Biology and Prognostic Implications of Bone Marrow Plasmacytoid Dendritic Cells in Multiple Myeloma Pathogenesis By Single-Cell RNA Sequencing

Introduction. In multiple myeloma (MM), plasmacytoid dendritic cells (pDCs) promote tumor growth, resistance to therapy-induced cytotoxicity, and an immunosuppressive microenvironment. Previously, we used RNA-seq to assess the response of MM cells to coculture with pDCs. To assess this interaction in vivo, we utilized single-cell RNA sequencing (scRNA-seq) of bone marrow (BM) cells from relapsed/refractory MM patients (n=3) and healthy donors (n=3) to investigate the gene expression of pDCs and normal or cancerous plasma cells.

Results. As expected, flow cytometry of fresh samples revealed that there were more pDCs in MM patients (~3% vs 0.2% of total MNCs, p < 0.05). After initial quality control, we had single-cell transcriptomic profiles for 42,946 cells (6 BM samples; median genes/cell ~ 1,329; Median UMI counts/cell ~3,588), which formed 33 cell clusters in graph-based clustering and UMAP analysis, and were further annotated to different cell types based on distinct set of markers for each. Quality control metrics were used to select cells with <10% of mitochondrial gene expression. Our analysis identified all the major cell types in the bone marrow microenvironment (e.g., B, T, NK, NKT, classical monocytes, pDCs, and myeloid dendritic cells including their subtypes). We identified pDCs by the expression of CD123, CD303/CLEC4C, CD304/NRP1, TCF4, ILT7/LILRA4, IRF8 and IRF4, and MM cells by the expression of Syndecan 1/CD138. A total of ~23000 transcripts were analyzed for each cell type using Limma, and we found that 10607 genes were upregulated and 1128 downregulated in pDCs from MM patients compared to healthy controls. Of note, only ~1200 and ~1672 genes were up- or downregulated, respectively, in myeloid dendritic cells, emphasizing the critical role of pDCs in MM pathogenesis.

1) We found that the toll-like receptor cascade (NES = -1.65) and interferon type I signaling (NES = -1.76) were downregulated in the pDCs of MM patients, which is consistent with our earlier data. 2) Both NRP1 (Log2FC: -1.105), which is implicated in several immunoregulatory roles including priming immune responses and type 1 interferon release by pDCs, and master transcription factor TCF4 (Log2FC: -0.661) (adj p < 0.05), which is an essential and specific regulator of pDC development, were downregulated in pDCs from MM patients. 3) We found that JCHAIN, which is the joining chain of multimeric IgA and IgM but has an undefined role in pDCs, was downregulated in the pDCs of MM patients (Log2FC: -0.934). 4) Several REACTOME gene sets were upregulated in the pDC of MM patients, including ClassA/1- Rhodopsin-like receptors (NES = 1.754) and CD22-mediated BCR regulation (NES = 1.81). 5) We further analyzed Siglec transcripts and found that CD22/SIGLEC2 (Log2FC: 3.14), CD169/SIGLEC1 (Log2FC: 2.075), and CD327/SIGLEC6 (Log2FC: 1.291) were upregulated in the pDCs of MM patients, which suggests that targeting hypersialylation may represent a novel approach to enhancing anti-MM immunity.

Conclusions Our results provide a comprehensive understanding of the immune changes in the pDCs of MM patients, which may help inform novel therapeutic strategies.

Chauhan:C4 Therapeutics: Current equity holder in publicly-traded company; Oncopeptides: Consultancy; Stemline Therapeutics: Consultancy. Anderson:Dynamic Cell Therapy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; NextRNA: Other: Scientific founder ; Raqia: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder ; OncoPep: Other: Scientific founder ; Starton: Membership on an entity's Board of Directors or advisory committees; Window: Membership on an entity's Board of Directors or advisory committees; Precision Biosciences: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.