Single-strand Break Repair Pathway Research Articles

Characterization of Two Novel PNKP Splice-Site Variants in a Proband With Microcephaly, Intellectual Disability, and Multiple Malformations.

Polynucleotide kinase phosphatase (PNKP), encoded by the PNKP gene, is a DNA processing enzyme involved in double-strand break and single-strand break repair pathways, which are essential for genome stability and for the correct development and maintenance of human nervous system. PNKP biallelic loss-of-function variants have been associated with a broad spectrum of neurological anomalies, ranging from congenital microcephaly with intellectual disability and seizures (MCSZ), to later onset forms of ataxia-oculomotor apraxia (AOA4) or peripheral neuropathy (CMT2B2). We report the atypical clinical manifestations of a patient with severe microcephaly, short stature, developmental delay, conductive hearing loss, and tracheoesophageal malformation, in the absence of seizures. Whole exome sequencing analysis identified two novel, compound heterozygous splice-site variants in the PNKP gene (NM_007254.4): c.1448+1G > A and c.199-8_199-5del. To demonstrate the effect of both variants on the splicing process and prove their pathogenicity, we performed a hybrid minigene assay, which successfully highlighted a deleterious impact on the transcript, particularly regarding the c.199-8_199-5del variant. The uncommon clinical features of the proband and the identification of two newly associated pathogenic variants add further evidence to the allelic and phenotypic heterogeneity of the PNKP locus.

Abstract 5858: Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer

Abstract Antibody drug conjugates (ADCs) are revolutionizing precision therapeutics in oncology. Sacituzumab Govitecan (SG), combining a Topoisomerase I (TOP1) inhibitor payload (SN38) and the tumor-selective antigen TROP2-targeting antibody hRS7, has demonstrated efficacy in challenging cancers including triple negative breast cancer (TNBC). Despite its promising clinical efficacy, the intricacies of sensitivity and resistance mechanisms to SG remain elusive. To systematically interrogate ADC sensitivity mechanisms, we employed genome-wide CRISPR-Cas9 knockout screening to discover novel regulators influencing SG sensitivity in human TNBC cells. Interactome analysis of top SG sensitizing hits showed clustered pathways of interest, including DNA repair/replication, mTORC1/metabolism, ubiquitin/proteolysis and endosome trafficking/sorting. A top druggable hit sensitizing to SG was PARP1, encoding the poly (ADP-ribose) polymerase 1 that is a key enzyme required for single-strand break repair pathways and for DNA replication fork stability. Furthermore, the synergistic lethality between SG and PARP1 was confirmed in experiments combining SG with PARP inhibitor treatment of TNBC cell lines. To selectively identify SG/ADC-specific genes and pathways, we performed a secondary round of CRISPR knockout and activation screens using a custom library comprising top hits discovered in the primary screen, treating cells in parallel with either SG or cytotoxic payload SN38 alone. Pathway analysis of genes selectively modulating sensitivity to SG but not SN38 revealed novel mediators involved in TROP2 turnover and recycling. We then performed validation and mechanistic studies to elucidate how specific genetic permutations regulate antibody delivery and sensitivity to SG. In summary, employing a systematic CRISPR screening method, we uncovered numerous novel pathways associated with both resistance and sensitization, playing a role in ADC delivery and target protein turnover. Our current focus involves prioritizing druggable genes and pathways, laying the foundation for proof-of-concept studies that will combine ADCs with specific targeted therapeutics, aiming to enhance tumor-killing efficacy. Citation Format: Bogang Wu, Sheng Sun, Nayana Thimmiah, Aiko Nagayama, James T. Coates, Ting Liu, Win Thant, Elena Bitman, David Li, John Doench, Aditya Bardia, Leif W. Ellisen. Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5858.

Abstract 1696: Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer

Abstract Antibody drug conjugates (ADC) are emerging as paradigm-changing precision therapeutics. Sacituzumab Govitecan (SG), which combines a Topoisomerase I (TOP1) inhibitor payload (SN38) with hRS7, an antibody targeting the tumor-selective antigen TROP2, is proving to be a successful cancer therapeutic for some highly refractory cancers including triple negative breast cancer (TNBC). However, mechanisms and biomarkers of sensitivity and resistance to SG remain poorly understood. Prior clinical trials have identified positive correlations between TROP2 expression and better outcome for metastatic TNBC patients receiving SG. Additionally, patient-derived specimen analyses by our group identified mutations in both TROP2 and TOP1 in post-progression metastatic lesions, suggesting multifaceted resistance mechanisms. To systematically interrogate ADC sensitivity mechanisms, we utilized genome-wide CRISPR-Cas9 knockout screening to identify novel regulators mediating SG sensitivity in human TNBC cells. Interactome analysis of top SG sensitizing hits showed clustered pathways of interest, including DNA repair/replication, mTORC1/metabolism, and endosome trafficking/sorting. The top druggable hit sensitizing to SG was PARP1, encoding the poly (ADP-ribose) polymerase 1 that is a key enzyme required for single-strand break repair pathways and for DNA replication fork stability. Conversely, the very top SG resistance-inducing hit in the genome was PARG, encoding the glycohydrolase that opposes PARP enzymatic activity. Furthermore, the synergistic lethality between TOP1 and PARP1 was confirmed in experiments combining SG with PARP inhibitor treatment of TNBC cell lines. In addition, to identify SG/ADC specific genes and pathways, we performed a secondary round of CRISPR screens using a custom library comprising top hits discovered in the primary screen, treating cells in parallel with either SG or cytotoxic payload SN38 alone. Pathway analysis of genes selectively modulating sensitivity to SG but not SN38 revealed novel mediators involved in TROP2 turnover and recycling. We then performed validation and mechanistic studies to elucidate how specific genetic permutations regulate antibody delivery and sensitivity to SG. In conclusion, through a systematic approach using CRISPR screening, we identified several novel resistance and sensitizing pathways that are implicated in ADC delivery and target protein turnover. We are currently prioritizing the druggable genes and pathways in preparation for proof-of-concept studies combining ADCs with select targeted therapeutics to achieve greater tumor killing efficacy. Citation Format: Bogang Wu, Sheng Sun, Nayana Thimmiah, Aiko Nagayama, James Coates, Win Thant, David Li, John Doench, Aditya Bardia, Leif Ellisen. Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1696.

