The Rationale for Combined Proteasome and Autophagy Inhibition in Multiple Myeloma Established Using Novel Translational Platforms
Authors: Shen John Paul Y.C. , Divakaran Sanjay , Ponduru Sridevi , Vogl Dan T. , Amaravadi Ravi K. , Bradner James E. 
Published: April 2020

Abstract
Multiple myeloma (MM) is selectively sensitive to perturbations of protein catabolism. Genetic evidence has identified a link between plasma cell differentiation and upregulation of the unfolded protein response. The effective, selective, cytotoxic effect of proteasome inhibition observed in MM therapy further validates pathways of protein degradation as therapeutic targets in this incurable disease. Upon proteasome inhibition, cancer cells utilize compensatory pathways such as aggresome formation and macroautophagy. Despite a remarkable increase in the granularity of knowledge surrounding autophagy in mammalian cell biology, the role of autophagy in MM cell survival has not been studied. Lysosome-dependent macroautophagy (autophagy) pathway is known to be up-regulated by chemical inhibition of the proteasome. Therefore we theorized that autophagy is a pathway of proteasome inhibitor resistance in MM, and that simultaneous inhibition of both the ubiquitin-proteasome system (UPS) and the autophagy pathway would result in synergistic toxicity in MM cells. We chose to explore this hypothesis utilizing a chemical genetic approach, with the goal of repurposing off-patent autophagy inhibitors for immediate translation in the context of human clinical investigation. We selected for study the combination of the only FDA-approved proteasome inhibitor, bortezomib, and the 4-aminoquinoline autophagy inhibitor, hydroxychloroquine (HCQ). Indeed, HCQ exhibits a dose-dependent cytotoxic effect on cultured human MM cells in vitro. Using high-throughput, high-content epifluorescence microscopy, we have demonstrated a dose-dependent increase in autophagosome number and average vesicle area in MM cells treated with HCQ consistent with inhibition of late autophagy. These findings were confirmed by electron microscopy. Autophagy inhibition by HCQ was verified by LC3 immunoblot with and without bafilomycin clamp. Gold-standard pulse-chase studies of HCQ verified inhibition of long-lived protein degradation in MM cells. Of note, in vivo studies of HCQ in a bioluminescent orthotopic xenograft model of MM failed to demonstrate a statistically evident disease response or prolongation of survival. Drawing on the aforementioned biological and clinical insights, we predicted that combination autophagy and proteasome inhibition would result in synergistic cytotoxicity. To explore multiple combinations of these classes of therapeutic agents, we devised a high-throughput translational platform for assessing synergy between therapeutic agents. In this model, library plates of compounds are established which may be “crossed” with each other by alternating plate orientation and delivering compounds to experimental assay microtiter plates by robotic pin transfer. Experimental measurements (i.e. cytotoxicity, protein content, post-translational modifications) are then processed in a semi-automated manner employing the median effect principle and illustrative graphical outputs. This platform was validated by modeling known, effective combination therapies in current clinical use or study in MM. We observe potent, synergistic toxicity from the combination of bortezomib and HCQ in both MM1.S and RPMI-8826 MM cell lines. Of note, the combination index observed is &lt;1 across the pharmacologically-achievable range of both compounds. As the bone marrow microenvironment is known to confer chemoresistance to MM cells, a co-culture assay was developed using high-content imaging to study microenvironmental interactions and MM biology with greater biological precision than established methods (i.e. thymidine uptake). Combined proteasome and autophagy inhibition exhibits synergistic cytotoxicity in this microenvironmental model, notably increasing activated caspase-3 in MM cells indicative of an apoptotic death. Based on these data we have initiated a Phase I clinical study at the University of Pennsylvania (D.V.), aimed at establishing the tolerability and pharmacodynamic effect of this combination in patients with proteasome-refractory MM.