Copy Number Amplification of DNA Damage Repair Pathways Potentiates Therapeutic Resistance in Cancer.

Rationale: Loss of DNA damage repair (DDR) in the tumor is an established hallmark of sensitivity to DNA damaging agents such as chemotherapy. However, there has been scant investigation into gain-of-function alterations of DDR genes in cancer. This study aims to investigate to what extent copy number amplification of DDR genes occurs in cancer, and what are their impacts on tumor genome instability, patient prognosis and therapy outcome.
Methods: Retrospective analysis was performed on the clinical, genomics, and pharmacogenomics data from 10,489 tumors, matched peripheral blood samples, and 1,005 cancer cell lines. The key discoveries were verified by an independent patient cohort and experimental validations.
Results: This study revealed that 13 of the 80 core DDR genes were significantly amplified and overexpressed across the pan-cancer scale. Tumors harboring DDR gene amplification exhibited decreased global mutation load and mechanism-specific mutation signature scores, suggesting an increased DDR proficiency in the DDR amplified tumors. Clinically, patients with DDR gene amplification showed poor prognosis in multiple cancer types. The most frequent Nibrin (NBN) gene amplification in ovarian cancer tumors was observed in 15 out of 31 independent ovarian cancer patients. NBN overexpression in breast and ovarian cancer cells leads to BRCA1-dependent olaparib resistance by promoting the phosphorylation of ATM-S1981 and homology-dependent recombination efficiency. Finally, integration of the cancer pharmacogenomics database of 37 genome-instability targeting drugs across 505 cancer cell lines revealed significant correlations between DDR gene copy number amplification and DDR drug resistance, suggesting candidate targets for increasing patient treatment response. Principal Conclusions: DDR gene amplification can lead to chemotherapy resistance and poor overall survival by augmenting DDR. These amplified DDR genes may serve as actionable clinical biomarkers for cancer management. © The author(s).

Introduction

Tumors with DNA damage repair (DDR) deficiency demonstrate sensitivity to genome-instability targeting chemotherapies through “synthetic lethality” 1-3. DDR targeting agents have shown promising benefit in the precision medicine for cancer 4-8. Poly ADP ribose polymerase inhibitor (PARPi) is the most remarkable example of such DDR targeting agents, and has shown success in both in vitro studies and cancer patients 9,10. This led to the FDA's consecutive approval of olaparib (2014) 11, rucaparib (2016) 12, niraparib (2017) 13, and talazoparib (2018) 14, for the treatment of advanced ovarian cancer and metastatic breast cancer patients with germline BRCA mutation.

Conversely, the restoration of DDR pathway function (e.g., revival of homology-dependent recombination (HDR) by loss of 53BP1 15 or REV7 16 in BRCA mutated cancer patients) introduces resistance to those DDR targeting agents. Despite the previously well-established connections between DDR loss-of-function and cancer development and treatment 17-19, how frequently the gain-of-function alterations in DDR pathways occur in cancer, and to what extent they affect the DNA damage repair clinical outcome and even drug response remain elusive. In this study, we aimed to characterize the landscape of copy number amplification across nine DDR pathways in cancer by integrating the multi-dimensional genomic data from primary cancer samples and cancer cell lines across 32 cancer types. By further integrating the DDR gene data with tumor mutation burden, mutation signature, clinical treatment information and cancer cell line pharmacogenomics data, we sought to determine the DDR gene amplifications' impacts on the tumor genome instability, patient prognosis and drug responses.

Methods

Characterization of DDR gene copy number amplification and overexpression across 32 cancer types

