Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

Antagonizing SET Augments the Effects of Radiation Therapy in Hepatocellular Carcinoma through Reactivation of PP2A-Mediated Akt Downregulation.

Increasing evidence suggests that SET functions as an oncoprotein and promotes cancer survival and therapeutic resistance. However, whether SET affects radiation therapy (RT)-mediated anticancer effects has not yet been explored. We investigated the impact of SET on RT sensitivity in hepatocellular carcinoma (HCC). Using colony and hepatosphere formation assays, we found that RT-induced proliferative inhibition was critically associated with SET expression. We next tested a novel SET antagonist, N4-(3-ethynylphenyl)-6,7-dimethoxy-N2-(4-phenoxyphenyl) quinazoline-2,4-diamine (EMQA), in combination with RT. We showed that additive use of EMQA significantly enhanced the effects of RT against HCC in vitro and in vivo. Notably, compared with mice receiving either RT or EMQA alone, the growth of PLC5 xenografted tumor in mice receiving RT plus EMQA was significantly reduced without compromising treatment tolerability. Furthermore, we proved that antagonizing SET to restore protein phosphatase 2A-mediated phospho-Akt (p-AKT) downregulation was responsible for the synergism between EMQA and RT. Our data demonstrate a new oncogenic property of SET and provide preclinical evidence that combining a SET antagonist and RT may be effective for treatment of HCC. Further investigation is warranted to validate the clinical relevance of this approach.

TYRO3 induces anti-PD-1/PD-L1 therapy resistance by limiting innate immunity and tumoral ferroptosis.

Immune checkpoint blockade therapy has demonstrated promising clinical outcomes for multiple cancer types. However, the emergence of resistance as well as inadequate biomarkers for patient stratification have largely limited the clinical benefits. Here, we showed that tumors with high TYRO3 expression exhibited anti-programmed cell death protein 1/programmed death ligand 1 (anti-PD-1/PD-L1) resistance in a syngeneic mouse model and in patients who received anti-PD-1/PD-L1 therapy. Mechanistically, TYRO3 inhibited tumor cell ferroptosis triggered by anti-PD-1/PD-L1 and facilitated the development of a protumor microenvironment by reducing the M1/M2 macrophage ratio, resulting in resistance to anti-PD-1/PD-L1 therapy. Inhibition of TYRO3 promoted tumor ferroptosis and sensitized resistant tumors to anti-PD-1 therapy. Collectively, our findings suggest that TYRO3 could serve as a predictive biomarker for patient selection and a promising therapeutic target to overcome anti-PD-1/PD-L1 resistance.

Epstein-Barr virus lytic transactivator Zta enhances chemotactic activity through induction of interleukin-8 in nasopharyngeal carcinoma cells.

Epstein-Barr virus (EBV)-associated, undifferentiated type of nasopharyngeal carcinoma (NPC) is characterized by intensive leukocyte infiltration. Interaction between the infiltrating cells and the tumor cells has been considered crucial for NPC development. Recruitment of the infiltrates can be directed by certain chemokines present in the NPC tissues. It is unknown whether and how EBV lytic infection regulates expression of the chemokines. Using an antibody array, we first found that several chemokines secreted from EBV-infected NPC cells are increased upon EBV reactivation into the lytic cycle, and interleukin-8 (IL-8) is the chemokine upregulated most significantly and consistently. Further studies showed that the EBV lytic transactivator Zta is a potent inducer of IL-8 in NPC cells, augmenting secreted and intracellular IL-8 proteins, as well as IL-8 RNA. Zta upregulates Egr-1, a cellular transcription factor that has been involved in upregulation of IL-8, but the Zta-induced IL-8 expression is independent of Egr-1. The ability of Zta to transactivate the IL-8 promoter is important for the induction of IL-8, and we have identified two Zta-responsive elements in the promoter. Zta can bind to these two elements in vitro and can also be recruited to the IL-8 promoter in vivo. DNA-binding-defective Zta mutants can neither activate the IL-8 promoter nor induce IL-8 production. In addition, Zta-expressing NPC cells exert enhanced chemotactic activity that is mainly mediated by IL-8. Since IL-8 may contribute to not only leukocyte infiltration but also multiple oncogenic processes, the present study provides a potential link between EBV lytic infection and pathogenesis of NPC.

Critical role of p53 in histone deacetylase inhibitor-induced Epstein-Barr virus Zta expression.

