DNA Damage Response Deficiency Research Articles

Abstract 5130: Phospho-proteomic dynamics reveal DNA damage and repair signatures of drug sensitivity

Abstract There are numerous drug development efforts of DNA damage response (DDR) targets, such as PARP, ATR, ATM, CHK1, where the ability to accurately identify responsive sub-populations for monotherapies and combinations is a major challenge. Patients are routinely assigned to these drugs based on BRCA mutation, or BRCAness profile, encompassing homologous recombination deficiencies (HRD) which do not always represent the responsive sub-populations. Since DNA replication and DNA repair mechanisms are dominated by signaling events, we hypothesized that HRD extends beyond the genomic status, and is manifested also in the level of protein expression and protein modification. To that end, we created a harmonized data atlas consisting of phenotypic drug sensitivity and basal and dynamic phosphoproteomics of multiple cell lines before and after treatment of multiple DDR drugs. Proteogenomic analysis of cell lines showed that some cell lines are sensitive to DDR drugs despite being DDR-proficient at the genomic level. Proteomic analysis of these cell lines showed that at least a third of them exhibit proteomic DDR deficiency, explaining DDR sensitivity beyond mutation status. We then utilized our proteogenomic atlas and computational platform to address challenging clinical needs, namely discovery of a predictive ATR biomarker and identifying potential combination treatments to overcome ATRi resistance. Using basal state and protein dynamics of ATR coupled with ATRi sensitivity, we developed a novel predictive ATR biomarker, and tested it using two independent validation datasets. Our ATR biomarker exhibited improved precision and recall compared to genomic biomarkers represented by mutations commonly used in the clinic, and was not confounded by mutations. In addition, integrated analysis of basal and dynamic data of multiple cell lines treated with ATRi, highlighted both known (e.g. ATM) and novel resistance mechanisms and suggested potential drug combinations. Altogether, we demonstrate here the benefit of utilizing (phospho)-proteomic data to tackle clinical challenges in DDR drug development, such as predictive biomarkers and combination strategies. We provide a proteomics-based framework that would enable future patient selection in DDR clinical trials. Citation Format: Gali Arad, Avital Hay-Koren, Nitzan Simchi, Dimitri Kovalerchik, Dina Daitchman, Amit Manor, Ofer Givton, Elyad Lezmi, Iris Alchanati, Yonatan Katzenelenbogen, Shay Herman, Alon Shtrikman, Galina Otonin, Eran Seger, Kirill Pevzner. Phospho-proteomic dynamics reveal DNA damage and repair signatures of drug sensitivity [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 5130.

Characterization of the signal transduction cascade for inflammatory gene expression in fibroblasts with ATM-ATR deficiencies after Ionizing radiation

Background and purposeIonizing radiation (IR) induces DNA double-strand breaks (DSBs), leading to micronuclei formation, which has emerged as a key mediator of inflammatory responses after IR. This study aimed to investigate the signaling cascade in inflammatory gene expression using fibroblasts harboring DNA damage response deficiency after exposure to IR. Materials and methodsMicronuclei formation was examined in human dermal fibroblasts derived from patients with deficiencies in ATM, ATR, MRE11, XLF, Artemis, or BRCA2 after IR. RNA-sequencing analysis was performed to assess gene expression, pathway mapping, and the balance of transcriptional activity using the transcription factor–based downstream gene expression mapping (TDEM) method developed in this study. ResultsDeficiencies in ATM, ATR, or MRE11 led to increased micronuclei formation after IR compared to normal cells. RNA-seq analysis revealed significant upregulation of inflammatory expression in cells deficient in ATM, ATR, or MRE11 following IR. Pathway mapping analysis identified the upregulation of RIG-I, MDA-5, IRF7, IL6, and interferon stimulated gene expression after IR. These changes were pronounced in cells deficient in ATM, ATR, or MRE11. TDEM analysis suggested the differential activation of STAT1/3–pathway between ATM and ATR deficiency. ConclusionEnhanced micronuclei formation upon ATM, ATR, or MRE11 deficiency activated the cGAS/STING, RIG-I-MDA-5-IRF7-IL6 pathway, resulting in its downstream interferon stimulated gene expression following exposure to IR. Our study provides comprehensive information regarding the status of inflammation-related gene expression under DSB repair deficiency after IR. The generated dataset may be useful in developing functional biomarkers to accurately identify patients sensitive to radiotherapy.

