A precision therapeutic strategy for hexokinase 1-null, hexokinase 2-positive cancers.

BACKGROUND: Precision medicine therapies require identification of unique molecular cancer characteristics. Hexokinase (HK) activity has been proposed as a therapeutic target; however, different hexokinase isoforms have not been well characterized as alternative targets. While HK2 is highly expressed in the majority of cancers, cancer subtypes with differential HK1 and HK2 expression have not been characterized for their sensitivities to HK2 silencing. METHODS: HK1 and HK2 expression in the Cancer Cell Line Encyclopedia dataset was analyzed. A doxycycline-inducible shRNA silencing system was used to examine the effect of HK2 knockdown in cultured cells and in xenograft models of HK1-HK2+ and HK1+HK2+ cancers. Glucose consumption and lactate production rates were measured to monitor HK activity in cell culture, and 18F-FDG PET/CT was used to monitor HK activity in xenograft tumors. A high-throughput screen was performed to search for synthetically lethal compounds in combination with HK2 inhibition in HK1-HK2+ liver cancer cells, and a combination therapy for liver cancers with this phenotype was developed. A metabolomic analysis was performed to examine changes in cellular energy levels and key metabolites in HK1-HK2+ cells treated with this combination therapy. The CRISPR Cas9 method was used to establish isogenic HK1+HK2+ and HK1-HK2+ cell lines to evaluate HK1-HK2+ cancer cell sensitivity to the combination therapy. RESULTS: Most tumors express both HK1 and HK2, and subsets of cancers from a wide variety of tissues of origin express only HK2. Unlike HK1+HK2+ cancers, HK1-HK2+ cancers are sensitive to HK2 silencing-induced cytostasis. Synthetic lethality was achieved in HK1-HK2+ liver cancer cells, by the combination of DPI, a mitochondrial complex I inhibitor, and HK2 inhibition, in HK1-HK2+ liver cancer cells. Perhexiline, a fatty acid oxidation inhibitor, further sensitizes HK1-HK2+ liver cancer cells to the complex I/HK2-targeted therapeutic combination. Although HK1+HK2+ lung cancer H460 cells are resistant to this therapeutic combination, isogenic HK1KOHK2+ cells are sensitive to this therapy. CONCLUSIONS: The HK1-HK2+ cancer subsets exist among a wide variety of cancer types. Selective inhibition of the HK1-HK2+ cancer cell-specific energy production pathways (HK2-driven glycolysis, oxidative phosphorylation and fatty acid oxidation), due to the unique presence of only the HK2 isoform, appears promising to treat HK1-HK2+ cancers. This therapeutic strategy will likely be tolerated by most normal tissues, where only HK1 is expressed.

Hexokinase 2 is targetable for HK1 negative, HK2 positive tumors from a wide variety of tissues of origin.

Although absent in most adult tissues, hexokinase 2 (HK2) is expressed in a majority of tumors and contributes to increased glucose consumption and to in vivo tumor 18F-FDG PET signaling. Methods: Both HK2 knockdown and knockout approaches were used to investigate the role of HK2 in cancer cell proliferation, in vivo xenograft tumor progression and 18F-FDG tumor accumulation. BioProfiler analysis monitored cell culture glucose consumption and lactate production; 18F-FDG PET/CT monitored in vivo tumor glucose accumulation. Cancer Cell Line Encyclopedia data were analyzed for HK1 and HK2 expression. Results: Neither cell proliferation in culture nor xenograft tumor progression are inhibited by HK2 knockdown or knockout in cancer cells that express HK1 and HK2. However, cancer subsets from a variety of tissues of origin express only HK2, but not HK1. In contrast to HK1+HK2+ cancers, HK2 knockdown in HK1-HK2+ cancer cells results in inhibition of cell proliferation, colony formation and xenograft tumor progression. Moreover, HK1KOHK2+ cancer cells are susceptible to HK2 inhibition, in contrast to their isogenic HK1+HK2+ parental cells. Conclusion: HK1 and HK2 expression are redundant in tumors; either can provide sufficient aerobic glycolysis for tumor growth; despite a reduction in 18F-FDG PET signal. Therapeutic HK2 inhibition is likely to be restricted to HK1-HK2+ tumor subsets, but stratification of tumors that express HK2, but not HK1, should identify tumors treatable with emerging HK2 specific inhibitors.

Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo.

UNLABELLED: Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1(HEP) mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1(HEP) mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1(HEP) mice. FX remained essential for Ad5 transduction in vivo in Ext1(HEP) mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study reopens the question of how adenovirus enters cells in vivo. IMPORTANCE: Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These results reopen the question of the identity of the Ad5 receptor in vivo and emphasize the necessity of demonstrating the nature of the receptor by genetic means, both for understanding Ad5 entry into cells in vivo and for optimization of Ad5 vectors as therapeutic agents.

Reversible suppression of cyclooxygenase 2 (COX-2) expression in vivo by inducible RNA interference.

Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), plays a critical role in many normal physiological functions and modulates a variety of pathological conditions. The ability to turn endogenous COX-2 on and off in a reversible fashion, at specific times and in specific cell types, would be a powerful tool in determining its role in many contexts. To achieve this goal, we took advantage of a recently developed RNA interference system in mice. An shRNA targeting the Cox2 mRNA 3'untranslated region was inserted into a microRNA expression cassette, under the control of a tetracycline response element (TRE) promoter. Transgenic mice containing the COX-2-shRNA were crossed with mice encoding a CAG promoter-driven reverse tetracycline transactivator, which activates the TRE promoter in the presence of tetracycline/doxycycline. To facilitate testing the system, we generated a knockin reporter mouse in which the firefly luciferase gene replaces the Cox2 coding region. Cox2 promoter activation in cultured cells from triple transgenic mice containing the luciferase allele, the shRNA and the transactivator transgene resulted in robust luciferase and COX-2 expression that was reversibly down-regulated by doxycycline administration. In vivo, using a skin inflammation-model, both luciferase and COX-2 expression were inhibited over 80% in mice that received doxycycline in their diet, leading to a significant reduction of infiltrating leukocytes. In summary, using inducible RNA interference to target COX-2 expression, we demonstrate potent, reversible Cox2 gene silencing in vivo. This system should provide a valuable tool to analyze cell type-specific roles for COX-2.