Search

Supplementary Fig. S1 from Intrinsically lower AKT, mammalian target of rapamycin, and hypoxia-inducible factor activity correlates with increased sensitivity to 2-deoxy-d-glucose under hypoxia in lung cancer cell lines

Down-regulation by small interfering RNA or absence of hypoxia-inducible factor (HIF-1α) has been shown to lead to increased sensitivity to glycolytic inhibitors in hypoxic tumor cells. In surveying a number of tumor types for differences in intrinsic levels of HIF under hypoxia, we find that the reduction of the upstream pathways of HIF, AKT, and mammalian target of rapamycin (mTOR) correlates with increased toxic effects of 2-deoxy-d-glucose (2-DG) in lung cancer cell lines when treated under hypoxia. Because HIF-1α translation is regulated by mTOR, we examined the effects of blocking mTOR under hypoxia with an analogue of rapamycin (CCI-779) in those cell lines that showed increased mTOR and AKT activity and found that HIF-1α down-regulation coincided with increased 2-DG killing. CCI-779, however, was ineffective in increasing 2-DG toxicity in cell lines that did not express HIF. These results support the hypothesis that although mTOR inhibition leads to the blockage of numerous downstream targets, CCI-779 increases the toxicity of 2-DG in hypoxic cells through down-regulation of HIF-1α. Overall, our findings show that CCI-779 hypersensitizes hypoxic tumor cells to 2-DG and suggests that the intrinsic expression of AKT, mTOR, and HIF in lung cancer, as well as other tumor types, may be important in dictating the decision on how best to use 2-DG alone or in combination with CCI-799 to kill hypoxic tumor cells clinically. [Mol Cancer Ther 2008;7(6):1506–13]

Chimeric antigen receptor (CAR) T-cell therapy remains limited to select centers that can carefully monitor adverse events. To broaden use of CAR T cells in community clinics and in a frontline setting, we developed a novel CD8+ CAR T-cell product, Descartes-08, with predictable pharmacokinetics for treatment of multiple myeloma. Descartes-08 is engineered by mRNA transfection to express anti-BCMA CAR for a defined length of time. Descartes-08 expresses anti-BCMA CAR for 1 week, limiting risk of uncontrolled proliferation; produce inflammatory cytokines in response to myeloma target cells; and are highly cytolytic against myeloma cells regardless of the presence of myeloma-protecting bone marrow stromal cells, exogenous a proliferation-inducing ligand, or drug resistance including IMiDs. The magnitude of cytolysis correlates with anti-BCMA CAR expression duration, indicating a temporal limit in activity. In the mouse model of aggressive disseminated human myeloma, Descartes-08 induces BCMA CAR-specific myeloma growth inhibition and significantly prolongs host survival (p

Immunoglobulin G (IgG) is the most abundant serum antibody and is a key determinant of the humoral immune response. Its structural characteristics and effector functions are modulated through the attachment of various sugar moieties called glycans. IgG N-glycome patterns change with the age of individual and in different diseases. Variability of IgG glycosylation within a population is well studied and is affected by a combination of genetic and environmental factors. However, global inter-population differences in IgG glycosylation have never been properly addressed. Here we present population-specific N-glycosylation patterns of whole IgG, analysed in 5 different populations totalling 10,482 IgG glycomes, and of IgG’s fragment crystallisable region (Fc), analysed in 2,530 samples from 27 populations sampled across the world. We observed that country of residence associates with many N-glycan features and is a strong predictor of monogalactosylation variability. IgG galactosylation also strongly correlated with the development level of a country, defined by United Nations health and socioeconomic development indicators. We found that subjects from developing countries had low IgG galactosylation levels, characteristic for inflammation and ageing. Our results suggest that citizens of developing countries may be exposed to country-specific environmental factors that can cause low-grade chronic inflammation and the apparent increase in biological age.

Unregulated growth and replication as well as an abnormal microenvironment, leads to elevated levels of stress which is a common trait of cancer. By inducing both energy and endoplasmic reticulum (ER) stress, 2-Deoxy-glucose (2-DG) is particularly well-suited to take advantage of the therapeutic window that heightened stress in tumors provides. Under hypoxia, blocking glycolysis with 2-DG leads to significant lowering of ATP resulting in energy stress and cell death in numerous carcinoma cell types. In contrast, under normoxia, 2-DG at a low-concentration is not toxic in most carcinomas tested, but induces growth inhibition, which is primarily due to ER stress. Here we find a synergistic toxic effect in several tumor cell lines in vitro combining 2-DG with fenofibrate (FF), a drug that has been safely used for over 40 years to lower cholesterol in patients. This combination induces much greater energy stress than either agent alone, as measured by ATP reduction, increased p-AMPK and downregulation of mtor. Inhibition of mtor results in blockage of GRP78 a critical component of the unfolded protein response which we speculate leads to greater ER stress as observed by increased p-eif2 alpha. Moreover, to avoid an insulin response and adsorption by the liver, 2-DG is delivered by slow-release pump yielding significant anti-tumor control when combined with FF. Our results provide promise for developing this combination clinically and others that combine 2-DG with agents that act synergistically to selectively increase energy and ER stress to a level that is toxic to numerous tumor cell types., NA

Type 1 diabetes (T1D) is an autoimmune disease characterized by destruction of pancreatic β cells. One of the promising therapeutic approaches in T1D is the transplantation of islets; however, it has serious limitations. To address these limitations, immunotherapeutic strategies have focused on restoring immunologic tolerance, preventing transplanted cell destruction by patients’ own immune system. Macrophage-derived chemokines such as chemokine-ligand-22 (CCL22) can be utilized for regulatory T cell (Treg) recruitment and graft tolerance. Stellate cells (SCs) have various immunomodulatory functions: recruitment of Tregs and induction of T-cell apoptosis. Here, we designed a unique immune-privileged microenvironment around implantable islets through overexpression of CCL22 proteins by SCs. We prepared pseudoislets with insulin-secreting mouse insulinoma-6 (MIN6) cells and human SCs as a model to mimic naive islet morphology. Our results demonstrated that transduced SCs can secrete CCL22 and recruit Tregs toward ​the implantation site in vivo. This study is promising to provide a fundamental understanding of SC-islet interaction and ligand synthesis and transport from SCs at the graft site for ensuring local immune tolerance. Our results also establish a new paradigm for creating tolerable grafts for other chronic diseases such as diabetes, anemia, and central nervous system (CNS) diseases, and advance the science of graft tolerance., Scientific and Technological Research Council of Turkey (TÜBİTAK)

