Abstract 642: TRAF2 mediates sensitivity to immunomodulatory drugs via NF-κB and ERK signaling in myeloma cells
Authors: Liu Jiye , Hideshima Teru , Xing Lijie , Wang Su , Zhou Wenrong , Ogiya Daisuke , Wen Kenneth , Tai Yu-Tzu , Cang Yong , Anderson Kenneth C. 
Published: September 2020

The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has improved patient outcome in multiple myeloma (MM); however, acquired resistance to IMiDs commonly underlies relapse of disease. Previous studies show that IMiDs bind to the CRL4CRBN ubiquitin ligase cereblon (CRBN) and promote proteasomal degradation of IKZF1 and IKZF3 followed by downregulation of c-Myc and IRF4, resulting in MM cell growth inhibition. Importantly, some MM cells show resistance to IMiDs despite harboring high CRBN expression levels.

Here we utilize genome-wide CRISPR-Cas9 knockout (KO) screening to identify novel mechanisms mediating resistance to IMiDs in MM. Among positively selected sgRNAs, we found three different sgRNAs targeting TRAF2 were enriched after IMiDs treatment. Individually KO of TRAF2 induced significant resistance of MM cells to Pom and Len treatment independent of CRBN-IKZF1/3 axis. TRAF2 KO cells show hyperactivation of the non-canonical NF-κB pathway and p52 KO re-sensitizes the cells to IMiDs treatment, suggesting that TRAF2 predominantly suppresses non-canonical NF-κB pathway, maintaining sensitivity to IMiDs in MM cells.

We next examined the relevance of TRAF2 downregulation in the context of the bone marrow (BM) microenvironment. Co-culture of MM cells with either BM stromal cells (BMSCs) or culture supernatants (BM-CS) confers resistance to IMiDs. Importantly, TRAF2 in MM cells is downregulated by BMSCs and BM-CS co-culture. TNF-α was elevated in BM-CS, and exogenous TNF-α triggered decreased TRAF2 expression in MM cells, associated with resistance to IMiDs. Taken together, these results suggest that IMiDs resistance in the BM microenvironment may be mediated, at least in part, by TRAF2 downregulation. As observed in the TRAF2 KO, exogenous TNF-α hyper-activated the noncanonical NF-κB pathway without affecting CRBN expression, further confirming a major role of TNF-α mediating protection against IMiDs-driven cytotoxicity.

Interestingly, we also found that TNF-α induces IMiDs resistance even in p52 KO cells, implicating additional mediating IMiDs resistance. Indeed, we found that ERK pathway was highly activated in TRAF2 KO MM cells, and confirmed that both BM-CS and TNF-α induced activation of ERK pathway in MM cells. Moreover, constitutive overexpression of phosphorylated-ERK-2 significantly reduced IMiDs-induced cytotoxicity. Finally, our in vitro and in vivo murine xenograft studies show that MEK inhibitor AZD6244 overcomes IMiDs resistance in TRAF2 KO MM cells or in BM-CS treated wild-type MM cells, indicating that MEK inhibitor can overcome IMiDs resistance in the BM microenvironment. Taken together, our studies therefore identify TRAF2 as a mediator of IMIDs sensitivity in MM cells independent of CRBN-IKZF1/3, and validate the therapeutic potential of MEK/ERK inhibitor to overcome IMiDs resistance.

Citation Format: Jiye Liu, Teru Hideshima, Lijie Xing, Su Wang, Wenrong Zhou, Daisuke Ogiya, Kenneth Wen, Yu-Tzu Tai, Yong Cang, Kenneth C. Anderson. TRAF2 mediates sensitivity to immunomodulatory drugs via NF-κB and ERK signaling in myeloma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 642.