Ataxia telangiectasia and Rad3-related (ATR) inhibition by VE-822 potently reversed 5-flourouracil resistance in colorectal cancer cells through targeting DNA damage response.

BACKGROUND: VE-822 is a novel inhibitor of ATR, a key kinase involved in the DNA damage response pathway. The role of ATR inhibition in reversing drug resistance in various cancer types has been investigated. Therefore, this study investigated the effects of ATR inhibition by VE-822 on reversing 5-fluorouracil (5-FU) resistance in colorectal cancer cell line (Caco-2). METHODS: Caco-2 and 5-FU resistance Caco-2 (Caco-2/5-FU) cells were treated with 5-FU and VE-822, alone and in combination. Cell proliferation and viability were assessed by MTT assay and Trypan Blue staining. P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) activities were measured by Rhodamine123 accumulation and uptake assay. The mRNA levels of P-gp, MRP-1, ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 1 (CHK1) were measured by qRT-PCR. Western blot was used to measure the protein levels of P-gp, MRP-1, γ-H2AX, ATR and CHK1 in cells. 8-Oxo-2'-deoxyguanosine (8-oxo-dG) levels were determined via ELISA. Apoptosis was evaluated by ELISA death assay, DAPI staining and lactate dehydrogenase (LDH) assay. RESULTS: The Caco-2/5-FU cells showed lower levels of 5-FU mediated proliferation inhibition in comparison to Caco-2 cells. VE-822 decreased the IC50 value of 5-FU on resistant cells. In addition, the expression levels and activity of P-gp and MRP-1 were significantly decreased in resistant cells treated with VE-822 (P < 0.05). The combination of 5-FU and VE-822 increased apoptosis in Caco-2/5-FU cells by downregulating CHK1 and ATR and upregulating γ-H2AX and 8-oxo-dG. CONCLUSION: The simultaneous treatment of resistant colorectal cancer cells with 5-FU and ATR inhibitor, VE-822, was demonstrated to be effective in reversing drug resistance and potentiating 5-FU mediated anticancer effects via targeting DNA damage.

RASSF1A promotes ATM signaling and RASSF1A methylation is a synthetic lethal marker for ATR inhibitors.

Aim: The mechanism of RASSF1A in DNA damage repair remains to be further clarified for applying to synthetic lethal strategy. Materials & methods: Eight esophageal cancer cell lines, 181 cases of esophageal dysplasia and 1066 cases of primary esophageal squamous cell carcinoma (ESCC) were employed. Methylation-specific PCR, the CRISPR/Cas9 technique, immunoprecipitation assay and a xenograft mouse model were used. Results: RASSF1A was methylated in 2.21% of esophageal dysplasia and 11.73% of ESCC. RASSF1A was also involved in DNA damage repair through activating Hippo signaling. Loss of RASSF1A expression sensitized esophageal cancer cell lines to ataxia telangiectasia mutated and rad3-related (ATR) inhibitor (VE-822) both in vitro and in vivo. Conclusion: RASSF1A methylation is a synthetic lethal marker for ATR inhibitors.

The ATR inhibitor berzosertib acts as a radio- and chemosensitizer in head and neck squamous cell carcinoma cell lines.

Alterations in the DNA damage response play a crucial role in radio- and chemoresistance of neoplastic cells. Activation of the Ataxia telangiectasia and Rad3-related (ATR) pathway is an important DNA damage response mechanism in head and neck squamous cell carcinoma (HNSCC). Berzosertib, a selective ATR inhibitor, shows promising radio- and chemosensitizing effects in preclinical studies and is well tolerated in clinical studies. The aim of this study was to elucidate the effect of berzosertib treatment in combination with radiation and cisplatin in HNSCC. The HNSCC cell lines Cal-27 and FaDu were treated with berzosertib alone and in combination with radiation or cisplatin. Cell viability and clonogenic survival were evaluated. The effect of combination treatment was evaluated with the SynergyFinder or combination index. Apoptosis was assessed via measurement of caspase 3/7 activation and migration was evaluated using a wound healing assay. Berzosertib treatment decreased cell viability in a dose-dependent manner and increased apoptosis. The IC50 of berzosertib treatment after 72 h was 0.25-0.29 µM. Combination with irradiation treatment led to a synergistic increase in radiosensitivity and a synergistic or additive decrease in colony formation. The combination of berzosertib and cisplatin decreased cell viability in a synergistic manner. Additionally, berzosertib inhibited migration at high doses. Berzosertib displays a cytotoxic effect in HNSCC at clinically relevant doses. Further evaluation of combination treatment with irradiation and cisplatin is strongly recommended in HNSCC patients as it may hold the potential to overcome treatment resistance, reduce treatment doses and thus mitigate adverse events.

VE-822 upregulates the deubiquitinase OTUD1 to stabilize FHL1 to inhibit the progression of lung adenocarcinoma.

