PARP Inhibitor Sensitivity Research Articles

Abstract 1752: BRCA-mediated tumorigenesis is origin and cell-type dependent

Abstract BRCA1 and BRCA2 mutations predispose to select cancers, yet the interplay between germline and somatic BRCA alterations in driving tumorigenesis and conferring drug sensitivity remain poorly understood. To determine which tumors are dependent on mutant BRCA, we integrated the prospective clinical sequencing of germline blood and matched tumor specimens from 17,152 advanced cancer patients with zygosity analysis, broader somatic molecular features, and treatment outcomes. Tumor lineage dictated BRCA dependence in cancers of both the 2.7% of carriers with germline pathogenic variants and the 1.8% of patients with somatic loss-of-function mutations in BRCA1 and BRCA2 across 38 affected cancer types. The rate of biallelic inactivation of mutant BRCA1/2 varied by mutational origin and tumor lineage. Consequently, BRCA-mediated phenotypes such as homologous recombination deficiency (HRD) were associated with BRCA1/2 mutations in a cell type- and zygosity-dependent manner. Phenotypic penetrance was greatest in tumors of high-risk cancer types and in tumors with biallelic inactivation of mutant BRCA, independent of its germline or somatic origin. Conversely, heterozygous BRCA mutations in other cancer types conferred no HRD phenotype. These lineage-specific differences among hallmarks of BRCA dependence also predicted differential response to PARP-inhibitor therapy. Collectively, only BRCA mutations in tumors of high BRCA penetrance had a strong selective pressure for somatic biallelic inactivation, conferred dose-dependent somatic phenotypic consequences, and PARP inhibitor sensitivity. In contrast, BRCA1/2-mutant patients with cancers not traditionally associated with BRCA susceptibility generally had tumorigenesis independent of mutant BRCA. Overall, mutant BRCA was a founding pathogenic event on which some tumors depended while in others it was likely a dispensable and biologically neutral passenger mutation unrelated to tumorigenesis. This difference was conditioned by lineage, mutational origin, and zygosity, an understanding of which requires integrated germline and somatic molecular characterization in cancer patients with implications for screening, disease pathogenesis, clinical trial design, and therapy. Citation Format: Philip Jonsson, Michael L. Cheng, Chaitanya Bandlamudi, Preethi Srinivasan, Shweta S. Chavan, Noah D. Friedman, Ezra Y. Rosen, Allison L. Richards, Nancy Bouvier, S. Duygu Selcuklu, Craig Bielski, Wassim Abida, Ahmet Zehir, Nikolaus Schultz, Mark T. Donoghue, Jose Baselga, Kenneth Offit, Marc Ladanyi, Eileen M. O’Reilly, Howard I. Scher, Zsofia K. Stadler, Mark E. Robson, David M. Hyman, Michael F. Berger, David B. Solit, Barry S. Taylor. BRCA-mediated tumorigenesis is origin and cell-type dependent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1752.

Abstract 1734: Crispr/Cas9 screening of Brca1 and Brca2 susceptibility genes in breast and ovarian cancer precursor cells can identify phenotypically different mutants with variable penetrance

