Our laboratory focuses on genome stability and cell cycle control with emphasis on three areas of research:
- Cell cycle checkpoints that respond to DNA damage and delay the onset of mitosis
- DNA repair and its coordination with cell cycle progression
- Regulation of bipolar mitotic spindle formation by NIMA-related kinases
Our experimental approaches are directly in large part by the use of the model system that is best suited for studying G2 and mitosis, the fission yeast Schizosaccharomyces pombe (Fig 1). The study of the cell cycle in this system was pioneered by Paul Nurse, and led to the discovery that the onset of mitosis is regulated by essentially identical mechanisms in this simple yeast as in human cells, being under the control of the cyclin-dependent kinase Cdc2 (Fig 2). We can therefore use the elegant genetics in the fission yeast system as a gene and pathway discovery tool, and these findings are then used to direct experiments in both cultured cells in vitro and in mouse tumor models. We aim not only to learn of the basic biology of these processes, but to use this information in the design of anti-cancer therapies targeted at particular tumor genotypes, especially those more refractory to conventional therapies.
More details on these projects, and recent publications from our lab are below. There are opportunities for Postdocs and Graduate Students to join these projects. For more information, contact Matthew O'Connell: matthew.oconnell@mssm.edu
Project details:
Cell cycle checkpoints that respond to DNA damage and delay the onset of mitosis
Cancer is a disease of the cell cycle. Tumor cells accumulate many genetic lesions that contribute to their molecular evolution as a tumor progresses, in large part due to the instability of their genomes. Defects in cell cycle checkpoints that function at the G1/S transition are almost universal in human tumors, and together with defects in DNA repair mechanisms lead to this genomic instability. A significant regulator of G1 checkpoints, p53, is mutated in over half of tumors. Such mutations also inhibit the ability of a cell to undergo DNA damage induced apoptosis, thus sending a damaged genome through S-phase. Alternative pathways that respond to DNA damage during G2 phase, and prevent entry into mitosis, are rarely mutated in cancer, suggesting they are essential for tumor cell viability. We aim to understand the biology of this G2 DNA damage checkpoint, and then utilize this information in the design and testing of novel anti-cancer therapies.
Much of our work has stemmed from an elucidation of the core regulatory events during the G2 checkpoint response in fission yeast, though we and many other labs have shown that these events also occur in human cells. Our contribution from this project has been to investigate the regulation and function of Chk1, which is the final effector kinase of this checkpoint. The transition from G2 into mitosis is under the control of the archetypal Cyclin-dependent kinase, Cdc2. Cdc2 is kept inactive in a G2 cell by inhibitory tyrosine phosphorylation by a tyrosine kinase known as Wee1. Once the decision to enter mitosis is made, this inhibitory phosphorylation is removed by a tyrosine phosphatase known as Cdc25. Chk1 prevents mitotic entry by acting as a "double-lock" - it both inhibits Cdc25 and activates Wee1 to ensure Cdc2 remains inactive in the face of receiving other signals to promote mitosis (Fig 3).
We have now moved on to ask how Chk1 is regulated - how is it activated, how is it inactivated, does it need to be inactivated for a cell to enter mitosis? Chk1 is activated by DNA damage, and this is achieved at least in part by its phosphorylation by a protein kinase known as Rad3 in fission yeast, and ATR in humans. However, we believe there is much more to Chk1 regulation than this event, albeit a critical one. Through extensive mutagenesis of Chk1 in yeast, and the study of analogous mutations in human Chk1, we have data pointing to both positive and negative regulation over Chk1 activity. We have instigated a series of genetic screens in yeast, utilizing the different Chk1 mutations we have generated, to identify such regulators. To date we have isolated 8 proteins that functionally and/or physically interact with Chk1 in yeast, and have cloned their human homologs. In some cases it appears these proteins regulate Chk1, whilst in others they appear to be new targets for Chk1 to phosphorylate. We are currently working to fully understand the nature of these interactions.
Knocking out Chk1 function renders cells hypersensitive to DNA damage, as entering mitosis prior to completing DNA repair is catastrophic, as cells fail to correctly segregate chromosomes, loose portions of chromosomes, etc. Combining the loss of p53 and Chk1 in the same cell is synergistic and renders cells extremely sensitive to DNA damage (Fig 4). This raises the possibility that inhibition of Chk1 may be an extremely efficient way to treat cancer, particularly those lacking p53. We are testing this hypothesis in a number of animal tumor models to determine the best combinations of tumor genotype, Chk1 inhibition and DNA damaging agents
DNA repair and its coordination with cell cycle progression
To achieve a meaningful checkpoint response, cell cycle progression must be coordinated with DNA repair. We isolated a hypomorphic mutation in a fission yeast gene, rad18, which rendered cells both unable to repair DNA damage, and also unable to maintain a checkpoint arrest (Fig 5). Rad18 was recently renamed Smc6. The Smc acronym comes from Structural Maintenance of Chromosomes. Two SMC complexes have been described, each of which contains two SMC proteins and a number of non-SMC proteins. These are the condensin, which is required for chromosome condensation, and the cohesin, which is required for sister chromatid cohesion. Rad18/Smc6 defines the third of these complexes, and includes another Smc family protein, Smc5, together with several other proteins.