BACKGROUND: The deubiquitinase ovarian tumor domain-containing 1 (OTUD1) has been considered as a tumor suppressor in many tumors, but there is minimal research on the role of OTUD1 in lung adenocarcinoma (LUAD) pathogenesis. METHODS: Bioinformatics analyses and western blot were applied for investigating OTUD1 expression in lung cancer and the drug that upregulated OTUD1. Kaplan-Meier analysis with log-rank test was used for survival analyses. IP-MS and co-IP were performed for identifying potential protein interactions with OTUD1. In vitro and in vivo assays were used for exploring the function of OTUD1 during the progression of LUAD. RESULTS: OTUD1 was dramatically downregulated in tumors and cell lines of human lung cancer. OTUD1 inhibited proliferation and migration of lung cancer cells in vitro. Moreover, OTUD1 inhibited growth of xenografts in nude mice and formation of primary lung tumors in urethane-induced lung cancer model. Mechanistically, we showed that OTUD1 deubiquitinated and stabilized FHL1. Furthermore, we listed and identified VE-822 as a candidate agonist for OTUD1. VE-822 inhibited proliferation of lung adenocarcinoma both in vitro and in vivo. CONCLUSION: These results indicated that the deubiquitinase OTUD1, which was upregulated by VE-822, inhibited the progression of LUAD in vitro and in vivo by deubiquitinating and stabilizing FHL1.

VE-822 Enhanced Cisplatin Chemotherapy Effects on Head and Neck Squamous Cell Carcinoma Drug-resistant Cells.

PURPOSE: The study aimed to assess the effect of p-ATR inhibitor VE-822 in the combination chemotherapy with cisplatin of head and neck squamous cell carcinoma and to explore the possible mechanism. METHODS: The DNA damage levels were determined by comet assay and western blot experiments in cisplatin-resistant and sensitive cell lines. The IC50 value changes after combination treatment with VE-822 in cisplatin sensitive and resistant cell lines were detected by the CCK-8 test. The effects of VE-822 combined with cisplatin on proliferation ability, colony formation ability, migration ability, cell apoptosis and cell cycle changes were observed in vitro. In vivo, the combination treatment effect was verified in the subcutaneous xenograft models of nude mice. Besides, the mechanism of VE-822 assisting cisplatin in chemotherapy was explored by comet assay, western blotting and immunohistochemical experiments. RESULTS: The increased expression of the p-ATR protein was related to the DNA damage repair pathway in head and neck squamous cell carcinoma cisplatin-resistant cells. VE-822 inhibited cell proliferation, colony formation and migration abilities and improved the cisplatin chemotherapeutic effects in subcutaneous xenograft models of nude mice by inhibiting the p-ATR expression and blocking DNA damage repair pathway. CONCLUSIONS: The p-ATR expression increased in head and neck squamous cell carcinoma cisplatinresistant cells. VE-822 significantly enhanced the therapeutic effect in cisplatin resistant head and neck squamous cell carcinoma by inhibiting p-ATR expression in vivo and in vitro.

ATR inhibition sensitizes liposarcoma to doxorubicin by increasing DNA damage.

Liposarcomas account for approximately 20% of all adult sarcomas and have limited therapeutic options outside of surgery. Inhibition of ataxia-telangiectasia and Rad3 related protein kinase (ATR) has emerged as a promising chemotherapeutic strategy in various cancers. However, its activation, expression, and function in liposarcoma remain unkown. In this study, we investigated the expression, function, and potential of ATR as a therapeutic target in liposarcoma. Activation and expression of ATR in liposarcoma was analyzed by immunohistochemistry, which was further explored for correlation with patient clinical characteristics. ATR-specific siRNA and the ATR inhibitor VE-822 were applied to determine the effect of ATR inhibition on liposarcoma cell proliferation and anti-apoptotic activity. Migration activity and clonogenicity were examined using wound healing and clonogenic assays. ATR (p-ATR) was overexpressed in 88.1% of the liposarcoma specimens and correlated with shorter overall survival in patients. Knockdown of ATR via specific siRNA or inhibition with VE-822 suppressed liposarcoma cell growth, proliferation, migration, colony-forming ability, and spheroid growth. Importantly, ATR inhibition significantly and synergistically enhanced liposarcoma cell line chemosensitivity to doxorubicin. Our findings support ATR as critical to liposarcoma proliferation and doxorubicin resistance. Therefore, the addition of ATR inhibition to a standard doxorubicin regimen is a potential treatment strategy for liposarcoma.

VE-822 mediated inhibition of ATR signaling sensitizes chondrosarcoma to cisplatin via reversion of the DNA damage response.

