Menin-inhibition boosts CAR-T cell therapy against NPM1 mutated and KMT2A-rearranged acute myeloid leukemia

Abstract

Abstract Inhibitors disrupting the Menin-KMT2A chromatin complex have emerged as an auspicious treatment for KMT2A-rearranged (KMT2A-r) leukemias. We previously discovered that NPM1-mutated (NPM1m) AML has a similar dependency on the interaction of menin with wild-type KMT2A and that menin inhibitors (men-i) suppress a MEIS1-driven leukemic gene expression program to induce differentiation (Kühn et al., Cancer Discovery 2016). Early clinical trials reported dramatic efficacy in heavily pretreated patients, leading to the FDA approval of the men-i revumenib for relapsed/refractory KMT2A-r ALs and priority review for NPM1m AML. Here, we discovered that men-i uniformly induces the expression of the immunotarget CLEC12A (also known as CLL-1) on both leukemia entities, thereby sensitizing these cells for chimeric antigen receptor (CAR)-based immunotherapy. Initially, RNAseq revealed CLEC12A to be consistently upregulated in KMT2A-r and NPM1m AML cells upon pharmacological inhibition with different men-i, including MI-503, ziftomenib, or VTP-50469 (close homologue to revumenib). These transcriptional changes translated into an increased protein surface expression as assessed by flow-cytometry (FC) and were also confirmed on primary NPM1m patient material. While CLEC12A is not bound by menin or KMT2A as evidenced by ChIP-seq, FC-assessment of various differentiation markers, including CD11b, were consistent with early monocytic differentiation as a potential mechanism of CLEC12A induction. To evaluate men-i as an AML priming treatment for CLEC12A-directed CAR-based immunotherapy, we first assessed men-i's effects on immune effector cell function. T cell proliferation was only mildly impaired upon long-term culture (d21) at very high concentrations, while NK cells moderately expanded. Men-i did not significantly impair the killing efficacy of non-transduced T or NK cells, and we did not detect significant immunophenotypic changes in the expression of various exhaustion markers (e.g. PD-1, TIM-3, LAG-3, TIGIT) by FC. Also, the IFN-γ response in T and NK cells was not impaired, and there were no significant changes in immune cell proportions as assessed by single-cell RNAseq of non-transduced T and NK cells following 4 days of men-i treatment compared to vehicle control. As these data indicated that men-i does not negatively affect immune effector cell functionality, we next engineered CLEC12A-directed CAR-T and CAR-NK cells containing a 4-1BB.CD3ζ stimulatory domain that showed effective killing of CLEC12A-expressing target cells in vitro. For combinatorial assessment, NPM1m OCI-AML3 were treated for 4 days with men-i before CAR-T or -NK cells were added in different Effector to Target (E:T) ratios. The combination of men-i with CLEC12A-directed CAR-treatment resulted in a significantly enhanced anti-leukemic activity, outperforming both single treatments across various donors. Notably, CLEC12A-directed CAR-T cells were similarly effective against an adaptive experimental model of men-i-resistant NPM1m OCI-AML3 cells that maintained high CLEC12A expression with continuous men-i treatment. The men-i and CAR-T cell combination was also effective in primary NPM1m AML cells upon six days of men-i with superior anti-leukemic activity over single therapy in half of the samples. Next, we confirmed that in vivo men-i treatment induced CLEC12A in two different NPM1m and KMT2A-r cell line-derived xenograft models. Combined in vivo men-i (14 days) and CLEC12A-directed CAR-T cell treatment resulted in significantly enhanced eradication of engrafted KMT2A-r MOLM-13 cells as assessed 9 days after CAR-T cell injection. To our knowledge, this is the first preclinical study demonstrating that men-i induces the immunotarget CLEC12A to overcome the limitations of CAR-based immunotherapy against AML. Combined men-i and CLEC12A-directed CAR-T cell treatment had dramatic anti-leukemic effects in vitro and in vivo and should soon be available for clinical investigation.