ALBERT

Description: 8-Aminoadenosine (8-NH2-Ado), a ribosyl nucleoside analog, in preclinical models of multiple myeloma inhibits phosphorylation of proteins in multiple growth and survival pathways, including Akt. Given that Akt controls the activity of mammalian target of rapamycin (mTOR), we hypothesized that 8-NH2-Ado would be active in mantle cell lymphoma (MCL), a hematological malignancy clinically responsive to mTOR inhibitors. In the current study, the preclinical efficacy of 8-NH2-Ado and its resulting effects on Akt/mTOR and extracellular-signal–regulated kinase signaling were evaluated using 4 MCL cell lines, primary MCL cells, and normal lymphocytes from healthy donors. For all MCL cell lines, 8-NH2-Ado inhibited growth and promoted cell death as shown by reduction of thymidine incorporation, loss of mitochondrial membrane potential, and poly (adenosine diphosphate-ribose) polymerase cleavage. The efficacy of 8-NH2-Ado was highly associated with intracellular accumulation of 8-NH2-adenosine triphosphate (ATP) and loss of endogenous ATP. Formation of 8-NH2-ATP was also associated with inhibition of transcription and translation accompanied by loss of phosphorylated (p-)Akt, p-mTOR, p-Erk1/2, p-phosphoprotein (p)38, p-S6, and p-4E-binding protein 1. While normal lymphocytes accumulated 8-NH2-ATP but maintained their viability with 8-NH2-Ado treatment, primary lymphoma cells accumulated higher concentrations of 8-NH2-ATP, had increased loss of ATP, and underwent apoptosis. We conclude that 8-NH2-Ado is efficacious in preclinical models of MCL and inhibits signaling of Akt/mTOR and Erk pathways.

Description: Abstract 1801 Chronic lymphocytic leukemia (CLL) is the most common leukemia seen in Western countries, primarily in the elderly, with a median age of diagnosis of 72 years. CLL is characterized by the aggressive accumulation of monoclonal peripheral (mature) CD5+ B cells in primary and secondary lymphoid tissues. Several classes of drugs currently exist to treat CLL and these include - nitrogen mustard alkylating agents, purine analogs, monoclonal antibodies, cyclin dependent kinase inhibitors, BTK and PI3K inhibitors. CLL can become resistant to existing therapies necessitating the need for identification of new targets and therapeutic strategies. Unique metabolic dependencies of cancer cells have been identified, further investigation of which could provide tumor cell specific targeting modalities. A myriad of tumor cells exhibit increased glucose uptake and metabolism of glucose via the in-efficient glycolytic pathway, a phenomenon first described by Warburg in the 1900's. Restriction of glucose utilization and metabolism has been shown to chemosensitize and/or elicit toxicity in a wide range of cancers. CLL metabolism is a relatively unexplored area. We sought to determine the dependency of CLL on glucose metabolism. As there are currently no CLL cell lines we used primary patient samples for these studies. CLL cells harvested from PBMCs were cultured in both glucose free and glucose containing media. Our results show that when CLL cells are cultured in these media, there is variation in sensitivity to glucose deprivation ranging from very sensitive to highly resistant (Figure 1). We have investigated possible resistance mechanisms and alternate sources of energy in CLL that could be responsible for maintaining viability even during glucose-withdrawal. We first investigated a role for glutamine. CLL cells sensitive to glucose withdrawal and cultured in the absence of glutamine, did not exhibit enhanced toxicity. These results suggest that cells resistant to glucose withdrawal were not maintaining viability via glutamine metabolism. The role of the mitochondrial metabolism was also investigated. We observed that when the CLL cells are cultured in the absence of glucose and are substituted with galactose, there is a rescue effect, with cell viability being restored. This rescue effect is also observed (although not to the same extent as with galactose) when the CLL cells in the absence of glucose are treated with 2-methylpyruvate (2MP). 2MP feeds directly into the mitochondria and can bypass the glycolytic pathway. CLL cells were also treated with metformin, which is complex-1 inhibitor, and we observed enhanced cell death. The results with galactose, 2MP and metformin all suggest that mitochondrial metabolism is an integral part of CLL metabolism, potentially playing a compensatory role upon glucose-withdrawal. The role of autophagy was also investigated and, using chloroquine we observed that autophagy was pro-survival in CLL patient samples. In summary we have observed varying sensitivity of CLL patient samples to glucose deprivation, and identified resistance mechanisms and alternative sources of energy in CLL cells. The reduction of extracellular glucose has been shown to induce resistance of normal cells to DNA damaging therapeutics, and enhancement of sensitivity in cancer cells, suggesting that glycolysis inhibition may expand the therapeutic window of currently used therapeutics. Targeting these unique metabolic dependencies in CLL could provide strategies to chemo-sensitize and target CLL more effectively with less toxicity. Figure 1: Variable sensitivity of CLL patient samples to 48-hour glucose free culture Figure 1:. Variable sensitivity of CLL patient samples to 48-hour glucose free culture Disclosures: No relevant conflicts of interest to declare.