Correlation of CCNE1 copy number levels with molecular profiles and outcomes for high grade serous ovarian and endometrial cancer (121)

<b>Objectives:</b> This study aimed to characterize <i>CCNE1</i> copy number (CN) in relation to other molecular characteristics of tumors, along with patient outcomes, to help guide future clinical trial design and potential novel targeted therapeutics development for these aggressive molecular subtypes of high-grade serous ovarian (HGSOC) and endometrial cancers (EMCA). <b>Methods:</b> Tumor samples were analyzed using Next-Gen sequencing (NGS) of the DNA (NextSeq, 592 genes or NovaSeq, whole-exome sequencing) and RNA (NovaSeq, whole-transcriptome sequencing) and immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). PD-L1 IHC used SP-142 (cut-off ≥1%). Microsatellite instability (MSI) was tested by fragment analysis, IHC, and NGS. Tumor mutation burden (TMB) was measured by totaling somatic mutations per tumor (high ≥10 mutations/MB). Relative abundance of immune-cell infiltrates was calculated by MCPCounter. <i>CCNE1</i> amplification (<i>CCNE1</i>^Amp) was defined as CN ≥6 and calculated by NGS/WES. Real-world overall survival (OS) was obtained from insurance claims data, and Kaplan-Meier estimates were calculated for molecularly defined patient cohorts. Statistical significance was determined using Chi-square and Wilcoxon rank-sum test, and p-values adjusted for multiple comparisons (q) to be <0.05. <b>Results:</b> <i>CCNE1</i> was amplified in 608/6306 (9.6%) of HGSOC, 214/2098 (10.2%) of serous EMCA, 191/749 (15.3%) of uterine carcinosarcomas (UCS), and 17/2053 (0.80%) of endometrioid EMCA. <i>TP53</i> mutations positively correlated with <i>CCNE1</i>^Amp. <i>CCNE1</i>^Amp HGSOC had fewer mutations in <i>BRCA1/2</i>, <i>ARID1A</i>, and <i>NF1</i> but increased CN amplification in other genes (Table 1). <i>PTEN</i> mutations were negatively correlated with <i>CCNE1</i>^Amp in UCS and endometrioid EMCA. There was no difference in immuno-oncology biomarkers (TMB, dMMR/MSI, PD-L1). However, increased <i>CCNE1</i> levels were associated with decreased fibroblasts in HGSOC and serous EMCA, decreased B cells in serous EMCA, and decreased CD3+ T cells in UCS. In HGSOC, <i>CCNE1</i>^Amp was associated with increased expression of cell-cycle checkpoint regulators, Fanconi anemia, homologous recombination, mismatch repair, and single-strand break repair pathway genes, but decreased expression of these genes in endometrioid EMCA. In tumors with >1 specimen serially submitted, there was a significant increase in <i>CCNE1</i> CN over time in 29% (49/169) of HGSOC and 23% of EMCA (36/158). There was no difference in <i>CCNE1</i> CN in tumors harboring <i>BRCA</i> reversion mutations (<i>n</i>=8) compared to wild-type (<i>n</i>=5110). <i>CCNE1</i>^Amp correlated with mRNA expression in HGSOC and EMCA. <i>CCNE1</i>^Amp was associated with worse OS after carboplatin-based treatment in HGSOC as well as in serous EMCA and UCS. <b>Conclusions:</b> <i>CCNE1</i>^Amp in HGSOC and EMCA is less common than previously identified. Both HGSOC and endometrioid EMCAs have distinct molecular characteristics of <i>CCNE1</i>^Amp tumors. <i>CCNE1</i> CN may increase with the emergence of treatment resistance. Finally, <i>CCNE1</i>^Amp tumors appear overall non-immunogenic.

Reconsidering the Chemical Nature of Strand Breaks Derived from Abasic Sites in Cellular DNA: Evidence for 3'-Glutathionylation.

