Abstract: 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.

Abstract: Imaging techniques based on fluorescence and bioluminescence have been important tools in visualizing tumor progression and studying the effect of drugs and immunotherapies on tumor immune microenvironment in animal models of cancer. However, transgenic expression of foreign proteins may induce immune responses in immunocompetent syngeneic tumor transplant models and augment the efficacy of experimental drugs. In this study, we show that the growth rate of Lewis lung carcinoma (LL/2) tumors was reduced after transduction of tdTomato and luciferase (tdTomato/Luc) compared to the parental cell line. tdTomato/Luc expression by LL/2 cells altered the tumor microenvironment by increasing tumor-infiltrating lymphocytes (TILs) while inhibiting tumor-induced myeloid-derived suppressor cells (MDSCs). Interestingly, tdTomato/Luc expression did not alter the response of LL/2 tumors to anti-PD-1 and anti-CTLA-4 antibodies. These results suggest that the use of tdTomato/Luc-transduced cancer cells to conduct studies in immune competent mice may lead to cell-extrinsic tdTomato/Luc-induced alterations in tumor growth and tumor immune microenvironment that need to be taken into consideration when evaluating the efficacy of anti-cancer drugs and vaccines in immunocompetent animal models.

Abstract: Multiple myeloma (MM) is a fatal plasma cell malignancy exhibiting enhanced glucose consumption associated with an aerobic glycolytic phenotype (i.e., the Warburg effect). We have previously demonstrated that myeloma cells exhibit constitutive plasma membrane (PM) localization of GLUT4, consistent with the dependence of MM cells on this transporter for maintenance of glucose consumption rates, proliferative capacity, and viability. The purpose of this study was to investigate the molecular basis of constitutive GLUT4 plasma membrane localization in MM cells. Findings We have elucidated a novel mechanism through which myeloma cells achieve constitutive GLUT4 activation involving elevated expression of the Rab-GTPase activating protein AS160_v2 splice variant to promote the Warburg effect. AS160_v2-positive MM cell lines display constitutive Thr642 phosphorylation, known to be required for inactivation of AS160 Rab-GAP activity. Importantly, we show that enforced expression of AS160_v2 is required for GLUT4 PM translocation and activation in these select MM lines. Furthermore, we demonstrate that ectopic expression of a full-length, phospho-deficient AS160 mutant is sufficient to impair constitutive GLUT4 cell surface residence, which is characteristic of MM cells. Conclusions This is the first study to tie AS160 de-regulation to increased glucose consumption rates and the Warburg effect in cancer. Future studies investigating connections between the insulin/IGF-1/AS160_v2/GLUT4 axis and FDG-PET positivity in myeloma patients are warranted and could provide rationale for therapeutically targeting this pathway in MM patients with advanced disease.

