IMiD® Immunomodulatory Agents Promote Apoptosis in Multiple Myeloma Cells through Induction of TRAIL Expression

Background: Immunomodulatory drugs exhibit several anti-Multiple Myeloma (MM) activities including: growth arrest, antiangiogenesis, inhibition of tumor necrosis factor-alpha signaling and induction of apoptosis. The mechanism of action of the IMiD® compounds lenalidomide and pomalidomide that are responsible for these effects are starting to become clear. IMiD® compounds directly bind Cereblon (CBRN), a substrate receptor of the cullin ring E3 ligase (CRL4), and induce CRL4^CRBN -mediated degradation of the zinc finger transcriptional repressors Aiolos and Ikaros by ubiquitin-mediated proteolysis. However, direct links between Aiolos or Ikaros degradation via the CRBN-Cul4 complex to the anti-MM activities of IMiD® drugs are poorly understood.

Methods and Results: IMiD® compound-induced apoptosis in MM cells is associated with extended duration of treatment and increased caspase-8 activity, but the precise mechanism of caspase-8 activation and delayed onset of apoptotic effects in response to treatment are unknown. Here we show that both lenalidomide and pomalidomide induce caspase-8, but not capsase-9 activity, in multiple MM cell lines after 3 days of treatment and apoptosis after 4 days without inducing significant changes in total caspase-8, c-FLIP (a casapse-8 inhibitory molecule) or caspase-3 gene expression. Bioinformatic analysis of public datasets revealed that that the pro-apoptotic protein TRAIL may be induced by IMiD® compound treatment in a CRBN dependent manner. We confirmed in multiple cell lines that IMiD® compound treatment or knockdown of Aiolos or Ikaros induces TRAIL gene expression. Furthermore, IMiD® compound treatment in pomalidomide resistant cells that lack CRBN expression failed to induce TRAIL gene expression suggesting TRAIL induction is regulated through the established IMiD® drug mediated CRBN-Aiolos/Ikaros pathway. ChIP-PCR analysis confirmed that TRAIL is a direct transcriptional target gene of Aiolos and Ikaros in MM cells. Incubation of cells with a TRAIL neutralizing antibody was sufficient to partially attenuate IMiD® induced apoptosis, thus confirming the role of TRAIL in IMiD® drug induced MM cell apoptosis. Furthermore, TRAIL secretion into the media of IMiD® compound treated cells was significantly increased only after 2 days treatment explaining the delayed induction of apoptosis in treated cells. Interestingly, MM cell lines that are intrinsically resistant to the apoptotic effects of IMiD® compounds, despite degrading Aiolos and Ikaros in response to drug, displayed a decrease in TRAIL sensitivity.

Conclusions: Taken together this data indicates that IMiD® drugs induce caspase-8 mediated apoptosis by directly inducing TRAIL gene expression via degradation of Aiolos and Ikaros. Additionally, this suggests that therapeutic interventions that can increase MM cell sensitivity to TRAIL might act synergistically with IMiD® compounds.