Description: Multiple myeloma is one of numerous malignancies characterized by increased glucose consumption, a phenomenon with significant prognostic implications in this disease. Few studies have focused on elucidating the molecular underpinnings of glucose transporter (GLUT) activation in cancer, knowledge that could facilitate identification of promising therapeutic targets. To address this issue, we performed gene expression profiling studies involving myeloma cell lines and primary cells as well as normal lymphocytes to uncover deregulated GLUT family members in myeloma. Our data demonstrate that myeloma cells exhibit reliance on constitutively cell surface-localized GLUT4 for basal glucose consumption, maintenance of Mcl-1 expression, growth, and survival. We also establish that the activities of the enigmatic transporters GLUT8 and GLUT11 are required for proliferation and viability in myeloma, albeit because of functionalities probably distinct from whole-cell glucose supply. As proof of principle regarding the therapeutic potential of GLUT-targeted compounds, we include evidence of the antimyeloma effects elicited against both cell lines and primary cells by the FDA-approved HIV protease inhibitor ritonavir, which exerts a selective off-target inhibitory effect on GLUT4. Our work reveals critical roles for novel GLUT family members and highlights a therapeutic strategy entailing selective GLUT inhibition to specifically target aberrant glucose metabolism in cancer.

Description: Background: Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) with a prevalence of approximately 15,000 cases in the United States. Although current therapeutics extend longevity, the median survival is 3 to 5 years warranting continued investigation for newer therapeutics. MCL is characterized by cells with enhanced proliferation combined with impaired apoptosis characteristic of indolent lymphomas. Therefore, therapeutic approaches targeting transcription, translation, or cellular bioenergetics may prove to be more effective than therapies targeting DNA replication. In addition, therapeutic strategies that exploit the altered cellular metabolism of tumor cells may be beneficial. Nucleoside analogues have been used extensively in the treatment of hematologic malignancies and are selective for tumor cells. Our laboratories have developed two purine nucleoside analogues i.e. 8-chloro-adenosine (currently in clinical trials) and a congener, 8-amino-adenosine (8-NH2-Ado), showing high efficacy for multiple myeloma, a slow growing plasma B-cell malignancy. Characterization of the mechanism of toxicity of 8-NH2-Ado in myeloma shows decreased RNA synthesis preceding decreased DNA synthesis, inactivation of Ser/Thr kinases, and reductions in intracellular ATP and glucose consumption. Based on this pleiotropic profile of cellular pathways involved in the execution of cell death by 8-NH2-Ado, we sought to determine its efficacy in MCL. Results: We determined toxicity of 8-NH2-Ado in a panel of MCL cell lines, including, JeKo-1, Mino and Granta 519. Viability was assessed by Annexin V/Dapi double staining after 24 hours of incubation with increasing concentrations of 8-NH2-Ado. All three cell lines demonstrated sensitivity to 8-NH2-Ado with JeKo-1 being the most sensitive (IC50 at 2 uM) followed by Mino and Granta 519. The induction of apoptosis correlated with cleavage of PARP and caspase activation and with decreases in cyclin D1 and Mcl-1 expression. JeKo-1 cells rapidly metabolized 8-NH2-Ado to 8-NH2-ATP. After 6 hrs of incubation, the 8-NH2-ATP intracellular concentration was more than 5 mM, and the ATP concentration was reduced by more than 50%. Additionally after 6 hrs of incubation, the rates of RNA and DNA synthesis were reduced by at least 60% based on [3H]uridine and [3H]thymidine incorporation assays. In an assessment of downstream signaling kinases, p38 and AKT were rapidly de-phosphorylated after 5 hrs of treatment. Because AKT controls cellular glucose consumption, we assessed effects on glucose consumption. In both the JeKo-1 and Granta 519 cells, we observed a similar reduction in glucose consumption; however, baseline glucose consumption in the less sensitive Granta 519 cells was higher. Conclusions: 8-NH2-Ado is highly toxic for the MCL cell lines tested. 8-NH2-Ado decreases Mcl-1 and cyclin D1 expression and decreases phosphorylation of AKT and p38 in both the JeKo-1 and Granta 519 cells. In the JeKo-1 cells, 8-NH2-Ado is metabolized to 8-NH2-ATP and decreases RNA/DNA synthesis and intracellular ATP. The early changes in cellular glucose consumption may facilitate 8-NH2-Ado induction of apoptosis. These pleiotropic features of 8-NH2-Ado in regulating cellular bioenergetics and induction of apoptosis may be particularly advantageous and warrant further investigation of 8-NH2- Ado for the treatment of MCL.

