Abstract 2912: The AP-1 transcription factor JunB promotes multiple myeloma cell proliferation, survival and drug resistance in the bone marrow microenvironment

Abstract

Abstract Introduction: The activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. In multiple myeloma (MM), the role of AP-1 is largely unknown. Experimental procedures: MM cell lines and primary tumor cells were co-cultured with primary bone marrow stromal cells (BMSCs) or BMSC lines. AP-1 expression was measured by western blot analysis and real-time PCR. AP-1 activation was determined using TransAM AP-1 assay kit. To investigate the upstream regulators of JunB, cytokine array and specific inhibitors were used followed by 3H-thymidine incorporation, western blot and TransAM AP-1 assays. To delineate the specific functional role of JunB in MM pathogenesis, we used pLKO.1- JunB shRNA (shJunB) and pLKO.1- scrambled shRNA (SCR) vectors for constitutive knockdown, as well as pMSCV-JunB-ER-IRES-GFP and empty vectors for inducible overexpression, together with 3H-thymidine incorporation, alamarBlue, flow cytometry and western blot analysis, as well as gene expression profiling (GEP). To evaluate the functional role of JunB in vivo, a MM xenograft mouse model was used. Results: Co-cultures of MM cells with BMSCs rapidly and strongly induced sustained expression and activation of JunB, but not of other AP-1 family members. Induction of JunB was predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, IL-6 stimulation of MM.1S cells resulted in rapid and strong upregulation of JunB. Conversely, anti-IL-6 receptor antibody tocilizumab blocked BMSC-induced JunB expression and activation. Pharmacologic inhibition identified the requirement of the MEK/ERK and NF-κB pathways for BMSC-induced JunB expression. Functionally, significant inhibition of proliferation was observed in MM cells carrying pLKO.1- shJunB, but not pLKO.1-SCR. Importantly, knockdown of other AP-1 family members had minor effects on MM cell proliferation. Moreover, GEP performed on MM.1S- shJunB cells co-cultured with BMSCs as well as data analysis of a patient cohort using Gene Set Enrichment Analysis (GSEA) suggested a key role for JunB in the regulation of Mcl-1 and c-Myc expression. Furthermore, knockdown of JunB overcame resistance of MM cells to dexamethasone. Conversely, 4-OHT treatment of MM cell lines transduced with JunB-ER but not control vector induced significant JunB/AP-1 luciferase activity and protected MM cells against bortezomib-induced apoptosis and ER stress. Confirming our in vitro data, preliminary results show significant inhibition of tumor growth in a xenograft mouse model inoculated with inducible Tet-shJunB-GFP but not Tet-SCR-GFP MM.1S cells upon treatment with doxycycline. Conclusion: Taken together, our data demonstrate for the first time an important and surprising role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. Citation Format: Fengjuan Fan, Sonia Vallet, Muhammad Hasan Bashari, Mostafa Jarahian, Eugenio Morelli, Dirk Hose, Latifa Bakiri, Claudia Ball, Hanno Glimm, Martin Sattler, Hartmut Goldschmidt, Giovanni Tonon, Pierfrancesco Tassone, Erwin F. Wagner, Dirk Jäger, Klaus Podar. The AP-1 transcription factor JunB promotes multiple myeloma cell proliferation, survival and drug resistance in the bone marrow microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2912.