Sarah James Hill - US grants

DESCRIPTION (provided by applicant): BRCA1 (B1) is a breast and ovarian tumor suppressor. Its best known function is in the repair of double strand breaks (DSB) by homologous recombination (HR). It can also function in other types of DNA damage repair (cf below), as well as in DNA damage checkpoints. However, there is limited in depth knowledge about any of the purported functions for B1or how it acts as a tumor suppressor. To address this problem, we purified B1-containing protein complexes from a human cell line and analyzed the complexes by mass spectrometry (MS). Our hope was that by better defining the B1 protein partner repertoire we might be able to better understand known B1 functions or potentially hypothesize new B1 functions based on the known functions of identified interactors. In this analysis, we repeatedly identified a protein called TONSL (aka NFKBIL2) as a B1 interactor. This protein has been suggested to function in the repair of collapsed replication forks. Recently, our group has shown that B1 functions in the repair of stalled replication forks. Given the similar suggested function for both proteins, and based on our preliminary analysis of the B1-TONSL interaction, we hypothesize that B1 recruits TONSL to collapsed replication forks to aid in the repair of DSB breaks arising at these sites. The four specific aims of this proposal wil focus on this hypothesis. The goal of specific aim 1 will be to perform a structure function analysis on the B1-TONSL interaction as a means of assessing, genetically, the hypothesis that B1 recruits TONSL to collapsed forks and that the B1- TONSL complex is involved in repair of these structures. The goal of aim 2 will be to understand an observation from our preliminary data. We find that after UV damage, B1 localizes at UV damage sites in virtually all S/G2 cells. However, TONSL co-localizes with B1 at these sites in only some of these cells. The goal of aim 2 is to determine why this is the case, which could shed light on how B1-TONSL complexes function. The goal of aim 3 is to search for and, if feasible, identify other members of the B1-TONSL subcomplex, by affinity purification and MS. By doing this we hope to identify subunits with known biochemical functions and then assess the contributions of these properties to B1-TONSL function and vice versa. This approach could further illuminate aspects of the function of this complex at collapsed forks and/or suggest new hypotheses for the function of this complex. The goal of aim 4 is to determine, with help from collaborators, whether TONSL and/or any other member of the B1-TONSL complex is a product of a breast cancer suppressor gene by searching for relevant mutations in whole genome sequence libraries of breast tumors. This work will be supervised by the candidate's sponsor in a program composed of laboratory-based research training leavened by attendance at relevant scientific meetings. Ideally, success in this effort will contribute to a better understanding of B1 function and how loss of B1 function leads to breast and/or ovarian cancer.