Abstract PR005: Identification of DNA Damage Repair genes controlling the immune landscape of breast tumors via spatial functional genomics

Abstract Immune checkpoint blockade (ICB) has revolutionized the field of cancer immunology, yet only 17-20% of patients who receive this treatment derive any clinical benefit. There is an unmet need to either identify the subset of patients who would respond to ICB or identify targets for combination therapies. By assessing frequently mutated pathways in these patients, we can identify potential biomarkers and candidates for combinatorial therapy. One such pathway is the DNA Damage Response (DDR) pathway. Faults in DDR pathways can lead to genome instability, higher tumor mutational burden (TMB), and accumulation of neoantigens in tumors. Consequently, tumors with one or more deficiencies in DDR (DDRd) have been shown to respond better to therapy and are associated with increased immune cell infiltration. However, previous work from our lab has shown that different gene alterations, even in the same DDR pathway, can polarize immune outcomes differently. To address this issue, we employed Perturb-map, a first-of-its-kind functional genomics approach, which utilizes protein barcodes (pro-Codes) to enable spatial resolution of CRISPR screens. We generated a Pro-Code/CRISPR library targeting frequently mutated DDR genes and introduced the library into 4T1 breast cancer cells, a syngeneic model of triple negative breast cancer. We introduced the cells into immunocompetent mice and performed multiplex imaging on metastatic lesions in the lung to spatially resolve each gene knockout tumor. We observed that loss of Rbbp8 and Polq led to larger tumors, whereas loss of Pms2 led to smaller tumors. Strikingly, Rbbp8 knockout tumors were also less infiltrated by CD4 T cells compared to control tumors. This system was also used to find therapeutic targets by performing synthetic lethality screens using mass cytometry. We transduced the library in isogenic Brca2 deficient 4T1 cells along with their wildtype (WT) counterparts and looked for enrichment or loss of pro-Codes across the different cell lines. These screens consistently identified known synthetic lethality partners like Parp1 in the Brca2 deficient cell lines and identified additional targets like Mre-11a, Bard1 and Rbbp8, which are being profiled further. Taken together, we have developed a scalable system to study the effect of DDR mutations on tumor cell intrinsic and extrinsic properties, which can not only help characterize immune outcomes but also identify potential therapeutic partners. This system proves to be a useful tool that can be leveraged to profile a number of different genes across multiple cancer types. Citation Format: Prerna Suri, Juhana Habib, Gurkan Mollaoglu, Luisanna Pia, Alessia Baccarini, Brian Brown, Robert Samstein. Identification of DNA Damage Repair genes controlling the immune landscape of breast tumors via spatial functional genomics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR005.

Abstract B031: Evaluating the consequence of homologous recombination deficiency in models of pediatric solid tumor

Abstract Homologous recombination deficiency (HRD) has been extensively studied in adult cancers where its association with sensitivity to Poly (ADP-ribose) polymerases (PARP) inhibitor sensitivity is well established. In these cancers, HRD is most frequently caused by biallelic inactivation of BRCA1/2 and tumors often exhibit characteristic genomic scars such as elevated HRD.sum score and specific mutational signatures derived from single nucleotide variants, insertions and deletions, and structural variants. Despite its clinical relevance, the therapeutic predictive value of HRD has not been systematically studied in pediatric cancers. Consequently, our team devised a strategy to identify and validate targetable deficiencies in DNA damage response (DDR) genes and assess genomic scars in pediatric tumors. Here, we report evaluation of HRD in a cohort of >200 patient-derived orthotopic xenograft models from the Childhood Solid Tumor Network (CSTN), a freely sharing resource established by our institution to accelerate the development of therapies for children with cancer. We provide an overview of our pipeline and discuss the landscape of genomic scars within our cohort. We profiled somatic and germline variants from over 500 DNA damage response genes and identified TP53 as the most frequently mutated gene with biallelic loss, whereas bi-allelic loss of BRCA1/2 was not found. Overall, our study suggests that HRD exists within solid tumors, but is driven by inactivation of genes other than BRCA1/2, resulting in a different set of genomic scars compared to those in adult cancers where HRD is common. Importantly, the CSTN contains models of pediatric solid tumors that are amenable to in vitro and in vivo interrogation, and therefore, will facilitate examination of the functional consequences of inactivated DDR genes, characterization of the unique genomic scar profiles underlying these inactivation events, and evaluation of novel therapeutic strategies. Citation Format: Evan Savage, Ke Xu, Jason Myer, Evadnie Rampersaud, Yawei Hui, Ti-Cheng Chang, Gang Wu, Christopher Tinkle, Anang Shelat. Evaluating the consequence of homologous recombination deficiency in models of pediatric solid tumor [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B031.

DNA damage response-related immune activation signature predicts the response to immune checkpoint inhibitors: from gastrointestinal cancer analysis to pan-cancer validation.

