ALBERT

Description: 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 Disclosures No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell–induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies.

Description: Cancer cells face enormous metabolic challenges to meet energetic and biosynthetic demands and, therefore, a hallmark of cancer cells is reprogrammed metabolic circuitry that results from oncogenic events selected during tumorigenesis to promote growth, survival and drug resistance. The increased metabolic demands in tumor cells are offset by autophagy - a basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components and potentially contributes to the growth of cancer cells. The proteasome is the catalytic core of the ubiquitin (Ub)-dependent proteolytic pathway that degrades short-lived and denatured proteins to maintain cell viability. Success of the proteasome inhibitor bortezomib has emerged as standard-of-care therapy for the invariably fatal malignancy MM to catapult the Ub+proteasome system (UPS) into a position of prominence in cancer biology and drug discovery, significant obstacles remain since many patients do not respond to bortezomib and those that do inevitably develop drug resistance through mechanisms that remain elusive. We discovered that in multiple myeloma (MM) cells, bortezomib triggers activation of AMPK - the master regulator of cellular energy metabolism. At physiologically-relevant concentrations, proteasome inhibitors further triggered AMPK-dependent autophagosome formation in myeloma cells that was directly and sequentially linked to apoptosis in drug-sensitive cells. However, in bortezomib-resistant cells, autophagosome formation and apoptosis were uncoupled to enhance drug resistance and the survival of tumor cells. Genetic knockout of the AMPK catalytic subunits in mouse embryonic fibroblasts reduced the effect of bortezomib on both autophagy and apoptosis. Similar effects were seen with genetic ablation of ULK1- a downstream substrate of AMPK - also required for bortezomib-induced autophagosome formation. Enforced expression of the autophagy-related gene Atg5 enhanced bortezomib-induced cell death while bortezomib treatment promoted ATG5 cleavage to yield a truncated, pro-apoptotic form. ATG5 participates in the initial steps of autophagy and was also cleaved by calpain to generate the truncated ATG5 form that translocates to the mitochondria, binds Bcl2 and induces mitochondrial outer membrane permeabilization (MOMP). We propose that ATG5 promotes the displacement of anti-apoptotic Bcl2 proteins from the pro-apoptotic Bcl2 family members, e.g., NOXA and PUMA, to facilitate MOMP - considered the point of commitment to cell death through the mitochondrial apoptotic pathway. Importantly, bortezomib in combination with the AMPK activators AICAR or metformin enhanced ATG5 cleavage and overcame resistance to proteasome inhibitors. The molecular events that regulate the complex interplay between autophagy and apoptosis as determinants of cell fate under physiologic and pathologic conditions have remained poorly understood. Since there is an urgent need to unravel the mechanism(s) of drug resistance and to develop more effective therapies, we propose pharmacologic repositioning of AMPK activators as a promising strategy to enhance the therapeutic efficacy of proteasome inhibitors to overcome drug resistance in the treatment of hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: The Ubiquitin (Ub)+Proteasome system (UPS) is a highly complex network that maintains cellular homeostasis through the selective turnover of targeted proteins. The proteasome serves as the catalytic core of the UPS to execute the efficient removal of ubiquitin-conjugated proteins. Pharmacologic inhibitors that exploit the pivotal role of the proteasome in cellular metabolism promote tumor cytotoxicity and yield durable clinical responses that have significantly improved survival of patients diagnosed with the invariably fatal plasma cell malignancy multiple myeloma (MM). However, while functional blockade of the proteasome has emerged as a successful anti-cancer strategy, drug resistance inevitably emerges through mechanisms that remain elusive. Since E3 Ub ligases target biologically-relevant proteins for proteasomal degradation and are frequently overexpressed in cancers, we hypothesized that altered E3 Ub ligase expression served as a mechanism of resistance to proteasome inhibitors (PIs). To address the role of individual E3s in myelomagenegesis, gene expression profiles of MM patient samples obtained prior to treatment were analyzed. Results indicated that Cullin-1 was overexpressed in MM patient tumor samples compared to normal or MGUS samples. Cullin-1 is an essential scaffolding component of multiple Skp1-Cullin-1-F-box protein E3 complexes that mediate the ubiquitination of proteins involved in cell cycle progression. Next. bone marrow-derived CD138+ plasma cells were obtained from MM patients that were then treated with the PI bortezomib. Samples were similarly probed to identify differences in E3 expression and to identify features that dictate therapeutic response. Again, Cullin-1 was overexpressed in samples from MM patients that did not respond to bortezomib but Cullin-1 was not overexpressed in samples from those patients that responded to bortezomib. Cullin-1 levels were also higher in bortezomib-resistant myeloma cell lines and drug-resistant cells were re-sensitized to bortezomib after short-hairpin-RNA-mediated Cul-1 