ATM Expression Research Articles

The Effect of Contrast Enhanced Abdominopelvic Magnetic Resonance Imaging on Expression and Methylation Level of ATM and AKT Genes.

ObjectiveTo evaluate the effect of contrast enhanced abdominopelvic magnetic resonance imaging (MRI), using a 3 Tesla scanner, on expression and methylation level of ATM and AKT genes in human peripheral blood lymphocytes.Materials and Methods In this prospective in vivo study, blood samples were obtained from 20 volunteer patients with mean age of 43 ± 8 years (range 32-68 years) before contrast enhanced MRI, 2 hours and 24 hours after contrast enhanced abdominopelvic 3 Tesla MRI. After separation of mononuclear cells from peripheral blood, using Ficoll-Hypaque, we analyzed gene expression changes of ATM and AKT genes 2 hours and 24 hours after MRI using quantitative reverse transcription polymerase chain reaction (qRT-PCR). We also evaluated methylation percentage of the above mentioned genes in before, 2 hours and 24 hours after MRI, using MethySYBR method. ResultsFold change analysis, in comparison with the baseline, respectively showed 1.1 ± 0.7 and 0.8 ± 0.5 mean of gene expressions in 2 and 24 hours after MRI for ATM, while the results were 1.4 ± 0.6 and 1.4 ± 1 for AKT (P>0.05). Methylation of the ATM gene promoter were 8.8 ± 1.5%, 9 ± 0.6% and 9 ± 0.8% in before contrast enhanced MRI, 2 and 24 hours after contrast enhanced MRI, respectively (P>0.05). Methylation of AKT gene promoter in before contrast enhanced MRI, 2 hours and 24 hours after contrast enhanced MRI was 5.4 ± 2.5, 5 ± 3.2, 4.9 ± 2.9 respectively (P>0.05).ConclusionContrast enhanced abdominopelvic MRI using 3 Tesla scanner apparently has no negative effect on the expression and promoter methylation level of ATM and AKT genes involved in the repair pathways of genome.

Induced-damages on preantral follicles by withanolide D, a potent chemotherapy candidate are not attenuated by melatonin

Withanolide D (WD) has been investigated as an antineoplastic drug. This study aimed to evaluate whether melatonin (MT) could attenuate toxic effects on preantral follicles enclosed in the ovarian cortex (experiment 1 - E1) or on isolated secondary follicles (experiment 2 - E2) exposed to WD. For E1, ovarian cortex was incubated for 48 h to: (1) α-MEM+; (2) α-MEM+ plus 6 μM WD; (3) α-MEM+ plus 3 mmol/L MT or (4) α-MEM+ plus WD and MT. For E2, secondary follicles were exposed for until 96 h in. (1) only to basic medium (α-MEM++/α-MEM++); (2) α-MEM++ plus 3 mmol/L MT (MT/MT); (3) α-MEM++ until 48 h, followed by more 48 h in 6 μM WD (α-MEM++/WD) or (4) a pre-exposure to MT for until 48 h, followed by more 48 h of exposure to WD plus MT (MT/MT + WD). The main results obtained showed that exposure to drugs caused damage to follicular morphology (WD or WD + MT) and diameter (WD) in the ovarian cortex or in isolated follicles. In pre-antral follicles in situ, ATM expression increased in the presence of WD, MT or association. As for the secondary follicles, ATM and γH2AX were immunostained in the granulosa and theca cells and oocytes in all treatments. TAp63α was immunostained in follicles included in the ovarian cortex and in isolated follicles. We conclude that melatonin did not provide protection and could have enhanced the toxic effect of WD to follicles surrounded or not by the ovarian cortex.

A Novel HCC Prognosis Predictor EEF1E1 Is Related to Immune Infiltration and May Be Involved in EEF1E1/ATM/p53 Signaling.

