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Correlation of biomarkers of homologous recombination repair deficiency with benefit of addition of berzosertib to gemcitabine. a SigMA identified a larger proportion of tumors positive for Sig3 compared to BRCA1/2 and homologous recombination repair (HRR) alterations. b HRR (including BRCA1/2) gene alterations identified in patient tumor specimens from the study. c Progression-free survival among patients with Signature 3 (Sig3) positive tumors. d Progression-free survival among patients with Sig3-negative tumors.
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In a trial of patients with high grade serous ovarian cancer (HGSOC), addition of the ATR inhibitor berzosertib to gemcitabine improved progression free survival (PFS) compared to gemcitabine alone but biomarkers predictive of treatment are lacking. Here we report a candidate biomarker of response to gemcitabine versus combined gemcitabine and ATR...
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... targeted NGS was performed in 57 patient tumor specimens and identified 14 (25%) patients with HRR mutated/ altered tumors (Fig. 2a). Of these, 11 (19%) patients had BRCA1/2-mutated tumors (7 BRCA1 and 4 BRCA2), 2 (4%) patients had tumors with BRIP1 mutations, and 1 (2%) patient had a tumor with a two-copy deletion of RAD51C (Fig. 2a, b). Using a previously developed and validated computational tool called SigMA (Signature Multivariate Analysis) 32,34 on the ...
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... targeted NGS was performed in 57 patient tumor specimens and identified 14 (25%) patients with HRR mutated/ altered tumors (Fig. 2a). Of these, 11 (19%) patients had BRCA1/2-mutated tumors (7 BRCA1 and 4 BRCA2), 2 (4%) patients had tumors with BRIP1 mutations, and 1 (2%) patient had a tumor with a two-copy deletion of RAD51C (Fig. 2a, b). Using a previously developed and validated computational tool called SigMA (Signature Multivariate Analysis) 32,34 on the OncoPanel sequencing data, we identified the presence of Sig3 in 23 (40%) of the patients, thereafter denoted as Sig3 positive. SigMA thus identified a larger proportion of tumors as HRR-deficient compared to ...
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... and validated computational tool called SigMA (Signature Multivariate Analysis) 32,34 on the OncoPanel sequencing data, we identified the presence of Sig3 in 23 (40%) of the patients, thereafter denoted as Sig3 positive. SigMA thus identified a larger proportion of tumors as HRR-deficient compared to BRCA1/2 and HRR gene mutations/alterations (Fig. 2a). Among BRCA1/2 wild-type tumors, both BRIP1-mutated tumors were Sig3 positive while the RAD51C deleted tumor was Sig3 ...
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... the benefit of the addition of berzosertib to gemcitabine was observed both among patients with tumors with HRR alterations/mutations and among those with tumors without HRR alterations (Supplement Fig. 1). Similarly, the benefit of the addition of berzosertib to gemcitabine was observed independently of Sig3 status (Fig. 2c, d). Taken together, these data indicate that these biomarkers of HRR deficiency were not associated with benefit of addition of berzosertib to gemcitabine. It is also important to note that although there was a trend that patients in the Sig3-negative subgroup responded better to gemcitabine compared to patients in the Sig3-positive ...
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... of HRR deficiency were not associated with benefit of addition of berzosertib to gemcitabine. It is also important to note that although there was a trend that patients in the Sig3-negative subgroup responded better to gemcitabine compared to patients in the Sig3-positive subgroup, this was not significant [HR 1.51 (90% CI 0.72-3.18)], Supplement Fig. 2. We also assessed for alterations of nucleotide excision repair (NER) in our dataset and found no tumors with such alterations 15,21 ...
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... of the association of response to gemcitabine with markers of HRR deficiency revealed a nonsignificant trend that patients in the Sig3-negative subgroup (reflective of HRR proficient tumors) responded better to gemcitabine compared to patients in the Sig3-positive subgroup (reflective of HRR-deficient tumors), Supplement Fig. 2. This observation is consistent with available literature in ovarian cancer suggesting that response to gemcitabine is at least equal (or even better) in patients with platinum-resistant disease (a setting enriched for HRR proficient tumors) compared to platinum sensitive disease (a setting enriched for HRR-deficient tumors) 36,37 ...
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... 71 Subsequently, further exploration must be conducted to accurately estimate gemcitabine's beneficial modulation of immune parameters from potential disadvantageous effects. Various investigations have sought biomarker correlates of gemcitabine treatment and patient outcomes, 175,176 and some studies have included hematological and immunological factors. 173,177 For example, Blomstrand et al. profiled blood proteins and cells in pancreatic cancer patients and evaluated whether any were prognostic for prolonged survival. ...
