Identification of a Cereblon-Independent Protein Degradation Pathway in Residual Myeloma Cells Treated with Immunomodulatory Drugs

Recent studies have shown that binding of immune modulatory drugs (IMiDs)^R  lenalidomide and pomalidomide (Pom) to the CRBN-CRL4 E3 ubiquitin ligase complex leads to degradation of IKZF1 and IKZF3, which in turn mediates anti-proliferative effects on myeloma cells and enhance IL2 expression by T cells. However as the drug-induced IKZF1 degradation is proteasome-mediated, this mechanism does not explain the apparent paradox that IMiDs mediate synergistic anti-myeloma effects with proteasome inhibitors in the clinic. In order to better understand how MM cells may resist potent IMiDs such as Pom, we examined the clonogenic growth properties of residual MM cells following Pom exposure. Surprisingly, residual MM cell lines and primary MM cells following Pom exposure exhibit paradoxically enhanced clonogenic growth in culture. Human MM cells persisting after Pom exposure also exhibit greater capacity for growth in vivo. Enhanced clonogenic growth was associated with increased expression of embryonal stem cell genes such as SOX2 and RNAi-mediated inhibition of SOX2 abrogated Pom-induced enrichment of clonogenic potential. We hypothesized that as with CBRN/IKZF1-mediated effects, the induction of ES / pluripotency genes in Pom-exposed cells may be related to degradation of repressors of gene transcription. Recent studies have identified MBD3 as a critical component of the pluripotency repressor complex. Pom-exposure led to ubiquitin and proteasome-mediated degradation of MBD3 and RNAi-mediated inhibition of MBD3 led to induction of pluripotency genes by MM cells. As expected, loss of CRBN abrogates the capacity of Pom to mediate degradation of IKZF1. Surprisingly, Pom-mediated degradation of MBD3, enrichment of ES genes and enhancement of clonogenic growth was independent of CRBN as it was observed in MM1s-R10R cells with genomic loss of CRBN, and was not impacted by RNAi-mediated inhibition of CRBN. RNAi-mediated inhibition of IKZF1 also does not lead to enrichment of ES genes and does not phenocopy the effects of MBD3 depletion. Residual MM cells in patients (n=2) treated with Pom also revealed marked depletion of MBD3 protein as well as enrichment of ES gene transcripts. Taken together, these data identify a CRBN/IKZF1-independent pathway for Pom-induced depletion of MBD3 and enrichment of pluripotency genes. Pre-exposure to proteasome inhibitor MG132 abrogated Pom-mediated loss of MBD3 and enrichment of ES genes. Similar results were obtained following treatment with Trichostatin A (TSA) and Romidepsin (FK228), which also inhibited Pom-induced depletion of MBD3. Therefore concurrent therapy of IMiDs with proteasome or HDAC inhibitors may mediate synergistic anti-tumor effects by abrogating adverse effects of Pom exposure on clonogenic growth of residual cells. In order to further dissect the underlying mechanism of Pom-induced degradation of MBD3 in MM cells, we hypothesized engagement of another E3 ligase complex other than CRBN. We show that Pom-induced degradation of MBD3 depends on TRIM27/29 E3 ligase complex, known to be overexpressed in MM plasma cells. Accordingly, RNAi-mediated downregulation of TRIM27/29 inhibits Pom-induced MBD3 depletion and enrichment of clonogenic growth.  In summary, these data identify a novel CRBN-independent, TRIM27/29 E3 ligase-dependent pathway for degradation of MDB3 engaged by Pomalidomide, which contributes to the biology of residual MM cells via enrichment of ES genes following exposure to these drugs in vitro/in vivo and provides a novel mechanism for observed synergy with proteasome and HDAC inhibitors. Targeting these pathways may be essential to enhance the therapeutic potential of IMiDs and minimize residual disease in MM.