One of the key factors that correlates with poor survival of patients with pancreatic cancer is the extent of hypoxic areas within the tumor tissue. The adaptation of pancreatic cancer cells to limited oxygen delivery promotes the induction of an invasive and treatment-resistant phenotype, triggering metastases at an early stage of tumor development, which resist in most cases adjuvant therapies following tumor resection. In this article, the authors summarize the evidence demonstrating the significance of hypoxia in pancreatic cancer pathogenesis and discuss the possible hypoxia-induced mechanisms underlying its aggressive nature. We then conclude with promising strategies that target hypoxia-adapted pancreatic cancer cells.

Despite recent advances in the identification of genomic alterations that lead to urothelial oncogenesis in vitro, patients with advanced urothelial carcinomas continue to have poor clinical outcomes. In the present review, we focus on targeted therapies that have yielded the most promising results alone or combined with traditional chemotherapy, including the antiangiogenesis agent bevacizumab, the human epidermal growth factor receptor 2 antibody trastuzumab, and the tyrosine kinase inhibitor cabozantinib. We also describe ongoing and developing clinical trials that use innovative approaches, including dose-dense scheduling of singular chemotherapy combinations, prospective screening of tumor tissues for mutational targets and biomarkers to predict chemosensitivity before the determination of the therapeutic regimen, and novel agents that target proteins in the immune checkpoint regulation pathway (programmed cell death protein 1 [PD-1] and anti-PD-ligand 1) that have shown significant potential in preclinical models and early clinical trials. New agents and targeted therapies, alone or combined with traditional chemotherapy, will only be validated through accrual to developing clinical trials that aim to translate these therapies into individualized treatments and improved survival rates in urothelial carcinoma.

SUMMARY Biliary tract carcinoma is a rare malignancy. We performed a comprehensive analysis of published prospective clinical trials in advanced biliary tract carcinoma in an attempt to identify active regimens in this setting. We searched PubMed and abstracts presented at the American Society of Clinical Oncology, Gastrointestinal Cancer Symposium, European Society of Medical Oncology and European Cancer Organization conferences for clinical trials in this disease. We found 83 trials. The effect of gemcitabine on overall survival benefit showed a strong trend (p = 0.014) and an improvement in progression-free survival (p = 0.003). Gemcitabine-based regimens containing 5-fluorouracil showed a trend toward an improved overall survival (p = 0.047) relative to platinum agents. Our findings support gemcitabine as the chemotherapy backbone for the treatment of patients with cholangiocarcinoma. Gemcitabine plus 5-fluorouracil combinations warrant further investigations.

Through the eons of time, out of all possible configurations, nature has selected glucose not only as a vital source of energy to sustain life but also as the molecule who's structure supplies the appropriate elements required for a cell to grow and multiply. This understanding, at least in part, explains the profound effects that the analog of glucose, 2-deoxy-d-glucose, has been shown to have on as common and widespread diseases as cancer, viral infection, aging-related morbidity, epilepsy, and others. This review is confined to summarizing some of the salient findings of this remarkable compound as they relate mainly to cancer.

Chimeric antigen receptor (CAR) T cells targeting BCMA are positioned to transform treatment of multiple myeloma (MM), and virally-generated anti-BCMA CAR T cells have shown impressive early stage clinical results. However, the safety risk/benefit, manufacturing constraints, and relevant patient populations of viral anti-BCMA CAR T have yet to be fully defined. Here we present preclinical characterization of an autologous mRNA-generated anti-BCMA CAR T cell product (Descartes-08) designed to reduce safety risk and broaden the fitness-for-use of anti-BCMA CAR T cell therapy. Descartes-08 are autologous CD8+ T cells that express anti-BCMA CAR on up to 90% of cells with duration of CAR expression for several days with subsequent reduction in expression to background approximately 1 week after their generation. The manufacturing process is clinically scalable with high purity and viability of Descartes-08 following cryopreservation. Descartes-08 undergo cytotoxic degranulation and produce cytokines IFNg, TNFα, IL-2, in response to multiple BCMA-expressing multiple myeloma target cell lines in an effector-to-target-ratio-dependent manner. Furthermore, Descartes-08 kills MM lines that are both resistant and sensitive to lenalidomide and pomalidomide, and/or MM cells that are grown in the presence of bone marrow stromal cells that clinically support MM survival. Moreover, Descartes-08 are highly cytotoxic against MM cells from both newly-diagnosed and relapsed patients. The magnitude of cytolytic and cytokine responses correlates with duration of anti-BCMA CAR expression and declines after 4 days, indicating a temporal limit in activity that is predicted to dramatically decrease the risk of severe cytokine release syndrome. In a mouse model of disseminated human MM, Descartes-08 shows CAR-specific suppression of myeloma that is maintained throughout the duration of treatment. Taken together, these results illustrate features of RNA-generated anti-BCMA CAR T cells that promise key clinical advantages, thereby supporting ongoing clinical development of Descartes-08 for treatment of MM (NCT03448978). Disclosures Kurtoglu: Cartesian Therapeutics: Employment. Zhang:Cartesian Therapeutics: Employment. Stewart:Cartesian Therapeutics: Employment. Anderson:Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder ; Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board.

