Defective DNA Damage Response Research Articles

Recent Progress in DNA Damage Response-Targeting PROTAC Degraders.

DNA damage response (DDR) defects in cells play a crucial role in tumor development by promoting DNA mutations. These mutations create vulnerabilities specific to cancer cells, which can be effectively targeted through synthetic lethality-based therapies. To date, numerous small molecule DDR inhibitors have been identified, and some of them have already been approved for clinical use. However, due to the complexity of the tumor microenvironment, mutations may occur in the amino acid residues of DDR targets. These mutations can affect the efficacy of small molecule inhibitors targeting DDR pathways. Therefore, researchers have turned their attention to next-generation DNA damage repair modulators, particularly those based on PROTAC technology. From this perspective, we overviewed the recent progress on DDR-targeting PROTAC degraders for cancer therapy. In addition, we also summarized the biological functions of different DDR targets. Finally, the challenges and future directions for DDR-target PROTAC degraders are also discussed in detail.

TRIM33 loss in multiple myeloma is associated with genomic instability and sensitivity to PARP inhibitors

Deletions of chromosome 1p (del(1p)) are a recurrent genomic aberration associated with poor outcome in Multiple myeloma (MM.) TRIM33, an E3 ligase and transcriptional co-repressor, is located within a commonly deleted region at 1p13.2. TRIM33 is reported to play a role in the regulation of mitosis and PARP-dependent DNA damage response (DDR), both of which are important for maintenance of genome stability. Here, we demonstrate that MM patients with loss of TRIM33 exhibit increased chromosomal instability and poor outcome. Through knockdown studies, we show that TRIM33 loss induces a DDR defect, leading to accumulation of DNA double strand breaks (DSBs) and slower DNA repair kinetics, along with reduced efficiency of non-homologous end joining (NHEJ). Furthermore, TRIM33 loss results in dysregulated ubiquitination of ALC1, an important regulator of response to PARP inhibition. We show that TRIM33 knockdown sensitizes MM cells to the PARP inhibitor Olaparib, and this is synergistic with the standard of care therapy bortezomib, even in co-culture with bone marrow stromal cells (BMSCs). These findings suggest that TRIM33 loss contributes to the pathogenesis of high-risk MM and that this may be therapeutically exploited through the use of PARP inhibitors.

Abstract 4573: Pre-clinical evaluation of small molecule CHK2 inhibitor, ART446, pre-IND candidate that enhances the olaparib and irinotecan

Abstract Background: Poly (ADP-ribose) polymerase (PARP) plays a pivotal role in DNA damage repair. Tumors with DNA Damage Response (DDR) defects exhibit increased susceptibility to drugs targeting PARP. The drugs induce DNA damage like Irinotecan (a Topoisomerase I inhibitor) are widely used in clinic as well. However, intrinsic resistance remains a significant obstacle, leading to frequent treatment failures. Chk2 is activated in response to these DNA damage inducing agents and hold the cell cycle. We developed pre-IND candidate ART446, with the aim of exploring its therapeutic potential in sensitizing DNA damage-targeting reagents. Results: In the radiometric HopSpot Kinase Assay, ART446 demonstrated significant inhibition of Chk2 (IC50, 9 nM). In HT-29 colon adenocarcinomas, co-treatment with SN38 and ART446 exhibited a synergistic therapeutic effect in vitro, with a subsequent in vivo tumor growth assay demonstrating a remarkable combined effect (TGI; Irinotecan, 46%; ART446, 16%; combined, 83.4%). Further extending our investigation to triple-negative breast adenocarcinoma MDA-MB-436, characterized by a BRCA1 mutation, we explored the sensitization potential of ART446 to PARP inhibitors. Strikingly, the combination therapy with a PARP inhibitor, Olaparib surpassed monotherapy outcomes, resulting in complete tumor growth inhibition (TGI: Olaparib, 73%; ART446, 20%; combined, 102%). Mechanistic insights revealed an increase in gamma H2AX levels by ART446 and a decrease in active forms of Chk2 which were activated by Olaparib affirming that Olaparib-induced Chk2 inhibition contributed to the sensitization effect. Conclusions: The pre-IND candidate ART446 exhibits promising potential in enhancing the therapeutic effects of Olaparib and Irinotecan. These findings provide a robust foundation for further clinical development, offering a strategic approach to overcome resistance in cancer treatment. Citation Format: Taegon Baik, Young Woo Kang, Ha Young Lee, Jin Whan Kim, A Hyeon Jeong, Jong Oh Kim, Woo-Young Kim. Pre-clinical evaluation of small molecule CHK2 inhibitor, ART446, pre-IND candidate that enhances the olaparib and irinotecan [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 4573.