Abstract BRCA1 and BRCA2 mutations are the most significant risk factors for breast and ovarian cancer identified so far. A recent analysis of breast/ovarian cancer risks in approx. 12,000 BRCA1 carriers and 7,000 BRCA2 carriers found that PTVs (protein truncating variants) in the central region of both genes are more associated with ovarian cancer development (Ovarian Cancer Cluster Regions (OCCRs)), while PTVs in the 5’ and 3’ regions are more associated with breast cancer development (Breast Cancer Cluster Regions (BCCRs)). We aimed to identify the functional consequences of different BRCA1/2 mutants by creating various PTVs in BCCR and OCCR regions of both genes in precursor cell types for Basal-like Breast Cancer (BLBC) and High-Grade Serous Ovarian Cancer (HGSOC). To this end, we created partially transformed models of breast and fallopian tube epithelial cells (MCF12A and FT282, respectively) via an overexpression of a hot-spot p53 mutation (R175H), frequently observed in BLBC and HGSOC. We then generated PTVs in MCF12Ap53R175H and FT282p53R175H cells in two regions of BRCA1 (exon 11 (OCCR) and 16 (BCCR)) and two regions of BRCA2 (exon 9 (BCCR) and 13 (OCCR)) using CRISPR/Cas9 system. We targeted the olfactory receptor OR10A4 as a negative control. We clonally derived cell lines and genotyped PTVs via molecular cloning and Sanger sequencing. We established models of MCF12Ap53R175H and FT282p53R175H cells carrying PTVs for almost all regions. Mostly heterozygous PTVs survived; except of BRCA1 BCCR PTVs, which were lethal in FT282p53R175H, while MCF12Ap53R175H cells survived homozygous PTVs in this region. Typically, all PTVs led to diminished growth, although FT282p53R175H with BRCA2 OCCR PTVs showed similar proliferation rate as control. Heterozygous BRCA1/2 mutations were sufficient to induce nonsense mediated decay of BRCA1/2 mRNA transcripts. BRCA1 OCCR PTVs also specifically increased processing of delta11q splice isoform, known to decrease PARP inhibitor and Cisplatin sensitivity in BRCA1 mutant cell lines. Consistently, derived BRCA1/2 mutant clones showed increased Cisplatin resistance. Immunofluorescence and flow cytometry confirmed decreased BRCA1 protein levels upon introduction of BRCA1 PTVs. Our studies demonstrate that different BRCA1/2 mutations have different phenotypic effects in breast and ovarian cancer precursor cells. We found that BRCA1 and BRCA2 BCCR PTVs in FT282p53R175H had more severe effect on survival and proliferation than OCCR PTVs. This suggests that OCCR PTVs may have survival advantage in FT282p53R175H cells, compared to BCCR PTVs. Next, we will perform high-throughput CRISPR/Cas9 screen of hundreds of BRCA1 and BRCA2 PTVs to functionally validate the genetic epidemiology data indicating the different disease risks for different germline mutations in these susceptibility genes. Citation Format: Justyna Kanska, Kruttika Dabke, Zachary Schwartz, Norma Rodriquez-Malave, Nikoo Safi, Simon Gayther. Crispr/Cas9 screening of Brca1 and Brca2 susceptibility genes in breast and ovarian cancer precursor cells can identify phenotypically different mutants with variable penetrance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1734.

Abstract 3772: Inter- and intra-tumoral variations in ASCL1, NEUROD1, and POU2F3 transcriptional programs underlie three distinct molecular subtypes of small cell lung cancers

Abstract Accounting for 15% of all lung cancer diagnoses, small cell lung cancer (SCLC) is an aggressive malignancy with dismal clinical outcomes, due in part to failure to define SCLC molecular subsets and identify the unique, targetable vulnerabilities therein. Recent data has begun to delineate these subsets by uncovering inter-tumoral heterogeneity in features such as DNA damage response, EMT, and neuroendocrine (NE) status. An integrated analysis of clinical samples to determine the implications of this heterogeneity broadly on SCLC classification has not yet been performed. Using RNAseq data from 81 SCLC tumor samples, we applied non-negative matrix factorization (NMF) which identified three clusters, each enriched for unique transcriptional programs driven by ASCL1 (30/81), NEUROD1 (24/81), or POU2F3 (27/81). These three genes encode transcription factors which define mutually exclusive NE-high, NE-low, and novel tuft cell variants of SCLC. Bulk RNAseq analyses of SCLC CTC-derived xenograft (CDX) models validated the clustering analysis in vivo. However, single-cell RNAseq from these same models reveals subtle evidence of intra-tumoral and intra-cellular heterogeneity in transcriptional programs that appear mutually exclusive in bulk analyses, suggesting plasticity among these variants. Guided by our NMF results, we performed RNAseq-based supervised clustering to classify each of 60 SCLC cell lines into ASCL1-driven, NEUROD1-driven, and POU2F3-driven clusters. Then, using reverse phase protein array data for these lines, we derived proteomic signatures for each cluster as follows: ASCL1-driven: E-cadherinhigh/TTF-1high/cMYClow, NEUROD1-driven: E-cadherinlow/TTF-1low/cMYChigh, and POU2F3-driven: E-cadherinhigh/TTF-1low/cMYChigh (ANOVA p<0.001 for each). Other targetable proteins vary significantly among these clusters including BCL-2 (lower in NEUROD1-driven; p<0.001) and PD-L1 (higher in ASCL1-driven; p=0.04). Correlating these clusters with IC50 values for over 500 drugs we find unique vulnerabilities. For example, POU2F3-driven lines were more sensitive to PARP inhibitors (PARPi), despite lacking biomarkers of PARPi sensitivity in SCLC (e.g. high SLFN11, low ATM). ASCL1-driven lines were more resistant to aurora kinase inhibitors (AURKi), as predicted given their lower expression of cMYC, a predictor of AURKi sensitivity in SCLC. Our data suggest that SCLC is subdivided on the basis of three unique transcriptional programs and that each subtype is characterized by diverse protein expression and drug responses. Single-cell analyses suggest, however, that multiple transcriptional programs may coexist within a single tumor or, even, a single cell, thus providing a potential novel mechanism for lineage switching and therapeutic resistance. Citation Format: Carl M. Gay, Lixia Diao, C. Allison Stewart, Yuanxin Xi, Robert J. Cardnell, Stephen G. Swisher, Jack A. Roth, Bonnie S. Glisson, Jing Wang, John V. Heymach, Lauren A. Byers. Inter- and intra-tumoral variations in ASCL1, NEUROD1, and POU2F3 transcriptional programs underlie three distinct molecular subtypes of small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3772.

