Single Cell RNA-Seq Analysis Revealed MIF/CD74 Pathway Determinants of BCMA CART Resistance in Relapsed/Refractory Multiple Myeloma

Anti-multiple myeloma B cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T-cell therapies has appeared as a significant treatment for relapsed/refractory multiple myeloma (R/R MM). Despite its positive therapeutic outcome, CAR-T resistance and relapse remain critical challenges. This study aims to uncover the molecular mechanisms of CART resistance by conducting single cell (sc) RNA-seq analysis on bone marrow (BM) samples from a R/R-MM patient undergoing BCMA CAR-T therapy clinical trials who finally relapsed 7 months later.

For scRNA-seq, 2 BM samples (total 19,682 cells) obtained from 2-time nodes (before CART infusion and remission) were integrated and annotated into 25 transcriptional cell clusters after dimension reduction. Through the Uniform Manifold Approximation and Projection (UMAP) result, cluster5 (C5), cluster7 (C7), cluster8 (C8), cluster9 (C9), and cluster20 (C20) were identified as tumor plasma cells. While C7, C8, C9, and C20 disappeared in the remission sample, indicating sensitivity to CAR-T therapy, C5 exhibited partial survival, suggesting resistance characteristics. This phenomenon suggested that C5 possessed primary resistance to BCMA CAR-T cell immunotherapy and may be the root cause of later relapse in this patient. InferCNV confirmed distinct heterogeneity in the tumor plasma cells of the resistant population, in which a healthy donor's BM sample from the GEO database GSE176131 was chosen to serve as the normal control. Differentially expressed genes (DEGs) analysis on the re-clustering of malignant plasma cells revealed significant post-treatment upregulation of MS4A1, HLA-DPB1, HLA-DRA, CD74, TMSB4X, IGHM, and IGLC2 in CAR-T resistance subpopulation.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the CART-resistant plasma cell subpopulations. Biological processes (BP), cellular components (CC), and molecular functions were predominantly associated with CD74. KEGG analysis demonstrated significant enrichment of the CD74-related pathway: antigen processing and presentation.

Cell-cell communication analysis was performed between tumor plasma cell subclusters and T lymphocytes using CellChat, revealing a notable reduction in the number of interaction pathways but an increase in strength after BCMA CAR-T treatment. Enhanced interactions were observed in the MHC-II, MIF, BTLA, CD40, CD86, MPZ, LT, SEMA4, BAFF, CD22, CD45, ITGB2, ICAM, ADGRE5, IL6, IFN-II, and GRN pathways. Notably, the CAR-T-resistant MM cell subpopulation exhibited intense interactions with T cells via the LTa1b2/LTBR and MIF/CD74 signaling pathways.

To validate these findings, co-culture experiments were conducted using the MM cell line H929 and BCMA CAR-T cells in vitro. Flow cytometry analysis demonstrated a significant upregulation of CD74 expression on H929 cell membranes at various effector/target(E:T) ratios, beginning after 12 hours. Moreover, when the E:T≥ 1:1, H929 cells co-cultured with CAR-T cells showed CD74 upregulation more significantly. Conversely, H929 cells co-cultured with mock T cells exhibited significant CD74 upregulation only when the E:T <1:1. Importantly, the high-expression of CD74 induced by CAR-T cells persisted and was still observable at 48h, whereas in mock T group the expression started decreasing significantly at 36h.

In summary, this study identified a MM cell subpopulation with upregulation of the MIF/CD74 pathway associated with BCMA CAR-T therapy resistance. These findings offer new insights into the molecular mechanisms underpinning BCMA CAR-T resistance, providing a foundation for potential therapeutic interventions.