PRIMUS-002: A multicentre, open-label, phase II study examining FOLFOX and nab-paclitaxel (FA) and nab-paclitaxel and gemcitabine (AG) as neoadjuvant therapy for (borderline) resectable pancreatic cancer (PC), focusing on biomarker and liquid biopsy development.

669 Background: There is increasing evidence suggesting benefit from a neoadjuvant approach to PC. However, the optimal regimen is unclear and will likely require a precision medicine approach. Platinum-containing regimens have shown survival benefit for PC, with occasional exceptional responders, but biomarkers (BM) of response are not well defined and treatment decisions are often based on patient performance status (PS) and co-morbidity. Tumors may show defective DNA damage response (DDR), conferring potential selective sensitivity to DNA-damaging agents (e.g. platinum) and newer targeted agents. We have shown that DDR deficiency (DDRd) is present in up to 20% of PC. This study aimed to exploit DDRd as a therapeutic vulnerability, with integrated analysis to define candidate BM for FA and AG response. Methods: Patients with resectable and borderline resectable PDAC patients who were deemed fit for potential surgery were recruited into the trial between April 2019 and July 2021. All had been molecularly profiled using the Precision-Panc Clinical Cancer Genome including a novel DDRd assay, and the transcriptome with longitudinal sampling (pre-, during, and post-treatment). Patients received either FA (nab-paclitaxel 150mg/m2 IV, oxaliplatin 85mg/m2, folinic acid 350mg flat dose, fluorouracil infusion 2400mg/m2 continuous IV infusion), or AG (nab-paclitaxel 125mg/m2, gemcitabine 1000 mg/m2) for 3 months, based on patient age and PS. The primary endpoint was disease progression (DP) during neoadjuvant therapy. Results: 31 patients in total were recruited into the trial unfortunately the trial was terminated early due to funding issues. The median age was 62 years old (31-73) 21 patients were male and 10 female. 7 resectable cases and 24 borderline resectable at diagnosis were included in the study. 26 patients were recruited to the FOLFOX-A arm and 5 to the AG arm. Following chemotherapy 6 patients had partial response, 17 stable disease and 6 progressive disease. 23 patients proceeded to surgery of which 6 the radical surgery was abandoned due to extent of malignancy The overall median survival time was 20.6 months with a 90% confidence interval of (12.9 months to 24.9 months). The median survival time was 23.7 months with 90% confidence interval of (12.9 months to 24.9 months) for FOLXFOX-A, and 20.5 months with a 90% confidence interval of (8.9 months to NA) for the AG arm Conclusions: We have demonstrated that these 2 regimes can be utilised in the neoadjuvant setting with surgery being undertaken safely following therapy (there was no surgical mortality and morbidity is in line of other international series). Further work is ongoing to identify potential biomarkers which may lead to better patient selection for a neoadjuvant approach. Clinical trial information: ISRCTN34129115 .

The metabolic basis of inherited neutropenias.

Neutrophils are the shortest-lived blood cells, which requires a prodigious degree of proliferation and differentiation to sustain physiologically sufficient numbers and be poised to respond quickly to infectious emergencies. More than 107 neutrophils are produced every minute in an adult bone marrow-a process that is tightly regulated by a small group of cytokines and chemical mediators and dependent on nutrients and energy. Like granulocyte colony-stimulating factor, the primary growth factor for granulopoiesis, they stimulate signalling pathways, some affecting metabolism. Nutrient or energy deficiency stresses the survival, proliferation, and differentiation of neutrophils and their precursors. Thus, it is not surprising that monogenic disorders related to metabolism exist that result in neutropenia. Among these are pathogenic mutations in HAX1, G6PC3, SLC37A4, TAFAZZIN, SBDS, EFL1 and the mitochondrial disorders. These mutations perturb carbohydrate, lipid and/or protein metabolism. We hypothesize that metabolic disturbances may drive the pathogenesis of a subset of inherited neutropenias just as defects in DNA damage response do in Fanconi anaemia, telomere maintenance in dyskeratosis congenita and ribosome formation in Diamond-Blackfan anaemia. Greater understanding of metabolic pathways in granulopoiesis will identify points of vulnerability in production and may point to new strategies for the treatment of neutropenias.

