Recombination Proficiency Research Articles

Safety and tolerability of olaparib combined with breast radiotherapy in patients with triple-negative breast cancer: Final results of the RADIOPARP phase 1 trial.

534 Background: Preclinical studies have demonstrated that triple-negative breast cancer (TNBC) cells were sensitive to PARP inhibitors when used as radiosensitizers. Combining PARP-inhibitors with radiotherapy may consequently enhance the biological effectiveness of the irradiation, leading to improved locoregional control in TNBC patients. We aimed to establish the appropriate dosing and the safety profile of Olaparib used as a radiosensitizer in combination with radiotherapy in TNBC patients with residual disease after neoadjuvant chemotherapy. Methods: RADIOPARP (NCT03109080) was a prospective phase I dose-escalation trial establishing the tolerance profile of Olaparib combined with breast radiotherapy in high-risk early TNBC patients. Inclusion criteria were resected TNBC with non-complete pathological response after neoadjuvant chemotherapy, or unresectable TNBC despite prior neoadjuvant chemotherapy. Olaparib was started seven days before irradiation and continued during radiotherapy. A time-to-event continual reassessment method was used to increase Olaparib through four increasing dose levels (50mg, 100mg, 150mg or 200mg twice a day) with a 25% maximum probability rate of dose-limiting toxicities (DLT). Radiotherapy delivered 50 Gy to the breast or to the chest wall, with or without lymph node irradiation. Toxicities were graded according to CTCAE (version 4.03). Homologous recombination (HR) proficiency status was genetically determined based on shallow whole genome sequencing. Results: Twenty-four TNBC patients were enrolled between 09/2017 and 11/2019. Olaparib was escalated to 200 mg twice a day without DLT and the MTD was not reached. With a median follow-up of 34 months, no late treatment-related grade ≥ 3 toxicity was observed, and the maximum observed toxicities were limited to grade 2 breast pain (n=2), fibrosis (n=2), deformity (n=1) and telangiectasia (n=1). Three-year OS and EFS were 83% [95% CI: 70%-100%] and 65% [51%-91%], respectively. HR proficiency status was not associated with OS and EFS. Conclusions: Olaparib used as a radiosensitizer in combination with radiotherapy in TNBC patients was well-tolerated. The MTD was not reached, and no significant late toxicity was reported. For future trials evaluating the anti-tumor efficacy of this combination, an Olaparib dose of 200 mg twice a day should be considered. Clinical trial information: NCT03109080.

Evaluation of homologous recombination deficiency biomarkers in patients with ovarian cancer treated with PARP inhibitors.

e17615 Background: Germline BRCA1/2 mutations are associated with response to Poly(ADP-ribose) polymerase inhibitors (PARPi). In addition to BRCA1/2 mutations, there are other Homologous Recombinations Deficiency (HRD) biomarker candidates already available in clinical practice including genome-wide loss-of-heterozygosity (gLOH) and myChoice score. Inconsistencies in biomarkers used in clinical trials with PARPi are a challenge to clinical interpretation. This study aims to compare clinically available HRD biomarkers in terms of benefits from PARPi. Methods: We performed database search for phase II or III randomized clinical trials comparing PARPi versus chemotherapy, and meta-analysis using generic inverse variance and a random-effects model. Patients were classified into three categories according to their HRD status: 1) BRCAmut (patients with BRCA1/2 mutation of germline or somatic origin), 2) non-BRCA HRD (patients BRCA wild-type (wt) with a different HRD biomarker - gLOH or myChoice); and 3) homologous recombination proficiency (HRP) (BRCAwt and without HRD biomarkers). From those that were BRCAwt, we compared myChoice+ with gLOH-high. Results: Eight studies (n = 4372 patients) with PARPi as first-line and recurrence settings were included. BRCAmut had PFS HR 0.29 (95%CI, 0.24-0.35), BRCAwt & HRD 0.43 (95%CI, 0.34-0.53) and HRP 0.74 (95%CI, 0.62-0.89). In a secondary analysis by HRD stratification method, patients with BRCAwt & myChoice > = 42 had PFS HR 0.43 (95%CI, 0.34-0.56), similar to patients with BRCAwt & gLOH-high with PFS HR 0.42 (95%CI, 0.28-0.62). Conclusions: Patients with BRCA mutations benefited the most from PARPi. From patients with BRCA1/2 wild-type, a similar benefit was found in patients with gLOH-high and those myChoice+, which was superior to HRP patients. The clinical development of further HRD biomarkers (i.e. Sig3, HRDetect) may help identify more patients who benefit from PARPi.