DNA damage response (DDR) deficiency has emerged as a prominent determinant of tumor immunogenicity. This study aimed to construct a DDR-related immune activation (DRIA) signature and evaluate the predictive accuracy of the DRIA signature for response to immune checkpoint inhibitor (ICI) therapy in gastrointestinal (GI) cancer. A DRIA signature was established based on two previously reported DNA damage immune response assays. Clinical and gene expression data from two published GI cancer cohorts were used to assess and validate the association between the DRIA score and response to ICI therapy. The predictive accuracy of the DRIA score was validated based on one ICI-treated melanoma and three pan-cancer published cohorts. The DRIA signature includes three genes (CXCL10, IDO1, and IFI44L). In the discovery cancer cohort, DRIA-high patients with gastric cancer achieved a higher response rate to ICI therapy than DRIA-low patients (81.8% vs. 8.8%; P < 0.001), and the predictive accuracy of the DRIA score [area under the receiver operating characteristic curve (AUC) = 0.845] was superior to the predictive accuracy of PD-L1 expression, tumor mutational burden, microsatellite instability, and Epstein-Barr virus status. The validation cohort demonstrated that the DRIA score identified responders with microsatellite-stable colorectal and pancreatic adenocarcinoma who received dual PD-1 and CTLA-4 blockade with radiation therapy. Furthermore, the predictive performance of the DRIA score was shown to be robust through an extended validation in melanoma, urothelial cancer, and pan-cancer. The DRIA signature has superior and robust predictive accuracy for the efficacy of ICI therapy in GI cancer and pan-cancer, indicating that the DRIA signature may serve as a powerful biomarker for guiding ICI therapy decisions.

SIOG2023-2-OA-103 - Prognostic Role of DNA Damage Response Deficiency in Older Patients With Colorectal Cancer

SIOG2023-2-OA-103 - Prognostic Role of DNA Damage Response Deficiency in Older Patients With Colorectal Cancer

Phase II study evaluating the efficacy of niraparib and dostarlimab (TSR-042) in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) patients.

6046 Background: Patients with recurrent and/or metastatic (R/M) HNSCC have poor overall survival. Immune checkpoint inhibitors (ICIs), specifically programmed death-1 (PD-1) inhibitors, result in durable responses in R/M HNSCC but &lt;20% benefit. Tumor mutational burden (TMB) correlates with ICI response. DNA pathway repair mutations have been reported in 17% of sporadic HNSCC and are associated with higher rates of TMB. Poly (ADP-ribose) polymerase (PARP) inhibition has demonstrated efficacy as a single agent in cancers with a DNA repair defect. We tested the combination of PARP inhibitor (niraparib) with the PD-1 inhibitor (dostarlimab) in R/M HNSCC patients (NCT04313504) with funding support from Tesaro/GSK. Methods: Patients with R/M HNSCC who failed at least one line of prior treatment including a PD-1 inhibitor and for whom no surgical or radiation curative options existed were eligible for the study. Patients received niraparib (200 mg daily) beginning one week prior to initiation of dostarlimab (500 mg q3wks x4 followed by 1000 mg q6wks). The primary endpoint was overall response rate (ORR) including complete response (CR), partial response (PR) and stable disease (SD) using RECIST v1.1. Fourteen patients were planned for the first stage and 10 additional patients for the second stage if ≥8 patients had a confirmed response. Safety was evaluated by CTCAE v5.0. Tumor PD-L1 immunohistochemistry was performed with 22c3 antibody and reported as combined positive score (CPS). TMB was considered high if ≥10Mut/Mb. Results: Ten of 24 planned patients were enrolled. Of the first 10 patients, 8 patients were evaluable by imaging and 1 patient clinically progressed on exam. Of the 9 evaluable patients, there were 0 CR, 1 PR, 1 SD, and 7 with progressive disease (PD). The trial was ended early for futility as it was not possible for at least 8 out of 14 patients to develop response. The patient with a PR remains on treatment past 24 months, was PD-L1 positive, had low TMB and no DDR deficiency. All patients had previously failed a PD-1 inhibitor. One patient had high TMB, 2 patients had a DDR, and 6 patients were PDL1 positive but 2 patients had insufficient tissue or indeterminate results for sequencing. Grade ≥ 3 adverse events occurred in 70% of patients with most common being hypertension (20%), hyponatremia (20%), and lung infection (20%). Conclusions: PARP-1 inhibition and PD-1 inhibition combined does not appear to enhance response over PD-1 inhibitor alone in R/M HNSCC who had previously failed PD-1 inhibitor treatment. However, a more focused population including those with DNA damage response (DDR) mutations and PD-1 treatment naïve may benefit. Clinical trial information: NCT04313504 .[Table: see text]

Investigation of synthetic lethality gene pairs in the DNA damage response pathway in response to immunotherapy in pan-cancer.