N6-Methylation of Adenosine of FZD10 mRNA Contributes to PARP Inhibitor Resistance.

Despite the high initial response rates to PARP inhibitors (PARPi) in BRCA-mutated epithelial ovarian cancers (EOC), PARPi resistance remains a major challenge. Chemical modifications of RNAs have emerged as a new layer of epigenetic gene regulation. N6-methyladenosine (m6A) is the most abundant chemical modification of mRNA, yet the role of m6A modification in PARPi resistance has not previously been explored. Here, we show that m6A modification of FZD10 mRNA contributes to PARPi resistance by upregulating the Wnt/β-catenin pathway in BRCA-mutated EOC cells. Global m6A profile revealed a significant increase in m6A modification in FZD10 mRNA, which correlated with increased FZD10 mRNA stability and an upregulation of the Wnt/β-catenin pathway. Depletion of FZD10 or inhibition of the Wnt/β-catenin sensitizes resistant cells to PARPi. Mechanistically, downregulation of m6A demethylases FTO and ALKBH5 was sufficient to increase FZD10 mRNA m6A modification and reduce PARPi sensitivity, which correlated with an increase in homologous recombination activity. Moreover, combined inhibition of PARP and Wnt/β-catenin showed synergistic suppression of PARPi-resistant cells in vitro and in vivo in a xenograft EOC mouse model. Overall, our results show that m6A contributes to PARPi resistance in BRCA-deficient EOC cells by upregulating the Wnt/β-catenin pathway via stabilization of FZD10. They also suggest that inhibition of the Wnt/β-catenin pathway represents a potential strategy to overcome PARPi resistance. SIGNIFICANCE: These findings elucidate a novel regulatory mechanism of PARPi resistance in EOC by showing that m6A modification of FZD10 mRNA contributes to PARPi resistance in BRCA-deficient EOC cells via upregulation of Wnt/β-catenin pathway.

Acquired Resistance of EGFR-Mutated Lung Cancer to Tyrosine Kinase Inhibitor Treatment Promotes PARP Inhibitor Sensitivity

SUMMARYLung cancers with oncogenic mutations in the epidermal growth factor receptor (EGFR) invariably acquire resistance to tyrosine kinase inhibitor (TKI) treatment. Vulnerabilities of EGFR TKI-resistant cancer cells that could be therapeutically exploited are incompletely understood. Here, we describe a poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor-sensitive phenotype that is conferred by TKI treatment in vitro and in vivo and appears independent of any particular TKI resistance mechanism. We find that PARP-1 protects cells against cytotoxic reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). Compared to TKI-naive cells, TKI-resistant cells exhibit signs of increased RAC1 activity. PARP-1 catalytic function is required for PARylation of RAC1 at evolutionarily conserved sites in TKI-resistant cells, which restricts NOX-mediated ROS production. Our data identify a role of PARP-1 in controlling ROS levels upon EGFR TKI treatment, with potentially broad implications for therapeutic targeting of the mechanisms that govern the survival of oncogene-driven cancer cells.

Efficacy and pharmacodynamics of niraparib in BRCA-mutant and wild-type intracranial triple-negative breast cancer murine models.

BackgroundDespite the poor prognosis of triple-negative breast cancer (TNBC) brain metastases, there are no approved systemic therapies. We explored the DNA-damaging poly(ADP-ribose) polymerase inhibitor (PARPi) niraparib in intracranial mouse models of breast cancer susceptibility protein (BRCA)-mutant TNBC.MethodsMice bearing intracranial human-derived TNBC cell lines (SUM149, MDA-MB-231Br, or MDA-MB-436) were treated with niraparib and monitored for survival; intracranial tissues were analyzed for PAR levels and niraparib concentration by mass spectrometry. RNASeq data of primary breast cancers using The Cancer Genome Atlas were analyzed for DNA damage signatures. Combined RAD51 and PARP inhibition in TNBC cell lines was assessed in vitro by colony-forming assays.ResultsDaily niraparib increased median survival and decreased tumor burden in the BRCA-mutant MDA-MB-436 model, but not in the BRCA-mutant SUM149 or BRCA-wild-type MDA-MB-231Br models despite high concentrations in intracranial tumors. RAD51 inhibitor B02 was shown to sensitize all cell lines to PARP inhibition (PARPi). In the analysis of BRCA-mutant primary human TNBCs, gene expression predictors of PARPi sensitivity and DNA repair signatures demonstrate widespread heterogeneity, which may explain the differential response to PARPi. Interestingly, these signatures are significantly correlated to RAD51 expression including PARPi sensitivity (R2 = 0.602, R2= 0.758).ConclusionsNiraparib penetrates intracranial tumor tissues in mouse models of TNBC with impressive single-agent efficacy in BRCA-mutant MDA-MB-436. Clinical evaluation of niraparib to treat TNBC brain metastases, an unmet clinical need desperate for improved therapies, is warranted. Further compromising DNA repair through RAD51 inhibition may further augment TNBC’s response to PARPi.

