Samuel K. McBrayer

Multiple myeloma exhibits novel dependence on GLUT4, GLUT8, and GLUT11: implications for glucose transporter-directed therapy

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.

Targeting Glucose Consumption and Autophagy in Myeloma with the Novel Nucleoside Analogue 8-Aminoadenosine

Multiple myeloma, an incurable plasma cell malignancy, is characterized by altered cellular metabolism and resistance to apoptosis. Recent connections between glucose metabolism and resistance to apoptosis provide a compelling rationale for targeting metabolic changes in cancer. In this study, we have examined the ability of the purine analogue 8-aminoadenosine to acutely reduce glucose consumption by regulating localization and expression of key glucose transporters. Myeloma cells counteracted the metabolic stress by activating autophagy. Co-treatment with inhibitors of autophagy results in marked enhancement of cell death. Glucose consumption by drug-resistant myeloma cells was unaffected by 8-aminoadenosine, and accordingly, no activation of autophagy was observed. However, these cells can be sensitized to 8-aminoadenosine under glucose-limiting conditions. The prosurvival autophagic response of myeloma to nutrient deprivation or to nucleoside analogue treatment has not been described previously. This study establishes the potential of metabolic targeting as a broader means to kill and sensitize myeloma and identifies a compound that can achieve this goal.

Developmental Regulation of Mitochondrial Apoptosis by c-Myc Governs Age- and Tissue-Specific Sensitivity to Cancer Therapeutics.

It is not understood why healthy tissues can exhibit varying levels of sensitivity to the same toxic stimuli. Using BH3 profiling, we find that mitochondria of many adult somatic tissues, including brain, heart, and kidneys, are profoundly refractory to pro-apoptotic signaling, leading to cellular resistance to cytotoxic chemotherapies and ionizing radiation. In contrast, mitochondria from these tissues in young mice and humans are primed for apoptosis, predisposing them to undergo cell death in response to genotoxic damage. While expression of the apoptotic protein machinery is nearly absent by adulthood, in young tissues its expression is driven by c-Myc, linking developmental growth to cell death. These differences may explain why pediatric cancer patients have a higher risk of developing treatment-associated toxicities.

Targeting the Warburg effect in hematological malignancies: from PET to therapy.

Purpose of review To highlight key studies providing rationale for and utility in targeting glycolysis for the treatment of hematological malignancies. Recent findings Several therapeutic strategies are capitalizing on the diagnostic utility of 18fluoro-deoxyglucose positron emission tomography that relies on increased glycolysis and glucose utilization in tumor cells. Although aerobic glycolysis was initially proposed by Warburg to be due to mitochondrial impairment, recent studies have shown a preferential switch to glycolysis in tumor cells with functional mitochondria. Increased glucose consumption can be advantageous for a tumor cell through stimulation of cellular biosynthetic, energetic, and pro-survival pathways. We now have a greater appreciation for the utilization of glucose in specific metabolic pathways that in some aspects can be complemented with other nutrients such as glutamine. Targeting glucose consumption for the treatment of hematological malignancies seems to be a promising field that will require characterization of tumor cell specific targets to inhibit glucose uptake and/or glycolysis. It is imperative to further our understanding of the tumor cell metabolome to target cellular bioenergetics in the treatment of cancer. Summary Targeting the glycolytic pathway for the treatment of hematological malignancies has sufficient rationale given the utility of 18fluoro-deoxyglucose positron emission tomography in diagnostic imaging. Further research is required in developing tumor cell specific therapeutics.

Expression and phosphorylation of the AS160_v2 splice variant supports GLUT4 activation and the Warburg effect in multiple myeloma.

BackgroundMultiple 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.FindingsWe 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.ConclusionsThis 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.

Integrative Gene Expression Profiling Reveals G6PD-Mediated Resistance to RNA-Directed Nucleoside Analogues in B-Cell Neoplasms

