Abstract LB-260: Identification of DTYMK and CHEK1 as therapeutic targets in LKB1 mutant non-small cell lung cancer
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Abstract The LKB1 tumor suppressor encodes a key metabolic sensor that integrates cell growth and metabolism. LKB1 is mutationally inactivated in multiple adult malignancies, including >20% of lung cancers, often simultaneously with activating KRAS mutations. LKB1 mutations are an important predictor of poor outcome and resistance to current therapeutic approaches. We employed an integrative approach to define novel therapeutic targets in Lkb1 mutant lung cancers. Matched cell lines from genetically engineered mouse models of cancer driven by activated Kras alone or in combination with Lkb1 deletion, were employed in high-throughput RNAi, kinase inhibitor, and metabolite screens. These screens identified knockdown of either Dtymk (deoxythymidylate kinase) or Chek1 (checkpoint kinase 1) as synthetically lethal with Lkb1 deficiency in both mouse and human lung cancer cell lines, and revealed that Lkb1 inactivation conferred marked sensitivity to treatment with CHEK1 inhibitors. Lkb1 deficient cells had a distinct metabolic profile, characterized by striking decreases in multiple nucleotide metabolites. Knockdown of DTYMK inhibited dTTP biosynthesis and, consequently, DNA synthesis, and knockdown of CHEK1 caused accumulation of DNA damage. We hypothesize that Lkb1 loss enhances dependence on these enzymes due to broad defects in nucleotide metabolism. Our studies support the development of therapies target DTYMK and CHEK1 in LKB1 mutant non-small cell lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-260. doi:1538-7445.AM2012-LB-260Keywords:
CHEK1
Synthetic Lethality
The protein kinase Chk1 is essential for the DNA damage checkpoint. Cells lacking Chk1 are hypersensitive to DNA-damaging agents such as UV light and gamma-irradiation because they fail to arrest the cell cycle when DNA damage is generated. Phosphorylation of Chk1 occurs after DNA damage and is dependent on the integrity of the DNA damage checkpoint pathway. We have tested whether a topoisomerase I inhibitor, camptothecin (CPT), generates DNA damage in the fission yeast Schizosaccharomyces pombe that results in Chk1 phosphorylation. We demonstrate that Chk1 is phosphorylated in response to CPT treatment in a time- and dose-dependent manner and that phosphorylation is dependent on an intact DNA damage checkpoint pathway. Furthermore, we show that cells must be actively dividing in order for CPT to generate a Chk1-responsive DNA damage signal. This observation is consistent with a model whereby the cytotoxic event caused by CPT treatment is the production of a DNA double-strand break resulting from the collision of a DNA replication fork with a trapped CPT-topoisomerase I cleavable complex. Cells lacking Chk1 are hypersensitive to CPT treatment, suggesting that the DNA damage checkpoint pathway can be an important determinant for CPT sensitivity or resistance. Finally, as a well-characterized, soluble agent that specifically causes DNA damage, CPT will allow a biochemical analysis of the checkpoint pathway that responds to DNA damage.
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801 DNA damage checkpoints are regulatory signal transduction pathways that allow cells to control DNA damage-induced cell cycle arrest, DNA repair, and cell death by apoptosis. Most human tumors arise from multiple genetic changes, including defective DNA damage checkpoints that provide potential targets for therapeutic use of DNA-damaging agents. One strategy to enhance the impact of these agents is to inhibit the checkpoint signaling in cancer cells. The checkpoint kinase 1 (Chk1) is one of the effector kinases that regulates cell cycle progression after DNA damage. Upon DNA damage, Chk1 is phosphorylated on Ser 345 and 317, and this phosphorylation is dependent on ATM (ataxia-telangiectasia mutated) or ATR (ATM and Rad 3-related). In addition to phosphorylation, DNA damage-dependent Chk1 activity could be modulated by other posttranslational modifications and protein-protein interaction. We have developed experimental procedures for identification of novel Chk1 posttranslational modifications and associated proteins. Using immunoprecipitation followed by mass spectroscopy analysis, we have found novel candidate Chk1-associated proteins. Potentially, interaction of these proteins with Chk1 could inhibit DNA damage-dependent Chk1 activation, leading to abrogation of checkpoint response and sensitization of cancer cells to DNA-damaging drugs.
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Checkpoint Kinase 2
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On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12. Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.
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Deletion of the Saccharomyces cerevisiae TOP3gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391–8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Δ strains. We show that top3Δ mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion, top3Δ strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Δ mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.
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The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology to replication factors PCNA and RF-C, respectively, suggesting that these factors promote checkpoint activation by “sensing” DNA damage directly. Mec1 kinase, however, phosphorylates the checkpoint protein Ddc2 in response to damage in the absence of all other known checkpoint proteins, suggesting instead that Mec1 and/or Ddc2 may act as the initial sensors of DNA damage. In this paper, we show that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break. Other forms of damage result in a greater number of Ddc1–GFP or Ddc2–GFP foci, in correlation with the number of damage sites generated, indicating that Ddc1 and Ddc2 are both recruited to sites of DNA damage. Interestingly, Ddc2 localization is severely abrogated in mec1 cells but requires no other known checkpoint genes, whereas Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1. Therefore, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. Further, we show that although Ddc1 remains strongly localized following checkpoint adaptation, many nuclei contain only dim foci of Ddc2–GFP, suggesting that Ddc2 localization may be down-regulated during resumption of cell division. Lastly, visualization of checkpoint proteins localized to damage sites serves as a useful tool for analysis of DNA damage in living cells.
