Alterations In DNA Repair Pathways Research Articles

Abstract A011: Activity of antibody-drug conjugates with radiation in preclinical bladder cancer models

Abstract Antibody-drug conjugates (ADCs) are transforming the treatment landscape in metastatic bladder cancer, but the role of ADCs in localized disease is uncertain. Radiation is a central component of trimodality therapy (TMT), a curative-intent treatment approach for muscle-invasive bladder cancer (MIBC) that includes concurrent radiation and cytotoxic chemotherapy. We investigated the safety and efficacy of combining novel ADCs with radiation in preclinical bladder cancer models. We observed additive cytotoxicity in vitro, and the combination of ADCs with conformal fractionated radiation was well-tolerated and resulted in improved tumor control compared to either agent alone in vivo. We also assessed the impact of DNA repair pathway alterations commonly observed in bladder tumors on sensitivity to ADCs alone and in combination with radiation and DNA repair targeted agents. Homologous recombination (HR) deficiency modestly sensitized bladder cancer cells to MMAE, the payload of the ADC enfortumab vedotin (EV), and potently sensitized cells to govitecan, the payload of the ADC sacitumuzab govitecan (SG). Conversely, nucleotide excision repair (NER) deficiency did not sensitize cells to either MMAE or govitecan. The combination of govitecan and PARP inhibition resulted in synergistic cell killing independent of HR or NER status. Together these data provide preclincal support for clinical efforts to combine ADCs with radiation in MIBC and identify rationale combination therapy approaches. Citation Format: Yuzhen Zhou, Surish Shanmugam, Vincent D'Andrea, Isabella Stelter, Dag Stormoen, Rea Chroneos, Timothy Hanlon, Ilana Epstein, Raie Bekele, William Anderson, Zoltan Szallasi, Joaquim Bellmunt, Kent Mouw. Activity of antibody-drug conjugates with radiation in preclinical bladder cancer models. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr A011.

HGG-46. PEDIATRIC RADIATION-INDUCED GLIOMA PATIENT SAMPLES AND NOVEL PATIENT-DERIVED MODELS REVEAL TRANSLATIONALLY RELEVANT TREATMENT SUSCEPTIBILITIES AND POTENTIAL GERMLINE RISK FACTORS

Abstract BACKGROUND Pediatric radiation-induced glioma (RIG) is an incurable secondary high-grade glioma (HGG) afflicting up to 4% of children who previously received cranial radiotherapy (RT) for other malignancies. RIG accounts for up to 10% of pediatric brain tumor deaths. It has no known genetic risk factors nor effective treatment and is uniformly fatal, partly due to extremely limited available models. Our recent molecular characterization of patient samples showed that while RIG and primary pediatric HGG share histological similarities, their genetic alterations and gene expression profiles differ. METHODS We obtained germline DNA sequencing from 28 RIG patients and 112 matched controls who received RT but did not develop RIG. In addition, we developed cell culture and murine orthotopic patient-derived xenograft (PDX) models from four RIGs. We conducted single-cell RNA-seq (scRNA-seq), drug screening/validation, and CRISPR functional screening on our novel models. RESULTS Germline mutations in TP53, PARP10, POLI, DCLRE1A, and MLH3 were more common in RIG patients than controls (p<0.002). Our matched cell culture and PDX models of RIG represent the genetic diversity of the disease. The PDX models form tumors in 30-120 days and closely recapitulate human RIG by histology and scRNA-seq. They show susceptibility across models to DNA-damaging treatments (etoposide, irinotecan, radiation), as well as MEK, PARP, and proteasome inhibition, with orthogonal supporting findings via CRISPR screening. Several two-treatment combinations also show efficacy and synergy across models, including radiation and trametinib in vivo (logrank p=0.029). Discussion: RIG is an understudied but major cause of pediatric CNS tumor mortality that differs genetically from primary pediatric HGG. Our findings suggest specific germline alterations in DNA repair pathways may put some patients at higher risk of developing RIG after cranial RT. We also introduce the first set of patient-derived RIG models in order to advance translational RIG research, with characterization of translationally relevant treatment susceptibilities.

Effect of ctDNA whole-exome sequencing in pancreatic adenocarcinoma on prognostic value, actionable alterations, and organ tropisms in metastatic disease.

