Metformin Suppresses the GRP78-Dependent Delivery of Aggresomes to Autophagosomes to Enhance the Anti-Myeloma Benefit of Proteasome Inhibition

Background:

Functional blockade of the ubiquitin (Ub)+proteasome system (UPS) using the small molecule bortezomib represents a remarkable bench-to-bedside success that has improved the outcome of patients diagnosed with multiple myeloma (MM). However, certain patients do not respond to bortezomib and those that do inevitably develop resistance through mechanisms that remain elusive and despite all currently available therapies, MM remains invariably fatal. While bortezomib disrupts protein homeostasis leading to tumor cell death but also activates pro-survival aggresome and autophagosome formation as compensatory protein clearance mechanisms to promote the emergence of drug resistance, tumor survival and disease relapse. While the therapeutic benefit of targeting the proteasome is unchallenged, more precise modalities that do not induce drug resistance are urgently needed. These studies were performed to identify pharmacologics that inhibited the autophagy pathway and to uncover their mechanisms of action as a means to synergistically enhance the cytotoxic effect of bortezomib for myeloma treatment.

Methods:

High-throughput screening (HTS) was performed to identify FDA-approved pharmacologic agents and bioactive molecules that prevented bortezomib-mediated induction of autophagosomes. Confocal microscopy combined with novel, dye-based fluorescent methods were used to visualize and quantitate aggresomes and autophagosomes. The effect of pharmacologics on aggresome and autophagosome formation was then determined. The effects of metformin, alone or combined with bortezomib were evaluated using MM cell lines, myeloma patient bone marrow-derived tumor cells and myeloma cells xenotransplanted into mouse models.

Results:

Bortezomib inhibits the UPS to promote the unwanted intracellular accumulation of ubiquitinated proteins that form aggregates dispersed throughout the cytoplasm. These aggregates are then coalesced into perinuclear aggresomes and subsequently disposed through autophagosome formation and the lysosomal pathway. Bortezomib treatment of myeloma cells increased levels of the endoplasmic reticulum (ER) molecular chaperone Glucose-Regulated Protein 78 (GRP78), a critical regulator of the unfolded protein response (UPR) and autophagosome formation. HTS revealed that the anti-diabetic biguanide metformin inhibited bortezomib-mediated autophagosome formation. Co-treatment with metformin and bortezomib increased the level of proteotoxic aggresomes but suppressed GRP78 and autophagosome formation. Metformin synergistically enhanced the anti-proliferative effect of bortezomib in myeloma cells and bone marrow-derived patient PCs. Co-treatment with metformin and proteasome inhibitors also overcame drug-resistance. shRNA-mediated knockdown of HSPA5, which encodes GRP78, similarly enhanced bortezomib-induced aggresome formation and blocked autophagosome formation. A GRP78 mutant that lacked the carboxy-terminal KDEL ER-retention motif also led to bortezomib-induced aggresomes without autophagosomes. Metformin and bortezomib together reduced myeloma xenotransplant growth in mice more effectively than either agent administered as single-agent.

Conclusions:

The ER chaperone GRP78 facilitates aggresome clearance by promoting autophagosome formation. Although bortezomib alone promotes GRP78 induction and aggresome formation, metformin suppresses GRP78 to uncouple the cytotoxic aggresomes from the autophagy pathway and to enhance the anti-myeloma effect of bortezomib. The results support the pharmacologic repositioning of metformin in combination with bortezomib as an effective anti-myeloma strategy. The molecular chaperone GRP78 holds promise as a target for cancer treatment as well as for neurodegenerative diseases in which intracellular protein aggregates are central to disease pathogenesis