Performance and clinical utility of homologous recombination deficiency (HRD) determined by genome-wide loss of heterozygosity (LOH).

e15025 Background: Homologous recombination deficiency (HRD) status plays an important role in identifying patients with ovarian cancer likely to benefit from Poly (ADP-ribose) polymerase inhibitor (PARPi) and platinum-based chemotherapies. Methods: ACTHRD is an NGS-based assay was designed to determine the homologous recombination deficiency (HRD) status based on the extent of genome-wide loss of heterozygosity (gLOH) and the detection of BRCA1/2 genes alteration. More than that, ACTHRD also assesses the mutational status of 22 homologous recombination repair (HRR) genes. We evaluated the performance and clinical utility of the ACTHRD and compared it with the FDA-approved companion test myChoice CDx. Results: Of the 36 specimens assayed, 24 were determined to be HRD, and 10 were determined to be homologous recombination repair proficient (HRP). The results showed high concordance with myChoice CDx with 100% positive percent agreement (PPA) and 90.91% negative percent agreement (NPA) in the HRD status estimation. Except one case showed HRD in the ACTHRD but showed homologous recombination proficiency (HRP) in the comparator panel. This patient responded to platinum-based chemotherapy and remained disease-free for up to 56 months. One BRCA1/2 wild-type patient was identified as HRD by myChoice CDx but not eligible for ACTHRD test due to low tumor cellularity experienced refractory 13 months after the initiation of the treatment. We further found that ovarian cancer patients identified as HRD by ACTHRD had significantly prolonged progression-free survival (PFS) after being treated with platinum-containing chemotherapies, regardless of BRCA1/2 mutational status. Conclusions: The ACTHRD assay provides high accuracy for identifying deleterious BRCA1/2 alterations and determining the HRD status of ovarian cancers. The analytical robustness of the test has been validated and indicates its suitability for clinical use. Notably, the prognosis data demonstrates that ACTHRD can predict eligible patients for platinum-based chemotherapy and may also be sensitive to PARPi treatment.

A Novel CDK4/6 and PARP Dual Inhibitor ZC-22 Effectively Suppresses Tumor Growth and Improves the Response to Cisplatin Treatment in Breast and Ovarian Cancer.

In recent years, three PARP inhibitors and three CDK4/6 inhibitors have been approved by the FDA for the treatment of recurrent ovarian cancer and advanced ER-positive breast cancer, respectively. However, the clinical benefits of the PARPi or CDK4/6i monotherapy are not as satisfied as expected and benefit only a fraction of patients. Current studies have shown therapeutic synergy for combinations of PARPi and CDK4/6i in breast and ovarian cancers with homologous recombination (HR) proficiency, which represents a new synthetic lethal strategy for treatment of these cancers regardless HR status. Thus, any compounds or strategies that can combine PARP and CDK4/6 inhibition will likely have great potential in improving clinic outcomes and in benefiting more patients. In this study, we developed a novel compound, ZC-22, that effectively inhibited both PARP and CDK4/6. This dual-targeting compound significantly inhibited breast and ovarian cancer cells by inducing cell cycle arrest and severe DNA damage both in vitro and in vivo. Interestingly, the efficacy of ZC-22 is even higher than the combination of PARPi Olaparib and CDK4/6i Abemaciclib in most breast and ovarian cancer cells, suggesting that it may be an effective alternative for the PARPi and CDK4/6i combination therapy. Moreover, ZC-22 sensitized breast and ovarian cancer cells to cisplatin treatment, a widely used chemotherapeutic agent. Altogether, our study has demonstrated the potency of a novel CDK4/6 and PARP dual inhibitor, which can potentially be developed into a monotherapy or combinatorial therapy with cisplatin for breast and ovarian cancer patients with HR proficiency.

Ploidy and recombination proficiency shape the evolutionary adaptation to constitutive DNA replication stress.

In haploid budding yeast, evolutionary adaptation to constitutive DNA replication stress alters three genome maintenance modules: DNA replication, the DNA damage checkpoint, and sister chromatid cohesion. We asked how these trajectories depend on genomic features by comparing the adaptation in three strains: haploids, diploids, and recombination deficient haploids. In all three, adaptation happens within 1000 generations at rates that are correlated with the initial fitness defect of the ancestors. Mutations in individual genes are selected at different frequencies in populations with different genomic features, but the benefits these mutations confer are similar in the three strains, and combinations of these mutations reproduce the fitness gains of evolved populations. Despite the differences in the selected mutations, adaptation targets the same three functional modules in strains with different genomic features, revealing a common evolutionary response to constitutive DNA replication stress.

Association of BRCA1/2, homologous recombination deficiency, and PD-L1 with clinical outcomes in patients receiving atezolizumab versus placebo combined with carboplatin, paclitaxel, and bevacizumab for newly diagnosed ovarian cancer: exploratory analyses of IMagyn050/GOG3015/ENGOT-ov39

