Microrna-29b Targets PSME4 to Inhibit Proteasome Activity and Prevent the Delivery of Aggresomes to Autophagosomes for Anti-Myeloma Benefit

Background:

Multiple myeloma (MM) is a malignancy of post-germinal center B-cells whose pathogenesis remains poorly understood and that remains invariably fatal. Evading apoptosis is a cancer hallmark that remains a serious obstacle in current treatment approaches and contributes to drug resistance. Despite the therapeutic benefit of the proteasome inhibitor bortezomib, drug resistance still accounts for the majority of tumor relapses and cancer-related deaths in MM patients. A precise understanding of the molecular mechanism(s) of resistance to bortezomib in myeloma is lacking. The goal of the present study was to identify and functionally characterize novel microRNAs (miRNAs) that inhibit the ubiquitin (Ub)+proteasome system (UPS) and also disrupt the aggresome-autophagy pathway for anti-myeloma benefit. 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.

Methods:

Myeloma cells were separately exposed to the proteasome inhibitors bortezomib, carfilzomib or ixazomib to generate cells with acquired drug resistance. Genome-wide, microarray-based profiling was then performed to identify miRNAs significantly deregulated in the drug-resistant myeloma cells relative to parental drug-naïve cells. Based upon the miRNA profiling, we identified a curated panel of miRNAs that were significantly deregulated in the drug-resistant cells. To determine the effect of individual miRNAs on myeloma cells, synthetic miRNA replacements or antagomirs were transfected into myeloma cells. The effect of the single miRNAs on the growth and drug resistance was then quantified using cell viability assays. Individual miRNA replacements or antagomirs 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 myeloma or 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 bone marrow-derived tumor cells relative to plasma cells obtained from healthy individuals. MiRNA-29b targets PSME4which 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 PSME4and the protein product PA200, a proteasome activator. The reduction of PA200 levels inhibited the proteasome’s chymotrypsin-like peptidase activity and the turnover of ornithine decarboxylase, a proteasome substrate degraded through Ub-independent mechanisms. Following proteasome inhibition, perinuclear aggregates of insoluble Ub-conjugated 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. Ub-conjugated proteins were also not detected in autophagosomes.

Conclusions:

MiRNA-29b replacement represents the first-in-class miRNA-based agent to inhibit proteasomes and to disrupt the efficient delivery of Ub-conjugate-containing aggresomes to the autophagy pathway. Taken together, our study highlights the potential of miRNA replacement therapy to synergistically enhance the anti-myeloma benefit of proteasome inhibitors.