Defects in DNA damage responses in SWI/SNF mutant cells and their impact on immune responses

The mammalian SWI/SNF chromatin remodelling complexes are commonly dysregulated in cancer. These complexes contribute to maintaining genome stability through a variety of pathways. Recent research has highlighted an important interplay between genome instability and immune signalling, and evidence suggests that this interplay can modulate the response to immunotherapy. Here, we review emerging studies where direct evidence of this relationship has been uncovered in SWI/SNF deficient cells. We also highlight genome maintenance activities of SWI/SNF that could potentially shape immune responses and discuss potential therapeutic implications.

ZMYM3 Mutations Cooperate with NOTCH1 Alterations, Reduce Histone H4 Acetylation and Promote Apoptosis Evasion in Chronic Lymphocytic Leukemia

Introduction: Recent next-generation sequencing (NGS) studies have identified up to 200 recurrently mutated genes in chronic lymphocytic leukemia (CLL), the vast majority of which appear mutated in <5% of CLL cases. Among the long list of CLL candidate driver genes is ZMYM3, a gene mutated in 2-4% of CLL patients. Since the impact of ZMYM3 mutations is unknown, we aimed to identify their clinical and biological consequences in CLL. Methods: 487 CLL patients were analyzed by targeted NGS using a customized panel of 54 CLL-related genes to characterize the mutational profile of ZMYM3 mutated patients ( ZMYM3 MUT) and the clinical impact of these variants. In addition, we introduced ZMYM3 truncating mutations into the CLL-derived HG3 cell line with two genetic backgrounds: wild-type (WT), and NOTCH1 mutated cells ( NOTCH1 MUT), to generate cells with either single ZMYM3 MUT, NOTCH1 MUT or combined ZMYM3 MUT NOTCH1 MUT. In these CLL models, we evaluated the impact of these alterations in gene expression, apoptosis, growth and DNA damage response (DDR). Results: A total of 32 ZMYM3 variants were identified in 30 CLL patients, most of which were loss-of-function mutations (24/32; 75%). These variants were distributed throughout the protein sequence, with a particular recurrent frameshift mutation occurring in 6 patients (p.P48fs) that was subsequently reproduced in our CLL model. Attending to their mutational profile, we identified that 70% (21/30) of ZMYM3 MUT patients harbored mutations in the NOTCH pathway, either in NOTCH1 (60%) or in negative regulators such as MED12, FBXW7 and SPEN, suggesting a cooperation between ZMYM3 dysfunction and NOTCH1 signaling mutations in CLL evolution. Interestingly, ZMYM3 MUT patients exhibited significantly shorter time to first treatment (TFT) than ZMYM3 WT cases (median: 35 vs 52 months; p=0.010). Furthermore, ZMYM3 mutations shortened TFT of early stage CLL patients, including Binet A (48 vs 108 months, p=0.002) or Rai 0/1 (48 vs 91 months, p=0.016), as well as in low-risk cytogenetics del(13q) patients (median: 45 vs 93 months; p=0.033). We next addressed the biological implications of ZMYM3 mutations and their co-occurrence with NOTCH1 mutations in HG3 cells. First, we performed RNA-sequencing and analyzed the number of differentially expressed genes (DEGs) (|fold change|>2, adjusted p-value<0.05). Comparing with WT cells, we detected 155 DEGs in ZMYM3 MUT and 83 DEGs in NOTCH1 MUT, whereas ZMYM3 MUT NOTCH1 MUT cells showed 690 DEGs,indicating that the combination of both mutations profoundly dysregulate gene expression. Immune regulation, apoptosis and DNA damage response were some of the biological processes dysregulated by ZMYM3 mutations (p<0.05). Furthermore, considering the suggested role of ZMYM3 in chromatin remodeling (Puente et al, Nature. 2015) and that ZMYM3 MUT cells showed altered expression of histone acetylation related genes (p<0.05), we identified that ZMYM3 mutations reduced global acetylation levels of histone H4 (p<0.05), which may underlie the ZMYM3-related changes in gene expression. To further define the functional effects of ZMYM3 mutations, we assessed in vitro their impact in the main dysregulated pathways identified by RNA-seq. First, we identified that ZMYM3 mutations impair DNA damage response, as reflected by the difficulties to arrest cell cycle in G2/M phase after irradiation. Moreover, we detected more γH2AX foci (p<0.05) in ZMYM3 MUTcells, indicating persistent DNA damage; and fewer RAD51 and BRCA1 foci than WT cells (p<0.001) after irradiation, suggesting defective DNA damage response. In parallel, we identified that ZMYM3 mutations promote apoptosis evasion, especially in the context of NOTCH1 mutations (p<0.001), which led to enhanced growth of ZMYM3 MUT NOTCH1 MUT cells (p<0.05). Of note, ZMYM3 MUT cells showed down-expression of caspases (-8, -7 and -3) and slightly higher levels of anti-apoptotic proteins (BCL2, MCL1 and BCL-XL), which may explain this apoptosis resistance. Conclusions: Mutations in ZMYM3 are mainly loss-of-function, associate with NOTCH1 signaling mutations and shorten TFT in CLL patients, suggesting that ZMYM3 mutational status may be a useful marker in the management of early stage CLL patients. Moreover, ZMYM3 mutations reduce histone H4 acetylation and cooperate with NOTCH1 mutations to dysregulate gene-expression, leading to impair DNA damage response and apoptosis evasion.

