Expression Of DNA Damage Response Proteins Research Articles

Abstract 1185: PRMT5 inhibition epigenetically regulates DNA damage response pathways in cancer cells and sensitizes to chemotherapy and PARP inhibition

Abstract Genetic defects of DNA damage response (DDR) pathways in cancer cells induce synthetic lethality with chemotherapy and PARP inhibitors. However, many patients with advanced cancers develop resistance to these therapies mainly due to circumvention and/or restoration of the inactivated DDR genes. In the present study, we demonstrate that pharmacological inhibition of PRMT5, the major type II protein arginine methyltransferase, epigenetically downregulates multiple genes involved in the DDR/DNA replication pathways, resulting in the sensitization of cancer cells to chemotherapy and PARP inhibition. Treatment with C220 or PRT543, potent and selective PRMT5 inhibitors, downregulates expression of DDR genes and/or proteins including BRCA1/2, ATM/ATR, CHK1/2, RAD51, POLD1/3, and PNKP in multiple types of cancer cells in vitro. Mechanistically, PRT543 promotes global alternative splicing (ΔPSI) changes, including intron retention and exon skipping. In particular, PRT543 significantly increases retained intron of POLD1 and PNKP genes, which are downstream of the spliceosome subunit SRSF1, a major protein substrate of PRMT5. Additionally, PRT543 increases intron retention of ATM and ATR genes. For BRCA1/2, CHK1/2 and RAD51, no changes in alternative splicing were observed, suggesting the involvement of other mechanisms such as the CLNS1A/H4R3me2s chromatin regulation pathway. The regulation of the DDR pathway by PRT543 is associated with increased DNA damage as determined by COMET and gamma H2AX analyses. Combining PRT543 with PARP inhibitors or DNA-alkylating agents demonstrates a potent synergistic interaction in both HR proficient and HR deficient cancer cell lines in vitro. Similar combination effects are observed in primary cultures of patient-derived breast cancer and high-grade serous ovarian cancer. Moreover, combination of PRT543 with PARP inhibition effectively inhibits the growth of HCC1569 CDX breast cancer in vivo. Further ex vivo and in vivo combination studies with PRT543 and PARP inhibitors or chemotherapy in genetically defined CDX and PDX models are ongoing. These studies not only reveal a novel mechanism of PRMT5 inhibition, but also suggest beneficial combination effects with other therapies, particularly in patients with tumors that are resistant to therapies that are dependent on DNA damage as their mechanism of action. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Koichi Ito, Jack Carter, Venkat Thodima, Youyou Zhang, Monisha Sivakumar, Mu Xu, Neha Bhagwat, Joseph Rager, Jacob Spruance, Lin Zhang, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRMT5 inhibition epigenetically regulates DNA damage response pathways in cancer cells and sensitizes to chemotherapy and PARP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1185.

Combination of Niraparib, Cisplatin and Twist Knockdown in Cisplatin-Resistant Ovarian Cancer Cells Potentially Enhances Synthetic Lethality through ER-Stress Mediated Mitochondrial Apoptosis Pathway.