2567 Background: The concept of synthetic lethality (SL) refers to the phenomenon where the simultaneous loss of function of two genes leads to cell death, while the loss of function of either gene alone has no effect. PARP inhibitors target poly ADP ribose polymerase (PARP) enzymes, which are involved in DNA repair, are the successful cases of utilizing synthetic lethality-based therapy to treat cancers with DNA damage response (DDR) deficiency. The prognosis for cancer patients already has a combination of synthetic lethal mutations especially in DDR pathway can vary and it is not yet fully understood how these mutations may impact the response to immunotherapy. Methods: SL gene combinations in the DDR pathway were screened out from SynLethDB database (http://histone.sce.ntu.edu.sg/SynLethDB/). The immunotherapy cohort (Samstein 2019, n=1661) and non-immunotherapy cohort (Zehir 2017, n=10336) with solid tumor were used to Kaplan-Meier analyze the correlation between SL gene combinations and prognosis. TMB scores were compared between DDR co-mutation group and non DDR co-mutation group. For investigating the associations between immune infiltrates and genetic features, TIMER 2.0 database ( http://timer.cistrome.org/ ) was conducted to estimation of immune infiltration levels on DDR mutation genes in pan-cancer. Results: 430 pairs of DDR mutation combinations were used to subgroup stratification, the DDR co-mutation group was associated with worse prognosis in non-immunotherapy cohort (HR=1.305, p&lt;0.0001) but better in immunotherapy cohort (HR=0.741,p&lt;0.0061). Interestingly, the TMB score in the DDR co-mutation group was significantly higher than non DDR co-mutation group (mean 50.03 vs 8.868, p&lt;0.0001) in immunotherapy cohort. The high-frequency mutation combinations (&gt;10%) were ATM-POLE(14.40%), ATM-ATR (13.60%), ATR-BRCA2 (13.60%), ATM-BRCA2 (12.00%) and BRCA2-RECQL4 (10.40%), respectively. Comparing common immune component with statistically significant under different DDR mutant genes, the ATM-POLE co-mutation tumor tended to have more CD4+ T cell infiltration while the ATM-ATR co-mutation tumor appeared to have more myeloid dendritic cell infiltration. Conclusions: The combinations of DDR SL mutation tumor might result to worse prognosis whereas these patients might benefit from immunotherapy. Our results will help to shed light on the complex interplay for the combinations of SL mutation in DDR pathway between cancer cells and the immune system and provide new insights into the development of effective cancer treatments.

Deep learning model accurately classifies metastatic tumors from primary tumors based on mutational signatures

Metastatic propagation is the leading cause of death for most cancers. Prediction and elucidation of metastatic process is crucial for the treatment of cancer. Even though somatic mutations have been linked to tumorigenesis and metastasis, it is less explored whether metastatic events can be identified through genomic mutational signatures, which are concise descriptions of the mutational processes. Here, we developed MetaWise, a Deep Neural Network (DNN) model, by applying mutational signatures as input features calculated from Whole-Exome Sequencing (WES) data of TCGA and other metastatic cohorts. This model can accurately classify metastatic tumors from primary tumors and outperform traditional machine learning (ML) models and a deep learning (DL) model, DiaDeL. Signatures of non-coding mutations also have a major impact on the model’s performance. SHapley Additive exPlanations (SHAP) and Local Surrogate (LIME) analyses identify several mutational signatures which are directly correlated to metastatic spread in cancers, including APOBEC-mutagenesis, UV-induced signatures, and DNA damage response deficiency signatures.

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.

Design, synthesis, and biological evaluation of pyrido[3,2-d]pyrimidine derivatives as novel ATR inhibitors

Targeting ataxia telangiectasia mutated and Rad3-related (ATR) kinase is being pursued as a new therapeutic strategy for the treatment of advanced solid tumor with specific DNA damage response deficiency. Herein, we report a series of pyrido[3,2-d]pyrimidine derivatives with potent ATR inhibitory activity through structure-based drug design. Among them, the representative compound 10q exhibited excellent potency against ATR in both biochemical and cellular assays. More importantly, 10q exhibited good liver microsomes stability in different species and also showed moderate inhibitory activity against HT-29 cells in combination treatment with the ATM inhibitor AZD1390. Thus, this work provides a promising lead compound against ATR for further study.

Abstract 1635: OKI-1546 is a selective, orally bioavailable inhibitor of CDK12 that causes tumor regression in multiple preclinical cancer models

Abstract Background: CDK12 regulates the expression of multiple genes in the DNA damage response (DDR) pathway by phosphorylating the C-terminal domain of RNA polymerase II (CTD pSer2), and it is a potential therapeutic target to exacerbate DNA damage and cell death in DDR-addicted, HER2-amplified, MYC-overexpressing, or EWS-FLI1-positive cancers. PARP inhibitor resistance is often driven by dysregulation of DDR signaling, and CDK12 inhibition may induce a state of DDR deficiency and increase the effectiveness of PARP inhibition. Materials and methods: Biochemical activity was assayed by ADP-glo (Promega), cellular proliferation was quantified by CellTiter-Glo (Promega), and CTD pSer2 was quantified by MSD immunoassay (Meso Scale Discovery). Kinase panels and in vivo studies are described in figure legends. Results: OKI-1546 has a biochemical IC50 of 15 nM against CDK12 and is highly selective for CDK12 and CDK13 when compared to other closely related CDKs; it is 649-, 81-, 11-, and 64-fold selective over CDKs 1, 2, 7, and 9, respectively. This selectivity was further confirmed via testing OKI-1546 against an affinity-based kinase panel. OKI-1546 potently inhibited proliferation of multiple cancer cell lines, with IC50 values that correlated with 50% inhibition of CTD pSer2. In CD-1 mice, OKI-1546 showed 77% oral bioavailability (%F) with a half-life of 2.7 hrs, and SD rats had 81 %F. Furthermore, a single 10 mg/kg dose reached plasma concentrations that surpassed the plasma protein binding-adjusted IC50 of CTD pSer2, which was confirmed in tumor-bearing mice showing 60% CTD pSer2 inhibition in tumors 4 hours after a single oral dose. That same dose translated into 119% tumor growth inhibition following 21 days of daily administration in the BRCA1 wild-type MDA-MB-231 tumor model and 121% tumor growth inhibition following 21 days of daily administration in the BRCA1-mutant MDA-MB-436 tumor model in mice. Four different PARP inhibitors synergistically combined with OKI-1546 to inhibit cell growth in multiple in vitro cancer models. Conclusion: OKI-1546 is a novel, covalent inhibitor of CDK12 and CDK13 that is potent, specific, orally available, and shows in vivo target engagement and tumor growth inhibition in preclinical cancer models. OKI-1546 showed activity in vivo as a single agent and cellular data suggest that inhibition of CDK12 has the potential to enhance the effects of other DDR-targeting therapeutic agents. Citation Format: Alexander A. Strait, David A. Mareska, Jyothi Mahadevan, Christie A. Schulte, Yevgeniy Izrayelit, Mark L. Boys, Richard Woessner. OKI-1546 is a selective, orally bioavailable inhibitor of CDK12 that causes tumor regression in multiple preclinical cancer 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 1635.

