Elucidating the Role of IRF4 in Malignant Plasma Cell Regeneration at Single Cell Resolution

Multiple myeloma (MM) is a fatal disease characterized by the abnormal growth of plasma cells. Despite advancements in treatment, many patients experience relapse, particularly those at high risk. Poor outcomes are attributed to the uncontrolled proliferation of malignant stem-like cells, however, the specific genetic factors driving myeloma stemness within the bone marrow niche are still unknown. These insights are crucial for understanding cancer stem cell biology in MM and enabling the development of novel therapeutic strategies targeting malignant stem cell generation in MM as well as other lymphoplasmacytic neoplasms. A potential candidate is interferon response factor-4 (IRF4), a transcription factor that regulates B-cell development and is critical for myeloma cell survival. This study investigated the contribution of IRF4 to drug resistance driven by the activation of key stem cell functional pathways at a single cell level, revealing novel mechanisms that connect IRF4 to myeloma regeneration.

First, to determine the extent to which IRF4 pathway activation drives stem cell gene expression and phenotypes in MM, we enforced IRF4 overexpression in human myeloma cell lines (HMCLs). We experimentally induced IRF4 using lentiviral vectors compared with negative vector controls as well as wild-type control cells. Functional drug resistance assays revealed that IRF4 upregulation (3-5 days) promoted intrinsic resistance of MM cells to standard-of-care drugs bortezomib and lenalidomide. Complementary whole transcriptome analysis 48 hours after transduction shows a small set of common differentially expressed genes between the three HMCLs. Subsequent gene set enrichment analyses revealed that these genes were enriched for pathways associated with mRNA splicing activity and protein translation - two functions that have previously been linked closely to normal or malignant hematopoietic stem cell function. The majority of IRF4 overexpression-associated genes were upregulated, suggesting that IRF4 turns on transcriptional programs that increase relative mRNA levels of associated genes, revealing mRNA splicing and protein translation as potential novel therapeutic vulnerabilities for MM. Finally, we extended these studies to investigate the molecular impact of IRF4 upregulation at a single cell level using 10X scRNA-seq.

To explore the clinical relevance of these findings and elucidate connections between IRF4 and stem cell gene expression in single cells from primary patients, we analyzed a public single-cell dataset from analysis of CD138⁺ cells from healthy and diseased bone marrow (BM) and generated new scRNA-seq clustering, creating more unified clusters compared to published results. We then characterized molecular alterations associated with expression of IRF4 in single cells and discovered that genes related to protein translation as well as a pro-survival transcriptional regulator ATF5, and the hematopoietic stem cell and MM associated cell surface antigen CD74 were correlated in IRF4⁺ cells. Subsequently, the cells were subdivided into four disease stages: MM, smoldering MM (SMM), monoclonal gammopathy of undetermined significance (MGUS) and normal BM (NBM). IRF4 was enriched in MM, SMM and MGUS compared to NBM, while genes CCND2 and IFI6 were found to have the highest fold-change in MM compared to NBM and were upregulated after IRF4 overexpression. These genes have been previously associated with translocations and expression in malignant plasma cells, respectively.

Together, these data show that overexpression of IRF4 induces malignant stem cell-associated gene expression programs and functional changes in MM cell drug sensitivity. Further evaluation of genes enriched in primary sample-derived IRF4-high versus low cell subpopulations provides essential new evidence supporting a role for IRF4 in risk for transformation of MM from SMM. Additionally, this research uncovers new candidate myeloma stem cell marker genes that were associated with IRF4 expression in MM (e.g. CD74) and identifies mRNA splicing pathways as upregulated in this subpopulation of MM cells. Together, these findings open new opportunities for therapeutic targeting in MM to block IRF4-driven lymphoid oncogenesis, for example with potent splicing modulator agents that are currently in development for other hematologic malignancies.