Brad Poore

Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer.

Significance There are no effective therapies currently available for advanced pancreatic cancer. We show that there are two populations of cancer cells within a pancreatic tumor that require targeting by different metabolic inhibitors for effective tumor control. Rapidly dividing cells use glutamine, and can be effectively killed by administration of a nanoparticle containing an inhibitor of glutamine metabolism. Hypoxic cells, which are slowly dividing cells, metabolize glucose and can be targeted by metformin, a drug used for the treatment of diabetes. Clinical trials are needed to determine whether combination therapy, with drugs that effectively block the metabolism of glutamine and glucose, improves the survival of patients with pancreatic cancer. Targeting glutamine metabolism via pharmacological inhibition of glutaminase has been translated into clinical trials as a novel cancer therapy, but available drugs lack optimal safety and efficacy. In this study, we used a proprietary emulsification process to encapsulate bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a selective but relatively insoluble glutaminase inhibitor, in nanoparticles. BPTES nanoparticles demonstrated improved pharmacokinetics and efficacy compared with unencapsulated BPTES. In addition, BPTES nanoparticles had no effect on the plasma levels of liver enzymes in contrast to CB-839, a glutaminase inhibitor that is currently in clinical trials. In a mouse model using orthotopic transplantation of patient-derived pancreatic tumor tissue, BPTES nanoparticle monotherapy led to modest antitumor effects. Using the HypoxCR reporter in vivo, we found that glutaminase inhibition reduced tumor growth by specifically targeting proliferating cancer cells but did not affect hypoxic, noncycling cells. Metabolomics analyses revealed that surviving tumor cells following glutaminase inhibition were reliant on glycolysis and glycogen synthesis. Based on these findings, metformin was selected for combination therapy with BPTES nanoparticles, which resulted in significantly greater pancreatic tumor reduction than either treatment alone. Thus, targeting of multiple metabolic pathways, including effective inhibition of glutaminase by nanoparticle drug delivery, holds promise as a novel therapy for pancreatic cancer.

Divergent paths for the selection of immunodominant epitopes from distinct antigenic sources

Immunodominant epitopes are few selected epitopes from complex antigens that initiate T cell responses. Here, to provide further insights into this process, we use a reductionist cell-free antigen processing system composed of defined components. We use the system to characterize steps in antigen processing of pathogen-derived proteins or autoantigens and we find distinct paths for peptide processing and selection. Autoantigen-derived immunodominant epitopes are resistant to digestion by cathepsins, whereas pathogen-derived epitopes are sensitive. Sensitivity to cathepsins enforces capture of pathogen-derived epitopes by Major Histocompatibility Complex class II (MHC class II) prior to processing, and resistance to HLA-DM-mediated-dissociation preserves the longevity of those epitopes. We show that immunodominance is established by higher relative abundance of the selected epitopes, which survive cathepsin digestion either by binding to MHC class II and resisting DM-mediated-dissociation, or being chemically resistant to cathepsins degradation. Non-dominant epitopes are sensitive to both DM and cathepsins and are destroyed.

Allosteric Glutaminase Inhibitors Based on a 1,4-Di(5-amino-1,3,4-thiadiazol-2-yl)butane Scaffold

A series of allosteric kidney-type glutaminase (GLS) inhibitors were designed and synthesized using 1,4-di(5-amino-1,3,4-thiadiazol-2-yl)butane as a core scaffold. A variety of modified phenylacetyl groups were incorporated into the 5-amino group of the two thiadiazole rings in an attempt to facilitate additional binding interactions with the allosteric binding site of GLS. Among the newly synthesized compounds, 4-hydroxy-N-[5-[4-[5-[(2-phenylacetyl)amino]-1,3,4-thiadiazol-2-yl]butyl]-1,3,4-thiadiazol-2-yl]-benzeneacetamide, 2m, potently inhibited GLS with an IC50 value of 70 nM, although it did not exhibit time-dependency as seen with CB-839. Antiproliferative effects of 2m on human breast cancer lines will be also presented in comparison with those observed with CB-839.

mTORC1 inhibition in pediatric low-grade glioma depletes glutathione and therapeutically synergizes with carboplatin