4149 Background: The dismal prognosis associated with pancreatic ductal adenocarcinoma (PDAC) needs a deeper understanding of progression mechanisms and personalized treatment. While the prognostic value of ctDNA has already been established, this study purposes to apply plasma whole-exome sequencing (WES) to identify molecular alterations beyond KRAS. This approach aims to predict survival, uncover progression mechanisms, and identify therapeutic targets. Methods: Eighty metastatic PDAC patients were prospectively recruited and divided into two consecutive cohorts: discovery (n=37) and validation (n=43). Tumor and plasma obtained at diagnosis were analyzed using WES. The variant allele frequency (VAF) of KRASmutations was quantified by ddPCR in plasma at baseline and at response from all patients (n=80). Results: Plasma WES identified at least one pathogenic variant across all individuals, which can be categorized into four pathways: oncogenic mechanisms, DNA repair, microsatellite instability and TGFb pathway alterations. 7 unique druggable mutations were detected in tissue and 11 in plasma. Other targetable mutations ( CDK12, NF1, BRCA2, and TSC2), were prevalent in plasma but absent in tissue. Patients with survival less than 11 months showed enrichment in cellular organization pathways and exclusive targetable CDK12 mutations. Longer survival cases exhibited exclusive mutations in DNA regulation and repair genes. Patients with liver metastasis displayed unique mutations not just in KRAS but also in the adaptive immune response pathway genes. Baseline plasma KRAS mutations detected by ddPCR was associated with worse progression-free survival. (HR=2.315, CI 95%= 1.027-5.217, p = 0.038; and HR= 3.022, CI 95% = 1.299 - 7.030, p= 0.007 in the discovery and validation cohort, respectively). A significant risk of progression (p = 0.006) was observed if KRAS VAF at response assessment did not decrease, at least, 84.75% in both cohorts. Conclusions: ctDNA WES unveils molecular signatures of rapid progression, potential actionable alterations, and liver metastasis-specific mutations in the adaptive immune response pathway. KRAS detection at baseline and its evolution during treatment emerge as prognostic biomarkers. [Table: see text]

P03.16.A ROLE OF BRD9 IN REGULATING RADIOTHERAPY INDUCED CHANGES IN CHROMATIN ARCHITECTURE OF GBM

Abstract Glioblastoma (GBM) is the most common, formidable, and aggressive type of adult brain tumor, with a poor prognosis. The standard treatment strategies are surgical resection, followed by radiotherapy and accompanying chemotherapy. However, 90% of patients suffer from recurrence of the disease due to the radiation resistance and chemotherapy resistance of glioblastoma cells, which exacerbate the success of the treatment, and thus, resistance remains one of the most important obstacles to overcome. The advances in molecular biology revealed that therapy resistance is related with alterations in DNA repair pathways as well as epigenetic changes governed by histone modifications and chromatin remodeling. Therefore, understanding the underlying epigenetic mechanisms that are involved in the DNA damage response has the potential to develop effective therapeutic strategies. In this manner, an epigenetic drug screen is conducted to identify novel epigenetic modifiers and their inhibitors as radiosensitizers. Bromodomain containing 9 (BRD9) was selected as target modifier while iBRD9 is selected as the radiosensitizer. Inhibition of BRD9 with a common probe, iBRD9 reduced cell viability and colony formation when combined with different doses of radiotherapy. Also, as an acetylated histone reader, inhibition of BRD9 increased the number of acetylated histones as shown by Western Blotting; where Belinostat treatment served as positive control. Currently, our efforts are directed towards understanding which genes are directly targeted by BRD9 in the presence and absence of radiotherapy. To this end, we have ectopically expressed Flag-tagged BRD9 and confirmed its expression by Western Blots. We showed that BRD9 was mainly localized to nucleus. Using this tool, we will perform ChIP sequencing and reveal radiotherapy induced changes in BRD9 binding sites.

Abstract 4327: MSI cancer associated DNA (TA)n-dinucleotide repeat expansions and implications for Werner synthetic lethality

Abstract Microsatellite instability (MSI) is caused by deficient DNA mismatch repair (MMR) and is a ubiquitous feature of cancer. Werner syndrome (WRN) helicase is involved in genome stability and DNA repair. We identified WRN as a synthetic-lethal target in dMMR/MSI cancers and highlighted WRN inhibition as a therapeutic option for dMMR/MSI cancers refractory to available therapies. A previously unappreciated genetic feature of dMMR/MSI cancer cells, DNA (TA)n-dinucleotide repeat expansions, were recently reported to cause vulnerability to WRN depletion. Our mechanistic understanding of TA-dinucleotide repeat expansion biology is limited, and their potential therapeutic implications are unclear. To investigate the landscape of these alterations in cancer, we inferred (TA)-dinucleotide repeat expansions by performing coverage analysis in a collection of hundreds of preclinical cancer models and human tumors (PCAWG) profiled by whole genome sequencing (WGS). We validated our findings in cancer cell lines and organoid cultures by performing long-read WGS. Furthermore, we investigated TA-expansions in single-cell-derived clones from human MSI tumors and cancer organoids. Finally, we inferred TA repeats in laser capture microdissection (LCMB)-derived samples obtained from patients affected by familial cancer predisposition syndromes. Our analysis unveils the landscape of TA-repeats alterations in a large collection of tumors and preclinical models, informing on the level of inter-tumor heterogeneity and their association with variable levels of WRN dependency. In addition, we investigated intra-patient tumor heterogeneity of TA-repeats length both within clonal organoids expanded from normal and neoplastic colorectal stem cells, and within different subclones derived from MSI cancer organoids. Furthermore, analysis of non-neoplastic and neoplastic tissues from patients affected by familial cancer predisposition syndromes revealed the pattern of TA-repeats expansions associated with various DNA-repair pathway alterations. Finally, we will discuss the clinical implications of our findings, as TA-repeats heterogeneity may affect sensitivity and resistance to the future generation of WRN inhibitors. Our data provide fresh insights into the inter and intra-tumoral heterogeneity of TA-dinucleotide repeat expansions in human cancers. These data contribute to understanding the role of MMR in cancer and exploiting Werner as a therapeutic target in cancer. Citation Format: Gabriele Picco, Shriram Bhosle, Maria Kalyva, Elena Grassi, Freddy Gibson, Angham Al Saedi, Sara Vieira, Mathijs Sanders, Livio Trusolino, Andrea Bertotti, Isidro Cortes-Ciriano, Mathew Garnett. MSI cancer associated DNA (TA)n-dinucleotide repeat expansions and implications for Werner synthetic lethality. [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 4327.