<h3>Objectives:</h3> Genomically unstable tumors, characterized by <i>BRCA1/2</i> alterations and homologous recombination deficiency (HRD), are hypothesized to be more mutated and potentially more sensitive to immune checkpoint inhibitors. To explore this hypothesis we analyzed outcomes in the IMagyn050 trial according to <i>BRCA1/2,</i> HRD, and PD-L1 status [Moore, ESMO 2020]. <h3>Methods:</h3> IMagyn050 (NCT03038100) is a double-blind randomized phase III trial evaluating the efficacy and safety of adding atezolizumab/placebo to carboplatin, paclitaxel, and bevacizumab (CPB) followed by maintenance bevacizumab plus atezolizumab/placebo. PD-L1 status was determined centrally using VENTANA SP142 (PD-L1+ defined as ≥1% tumor-infiltrating immune cells expressing PD-L1). Deleterious tumor germline/somatic <i>BRCA1/2</i> alterations (<i>BRCA1/2</i>m), genomic loss of heterozygosity (gLOH), tumor mutation burden (TMB), and microsatellite instability (MSI) were evaluated using the FoundationOne assay (Foundation Medicine, Inc., Cambridge, MA). HRD and homologous recombination proficiency (HRP) were defined as gLOH ≥16% and <16%, respectively, regardless of <i>BRCA1/2</i>m status. Potential associations between clinical outcome (RECIST-defined progression-free survival [PFS]) and <i>BRCA1/2,</i> HRD, and PD-L1 status were evaluated using standard correlation analyses and Kaplan-Meier estimates. <h3>Results:</h3> Among 1301 randomized patients, samples from 1050 were evaluable for <i>BRCA1/2</i> (22% were <i>BRCA1/2</i>m, 78% <i>BRCA1/2</i> nonmutant) and 980 were evaluable for gLOH (46% were HRD, 54% HRP). Median TMB was similarly low in <i>BRCA1/2</i>m and <i>BRCA1/2</i> nonmutant (3.78 vs 2.52 Mut/Mb, respectively), and HRD and HRP (3.78 vs 2.52 Mut/Mb, respectively) tumors. Only 3% (29/1024) of evaluable tumors had TMB ≥10 Mut/Mb and 0.3% (3/1022) were MSI-high (1 mixed, 1 undifferentiated, 1 other). All high-grade serous cases were MS-stable. PFS prognosis (assessed in the placebo + CPB arm) was improved in patients with <i>BRCA1/2</i>m (hazard ratio [HR] 0.62, 95% CI 0.46–0.84) and HRD (HR 0.63, 95% CI 0.49–0.80) tumors. There was a suggested association between PD-L1+ and HRD (HRD prevalence: 40% vs 25% in PD-L1+ vs PD-L1– subgroups, respectively; exploratory Fisher's exact test p=0.0001) but not with <i>BRCA1/2</i>m (<i>BRCA1/2</i>m prevalence: 21% vs 14%, respectively; exploratory Fisher exact test p=0.064). Adding atezolizumab to CPB did not improve PFS, irrespective of <i>BRCA1/2</i> or HRD status (table). In the PD-L1+ population, HRs were similar in <i>BRCA1/2</i>m and <i>BRCA1/2</i> nonmutant subgroups, and in HRD and HRP subgroups. In the PD-L1– population, atezolizumab did not improve PFS. <h3>Conclusions:</h3> We showed that the majority of ovarian tumors have low TMB scores regardless of <i>BRCA1/2</i> or HRD. Neither <i>BRCA1/2</i>m nor HRD was associated with greater clinical benefit from adding atezolizumab to CPB. PD-L1 status was more reliably associated with numerically longer PFS with atezolizumab + CPB. This is the first randomized double-blind trial in ovarian cancer to demonstrate that genomic instability triggered by <i>BRCA1/2</i>m or HRD does not improve sensitivity to immune checkpoint inhibitors.

DDX11 loss causes replication stress and pharmacologically exploitable DNA repair defects

DDX11 encodes an iron-sulfur cluster DNA helicase required for development, mutated, and overexpressed in cancers. Here, we show that loss of DDX11 causes replication stress and sensitizes cancer cells to DNA damaging agents, including poly ADP ribose polymerase (PARP) inhibitors and platinum drugs. We find that DDX11 helicase activity prevents chemotherapy drug hypersensitivity and accumulation of DNA damage. Mechanistically, DDX11 acts downstream of 53BP1 to mediate homology-directed repair and RAD51 focus formation in manners nonredundant with BRCA1 and BRCA2. As a result, DDX11 down-regulation aggravates the chemotherapeutic sensitivity of BRCA1/2-mutated cancers and resensitizes chemotherapy drug-resistant BRCA1/2-mutated cancer cells that regained homologous recombination proficiency. The results further indicate that DDX11 facilitates recombination repair by assisting double strand break resection and the loading of both RPA and RAD51 on single-stranded DNA substrates. We propose DDX11 as a potential target in cancers by creating pharmacologically exploitable DNA repair vulnerabilities.

Cost-effectiveness of Maintenance Therapy Based on Molecular Classification Following Treatment of Primary Epithelial Ovarian Cancer in the United States