Poly (ADP-ribose) polymerase 1 inhibitors (PARPi) are used to treat recurrent ovarian cancer (OC) patients due to greater survival benefits and minimal side effects, especially in those patients with complete or partial response to platinum-based chemotherapy. However, acquired resistance of platinum-based chemotherapy leads to the limited efficacy of PARPi monotherapy in most patients. Twist is recognized as a possible oncogene and contributes to acquired cisplatin resistance in OC cells. In this study, we show how Twist knockdown cisplatin-resistant (CisR) OC cells blocked DNA damage response (DDR) to sensitize these cells to a concurrent treatment of cisplatin as a platinum-based chemotherapy agent and niraparib as a PARPi on in vitro two-dimensional (2D) and three-dimensional (3D) cell culture. To investigate the lethality of PARPi and cisplatin on Twist knockdown CisR OC cells, two CisR cell lines (OV90 and SKOV3) were established using step-wise dose escalation method. In addition, in vitro 3D spheroidal cell model was generated using modified hanging drop and hydrogel scaffolds techniques on poly-2-hydroxylethly methacrylate (poly-HEMA) coated plates. Twist expression was strongly correlated with the expression of DDR proteins, PARP1 and XRCC1 and overexpression of both proteins was associated with cisplatin resistance in OC cells. Moreover, combination of cisplatin (Cis) and niraparib (Nira) produced lethality on Twist-knockdown CisR OC cells, according to combination index (CI). We found that Cis alone, Nira alone, or a combination of Cis+Nira therapy increased cell death by suppressing DDR proteins in 2D monolayer cell culture. Notably, the combination of Nira and Cis was considerably effective against 3D-cultures of Twist knockdown CisR OC cells in which Endoplasmic reticulum (ER) stress is upregulated, leading to initiation of mitochondrial-mediated cell death. In addition, immunohistochemically, Cis alone, Nira alone or Cis+Nira showed lower ki-67 (cell proliferative marker) expression and higher cleaved caspase-3 (apoptotic marker) immuno-reactivity. Hence, lethality of PARPi with the combination of Cis on Twist knockdown CisR OC cells may provide an effective way to expand the therapeutic potential to overcome platinum-based chemotherapy resistance and PARPi cross resistance in OC.

LINCS gene expression signature analysis revealed bosutinib as a radiosensitizer of breast cancer cells by targeting eIF4G1.

Radioresistance is the predominant cause for radiotherapy failure and disease progression, resulting in increased breast cancer‑associated mortality. Using gene expression signature analysis of the Library of Integrated Network‑Based Cellular Signatures (LINCS) and Gene Expression Omnibus (GEO), the aim of the present study was to systematically identify potential candidate radiosensitizers from known drugs. The similarity of integrated gene expression signatures between irradiated eukaryotic translation initiation factor 4γ1(eIF4G1)‑silenced breast cancer cells and known drugs was measured using enrichment scores(ES). Drugs with positive ES were selected as potential radiosensitizers. The radiosensitizing effects of the candidate drugs were analyzed in breast cancer cell lines (MCF‑7, MX‑1 and MDA‑MB‑231) using CCK‑8 and colony formation assays following exposure to ionizing radiation. Cell apoptosis was measured using flow cytometry. The expression levels of eIF4G1 and DNA damage response (DDR) proteins were analyzed by western blotting. Bosutinib was identified as a promising radiosensitizer, as its administration markedly reduced the dosage required both for the drug and for ionizing radiation, which may be associated with fewer treatment‑associated adverse reactions. Moreover, combined treatment of ionizing radiation and bosutinib significantly increased cell killing in all three cell lines, compared with ionizing radiation or bosutinib alone. Among the three cell lines, MX‑1 cells were identified as the most sensitive to both ionizing radiation and bosutinib. Bosutinib markedly downregulated the expression of eIF4G1 in a dose‑dependent manner and also reduced the expression of DDR proteins (including ATM, XRCC4, ATRIP, and GADD45A). Moreover, eIF4G1 was identified as a key target of bosutinib that may regulate DNA damage induced by ionizing radiation. Thus, bosutinib may serve as a potential candidate radiosensitizer for breast cancer therapy.

Abstract 4125: Investigating determinants of sensitivity to ATR inhibition, as predictive biomarkers to single agent ATR inhibitor, VE-821, in ovarian cancer