The hydrolytic loss of coding bases from cellular DNA is a common and unavoidable reaction. The resulting abasic sites can undergo β-elimination of the 3'-phosphoryl group to generate a strand break with an electrophilic α,β-unsaturated aldehyde residue on the 3'-terminus. The work reported here provides evidence that the thiol residue of the cellular tripeptide glutathione rapidly adds to the alkenal group on the 3'-terminus of an AP-derived strand break. The resulting glutathionylated adduct is the only major cleavage product observed when β-elimination occurs at an AP site in the presence of glutathione. Formation of the glutathionylated cleavage product is reversible, but in the presence of physiological concentrations of glutathione, the adduct persists for days. Biochemical experiments provided evidence that the 3'-phosphodiesterase activity of the enzyme apurinic/apyrimidinic endonuclease (APE1) can remove the glutathionylated sugar remnant from an AP-derived strand break to generate the 3'OH residue required for repair via base excision or single-strand break repair pathways. The results suggest that a previously unrecognized 3'glutathionylated sugar remnant─and not the canonical α,β-unsaturated aldehyde end group─may be the true strand cleavage product arising from β-elimination at an abasic site in cellular DNA. This work introduces the 3'glutathionylated cleavage product as the major blocking group that must be trimmed to enable repair of abasic site-derived strand breaks by the base excision repair or single-strand break repair pathways.

Formation and Repair of an Interstrand DNA Cross-Link Arising from a Common Endogenous Lesion.

Interstrand DNA cross-links (ICLs) are cytotoxic because they block the strand separation required for read-out and replication of the genetic information in duplex DNA. The unavoidable formation of ICLs in cellular DNA may contribute to aging, neurodegeneration, and cancer. Here, we describe the formation and properties of a structurally complex ICL derived from an apurinic/apyrimidinic (AP) site, which is one of the most common endogenous lesions in cellular DNA. The results characterize a cross-link arising from aza-Michael addition of the N2-amino group of a guanine residue to the electrophilic sugar remnant generated by spermine-mediated strand cleavage at an AP site in duplex DNA. An α,β-unsaturated iminium ion is the critical intermediate involved in ICL formation. Studies employing the bacteriophage φ29 polymerase provided evidence that this ICL can block critical DNA transactions that require strand separation. The results of biochemical studies suggest that this complex strand break/ICL might be repaired by a simple mechanism in which the 3'-exonuclease action of the enzyme apurinic/apyrimidinic endonuclease (APE1) unhooks the cross-link to initiate repair via the single-strand break repair pathway.

Analysis of Base Excision and Single-Strand Break Repair Activities in Trypanosomatid Extracts.

Cellular DNA is inherently unstable, subject to both spontaneous hydrolysis and attack by a range of exogenous and endogenous chemicals as well as physical agents such as ionizing and ultraviolet radiation. For parasitic protists, where an inoculum of infectious parasites is typically small and natural infections are often chronic with low parasitemia, they are also vulnerable to DNA damaging agents arising from innate immune defenses. The majority of DNA damage consists of relatively minor changes to the primary structure of the DNA, such as base deamination, oxidation, or alkylation and scission of the phosphodiester backbone. Yet these small changes can have serious consequences, often being mutagenic or cytotoxic. Cells have therefore evolved efficient mechanisms to repair such damage, with base excision and single strand break repair playing the primary role here. In this chapter we describe a method for analyzing the activity from cell extracts of various enzymes involved in the base excision and single strand break repair pathways of trypanosomatid parasites.