The deubiquitylating enzyme USP15 regulates homologous recombination repair and cancer cell response to PARP inhibitors

Poly-(ADP-ribose) polymerase inhibitors (PARPi) selectively kill breast and ovarian cancers with defects in homologous recombination (HR) caused by BRCA1/2 mutations. There is also clinical evidence for the utility of PARPi in breast and ovarian cancers without BRCA mutations, but the underlying mechanism is not clear. Here, we report that the deubiquitylating enzyme USP15 affects cancer cell response to PARPi by regulating HR. Mechanistically, USP15 is recruited to DNA double-strand breaks (DSBs) by MDC1, which requires the FHA domain of MDC1 and phosphorylated Ser678 of USP15. Subsequently, USP15 deubiquitinates BARD1 BRCT domain, and promotes BARD1-HP1γ interaction, resulting in BRCA1/BARD1 retention at DSBs. USP15 knockout mice exhibit genomic instability in vivo. Furthermore, cancer-associated USP15 mutations, with decreased USP15-BARD1 interaction, increases PARP inhibitor sensitivity in cancer cells. Thus, our results identify a novel regulator of HR, which is a potential biomarker for therapeutic treatment using PARP inhibitors in cancers.

DNA Replication Vulnerabilities Render Ovarian Cancer Cells Sensitive to Poly(ADP-Ribose) Glycohydrolase Inhibitors

SummaryInhibitors of poly(ADP-ribose) polymerase (PARP) have demonstrated efficacy in women with BRCA-mutant ovarian cancer. However, only 15%–20% of ovarian cancers harbor BRCA mutations, therefore additional therapies are required. Here, we show that a subset of ovarian cancer cell lines and ex vivo models derived from patient biopsies are sensitive to a poly(ADP-ribose) glycohydrolase (PARG) inhibitor. Sensitivity is due to underlying DNA replication vulnerabilities that cause persistent fork stalling and replication catastrophe. PARG inhibition is synthetic lethal with inhibition of DNA replication factors, allowing additional models to be sensitized by CHK1 inhibitors. Because PARG and PARP inhibitor sensitivity are mutually exclusive, our observations demonstrate that PARG inhibitors have therapeutic potential to complement PARP inhibitor strategies in the treatment of ovarian cancer.

The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair.

P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.

CCDC6 and USP7 expression levels suggest novel treatment options in high-grade urothelial bladder cancer

BackgroundThe muscle invasive form of urothelial bladder cancer (UBC) is a deadly disease. Currently, the therapeutic approach of UBC is mostly based on surgery and standard chemotherapy.Biomarkers to establish appropriate drugs usage are missing. Deficiency of the tumor suppressor CCDC6 determines PARP-inhibitor sensitivity. The CCDC6 levels are modulated by the deubiquitinase USP7. In this work we scored CCDC6 and USP7 expression levels in primary UBC and we evaluated the expression levels of CCDC6 in correlation with the effects of the PARP-inhibitors combined with the USP7 inhibitor, P5091, in vitro. Since PARP-inhibitors could be enhanced by conventional chemotherapy or DNA damage inducers, we tested the new agent RRx-001, able to induce DNA damage, to prove the benefit of combined treatments in bladder cancer cells.MethodsThe J82, T24, 5637 and KU-19-19 bladder cancer cells were exposed to USP7 inhibitor P5091 in presence of cycloheximide to analyse the CCDC6 stability. Upon the CCDC6 degradation induced by P5091, the cells sensitivity to PARP-inhibitor was evaluated by cell viability assays. The ability of the DNA damage inducer RRx-001 to modulate CCDC6 protein levels and H2AX phosphorylation was detected at immunoblot. The combination of USP7 inhibitor plus RRx-001 enhanced the PARP-inhibitor sensitivity, as evaluated by cell viability assays. The results of the scores and correlation of CCDC6 and USP7 expression levels obtained by UBC primary biopsies staining were used to cluster patients by a K-mean cluster analysis.ResultsP5091 determining CCDC6 degradation promoted bladder cancer cells sensitivity to PARP-inhibitor drugs. RRx-001, by inducing DNA damage, enhanced the effects of the combined treatment. The immunohistochemical staining of both CCDC6 and USP7 proteins allowed to cluster the high grade (G3) UBC patients, on the basis of CCDC6 expression levels.ConclusionsIn high grade UBC the identification of two clusters of patients based on CCDC6 and USP7 expession can possibly indicate the use of PARP-inhibitor drugs, in combination with USP7 inhibitor in addition to the DNA damage inducer RRx-001, that also acts as an immunomodulatory agent, offering novel therapeutic strategy for personalized medicine in bladder cancer patients.