Abstract Introduction: ATR is a key kinase in the DNA damage response (DDR) that signals replication stress to the S and G2/M checkpoints. Loss of G1 checkpoint control is almost ubiquitous in cancer cells, which, coupled with frequent activation of oncogenes makes them more reliant on their S and G2/M checkpoints. Therefore, ATR is an attractive anti-cancer target. Several determinants of sensitivity to ATR inhibitors (ATRi) have been identified in the literature, such as ATM, TP53, cyclin E and ARID1A. In high grade serous ovarian cancer, almost 100% of all tumours have lost TP53 function, around 20% have activation of cyclin E, and ARID1A mutations are found in nearly 50% of all clear cell ovarian cancers. Therefore, ATR inhibition may be a successful treatment strategy in ovarian cancer. Experimental Procedures: ATRi, VE-821, the preclinical candidate of VX-970 now known as M6620, was used throughout these experiments. The inhibition of ATR activity and the cytotoxicity of VE-821 was assessed in 13 ovarian cancer cell lines and related to the protein and mRNA expression of DDR proteins, oncogenes and other known determinants of sensitivity to ATRi. Growth inhibition by VE-821 was determined in patient derived primary cultures of malignant ovarian cancer ascites cells. Results: Ovarian cancer cell lines and patient samples display a range of sensitivities to VE-821. UWB1.289 cells lacking BRCA1 were the most sensitive to VE-821 (LC50 0.64 µM ± 0.06) and 1.5 times more sensitive than its BRCA1 corrected derivative, confirming previous reports of BRCA1 determining sensitivity to ATRi. NIH-OVCAR3 cells were the least sensitive to VE-821 (LC50 17.87 ± 1.86 µM) and CAOV3 were the second most sensitive to VE-821 (LC50 0.78 ± 0.55 µM), which did not correlate with ATR inhibition or expression of DDR proteins, oncogenes or other known determinants of sensitivity. There was a range of sensitivities to VE-821 in the 7 patient samples analysed to date, with the GI50 ranging from 0.78 µM to 29.08 µM, with no correlation between the growth rates of the sample to its sensitivity. Conclusions: These data show that determinants of sensitivity to ATRi, identified in isogenic cell line pairs, may not translate to the more complex molecular pathology of cancer cells where several determinants of sensitivity and resistance may be present simultaneously. More complex pathway analysis or functional readouts e.g. of replication stress, may be needed as predictive biomarkers for ATRi sensitivity. Citation Format: Alice Mary Bradbury, Frank T. Zenke, Yvette Drew, Nicola J. Curtin. Investigating determinants of sensitivity to ATR inhibition, as predictive biomarkers to single agent ATR inhibitor, VE-821, in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4125.

ATR Inhibition Potentiates PARP Inhibitor Cytotoxicity in High Risk Neuroblastoma Cell Lines by Multiple Mechanisms.

Background: High risk neuroblastoma (HR-NB) is one the most difficult childhood cancers to cure. These tumours frequently present with DNA damage response (DDR) defects including loss or mutation of key DDR genes, oncogene-induced replication stress (RS) and cell cycle checkpoint dysfunction. Aim: To identify biomarkers of sensitivity to inhibition of Ataxia telangiectasia and Rad3 related (ATR), a DNA damage sensor, and poly (ADP-ribose) polymerase (PARP), which is required for single strand break repair. We also hypothesise that combining ATR and PARP inhibition is synergistic. Methods: Single agent sensitivity to VE-821 (ATR inhibitor) and olaparib (PARP inhibitor), and the combination, was determined using cell proliferation and clonogenic assays, in HR-NB cell lines. Basal expression of DDR proteins, including ataxia telangiectasia mutated (ATM) and ATR, was assessed using Western blotting. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS, respectively. RS and homologous recombination repair (HRR) activity was also measured by γH2AX and Rad51 foci formation using immunofluorescence. Results: MYCN amplification and/or low ATM protein expression were associated with sensitivity to VE-821 (p < 0.05). VE-821 was synergistic with olaparib (CI value 0.04–0.89) independent of MYCN or ATM status. Olaparib increased H2AXS129 phosphorylation which was further increased by VE-821. Olaparib-induced Rad51 foci formation was reduced by VE-821 suggesting inhibition of HRR. Conclusion: RS associated with MYCN amplification, ATR loss or PARP inhibition increases sensitivity to the ATR inhibitor VE-821. These findings suggest a potential therapeutic strategy for the treatment of HR-NB.

Abstract 4146: Selinexor synergizes with DNA damaging agents through down-regulation of key DNA damage response genes

Abstract Background: Selinexor is a first-in-class, orally bioavailable SINE (Selective Inhibitor of Nuclear Export) compound currently in phase 1/2 clinical trials of patients with solid and hematological malignancies. SINE compounds bind exportin 1 (XPO1/CRM1) and inhibit nuclear export, resulting in nuclear retention of major tumor suppressor proteins (TSPs). We previously found through Reverse Phase Protein Array analysis that selinexor treatment alters the level of key DNA damage response (DDR) proteins. We also observed enhanced anti-tumor activity when combining selinexor with standard DNA damaging agents (DDA) in pre-clinical models and clinical studies. We hypothesize that a reduction in DDR protein expression by selinexor drives the synergy observed in combination with DDAs. Methods: Solid and hematological cancer cell lysates treated with selinexor +/- DDAs (cisplatin, doxorubicin, docetaxel and gemcitabine) were analyzed by immunoblotting and qPCR. Comet assay and γH2A.X staining were analyzed by immunofluorescence to evaluate DNA damage. Mice bearing MDA-MB-231 (triple negative breast cancer) tumors were treated with sub-toxic doses of selinexor (2.5 mg/kg), cisplatin (4 mg/kg) or docetaxel (4 mg/kg) alone or in combination. Tumor growth and mouse weight were followed for 25 days. Results: Selinexor reduced the expression of DDR mRNA and proteins involved in single-stranded (SSB) and double-stranded break (DSB) repair as early as 2 hours post treatment. We observed a dose-dependent correlation between the degree of DDR protein reduction and selinexor cytotoxicity in vitro. Selinexor prevented DDR activation following exposure to DDAs and led to synergistic cell death. Treatment of cells with selinexor plus DDA showed enhanced activity in comet assays and an increase in γH2A.X staining when compared to treatment with the single agents. Exposure to selinexor 6 - 24 hours after DDA treatment inhibited the DDR mechanism and was more synergistic than the opposite order (Sel → DDA) or simultaneous treatment. In a MDA-MB-231 xenograft, treatment with selinexor, docetaxel (SSB) or cisplatin (DSB) alone resulted in 66.7%, 51.5% or 26.6% TGI, respectively when compared to vehicle control whereas sequential treatment of docetaxel or cisplatin followed by selinexor resulted in 93.9% TGI or 9.6% tumor regression, respectively. No significant weight loss was seen in any of the groups by the end of the study. IHC staining and immunoblot analysis of tumor tissue and sections showed a reduction of DDR proteins. Conclusion: Selinexor synergizes with DDAs by increasing both SSB and DSB through inhibition of relevant DDR proteins. This data supports clinical development of selinexor in combination with standard chemotherapeutic agents in patients with solid and hematological malignancies. The results also suggest that sequential treatment of patients with DDAs followed by selinexor might be more beneficial. Citation Format: Trinayan Kashyap, Christian Argueta, Boris Klebanov, Oscar Gonzalez, Erkan Baloglu, Yosef Landesman, Margaret Lee, Humphrey Gardner, Sharon Shacham, William Senapedis. Selinexor synergizes with DNA damaging agents through down-regulation of key DNA damage response genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4146. doi:10.1158/1538-7445.AM2017-4146

SEI1 induces genomic instability by inhibiting DNA damage response in ovarian cancer

Previous studies have shown that the oncogene SEI1 is highly expressed in ovarian carcinomas, and promoting genomic instability. However, the molecular mechanism of SEI1 in promoting genomic instability remains unclear. We observed SEI1 overexpression in 30 of 46 cases of ovarian cancer compared to non-tumor tissues and the overexpression of SEI1 was positively associated with the tumor FIGO stage. Our functional studies revealed that overexpression of SEI1 could induce genomic instability and increased DNA strand breaks. In contrast, SEI1 co-localized with γH2AX and phosphorylated ATM and DNAPKcs in the nucleus. Furthermore, we found that overexpression of SEI1 induced translocation of the SEI1 protein from the cytoplasm to the nucleus; ATM and DNAPKcs were associated with the cytoplasm-to-nucleus translocation of SEI1. To further prove the correlation between the DNA damage response (DDR) and SEI1, we knocked down SEI1 expression in SEI1-transfected ovarian cancer cell lines. The expression of DDR proteins was significantly downregulated, and the number of micronuclei was significantly decreased. Together, these results define a new mechanism of SEI1 in the regulation of genomic stability and in the malignant progression of ovarian cancer.

A high resolution genomic portrait of bladder cancer: correlation between genomic aberrations and the DNA damage response

One major challenge in cancer research is to understand the complex interplay between the DNA damage response (DDR), genomic integrity, and tumor development. To address these issues, we analyzed 43 bladder tumor genomes from 22 patients using single nucleotide polymorphism (SNP) arrays, and tissue expression of multiple DDR proteins, including Timeless and its interaction partner Tipin. The SNP profiles confirmed and extended known copy number alterations (CNAs) at high resolution, showed clustering of CNAs at nine common fragile sites, and revealed that most metachronous tumors were clonally related. The occurrence of many novel uniparental disomy regions (UPDs) was of potential functional importance in some tumors because UPDs spanned mutated FGFR3 and PIK3CA alleles, and also homozygous deletion of the CDKN2A tumor suppressor locus. The DDR signaling as evaluated by phospho-epitope-specific antibodies against Ser139-phosphorylated H2A histone family member X (γH2AX), ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR) was commonly activated in tumors with both moderate and high extent of accumulated genomic aberrations, the latter tumors showing a more frequent loss of ATM expression. Strikingly, the tumor genomes exhibiting the most complex alterations were associated with a high Ki67-proliferation index, abundant Timeless but not Tipin expression, aberrant p53 expression, and homozygous CDKN2A deletions. Of clinical relevance, evaluation of a tissue microarray (TMA; n=319) showed that abundant Timeless expression was associated with risk of progression to muscle-invasive disease (P<0.0005; hazard ratio, 2.4; 95% confidence interval, 1.6-3.8) and higher T stage (P<0.05). Univariate analysis confirmed this association (P=0.006) in an independent cohort (n=241) but statistical significance was not reached in a multivariate model. Overall, our results are consistent with DDR activation preceding the accumulation of genomic aberrations. Tumors with extensive genomic rearrangements were associated with inactivation of CDKN2A, excessive proliferation, and robust Timeless expression, the latter also correlating with the risk of disease progression. Moreover, we provide evidence to suggest that UPDs likely contribute to bladder tumorigenesis.

Prognostic implications of the DNA damage response pathway in glioblastoma

Genomic instability and resistance to genotoxic therapies for glioblastoma (GBM) suggest aberrant DNA damage response (DDR), since DDR maintains the genomic integrity against genotoxic insults including anti-tumor therapies. To elucidate the biological and clinical meaning of DDR in GBM, we retrospectively investigated the immunohistochemical expression of DDR proteins (ATM, Chk1, Chk2, TopBP1, Rad17, p53, Nbs1, MDC1, γH2AX and RPA1) in 69 GBM surgical samples and their relation with GBM patient survival. Remarkably, higher expression of ATM revealed significantly longer overall survival (OS) and progression-free survival (PFS) (p<0.05). Upon multivariate analysis, expression level of ATM was an independent factor for longer OS (p=0.020) and longer PFS (p=0.019). Since ATM induces cell cycle arrest or apoptosis through cell cycle regulators in response to genotoxic insults, these results indicate that aberrant DDR signaling through ATM in GBM may be associated with resistance to genotoxic anti-tumor therapeutics. Conclusively, we emphasize that the identification of DDR machinery, which can be involved in unstable genomic status or genotoxic therapies in GBM, is very important to predict patient outcome.