Description: Multiple myeloma (MM) is a commonly occurring hematologic malignancy in the United States with poor prognosis. Among all treatments, proteasome inhibitor (PI) based regimens have been a major breakthrough for patients' outcomes. Available PIs all target 20S proteasome core complex, and the duration of response is limited by toxicity and resistance development. Until now, the underlying mechanism of drug resistance remains unclear. The proteasome is the major proteolytic machinery in protein homeostasis which is pivotal for myeloma cell survival. A functional proteasome consists of 20S proteasome core particle with regulatory particle on one or both ends. There are 3 types of proteasome regulators that could activate a 20S proteasome, PA700 (19S), 11S REG (PA28) and PA200. The 11S REG (PA28) protein family consists of three members, α, β, and γ. PA28 α/β are IFN-γ inducible and with higher expression in antigen presenting cells. Currently, the function of 11S subunit remains largely unknown. Our analysis of plasma cells from MM patients and healthy donors has demonstrated that expression of 11S proteasome is higher in myeloma cells than normal plasma cells and progressively upregulated with disease progression. To further identify the function of 11S proteasome especially PA28α in MM, we generate PA28α knockdown stable MM cell lines. We have found that knockdown of PA28α inhibits MM cell growth and proliferation, also induces myeloma cell resistance to PIs. The mechanism of PI resistance is different from knocking down of 19S or 20S proteasome subunits. Silencing of PA28α inhibits proteasome activity and decreases proteasome work load concurrently, resulting in a favorable proteasome load vs capacity ratio. Altogether, in this report, we describe the function of PA28α in MM cells, also provide novel insights into regulating PIs sensitivity through modulation of the 11S proteasome subunit PA28α. Disclosures Hofmeister: Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Kaufman:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Incyte: Consultancy; Celgene: Consultancy; Winship Cancer Institute of Emory University: Employment; AbbVie: Consultancy; Takeda: Consultancy; TG Therapeutics: Consultancy. Nooka:Amgen: Honoraria, Other: advisory board participation; GSK: Honoraria, Other: advisory board participation; Celgene: Honoraria, Other: advisory board participation; Takeda: Honoraria, Other: advisory board participation; Spectrum pharmaceuticals: Honoraria, Other: advisory board participation; BMS: Honoraria, Other: advisory board participation; Janssen: Honoraria, Other: advisory board participation; Adaptive technologies: Honoraria, Other: advisory board participation. Boise:Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Lonial:Takeda: Consultancy, Research Funding; Amgen: Consultancy; BMS: Consultancy; Janssen: Consultancy, Research Funding; GSK: Consultancy; Karyopharm: Consultancy; Genentech: Consultancy; Celgene Corporation: Consultancy, Research Funding.

Description: Abstract 1850 Multiple Myeloma (MM) is an incurable heterogeneous B cell malignancy characterized by hyperdiploidy and/or aberrant chromosomal translocations warranting strategies that can target diverse molecular subtypes. One such unifying feature in MM entails an increased avidity for glucose that forms the basis for clinical imaging of myeloma by 18fluoro-deoxyglucose positron emission tomography. We are however not yet able to target abnormal glucose utilization for therapy. Given that glucose entry is a key rate-limiting step in glycolysis we performed an unbiased multi-cell line gene-expression profiling study combined with functional knock-down experiments to determine critical glucose transporters facilitating glucose entry in myeloma. These studies revealed a critical dependence of myeloma on the insulin-responsive glucose transporter GLUT4, and GLUTs 8 and 11. Our data demonstrate that myeloma cells exhibit a significant reliance on constitutively cell surface-localized GLUT4 for basal glucose consumption and viability. The functional significance of these transporters was further bolstered by detection of similar patterns of expression in myeloma patient samples. We also tested the therapeutic utility of targeting plasma-membrane localized GLUT4 in MM cell lines and patient samples with the FDA approved HIV protease inhibitor ritonavir that has an off-target inhibitory effect on GLUT4, providing proof of principle that GLUTs can be targeted for therapeutic benefit. In this study we have investigated the mechanism by which GLUT4 suppression elicits cell death in myeloma. GLUT4 contributes to a significant fraction of glucose entry in myeloma while GLUTs 8 and 11 appear to have a significant impact on myeloma cell viability relating to alternative enigmatic subcellular functions not reliant on glucose entry per se. To further elucidate the mechanisms associated with cell death upon glucose deprivation in response to GLUT4 suppression, we examined expression levels of key apoptotic effectors in three myeloma cell lines expressing GLUT 1, 4, 8 or 11 shRNAs. Suppression of GLUT4 appears to selectively engage an apoptotic cascade involving