Single-Cell RNA Analysis of Bone Marrow Mononuclear Cells Reveals Abnormal Activation of T Cells and Monocytes, and Identifies CD35 As a Novel Biomarker and Therapeutic Target in Waldenstrom Macroglobulinemia

Background:

Waldenström macroglobulinemia (WM) is an indolent B-cell malignancy for which there is currently no cure. The underlying molecular mechanisms and the impact of the immunosuppressive microenvironment on WM oncogenesis remain still incompletely understood.

Methods:

We utilized single-cell RNA sequencing combined with BCR and TCR sequencing to analyze bone marrow samples from 7 healthy donors (HD) and 26 newly diagnosed WM patients. In vitro experiments were performed using antibodies to validate the function of the novel WM biomarker CD35 in BCWM.1 and MWCL-1 cell lines.

Results:

Our findings highlighted significant differences in the immune microenvironment between WM patients and healthy donors. In WM patients, we observed a marked reduction in CD8⁺ naïve T cells and an increase in CD8⁺ effector T cells compared to healthy donors. Monocyte proportions were also lower in WM patients. Several stages of normal B cells, including pre-B cells, naïve B cells, and memory B cells, were significantly reduced in WM patients. Notably, clonal B cells were predominantly found in CXCR4^mut patients, while clonal plasma cells were more common in CXCR4^wt patients.

We employed non-negative matrix factorization to characterize T cells and monocytes, identifying key signatures based on their top 10 genes. Four T cell signatures were identified: T-sig1, enriched in CD4⁺ T cells; T-sig2, enriched in CD8⁺ memory and effector T cells; T-sig3, representing the TCR activation pathway and significantly elevated in WM patients; and T-sig4, enriched in CD8⁺ naïve T cells. There were no significant differences in T-sig3 between CXCR4^mut and CXCR4^wt patients.

By defining T cell exhaustion and cytotoxicity scores, we found a significant reduction in the cytotoxic capacity of CD8⁺ T cells and an increase in exhaustion scores in WM patients, with no significant differences between CXCR4^mut and CXCR4^wt patients. Additionally, four monocyte signatures were identified. M-sig2, associated with cellular activation, was significantly increased in WM patients. M-sig4, enriched for MHC II-related molecules, was significantly reduced in CD14⁺ monocytes, indicating a possible enrichment of CD14⁺HLA-DR^-/low MDSCs in WM patients. The pro-inflammatory M-sig1 was significantly increased, whereas the anti-inflammatory M-sig3 was significantly decreased in WM patients.

To identify new biomarkers for WM tumor cells, we used logistic regression to create a classifier distinguishing WM tumor cell (clonal B cells and clonal plasma cells) from normal counterparts (memory B cells and normal plasma cells) in healthy donors, achieving an AUC of 0.99. This analysis revealed the top 20 differentially expressed genes (DEGs) between tumor and normal cells.

Patient-specific transcriptional changes were further discovered by comparing tumor cells to normal cells from the same samples. Among the top 20 DEGs, CR1 (CD35) had the highest AUC for distinguishing tumor cells from normal cells in patients. This finding was validated using an additional bulk-RNA dataset, which showed significantly reduced CD35 expression in WM.

Given the role of CD35 in inhibiting BCR signaling pathways, we treated WM cell lines with CD35 antibodies. The CD35 antibody significantly inhibited tumor cell proliferation, with IC50 values for BCWM.1 cells ranging from 8.34-26.52μg/mL and for MWCL-1 cells from 5.67-12.04μg/mL. Flow cytometry demonstrated that CD35 antibodies induced apoptosis in WM tumor cells in a dose-dependent manner.

Conclusion:

Our study reveals that T cells and monocytes in the WM bone marrow microenvironment are hyperactivated but exhibit reduced cytotoxic function. We identified CD35 as a robust marker for distinguishing WM tumor cells from normal B-cell counterparts. CD35 targeting antibodies induced robust apoptosis and/or significantly inhibited the proliferation of WM tumor cells, supporting the investigation of CD35 as a promising biomarker and therapeutic target for WM.

Sarosiek:ADC Therapeutics: Research Funding; Cellectar Biosciences: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding. Castillo:AbbVie: Consultancy, Research Funding; Janssen: Consultancy; AstraZeneca: Consultancy, Research Funding; Cellectar Biosciences: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Mustang Bio: Consultancy; LOXO: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy. Treon:Janssen: Honoraria, Research Funding; Eli Lilly: Research Funding; AbbVie/Pharmacyclics: Honoraria, Research Funding; BeiGene, Inc.: Honoraria, Research Funding; Parexel: Honoraria, Research Funding.