Single-cell analysis of the multiple myeloma microenvironment after gamma-secretase inhibition and CAR T-cell therapy

• Inhibition of gamma-secretase-mediated shedding of BCMA from the surface of myeloma tumor cells may improve BCMA-targeted therapies

• Single-cell analysis reveals transcriptional and epigenetic changes in the immune microenvironment after gamma-secretase inhibition

Chimeric antigen receptor (CAR) T-cells and bispecific antibodies (BsAb) targeting B-cell maturation antigen (BCMA) have significantly advanced the treatment of relapsed and refractory multiple myeloma (MM). Resistance to BCMA-targeting therapies, nonetheless, remains a significant challenge. BCMA shedding by gamma-secretase is a known resistance mechanism, and preclinical studies suggest that inhibition may improve anti-BCMA therapy. Leveraging a phase I clinical trial of the gamma-secretase inhibitor (GSI), crenigacestat, with anti-BCMA CAR T-cells (FCARH143), we utilized single-nuclei RNA sequencing and Assay for Transposase-Accessible Chromatin (ATAC) sequencing to characterize the effects of GSI on the tumor microenvironment. The most significant impacts of GSI involved effects on monocytes, which are known to promote tumor growth. In addition to observing a reduction in the frequency of non-classical monocytes, we also detected significant changes in gene expression, chromatin accessibility, and inferred cell-cell interactions following exposure to GSI. Although many genes with altered expression are associated with gamma-secretase-dependent signaling, such as Notch, other pathways were affected, indicating GSI has far-reaching effects. Finally, we detected monoallelic deletion of the BCMA locus in some patients with prior exposure to anti-BCMA therapy, which significantly correlated with reduced progression-free survival (median PFS 57 days versus 861 days). GSIs are being explored in combination with the full spectrum of BCMA targeting agents, and our results reveal widespread effects of GSI on both tumor and immune cell populations, providing insight into mechanisms for enhancing BCMA-directed therapies.