knockdown. Cells overexpressing Cullin-1 displayed increased NF-Kappa B activity and a reduced sensitivity to bortezomib-induced apoptosis as demonstrated by staining for annexin-positivity. The effect of Cullin-1 expression level on the sensitivity to bortezomib treatment was determined using MM xenografts in athymic SCID mice. We have used a biologically-supervised, microarray-based approach to identify Cul-1 overexpression in a subset of myeloma patients and correlated expression with clinical resistance to bortezomib. Functional studies demonstrated that Cullin-1 sustains activation of the NF-Kappa B pathway and decreases cellular response to bortezomib in vitro in human cell lines. We conclude that Cullin-1 attenuates bortezomib anti-MM activity by maintaining NF-Kappa B signaling and hence promoting tumor survival. Engineered overexpression or shRNA-mediated inactivation of Cullin-1 modulated the cytotoxic effect of bortezomib to further recapitulate the premise that tumor genomics dictate therapeutic response. Cullin-1 is a novel effector within the UPS that offers promise as an oncologic target either alone or in synergistic combination with proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Multiple myeloma (MM) is a heterogeneous diseaseand there is an increased need for more accurate risk classification methods to improve treatment decision-making because of its high impact on clinical outcomes. Here, we demonstrate evidence to support the prognostic value of non-coding RNAs (ncRNAs) as newly discovered genetic biomarkers of drug-resistant and/or high-risk forms of MM. NcRNAs, e.g., long ncRNAs (lncRNAs) and microRNAs (miRNAs), act as positive or negative regulators of gene expression to control cell proliferation, apoptosis and drug resistance. NcRNAs have been shown to play a role in both solid and hematological tumors. Stratification of MM based upon cytogenetic abnormalities and protein-coding gene signatures does not adequately correlate with the depth and durability of response to novel agents such as bortezomib. Therefore, ncRNAs as new class of molecular effectors may enhance the basic understanding of myelomagenesis and provide better stratification of myeloma subtypes. To investigate the role of ncRNAs in resistance to proteasome inhibitors (PIs), we compared global ncRNA profiling in drug-naïve cells to cells with acquired resistance to the PIs bortezomib, carfilzomib and ixazomib. We hypothesized that ncRNAs commonly deregulated in the 3 resistant cell lines would yield a ncRNA signature and novel therapeutic targets. Experimental Procedures: RPMI 8226 cells resistant to PIs were generated through successive exposure to bortezomib, carfilzomib or ixazomib over a period of 6 months. Total RNA was isolated and genome-wide ncRNA expression profiling was performed using Affymetrix3.0 microarray chips that contained nearly 40,000 miRNA and 13,300 lncRNA probes. NcRNA expression profiles from drug-resistant cells were compared to that of drug- naïve parental cells treated with vehicle alone using the same treatment algorithm. Housekeeping genes were used for log expression normalization. MM patients' bone marrow aspirates were obtained from patients after University of Cincinnati Institutional Review Board approval. Results: Bioinformatic analysis of the ncRNA profiles identified a panel of 87 lncRNAs and ~40 miRNAs that were significantly (〉100-fold) deregulated in all three drug-resistant cell lines relative to drug- naïve parental cells. Strikingly, ~90% of the deregulated lncRNAs exhibited a similar expression pattern in all 3 PI-resistant cell lines. Twenty lncRNAs were deregulated 〉 1000-fold in all 3 resistant cell lines (Figure 1). RPMI 8226 cells carry a chromosomal (14,16) translocation. Interestingly, none of the deregulated lncRNAs detected here localized to chromosome 14 or 16, suggestive of a cytogenetic-independent mechanism of drug resistance. The lncRNA COL4A-2A was upregulated 〉5,000-fold in resistant cells and displayed extensive sequence complementarity to miRNA-29 that was downregulated in resistant cells. Also, our microarray-based studies have identified ncRNAs deregulated in MM patient tumor samples relative to normal plasma cells from healthy age-matched individuals. A significant number of the deregulated ncRNAs between drug- naïve and drug resistant cells were also deregulated in normal plasma cells relative to myeloma cells. Studies are correlating the ncRNA patterns seen in drug-sensitive and drug-resistant cell lines with ncRNA patterns obtained from malignant plasma cells of patients currently receiving bortezomib-based therapy. Updated results to correlate ncRNA expression with myeloma patient response to bortezomib will be presented.Conclusions: Taken together, we have identified a curated panel of ncRNAs deregulated in common within myeloma cells generated with acquired resistant to three different clinically-relevant proteasome inhibitors. Ongoing studies will correlate ncRNA expression patterns from resistant cells with patterns generated from patients with monoclonal gammopathy of unknown significance (MGUS), Smoldering MM, newly diagnosed MM, refractory disease and plasma cell leukemia. In addition, ncRNA patterns will be generated based upon MM patient response to bortezomib. Further investigation is warranted to shed light on the role of these ncRNAs in the development of MM, to identify their targets and to define their role in drug resistance. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.