BackgroundEEF1E1 has been reported to play a role in ovarian cancer, breast cancer, non-small cell lung cancer and other cancers, but its role and mechanism in hepatocellular carcinoma (HCC) are still unknown.MethodsEEF1E1 expression in human HCC was analyzed via the GTEx and TCGA database. Logistic regression analysis was used to analyze the clinicopathological correlation of EEF1E1 expression. The correlation between EEF1E1 expression and patients’ prognosis was analyzed in HCC, shown by forest plots, nomogram and Kaplan–Meier curves. Hazard ratio (HR) with 95% confidence intervals and log-rank p-value were calculated via multivariate/univariate survival analyses. Moreover, the correlation between EEF1E1 and tumor immune infiltration was analyzed using the gsva package with the ssgsea algorithm. Pearson correlation was used to investigate the correlation between EEF1E1 expression and p53 pathway genes expression. Two third-party databases were used to validate the content of EEF1E1 protein and mRNA expression patterns and prognosis analysis. The immunohistochemistry and multiplex immunohistochemistry was used to verify the bio-informatics results.ResultsEEF1E1 mRNA and protein expression in tumor was statistically higher than normal (EEF1E1 mRNA: p < 0.001; EEF1E1 protein: p < 0.01). Results from paired t-test (cancer and adjacent tissues) exhibited consistent trend (t = 7.572, p < 0.001). Immunohistochemistry showed that EEF1E1 is highly expressed in cancer. The expression of EEF1E1 was positively correlated with body weight, BMI, tumor status, vascular invasion, AFP, logistic grade, T stage and pathological stage. The univariate Cox model revealed that high EEF1E1 expression was strongly associated with worse OS (HR=2.581; 95% CI: 1.782-3.739; p < 0.001), as was T stage, pathologic stage, Histologic grade. High EEF1E1 expression was the only independent prognostic factor associated with OS (HR=2.57; 95% CI: 1.715-3.851; p < 0.001) in the multivariate analysis. EEF1E1 was significantly correlated with various immune cells, including cytotoxic cells, DC, macrophages, neutrophils, NK cd56bright, TFH, Tgd, Th17, Th2, Treg. Multiplex immunohistochemistry showed that the EEF1E1 protein level is positively correlated to the CD3, CD4, PD1 and is negatively correlated to the CD8. The expression level of EEF1E1 in HCC was significantly correlated with the key genes involved in the p53 pathway. The expression of EEF1E1, ATM, p53 and CASPASE3 in HCC tissues was significantly higher than that in adjacent tissues.ConclusionEEF1E1 is highly expressed in cancer tissues in HCC. EEF1E1’s high expression is significantly correlated with worse prognosis and immune cell infiltration of HCC. EEF1E1 may be participating in EEF1E1/ATM/p53 signaling pathway in HCC.

Abstract 2044: Genomic scar score: a robust model to predict recombination repair deficient based on genomic instability

Abstract Background: Clinical benefit of PARP inhibitors or Platinum-based chemotherapy has been observed and well proved in BRCA mutation patients. However, there are other mechanisms to induce recombination repair deficient (HRD), such as BRCA methylation, ATM expression loss; HRR gene mutation in addition to BRCA mutation. Whether these complicated HRD events or phenotype could be measured by a unified model to accurately identify HRD positive patients is important. Methods: In this study, we use support vector machine method to build genomic scar (GS) model to predict HRD events, which takes different types of chromosomal copy number into consideration to evaluate genomic instability. The accuracy of GS model was tested by different NGS platform from WGS to two captured SNP Panel. GS status association with Cisplatin-based chemotherapy was also preliminarily validated. Results: GS model displayed more than 95% accuracy to detect BRCA-ness events and is also able to enrich different types of HRR dysfunction, such as HRR-related mutation or BRCA1 promoter methylation. Moreover, GS model can identify patient benefits from Cisplatin-based chemotherapy, and GS score (GSS) high group had longer progression free survival (PFS) than GSS low group (11 vs 8.5 months, HR=0.5, P=0.05). Meanwhile, GSS showed the high concordance among different NGS platform including WGS; two captured SNP Panels, which implied the high robustness of GS model. Conclusion: GS was a robust model to predict HRD and had the clinical potential to enrich patients who will response to PARP inhibitors and/or Platinum-based chemotherapy. Citation Format: Hao Wen, Shuang Yang, Xuejun Chen, Ying Weng, Changbin Zhu, Li Ruan, Hua Dong, Xiaohua Wu. Genomic scar score: a robust model to predict recombination repair deficient based on genomic instability [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 2044.

IDH1/2 -Mutated Acute Myeloid Leukemia Has Impaired DNA Damage Response and Is Sensitive to Monotherapy with the PARP Inhibitior Olaparib

