Chromodomain Helicase DNA-Binding Protein 7 Is Suppressed in the Perinecrotic/Ischemic Microenvironment and Is a Novel Regulator of Glioblastoma Angiogenesis.

Tumorigenic and non-neoplastic tissue injury occurs via the ischemic microenvironment defined by low oxygen, pH, and nutrients due to blood supply malfunction. Ischemic conditions exist within regions of pseudopalisading necrosis, a pathological hallmark of glioblastoma (GBM), the most common primary malignant brain tumor in adults. To recapitulate the physiologic microenvironment found in GBM tumors and tissue injury, we developed an in vitro ischemic model and identified chromodomain helicase DNA-binding protein 7 (CHD7) as a novel ischemia-regulated gene. Point mutations in the CHD7 gene are causal in CHARGE syndrome (a developmental disorder causing coloboma, heart defects, atresia choanae, retardation of growth, and genital and ear anomalies) and interrupt the epigenetic functions of CHD7 in regulating neural stem cell maintenance and development. Using our ischemic system, we observed microenvironment-mediated decreases in CHD7 expression in brain tumor-initiating cells and neural stem cells. Validating our approach, CHD7 was suppressed in the perinecrotic niche of GBM patient and xenograft sections, and an interrogation of patient gene expression datasets determined correlations of low CHD7 with increasing glioma grade and worse patient outcomes. Segregation of GBM by molecular subtype revealed a novel observation that CHD7 expression is elevated in proneural versus mesenchymal GBM. Genetic targeting of CHD7 and subsequent gene ontology analysis of RNA sequencing data indicated angiogenesis as a primary biological function affected by CHD7 expression changes. We validated this finding in tube-formation assays and vessel formation in orthotopic GBM models. Together, our data provide further understanding of molecular responses to ischemia and a novel function of CHD7 in regulating angiogenesis in both neoplastic and non-neoplastic systems. Stem Cells 2019;37:453-462.

Preclinical evaluation of oncolytic δγ(1)34.5 herpes simplex virus expressing interleukin-12 for therapy of breast cancer brain metastases.

The metastasis of breast cancer to the brain and central nervous system (CNS) is a problem of increasing importance. As improving treatments continue to extend patient survival, the incidence of CNS metastases from breast cancer is on the rise. New treatments are needed, as current treatments are limited by deleterious side effects and are generally palliative. We have previously described an oncolytic herpes simplex virus (HSV), designated M002, which lacks both copies of the γ(1)34.5 neurovirulence gene and carries a murine interleukin 12 (IL-12) expression cassette, and have validated its antitumor efficacy in a variety of preclinical models of primary brain tumors. However, M002 has not been yet evaluated for use against metastatic brain tumors. Here, we demonstrate the following: both human breast cancer and murine mammary carcinoma cells support viral replication and IL-12 expression from M002; M002 replicates in and destroys breast cancer cells from a variety of histological subtypes, including "triple-negative" and HER2 overexpressing; M002 improves survival in an immunocompetent model more effectively than does a non-cytokine control virus. Thus, we conclude from this proof-of-principle study that a γ(1)34.5-deleted IL-12 expressing oncolytic HSV may be a potential new therapy for breast cancer brain metastases.

Engineered herpes simplex viruses efficiently infect and kill CD133+ human glioma xenograft cells that express CD111.

Oncolytic herpes simplex viruses (HSV) hold promise for therapy of glioblastoma multiforme (GBM) resistant to traditional therapies. We examined the ability of genetically engineered HSV to infect and kill cells that express CD133, a putative marker of glioma progenitor cells (GPC), to determine if GPC have an inherent therapeutic resistance to HSV. Expression of CD133 and CD111 (nectin-1), the major entry molecule for HSV, was variable in six human glioma xenografts, at initial disaggregation and after tissue culture. Importantly, both CD133+ and CD133- populations of glioma cells expressed CD111 in similar relative proportions in five xenografts, and CD133+ and CD133- glioma cell subpopulations were equally sensitive to killing in vitro by graded dilutions of wild-type HSV-1(F) or several different gamma(1)34.5-deleted viruses. GPC did not display an inherent resistance to HSV. While CD111 expression was an important factor for determining sensitivity of glioma cells to HSV oncolysis, it was not the only factor. Our findings support the notion that HSV will not be able to effectively enter, infect, and kill cells in tumors that have low CD111 expression (<20%). However, virotherapy with HSV may be very effective against CD111+ GPC resistant to traditional therapies.

Pharmacologic manipulations of mitochondrial membrane potential (DeltaPsim) selectively in glioma cells.

Metabolic control theory applies principles of bioenergetics for the control or management of complex diseases. Since metabolism is a general process underlying all biologic phenotypes, changes in metabolism can potentially modify phenotype. Therefore, it is reasonable to assume that experimental modulation of the availability of cellular energy can potentially alter cell phenotypes and cell functions critical to tumor progression including cell division. The purpose of this study was to determine if OMX-2, a methylquinone system designed to shuttle electrons from mitochondrial complexes, was able to target mitochondria in cancer cells and trigger cell death. Using flow cytometry, cell viability assays, and ATP measurements, we found that OMX-2 differentially decreased DeltaPsim without triggering cell death. In contrast, known blockers of the Electron Transport Chain (ETC) decreased DeltaPsim and triggered cell death. When normal cells were treated with OMX-2, neither DeltaPsim or cell death was triggered. Furthermore, OMX-2 modulated intracellular ATP and decreased cell numbers of glioma cells. Cell cycle analysis indicated that OMX-2 induced a reversible cell cycle arrest in G1/S. Finally, impairment of glycolysis by 2-Deoxyglucose (2-DOG) acted synergistically with OMX-2 to trigger cell death. Overall, these results indicate that it is possible to selectively target cancer cells by decreasing DeltaPsim and induced cell cycle arrest without triggering cell death. Moreover, pharmacological approaches designed to act on both glycolysis and oxidative phosphorylation can be considered as a new approach to selectively kill cancer cells.