RNA-Seq gene expression, somatic mutation and somatic copy number alteration (SCNA) of 80 “core-list” from 276 “full-list” DNA Damage Repair (DDR) genes 20 in 10,489 primary tumors were obtained from the TCGA PanCancerAtlas 21 cohort consisting of tumor patients across 32 cancer types. The copy number segmentation data (SCNA score) were obtained from the Circular Binary Segmentation (CBS) algorithm 22, and the GISTIC (Genomic Identification of Significant Targets in Cancer) calls comprising -2 (deletion), -1 (loss), 0 (diploid), 1 (gain), and 2 (amplification) were made using GISTIC2.0 20. mRNA expressions data and copy number alterations of the 80 core DDR genes across 1,005 cancer cell lines were downloaded from the Genomics of Drug Sensitivity in Cancer (GDSC) database 23. Genes with over 5% of samples harboring GISTIC call = -2 or 2 in more than two cancer types were defined as recurrently copy number deleted or amplified. A pathway is labeled as significantly amplified in one sample if at least one gene in the pathway showed amplification in the sample with a false discovery rate (FDR) < 0.25 (see Supplementary Methods). Spearman's rank correlation coefficient was used to detect the correlation between gene expression and copy number alteration for each gene in the cell lines and patient samples respectively. Gene Set Enrichment Analysis (GSEA) 24 was performed to further interpret the association between DDR gene amplification and mRNA overexpression (see Supplementary Methods).

Assessment of the relationship between tumor genome stability and DDR gene copy number amplification in the TCGA patient samples

The tumor genome stability information, including mutation burden and mutation signature scores for the PanCancerAtlas patients was obtained from The Cancer Genome Atlas (TCGA) database 21. Two-sample t-test was used to show the difference in mutation burden/mutation signature scores between samples containing copy number amplifications (GISTIC call =2) of a specific gene vs. the other samples. GSEA analysis was performed on the gene list ranked by the correlation between the gene copy number and mutation burden to determine whether DDR pathways are enriched in the top genes whose copy number gain/amplification could decrease genome instability.

Chemotherapy and radiotherapy information of cancer patients and survival analysis

The raw clinical data of 10,237 TCGA patients across 33 cancer types were obtained from the Genomic Data Commons (GDC). Chemotherapy, radiotherapy and patient survival information were extracted from the raw TCGA clinical data as we previously reported 25 (see Supplementary Methods). Overall survival rates were estimated by Kaplan-Meier curves between patients with or without specific gene copy number amplification/gain (CNAmp; GISTIC calls = 1 and 2) versus others and compared in the specific cancer types using a Cox regression model stratified by the DDR gene SNCA score.

Association analysis between DDR gene copy number alteration and cell line drug response

Drug response data of 37 genome-instability targeting drugs across 1,005 cancer cell lines were downloaded from the GDSC database 23 (see Supplementary Methods) and processed as in our previous report 25. Five hundred and five cell lines with multi-dimensional pharmacogenomics data available were retained for the following analysis. A logarithmic transformed half maximal inhibitory concentration (IC50) value was used to indicate the drug response in each cell line. Correlation between gene copy number alteration and treatment response to each drug was calculated by Spearman's rank correlation coefficient. The difference between drug responses in the cell lines bearing different DDR gene copy numbers was determined by Wilcoxon rank-sum test.

Laboratory Methods

The copy number and protein expression of NBN were determined in formalin fixed paraffin embedded (FFPE) ovarian cancer/para-cancerous tissues from 31 serous epithelial ovarian cancer patients seen in the Department of Gynecological Surgery in the Obstetrics & Gynecology Hospital of Fudan University, by digital droplet PCR (ddPCR) and immunohistochemistry respectively. Written informed consent was obtained from all participants. This study obtained institutional review board approval for the characterization of these molecular features of tumor samples from each patient.

Human breast cancer cell line MCF-7 was a kind gift from Dr. Shilpa Sant. Human ovarian cancer cell lines OVCAR4 and SK-OV3 were purchased from Charles River Laboratories (Frederick, MD) and American Type Culture Collection (ATCC) (Manassas, VA). HA-NBN was stably overexpressed in the cell lines by lentiviral infection. NBN, BRCA1, or ATM was knocked down by siRNA in the cell lines. The cell lines were treated with olaparib (LC laboratories, #O-9201) or cisplatin (Selleckchem, #S1166) for drug response assay, or (S)-(+)-camptothecin (Sigma-Aldrich, #C9911) for double-strand break induction, before the cellular phenotype assays. Cell viability was determined by MTT assay using the CellTiter cell proliferation assay kit (Promega, #G4100), or clonogenic assay by crystal violet staining. Homology-dependent recombination efficacy was determined by RAD51 foci formation assay by immunofluorescence-based foci counting. The in-vivo tumor model was developed using nude mice by subcutaneous injection of SK-OV3 cancer cells with or without NBN overexpression. After administration of cisplatin, olaparib, or saline by intraperitoneal injection, the tumor drug response was assessed as tumor weight [drug treatment]/tumor weight [saline treatment]. Detailed information for laboratory experiments is provided in the Supplementary Methods.