53BP1 as a potential predictor of response in PARP inhibitor-treated homologous recombination-deficient ovarian cancer

ObjectivePoly(ADP-ribose) polymerase (PARP) inhibitors have shown substantial activity in homologous recombination- (HR-) deficient ovarian cancer and are undergoing testing in other HR-deficient tumors. For reasons that are incompletely understood, not all patients with HR-deficient cancers respond to these agents. Preclinical studies have demonstrated that changes in alternative DNA repair pathways affect PARP inhibitor (PARPi) sensitivity in ovarian cancer models. This has not previously been assessed in the clinical setting. MethodsClonogenic and plasmid-based HR repair assays were performed to compare BRCA1-mutant COV362 ovarian cancer cells with or without 53BP1 gene deletion. Archival biopsies from ovarian cancer patients in the phase I, open-label clinical trial of PARPi ABT-767 were stained for PARP1, RAD51, 53BP1 and multiple components of the nonhomologous end-joining (NHEJ) DNA repair pathway. Modified histochemistry- (H-) scores were determined for each repair protein in each sample. HRD score was determined from tumor DNA. Results53BP1 deletion increased HR in BRCA1-mutant COV362 cells and decreased PARPi sensitivity in vitro. In 36 women with relapsed ovarian cancer, responses to the PARPi ABT-767 were observed exclusively in cancers with HR deficiency. In this subset, 7 of 18 patients (39%) had objective responses. The actual HRD score did not further correlate with change from baseline tumor volume (r = 0.050; p = 0.87). However, in the HR-deficient subset, decreased 53BP1 H-score was associated with decreased antitumor efficacy of ABT-767 (r = −0.69, p = 0.004). ConclusionDifferences in complementary repair pathways, particularly 53BP1, correlate with PARPi response of HR-deficient ovarian cancers.

Prospect for Application of PARP Inhibitor in Patients with HER2 Negative Breast Cancer.

Human epidermal growth factor receptor (HER2) negative metastatic breast cancer (BC) accounts for 73% of BC. The molecular analysis of this disease is essential for potential options for targeted therapy. Several promising clinical strategies are being evaluated which includes endocrine therapy, modified chemotherapy, angiogenesis inhibitors, immune checkpoint inhibitors, and anti-androgens. New therapeutic approaches are being developed that target BC patients with germline mutations in either BRCA1, BRCA2 as well as BRCAness, a condition in which tumors have molecular similarity to BRCA-mutated tumors. Poly (ADP-ribose) polymerase inhibitors (PARPi) which are effective therapy in germline BRCA1 and BRCA2 mutations, are also observed to be effective in somatic mutations. Germline mutations in the homologous recombination pathway genes could also contribute to PARPi sensitivity. PARPi act as chemo- and radio-sensitizers by limiting the DNA-damage response and potentiating the activity of chemo- and radio-therapy when used alone or in combination with chemotherapy. Apart from PARPi as monotherapy, additional researches are ongoing in combination with cytotoxic chemotherapeutics and targeted agents in HER2 negative BC. This review aims at the most recent developments in the targeted therapy, summarizes the recent clinical trials outcomes, along with the overview of ongoing clinical trials in HER2 negative patients with BRCA1/2 mutations and sporadic tumors with BRCAness.

Direct Ex Vivo Observation of Homologous Recombination Defect Reversal After DNA-Damaging Chemotherapy in Patients With Metastatic Breast Cancer.

