The Weiss Lab is currently conducting several distinct research projects centered on (1) DNA damage response (DDR) mechanisms (2) ribonucleotide reductase and the regulation of nucleotide biosynthesis; and (3) metabolic alterations in cancers.
DNA DAMAGE RESPONSE MECHANISMS AND THEIR ROLES IN ORGANISMAL DEVELOPMENT AND DISEASE
A. Hus1 and mammalian DNA damage checkpoints.
DNA damage checkpoint mechanisms respond to genome damage by inducing cell cycle arrest and promoting DNA repair. There are two main mammalian checkpoint pathways that respond to distinct DNA lesions. One pathway responds primarily to double-stranded DNA breaks (DSB) and centers on the large protein kinase ATM, which is encoded by the gene mutated in the human genomic instability syndrome Ataxia-Telangiectasia. A second major pathway is headed by the related kinase ATR and responds to regions of ssDNA that arise at processed DNA lesions and stalled replication forks. Optimal signaling by Atr requires a trimer of RAD9, RAD1, and HUS1 (the 9-1-1 complex), which is independently recruited to DNA lesions. The 9-1-1 complex shares structural similarity with the toroidal sliding clamp proliferating cell nuclear antigen (PCNA) and is loaded onto DNA at primer-template junctions by the RAD17 clamp loader complex. Like PCNA, the 9-1- 1 complex acts as a molecular scaffold, stabilizing higher order complexes involving ATR and its activator TOPBP1. The 9-1-1 complex also has a unique role among checkpoint proteins as it also appears to directly participate in DNA repair by associating with various repair proteins, although physiological significance of these interactions is unknown in most cases.
1. Effects of partial Hus1 impairment on carcinogenesis.
2. Genetic interactions between Hus1 and other genome maintenance genes.
3. Roles for the 9-1-1 complex in meiosis.
4. Molecular mechanisms of genome maintenance by HUS1.
B. DNA damage response mechanisms in Testicular Germ Cell Tumors.
Testicular germ cell tumors (TGCTs) are the most common cancers in young men, and are unusual among solid tumors in that they (1) do not show markers of an active DDR at early stages, (2) rarely contain mutations in DDR genes like p53 and Atm, and (3) show remarkably high cure rates following treatment with DNA-damaging chemotherapies. In order to test how DDR function impacts the origins and treatment sensitivity of testicular cancers, we generated a novel mouse model of TGCT featuring metastatic, chemosensitive malignancies. Continued analysis of this model holds promise for elucidating the molecular determinants of cancer susceptibility and therapeutic sensitivity in germ cell malignancies, with important implications for the prevention and treatment of a variety of cancers.
REGULATION OF RIBONUCLEOTIDE REDUCTASE AND ITS IMPACT ON GENOMIC INTEGRITY
The cellular concentration of deoxyribonucleotide triphosphates (dNTPs), the basic building blocks of DNA, has a major impact on genomic stability. An adequate supply of nucleotides is needed for both DNA replication and repair. However, excess nucleotides lower the fidelity of DNA replication, leading to mutations that could impair cell fitness or lead to neoplastic transformation. The rate limiting step in the de novo biosynthesis of dNTPs is catalyzed by the enzyme ribonucleotide reductase (RNR). RNR enzyme activity is tightly controlled by a number of elaborate regulatory mechanisms, including allosteric control, protein levels of the ß subunit, post-translational modifications, and subcellular localization. Although RNR is a major determinant of genomic integrity, much remains to be determined regarding the impact of RNR deregulation in mammals.
1. A mouse model for lung cancer based on RNR overexpression.
2. Regulation of RNR by allosteric control.
METABOLIC ALTERATIONS IN CANCER
A. Sirtuin enzymes as targets for cancer therapy.
The silent information regulator (Sirtuin) family is a group of seven enzymes (SIRT1-7) that regulates cellular metabolism and influences aging and age-associated disease. In collaborative studies, we are investigating the functions of two sirtuins in cancers. The first, the mitochondrial sirtuin SIRT5, was recently shown by our collaborator Dr. Hening Lin and colleagues to act preferentially on succinyl and malonyl modifications on target proteins. We currently are investigating how SIRT5 impacts cancer metabolism and whether SIRT5 dysfunction affects malignant transformation and tumorigenesis. In an additional collaboration with the Lin Laboratory, we are testing the tumor suppressing activity of small molecule inhibitors of SIRT2, a deacetylase that regulates tubulin and other substrates and like SIRT5 is required for cancer cell proliferation in vitro.
B. DDR mechanisms and non-alcoholic fatty liver disease (NAFLD).
NAFLD is a prevalent but poorly understood disease that can progress to steatohepatitis and hepatocellular carcinoma. We have investigated the role of the DDR in NALFD pathogenesis and previously reported that targeted inactivation of the DDR kinase ATM induces apoptosis in response to diet-induced hepatic oxidative stress and thereby promotes liver fibrosis. We currently are extending these findings by assessing the combined effects of high fat diet feeding and other DDR pathway defects.