Introduction: Somatic mutations in the genes encoding for isocitrate dehydrogenase 1 and 2 (IDH1/2MUT) occur in ~20% of patients with myeloid neoplasms, including acute myeloid leukemia (AML). IDH1/2MUT enzymes produce D -2-hydroxyglutarate (D -2HG), the accumulation of which is implicated in oncogenesis by inhibition of various enzymes, including TET2. IDH1/2MUT sensitize solid tumor cells to cytotoxic therapy via downregulation of the DNA repair gene ATM and this is linked to increased sensitivity to the PARP inhibitor olaparib. The sensitivity of IDH1/2MUT AML to olaparib has not yet been investigated.Methods: Peripheral blood and bone marrow samples were obtained from IDH1/2MUT and wild-type (IDH1/2WT)AML patients and brought in culture. ATM protein expression was investigated using western blots. AML cells were treated with 0-6 Gy irradiation, 0-50 nM daunorubicin or 0-25 µM olaparib and therapy response was determined in colony formation assays, where cells were seeded at a density of 1*104-1*106 cells/ml, depending on the concentration of the cytotoxic agent, in 3 ml Methocult methylcellulose medium. DNA double-strand breaks (DSB) were determined using immunofluorescence stainings against γ-H2AX. The aforementioned experiments were performed in the presence or absence of 1 µM IDH1/2MUT inhibitor AGI-5198 or AGI-6780, 10 mM D -2HG and/or an siRNA directed against ATM.Results:IDH1/2MUT AML cells were sensitized to treatment with daunorubicin or IR compared to IDH1/2WT and TET2-/- AML cells (Fig 1A-B). Pretreatment with an IDH1/2MUT inhibitor (Fig 1C) protected IDH1/2MUT AML cells (Fig 1D-G) but not IDH1/2WT AML (Fig 1H-I) against subsequent treatment with daunorubicin or IR. As mechanistic evidence for enhanced therapy responses, IDH1/2MUT AML cells had decreased ATM protein expression relative to IDH1/2WT and TET2-/- primary AML cells and incubation with an IDH1/2MUT inhibitor restored ATM expression in IDH1/2MUT AML cells (Fig 2A). In TCGA data, ATM mRNA expression was also severely decreased in IDH1MUT AML and moderately decreased in IDH2MUT and TET2-/- AML (Fig 2B). Consistent with an impaired DNA damage response owing to ATM repression, IDH1/2MUT primary human AML cells had more ɣ-H2AX+ foci (which recognize DNA DSBs) than IDH1/2WT AML cells after IR or daunorubicin treatment and also in steady-state conditions without treatment with a cytotoxic agent. Moreover, pretreatment with an IDH1/2MUT inhibitor prior to treatment with IR or daunorubicin diminished the difference in the number of ɣ-H2AX+ foci between IDH1/2MUT and IDH1/2WT AML cells (Fig 2C), corroborating with restored ATM expression after incubation with an IDH1/2MUT inhibitor in IDH1/2MUT cells. The impairments in DNA damage repair in IDH1/2MUT AML cells were associated with a sensitization of IDH1/2MUT AML cells to olaparib compared to IDH1/2WT AML cells (Fig 2D). Furthermore, IDH1/2MUT inhibitors protected IDH1/2MUT AML cells, but not IDH1/2WT AML cells, against olaparib, providing additional mechanistic evidence (Fig 2D). ATM knockdown using siRNA in IDH1MUT, IDH2MUT and IDH1/2WT AML cells did not affect the sensitivity of IDH1/2MUT AML cells to daunorubicin or IR (Fig 3A-D) but sensitized IDH1/2WT AML cells to these treatments (Fig 3E-F), which in agreement with ATM already being repressed in IDH1/2MUT AML cells (Fig 2A). After pretreatment with D -2HG, untransfected IDH1/2WT AML cells were sensitized to daunorubicin or IR, but not when ATM was knocked down (Fig 3E-F), showing a mechanistic link between ATM and D -2HG. While IDH1/2MUT inhibitors protected untransfected IDH1/2MUT AML cells against daunorubicin or IR (Fig 1D-G), IDH1/2MUT inhibitors could not protect IDH1/2MUT AML cells when ATM was knocked down (Fig 3G-H). These findings, combined with related earlier findings in IDH1MUT solid tumors, are summarized in a model shown in Fig 3I.Discussion:IDH1/2MUT AML cells have an impaired DNA damage response via repression of ATM, which is associated with sensitization of IDH1/2MUT AML cells to the PARP inhibitor olaparib. Furthermore, the sensitization of IDH1/2MUT AML cells to olaparib is reversed when olaparib is combined with an IDH1/2MUT inhibitor.Olaparib, either as monotherapy but preferably combined with a DNA-damaging agent, is thus effective in exploiting ineffective DNA damage repair in IDH1/2MUT AML cells and may represent a possible effective future therapy for IDH1/2MUT AML. [Display omitted] DisclosuresMakishima:Yasuda Medical Foundation: Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees.

Decreased ATM Protein Expression Is Substantiated with PTEN Loss in Defining Aggressive Phenotype of Prostate Cancer Associated with Lethal Disease

BackgroundAtaxia Telangiectasia Mutated (ATM) serine/threonine protein kinase is a known tumor suppressor, involved in DNA damage repair. It has prognostic and predictive therapeutic implications and is associated with aggressive prostate cancer (PCa). ObjectiveTo investigate the prognostic value of ATM protein expression in PCa patients and assessed the combined value of ATM, ERG, and PTEN status. Design, setting, and participantsThis study consisted of 303 patients with incidental, locally advanced, and castrate-resistant PCa by transurethral resection of the prostate (TURP). Outcome measurements and statistical analysisTURP samples from 303 PCa patients were assessed by immunohistochemistry (IHC for ATM, ERG, and PTEN. Individual and combined marker status were correlated with International Society of Urological Pathology Gleason grade group, overall survival (OS), and PCa-specific mortality (PCSM). Results and limitationsDecreased ATM expression (negative/weak intensity) occurred in 164/303 (54.1%) patients, and was associated with shorter OS and higher PCSM (p = 0.015 and p = 0.001, respectively). Negative/weak ATM expression was significantly associated with PCSM with a hazard ratio of 2.09 (95% confidence interval 1.34–3.27, p = 0.001). Assessment of Combined ATM/PTEN expression showed improved prognostic power to predict OS and PCSM, independent of Gleason grade groups. ConclusionsDecreased ATM protein expression is associated with poor outcomes in advanced PCa patients. Patients with combined low ATM/PTEN negative expression are at the highest risk for reduced OS and PCSM. Assessing the combined status of ATM/PTEN by IHC in PCa patients may aid in risk stratification relative to OS and PCSM. Moreover, since ATM plays an integral role in DNA damage response pathways, future studies will enhance our understanding of how outcomes of patients with altered ATM and PTEN expression can be improved further with poly-ADP ribose polymerase inhibitors (PARPi), combinations of PARPi and androgen receptor–targeted therapies, as well as platinum-based chemotherapies. Patient summaryLower ATM intensity is associated with increased cancer-specific mortality in prostate cancer patients. Patients with lower ATM and PTEN negative expression showed decreased overall survival and increased cancer mortality compared with controls.

Circulating tumor DNA (ctDNA) mutations may predict treatment response in extensive-stage small cell lung cancer (ES-SCLC) treated with talazoparib and temozolomide (TMZ).

8564 Background: Poly (ADP-ribose) polymerase (PARP) inhibition in combination with TMZ is a promising treatment strategy for ES-SCLC. In SCLC models, talazoparib, a potent PARP inhibitor, exhibits cytotoxic effects by inhibiting PARP proteins 1/2 and trapping PARP on DNA while TMZ potentiates antitumor response by contributing to genomic instability (Wainberg 2016). Prior ctDNA studies in SCLC have suggested that treatment precipitates the appearance of DNA repair alterations (Nong 2018), but it is unknown whether homologous recombination deficiency (HRD) predicts for treatment response with this combination. Methods: Patients (pts) with relapsed or refractory ES-SCLC were treated with oral talazoparib 0.75 mg daily on 28-day cycles and oral TMZ 37.5 mg/m2 on days 1-5 in a phase 2 clinical trial (UCLA/TRIO-US L-07, NCT03672773). ctDNA was collected and assessed based on allele frequency and plasma copy number at baseline and every 8 weeks during treatment with the Guardant360 assay (Redwood City, CA). HRD was defined as a deletion or missense mutation known or likely to result in aberrant expression of ATM or BRCA1/2 (other HRD genes not detected by assay). Response to treatment was defined by RECIST 1.1 criteria. Fisher’s exact tests were used to compare proportions of patients with P-values &lt; 0.05 considered statistically significant ( www.r-project.org , Vienna, AU). Results: For 15 evaluable pts in the first Simon stage of this trial, 45 ctDNA samples were collected. The most common baseline genetic alterations were mutations in TP53 (14 pts), BRCA2 (5 pts), ATM (4 pts), and RB1 (3 pts). Of those with &gt; 1 ctDNA timepoint collected, 10/11 (90.9%) pts had ≥1 new mutation (range 1-19) detected after receiving treatment (range 8-35 weeks), most commonly in ATM (5 pts). Overall, 5 pts had confirmed partial responses (PR), 7 had stable disease, and 3 had progressive disease. Disease control (DC) was associated with the presence of new mutations (P = 0.022) and was more common in those with HRD, with DC in 9/10 (90.0%) HRD pts vs 3/5 (60.0%) pts without HRD. All those with PRs experienced a ctDNA nadir at 8 weeks of treatment with nearly all (4/5, 80.0%) exhibiting HRD, 2 at baseline and 2 at 8 weeks of treatment. Conclusions: Mutations in DNA repair genes occur on treatment with talazoparib and TMZ and may associate with disease control. With a response rate of 33% in the first Simon stage of this trial, the TRIO-US L-07 trial exploring the combination of talazoparib and TMZ will be assessed in 13 additional patients, after which additional ctDNA analyses will be performed on the cohort as a whole. Clinical trial information: NCT03672773.

Increased DNA repair capacity augments resistance of glioblastoma cells to photodynamic therapy

Photodynamic therapy (PDT) is a clinically approved cancer therapy of low invasiveness. The therapeutic procedure involves administering a photosensitizing drug (PS), which is then activated with monochromatic light of a specific wavelength. The photochemical reaction produces highly toxic oxygen species. The development of resistance to PDT in some cancer cells is its main limitation. Several mechanisms are known to be involved in the development of cellular defense against cytotoxic effects of PDT, including activation of antioxidant enzymes, drug efflux pumps, degradation of PS, and overexpression of protein chaperons. Another putative factor that plays an important role in the development of resistance of cancer cells to PDT seems to be DNA repair; however, it has not been well studied so far. To explore the role of DNA repair and other potential novel mechanisms associated with the resistance to PDT in the glioblastoma cells, cells stably resistant to PDT were isolated from PDT sensitive cells following repetitive PDT cycles. Duly characterization of isolated PDT-resistant glioblastoma revealed that the resistance to PDT might be a consequence of several mechanisms, including higher repair efficiency of oxidative DNA damage and repair of DNA breaks. Higher activity of APE1 endonuclease and increased expression and activation of DNA damage kinase ATM was demonstrated in the U-87 MGR cell line, suggesting and proving that they are good targets for sensitization of resistant cells to PDT.

Downregulation of ATM and BRCA1 Predicts Poor Outcome in Head and Neck Cancer: Implications for ATM-Targeted Therapy.

ATM and BRCA1 are DNA repair genes that play a central role in homologous recombination repair. Alterations of ATM and BRCA1 gene expression are found in cancers, some of which are correlated with treatment response and patient outcome. However, the role of ATM and BRCA1 gene expression in head and neck cancer (HNC) is not well characterized. Here, we examined the prognostic role of ATM and BRCA1 expression in two HNC cohorts with and without betel quid (BQ) exposure. The results showed that the expression of ATM and BRCA1 was downregulated in BQ-associated HNC, as the BQ ingredient arecoline could suppress the expression of both genes. Low expression of either ATM or BRCA1 was correlated with poor overall survival (OS) and was an independent prognostic factor in multivariate analysis (ATM HR: 1.895, p = 0.041; BRCA1 HR: 2.163, p = 0.040). The combination of ATM and BRCA1 expression states further improved on the prediction of OS (HR: 4.195, p = 0.001, both low vs. both high expression). Transcriptomic analysis showed that inhibition of ATM kinase by KU55933 induced apoptosis signaling and potentiated cisplatin-induced cytotoxicity. These data unveil poor prognosis in the HNC patient subgroup with low expression of ATM and BRCA1 and support the notion of ATM-targeted therapy.

FLYWCH1, a Multi-Functional Zinc Finger Protein Contributes to the DNA Repair Pathway.

Over recent years, several Cys2-His2 (C2H2) domain-containing proteins have emerged as critical players in repairing DNA-double strand breaks. Human FLYWCH1 is a newly characterised nuclear transcription factor with (C2H2)-type zinc-finger DNA-binding domains. Yet, our knowledge about FLYWCH1 is still in its infancy. This study explores the expression, role and regulation of FLYWCH1 in the context of DNA damage and repair. We provide evidence suggesting a potential contribution of FLYWCH1 in facilitating the recruitment of DNA-damage response proteins (DDRPs). We found that FLYWCH1 colocalises with γH2AX in normal fibroblasts and colorectal cancer (CRC) cell lines. Importantly, our results showed that enforced expression of FLYWCH1 induces the expression of γH2AX, ATM and P53 proteins. Using an ATM-knockout (ATMKO) model, we indicated that FLYWCH1 mediates the phosphorylation of H2AX (Ser139) independently to ATM expression. On the other hand, the induction of DNA damage using UV-light induces the endogenous expression of FLYWCH1. Conversely, cisplatin treatment reduces the endogenous level of FLYWCH1 in CRC cell lines. Together, our findings uncover a novel FLYWCH1/H2AX phosphorylation axis in steady-state conditions and during the induction of the DNA-damage response (DDR). Although the role of FLYWCH1 within the DDR machinery remains largely uncharacterised and poorly understood, we here report for the first-time findings that implicate FLYWCH1 as a potential participant in the DNA damage response signaling pathways.

Abstract PS10-06: Comparative analysis of anti-proliferative effects and gene profiling of lapatinib, neratinib, and tucatinib