PROJECT SUMMARY Genomic analysis of high grade serous ovarian cancer (HGSC) has revealed that up to 50% of HGSCs harbor a genomic alteration in a DNA damage repair (DDR) gene, mostly in the BRCA repair pathway. Functional profiling of DDR capacity of patient derived HGSC organoids has revealed that over 60% of HGSCs harbor defects in the stalled replication fork protection DDR pathway. Further analysis of these HGSC organoids reveals that in patients who receive neoadjuvant chemotherapy, a tumor which initially had a replication fork protection defect and was carboplatin sensitive can be induced by the neoadjuvant chemotherapy to undergo replication fork stabilization and become carboplatin resistant. Transcriptional analysis of the post-neoadjuvant fork stable organoids reveals that these cultures have undergone an epithelial-mesenchymal transition. Based on these results, the hypothesis is that defects in the stalled replication fork protection DDR pathway are a fundamental molecular defect in HGSC that when perturbed by treatment with neoadjuvant carboplatin can lead to loss of the defect at the molecular level, alterations in the overall state of the tumor cells, and changes in the way the tumor cells interact with the surrounding stroma and immune cells, all of which combine to create a broadly therapy resistant tumor microenvironment. The research challenge I will pursue is to dissect the evolving mechanisms by which tumor cells which originally harbor fork protection defects and are induced to lose the defect interact with the surrounding stroma and immune cells and how these changing interactions might be manipulated to prevent a deeply resistant tumor microenvironment. The work will utilize a novel HGSC organoid co-culture system which will be highly innovative for the field because it will allow real time assessment of the interactions between DDR defective or altered tumor cells and the surrounding normal cells over time and treatment. Approach: The goal of Aim 1 is to generate growth conditions for co-cultures of fork unstable platinum sensitive organoids with patient matched intra-tumoral stromal cells and all immune cells (T cells, B cells, NK cells, dendritic cells, macrophages, and neutrophils). The goal of Aim 2 will be to generate isogenic pairs of the organoids from Aim 1 that are fork stable and unstable and then study how the interaction of the tumor cells with the surrounding stromal cells changes in the organoid co-culture system as the tumor cells undergo selective fork stabilization. The goal of Aim 3 is to utilize the isogenic pairs of fork unstable and stable organoids from Aim 2 in the co-culture system and determine how the interaction of the tumor cells with the surrounding immune cells changes as the tumor cells undergo fork stabilization and how this may alter response to immuno-oncologic agents. This work will have major impact in HGSC because it will help understand the evolution of the tumor-normal cell interaction as the tumor cell fork protection defects are altered and stressed. This may allow for the development of rational combination therapies that simultaneously target the tumor cell defects and also prevent problematic tumor-normal cell interactions.

PROJECT SUMMARY Genomic analysis suggests that up to 50% of High Grade Serous Ovarian Cancers (HGSCs) harbor a genomic alteration that might confer a DNA damage repair defect, making therapies that target such defects potential treatment options. There is no method to predict which patients will respond to such therapies, which is a major problem in the field. Preliminary data indicate that patient derived HGSC organoids may be a faithful model system in which to perform functional assays to predict patient therapeutic response. Data from a limited analysis of HGSC organoids suggest that stalled replication fork protection defects are more common than homologous recombination defects in HGSC and that more patients may benefit from the wider array of therapies available to target such defects, including carboplatin, gemcitabine, ATR, WEE1, and CHK1 inhibitors. The goal of this mentored research career development proposal is to utilize patient derived HGSC organoid cultures to understand the prevalence, mechanisms, and therapeutic relevance of stalled replication fork protection defects in HGSC. The proposed research studies encompass multiple disciplines including molecular biology, DNA sequencing, and animal modeling which will help investigate the role of stalled replication fork protection defects in HGSC and also provide a well-rounded career development strategy for becoming scientifically independent through execution of the following specific aims: Specific Aim 1: Assess the prevalence of fork protection defects in HGSC and whether fork protection defects predicted by HGSC organoid functional assays lead to therapeutic sensitivity to carboplatin and ATR, WEE1, and CHK1 inhibitors. This will be accomplished by generating organoids from patients being treated with carboplatin and ATR, WEE1, and CHK1 inhibitors, performing functional assays to assess stalled fork protection capacity and therapeutic sensitivity of the organoids in parallel to sequencing analysis, and comparing organoid and patient outcomes. Specific Aim 2: Uncover mechanisms leading to fork instability in the organoids and whether different mechanisms of fork protection defects lead to differing sensitivities to the above agents. This will be accomplished using molecular and cellular biology analysis of specific pathways in the organoids. Specific Aim 3: In vivo validation of in vitro mechanisms of stalled replication fork protection defects leading to therapeutic responses in organoid xenograft models of HGSC. This will be accomplished by generating mouse models using select organoids from aim 2 and testing them for therapeutic responses to agents used in aim 2. The career development award candidate is an MD/PhD clinically trained in anatomic pathology. The proposed research will occur at Dana-Farber Cancer Institute under the mentorship of Dr. Alan D'Andrea. The candidate will utilize the additional training provided by this award to facilitate her ultimate career goal of becoming an independent physician scientist and leader in the field of ovarian cancer.