Synergistic Activities of Proteasome Inhibitors and Immunomodulatory Drugs in Multiple Myeloma Converge in the Unfolded Protein Response

Introduction Proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs) reflect synergistic treatment modalities in multiple myeloma (MM) and their combination marks the backbone of approved first line therapies. Although clinically established, the molecular mechanisms, how IMiDs convey synergistic anti-myeloma activity in combination with PIs, remain elusive. The currently most anticipated mode of action of IMiDs is the redirection of the CRBN E3 ubiquitin ligase towards the transcription factors IKZF1/3 leading to their ubiquitin-dependent degradation, which creates a paradoxon as the combination with PIs would be expected to antagonize this effect of IMiDs by preventing proteasomal degradation of those CRBN neosubstrates. Previously, we described CRBN as an HSP90 co-chaperone with a central role in the quality control of transmembrane proteins, a function compromised by IMiDs mediating anti-MM activity. Against this background, we investigated whether excessive accumulation of unfolded proteins and the induction of the unfolded protein response (UPR) contributes to the synergism of PIs and IMiDs.

Methods Different MM cell lines as well as primary patient-derived CD138+ MM cells extracted from newly diagnosed MM (NDMM) or relapsed/refractory MM (RRMM) patients were exposed to single agent treatment with bortezomib (PI), lenalidomide (IMiD) or a combination of both. Functionally, UPR signaling was studied on transcriptional and on protein level using qPCR and Western Blot analyses. The intracellular accumulation of misfolded proteins was analyzed by FACS and confocal microscopy.

Results We subjected IMiD-sensitive and -resistant MM cell lines as well as primary patient-derived CD138+ MM cells to single treatments with bortezomib, lenalidomide or the combination of both. Bortezomib treatment, as expected, triggered an acute activation of the UPR. Strikingly, we also observed UPR induction upon single lenalidomide exposure. This effect was significantly enhanced upon the combination of both agents as evidenced by intracellular accumulation of misfolded proteins, phosphorylation of ER stress sensors and activation of UPR-specific transcriptional signatures, ultimately resulting in increased induction of intrinsic apoptosis. This suggests that the synergistic anti-myeloma activity of PIs and IMiDs arises from the collaborative activation of the UPR through the simultaneous disruption of the HSP90 co-chaperone function of CRBN and the protein degradation machinery. Of note, the observed UPR phenotype upon lenalidomide treatment was neither evident in IMiD-resistant MM cell lines nor RRMM patients. To systematically validate these findings, we set up a multi-omics screening approach (RNASeq and mass spectrometric analysis) comparing differentially expressed genes or proteins upon lenalidomide treatment in an IMiD-sensitive or -resistant MM setting.

Conclusion We propose that the synergism of PI and IMiDs in MM patients relies, at least in part, on the collaborative effect of both drugs on the induction of toxic protein aggregates and the activation of the UPR.

No relevant conflicts of interest to declare.