Sharon Cantor - US grants

[unreadable] DESCRIPTION (provided by applicant): Chemoresistance is a major problem in cancer therapy, but it is largely unclear how cancer cells become resistant. Deficiency in the mismatch-repair (MMR) proteins allows cells to resist toxic chemotherapeutic agents such as DNA methylators. However, the distinct MMR function that sensitizes cells to DNA methylation is not clearly defined given the multiple MMR functions. MMR proteins function to repair base mismatches after DNA replication, inhibit recombination between non-identical DNA sequences, as well as activate both checkpoint and apoptotic responses following DNA damage. Separation-of- function mutants, suggest that resistance to DNA methylation is dependent on a disrupted checkpoint. Recently, we established that both MMR proteins of the MutL1 complex (MLH1/PMS2) and BACH1/FANCJ (BRCA1-associated C- terminal helicase/Fanconi Anemia complementation group J) are required for checkpoint signaling. Specifically, we identified that BACH1 binds directly to MLH1 and that a mutant version of BACH1 ablated for MLH1 binding failed to elicit an interstrand crosslink (ICL)-induced checkpoint response. Since ICLs activate the intra S-phase checkpoint, and both MLH1 and BACH1 have been shown independently to function in the intra S-phase checkpoint, this checkpoint likely requires the formation of a BACH1/MutL1 complex. We will test this possibility directly. In addition, we will determine whether BACH1 also participates in the DNA-methylation-induced G2/M accumulation checkpoint similar to MutL1. Consistent with a role for BACH1 in the DNA methylation-induced response, our lab has shown that similar to MutL1 deficient cells, BACH1 deficient cells are resistant to DNA methylation. In contrast, BRCA1 deficient cells are sensitive to DNA methylation, suggesting that BACH1 uniquely functions in the DNA methylati1n response. We propose to dissect the role of a BACH1/MutL1 complex in both checkpoint and repair functions. We will determine whether the formation of an intact complex is required to restore chemosensitivity to resistant null BACH1 or MutL1 cells. Defining the function of the BACH1/MutL1 complex ideally will provide insight towards restoring chemosensitivity to cancer cells. Along these lines, we will test whether manipulation of the recombination function of the BACH1/BRCA1 complex will uniquely sensitizes MMR deficient cells to chemotherapies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Ovarian cancer is the most common cause of cancer death from gynecologic tumors in the United States. While some ovarian tumors initially respond to first line chemotherapy, the majority of advanced-stage ovarian cancers develop chemoresistance and patients succumb to their disease. Without a full molecular understanding of how resistance is mediated, even promising new therapies could ultimately fail. Thus, we propose to define these mechanisms and develop tools to anticipate when resistance to chemotherapy will occur. This knowledge will be critical to guide therapy choice and will provide insight into new therapies targeting the mechanisms of resistance. Our innovative idea was to identify mechanisms of chemoresistance in ovarian cancer by an unbiased screen. Indeed, our survival based genome-wide shRNA screen enabled us to identify genes, whose loss is essential for the cancer cells to resist cisplatin therapy. Significantly, several of these genes ae mutated or lost in ovarian cancer and associated with patient response to therapy indicating that our screening strategy was a success. Now, with these genes in hand, our proposed research plan in Aim 1 is to further validate genes in vitro and in vivo. In Aim 2, we will define how depletion of these genes confers resistance. Not only will we learn how loss of these genes unleashes resistance in ovarian cancer, but also these studies will help identify what resistance pathways should be inhibited to enhance the efficacy of therapy. In particular, we will test the hypothesis that genes whose loss confers cisplatin resistance also confer a drug tolerant state (DTS), in which chromatin modifications and DNA damage responses are reduced. Thus, we will determine if knocking down validated gene candidates induces a DTS, or, conversely, ectopic expression of validated gene candidates reverses a DTS. Because epigenetic drugs that preserve chromatin modifications and restore a robust DNA damage response sensitize cells in a DTS, we will test whether epigenetic drugs will re-sensitize cisplatin resistant ovarian cancer cells. Finally, in Aim 3, we will test the hypothesis that genes identified in our screen will be useful as biomarkers. Given that resistance uniquely requires that these genes be depleted, low expression of these genes in ovarian tumors could predict poor response to cisplatin. Thus, it will be critical not only to determine whether these genes are tumor suppressors disrupted in ovarian cancer, but also whether these genes provide a signature predicting cisplatin response in patients, ideas we will test with outstanding reagents and expertise provided by our clinical collaborators. Altogether, the research plan proposed here will provide opportunities to define, detect (biomarkers), and ultimately disable compensatory mechanisms in ovarian cancer.

