DNA Damage Checkpoint Response Research Articles

Pharmacological Inhibition of the DNA Damage Checkpoint Prevents Radiation-Induced Oocyte Death.

Ovarian function is directly correlated with survival of the primordial follicle reserve. Women diagnosed with cancer have a primary imperative of treating the cancer, but since the resting oocytes are hypersensitive to the DNA-damaging modalities of certain chemo- and radiotherapeutic regimens, such patients face the collateral outcome of premature loss of fertility and ovarian endocrine function. Current options for fertility preservation primarily include the collection and cryopreservation of oocytes or in vitro-fertilized oocytes, but this necessitates a delay in cancer treatment and additional assisted reproductive technology procedures. Here, we evaluated the potential of pharmacological preservation of ovarian function by inhibiting a key element of the oocyte DNA damage checkpoint response, checkpoint kinase 2 (CHK2; CHEK2). Whereas nonlethal doses of ionizing radiation (IR) eradicate immature oocytes in wild-type mice, irradiated Chk2-/- mice retain their oocytes and, thus, fertility. Using an ovarian culture system, we show that transient administration of the CHK2 inhibitor 2-(4-(4-chlorophenoxy)phenyl)-1H-benzimidazole-5-carboxamide-hydrate ("CHK2iII") blocked activation of the CHK2 targets TRP53 and TRP63 in response to sterilizing doses of IR, and preserved oocyte viability. After transfer into sterilized host females, these ovaries proved functional and readily yielded normal offspring. These results provide experimental evidence that chemical inhibition of CHK2 is a potentially effective treatment for preserving the fertility and ovarian endocrine function of women exposed to DNA-damaging cancer therapies such as IR.

Abstract 2026: CHK1 inhibition synergizes with cisplatin to promote mitotic cell death via activation of caspase 2 and downregulation of E2F1 in small cell lung cancer

Abstract Platinum-based chemotherapy remains the standard treatment for patients with small cell lung cancer (SCLC), one of the deadliest cancers, but the benefit of such therapy is often hampered by rapid emergence of drug resistance. Thus far, there is no targeted therapy available for SCLC, mainly due to the lack of targetable oncogenic driver alterations; nevertheless, over 90% of SCLC tumors harbor inactivating mutations in tumor suppressor p53, an important checkpoint regulator for the control of cell cycle progression in the event of DNA damage. SCLC cells with p53-deficiency heavily rely on the checkpoint kinase 1 (CHK1), a key checkpoint regulator, to prevent the progression of cell cycle through G2/M in the presence of damaged DNA. Using CHK1 inhibitors and siRNA knockdown approaches, we explored the potential therapeutic benefit of CHK1 inhibition in SCLC models in combination with cisplatin, a DNA damaging agent. We demonstrated that CHK1 inhibition was able to release G2/M cell cycle arrest induced by cisplatin, and synergized with cisplatin to induce mitotic cell death especially in the p53-deficient SCLC cell lines. CHK1 inhibition activated caspase 2 and downregulated the expression of E2F1 transcription factor in these cells. We showed that activation of caspase 2 was required for the mitotic cell death induced by the combination of cisplatin and CHK1 inhibition. Interestingly, the upregulation of E2F1 induced by CHK1 inhibition was mediated in part by caspase 2, and E2F1 played a role in the DNA damage checkpoint control as knockdown of E2F1 enhanced cytotoxicity induced by cisplatin or CHK1 inhibitor in GLC4 cells. However, ectopic overexpression of E2F1 also led to increase of phospho-histone H3, γH2AX and cleaved PARP, and significantly enhanced cisplatin cytotoxicity, indicating that proper dosage of E2F1 is critical for DNA damage checkpoint response. These data provide a mechanistic insight of CHK1-mediated DNA damage checkpoint control and the anticancer effect of CHK1 inhibition against SCLC. In tumor samples derived from SCLC patients treated with platinum based chemotherapy, we found that high expression of CHK1 and E2F1 significantly correlated with poorer overall survival, further suggesting a link between CHK1 and E2F1 and potentially collaborative roles of these two factors in DNA damage checkpoint response. Moreover, we showed that CHK1 inhibition resensitized cisplatin-resistant cells to cisplatin treatment and induced mitotic cell death via caspase activation in these cells. These data indicate that CHK1 inhibition may enhance the benefit of cisplatin treatment for SCLC and overcome cisplatin resistance. We are presently confirming the in vitro results of CHK1 inhibition in combination with cisplatin treatment in xenograft tumor models in athymic nude mice. Citation Format: Wei-Hsun Hsu, Xiaoliang Zhao, In-Gyu Kim, Guanhua Rao, Justine McCutcheon, Shuo-Tse Hsu, Afshin Dowlati, Yu-Wen Zhang, Giuseppe Giaccone. CHK1 inhibition synergizes with cisplatin to promote mitotic cell death via activation of caspase 2 and downregulation of E2F1 in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2026. doi:10.1158/1538-7445.AM2017-2026