PF663 TRANSCRIPTION ALTERATION OF DNA REPAIR GENES IN PHILADELPHIA CHROMOSOME NEGATIVE MYELOPROLIFERATIVE NEOPLASMS

Background: In Philadelphia chromosome negative myeloproliferative neoplasms (Ph-neg MPN), curative therapeutic options are still limited. The concept of synthetic lethality provides an alternative strategy to overcome inherent treatment resistance by exploiting tumor-specific vulnerabilities. Altered DNA repair pathways have been implicated in the pathogenesis of myeloproliferative neoplasms (MPN). Aims: Based on these results, the aim of our study was to investigate whether transcriptional alterations of genes involved in DNA repair pathways are present in cells from patients with Ph-neg MPN. Methods: 57 patients from the Aachen MPN-registry of patients with classical, Ph-neg MPN (essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF)) and 13 controls were included in our study after written informed consent. Differential expression of 11 genes of DNA repair pathways (Homologous recombination repair pathway (HRR); non homologous end joining pathway (NHEJ) and single strand break repair pathway (SSB)) were analyzed by qRT-PCR. Gene expression data (each gene in % of GAPDH as housekeeping gene) of each entity was compared to expression levels of normal controls using Mann-Whitney U test (IBM SPSS statistics 23; Prism 7 for Windows, Version 7.04, © GraphPad Software inc.). Results: The final analysis included 58 patient samples (17 PV, 15 ET, 14 MF [9 primary MF, 5 secondary MF] and 12 controls). JAK2 mutational status was analyzed in all samples and JAK2 V617F mutations were detected in 94% of PV, 67% of ET and 86% of MF cases. Gene expression data (Figure 1 A+B) of ET samples revealed statistically significant differences in expression compared to controls in the following genes: in the genes affecting the base excision repair, LIG3 showed lower expression (p < 0.05) compared to controls. In the genes of the NHEJ pathway, a statistically significant higher expression was observed for PRKDC (p < 0.05) and XRCC5 (p < 0.01) and for genes of the HRR upregulation was detected for BRCA1 (p < 0.05) and for BRCA2 (p < 0.001). In PV, LIG3 and PRKDC (SBB) expression was lower compared to controls (Figure 1 C+D). Expression of RAD52 was decreased, whereas no differences were detected for BRCA1, BRCA2, RPA1 and RPA2 (HRR), which was different from ET and MF. No alterations in gene expression involved in NHEJ were observed. MF samples showed a markedly different expression pattern compared to the other two entities (Figure 1 E+F). In fact, 9 out of the 11 analyzed genes revealed a significant downregulation, only BRCA1 and BRCA2 expression was not altered. So none of the genes of the NHEJ and the SSB showed expression levels comparable to healthy controls. Furthermore, in ET and MF samples we analyzed differences in gene expression in JAK2 positive and negative samples but detected no significant differences.Summary/Conclusion: In conclusion, our study demonstrates significant alterations in expression of genes involved in DNA-repair in Ph-neg MPN compared to controls as well as marked differences among the three entities. In MF, genes of all analyzed DNA repair mechanisms were significantly downregulated. Although further studies are needed, our data support the relevance of targeting DNA repair pathways and suggest that synthetic lethality may offer a potential new approach in the therapy of Ph-neg MPN.

CRISPR/Cas9-Generated Models Uncover Therapeutic Vulnerabilities of Del(11q) Chronic Lymphocytic Leukemia Cells to Dual BCR and PARP Inhibition

Chromosome 11q22.3 deletion (del(11q)) is one of the most common cytogenetic alterations in CLL and usually involves both ATM and BIRC3 genes. Concomitant mutations in ATM and/or BIRC3 in the remaining allele have been associated with poor survival. Despite the encouraging efficacy of novel agents targeting BCR and BCL2 pathways, del(11q) patients still have an inferior outcome and the development of resistance to these drugs has been increasingly reported. We therefore investigated the functional impact of del(11q) together with loss-of-function mutations in ATM and/or BIRC3 and whether CLLs harboring these alterations could benefit from novel combinatorial therapies.To address these questions, we used the CRISPR/Cas9 system to generate an isogenic CLL cell line to model del(11q) derived from HG3 cells by introducing two guide RNAs targeting the 11q22.1 and 11q23.3 regions. The presence of a monoallelic deletion (size ~17 Mb) was confirmed in 100% of the cells by FISH. Truncating mutations in ATM and/or BIRC3 were introduced in the remaining allele, generating HG3 del(11q) ATMKO, del(11q) BIRC3KO and del(11q) ATMKOBIRC3KO (three clones per condition). In addition, single ATMKO and BIRC3KO mutations, or the combination of both, were introduced into both HG3 and MEC1 CLL-derived cells (three clones per condition).Functional in vitro studies revealed that del(11q) BIRC3KO cells had increased growth rates compared to del(11q) BIRC3WT clones (P<0.01). Similar results were observed in HG3 and MEC1 BIRC3KO cells (P<0.01; P<0.05). Moreover, biallelic inactivation of BIRC3 in del(11q) cells resulted in cytoplasmic stabilization of NF-kB-inducing kinase (NIK), leading to higher nuclear NF-kB2 (p52) activation (P<0.01) as measured by ELISA. In parallel, we analyzed the DNA damage response (DDR) of these cells, and showed that del(11q) ATMKO cells displayed reduced pH2AX levels (P<0.001) and an accumulation of unrepaired double strand breaks (DSB) (P<0.001) after irradiation, as determined by comet assays. Consistently, in vivo subcutaneous xenografts showed that HG3 ATMKOBIRC3KO tumors presented proliferative advantage, higher p52 levels and greater genomic and mitotic instability than HG3WT tumors, indicating a more aggressive phenotype.We next assessed the response of these CRISPR/Cas9-edited CLL cell lines to therapy. Of note, only TP53KO clones (also generated by CRISPR/Cas9), and not del(11q) BIRC3KO cells, showed resistance to fludarabine (mean IC50 16.9 uM vs. 4.1 uM; mean apoptotic cells (5 uM) 5.5% vs. 22.5%; P<0.05). Moreover, del(11q) cells were slightly more resistant to ibrutinib (IBRU) treatment compared to WT cells (mean IC50 10 uM vs. 3.7 uM; P<0.05). Interestingly, exploiting the DDR deficiencies underlying del(11q) by targeting the single strand break repair pathway with the PARP inhibitor olaparib (OLA), del(11q) ATMKO cells were not able to proliferate even 12 days after treatment (3 uM), independently of the mutational status of BIRC3 (P<0.01). In vivo intravenous HG3-derived xenografts (N=20) showed that OLA (100 mg/kg) reduced hCD45+ cells in the peripheral blood (P<0.01) and significantly improved survival of del(11q) ATMKOBIRC3KO xenografted mice (P<0.01).Moreover, IBRU potentiated the effects of OLA in cell viability (72h) in all the del(11q) clones (combination indexes 0.69-0.85), leading to an increased necrotic cell death, as shown by annexin V/PI staining (P<0.001) and HMGB1 release. Remarkably, we found that IBRU caused downregulation of the DNA repair protein RAD51, leading to impaired RAD51 foci formation in DSB lesions (P<0.01). Consistently, IBRU (1 uM) reduced the homologous recombination (HR) repair efficiency in HG3 cells (P=0.001), as determined by an HR-reporter construct. This IBRU-dependent impairment of HR repair could explain the synergistic effects with OLA by synthetic lethality.In conclusion, we demonstrate that del(11q) CLL cells with biallelic inactivation of BIRC3 and ATM show enhanced proliferation through activation of the non-canonical NF-kB pathway, and accumulation of DNA damage contributing to genomic instability. We show that these defects on the DDR can be therapeutically targeted by synthetic lethal approaches using PARP inhibitors either alone or in combination with BCR inhibitors, providing a rationale for the study of this combination in relapsed del(11q) CLL patients.PI15/01471 SA085U16 JCyL-MQ FEHH-MH DisclosuresGarcía-Tuñón:Novartis: Research Funding. Wu:Neon Therapeutics: Equity Ownership.