Although the development of immunotherapies has been revolutionary in the treatment of several cancers, many cancer types remain unresponsive to immune‐based treatment and are largely managed by chemotherapy drugs. However, chemotherapeutics are not infallible and are frequently rendered ineffective as resistance develops from prolonged exposure. Recent investigations have indicated that some chemotherapy drugs have additional functions beyond their normative cytotoxic capacity and are in fact immune‐modifying agents. Of the pharmaceuticals with identified immune‐editing properties, gemcitabine is well‐studied and of interest to clinicians and scientists alike. Gemcitabine is a chemotherapy drug approved for the treatment of multiple cancers, including breast, lung, pancreatic, and ovarian. Because of its broad applications, relatively low toxicity profile, and history as a favorable combinatory partner, there is promise in the recharacterization of gemcitabine in the context of the immune system. Such efforts may allow the identification of suitable immunotherapeutic combinations, wherein gemcitabine can be used as a priming agent to improve immunotherapy efficacy in traditionally insensitive cancers. This review looks to highlight documented immunomodulatory abilities of one of the most well‐known chemotherapy agents, gemcitabine, relating to its influence on cells and proteins of the immune system.
... HRD score was defined as an unweighted sum of three signatures: Number of telomeric Allelic Imbalances (NtAI), Large-scale State Transitions (LST), and loss of heterozygosity (LOH) ( 16 ). Replication Stress (RS) was defined as previously described ( 17 ). In brief, tumors classified as RS-high included at least one of the following alterations ( CCNE1 amplification, MYC and MYCL1 amplification, KRAS amplification, NF1 mutation, RB1 two-copy loss, CDKN2A two-copy loss, ERBB2 amplification) whereas tumors without these features were classified as R S-low. ...
Genome maintenance is an enabling characteristic that allows neoplastic cells to tolerate the inherent stresses of tumorigenesis and evade therapy-induced genotoxicity. Neoplastic cells also deploy many mis-expressed germ cell proteins termed Cancer Testes Antigens (CTAs) to promote genome maintenance and survival. Here, we present the first comprehensive characterization of the DNA Damage Response (DDR) and CTA transcriptional landscapes of endometrial cancer in relation to conventional histological and molecular subtypes. We show endometrial serous carcinoma (ESC), an aggressive endometrial cancer subtype, is defined by gene expression signatures comprising members of the Replication Fork Protection Complex (RFPC) and Fanconi Anemia (FA) pathway and CTAs with mitotic functions. DDR and CTA-based profiling also defines a subset of highly aggressive endometrioid endometrial carcinomas (EEC) with poor clinical outcomes that share similar profiles to ESC yet have distinct characteristics based on conventional histological and genomic features. Using an unbiased CRISPR-based genetic screen and a candidate gene approach, we confirm that DDR and CTA genes that constitute the ESC and related EEC gene signatures are required for proliferation and therapy-resistance of cultured endometrial cancer cells. Our study validates the use of DDR and CTA-based tumor classifiers and reveals new vulnerabilities of aggressive endometrial cancer where none currently exist.
... RS can be caused by any events disrupting DNA replication but is traditionally defined as the slowing or stalling of replication-fork progression and/or DNA replication [7][8][9] . HGSOC cells have changes in many genes involved in DNA replication, e.g., universal TP53 mutations with consequently defective G1/S cellcycle regulation 3 , CCNE1 amplification, and loss of RB1 and NF1 10 . As a result, HGSOC cells highly depend on other S-and G2/M checkpoint pathways, e.g., Ataxia Telangiectasia and Rad3-related kinase (ATR)/ checkpoint kinase 1 (CHK1) signaling for DNA replication 11 . ...
... Those conflicting observations suggest that a singlebiomarker selection to target a specific population is challenging because RS is a dynamic process and there is no clear consensus about RS-related biomarkers for predicting response to ATR/CHK1 blockade. Another effort includes investigating gene signatures potentially reflective of high-RS 10,23 . The phase 2/3 clinical trial of gemcitabine alone versus gemcitabine and ATR inhibitor (ATRi) berzosertib combination in HGSOC suggested a high-RS molecular signature (MYC, MYCL1, ERBB2 and KRAS amplifications, RB1 or CDKN2A two-copy loss, or NF1 mutations) may predict gemcitabine response in patients with a high-RS background, while those with a low-RS may benefit from a combination strategy 10 . ...
... Another effort includes investigating gene signatures potentially reflective of high-RS 10,23 . The phase 2/3 clinical trial of gemcitabine alone versus gemcitabine and ATR inhibitor (ATRi) berzosertib combination in HGSOC suggested a high-RS molecular signature (MYC, MYCL1, ERBB2 and KRAS amplifications, RB1 or CDKN2A two-copy loss, or NF1 mutations) may predict gemcitabine response in patients with a high-RS background, while those with a low-RS may benefit from a combination strategy 10 . Collectively, those data suggest that additional efforts are needed to define RS and its clinical utility as a predictive biomarker. ...
The multi-cohort phase 2 trial NCT02203513 was designed to evaluate the clinical activity of the CHK1 inhibitor (CHK1i) prexasertib in patients with breast or ovarian cancer. Here we report the activity of CHK1i in platinum-resistant high-grade serous ovarian carcinoma (HGSOC) with measurable and biopsiable disease (cohort 5), or without biopsiable disease (cohort 6). The primary endpoint was objective response rate (ORR). Secondary outcomes were safety and progression-free survival (PFS). 49 heavily pretreated patients were enrolled (24 in cohort 5, 25 in cohort 6). Among the 39 RECISTv1.1-evaluable patients, ORR was 33.3% in cohort 5 and 28.6% in cohort 6. Primary endpoint was not evaluable due to early stop of the trial. The median PFS was 4 months in cohort 5 and 6 months in cohort 6. Toxicity was manageable. Translational research was an exploratory endpoint. Potential biomarkers were investigated using pre-treatment fresh biopsies and serial blood samples. Transcriptomic analysis revealed high levels of DNA replication-related genes ( POLA1 , POLE , GINS3 ) associated with lack of clinical benefit [defined post-hoc as PFS < 6 months]. Subsequent preclinical experiments demonstrated significant cytotoxicity of POLA1 silencing in combination with CHK1i in platinum-resistant HGSOC cell line models. Therefore, POLA1 expression may be predictive for CHK1i resistance, and the concurrent POLA1 inhibition may improve the efficacy of CHK1i monotherapy in this hard-to-treat population, deserving further investigation.
... Last, selected chemotherapeutics such as gemcitabine, an antimetabolite that inhibits DNA synthesis, also achieved high efficacy and synergy when combined with ATR and ATM inhibitors ( Fig. 2a, b). While the synergistic relationship between ATRi and gemcitabine has been reported before 30 , we note that similar relationships between gemcitabine and either DNA-PKi or ATMi have not been reported before, to our knowledge. Overall, the dataset shows a low Pearson's correlation of 0.2 (p < 1e−22) between efficacy and synergy, which, while well within the range of values observed in previous studies 31,32 , highlights the need of analyzing both measures of response independently. ...
One fundamental principle that underlies various cancer treatments, such as traditional chemotherapy and radiotherapy, involves the induction of catastrophic DNA damage, leading to the apoptosis of cancer cells. In our study, we conduct a comprehensive dose-response combination screening focused on inhibitors that target key kinases involved in the DNA damage response (DDR): ATR, ATM, and DNA-PK. This screening involves 87 anti-cancer agents, including six DDR inhibitors, and encompasses 62 different cell lines spanning 12 types of tumors, resulting in a total of 17,912 combination treatment experiments. Within these combinations, we analyze the most effective and synergistic drug pairs across all tested cell lines, considering the variations among cancers originating from different tissues. Our analysis reveals inhibitors of five DDR-related pathways (DNA topoisomerase, PLK1 kinase, p53-inducible ribonucleotide reductase, PARP, and cell cycle checkpoint proteins) that exhibit strong combinatorial efficacy and synergy when used alongside ATM/ATR/DNA-PK inhibitors.
... Given the challenge of predicting the functional impact of clinically observed ATM alterations, several recent efforts in other tumor types have focused on developing an ATM IHC assay to identify tumors with loss of ATM protein (31)(32)(33). IHC is an attractive method because it has the potential to identify functional ATM deficiency conferred by a wide variety of genetic or epigenetic alterations, and ATM IHC has been performed in several PARP and ATR inhibitor trials (11,34,35). However, ATM IHC has not been investigated specifically in bladder cancer. ...
Ataxia-telangiectasia mutated (ATM) plays a central role in the cellular response to DNA damage and ATM alterations are common in several tumor types including bladder cancer. However, the specific impact of ATM alterations on therapy response in bladder cancer is uncertain. Here, we combine preclinical modeling and clinical analyses to comprehensively define the impact of ATM alterations on bladder cancer. We show that ATM loss is sufficient to increase sensitivity to DNA-damaging agents including cisplatin and radiation. Furthermore, ATM loss drives sensitivity to DNA repair–targeted agents including poly(ADP-ribose) polymerase (PARP) and Ataxia telangiectasia and Rad3 related (ATR) inhibitors. ATM loss alters the immune microenvironment and improves anti-PD1 response in preclinical bladder models but is not associated with improved anti-PD1/PD-L1 response in clinical cohorts. Last, we show that ATM expression by immunohistochemistry is strongly correlated with response to chemoradiotherapy. Together, these data define a potential role for ATM as a predictive biomarker in bladder cancer.
... HRD score was derived as an unweighted sum of three signatures: Number of telomeric Allelic Imbalances (NtAI), Large-scale State Transitions (LST), and HRD-LOH 68 . Replication Stress (RS) was derived as previously described 69 . In brief, tumors classified as RS-high included at least one of the following alterations (CCNE1 amplification, MYC and MYCL1 amplification, KRAS amplification, NF1 mutation, RB1 two-copy loss, CDKN2A two-copy loss, ERBB2 amplification) whereas tumors without these features were classified as RS-low. ...
Genome maintenance is an enabling characteristic that allows neoplastic cells to tolerate the inherent stresses of tumorigenesis and evade therapy-induced genotoxicity. Neoplastic cells also deploy mis-expressed germ cell proteins termed Cancer Testes Antigens (CTAs) to promote genome maintenance and survival. Here, we present the first comprehensive characterization of the DNA Damage Response (DDR) and CTA transcriptional landscapes of endometrial cancer in relation to conventional histological and molecular subtypes. We show endometrial serous carcinoma (ESC), an aggressive endometrial cancer subtype, is defined by gene expression signatures comprising members of the Replication Fork Protection Complex (RFPC) and Fanconi Anemia (FA) pathway and CTAs with mitotic functions. DDR and CTA-based profiling also defines a subset of highly aggressive endometrioid endometrial carcinomas (EEC) with poor clinical outcomes that share similar profiles to ESC yet have distinct characteristics based on conventional histological and genomic features. Using an unbiased CRISPR-based genetic screen and a candidate gene approach, we confirm that DDR and CTA genes that constitute the ESC and related EEC gene signatures are required for proliferation and therapy-resistance of cultured endometrial cancer cells. Our study validates the use of DDR and CTA-based tumor classifiers and reveals new vulnerabilities of aggressive endometrial cancer where none currently exist.
... 14,61,62 Increased replication stress has also been linked to ovarian cancer, and recent studies have looked for ways to target this through new therapeutic strategies for ovarian cancer. [63][64][65] The compromised ability of FTEs to effectively handle stalled replication forks and consequently accumulate DNA damage upon replication stress renders them acutely vulnerable to genomic instability. We propose that this susceptibility positions FTEs as significant contributors to the initiation and progression of ovarian cancer. ...
Clinical and molecular evidence indicates that high-grade serous ovarian cancer (HGSOC) primarily originates from the fallopian tube, not the ovarian surface. However, the reasons for this preference remain unclear. Our study highlights significant differences between fallopian tube epithelial (FTE) and ovarian surface epithelial (OSE) cells, providing the molecular basis for FTEs as site of origin of HGSOC. FTEs, unlike OSEs, exhibit heightened replication stress (RS), impaired repair of stalled forks, ineffective G2/M checkpoint, and increased tumorigenicity. BRCA1 heterozygosity exacerbates these defects, resulting in RS suppression haploinsufficiency and an aggressive tumor phenotype. Examination of human and mouse sections reveals buildup of the RS marker 53BP1 primarily in the fallopian tubes, particularly at the fimbrial ends. Furthermore, menopausal status influences RS levels. Our study provides a mechanistic rationale for FTE as the site of origin for HGSOC, investigates the impact of BRCA1 heterozygosity, and lays the groundwork for targeting early HGSOC drivers.
... Authors identified replication stress as an important biomarker of response to gemcitabine. Patients with high replication stress (defined as at least one genomic alteration due to the dysregulated RB pathway and/or oncogene-induced replication stress) [109]. Single-agent activity of the oral ATR inhibitor RP3500 in advanced OC was recently demonstrated in the phase I TRESR trial, with an ORR of 25% in patients with synthetic lethal genomic alterations [110]. ...
Ovarian cancer is the leading cause of gynecological cancer death. Improved understanding of the biologic pathways and introduction of poly (ADP-ribose) polymerase inhibitors (PARPi) during the last decade have changed the treatment landscape. This has improved outcomes, but unfortunately half the women with ovarian cancer still succumb to the disease within 5 years of diagnosis. Pathways of resistance to PARPi and chemotherapy have been studied extensively, but there is an unmet need to overcome treatment failure and improve outcome. Major mechanisms of PARPi resistance include restoration of homologous recombination repair activity, alteration of PARP function, stabilization of the replication fork, drug efflux, and activation of alternate pathways. These resistant mechanisms can be targeted to sensitize the resistant ovarian cancer cells either by rechallenging with PARPi, overcoming resistance mechanism or bypassing resistance pathways. Augmenting the PARPi activity by combining it with other targets in the DNA damage response pathway, antiangiogenic agents and immune checkpoint inhibitors can potentially overcome the resistance mechanisms. Methods to bypass resistance include targeting non-cross-resistant pathways acting independent of homologous recombination repair (HRR), modulating tumour microenvironment, and enhancing drug delivery systems such as antibody drug conjugates. In this review, we will discuss the first-line management of ovarian cancer, resistance mechanisms and potential strategies to overcome these.
... The ATRi berzosertinib has been combined with gemcitabine in a phase II trial in patients with recurrent EOC and a platinum-free interval < 6 months (n = 70). PFS was improved in the experimental arm and the greatest benefit in PFS was obtained in patients with a platinum-free interval ≤ 3 months (PFS 27.7 weeks vs. 9 weeks, HR 0.29; 90%CI 0.12 to 0.71) [105] . A preplanned exploratory analysis revealed a better response trend with gemcitabine alone in patients in the Signature 3-negative subgroup (reflective of HRp tumors) compared to patients in the Signature3-positive subgroup (reflective of HRD tumor) [106] . ...
Ovarian cancer is the most lethal gynecologic cancer. Optimal cytoreductive surgery followed by platinum-based chemotherapy with or without bevacizumab is the conventional therapeutic strategy. Since 2016, the pharmacological treatment of epithelial ovarian cancer has significantly changed following the introduction of the poly (ADP-ribose) polymerase inhibitors (PARPi). BRCA1/2 mutations and homologous recombination deficiency (HRD) have been established as predictive biomarkers of the benefit from platinum-based chemotherapy and PARPi. While in the absence of HRD (the so-called homologous recombination proficiency, HRp), patients derive minimal benefit from PARPi, the use of the antiangiogenic agent bevacizumab in first line did not result in different efficacy according to the presence of homologous recombination repair (HRR) genes mutations. No clinical trials have currently compared PARPi and bevacizumab as maintenance therapy in the HRp population. Different strategies are under investigation to overcome primary and acquired resistance to PARPi and to increase the sensitivity of HRp tumors to these agents. These tumors are characterized by frequent amplifications of Cyclin E and MYC, resulting in high replication stress. Different agents targeting DNA replication stress, such as ATR, WEE1 and CHK1 inhibitors, are currently being explored in preclinical models and clinical trials and have shown promising preliminary signs of activity. In this review, we will summarize the available evidence on the activity of PARPi in HRp tumors and the ongoing research to develop new treatment options in this hard-to-treat population.
... It has been proposed that PARP1 inhibition is required to induce DNA damage [82] . Moreover, inhibition of PARP2 has been linked to suppression of erythropoiesis [83] , and has been suggested to be a driver of the hematologic toxicity associated with the currently approved PARP inhibitors [84] . The first nextgeneration PARPi to enter clinical trials AZD5305 is a highly potent and selective PARP1 inhibitor [84] . ...
... Moreover, inhibition of PARP2 has been linked to suppression of erythropoiesis [83] , and has been suggested to be a driver of the hematologic toxicity associated with the currently approved PARP inhibitors [84] . The first nextgeneration PARPi to enter clinical trials AZD5305 is a highly potent and selective PARP1 inhibitor [84] . AZD5305 is being studied in the ongoing PETRA study (NCT04644068), a phase I/II study of AZD5305 in patients with tumors harboring mutations in key DDR genes. ...
Epithelial ovarian cancer (EOC) is treated in the first-line setting with combined platinum and taxane chemotherapy, often followed by a maintenance poly (ADP-ribose) polymerase inhibitor (PARPi). Responses to first-line treatment are frequent. For many patients, however, responses are suboptimal or short-lived. Over the last several years, multiple new classes of agents targeting DNA damage response (DDR) mechanisms have advanced through clinical development. In this review, we explore the preclinical rationale for the use of ATR inhibitors, CHK1 inhibitors, and WEE1 inhibitors, emphasizing their application to chemotherapy-resistant and PARPi-resistant ovarian cancer. We also present an overview of the clinical development of the leading drugs in each of these classes, emphasizing the rationale for monotherapy and combination therapy approaches.