Abstract 5355: Elraglusib response prediction and mechanistic discovery using iterative machine learning

Abstract Elraglusib is a selective GSK-3 inhibitor that has activity on a broad array of neoplasms as well as their immune microenvironment. However the determinants of tumor response have not been elucidated. Here, we use genomic panels of cancer driver genes to predict patient response to elraglusib therapy as measured by RECIST criteria. Patient response in the Phase I 1801 trial (NCT03678883) was matched to genomic data from patient tumors, with 80 cases used to train Machine Learning models and 26 cases reserved as a test set for model evaluation. When predicting response from mutation status alone our models reached a modest accuracy of 65% in the test set, which we attributed to the sparsity of genomic data. To remedy this, we engineered pathway-based features to provide combinatorial information while ensuring feature stability. To select the optimal combination of features, we designed an iterative design process that first randomized feature combinations and then created refined models from the highest performing pathway features. This resulted in several final models with greater than 88% accuracy. We retained several models of similar accuracy to more comprehensively identify potential biomarkers and combinatorial relationships. These models were then interpreted using SHapley Additive exPlanations (SHAP) where we were able to identify highly predictive features for both the entire pan-cancer cohort and specific histologies. We found the feature constructed from the Reactome pathway “Chromatin Modifying Enzymes” frequently occurred in high performing models with a high-ranking SHAP value. This feature included genes such as SMARCA4, HDAC2, and KDM5A, and was negatively associated with patient response. Another frequently observed high-ranking pathway feature with a negative response association was based on the “Innate Immune System” Reactome pathway. Many of the features highlighted in our models were negatively associated with elraglusib response and thus could be general markers of poor prognosis. To address this, we altered our pipeline to test only positively associated features which substantially reduced our feature pool to only 285 features but still produced models with accuracies of up to 81%. Analysis of key features used by these models identified several positive markers of elraglusib including mutations in the POLE gene, which previously has been linked to DNA-damage response deficiency and anti-tumor immune response. In sum, we forecast elraglusib response for a variety of tumor histologies and simultaneously reveal potential mechanisms of elraglusib sensitivity and biological action. In future work we will investigate the utility of using POLE and other identified biomarker candidates to predict the likelihood of patient response to elraglusib, as well as whether our machine learning models will be an effective tool to guide patient stratification. Citation Format: Joseph McDermott, Taylor Weiskittel, Daniel Billadeau, Benedito Carneiro, Hu Li, Andrew Mazar. Elraglusib response prediction and mechanistic discovery using iterative machine learning. [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 5355.

DNA Repair Deficiency Regulates Immunity Response in Cancers: Molecular Mechanism and Approaches for Combining Immunotherapy

Defects in DNA repair pathways can lead to genomic instability in multiple tumor types, which contributes to tumor immunogenicity. Inhibition of DNA damage response (DDR) has been reported to increase tumor susceptibility to anticancer immunotherapy. However, the interplay between DDR and the immune signaling pathways remains unclear. In this review, we will discuss how a deficiency in DDR affects anti-tumor immunity, highlighting the cGAS-STING axis as an important link. We will also review the clinical trials that combine DDR inhibition and immune-oncology treatments. A better understanding of these pathways will help exploit cancer immunotherapy and DDR pathways to improve treatment outcomes for various cancers.

