 cells with S1PR modulators and FLT3 inhibitors transcriptionally downregulated Sphk1 and inactivated downstream AKT, p70S6K and BAD. Constitutive Sphk1 expression in G12D NRAS-mutated MOLM-14 cells abrogated AKT, p70S6K and BAD inactivation and FLT3 inhibitor resensitization by gilteritinib and FTY720 combination treatment. FTY720 co-treatment overcame gilteritinib resistance of G12D NRAS-mutated MOLM-14 cells in vivo, evidenced by decreased luminescence (p=0.015) and prolonged survival (p=0.0035), comparing gilteritinib and FTY720 to single-agent gilteritinib.

Conclusions The S1PR agonists fingolimod (FTY720) and mocravimod (KRP203) resensitize FLT3-ITD AML cells harboring G12D, G12S, Q61K, and Q61H, but not G12C, NRAS mutations to FLT3 inhibitors. The data support potential clinical efficacy of combination regimens with these clinically applicable drugs to overcome FLT3 inhibitor resistance driven by most NRAS mutations.

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2025
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