DNA-PKcs promotes chromatin decondensation to facilitate initiation of the DNA damage response.

The DNA damage response (DDR) encompasses the cellular response to DNA double-stranded breaks (DSBs), and includes recognition of the DSB, recruitment of numerous factors to the DNA damage site, initiation of signaling cascades, chromatin remodeling, cell-cycle checkpoint activation, and repair of the DSB. Key drivers of the DDR are multiple members of the phosphatidylinositol 3-kinase-related kinase family, including ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). ATM and ATR modulate multiple portions of the DDR, but DNA-PKcs is believed to primarily function in the DSB repair pathway, non-homologous end joining. Utilizing a human cell line in which the kinase domain of DNA-PKcs is inactivated, we show here that DNA-PKcs kinase activity is required for the cellular response to DSBs immediately after their induction. Specifically, DNA-PKcs kinase activity initiates phosphorylation of the chromatin factors H2AX and KAP1 following ionizing radiation exposure and drives local chromatin decondensation near the DSB site. Furthermore, loss of DNA-PKcs kinase activity results in a marked decrease in the recruitment of numerous members of the DDR machinery to DSBs. Collectively, these results provide clear evidence that DNA-PKcs activity is pivotal for the initiation of the DDR.

Transposon Insertion Mutagenesis in Mice for Modeling Human Cancers: Critical Insights Gained and New Opportunities.

Transposon mutagenesis has been used to model many types of human cancer in mice, leading to the discovery of novel cancer genes and insights into the mechanism of tumorigenesis. For this review, we identified over twenty types of human cancer that have been modeled in the mouse using Sleeping Beauty and piggyBac transposon insertion mutagenesis. We examine several specific biological insights that have been gained and describe opportunities for continued research. Specifically, we review studies with a focus on understanding metastasis, therapy resistance, and tumor cell of origin. Additionally, we propose further uses of transposon-based models to identify rarely mutated driver genes across many cancers, understand additional mechanisms of drug resistance and metastasis, and define personalized therapies for cancer patients with obesity as a comorbidity.

Chronic liver injury alters driver mutation profiles in hepatocellular carcinoma in mice.

Most hepatocellular carcinomas (HCCs) develop in a chronically injured liver, yet the extent to which this microenvironment promotes neoplastic transformation or influences selective pressures for genetic drivers of HCC remains unclear. We sought to determine the impact of hepatic injury in an established mouse model of HCC induced by Sleeping Beauty transposon mutagenesis. Chemically induced chronic liver injury dramatically increased tumor penetrance and significantly altered driver mutation profiles, likely reflecting distinct selective pressures. In addition to established human HCC genes and pathways, we identified several injury-associated candidates that represent promising loci for further study. Among them, we found that FIGN is overexpressed in human HCC and promotes hepatocyte invasion. We also validated Gli2's oncogenic potential in vivo, providing direct evidence that Hedgehog signaling can drive liver tumorigenesis in the context of chronic injury. Finally, we show that a subset of injury-associated candidate genes identifies two distinct classes of human HCCs. Further analysis of these two subclasses revealed significant trends among common molecular classification schemes of HCC. The genes and mechanisms identified here provide functional insights into the origin of HCC in a chronic liver damage environment. CONCLUSION: A chronically damaged liver microenvironment influences the genetic mechanisms that drive hepatocarcinogenesis. (Hepatology 2018;67:924-939).

Conditionally replicative adenovirus as a therapy for malignant peripheral nerve sheath tumors.

Oncolytic adenoviruses (Ads) stand out as a promising strategy for the targeted infection and lysis of tumor cells, with well-established clinical utility across various malignancies. This study delves into the therapeutic potential of oncolytic Ads in the context of neurofibromatosis type 1 (NF1)-associated malignant peripheral nerve sheath tumors (MPNSTs). Specifically, we evaluate conditionally replicative adenoviruses (CRAds) driven by the cyclooxygenase 2 (COX2) promoter, as selective agents against MPNSTs, demonstrating their preferential targeting of MPNST cells compared with non-malignant Schwann cell control. COX2-driven CRAds, particularly those with modified fiber-knobs exhibit superior binding affinity toward MPNST cells and demonstrate efficient and preferential replication and lysis of MPNST cells, with minimal impact on non-malignant control cells. In vivo experiments involving intratumoral CRAd injections in immunocompromised mice with human MPNST xenografts significantly extend survival and reduce tumor growth rate compared with controls. Moreover, in immunocompetent mouse models with MPNST-like allografts, CRAd injections induce a robust infiltration of CD8+ T cells into the tumor microenvironment (TME), indicating the potential to promote a pro-inflammatory response. These findings underscore oncolytic Ads as promising, selective, and minimally toxic agents for MPNST therapy, warranting further exploration.

Sleeping Beauty Insertional Mutagenesis Reveals Important Genetic Drivers of Central Nervous System Embryonal Tumors.

Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty (SB) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB-driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10, and Foxr2. Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo. We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. SIGNIFICANCE: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET.

Sleeping Beauty Insertional Mutagenesis in Mice Identifies Drivers of Steatosis-Associated Hepatic Tumors.

Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways. The protein kinase A/cyclic AMP signaling pathway was altered frequently in mouse and human steatosis-associated HCC. We found that activated PKA expression drove steatosis-specific liver tumorigenesis in a mouse model. Another candidate HCC driver, the N-acetyltransferase NAT10, which we found to be overexpressed in human steatosis-associated HCC and associated with decreased survival in human HCC, also drove liver tumorigenesis in a steatotic mouse model. This study identifies genes and pathways promoting HCC that may represent novel targets for prevention and treatment in the context of hepatic steatosis, an area of rapidly growing clinical significance. Cancer Res; 77(23); 6576-88. ©2017 AACR.