This phase I trial was initiated to evaluate the safety, pharmacokinetics (PK) and maximum tolerated dose (MTD) of the glycolytic inhibitor, 2-deoxy-D-glucose (2DG) in combination with docetaxel, in patients with advanced solid tumors.A modified accelerated titration design was used. 2DG was administered orally once daily for 7 days every other week starting at a dose of 2 mg/kg and docetaxel was administered intravenously at 30 mg/m(2) for 3 of every 4 weeks beginning on day 1 of week 2. Following the completion of dose escalation, cohorts of patients were then treated with 2DG for 21 days or every day of each 4-week cycle for up to 12 cycles.Thirty-four patients were enrolled: 21 on every other week, 6 on a 21 of 28-day cycle and 7 on the continuous 2DG dosing schedule. There were no dose-limiting toxicities which met the MTD criteria. The most common adverse events were fatigue, sweating, dizziness and nausea mimicking the hypoglycemic symptoms expected from 2DG administration. Therefore, 63 mg/kg was selected as the clinically tolerable dose. The most significant adverse effects noted at 63-88 mg/kg doses were reversible hyperglycemia (100 %), gastrointestinal bleeding (6 %) and reversible grade 3 QTc prolongation (22 %). Eleven patients (32 %) had stable disease, 1 patient (3 %) partial response and 22 patients (66 %) progressive disease as their best response. There was no PK interaction between 2DG and docetaxel.The recommended dose of 2DG in combination with weekly docetaxel is 63 mg/kg/day with tolerable adverse effects.

Dependence on Bcl-2 proteins is a common feature of cancer cells and provides a therapeutic opportunity. ABT-737 is an antagonist of antiapoptotic Bcl-2 proteins and therefore is a good predictor of Bcl-xL/Bcl-2 dependence. Surprisingly, analysis of Mcl-1–dependent multiple myeloma cell lines revealed codependence on Bcl-2/Bcl-xL in half the cells tested. Codependence is not predicted by the expression level of antiapoptotic proteins, rather through interactions with Bim. Consistent with these findings, acquired resistance to ABT-737 results in loss of codependence through redistribution of Bim to Mcl-1. Overall, these results suggest that complex interactions, and not simply expression patterns of Bcl-2 proteins, need to be investigated to understand Bcl-2 dependence and how to better use agents, such as ABT-737.

To study the feasibility of using 2-deoxy-D-glucose (2-DG)-labeled gold nanoparticle (AuNP-DG) as a computed tomography (CT) contrast agent with tumor targeting capability through in vitro experiments.Gold nanoparticles (AuNP) were fabricated and were conjugated with 2-deoxy-D-glucose. The human alveolar epithelial cancer cell line, A-549, was chosen for the in vitro cellular uptake assay. Two groups of cell samples were incubated with the AuNP-DG and the unlabeled AuNP, respectively. Following the incubation, the cells were washed with sterile PBS to remove the excess gold nanoparticles and spun to cell pellets using a centrifuge. The cell pellets were imaged using a microCT scanner immediately after the centrifugation. The reconstructed CT images were analyzed using a commercial software package.Significant contrast enhancement in the cell samples incubated with the AuNP-DG with respect to the cell samples incubated with the unlabeled AuNP was observed in multiple CT slices.Results from this study demonstrate enhanced uptake of 2-DG-labeled gold nanoparticle by cancer cells in vitro and warrant further experiments to study the exact molecular mechanism by which the AuNP-DG is internalized and retained in the tumor cells.

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10 muM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (Delta Psi m). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21(Waf1/Cip1) was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p < 0.05) and extended tumor doubling time (p < 0.001). Our observations suggest that GHRH antagonist JMR-132 exerts its antiproliferative effect on experimental colon cancer cells through p21(Waf1/Cip1) mediated S-phase arrest along with apoptosis involving the intrinsic pathway.

Multiple myeloma (MM) cells continuously secrete large amounts of immunoglobulins that are folded in the endoplasmic reticulum (ER) whose function depend on the Ca(2+) concentration inside its lumen. Recently, it was shown that the ER membrane leaks Ca(2+) that is captured and delivered back by mitochondria in order to prevent its loss. Thus, we hypothesized that the highly active and abundant ER in MM cells results in greater Ca(2+)-regulation by mitochondria which would render them sensitive to mitochondrial inhibitors. Here, we indeed find that Ca(2+) leak is greater in 3 MM, when compared to 2 B-cell leukemia cell lines. Moreover, this greater leak in MM cells is associated with hypersensitivity to various mitochondrial inhibitors, including CCCP. Consistent with our hypothesis, CCCP is more potent in inducing the unfolded protein response marker, CHOP/GADD153 in MM versus B-cell leukemia lines. Additionally, MM cells are found to be significantly more sensitive to clinically used fenofibrate and troglitazone, both of which were recently shown to have inhibitory effects on mitochondrial function. Overall, our results demonstrate that the unusually high ER activity in MM cells may be exploited for therapeutic benefit through the use of mitochondrial inhibitors including troglitazone and fenofibrate.

Down-regulation by small interfering RNA or absence of hypoxia-inducible factor (HIF-1α) has been shown to lead to increased sensitivity to glycolytic inhibitors in hypoxic tumor cells. In surveying a number of tumor types for differences in intrinsic levels of HIF under hypoxia, we find that the reduction of the upstream pathways of HIF, AKT, and mammalian target of rapamycin (mTOR) correlates with increased toxic effects of 2-deoxy-d-glucose (2-DG) in lung cancer cell lines when treated under hypoxia. Because HIF-1α translation is regulated by mTOR, we examined the effects of blocking mTOR under hypoxia with an analogue of rapamycin (CCI-779) in those cell lines that showed increased mTOR and AKT activity and found that HIF-1α down-regulation coincided with increased 2-DG killing. CCI-779, however, was ineffective in increasing 2-DG toxicity in cell lines that did not express HIF. These results support the hypothesis that although mTOR inhibition leads to the blockage of numerous downstream targets, CCI-779 increases the toxicity of 2-DG in hypoxic cells through down-regulation of HIF-1α. Overall, our findings show that CCI-779 hypersensitizes hypoxic tumor cells to 2-DG and suggests that the intrinsic expression of AKT, mTOR, and HIF in lung cancer, as well as other tumor types, may be important in dictating the decision on how best to use 2-DG alone or in combination with CCI-799 to kill hypoxic tumor cells clinically. [Mol Cancer Ther 2008;7(6):1506–13]

