ATR Inhibitor VE-821 Research Articles

The ATR inhibitor VE-821 increases the sensitivity of gastric cancer cells to cisplatin

BackgroundChemoresistance is a common event after cancer chemotherapy, including gastric cancer (GC). Cisplatin has been reported to induce the DNA damage response (DDR), thus leading to chemoresistance. VE-821, a specific inhibitor of ATR, has been proven to suppress a variety of solid malignancies effectively. Our study aimed to explore the effect of VE-821 on enhancing the chemical sensitivity to cisplatin and clarify the potential molecular mechanisms. MethodsCell viability and apoptosis of MKN-45 and AGS were measured by CCK8 and flow cytometry assay respectively. Western blotting was used to detect the expression of target proteins. TCGA database was used to analyze the correlation between the ATR expression with the prognosis of GC patients. The viability of GC organoids was detected by Cell Titer Glo (CTG) through luminescence. ResultsCisplatin inhibited the proliferation and induced apoptosis of GC cells with a relatively high IC50 value, and increased the phosphorylation levels of ATR-CHK1 and H2AX. VE-821 achieved the same effects but by downregulating the phosphorylation levels of the ATR-CHK1 pathway. Besides, higher ATR expression in GC tissues was positively correlated with higher pathological stage in GC patients. Interestingly, ATR inhibition reversed cisplatin-induced STAT3 activation and enhanced H2AX levels. Moreover, VE-821 significantly sensitized GC cells to cisplatin, and these two drugs had synergistic effects in GC cell lines, organoids, and in vivo. ConclusionOur results suggested VE-821 sensitized GC cells to cisplatin via reversing DDR activation. And VE-821 treatment may be a promising therapeutic strategy for GC patients with cisplatin resistance.

Synergistic anticancer activity of combined ATR and ribonucleotide reductase inhibition in Ewing's sarcoma cells.

Ewing's sarcoma is a highly malignant childhood tumour whose outcome has hardly changed over the past two decades despite numerous attempts at chemotherapy intensification. It is therefore essential to identify new treatment options. The present study was conducted to explore the effectiveness of combined inhibition of two promising targets, ATR and ribonucleotide reductase (RNR), in Ewing's sarcoma cells. Effects of the ATR inhibitor VE821 in combination with the RNR inhibitors triapine and didox were assessed in three Ewing's sarcoma cell lines with different TP53 status (WE-68, SK-ES-1, A673) by flow cytometric analysis of cell death, mitochondrial depolarisation and cell cycle distribution as well as by caspase 3/7 activity determination, by immunoblotting and by real-time RT-PCR. Interactions between inhibitors were evaluated by combination index analysis. Single ATR or RNR inhibitor treatment produced small to moderate effects, while their combined treatment produced strong synergistic ones. ATR and RNR inhibitors elicited synergistic cell death and cooperated in inducing mitochondrial depolarisation, caspase 3/7 activity and DNA fragmentation, evidencing an apoptotic form of cell death. All effects were independent of functional p53. In addition, VE821 in combination with triapine increased p53 level and induced p53 target gene expression (CDKN1A, BBC3) in p53 wild-type Ewing's sarcoma cells. Our study reveals that combined targeting of ATR and RNR was effective against Ewing's sarcoma in vitro and thus rationalises an in vivo exploration into the potential of combining ATR and RNR inhibitors as a new strategy for the treatment of this challenging disease.

The ATR Inhibitor VE-821 Enhances the Radiosensitivity and Suppresses DNA Repair Mechanisms of Human Chondrosarcoma Cells.

To overcome the resistance to radiotherapy in chondrosarcomas, the prevention of efficient DNA repair with an additional treatment was explored for particle beams as well as reference X-ray irradiation. The combined treatment with DNA repair inhibitors-with a focus on ATRi VE-821-and proton or carbon ions irradiation was investigated regarding cell viability, proliferation, cell cycle distribution, MAPK phosphorylation, and the expression of key DNA repair genes in two human chondrosarcoma cell lines. Pre-treatment with the PARPis Olaparib or Veliparib, the ATMi Ku-55933, and the ATRi VE-821 resulted in a dose-dependent reduction in viability, whereas VE-821 has the most efficient response. Quantification of γH2AX phosphorylation and protein expression of the DNA repair pathways showed a reduced regenerative capacity after irradiation. Furthermore, combined treatment with VE-821 and particle irradiation increased MAPK phosphorylation and the expression of apoptosis markers. At the gene expression and at the protein expression/phosphorylation level, we were able to demonstrate the preservation of DNA damage after combined treatment. The present data showed that the combined treatment with ATMi VE-821 increases the radiosensitivity of human chondrosarcoma cells in vitro and significantly suppresses efficient DNA repair mechanisms, thus improving the efficiency of radiotherapy.