Project Summary: Great progress has been made in uncovering the proteins and pathways that function in the replication stress response. In particular, the hereditary breast cancer genes, as well as genes mutated in Fanconi anemia (FA) function in the replication stress response. It is now understood that loss of their function in the replication stress response contributes to the sensitivity of associated tumors to chemotherapies, such as cisplatin. However, the distinct functions for the BRCA-FA proteins are largely unknown. Here, we propose to analyze DNA replication fork dynamics, replisome components, and identify patient mutations that have specific defects in the replication stress response. To define how a cell transitions from defective to dysregulated replication, we have engineered cells expressing different mutant versions of the BRCA1-associated FANCJ also mutated in breast/ovarian cancer and FA. Similar to BRCA1, we have uncovered that FANCJ functions to protect replication forks from collapse. We also found that this FANCJ fork protection function requires its direct interaction with the mismatch repair (MMR) protein, MLH1. This finding provides insight as to why cells lacking the FANCJ-MLH1 interaction fail to recover from replication stress. We have also identified putative gain-of-function FANCJ mutants, such as the BRCA1-interaction defective mutant, that circumvent replication stress, keep forks intact, and confer hyper- resistance to replication stress inducing agents. In Aim 1, we will seek to define how FANCJ interactions direct DNA replication fork dynamics. Given that FANCJ localizes to replication forks, displaces proteins, and unwinds DNA, we hypothesize that disrupted vs dysregulated replication will reflect not only changes in DNA structures, but also the proteins found at DNA replication forks. In Aim 2, we will seek to determine how FANCJ contributes to the composition of the replisome in both unchallenged and at stressed replication forks. Replication stress induces changes to FANCJ protein interactions and post-translation modifications. Some of these changes occur at sites we found to be mutated in cancer patients. In Aim 3, we will seek to generate FANCJ mutants resistant to replication stress induced changes to uncover mechanisms regulating FANCJ function that are lost in cancer. Collectively, by defining how cells succumb to- or survive- toxic DNA damage that normally interferes with replication, we will gain insight towards mechanisms transitioning cells from defective to dysregulated replication in cancer. !

The goal of this grant is to harness a new understanding of vulnerabilities in tumors with mutations in the hereditary breast cancer genes. We have found that cells deficient in the BRCA-pathway genes, fail to properly respond to DNA replication perturbations (stress) and consequently replication is not restrained properly and ssDNA regions (gaps) develop. We find that when gaps are present, BRCA cancer cells are sensitive to therapy and when gaps are avoided, resistance occurs. Our findings that gaps are fundamental to therapy response is a paradigm shift in the current framework that proposes that persistent DNA breaks and fork degradation is the cause of sensitivity. Thus, we propose to employ state-of-the-art experiments to map the molecular determinants of this BRCA pathway fork restraint function. Moreover, will identify the gap making machinery that is critical for therapy response and the gap avoidance machinery that is critical to therapy resistance. Lastly, we will re- examine models of therapy resistance previously attributed to restored DNA repair and fork protection and determine if gap suppression is instead the fundamental resistance mechanism. Collectively, these proposed studies will identify how cancer cells succumb to and eventually gain resistance to chemotherapy and provide valuable insight towards biomarkers predicting resistance and drugs that prevent resistance.

Abstract The overall goal of this proposal is to understand and target cancer cell addictions to stress- tolerance pathways. We have discovered that cancer cells maintain proliferation by engaging in a pathway known as translesion synthesis (TLS). By employing TLS, cancer cells are able to replicate in a continuous manner and subvert the replication stress response. Here, we propose to implement state-of-the-art assays to analyze how cancer cells alter DNA replication fork dynamics and replisome components to promote TLS. By defining the core TLS machinery, we will seek to identify biomarkers and novel targets of TLS. Importantly, we have developed a small molecule inhibitor of TLS that selectively halts DNA replication in several cancer cell lines that are dependent on TLS. Moreover, we found that the colony forming potential of TLS-dependent cancer cells is dramatically reduced upon inhibition of TLS. Thus, we propose to identify the scope of TLS dependent cancers using cell screening and data base analysis. In addition, to fully further develop the therapeutic potential of TLS inhibition, we propose to measure and improve the anti- cancer potential of our lead small molecules. Collectively, these proposed studies will identify how cancer cells engage TLS and how best to block TLS to selectively target cancer.