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    Myeloma Genomics and Microenvironment and immune profiling

    Category: Myeloma Genomics and Microenvironment and immune profiling

    Single-cell RNA-seq studies underline the influence at baseline of the BM immune composition in promoting resistance to CAR-T therapies in Multiple Myeloma

    (PA-207) Single-cell RNA-seq Studies Underline the Influence at Baseline of the BM Immune Composition in Promoting Resistance to CAR-T Therapies in Multiple Myeloma

    Friday, September 19, 2025
    Introduction: BCMA- and GPRC5D-directed chimeric antigen receptor T-cell (CAR-T) therapies induce high response rates in multiple myeloma (MM) patients. However, clinical relapses occur in most cases. While the quality of T cells is known to influence responses to CAR-T, the impact of the bone marrow (BM) niche on patients’ outcomes is not fully known. In this study, we used single-cell transcriptomics analysis to evaluate how at baseline the composition of the immune microenvironment correlates with response to CAR-T therapies.

    Methods: BM aspirates were collected from MM patients treated with anti-BCMA (n=4) or -GPRC5D (n=4) CAR-T. Patients were defined as responders (R; n=4) if achieved ≥ partial response (PR) for at least 4 months. Following Ficoll separation and magnetic sorting the CD138- immune and CD138+ MM cells were isolated and used to perform unbiased single-cell mRNA profiling (10x GemCode, NextSeq 1000). CellRanger and Seurat were used for sample de-multiplexing, barcode processing, single-cell 3′ gene counting, and data analysis. A total of 36,450 cells were annotated based on their gene expression signature using scGate and ProjecTILs packages.

    Results: We first compared the baseline immunome profiling of CD138- cells obtained from R and NR. Notably, in NR we observed a highly inflammatory microenvironment characterized by an enrichment of dysfunctional NK cells (CCL3+, CX3CR1+, HAVCR2+, GZMH+) and a higher number of tolerogenic cDC1 (IDO1+) cells together with pDC and monoDC with inflammatory features. In contrast, an activated innate immune microenvironment with significant enrichment of highly cytotoxic NK cells (GZMK+, CXCR4+, CD160+, NCR3+) and an increased number of cDC1 and cDC2 cells promoting type 1 and 2 immune responses were observed in R. Among the T cells in NR we identified an increased number of regulatory CD4 T cells (TIGIT+, FOXP3+, ICOS+), dysregulated CD4 Th17 cells (CTLA4+, CD28+, GZMA+, IFNG+), and exhausted CD8 T cells (HAVCR2+, LAG3+, TIGIT+, TOX+) contributing to a dysfunctional microenvironment. In contrast, enrichment of naïve and effector memory T cells was observed in R to support the cytotoxicity and persistence of  CAR-T cells. Lastly, the analysis of the MM cells transcriptome of NR confirmed the expression of BCMA and GPRC5D on tumor cells and excluded antigenic escape as a cause of resistance. Analysis of cell-cell interactions between tumor and immune cells using CellChat is currently ongoing and will be updated at the meeting.

    Conclusions: In conclusion, through single-cell transcriptomic studies we have characterized the BM niche of patients treated with CAR-T cells and identified in NR a tolerogenic, dysfunctional, and pro-inflammatory BM microenvironment that promotes tumor progression. Our findings underline the influence of the BM immune composition in promoting resistance to CAR-T and support the integration of novel therapeutic strategies able to overcome the negative effects of BM niche and improve the anti-tumor effect of CAR-T in MM.