Abstract 1411: Utilizing 7-azaindoles, 2,7-diazaindoles, and 1H-pyrazoles as core structures for novel cancer chemosensitizers

Abstract A quickly developing chemoresistance is the hallmark of the standard treatment failure and the poor patient prognosis. Resistance is often connected to the overexpression of the specific kinases that are involved in DNA damage response. Contrary, their inhibition could lead to augmented cancer cell sensitization to conventional approach. Among them, ataxia-telangiectasia and Rad3-related kinase (ATR), the major replication stress responder, is one of the most attractive target. Within this study our aim was to find novel chemosensitizing agents for the cancer treatment employment. Inspired by clinical candidates targeting ATR, we designed and prepared large library of 40 novel compounds utilizing 7-azaindoles, 2,7-diazaindoles, and 1H-pyrazoles as core structures. Initially, our presumption to have ATR inhibitors were confirmed by molecular dynamic study. All the compounds alone or in combination with cisplatin were screened against panel of nine cancer cell lines and one healthy cell line. The results were compared with well-known ATR inhibitor VE-821. Several compounds significantly inhibit the cell viability and some were able to potentiate cisplatin effect. From structure-activity relationship point of view, 7-azaindoles were the most cytotoxic compounds when substituted in position 3 and 5. Contrary, 2,7-diazaindoles expressed the most potent chemosensitizing capabilities. 1H-Pyrazoles were the least potent compounds within this screen. Interestingly, the status of tumor suppressor protein p53 (defect in G1) did not significantly influence chemosensitization or cytotoxic effect. Three highlighted compounds 3, 22, and 29 were selected for Broad oncology panel screening containing 104 kinases which could be potentially targeted. Only compound 29, the 2,7-diazaindole representative, showed ATR inhibitory efficacy with the IC50 around 10 µM. In contrast, the compound 22, 7-azaindole and the most cytotoxic one, expressed the multi-kinase activity with the substantial inhibitory capability against vascular endothelial growth factor receptor 3 protein (VEGFR-3). VEGFR-3 is also very attractive anticancer target with several inhibitors already approved. Finally, several compounds showed chemosensitizing potencies for temozolomide against secondary astrocytoma cell line. In overall, we showed that both, 7-azaindoles, and 2,7-diazaindoles, could be effectively utilized as novel anticancer agents. Whereas 2,7-diazaindoles could become efficient ATR inhibitors, 7-azaindoles have potential as antiangiogenics, VEGFR-3 inhibitors. Besides, both structures could be used for a glioblastoma treatment as temozolomide sensitizers. More research is currently ongoing to fully exploit potential of these compounds. Citation Format: Lukas Gorecki, Darina Muthna, Sara Merdita, Martin Andrs, Tomas Kucera, Tereza Kobrlova, Martina Rezacova, Jan Korabecny. Utilizing 7-azaindoles, 2,7-diazaindoles, and 1H-pyrazoles as core structures for novel cancer chemosensitizers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1411.

Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing\u2019s sarcoma cells

IntroductionEwing's sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades. In this study, combination treatments of the HSP90 inhibitor AUY922 with either the ATR inhibitor VE821 or the ATM inhibitor KU55933 were investigated for their effectiveness in Ewing's sarcoma cells.MethodsEffects were determined in p53 wild-type and p53 null Ewing's sarcoma cell lines by flow cytometric analyses of cell death, mitochondrial depolarization and cell-cycle distribution as well as fluorescence and transmission electron microscopy. They were molecularly characterized by gene and protein expression profiling, and by quantitative whole proteome analysis.ResultsAUY922 alone induced DNA damage, apoptosis and ER stress, while reducing the abundance of DNA repair proteins. The combination of AUY922 with VE821 led to strong apoptosis induction independent of the cellular p53 status, yet based on different molecular mechanisms. p53 wild-type cells activated pro-apoptotic gene transcription and underwent mitochondria-mediated apoptosis, while p53 null cells accumulated higher levels of DNA damage, ER stress and autophagy, eventually leading to apoptosis. Impaired PI3K/AKT/mTOR signaling further contributed to the antineoplastic combination effects of AUY922 and VE821. In contrast, the combination of AUY922 with KU55933 did not produce a cooperative effect.ConclusionOur study reveals that HSP90 and ATR inhibitor combination treatment may be an effective therapeutic approach for Ewing's sarcoma irrespective of the p53 status.

NFB-01. FUNCTIONAL CHARACTERIZATION OF ATRX LOSS IN NF1-ASSOCIATED GLIOMA AND MPNST

To identify the biologic relevance of ATRX loss in NF1-associated gliomagenesis, we studied the effects of Atrx loss using four previously characterized Nf1+/-Trp53+/- murine glioma lines. Lines 130G#3 and 158D#8 (corresponding to grade IV and III gliomas, respectively) displayed preserved ATRX protein expression compared to NIH-3T3 cells. We studied the effects of Atrx knockdown in these two lines in the presence and absence of the TERT inhibitor, BIRBR1532. Using a telomere-specific FISH assay, we identified increased signal intensity after Atrx knockdown, only in the presence of the TERT inhibitor. These features are reminiscent of ALT, although there were no significant alterations in cell growth. Next, we studied the effect of ATRX loss in MPNST lines ST88-14, NF90-8, STS-26T. These cell lines all expressed ATRX and DAXX. However, STS-26T contained a TERT promoter mutation and ST88-14 had a known SNP in the TERT promoter, while NF90-8 had no alterations. ATRX siRNA knockdown showed no significant effects in cell proliferation or apoptosis. However, ATRX knockdown resulted in rare ultra-bright foci, indicative of ALT. Next, we studied the in vitro effect of the ATR inhibitor VE-821 in MPNST cell lines. Only NF90-8 (lacking TERT alterations) demonstrated a decrease in growth after ATRX knockdown and VE-821 treatment. However, ATRX knockdown alone did not affect sensitivity to carboplatin. Our findings further support a role for ATRX loss with subsequent ALT activation in a biologic subset of NF1-associated malignancies, thereby opening an opportunity for therapeutic targeting of these aggressive tumors using specific classes of drugs.