Abstract PD9-06: Histopathological and molecular immune landscape and DNA damage response signatures to predict response to carboplatin and docetaxel in TNT trial TNBC cohort

Abstract Background The TNT trial (NCT00532727) showed no evidence of carboplatin (C) superiority over docetaxel (D) overall in metastatic triple negative breast cancers (TNBC), but a C benefit was observed in the pre-specified sub-group analysis in patients with a gBRCA1/2 mutation (Tutt et al, Nat Med 2018). Given only ~30% of patients have a gBRCA1/2 mutation, broader predictive biomarkers of response are needed. In this cohort we previously found that DNA Damage Response (DDR) signatures were associated with improved C response in chemotherapy (CT) naïve patients only (Tovey et al, ASCO 2020). Since DDR activities influence tumour immune-microenvironment, we explored the predictive ability of immune cell markers and performed integrative analyses on multi-omics features to identify novel TNBC subgroups. Patients and Methods Tumour infiltrating lymphocytes (TILs) were evaluated on haematoxylin and eosin stained primary tumour (PT) slides for 222/376 TNT patients. Formalin-fixed paraffin-embedded PT tissues from 186/376 TNT patients were successfully profiled using total RNA-sequencing. Matched recurrence (REC) was also sequenced for 13 patients. Twenty-five immune signatures were assessed. Logistic regression and restricted mean progression free survival (PFS) were applied to delineate the relationship of these features with treatment outcomes. Random forest clustering of multi-omics DDR and immune biology markers, including gene expression signatures and mutation/methylation status, was applied to identify subgroups. We further molecularly characterised these clusters through supervised clustering of 693 gene expression “modules” (sets of co-expressed genes), immune cell deconvolution and genomic scars. Results Immune gene expression signatures and TILs were highly correlated. Average immune infiltration based on ConsensusTME was lower in mutated/methylated tumours compared with BRCA1 wildtype tumours (p=0.04). Immune signature score markers decreased from PT to REC, demonstrating a dynamic immune microenvironment. In the overall population and when restricting to prior CT treated patients, high immune infiltration (gene expression based &amp; TILs) was associated with response to D while C response rates were not associated with immune scores (interaction p-values&amp;lt; 0.05). This did not translate to a PFS benefit. Multi-omics clustering identified 6 biological subgroups including immune enriched, immune depleted, DDR deficient and proficient clusters as well as 2 small clusters with no obvious distinguishing features. Immune enriched TNBC were predominantly basal-like immune activated with high B-cell/T-cell diversity. Immune depleted TNBC showed higher activity of proliferation and DDR pathway modules. DDR proficient tumours had low expression of immune markers and enrichment for ESR1/PGR expression, markers of extra cellular formation, cell structure, lipid metabolism and proliferation. The DDR deficient cluster was enriched for proliferation and demonstrated high number of TILs despite no apparent enrichment for gene expression-based immune modules. In the prior CT treated cohort, the immune enriched cluster had preferential response to D (62.5% (D) vs. 29.4% (C); p=0.02). The immune depleted cluster had preferential response to C (8.0% (D) vs. 40.0% (C); p=0.01). Numbers were too small to assess differential response within the other clusters or in the CT naïve cohort. Conclusions Tumours with high immune features have high response to D while those with low immune features have preferential response to C in advanced TNBC. Combining multi-omics markers of DDR deficiency and immune biology can identify clusters of patients with distinct biological profiles and differential treatment specific response rates. Citation Format: Holly Tovey, Orsolya Sipos, Katherine A Hoadley, Joel S Parker, Jelmar Quist, Sarah Kernaghan, Lucy Kilburn, Roberto Salgado, Sherene Loi, Richard D Kennedy, Ioannis Roxanis, Patrycja Gazinska, Sarah E. Pinder, Judith Bliss, Charles M. Perou, Syed Haider, Andrew Tutt, Anita Grigoriadis, Maggie Chon U Cheang. Histopathological and molecular immune landscape and DNA damage response signatures to predict response to carboplatin and docetaxel in TNT trial TNBC cohort [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-06.

The potential of PARP inhibitors in targeted cancer therapy and immunotherapy.

DNA damage response (DDR) deficiencies result in genome instability, which is one of the hallmarks of cancer. Poly (ADP-ribose) polymerase (PARP) enzymes take part in various DDR pathways, determining cell fate in the wake of DNA damage. PARPs are readily druggable and PARP inhibitors (PARPi) against the main DDR-associated PARPs, PARP1 and PARP2, are currently approved for the treatment of a range of tumor types. Inhibition of efficient PARP1/2-dependent DDR is fatal for tumor cells with homologous recombination deficiencies (HRD), especially defects in breast cancer type 1 susceptibility protein 1 or 2 (BRCA1/2)-dependent pathway, while allowing healthy cells to survive. Moreover, PARPi indirectly influence the tumor microenvironment by increasing genomic instability, immune pathway activation and PD-L1 expression on cancer cells. For this reason, PARPi might enhance sensitivity to immune checkpoint inhibitors (ICIs), such as anti-PD-(L)1 or anti-CTLA4, providing a rationale for PARPi-ICI combination therapies. In this review, we discuss the complex background of the different roles of PARP1/2 in the cell and summarize the basics of how PARPi work from bench to bedside. Furthermore, we detail the early data of ongoing clinical trials indicating the synergistic effect of PARPi and ICIs. We also introduce the diagnostic tools for therapy development and discuss the future perspectives and limitations of this approach.