Biomarkers that predict response to poly (ADP-ribose) polymerase inhibitors (PARPis) are required to detect PARPi sensitivity beyond germline BRCA-mutated (gBRCAm) cancers and PARPi resistance among reverted gBRCAm cancers. Therefore, we previously developed the Repair Capacity (RECAP) test, a functional homologous recombination (HR) assay that exploits the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh primary breast cancer tissue. The aim of the current study was to validate the feasibility of this test on histologic biopsy specimens from metastatic breast cancer and to explore the utility of the RECAP test as a predictive tool for treatment with DNA-damaging agents, such as PARPis. Fresh tissue biopsies from easily accessible metastatic lesions from patients with locally advanced or metastatic breast cancer were irradiated with 5 Gy and cultured for 2 hours followed by detection of RAD51 foci presence (HR proficient) or absence (HR deficient [HRD]). HRD biopsy specimens as well as platinum/PARP-resistant specimens were subjected to BRCA1/2 sequencing. RECAP had a success rate of 93% on biopsy specimens from metastatic breast cancer lesions (n = 44). Although HRD was detected in 13 (32%) of 41 specimens, only five showed a gBRCAm. In three patients with gBRCAm, post-treatment RECAP tests showed HR phenotype reversion after in vivo progressive disease on platinum and PARPi treatment, which was explained in one patient by a secondary BRCA1 mutation. The RECAP test, which reflects real-time HR status regardless of BRCA mutations, is feasible in metastatic breast cancer biopsy specimens. Compared with gBRCA analysis, it may identify twice as many candidates for PARPi treatment.

The F-Box Domain-Dependent Activity of EMI1 Regulates PARPi Sensitivity in Triple-Negative Breast Cancers

The BRCA1-BRCA2-RAD51 axis is essential for homologous recombination repair (HRR) and is frequently disrupted in breast cancers. PARP inhibitors (PARPis) are used clinically to treat BRCA-mutated breast tumors. Using a genetic screen, we identified EMI1 as a modulator of PARPi sensitivity in triple-negative breast cancer (TNBC) cells. This function requires the F-box domain of EMI1, through which EMI1 assembles a canonical SCF ubiquitin ligase complex that constitutively targets RAD51 for degradation. In response to genotoxic stress, CHK1-mediated phosphorylation of RAD51 counteracts EMI1-dependent degradation by enhancing RAD51's affinity for BRCA2, leading to RAD51 accumulation. Inhibition of RAD51 degradation restores HRR in BRCA1-depleted cells. Human breast cancer samples display an inverse correlation between EMI1 and RAD51 protein levels. A subset of BRCA1-deficient TNBC cells develop resistance to PARPi by downregulating EMI1 and restoring RAD51-dependent HRR. Notably, reconstitution of EMI1 expression reestablishes PARPi sensitivity both in cellular systems and in an orthotopic mouse model.

The ASCIZ-DYNLL1 axis promotes 53BP1-dependent non-homologous end joining and PARP inhibitor sensitivity

53BP1 controls a specialized non-homologous end joining (NHEJ) pathway that is essential for adaptive immunity, yet oncogenic in BRCA1 mutant cancers. Intra-chromosomal DNA double-strand break (DSB) joining events during immunoglobulin class switch recombination (CSR) require 53BP1. However, in BRCA1 mutant cells, 53BP1 blocks homologous recombination (HR) and promotes toxic NHEJ, resulting in genomic instability. Here, we identify the protein dimerization hub—DYNLL1—as an organizer of multimeric 53BP1 complexes. DYNLL1 binding stimulates 53BP1 oligomerization, and promotes 53BP1’s recruitment to, and interaction with, DSB-associated chromatin. Consequently, DYNLL1 regulates 53BP1-dependent NHEJ: CSR is compromised upon deletion of Dynll1 or its transcriptional regulator Asciz, or by mutation of DYNLL1 binding motifs in 53BP1; furthermore, Brca1 mutant cells and tumours are rendered resistant to poly-ADP ribose polymerase (PARP) inhibitor treatments upon deletion of Dynll1 or Asciz. Thus, our results reveal a mechanism that regulates 53BP1-dependent NHEJ and the therapeutic response of BRCA1-deficient cancers.

USP1 Is Required for Replication Fork Protection in BRCA1-Deficient Tumors

BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.

An Integrated Genomics Analysis Identifies Recurrent Gain- and Loss-of-Function Events and in Distinct Subtypes of Multiple Myeloma