Abstract 546: PARG and WEE1 inhibition in GI cancers: A novel synergistic, targeted therapeutic approach

Abstract Colorectal carcinomas (CRC) and pancreatic ductal adenocarcinomas (PDAC) are the 2nd and 3rd leading causes of cancer-related deaths in the U.S. respectively. Recent work from our lab highlighted Poly ADP-Ribose Glycohydrolase (PARG) as a target in PDAC, and PARG inhibition (PARGi) sensitized PDAC cells to the DNA damaging agents, 5-FU and oxaliplatin. Other labs have shown that PARG inhibition causes replication stress in cancer cells. CRC and PDAC cells, while cellularly different, share a high frequency of KRAS and P53 inactivating mutations. High levels of replication stress (RS) has also been well documented in these tumor types. Targeting DNA Damage Response Kinases (DDRKs) in combination with chemotherapy (mostly DNA damage inducers like 5-FU, oxaliplatin, etc.) has gained recent interest in solid tumors but overlapping toxicities and tolerability have been issues clinically. Accordingly, we hypothesized that combined inhibition of PARG and DDRKs could yield synergistic effects in tumors with high replication stress while alleviating adverse events seen in other combination strategies. We determined in drug sensitivity assays that PARGi did not synergize with either the CHK1 inhibitor prexasertib or the ATR inhibitor VE-821 in our cell lines but did synergize with Wee1i (AZD1775) in both PDAC and CRC cell lines (Comb. Analysis, p<.05). Additionally, we assessed long-term cell survival using colony formation assays, which yielded a dramatic reduction in cell survival for Panc1, MiaPaCa-2, CFPAC1 (PDAC cells), HCT116 (CRC cells) and SW480 (CRC cells) cell lines with the combination of PARGi and Wee1i as compared to single-agent controls (p<.001). To validate the targeted synergy between AZD1775 and PARGi, we utilized genetic models of PARG and Wee1 silencing, including CRISPR PARG knockout cell lines, siRNA, and shRNA knockdown models. Genetic knockdown of Wee1 and PARG in PDAC and CRC cell lines yielded strikingly similar results in colony formation assays, with 2 and 12-fold changes respectively compared to Wee1i alone (P-value < .0001). HCT116 CRISPR PARG knockouts yielded a 5-fold difference in Wee1i sensitivity in colony formation assays. Mechanistically, western blot analysis demonstrated PARGi and Wee1i led to >2-fold increases in PARylation, yH2AX, and cleaved-caspase 3 as compared to single-agent controls. In conclusion, combined PARG - Wee1 inhibition is a novel targeting strategy in PDAC and CRC tumors that results in decreased cell viability. We are currently testing the efficacy of AZD1775 in PARG CRISPR-knockout tumors in in vivo xenograft models. Ongoing studies are focused on assessing the effect of Wee1i + PARGi on chromosomal aberrations, disruption in replication fork dynamics via DNA fiber labeling, and cell cycle dependencies. Citation Format: Lebaron C. Agostini, Aditi Jain, Michael Pishvaian, Charles Yeo, Jonathan Brody. PARG and WEE1 inhibition in GI cancers: A novel synergistic, targeted therapeutic approach [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 546.

