TRAF2 Mediates Sensitivity to Immunomodulatory Drugs in the Bone Marrow Microenvironment
Authors: Liu Jiye , Hideshima Teru , Xing Lijie , Wang Su , Ogiya Daisuke , Samur Mehmet K. , Munshi Nikhil C. , Richardson Paul G. , Wen Kenneth , Tai Yu-Tzu , Cang Yong , Anderson Kenneth 
Published: February 2021

The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has improved patient outcome in multiple myeloma (MM). Previous studies have shown that IMiDs directly bind cereblon (CRBN), a substrate adaptor of Cullin4 Ring Ligase (CRL4), and activate CRL4CRBN ligase, thereby selectively targeting two B cell transcription factors IKZF1 and IKZF3 for ubiquitylation and proteasomal degradation. We have shown that IMiDs also directly bind and inhibit TP53-regulating kinase activity, resulting in MM cell growth inhibition. Importantly, IMiDs have achieved additive and synergistic anti-MM activity when combined with proteasome inhibitors and monoclonal antibodies in vitro, and are now utilized in combinations to treat newly diagnosed MM. However, development of resistance to IMiDs commonly underlies relapse of disease.
To delineate mechanisms of IMiDs resistance, the majority of previous studies have focused on CRBN. Although downregulation or mutations in CRBN can be associated with IMiDs resistance, MM cells can manifest resistance without any CRBN dysfunction, indicating alternative mechanisms of IMiDs resistance. We and others have shown a critical role of the BM microenvironment in MM pathogenesis: secretion of soluble factors from cellular components activates intracellular signaling pathways that promote MM cell migration, proliferation, survival, and drug resistance. To date, however, the mechanisms whether tumor microenvironment mediates IMiDs resistance have not been fully delineated.
In our genome-wide CRISPR-Cas9 knockout screening, we have validated that CSN9 signalosome complex regulates sensitivity to IMiDs by modulating CRBN expression. Our screen also identified TRAF2, a member of the TNF receptor associated factor protein family, to regulate IMiDs sensitivity. To confirm modulation of IMiDs sensitivity by TRAF2, we here individually cloned TRAF2 sgRNAs into LentiCRISPRv2 vector, and then re-introduced them into MM cells. As expected, TRAF2-KO MM cells acquired significant resistance to Pom and Len treatment. Importantly, TRAF2 KO showed no effect on CRBN expression; moreover, IMiDs treatment of TRAF2 KO MM cells still triggered IKZF1 and IKZF3 degradation associated with downregulation of IRF4, the main effector of MM cell survival. Importantly, we observed that TRAF2 KO MM cells expressed high levels of p-ERK. We and others have shown that the bone marrow (BM) microenvironment activates ERK signaling pathway and plays a crucial role in drug resistance, and here showed that co-culture of MM cells with BM stromal cells (BMSCs) or culture supernatants (SC-sup) confers resistance to IMiDs, associated with downregulation of TRAF2. These results suggest that IMiDs resistance in the BM microenvironment may be mediated by ERK signaling pathway induced by soluble factors.
AZD6244 is a potent and highly selective MEK inhibitor, and we next investigated whether AZD6244 inhibits ERK1/2 activation and overcome IMiDs resistance in TRAF2 KO MM cells. Importantly, addition of AZD6244 overcame resistance to IMiDs in both TRAF2 KO MM cells and SC-sup treated TRAF2 wild-type (WT) cells. In our human MM murine xenograft model, we observed that TRAF2 WT MM cells were sensitive to Pom treatment in vivo, while TRAF2 inducible KD MM cells demonstrated resistance. Importantly, the combination of AZD6244 and Pom significantly reduced growth even of TRAF2 KD MM cells, indicating that MEK inhibitor can inhibit activation of ERK1/2 and overcome IMiDs resistance in vivo. Finally, we confirmed that the number of patient samples with activated ERK pathway was significantly enriched at relapse while on lenalidomide maintenance therapy compared to diagnosis (BIOCARTA ERK pathway analysis 67% vs 39%, respectively, FDR&lt;0.05), implicating ERK activity in clinical resistance to IMiDs.
In summary, we have identified and validated TRAF2 as a CRBN-IKZF1/3 axis-independent regulator of BM microenvironment-mediated sensitivity to IMiDs. These studies not only identify a novel mechanism of IMiDs resistance in the tumor microenvironment, but also provide the preclinical rationale for combining inhibitors of MEK/ERK signaling with IMiDs to overcome IMiDs resistance and improve patient outcome.

Disclosures
Munshi: Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Anderson:Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.