Results

A systematic analysis revealed that DDR genes are significantly amplified and overexpressed in cancer patients

We focused on scrutinizing the copy number amplification alterations of 80 “core” DNA damage repair (DDR) genes 20 composing nine major DDR pathways, including Base Excision Repair (BER), Direct Repair (DR), Fanconi Anemia (FA), Homology-Dependent Recombination (HDR), Mismatch Repair (MMR), Non-homologous End Joining (NHEJ), Nucleotide Excision Repair (NER), Translesion Synthesis (TLS), Damage Sensors and Others. Consistent with previous studies 20, our mutation analysis did not identify recurrent gain-of-function point mutations in the DDR genes (data not shown).

Intriguingly, we observed recurrent DDR gene copy number amplifications/gains (CNAmps) among the 10,489 TCGA cancer samples across 32 tumor types (Figure , 1B, , and ). Among the nine DDR pathways, pathways responsible for the double strand break (DSB) restoration (HDR and NHEJ), as well as the damage sensors (FDR = 0.13) were significantly amplified across the pan-cancer cohort (FDR < 0.25). The HDR pathway, as the most prevalently amplified DDR pathway (76.8% of pan-cancer), was significantly amplified over 18 cancer types including lung adenocarcinoma (LUAD, 464 [90.8%] of 511), rectum adenocarcinoma (READ, 142 [91.6%] of 155), ovarian serous cystadenocarcinoma (OV, 544 [96.8%] of 562), lower grade glioma (LGG, 307 [60.2%] of 510)/glioblastoma (GBM, 526 [92.1%] of 571) and breast invasive carcinoma (BRCA, 948 [88.6%] of 1070) (FDR < 0.25). We observed that cancer types sharing similar tissue origins or carcinogenic risk factors harbor similar DDR gene CNAmp patterns, such as seen in colon adenocarcinoma (COAD) and READ (r = 0.97, P = 6.2×10-52), head and neck squamous carcinoma (HNSC) and esophageal carcinoma (ESCA) (r = 0.83, P = 2.9×10-21), and liver hepatocellular carcinoma (LIHC) and cholangiocarcinoma (CHOL) (r = 0.83, P = 9.3×10-22) ().

On the individual gene level, 13 of the 80 core DDR genes showed significantly recurrent amplification among multiple cancer types (genes amplified in over 5% of samples in more than two cancer types, see Supplementary Methods). In contrast, only 3 DDR genes showed significantly recurrent deletion under the same criteria (). The most frequently amplified genes across the Pan-Cancer Atlas were NBN (n = 4,275, 40.8%), EXO1 (n = 3,714, 35.4%), PARP1 (n = 3,695, 35.2%), PRKDC (n = 3,650, 34.8%) and POLB (n = 2,794, 26.6%) (Figure ). All of the 13 recurrently amplified DDR genes exhibited significant overexpression in the amplified tumors (P < 10-20, ). Gene Set Enrichment Analysis (GSEA) revealed that overexpression of all of the nine DDR pathways in the tumors is significantly driven by their CNAmp (FDR < 0.1) (Figure ). To further validate the DDR pathways' CNAmp and their overexpression in cancer, we investigated the DNA copy number and mRNA expression data of 1,005 cancer cell lines from the Genomics of Drug Sensitivity in Cancer (GDSC) database 23. The overexpression of all 13 of the recurrently amplified DDR genes was significantly driven by copy number amplification in the cancer cell lines ( and and S3).