INTRODUCTION: Cisplatin has been reported to elicit the DNA damage response (DDR) via activation of the ATR-Chk1 pathway, which in turn contributes to the induction of cisplatin resistance. Inhibition of ATR-Chk1 signaling reverses cisplatin resistance in some cancers. However, the influence of inhibiting ATR-Chk1 signaling on cisplatin resistance in chondrosarcoma cancer has not been reported. MATERIALS AND METHODS: We compared the expression levels of ATR kinases in human nasopharyngeal carcinoma, choriocarcinoma and chondrosarcoma cell lines. We inhibited ATR kinase function with VE-822, a selective ATR inhibitor, and suppressed ATR kinase expression with shRNA. Western blotting, the CCK-8 assay, cell cycle distribution assay and apoptosis analysis were used to study the influence of inhibiting ATR-Chk1 signaling on reversing cisplatin resistance in chondrosarcoma cell lines. RESULTS: We found that chondrosarcoma cells expressed very low basal levels of phosphorylated ATR, but cisplatin treatment induced the activation of ATR-Chk1 signaling in a dose- and time-dependent manner, suggesting the induction of DDR. As expected, ATR inhibition with VE-822 reversed cisplatin-induced DDR and enhanced cisplatin-induced activation of H2AX, which is an important marker of DNA damage. Meanwhile, ATR inhibition by RNA interference also reversed DDR and promoted DNA damage. Furthermore, both pharmacological and molecular inhibition of ATR accelerated cisplatin-induced inhibition of cell proliferation and cell death. CONCLUSION: Our results suggested that inhibiting ATR activation promoted cisplatin-induced cell death via reversion of DDR, and VE-822 may be a valuable strategy for the prevention of cisplatin resistance in chondrosarcoma.

Inhibition of Wee1 sensitizes AML cells to ATR inhibitor VE-822-induced DNA damage and apoptosis.

Resistance to standard induction therapy and relapse remain the primary challenges for improving therapeutic effects in acute myeloid leukemia (AML); thus, novel therapeutic strategies are urgently required. Ataxia telangiectasia and Rad3-related protein (ATR) is a key regulator of different types of DNA damage, which is crucial for the maintenance of genomic integrity. The ATR-selective inhibitor VE-822 has proper solubility, potency, and pharmacokinetic properties. In this study, we investigated the anti-leukemic effects of VE-822 alone or combined with Wee1-selective inhibitor AZD1775 in AML cells. Our results showed that VE-822 inhibited AML cell proliferation and induced apoptosis in a dose-dependent manner. AZD1775 significantly promoted VE-822-induced inhibition of AML cell proliferation and led to a decreased number of cells in the G2/M phase. VE-822 and AZD1775 decreased the protein levels of ribonucleotide reductase M1 (RRM1) and M2 (RRM2) subunits, key enzymes in the synthesis of deoxyribonucleoside triphosphate, which increased DNA replication stress. VE-822 combined with AZD1775 synergistically induced AML cell apoptosis and led to replication stress and DNA damage in AML cell lines. Our study demonstrated that AZD1775 synergistically promotes VE-822-induced anti-leukemic activity in AML cell lines and provides support for clinical research on VE-822 in combination with AZD1775 for the treatment of AML patients.

Small-molecule inhibitors of Ataxia Telangiectasia and Rad3 related kinase (ATR) sensitize lymphoma cells to UVA radiation.

BACKGROUND: Psoralen plus ultraviolet A (PUVA) photochemotherapy is a combination treatment used for inflammatory and neoplastic skin diseases such as mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma (CTCL). However, 30% of MF patients do not respond sufficiently to PUVA and require more aggressive therapies. OBJECTIVE: The aim of this project was to investigate whether inhibition of Ataxia Telangiectasia and Rad3 related kinase (ATR) may enhance efficacy of phototherapy. METHODS: CTCL cell lines (MyLa2000, SeAx and Mac2a) served as in vitro cell models. ATR and Chk1 were inhibited by small molecule antagonists VE-821, VE-822 or Chir-124, or by small interfering RNAs (siRNAs). Cell cycle and viability were assessed by flow cytometry. RESULTS: Small molecule inhibitors of ATR and Chk1 potently sensitized all cell lines to PUVA and, importantly, also to UVA, which by itself did not cause apoptotic response. VE-821/2 blocked ATR pathway activation and released the cells from the G2/M block caused by UVA and PUVA, but did not affect apoptosis caused by other chemotherapeutics (etoposide, gemcitabine, doxorubicine) or by hydrogen peroxide. Knockdown of ATR and Chk1 with siRNA also blocked the ATR pathway and released the cells from G2/M block but did not sensitize the cells to UVA as observed with the small molecule inhibitors. The latter suggested that the synergism between VE-821/2 or Chir-124 and UVA was not solely caused by specific blocking of ATR kinase but also ATR-independent photosensitization. This hypothesis was further verified by administrating VE-821/2 or Chir-124 before and after UVA irradiation, as well as comparing their activity with other ATR and Chk1 inhibitors (AZD6738 and MK8776). We found that only VE-821/2 and Chir-124 kinase inhibitors had synergistic effect with UVA, and only if applied before treatment with UVA. CONCLUSION: Small molecule ATR and Chk1 inhibitors potently sensitize lymphoma cells to UVA radiation and induce a prominent apoptotic response. Interestingly, this effect is due to the dual (kinase inhibiting and photosensitizing) mode of action of these compounds.