RAR Gamma Activation Sensitizes Human Myeloma Cells to Carfilzomib Treatment through OAS-RNase L Innate Immune Pathway

Proteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz) kill MM cells by disrupting the degradation of misfolded proteins, presumably derived from high-level immunoglobulin production, which provides an appealing explanation for why MM cells are so uniquely sensitive to PIs. However, relapses are frequent and acquired resistance to PI treatment emerges in most patients. Therefore, identifying novel and safe drugs overcoming PI resistance in MM will aid in chemo-(re)sensitization, reducing PI-induced side effects, and maximizing the outcomes of PI therapy.

Here we performed a high-throughput screen of 1855 FDA-approved drugs (Figure 1) and identified all-trans retinoic acid (ATRA), a classic pan-retinoic acid receptor (RAR) agonist used successfully to treat acute promyelocytic leukemia (APL), as a potent drug that enhanced MM sensitivity to Cfz-induced cytotoxicity and re-sensitized Cfz-resistant MM cells to Cfz in vitro. To determine which RARs are important for ATRA enhancement of Cfz-induced apoptosis in MM, esiRNAs of RARs for knocking down RARs and selective agonists of RARs were used in Cfz-treated MM cells. We identified that RARγ activation is important for ATRA sensitizing MM cells to Cfz treatment.

To determine which signaling pathways are involved in ATRA-treated MM cells, gene-profiling data analysis of Cfz- versus ATRA+Cfz-treated MM cells was performed and real-time PCR was used to validate the microarray results. We found that ATRA treatment activated IFN-β response pathway (Figure 2), leading to upregulated expression of IRF1 and OAS1-3. Interestingly, similar to ATRA, IFN-β, which alone did not induce MM apoptosis, enhanced Cfz-induced MM cell apoptosis. Furthermore, using RNA integrity assay and dsRNA detection assay, we identified that ATRA treatment elevated the expression of OASs, which synthesized 2-5A upon binding to dsRNA induced by Cfz and resulted in cellular RNA degradation by RNase L and cell death (Figure 3). By knocking down each gene of OAS1-3, we demonstrated that OAS1 is essential for ATRA to sensitize MM cells to Cfz-induced apoptosis.

Furthermore, we determined the impact and significance of RARγ and OAS1 in human MM pathogenesis and drug response by analyzing the gene-profiling data of 264 MM patients from Mulligan et al. datasets and 1,143 MM patients from MMRF coMMpass study IA13. In support of these findings, analyses of the large patient's gene-profiling datasets showed a strong and positive correlation between RARγ and OAS1 expression and patient’s response to PI treatment (Figure 4).

Finally, BMS961, a selective RARγ agonist, similar to ATRA, could also (re)sensitize MM cells to Cfz in vitro, and both ATRA and BMS961 significantly enhanced the therapeutic effects of Cfz in established MM in vivo (Figure 5). Thus, this study highlights the potential for RARγ agonists to sensitize MM and overcome MM resistance to Cfz treatment in patients.