BACKGROUND Pediatric low-grade glioma (pLGG) often initially responds to front-line therapies such as carboplatin, but more than 50% of treated tumors eventually progress and require additional therapy. With the discovery that pLGG often contains mammalian target of rapamycin (mTOR) activation, new treatment modalities and combinations are now possible for patients. The purpose of this study was to determine if carboplatin is synergistic with the mTOR complex 1 inhibitor everolimus in pLGG. METHODS We treated 4 pLGG cell lines and 1 patient-derived xenograft line representing various pLGG genotypes, including neurofibromatosis type 1 loss, proto-oncogene B-Raf (BRAF)-KIAA1549 fusion, and BRAFV600E mutation, with carboplatin and/or everolimus and performed assays for growth, cell proliferation, and cell death. Immunohistochemistry as well as in vivo and in vitro metabolomics studies were also performed. RESULTS Carboplatin synergized with everolimus in all of our 4 pLGG cell lines (combination index <1 at Fa 0.5). Combination therapy was superior at inhibiting tumor growth in vivo. Combination treatment increased levels of apoptosis as well as gamma-H2AX phosphorylation compared with either agent alone. Everolimus treatment suppressed the conversion of glutamine and glutamate into glutathione both in vitro and in vivo. Exogenous glutathione reversed the effects of carboplatin and everolimus. CONCLUSIONS The combination of carboplatin and everolimus was effective at inducing cell death and slowing tumor growth in pLGG models. Everolimus decreased the amount of available glutathione inside the cell, preventing the detoxification of carboplatin and inducing increased DNA damage and apoptosis.

Abstract 3549: C-MYC sensitizes GBM with primitive features to glutamine metabolism disruption

Glioblastoma (GBM) is among the most lethal of known human cancers, with a median survival of less than 15 months. The highly infiltrative nature and genetic heterogeneity of GBM renders treatment difficult. Therefore, better and more targeted therapies are needed for patients with GBM. There is a new WHO subset of GBM that contains primitive neuronal components (GBM-PNC). These tumors can arise from a histologically classic GBM, and often the GBM-PNC portions of the tumor contain C-MYC or N-MYC amplifications. High MYC expression is known to alter cellular metabolism, increasing reliance on glutamine, which may create opportunities for therapeutic intervention. We hypothesized that depriving GBM-PNC cells of glutamine using metabolic inhibitors would suppress growth and tumorigenicity. To create genetically appropriate GBM-PNC models, we derived cortex (CTX) human neural stem cells and transformed them through lentiviral expression of mutant p53, constitutively-active AKT and hTERT. Transformed neurospheres were then lentivirally transduced with either C-MYC or BMI1. These models formed aggressive tumors in mice and recapitulated the histological features of GBM with expression of NESTIN, GFAP, and MAP2. When treated with the glutamine metabolic inhibitors DON or Acivicin, transformed neurospheres that expressed C-MYC had decreased cellular proliferation (BrdU incorporation, P Citation Format: Brad Andrew Poore, Isabella Taylor, Jeffrey Rubens, Allison Hanaford, Micah Maxwell, Charles Eberhart, Eric Raabe. C-MYC sensitizes GBM with primitive features to glutamine metabolism disruption [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3549. doi:10.1158/1538-7445.AM2017-3549

Glucose-Independent Glutamine-Driven TCA Cycle in Cancer Cells

Although genetic alterations are ultimately believed to cause cancer, they can also lead to dysregulation of genes encoding metabolic enzymes that enables cancer cells to proliferate and metastasize. Even though the tricarboxylic acid (TCA) cycle is considered an oxidation pathway for glucose metabolism, Le et al. have demonstrated otherwise. Using stable isotope-resolved metabolomics to resolve 13C and 15N from labeled glucose or glutamine, they found that B-cell cancers maintain their TCA cycle solely relying on glutamine when glucose is withdrawn. This glucose-independent TCA cycle allows cancer cells to maintain its bioenergetics, catabolic, and anabolic needs under glucose limited conditions. The effect is so profound that certain cancer cells become addicted and cannot proliferate without glutamine, even in the presence of glucose. Due to glutamine’s emerging role in cancer, targeting the glutaminolysis pathway is a promising new approach to cancer therapy. Moreover, the results of their research demonstrated that inhibition of glutaminase, an enzyme that converts glutamine to glutamate, slows B-cell cancer growth. The ability of B-cell cancer cells to reprogram their metabolism by using glutamine instead of glucose to adapt to the nutrient availability in the tumor microenvironment confers a selective advantage for cancer cell survival and proliferation. This knowledge gives researchers a critical means by which to exploit the metabolic adaptations of these cancer cells and develop new cancer therapies.