CHEK2 gene in metastatic prostate cancer: A cordial check on DNA damage repair pathway

Mutation in homologous recombination repair (HRR) pathway is well established in ovarian cancers. Multiple trials have shown variable efficacy in prostate cancers. Mutations other than BRCA1/BRCA2 have been recently reported in prostate cancers. We describe a unique case of an elderly male with metastatic castration-resistant prostate cancer. He responded to hormonal therapy for 6 months but later progressed. Chemotherapy with docetaxel produced severe side effects in the patient and, thus, was discontinued. Next-generation sequencing was performed on tissue sample which selected a pathogenic mutation (TIER 1) in CHEK2 gene, which is a missense mutation, leading to loss of function of protein. CHEK2 is a cell cycle regulator and a tumor suppressor gene mediating homologous recombinant DNA repair pathway and genetic alterations in it makes tumor susceptible to newer targeted therapies.

Targeted Inhibition of DNA-PKcs, ATM, ATR, PARP, and Rad51 Modulate Response to X Rays and Protons.

Small molecule inhibitors are currently in preclinical and clinical development for the treatment of selected cancers, particularly those with existing genetic alterations in DNA repair and DNA damage response (DDR) pathways. Keen interest has also been expressed in combining such agents with other targeted antitumor strategies such as radiotherapy. Radiotherapy exerts its cytotoxic effects primarily through DNA damage-induced cell death; therefore, inhibiting DNA repair and the DDR should lead to additive and/or synergistic radiosensitizing effects. In this study we screened the response to X-ray or proton radiation in cell lines treated with DDR inhibitors (DDRis) targeting ATM, ATR, DNA-PKcs, Rad51, and PARP, with survival metrics established using clonogenic assays. We observed that DDRis generate significant radiosensitization in cancer and primary cells derived from normal tissue. Existing genetic defects in cancer cells appear to be an important consideration when determining the optimal inhibitor to use for synergistic combination with radiation. We also show that while greater radiosensitization can be achieved with protons (9.9 keV/µm) combined with DDRis, the relative biological effectiveness is unchanged or in some cases reduced. Our results indicate that while targeting the DDR can significantly radiosensitize cancer cells to such combinations, normal cells may also be equally or more severely affected, depending on the DDRi used. These data highlight the importance of identifying genetic defects as predictive biomarkers of response for combination treatment.

VHL and DNA damage repair pathway alterations as potential clinical biomarkers for first-line TKIs in metastatic clear cell renal cell carcinomas

PurposeVascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) are being used for the first-line treatment of metastatic clear cell renal cell carcinoma (mccRCC). Here, we set out to explore associations between genomic statuses, gene expression clusters and clinical outcomes of mccRCCs upon the application of VEGFR-TKIs.MethodsA retrospective study of 56 patients with mccRCC who received first-line VEGFR-TKIs and who underwent genomic profiling and whole transcriptome sequencing was conducted. Survival analysis was carried out using log-rank tests and Cox regression analyses, and Kaplan–Meier curves were plotted. Clustering was performed using the K-means method.ResultsAmong the 56 patients tested, 17 harbored DNA Damage and Repair (DDR) pathway alterations and 35 VHL mutations. The median progression-free survival (PFS) rates for the DDR and VHL alteration groups were 18 and 18 months, respectively, compared with 14 and 10 months for the nonmutant groups. DDR mutations, VHL mutations and co-mutations were identified as prognostic biomarkers of a longer PFS (p = 0.017, 0.04, 0.014). K-means clustering of expressed transcripts revealed three clusters of 40 patients: C_1, C_2 and C_3. The C_1 cluster exhibited the best PFS and objective response rate (ORR) to TKI therapy, with the highest proportion of DDR and VHL mutations. Further analysis of the tumor immune environment revealed that the C_1 cluster was enriched in activated CD8 T cells and effector CD4 T cells, whereas the C_2 cluster was enriched in eosinophils, mast cells and DC cells and, thus, in immunosuppressive cells.ConclusionsWe found that patients with mccRCC harboring DDR and VHL alterations were more likely to benefit from first-line VEGF-TKI systemic therapy than patients with wild-type disease. In addition, we found that a three-cluster prognostic model based on gene expression can predict PFS and ORR, which was well-matched with activated TIL infiltration.