Abstract Introduction: Human epidermal growth factor 2 (HER2/ERBB2) is frequently amplified or mutated across various cancer types. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. All three TKIs bind and inhibit the kinase domain of HER2 but differ both in the mechanism of binding and in specificity for other HER family members. Direct comparisons to differentiate the pre-clinical efficacy of the three TKIs have been limited to small-scale studies and novel biomarkers of response to further define appropriate patient populations are required. Methods: In this study, the anti-proliferative effects of the three TKIs were compared using a 115-cancer cell line panel, including 12 breast cancer cell lines and 22 cell lines harbouring point mutations or amplifications of EGFR, HER2, or HER3. Hierarchical clustering analysis was carried out to compare the IC50 “fingerprint” of the three TKIs to 168 other anti-cancer agents. Novel markers of TKI sensitivity and resistance were identified through cross-analysis of each drug response profile with mutation, copy number variation, and gene expression data. Results: All three TKIs were effective against HER2-positive breast cancer models; neratinib showed the most potent activity, followed by tucatinib and lapatinib respectively (Table 1). Neratinib displayed the greatest anti-proliferative activity in HER2-mutant and EGFR-mutant cell lines. Clustering analysis revealed that the anti-proliferative profile of tucatinib was most similar to trastuzumab, while neratinib and lapatinib were most like other HER family inhibitors. Mutation and gene expression analysis identified potential markers of response for each TKI. High expression of four genes (HER2, VTCN1, CDK12, and RAC1) correlated with response to all three TKIs. DNA damage repair genes were significantly associated with resistance to the HER2-targeted TKIs. BRCA2 mutation was correlated with neratinib and tucatinib response, and high gene expression of ATM, BRCA2, and BRCA1 were all associated with neratinib resistance. Conclusions: Neratinib was the most effective HER2-targeted TKI against HER2-amplified, -mutant, and EGFR-mutant cell lines. This analysis revealed possible mechanisms that may be exploited using combinatorial strategies involving CDK inhibitors, immunotherapies, and targeting DNA repair pathways. Table: IC50 values for neratinib, lapatinib, and tucatinib in the HER2+ breast cancer cell linesIC50 values (nM)Cell linesNeratinibLapatinibTucatinibAU-56520294125BT-4745926229HCC195413814262122MDA-MB-453306228445928SKBR3715222 Citation Format: Neil T Conlon, Jeffrey J Kooijman, Suzanne JC van Gerwen, Winfried R Mulder, Guido JR Zaman, Irmina Diala, Lisa D Eli, Alshad S Lalani, John Crown, Denis Collins. Comparative analysis of anti-proliferative effects and gene profiling of lapatinib, neratinib, and tucatinib [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-06.

Comparative analysis of drug response and gene profiling of HER2-targeted tyrosine kinase inhibitors

BackgroundHuman epidermal growth factor 2 (HER2/ERBB2) is frequently amplified/mutated in cancer. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. Direct comparisons of the preclinical efficacy of the TKIs have been limited to small-scale studies. Novel biomarkers are required to define beneficial patient populations.MethodsIn this study, the anti-proliferative effects of the three TKIs were directly compared using a 115 cancer cell line panel. Novel TKI response/resistance markers were identified through cross-analysis of drug response profiles with mutation, gene copy number and expression data.ResultsAll three TKIs were effective against HER2-amplified breast cancer models; neratinib showing the most potent activity, followed by tucatinib then lapatinib. Neratinib displayed the greatest activity in HER2-mutant and EGFR-mutant cells. High expression of HER2, VTCN1, CDK12, and RAC1 correlated with response to all three TKIs. DNA damage repair genes were associated with TKI resistance. BRCA2 mutations were correlated with neratinib and tucatinib response, and high expression of ATM, BRCA2, and BRCA1 were associated with neratinib resistance.ConclusionsNeratinib was the most effective HER2-targeted TKI against HER2-amplified, -mutant, and EGFR-mutant cell lines. This analysis revealed novel resistance mechanisms that may be exploited using combinatorial strategies.

N6-methyladenosine regulates ATM expression and downstream signaling.

N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNAs, which plays an important role in regulating multiple biological processes. ATM is a major protein kinase that regulates the DNA damage response. Here, we identified that ATM is a m6A-modificated gene. METTL3 (a m6A “writer”) and FTO (a m6A “eraser”) oppositely regulated ATM expression and its downstream signaling. Mechanically, m6A “readers” YTHDFs and eIF3A suppressed ATM expression in the post-transcriptional levels. We also revealed the oncogenic potential of METTL3 and YTHDF1 related to ATM modulation. This is the first report that ATM, a master in the DNA damage response, is modified by m6A epigenetic modification, and METTL3 disrupts the ATM stability via m6A modification, thereby affecting the DNA-damage response.

Study on mechanism of matrine in treatment of COVID-19 combined with liver injury by network pharmacology and molecular docking technology

The aim of the present study was to investigate the pharmacological mechanism of matrine in treatment of COVID-19 combined with liver injury. Potential targets related to matrine, COVID-19 and liver injury were identified from several databases. We constructed PPI network and screened the core targets according to the degree value. Then, GO and KEGG enrichment were carried out. Molecular docking technology was used to verify the affinity between matrine and the crystal structure of core target protein. Finally, real-time RT-PCR was used to detect the effects of matrine on hub gene expression in liver tissue of liver injury mice and lung tissue of lung injury mice to further confirm the results of network pharmacological analysis. The results show that six core targets including AKT1, TP53, TNF, IL6, BCL2L1 and ATM were identified. The potential therapeutic mechanism of matrine on COVID-19 combined with liver injury is closely related to regulate antiviral process, improve immune system and regulate the level of inflammatory factors. Molecular docking showed that matrine could spontaneously bind to the receptor protein and had strong binding force. Real-time RT-PCR demonstrated that matrine could significantly reduce the expression of AKT1, TP53, TNF, IL6 and ATM in mice with liver injury or lung injury (P < 0.05), and increase the expression of BCL2L1 to a certain extent (P > 0.05). Our results indicate that matrine can achieve simultaneous intervention of COVID-19 combined with liver injury by multi-dimensional pharmacological mechanism.

Impaired endoplasmic reticulum-mitochondrial signaling in ataxia-telangiectasia.

SummaryThere is evidence that ATM mutated in ataxia-telangiectasia (A-T) plays a key role in protecting against mitochondrial dysfunction, the mechanism for which remains unresolved. We demonstrate here that ATM-deficient cells are exquisitely sensitive to nutrient deprivation, which can be explained by defective cross talk between the endoplasmic reticulum (ER) and the mitochondrion. Tethering between these two organelles in response to stress was reduced in cells lacking ATM, and consistent with this, Ca2+ release and transfer between ER and mitochondria was reduced dramatically when compared with control cells. The impact of this on mitochondrial function was evident from an increase in oxygen consumption rates and a defect in mitophagy in ATM-deficient cells. Our findings reveal that ER-mitochondrial connectivity through IP3R1-GRP75-VDAC1, to maintain Ca2+ homeostasis, as well as an abnormality in mitochondrial fusion defective in response to nutrient stress, can account for at least part of the mitochondrial dysfunction observed in A-T cells.

Chromosomal abnormalities and dysregulated DNA repair gene expression in farmers exposed to pesticides

Exposure to pesticides is considered a major factor underlying increased risk of hematological disorders in agricultural workers due to its carcinogenic potential. However, genotoxic impact of pesticides in DNA integrity of bone marrow stem cells (BMSC) of farmers exposed is not yet well known. We evaluated presence of chromosomal abnormalities (CA) and mRNA expression of DNA repair targets (ATM, BRCA1, BRCA2, RAD51, XRCC5, XRCC6, LIG4, CSA, CSB, XPA, XPC, XPG) in 90 bone marrow samples of farmers divided into three groups: commercial farming (CF), family farming (FF) and organic farming (OF). Our results showed that farmers in CF (72.7 %) and FF (27.3 %) groups had significantly higher values of CA when compared to OF group (0.0 %; p = 0.003). CF showed lower XPG (p = 0.008), CSA (p < 0.001), ATM (p = 0.036) and LIG4 (p = 0.004) mRNA expression than OF. FF presented lower XPG (p = 0.012) and LIG4 (p = 0.004) expression than OF. CF + FF individual with ≥12 years of exposure to pesticides showed decreased mRNA expression of XPC (p = 0.001), XPG (p = 0.010), CSB (p = 0.05), ATM (p = 0.030) and LIG4 (p = 0.044) than those who have been exposed for <12 years. CF + FF with CA showed a lower expression of BRCA2 when compared to CF + FF group without CA (p = 0.007). These results highlight that genotoxic exposure to pesticides negatively affects expression profile of important DNA repair genes in BMSC, favoring irreparable chromosomal lesions.

Chloroquine reverses chemoresistance via upregulation of p21WAF1/CIP1 and autophagy inhibition in ovarian cancer

Overcoming drug-resistance is a big challenge to improve the survival of patients with epithelial ovarian cancer (EOC). In this study, we investigated the effect of chloroquine (CQ) and its combination with cisplatin (CDDP) in drug-resistant EOC cells. We used the three EOC cell lines CDDP-resistant A2780-CP20, RMG-1 cells, and CDDP-sensitive A2780 cells. The CQ-CDDP combination significantly decreased cell proliferation and increased apoptosis in all cell lines. The combination induced expression of γH2AX, a DNA damage marker protein, and induced G2/M cell cycle arrest. Although the CQ-CDDP combination decreased protein expression of ATM and ATR, phosphorylation of ATM was increased and expression of p21WAF1/CIP1 was also increased in CQ-CDDP-treated cells. Knockdown of p21WAF1/CIP1 by shRNA reduced the expression of γH2AX and phosphorylated ATM and inhibited caspase-3 activity but induced ATM protein expression. Knockdown of p21WAF1/CIP1 partly inhibited CQ-CDDP-induced G2/M arrest, demonstrating that knockdown of p21WAF1/CIP1 overcame the cytotoxic effect of the CQ-CDDP combination. Ectopic expression of p21WAF1/CIP1 in CDDP-treated ATG5-shRNA/A2780-CP20 cells increased expression of γH2AX and caspase-3 activity, demonstrating increased DNA damage and cell death. The inhibition of autophagy by ATG5-shRNA demonstrated similar results upon CDDP treatment, except p21WAF1/CIP1 expression. In an in vivo efficacy study, the CQ-CDDP combination significantly decreased tumor weight and increased expression of γH2AX and p21WAF1/CIP1 in A2780-CP20 orthotopic xenografts and a drug-resistant patient-derived xenograft model of EOC compared with controls. These results demonstrated that CQ increases cytotoxicity in combination with CDDP by inducing lethal DNA damage by induction of p21WAF1/CIP1 expression and autophagy inhibition in CDDP-resistant EOC.

