Mitochondrial dysfunction is an underlying pathology in numerous diseases. Delivery of diagnostic and therapeutic cargo directly into mitochondria is a powerful approach to study and treat these diseases. The triphenylphosphonium (TPP+) moiety is the most widely used mitochondriotropic carrier. However, studies have shown that TPP+ is not inert; TPP+ conjugates uncouple mitochondrial oxidative phosphorylation. To date, all efforts toward addressing this problem have focused on modifying lipophilicity of TPP+-linker-cargo conjugates to alter mitochondrial uptake, albeit with limited success. We show that structural modifications to the TPP+ phenyl rings that decrease electron density on the phosphorus atom can abrogate uncoupling activity as compared to the parent TPP+ moiety and prevent dissipation of mitochondrial membrane potential. These alterations of the TPP+ structure do not negatively affect the delivery of cargo to mitochondria. Results here identify the 4-CF3-phenyl TPP+ moiety as an inert mitochondria-targeting carrier to safely target pharmacophores and probes to mitochondria.

The chronically proliferative cancer phenotype requires metabolic reprogramming to meet the increased energy and biosynthetic demands of rapid cell division. This results in increased glucose uptake and usage (the Warburg effect). Cancer cells that undergo the Warburg effect may also become reliant on glutamine, a term called ‘glutamine addiction’. Recent studies in ovarian cancer suggest that highly invasive ovarian cancer cells show a remarkable dependence on glutamine, hence implicating glutamine metabolism in metastasis. Glutaminase is the first enzyme that is involved in glutaminolysis and it catalyzes the rate-limiting conversion of glutamine to glutamate and ammonia. There are two genes that code for glutaminase in the human genome, glutaminase 1 (GLS1), and glutaminase 2 (GLS2). GLS2 is associated with cell differentiation whereas GLS1 expression is up-regulated in cancer. Hence, GLS1 had been proposed to be better adapted to meet the altered metabolic needs of the tumor phenotype. GLS1 can be alternatively spliced into two isoforms, KGA and GAC. Although both KGA and GAC have been implicated in cancer cell metabolism, there is still controversy over the actual isoform that is most important for tumorigenesis. Whereas the KGA isoform is repressed by miR-23, there is no documented miRNA repression of GAC suggesting that GAC is more tumorigenic. Using next-generation sequencing, we recently identified an unannotated novel KGA transcript with a truncated 3′ untranslated region (3′UTR) which is more stable than the traditionally known KGA transcript. Our goal is to determine the expression profile of different glutaminase isoforms in the highly lethal gynecological malignancy, ovarian cancer, and their role in tumorigenesis and metastasis. We have developed amplicons to measure total GLS1 and GLS2 transcripts using quantitative real time PCR (qRT-PCR). Although we unexpectedly detected both GLS2 and GLS1 transcripts, the mRNA levels of GLS2 were significantly lower than those of GLS1. To determine the specific GLS1 isoforms expressed we used isoform specific amplicons and detected both GAC and KGA using qRT-PCR. Western blot analysis was able to detect both GAC and KGA protein in one cell line only suggesting that the KGA mRNA we detected was subject to miR-23 repression. Inhibition of total GLS1 activity with a glutaminase inhibitor resulted in decreased colony formation as shown by soft agar assays. In conclusion, the expression profile of different GLS1 isoforms in highly invasive ovarian cancer supports the role of glutamine metabolism in maintaining the metastatic phenotype. This makes glutamine metabolism a viable therapeutic target for metastatic ovarian cancer. Citation Format: Chioniso P. Masamha, Patrick LaFontaine, Bettine E. Gibbs. Deciphering the dynamics of alternative pre-mRNA processing of glutaminase in metastatic ovarian cancer [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 5418. doi:10.1158/1538-7445.AM2017-5418

Mantle Cell Lymphoma (MCL) is an aggressive B‐cell non‐Hodgkin's lymphoma (NHL) subtype that accounts for 7% of all NHL cases. Upon relapse, standard salvage regimens are ineffective and the disease is considered clinically incurable. Hence, novel therapeutic strategies that effectively target deregulated pathways that contribute to MCL pathogenesis are warranted. The hallmark of MCL is a chromosomal translocation t (11:14) event that results in the constitutive expression of the G1‐phase cell cycle regulatory oncogene, cyclin D1 (CCND1), resulting in uncontrolled cell proliferation. Cyclin D1 binds to and activates cyclin dependent kinases 4 and 6 (CDK4/6) and the complex phosphorylates the tumor suppressor gene product retinoblastoma (RB), a negative regulator of the cell cycle. MCL patients whose tumors express cyclin D1 have reduced survival. Uncontrolled cell proliferation results in metabolic alterations of tumor cells to support the energy and biosynthetic needs of unregulated cell growth. Adaptations by malignant cells involve changes in the expression of genes involved in metabolism. This results in both increased glucose and glutamine uptake and metabolism in tumor cells. Recent studies in metabolically reprogrammed tumor cells have shown increased levels of glutaminase, the enzyme involved in the first steps of glutamine metabolism. We hypothesize that molecular targeting of proliferation and glutamine metabolism will induce synergistic tumor cell death. In vitro studies with a MCL cell line were completed using Palbociclib, a potent and selective inhibitor of CDK4/6 which was recently approved by the FDA for the treatment of HR+/HER2− metastatic breast cancer. MTT assays were also carried out using BPTES which inhibits enzymatic activity by binding to the glutaminase tetramer, stabilizing it in an inactive conformation. Cell viability assays show that treatment of MCL cells with Palbociclib resulted in a dose dependent reduction in cell viability. Treatment with 5μM BPTES alone resulted in a slight reduction in cell viability. However, treatment with 5μM BPTES sensitizes cells to treatment with different concentrations of Palbociclib. Studies are ongoing using different concentrations of BPTES with a series of concentrations of Palbociclib to determine the synergistic effects of the combination treatment. As studies continue, quantification of synergy will be relayed using the Chou‐Talalay method with computerized simulation. Preliminary cell cycle assays using flow cytometry show that treatment of cells with Palbociclib induce G1‐phase cell cycle arrest. Establishing a treatment regimen that achieves a synergistic therapeutic effect may allow for integration of a novel molecular targeted approach to treating MCL.Support or Funding InformationButler University Holcomb research award, AFPE Gateway to Research awardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.