PD-L1 immune cell expression levels and clinical outcomes in advanced triple-negative breast cancer treated with atezolizumab.

e13066 Background: Treatment with immune checkpoint inhibitors (ICI) has become a standard of care for metastatic PD-L1 positive TNBC patients and PD-L1 has emerged as an important predictive biomarker. Predictive differences of PD-L1 were found in response to immunotherapy in different studies. Alterations in DNA repair pathways (aDDR) have also been investigated as a predictive biomarker for ICI due to the increased mutations and neoantigen production. Real-world data regarding outcomes of treatment of metastatic (m)TNBC patients (pts) with ICI and predictive value of PD-L1 and aDDR is lacking. In this study PD-L1 and aDDR were evaluated for association with clinical benefit of treatment with atezolizumab and nab-paclitaxel (A+nP) in PD-L1 positive mTNBC patients treated in routine clinical practice. Methods: Medical records from 41 PD-L1 positive mTNBC patients consecutively treated with A+nP at Oncology Institute Ljubljana between April 2019 and October 2021 were collected for the present study. PD-L1 expression was assessed on immune cells (IC) as percentage of tumor area using VENTANA SP142 PD-L1 IHC assay (<1% [PD-L1 negative] vs. ≥1% [PD-L1 positive). Patients were divided into 2 subgroups based on PD-L1 status, high (PD-L1≥ 5%) and low (PD-L1 ≥1% and <5%). Germline alteration status in DDR genes was determined on blood samples. We used Kaplan-Meier survival curves to calculate median (m) PFS and OS . The prognostic impact of PD-L1exspresion levels and aDDR was calculated using Cox model. Results: Median follow up of the study was 22 months. Majority of patients had low PD-L1 status (25/41, 61%) and 54% of evaluable patients (15/28) were found to harbor deleterious alterations in DDR genes, of them BRCA1 in 9 pts, BRCA2 in 2 pts, and PALB2, CHEK2, CHD1, RAD51D in 1 pts. Median PFS in the entire cohort was 8.26 months; 95% CI (3.54-12.98) and mOS 20.49 months; 95% CI (11.3-29.7). Significant survival benefit was observed among patients with high PD-L1 status treated with A+nP. No difference in survival was found in patients harbouring germline mutations (aDDR positive) versus without mutations (Table). Conclusions: PD-L1 positive mTNBC patients with high PD-L1 status (≥5%) had almost three times less risk of progression and death compared to low PD-L1 status (≥1% and <5%). Presence of aDDR showed no effect on disease outcome. [Table: see text]

Associations between genomic instability score (GIS), molecular characterization, and clinical outcomes in high grade serous ovarian cancers (HGSOC).

e17596 Background: To describe the molecular profiles of tumors with a positive GIS status (GI+) in patients with newly diagnosed BRCA wild-type HGSOC and to examine possible associations between genomic mutations and survival outcomes in these patients. Methods: Genetic testing results of patients who underwent both somatic BRCA1/BRCA2 and germline multigene panel testing between 1/1/2015 and 12/1/2020 were prospectively collected. Patients had multigene panel germline testing in tandem with Myriad somatic BRCA1/2 testing and GIS assay. Expanded somatic NGS testing (108 gene panel) was then performed on GIS+ (GIS ≥42) tumor samples from women without somatic or germline BRCA mutations. The primary outcomes were the type and frequency of genomic mutations, which were grouped according to the following pathways: DNA-repair related (separated into homologous recombination (HR), Fanconi anemia (FA), other DNA repair related pathway) or non-DNA repair related. GIS and molecular profiles of tumors from patients who recurred versus those who did not recur were compared. Results: 143 women with HGSOC underwent both germline and somatic testing. 18/143 (12.5%) tumors with negative germline multigene panel and negative somatic BRCA1/2 testing were GIS+. 17/18 (94.4%) GIS+ tumors had pathogenic TP53 mutations. While all samples had at least one DNA repair related variant, only 22.2% had an HR-associated variant, and 16.7% had an FA variant. At time of analysis, 10 patients had recurred and 8 patients had not recurred. Median PFS of those who recurred was 15.8 months (range 4.8-30.1 months). Patients who recurred had lower GIS (median 47.5 vs. 69.5, p = 0.04), but they had no appreciable differences in gene or pathway alterations when compared to those who did not recur. Conclusions: Known pathogenic alterations in DNA repair pathways commonly associated with HR were rare in this cohort of BRCA negative HGSOC patients with GIS+ tumors. However, there was a high frequency of other DNA repair pathway alterations. Further investigation of the molecular signatures associated with recurrence are needed to identify potential novel targets. Lower GIS in this cohort were associated with higher risk of recurrence, although this finding must be validated in the setting of larger studies. [Table: see text]

Artificial intelligence to identify genetic alterations in conventional histopathology.