DNA Damage Response and Mismatch Repair Gene Defects in Advanced and Metastatic Prostate Cancer.

Alterations in DNA damage response (DDR) and related genes are present in up to 25% of advanced prostate cancers (PCa). Most frequently altered genes are involved in the homologous recombination repair, the Fanconi anemia, and the mismatch repair pathways, and their deficiencies lead to a highly heterogeneous spectrum of DDR-deficient phenotypes. More than half of these alterations concern non- BRCA DDR genes. From a therapeutic perspective, poly-ADP-ribose polymerase inhibitors have demonstrated robust clinical efficacy in tumors with BRCA2 and BRCA1 alterations. Mismatch repair-deficient PCa, and a subset of CDK12-deficient PCa, are vulnerable to immune checkpoint inhibitors. Emerging data point to the efficacy of ATR inhibitors in PCa with ATM deficiencies. Still, therapeutic implications are insufficiently clarified for most of the non- BRCA DDR alterations, and no successful targeted treatment options have been established.

Effect of ubiquitin protease system on DNA damage response in prostate cancer (Review).

Genomic instability is an essential hallmark of cancer, and cellular DNA damage response (DDR) defects drive tumorigenesis by disrupting genomic stability. Several studies have identified abnormalities in DDR-associated genes, and a dysfunctional ubiquitin-proteasome system (UPS) is the most common molecular event in metastatic castration-resistant prostate cancer (PCa). For example, mutations in Speckle-type BTB/POZ protein-Ser119 result in DDR downstream target activation deficiency. Skp2 excessive upregulation inhibits homologous recombination repair and promotes cell growth and migration. Abnormally high expression of a deubiquitination enzyme, ubiquitin-specific protease 12, stabilizes E3 ligase MDM2, which further leads to p53 degradation, causing DDR interruption and genomic instability. In the present review, the basic pathways of DDR, UPS dysfunction, and its induced DDR alterations mediated by genomic instability, and especially the potential application of UPS and DDR alterations as biomarkers and therapeutic targets in PCa treatment, were described.

Runx1-R188Q germ line mutation induces inflammation and predisposition to hematologic malignancies in mice

AbstractGerm line mutations in the RUNX1 gene cause familial platelet disorder (FPD), an inherited disease associated with lifetime risk to hematopoietic malignancies (HM). Patients with FPD frequently show clonal expansion of premalignant cells preceding HM onset. Despite the extensive studies on the role of RUNX1 in hematopoiesis, its function in the premalignant bone marrow (BM) is not well-understood. Here, we characterized the hematopoietic progenitor compartments using a mouse strain carrying an FPD-associated mutation, Runx1R188Q. Immunophenotypic analysis showed an increase in the number of hematopoietic stem and progenitor cells (HSPCs) in the Runx1R188Q/+ mice. However, the comparison of Sca-1 and CD86 markers suggested that Sca-1 expression may result from systemic inflammation. Cytokine profiling confirmed the dysregulation of interferon-response cytokines in the BM. Furthermore, the expression of CD48, another inflammation-response protein, was also increased in Runx1R188Q/+ HSPCs. The DNA-damage response activity of Runx1R188Q/+ hematopoietic progenitor cells was defective in vitro, suggesting that Runx1R188Q may promote genomic instability. The differentiation of long-term repopulating HSCs was reduced in Runx1R188Q/+ recipient mice. Furthermore, we found that Runx1R188Q/+ HSPCs outcompete their wild-type counterparts in bidirectional repopulation assays, and that the genetic makeup of recipient mice did not significantly affect the clonal dynamics under this setting. Finally, we demonstrate that Runx1R188Q predisposes to HM in cooperation with somatic mutations found in FPDHM, using 3 mouse models. These studies establish a novel murine FPDHM model and demonstrate that germ line Runx1 mutations induce a premalignant phenotype marked by BM inflammation, selective expansion capacity, defective DNA-damage response, and predisposition to HM.

Design, Synthesis, and Biological Evaluation of Potent and Selective Inhibitors of Ataxia Telangiectasia Mutated and Rad3-Related (ATR) Kinase for the Efficient Treatment of Cancer.

