Abstract
Introduction: Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy, in which nearly all patients are expected to relapse and undergo multiple lines of treatment over the course of their disease. By the time patients receive CAR-T cell therapy, they have typically been exposed to numerous prior therapies, including high-dose melphalan (HDM). Chemotherapeutic agents that cause genotoxic stress, such as HDM, can induce cellular senescence in normal cells. More recently, it has been demonstrated that tumor cells can also exhibit features of therapy-induced senescence (TIS). TIS in tumor cells is associated with distinct gene expression patterns that have been implicated in resistance to therapy and relapse. To investigate whether TIS is activated in MM cells following exposure to chemotherapies, particularly in the context of late-stage disease, we performed an integrated analysis of single-cell RNA sequencing (scRNA-seq) datasets.
Methods: We integrated scRNA-seq data from bone marrow plasma cells and MM cells using the following three sources: 1) Boiarsky, et al. (GSE193531, dbGaP: phs001323.v3.p1), which includes healthy donors, MGUS, SMM, and newly diagnosed MM (NDMM) patients; 2) Dhodapkar, et al. (GSE210079), which includes late-stage MM patients pre- and post-CAR-T cell therapy; and 3) newly generated scRNA-seq data from healthy donor (N=3) and late-stage MM patients pre- (N=10) and post- (N=2) CAR-T cell therapy. Non-plasma cells were excluded based on canonical plasma cell markers.
We used Seurat v4.3 for data integration, employing canonical correlation analysis (CCA) to define anchors using the FindIntegrationAnchors and IntegrateData functions. Integration quality across datasets was validated by the consistent clustering of healthy plasma cells and expected expression patterns of chromosomal translocation markers CCND1 in t(11;14) cases and FGFR3 in t(4;14) cases, as previously described (PMID: 36396631).
To evaluate senescence, we applied single-cell gene set enrichment analysis (scGSEA) using previously published gene sets (PMID: 40164720): SenUp (Senescence Upregulated), SenGA (Senescence Growth Arrest), and SCAPs (Senescent Cell Anti-apoptosis Pathways). We also curated a Plasma Cell Senescence (PCSen) gene set, composed of senescence-related genes that were differentially upregulated in pre-malignant plasma cells across two independent datasets (GSE5900, GSE47552).A normal plasma cell gene set was also generated, comprising genes differentially expressed between healthy and abnormal plasma cells in GSE193531.
Results:In the integrated dataset, scGSEA revealed significantly higher enrichment of PCSen, SenUp, SenGA, and SCAPs in late-stage MM cells (both pre- and post-CAR-T cell therapy) compared to NDMM and healthy donors (p<0.05, Kruskal-Wallis with Dunn's posttest). Conversely, the Normal gene set was significantly enriched in healthy plasma cells relative to MM (p<0.05). Expression of CDKN2A, which encodes the senescence marker p16, was also increased in late-stage MM relative to NDMM or healthy plasma cells. Notably, in our newly generated scRNA-seq samples collected within 6 months before CAR-T therapy, we observed a significant inverse correlation between senescence marker expression and progression free survival (PFS) post-CAR-T, suggesting that MM cells exhibiting TIS may be more resistant to T cell mediated cytotoxicity. Supporting this, preliminary in vitro experiments showed that MM cells treated with HDM displayed reduced sensitivity to T cell-mediated cytotoxicity, consistent with a senescence-associated resistant phenotype.
Conclusion: Our findings demonstrate that late-stage MM cells exhibit transcriptional signatures consistent with therapy-induced senescence, potentially driven by prior exposure to chemotherapy. This senescent phenotype may contribute to resistance to immunotherapies, including CAR-T cells. These data highlight senescence as a mechanism of resistance and a potential biomarker for predicting CAR-T outcomes. Further studies are needed to explore senescence-targeted strategies to enhance immunotherapeutic efficacy in MM.