The tumor suppressor gene p53 plays a central role in the maintenance of normal cell growth and genetic integrity, while its impact on the Epstein-Barr virus (EBV) life cycle remains elusive. We found that p53 is important for histone deacetylase inhibitor-induced EBV lytic gene expression in nasopharyngeal carcinoma cells. Restoration of p53 in p53-null, EBV-infected H1299 cells augments the potential for viral lytic cycle initiation. Evidence from reporter assays demonstrated that p53 contributes to the expression of the immediate-early viral Zta gene. Further analysis indicated that the DNA-binding ability of p53 and phosphorylation of Ser392 may be critical. This study provides the first evidence that p53 is involved in the regulation of EBV lytic cycle initiation.

Palbociclib enhances radiosensitivity of hepatocellular carcinoma and cholangiocarcinoma via inhibiting ataxia telangiectasia-mutated kinase-mediated DNA damage response.

AIM: Palbociclib is an oral cyclin-dependent kinase 4/6 inhibitor, which is efficacious in treating breast cancer. Currently, there are numerous active clinical trials testing palbociclib alone or in combination with other medications for treating various types of malignancies. Here, we evaluated the anti-cancer effect of palbociclib in combination with radiation therapy (RT) for treating human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) and addressed the molecular mechanism behind the combination therapy. METHODS: Immunofluorescence staining of γH2AX or 53BP1 was used to determine the effect of palbociclib on double-strand break (DSB) repair. Clonogenic assays, sphere formation and cell death ELISA were performed to study the sensitising effect of palbociclib on radiation-induced cytotoxicity. Signal alteration in DSB repair pathways was examined by Western blot analysis. Finally, we evaluated the in vivo anti-cancer activity and the associated molecular events of the combination therapy in a preclinical HCC xenograft model. RESULTS: Palbociclib affected the kinetics of DNA repair and enhanced the radiation sensitivity of HCC and CCA cells. Importantly, we found that palbociclib inhibits ataxia telangiectasia-mutated (ATM) kinase, the key upstream kinase responding to RT-induced DSBs. Furthermore, we showed that the inhibitory effect of palbociclib on RT-induced ATM kinase activation is mediated by protein phosphatase 5 (PP5). Both in vitro and in vivo investigations revealed that the inhibition of the PP5-ATM axis by palbociclib after DNA damage is responsible for the synergism between palbociclib and RT. CONCLUSION: Our findings provide a novel combination strategy against liver cancer cells. Clinical trials using palbociclib as an adjuvant in RT are warranted.

STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion.

Enriched PD-L1 expression in cancer stem-like cells (CSCs) contributes to CSC immune evasion. However, the mechanisms underlying PD-L1 enrichment in CSCs remain unclear. Here, we demonstrate that epithelial-mesenchymal transition (EMT) enriches PD-L1 in CSCs by the EMT/ß-catenin/STT3/PD-L1 signaling axis, in which EMT transcriptionally induces N-glycosyltransferase STT3 through ß-catenin, and subsequent STT3-dependent PD-L1 N-glycosylation stabilizes and upregulates PD-L1. The axis is also utilized by the general cancer cell population, but it has much more profound effect on CSCs as EMT induces more STT3 in CSCs than in non-CSCs. We further identify a non-canonical mesenchymal-epithelial transition (MET) activity of etoposide, which suppresses the EMT/ß-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear ß-catenin reduction, leading to PD-L1 downregulation of CSCs and non-CSCs and sensitization of cancer cells to anti-Tim-3 therapy. Together, our results link MET to PD-L1 stabilization through glycosylation regulation and reveal it as a potential strategy to enhance cancer immunotherapy efficacy.

Targeting PKCδ as a Therapeutic Strategy against Heterogeneous Mechanisms of EGFR Inhibitor Resistance in EGFR-Mutant Lung Cancer.

Multiple mechanisms of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been identified in EGFR-mutant non-small cell lung cancer (NSCLC); however, recurrent resistance to EGFR TKIs due to the heterogeneous mechanisms underlying resistance within a single patient remains a major challenge in the clinic. Here, we report a role of nuclear protein kinase Cδ (PKCδ) as a common axis across multiple known TKI-resistance mechanisms. Specifically, we demonstrate that TKI-inactivated EGFR dimerizes with other membrane receptors implicated in TKI resistance to promote PKCδ nuclear translocation. Moreover, the level of nuclear PKCδ is associated with TKI response in patients. The combined inhibition of PKCδ and EGFR induces marked regression of resistant NSCLC tumors with EGFR mutations.

Eradication of Triple-Negative Breast Cancer Cells by Targeting Glycosylated PD-L1.