In tumor cells growing under hypoxia, inhibiting glycolysis with 2-deoxy-d-glucose (2-DG) leads to cell death, whereas under normoxic conditions cells similarly treated survive. Surprisingly, here we find that 2-DG is toxic in select tumor cell lines growing under normal oxygen tension. In contrast, a more potent glycolytic inhibitor, 2-fluorodeoxy-d-glucose, shows little or no toxicity in these cell types, indicating that a mechanism other than inhibition of glycolysis is responsible for their sensitivity to 2-DG under normoxia. A clue to this other mechanism comes from previous studies in which it was shown that 2-DG interferes with viral N-linked glycosylation and is reversible by exogenous addition of mannose. Similarly, we find that 2-DG interferes with N-linked glycosylation more potently in the tumor cell types that are sensitive to 2-DG under normoxia, which can be reversed by exogenous mannose. Additionally, 2-DG induces an unfolded protein response, including up-regulation of GADD153 (C/EBP-homologous protein), an unfolded protein response–specific mediator of apoptosis, more effectively in 2-DG–sensitive cells. We conclude that 2-DG seems to be toxic in select tumor cell types growing under normoxia by inhibition of N-linked glycosylation and not by glycolysis. Because in a phase I study 2-DG is used in combination with an anticancer agent to target hypoxic cells, our results raise the possibility that in certain cases, 2-DG could be used as a single agent to selectively kill both the aerobic (via interference with glycosylation) and hypoxic (via inhibition of glycolysis) cells of a solid tumor. [Mol Cancer Ther 2007;6(11):3049–58]

Through the eons of time, out of all possible configurations, nature has selected glucose not only as a vital source of energy to sustain life but also as the molecule who's structure supplies the appropriate elements required for a cell to grow and multiply. This understanding, at least in part, explains the profound effects that the analog of glucose, 2-deoxy-d-glucose, has been shown to have on as common and widespread diseases as cancer, viral infection, aging-related morbidity, epilepsy, and others. This review is confined to summarizing some of the salient findings of this remarkable compound as they relate mainly to cancer.

Although cisplatin is the drug of choice in treating lung cancer patients, relapse and resistance is a common drawback to its clinical effectiveness. Based on cisplatin's reported ability to interfere with numerous cellular components, including mitochondria, we probed alterations in metabolism in cisplatin-resistant tumor cell lines to reveal targets for overcoming this important form of resistance.Cisplatin-resistant lung and ovarian cancer cell lines were used to evaluate the efficacy of metabolic inhibitors for selectively targeting cisplatin-resistant cells under varying oxygen conditions.Three cisplatin-resistant cancer cell lines expressed lower HKII protein when compared to the respective cisplatin-sensitive cancer cell lines from which they were derived. Under anaerobic and hypoxic conditions, treatment with the glycolytic inhibitors 2-deoxyglucose (2-DG) and 2-fluorodeoxyglucose (2-FDG) correlated with increased cytotoxicity and more pronounced decreases in lactate production in cisplatin-resistant cells, indicating a greater blockage of glycolysis. Knockdown of HKI or HKII with siRNA in the parental lung cancer cell lines led to increased 2-FDG-induced cell death under anaerobic conditions. Under normal oxygen conditions, blockage of either fatty acid oxidation or deprivation of glutamine resulted in cell death in cisplatin-resistant lung cancer cell lines.Altered hexokinase levels in cisplatin-resistant cancer cell lines leads to increased sensitivity to glycolytic inhibition under anaerobic conditions, whereas under normoxic conditions, blockage of either fatty acid oxidation or deprivation of glutamine leads to cell death. These findings may be clinically applicable when considering cisplatin resistance.

As tumors evolve, they upregulate glucose metabolism while also encountering intermittent periods of glucose deprivation. Here, we investigate mechanisms by which pancreatic cancer cells respond to therapeutic (2-deoxy-D-glucose, 2-DG) and physiologic (glucose starvation, GS) forms of glucose restriction.From a tumor cell line (1420) that is unusually sensitive to 2-DG under normoxia, low (14DG2)- and high (14DG5)-dose resistant cell lines were selected and used to probe the metabolic pathways involved with their response to different forms of glucose deprivation.Muted induction of the unfolded protein response was found to correlate with resistance to 2-DG. Additionally, 14DG2 displayed reduced 2-DG uptake, while 14DG5 was cross-resistant to tunicamycin, suggesting it has enhanced ability to manage glycosylation defects. Conversely, 2-DG-resistant cell lines were more sensitive than their parental cell line to GS, which coincided with lowered levels of glycogen phosphorylase (PYGB) and reduced breakdown of glycogen to glucose in the 2-DG-resistant cell lines. Moreover, by inhibiting PYGB in the parental cell line, sensitivity to GS was increased.Overall, the data demonstrate that the manner in which glucose is restricted in tumor cells, i.e., therapeutic or physiologic, leads to differential biological responses involving distinct glucose metabolic pathways. Moreover, in evolving tumors where glucose restriction occurs, the identification of PYGB as a metabolic target may have clinical application.

Inhibition of glucose metabolism has recently become an attractive target for cancer treatment. Accordingly, since 2-deoxyglucose (2-DG) competes effectively with glucose, it has come under increasing scrutiny as a therapeutic agent. The initial response of tumor cells to 2-DG is growth inhibition, which is thought to conserve energy and consequently protect cells from its ATP-lowering effects as a glycolytic inhibitor. However, since 2-DG also mimics mannose and thereby interferes with N-linked glycosylation, the question is raised of how this sugar analog inhibits tumor cell growth and whether the mechanism by which it protects cells can be manipulated to convert 2-DG-induced growth inhibition to cell death.Cell growth and death were measured via counting viable and dead cells based on trypan blue exclusion. Markers of ATP reduction and the unfolded protein response (UPR) were detected by Western blot. Protein functions were manipulated through chemical compounds, siRNA and the use of gene-specific wild-type and knock-out mouse embryonic fibroblasts (MEFs).At 2-DG concentrations that can be achieved in human plasma without causing significant side effects, we find (a) It induces growth inhibition predominantly by interference with glycosylation, which leads to accumulation of unfolded proteins in the endoplasmic reticulum activating the UPR; (b) Inhibition of PERK (but not ATF6 or IRE1), a major component of the UPR, leads to conversion of 2-DG-induced growth inhibition to cell death and (c) secondarily to PERK, inhibition of GCN2, a kinase that is activated in response to low intracellular glutamine, increases 2-DG's cytotoxic effects in PERK -/- MEFs.Overall, these findings present a novel anticancer strategy that can be translated into therapeutic gain as they uncover the metabolic target PERK, and to a lesser degree GCN2, that when inhibited convert 2-DG's static effect to a toxic one in tumor cells growing under normoxia.