The SF3B1 K700E Mutation Induces R-Loop Accumulation and Associated DNA Damage

The myelodysplastic syndromes (MDS) are common myeloid malignancies. Mutations in genes involved in pre-mRNA splicing (SF3B1, SRSF2, U2AF1 and ZRSR2) are the most common mutations found in MDS. There is evidence that some spliceosomal components play a role in the maintenance of genomic stability. Splicing is a transcription coupled process; splicing factor mutations affect transcription and may lead to the accumulation of R-loops (RNA-DNA hybrids with a displaced single stranded DNA). Mutations in the splicing factors SRSF2 and U2AF1 have been recently shown to increase R-loops formation in leukemia cell lines, resulting in increased DNA damage, replication stress and activation of the ATR-Chk1 pathway. SF3B1 is the most frequently mutated splicing factor gene in MDS, but a role for mutated SF3B1 in R-loop accumulation and DNA damage has not yet been reported in hematopoietic cells. We have investigated the effects of the common SF3B1 K700E mutation on R-loop formation and DNA damage response in MDS and leukemia cells. R-loop signals and the DNA damage response were measured by immunofluorescence staining using S9.6 and anti-γ-H2AX antibodies respectively. Firstly, we studied K562 (myeloid leukemia) cells with the SF3B1 K700E mutation and isogenic SF3B1 K700K wildtype (WT) K562 cells. K562 cells with SF3B1 mutation showed a significant increase in the number of S9.6 foci [Fold change (FC) 2.01, p<0.001] and in the number of γ-H2AX foci (FC 2.32, p<0.001), indicating increased R-loops and DNA damage, compared to SF3B1 WT K562 cells. Moreover, we observed increased Chk1 phosphorylation at Ser345, a hallmark of activation of the ATR pathway, in SF3B1 mutant K562 cells. Next, we analyzed induced pluripotent stem cells (iPSCs) that we generated from the bone marrow cells of one SF3B1 mutant MDS patient and of one healthy control. A significant increase in R-loops and DNA damage response was observed in an iPSC clone harboring SF3B1 mutation compared to another iPSC clone without SF3B1 mutation obtained from same MDS patient (S9.6 mean fluorescence intensity - FC 1.72, p<0.001; γ-H2AX foci - FC 1.34, p=0.052) and to iPSCs from the healthy control (S9.6 mean fluorescence intensity - FC 1.53, p<0.001; γ-H2AX foci - FC 1.61, p=0.006). In addition, bone marrow CD34+ cells from a SF3B1 mutant MDS patient showed increased R-loops (as measured by number of S9.6 foci) compared to CD34+ cells from a MDS patient without splicing factor mutations (FC 1.9) and from a healthy control (FC 2.6). To investigate whether the observed DNA damage and ATR activation in SF3B1 mutant K562 cells result from induced R-loops, we overexpressed RNASEH1 (encoding an enzyme that degrades the RNA in RNA:DNA hybrids) to resolve R-loops in these cells. RNASEH1 overexpression significantly reduced the number of S9.6 (FC 0.51, p<0.001) and γ-H2AX foci (FC 0.63, p=0.035) in SF3B1 mutant K562 cells compared to SF3B1 WT K562 cells. RNASEH1 overexpression also resulted in decreased Chk1 phosphorylation, indicating suppression of ATR pathway activation in SF3B1 mutant K562 cells. To determine the functional importance of ATR activation associated with SF3B1 mutation, we evaluated the sensitivity of SF3B1 mutant cells towards the ATR inhibitor VE-821. SF3B1 mutant K562 cells showed preferential sensitivity towards VE-821 compared to SF3B1 WT K562 cells. Chk1 is a critical substrate of ATR, and we next investigated the effects of Chk1 inhibition in SF3B1 mutant cells. Interestingly, SF3B1 mutant K562 cells demonstrated preferential sensitivity towards the Chk1 inhibitor UCN-1 (IC50 61.8 nM) compared to SF3B1 WT K562 cells (IC50 267 nM), suggesting that ATR activation is important for the survival of SF3B1 mutant cells. SF3B1 mutant K562 cells were preferentially sensitive to the splicing modulator Sudemycin D6 (IC50 53.2 nM) compared to SF3B1 WT K562 cells (IC50 130.7 nM). The effects of VE-821 and UCN-1 on SF3B1 mutant K562 cells were enhanced by Sudemycin D6 (Combination index <1), indicating synergy. In summary, our results show that the SF3B1 mutation leads to accumulation of R-loops and associated DNA damage resulting in activation of the ATR pathway in MDS and leukemia cells. Thus different mutated splicing factors have convergent effects on R-loop elevation leading to DNA damage. Moreover, our data suggest that Chk1 inhibition, alone or in combination with splicing modulators, may represent a novel therapeutic strategy to target SF3B1 mutant cells. Disclosures Schuh: Janssen: Speakers Bureau; Verastem: Speakers Bureau; Kite: Speakers Bureau; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau; Bristol-Myers Squibb: Research Funding; AbbVie: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau. Wiseman:Novartis, Celgene: Consultancy, Honoraria.

Inhibition of ATR Overcomes Chemotherapy Resistance in p53 Deficient Myeloma Cells