Abstract OT2-01-13: A phase 3, open-label, randomized, 2-arm international study of the oral dual PARP inhibitor talazoparib in germline BRCA mutation subjects with locally advanced and/or metastatic breast cancer (EMBRACA)

Abstract Background: Cancer cells with deleterious mutations in breast cancer susceptibility genes 1 and 2 (BRCA1/2) are deficient in the DNA double-strand break repair mechanism, rendering them highly dependent on the single-strand break repair pathway, regulated by poly(ADP-ribose) polymerase (PARP). Inhibition of PARP results in synthetic lethality in cells with a BRCA1/2 mutation because of accumulation of irreparable DNA damage; PARP inhibitors have the potential to be selectively toxic for BRCA-mutated cells. In addition to catalytic inhibition, it has been shown that some PARP inhibitors induce PARP trapping at sites of DNA damage. The capacity to trap PARP-DNA complexes varies widely across different PARP inhibitors and is not correlated with PARP catalytic inhibition. Preclinical models have shown trapping PARP on DNA is more potent at inducing cancer cell death than enzymatic inhibition of PARP alone. Talazoparib is a dual-mechanism PARP inhibitor that both inhibits the PARP enzyme and effectively traps PARP on DNA, preventing DNA damage repair and resulting in cell death in BRCA1/2-mutated cells. In preclinical studies, talazoparib at nanomolar concentrations showed the highest efficiency at trapping PARP-DNA complexes relative to other PARP inhibitors. In a previous phase 1/2 clinical study, talazoparib as monotherapy (1 mg once daily) resulted in a 50% response rate and an 86% clinical benefit rate at 24 weeks in 14 patients with a germline BRCA1/2 mutation and advanced breast cancer (aBC). Methods: This open-label, randomized, 2-arm, international phase 3 trial (EMBRACA) compares the efficacy and safety of talazoparib with protocol-specific physician's choice (capecitabine, eribulin, gemcitabine or vinorelbine) in patients with aBC. The primary objective is progression-free survival by central imaging. Secondary objectives are objective response rate, overall survival, safety and pharmacokinetics of talazoparib. Exploratory objectives include health-related quality of life measurements and biomarker research in blood and tumor samples that may permit characterization of mechanisms involved in tumor sensitivity and resistance to talazoparib. Key patient eligibility criteria include aged ≥18 years with histologically/cytologically confirmed breast cancer; locally advanced and/or metastatic disease appropriate for systemic single-agent cytotoxic chemotherapy; deleterious or pathogenic germline BRCA1/2 mutations by central laboratory; ≤3 prior cytotoxic chemotherapy regimens for advanced disease (prior platinum is allowed provided patients did not relapse within 6 months in the adjuvant setting or did not progress on platinum therapy); prior treatment with a taxane and/or anthracycline unless medically contraindicated; and ECOG performance status ≤2. Patients (N=429) will be randomized 2:1 to receive either talazoparib capsules (1 mg/day, 21-day cycles) or physician's choice treatment. This trial is currently enrolling patients from the USA, Europe, Israel, Ukraine, Russia, Korea, Australia, Taiwan and Brazil (NCT01945775). This study is funded by Medivation, Inc. Citation Format: Litton J, Ettl J, Hurvitz SA, Mina LA, Rugo HS, Lee K-H, Yerushalmi R, Woodward N, Goncalves A, Moreno F, Roche H, Im Y-H, Martin M, Bhattacharya S, Peterson A, Hannah A, Eiermann W, Blum J. A phase 3, open-label, randomized, 2-arm international study of the oral dual PARP inhibitor talazoparib in germline BRCA mutation subjects with locally advanced and/or metastatic breast cancer (EMBRACA) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT2-01-13.