Ataxia telangiectasia mutated and Rad3-related (ATR), a vital member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, plays a critical role in the DNA damage response (DDR). Tumor cells with a loss of DDR function or defects in the ataxia telangiectasia mutated (ATM) gene are generally more dependent on ATR for survival, suggesting that ATR is an attractive anticancer drug target based on its synthetic lethality. Herein, we present a potent and highly selective ATR inhibitor, ZH-12 (IC50 = 0.0068 μM). It showed potent antitumor activity as a single agent or in combination with cisplatin in the human colorectal adenocarcinoma LoVo tumor xenograft mouse model. Overall, ZH-12 may be a promising ATR inhibitor based on the principle of synthetic lethality and deserves further in-depth study.

Chromosomal instability and its effect on cell lines.

Cancer cell lines are invaluable model systems for biomedical research because they provide an almost unlimited supply of biological materials. However, there is considerable skepticism regarding the reproducibility of data derived from these in vitro models. Chromosomal instability (CIN) is one of the primary issues associated with cell lines, which can cause genetic heterogeneity and unstable cell properties within a cell population. Many of these problems can be avoided with some precautions. Here we review the underlying causes of CIN, including merotelic attachment, telomere dysfunction, DNA damage response defects, mitotic checkpoint defects and cell cycle disturbances. In this review we summarize studies highlighting the consequences of CIN in various cell lines and provide suggestions on monitoring and controlling CIN during cell culture.

Abstract CT268: Targeting replication stress and chemotherapy resistance with a combination of sacituzumab govitecan and berzosertib: A phase I clinical trial

Abstract Despite provocative preclinical results, dose-limiting toxicities have precluded rational combinations of cytotoxic chemotherapies that increase DNA damage with DNA damage response (DDR) inhibitors. We hypothesized that tumor-targeted delivery of cytotoxic chemotherapy might enable tolerable and active combinations with DDR inhibitors. We conducted a phase I clinical trial combining ataxia telangiectasia and Rad3-related (ATR) inhibitor berzosertib with sacituzumab govitecan, a trophoblast cell surface antigen 2 (Trop-2) directed antibody drug conjugate (ADC) that delivers high tumoral concentrations of topoisomerase 1 (TOP1) inhibitor SN-38. Depletion of ATR, the main transducer of replication stress is synthetically lethal with double-strand breaks (DSB) generated by TOP1 inhibitors. Patients with DDR gene-mutated or high replication stress solid tumors were enrolled since such tumors are particularly susceptible to ATR inhibition. Primary end point was identification of the maximum tolerated dose of the combination. Efficacy and pharmacodynamics were secondary end points. Using 3 + 3 dose escalation, sacituzumab govitecan (8-10 mg/m2, days 1, 8) and berzosertib (140-210 mg/m2, days 2, 9) were administered to 12 patients across three dose levels in 21-day cycles. The combination was well tolerated, with improved safety profile over conventional chemotherapy-based combinations, which allowed dose escalation to the highest planned dose level. There were no dose limiting toxicities. Common treatment-related adverse events (TRAE) were neutropenia (41.7%), diarrhea (50%), and fatigue (50%). Grade 3 TRAEs occurred in 58.3% of patients and included neutropenia (25%) and diarrhea (8.3%). There were no instances of febrile neutropenia or clinically significant grade 4 TRAEs. Pharmacodynamic studies showed evidence of ATR inhibition and enhanced DNA DSB in response to the combination. While no tumor responses were seen in three patients with DDR defects including BRCA1 and ATM mutations, two patients with neuroendocrine prostate cancer, a highly aggressive subtype of prostate cancer, showed partial or metabolic responses. A patient with EGFR-transformed small cell lung cancer (SCLC) also experienced partial response, together yielding objective responses in 3 of 12 evaluable patients (25%). Ongoing phase II expansion cohorts are evaluating efficacy of sacituzumab govitecan 10mg/m2 and berzosertib 210mg/m2 in patients with SCLC, extra-pulmonary small cell cancers, and DDR-mutated solid tumors. ADC-based delivery of cytotoxic payload represents a new therapeutic paradigm to extend the benefit of DDR inhibitors to target replication stress and chemotherapy resistance, with minimal added toxicities. Clinical trial information: NCT04826341 Citation Format: Melissa L. Abel, Nobukyuki Takahashi, Parth Desai, Cody Peer, Christophe Redon, Samantha Nichols, Rasa Vilimas, Min-Jung Lee, Sunmin Lee, Linda Sciuto, Danielle Pinkiert, Meenakshi Shelat, Chante Graham, Seth Steinberg, William D. Figg, Mirit Aladjem, Jane Trepel, Yves Pommier, Anish Thomas. Targeting replication stress and chemotherapy resistance with a combination of sacituzumab govitecan and berzosertib: A phase I clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT268.