Protein glycosylation provides proteomic diversity in regulating protein localization, stability, and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression. In the study of immune receptor glycosylation, we showed that EGF induces programmed death ligand 1 (PD-L1) and receptor programmed cell death protein 1 (PD-1) interaction, requiring ß-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation, drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy.

PARP Inhibitor Upregulates PD-L1 Expression and Enhances Cancer-Associated Immunosuppression.

Purpose: To explore whether a cross-talk exists between PARP inhibition and PD-L1/PD-1 immune checkpoint axis, and determine whether blockade of PD-L1/PD-1 potentiates PARP inhibitor (PARPi) in tumor suppression.Experimental Design: Breast cancer cell lines, xenograft tumors, and syngeneic tumors treated with PARPi were assessed for PD-L1 expression by immunoblotting, IHC, and FACS analyses. The phospho-kinase antibody array screen was used to explore the underlying mechanism of PARPi-induced PD-L1 upregulation. The therapeutic efficacy of PARPi alone, PD-L1 blockade alone, or their combination was tested in a syngeneic tumor model. The tumor-infiltrating lymphocytes and tumor cells isolated from syngeneic tumors were analyzed by CyTOF and FACS to evaluate the activity of antitumor immunity in the tumor microenvironment.Results: PARPi upregulated PD-L1 expression in breast cancer cell lines and animal models. Mechanistically, PARPi inactivated GSK3ß, which in turn enhanced PARPi-mediated PD-L1 upregulation. PARPi attenuated anticancer immunity via upregulation of PD-L1, and blockade of PD-L1 resensitized PARPi-treated cancer cells to T-cell killing. The combination of PARPi and anti-PD-L1 therapy compared with each agent alone significantly increased the therapeutic efficacy in vivoConclusions: Our study demonstrates a cross-talk between PARPi and tumor-associated immunosuppression and provides evidence to support the combination of PARPi and PD-L1 or PD-1 immune checkpoint blockade as a potential therapeutic approach to treat breast cancer. Clin Cancer Res; 23(14); 3711-20. ©2017 AACR.

A tumor vessel-targeting fusion protein elicits a chemotherapeutic bystander effect in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease characterized by a prominent desmoplastic stroma that may constrain tumor progression but also limit the access of therapeutic drugs. In this study, we explored a tumor-targeting strategy that enlists an engineered anti-angiogenic protein consisting of endostatin and cytosine deaminase linked to uracil phosphoribosyltransferase (EndoCD). This protein selectively binds to tumor vessels to compromise tumor angiogenesis and converts the non-toxic 5-fluorocytosine (5-FC) to the cytotoxic 5-fluorouracil to produce a chemotherapeutic bystander effect at the pancreatic tumor site. We found that resveratrol increased the protein stability of EndoCD through suppression of chymotrypsin-like proteinase activity and synergistically enhances EndoCD-mediated 5-FC-induced cell killing. In various PDAC mouse models, the EndoCD/5-FC/resveratrol regimen decreased intratumoral vascular density and stroma formation and enhances apoptosis in tumors cells as well as in surrounding endothelial, pancreatic stellate, and immune cells, leading to reduced tumor growth and extended survival. Thus, the EndoCD/5-FC/resveratrol combination may be an effective treatment option for PDAC.

Severe Hepatitis Promotes Hepatocellular Carcinoma Recurrence via NF-κB Pathway-Mediated Epithelial-Mesenchymal Transition after Resection.

PURPOSE: Surgical resection is considered as a curative treatment modality for hepatocellular carcinoma; however, the incidence of postoperative tumor recurrence is high, leading to worse patient survival. Persistent hepatitis (inflammation) is one of the risk factors of tumor recurrence after surgical resection. The aim of this study is to investigate the underlying mechanisms linking liver inflammation to hepatocellular carcinoma progression. EXPERIMENTAL DESIGN: In this study, we used a cytokine array to identify important cytokines whose levels are increased in liver microenvironment with severe hepatitis. We evaluated the morphologic changes, migration and invasion ability, and signal transduction in hepatocellular carcinoma cells with or without inflammatory cytokine in vitro Finally, we analyzed the NF-κB signal pathway in tumor specimens from 232 patients with hepatocellular carcinoma by immunohistochemical staining. RESULTS: The proinflammatory cytokine TNFα was increased in the peritumoral microenvironment and contributed to tumor recurrence and metastasis. Specifically, TNFα promoted hepatocellular carcinoma cancer cell migration, invasion, and epithelial-mesenchymal transition (EMT) by upregulating the transcriptional regulator, Snail. We identified Snail as a direct target gene downstream of the TNFα-mediated canonical NF-κB activation. In addition, tumor recurrence-free survival of hepatocellular carcinoma patients correlated negatively with high p65 and Snail expression and positively with high E-cadherin expression. CONCLUSIONS: Our results establish a signaling axis that explains how inflammatory tumor microenvironment promotes hepatocellular carcinoma recurrence and metastasis. These findings suggest that controlling liver inflammation and/or targeting NF-κB-mediated Snail expression may be a potential therapeutic strategy to prevent hepatocellular carcinoma recurrence after hepatectomy.

Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity.

Extracellular interaction between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) leads to tumour-associated immune escape. Here we show that the immunosuppression activity of PD-L1 is stringently modulated by ubiquitination and N-glycosylation. We show that glycogen synthase kinase 3ß (GSK3ß) interacts with PD-L1 and induces phosphorylation-dependent proteasome degradation of PD-L1 by ß-TrCP. In-depth analysis of PD-L1 N192, N200 and N219 glycosylation suggests that glycosylation antagonizes GSK3ß binding. In this regard, only non-glycosylated PD-L1 forms a complex with GSK3ß and ß-TrCP. We also demonstrate that epidermal growth factor (EGF) stabilizes PD-L1 via GSK3ß inactivation in basal-like breast cancer. Inhibition of EGF signalling by gefitinib destabilizes PD-L1, enhances antitumour T-cell immunity and therapeutic efficacy of PD-1 blockade in syngeneic mouse models. Together, our results link ubiquitination and glycosylation pathways to the stringent regulation of PD-L1, which could lead to potential therapeutic strategies to enhance cancer immune therapy efficacy.

Deubiquitination and Stabilization of PD-L1 by CSN5.

Pro-inflammatory cytokines produced in the tumor microenvironment lead to eradication of anti-tumor immunity and enhanced tumor cell survival. In the current study, we identified tumor necrosis factor alpha (TNF-α) as a major factor triggering cancer cell immunosuppression against T cell surveillance via stabilization of programmed cell death-ligand 1 (PD-L1). We demonstrated that COP9 signalosome 5 (CSN5), induced by NF-κB p65, is required for TNF-α-mediated PD-L1 stabilization in cancer cells. CSN5 inhibits the ubiquitination and degradation of PD-L1. Inhibition of CSN5 by curcumin diminished cancer cell PD-L1 expression and sensitized cancer cells to anti-CTLA4 therapy.

AKT1 Inhibits Epithelial-to-Mesenchymal Transition in Breast Cancer through Phosphorylation-Dependent Twist1 Degradation.

Epithelial-to-mesenchymal transition (EMT) is an essential physiologic process that promotes cancer cell migration, invasion, and metastasis. Several lines of evidence from both cellular and genetic studies suggest that AKT1/PKBα, but not AKT2 or AKT3, serves as a negative regulator of EMT and breast cancer metastasis. However, the underlying mechanism by which AKT1 suppresses EMT remains poorly defined. Here, we demonstrate that phosphorylation of Twist1 by AKT1 is required for ß-TrCP-mediated Twist1 ubiquitination and degradation. The clinically used AKT inhibitor MK-2206, which possesses higher specificity toward AKT1, stabilized Twist1 and enhanced EMT in breast cancer cells. However, we discovered that resveratrol, a naturally occurring compound, induced ß-TrCP-mediated Twist1 degradation to attenuate MK-2206-induced EMT in breast cancer cells. Taken together, our findings demonstrate that resveratrol counteracts the unexpected metastatic potential induced by anti-AKT therapy and therefore suggest that the addition of resveratrol to an anti-AKT therapeutic regimen may provide extra support for limiting EMT.

EGFR Signaling Enhances Aerobic Glycolysis in Triple-Negative Breast Cancer Cells to Promote Tumor Growth and Immune Escape.

Oncogenic signaling reprograms cancer cell metabolism to augment the production of glycolytic metabolites in favor of tumor growth. The ability of cancer cells to evade immunosurveillance and the role of metabolic regulators in T-cell functions suggest that oncogene-induced metabolic reprogramming may be linked to immune escape. EGF signaling, frequently dysregulated in triple-negative breast cancer (TNBC), is also associated with increased glycolysis. Here, we demonstrated in TNBC cells that EGF signaling activates the first step in glycolysis, but impedes the last step, leading to an accumulation of metabolic intermediates in this pathway. Furthermore, we showed that one of these intermediates, fructose 1,6 bisphosphate (F1,6BP), directly binds to and enhances the activity of the EGFR, thereby increasing lactate excretion, which leads to inhibition of local cytotoxic T-cell activity. Notably, combining the glycolysis inhibitor 2-deoxy-d-glucose with the EGFR inhibitor gefitinib effectively suppressed TNBC cell proliferation and tumor growth. Our results illustrate how jointly targeting the EGFR/F1,6BP signaling axis may offer an immediately applicable therapeutic strategy to treat TNBC.