Coping with DNA Double-Strand Breaks via ATM Signaling Pathway in Bovine Oocytes.

As a common injury almost all cells face, DNA damage in oocytes—especially double-strand breaks (DSBs), which occur naturally during the first meiosis phase (meiosis I) due to synaptic complex separation—affects the fertilization ability of oocytes, instead of causing cancer (as in somatic cells). The mechanism of oocytes to effectively repair DSB damage has not yet been clearly studied, especially considering medically induced DSBs superimposed on naturally occurring DSBs in meiosis I. It was found that maturation rates decreased or increased, respectively corresponding with overexpression or interference of p21 in bovine oocytes. At the same time, the maturation rate of bovine oocytes decreased with a gradual increase in Zeocin dose, and the p21 expression in those immature oocytes changed significantly with the gradual increase in Zeocin dose (same as increased DSB intensity). Same as p21, the variation trend of ATM expression was consistent with the gradual increase in Zeocin dose. Furthermore, the oocytes demonstrated tolerance to DSBs during meiosis I, while the maturation rates decreased when the damage exceeded a certain threshold; according to which, it may be that ATM regulates the p53–p21 pathway to affect the completion of meiosis. In addition, nonhomologous recombination and cumulus cells are potentially involved in the process by which oocytes respond to DSB damage.

LncRNA HOTAIR Promotes DNA Repair and Radioresistance of Breast Cancer via EZH2.

Mounting evidence shows that long noncoding RNAs play important roles in human diseases and radioresistance could be an important factor for tumor recurrence. We observed that HOX antisense intergenic RNA (HOTAIR) expression increased in invasive ductal carcinoma (IDC) tissue. The irradiation effect of HOTAIR was detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, cell cycle and apoptosis analysis, and xenografts in nude mice. Western blot, RNA pulldown assay, and RNA immunoprecipitation were used for mechanistic studies. HOTAIR, upregulated in IDC of breast cancer tissue, could promote breast cancer cell proliferation by regulating cell cycle and apoptosis. Overexpression of HOTAIR promoted DNA damage repair factors KU70, KU80, DNA-PKs, and ATM expression, and these could be impeded by small molecular inhibitors of enhancer of zeste homolog 2 (EZH2). Meanwhile, we found that HOTAIR could facilitate recruitment of EZH2 to the Avian Myelocytomatosis Viral Oncogene Homolog (MYC) promoter. HOTAIR is an important modulator not only to the prognostic of breast cancer, but also a good marker for radiotherapy.

Effects of miR-203a targeting and its target gene on lymph node metastasis of breast cancer

Objective: To study the expression and correlation of mir-203a and its target gene ATM in breast cancer tissues, so as to provide theoretical basis for the pathogenesis of breast cancer, especially lymph node metastasis. Methods: Thirty paired breast cancer and paracancer normal tissues were collected, and RT-qPCR was used to detect the relative expression levels of mir-203a and ATM in the samples of the two groups. Correlation analysis was conducted for mir-203a and ATM, and correlation analysis was conducted for the pathological characteristics, so as to compare whether there were statistical differences between mir-203a and ATM in lymph node metastasis and non-metastasis. Results: Compared with normal paracancer tissues, the expression level of mir-203a in breast cancer tissues was significantly increased (P＜0.01), and the expression level of ATM was significantly decreased (P＜0.01), showing a significant negative correlation between the two tissues (r=-0.847,P＜0.01).The expression level of mir-203a and ATM was significantly correlated with lymph node metastasis and different clinical stages (P＜0.05). The expression level of mir-203a in the group with lymph node metastasis were significantly lower than that in the group without lymph node metastasis (P＜0.05), and the expression of ATM in the group with lymph node metastasis was significantly higher than that in the group without lymph node metastasis (P＜0.01). Conclusion: The overexpression of mir-203a in the early stage of breast cancer may inhibit the expression of its target gene ATM, which may be a protective mechanism to regulate the proliferation,metastasis and invasiveness of tumor cells. In the middle and late stage, mir-203a is down-regulated and the ATM gene is up-regulated, which may be involved in lymph node metastasis.