There are large randomized clinical trials-SOLO-1 (Olaparib Maintenance Monotherapy in Patients With BRCA Mutated Ovarian Cancer Following First Line Platinum Based Chemotherapy [December 2018]), PRIMA (A Study of Niraparib Maintenance Treatment in Patients With Advanced Ovarian Cancer Following Response on Front-Line Platinum-Based Chemotherapy [September 2019]), and PAOLA-1 (Platine, Avastin and Olaparib in 1st Line [December 2019])-reporting positive efficacy results for maintenance regimens for women with primary, advanced epithelial ovarian cancer. The findings resulted in approval by the US Food and Drug Administration of the treatments studied as of May 2020. However, there are pressing economic considerations given the many eligible patients and substantial associated costs. To evaluate the cost-effectiveness of maintenance strategies for patients with (1) a BRCA variant, (2) homologous recombination deficiency without a BRCA variant, or (3) homologous recombination proficiency. In this economic evaluation of the US health care sector using simulated patients with primary epithelial ovarian cancer, 3 decision trees were developed, one for each molecular signature. The maintenance strategies evaluated were olaparib (SOLO-1), olaparib-bevacizumab (PAOLA-1), bevacizumab (PAOLA-1), and niraparib (PRIMA). Base case 1 assessed olaparib, olaparib-bevacizumab, bevacizumab, and niraparib vs observation of a patient with a BRCA variant. Base case 2 assessed olaparib-bevacizumab, bevacizumab, and niraparib vs observation in a patient with homologous recombination deficiency without a BRCA variant. Base case 3 assessed olaparib-bevacizumab, bevacizumab, and niraparib vs observation in a patient with homologous recombination proficiency. The time horizon was 24 months. Costs were estimated from Medicare claims, wholesale acquisition prices, and published sources. Probabilistic sensitivity analyses with microsimulation were then conducted to account for uncertainty and assess model stability. One-way sensitivity analyses were also performed. The study was performed from January through June 2020. Incremental cost-effectiveness ratios (ICERs) in US dollars per progression-free life-year saved (PF-LYS). Assuming a willingness-to-pay threshold of $100 000/PF-LYS, none of the drugs could be considered cost-effective compared with observation. In the case of a patient with a BRCA variant, olaparib was the most cost-effective (ICER, $186 777/PF-LYS). The third-party payer price per month of olaparib would need to be reduced from approximately $17 000 to $9000 to be considered cost-effective. Olaparib-bevacizumab was the most cost-effective in the case of a patient with homologous recombination deficiency without a BRCA variant (ICER, $629 347/PF-LYS), and bevacizumab was the most cost-effective in the case of patient with homologous recombination proficiency (ICER, $557 865/PF-LYS). Even at a price of $0 per month, niraparib could not be considered cost-effective as a maintenance strategy for patients with homologous recombination proficiency. The findings of this study suggest that, at current costs, maintenance therapy for primary ovarian cancer is not cost-effective, regardless of molecular signature. For certain therapies, lowering the drug price alone may not make them cost-effective.

Abstract 6280: CDK4/6 inhibitor-mediatated radiosensitization of estrogen receptor positive breast cancer

Abstract Purpose: Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors like palbociclib, ribociclib, and abemaciclib have improved progression free survival in patients with metastatic, estrogen receptor positive (ER+) breast cancer, acquired resistance to these drugs limits their efficacy. Despite promising new studies defining the utility of CDK4/6 inhibitors in the upfront, non-metastatic setting, there is limited data available on the effects of concurrent CDK4/6 inhibition and radiation (RT). Methods: Transcriptomic and proteomic expression data was used to quantify changes in RNA and protein expression in ER+ breast cancer cell lines (MCF-7, T47D) after short term (16 hour) CDK4/6 inhibition. Proliferation assays were used to determine the half-maximal inhibitory concentration (IC50) of palbociclib, ribociclib, and abemaciclib. Clonogenic survival assays were performed to calculate the radiation enhancement ratio (rER) and the surviving fraction at 2 Gy for each treatment. Homologous recombination (HR) proficiency was assessed using RAD51 and γH2AX foci formation and a pYFP reporter was used to assess non-homologous end joining (NHEJ) efficiency. Western blots were used to quantify protein expression. MCF-7 xenografts were used to study the efficacy of combination (palbociclib + RT) therapy in vivo. MCF-7 and T47D cell lines with acquired resistance to CDK4/6 inhibition (IC50 &amp;gt;1uM) were used for comparison in all assays. Results: Transcriptomic and proteomic analyses identified changes in expression of DNA damage response mediators and cell cycle machinery with short term CDK4/6 inhibition. Palbociclib significantly radiosensitized ER+ cell lines at concentrations at or below the IC50 value in clonogenic survival assays (MCF-7 rER: 1.22-1.52, T47D rER: 1.23-1.50) and led to a decrease in the surviving fraction of cells at 2 Gy (p &amp;lt; 0.001). Similar results were observed in ribociclib- (rER: 1.08 - 1.68) and abemaciclib-treated (rER: 1.19 - 2.05) cells. MCF-7 and T47D cells treated with CDK4/6 inhibition and RT showed a decrease in RAD51 foci formation, suggesting a decrease in HR efficiency (p &amp;lt; 0.001). However, CDK4/6 inhibition did not affect NHEJ efficiency (p &amp;gt; 0.05). CDK4/6 inhibition + RT led to a decrease in expression of protein expression of HR meditators like p-CHK1 but did not affect phosphorylation of NHEJ proteins like pKu80/pKu70. Cells with acquired resistance to CDK4/6 inhibition did not demonstrate radiosensitization (MCF-7 rER: 0.93 - 1.03, T47D rER: 0.96 - 1.11) or changes in RAD51 foci formation with combination treatment. Conclusions: Our data suggests that CDK4/6 inhibitor-mediated radiosensitization may be effective in ER+ breast cancers prior to the development of CDK4/6 inhibitor resistance. These studies provide preclinical rationale to test CDK4/6 inhibition + RT in women with locally-advanced ER+ breast cancer at high risk for locoregional recurrence. Citation Format: Andrea M. Pesch, Nicole Hirsh, Benjamin C. Chandler, Anna R. Michmerhuizen, Cassandra L. Ritter, Marlie Androsiglio, Kari Wilder-Romans, Meilan Liu, Christina L. Gersch, Jose M. Larios, James M. Rae, Corey W. Speers. CDK4/6 inhibitor-mediatated radiosensitization of estrogen receptor positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6280.