Precision oncology relies on the identification of targetable molecular alterations in tumor tissues. In many tumor types, a limited set of molecular tests is currently part of standard diagnostic workflows. However, universal testing for all targetable alterations, especially rare ones, is limited by the cost and availability of molecular assays. From 2017 to 2021, multiple studies have shown that artificial intelligence (AI) methods can predict the probability of specific genetic alterations directly from conventional hematoxylin and eosin (H&E) tissue slides. Although these methods are currently less accurate than gold standard testing (e.g. immunohistochemistry, polymerase chain reaction or next-generation sequencing), they could be used as pre-screening tools to reduce the workload of genetic analyses. In this systematic literature review, we summarize the state of the art in predicting molecular alterations from H&E using AI. We found that AI methods perform reasonably well across multiple tumor types, although few algorithms have been broadly validated. In addition, we found that genetic alterations in FGFR, IDH, PIK3CA, BRAF, TP53, and DNA repair pathways are predictable from H&E in multiple tumor types, while many other genetic alterations have rarely been investigated or were only poorly predictable. Finally, we discuss the next steps for the implementation of AI-based surrogate tests in diagnostic workflows. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Evaluation of tumor genomic variants in Japanese patients with metastatic castration-sensitive prostate cancer (mCSPC) treated with apalutamide: Protocol of a phase 4 study (CUARTET).

TPS196 Background: Apalutamide is an oral non-steroidal, potent, and highly selective androgen receptor antagonist. Alterations in androgen signaling, DNA repair, and other pathways impact on castration sensitivity and outcomes of androgen antagonists. Currently, there is limited data on the types or effects of variants in prostate cancer (PC)-related genes among Japanese mCSPC (castration-naïve or castration-sensitive) patients treated with apalutamide. Methods: CUARTET is a collaborative phase 4 study with a primary objective of evaluating genomic variants in 73 PC driver genes during apalutamide treatment and hence identify potential biomarkers. Men aged ≥20 years with mCSPC scheduled to start apalutamide at 20 participating institutions will be eligible. Patients previously treated with abiraterone, docetaxel, enzalutamide, apalutamide or darolutamide, are ineligible, as are those who received androgen-deprivation therapy or combined androgen blockade for >6 months before registration or neoadjuvant/adjuvant hormonal therapy for >36 months. The primary endpoint is the changes in genomic variants of 73 PC driver genes between pre- and post-treatment of apalutamide. Secondary endpoints, stratified by baseline genomic variants, include the proportion of patients with nadir prostate–specific antigen (PSA) ≤0.2 ng/mL, PSA–progression-free survival (PFS), PFS, overall survival, time to castration resistance, and PFS2. Other planned outcomes include safety, incidence/severity of apalutamide-related skin rash stratified by single nucleotide polymorphisms or human leukocyte antigen typing, and clinical outcomes in patients with only distant lymph node metastases. The first patient was enrolled in December 2020, and it is expected to enroll 100 patients during the 1.5-year registration period. Eighty-one patients have been enrolled in this study by July 2021. The patients will be followed up for a maximum of 4.5 years. Interim analyses are planned after the last patient has been registered. Results of CUARTET are expected to reveal the genomic variants, including those in androgen signaling-related genes, that are potentially involved in acquired resistance to apalutamide. The results will reveal biomarkers to aid treatment decision making for mCSPC patients, including the treatment options following apalutamide. Clinical trial information: NCT04601441.

