Bcl6 as a Novel Therapeutic Target in Multiple Myeloma (MM).
Authors: Hideshima Teru , Mitsiades Constantine S. , Ikeda Hiroshi , Chauhan Dharminder , Raje Noopur , Gorgun Gullu , Fulciniti Mariateresa , Cirstea Diana , Perron Giulia , Santo Loredana , Hideshima Ayaka , Podar Klaus , Munshi Nikhil C. , Richardson Paul , Carrasco Daniel R. , Anderson Kenneth C. 
Published: October 2019

Abstract
Abstract 295
BCL6 (B- cell lymphoma 6) is a proto-oncogene encoding a transcriptional repressor and regulates germinal center B cell differentiation. It is deregulated by chromosomal translocations or aberrant somatic hypermutation in a subset of diffuse large B cell lymphomas (Polo, JM. et al. PNAS, 2007). Importantly, small peptide inhibitor of BCL6 mediates cytotoxicity in primary human DLBCL cells both in vitro and in vivo, without effecting normal lymphoid tissue (Cerchietti, LC. et al. Blood, 2009). In this study, we examined modulation of Bcl6 expression and its sequelae in human MM cells in the context of bone marrow (BM) microenvironment.
By Western blot analysis, constitutive expression of Bcl6 was either undetectable or very weakly expressed in all MM cell lines (MM.1S, MM.1R, RPMI8226, RPMI-LR5 H929, OPM1 and OPM2) except U266 cells. Importantly, however, Bcl6 expression was markedly upregulated by co-culture of MM cells with bone marrow stromal cells (BMSCs). Since U266 has high baseline phopho-STAT3 and BMSC-induced Bcl6 upregulation in other MM cell lines was associated with phosphorylation of STAT3, we hypothesized that JAK/STAT3 signaling might mediate Bcl6 expression in MM cells in the context of BM microenvironment. Indeed, anti-IL-6 neutralizing Ab significantly blocked BMSC co-culture-induced Bcl6 expression. To assess the clinical relevance of Bcl6 expression observed in MM cell lines, we examined its expression in patient MM cells by tissue microarray immunohistochemical analysis, confirming that Bcl6 was strongly positive within the nucleus in all cases. To obtain direct evidence that IL-6 triggered Bcl6 expression, we cultured MM cells with recombinant human IL-6; as expected, IL-6 strongly triggered Bcl6 expression in MM cell lines in a dose- and time-dependent fashion whereas other cytokines (ie, IGF-1, VEGF, IL-3) did not. Importantly, Bcl6 was also induced by IL-6 in primary tumor cells from MM patients. Real time RT-PCR confirmed that both BMSC co-culture and IL-6 treatment upregulated expression of Bcl6 mRNA. Furthermore, oncostatin-M, which also induces phospho-STAT3, similarly upregulated Bcl6. Conversely, pan-JAK inhibitor AG490 and STAT3 siRNA markedly downregulated Bcl6 expression in U266 cells, associated with downregulation of phospho-STAT3. Taken together, these results indicate that gp130 family member cytokines upregulate Bcl6 expression in MM cells via JAK/STAT3 signaling. We also found that TNFα also triggered Bcl6 in MM cell lines (MM.1S, RPMI8226, OPM1 and OPM2) and primary MM tumor cells; however, TNFα-induced upregulation of Bcl6 was independent on STAT3 activation. Importantly, IKKβ inhibitor MLN120B significantly reduced Bcl6 expression in MM cells triggered by TNFα. Taken together, these results suggested that TNFα-induced Bcl6 expression is mediated via the canonical NF-κB signaling pathway. Finally, to examine the biologic significance of Bcl6 inhibition in MM cells, we downregulated Bcl6 expression using lentiviral shRNA, in the presence or absence of IL-6. Importantly, downregulation of Bcl6 significantly decreased the number of MM cells, associated with decreased S and G2/M phase on cell cycle analysis. Our results therefore suggest that Bcl-6 expression is modulated via both JAk/STAT3 and NF-κB pathways in MM cells. Therefore Bcl6 represents a novel therapeutic target in MM.since targeting these cascades could downregulate Bcl-6 expression, and inhibit growth of MM cells in the bone marrow milieu.

Disclosures:
Raje: Millennium: Speakers Bureau. Munshi:: . Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millennium: Research Funding.