The nucleoside analogues 8-amino-adenosine and 8-chloro-adenosine have been investigated in the context of B-lineage lymphoid malignancies by our laboratories due to the selective cytotoxicity they exhibit toward multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL) cell lines and primary cells. Encouraging pharmacokinetic and pharmacodynamic properties of 8-chloro-adenosine being documented in an ongoing Phase I trial in CLL provide additional impetus for the study of these promising drugs. In order to foster a deeper understanding of the commonalities between their mechanisms of action and gain insight into specific patient cohorts positioned to achieve maximal benefit from treatment, we devised a novel two-tiered chemoinformatic screen to identify molecular determinants of responsiveness to these compounds. This screen entailed: 1) the elucidation of gene expression patterns highly associated with the anti-tumor activity of 8-chloro-adenosine in the NCI-60 cell line panel, 2) characterization of altered transcript abundances between paired MM and MCL cell lines exhibiting differential susceptibility to 8-amino-adenosine, and 3) integration of the resulting datasets. This approach generated a signature of seven unique genes including G6PD which encodes the rate-determining enzyme of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase. Bioinformatic analysis of primary cell gene expression data demonstrated that G6PD is frequently overexpressed in MM and CLL, highlighting the potential clinical implications of this finding. Utilizing the paired sensitive and resistant MM and MCL cell lines as a model system, we go on to demonstrate through loss-of-function and gain-of-function studies that elevated G6PD expression is necessary to maintain resistance to 8-amino- and 8-chloro-adenosine but insufficient to induce de novo resistance in sensitive cells. Taken together, these results indicate that G6PD activity antagonizes the cytotoxicity of 8-substituted adenosine analogues and suggests that administration of these agents to patients with B-cell malignancies exhibiting normal levels of G6PD expression may be particularly efficacious.

HIF activation causes synthetic lethality between the VHL tumor suppressor and the EZH1 histone methyltransferase.

Dysregulated H3K27 demethylase activity in pVHL-deficient cells increases dependence on the EZH1 H3K27 methyltransferase. Renal cancer’s loss is patients’ gain Clear cell renal cell carcinoma is the most common form of kidney cancer, and it is typically linked to the loss of von Hippel–Lindau protein (pVHL), a tumor suppressor. This loss results in activation of genes that are normally induced by hypoxia, including some histone demethylases. Chakraborty et al. discovered that increased H3K27 demethylase activity renders pVHL-deficient cells hyperdependent on an opposing H3K27 methyltransferase, EZH1, for survival. Inhibition of EZH1 is lethal for these cells. Inactivation of the von Hippel–Lindau tumor suppressor protein (pVHL) is the signature lesion in the most common form of kidney cancer, clear cell renal cell carcinoma (ccRCC). pVHL loss causes the transcriptional activation of hypoxia-inducible factor (HIF) target genes, including many genes that encode histone lysine demethylases. Moreover, chromatin regulators are frequently mutated in this disease. We found that ccRCC displays increased H3K27 acetylation and a shift toward mono- or unmethylated H3K27 caused by an HIF-dependent increase in H3K27 demethylase activity. Using a focused short hairpin RNA library, as well as CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) and a pharmacological inhibitor, we discovered that pVHL-defective ccRCC cells are hyperdependent on the H3K27 methyltransferase EZH1 for survival. Therefore, targeting EZH1 could be therapeutically useful in ccRCC.

Autochthonous tumors driven byRb1loss have an ongoing requirement for the RBP2 histone demethylase

Significance Developing therapeutic strategies for tumors driven by tumor suppressor gene inactivation, as opposed to oncogene activation, represents a significant challenge in oncology. While restoration of tumor suppressor functionality is generally not feasible, inhibiting proteins that act downstream of lost tumor suppressors represents one strategy to overcome this challenge. In this study, we applied this concept to the tumor suppressor gene retinoblastoma 1 (RB1). The RB1 gene product, pRB, associates with the RBP2 histone demethylase and RBP2 is deregulated in RB1-null cancers. Here, we show that genetic ablation of RBP2 in established, autochthonous pRB-defective murine tumors retards their growth and enhances mouse survival. Our findings provide a further rationale for the development and testing of pharmacological RBP2 inhibitors for cancer treatment. Inactivation of the retinoblastoma gene (RB1) product, pRB, is common in many human cancers. Targeting downstream effectors of pRB that are central to tumorigenesis is a promising strategy to block the growth of tumors harboring loss-of-function RB1 mutations. One such effector is retinoblastoma-binding protein 2 (RBP2, also called JARID1A or KDM5A), which encodes an H3K4 demethylase. Binding of pRB to RBP2 has been linked to the ability of pRB to promote senescence and differentiation. Importantly, genetic ablation of RBP2 is sufficient to phenocopy pRB’s ability to induce these cellular changes in cell culture experiments. Moreover, germline Rbp2 deletion significantly impedes tumorigenesis in Rb1+/− mice. The value of RBP2 as a therapeutic target in cancer, however, hinges on whether loss of RBP2 could block the growth of established tumors as opposed to simply delaying their onset. Here we show that conditional, systemic ablation of RBP2 in tumor-bearing Rb1+/− mice is sufficient to slow tumor growth and significantly extend survival without causing obvious toxicity to the host. These findings show that established Rb1-null tumors require RBP2 for growth and further credential RBP2 as a therapeutic target in human cancers driven by RB1 inactivation.