suppression of MCL-1 and BCL-xL in addition to an increase in pro-apoptotic BAX expression. We do not detect up-regulation of PUMA or NOXA that have previously been demonstrated to play a role in glucose-deprivation induced cell death in other cell types. The suppression of MCL-1 is associated with an upstream activation of GSK-3. Expression of an MCL-1 ubiquitination-resistant mutant prevents reduction of MCL-1 protein upon GLUT4 knockdown, and reverses the toxicity elicited by GLUT4 suppression. In addition we do not detect the appearance of the cleaved pro-apoptotic fragment of MCL-1. These observations suggest glucose-deprivation induced cell death in response to GLUT4 reduction is mediated by ubiquitination and proteasomal degradation of MCL-1. In sum we have discovered a novel myeloma specific method to target glucose entry and elicit apoptosis via suppression of MCL-1 expression. MCL-1 is a key effector of chemo-resistance in MM with levels of expression correlating to disease severity. Therefore strategies that can target MCL-1 will have significant utility in treatment of this fatal malignancy. These observations provide further rationale for the development of GLUT4-specific biologics to target aberrant glucose metabolism in myeloma and potentially other glucose-driven cancers. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4016 Multiple Myeloma (MM) is an incurable plasma cell malignancy accounting for 11,000 deaths annually in the US and 20% of deaths from all hematological malignancies. MM is one of myriad malignancies exhibiting enhanced glucose consumption associated with an aerobic glycolytic phenotype (i.e. the Warburg effect). We have recently published a study defining key glucose transporters responsible for facilitating glucose entry in myeloma and observed that MM cells exhibit reliance on constitutively cell surface-localized GLUT4 for basal glucose consumption. MM cells cultured in the absence of glucose or upon GLUT4 suppression exhibit either overt apoptosis (sensitive phenotype) or growth arrest (resistant phenotype). To further demonstrate the clinical utility of targeting GLUT4 for MM therapy we tested a HIV protease inhibitor ritonavir that has an off-target inhibitory effect on GLUT4. Treatment of MM cells with physiologically achievable concentrations of ritonavir blocks glucose entry resulting in MM cell death or growth suppression. The objective of this study was to define those metabolic pathways in resistant MM cell lines which compensate for the decrease in cellular glucose and protect the cells from toxicity caused by glucose-deprivation or GLUT4 suppression. We hypothesized that resistant cells (i.e. JJN3) cultured in the absence of glucose revert to mitochondrial metabolism or autophagy to prevent cell death. The autophagy inhibitor chloroquine did not sensitize resistant cells cultured in the absence of glucose, ruling out autophagy as a source of compensatory metabolites. We next established that the mitochondrial substrates 2-methyl pyruvate or galactose rescue sensitive cells from toxicity caused by culture in the lack of glucose (Fig. 1A). These results suggest the potential for mitochondrial metabolism to rescue cell death ensuing upon inhibition of glycolysis. We therefore treated the resistant JJN3 cells with mitochondrial inhibitors metformin or rotenone to determine if we could elicit toxicity upon glucose-withdrawal. Indeed, resistant cells were sensitized to glucose-withdrawal upon treatment with complex 1 inhibitors metformin (Figure 1B) or rotenone. Metformin treatment alone had a minimal impact on the viability of resistant cells cultured in the presence of glucose (Fig. 1B). To simulate glucose-withdrawal we tested the ability of ritonavir (HIV protease inhibitor that has an off target inhibitory effect on GLUT4) to elicit toxicity in combination with metformin in the resistant cells. Our studies revealed that addition of ritonavir with metformin synergizes to elicit toxicity in resistant cells (Fig. 1C). This synergy was also observed in additional MM cell lines (KMS11 and U266). The biguanide metformin is the most commonly prescribed anti-hyperglycemic drug for the treatment of Type II diabetes. Epidemiologic studies have correlated metformin with a reduced risk of cancer in diabetics earning the drug recognition as a possible anti-neoplastic agent for various types of malignancies. This combinatorial regimen of ritonavir and metformin was not toxic in normal PBMC. The selective tumor specific synthetic lethality induced in ritonavir treated resistant cells upon metformin treatment is detected at doses that are clinically achievable with both compounds. We have initiated studies to evaluate this combination in mouse models of myeloma and patient samples. HIV patients chronically treated with ritonavir who exhibit diabetic symptoms have been treated with metformin indicating this combination treatment is well tolerated in humans. Our studies reveal a potent combinatorial regimen involving repurposed, FDA approved, ritonavir and metformin for the treatment of MM and potentially other glucose-driven malignancies. Disclosures: No relevant conflicts of interest to declare.