Tumors with DDR gene CNAmp exhibit decreased tumor genome instability and reduced mutational signature scores

With the observation of significantly recurrent overexpression and amplification of DDR genes in both primary tumors and cancer cell lines, we wondered if CNAmp and overexpression of DDR genes would increase the DDR function in tumor cells. In this regard, we investigated whether there was a difference in the mutation burdens 20 between the tumors with or without DDR gene CNAmp. This analysis revealed that tumors harboring CNAmp of 11 individual DDR genes (4 of which are recurrently amplified among multiple cancer types, UBE2T, PARP1, PRKDC, and RAD52) exhibited significantly reduced mutation burden versus those without CNAmp of these 11 DDR genes (Figure ), suggesting that the amplification of DDR genes might lead to an increased DDR function in those tumors. For example, the amplification of the BER pathway, including the genes UNG, POLE, TDG, and PARP1, is prominently correlated with genome stability in the OVs, as tumors with a stable genome were significantly enriched in the BER pathway gene amplified sample set (NES = 1.802, FDR = 0.007) (Figure ).

Cancer type-specific mutation burden analysis at the gene level further confirmed the association between DDR gene CNAmp and increased tumor genome stability (Figure and ). For instance, POLE is a gene involved in the BER pathway. Its loss-of-function mutations have been established to cause a hyper-mutator phenotype in multiple cancer types 26. In our study, we found that POLE amplified tumor samples exhibited 50% reduced mutation burden than tumors without POLE amplification in bladder urothelial carcinoma (BLCA) and OV (Figure ), suggesting that the gain-of-function POLE CNAmp may lead to an increased DDR function in cancer.

In addition to using the mutation burden as an indicator for global genome instability, we further compared the mechanistic specific mutation spectrum between the DDR gene amplified tumors and other tumor samples to determine if DDR gene CNAmp could alter the recurrent mutations accumulated under specific mutation sources and mutation mechanisms 27,28. When considering all the 21 previously defined mutation signatures 28, including smoking-, UVB exposure-, and POLE deficiency-induced mutation signatures, we observed that tumors with DDR gene amplification have a significantly lower incidence of the aforementioned DNA damage (Figure and ). For example, LUSCs, COADs, and OVs with PARP1 amplification have a significant reduction in the smoking-induced mutation signature (LUSC, fold change < 10-3, P = 3.09×10-23; COAD, fold change < 10-3, P = 1.74×10-19; OV, fold change = 0.45, P = 0.01) (Figure ), which indicates a critical role for the error-free BER pathway genes in amending the smoking-induced DNA lesions 29. Tumors bearing amplification of FA pathway genes (FANCB, FANCC and FANCM in LGG and UBE2T in GBM) and HDR genes (ATM, CHECK1, MRE11A in GBM and GEN1 in skin cutaneous melanoma [SKCM]) showed significantly reduced temozolomide-induced signature score (Figure ), indicating the critical role of DSB-associated recombinational repair in attenuating alkylating agent-induced genome lesions 30,31. These observations suggest that the CNAmp of DDR genes would restore the DDR function in tumor cells, thus alleviating the genome lesions and maintaining genome stability in the tumor.

DDR gene CNAmp in the tumor is significantly correlated with poor cancer patient survival