Dynamics of DNA damage response markers, RAD51 and XRCC1, throughout the menstrual cycle and pregnancy in healthy women. Increment during pregnancy and relation to a low titer of antinuclear antibodies.

Antinuclear antibodies (ANA) potentially play a role in the pathogenesis of connective tissue diseases (CTDs); however, they are also detected in healthy individuals. Our understanding of the physiological and pathological origin of ANA is incomplete. Both; female reproductive processes and deficiencies in DNA damage response (DDR) have been independently associated with ANA production. However, the link between these two factors and the presence of ANA is unclear METHOD OF STUDY: A cohort study was conducted on 87 non-pregnant and 51 healthy pregnant women. ANA and the DDR markers X-ray repair cross-complementing 1 (XRCC1) and recombinase (RAD51) were quantified in sera by enzyme-linked immunosorbent assay. Additionally, ANA was detected by indirect immunofluorescence in HEp-2 cells in 1:40 diluted sera during the three phases of the menstrual cycle in non-pregnant women and every trimester in pregnant women RESULTS: The prevalence of ANA+ in healthy women was 26.1%, with no significant differences between pregnant and non-pregnant women. ANA and DDR marker levels did not vary among the three phases of the menstrual cycle or the three trimesters of pregnancy. However, DDR marker levels were higher in pregnant women than in non-pregnant women (p&lt;.001) and were found to be higher in ANA+ women (XRCC1, p=.025; RAD51, p=.006) CONCLUSIONS: The menstrual cycle and pregnancy did not influence the levels of DDR markers or ANA in healthy women; however, the DDR was higher in pregnancy and ANA+ women. The results suggest a potential role of DDR in the pathophysiology of ANA.

Abstract CT006: Phase Ib expansion trial of the safety and efficacy of the oral ataxia telangiectasia and Rad3-related (ATR) inhibitor elimusertib in advanced solid tumors with DNA damage response (DDR) defects

Abstract Introduction: ATR kinase is a critical component of DDR machinery and is activated by DNA damage or replication stress. Elimusertib is a selective ATR inhibitor with promising antitumor activity in pts with advanced solid tumors with ataxia telangiectasia mutated (ATM) protein loss and/or ATM putative deleterious alterations (Yap et al. Cancer Discov 2021). We report here the safety and efficacy of elimusertib in expansion cohorts of pts with a range of cancer types and DDR deficiencies and/or ATM loss. We also explored an alternative dose schedule in pts with ATM aberrations. Methods: Pts with advanced solid tumors resistant or refractory to standard treatment were screened for pathogenic DDR defects by next-generation sequencing or ATM protein loss by immunohistochemistry (IHC) analysis of baseline tumor tissue. Pts were assigned to cohorts: colorectal cancer (CRC); castration-resistant prostate cancer (CRPC); HER2− breast cancer (BC); gynecologic (GYN, mainly ovarian and endometrial); and advanced cancers with ATM IHC loss. Pts were treated with elimusertib 40 mg twice daily (BID) 3 days on/4 days off (3 on/4 off). A schedule of 3 on/11 off was also explored by dose escalation in pts with ATM loss or inactivating mutation. Results: 143 pts received ≥1 dose of elimusertib 40 mg BID 3 on/4 off, including 24 CRC, 19 CRPC, 19 BC, 45 GYN, and 36 ATM loss. 56% of pts had ≥4 prior therapy lines. Drug-related grade 3 and 4 treatment-emergent adverse events (TEAEs) were observed in 69% and 15% of pts, respectively, mainly anemia, leukopenia/neutropenia, and thrombocytopenia leading to dose modification but not withdrawal. 32 pts were treated 3 on/11 off with doses from 60 to 120 mg BID. Compared with 3 on/4 off, drug-related grade ≥3 hematologic TEAEs and dose modifications improved at lower doses, with 80 mg 3 on/11 off determined as the recommended dose. In pts receiving elimusertib 3 on/4 off, 5 RECIST partial responses were observed: 1 in ovarian (BRCA1 heterozygous mutation), 1 in BC (BRCA2 mutation), and 3 in ATM IHC loss: 2 CRPC pts both with ATM mutations (1 heterozygous, 1 homozygous), including 1 with FANCA mutation; 1 esophageal with ATM mutation. In pts with ATM IHC loss, objective response rate (ORR) was 9% and disease control rate (DCR) was 65%. A similar ORR was seen on 3 on/11 off in pts with ATM aberrations. In GYN pts, ORR was 2.3% and DCR was 73%. Conclusions: Elimusertib monotherapy demonstrated clinical activity in pts with DDR defects. Overall safety was manageable and hematologic toxicity improved on the 3 on/11 off schedule. Further biomarker analysis is underway to identify potential gene signatures associated with response. Clinical development of elimusertib in combination with checkpoint inhibitors and chemotherapy is ongoing (NCT04095273, NCT04514497). Citation Format: Timothy A. Yap, David S. Tan, Anastasios Stathis, Geoffrey I. Shapiro, Satoru Iwasa, Markus Joerger, Jingsong Zhang, Ruth Plummer, Michael Sawyer, Aaron C. Tan, Vincent Castonguay, Nashat Gabrail, Nobuaki Matsubara, Gary Wilkinson, Matthias Ludwig, Yinghui Zhou, Claudia Merz, Joseph Hreiki, Neelesh Sharma, Johan deBono. Phase Ib expansion trial of the safety and efficacy of the oral ataxia telangiectasia and Rad3-related (ATR) inhibitor elimusertib in advanced solid tumors with DNA damage response (DDR) defects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT006.

