Novel Therapeutic Strategies to Target Protein Quality Control Compartments in Multiple Myeloma

Background:

Precisely regulated quality control systems maintain protein homeostasis and cell viability. Cells have developed strategies to cope with defective proteins. Molecular chaperones refold aberrant proteins to restore their native conformation, but, if these proteins cannot be repaired, they are rapidly destroyed by the ubiquitin (Ub)+proteasome system (UPS). Functional blockade of the UPS leads to the accumulation of proteotoxic Ub-conjugates and has been exploited for therapeutic gain in multiple myeloma (MM) treatment. While the therapeutic benefit of proteasome inhibitors remains unchallenged, cancer cells adapt through the induction of alternate compensatory protein clearance mechanisms, e.g., aggresomes, autophagosomes to promote tumor survival, treatment failure and relapse, Here, we investigated the link between proteasome inhibition and formation of the newly discovered JUxtaNuclear Quality control compartments-JUNQ. These structures are spatially distinct from aggresomes and autophagosomes and contain Ub-conjugates, proteasomes, the heat shock protein (HSP)104 and other components. Here, we investigated the role of the molecular chaperone glucose-regulated protein (GRP78), which is required for ER integrity, on these compensatory protein clearance mechanisms.

Methods:

Myeloma cells were treated with the proteasome inhibitor bortezomib. Immunofluorescence staining, confocal microscopy and multi-level imaging detected the accumulation and co-localization of Ub-pathway substrates, proteasomes and the molecular chaperones GRP78 and HSP104. Highly specific dye-based methods were used to detect and quantitate aggresomes and autophagosomes in these cells and to determine the drug effects. The experiments were performed using drug naïve and drug-resistant MM cell lines as well as bone marrow-derived myeloma patient tumor samples. In addition, a highly specialized human embryonic kidney HEK-293 cell line was employed that expressed a green fluorescent reporter (GFPu) that was a Ub-proteasome system substrate. The GRP78-expressing gene HSPA5was silenced using siRNA in the HEK cells.

Results:

GRP78 has an obligatory role in autophagosome formation. HSPA5, which encodes GRP78, was silenced in HEK cells to determine the role of GRP78 on protein quality control compartments. GRP78-knockout cells were tretaed with bortezomib to induce aggresomes but autophagosome formation was significantly inhibited. Bortezomib treatment also promoted the accumulation of the GFPu substrate that co-localized with Ub, proteasomes and HSP104. Importantly, in GRP78-deficient cells, HSP104 was upregulated even in the absence of bortezomib. At early time points, the fluorescent intensity of the GFPu reporter co-localized with HSP104 and was much higher than that seen in the control cells. Also, Ub and proteasome levels were higher and co-localized with GFPu and HSP104, consistent with the formation of JUNQ compartments. However, at later time points, green fluorescent intensity was dispersed throughout the cytoplasm with minimal co-localization with HSP104 to suggest that in GRP78-deficient cells those compartments did not remain stable. The addition of bortezomib at physiologically-relevant concentrations (10nM) to myeloma cells and MM patient tumor cells led to the similar co-localization of HSP104 with Ub-conjugated proteins and proteasomes. Importantly, HSP104 levels and the intensity of HSP104 co-localization with proteasomes and Ub was much higher in myeloma cells resistant to each of proteasome inhibitors bortezomib, carfilzomib or ixazomib than that seen in drug-sensitive cells.

Conclusions:

Taken together, the results demonstrate that GRP78 suppression inhibits autophagosomes and destabilizes the formation of HSP104-containing structures that are consistent with JUNQ. Using myeloma as a clinically-relevant cancer model, we demonstrate that physiologically-relevant concentrations of an FDA-approved proteasome inhibitor induce these newly discovered protein quality control compartments. JUNQ may serve as the temporary storage site for Ub-proteins that cannot be folded or degraded. Genetic or pharmacologic inhibition of GRP78 may reduce JUNQ formation but does not inhibit aggresome formation with therapeutic potential to enhance the benefit of proteasome inhibition and to overcome drug resistance in MM.