To investigate whether DDR gene amplification is clinically relevant, we analyzed the correlation between patient overall survival and DDR gene CNAmp in each cancer type. As shown in Figure and , the CNAmp of core DDR genes exhibited a broad correlation with unfavorable survival of cancer patients. For example, PMS2 is a critical component of the MutL alpha heterodimer for the initiation of mismatch repair 32, 33. Amplification of the PMS2 gene is frequently found in glioblastoma multiforme (GBM, 454 [79.5%] of 571), lower grade glioma (LGG, 134 [22.4%] of 510), HNSC (205 [39.7%] of 517) and OV (132 [23.5%] of 562). Those patients with PMS2 gene amplification have significantly shorter survival compared to non-CNAmp patients in each cancer type (GBM, HR = 1.53, 95% CI 1.22 to 1.92, P = 1.98×10-4; LGG, HR = 2.32, 95% CI 1.69 to 3.19, P = 2.33×10-7; HNSC, HR = 1.58, 95% CI 1.24 to 2.02, P = 2.22×10-4; OV, HR = 1.42, 95% CI 1.12 to 1.79, P = 3.48×10-3) (Figure ). Another recurrently amplified DDR gene, POLM, plays an essential role in NHEJ repair for DSB 34, 35. Poor survival was observed in patients with POLM amplification in multiple cancer types (HNSC: CNAmp frequency = 36.4% [188 of 517], HR = 1.40, 95% CI 1.09 to 1.81, P = 9.17×10-3; LGG: CNAmp frequency = 23.5% [120 of 510], HR = 2.47, 95% CI 1.78 to 3.44, P = 6.83×10-3; and LUAD: CNAmp frequency = 51.9% [265 of 511], HR = 1.52, 95% CI 1.15 to 2.03, P = 3.83×10-3) (Figure ). PRKDC amplification also significantly correlated with poor patient survival in multiple cancer types (sarcoma [SARC]: CNAmp frequency = 35.2% [89 of 253], HR = 2.15, 95% CI 1.46 to 3.18, P = 1.12×10-4; uterine corpus endometrial carcinoma [UCEC]: CNAmp frequency = 31.5% [165 of 523], HR = 1.72, 95% CI 1.21 to 2.44, P = 2.50×10-3) (Figure ). Results for more DDR genes can be found in .

Integrated evidence revealed that NBN CNAmp induces cisplatin and PARP inhibitor resistance in breast and ovarian cancer

Previous studies have demonstrated that restored DDR function leads to chemotherapy resistance and thus poor patient survival 15, 36, 37. The observation of significant positive correlations between DDR gene CNAmp and reduced tumor mutation burden, mechanism specific mutation signatures, and poor patient survival lead to our hypothesis that CNAmp of these DDR genes may cause poor patient survival by augmenting DDR function and consequently chemotherapy resistance in the tumor. Among the recurrent DDR gene CNAmps, NBN CNAmp is the most prominent molecular event that occurs in over 40% of patients across 16 cancer types. NBN's overexpression is highly driven by its CNAmp in both primary tumors (TCGA database, P = 2.50×10-60) and cancer cell lines (GDSC database, P = 3.94×10-5), which was also observed in two independent serous ovarian carcinoma studies (Etemadmoghadam D, et al.38, 39, P = 2.45×10-7; Ducie J, et al.40, P < 10-5) (). Moreover, NBN CNAmp is most prominently correlated with poor overall survival in OV patients (HR = 1.36, 95% CI 1.13 to 1.64, P = 9.62×10-4) (Figure ). To corroborate NBN's CNAmp and overexpression in ovarian cancer, we further experimentally quantified NBN gene copy number and protein expression in an independent cohort of 31 serous ovarian cancer samples using droplet digital PCR and immunohistochemistry respectively. These assays independently validated that NBN protein overexpression is highly associated with its CNAmp (Fisher's exact test, P = 0.012) (Figure ).

The NBN gene encodes Nibrin (P95), a component of the MRE11-RAD50-NBN (MRN) complex that has been identified as a crucial player in the DSB end processing and HDR repairing processes 41. Since platinum-based drugs and PARP inhibitors have been extensively used as the DSB-targeting chemotherapy of ovarian cancer, we performed a correlation analysis between NBN copy number alterations and the drug treatment response to cisplatin and PARPis across 505 cancer cell lines from the GDSC database. This analysis revealed that NBN copy number is highly correlated with cellular viability responses to cisplatin treatment (rho = 0.25, P = 1.60×10-3), which is the most commonly used chemotherapy for OV patients. Moreover, the cancer cell lines with NBN amplification showed significantly increased resistance to PARP inhibitors olaparib and veliparib (Figure ). We thus overexpressed NBN in OVCAR4 and MCF-7 cells. MTT assay and clonogenic assay indicated that NBN over-expression can significantly increase resistance to cisplatin and olaparib in MCF-7 (Figure , E, and F) and OVCAR4 (Figure , H, and I) cells. The increased resistance to cisplatin and olaparib after NBN overexpression was also confirmed in vivo in a xenograft drug response mouse model (cisplatin, P = 0.019; olaparib, P = 0.032) (Figure ). Consistently, NBN depletion significantly sensitized these cancer cells to cisplatin or olaparib treatment (Figure and ). In accordance with its regulation of cell line response to cisplatin and olaparib treatments, NBN overexpression significantly augmented HDR proficiency as indicated by increased RAD51 foci formation after DSB induction by camptothecin (Figure ) and reduced γH2AX phosphorylation after cisplatin or olaparib treatment (Figure ) in both MCF-7 and OVCAR4 cells.