Abstract 841: UTR analysis identifies Musashi1 as a translational regulator of radiation-induced Golgi-related gene expression

Abstract The cellular radioresponse is regulated by post-translational modification of constitutively expressed proteins and by changes in gene expression. Although the role of constitutively expressed proteins, such as those involved in the DNA damage response and cell cycle checkpoints, is well-understood in the context of the radioresponse, radiation-induced changes in gene expression are not as clearly defined. We have previously demonstrated that glioblastoma stem-like cells (GSCs) alter their gene expression at the translational level in response to ionizing radiation (IR). In particular, the NSC11 GSC line demonstrated thousands of genes differentially translated at one hour after a dose of 2 Gy, while considerably fewer were modified at the transcriptome level. Furthermore, there was little overlap between the transcriptome and the translatome. Genes undergoing translational regulation in response to IR fell into distinct functional categories, including mitochondrial structure and function, Golgi function, and translational regulation. Here, we aimed to elucidate the mechanism by which IR induces translational changes in the NSC11 GSC line. NSC11 cells were either exposed to 2 Gy or mock irradiated, and polysomes were isolated by sucrose gradient fractionation one hour later. Polysome-bound mRNA was purified and analyzed by microarray. The 5’ and 3’ untranslated regions (UTRs) of the top 75 up- and down-regulated genes were analyzed for the presence of regulatory elements. The most frequently occurring element in the UTRs of both the top up- and down-regulated genes was the Musashi1 binding site. Musashi1 is an RNA binding protein that typically binds to 3’ UTRs to regulate mRNA translation. It is a neural stem cell marker, and its expression is correlated with the grade of malignancy in glioma. GO term analysis of known Musashi1 target genes that were translationally regulated in response to IR revealed an enrichment of genes involved in Golgi function and vesicular trafficking. Knockdown of Musashi1 prevented IR-induced Golgi dispersal in NSC11 cells. Musashi1 knockdown also radiosensitized NSC11 cells, indicating a role for the protein in the cellular radioresponse. Together, these data demonstrate that in the NSC11 GSC line, Musashi1 contributes to radiation-induced translational regulation, specifically in the context of Golgi-related genes, and suggests that Musashi1 is a novel target for radiosensitization. Citation Format: Stacey L. Lehman, John W. O'Neill, Kevin Camphausen, Philip J. Tofilon. UTR analysis identifies Musashi1 as a translational regulator of radiation-induced Golgi-related gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 841. doi:10.1158/1538-7445.AM2017-841

Abstract 4733: Dysregulation of CUL4A and CUL4B ubiquitin ligases in lung cancer

Abstract Background: The Cullin-RING ubiquitin ligases 4 (CRL4) is implicated in controlling cell cycle, DNA damage repair and checkpoint response based on studies employing cell lines and mouse models. CRL4 proteins, including CUL4A and CUL4B are often highly accumulated in human malignancies. Elevated CRL4 attenuates DNA damage repair and increases genome instability that is believed to facilitate tumorigenesis. However, this has yet to be evaluated in human cancer patients. Methods: 352 lung cancer and 62 benign lung specimens of Asian origin were constructed into tissue microarrays (TMAs) of four distinct lung cancer subtypes. Expression of CUL4A, CUL4B and their substrates was detected by immunohistochemistry (IHC), and analyzed statistically for their prognostic value and association with DNA damage response and genomic instability. Results: Both CUL4A and CUL4B are overexpressed in the majority of lung carcinomas (PCUL4A<0.001, PCUL4B<0.001), and significantly associated with tumor size (PCUL4A<0.001, PCUL4B=0.002), lymphatic invasion (PCUL4A=0.004, PCUL4B<0.001), metastasis (PCUL4A=0.019, PCUL4B=0.006), and advanced TNM stage (PCUL4A<0.001, PCUL4B<0.001), which parallels gene amplification and abnormal activation of the canonical Wnt signaling. Moreover, overexpression of CUL4A, but not CUL4B, is significantly associated with tobacco smoking (P=0.01), and inversely correlated with XPC and p21, both of which are substrates of CUL4A (PCUL4A=0.019, PCUL4B=0.006). Higher levels of CUL4A or CUL4B are significantly associated with patients’ OS (PCUL4A<0.001, PCUL4B<0.001) and PFS (PCUL4A<0.001, PCUL4B=0.001). Conclusion: Our findings revealed that CUL4A and CUL4B are differentially associated with etiological factors for pulmonary malignancies and are independent prognostic markers for survival of distinct lung cancer subtypes. Citation Format: Hui Li, Lei Jia, Fan Yan, Pengbo Zhou. Dysregulation of CUL4A and CUL4B ubiquitin ligases in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4733. doi:10.1158/1538-7445.AM2017-4733

Tumor-initiating cells contribute to radiation resistance in primary human renal clear cell carcinomas by activating the DNA damage checkpoint response.