In MM, as well as in most hematological malignancies, deficiency in p53 pathway (mainly because of TP53 deletion and/or mutation) is associated with resistance to treatments (Tessoulin Blood Reviews 2017; 31:251). Recent clinical studies have shown that deletion or mutation of TP53 are the most adverse prognostic values for patients (Thakurta Blood 2019;133:1217). Although these patients are easily identified, there is no dedicated therapies for them. p53 pathway is central for homeostasis and cell adaptation/response to many stresses, including DNA repair orchestration and survival regulation. In p53 deficient cells, DNA damaging drugs don't induce massive apoptosis and cells escape to death. In normal cells, DNA damaging drugs induce cell cycle arrest and DNA repair, mainly orchestrated by p53 target genes. Cell cycle arrest in S phase, which is critical for allowing homologous DNA repair, is activated by cell cycle check-point inhibitor such as Chk1, an ATR target. In p53 deficient cells, inhibiting check point inhibitor using ATR inhibitor should allow DNA damaged cells to progress into cell cycle despite the lack of repair and in fine induce replicative/mitotic catastrophe. The aim of this study was to assess whether inhibiting ATR in p53 deficient myeloma cells could overcome chemotherapy resistance. ATR inhibitor, VE-821, was assessed in 13 human myeloma cell lines (HMCLs) alone and in combination with DNA damaging agents, CX5461, a G quadruplex inhibitor, or melphalan, the « myeloma » alkylating drug. The HMCL cohort included 8 HMCLs, 5 TP53Abn and 5 TP53wt. Cell viability was assessed using Cell Titer Glo assay or using flow cytometry (loss of AnnexinV or CD138 staining in HMCLs or primary myeloma cells, respectively). In our cohort of 13 HMCLs and by contrast to previous results, CX5461 was more efficient in TP53wt than in TP53abn HMCLs (mean of death at 0.5mM was 43% versus 24%, p=0.04). Melphalan was also more potent in TP53wt than in TP53abn HMCLs (LD50 values were 26 mM versus 10 mM, p=0.008). By contrast, ATR inhibitor VE-821 (2.5mM) was efficient in both types of HMCLs (mean of death in TP53wt was 45% and 28% in TP53abn HMCLs, p=0.6). Combination of CX5641 (0.5mM) with VE-821 (2.5mM) was more efficient than each drug alone and efficacy was not dependent on TP53 status (mean of death in TP53wt was 69% versus 56% in TP53abn HMCLs, p=0.6): interestingly, combo was efficient in all TP53abn HMCLs, being either additive (n=5) or even synergistic (n=3). By contrast, combo was not efficient in all TP53wt HMCLs (either additive or antagonist). Combination of melphalan (10 mM) with VE-821 (2.5mM) was also synergistic in TP53abn HMCLs (mean of cell death was 9% with melphalan and 73% for combo, p<0.05). Preliminary results of combos in 6 consecutive primary samples with MM or plasma cell leukemia (3 TP53wt and 3 TP53abn) demonstrated efficacy. Indeed, in the 3 TP53abn samples, both CX5641/VE-821 and melphalan/VE-821 combos displayed synergism or additivity: median of expected values versus observed values was 61% versus 74% for CX5641/VE-821, and 98% versus 89% for melphalan/VE-821, respectively. In the 3 TP53wt samples, combos displayed additivity or antagonism: median of expected versus observed values was 15% versus 15% for CX5641/VE-821, and 100% versus 62% for melphalan/VE-821, respectively. In normal peripheral blood cells (n=2), both combos were not cytotoxic (mean values of cell death were 0% with CX5641/VE-821 and 3% with melphalan/VE-821). To decipher the molecular pathway involved in cell response, we monitored cell cycle using BrdU/IP assay, replicative stress response using Chk1 phosphorylation and DNA double strand breaks using Comets assays in 3 TP53abn HMCLs. At 24h, CX5641 induced an increase of cells in S (mean of increase 12%) and G2M phases (11%), while VE-821 didn't modify cell cycle. Combination of CX5641 with VE-821 induced a dramatic increase of cells in G2M (20%) (and in subG2 phase), and a decrease of cells in S phase (10%), indicating that cells blocked in S phase by CX5641 were released by VE-821.CX5641 induced Chk1 phosphorylation, which was inhibited by addition of VE-821, confirming the CX5641/ATR/Chk1 signaling. Finally, CX5641 and VE-821 induced comets, confirming irreversible DNA double strand breaks. All these results show that inhibition of ATR after inducing DNA damage in TP53abn myeloma cells efficiently induces cell death, while preserving normal cells. Disclosures Moreau: Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

P1.14-41 The Combination of the PARP Inhibitor Olaparib and the ATR Inhibitor VE-821 Selectively Targets ATM-Deficient Lung Cancer Cells

The driving principle behind precision medicine is to specifically target genetic variations that arise in tumorigenesis while leaving normal cells unaffected. Mutations in Ataxia Telangiectasia Mutated (ATM) may offer such a therapeutic target. ATM is mutated in approximately 12% of lung cancers and up to 40% of lung adenocarcinoma have been reported to lack ATM protein expression. ATM is an apex signaling kinase that responds to DNA-double strand breaks, playing a direct role in DNA repair as well as the initiation of signaling cascades that can lead to cell cycle arrest and apoptosis. We asked whether ATM-deficient human lung cancer cells are sensitive to the poly-ADP ribose polymerase (PARP) inhibitor olaparib, and investigated the mechanism of action of olaparib in these cells. We analyzed drug sensitivity for 61 lung adenocarcinoma cell lines from the Genomics of Drug Sensitivity in Cancer (GDSC) project and deleted ATM from lung adenocarcinoma A549 cells using CRISPR/Cas9. We determined the effects on cell viability using trypan blue exclusion and clonogenic survival assays. To investigate the mechanism of sensitivity of ATM-deficient cells to PARP and ATR inhibitors we used flow cytometry and cell viability assays as above. We observed a positive correlation between olaparib IC50 values and ATM mRNA expression. ATM mutant cell lines were more sensitive to olaparib compared to ATM wild-type cell lines or cell lines with amplified ATM. Additionally, ATM-deficient lung cancer cells were sensitive to olaparib, as are lung cancer cells (A549) with CRISPR/Cas9 deletion of ATM. Mechanistically, olaparib caused the temporary and reversible accumulation of G2 phase cells in ATM-deficient cells which manifested as a decrease in proliferation in both the trypan blue exclusion assay and clonogenic survival assay. Olaparib did not induce cell death in ATM-deficient cells, however cell death was induced when olaparib was used in combination with the ATR inhibitor VE-821. We show that olaparib acts as a cytostatic agent in ATM-deficient lung cancer cells, inducing a reversible and temporary growth arrest in G2 phase. Only when combined with the ATR inhibitor VE-821 was cell death observed and only in ATM-deficient cells. Our data suggest that patients with ATM-deficient lung cancer could benefit from combinatorial treatment with PARP and ATR inhibitors.

