Enhanced Survival and Adherence of Melphalan-Induced Senescent-like Dormant Multiple Myeloma Cells Co-Cultured with Bone Marrow Stromal Cells

Abstract

Introduction: Multiple myeloma (MM) is an incurable plasma cell cancer and the second most common hematological malignancy in the United States. Therapeutic advances have greatly improved patient survival over the last decade; however, all patients are expected to relapse. New treatments are critically needed to overcome relapse and provide durable response. There is evidence that dormant MM cells exist in the bone marrow, where they exhibit reduced proliferative capacity and resistance to treatment. These cells are present over extended periods of time and may regain proliferative capacity to drive disease relapse. Tumor dormancy shares many features with senescence, a state of stress-induced growth arrest. Chemotherapies, including those used in the standard treatment of multiple myeloma such as melphalan, have been shown to cause therapy-induced senescence in a variety of tissues. Dormant MM cells have been shown to be maintained in their non-proliferative state by interactions with bone lining cells. Interestingly, matrix signaling has also been shown to protect against chemotherapy-induced apoptosis in an in vitro ovarian cancer organoid model. Taken together, this suggests that interaction of MM cells with the bone niche maintains both growth arrest and survival. We hypothesize that therapy-induced senescence drives dormancy in MM cells that is dependent on interactions with the mesenchymal bone niche. Methods: GFP-expressing 5TGM1 mouse MM cells were treated with vehicle or melphalan for 48 hours and then cultured in the presence or absence of ex vivo mouse bone marrow stromal cells (BMSCs). Cells were maintained for 12 additional days to allow for the development of a senescent phenotype after the initial stress response. During this period, cells were imaged and quantified during media replacements to assess adherence/survival. Cells were then fixed and assessed for nuclear foci, phospho-γH2AX, and telomeres by immunofluorescence/fluorescence in-situ hybridization (IF/FISH). Results: Melphalan-treated 5TGM1 cells cultured with BMSCs were significantly (p<0.0001) growth arrested over the 2-week monitoring period (mean cumulative doublings: 1.26 ± 0.179) compared to vehicle-treated cells (5.84 ± 0.122). Co-culture with BMSCs significantly (p<0.0001) enhanced melphalan-treated 5TGM1 cell adherence (mean percent of total cells washed-off: 21.7% ± 4.2%) compared to culture without BMSCs (92.2% ± 4.3%) during media replacement. Of note, melphalan-treated 5TGM1 cells did not survive in the absence of BMSCs after a week in culture. Melphalan-treated 5TGM1 cells also exhibited significantly greater (p<0.001) adherence with BMSCs (mean percent of total cells washed-off: 21.7% ± 4.2%) than vehicle-treated cells (48.9% ± 5.1%). Melphalan-treated 5TGM1 cells were significantly (p<0.0001) larger (median area in square microns: 63.96 [7.66-954.05]) than vehicle-treated cells (44.43 [7.66-434.32]) and exhibited both heterochromatin foci and persistent DNA damage foci associated with telomeres, all of which are markers of cellular senescence. Conclusions: Our findings suggest that interactions with BMSCs promote the survival of chemotherapy-treated MM tumor cells. The enhanced adherence of these melphalan-treated MM cells also suggests that current methods for minimal residual disease assessment may be inadequate, as rare populations of MM cells adhered to the marrow stroma may not be captured by routine bone marrow aspiration. These cells exhibit features of therapy-induced senescence which may be key to MM dormancy. Thus, targeting senescence survival pathways via senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent disease relapse.