Pathological RANK Signaling in Combination with TCL1 Drives a Multiple Myeloma-like Disease in Mice

Abstract

Receptor activator of nuclear factor-kappa B (RANK) and its ligand, RANKL, play an important role in different cancer entities, including B cell malignancies (Renema et al, 2016). In diffuse large B cell lymphoma, recurrent RANK mutations were detected. When expressed in murine B cells, RANK(K240E) leads to the development of chronic lymphocytic leukemia (CLL; Alankus et al. 2021). In addition, the RANK/RANKL signaling pathway is also relevant for multiple myeloma (MM) niche formation and disease progression (Raje et al. 2019). To study the consequences of constitutive activation of RANK signaling in combination with the oncogene TCL1, we crossed our RANK(K240E) transgenic mouse model with the TCL1 mouse model to obtain Eµ-TCL1*RANK(K240E)*CD19Cre mice (TC-RK) mice and followed disease burden. When we analyzed the symptom-free survival, we observed that TC-RK mice have a reduced life span with a median survival of 30.1 weeks as compared to >50 weeks in TC mice. Flow cytometric analysis of terminally ill TC-RK mice revealed a strong accumulation of a RANK expressing, CD19-negative population in the spleen, bone marrow (BM) and liver of TC-RK mice, with up to 80-90% of lymphocytes harvested from these organs. Apart from their increased cell size, those RANK+CD19- cells showed strong expression of CD138 and reduced surface expression of B220, and no surface IgM and IgD compared to RANK+CD19+ cells, suggesting they have differentiated to plasma cells. Surprisingly, we observed low levels of CLL cells in the peripheral blood and the spleen, although RANK(K240E) and TCL1 alone induce a CLL-like disease in mice. However, similar to what is observed in human plasma cell disorders, including MM, serum electrophoresis of diseased TC-RK mice revealed sharp clonal spikes in the gamma-fraction, which were not detected in any RK- or TC-derived control serum. Furthermore, plasma cells of TC-RK mice readily engrafted and promoted monoclonal disease development in immunocompromised mice. This data strongly suggests that the cooperation of TCL1 with RANK(K240E) signaling promotes the transformation of plasma cells and implicates a direct role of B-cell intrinsic RANK signaling in MM pathogenesis. To gain insight into the pathogenesis of the MM-like disease in the TC-RK mice, we next examined whether RANK and/or TCL1 expression impacts the plasma cell differentiation capacity in vitro. Ex vivo stimulation with suboptimal LPS concentration revealed an increased differentiation into plasma cells in both, RK and TC-RK derived B cells, as compared to CD19Cre and TC mice. This indicates that active RANK signaling alone is sufficient to promote survival and plasma cell differentiation in vitro. In line, 6-week-old TC-RK and RK mice showed an increased plasma cell population in the spleen, but not in the BM, compared to TC and CD19Cre mice. At 20 weeks of age, only TC-RK mice showed a strong accumulation of plasma cells in the BM (29.9%±11.2, n≥4), spleen (29.7%±8.5, n≥4), and liver (11.7%±6, n≥4), suggesting that only the TC-RK expressing malignant cells disseminate and localize to extra-medullary sites at an advanced stage of disease, mimicking MM. We are currently analyzing bulk and single cell RNA sequencing data to gain insight into the pathways contributing to the disease pathogenesis in this model. To study the relevance of RANK expression in human myeloma cells, we analyzed RANK protein in levels in MM cell lines and observed comparable levels to CLL cell lines, that depend on RANKL for their in vivo progression. Denosumab, a monoclonal antibody blocking RANKL, is used in MM patients to reduce bone turnover. Notably, blocking RANKL in preclinical models reduces MM disease burden (Pearse et al. 2001) and anti-myeloma effects of denosumab have been discussed in MM patient subsets (Terpos et al. 2021). We are therefore currently testing the effects of anti-RANKL treatment on MM cells and evaluating, whether denosumab treatment sensitizes MM cells to other therapies. Taken together, our data implicates a direct role of B-cell intrinsic RANK signaling in MM pathogenesis. Further understanding of the molecular effects of RANK/RANKL in MM should help to optimize treatment combinations to reduce bone disease and target the malignant cells effectively, in order to provide more effective treatments to this deadly disease.