Oncogenic Functions of Gli1 in Pancreatic Adenocarcinoma Are Supported by Its PRMT1-Mediated Methylation.

The oncogenic transcription factor Gli1 is a critical effector in the Hedgehog (Hh) pathway, which is necessary for the development and progression of pancreatic ductal adenocarcinoma (PDAC). Although TGFß and K-Ras are known regulators of Gli1 gene transcription in this setting, it is not understood how Gli1 functional activity is regulated. Here, we report the identification of Gli1 as a substrate for the protein arginine N-methyltransferase PRMT1 in PDAC. We found that PRMT1 methylates Gli1 at R597, promoting its transcriptional activity by enhancing the binding of Gli1 to its target gene promoters. Interruption of Gli1 methylation attenuates oncogenic functions of Gli1 and sensitizes PDAC cells to gemcitabine treatment. In human PDAC specimens, the levels of both total Gli1 and methylated Gli1 were correlated positively with PRMT1 protein levels. Notably, PRMT1 regulated Gli1 independently of the canonical Hh pathway as well as the TGFß/Kras-mediated noncanonical Hh pathway, thereby signifying a novel regulatory mechanism for Gli1 transcriptional activity. Taken together, our results identified a new posttranslational modification of Gli1 that underlies its pivotal oncogenic functions in PDAC. Cancer Res; 76(23); 7049-58. ©2016 AACR.

Proteasome inhibition enhances the killing effect of BikDD gene therapy.

BikDD, a phosphorylation-mimic mutant of pro-apoptotic protein Bik, elicits strong apoptosis in cancer cells when introduced via an expression platform termed VP16-GAL4-WPRE integrated systemic amplifier (VISA) under the control of a cancer-specific promoter both in vitro and in vivo. C-VISA-BikDD expression plasmid encapsulated in liposomes is currently in the process to initiate a phase I clinical trial for pancreatic cancer. In this study, we report a potential combination approach of BikDD with proteasome inhibitors on the basis of our findings that exogenously expressed BikDD protein undergoes proteasome-mediated degradation via both ubiquitin-dependent and -independent pathways. Inhibition of proteasome increases the protein stability of BikDD, enhancing the apoptotic effect of BikDD. Hence, high proteasome activity may be a mechanism by which intrinsic and acquired resistance occurs in BikDD gene therapy, and a combination therapy with current clinically approved proteasome inhibitor may overcome resistance.

The Hippo Coactivator YAP1 Mediates EGFR Overexpression and Confers Chemoresistance in Esophageal Cancer.

PURPOSE: Esophageal cancer is an aggressive malignancy and often resistant to therapy. Overexpression of EGFR has been associated with poor prognosis of patients with esophageal cancer. However, clinical trials using EGFR inhibitors have not provided benefit for patients with esophageal cancer. Failure of EGFR inhibition may be due to crosstalk with other oncogenic pathways. EXPERIMENTAL DESIGN: In this study, expression of YAP1 and EGFR were examined in EAC-resistant tumor tissues versus sensitive tissues by IHC. Western blot analysis, immunofluorescence, real-time PCR, promoter analysis, site-directed mutagenesis, and in vitro and in vivo functional assays were performed to elucidate the YAP1-mediated EGFR expression and transcription and the relationship with chemoresistance in esophageal cancer. RESULTS: We demonstrate that Hippo pathway coactivator YAP1 can induce EGFR expression and transcription in multiple cell systems. Both YAP1 and EGFR are overexpressed in resistant esophageal cancer tissues compared with sensitive esophageal cancer tissues. Furthermore, we found that YAP1 increases EGFR expression at the level of transcription requiring an intact TEAD-binding site in the EGFR promoter. Most importantly, exogenous induction of YAP1 induces resistance to 5-fluorouracil and docetaxcel, whereas knockdown of YAP1 sensitizes esophageal cancer cells to these cytotoxics. Verteporfin, a YAP1 inhibitor, effectively inhibits both YAP1 and EGFR expression and sensitizes cells to cytotoxics. CONCLUSIONS: Our data provide evidence that YAP1 upregulation of EGFR plays an important role in conferring therapy resistance in esophageal cancer cells. Targeting YAP1-EGFR axis may be more efficacious than targeting EGFR alone in esophageal cancer.

Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response.

Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition.