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Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors Wenchao Ji b, Xiang Weng c, Danhua Xu b, Shufan Cai b, Ling Ding a, * a
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang Province, China Department of pharmacy, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China c Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 October 2019 Accepted 6 November 2019 Available online xxx
Lung cancer is the leading cause of cancer death worldwide. PARP inhibitors have become a new line of cancer therapy and a successful demonstration of the synthetic lethality concept. The mechanism and efficacy of PARP inhibitors have been well studied in some cancers, especially homologous recombination (HR)-deficient ovarian cancer and breast cancer, yet such studies are still relatively fewer in lung cancer. Here we found that HR genes are frequently mutated in lung cancer patients, exposing a window for targeted therapies by PARP inhibitors. We depleted BRCA1 and BRCA2 in non-small cell lung cancer (NSCLC) cancer cells and found these cells are hypersensitive to the PARP inhibitor olaparib in cell viability and clonogenic survival assays. Olaparib specifically induces apoptosis in A549 cells with BRCA1 or BRCA2 depletion, as determined by positive Annexin-V staining. In addition, we show that A549 cells with ATM shRNA knockdown are also hypersensitive to Olaparib. In summary, our data support the potential use of PARP inhibitors in NSCLC with HR deficiency. © 2019 Published by Elsevier Inc.
Keywords: Lung cancer Homologous recombination PARP inhibitor BRCA ATM
1. Introduction Lung cancer is the most common type of solid tumors in China, and the 5-year survival for lung cancer in China is only 19% [1]. Lung cancer also remains the most commonly diagnosed cancer type and the leading cause of cancer death world-wide (11.6% of total cases and 18.4% of the total cancer deaths, respectively) [2]. In the United States, nearly 55 per 100,000 people was diagnosed with lung or bronchus cancer each year, and they are responsible for 23.5% of cancer-related death, putting lung cancer the leading cause of cancer-related mortality in the United States (National Cancer Institute, USA). Despite huge efforts that were invested in basic and clinical research, the 5-year survival rate for lung cancer has not changed significantly in the past two decades (15.8% in 2000 vs. 19.4% in 2019, National Cancer Institute, USA). Approximately 80% of all lung cancers are non-small cell lung cancer (NSCLC) (American Cancer Society, Cancer Facts & Figures 2019). The standard of care for advanced NSCLC is platinumbased doublet, generally cisplatin or carboplatin-based [3].
* Corresponding author. College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang Province, 310058, China. E-mail address: [email protected] (L. Ding).
Although patients generally response, the outcome is not very promising. Targeted therapy such as epidermal growth factor receptor (EGFR) tyrosine kinase (EGFR-TK) inhibitors have been shown to improve the progress free survival (PFS) of NSCLC, yet the drug can only be used for EGFR-driven lung cancers, which only account for a fraction of all lung cancer patients. In addition, drug resistance to those therapies have become a challenge [4e6]. New therapies for effective lung cancer treatment are urgently needed. Because lungs are the most exposed organ to exogenous DNA damaging agents such as smoking and air pollution, lung cancers normally contain high mutation loads [7]. Sequencing of the lung cancer cell lines reveals that the average mutation rate of lung cancers was 4.2 per megabase [8]. The highly mutated tumor suppressing gene such as TP53 and proto-oncogenes such as EGFR, ALK and ROS1 have been widely studied [9,10]. Increasing number of studies on mutations in DNA repair genes are undertaking, in an attempt to find DNA repair defects that can be targeted for precision medicine. Homologous repair (HR) genes have been implicated in lung cancer survival after treatment, although mixed clinical outcomes are associated with BRCA1 mutations. Margeli, Taron and their colleagues show that lower expression of BRCA1 predicts a better outcome in lung cancer patients [11,12]. Based on the predictive value of BRCA1 expression, low expression of BRCA1 was
https://doi.org/10.1016/j.bbrc.2019.11.050 0006-291X/© 2019 Published by Elsevier Inc.
Please cite this article as: W. Ji et al., Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.050
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used as a biomarker in a Trabectedin clinical trial in platinum refractory NSCLC patients [13]. However, Choi et al. show later that low expression of BRCA1pS1423 or ERCC1 was significantly associated with worth outcome [14]. In addition, mutations in ataxia telangiectasia mutated (ATM) is associated with increased onset rates of lung cancer [15]. Less is known about how BRCA2 (another critical HR gene) mutations or expression affects the outcomes of lung cancer patients. In summary, more mechanistic study of those DNA repair genes in lung cancer is needed in order to harness it for targeted therapy. The poly-(ADP-ribose) polymerase (PARP) inhibitors are a new line of chemotherapy that showed remarkable efficacy in ovarian cancer, especially in those with BRCA mutations [16]. PARP family of enzymes play critical roles in DNA single-strand break (SSB) repair, and inhibition of which leads to the generation of double-strand breaks (DSB) [17]. The deleterious double-strand breaks are repair mostly by homologous recombination (HR) during S and G2/M phases of the cell cycle. Inhibition of PARP in HR deficient cells causes accumulation of DSBs that eventually leads to apoptosis of cells [18]. Therefore, inhibition of PARP and HR deficiency are synthetically lethal [19,20]. This synthetical lethality relationship led to the discovery of PARP inhibitors, effective chemicals that specifically kill HR deficient cancer cells, and this line of drugs have been huge success. Till date, four PARP inhibitors have been approved by FDA for treatment of ovarian cancer and breast cancer, namely Olaparib, Niraparib, Rucaparib and Talazoparib, based on their outstanding performance in clinical trials [21e24]. Despite the huge success, the application for PARP inhibitors are still very limited. Theoretically, this synthetic lethality should be conserved in other cancer types. Yet, experimental and clinical studies are still lacking to extend PARP inhibitors to other cancer types. Although PARP inhibitors have been successfully used in ovarian and breast cancers, a lot remains to be done before their potential use in lung cancer. Here, we show that homologous recombination genes are frequently mutated in non-small cell lung cancer, including the critical HR genes BRCA1, BRCA2, and ATM. BRCA1, BRCA2 and ATM depleted lung cancer cells are hypersensitive to PARP inhibitors. These HR-deficient (but not HR-proficient) lung cancer cells are apoptotic after exposure to the PARP inhibitor olaparib. Our data shown in this study strengthen the idea of using PARP inhibitor as targeted therapy for HR-deficient lung cancer. 2. Results 2.1. HR genes are frequently mutated in lung cancer We have explored the mutation rates of the key HR DNA repair genes in lung cancer using bioinformatic tools and publicly available databases. By analyzing mutation data in 1962 samples in 8 lung cancer studies in cBioportal (www.cbioportal.org), including 4 non-small cell lung cancer (NSCLC) and 4 small cell lung cancer (SCLC) studies, and we found the HR genes are highly mutated in lung cancer patients, in both NSCLC and SCLC cancer patients (Fig. 1 and S1). 23 HR genes were used as queries in this analysis, including key HR genes such as BRCA1, BRCA2, ATM, RAD51, RAD52, and MRE11 (Table S1). HR genes were altered in about 30% of all lung cancers in total, and almost all of those were gene mutations in SCLC and majority of those were mutations and deletions in NSCLC (Fig. 1A). Further analysis revealed that mutations and deletions in BRCA1, BRCA2 and ATM accounts about half of all HR alterations (14% for NSCLC and 12% for SCLC, Fig. 1B). In NSCLC, BRCA1, BRCA2 and ATM mutations and deletions accounts for 3%, 4.5%, and 7%, respectively. In SCLC, BRCA1, BRCA2 and ATM mutations accounts for 5.5%, 3.5%, and 4.5%, respectively (Fig. S1A). In additional, we found that low ATM expression is associated with worth overall
survival of lung cancer patients (Fig. S1B). These data suggest that HR genes are frequently mutated in lung cancer, and that targeting HR-deficient lung cancer is clinically relevant, especially given the high volume of cancer patients. 2.2. BRCA1/2-deficient NSCLC cells are hypersensitive to PARP inhibitors PARP inhibitors are well-known to kill ovarian and breast cancer cells that are deficient in the HR pathway, a synthetic lethality relationship that has been successfully translated to ovarian cancer treatment. In order to study whether this synthetic lethality relationship also exists in lung cancer cells, we knocked down the HR genes in the NSCLC cell line A549, and asked whether knockdown of these genes leads to sensitivity to PARP inhibitors. First, we depleted BRCA1 using siRNA transfection. Western blots show that BRCA1 was successfully knocked down at protein level with high efficiency (Fig. 1C). The knockdown of BRCA1 in A549 cells was also confirmed by the cisplatin sensitivity of these cells (Fig. S2A), a classic drug sensitivity of HR-deficient cells [25]. Next, we tested whether cells with BRCA1 depletion were sensitive to the PARP inhibitor olaparib (AZD2881), using the CellTiter-Glo (CTG) luminescent cell viability assay. We found that cells transfected with BRCA1 siRNA were much more sensitive to olaparib than the control siRNA transfected cells (Fig. 1D). To confirm these results, we performed clonogenic survival assays. Consistent with the CTG assays, knockdown of BRCA1 resulted in hypersensitivity of A594 cells to olaparib. For example, 1 mM of olaparib almost totally killed BRCA1-depleted cells while the control siRNA treated cells were not impacted (Fig. 1E and F). These data demonstrated that BRCA1 deficient NSCLC cancer cells are hypersensitive to PARP inhibitor olaparib. Next, we performed same sets of experiments in BRCA2depleted A549 cells. Similar results were observed when we compared siBRCA2-and siControl-treated cells (Fig. 2). BRCA2 was successfully knocked down in A549 cells (Fig. 2A) and BRCA2 knockdown cells were sensitive to cisplatin as expected (Fig. S2B). BRCA2 knockdown cells were more sensitive to olaparib than control counterparts in CTG assays (Fig. 2B), and the same results were observed in clonogenic survival assays (Fig. 2C and D). However, we also noticed that BRCA2-depleted cells were less sensitive to olaparib than BRCA1 depleted cells (compare Figs. 2B to Fig. 1D), which may be explained by the more critical role of BRCA1 in HR. Those results suggest that PARP inhibitors could effectively kill BRCA1/BRCA2 deficient cells while spare the wild type control cells. 2.3. ATM knockdown sensitizes A549 cells to PARP inhibitor ATM is a master regulator of DNA double-strand break repair, which influences the repair efficiency of both HR and NHEJ. Since ATM is highly mutated in NSCLC, we next tested if knockdown of ATM is synthetically lethal with PARP inhibition. ATM was successfully knocked down at protein level by shRNA after puromycin selection of lentivirus infected cells, as shown by the absence of ATM band in Western blot after puromycin selection (Fig. 3A). We next tested olaparib sensitivity using CTG cell viability assay and clonogenic survival assay. A549 with shATM knock down was clearly hypersensitive in CTG assay (Fig. 3B). Consistently, ATMdepleted A549 cells were hypersensitive to olaparib when tested in clonogenic survival assay (Fig. 3C). Quantification of the images in clonogenic assays clear show that same conclusion: ATMdepleted cells were hypersensitive to the PARP inhibitor olaparib (Fig. 3D). These results show that ATM deficient NSCLC cells are also sensitive to PARP inhibitors and indicate that PARPi may be used in NSCLC using ATM status as a biomarker.
Please cite this article as: W. Ji et al., Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.050
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Fig. 1. Gene alteration rates of homologous recombination genes in NSCLC and SCLC patients and olaparib sensitivity of BRCA1-deficient NSCLC cells. (A) 23 well-known homologous recombination genes were entered as queries for genes alterations in lung cancer studies (8 studies, 4 for NSCLC and 4 for SCLC). A list of the queried HR genes can be found in Supplemental Table 1. The total sample size is 1962. (B) The alteration frequency of BRCA1, BRCA2 and ATM genes was analyzed together in NSCLC and SCLC. 12e14% of the three genes were altered in total, which were almost exclusively mutation and deletions. (C) A Western blot image shows knockdown of BRCA1. Two independent siRNA were used. (D) PARP inhibitor (olaparib) sensitivity of BRCA1-depleted A549 in CellTiter-Glo cell viability assay (CTG). (E) Clonogenic survival assays show that siBRCA1 A549 cells were hypersensitive to olaparib. (F) Quantification of clonogenic survival assays shown in (E). Mean and S.D. of 6 replicates were shown for CTG assays in (D). For clonogenic survival assays in (E-F), representative images of three independent experiments were shown and quantification are mean and standard deviation (S.D.).
2.4. PARP inhibitor induces apoptosis in BRCA1 and BRCA2 depleted NSCLC PARP inhibitors are known to induce apoptosis in BRCA-deficient breast cancer cells [18]. We next asked whether PARP inhibitors also cause apoptosis in BRCA-deficient NSCLC. A549 cells were first transfected with siRNA for 48 h and then treated with olaparib for 48 h. Cells were then stained with FITC-conjugated anti-Annexin V antibody for flow cell cytometry detection. We found that olaparib specifically induced apoptosis in siBRCA1-treated but not siControltreated A549 cells (Fig. 4A and B). When we treated siBRCA1-treated cells with 1 mM of olaparib, 17.7% of cells were apoptotic with positive Annexin V staining, while only 3% of siControl-treated cells were apoptotic. Similar results were observed in siBRCA2-treated cells. Olaparib specifically induced apoptosis in BRCA2-depleted cells but not in control cells (Fig. 4C and D). Consistent with the olaparib sensitivity data, siBRCA2 cells were less apoptotic compared to siBRCA1 cells upon olaparib treatment (13.2% v.s. 17.7% at 1 mM olaparib). These data suggest that olaparib specifically induces apoptosis in BRCA-deficient cancer cells, explaining why olaparib selectively kills BRCA-deficient cells.
3. Discussion Lung cancer is the leading cause of cancer death in China and world-wide, the 5-year survival rate of which has not changed much in the past 20 years. New and more effective therapeutics is needed imminently. PARP inhibitors have been proved highly effective in ovarian and breast cancers but not in other cancers, including lung cancer. Here, we study the synthetic lethality relationship between HR deficiency and PARP inhibition in non-small cell lung cancer cells. We found that HR genes, including BRCA1, BRCA2 and ATM are frequently mutated or deleted in NSCLC, which can potentially be biomarkers for using PARP inhibitors. Experimentally, we depleted BRCA1, BRCA2 and ATM in NSCLC cell line, and found cells lacking those HR proteins are hypersensitive to olaparib. Mechanistically, PARP inhibitors induces apoptosis in those HR-deficient cells. Our results suggest that NSCLC with HRdeficiency are hypersensitive PARP inhibitors and indicate that PARP inhibitors could be a potential treatment for NSCLC with mutations in HR genes. PARP inhibitors are arguably the most successful example of synthetic lethality. Since the discovery of the synthetic lethality
Please cite this article as: W. Ji et al., Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.050
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Fig. 2. BRCA2 deficient NSCLC were sensitive to PARP inhibitor olaparib. (A) A Western blot image shows knockdown of BRCA2 using two different siRNA. (B) Olaparib sensitivity of BRCA2-depleted A549 cells in CTG assays. (CeD) Representative images and quantification of clonogenic assays of A549 cells treated with siCtrl or siBRCA2 and increasing concentration of olaparib. Mean and SD of 6 replicates were shown for CTG assays in (B). For clonogenic survival assays (CeD), representative images of three independent experiments were shown and quantification are mean and S.D.
Fig. 3. Knockdown of ATM by shRNA sensitizes lung cancer cells to PARP inhibitor olaparib. (A) A Western blot image shows that ATM was knocked down by shRNA in A549 cells. (B) A549 cells with ATM shRNA was hypersensitive to olaparib in CTG assays. (C) Representative images of clonogenic assays in shScramble and shATM infected A549 cells after olaparib treatment. (D) Quantification of the clonogenic survival assays in (C). Data shown in B and D are mean and SD from three independent experiments.
relationship between PARP and BRCA genes in 2005 [19,26], rapid progress has been made for this class of drugs; within less than 10 year, the first PARP inhibitor olaparib (AstraZeneca) was approved in late 2014 for BRCA-mutated ovarian cancer [27]. PARP inhibitors
showed remarkable efficacy in ovarian cancers [21e23,28]. More recently, PARP inhibitors also show good response in breast cancer and pancreatic cancers and subsequently approved for these two cancer types [29,30]. In principal, any cancer cells that are deficient
Please cite this article as: W. Ji et al., Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.050
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Fig. 4. Olaparib specifically induces apoptosis in BRCA1/BRCA2-depleted A549 cells. (A) Flow cell cytometry shows the FITC (Annexin V) positive cells after 1 mM olaparib treatment in siCtrl or siBRCA1 treated A549 cells. (B) Quantification of Annexin V-FITC positive cells after 1 mM olaparib treatment in siCtrl or siBRCA1 treated A549 cells. (C) Flow cell cytometry shows the FITC (Annexin V) positive cells after 1 mM olaparib treatment in siCtrl or siBRCA2 treated A549 cells. (D) Quantification of Annexin V-FITC positive cells after 1 mM olaparib treatment in siCtrl or siBRCA2 treated A549 cells. A549 cells were transfected with siControl or siBRCA1 (A-B) or siBRCA2 (CeD) for 48 h, and cells were treated olaparib for additional 48 h. Cells were stained with anti-AnnexinV antibody and PI for apoptosis analysis. Stained cells were analyzed by flow cell cytometry. Data shown in B and D are mean and S.D. from three measurements.
in HR repair of DNA double-strand breaks should be sensitive to PARP inhibitors, given HR of double-strand breaks and PARPmediated single-strand break repair are highly conserved mechanisms in eukaryotes. On the other hand, using PARP inhibitors in lung cancer is still under debate [31]. Although some progress in clinic [32], mixed outcomes were reported with BRCA mutations [11,12,14]. More basic research in the PARP and HR synthetic lethality relationship is needed to support the use of PARP inhibitors in lung cancer. Our results strengthened the idea that HR-deficient cancer cells, including loss of BRCA1, BRCA2, or ATM, are hypersensitive to PARP inhibitors. Although the mutation/deletion rate of individual gene is not very high, the total mutation rate of the three HR genes is as high as 14%. Given the high volume of lung cancer patients (mostly NSCLC), there are a great number of patients who will benefit from PARP inhibitors treatment, if they are successfully used. Our study supports the effectiveness of PARP inhibitors in HR-deficient lung cancer. Our study also implies that BRCA1, BRCA2, ATM status can be biomarkers for using PARP inhibitors in lung cancer. To further develop this hypothesis, other NSCLC cell lines and patient-derived cell lines with such HR deficiency should be tested, and animal studies should be followed. It will also interesting to study whether NSCLC with mutations in other HR genes (such as RAD51 and Fanconi Anemia genes) are sensitive to PARP inhibitors.
4. Materials and methods 4.1. Cell lines The NSCLC cell line A549 were purchased from American Type Culture Collection (ATCC). A549 cells were cultured in DMEM with 10% FBS and 1% penicillin and streptomycin. Cell were cultured at 37 C with 5% CO2.
4.2. siRNA knockdown Cells were transfected with siRNA using Lipofectamine RNAiMAX (Life Technologies) in Opti-MEM (Life Technologies) following manufacturer’s instruction. All siRNAs used in this study were purchased from Qiagen. BRCA1_2 FlexiTube siRNA (SI00096313) was used as siBRCA1#1 and Hs_BRCA1_15 FlexiTube siRNA (SI02664368) as siBRCA1 #2. Hs_BRCA2_1 FlexiTube siRNA (SI00000966) was used as siBRCA2 #1 and Hs_BRCA2_3 FlexiTube siRNA (SI00000980) was used as siBRCA2 #2. Control siRNA was All Stars Negative control from QIAGEN (Cat#1027281). ATM-shRNA construct pLV.ATMi was a gift from Didier Trono (Addgene plasmid#14542), the sense oligo sequence used for cloning was 50 GATCCCCGGA TTTGCGTATT ACTCAGTTCA AGAGACTGAG TAATACGCAA ATCCTTTTTG GAAA-3’ [33].
4.3. Western blot The BRCA1 antibody was purchased from Cell Signaling Technology (#9010). The anti-BRCA2 antibody was purchased from Abcam (ab123491). Anti-ATM Mouse mAb (11G12) was purchased from Cell Signaling Technology (#92356S). Beta-Actin was purchased from Cell Signaling Technology (#3700S). The IRDye680RDlabeled Goat-anti-Rabbit and IRDye800CW-labeled Goat-antiMouse secondary antibodies were purchased from LI-COR Biosciences. To prepare whole-cell extracts, cells were washed twice with ice-cold PBS and lysed in2x Laemmli Sample Buffer (Bio-Rad) with DTT directly. Samples were sonicated and subjected to SDSPAGE and transferred to a nitrocellular membrane. Membranes were blocked LI-COR Block for 1 h and then incubated with primary antibody overnight at 4 C with rotation. The membranes were incubated with by LI-COR secondary antibodies, washed with PBST, and scanned on LI-COR imager.
Please cite this article as: W. Ji et al., Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.050
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4.4. CellTiter-Glo cell viability assay 1000 cells per well were seeded in a 96-well plate the day before drugs were added. Olaparib was purchased from SelleckChem (a.k.a. AZD2281, Cat#S1060) and dissolved in DMSO. Cells were incubated with olaparib for 6 days (4 days for cisplatin). After drug treatment, cell viability was measured using the CellTiter-Glo luminescent assay (Promega) following manufacturer’s instructions. 4.5. Clonogenic survival assay A594 cancer cells were transfected with siRNA targeting BRCA1 or BRCA2 for 48 h, Lipofectamine RNAiMAX (Life Technologies). For ATM shRNA knockdown, cells were infected with lentivirus with shATM and selected by puromycin. After gene knowckdown, cells were trypsinized, counted, and 1,000 cells were seeded onto 6-well plates. Next day, cells were treated with different doses of olaparib. Cells were allowed to grow for 12 days to form colonies. The dishes were then stained with 0.5% of crystal violet in methanol for 1 h and destained with water. The destained plates were dried and the plates were imaged. The images obtained were analyzed by ImageJ by measuring the area coverage of colonies.
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4.6. Flow cell cytometry A594 cancer cells were transfected with siRNA targeting BRCA1 or BRCA2 using Lipofectamine RNAiMAX (Life Technologies). 48 h later, cells were treated with olparib for additional 48 h. After drug treatent, cells were trypsinized, washed with PBS twice, and stained with Annexin V antibody and PI by using the Annexin VFITC Apoptosis Staining/Detection Kit (abcam #14085). The stained cells were immediately analyzed by flow cell cytometry for Annexin V-FITC positive cells.
[14]
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Author contributions
[19]
WJ and LD concepted this study. WJ, XW, DX, and SC performed the experiments and analyzed the data. WJ and LD wrote the paper. All authors were involved in critical discussions and editing of the manuscript.
[20] [21] [22]
Declaration of competing interest [23]
The authors declare no conflict of interest. Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 81773754 to L. Ding). This was also supported by Zhejiang Provincial Natural Science Foundation (LY15H310005).
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.050. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.050. References [1] H. Zeng, et al., Changing cancer survival in China during 2003-15: a pooled
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Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors Wenchao Ji b, Xiang Weng c, Danhua Xu b, Shufan Cai b, Ling Ding a, * a
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang Province, China Department of pharmacy, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China c Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 October 2019 Accepted 6 November 2019 Available online xxx
Lung cancer is the leading cause of cancer death worldwide. PARP inhibitors have become a new line of cancer therapy and a successful demonstration of the synthetic lethality concept. The mechanism and efficacy of PARP inhibitors have been well studied in some cancers, especially homologous recombination (HR)-deficient ovarian cancer and breast cancer, yet such studies are still relatively fewer in lung cancer. Here we found that HR genes are frequently mutated in lung cancer patients, exposing a window for targeted therapies by PARP inhibitors. We depleted BRCA1 and BRCA2 in non-small cell lung cancer (NSCLC) cancer cells and found these cells are hypersensitive to the PARP inhibitor olaparib in cell viability and clonogenic survival assays. Olaparib specifically induces apoptosis in A549 cells with BRCA1 or BRCA2 depletion, as determined by positive Annexin-V staining. In addition, we show that A549 cells with ATM shRNA knockdown are also hypersensitive to Olaparib. In summary, our data support the potential use of PARP inhibitors in NSCLC with HR deficiency. © 2019 Published by Elsevier Inc.
Keywords: Lung cancer Homologous recombination PARP inhibitor BRCA ATM
1. Introduction Lung cancer is the most common type of solid tumors in China, and the 5-year survival for lung cancer in China is only 19% [1]. Lung cancer also remains the most commonly diagnosed cancer type and the leading cause of cancer death world-wide (11.6% of total cases and 18.4% of the total cancer deaths, respectively) [2]. In the United States, nearly 55 per 100,000 people was diagnosed with lung or bronchus cancer each year, and they are responsible for 23.5% of cancer-related death, putting lung cancer the leading cause of cancer-related mortality in the United States (National Cancer Institute, USA). Despite huge efforts that were invested in basic and clinical research, the 5-year survival rate for lung cancer has not changed significantly in the past two decades (15.8% in 2000 vs. 19.4% in 2019, National Cancer Institute, USA). Approximately 80% of all lung cancers are non-small cell lung cancer (NSCLC) (American Cancer Society, Cancer Facts & Figures 2019). The standard of care for advanced NSCLC is platinumbased doublet, generally cisplatin or carboplatin-based [3].
* Corresponding author. College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang Province, 310058, China. E-mail address: [email protected] (L. Ding).
Although patients generally response, the outcome is not very promising. Targeted therapy such as epidermal growth factor receptor (EGFR) tyrosine kinase (EGFR-TK) inhibitors have been shown to improve the progress free survival (PFS) of NSCLC, yet the drug can only be used for EGFR-driven lung cancers, which only account for a fraction of all lung cancer patients. In addition, drug resistance to those therapies have become a challenge [4e6]. New therapies for effective lung cancer treatment are urgently needed. Because lungs are the most exposed organ to exogenous DNA damaging agents such as smoking and air pollution, lung cancers normally contain high mutation loads [7]. Sequencing of the lung cancer cell lines reveals that the average mutation rate of lung cancers was 4.2 per megabase [8]. The highly mutated tumor suppressing gene such as TP53 and proto-oncogenes such as EGFR, ALK and ROS1 have been widely studied [9,10]. Increasing number of studies on mutations in DNA repair genes are undertaking, in an attempt to find DNA repair defects that can be targeted for precision medicine. Homologous repair (HR) genes have been implicated in lung cancer survival after treatment, although mixed clinical outcomes are associated with BRCA1 mutations. Margeli, Taron and their colleagues show that lower expression of BRCA1 predicts a better outcome in lung cancer patients [11,12]. Based on the predictive value of BRCA1 expression, low expression of BRCA1 was
https://doi.org/10.1016/j.bbrc.2019.11.050 0006-291X/© 2019 Published by Elsevier Inc.
Please cite this article as: W. Ji et al., Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.050
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used as a biomarker in a Trabectedin clinical trial in platinum refractory NSCLC patients [13]. However, Choi et al. show later that low expression of BRCA1pS1423 or ERCC1 was significantly associated with worth outcome [14]. In addition, mutations in ataxia telangiectasia mutated (ATM) is associated with increased onset rates of lung cancer [15]. Less is known about how BRCA2 (another critical HR gene) mutations or expression affects the outcomes of lung cancer patients. In summary, more mechanistic study of those DNA repair genes in lung cancer is needed in order to harness it for targeted therapy. The poly-(ADP-ribose) polymerase (PARP) inhibitors are a new line of chemotherapy that showed remarkable efficacy in ovarian cancer, especially in those with BRCA mutations [16]. PARP family of enzymes play critical roles in DNA single-strand break (SSB) repair, and inhibition of which leads to the generation of double-strand breaks (DSB) [17]. The deleterious double-strand breaks are repair mostly by homologous recombination (HR) during S and G2/M phases of the cell cycle. Inhibition of PARP in HR deficient cells causes accumulation of DSBs that eventually leads to apoptosis of cells [18]. Therefore, inhibition of PARP and HR deficiency are synthetically lethal [19,20]. This synthetical lethality relationship led to the discovery of PARP inhibitors, effective chemicals that specifically kill HR deficient cancer cells, and this line of drugs have been huge success. Till date, four PARP inhibitors have been approved by FDA for treatment of ovarian cancer and breast cancer, namely Olaparib, Niraparib, Rucaparib and Talazoparib, based on their outstanding performance in clinical trials [21e24]. Despite the huge success, the application for PARP inhibitors are still very limited. Theoretically, this synthetic lethality should be conserved in other cancer types. Yet, experimental and clinical studies are still lacking to extend PARP inhibitors to other cancer types. Although PARP inhibitors have been successfully used in ovarian and breast cancers, a lot remains to be done before their potential use in lung cancer. Here, we show that homologous recombination genes are frequently mutated in non-small cell lung cancer, including the critical HR genes BRCA1, BRCA2, and ATM. BRCA1, BRCA2 and ATM depleted lung cancer cells are hypersensitive to PARP inhibitors. These HR-deficient (but not HR-proficient) lung cancer cells are apoptotic after exposure to the PARP inhibitor olaparib. Our data shown in this study strengthen the idea of using PARP inhibitor as targeted therapy for HR-deficient lung cancer. 2. Results 2.1. HR genes are frequently mutated in lung cancer We have explored the mutation rates of the key HR DNA repair genes in lung cancer using bioinformatic tools and publicly available databases. By analyzing mutation data in 1962 samples in 8 lung cancer studies in cBioportal (www.cbioportal.org), including 4 non-small cell lung cancer (NSCLC) and 4 small cell lung cancer (SCLC) studies, and we found the HR genes are highly mutated in lung cancer patients, in both NSCLC and SCLC cancer patients (Fig. 1 and S1). 23 HR genes were used as queries in this analysis, including key HR genes such as BRCA1, BRCA2, ATM, RAD51, RAD52, and MRE11 (Table S1). HR genes were altered in about 30% of all lung cancers in total, and almost all of those were gene mutations in SCLC and majority of those were mutations and deletions in NSCLC (Fig. 1A). Further analysis revealed that mutations and deletions in BRCA1, BRCA2 and ATM accounts about half of all HR alterations (14% for NSCLC and 12% for SCLC, Fig. 1B). In NSCLC, BRCA1, BRCA2 and ATM mutations and deletions accounts for 3%, 4.5%, and 7%, respectively. In SCLC, BRCA1, BRCA2 and ATM mutations accounts for 5.5%, 3.5%, and 4.5%, respectively (Fig. S1A). In additional, we found that low ATM expression is associated with worth overall
survival of lung cancer patients (Fig. S1B). These data suggest that HR genes are frequently mutated in lung cancer, and that targeting HR-deficient lung cancer is clinically relevant, especially given the high volume of cancer patients. 2.2. BRCA1/2-deficient NSCLC cells are hypersensitive to PARP inhibitors PARP inhibitors are well-known to kill ovarian and breast cancer cells that are deficient in the HR pathway, a synthetic lethality relationship that has been successfully translated to ovarian cancer treatment. In order to study whether this synthetic lethality relationship also exists in lung cancer cells, we knocked down the HR genes in the NSCLC cell line A549, and asked whether knockdown of these genes leads to sensitivity to PARP inhibitors. First, we depleted BRCA1 using siRNA transfection. Western blots show that BRCA1 was successfully knocked down at protein level with high efficiency (Fig. 1C). The knockdown of BRCA1 in A549 cells was also confirmed by the cisplatin sensitivity of these cells (Fig. S2A), a classic drug sensitivity of HR-deficient cells [25]. Next, we tested whether cells with BRCA1 depletion were sensitive to the PARP inhibitor olaparib (AZD2881), using the CellTiter-Glo (CTG) luminescent cell viability assay. We found that cells transfected with BRCA1 siRNA were much more sensitive to olaparib than the control siRNA transfected cells (Fig. 1D). To confirm these results, we performed clonogenic survival assays. Consistent with the CTG assays, knockdown of BRCA1 resulted in hypersensitivity of A594 cells to olaparib. For example, 1 mM of olaparib almost totally killed BRCA1-depleted cells while the control siRNA treated cells were not impacted (Fig. 1E and F). These data demonstrated that BRCA1 deficient NSCLC cancer cells are hypersensitive to PARP inhibitor olaparib. Next, we performed same sets of experiments in BRCA2depleted A549 cells. Similar results were observed when we compared siBRCA2-and siControl-treated cells (Fig. 2). BRCA2 was successfully knocked down in A549 cells (Fig. 2A) and BRCA2 knockdown cells were sensitive to cisplatin as expected (Fig. S2B). BRCA2 knockdown cells were more sensitive to olaparib than control counterparts in CTG assays (Fig. 2B), and the same results were observed in clonogenic survival assays (Fig. 2C and D). However, we also noticed that BRCA2-depleted cells were less sensitive to olaparib than BRCA1 depleted cells (compare Figs. 2B to Fig. 1D), which may be explained by the more critical role of BRCA1 in HR. Those results suggest that PARP inhibitors could effectively kill BRCA1/BRCA2 deficient cells while spare the wild type control cells. 2.3. ATM knockdown sensitizes A549 cells to PARP inhibitor ATM is a master regulator of DNA double-strand break repair, which influences the repair efficiency of both HR and NHEJ. Since ATM is highly mutated in NSCLC, we next tested if knockdown of ATM is synthetically lethal with PARP inhibition. ATM was successfully knocked down at protein level by shRNA after puromycin selection of lentivirus infected cells, as shown by the absence of ATM band in Western blot after puromycin selection (Fig. 3A). We next tested olaparib sensitivity using CTG cell viability assay and clonogenic survival assay. A549 with shATM knock down was clearly hypersensitive in CTG assay (Fig. 3B). Consistently, ATMdepleted A549 cells were hypersensitive to olaparib when tested in clonogenic survival assay (Fig. 3C). Quantification of the images in clonogenic assays clear show that same conclusion: ATMdepleted cells were hypersensitive to the PARP inhibitor olaparib (Fig. 3D). These results show that ATM deficient NSCLC cells are also sensitive to PARP inhibitors and indicate that PARPi may be used in NSCLC using ATM status as a biomarker.
Please cite this article as: W. Ji et al., Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.050
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Fig. 1. Gene alteration rates of homologous recombination genes in NSCLC and SCLC patients and olaparib sensitivity of BRCA1-deficient NSCLC cells. (A) 23 well-known homologous recombination genes were entered as queries for genes alterations in lung cancer studies (8 studies, 4 for NSCLC and 4 for SCLC). A list of the queried HR genes can be found in Supplemental Table 1. The total sample size is 1962. (B) The alteration frequency of BRCA1, BRCA2 and ATM genes was analyzed together in NSCLC and SCLC. 12e14% of the three genes were altered in total, which were almost exclusively mutation and deletions. (C) A Western blot image shows knockdown of BRCA1. Two independent siRNA were used. (D) PARP inhibitor (olaparib) sensitivity of BRCA1-depleted A549 in CellTiter-Glo cell viability assay (CTG). (E) Clonogenic survival assays show that siBRCA1 A549 cells were hypersensitive to olaparib. (F) Quantification of clonogenic survival assays shown in (E). Mean and S.D. of 6 replicates were shown for CTG assays in (D). For clonogenic survival assays in (E-F), representative images of three independent experiments were shown and quantification are mean and standard deviation (S.D.).
2.4. PARP inhibitor induces apoptosis in BRCA1 and BRCA2 depleted NSCLC PARP inhibitors are known to induce apoptosis in BRCA-deficient breast cancer cells [18]. We next asked whether PARP inhibitors also cause apoptosis in BRCA-deficient NSCLC. A549 cells were first transfected with siRNA for 48 h and then treated with olaparib for 48 h. Cells were then stained with FITC-conjugated anti-Annexin V antibody for flow cell cytometry detection. We found that olaparib specifically induced apoptosis in siBRCA1-treated but not siControltreated A549 cells (Fig. 4A and B). When we treated siBRCA1-treated cells with 1 mM of olaparib, 17.7% of cells were apoptotic with positive Annexin V staining, while only 3% of siControl-treated cells were apoptotic. Similar results were observed in siBRCA2-treated cells. Olaparib specifically induced apoptosis in BRCA2-depleted cells but not in control cells (Fig. 4C and D). Consistent with the olaparib sensitivity data, siBRCA2 cells were less apoptotic compared to siBRCA1 cells upon olaparib treatment (13.2% v.s. 17.7% at 1 mM olaparib). These data suggest that olaparib specifically induces apoptosis in BRCA-deficient cancer cells, explaining why olaparib selectively kills BRCA-deficient cells.
3. Discussion Lung cancer is the leading cause of cancer death in China and world-wide, the 5-year survival rate of which has not changed much in the past 20 years. New and more effective therapeutics is needed imminently. PARP inhibitors have been proved highly effective in ovarian and breast cancers but not in other cancers, including lung cancer. Here, we study the synthetic lethality relationship between HR deficiency and PARP inhibition in non-small cell lung cancer cells. We found that HR genes, including BRCA1, BRCA2 and ATM are frequently mutated or deleted in NSCLC, which can potentially be biomarkers for using PARP inhibitors. Experimentally, we depleted BRCA1, BRCA2 and ATM in NSCLC cell line, and found cells lacking those HR proteins are hypersensitive to olaparib. Mechanistically, PARP inhibitors induces apoptosis in those HR-deficient cells. Our results suggest that NSCLC with HRdeficiency are hypersensitive PARP inhibitors and indicate that PARP inhibitors could be a potential treatment for NSCLC with mutations in HR genes. PARP inhibitors are arguably the most successful example of synthetic lethality. Since the discovery of the synthetic lethality
Please cite this article as: W. Ji et al., Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.050
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Fig. 2. BRCA2 deficient NSCLC were sensitive to PARP inhibitor olaparib. (A) A Western blot image shows knockdown of BRCA2 using two different siRNA. (B) Olaparib sensitivity of BRCA2-depleted A549 cells in CTG assays. (CeD) Representative images and quantification of clonogenic assays of A549 cells treated with siCtrl or siBRCA2 and increasing concentration of olaparib. Mean and SD of 6 replicates were shown for CTG assays in (B). For clonogenic survival assays (CeD), representative images of three independent experiments were shown and quantification are mean and S.D.
Fig. 3. Knockdown of ATM by shRNA sensitizes lung cancer cells to PARP inhibitor olaparib. (A) A Western blot image shows that ATM was knocked down by shRNA in A549 cells. (B) A549 cells with ATM shRNA was hypersensitive to olaparib in CTG assays. (C) Representative images of clonogenic assays in shScramble and shATM infected A549 cells after olaparib treatment. (D) Quantification of the clonogenic survival assays in (C). Data shown in B and D are mean and SD from three independent experiments.
relationship between PARP and BRCA genes in 2005 [19,26], rapid progress has been made for this class of drugs; within less than 10 year, the first PARP inhibitor olaparib (AstraZeneca) was approved in late 2014 for BRCA-mutated ovarian cancer [27]. PARP inhibitors
showed remarkable efficacy in ovarian cancers [21e23,28]. More recently, PARP inhibitors also show good response in breast cancer and pancreatic cancers and subsequently approved for these two cancer types [29,30]. In principal, any cancer cells that are deficient
Please cite this article as: W. Ji et al., Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.050
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Fig. 4. Olaparib specifically induces apoptosis in BRCA1/BRCA2-depleted A549 cells. (A) Flow cell cytometry shows the FITC (Annexin V) positive cells after 1 mM olaparib treatment in siCtrl or siBRCA1 treated A549 cells. (B) Quantification of Annexin V-FITC positive cells after 1 mM olaparib treatment in siCtrl or siBRCA1 treated A549 cells. (C) Flow cell cytometry shows the FITC (Annexin V) positive cells after 1 mM olaparib treatment in siCtrl or siBRCA2 treated A549 cells. (D) Quantification of Annexin V-FITC positive cells after 1 mM olaparib treatment in siCtrl or siBRCA2 treated A549 cells. A549 cells were transfected with siControl or siBRCA1 (A-B) or siBRCA2 (CeD) for 48 h, and cells were treated olaparib for additional 48 h. Cells were stained with anti-AnnexinV antibody and PI for apoptosis analysis. Stained cells were analyzed by flow cell cytometry. Data shown in B and D are mean and S.D. from three measurements.
in HR repair of DNA double-strand breaks should be sensitive to PARP inhibitors, given HR of double-strand breaks and PARPmediated single-strand break repair are highly conserved mechanisms in eukaryotes. On the other hand, using PARP inhibitors in lung cancer is still under debate [31]. Although some progress in clinic [32], mixed outcomes were reported with BRCA mutations [11,12,14]. More basic research in the PARP and HR synthetic lethality relationship is needed to support the use of PARP inhibitors in lung cancer. Our results strengthened the idea that HR-deficient cancer cells, including loss of BRCA1, BRCA2, or ATM, are hypersensitive to PARP inhibitors. Although the mutation/deletion rate of individual gene is not very high, the total mutation rate of the three HR genes is as high as 14%. Given the high volume of lung cancer patients (mostly NSCLC), there are a great number of patients who will benefit from PARP inhibitors treatment, if they are successfully used. Our study supports the effectiveness of PARP inhibitors in HR-deficient lung cancer. Our study also implies that BRCA1, BRCA2, ATM status can be biomarkers for using PARP inhibitors in lung cancer. To further develop this hypothesis, other NSCLC cell lines and patient-derived cell lines with such HR deficiency should be tested, and animal studies should be followed. It will also interesting to study whether NSCLC with mutations in other HR genes (such as RAD51 and Fanconi Anemia genes) are sensitive to PARP inhibitors.
4. Materials and methods 4.1. Cell lines The NSCLC cell line A549 were purchased from American Type Culture Collection (ATCC). A549 cells were cultured in DMEM with 10% FBS and 1% penicillin and streptomycin. Cell were cultured at 37 C with 5% CO2.
4.2. siRNA knockdown Cells were transfected with siRNA using Lipofectamine RNAiMAX (Life Technologies) in Opti-MEM (Life Technologies) following manufacturer’s instruction. All siRNAs used in this study were purchased from Qiagen. BRCA1_2 FlexiTube siRNA (SI00096313) was used as siBRCA1#1 and Hs_BRCA1_15 FlexiTube siRNA (SI02664368) as siBRCA1 #2. Hs_BRCA2_1 FlexiTube siRNA (SI00000966) was used as siBRCA2 #1 and Hs_BRCA2_3 FlexiTube siRNA (SI00000980) was used as siBRCA2 #2. Control siRNA was All Stars Negative control from QIAGEN (Cat#1027281). ATM-shRNA construct pLV.ATMi was a gift from Didier Trono (Addgene plasmid#14542), the sense oligo sequence used for cloning was 50 GATCCCCGGA TTTGCGTATT ACTCAGTTCA AGAGACTGAG TAATACGCAA ATCCTTTTTG GAAA-3’ [33].
4.3. Western blot The BRCA1 antibody was purchased from Cell Signaling Technology (#9010). The anti-BRCA2 antibody was purchased from Abcam (ab123491). Anti-ATM Mouse mAb (11G12) was purchased from Cell Signaling Technology (#92356S). Beta-Actin was purchased from Cell Signaling Technology (#3700S). The IRDye680RDlabeled Goat-anti-Rabbit and IRDye800CW-labeled Goat-antiMouse secondary antibodies were purchased from LI-COR Biosciences. To prepare whole-cell extracts, cells were washed twice with ice-cold PBS and lysed in2x Laemmli Sample Buffer (Bio-Rad) with DTT directly. Samples were sonicated and subjected to SDSPAGE and transferred to a nitrocellular membrane. Membranes were blocked LI-COR Block for 1 h and then incubated with primary antibody overnight at 4 C with rotation. The membranes were incubated with by LI-COR secondary antibodies, washed with PBST, and scanned on LI-COR imager.
Please cite this article as: W. Ji et al., Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.050
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4.4. CellTiter-Glo cell viability assay 1000 cells per well were seeded in a 96-well plate the day before drugs were added. Olaparib was purchased from SelleckChem (a.k.a. AZD2281, Cat#S1060) and dissolved in DMSO. Cells were incubated with olaparib for 6 days (4 days for cisplatin). After drug treatment, cell viability was measured using the CellTiter-Glo luminescent assay (Promega) following manufacturer’s instructions. 4.5. Clonogenic survival assay A594 cancer cells were transfected with siRNA targeting BRCA1 or BRCA2 for 48 h, Lipofectamine RNAiMAX (Life Technologies). For ATM shRNA knockdown, cells were infected with lentivirus with shATM and selected by puromycin. After gene knowckdown, cells were trypsinized, counted, and 1,000 cells were seeded onto 6-well plates. Next day, cells were treated with different doses of olaparib. Cells were allowed to grow for 12 days to form colonies. The dishes were then stained with 0.5% of crystal violet in methanol for 1 h and destained with water. The destained plates were dried and the plates were imaged. The images obtained were analyzed by ImageJ by measuring the area coverage of colonies.
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4.6. Flow cell cytometry A594 cancer cells were transfected with siRNA targeting BRCA1 or BRCA2 using Lipofectamine RNAiMAX (Life Technologies). 48 h later, cells were treated with olparib for additional 48 h. After drug treatent, cells were trypsinized, washed with PBS twice, and stained with Annexin V antibody and PI by using the Annexin VFITC Apoptosis Staining/Detection Kit (abcam #14085). The stained cells were immediately analyzed by flow cell cytometry for Annexin V-FITC positive cells.
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Author contributions
[19]
WJ and LD concepted this study. WJ, XW, DX, and SC performed the experiments and analyzed the data. WJ and LD wrote the paper. All authors were involved in critical discussions and editing of the manuscript.
[20] [21] [22]
Declaration of competing interest [23]
The authors declare no conflict of interest. Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 81773754 to L. Ding). This was also supported by Zhejiang Provincial Natural Science Foundation (LY15H310005).
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.050. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.050. References [1] H. Zeng, et al., Changing cancer survival in China during 2003-15: a pooled
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Please cite this article as: W. Ji et al., Non-small cell lung cancer cells with deficiencies in homologous recombination genes are sensitive to PARP inhibitors, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.050