Abstract B15: Targeting SQSTM1/p62 induces cargo-loading failure and converts autophagy to apoptosis via NBK/Bik in human multiple myeloma cells.

Purpose: The purpose of this study was to determine whether transcriptional repression of the adaptor protein SQSTM1/p62, which plays a critical role in recognizing/loading cargo (e.g., malfolded proteins) into autophagosomes for lysosomal degradation, could potentiate multiple myeloma (MM) cell death in response to agents (e.g., BH3-mimetics) that elicit a cytoprotective autophagic response. Methods: To this end, various multiple myeloma cell lines (e.g., U266, RPMI8226, and bortezomib-resistant PS-R U266 cells) were exposed to the pan BH3-mimetic obatoclax (Teva) in the presence or absence of pan-CDK inhibitors e.g., flavopiridol (Telero), dinaciclib (Merck), after which the induction of autophagy (LC3-II expression, GFP-LC3 puncta by confocal microscopy) and apoptosis (Annexin V staining by flow cytometry) were monitored. Autophagic flux was determined using cells transfected with a pBABE mCherry-EGFP-LC3B plasmid, followed by observation of autophagosomes and autolysosomes. Accumulation of ubiquitinated protein aggregates was assessed by filter trap (retardation) assays and immunofluorescent staining using an anti-ubiquitin antibody. The functional significance of perturbations in protein expression (e.g., p62, CDK9, cyclin T, Bik) was determined using cells in which target gene expression was down-regulated by shRNA. Alterations in the appearance of autophagic vacuoles (AV) were observed using electron microscopy. Lysosome biogenesis was analyzed by LysoTracker Probe. Gene expression was determined by quantitative real-time PCR (qPCR), and protein-protein interactions characterized by co-immunoprecipitation. The in vivo effects of combined treatment were assessed in mouse MM flank xenograft models. Results: Here we report that whereas p62 levels fluctuated in a time-dependent manner during autophagy induced by obatoclax, inhibition (e.g., by CDK inhibitors administered at sub-micromolar concentrations) or knockdown of CDK9 or cyclin T1 transcriptionally suppressed p62 expression, but did not affect autophagic flux or modify lysosome biogenesis. These interventions, or shRNA directly targeting SQSTM1/p62, resulted in cargo-loading failure and inefficient autophagy, phenomena recently described in Huntington9s disease neurons. Inefficient autophagy was characterized by the formation of “empty” AVs accompanied by the accumulation of ubiquitinated proteins, presumably due to failure of loading into autophagosomes for degradation. These events led to accumulation of the BH3-only protein NBK/Bik on ER, most likely by blocking loading and autophagic degradation of Bik. Whereas Bik up-regulation was further enhanced by disruption of distal autophagic events (e.g., autophagosome maturation) by chloroquine or Lamp2 shRNA, it was substantially diminished by inhibition of autophagy initiation (e.g., by shRNA targeting Ulk1, Beclin-1, or Atg5, or pharmacologically by 3-MA or spautin-1), arguing that Bik accumulation primarily stems from inefficient autophagy. Moreover, NBK/Bik knock-down markedly attenuated apoptosis in vitro and in in vivo. Finally, similar interactions between a CDK inhibitor and obatoclax were observed in vivo in MM mouse models. Together, these findings identify a novel cross-link between autophagy and apoptosis, wherein targeting p62 converts cytoprotective autophagy to an inefficient form due to cargo loading failure, leading to NBK/Bik accumulation which triggers apoptosis. Conclusions: Collectively, these findings highlight a novel approach to target the cargo-loading process during cytoprotective autophagy by the transcriptional down-regulation of the adaptor protein p62, rather than direct inhibition of autophagy at either early or late stages (e.g., by chloroquine). Together, these findings provide new mechanistic insights into and a theoretical foundation for developing a autophagy-targeting therapeutic strategy based on induction of inefficient autophagy in MM. Citation Format: Yun Dai, Shuang Chen, Liang Zhou, Yu Zhang, Yun Leng, Xin-Yan Pei, Hui Lin, Richard Jones, Orlowski Robert, Steven Grant. Targeting SQSTM1/p62 induces cargo-loading failure and converts autophagy to apoptosis via NBK/Bik in human multiple myeloma cells. [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 B15.