Dbait : un concept innovant pour inhiber la réparation de l’ADN et contribuer aux traitements des cancers

The ability of cancer cells to recognize damage and initiate DNA repair is an important mechanism for therapeutic resistance. The use of inhibitors of DNA damage repair or signaling pathways appears to provide a unique opportunity for targeting genetic differences between tumor and normal cells. In this review, we firstly describe the main DNA lesions induced by the different treatments and the pathways involved in their repair. Then we review the mechanism of action and applications of an innovative DNA repair inhibitor: Dbait (and its clinical form DT01). Dbait/DT01 consists of 32 bp deoxyribonucleotides forming an intramolecular DNA double helix that mimics DNA lesions. They act as a bait for DNA damage signaling enzymes, the polyadenyl-ribose polymerase (PARP), and the DNA-dependent kinase (DNA-PK), inducing a "false" DNA damage signal and ultimately inhibiting recruitment at the damage site of many proteins involved in double-strand break and single-strand break repair pathways. Preclinical studies have demonstrated the capacity of Dbait/DT01 to improve the efficiency of (i) chemotherapy in colorectal cancer or hepatocellular carcinoma models, (ii) radiofrequency ablative in colorectal cancer liver metastases models, and (iii) radiotherapy in xenografted mice with head & neck squamous cell carcinoma, glioblastoma and melanoma. Following this good preclinical results, we performed a first-in-human phase 1-2a study evaluating the safety and efficacy of the combination of DT01 with radiotherapy for the treatment of skin metastases of melanoma. Twenty-three patients were included. No dose-limiting toxicity was observed. An objective response was observed in 59% lesions, including 30% complete responses. This first promising clinical efficacy provides future potential interesting clinical development of Dbait/DT01 with various anticancer treatments.

Abstract OT1-03-17: ABRAZO: An international phase 2 (2-stage, 2-cohort) study of the oral PARP inhibitor talazoparib (BMN 673) in BRCA mutation subjects with locally advanced and/or metastatic breast cancer

Abstract Background: Cancer cells with deleterious mutations in breast cancer susceptibility genes 1 and 2 (BRCA1/2) are deficient in the DNA double-strand break repair mechanism, rendering them highly dependent on the single-strand break repair pathway, which is initiated by poly-(ADP-ribose) polymerase (PARP) [1-3]. In cells with deleterious BRCA1/2 mutations, PARP inhibition is synthetically lethal because of accumulation of irreparable DNA damage [1-3]. Talazoparib (BMN 673) exhibits a novel two-pronged approach in treating BRCA1/2-mutant tumors: 1) potent catalytic inhibition of the PARP enzyme; and 2) trapping of PARP at sites of DNA damage [4-7]. The capacity to trap PARP-DNA complexes varies widely across PARP inhibitors and is not correlated with catalytic inhibition potency [4-7]. In preclinical models, trapping PARP on DNA is more potent at inducing cancer cell death than enzymatic inhibition of PARP alone [4,7]. Talazoparib is the most potent clinical-stage PARP inhibitor tested to date with the highest efficacy at trapping PARP-DNA complexes [7]. Talazoparib has shown single-agent antitumor efficacy in several solid tumor types and was generally well tolerated in a phase 1/2 clinical study [8]. Methods: This 2-stage, 2-cohort, phase 2 international study (ABRAZO) evaluates the safety and efficacy of talazoparib in patients with a deleterious germline BRCA1 or BRCA2 mutation with locally advanced and/or metastatic breast cancer. Eligible subjects will be assigned to one of two cohorts based on prior chemotherapy for metastatic disease. Cohort 1 (n=70) includes patients with a complete response (CR) or partial response (PR) to platinum-containing regimens for metastatic disease. Cohort 2 (n=70) includes patients who have received &amp;gt;2 prior chemotherapy regimens in the metastatic setting but have not had prior platinum therapy for locally advanced or metastatic disease (prior adjuvant or neoadjuvant therapy with a platinum is allowed). The primary objective is objective response rate (ORR). Secondary objectives include clinical benefit response (CBR) rate, duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Health-related quality of life (QoL) assessments are an exploratory objective. Eligible subjects will receive oral talazoparib (1 mg/day, 21-day cycles) until disease progression or unacceptable toxicity. This trial is currently enrolling patients from the United States and Europe (NCT02034916). This study is funded by BioMarin Pharmaceutical Inc.

