Molecular Sequaele of Activin A-Dependent Osteoblast Inhibition in Myeloma.
Authors: Vallet Sonia , Mukherjee Siddhartha , Vaghela Nileshwari , Hideshima Teru , Fulciniti Mariateresa , Pozzi Samantha , Santo Loredana , Cirstea Diana , Rahemtullah Aliyah , Attar Eyal C. , Guimaraes Alexander R , Chauhan Dharminder , Xie Wanling , Patel Chirayu , Schoonmaker Jesse , Churchill Michael , Patel Kishan , Weller Edie , Munshi Nikhil C. , Seehra Jasbir , Anderson Kenneth C. , Scadden David T. , Raje Noopur 
Published: October 2019

Abstract
Abstract 1789
Poster Board I-815
Understanding the pathogenesis of cancer-related bone disease is critical to the discovery of new therapies. The development of osteolytic lesions in multiple myeloma (MM) results from unopposed osteoclast activity due to decreased osteoblast (OB) function. We recently demonstrated that activin A contributes to MM-mediated OB inhibition. The availability of a clinical-grade activin A inhibitor that reduces tumor burden by restoring the bone structure in vivo, underscores the relevance of this pathway in the development of MM-bone disease. Here, we characterize the signaling pathway mediating OB inhibition by activin A. Activin A secretion by bone marrow stromal cells (BMSC) is enhanced in the presence of MM cells which was completely abrogated and using a transwell system. To identify the receptors and pathways involved in activin A secretion, we used neutralizing antibody against several integrins, CD40 and osteopontin. Only VLA4 neutralizing antibodies partially inhibited activin secretion by about 20% (range 10-30%, p<0.05). Since activin A promoter contains a cJUN-binding sequence, we explored the relations between JNK and activin. We show that upon MM cell binding to BMSC, stromal-derived JNK is phosphorylated and SP600125 (20 μM), a specific JNK inhibitor, impairs activin A secretion by BMSC both at basal conditions and in the presence of MM (40% and 54%, respectively; p<0.01). These results suggest that activin secretion requires JNK activation induced by cell-to-cell contact. We next investigated the mechanism underlying activin-mediated OB inhibition by assessing the activity of several signaling pathways critical to OB differentiation. First, we showed that activin A induces SMAD2 phosphorylation, while no effects were noted on SMAD1, β-catenin, pP65 and pERK. Then, using a doxycycline-inducible β-catenin system, we demonstrate that β-catenin activation overcomes activin A inhibition of OB differentiation. These results suggest that activin/SMAD2 and β-catenin modulate OB differentiation by affecting a common downstream target. We next evaluated the expression of two candidates, the transcription factors RUNX2 and DLX5. Only DLX5 expression was downregulated by exogenous activin A (5 fold decrease at 96 hours, p< 0.01). Specific inhibition of SMAD2 via shRNA-mediated knock-down upregulated basal DLX5 and ALP mRNA expression (1.5 and 4.7 fold increase respectively, p<0.05), and partially overcame activin A inhibitory effects. In turn, DLX5 knock-down abrogated OB differentiation without additive effects by exogenous activin A. The clinical relevance of DLX5 was confirmed by the strong correlation between its expression levels in BM biopsies from MM patients and activin A levels in BM serum. Finally, we demonstrate by in vitro and in-vivo studies that MM-mediated DLX5 inhibition is restored by treatment with RAP-011, the specific activin inhibitor. In conclusion, we show that MM cell engagement of BMSC enhances activin A secretion via adhesion-mediated JNK activation and activin A, in turn, inhibits osteoblast differentiation via SMAD2-dependent DLX5 downregulation. This study identifies a novel pathway relevant to OB differentiation and amenable to drug targeting.

Disclosures
Chauhan: Nereus Pharmaceuticals, Inc: Consultancy. Seehra:Acceleron Pharma: Employment. Anderson:Celgene : Research Funding; Novartis: Research Funding; Millennium: Research Funding. Scadden:Fate Therapeutics: Consultancy. Raje:Astrazeneca, Novartis, Celgene: Research Funding.