Therapy Induced Senescence Is Associated with a Myeloid Gene Signature in Multiple Myeloma Plasma Cells

Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy. Despite therapeutic advances that have vastly improved survival, all patients are expected to relapse. Current standard of care includes autologous stem cell transplant (ASCT) with high-dose melphalan (HDM) administered as a conditioning agent. Cytotoxic chemotherapies such as melphalan have been shown to drive therapy-induced senescence (TIS) in several tissues. We hypothesize that HDM activates TIS pathways in surviving MM cells and that this may correlate with patient outcomes post-ASCT.

We developed an in vitro HDM model using 5TGM1 mouse MM cells treated with vehicle (Veh) or HDM (10µM, IC90) for 6h, followed by co-culture with primary mouse bone marrow stromal cells. Image analysis was performed to quantify proliferation by cumulative population doublings (CPDs) over 10 days, after which 5TGM1 cells were evaluated for cell size, telomere-associated DNA damage foci (TAFs), sensitivity to senolysis, and senescence gene expression (scRNA-seq and RT-qPCR). The effect of HDM on CPDs and cell size was also evaluated in human MM cell lines RPMI-8226 and H929. scRNA-seq was used to analyze CD138⁺ PCs from MM patients at various timepoints post-ASCT. Longitudinal MM patient iliac crest biopsies (diagnosis, post-ASCT; N=20) were stained for CD138⁺ PCs; PC burden was quantified by AI-assisted histology. Lastly, we established an in vivo model of induction therapy (bortezomib) followed by HDM/SCT in Vk*MYC mouse MM engrafted C57BL/6 mice. Tumor burden was tracked by serum protein electrophoresis for M-spike using submandibular bleeds. Bone marrow was then flushed from long bones and analyzed by flow cytometry and immunofluorescence.

HDM-5TGM1 showed decreased CPDs (HDM 0.32±0.31, Veh 6.72±0.69, p<0.0001, N=3), increased size (median pixel size: HDM 413.2±99.98, Veh 207±8.88, p<0.05, N=3), and increased TAF⁺ cells (Percent TAF⁺ cells: HDM 34.77%±10.74%, Veh 11.41%±1.58%, p<0.05, N=3) consistent with known TIS features. Preliminary assessment (N=1) of human MM cell lines showed similar growth arrest (RPMI-8226 CPDs: HDM 0.058±0.101, Veh 4.185±0.358; H929 CPDs: HDM 0±0, Veh 4.61±0.243) and size increase (RPMI-8226 mean pixel size: HDM 804.1±6.048, Veh 526.9±2.115; H929 mean pixel size: HDM 371.2±26.95, Veh 130.3±41.43), suggesting a translatable mechanism. HDM-5TGM1 cells were more sensitive to senolysis by dasatinib+quercetin (viability: HDM 54.9%±0.09%, Veh 76.0%±0.05%, N=2). scRNA-seq analysis showed that HDM-5TGM1 were enriched for gene sets related to senescence pathways, including the published set SenMayo (FDR<0.25). HDM-5TGM1 cells also exhibited myeloid gene expression consistent with previous publications on MM dormancy. RT-qPCR confirmed significant increases in HDM-5TGM1 (p<0.001, N=3) in senescence markers (Cdkn1a, Cdkn1c, Glb1), senescent cell anti-apoptosis pathway genes (SCAPs, Bcl2l1), senescence associated secretory phenotype (SASP) genes (Ccl5, Icam1, Mmp13), and myeloid markers (Axl, Fcer1g, Mpeg1). scRNA-seq of patient CD138⁺ PCs confirmed a myeloid-like cluster that gained prominence in relapsed patients with resistant disease.

CD138⁺ PCs were detected in patient bone biopsies post-ASCT. Interestingly, patients who relapsed (≤3 years, N=9) had a greater percent reduction in PC burden from diagnosis to post-ASCT when compared to patients that did not relapse (N=11) in that time (p=0.02). Further, CD138⁺ PC burden post-ASCT positively correlated with time in remission in relapsed patients (R=0.697, p=0.03).

Bortezomib reduced serum M-spike in Vk*MYC-engrafted mice (p=0.0003); M-spike was further reduced by HDM/SCT compared to Veh-treated mice (p=0.0007). Flow cytometry confirmed a decrease in Vk*MYC cells after HDM/SCT (p=0.0043), and the percentage of Vk*MYC cells with greater size and complexity was increased compared to Veh-treated mice (p=0.0043), consistent with our in vitro data. Vk*MYC cells from HDM-treated mice also trended for more TAF⁺ cells (>3 TAFs) compared to Veh-treated mice (p=0.0714).

Our findings suggest that HDM can induce a TIS and myeloid signature in surviving MM cells. Greater MM burden post-ASCT appears to be associated with longer durable response, again suggesting that these cells may be in growth arrest consistent with TIS. Finally, senolytics may be a novel approach to eliminate surviving MM cells and prevent relapse.

Kourelis:Novartis: Research Funding; Pfizer: Research Funding. Lin:Janssen: Consultancy, Research Funding; Legend: Consultancy; Celgene: Consultancy, Research Funding; Sanofi: Consultancy; NexImmune: Membership on an entity's Board of Directors or advisory committees; Caribou: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Regeneron: Consultancy; Genentech: Consultancy.