Abstract OT1-03-16: EMBRACA: A phase 3, open-label, randomized, parallel, 2-arm international study of the oral PARP inhibitor talazoparib (BMN 673) versus physician's choice in BRCA mutation subjects with locally advanced and/or metastatic breast cancer

Abstract Background: Cancer cells with deleterious mutations in breast cancer susceptibility genes 1 and 2 (BRCA1/2) are deficient in the DNA double-strand break repair mechanism, rendering them highly dependent on the single-strand break repair pathway, which is initiated by poly-(ADP-ribose) polymerase (PARP) [1-3]. In cells with deleterious BRCA1/2 mutations, PARP inhibition is synthetically lethal because of accumulation of irreparable DNA damage [1-3]. Talazoparib (BMN 673) exhibits a novel two-pronged approach in treating BRCA1/2-mutant tumors: 1) potent catalytic inhibition of the PARP enzyme; and 2) trapping of PARP at sites of DNA damage [4-7]. The capacity to trap PARP-DNA complexes varies widely across PARP inhibitors and is not correlated with catalytic inhibition potency [4-7]. In preclinical models, trapping PARP on DNA is more potent at inducing cancer cell death than enzymatic inhibition of PARP alone [4,7]. Talazoparib is the most potent clinical-stage PARP inhibitor tested to date with the highest efficacy at trapping PARP-DNA complexes [7]. Talazoparib has shown single-agent antitumor efficacy in several solid tumor types and was generally well tolerated in a phase 1/2 clinical study [8]. Methods: This open-label, randomized, parallel, 2-arm, phase 3 international trial (EMBRACA) compares the safety and efficacy of talazoparib with physician's choice (capecitabine, eribulin, gemcitabine, or vinorelbine) in patients with advanced breast cancer. The primary objective is progression free survival (PFS). Secondary objectives include objective response rate (ORR), overall survival (OS), and safety. Exploratory objectives include duration of response (DOR) for objective responders and health-related quality of life measurements. Subject eligibility includes age ≥18 years with histologically/cytologically confirmed breast carcinoma, locally advanced and/or metastatic disease, germline BRCA1/2 mutations, ≤2 prior chemotherapy-inclusive regimens for locally advanced and/or metastatic disease, prior treatment with a taxane and/or anthracycline, and Eastern Cooperative Oncology Group (ECOG) performance status ≤1. Subjects (n=429) will be randomized 2:1 to receive either talazoparib oral capsules (1 mg/day, 21-day cycles) or physician's choice treatment. This trial is currently enrolling patients from the United States, Asia/Pacific, Europe, Israel, and South America (NCT01945775). This study is funded by BioMarin Pharmaceutical Inc.

Substrate Rescue of DNA Polymerase β Containing a Catastrophic L22P Mutation

DNA polymerase (pol) β is a multidomain enzyme with two enzymatic activities that plays a central role in the overlapping base excision repair and single-strand break repair pathways. The high frequency of pol β variants identified in tumor-derived tissues suggests a possible role in the progression of cancer, making the determination of the functional consequences of these variants of interest. Pol β containing a proline substitution for leucine 22 in the lyase domain (LD), identified in gastric tumors, has been reported to exhibit severe impairment of both lyase and polymerase activities. Nuclear magnetic resonance (NMR) spectroscopic evaluations of both pol β and the isolated LD containing the L22P mutation demonstrate destabilization sufficient to result in LD-selective unfolding with minimal structural perturbations to the polymerase domain. Unexpectedly, addition of single-stranded or hairpin DNA resulted in partial refolding of the mutated lyase domain, both in isolation and for the full-length enzyme. Further, formation of an abortive ternary complex using Ca2+ and a complementary dNTP indicates that the fraction of pol β(L22P) containing the folded LD undergoes conformational activation similar to that of the wild-type enzyme. Kinetic characterization of the polymerase activity of L22P pol β indicates that the L22P mutation compromises DNA binding, but nearly wild-type catalytic rates can be observed at elevated substrate concentrations. The organic osmolyte trimethylamine N-oxide (TMAO) is similarly able to induce folding and kinetic activation of both polymerase and lyase activities of the mutant. Kinetic data indicate synergy between the TMAO cosolvent and substrate binding. NMR data indicate that the effect of the DNA results primarily from interaction with the folded LD(L22P), while the effect of the TMAO results primarily from destabilization of the unfolded LD(L22P). These studies illustrate that substrate-induced catalytic activation of pol β provides an optimal enzyme conformation even in the presence of a strongly destabilizing point mutation. Accordingly, it remains to be determined whether this mutation alters the threshold of cellular repair activity needed for routine genome maintenance or whether the “inactive” variant interferes with DNA repair.

Patching and Single-Strand Ligation in Nonhomologous DNA End Joining Despite Persistence of a Closely Opposed 3′-Phosphoglycolate-Terminated Strand Break

Previous work showed that in human nuclear extracts, double-strand break substrates bearing partially complementary (-ACG) 3'-phosphoglycolate (PG)-terminated 3' overhangs are joined by a mechanism involving annealing of the terminal CG dinucleotides, PG removal, single-base gap filling and ligation. However, in these extracts only a minority of the breaks are rejoined, and most of the 3'-PG termini remain intact even after several hours. To determine whether the presence of a persistent 3'-PG prevents patching and ligation of the opposite strand, a substrate was constructed with two -ACG overhangs, one PG-terminated and one hydroxyl-terminated. after incubation in HeLa cell nuclear extracts, two major repair products of similar yield were formed: a fully repaired duplex and a nicked duplex in which the initial 3'-PG terminus remained intact. These results indicate that patching and ligation can proceed to completion in the unmodified strand despite persistence of the 3'-PG-terminated break in the opposite strand. The break in the PG-containing strand could then presumably be rejoined by a single-strand break repair pathway.