It has been well documented that the MRN complex interacts with ATM during the initial stage of HDR 42. Our analysis also found a strong association between NBN amplification and AZD7762 (inhibitor for ATM substrate CHECK1/2) resistance (Figure ). After DSB induction by camptothecin, MCF-7 cells overexpressing NBN showed an increased level of ATM phosphorylation (S1981) compared with the control cell line (Figure ), suggesting that NBN CNAmp's induction of chemotherapy resistance may be mediated by ATM phosphorylation. ATM phosphorylation is the critical step for the activation of downstream HDR proteins including BRCA1 43, 44. Further siRNA treatment of either the ATM or the BRCA1 gene alleviates the NBN overexpression induced drug (e.g., cisplatin and olaparib) resistance phenotype in MCF-7 cells (Figure , E), which unequivocally demonstrates that the NBN CNAmp induced drug-resistant phenotype is mediated through the ATM-HDR activation.

Pharmacogenomics analysis unveiled an overall significant correlation between DDR gene CNAmps and genome-instability targeting drug resistance

Resistance to chemotherapy is one of the major barriers to improving cancer survival. One-third of the current FDA approved anti-cancer drugs are targeting genome instability or DNA replication 45. Encouraged by the experimental validation that NBN CNAmp leads to cisplatin and olaparib resistance, we wondered if other DDR gene CNAmps could lead to poor patient prognosis through prompting resistance to genome instability targeting chemotherapy. In this regard, we further integrated the copy number alterations data across 505 cancer cell lines and their responses to 37 genome-instability targeting drugs (i.e., 23 DNA-damaging drugs and 14 cell cycle/TP53 targeting agents) in GDSC. This analysis revealed the landscape of DDR gene CNAmps and response to genome-instability targeting drugs including 468 significant associations between the 80 DDR CNAmps and the 37 genome-instability targeting drugs (Figure ). Among the 468 significant associations, 430 (92%) significant positive correlations indicated that DDR gene CNAmp lead to drug resistance (), suggesting that the CNAmp of DDR genes might induce a resistant phenotype to chemotherapy targeting genome-instability.

Gene-level analysis revealed that CNAmp of FANCM, the pivotal component of the Fanconi anemia (FA) pathway that relieves the DNA inter-strand cross-link 46, has significant correlations with cellular responses to DSB inducing agents such as topoisomerase inhibitor (camptothecin, P = 5.54×10-3), CHECK1/2 inhibitor (AZD7762, P = 1.90×10-5), and PARP inhibitors (olaparib: P = 3.34×10-3, veliparib: P = 1.29×10-4) (), which is consistent with previous studies showing that FANCM is intensively involved in the DDR response and regulates PARPi sensitivity 47,48.

On the pathway level, the cancer cell lines with CNAmp of DDR genes across the BER (5 out of 10 genes), FA (3 out of 8 genes) and HDR (7 out of 21 genes) pathways exhibited significantly higher IC50s to camptothecin (P < 0.05). Camptothecin (Irinotecan) is a topoisomerase inhibitor that induces lethal replication fork collisions between advancing replication forks during S-phase DNA replication. The BER, FA and HDR pathways are critical for the rescue of the stalled replication fork 49-52, which provides a mechanistic explanation for the correlation between DDR pathway amplification and Irinotecan resistance.