Discovery of Thieno[3,2-d]pyrimidine derivatives as potent and selective inhibitors of ataxia telangiectasia mutated and Rad3 related (ATR) kinase

The ataxia telangiectasia mutated and rad3-related (ATR) kinase regulates the DNA damage response (DDR), which plays a critical role in the ATR-Chk1 signaling pathway. ATR inhibition can induce synthetic lethality (SL) with several DDR deficiencies, making it an attractive drug target for cancers with DDR defects. In this study, we developed a series of selective and potent ATR inhibitors with a thieno[3,2-d]pyrimidine scaffold using a hybrid design. We identified compound 34 as a representative molecule that inhibited ATR kinase with an IC50 value of 1.5 nM and showed reduced potency against other kinases tested. Compound 34 also exhibited potent antiproliferative effects against LoVo cells and SL effects against HT-29 cells. Moreover, compound 34 demonstrated good pharmacokinetic properties, in vivo antitumor efficacy, and no obvious toxicity in the LoVo xenograft tumor model. Therefore, compound 34 is a promising lead compound for drug development to combat specific DDR deficiencies in cancer patients.

Abstract 6111: Design of a small molecule screening assay to detect DNA trapping of PARP1/2

Abstract Poly(ADP-ribose) polymerase (PARP) inhibitors are currently used in the clinic for the treatment of tumors with a defective DNA damage response (DDR). When PARP1 or PARP2 binds damaged DNA, it adds poly(ADP-ribose) chains to its own backbone and to other DDR proteins, which recruits and activates them. PARylated PARP1/2 next detaches from the DNA so that the other PARylated proteins can initiate the repair process. It has been observed that some PARP inhibitors prevent PARP1/2 from dissociating the DNA. The continuous presence of PARP at the site of damage prevents repair and blocks replication, leading to cell death. Therefore, drugs that trap PARP1/2 to the DNA tend to be significantly more cytotoxic than other PARP inhibitors, which is highly desirable. This study describes the design and optimization of novel PARPtrap assays to specifically assess the ability of a drug to trap PARP onto DNA. The assay is based on principles of fluorescence polarization and uses fluorescently labeled DNA probes that are excited by polarized light and emit light with a degree of polarization that is proportional to the rate of molecular rotation. The free DNA probes that rotate fast have low fluorescence polarization (FP), but high FP when are bound to PARP1 or PARP2. When NAD+ is added, the PARylated enzymes detach from the probe, reducing FP levels. If a PARP inhibitor is added, the inhibitor’s trapping ability increases FP in a dose-dependent manner. Proof-of-principle titration of known PARP-trapping inhibitors (Talazoparib, AZD305, Olaparib and Veliparib) was performed to validate the assay. We observed that: i)The known relative trapping efficacies of Talazoparib, Olaparib and Veliparib were similar to known relative efficacies. ii)Talazoparib, Olaparib and Veliparib had similar trapping efficacy against PARP1 and PARP2, as measured by their EC50, whereas AZD305 was 1,000 times more efficient at trapping PARP1 than it was PARP2, demonstrating selectivity between PARP1 and PARP2. iii)AZD305 displayed as efficient DNA trapping activity toward PARP1 as best-in-class Talazoparib. In summary, we have designed an innovative PARPtrap assay designed for the high throughput screening of small molecule libraries to specifically identify or compare inhibitors that are capable of trapping PARP1 and/or PARP2 onto DNA. Citation Format: Kasia Zientara-Rytter, Veronique T. Baron, Junguk Park, Pavel Shashkin, Henry Zhu. Design of a small molecule screening assay to detect DNA trapping of PARP1/2 [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 6111.

