Single-Cell Proteomic Analysis Reveals Menin Inhibition-Induced Proteomic Alterations in AML Patients Treated with Revumenib

Menin inhibition is a promising therapeutic approach for acute myeloid leukemia (AML) with certain genetic alterations including KMT2A rearrangements or NPM1 mutations. The first-in-human Phase 1 clinical trial of revumenib (SNDX-5613) in patients with R/R AML showed promising results and induced complete remission in patients who refractory to multiple lines to therapy. These findings suggest that revumenib could provide a new therapeutic option for patients with AML with KMT2A and NPM1 genetic alterations.

AML is characterized by the existence of distinct differentiation hierarchies, a phenomenon substantially contributing to disease heterogeneity. AML hierarchies exhibit distinct expressions of phenotypic profiles, apoptotic networks, signaling pathways, and metabolic profiles, leading to varying sensitivity towards targeted therapies, resulting in differential sensitivity to targeted therapies. Revumenib induces AML differentiation and thus shift the balance between AML hierarchies, resulting in drastic alterations in the overall AML proteomic profiles and enrichment of distinct leukemia phenotypes with differential sensitivity to targeted therapies. Detailed interrogation of revumenib-induced proteomic alterations could permit precise targeting of persistent cells and development of data-driven combinatorial approaches to achieve higher response rates. Moreover, the distinct combinations of genetic alterations can impact the efficacy of revumenib-induced differentiation and therapeutic outcomes. Thus, to better understand these complexities we performed multimodal single-cell (sc) analysis of AML landscape in R/R AML treated with revumenib (n=4) in Phase 1. We utilized two leukemia-focused 51-parameter CyTOF panels to analyze pre and post-treatment samples from R/R AML patients treated with revumenib. Sc proteogenomic profiling was performed using MissionBio platform using a panel covering 37 genes with recurrent AML-relevant mutations. UMAP and clustering analyses of CyTOF data identified AML blasts, unveiled quantitative changes in cell composition, and delineated proteomic shifts induced under revumenib therapy pressure. We observed enrichment of CD11b+/-CD68+, more differentiated monocytic cells situated at close proximity to AML blasts and depletion of immature AML blasts. Through unsupervised analysis, we discovered discrete AML differentiation hierarchies, delineated by a differentiation continuum that initiates with immature AML blasts (CD34+/-c-Kit+/-), progresses through intermediate AML blasts (CD68+/CD11b-/CD14-), and culminates with more differentiated monocytic cells (CD68++/CD11b+/CD14+/-). Notably, intermediate AML blasts and monocytic cells were enriched in post-treatment samples. Simultaneous ScDNA+protein analysis showed enrichment of differentiated AML blasts with monocytic/dendritic cell phenotype and harboring AML mutations. Differentiated AML blasts expressed higher levels of MCL1 and BCL-xL and upon differentiation we observed gradual downregulation of BCL2. Importantly, intermediate and more differentiated AML blasts under revumenib therapy expressed high levels of p53 protein, a feature that can partly attributed to NPM1 mutations. Sc proteomic analysis delineated proteomic shifts elicited by revumenib and identified potential therapeutic partners, by targeting MCL1, BCL-xL and MDM2, that could be used in combination with revumenib to improve therapeutic efficacy.

We next performed differential expression analysis by comparing the proteomic profiles of pre- and post-therapy samples. We observed suppression of less differentiated AML phenotypic profiles (c-Kit, CD123, CD34) along with MEIS1 and PBX3. Importantly, we also observed significant alterations in immune landscape, suggesting that revumenib could rewire Myeloid-to-T and NK cell interaction/communication by inducing proteomic shifts and driving AML blast differentiation. Further analysis of an expanded cohort of R/R AML patients is currently underway.

In conclusion, multimodal sc analysis revealed profound proteomic alterations induced by revumenib and identified potential therapeutic targets. These findings present new avenues for optimizing combinatorial treatment regimens, enhancing revumenib's efficacy, and illuminating survival mechanisms in persistent, differentiated AML blasts.