Abstract: Introduction: Multiple Myeloma (MM), the second most common blood cancer, is a clonal disease of long-lived plasma cells (PC) that is currently incurable. Symptomatic myeloma-defining events include hyper calcemia, renal dysfunction, anemia, and bone disease (CRAB criteria). Renal dysfunction is primarily due to free light chain (FLC) precipitation with uromodulin in the distal tubules resulting in light chain cast nephropathy (LCCN). Renal dysfunction may have a negative impact on patient outcomes as it can influence the ability to optimally treat patients, and in some may result in hemodialysis dependence. Therefore, understanding the factors that can contribute to renal dysfunction could allow for early intervention to prevent associated morbidity. Methods: To determine whether certain proteins or genetic mutations in MM patients are associated with renal failure, we analyzed data from the Multiple Myeloma Research Foundation's CoMMpass Study (NCT01454297, Interim Analysis 16) using creatinine levels as a surrogate for renal dysfunction ( & gt;176 μmol/L indicates renal failure). We correlated creatinine with other measures of renal dysfunction (BUN), serum protein levels (total protein, M-protein, IgG, IgA, IgM, IgL-kappa, IgL-lambda), as well as structural changes including t(11;14), t(12;14), t(14;16), t(14;20), t(6;14), t(8;14), hyperdiploidly, 1q21 gains and 17p13 loss as determined by whole genome sequencing (WGS). We used whole exosome sequencing (WES) data to assess if common mutations (KRAS, NRAS, BRAF, DIS3, FAM46C) were associated with renal dysfunction. The numbers in each comparison differed based on the availability of WGS (structural events: 621 vs. 96) or WES (SNVs: 1003 vs. 119). To study gene expression we focused on the patients with the highest and lowest creatinine levels (70/group). Since there was a sex bias in these groups we included a covariate for sex to determine gene expression changes independent of sex. Finally, we analyzed RNAseq analysis for expression of light chain variable regions to determine if there was an association of light chain usage with renal dysfunction. Results: We initially correlated BUN, serum protein levels and structural events with creatinine levels, but only found a significant correlation with BUN levels (Pearson R=0.7275, P= & lt;0.0001, N=816). We next performed a contingency analysis comparing genetic events in patients with creatinine & gt;176 μmol/L versus those & lt;176 μmol/L. While there were no differences in structural events between those with normal vs. elevated creatinine, we did observe a significant increase in BRAF mutations in patients with high creatinine (Fisher's exact test, P=0.0093). We also observed a trend towards significance with KRAS mutations (Fisher's exact test P=0.0930). We examined light chain variable region usage but found no association with high creatinine levels. We expanded this analysis to all genes, and found 110 genes differentially expressed with renal dysfunction, which were enriched for genes involved in inflammatory responses (AIF1, LY96, LYVE1, MPEG1, SELL1, STAB1, TNFRSF12A) and metabolism (COX7A2, FOLR2, MT-ND1, MT-ND3). Conclusions: Our data are consistent with previous studies with respect to the lack of association of renal dysfunction with serum proteins and translocations. However, we observed interesting associations with BRAF mutations and a trend with KRAS mutations, suggesting a role in disease pathogenesis beyond driving tumorigenesis. Additionally, we detected expression changes with several genes involved in inflammatory responses. We cannot conclude if this is due to inflammation related to acute kidney disease or if the myeloma plasma cells expressing these genes are more likely to produce FLC that result in LCCN. Finally, we did not observe a variable chain usage bias, however our study is limited to expression and therefore points to the likelihood that somatic hypermutation plays an important role in whether a light chain can lead to renal disease. Taken together, these data suggest the presence of genetic and biological differences in myeloma cells associated with renal dysfunction and provide insights into identifying patients that could develop renal disease. ACKNOWLEDGEMENTS This work was supported through the STEP-UP HS program from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health, R25DK113659 Disclosures Joseph: GSK: Honoraria; BMS: Research Funding; Takeda: Research Funding; Karyopharm: Honoraria. Hofmeister: Sanofi: Other: National PI for CST; PI or co-PI IST; BMS/Celgene: Other: National PI for CST; PI or co-PI IST; Local PI of CST; Nektar Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Local PI of CST; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Local PI of CST; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Other: Local PI of CST; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Oncolytics: Other: National PI for CST; PI or co-PI IST; Takeda: Other: Local PI of CST; Genzyme: Membership on an entity's Board of Directors or advisory committees; Myeloma360: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; BioAscend: Other: CME speaker; Philips Gilmore: Other: CME speaker; Non-pharma speaker for education, research, marketing; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Non-CME speaker; BlueBird Bio: Other: Non-CME speaker; Aptitude Health: Other: Non-pharma speaker for education, research, marketing; Verascity: Other: Non-pharma speaker for education, research, marketing; TRM Oncology: Other: Non-pharma speaker for education, research, marketing; DAVA Oncology: Other: Non-pharma speaker for education, research, marketing; Medscape: Other: Non-pharma speaker for education, research, marketing; Ohio State University: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: IP rights, Patents & Royalties. Kaufman: Genentech, AbbVie, Janssen: Consultancy, Research Funding; Incyte, celgene: Consultancy; Tecnofarma SAS, AbbVie: Honoraria; Amgen: Research Funding; BMS: Consultancy, Research Funding; Fortis Therapeutics: Research Funding; Heidelberg Pharma: Research Funding; Incyte, TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Novartis: Research Funding; Roche/Genetech, Tecnopharma: Consultancy, Honoraria; Sutro, Takeda: Research Funding. Lonial: AMGEN: Consultancy, Honoraria; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Merck: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding. Nooka: Takeda: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Other: Travel expenses; Amgen: Consultancy, Research Funding; Karyopharm Therapeutics: Consultancy; Bristol-Myers Squibb: Consultancy; Adaptive technologies: Consultancy; Sanofi: Consultancy; Oncopeptides: Consultancy; Janssen Oncology: Consultancy, Research Funding. Boise: AstraZeneca: Consultancy, Research Funding; Abbvie: Consultancy.

Abstract: 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.