Antileukemic activity of novel adenosine derivatives

The present study investigated the effect of cladribine (CLA) and six of its derivatives containing a formamidine group at position 6 (CLA-FDM, CLA-FPAZ, CLA-FPIR, CLA-FPIP, CLA-FHEX, and CLA-FMOR) on acute promyelocytic, lymphoblastic, and acute monocytic leukemia cells. The role of ATR kinase in deoxycytidine kinase (dCK) activation in response to DNA damage was assessed. The presence of DNA lesions was assessed by measurement phosphorylation of H2AX and by using the alkaline comet assay with proteinase K post-treatment following assessment of the cell cycle. Apoptotic events such as alterations in intracellular calcium concentration, caspase-3/7 activity and increased sub-G1 cell population were measured. CLA derivatives were highly effective against leukemic cells, showing high cytotoxicity, causing DNA fragmentation, and inducing DNA-protein cross-links in leukemic cells. CLA-FMOR showed the highest efficacy. CLA derivatives increased the levels of intracellular calcium ions, caspase-3/7 and the percentage of sub-G1 apoptotic cells and blocked cells in the S phase of the cell cycle to a greater extent than free CLA. The selective ATR inhibitor VE-821 significantly suppressed the increase in dCK activity and decreased basal dCK activity. The present results suggested that ATR kinase controls dCK activity in response to synthetic CLA derivatives.

Linking hypoxia, DNA damage and proliferation in multicellular tumor spheroids

BackgroundMulticellular Tumor Spheroids are frequently used to mimic the regionalization of proliferation and the hypoxic environment within avascular tumors. Here we exploit these features to study the activation of DNA damage repair pathways and their correlation to developing hypoxia.MethodsActivation of DNA damage repair markers, proliferation, cell death, glycogen accumulation and developing hypoxia were investigated using immunofluorescence, immuno-histochemistry, EdU incorporation, Western blots, COMET assays, and pharmacological agents in A673 Ewing sarcoma spheroids and monolayer cultures.ResultsDNA damage marker γ-H2AX is observed in the hypoxic, peri-necrotic region of growing spheroids. While most proliferating cells are seen on the spheroid surface, there are also a few Ki-67 positive cells in the hypoxic zone. The hypoxia-induced phosphorylation of H2AX to form γ-H2AX in spheroids is attenuated by the ATM inhibitor KU55933, but not the ATR inhibitor VE-821.ConclusionTumor spheroids mimic tumor microenvironments such as the anoxic, hypoxic and oxic niches within solid tumors, as well as populations of cells that are viable, proliferating, and undergoing DNA damage repair processes under these different micro-environmental conditions. ATM, but not ATR, is the primary kinase responsible for γ-H2AX formation in the hypoxic core of A673 spheroids. Spheroids could offer unique advantages in testing therapeutics designed to target malignant cells that evade conventional treatment strategies by adapting to the hypoxic tumor microenvironment.

Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities.

SummaryATR and CHK1 maintain cancer cell survival under replication stress and inhibitors of both kinases are currently undergoing clinical trials. As ATR activity is increased after CHK1 inhibition, we hypothesized that this may indicate an increased reliance on ATR for survival. Indeed, we observe that replication stress induced by the CHK1 inhibitor AZD7762 results in replication catastrophe and apoptosis, when combined with the ATR inhibitor VE-821 specifically in cancer cells. Combined treatment with ATR and CHK1 inhibitors leads to replication fork arrest, ssDNA accumulation, replication collapse, and synergistic cell death in cancer cells in vitro and in vivo. Inhibition of CDK reversed replication stress and synthetic lethality, demonstrating that regulation of origin firing by ATR and CHK1 explains the synthetic lethality. In conclusion, this study exemplifies cancer-specific synthetic lethality between two proteins in the same pathway and raises the prospect of combining ATR and CHK1 inhibitors as promising cancer therapy.

A crucial role for ATR in the regulation of deoxycytidine kinase activity

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme for salvage of deoxynucleosides and activation of numerous anticancer and antiviral nucleoside analogs. dCK activity is enhanced in response to several genotoxic treatments, which has been correlated with an increase of dCK phosphorylation at Ser-74. ATM was recently identified as the kinase responsible for Ser-74 phosphorylation and dCK activation after ionizing radiation (IR). Here, we investigated the role of ATM and the related kinase ATR in dCK activation induced by other types of DNA damage. Using ATM-deficient cells or the ATM inhibitor KU-60019, we found that ATM was not required for dCK activation caused by UV light, aphidicolin, cladribine, and unexpectedly also IR. On the other hand, the selective ATR inhibitor VE-821 significantly reduced up-regulation of dCK activity induced by these genotoxic agents, though not IR, and also down-regulated basal dCK activity. A role for ATR in the control of dCK activity was confirmed by using ATR siRNA and ATR-Seckel cells. ATR was also found to directly phosphorylate dCK at Ser-74 in vitro. Further studies revealed that ATR, which is also activated in response to IR, although later than ATM, was responsible for IR-induced dCK activation in ATM-deficient cells or in the presence of KU-60019. Overall, our results demonstrate that ATR controls basal dCK activity and dCK activation in response to replication stress and indicate that ATR can activate dCK after IR if ATM is lacking or inhibited.