Abstract A35: Targeting the IDH1-mediated metabolic-epigenetic axis in cyclin E-high ovarian cancer

Abstract Cyclin E is an oncogene that is overexpressed in approximately 20% of high-grade serous ovarian carcinomas (HGSOC) and is associated with worse survival and resistance to DNA-damaging agents due to homologous recombination (HR) proficiency. Additionally, cyclin E is considered an initiating factor in the transformation of fallopian tube cells, the cell of origin of HGSOC. In normal cells, increased cyclin E induces replication stress and subsequent DNA damage that leads to cellular senescence, a state of cell cycle arrest. Previous studies demonstrated that cyclin E overexpression in fallopian tube increases expression of DNA damage response (DDR) genes. This presumably allows for tolerance of replication stress, repair of DNA damage, and bypass of senescence. However, the mechanism by which cyclin E-high cells bypass senescence and increase DDR gene expression remains unclear. We previously published that the TCA cycle enzyme isocitrate dehydrogenase I (IDH1) is high in HGSOC cells compared to fallopian tube. Importantly, IDH1 is critical for HGSOC cell proliferation as knockdown or inhibition of IDH1-induced senescence through increased repressive histone H3K9me2 at multiple proliferation-promoting gene loci. Excitingly, we observed a positive correlation between high cyclin E expression and high IDH1 in both fallopian tube and HGSOC cells. Indeed, overexpression of cyclin E increased IDH1 expression in both fallopian tube and HGSOC cells. Therefore, we hypothesized that upregulation of IDH1 in cyclin E high cells increases DDR gene expression by altering the epigenetic landscape and thereby suppressing senescence induction. Our data demonstrate that IDH1 overexpression in fallopian tube phenocopies cyclin E overexpression by increasing expression of several DDR genes, including BRCA2. Indeed, knockdown of IDH1 in cyclin E-high cells decreased DDR gene expression, demonstrating that IDH1 is necessary for DDR gene expression in cyclin E-high cells. Mechanistically, we determined that this is through a metabolic-epigenetic axis, which regulates occupancy of repressive H3K9 methylation at DDR gene loci. Finally, to determine whether the alteration in DDR gene expression is functionally relevant, we treated cyclin E-high PARPi-resistant cells with an IDH1 inhibitor. While neither inhibitor affected proliferation of cyclin E-high HGSOC cells alone, the combination was highly synergistic, suggesting that the IDH1 inhibitor converted HR-proficient cyclin E-high cells to an HR-deficient phenotype. Together, our data suggest that IDH1-mediated metabolism affects the epigenome in cyclin E-high cells, which contributes to both fallopian tube transformation and HR proficiency. Targeting IDH1 with currently FDA-approved inhibitors may therefore be a rational therapeutic strategy for cyclin E-high HGSOC patients. Citation Format: Erika S. Dahl, Raquel Buj, Qingyuan Jia, Kelly E. Leon, Katherine M. Aird. Targeting the IDH1-mediated metabolic-epigenetic axis in cyclin E-high ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A35.

Methylation of the central transcriptional regulator KLF4 by PRMT5 is required for DNA end resection and recombination

Cell fitness and survival upon exposure to DNA damage depends on the repair of DNA lesions. Interestingly, cellular identity does affect and finetunes such response, although the molecular basis of such differences between tissues and cell types is not well understood. Thus, a possibility is that DNA repair itself is controlled by the mechanisms that govern cell identity. Here we show that the KLF4, involved in cellular homeostasis, proliferation, cell reprogramming and cancer development, directly regulates resection and homologous recombination proficiency. Indeed, resection efficiency follows KLF4 protein levels, i.e. decreases upon KLF4 downregulation and increases when is overexpressed. Moreover, KLF4 role in resection requires its methylation by the methyl-transferase PRMT5. Thus, PRMT5 depletion not only mimics KLF4 downregulation, but also showed an epistatic genetic relationship. Our data support a model in which the methylation of KLF4 by PRMT5 is a priming event required to license DNA resection and homologous recombination.