Abstract 6180: Antitumor activity of the ATR inhibitor Elimusertib in patient-derived xenograft models with DNA damage response pathway alterations

Abstract Introduction: The ataxia telangiectasia and RAD3-related (ATR) kinase is a key component of the DNA damage response (DDR) and functions in conjunction with ataxia telangiectasia mutated (ATM). ATM loss or functional deficiency may enhance reliance on ATR signaling. Preclinical and clinical studies have pointed to a potential synthetic lethality of ATR inhibition and ATM loss. In this study, we sought to further elaborate potential candidates for ATR inhibition by testing the anti-tumor effect of elimusertib (BAY 1895344) in patient-derived tumor xenograft (PDX) models with DDR alterations including ATM loss. Methods: Patient derived xenograft (PDX) models were implanted into athymic nu/nu mice. Once tumors reached an appropriate size of approximately 200-400mm3 treatments were started. Mice were randomized (N= 3-8) to different treatment arms for assessment of anti-tumor activity of elimusertib as monotherapy and in combination therapy with copanlisib. Monotherapy treatment was performed with 20 mg/kg and 40 mg/kg doses, applied twice daily for 3 days with 4 days treatment break (BID 3 days on/4 days off). Efficacy read-out was either Event-Free Survival (EFS-2), defined as time for doubling of tumor volume relative to baseline, or response based on RECIST criteria:(partial response [PR]= >30% decrease, stable disease [SD]= between 20% increase and 30% decrease, and progressive disease [PD]= >20% increase). For assessment of pharmacodynamic effects, tumors were harvested 10 days after treatment start and analyzed by immunohistochemistry (IHC) and RPPA (Reverse Phase Proteomics Array). Results: Elimusertib was tested in 21 PDX models with various DDR alterations. Of those 11 showed a statistically significant prolongation of event free survival compared to control. In 4 PDX models elimusertib reached a partial response (PR), and another 4 models showed stable disease (SD). Amongst the 5 models with ATM protein Ioss (IHC), one was PR and another one SD. In 3 of 5 PARP inhibitor resistant there was statistically significant prolongation of EFS upon elimusertib treatment vs control. We found an increase in DNA damage in tumors, as indicated by increased level of ƳH2AX (on IHC and RPPA). We also detected an increase in PI3K/mTOR pathway signaling as determined by p-MTOR and pS6 (RPPA) in 2 of 4 models (PR and PD model) with different sensitivity to ATRi and PARPi alone. We tested the combination of elimusertib with the PI3K inhibitor copanlisib achieved a statistically significant increase of EFS compared to monotherapy in 3 of 11 models tested. Conclusion ATR inhibition shows potent monotherapy activity in selected PDX models that harbor DDR defects, including models with intrinsic and acquired PARPi resistance. Further work is needed to identify markers predicting sensitivity or resistance that providing rationale for synergistic combination therapies. Citation Format: Kaushik Varadarajan, Christian X. Pico, Kurt Evans, Maria G. Raso, Yasmeen Rizvi, Xiaofeng Zheng, Timothy P. Diperi, Bailiang Wang, Stephen Scott, Ming Zhao, Argun Akcakanat, Antje M. Wengner, Timothy A. Yap, Funda Meric-Bernstam. Antitumor activity of the ATR inhibitor Elimusertib in patient-derived xenograft models with DNA damage response pathway alterations. [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 6180.

Abstract 311: Optimization of treatment schedule for the combination therapy of ATR inhibitor elimusertib and PARP inhibitor niraparib in preclinical tumor models

Abstract Background: Ataxia telangiectasia and Rad3-related (ATR) kinase and poly-(ADP-ribose) polymerase (PARP) are central components of the DNA damage response (DDR) machinery that act via distinct DNA repair pathways whereby combined inhibition of ATR and PARP is expected to be highly synergistic. Elimusertib is a potent and selective ATR inhibitor currently in phase I development in patients with advanced solid tumors carrying DDR defects. Niraparib is an approved PARP inhibitor for the treatment of ovarian cancer. Several different dosing schedules of a combination treatment with elimusertib and niraparib were explored in preclinical tumor model systems to identify a dose regimen that optimally balances safety and efficacy for translation into clinical studies. Methods: Preclinical human mCRPC xenograft model 22RV1 in male immunocompromised (NMRI nude) mice was used. Multiple variations of treatment duration and sequence of drug application were investigated. Both drugs were used at their MTD in the respective combination dosing regimen. Antitumor activity was assessed by tumor area measurement, and safety/tolerability was determined by body weight change and evaluation of hematological parameters. Results: Preclinical data demonstrated strong synergistic antitumor activity resulting from combined blockade of ATR and PARP. In vitro and in vivo data revealed that concomitant inhibition of both ATR and PARP mediated DNA repair is required for maximal synergy. 13 different combination dosing schedules were tested in vivo. Elimusertib was dosed between 20 and 40 mg/kg BID for 3 days on/4 days off, or 3 days on/11 days off. Niraparib was dosed QD continuously or using various intermittent schedules. Overall, best efficacy and tolerability was achieved when PARPi and ATRi were applied concurrently using a discontinuous schedule. The condensed treatment time of both drugs and the extended treatment-free period demonstrated better mitigation of hematological effects, reducing the treatment impact on red blood cell counts in peripheral blood. Conclusion: In preclinical in vitro and in vivo studies it was demonstrated that concurrent inhibition of ATR and PARP resulted in higher tumor cell cytotoxicity than any sequential blockade of ATR and PARP. Evaluating different treatment regimens of combined elimusertib and niraparib indicated that applying both drugs using a discontinuous schedule to balance efficacy and safety in a preclinical mCRPC xenograft model produces an optimal therapeutic window. A non-randomized, open-label trial evaluating the safety and efficacy of elimusertib in combination with niraparib for the treatment of patients with advanced solid tumors is currently ongoing (NCT04267939). Citation Format: Antje Margret Wengner, Gerhard Siemeister, Bart Ploeger, Lisa Ehresmann, Nils Guthof, Gary Wilkinson. Optimization of treatment schedule for the combination therapy of ATR inhibitor elimusertib and PARP inhibitor niraparib in preclinical tumor models [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 311.

