Novel Mechanisms Underlying IMiDs Resistance in Multiple Myeloma: Therapeutic Implications

Introduction

Immunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind to the CRL4^CRBN ubiquitin ligase and trigger proteasomal degradation of IKZF1/3 and cytotoxicity of multiple myeloma (MM) cells. Previous studies show that downregulation of CRL4^CRBN abrogates sensitivity to IMiDs in MM cell lines. Clinical studies, show that IMiDs achieve better clinical responses in MM patients when combined with proteasome inhibitor bortezomib, which would in principle inhibit proteasomal degradation of IMiDs-targeted proteins. Moreover, some MM cells are resistant to IMiDs treatment despite harboring high CRBN expression levels. Thus, the molecular mechanisms of intrinsic resistance to IMiDs are not yet fully understood. In this study, we found that dual roles of IMiDs in activating CRL4^CRBN and a mechanism of action for combination therapy of IMiDs with bortezomib. We also show that non-canonical NF-κB pathway, independent of CRBN, is involved in the IMiDs resistance in MM.

Methods and Results

To study the molecular mechanisms underlying IMiDs resistance, we first performed genome-wide knockout screening in IMiDs-sensitive MM.1S cells, using a CRISPR-Cas9 GeCKOv2 library containing 6 unique sgRNAs against each of 19,050 genes and 4 sgRNAs against each of 1,864 miRNAs. Twenty-eight genes and one miRNA were identified which were associated with resistance to IMiDs. All six sgRNAs targeting CRBN were also identified, consistent with CRBN as the direct target of IMiDs and required for their MM cytotoxicity. Among these validated genes, 7 of 9 components of the COP signalosome (CSN) subunits were found to be required for MM cell sensitivity to IMiDs. Deletion of any of these CSN subunits in MM cells led to a dramatic decrease of CRBN protein level thereby inhibiting IKZF1/3 degradation induced by IMiDs.

To investigate whether the CSN regulates CRBN level through the ubiquitin-proteasome pathway, we carried out ubiquitination assays and found that CRBN can be degraded by proteasomes via cullin E3, rather than CRL4, ubiquitin ligase. Moreover, we identified the interaction of CRBN with SCF^Fbxo7 complex, and demonstrated that CRBN interacted with both CRL4 and SCF^Fbxo7 with distinct domains. To determine if CRBN is a SCF^Fbxo7 substrate, we examined the ubiquitination and degradation of CRBN after altering SCF^Fbxo7 complex. CRBN, when unbound to CRL4, is ubiquitinated by SCF^Fbxo7, followed by proteasomal degradation. Importantly, deletion of Cul1 or Fbxo7 completely restored the CRBN protein in CSN2 or CSN6 knockout MM cells to levels found in wild type cells, as well as re-sensitized these CSN mutant MM cells to pomalidomide-induced cytotoxicity.

Since CRBN protein turnover is controlled by a SCF ubiquitin ligase, we next tested whether proteasome inhibitor, in addition to their inherent anti-MM activities, might similarly enhance IMiDs-induced MM cell cytotoxicity. As expected, sequential treatment with bortezomib and pomalidomide primed MM cells to pomalidomide-induced downregulation of IKZF3 and cell death, associated with synergistic cytotoxicity. We also found that lenalidomide potently inhibited the ubiquitination of CRBN, which was abrogated by loss of lenalidomide binding on CRBN, downregulation of Fbxo7, or Cul1 expression. These data suggest that IMiDs stabilize the CRBN protein both by shifting its assembly with the SCF and CRL4 complexes and by directly blocking its ubiquitination and degradation targeted by SCF^Fbxo7.

From the genome-scale CRISPR-Cas9 knockout screening, we also found that knock-out of TRAF2, a member of the TNF receptor associated factor protein family, conferred resistance of MM cells to IMiDs treatment, without effecting CRBN level and degradation of IKZF1/3 induced by IMiDs. TRAF2 -KO MM cells displayed increased processing of p100 NF-κB2 protein to p52, resulting in hyperactivation of the non-canonical NF-κB pathway. This finding suggests that the non-canonical NF-kB pathway may be involved in the intrinsic resistance of MM to IMiDs.

Conclusions

Taken together, our findings therefore identify novel mechanisms of IMiDs resistance, involving the regulation of CRBN and the non-canonical NF-κB pathway, and provide a mechanistic framework to inform combination therapy strategies to overcome IMiDs resistance in MM.