Isomerization of BRCA1–BARD1 promotes replication fork protection

The integrity of genomes is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi anaemia proteins protect stalled replication forks from degradation by nucleases, through pathways that involve RAD51. The contribution and regulation of BRCA1 in replication fork protection, and how this role relates to its role in homologous recombination, is unclear. Here we show that BRCA1 in complex with BARD1, and not the canonical BRCA1-PALB2 interaction, is required for fork protection. BRCA1-BARD1 is regulated by a conformational change mediated by the phosphorylation-directed prolyl isomerase PIN1. PIN1 activity enhances BRCA1-BARD1 interaction with RAD51, thereby increasing the presence of RAD51 at stalled replication structures. We identify genetic variants of BRCA1-BARD1 in patients withcancer that exhibit poor protection of nascent strands but retain homologous recombination proficiency, thus defining domains of BRCA1-BARD1 that are required for fork protection and associated with cancer development. Together, these findings reveal a BRCA1-mediated pathway that governs replication fork protection.

Abstract CT050: Expansion cohort of Phase I study of oral sapacitabine and oral seliciclib in patients with metastatic breast cancer and BRCA1/2 mutations

Abstract Introduction: Sapacitabine, a nucleoside analog, and seliciclib, a cyclin-dependent kinase 2/9 inhibitor, constitute a novel oral regimen aimed at augmenting DNA damage and impairing cell cycle checkpoints. The initial phase I cohort investigating this combination demonstrated a 25% response rate in BRCA carriers. Hence, we developed an expansion cohort to assess the safety and efficacy of this regimen in patients with metastatic breast cancer and BRCA1/2 mutations. Methods: We enrolled 20 patients with HER2-negative metastatic breast cancer and germline or somatic BRCA1/2 mutations, who were treated with sapacitabine 50 mg twice daily for days 1-7 followed by seliciclib 800 mg twice daily for days 8-10 of a 21-day cycle. Baseline or archival biopsies underwent RAD51 immunohistochemistry to assess for functional homologous recombination proficiency. Available tissue was sent for whole exome and transcriptome sequencing, and pre- and post-treatment blood was submitted for cell-free DNA sequencing to assess for genomic correlates of response. Results: Participants received a median of 2 prior lines of chemotherapy for metastatic disease. Of the 9 patients who received a prior platinum agent, 6 progressed on this therapy. In addition to chemotherapy, 7 patients received and progressed on a prior PARP inhibitor. The overall response rate for sapacitabine and seliciclib in this cohort was 10%, consisting of 2 patients with partial responses lasting 4.7 and 9.0 months, respectively. The clinical benefit rate (CR + PR + SD ≥ 6 months) was 30%, and durations of stable disease ≥ 6 months ranged from 7.4 to 11.7 months. For all patients, median PFS was 3.7 months. The most frequent grade 3/4 adverse events were neutropenia (25% of patients), transaminitis (20%), and rash (10%). No patients who progressed on prior PARP inhibitor therapy and 6 of 13 patients (46%) with no history of PARP inhibitor resistance experienced clinical benefit (p = 0.052 by Fisher’s exact test). In contrast, 1 of 6 patients (17%) who progressed on prior platinum chemotherapy and 5 of 14 patients (36%) with no history of platinum resistance experienced clinical benefit (p = 0.61 by Fisher’s exact test). Notably, the tumors of some resistant patients harbored BRCA reversion mutations. Additional genomic analyses and RAD51 immunohistochemistry will be presented. Conclusions: The combination of sapacitabine and seliciclib was safe and led to durable clinical benefit in some patients with metastatic breast cancer and BRCA1/2 mutations. Prior progression on PARP inhibitors predicted resistance to this combination, associated in some cases with BRCA reversion mutations. Based on these results, the combination of sapacitabine and the PARP inhibitor olaparib is now being investigated in patients with PARP-naïve metastatic HER2-negative breast cancer and germline BRCA1/2 mutations. Citation Format: Tanya Keenan, David Liu, Haitham Elmarakeby, Daniel Stover, Bose Kochupurakkal, Adam Tracy, Elaine Danielczyk, Leilani Anderson, Chelsea Andrews, Brendan Reardon, Beth Overmoyer, Eric Winer, Daniella Zheleva, Judy Chiao, David Blake, Eliezer Van Allen, Geoffrey I. Shapiro, Sara Tolaney. Expansion cohort of Phase I study of oral sapacitabine and oral seliciclib in patients with metastatic breast cancer and BRCA1/2 mutations [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 CT050.

Association of colon cancer (CC) molecular signatures with prognosis and oxaliplatin prediction-benefit in the MOSAIC Trial (Multicenter International Study of Oxaliplatin/5FU-LV in the Adjuvant Treatment of Colon Cancer).