Background During tumor angiogenesis, endothelial cells (ECs) are engaged in a number of energy consuming biological processes, such as proliferation, migration, and capillary formation. Since glucose uptake and metabolism are increased to meet this energy need, the effects of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on in vitro and in vivo angiogenesis were investigated. Methodology/Principal Findings In cell culture, 2-DG inhibited EC growth, induced cytotoxicity, blocked migration, and inhibited actively forming but not established endothelial capillaries. Surprisingly, 2-DG was a better inhibitor of these EC properties than two more efficacious glycolytic inhibitors, 2-fluorodeoxy-D-glucose and oxamate. As an alternative to a glycolytic inhibitory mechanism, we considered 2-DG's ability to interfere with endothelial N-linked glycosylation. 2-DG's effects were reversed by mannose, an N-linked glycosylation precursor, and at relevant concentrations 2-DG also inhibited synthesis of the lipid linked oligosaccharide (LLO) N-glycosylation donor in a mannose-reversible manner. Inhibition of LLO synthesis activated the unfolded protein response (UPR), which resulted in induction of GADD153/CHOP and EC apoptosis (TUNEL assay). Thus, 2-DG's effects on ECs appeared primarily due to inhibition of LLOs synthesis, not glycolysis. 2-DG was then evaluated in two mouse models, inhibiting angiogenesis in both the matrigel plug assay and the LHBETATAG transgenic retinoblastoma model. Conclusions/Significance In conclusion, 2-DG inhibits endothelial cell angiogenesis in vitro and in vivo, at concentrations below those affecting tumor cells directly, most likely by interfering with N-linked glycosylation rather than glycolysis. Our data underscore the importance of glucose metabolism on neovascularization, and demonstrate a novel approach for anti-angiogenic strategies.

The purpose of this study was to investigate the feasibility of using a 2-deoxy-d-glucose (2-DG) labeled gold nanoparticle (AuNP-2-DG) as a functionally targeted computed tomography (CT) contrast agent to obtain high-resolution metabolic and anatomic information of tumor in a single CT scan. Gold nanoparticles (AuNPs) were fabricated and were conjugated with 1-DG or 2-DG. 1-DG provides an excellent comparison since it is known to interfere with the ability of the glucose transporter to recognize the sugar moiety. The human alveolar epithelial cancer cell line, A-549, was chosen for the in vitro cellular uptake assay. Three groups of cell samples were incubated with the 1-DG or 2-DG labeled AuNP and the unlabeled AuNP. Following the incubation, the cells were washed with sterile phosphate buffered saline to remove the excess AuNPs and spun using a centrifuge. The cell pellets were imaged using a microCT scanner immediately after the centrifugation. Internalization of AuNP-2-DG is verified using transmission electron microscopy imaging. Significant contrast enhancement in the cell samples incubated with the AuNP-2-DG with respect to the cell samples incubated with the unlabeled AuNP and the AuNP-1-DG was observed in multiple CT slices. Results from our in vitro experiments suggest that the AuNP-2-DG may be used as a functional CT contrast agent to provide high-resolution metabolic and anatomic information in a single CT scan. These results justify further in vitro and in vivo experiments to study the feasibility of using the AuNP-2-DG as a functional CT contrast agent in radiation therapy settings.

The glucose analog and glycolytic inhibitor 2-deoxy-d-glucose (2-DG), which is currently under clinical evaluation for targeting cancer cells, not only blocks glycolysis thereby reducing cellular ATP, but also interferes with N-linked glycosylation, which leads to endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). Both bioenergetic challenge and ER stress have been shown to activate autophagy, a bulk cellular degradation process that plays either a pro- or anti-death role. Here, we investigate which pathway 2-DG interferes with that activates autophagy and the role of this process in modulating 2-DG-induced toxicity. Pancreatic cancer cell line 1420, melanoma cell line MDA-MB-435 and breast cancer cell line SKBR3 were used to investigate the relationship between induction by 2-DG treatment of ER stress/UPR, ATP reduction and activation of autophagy. ER stress/UPR (Grp78 and CHOP) and autophagy (LC3B II) markers were assayed by immunoblotting, while ATP levels were measured using the CellTiter-Glo Luminescent Cell Viability Assay. Autophagy was also measured by immunofluorescence utilizing LC3B antibody. Cell death was detected with a Vi-Cell cell viability analyzer using trypan blue exclusion. In the three different cancer cell lines described earlier, we find that 2-DG upregulates autophagy, increases ER stress and lowers ATP levels. Addition of exogenous mannose reverses 2-DG-induced autophagy and ER stress but does not recover the lowered levels of ATP. Moreover, under anaerobic conditions where 2-DG severely depletes ATP, autophagy is diminished rather than activated, which correlates with lowered levels of the ER stress marker Grp78. Additionally, when autophagy is blocked by siRNA, cell sensitivity to 2-DG is increased corresponding with upregulation of ER stress-mediated apoptosis. Similar increased toxicity is observed with 3-methyladenine, a known autophagy inhibitor. In contrast, rapamycin which enhances autophagy reduces 2-DG-induced toxicity. Overall, these results indicate that the major mechanism by which 2-DG stimulates autophagy is through ER stress/UPR and not by lowering ATP levels. Furthermore, autophagy plays a protective role against 2-DG-elicited cell death apparently by relieving ER stress. These data suggest that combining autophagy inhibitors with 2-DG may be useful clinically.