MTR-03ATRX REDUCTION PROMOTES ALKYLATING AGENT RESISTANCE AND XENOGRAFT GROWTH IN A SUBSET OF GLIOMAS

MTR-03. ATRX REDUCTION PROMOTES ALKYLATING AGENT RESISTANCE AND XENOGRAFT GROWTH IN A SUBSET OF GLIOMAS Ming Yuan, Ping An, Kah Suan Lim, Isabella Taylor, Fausto Rodriguez, Eric Raabe, and Charles Eberhart; Johns Hopkins University, Baltimore, MD, USA BACKGROUND:Loss ofATRXfunctionhas been implicated inalternative lengthening of telomeres (ALT) and is common in IDH mutant astrocytoma of varying grades. However, the functional effects of ATRX loss on glioma growth are not clear, although it has been suggested that it can modulate response to ATR inhibitors such as VE-821. METHODS: We evaluated the ALT-positive GBM neurosphere line JHH-GBM14, which retains ATRX expression in a subset of cells, the ALT-negative neurosphere line 040821 and LN229 cells. Two distinct shRNA were used to suppress ATRX protein expression. RESULTS: ATRX knockdown had no effect on the overall in vitro growth of the three lines, but GBM14 cells became resistant to temozolamide and hydroxyurea after ATRX knockdown, while the two ALT-negative lines did not. A modest, non-significant increase in radiation resistance was also noted. Sensitivity to the ATR inhibitor VE-821 was not affected by decreased ATRX levels in any of the three lines. Interestingly, clonogenic growth of GBM14, theexpression of several stemcellmarkers, aswellas its ability togenerate large flank and intracranial xenografts was increased by ATRX knockdown, while these effects were not seen in 040821 GBM neurospheres. CONCLUSIONS: While ATRX knockdown had little effect on in vitro tumor growth, it dramatically affected the treatment response, clonogenicity and in vivo growth of ALT-positive GBM14 cells, but not two GBM models lacking ALT. These data suggest that in some GBM, ATRX can modulate growth and treatment sensitivity as well as affecting telomere biology. Neuro-Oncology 17:v124–v129, 2015. doi:10.1093/neuonc/nov219.3 Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2015.

Common cancer-associated imbalances in the DNA damage response confer sensitivity to single agent ATR inhibition.

ATR is an attractive target in cancer therapy because it signals replication stress and DNA lesions for repair and to S/G2 checkpoints. Cancer-specific defects in the DNA damage response (DDR) may render cancer cells vulnerable to ATR inhibition alone. We determined the cytotoxicity of the ATR inhibitor VE-821 in isogenically matched cells with DDR imbalance. Cell cycle arrest, DNA damage accumulation and repair were determined following VE-821 exposure.Defects in homologous recombination repair (HRR: ATM, BRCA2 and XRCC3) and base excision repair (BER: XRCC1) conferred sensitivity to VE-821. Surprisingly, the loss of different components of the trimeric non-homologous end-joining (NHEJ) protein DNA-PK had opposing effects. Loss of the DNA-binding component, Ku80, caused hypersensitivity to VE-821, but loss of its partner catalytic subunit, DNA-PKcs, did not. Unexpectedly, VE-821 was particularly cytotoxic to human and hamster cells expressing high levels of DNA-PKcs. High DNA-PKcs was associated with replicative stress and activation of the DDR. VE-821 suppressed HRR, determined by RAD51 focus formation, to a greater extent in cells with high DNA-PKcs.Defects in HRR and BER and high DNA-PKcs expression, that are common in cancer, confer sensitivity to ATR inhibitor monotherapy and may be developed as predictive biomarkers for personalised medicine.

VE-821, an ATR inhibitor, causes radiosensitization in human tumor cells irradiated with high LET radiation.

BackgroundHigh linear energy transfer (LET) radiation such as carbon ion particles is successfully used for treatment of solid tumors. The reason why high LET radiation accomplishes greater tumor-killing than X-rays is still not completely understood. One factor would be the clustered or complex-type DNA damages. We previously reported that complex DNA double-strand breaks produced by high LET radiation enhanced DNA end resection, and this could lead to higher kinase activity of ATR protein recruited to RPA-coated single-stranded DNA. Although the effect of ATR inhibition on cells exposed to low LET gamma-rays has recently been reported, little is known regarding the effect of ATR inhibitor on cells treated with high LET radiation. The purpose of this study is to investigate the effects of the ATR inhibitor VE-821 in human tumor and normal cells irradiated with high LET carbon ions.FindingsHeLa, U2OS, and 1BR-hTERT (normal) cells were pre-treated with 1 μM VE-821 for 1 hour and irradiated with either high LET carbon ions or X-rays. Cell survival, cell cycle distribution, cell growth, and micronuclei formation were evaluated. VE-821 caused abrogation of G2/M checkpoint and forced irradiated cells to divide into daughter cells. We also found that carbon ions caused a higher number of multiple micronuclei than X-rays, leading to decreased cell survival in tumor cells when treated with VE-821, while the survival of irradiated normal cells were not significantly affected by this inhibitor.ConclusionsATR inhibitor would be an effective tumor radiosensitizer with carbon ion irradiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0464-y) contains supplementary material, which is available to authorized users.