We have identified several other mutations in fission yeast smc6 that also have the dual repair and checkpoint deficiency, as well as mutations in one other protein of the complex. We are now concentrating on the relationship between Smc6 function and Chk1 signaling. From here the two projects intersect and enable us to learn about each of these events. Initial observations indicate that smc6 mutants fail to arrest despite Chk1 activation. Our model is that the primary defect in smc6 mutants is at the level of chromatin organization for DNA repair, and that the cell actually monitors ongoing repair during a checkpoint, rather than DNA damage per se.
These mutations in smc6 also enable us to ask questions about the function of this complex in the absence of exogenous DNA damage. We presume that these are either identical or at least highly related, and that the effects of irradiation take the requirement for this complex to a higher level required for viability.
We have also identified the human homologs of these genes, and are exploring function with the analysis of mutants and by gene silencing, and the effects of this on chromosome dynamics and cell cycle progression.
Regulation of bipolar mitotic spindle formation by NIMA-related kinases
Mitosis sees a massive reorganization of cellular architecture, the formation of a mitotic spindle, and the condensation and segregation of chromosomes. It is essential that the segregation of chromosomes be balanced, so that both daughter cells obtain an identical genome. The regulation of this amazingly complicated process is under the control of a number of protein kinases, such as the Aurora and Polo families, that are highly conserved both functionally and physically from yeast to man. A third family, the NRK - NIMA-Related Kinases - are a third and important, though poorly understood family and we aim to define how these proteins regulate mitotic progression.
NIMA is a protein kinase that regulates mitotic entry in the fungus Aspergillus nidulans. Homologs of NIMA have now been identified in a number of species, including man. Unlike most other mitotic regulators, however, the function of this protein kinase remains poorly understood. We cloned the fission yeast NIMA homolog, fin1, and have been investigating its function in this system.
We have found that fin1 is required for the formation of a functional bipolar mitotic spindle that is required for accurate chromosome segregation. A large number of genetic interactions place fin1 function in time (mitosis) and in space (the spindle poles) (Fig 6). Moreover, Fin1 is localized to the poles of the spindle during mitosis, and is significantly up-regulated as the cells pass from metaphase to anaphase.
We are now investigating the molecular events controlled by Fin1 to ensure faithful chromosome segregation. It is not entirely clear at this stage as to which of several NIMA-related kinases are the "real" NIMA In human cells, if indeed this is limited to only one protein. However, by studying the function of the related fin1 in what is the best genetic system to study mitosis, and learning molecular details of how it exerts its effects, we aim to understand similar events in human cells. These findings may be very important in understanding chromosome aberrations in human cancers.
Specific Clinical/Research Interest: Regulation of the cell cycle; control over genomic stability and chromosome dynamics; the design of novel strategies for anti-cancer therapies
Current Students: PhD: Claudia Tapia-Alveal, Karen Kuntz; MD/PhD: Kirstin Bass
Postdoctoral Fellows: Teresa Mateo-Calonge, Emily Outwin
Summary of Research Studies:
Perhaps the most fundamental process in biology is that by which one cell becomes two. Our lab studies the control of the cell division cycle, and the signaling pathways, called checkpoints, which respond to chromosomal damage and prevent cell cycle progression until that damage is repaired. Such checkpoints function throughout the cell cycle. Those working in G1 phase to prevent the replication of damaged DNA are almost invariably mutated or inactivated in cancers. These defects contribute not only to the instability of tumor cell genomes, but can also knock-out pro-apoptotic pathways, rendering tumors resistant to treatment. Those functioning in G2 phase to prevent commitment to mitosis are, however, virtually always intact and appear to be required for the viability of tumor cells that lack G1 checkpoints. Our research is geared to dissect the molecular and cell biology of G2 checkpoints, and aims to use this knowledge in the design and testing of targeted anti-cancer th! erapies. We utilize a wide range of experimental systems including yeast, human cells in culture, and mouse models of cancer, and take genetic, biochemical, molecular and cell biological approaches. We are currently focuses our efforts into two areas of research: (1) the regulation and function of a checkpoint effector protein kinase, Chk1, and its suitability as a target in anti-cancer therapy; and (2) control of chromosome dynamics in the coordination of DNA repair and cell cycle progression.
