Ramani Dinavahi

Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression

As the result of genetic alterations and tumor hypoxia, many cancer cells avidly take up glucose and generate lactate through lactate dehydrogenase A (LDHA), which is encoded by a target gene of c-Myc and hypoxia-inducible factor (HIF-1). Previous studies with reduction of LDHA expression indicate that LDHA is involved in tumor initiation, but its role in tumor maintenance and progression has not been established. Furthermore, how reduction of LDHA expression by interference or antisense RNA inhibits tumorigenesis is not well understood. Here, we report that reduction of LDHA by siRNA or its inhibition by a small-molecule inhibitor (FX11 [3-dihydroxy-6-methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylic acid]) reduced ATP levels and induced significant oxidative stress and cell death that could be partially reversed by the antioxidant N-acetylcysteine. Furthermore, we document that FX11 inhibited the progression of sizable human lymphoma and pancreatic cancer xenografts. When used in combination with the NAD+ synthesis inhibitor FK866, FX11 induced lymphoma regression. Hence, inhibition of LDHA with FX11 is an achievable and tolerable treatment for LDHA-dependent tumors. Our studies document a therapeutical approach to the Warburg effect and demonstrate that oxidative stress and metabolic phenotyping of cancers are critical aspects of cancer biology to consider for the therapeutical targeting of cancer energy metabolism.

Conditional Deletion of c-myc Does Not Impair Liver Regeneration

The oncogene c-myc encodes a transcription factor that has long been considered essential to liver regeneration, the process by which fully differentiated hepatocytes proliferate in an attempt to maintain a normal functional mass in response to hepatic injury. Experimental liver regeneration can be induced upon 70% partial hepatectomy and is accompanied by an increase in c-myc expression accompanying the synchronous entry of remaining hepatocytes into the cell cycle. Because liver regeneration is an essential process for achieving liver homeostasis, therapies directed at reducing MYC expression in hepatocellular carcinoma are fraught with the theoretical possibility of injuring adjacent noncancerous liver cells, thereby restricting the livers normal regenerative response to injury. To determine if intact c-myc is required for liver regeneration, we reduced hepatic c-myc in c-myc(fl/fl) mice using an adenoviral vector that expresses Cre recombinase. Despite a 90% decrease in hepatic expression of c-myc, restoration of liver mass 7 days later was not compromised. Reconstituted liver retained the same decrease in hepatic c-myc, indicating that hepatocytes deficient in c-myc were able to proliferate in response to partial hepatectomy. Although c-myc is required for embryonic development, our findings indicate that it is not required for the maintenance of the adult liver.

Abstract 2957: Inhibition of glutaminase induces slows tumor growth cell autonomously and promotes survival in a MYC driven hepatocellular carcinoma mouse model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Glutamine catabolism has been shown to be critical in many cancer types, particularly in MYC dependent tumor types. Given that MYC dependent cell lines have been shown to be dependent on glutamine for growth and survival, we hypothesize that inhibiting glutamine metabolism could slow the growth of MYC driven tumors in vivo. Small molecule (BPTES) inhibition of kidney-type glutaminase (GLS) is being developed as an anti-cancer therapy, yet basic questions remained unanswered about glutamine inhibition. The BPTES insensitive liver-type glutaminase (GLS2) has been proposed to act as a tumor suppressor in liver cancer cell lines. We have found that hepatocellular carcinomas downregulate GLS2 and upregulate GLS compared to surrounding tissue in both humans and mice, leaving hepatocellular carcinomas potentially sensitive to GLS inhibition. BPTES slows the growth of hepatocellular carcinoma cell lines in vitro. Here were report the first study that demonstrates the ability of BPTES to slow tumor progression in genetic cancer models in immunocompetent mice, showing the BPTES prolongs survival of mice in a MYC driven hepatocellular carcinoma model. However, we could not determine from these data if the anti-cancer effects of GLS inhibition in vivo were systemic or cell autonomous, causing us to switch to a xenograft system. Using prostate and lymphoma cell lines overexpressing the BPTES resistant GLS K325A mutant, we show that BPTES functions through on-target inhibition of GLS in vitro. We then used a p493-6 B-cell lymphoma cell line to determine the cell autonomous effects of BPTES in vivo. P493-6 xenograft growth was slowed by BPTES treatment, while p493 xenografts overexpressing either wild type GLS or BPTES resistant GLS K325A were not slowed by BPTES treatment in vivo. We then set out to develop an alternative method to perform tumor specific knock down GLS in vivo to obtain genetic evidence of the role of GLS in tumor cells, developing a human specific vivo-morpholino that targets GLS for nonsense-mediated decay. Consistent with the effect of BPTES on tumor cells, the human GLS targeting vivo-morpholino slowed p493-6 xenograft growth in vivo. As the vivo-morpholino knockdown of GLS was xenograft specific, it demonstrates that GLS inhibition slows cancer growth through cancer cell autonomous methods and not though systemic changes that alter the tumor niche. In summary, we find that BPTES exhibits on target GLS inhibition in vivo to prolong survival in mouse hepatocellular carcinoma mouse models. Citation Format: Zachary Stine, Yan Xiang, Jinsong Xia, Ping Gao, Ramani Dinavahi, Chi V. Dang. Inhibition of glutaminase induces slows tumor growth cell autonomously and promotes survival in a MYC driven hepatocellular carcinoma mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2957. doi:10.1158/1538-7445.AM2014-2957