Abstract LB039: VHL-deficient renal cell carcinoma displays defective ATM activation and sensitivity to ATR inhibition

Abstract Introduction: Clear cell renal cell carcinoma (ccRCC) displays genomic instability, but lacks mutations in canonical DNA damage response (DDR) genes. Bi-allelic loss of VHL is a near ubiquitous, truncal event in ccRCC development. Herein, we performed preclinical and clinical analysis to understand how VHL loss impacts DDR pathway activity and how specific DDR deficiencies (DDR-D) may be targeted in ccRCC. Methods: We performed DNA whole exome sequencing (WES) and reverse phase protein array (RPPA) early stage ccRCC tumors. All patients were consented under an IRB-approved protocol for the use of tissue. Transcriptomic and protein analyses of select DDR pathways was performed on in silico data (TCGA). DDR functional assays and ATR inhibitor treatment were performed on preclinical cell line models, and in vivo ATR inhibitor treatment in ccRCC xenografts with varying VHL status. Results: Bi-allelic loss of VHL via mutation in one allele and copy number loss of chromosome 3p was observed in 13/13 patient tumors. Mutations in other genes associated with ccRCC and/or canonical DDR genes were not observed. Despite VHL loss being the only driver mutation, these early stage tumors displayed tumor mutational burden (TMB) similar to ccRCC across all tumor stages (1.1 ± 0.5 mut/Mb). Proteomic analysis of patient tumor versus normal tissue (N=12) revealed significantly different (P&amp;lt;0.05) mean expression level of 218 proteins, including significantly lower phospho-ATM at the autophosphorylated S1981 site and lower phospho-CHK2 at the downstream T68 site. VHL-/- cells display defective binding to and repressed activation of Tip60 (KAT5), a key acetyltransferase that activates ATM kinase and aids in the selection of DNA repair pathways following damage. Lastly, treatment with ATRi (VE-822) of 786-O and 786-VHL overexpressing tumor xenografts showed ATRi was safe and significantly inhibited tumor growth preferentially in the 786-O VHL null model. Conclusions: Here we show in patient tissue and preclinical models that VHL biallelic loss results in genomic instability and functional impairment of ATM kinase activity; furthermore, we provide preclinical evidence for selectively targeting the ATR signaling cascade in patients with VHL-deficient kidney cancer. Clinical trials of ATR inhibitor based therapy in patients with advanced cancer, including ccRCC, are underway (e.g. NCT04266912). Citation Format: Patrick G. Pilie, Lijun Zhou, Yan-Ting Zhang, Christine Peterson, Daniel McGrail, Yang Peng, Guang Peng, Xiande Liu, Xuesong Zhang, Eric Jonasch. VHL-deficient renal cell carcinoma displays defective ATM activation and sensitivity to ATR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB039.

Mutational landscape of DNA damage response deficiency-related genes and its association with immune biomarkers in esophageal squamous cell carcinoma.

e16100 Background: Esophageal squamous cell carcinoma (ESCC) has limited effective treatment strategies. DNA damage response (DDR) genes are of therapeutic interest in multiple cancer types. This study aimed to depict the landscape of DDR mutations in ESCC and evaluate the association between DDR mutations and known immunotherapy biomarkers. Methods: We recruited 250 Chinese patients with ESCC and performed next-generation sequencing. The DDR status definition was based on the mutational status of any of the 25 DDR core genes. A total of 107 patients underwent a PD-L1 examination. PDL1-H was defined as CPS ≥10. Results: Among the 250 patients, 73 (29.2%) harbored at least one DDR gene mutation and were defined as DDR-mut. Among the six functional DDR pathways, homologous recombination (HR) (12.4%, 31/250) and others (11.6%, 29/250) were the most frequently mutated. DDR-mut patients were significantly associated with higher tumor mutational burden than those in the DDR-wt group ( P = 7.4e-07). Patients with PDL1-H accounted for 21.2% (36/107) of the patients. PDL1-H was more prevalent in DDR-mut than DDR-wt, although the P value did not reach a significant level (40.5% vs. 30%, P = 0.29). Further analysis revealed that BRCA1, one of the most frequently mutated genes in the HR pathway, was significantly associated with PDL1-H ( P = 0.01). Conclusions: Our data revealed a subset of patients with ESCC harbored DDR gene mutations. Patients with these DDR gene mutations are significantly associated with immune biomarkers, implying the potential feasibility of combining DDR agents with immunotherapy in patients with DDR deficiency.