Abstract 1707: Accelerated proteasome turnover as a potential mechanism of acquired resistance to carfilzomib in BxPC3 pancreatic cancer cells

Abstract

Background: The addition of proteasome inhibitors bortezomib and carfilzomib to the multiple myeloma therapy has drastically improved the outcome of patients. However, drug resistance remains a major obstacle in proteasome inhibitor therapy. Several resistance mechanisms have been proposed in vitro, but have not been identified as contributing factors to proteasome inhibitor resistance observed in the clinic. Thus, we set out to investigate previously unexplored resistance mechanisms using BxPC3 pancreatic cancer cells with acquired resistance to carfilzomib. Methods and Results: We established carfilzomib-resistant BxPC3 cells (BxPC3/Cfz) by adapting the parental BxPC3 (human pancreatic cancer) cells to escalating drug concentrations over time. Using cell viability assays, we confirmed that BxPC3/Cfz cells have an increased IC50 values (10-fold) for carfilzomib compared to its parental control. The proteasome activity of resistant and parental BxPC3 cells was measured in vitro using the fluorogenic peptide substrate Suc-LLVY-AMC. Our results showed that majority of the proteasome activity in BxPC3/Cfz cells was restored within 96 hours following initial drug treatment. Decreased drug permeability and enhanced metabolism were ruled out as potential contributors to the lack of activity inhibition. This was based on the lack of efflux transporter upregulation or differences in the rate of drug degradation in the resistant cells compared to parental controls. Given that the recovery of proteasome activity in BxPC3/Cfz cells is much faster than the reported proteasome half-life of 5-12 days, we further examined whether accelerated recovery of proteasome activity in BxPC3/Cfz cells is associated with the development of acquired resistance. To monitor the rate of proteasome degradation, we utilized an activity-based probe to label proteasomes inside cells and in-gel fluorescence assays for visualization. Using a similar approach, we also measured the rate of proteasome synthesis by western blotting analysis of unlabeled proteasome subunits. Conclusion: Here, we report that the acquired resistance to carfilzomib in BxPC3 cells does not involve previously reported resistance mechanisms, but may arise from accelerated proteasome turnover. In order to further validate our findings as a novel mechanism determining cellular sensitivity to proteasome inhibition, we are currently assessing whether cancer cells that are intrinsically resistant to proteasome inhibition have increased rates of proteasome turnover compared to sensitive cells. Taken together, our findings may provide previously unaddressed insights into cancer resistance to proteasome inhibition. Citation Format: Lin Ao, Jieun Park, Di Hu, Hyun Young Jeong, Kyung Bo Kim, Wooin Lee. Accelerated proteasome turnover as a potential mechanism of acquired resistance to carfilzomib in BxPC3 pancreatic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1707. doi:10.1158/1538-7445.AM2014-1707