Restoration of Bone Balance Via Activin a Inhibition Results in Anti-Myeloma Activity
Authors: Vallet Sonia , Mukherjee Siddhartha , Vaghela Nileshwari , Pozzi Samantha , Santo Loredana , Cirstea Diana , Fulciniti Mariateresa , Rahemtullah Aliyah , Attar Eyal , Guimaraes Alexander R , Xie Wanling , Patel Chirayu , Schoonmaker Jesse , Weller Edie , Hideshima Teru , Munshi Nikhil C. , Seehra Jas , Scadden David T. , Anderson Kenneth C , Raje Noopur 
Published: October 2019

Abstract
A distinct feature of multiple myeloma (MM) is the tight interaction between malignant plasma cells and their bone microenvironment, creating a niche suitable for MM growth. In particular, MM cells inhibit osteoblast (OB) differentiation and stimulate osteoclast (OC) function, resulting in imbalanced bone remodeling and osteolytic bone disease. Here we studied a novel cytokine, activin A, identified from a broad range of cytokines, in the development of MM bone disease. We next asked whether activin A inhibition could restore bone balance and suppress tumor growth. Activin, a member of the TNF-α superfamily, is a pleiotropic cytokine involved in bone remodeling. Here, we observed, that MM patients with multiple osteolytic lesions had a 4-fold increase in activin A expression levels in bone marrow plasma compared to MM patients with one or less osteolytic lesions and non-MM patients (average 123.6 ± 136 vs 26.4 ± 21.4 vs 30.6 ± 25.1 pg/ml respectively, p<0.05). Interestingly, our data demonstrate that the main source of activin in the MM niche are bone marrow stromal cells (BMSCs), followed by OCs, and OBs (average levels in 72h culture supernatant are 1884, 1300, 299 pg/ml, respectively). In contrast, MM cells did not secrete activin, but stimulated its secretion in coculture by BMSC (by 1.3 to 2 fold increase). Activin A stimulated OC differentiation in synergy with RANKL and M-CSF via induction of a three-fold increase in precursor cell proliferation. Moreover, activin A had a potent inhibitory effect on OB differentiation as verified by ALP activity (reduced by 30% compared to control, p<0.05) and OB function, assessed with alizarin red (80% inhibition, p< 0.01). To test the role of targeting activin A with therapeutic intent, we used both a neutralizing antibody and a soluble receptor fusion, RAP-011 (Acceleron Pharma Inc., Cambridge). In effect, both strategies enhanced OB differentiation and activity (5 fold increase in calcium deposition at day 21, p<0.05). This was confirmed by quantitative-PCR analysis of ALP and osteocalcin gene expression. Importantly, RAP-011 promoted OB differentiation even in the presence of INA6 MM cells and reversed the inhibitory effects of the stroma-dependent MOLP5 MM cells as well as patient derived MM cells on OB. Enhanced OB differentiation by RAP-011 resulted in inhibition of MM cell proliferation compared to BMSCs and mature OB. These data thus suggest that manipulating the bone niche may result in reduced tumor growth. To further verify if these results translated in reduced tumor growth in vivo, we used the SCID-hu mouse model consisting of INA6 MM cells injected in a subcutaneously implanted fetal human bone. RAP-011 treatment resulted in a decrease in the number of osteolytic lesions assessed by CT imaging accompanied by improved bone density. These effects translated in reduced MM cell growth, analyzed by soluble human IL6 receptor levels, in the treated group compared to the control (p<0.02). These data therefore suggest that activin A is involved in development of MM bone disease and can be effectively targeted by a novel clinical grade compound. RAP-011 demonstrated bone-anabolic effects via inhibition of OC formation and stimulation of OB differentiation resulting in restoration of bone balance within the MM niche, which translated in in-vivo inhibition of MM cell growth. These effects of RAP-011 support the use of the human ACE-011 as an attractive approach for the treatment of MM.