The Molecular and Cellular Basis of Hutchinson-Gilford Progeria Syndrome and Potential Treatments.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, autosomal-dominant, and fatal premature aging syndrome. HGPS is most often derived from a de novo point mutation in the LMNA gene, which results in an alternative splicing defect and the generation of the mutant protein, progerin. Progerin behaves in a dominant-negative fashion, leading to a variety of cellular and molecular changes, including nuclear abnormalities, defective DNA damage response (DDR) and DNA repair, and accelerated telomere attrition. Intriguingly, many of the manifestations of the HGPS cells are shared with normal aging cells. However, at a clinical level, HGPS does not fully match normal aging because of the accelerated nature of the phenotypes and its primary effects on connective tissues. Furthermore, the epigenetic changes in HGPS patients are of great interest and may play a crucial role in the pathogenesis of HGPS. Finally, various treatments for the HGPS patients have been developed in recent years with important effects at a cellular level, which translate to symptomatic improvement and increased lifespan.

Combination of EphA2- and Wee1-Targeted Therapies in Endometrial Cancer.

EphA2 tyrosine kinase is upregulated in many cancers and correlated with poor survival of patients, including those with endometrial cancer. EphA2-targeted drugs have shown modest clinical benefit. To improve the therapeutic response to such drugs, we performed a high-throughput chemical screen to discover novel synergistic partners for EphA2-targeted therapeutics. Our screen identified the Wee1 kinase inhibitor, MK1775, as a synergistic partner to EphA2, and this finding was confirmed using both in vitro and in vivo experiments. We hypothesized that Wee1 inhibition would sensitize cells to EphA2-targeted therapy. Combination treatment decreased cell viability, induced apoptosis, and reduced clonogenic potential in endometrial cancer cell lines. In vivo Hec1A and Ishikawa-Luc orthotopic mouse models of endometrial cancer showed greater anti-tumor responses to combination treatment than to either monotherapy. RNASeq analysis highlighted reduced cell proliferation and defective DNA damage response pathways as potential mediators of the combination's effects. In conclusion, our preclinical findings indicate that Wee1 inhibition can enhance the response to EphA2-targeted therapeutics in endometrial cancer; this strategy thus warrants further development.

Systematic characterization of Brassica napus UBC13 genes involved in DNA-damage response and K63-linked polyubiquitination

BackgroundUbc13 is the only known ubiquitin conjugating enzyme (Ubc/E2) dedicated to promoting Lys (K)63-linked polyubiquitination, and this process requires a Ubc/E2 variant (UEV). Unlike conventional K48-linked polyubiquitination that targets proteins for degradation, K63-linked polyubiquitination, which is involved in several cellular processes, does not target proteins for degradation but alter their activities.ResultsIn this study we report the identification and functional characterization of 12 Brassica napus UBC13 genes. All the cloned UBC13 gene products were able to physically interact with AtUev1D, an Arabidopsis UEV, to form stable complexes that are capable of catalyzing K63-linked polyubiquitination in vitro. Furthermore, BnUBC13 genes functionally complemented the yeast ubc13 null mutant defects in spontaneous mutagenesis and DNA-damage responses, suggesting that BnUBC13s can replace yeast UBC13 in mediating K63-linked polyubiquitination and error-free DNA-damage tolerance.ConclusionCollectively, this study provides convincing data to support notions that B. napus Ubc13s promote K63-linked polyubiquitination and are probably required for abiotic stress response. Since plant Ubc13-UEV are also implicated in other developmental and stress responses, this systematic study sets a milestone in exploring roles of K63-linked polyubiquitination in this agriculturally important crop.