3503 Background: MOSAIC and C-07 showed that oxaliplatin (OX) added to 5-fluorouracil plus leucovorin significantly improved disease free survival (DFS). However, OX is associated with neurotoxicity and the vast majority of patients do not receive OX-benefit; highlighting the importance of an OX-benefit predictor. In C-07, colon tumors with a CRCA (Colorectal Cancer Assigner) stem-like subtype were associated with a poor prognosis and no OX-benefit, but stage III patients with an enterocyte subtype did. We tested the association of CRCA and CMS molecular subtypes with prognosis and OX-benefit in stage III MOSAIC patients. Also, recombination proficiency scores (RPS) were tested for the same associations. RPS scores quantify the efficiency of DNA-damage repair. Low RPS have been associated with inferior overall survival (OS) in non-small cell lung carcinoma patients treated with surgery alone but better OS for patients who received surgery plus chemotherapy. Methods: Gene expression profiles from 590 stage III pts with follow up were successfully profiled by a custom designed nCounter code set. CRCA subtypes were determined by a locked down algorithm based on a re-estimated centroid using 72 genes (Song et al 2016). CMS subtypes were determined by modified single sample predictor (SSP) using 84 genes. RPS scores were determined as previously described by Pitroda et al (2014). Signature predictions were made while blinded to clinical outcome and signature performance was evaluated while blinded to gene expression. Results: The stem-like subtype was associated with a very poor prognosis (Stem-like vs others HR=1.56, p&lt;0.01) and no OX-benefit. CRCA and CMS subtypes did not associate with OX-benefit. Using a median cut point, stage III patients with low RPS scores received significant OX-benefit (HR=0.67, p=0.033, N=290) and patients with high RPS scores did not (HR=1.2, p=0.32, N=300) with significant interaction p=0.025. Conclusions: The observation in C-07, that the stem-like subtype has the poorest prognosis and did not receive OX-benefit, was validated in MOSAIC, identifying patients who need new therapies. RPS scores may help to identify the subset of patients with OX-benefit in stage III CC, confirmation of this observation is currently being investigated in C-07. Support: PA DoH; NSABP; Sanofi.

Low Recombination Proficiency Score (RPS) Predicts Heightened Sensitivity to DNA-Damaging Chemotherapy in Breast Cancer.

Purpose: Molecular-based cancer tests have been developed to augment the standard clinical and pathologic features used to tailor treatments to individual breast cancer patients. Homologous recombination (HR) repairs double-stranded DNA breaks and promotes tolerance to lesions that disrupt DNA replication. Recombination Proficiency Score (RPS) quantifies HR efficiency based on the expression of four genes involved in DNA damage repair. We hypothesized low RPS values can identify HR-deficient breast cancers most sensitive to DNA-damaging chemotherapy.Experimental Design: We collected pathologic tumor responses and tumor gene expression values for breast cancer patients that were prospectively enrolled on clinical trials involving preoperative chemotherapy followed by surgery (N = 513). We developed an algorithm to calculate breast cancer-specific RPS (RPSb) values on an individual sample basis.Results: Low RPSb tumors are approximately twice as likely to exhibit a complete pathologic response or minimal residual disease to preoperative anthracycline-based chemotherapy as compared with high RPSb tumors. Basal, HER2-enriched, and luminal B breast cancer subtypes exhibit low RPSb values. In addition, RPSb predicts treatment responsiveness after controlling for clinical and pathologic features, as well as intrinsic breast subtype.Conclusions: Overall, our findings indicate that low RPS breast cancers exhibit aggressive features at baseline, but they have heightened sensitivity to DNA-damaging chemotherapy. Low RPSb values in basal, HER2-enriched, and luminal B subtypes provide a mechanistic explanation for their clinical behaviors and genomic instability. RPSb augments standard clinical and pathologic features used to tailor treatments, thereby enabling more personalized treatment strategies for individual breast cancer patients. Clin Cancer Res; 23(15); 4493-500. ©2017 AACR.

Abstract 2796: Development of a RAD51-based assay for determining homologous recombination proficiency and PARP inhibitor sensitivity

Abstract Homologous recombination (HR) repair deficiency confers sensitivity to inhibitors of poly(ADP-ribose) polymerase (PARP). To date, the identification of tumors with impaired HR has relied on genomic features, including mutational signature, LOH-based HRD assays or gene expression analyses defining ‘BRCAness’. These tests analyze history of the tumor rather than providing a functional assessment of HR status at the time of diagnosis. Therefore, development of a functional assay for HR status in tumors is essential to make accurate treatment decisions. Here, we describe a RAD51-based immunohistochemical (IHC) assay that identifies HR status. We first screened commercial anti-RAD51 antibodies and identified a monoclonal antibody that detects RAD51 foci in HR-competent normal fibroblasts and shows no evidence of foci in HR-deficient (BRCA2-/-) VU423 fibroblasts after γ-irradiation. Conditions for detecting RAD51 foci in FFPE samples were identified using HR-deficient and HR-proficient triple-negative breast cancer cell lines. HR-deficient, PARP inhibitor-sensitive cell lines exhibited high levels of nuclear RAD51 and no evidence of foci, whereas HR-proficient, PARP inhibitor-resistant cells had low levels of nuclear RAD51 and foci. This result was confirmed in a BRCA1-mutated, PARP inhibitor-sensitive PDX model, where there was no evidence of foci although RAD51 levels were high. We further evaluated the pattern of RAD51 staining in 13 high-grade serous ovarian cancer (HGSOC) PDX models, for which sensitivity to olaparib had been characterized. The only olaparib-sensitive model demonstrated complete absence of RAD51 staining. Among the other 12 olaparib-resistant models, RAD51 foci were detectable, both before and after irradiation. The presence or absence of RAD51 foci correlated with olaparib sensitivity and not with BRCA mutation status. Therefore, tumors that are HR-deficient and PARP inhibitor-sensitive are characterized by either high RAD51 nuclear staining without foci, or absence of RAD51 staining. To validate these findings, we analyzed RAD51 staining patterns in a cohort of 50 primary HGSOCs from patients subsequently treated with platinum-based chemotherapy. Among these 50 samples, 45 demonstrated either RAD51 nuclear staining without foci or an absence of RAD51 staining. Five samples demonstrated RAD51 staining with foci. The median survivals of these groups were 6.1 and 1.5 years, respectively. In conclusion, we have developed a robust IHC assay for determining the functional HR-status in tumor samples. Further work will be required to determine if the staining patterns observed predict PARP inhibitor sensitivity among primary patient samples. Funded by a Biomarker Supplement to UM1 CA186709, NIH Grant P50 CA168504, SU2C Ovarian Cancer Dream Team and BCRF grant. Citation Format: Bose S. Kochupurakkal, Kalindi Parmar, Jean-Bernard Lazaro, Christine Unitt, Qing Zeng, Hunter Reavis, Chirag Ganesa, Shan Zhou, Joyce Liu, Sangeetha Palakurthi, Kyle Strickland, Brooke Howitt, Panagiotis Konstantinopoulos, Paul Kirschmeier, Joseph Geradts, Ronny Drapkin, Ursula Matulonis, Alan D'Andrea, Geoffrey Shapiro. Development of a RAD51-based assay for determining homologous recombination proficiency and PARP inhibitor sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2796. doi:10.1158/1538-7445.AM2017-2796