An unexpected similarity between cancer and cardiac muscle cells in their sensitivity to anthracyclines and delocalized lipophilic cations (DLC) prompted a series of studies in which it was shown that the positive charge of these compounds is central to their selective accumulation and toxicity in these two distinct cell types. An initial finding to explain this phenomenon was that cancer and cardiac muscle cells exhibit high negative plasma membrane potentials resulting in increased uptake of these agents. However, the p-glycoprotein efflux pump was shown to be another factor underlying differential accumulation of these compounds, since it recognizes positively charged drugs and thereby actively reduces their intracellular concentrations. The delocalized positive charge and lipophilicity of DLCs leads to their retention and inhibition of ATP synthesis in mitochondria. Years later it was realized that cancer cells in the hypoxic portions of solid tumors were similar to those treated with DLCs in relying mainly on anaerobic metabolism for survival and could thus be targeted with a glycolytic inhibitor, 2-deoxy-D-glucose (2-DG). This hypothesis has lead to a Phase I clinical trial in which 2-DG is used to selectively kill the hypoxic tumor cell population which are resistant to standard chemotherapy or radiation.

The dependence of hypoxic tumor cells on glycolysis as their main means of producing ATP provides a selective target for agents that block this pathway, such as 2-deoxy-D-glucose (2-DG) and 2-fluoro-deoxy-D-glucose (2-FDG). Moreover, it was demonstrated that 2-FDG is a more potent glycolytic inhibitor with greater cytotoxic activity than 2-DG. This activity correlates with the closer structural similarity of 2-FDG to glucose than 2-DG, which makes it a better inhibitor of hexokinase, the first enzyme in the glycolytic pathway. In contrast, because of its structural similarity to mannose, 2-DG is known to be more effective than 2-FDG in interfering with N-linked glycosylation. Recently, it was reported that 2-DG, at a relatively low dose, is toxic to certain tumor cells, even under aerobic conditions, whereas 2-FDG is not. These results indicate that the toxic effects of 2-DG in selected tumor cells under aerobic conditions is through inhibition of glycosylation rather than glycolysis. The intention of this minireview is to discuss the effects and potential clinical impact of 2-DG and 2-FDG as antitumor agents and to clarify the differential mechanisms by which these two glucose analogues produce toxicity in tumor cells growing under anaerobic or aerobic conditions.

Hypoxic regions within solid tumors harbor cells that are resistant to standard chemotherapy and radiotherapy. Because oxygen is required to produce ATP by oxidative phosphorylation, under hypoxia, cells rely more on glycolysis to generate ATP and are thereby sensitive to 2-deoxy-d-glucose (2-DG), an inhibitor of this pathway. Universally, cells respond to lowered oxygen tension by increasing the amount of glycolytic enzymes and glucose transporters via the well-characterized hypoxia-inducible factor-1 (HIF). To evaluate the effects of HIF on 2-DG sensitivity, the following three models were used: (a) cells treated with oligomycin to block mitochondrial function in the presence (HIF+) or absence (HIF−) of hypoxia, (b) cells treated with small interfering RNA specific for HIF-1α and control cells cultured under hypoxia, and (c) a mutant cell line unable to initiate the HIF response and its parental HIF+ counterpart under hypoxic conditions. In all three models, HIF increased resistance to 2-DG and other glycolytic inhibitors but not to other chemotherapeutic agents. Additionally, HIF reduced the effects of 2-DG on glycolysis (as measured by ATP and lactate assays). Because HIF increases glycolytic enzymes, it follows that greater amounts of 2-DG would be required to inhibit glycolysis, thereby leading to increased resistance to it under hypoxia. Indeed, hexokinase, aldolase, and lactate dehydrogenase were found to be increased as a function of HIF under the hypoxic conditions and cell types we used; however, phosphoglucose isomerase was not. Although both hexokinase and phosphoglucose isomerase are known to interact with 2-DG, our findings of increased levels of hexokinase more likely implicate this enzyme in the mechanism of HIF-mediated resistance to 2-DG. Moreover, because 2-DG is now in phase I clinical trials, our results suggest that glycolytic inhibitors may be more effective clinically when combined with agents that inhibit HIF. [Mol Cancer Ther 2007;6(2):732–41]

Meeting abstracts Tremelimumab is a fully human monoclonal antibody that binds to CTLA-4 expressed on the surface of activated T lymphocytes and results in inhibition of B7-CTLA-4-mediated down regulation of T cell activation. Both transcatheter arterial chemoembolization (TACE) and radiofrequency

Although cisplatin is the drug of choice in treating lung cancer patients, acquired resistance appears to be a common and serious drawback to its effectiveness in the clinic. Using two pairs of cisplatin sensitive and resistant lung cancer cell lines developed in vitro, we have found significantly lowered hexokinase II (HKII) levels in both resistant cell lines. As a consequence of lowered HKII, these cell lines, when grown under anaerobic conditions, are markedly more sensitive to the glycolytic inhibitor 2-deoxy-D-glucose (2DG) than their parental cell lines from which they were derived. This was our first indication that lowered HKII levels in these cisplatin resistant cells has metabolic consequences in different environmental conditions. Thus, it appears that a fundamental difference in glucose metabolism is associated with resistance to cisplatin in these cell lines. It is the aim of this study to explore whether reduced HKII is a common occurrence in cisplatin resistant cells and whether these differences uncover metabolic targets that can be exploited for therapeutic gain. Much like the Atkins diet, when carbohydrates are lowered or glycolysis is inhibited, other sources of energy such as fatty acids and amino acids can be used to maintain cell viability. A trypan blue cytotoxicity assay was used to determine the sensitivity of our cisplatin resistant cell lines to a pharmacological inhibitor, etomoxir, toward fatty acid oxidation. Thus, we tested and found that one of these cisplatin resistant cell lines is sensitive to interference with fatty acid oxidation. Next, we placed our cell lines in glutamine free medium, and found that the second cisplatin resistant cell line is sensitive to glutamine deprivation. Moreover, when lactate production was measured in anaerobic conditions, cisplatin resistant cell lines produced less lactate when challenged with the glycolytic inhibitor, 2DG, than their cisplatin sensitive parental cell lines. Overall, our results indicate that lowered HKII levels in two cisplatin resistant cell lines appear to be associated with a decreased reliance on glucose metabolism, with a shift to either fatty acid oxidation or glutaminolysis. Moreover, these cisplatin resistant cells are sensitive to glycolytic inhibition when placed in oxygen-deprived conditions. Thus, these new metabolic targets may be exploited for therapeutic gain in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4087. doi:10.1158/1538-7445.AM2011-4087