Abstract 2799: Unexpected sustained hypoxic glutamine metabolism and therapeutic opportunities

Rationale: As the result of genetic alterations and tumor hypoxia, cancer cells re-program metabolism to meet increased energy demand for enhanced anabolism, cell proliferation, and protection from oxidative damage and cell death signals. Whereas glycolysis and its regulation by the MYC oncogene product (Myc) have been intensively studied, glutaminolysis and anapleurosis, especially under hypoxia, have not been defined. Objectives: We sought to determine whether glutamine, a key source of nitrogen and anabolic carbon skeleton for mammalian cells, is still utilized under hypoxic condition and how it is regulated by MYC. Methods: We used NMR and FT-ICR-MS to resolve 13 C and 15 N labeling patterns of metabolites derived from labeled glutamine or glucose in the P493 human B lymphoma model that bear a tetracycline-repressible MYC, under normoxic and hypoxic conditions. Levels of specific enzymes, which are involved in glucose and glutamine metabolism, were determined by immunoblotting and LC-MRM-MS and compared with the metabolomic profiles. Measurements and Main Results: Myc regulates almost the entire glycolytic pathway. As expected for hypoxia, many glycolytic enzymes levels and lactate production were elevated, and 13 C- glucose catabolism by the TCA cycle was significantly diminished. Myc elevated glutaminase (GLS, which converts glutamine to glutamate and NH 4 + ), the glutamine transporter SLC1A5, and the transaminases GOT1 and GPT2, suggesting that transamination is favored with Myc activation. While GLS-mediated NH 4 + release persisted under hypoxia, the Myc-induced transaminases favor the production of α-ketoglutarate without additional NH 4 + production. The 13 C labeling pattern of Ala is consistent with active mitochondrial GPT2 that transaminates 13 C-pyruvate derived from 13 C- glucose. Given that mitochondrial function is generally believed to be diminished by hypoxia, glutamine metabolism unexpectedly persisted in hypoxia with continued oxidation of glutamine carbons by the TCA cycle. The key enzymes and main products of 13 C labeled glutamine were not decreased by hypoxia, and glutamine contributed to substantial de novo glutathione production in hypoxia. Because glutamine metabolism is sustained in hypoxia, which is pervasive in the tumor microenvironment, we sought to target glutaminase in vivo. We found that small molecule inhibition of glutaminase could diminish lymphoma and pancreatic cancer xenograft growth in vivo. Conclusions: Our studies reveal a previously unsuspected sustained glutamine metabolism in hypoxia. Glutamine contributes to TCA cycle carbons as well as enhances glutathione synthesis in hypoxia, suggesting that cancer cells reprogram metabolism via both glucose and glutamine to adapt to the tumor microenvironment. Our findings indicate that key nodal points in glutamine metabolism in addition to those in glycolysis could be targeted for therapy. 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 2799. doi:10.1158/1538-7445.AM2011-2799