Abstract A18: MicroRNA-29b replacement inhibits proteasomes and disrupts the aggresome-autophagy pathway to enhance the antimyeloma benefit of bortezomib.

Purpose: The goal of the present study was to identify and functionally characterize novel microRNAs (miRNAs) that inhibit the ubiquitin+proteasome system (UPS) and also disrupt the aggresome+autophagy pathway for anti-myeloma benefit. Background: Evading apoptosis is a cancer hallmark that remains a serious obstacle in current treatment approaches. Drug resistance accounts for the majority of tumor relapses and cancer-related deaths. Functional blockade of the UPS using targeted therapeutics has translated into clinical success and transformed the management of MM. However, drug resistance emerges through induction of the aggresome+autophagy pathway as a compensatory protein clearance mechanism leading to treatment failure, disease relapse and ultimately fatal outcome. Experimental Procedures: Myeloma cells were exposed to the proteasome inhibitors bortezomib, carfilzomib or ixazomib to generate cells with acquired drug resistance. Genome-wide, microarray-based profiling was performed to identify miRNAs significantly deregulated in the drug-resistant myeloma cells relative to parental drug-naive cells. Based upon the miRNA profiling, we identified a curated panel of miRNAs that were significantly reduced in drug-resistant cells. To determine the effect of individual miRNAs, synthetic miRNA replacements were transfected into myeloma cells. Individual miRNA replacements were also transfected into human embryonic kidney (HEK) cells that expressed a short-lived green fluorescent protein that is degraded by the proteasome. The effect of individual miRNAs on the UPS was then quantitated in HEK cells by confocal microscopy and fluorescent imaging. Results: MiRNA-29b was significantly downregulated in the myeloma cells generated with acquired resistance to bortezomib, carfilzomib and ixazomib relative to the parental cells. MiRNA-29b was also downregulated in MM patient tumor cells relative to plasma cells form healthy individuals. MiRNA-29b targeted PSME4 which encodes the proteasome activator PA200. Transfection of synthetically-engineered miRNA-29b replacements reduced the viability of myeloma cells and patient tumor cells and synergistically enhanced the cytotoxic effect of proteasome inhibitors. The miRNA-29b replacement also reduced the growth of myeloma xenografts in mouse models in vivo. MiRNA-29b replacements decreased expression of PSME4 and the protein product PA200, a proteasome activator. Reduction in PA200 reduced the proteasome9s chymotrypsin-like peptidase activity and inhibited ornithine decarboxylase turnover, a proteasome substrate degraded through Ub-independent mechanisms. Following proteasome inhibition, perinuclear aggregates of insoluble ubiquitinated proteins, termed aggresomes, accumulate and are degraded by the autophagy pathway. Importantly, in contrast to bortezomib, miRNA-29b replacement inhibited proteasome activity but did not induce the formation of perinuclear aggresomes or autophagosomes. Rather, immunofluorescence studies indicated that miRNA-29b replacement promoted the accumulation of small, ubiquitinated protein aggregates that were dispersed throughout the cytoplasm but were not transported by microtubules and were not coalesced into mature aggresomes. The ubiquitinated proteins also were not detected in autophagosomes. Conclusions: MiRNA-29b replacement represents the first-in-class miRNA-based agent to inhibit proteasomes and uncouple the UPS from the aggresome-autophagy pathway. Taken together, our study highlights the potential of miRNA replacement therapy to synergistically enhance the anti-myeloma effect of PIs. Citation Format: Sajjeev Jagannathan, Nikhil Vad, Mohamed Abdel-Malek, Ehsan Malek, James Driscoll. MicroRNA-29b replacement inhibits proteasomes and disrupts the aggresome-autophagy pathway to enhance the antimyeloma benefit of bortezomib. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A18.