Chang, Xiu-Bao

Abstract Abstract 1807 We have recently demonstrated that cereblon (CRBN) mediates the direct anti-myeloma activity of immunomodulatory drugs (IMiDs). However, the genes/pathways downstream of CRBN associated with anti-myeloma activity remain unclear. We, and others, identified interferon regulatory factor 4 (IRF4) as one of the downstream targets of CRBN-associated signaling. Both lenalidomide treatment and CRBN knockdown downregulate IRF4. IRF4 levels return to baseline in IMiD resistant cells surviving CRBN silencing. To determine whether IMiD-induced IRF4 downregulation is critical to anti-MM activity, we overexpressed IRF4 in two IMiD-sensitive human MM cell lines (HMCLs), KMS11 and MM1.S, followed by lenalidomide treatment. Lenalidomide-induced cytotoxicity was greatly impaired in both HMCLs overexpressing IRF4 compared with the control virus infected cells. Further analysis indicated that IRF4 over-expression does not completely prevent lenalidomide-induced growth arrest, but reduces cell death by 70% after lenalidomide treatment. Immunoblotting analysis of KMS11 cells indicated that IRF4 over-expression blocks lenalidomide-induced activation of caspase 8, reduces up-regulation of p21waf and increases CDK6 expression but does not significantly affect lenalidomide-induced MYC down-regulation. Although cereblon and IRF4 are broadly expressed in MM, baseline levels of expression are only weakly correlated (r=0.22) in primary MM patient gene expression analysis. Gene expression studies revealed statistical changes in 1,368 genes when comparing high versus low CRBN expression in primary myeloma samples. Interestingly genes associated with high CRBN expression included cyclin D2, SOCS3 and IL4 while genes associated with low cereblon expression included cyclin D1, FRZB and CD200. In order to understand how CRBN is connected with downstream anti-myeloma signaling, a structure-function study was performed to determine which CRBN domain is required for lenalidomide-induced IRF4 down-regulation and cytotoxicity. Lentiviral constructs expressing wild-type CRBN and a series of mutated CRBN were generated, including mutations at thalidomide binding site (Y384A/W386A), deletion of DDB1 binding region (ΔMid) and truncations at N-terminal and C-terminal. Lentiviruses from these constructs were used to infect IMiDs resistant HMCLs, OCI-MY5 and MM1.S res. Both of these cell lines have very low endogenous CRBN expression and they became sensitive to lenalidomide after introduction of wild-type CRBN. Conversely, introduction of CRBN with mutated thalidomide-binding site or with DDB1 binding region depletion failed to mediate lenalidomide toxicity and down-regulation of IRF4. OCI-MY5 cells expressing either N-terminal or C-terminal truncated CRBN showed substantial reduced responses (more than 50%) to lenalidomide compared with wild-type CRBN expressing cells. Deletion of only 20–30 amino acids at either ends of CRBN greatly impaired the protein function, suggesting that protein folding might be important for CRBN-mediated IMiD response. Our data indicate that IMiD induced myeloma cytotoxicity is largely mediated by modifying CRBN associated E3 ubiqutin ligase and subsequent IRF4 downregulation, suggesting the CRBN-IRF4 axis is a potential target for development of new anti-myeloma drugs. Disclosures: Schmidt: Karyopharm: Research Funding. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Immunomodulatory drugs (IMiDs) are profoundly active compounds in the treatment of patients with multiple myeloma (MM). However, despite the fact that treatment with IMiDs has dramatically improved survival for patients with MM, the majority of MM patients develop IMiDs resistance over time. We have found that expression of functional cereblon is required for IMiDs′ action. In addition, it has been reported that cells expressing high levels of cereblon are resistant to proteasome inhibitor, implying that patients with high levels of cereblon should be resistant to proteasome inhibitor. If the above conclusions are correct, cereblon could be considered as a biomarker to determine which standard regimens should be used to treat patients with MM. Unfortunately, the conclusions mentioned above have not been clinically confirmed. In order to confirm these conclusions, we have generated three highly specific mouse monoclonal antibodies (mAbs) against full-length human cereblon. These mAbs can be used to do western blot, immunoprecipitation and immunohistochemistry staining. In addition, their epitopes have been precisely determined and the peptides covering their epitopes completely blocked the antibody binding to cereblon in western blot analysis or in immunohistochemistry staining of MM patients′ specimens.

Abstract Immunomodulatory drugs (IMiDs) are therapeutically active compounds that bind to the E3 ubiquitin ligase substrate recruiter cereblon (CRBN) and induce many cellular responses, including cytotoxicity. We had found that the expression of CRBN in multiple myeloma (MM) cells provided the basis for their responses to IMiDs. In this report, we have identified argonaute 2 (AGO2) as a new member of CRBN-downstream binding protein and found that: 1) the steady-state levels of CRBN in IMiD-sensitive cells are significantly higher than in IMiD-resistant cells; 2) the steady-state levels of AGO2 in IMiD-sensitive cells are significantly less compared to IMiD-resistant cells, suggesting that CRBN plays a role in regulating AGO2. AGO2 is a component of microRNA (miRNA)-induced silencing complexes and plays an important role in miRNA maturation and function. The matured miRNAs are considered as key regulators of almost all cellular pathways. Treatment of IMiD-sensitive MM cells with lenalidomide, an IMiD, significantly increased CRBN, decreased both AGO2 and miRNAs and inhibited cell growth. In contrast, although treatment of IMiD-resistant MM cells with lenalidomide moderately increased CRBN and decreased both AGO2 and miRNAs, this treatment did not significantly inhibit cell growth. However, silencing of AGO2 with small hairpin RNA significantly decreased both AGO2 and miRNAs and inhibited cell growth regardless of whether the cells are sensitive or resistant to IMiDs. Therefore, AGO2 could be considered as a new drug target for overcoming IMiDs resistance in MM cells. Citation Format: Qinqin Xu, Yuexian Hou, Paul Langlais, Patrick Erickson, James Zhu, Changxin Shi, Moulun Luo, Yuanxiao Zhu, Lawrence Mandarino, Keith Stewart, Xiu-Bao Chang. Expression of CRBN binding protein AGO2 plays important roles for myeloma cell growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3625. doi:10.1158/1538-7445.AM2015-3625