Patients with high grade serous ovarian carcinoma (HGSOC) have poor prognosis due to a combination of factors including the lack of effective early detection and new therapeutic regimens. Approximately 15-20% of all epithelial ovarian cancers harbor germ line or somatic mutations in BRCA1/2. Current standard of care for BRCA1/2 mutated ovarian cancer patients is platinum-based chemotherapy. Despite their striking sensitivity, a substantial fraction of patients eventually develop resistance to platinum therapy. Recently, poly-ADP ribose polymerase inhibitors (PARP inhibitor) have emerged as a potent antitumor agent against BRCA1/2-mutated HGSOCs. A significantly large fraction of patients despite an initial response to therapy eventually develop resistance. Currently very few clinically relevant and actionable mechanisms of resistance to chemotherapy in HGSOC patients have been identified.
Overwhelming evidence indicates therapy resistance might be a cause of de-novo mutations selected for during tumor development. Previous work from our lab, focused on a genome wide CRISPR-cas9 loss-of-function screen in BRCA1 and BRCA2 mutated carcinoma, has identified several genes in the ribonucleotide excision repair (RER) pathway as factors in mediating resistance to platinum therapy and PARP inhibitors in BRCA2 mutated ovarian carcinoma. However, the intricate details involved in mediating this response are currently unstudied.
We hypothesize that loss of genes in the ribonucleotide excision repair pathway circumvent DNA damage response pathway in BRCA2 mutated carcinoma towards increasing genome stability and are hence responsible for chemo resistance and worse outcome in patients.
Aim 1: To determine whether RNaseH2A plays a role in double strand break repair pathway choice in BRCA2 mutant cells.
BRCA2 loss leads to a loss of DSB repair by homologous recombination and an increase in double strand break repair by the highly toxic single strand annealing (SSA) repair pathway. We will determine whether RNaseH2A knockout in BRCA wild-type and BRCA2-mutated cells alters repair pathway choice after induction of double strand breaks by irradiation and DNA damaging agents (e.g. cisplatin and olaparib).
Aim 2: To determine whether loss of RNaseH2A affects genome stability in BRCA2 mutant cells.
BRCA2 loss leads to chromosomal instability and an increase in sister chromatid recombination. BRCA2 is also critical for R-loop processing and loss of BRCA2 is associated with an increase in R-loops and genome instability. Additionally, BRCA2 plays a crucial role in replication fork progression and loss of BRCA2 leads to an increase in stalled replication forks, which are a source of genome instability. We will determine if loss of RNaseH2A increases the genomic stability of BRCA2 mutant cells with and without DNA damage.
Aim 3: To determine RNaseH2A interacting partners in BRCA wild-type and BRCA1 and BRCA2 mutant cells.
Based on results from our CRISPR screen, loss of RNaseH2A selectively causes resistance in BRCA2 mutant cells, but not in BRCA1 mutant cells. Identifying RNaseH2A interacting partners in distinct genetic background will better elucidate the mechanistic details leading to resistance in BRCA2 mutant cells. We will perform immunoprecipitation followed by mass spectrometry to identify RNaseH2A binding proteins.
BRCA2 mutations are prevalent in ovarian, breast and pancreatic cancer. PARP inhibitors are currently in clinical trials for use in pancreatic cancer and are FDA approved for breast and ovarian cancer patients with germline BRCA1/BRCA2 mutations. However, since the response rate is low, identifying mechanisms of resistance is increasingly important. Collectively, results from the studies outlined in this proposal will help identify a novel mechanism of resistance which can help design better therapies for patients with a BRCA2 mutation.
Background and significance
Ovarian cancer is the fifth leading cause of mortality in women and the most lethal of gynecologic tumors. 5-year survival rate for patients diagnosed with HGSOC is approximately 25%, with complete remission seen in very few cases. BRCA1/2 breast and ovarian cancer susceptibility genes encode proteins that are key components of the homologous recombination pathway and are essential for accurate double strand break (DSB) repair. Nearly 50% of all ovarian cancers harbor germline and/or somatic loss-of-function mutations in genes that function in the high-fidelity homologous recombination repair pathway. Beyond their role in homologous recombination, BRCA1 and BRCA2 also play a crucial role in maintaining genomic integrity by protecting replication forks from nucleolytic degradation.
Treatment of patients with BRCA1/2 mutated high-grade serous ovarian carcinomas (HGSOCs), a particularly aggressive ovarian cancer subtype, with double strand DNA break (DSB)-inducing agents [e.g. platinum and Poly(ADP-ribose) polymerase (PARP) inhibitors] results in an improved therapeutic outcome. Platinum analogues (cisplatin and carboplatin) that induce inter-strand and intra-strand crosslinks (ICL) are standard of care therapy to treat ovarian cancer. Repair of ICL is dependent on the homologous recombination repair (HR) pathway. HR-deficient tumors exhibit a striking sensitivity to platinum-based agents due to the underlying defect in HR-mediated double strand break repair. However, despite the initial response to platinum therapy, a large fraction of tumors develop resistance.