BACKGROUND: Cancer cells are more glycolytic than normal cells and thereby take up more glucose, as demonstrated by the positron emission tomography (PET) scan. This can be exploited for cancer therapy using glycolytic inhibitors such as 2-deoxy-D-glucose (2DG). Due to its similarity to mannose, 2DG also interferes with N-linked glycosylation resulting in an unfolded protein response (UPR), which slows growth of tumors. Additionally, 2DG inhibits productive infection of enveloped viruses, e.g. herpesviruses, due to its interference with glycosylation of viral proteins. Kaposi's sarcoma (KS) is a cancer that is caused by infection of a gammaherpesvirus; KS might be particularly vulnerable to 2DG, given the chemical's properties as both an anti-cancer and an anti-viral agent. We are investigating the mechanisms and their resulting effects by which cell culture models for KS are sensitive to 2DG treatment in the context of viral replication. DESIGN: Two models of herpesvirus infection were used for 2DG treatment in vitro: KS-associated herpesvirus (KSHV) and murine herpesvirus-68 (MHV-68). Human embryonic kidney cells latently infected with a recombinant KSHV genome (HEK293rKSHV.219) were induced to lytic reactivation upon addition of sodium butyrate. Murine embryonic fibroblasts were infected de novo with MHV-68 to cause lytic replication. In each model, cells were treated with nontoxic doses of 2DG. RNA and protein levels were measured at 24 hours treatment; cell proliferation and viral copy number were measured at 72 hours. RESULTS: In two different models of gammaherpesvirus infection we find that 2DG inhibits expression of early and late viral lytic genes, as well as interferes with the ability of the host to produce infectious virions. While 2DG is used at nontoxic concentrations, the compound inhibits cell proliferation. Treatment with inhibitors of glycolysis–but not glycosylation–does not significantly decrease viral copy number or infectious particles released from infected cells. Furthermore, 2DG is correlated with UPR induction by the phosphorylation of the alpha subunit of eukaryotic translation initiation factor-2 as well as up-regulation of the endoplasmic reticulum (ER) chaperone glucose-regulated protein-78 at both the RNA and protein levels. CONCLUSION: Overall, these results indicate that a major mechanism by which 2DG inhibits viral replication might be through ER stress/UPR. Furthermore, 2DG action takes place early in the viral lytic cycle, preventing the induction of viral genes, which correlates to the inability to produce mature viral particles. These data suggest that 2DG may be clinically beneficial in the treatment of herpesvirus-induced cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2725. doi:10.1158/1538-7445.AM2011-2725

Background: Cancer cells have greater aerobic glycolysis than normal cells and therefore take up more glucose, as demonstrated by the positron emission tomography (PET) scan. This phenomenon can be exploited for cancer therapy using glycolytic inhibitors such as 2-deoxy-glucose (2-DG). Due to its structural similarity to mannose, 2-DG also interferes with N-linked glycosylation, a co-translational modification that directs protein folding. This obstruction triggers an unfolded protein response (UPR), which kills certain tumor types grown under normoxia, for example pancreatic cancer cell line, 1420 (ATCC Mia Paca-2). Since 2-DG is in clinical trials, we are investigating mechanisms by which tumor cells have acquired or are intrinsically resistant to 2-DG. By continuous 2-DG treatment of 1420 cells, we isolated a two-fold resistant variant cell line, 14DG2, and compared it to pancreatic cell line, 1469 (ATCC Panc-1), that intrinsically displays resistance to 2-DG. 2-DG has often been compared to and used as a substitute for glucose deprivation which can also lead to UPR. Glucose deprivation is common in tumor development since cells can outgrow the nutrient supply and may lack functional vasculature. Thus, we investigated the effects of glucose starvation on the same cell lines and compared our results to those where 2-DG induced UPR. Results: 2-DG resistant cell lines, 1469 and 14DG2, display greater sensitivity to glucose deprivation than 2-DG sensitive cell line 1420 which indicates that 2-DG treatment is not an adequate substitute for glucose deprivation. The resistant cell lines show lower protein levels of glucose transporters and/or hexokinase II, which correlates with their decreased uptake of radioactively-labeled 2-DG. We can deduce that these cells also take up less glucose and this may be the reason for their increased sensitivity to glucose starvation conditions. 2-DG resistant cell lines display higher basal levels of UPR folding chaperone Grp78 when measured by western blot. Using siRNA or versipelostatin to inhibit Grp78, a dramatic increase in 2-DG cytotoxicity in the two resistant cell lines was observed suggesting that the resistant cell lines are prepared to cope with ER stress more so than the sensitive cell line. Interestingly, these cell lines also showed resistance to tunicamycin but not to other ER stressors i.e. thapsigargin, brefeldin and velcade. Thus, it appears that 2-DG resistance in these cell lines is involved with processing of oligosaccharide and/or glycosylation interference. Conclusions: The manner in which glucose is restricted in tumor cells, i.e. 2-DG treatment or glucose deprivation, dictates resistance to these two different conditions. Resistance to 2-DG, which may arise in the clinical use of this sugar analog, can be overcome by reducing the effectiveness of the unfolded protein response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4064. doi:10.1158/1538-7445.AM2011-4064

