MGUS and SMM Plasma Cells Exhibit a Senescence-like Phenotype and Accumulation of Transposable Elements That May Contribute to Disease Progression

Multiple myeloma (MM) is preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). While MGUS/SMM are considered benign and non-proliferative, many of the same oncogenes and chromosomal abnormalities in MM plasma cells (PCs) are also present in MGUS and SMM. Since oncogenic stress induces senescence growth arrest to prevent tumorigenesis, we hypothesized that MGUS and SMM PCs may be in a senescent-like state.

Our analysis of previously published human gene array datasets (GSE5900 and GSE47552) revealed that compared to healthy PCs, MGUS/SMM PCs had significant increases in the expression of senescence markers CDKN1A (GSE5900: LogFC=1.59/1.44; GSE47552: LogFC=1.08/1.34, FDR<0.05) and GADD45A (GSE5900: 1.32/1.76; GSE47552: 2.75/3.33, FDR<0.05). We customized senescence phenotyping gene sets by compiling curated gene lists to test against two published senescence datasets (GSE66236 and GSE109700) and selected genes significantly induced in both senescence datasets. Gene Set Enrichment Analysis (GSEA) on the GSE5900 dataset showed significant enrichment in MGUS/SMM PCs (q<0.25) of our customized gene sets 'Senescence Up', 'Senescence Growth Arrest', 'Senescent Cell Anti-Apoptosis Pathways', and 'Senescence Down'. The gene set 'Inflammatory Senescence-Associated Secretory Phenotype (SASP)’ was significantly enriched only in SMM PCs.

We next evaluated the KaLwRij MGUS mouse model. 10-mo-old female KaLwRij mice were treated with placebo (N=6) or senolytics (dasatinib+quercetin, D+Q, N=7). D+Q treatment significantly reduced PCs compared to placebo, while restoring B cell number and functional gene expression. PCs isolated from D+Q-treated KaLwRij mice exhibited significant reductions in Myc and Il1b gene expression versus placebo (p<0.05) and a trend towards reduced Trp53 and Rel expression (p=0.13/0.11), supporting that senescent PCs are targeted by D+Q. Single-cell RNA-sequencing and single-sample (ss)GSEA of 24-mo-old KaLwRij PCs revealed a PC subset enriched for the 'Plasma Cell Senescence’ gene set, which included genes from our custom senescence gene sets that were differentially expressed in both human PCs datasets GSE5900 and GSE47552.

Notably, PCs in this subset showed enrichment for 'Inflammatory SASP’ (median: 15.26% [range: 15.24-16.32] of the senescence-enriched PCs) and 'Interferon (IFN) SASP’ (median: 39.36% [33.44-43.4]) gene sets. The IFN-SASP is characteristic of late senescence, driven by the accumulation of transposable elements and activation of cytosolic DNA sensing pathways. In both GSE5900 and GSE47552 datasets, we found significant enrichment (q<0.25) of 'IFN-SASP’ in SMM but not MGUS PCs. Further, whole transcriptome RNA-sequencing of PCs collected from consenting patients (Mayo Clinic IRB 521-93) showed that SMM PCs had increased expression of LINE1 retrotransposon L1HS in relation to normal PCs (log₂FC = 0.95, FDR<0.1), but not PCs from MGUS (log₂FC =-0.07, FDR=0.83) or MM (log₂FC = 0.33, FDR=0.5). Immunostaining confirmed increased cytosolic DNA:RNA hybrids in SMM (median frequency of cells with mean fluorescence intensity > 0.04 intensity units = 33%[8.3-45.8], N=5) versus MGUS (16.4%[9-32.4], N=4), MM (4.4%[0.2-17.6], N=5), and healthy PCs (1.5%[0.8-3.5], N=5), which is consistent with cytosolic DNA-mediated activation of the IFN SASP in SMM PCs. When these same patients were redistributed according to disease progression over follow-up time, expression of L1HS was higher in patients with progressing SMM and newly diagnosed MM (NDMM) patients who had recently progressed from SMM (log₂FC = 1.12, FDR<0.1) versus patients with stable MGUS (log₂FC = 0.2, FDR=0.56) or advanced MM (log₂FC = 0.45, FDR=0.38). Increased DNA:RNA staining was observed in progressing SMM PCs (median frequency = 41%[33-45.8], N=3), but the results for NDMM patients who progressed from SMM (4.4%[0.6-17.6], N=3) and advanced MM (3%[0.2-5.6], N=2) were comparable to healthy or stable patients (3.5%[0.8-32.6], N=9).

These data demonstrate that MGUS and SMM PCs exhibit senescence features, suggest mechanisms that may contribute to MM tumorigenesis, and show that pharmacological ablation of senescent cells may prevent progression from MGUS/SMM to MM.

Kourelis:Novartis: Research Funding.