Abstract PD3-06: The SRC family kinase, YES1, controls chromosomal stability and promotes growth of triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a collection of heterogeneous diseases with limited therapeutic options primarily involving cytotoxic chemotherapy. The distinct molecular profile and increased chromosomal instability (CIN) of TNBC make it a difficult disease to treat. While many patients initially respond to treatment, resistance is common, resulting in poor patient outcomes. Thus identifying vulnerabilities in this disease is necessary to improve TNBC patient outcomes. To identify potential therapeutic targets in this disease, we focused on Src Family Kinases (SFKs). The SFK family is comprised of 9 non-receptor tyrosine kinases that interact with upstream signaling partners to regulate cell phenotypes such as adhesion, motility, survival and mitosis. Five SFKs are overexpressed in breast cancer, including Src, the founding member of the family. Within the basal breast cancer subtype, one of the most highly overexpressed SFKs is YES1. High YES1 expression is also associated with poorer outcomes in TNBC patients when compared to tumors with low YES1 expression. We have found that TNBC cells are reliant on sustained YES1 expression for viability, growth, cell cycle progression, and maintenance of genomic stability. Transiently silencing YES1 causes a significant decrease in cell growth and increase in apoptosis. Furthermore, loss of YES1 expression induces features of whole chromosomal instability (w-CIN), including micronucleation, multinucleation, and dysmorphic nuclei. An increase in γ-H2AX positive staining was also noted in cells with decreased YES1 expression, indicating an accumulation of double strand DNA breaks (DSB) that are indicative of structural chromosomal instability (s-CIN). These findings were recapitulated using several pan-SFK inhibitors, including FDA approved agents dasatinib and saracatinib. Loss of YES1 function also results in perturbed cell cycle progression, particularly a G2/M delay. RNA-sequencing and Reverse Phase Protein Array (RPPA) revealed alterations in mitotic, replication stress, and DNA repair pathways following the loss of YES1. These data demonstrate that YES1 is a previously understudied signaling component of mitotic- and DNA damage-regulating pathways in TNBC. More broadly, they suggest that YES1 may be a therapeutically targetable vulnerability that could be paired with other DNA damaging or mitotic inhibitors to improve treatment efficacy in TNBC. Current SFK-targeted agents globally impact all SFKs. The data presented here support efforts to identify a YES1 selective inhibitor that should be more specific for treating TNBC with less toxicity that pan-SFK drugs. Citation Format: Katrina Piemonte, Elyse Donaubauer, Ruth Keri. The SRC family kinase, YES1, controls chromosomal stability and promotes growth of triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-06.

Circulating tumour DNA reveals genetic traits of patients with intraductal carcinoma of the prostate.

To investigate the genetic alterations of patients with prostate cancer (PCa) with and without intraductal carcinoma of the prostate (IDC-P). We performed targeted sequencing of plasma cell-free DNA on 161 patients with prostate adenocarcinoma (PAC) with IDC-P and 84 without IDC-P. Genomic alterations were compared between these two groups. The association between genetic alterations and patients' survival outcomes was also explored. We identified that 29.8% (48/161) and 21.4% (18/84) of patients with and without IDC-P harboured genomic alterations in DNA repair pathways, respectively (P = 0.210). Pathogenic germline DNA repair alterations were frequently detected in IDC-P carriers compared to IDC-P non-carriers (11.8% [19/161] vs 2.4% [two of 84], P = 0.024). Germline BReast CAncer type 2 susceptibility protein (BRCA2) and somatic cyclin-dependent kinase 12 (CDK12) defects were specifically identified in IDC-P carriers relative to PAC (BRCA2: 8.7% [14/161] vs 0% and CDK12: 6.8% [11/161] vs 1.2% [one of 84]). Patients with IDC-P had a distinct androgen receptor (AR) pathway alteration, characterised by an enrichment of nuclear receptor corepressor 2 (NCOR2) mutations compared with patients with pure PAC (21.1% [34/161] vs 6.0% [five of 84], P = 0.004). Increased AR alterations were detected in patients harbouring tumours with an IDC-P proportion of ≥10% vs those with an IDC-P proportion of <10% (6.4% [five of 78] vs 18.1% [15/83], P = 0.045). For IDC-P carriers, tumour protein p53 (TP53) mutation was associated with shorter castration-resistant-free survival (median 10.9 vs 28.9 months, P = 0.026), and BRCA2 alteration was related to rapid prostate-specific antigen progression for those receiving abiraterone treatment (median 9.1 vs 11.9 months, P = 0.036). Our findings provide genomic evidence explaining the aggressive phenotype of tumours with IDC-P, highlighting the potential therapeutic strategies for this patient population.

DNA damage repair in glioblastoma: current perspectives on its role in tumour progression, treatment resistance and PIKKing potential therapeutic targets.

The aggressive, invasive and treatment resistant nature of glioblastoma makes it one of the most lethal cancers in humans. Total surgical resection is difficult, and a combination of radiation and chemotherapy is used to treat the remaining invasive cells beyond the tumour border by inducing DNA damage and activating cell death pathways in glioblastoma cells. Unfortunately, recurrence is common and a major hurdle in treatment, often met with a more aggressive and treatment resistant tumour. A mechanism of resistance is the response of DNA repair pathways upon treatment-induced DNA damage, which enact cell-cycle arrest and repair of DNA damage that would otherwise cause cell death in tumour cells. In this review, we discuss the significance of DNA repair mechanisms in tumour formation, aggression and treatment resistance. We identify an underlying trend in the literature, wherein alterations in DNA repair pathways facilitate glioma progression, while established high-grade gliomas benefit from constitutively active DNA repair pathways in the repair of treatment-induced DNA damage. We also consider the clinical feasibility of inhibiting DNA repair in glioblastoma and current strategies of using DNA repair inhibitors as agents in combination with chemotherapy, radiation or immunotherapy. Finally, the importance of blood-brain barrier penetrance when designing novel small-molecule inhibitors is discussed.