Abstract 2849: The combination of the PARP inhibitor talazoparib (BMN 673) with the ATR inhibitor VE-821 overcomes the drug resistance of Schlafen 11-deficient cells

Abstract Poly(ADP-ribose)polymerases (PARP) inhibitors work as catalytic inhibitors as well as PARP poisons by trapping PARP-DNA complexes (Murai et al., Cancer Res. 2012: 72:5588-99), and talazoparib is the most potent PARP inhibitor to date in terms of cytotoxicity and PARP trapping (Murai et al., Mol. Cancer Ther. 2014:13:433-43). Nevertheless, mining the NCI60 data shows that half of the 60 cell lines are tolerant to talazoparib. To elucidate the mechanisms of such tolerance, we searched for genes with expression levels correlated to talazoparib sensitivity using CellMiner (http://discover.nci.nih.gov/cellminer/). Schlafen 11 (SLFN11) turned out to be one of the most positively correlated genes whose expression is correlated with cellular sensitivity to talazoparib. Using genetically altered cells, we established that SLFN11 inactivation confers high resistance to talazoparib, revealing that SLFN11 inactivation, which is common in cancer cells (Zoppoli et al. PNAS 2014: 109:15030-5), is a novel mechanism of resistance for PARP inhibitors. Based on genetically engineered CRISPR/casp9 SLFN11-knockout cell lines and cell cycle analyses, we found that the parent SLFN11-proficient cells arrest in S phase without DNA replication, while SLFN11-knock out cells maintain their replication until entering G2 phase in the presence of talazoparib. Although ATR is the key regulator of intra-S phase check point, the addition of the ATR inhibitor (VE-821) (Josse et al., Cancer Res. 2014) does not rescue the replication arrest in SLFN11-proficient cells, suggesting the existence of a SLFN11-dependent replication arrest mechanism independently of ATR. On the other hand, the addition of the ATR inhibitor in SLFN11-knock out cells alters the arresting point from G2 to S phase, and eventually increases dead cells. This is probably because the prolonged replication block leads to the collapse of replication forks, resulting in DNA double-strand breaks. Consistent to these findings, the combination of the ATR inhibitor with talazoparib synergistically sensitizes SLFN11-knock out cells more efficiently than the parent SLFN11-proficient cells. Together, these results demonstrate that the combination of the ATR inhibitor with talazoparib can overcome the resistance of SLFN11-deficient cells to talazoparib. Citation Format: Junko Murai, Yves Pommier. The combination of the PARP inhibitor talazoparib (BMN 673) with the ATR inhibitor VE-821 overcomes the drug resistance of Schlafen 11-deficient cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2849. doi:10.1158/1538-7445.AM2015-2849

ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase i inhibitors by disabling DNA replication initiation and fork elongation responses.

Camptothecin and its derivatives, topotecan and irinotecan, are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress, came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821 confirmed marked antiproliferative synergy with camptothecin and even greater synergy with LMP-400. Single-cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970, enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof of principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials.

Abstract 794: The ATR inhibitor VE-821 in combination with the novel topoisomerase I inhibitor LMP-400 selectively kills cancer cells by disabling DNA replication initiation and fork elongation

Abstract Camptothecin, a specific topoisomerase I inhibitor is a potent anticancer drug, especially against solid tumors. This agent produces well-characterized double-strand breaks upon collision of replication forks with topoisomerase I cleavage complexes. In an attempt to improve its efficacy, we conducted a synthetic lethal siRNA screening using a library that targets nearly 7000 human genes. Depletion of ATR, the main transducer of replication stress-induced DNA damage response exacerbated cytotoxic response to both camptothecin and the indenoisoquinoline LMP-400, a novel class of topoisomerase inhibitors in clinical trial. Inhibition of ATR by the recently developed specific inhibitor VE-821 induced synergistic antiproliferative activity when combined with either topoisomerase inhibitor. Cytotoxicity induced by the combination with LMP-400 was greater than with camptothecin. Using single cell analysis and DNA fiber spread, we show that VE-821 abrogated the S-phase checkpoint, restored origin firing and replication fork progression. Moreover, the combination of a topoisomerase inhibitor with VE-821 inhibited the phosphorylation of ATR and ATR-mediated Chk1 phosphorylation but strongly induced γH2AX. Single cell analysis revealed that γH2AX pattern changed overtime from well-defined focus to a pan-nuclear staining. The change in γH2AX pattern can be useful as a predictive biomarker to evaluate the efficacy of therapy. The key implication of our work is the clinical rationale it provides to evaluate the combination of indenoisoquinoline topoisomerase I inhibitors with ATR inhibitors. Citation Format: Rozenn Jossé, Scott E. Martin, Rajarshi Guha, Pinar Ormanoglu, Thomas Pfister, Joel Morris, James H. Doroshow, Yves Pommier. The ATR inhibitor VE-821 in combination with the novel topoisomerase I inhibitor LMP-400 selectively kills cancer cells by disabling DNA replication initiation and fork elongation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 794. doi:10.1158/1538-7445.AM2014-794