Since 2014, three Poly-ADP ribose polymerase inhibitors (PARP inhibitors): Olaparib, Rucaparib, Niraparib have gained accelerated FDA approval for monotherapy treatment in epithelial ovarian cancer (EOC) patients. PARP1 is a critical enzyme for base excision repair (BER) pathway. Predicted model for PARP inhibitor dependent response observed in BRCA mutated EOC tumors has been attributed to the synthetic lethality observed upon loss of PARP activity and homologous recombination deficiency. Several other theories including PARP trapping at DNA damage sites have also been proposed to explain the cytotoxic effects of PARP inhibitors. Despite the predicted PARP inhibitor-induced synthetic lethality, in a study conducted by AstraZeneca, of the 137 patients with BRCA mutations, ~66% patients did not respond to PARP inhibitor Olaparib, suggesting that de novo and acquired PARP inhibitor resistance is a significant clinical problem in homologous recombination-defective HGSOCs.
Resistance mechanism to platinum therapy and PARP inhibitors
Our current understanding of how drug-responsive cancer cells become refractory to further treatment is nascent. The mechanisms that promote drug resistance, be it de novo or acquired, are complex and include genetic as well as epigenetic mechanisms. Considering that PARP inhibitors have only been in clinic for the past 7 years, there has been very limited investigations of the mechanisms of resistance. With the increasing use of PARP inhibitor in the clinic, the population of patients with treatment resistant disease is increasing.
One important mechanism for PARP inhibitor resistance that has emerged from in-vitro studies in patient derived cell lines and from sequencing BRCA mutant tumors is the restoration of HR through reversion mutations in the BRCA1 or BRCA2 genes , . However, the frequency of this event in a patient population is not known and might not represent a significant percentage. Loss of the non-homologous end joining (NHEJ) protein 53BP1 restores homologous recombination in BRCA1-deficient cells . Replication fork stabilization by regulating access of nucleases at stalled replication forks has recently been identified as a resistance mechanism in BRCA2 mutated cells. Although these observations are interesting from a mechanistic standpoint, their clinical relevance remains unclear.
A systematic and comprehensive investigation of the mechanisms underlying chemotherapy resistance to identify factors and pathways that can be targeted to overcome resistance to platinum and PARP inhibitor in homologous recombination -defective HGSOC patients is an unmet need of high clinical importance.
Loss of BRCA1 and BRCA2 is predicted to confer sensitivity to PARP inhibitor. However, the genomic landscape for BRCA1 and BRCA2 mutant tumors are strikingly different. Several groups have reported distinct chromosomal aberrations and mutational signature associated with loss of genes in the homologous recombination pathway, hence it is plausible that the mechanisms of resistance to platinum therapy and PARP inhibitor in BRCA1- and BRCA2- mutated carcinomas are dissimilar. Since homologous recombination is not restored upon BRCA2 loss, identifying the mechanism of resistance in BRCA2 mutated carcinomas has been particularly challenging.
Our group has conducted an unbiased CRISPR-cas9 knockout screen in three BRCA1 and BRCA2 mutant- patient derived HGSOC cell lines to identify genes, loss of which would result in resistance to platinum analogue cisplatin and PARP inhibitor Olaparib. Pathway analysis of top hits from the screen identified genes in several pathways in particular apoptosis, transcription regulation, immune response, histone acetylation regulation and BMP signaling to be involved in resistance. More importantly, among the top hits we identified sgRNA targeting genes in the ribonucleotide excision repair (RER) pathway to be enriched in the drug resistant population exclusively in BRCA2 mutant cells.
Ribonucleotide excision repair pathway is one of the major repair pathways responsible for maintaining genome stability by removing the aberrantly inserted ribonucleotides from the cellular genome. Approximately for every 103 dNTP’s (deoxy ribonucleotide) that are incorporated into the replicating DNA, 1 rNTP (ribonucleotide) is incorporated in error. RNaseH2A recognizes and hydrolyzes rNTP within a DNA strand creating a nick which is fixed by the coordinate action of Polymerase delta (Pol δ) and FEN1 mediating replacement of nucleotides in the nicked strand and mediating cleavage of the 5’- overhang followed by ligation of the nick by Ligase1. Currently, the role of RER in DNA damage response and repair is unknown. We hypothesize that dysregulation of RER pathway specifically complements DNA damage response and repair in homologous recombination deficient BRCA2 mutated carcinomas.
Innovation and significance
There is an unmet need to identify clinically relevant mechanisms of resistance increasing population of HGSOC patients in the clinic with resistance to therapy. The intended objective of this proposal is to study the role of ribonucleotide excision repair pathway in mediating chemo resistance in BRCA2 mutated carcinomas. Additionally, our studies will uncover a previously unknown role for RNaseH2A in double strand break repair. Furthermore, BRCA2 mutations predisposes patients to an increased risk of breast and pancreatic cancer. PARP inhibitor Olaparib recently received FDA approval for treatment in metastatic germline BRCA mutated breast cancer and is currently in clinical trials for patients with metastatic germline BRCA mutated pancreatic cancer. Studying the mechanism will improve our understanding of genome stabilizing pathways in BRCA2 mutated carcinomas which will be critical in improving therapeutic care for patients.