Abstract PO-044: Exploiting a radiotherapy induced immunologic double bind in advanced prostate cancer

Abstract An evolutionary double bind occurs in cancer when resistance to a commonly used therapy increases the sensitivity to a second therapy. Typically, the second therapy is most effective in the recurrent setting and less effective when given up front. There is a tremendous need for novel second-line therapies able to exploit these new sensitivities. Prostate cancer remains one of the most treatable cancers, however, advanced disease is typically metastatic and currently incurable. Radiation therapy (RT) is often used in the resistant setting. Given locally, RT induces double-strand DNA breaks in tumor cells. This results in cellular stress responses that either trigger apoptosis and cell death, or DNA repair and cell survival. Radiosensitivity is related to the DNA repair capabilities of a tumor. Activation of DNA damage response pathways, which are mediated by ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR), induces changes in surviving cancer cells. Most notably, it has been shown that RT, as well as other DNA damaging agents, can increase natural killer cell (NK) ligand expression on tumor cells. Here we utilize an isogenic cell line model of radioresistant prostate cancer. Our pre-clinical model shows that alterations in DNA repair pathways required to maintain resistance to RT correlate with changes in NK-cell ligands, including: major histocompatibility complex class I chain-related protein A/B (MICA/B), Nectin-2 (CD112), and poliovirus receptor (PVR). This results in a 2-fold increase in sensitivity to NK-cell mediated killing in the radiation resistant cell line. An “evolutionary game assay” was performed by preparing mixtures of parental and radiation-resistant cell lines at 4 different ratios: 100:0, 90:10, 10:90 and 0:100. This was repeated for six treatment scenarios: untreated, 6Gy radiation, 1:1 NK cells, 5:1 NK cells, 6Gy with 1:1 NK cells, and 6Gy with 5:1 NK cells. Comparison of monotypic growth rates to coculture mixtures of parental and radiation-resistant cell lines enable quantification of competitive interactions through the use of evolutionary math models. This assay quantifies effective fitness (growth rate) to experimentally determine the frequency-dependent gradient of selection. Preliminary results indicate the emergence of three qualitatively unique evolutionary scenarios: 1) dominance of resistance (radiation therapy), 2) dominance of sensitive (5:1 NK), and 3) coexistence (untreated; 1:1 NK). These results suggest the existence of an evolutionary double-bind when NK-cell administration follows radiation. This was experimentally validated and confirmed by repeating the evolutionary assay with sequential radiation and NK therapy. Importantly, there is a minimum threshold of NK cells required to achieve the double-bind. Direct measurement of the fitness landscape underlying radiation and NK treatment enabled the discovery and validation of an evolutionary strategy to re-sensitize tumors after radiation treatment. Citation Format: Kimberly Luddy, Jeffrey West, Mark Robertson-Tessi, Alexander Anderson, Laure Marignol, Robert Gatenby, Cliona O'Farrelly. Exploiting a radiotherapy induced immunologic double bind in advanced prostate cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-044.

High-risk gene expression profiles for colorectal liver metastases

Presenter: Michael Turgeon MD | Emory University Background: Over half of patients with colorectal cancer develop liver metastases with a minority of patients considered resectable at presentation. While there has been significant progress in the development of new biologics for the treatment of colorectal liver metastases (CRLM), only a small proportion of patients harbor actionable mutations. Furthermore, gene expression patterns of liver-specific metastases remain unclear. Our aim was to identify high-risk gene expression profiles for patients with CRLM to better inform prognosis and the development of novel targeted therapies. Methods: We examined 53 formalin-fixed paraffin-embedded (FFPE) colorectal liver metastasis samples from patients who underwent complete metastasectomy from 2009-2017 at a single institution. Immune cell infiltration was confirmed with CD45 antibody staining via immunohistochemistry (IHC). Targeted mRNA expression profiling was performed with extracted RNA using the NanoString Immune Oncology (IO360) 750-gene panel. Expression counts were normalized by the geometric mean of the positive-control and the most stable housekeeping genes were selected using the geNorm algorithm followed by log2 transformation. The differential expression analysis with fold changes and p-values were calculated using the NanoString methodology. Groups of interest were dichotomized based on synchronous versus metachronous disease and risk profiles. Risk profiles were based on median and quartile overall survival (OS) cut-points. “High-risk” was considered OS below the median or 25th percentile, while “low-risk” was OS above the median, above the 75th percentile, or no death due to disease. Results: Of 53 patients, median overall survival was 58 months. Median follow-up was 36 months. Using the NanoString IO360 panel, we detected no differentially expressed genes between synchronous (n=28) and metachronous (n=25) CRLM. Considering risk profiles, four differentially expressed genes were identified comparing tumors from high-risk (n=4) versus low-risk (n=18) patients based on a median OS cut-point of 45 months. Pathway analysis indicated these gene expression signatures were associated with antigen presentation, DNA repair, and collagen synthesis. When comparing the 25th percentile (high-risk n=2) to the 75th percentile (low-risk n=16), a total of 68 down-regulated genes were detected that were primarily involved in the antigen presentation pathways. Conclusion: Patients with colorectal liver metastases with poor overall survival may have a distinct, “high-risk” gene expression profile that manifests in alterations in antigen presentation, DNA repair, and collagen synthesis pathways. The identified genes serve as promising molecular markers for prognostication and for the development of novel targeted therapies. Next steps include NanoString analysis with an expanded cohort, immunohistochemistry to validate protein expression, and modulation of gene expression in cell lines.