Low Recombination Proficiency Score (RPS) in Breast Cancer Predicts Malignant Potential and Heightened Sensitivity to DNA-damaging Therapies

Low Recombination Proficiency Score (RPS) in Breast Cancer Predicts Malignant Potential and Heightened Sensitivity to DNA-damaging Therapies

A zinc finger nuclease assay to rapidly quantitate homologous recombination proficiency in human cell lines

Homologous recombination (HR) is a cellular mechanism for accurate repair of double-strand DNA breaks, often deregulated in cancer. Development of novel cancer therapeutics targeting HR pathways would benefit from a quantitative and rapid means of measuring HR. Here, we describe a zinc finger nuclease (ZFN) assay that can quantify HR. Knockdown of BRCA1 or inactivation of BRCA2 decreased HR activity in cells, whereas gene restoration induced activity 8-fold. HR activity was also reflective of BRCA1/2 status in cells with known endogenous mutations.

Molecular determinants responsible for recognition of the single-stranded DNA regulatory sequence, χ, by RecBCD enzyme

The RecBCD enzyme is important for both restriction of foreign DNA and recombinational DNA repair. Switching enzyme function from the destructive antiviral state to the productive recombinational state is regulated by the recombination hotspot, χ (5'-GCTGGTGG-3'). Recognition of χ is unique in that it is recognized as a specific sequence within single-stranded DNA (ssDNA) during DNA translocation and unwinding by RecBCD. The molecular determinants of χ recognition and the subsequent alteration in function are unknown. Consequently, we mutated residues within the RecC subunit that comprise a channel where ssDNA is thought to be scanned for a χ sequence. These mutants were characterized in vivo with regard to χ recognition, UV-sensitivity, phage degradation, and recombination proficiency. Of 38 residues mutated, 11 were previously undescribed mutations that altered χ recognition. The mutants fell into two classes: five that failed to respond to χ, and six that suggested a relaxed specificity for χ recognition. The location of the first set of mutations defines a recognition structure responsible for sequence-specific binding of ssDNA. The second set defines a highly conserved structure, linked to the recognition structure, which we hypothesize regulates conversion of RecBCD from a molecular machine that destroys DNA to one that repairs it. These findings offer insight into the evolution of enzymes with alternate χ recognition specificities.

Genetic characterization of Escherichia coli RecN protein as a member of SMC family of proteins

The proteins of SMC family are characterized by having Walker A and B sites. The Escherichia coli RecN protein is a prokaryotic member of SMC family that involved in the induced excision of Tn10 and the repair of the DNA double strand breaks. In this work, the Walker A nucleotide binding site of the E. coli RecN protein was mutated by changing the highly conserved lysine residue 35 to the aspartic acid (D), designated as recNK35D. Reverse genetics was utilized to delete the entire recN gene (ΔrecN108) or introduce the recNK35D gene into the E. coli chromosomal DNA. The recNK35D cells showed decrease in the frequency of excision of Tn10 from gal76::Tn10 after treatment with mitomycin C compared to recN+ cells. The ΔrecN108 cells showed an un-induced increase in the frequency of Tn10 excision from gal76::Tn10 in rec+ background while, recBC sbcBC ΔrecN108 cells are completely deficient in Tn10 excision. The recombination proficiency is reduced in cells carrying recBC sbcBC cells in addition recNK35D mutation. We observed that the Walker A nucleotide binding site is important for the RecN protein. Strains that deleted recN gene are recombination deficient and more sensitive to mitomycin C than strains carrying recNK35D.