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Abstract Background: KRAS is among the most commonly mutated human cancer genes, and is mutated in approximately 20% of human tumors. In colorectal cancer KRAS mutation is a predictor of anti-EGFR treatment resistance and different KRAS mutations can lead to different anti-EGFR treatment responses. Despite this, KRAS has proven difficult to target with small molecule inhibitors. Identifying synthetic lethal interactions that occur only in the context of tumor specific KRAS mutations represents one approach that could be used to target tumor cells. Material and methods: To identify KRAS synthetic lethal interactions we have generated functional profiles of constitutively activated KRAS mutant cancers, using an integrated approach that involves cell viability screening. We used a panel of isogenic tumor cell lines carrying different KRAS mutations (G12D, G12S, G12V and G13D) where we performed siRNA screens using a library targeting kinases and kinase-related and tumor suppressor genes. The validation of the siRNA hits was performed in a panel of non-isogenic colorectal and pancreatic cell lines to assess the generality of the effect. By determining the effect on cell viability of each siRNA in each cell model we were able to identify KRAS mutation specific effects. Results: By using high throughput functional viability profiling in KRAS mutant models we have been able to identify a series of genetic dependencies specific for tumor cells with oncogenic KRAS mutations. We have also some exciting preliminary results with small molecule inhibitors, which target some of the genes that we identified as being critical for the survival of KRAS mutant cancers. Conclusion: Using functional viability profiling of a series of KRAS isogenic cell lines we have now identified and confirmed several KRAS synthetically lethal candidate genes. Our subsequent work will focus on dissecting of the molecular mechanisms responsible for these KRAS mutant selective effects, and possible combinations with other drugs clinically available. Citation Format: Sara Costa-Cabral, Eliana Marinari, Marieke Aarts, Jenna L. Riffell, Christopher Torrence, Christopher L. Lord, Alan Ashworth. Using KRAS synthetic lethality to design novel therapeutic approaches to cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B24.
Synthetic Lethality
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The checkpoint kinases Chk1 and Chk2 are central to the induction of cell cycle arrest, DNA repair, and apoptosis as elements in the DNA-damage checkpoint. We report here that in several human tumor cell lines, Chk1 and Chk2 control the induction of the p53 related transcription factor p73 in response to DNA damage. Multiple experimental systems were used to show that interference with or augmentation of Chk1 or Chk2 signaling strongly impacts p73 accumulation. Furthermore, Chk1 and Chk2 control p73 mRNA accumulation after DNA damage. We demonstrate as well that E2F1 directs p73 expression in the presence and absence of DNA damage. Chk1 and Chk2, in turn, are vital to E2F1 stabilization and activity after genotoxic stress. Thus, Chk1, Chk2, E2F1, and p73 function in a pathway mediating p53-independent cell death produced by cytotoxic drugs. Since p53 is often obviated through mutation as a cellular port for anticancer intervention, this pathway controlling p53 autonomous pro-apoptotic signaling is of potential therapeutic importance.
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E2F1
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K-RAS accounts for 90% of RAS mutations in lung adenocarcinomas, the most commonly mutated oncogene in NSCLC, with mutations detected in about 25% of all tumors. Direct inhibition of KRAS has proven clinically challenging. So far, no successful targeted therapy has been developed and remains an elusive target for cancer therapy. Despite significant efforts, currently there are no drugs directly targeting mutated KRAS. Thus, new strategies have emerged for targeting RAS including the use of synthetic lethality. A specific knowledge of individual tumor molecular abnormalities that result in oncogene-specific "synthetic lethal" interactions will allow the rationale to combine promising targeted therapies for KRAS-mutated NSCLC. In this article, we review the new approach based on testing drugs or combinations of agents that work downstream of activated K-RAS.
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Targeted Therapy
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A damage is a general event in the life of cells and may lead to mutation, cancer, and cell/organ death. DNA damage occurring in different phases of cell cycle can activate different damage checkpoint pathways to halt the progress of cell cycle in order to provide time for DNA damage repair. If DNA damage cannot be repaired, cellular apoptosis may be induced. Therefore, DNA damage checkpoint is of great significance for cell survival after DNA damage. This article summarizes recent research on DNA damage responses, including DNA damage checkpoint, DNA damage repair, transcriptional response, and cell apoptosis. We focus on how the DNA damage checkpoint pathway is activated after DNA damage, as well as the functional mechanism of the DNA damage checkpoint pathway. The review aims to help readers understand the great significance of DNA damage checkpoint pathway, providing a theoretical basis for its application in radiotherapy and chemotherapy for cancer.
Key words:
DNA damage; Checkpoint pathway; Cell cycle; Signal transduction; Cell apoptosis
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On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12. Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.
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Checkpoint Kinase 2
DNA re-replication
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