Background. Acute myeloid leukemia (AML) remains a clinical challenge due to high relapse rates and limited durable treatment options, especially in older or unfit patients. Among the most frequent genetic alterations in AML are NPM1 mutations (NPM1c) and KMT2A/MLL rearrangements (KMT2Ar), both of which are highly dependent on menin for inducing leukemogenesis. While small-molecule menin inhibitors have shown early clinical promise in these subtypes, resistance—particularly via MEN1 mutations—limits their long-term efficacy. We previously developed a TCR-mimetic CAR T cell targeting the NPM1c-derived neoepitope (AIQ) and a CD70-specific CAR T cell that leverages the restricted expression of CD70 on AML blasts. We hypothesized that combining NPM1-CAR or CD70-CAR T cells with menin inhibition could enhance anti-leukemic efficacy and suppress the emergence of resistance.

Methods. NPM1c-specific CAR T cells were generated using our previously published TCR-mimetic construct targeting the AIQ neoepitope-HLA-A2 complex (Xie et al., Nat Biomed Eng, 2022), while the CD70 CAR was developed using the ectodomain of CD27 (the ligand for CD70). Both constructs incorporated 4-1BB and CD3ζ signaling domains. CAR T cells were generated via VSVG lentiviral transduction of healthy donor T cells. The menin inhibitor revumenib (SNDX-5613) was used for in vitro and in vivo experiments. AML cell lines—including NPM1-mutant OCI-AML3 and KMT2Ar cell lines MV4;11 and MOLM13—or PDX samples were cultured with revumenib to assess changes in MHC-I and CD70 expression via flow cytometry and RNA-seq. Cytotoxicity assays were performed with and without revumenib across multiple effector:target (E:T) ratios. In vivo efficacy and safety were evaluated in NOD Scid Gamma (NSG) mice engrafted with either NPM1-mutant HLA-A2⁺ PDX or luciferase-recombinant MV4;11 (with or without MEN1 mutant mix). Mice were treated with: (a) vehicle, (b) revumenib chow (0.033–0.1% starting on day 4 after tumor cells injection), (c) a single dose of CAR T cells (5–7 × 10⁶ on day 4 after tumor cell injection), or (d) the combination of CAR T cells and revumenib chow. Tumor burden (BLI), circulating blasts (flow cytometry), CAR T cell persistence, and survival were monitored.

Results. After 5 days of incubation, revumenib did not impair CAR T cell viability, proliferation, or IFN-γ secretion, despite MEN1 expression in T cells. Next, we evaluated the combination of revumenib plus NPM1 and CD70-CAR T against the above-mentioned AML cell lines at different E:T ratios to assess killing capacity. Revumenib (250 nM, 4 days) significantly augmented NPM1-CAR T cell cytotoxicity against OCI-AML3 across all E:T ratios, with lysis increasing from 41% to 96% at 5:1 (p < 0.05). Similarly, revumenib (50 nM for 4 days) significantly enhanced the cytotoxicity of CD70-CAR T cells against MV4;11 cells, increasing lysis from 18% to 65% at a 0.5:1 E:T ratio (p < 0.05). In both cases, CAR T cell efficacy was maintained regardless of menin mutation status. In NSG mice bearing NPM1-mutant PDX AML, revumenib (0.033% chow) combined with NPM1c-CAR T cells was safe and extended survival beyond 160 days, compared to median survival of 28 (PDX alone, p < 0.05), 54 (CAR T, p < 0.05), and 84 days (revumenib, p< 0.05). In parallel, NSG mice bearing a mixed population of luciferase+ MV4;11 and MEN1-mutant MV4;11 cells (80:20 ratio) exhibited significantly improved tumor control when treated with CD70-CAR T cells plus revumenib (0.033% chow), relative to CD70-CAR T cells alone. Survival outcomes are currently being evaluated. Notably, CD8⁺ T cells dominated the early CAR T cell response (day 14), while CD4⁺ T cells became more prominent at later stages (day 70). This temporal shift, along with enhanced anti-leukemic activity observed with the combination therapies, may be partly explained by revumenib-induced upregulation of HLA class I and CD70 expression in AML cells.

Conclusions. These findings highlight a novel, rational combination strategy that leverages menin inhibition to potentiate the efficacy of NPM1c- and CD70-specific CAR T cells while simultaneously overcoming resistance in genetically defined AML subtypes. Our preclinical data strongly support clinical translation of this approach, particularly in patients with NPM1-mutant or KMT2Ar AML.

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2025
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