Abstract 1183: Metabolic characterization of follicular lymphoma transformation

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Follicular lymphoma (FL), a form of non-Hodgkin lymphoma, is the second most common form of B-cell lymphoma and remains incurable in the majority of cases despite recent advances in therapy. Following an indolent phase, 50% of patients suffer disease transformation to an aggressive form of lymphoma (transformed FL; tFL). This dramatic switch in disease behavior typically culminates in rapid deterioration and patient demise. Accordingly, much effort has been focused on understanding the genetics of transformation and has resulted in the identification of key genetic lesions (e.g. MYC activation, loss of p53, cell cycle controls, activation of NFKB (TNFAIp3/A20)). However, exactly how tumor metabolism, which is altered by these genetic lesions, contributes to disease aggressiveness is not known and therefore the metabolic changes that occur upon FL transformation are poorly understood. We have developed a murine model of FL transformation that recapitulates key genetic and pathological aspects of human FL (Oricchio et al, 2011). This model allows us to study the genetics and metabolism of FL transformation. Specifically, we will systematically define the metabolic profiles of genetically defined murine lymphomas that represent different stages of transformation and cross-compare metabolic changes to primary patient samples. This ensures focus on clinically relevant changes and provides metabolic annotation of distinct tumor genotypes. This study builds on the complementary strengths of the Le lab (Johns Hopkins) in cancer metabolism and the Wendel lab (MSKCC) in modeling FL genetics. Our study shows that MYC transformed FL exhibits a distinct metabolic profile as compared to indolent follicular lymphoma or normal spleen. The most altered pathways examined thus far are glycolysis and amino acid metabolism. We believe that the transformed lymphoma is more reliant upon glycolysis and may be exhibiting aerobic glycolysis, also known as the Warburg Effect. This is substantiated from previous experiments in which a Burkitt lymphoma line, also overexpressing MYC, was found to have altered glycolytic intermediates levels. We next plan to examine the metabolic profile of other transformed follicular lymphoma models of known genetic backgrounds. We will also cross validate the transformed mouse models with follicular lymphoma patient samples of known genetic backgrounds to identify potential metabolic markers. Citation Format: Brad Poore, Ana Ortega-Molina, Christopher Nguyen, Liang Zhao, Anne Blackwell, Thomas Hartung, Hans-Guido Wendel, Anne Le. Metabolic characterization of follicular lymphoma transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1183. doi:10.1158/1538-7445.AM2015-1183

Abstract 852: Novel role and mechanism of the LDHA protein as a survival factor for cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Rationale: Lactate dehydrogenase A (LDHA), which catalyzes the conversion of pyruvate to lactate, serves as a poor prognostic serum marker for many different types of human cancers; however, it is not known whether this is simply the result of LDHA being released from dying cancer cells in patients with high tumor burden or whether LDHA could also be a signaling molecule that portends poor clinical outcome. Based on the observation that extracellular LDHA was identified as the key survival factor for cardiomyocytes exposed to oxidative stress, we sought to determine whether extracellular LDHA can act as a cancer cell survival factor and hence contributes to poor clinical outcome. We reasoned that LDHA released from the damaged cells could activate intracellular signaling pathways in surrounding tumor cells by means of membrane receptors, ultimately protecting those surrounding tumor cells from oxidative stress-induced cell death. Methods: To investigate our hypothesis, recombinant LDHA and its isoform LDHB was produced by transforming E. coli strain BL21(DE3) with pET-SUMO expression vector (Clontech) containing full-length human LDHA or LDHB cDNA. The resulting recombinant proteins were purified using TALON Metal Affinity Resin and used for 16h pre-treatment of Ramos human Burkitts lymphoma and A6L human pancreatic cancer cells cultured in full serum containing medium. The amount of recombinant proteins relative to serum albumin is estimated to be less 1% and hence any effects of LDHA could not be simply due to non-specific protein concentration effect. Following the pre-treatment procedure, these cancer cells were subjected to oxidative stress induced by hydrogen peroxide (H2O2) at 100 μM for 24h. Cells were then counted in a hemacytometer using trypan blue dye to exclude dead cells. Results: We observed that while LDHB had no effect on cell death induced by H2O2 as compared to the non-pretreatment group, LDHA can act as a protective factor against oxidative stress induced by H2O2, and ultimately cell death. We also investigated the mechanism involved in the protective role of LDHA by using recombinant LDHA as a molecular probe against membrane proteins that were extracted from cancer cells. Conclusion: Our study defines the novel role and mechanism of LDHA protein as a survival factor in cancer cells, in addition to its enzymatic activity, which may explain the prognostic significance of extracellular LDHA. Furthermore, this study can lead to the identification of new membrane receptor(s) for LDHA which may provide new targets for cancer therapy. Citation Format: Brad Poore, Joshua Kee Park, Iman Afif, Laura Shingleton, Chi Van Dang, Anne Le. Novel role and mechanism of the LDHA protein as a survival factor for cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 852. doi:10.1158/1538-7445.AM2013-852