Genomic profiling of platinum-resistant ovarian cancer: The road into druggable targets.

Ovarian cancer is the most lethal gynecologic cancer. High-grade serous carcinoma (HGSC) is the most frequent histologic subtype and while it is a highly platinum-sensitive cancer at initial treatment, nearly 90 % of stage IIIC patients recur in 5 years and eventually become resistant to platinum treatment. Historically, the definition of platinum-resistant disease is based on the time interval between last platinum therapy and recurrence shorter than 6 months. Nowadays the use of sophisticated imaging techniques and serum markers to detect recurrence makes the accuracy of this clinical definition less clear and even more debatable as we begin to better understand the molecular landscape of HGSC and markers of platinum resistance and sensitivity. HGSC is characterized by a low frequency of recurrent mutations, great genomic instability with widespread copy number variations, universal TP53 mutations, and homologous recombination deficiency in more than 50 % of cases. Platinum agents form DNA adducts and intra- and inter-strand cross-links in the DNA. Most of DNA repair pathways are involved at some point in the repair of platinum induced DNA damaging, most notably homologous recombination, Fanconi Anemia, and nucleotide excision repair pathways. Mechanisms of platinum resistance are related mostly to the limitation of platinum-DNA adduct formation by changing cellular pharmacology, and to the prevention of cell death after DNA damage due to alterations in DNA repair pathways and cell cycle regulation. Understanding these mechanisms of sensitivity and resistance may help to define the utility of platinum re-challenge in each situation and guide new therapeutic opportunities. Moreover, the discovery of mechanisms of synthetic lethality related to alterations in DNA repair and cell cycle regulation pathways has opened up a new avenue for drug therapy in the last decade. In the present article, we review pathways involved in platinum-induced DNA damage repair and their relationship with genomic alterations present in HGSC. Moreover, we report new treatment strategies that are underway to target these alterations.

Inhibition of DNA Repair in Cancer Therapy: Toward a Multi-Target Approach.

Alterations in DNA repair pathways are one of the main drivers of cancer insurgence. Nevertheless, cancer cells are more susceptible to DNA damage than normal cells and they rely on specific functional repair pathways to survive. Thanks to advances in genome sequencing, we now have a better idea of which genes are mutated in specific cancers and this prompted the development of inhibitors targeting DNA repair players involved in pathways essential for cancer cells survival. Currently, the pivotal concept is that combining the inhibition of mechanisms on which cancer cells viability depends is the most promising way to treat tumorigenesis. Numerous inhibitors have been developed and for many of them, efficacy has been demonstrated either alone or in combination with chemo or radiotherapy. In this review, we will analyze the principal pathways involved in cell cycle checkpoint and DNA repair focusing on how their alterations could predispose to cancer, then we will explore the inhibitors developed or in development specifically targeting different proteins involved in each pathway, underscoring the rationale behind their usage and how their combination and/or exploitation as adjuvants to classic therapies could help in patients clinical outcome.

DNA Repair and Ovarian Carcinogenesis: Impact on Risk, Prognosis and Therapy Outcome.

There is ample evidence for the essential involvement of DNA repair and DNA damage response in the onset of solid malignancies, including ovarian cancer. Indeed, high-penetrance germline mutations in DNA repair genes are important players in familial cancers: BRCA1, BRCA2 mutations or mismatch repair, and polymerase deficiency in colorectal, breast, and ovarian cancers. Recently, some molecular hallmarks (e.g., TP53, KRAS, BRAF, RAD51C/D or PTEN mutations) of ovarian carcinomas were identified. The manuscript overviews the role of DNA repair machinery in ovarian cancer, its risk, prognosis, and therapy outcome. We have attempted to expose molecular hallmarks of ovarian cancer with a focus on DNA repair system and scrutinized genetic, epigenetic, functional, and protein alterations in individual DNA repair pathways (homologous recombination, non-homologous end-joining, DNA mismatch repair, base- and nucleotide-excision repair, and direct repair). We suggest that lack of knowledge particularly in non-homologous end joining repair pathway and the interplay between DNA repair pathways needs to be confronted. The most important genes of the DNA repair system are emphasized and their targeting in ovarian cancer will deserve further attention. The function of those genes, as well as the functional status of the entire DNA repair pathways, should be investigated in detail in the near future.