Polynucleotide kinase as a potential target for enhancing cytotoxicity by ionizing radiation and topoisomerase I inhibitors.

The cytotoxicity of many antineoplastic agents is due to their capacity to damage DNA and there is evidence indicating that DNA repair contributes to the cellular resistance to such agents. DNA strand breaks constitute a significant proportion of the lesions generated by a broad range of genotoxic agents, either directly, or during the course of DNA repair. Strand breaks that are caused by many agents including ionizing radiation, topoisomerase I inhibitors, and DNA repair glycosylases such as NEIL1 and NEIL2, often contain 5'-hydroxyl and/or 3'-phosphate termini. These ends must be converted to 5'-phosphate and 3'-hydroxyl termini in order to allow DNA polymerases and ligases to catalyze repair synthesis and strand rejoining. A key enzyme involved in this end-processing is polynucleotide kinase (PNK), which possesses two enzyme activities, a DNA 5'-kinase activity and a 3'-phosphatase activity. PNK participates in the single-strand break repair pathway and the non-homologous end joining pathway for double-strand break repair. RNAi-mediated down-regulation of PNK renders cells more sensitive to ionizing radiation and camptothecin, a topoisomerase I inhibitor. Structural analysis of PNK revealed the protein is composed of three domains, the kinase domain at the C-terminus, the phosphatase domain in the centre and a forkhead associated (FHA) domain at the N-terminus. The FHA domain plays a critical role in the binding of PNK to other DNA repair proteins. Thus each PNK domain may be a suitable target for small molecule inhibition to effectively reduce resistance to ionizing radiation and topoisomerase I inhibitors.

Cytotoxicity and mutagenicity of endogenous DNA base lesions as potential cause of human aging

Endogenous factors constitute a substantial source of damage to the genomic DNA. The type of damage includes a number of different base lesions and single- and double-strand breaks. Unrepaired DNA damage can give rise to mutations and may cause cell death. A number of studies have demonstrated an association between aging and the accumulation of DNA damage. This may be attributed to reduced DNA repair with age, although this is apparently not a general feature for all types of damage and repair mechanisms. Therefore, detailed studies that improve our knowledge of DNA repair systems as well as mutagenic and toxic effects of DNA lesions will help us to gain a better insight into the mechanisms of aging. The aim of this review is to provide a brief description of cytotoxic and mutagenic endogenous DNA lesions that are mainly repaired by base excision repair and single-strand break repair pathways and to discuss the potential role of DNA lesions and DNA repair dysfunction in the onset of human aging.

Substrate Specificity of Tyrosyl-DNA Phosphodiesterase I (Tdp1)

Tyrosyl-DNA phosphodiesterase I (Tdp1) hydrolyzes 3'-phosphotyrosyl bonds to generate 3'-phosphate DNA and tyrosine in vitro. Tdp1 is involved in the repair of DNA lesions created by topoisomerase I, although the in vivo substrate is not known. Here we study the kinetic and binding properties of human Tdp1 (hTdp1) to identify appropriate 3'-phosphotyrosyl DNA substrates. Genetic studies argue that Tdp1 is involved in double and single strand break repair pathways; however, x-ray crystal structures suggest that Tdp1 can only bind single strand DNA. Separate kinetic and binding experiments show that hTdp1 has a preference for single-stranded and blunt-ended duplex substrates over nicked and tailed duplex substrate conformations. Based on these results, we present a new model to explain Tdp1/DNA binding properties. These results suggest that Tdp1 only acts upon double strand breaks in vivo, and the roles of Tdp1 in yeast and mammalian cells are discussed.

Association of XRCC1 and tyrosyl DNA phosphodiesterase (Tdp1) for the repair of topoisomerase I-mediated DNA lesions

DNA topoisomerase I (Top1) is converted into a cellular poison by camptothecin (CPT) and various endogenous and exogenous DNA lesions. In this study, we used X-ray repair complementation group 1 (XRCC1)-deficient and XRCC1-complemented EM9 cells to investigate the mechanism by which XRCC1 affects the cellular responses to Top1 cleavage complexes induced by CPT. XRCC1 complementation enhanced survival to CPT-induced DNA lesions produced independently of DNA replication. CPT-induced comparable levels of Top1 cleavage complexes (single-strand break (SSB) and DNA–protein cross-links (DPC)) in both XRCC1-deficient and XRCC1-complemented cells. However, XRCC1-complemented cells repaired Top1-induced DNA breaks faster than XRCC1-deficient cells, and exhibited enhanced tyrosyl DNA phosphodiesterase (Tdp1) and polynucleotide kinase phosphatase (PNKP) activities. XRCC1 immunoprecipitates contained Tdp1 polypeptide, and both Tdp1 and PNKP activities, indicating a functional connection between the XRCC1 single-strand break repair pathway and the repair of Top1 covalent complexes by Tdp1 and PNKP.