Consistent with our previous observation, HDR pathway CNAmp is associated with increased IC50 of PARPi. Six (29% of 21) HDR genes (NBN, GEN1, BARD1, RAD50, BRCA1, and BRIP1) showed significant positive correlations between the gene copy number alterations and cell line responses to veliparib and olaparib treatments respectively (P < 0.05) (). Together with our previous experimental phenotype validation of NBN overexpression in the cancer cell lines, this study further suggests that the observed significant correlation between DDR gene CNAmp and poor patient survival maybe attributed to increased DDR function and chemotherapy resistance in tumors with DDR gene CNAmps.

Discussion

In the current study, by integrating multi-dimensional genomics and clinical data in cancer patients and cancer cell lines, we demonstrated that DNA damage repair (DDR) genes' copy number amplification/gain (CNAmp) and overexpression not only recurrently occur across 32 cancer types, but also lead to elevated DNA repair capacity and increased chemotherapy resistance. To the best of our knowledge, this is the first study systematically depicting the DDR pathways copy number amplification landscape and their clinical consequences in cancer.

DDR pathway deficiencies have been well-established as drug-actionable targets for cancer therapy 53,54. However, frequent CNAmp of DDR genes, which may play critical roles in the therapeutic resistance of cancer, have long been neglected in cancer study and treatment. Previous studies reported that the restoration of homology-dependent recombination (HDR) function by somatic reversion of germline BRCA1/2 mutations confers platinum and PARPi resistance in ovarian cancer 37. In our study, we have unveiled a general connection between DDR gene CNAmps, increased genome integrity, and poor cancer patient survival. Our analysis revealed that the DDR genes are significantly overexpressed in tumors with DDR CNAmps, and correlates with the reduced genome instability across 32 cancer types. Since the genome instability has been intensively reported as a prognosis marker for the cancer patient 55-57, we further adjusted the survival analysis using the genome instability data and confirmed that the strong connection between DDR gene amplification and poor patient survival still stood (data not shown).

Consistent with their association with poor prognosis, DDR gene amplification were shown to be correlated with anti-cancer drug resistance to 37 genome instability-targeting drugs across 505 cancer cell lines. We have further experimentally validated that overexpression of NBN, the most frequently amplified DDR gene in the pan-cancer cohort; can directly induce the olaparib resistance in both breast and ovarian cancer cell lines through activating the HDR pathway. Whether the NBN overexpression induced cisplatin and olaparib resistance is specifically mediated by BRCA1 will be studied in the future investigation. Our study has not only provided a panorama of DDR genes and anti-cancer drugs interactions, but also a novel molecular mechanism for the intrinsic resistance of genome-instability targeting chemotherapy. Note that all the 10,489 tumor samples and 505 cancer cell lines in this study are chemotherapy naïve, which means that the DDR gene CNAmps have already existed in some tumors before chemotherapy. It is possible that some patients have a subclone of cancer cells with DDR gene CNAmp in their primary tumors. Those patients will initially respond to DDR targeting drugs, and later on develop resistance to the drug after a DDR gene CNAmp subclone starts to expand and becomes the major clone under drug selection pressure 58. One limitation of our study was that we were unable to determine if DDR gene CNAmp occurs in all cancer cells or only a subclone within a tumor. Further study harnessing single-cell sequencing technology 59, 60, and clinical follow-ups are required to comprehensively decipher the roles of DDR gene CNAmp events in cancer development and drug resistance.

DDR gene CNAmps may also serve as reliable biomarkers for cancer precision therapy. Tremendous efforts have been invested to develop clinical actionable methods to measure DDR function and thus predict clinical outcome and chemotherapy response in various cancer types. Those methods include HDR assay, DDR gene-expression profiling, and HDR protein quantification 61-63. However, poor technical reproducibility and inconsistent results with current DDR signaling models raise the translational barrier for these conceivable markers 64, 65. In this study, we have confirmed the NBN CNAmp by digital droplet PCR and its strong correlation with gene overexpression in serous ovarian cancer samples. These findings suggest that the copy number quantification of DDR genes at the DNA level can be a promising biomarker for beneficiary identification, response evaluation and prediction of genome instability-targeting therapy, although the clinical feasibility awaits validation from further clinical trials.

Supplementary figures and tables.

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