Oncogenic R132 IDH1 Mutations Limit NADPH for De Novo Lipogenesis through (D)2-Hydroxyglutarate Production in Fibrosarcoma Sells

SUMMARY Neomorphic mutations in NADP-dependent isocitrate dehydrogenases (IDH1 and IDH2) contribute to tumorigenesis in several cancers. Although significant research has focused on the hypermethylation phenotypes associated with (D)2-hydroxyglutarate (D2HG) accumulation, the metabolic consequences of these mutations may also provide therapeutic opportunities. Here we apply flux-based approaches to genetically engineered cell lines with an endogenous IDH1 mutation to examine the metabolic impacts of increased D2HG production and altered IDH flux as a function of IDH1 mutation or expression. D2HG synthesis in IDH1-mutant cells consumes NADPH at rates similar to de novo lipogenesis. IDH1-mutant cells exhibit increased dependence on exogenous lipid sources for in vitro growth, as removal of medium lipids slows growth more dramatically in IDH1-mutant cells compared with those expressing wild-type or enzymatically inactive alleles. NADPH regeneration may be limiting for lipogenesis and potentially redox homeostasis in IDH1-mutant cells, highlighting critical links between cellular biosynthesis and redox metabolism.

Abstract 1268: Aberrant plasma membrane localization of GLUT4 sustains the glycolytic phenotype in multiple myeloma

The incurable plasma cell malignancy multiple myeloma (MM) is characterized by the progressive development of chemoresistance, thus warranting the design of new therapeutic strategies tailored to unique molecular mechanisms driving tumor propagation. One aspect of myeloma pathogenesis which has not been exploited therapeutically is an abnormal avidity for glucose. Inhibition of glucose metabolism has broad applicability in cancer and has demonstrated in vitro potency; unfortunately, previous attempts focusing on hexokinase inhibition have been largely unsuccessful. Targeting the upstream process of glucose transport has not been a major area of investigation due to the common association between GLUT1 and cancer and the importance of GLUT1 in many normal tissues. However, the specific glucose transporters responsible for maintaining the glycolytic phenotype in myeloma have not been identified, leaving open the possibility that family members other than GLUT1 play important roles in this cellular context. Therefore, we undertook an unbiased real time PCR-based screen of GLUT gene expression profiles in myeloma cell lines and control B lymphocytes. GLUTs 4, 8, and 11 exhibit widespread overexpression in MM cells with no significant difference noted in GLUT1 levels. Examination of microarray studies of MM patient samples and western blot analyses of MM cell lines and normal B cells confirm the upregulation of GLUT8 and GLUT11 in clinical specimens and at the protein level. However, we did not find further evidence for GLUT4 overexpression. After evaluating the subcellular localization of GLUT4 through microscopy, we determined that a substantial fraction of GLUT4 protein is constitutively mislocalized to the plasma membrane in myeloma cells, an event normally regulated by insulin in non-malignant tissues. Indeed, cell fractionation studies demonstrate a dramatic increase in plasma membrane GLUT4 content in MM cell lines relative to PBMC. Lentiviral transduction of GLUT4-targeted shRNA verifies the functional relevance of GLUT4 activity, as MM cells with reduced GLUT4 expression exhibit dramatic deficiencies in glucose transport and lactate production rates, which leads to growth inhibition and significant cell death. Similar studies with a GLUT1-targeted shRNA reveal comparable trends but less potent effects. Next, we investigated a class of HIV protease inhibitors which elicit off-target inhibitory effects on GLUT4 in vivo. Treatment with these compounds recapitulates the effects of GLUT4 knockdown, thus supporting the relevance of GLUT4 in myeloma. These studies represent the first time a prominent, functional role for GLUT4 has been demonstrated in any cancer and highlight a therapeutic strategy involving the repositioning of an FDA-approved class of drugs to target myeloma cell metabolism. 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 1268. doi:10.1158/1538-7445.AM2011-1268