Multiple myeloma (MM) is a pathological description for a plasma cell malignancy that exhibits a high degree of genetic diversity between patients. Whole genome analysis methods have elucidated multiple distinct subtypes of disease, however, we have a very limited understanding of what drives each independent subtype. To address this issue, we developed a comprehensive analysis approach to identify MM subtypes and associated genetic features in untreated patient samples from the MMRF CoMMpass study (NCT01454297). In this study we attempt to characterize each sample using whole genome (WGS), exome (WES), and RNA (RNAseq) sequencing. For this analysis we have limited the baseline cohort from the 982 patients with at least one assay completed to the 591 which are completely characterized.Most cohort level analyses focus on specific genetic aberrations such as copy number (CN) (eg. GISTIC) or somatic mutations (eg. MutSig) to identify genes which are important in the cancer. However, it is well known that cancer cells will dysregulate genes and pathways through a diverse array of mechanisms. We developed an integrated model, leveraging WGS, WES, and RNAseq data, that simultaneously considers; translocations, inversions, CN, copy neutral loss-of-heterozygosity, somatic mutations, constitutional inherited gene defects, gene expression, and in-frame genomically validated gene fusions; in a comprehensive analysis to aid in the discovery of putative oncogenes or tumor suppressor genes.We first defined two independent subtype classes within the cohort using consensus clustering on the gene expression and copy number data, which identified 12 and 14 subtypes, respectively. We also identified 59 significantly mutated genes using MutSig with a robust resampling approach. Then using our integrated approach we identified 64 putative loss-of-function (LOF) genes which have complete, bi-allelic loss, through anyone of 11 possible combinations of genetic defects in at least 1% of the cohort. These include expected tumor suppressors; TRAF3 (10.1%), DIS3 (6.9%), FAM46C (5.1%), TP53 (4.1%), RB1 (3.2%), and NF1 (1.0%). On chromosome 13 alone we identified 17 LOF genes with LOF events occuring in 15.4% of patients with the most common genes being PSPC1, BRCA2, RB1, DIS3, and TGDS. This process also identified LOF events in genes associated with 5-FU sensitivity, DPYD, and PARP inhibitor sensitivity, BRCA1, BRCA2, and STAG2 suggesting some patients may benefit from these agents. We identified 19 putative gain-of-function (GOF) genes, which had recurrent mutations at the same amino acid position, high level CN gains (6 or more copies), overexpression associated with a structural rearrangement, or inframe fusion transcripts in at least 1% of the cohort. These include expected oncogenes; NRAS, KRAS, BRAF, and myeloma specific genes like WHSC1. Interestingly this approach identified a series of genes that were not detected by MutSig such as IRF4, HIST1H1E, DUSP2, and MAP3K14 plus a number with recurrent suspect activating mutations; BMP2K, PTPN11, ALOX15, and ANKLE1.Few subtypes of MM have defined genetic events outside of gene expression subtypes defined by the common IgH translocations. Comparing genes identified in our LOF and GOF analysis with our gene expression or CN defined subtypes identified a number of associations. For instance, in the MS gene expression subtype, characterized by t(4;14), we detected the expected associations with WHSC1 and FGFR3 along with a significant enrichment of TRAF3 and DIS3 LOF events and an under representation of NRAS mutations. A gene expression group associated with a hyperdiploid karyotype and 1q gains has significantly fewer NRAS and KRAS GOF events but is enriched for TRAF3 LOF events. GOF events in IRF4 were exclusively found in the CD2 gene expression groups associated with cyclin D translocations. Interestingly, the LOF events in TGDS were exclusively found in a single CN defined subtype characterized by a hyperdiploid karyotype with 1q gains but lacking chromosome 11 trisomies. Finally, the gene expression defined proliferation (PR) subtype, which exhibits poor OS (HR = 3.996, 95% CI = 2.632 - 6.067, p < 0.001) is highly enriched for bi-allelic LOF events in RB1 and MAX. This finding provides the first genetic explanation for why patients with such diverse genetic backgrounds cluster in this high risk subtype. DisclosuresLonial:Amgen: Research Funding.

Targeting an RNaseH2 Defect in Chronic Lymphocytic Leukaemia with PARP Inhibitors