Abstract 4054 The inappropriate activation of oncogenes can result in the up-regulation of pro-apoptotic signals often in the form of BH3-only proteins such as Bim, Noxa or Puma. This renders cells dependent on anti-apoptotic proteins including BCL-2, BCL-xL and MCL-1. Thus cancer cells would be predicted to be more susceptible to inhibition of BCL-2 family proteins, prompting the development and testing of several small molecule inhibitors of this class of proteins. Multiple myeloma is a plasma cell malignancy of the bone marrow and like normal plasma cells, myeloma plasma cells express MCL-1. A search of gene expression profile data from normal plasma cells (n=22), MGUS plasma cells (n=12), plasma cells from patients with asymptomatic (smoldering) myeloma (n=44) or newly diagnosed multiple myeloma (n=538) revealed no significant difference in MCL-1 mRNA expression associated with progression of disease. We then determined MCL-1 dependence through the introduction of siRNA in 4 MM cell lines and consistent with previous findings using anti-sense oligonucleotides in additional lines, we demonstrated that all cell lines tested were MCL-1 dependent. However using the BCL-2/BCL-xL/BCL-w-selective inhibitor ABT-737 we found that 3 of the 6 MCL1-dependent cell lines tested were sensitive (IC50 for Annexin V-FITC positive at 24 hrs of 300 – 600 nM) and therefore also dependent on BCL-xL/BCL-2. Taken together this is the first formal demonstration that cells can be co-dependent on multiple Bcl-2 family members. We have previously reported that ABT-737 sensitivity, and what we now refer to as co-dependence on MCL-1 and BCL-xL/BCL-2, is determined by the distribution of BIM on the anti-apoptotic BCL-2 proteins in these cells. We have now expanded these findings to patient samples that displayed sensitivity to ABT-737 that is similar to what we have observed in the co-dependent cell line MM.1s. Consistent with these findings co-immunoprecipation revealed BIM binding predominantly to BCL-xL. Additionally we have now demonstrated that BIM binding is not simply controlled by the expression levels of BCL-xL or MCL-1 as enforced over-expression of each protein could alter the sensitivity of co-dependent cell lines to ABT-737 but did not alter the initial distribution of BIM amongst these proteins. These data suggest that additional factors regulate the association of BIM with anti-apoptotic BCL-2 proteins. These factors could include differences in cellular localization of these proteins as well as differences in post-translational modifications of either pro- or anti-apoptotic BCL-2 family proteins. Disclosures: Boise: University of Chicago: Patents & Royalties.

Based on emerging evidence that oncogenes direct increased glucose metabolism in tumor cells, the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG) appears to be a promising drug to selectively target cancer cells. 2-DG however, not only blocks glycolysis and reduces cellular ATP production but also interferes with N-linked glycosylation, thereby inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Both bioenergetic challenge and ER stress/UPR have been shown to activate autophagy, a bulk cellular degradation process that plays either a pro- or anti-death role. This led us to investigate the following, 1) which pathway that 2-DG interferes with is responsible for stimulating autophagy, and 2) the role of autophagy in modulating 2-DG-induced cellular responses and toxicity. In four cancer cell lines (1420, MDA-MB-435, SKBR3 and T98G) we find that 2-DG upregulates autophagy (LC3B II), increases the ER stress/UPR markers, Grp78 and CHOP, and lowers ATP levels. Addition of exogenous mannose reverses 2-DG-induced autophagy and expression of GRP78 and CHOP but does not recover the lowered levels of ATP. Additionally, when autophagy is either blocked or enhanced by 3-methyladenine (3-MA) or rapamycin, respectively, 3-MA increases whereas rapamycin reduces cancer cell sensitivity to 2-DG. In support of these results, knock down of autophagy-related gene 7 (Atg7) with small interfering RNAs (siRNAs) increases sensitivity to 2-DG in 1420 cells, which correlates with increases in the ER stress/UPR markers as well as the apoptotic marker, cleaved caspase 3. Overall, these results indicate that the major mechanism by which 2-DG stimulates autophagy is through ER stress/UPR, and autophagy protects cells from 2-DG-induced toxicity through relief of ER stress. Thus, combining autophagy inhibitors with 2-DG may be useful clinically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4840.

Abstract 2851 Poster Board II-827 ABT-737 and its orally active analog ABT-263 are Bcl-2-family inhibitors that are currently in clinical trials for a variety of cancers including hematological malignancies such as multiple myeloma. Previously, we reported that the sensitivity of multiple myeloma cell lines to ABT-737 correlates with the interactions, but not the expression, of Bcl-2 proteins. Analysis of 6 multiple myeloma cell lines revealed that expression of Bcl-2 proteins did not correlate with sensitivity, however the sensitive cells (8226/S, MM.1S and KMS-11) have a substantial amount of their pro-apoptotic Bcl-2 protein, Bak, bound to Bcl-xL. On the other hand, in the insensitive cell lines (U266, KMS-11 and OPM2), Bak was found to be associated with Mcl-1, a family member that does not bind ABT-737 and thereby confers resistance to this drug. Furthermore, we also showed that release of the BH3-only protein Bim by ABT-737 from Bcl-xL and Bcl-2 also contributes to cell death in 8226/S and MM.1S. The purpose of the current study is to further investigate the role of Bim in ABT-737-induced cell death in the multiple myeloma lines. Similar to Bak, a substantial amount of Bim is bound to Bcl-xL and Bcl-2 in the ABT-737-sensitive cell lines, MM.1S and KMS-18, while in the insensitive cell lines, it is highly bound to Mcl-1. Surprisingly, in the ABT-737-sensitive 8226/S cells, Bim appears to bind to Mcl-1. However in these cells, ABT-737 treatment resulted in upregulation of Noxa, which is a BH3-only protein that binds Mcl-1 and can release Bim. Taken together these data suggest that although binding of Bim to Mcl-1 may confer resistance to ABT-737, in certain cell types this treatment could also induce Noxa expression that antagonizes Mcl-1-mediated resistance. Consistent with this hypothesis, Mcl-1 overexpression as well as knockdown of Noxa expression significantly protected 8226/S cells from ABT-737-induced cell death while they had no effect in MM.1S cells. To further demonstrate the role of Bim in ABT-737-induced cell death, ABT-resistant 8226/S, KMS-11, KMS-18 and U266 cell lines were generated. In the resistant cell lines of 8226/S and KMS-18, Bim is exclusively bound to Mcl-1, which was overexpressed as compared to the parental cells. Bak binding was not affected by acquisition of ABT-737 resistance. This result is in agreement with the findings that interaction of Bim and Mcl-1 confers resistance to ABT-737. On the other hand, in ABT-resistant U266 and KMS-11 cell lines, Bim expression was down-regulated while Mcl-1 levels were not changed. Thus, it appears that in cells where Bim is already bound to Mcl-1, further resistance is achieved by down-regulating the expression of this BH3-only protein. Overall, these results suggest that the complex interactions between Bcl-2 proteins need to be investigated in order to understand how multiple myeloma cells may respond to ABT-737 treatment. Disclosures: Boise: University of Chicago: Patents & Royalties.