The therapeutic exploitation of molecular defects within the DNA damage response (DDR) in tumour cells has become an important treatment paradigm. ‘Synthetic lethality’ relies on pharmacological inhibition of pathways upon which DDR-deficient tumour cells have become dependent for their survival. This induces an intolerable level of unrepaired DNA damage in the tumour cells resulting in cell death, whilst sparing DDR-proficient normal cellsDeletion of 13q14 is a frequent, early event in the pathogenesis of CLL. Alongside well-described tumour suppressor genes this genomic region also encompasses the DDR gene, RNASEH2B, which encodes a subunit of the heterotrimeric enzymatic complex, RNaseH2. This complex is a principal component of ribonucleotide excision repair (RER), a DDR pathway that removes ribonucleotides incorporated into DNA by error prone DNA repair polymerases. If unremoved, these DNA-incorporated ribonucleotides lead to DNA damage, chromosome instability and mutagenesis (Reijns et al, Cell. 2012;149:1008).We recently reported a synthetically lethal interaction between the functional loss of RNaseH2 enzymatic complex and PARP inhibition (Zimmerman et al, Nature 2018, 559:285). We observed that inactivation of any of the three RNAseH2 subunits (A,B,C) leads to loss of enzymatic activity of this complex and also that primary CLL tumours with 13q14 deletion involving the RNASEH2B locus are sensitive to PARP inhibitors (PARPi) in vitro.In light of these preliminary observations, we addressed the following questions: a) Do monoalleic and biallelic RNASEH2B deletions have equal consequences for RNAseH2 enzymatic activity and sensitivity to PARP inhibition in CLL? d) Can loss of RNAseH2 activity be caused by an alternative mechanism, such as mutations in RNASEH2B? c) Can the PARPi sensitivity of RNaseH2-deficient CLLs be demonstrated in vivo, in patient-derived xenografts? d) Is PARP inhibition an option for RNAseH2 deficient tumours with limited response to other treatments?Analysis of 100 primary CLL tumours through a combination of multiplex ligation-dependent probe amplification (MLPA), CGH microarrays and Sanger sequencing identified 29 tumours with monoallelic and 14 with biallelic RNASEH2B deletions. None of the analysed tumours had mutations in RNASEH2B. Increased levels of genomic ribonucleotides were confirmed in all RNASEH2B deleted tumours by two complementary methods: alkaline gel electrophoresis and DNA nick translation. We found that the RNaseH2 enzymatic defect and sensitivity to PARP inhibition were evident in all RNASEH2B deleted tumours, but were more profound in those harbouring biallelic deletion compared to tumours that have lost only one RNASEH2B allele. Furthermore, sensitivity to PARP inhibitors was dependent on PARP-trapping capacity and therefore cytotoxicity was most prominent in response to PARP-inhibitors with a potent PARP trapping capacity such as talazoparib. In vivo experiments revealed similar trends, with CLL xenografts derived from tumours with biallelic RNASEH2B deletion being differentially sensitive to Talazoparib. Notably, the PARP inhibition sensitivity of RNAseH2-deficient primary CLLs was independent of patients' response to different treatments.In summary, we conclude that the RNASEH2B loss associated with 13q14 deletion represents a frequent cause of RNaseH2 enzymatic defect that renders primary CLL tumours sensitive to PARP-trapping inhibitors. Our findings expand the range of molecular defects in CLL that are amenable to treatment with clinically applicable PARP inhibitors and may have implications for the management of patients with limited response to other treatments. DisclosuresNo relevant conflicts of interest to declare.

Targeting homologous repair deficiency in breast and ovarian cancers: Biological pathways, preclinical and clinical data.

Mutation or epigenetic silencing of homologous recombination (HR) repair genes is characteristic of a growing proportion of triple-negative breast cancers (TNBCs) and high-grade serous ovarian carcinomas. Defects in HR lead to genome instability, allowing cells to acquire the multiple genetic alterations essential for cancer development. However, this deficiency can also be exploited by using DNA damaging agents or by targeting compensatory repair pathways. A noteworthy example is treatment of TNBC and epithelial ovarian cancer harboring BRCA1/2 germline mutations using platinum salts and/or PARP inhibitors. Dramatic responses to PARP inhibitors may support a wider use in the HR-deficient population beyond those with mutated germline BRCA1 and 2. In this review, we discuss HR deficiency hallmarks as predictive biomarkers for platinum salt and PARP inhibitor sensitivity for selecting patients affected by TNBC or epithelial ovarian cancer who could benefit from these therapeutic options.

Prediction of DNA Repair Inhibitor Response in Short-Term Patient-Derived Ovarian Cancer Organoids.

Based on genomic analysis, 50% of high-grade serous ovarian cancers (HGSC) are predicted to have DNA repair defects. Whether this substantial subset of HGSCs actually have functional repair defects remains unknown. Here, we devise a platform for functional profiling of DNA repair in short-term patient-derived HGSC organoids. We tested 33 organoid cultures derived from 22 patients with HGSC for defects in homologous recombination (HR) and replication fork protection. Regardless of DNA repair gene mutational status, a functional defect in HR in the organoids correlated with PARP inhibitor sensitivity. A functional defect in replication fork protection correlated with carboplatin and CHK1 and ATR inhibitor sensitivity. Our results indicate that a combination of genomic analysis and functional testing of organoids allows for the identification of targetable DNA damage repair defects. Larger numbers of patient-derived organoids must be analyzed to determine whether these assays can reproducibly predict patient response in the clinic.Significance: Patient-derived ovarian tumor organoids grow rapidly and match the tumors from which they are derived, both genetically and functionally. These organoids can be used for DNA repair profiling and therapeutic sensitivity testing and provide a rapid means of assessing targetable defects in the parent tumor, offering more suitable treatment options. Cancer Discov; 8(11); 1404-21. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333.