The use of radiotherapy in patients with clear cell renal carcinoma (ccRCC) is predominantly limited to palliation of metastases or control of local growth, because ccRCC cells readily develop radioresistance. The mechanisms underlying ccRCC resistance remain elusive. The present study demonstrated that ccRCC cells that survive fractionated radiation treatment display tumor-initiating cell (TIC) characteristics, such as high self-renewal and tumorigenic capacities, and overexpress stemness genes. ccRCC cells that survived fractionated radiation exhibited increased activation of the DNA damage checkpoint response and G2/M phase arrest compared with sham-irradiated cells. The results of the present study suggest that ionizing radiation destroys the bulk of tumor cells within ccRCC, but spares TICs; this subpopulation confers ccRCC radioresistance and may cause tumor recurrence or relapse following radiotherapy. Furthermore, these findings indicate that the DNA damage checkpoint response may serve as a potential therapeutic target for overcoming resistance of TICs in patients with ccRCC.

Ras-MEK Signaling Mediates a Critical Chk1-Dependent DNA Damage Response in Cancer Cells.

Cancer cell line profiling to identify previously unrecognized kinase dependencies revealed a novel nonmutational dependency on the DNA damage response checkpoint kinase Chk1. Although Chk1 is a promising therapeutic target in p53-deficient cancers, we found that Ras-MEK signaling engages Chk1 in a subset of osteosarcoma, ovarian, and breast cancer cells to enable their survival upon DNA damage, irrespective of p53 mutation status. Mechanistically, Ras-MEK signaling drives Chk1 expression and promotes cancer cell growth that produces genotoxic stress that requires Chk1 to mediate a response to the consequent DNA damage. Reciprocally, Chk1 engages a negative feedback loop to prevent hyperactivation of Ras-MEK signaling, thereby limiting DNA damage. Furthermore, exogenous DNA damage promotes Chk1 dependency, and pharmacologic Chk1 inhibition combined with genotoxic chemotherapy potentiates a DNA damage response and tumor cell killing. These findings reveal a mechanism-based diagnostic strategy to identify cancer patients that may benefit from Chk1-targeted therapy. Mol Cancer Ther; 16(4); 694-704. ©2017 AACR.

NBS1 Phosphorylation Status Dictates Repair Choice of Dysfunctional Telomeres

Telomeres employ TRF2 to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), thereby repressing ATM-dependent DNA damage checkpoint responses. How TRF2 prevents MRN activation at dysfunctional telomeres is unclear. Here, we show that the phosphorylation status of NBS1 determines the repair pathway choice of dysfunctional telomeres. The crystal structure of the TRF2-NBS1 complex at 3.0Å resolution shows that the NBS1 429YQLSP433 motif interacts specifically with the TRF2TRFH domain. Phosphorylation of NBS1 serine 432 by CDK2 in S/G2 dissociates NBS1 from TRF2, promoting TRF2-Apollo/SNM1B complex formation and the protection of leading-strand telomeres. Classical-NHEJ-mediated repair of telomeres lacking TRF2 requires phosphorylated NBS1S432 to activate ATM, while interaction of de-phosphorylated NBS1S432 with TRF2 promotes alternative-NHEJ repair of telomeres lacking POT1-TPP1. Our work advances understanding of how the TRF2TRFH domain orchestrates telomere end protection and reveals how the phosphorylation status of the NBS1S432 dictates repair pathway choice of dysfunctional telomeres.

Inhibition of Chk1 with the small molecule inhibitor V158411 induces DNA damage and cell death in an unperturbed S-phase.

Chk1 kinase is a critical component of the DNA damage response checkpoint and Chk1 inhibitors are currently under clinical investigation. Chk1 suppresses oncogene-induced replication stress with Chk1 inhibitors demonstrating activity as a monotherapy in numerous cancer types. Understanding the mechanism by which Chk1 inhibitors induce DNA damage and cancer cell death is essential for their future clinical development. Here we characterize the mechanism by which the novel Chk1 inhibitor (V158411) increased DNA damage and cell death in models of human cancer. V158411 induced a time- and concentration-dependent increase in γH2AX-positive nuclei that was restricted to cells actively undergoing DNA synthesis. γH2AX induction was an early event and correlated with activation of the ATR/ATM/DNA-PKcs DNA damage response pathways. The appearance of γH2AX positive nuclei preceded ssDNA appearance and RPA exhaustion. Complete and sustained inhibition of Chk1 kinase was necessary to activate a robust γH2AX induction and growth inhibition. Chk1 inhibitor cytotoxicity correlated with induction of DNA damage with cells undergoing apoptosis, mitotic slippage and DNA damage-induced permanent cell cycle arrest. We identified two distinct classes of Chk1 inhibitors: those that induced a strong increase in γH2AX, pChk1 (S317) and pRPA32 (S4/S8) (including V158411, LY2603618 and ARRY-1A) and those that did not (including MK-8776 and GNE-900). Tumor cell death, induced through increased DNA damage, coupled with abrogation of cell cycle checkpoints makes selective inhibitors of Chk1 a potentially useful therapeutic treatment for multiple human cancers.

Mdc1 modulates the interaction between TopBP1 and the MRN complex during DNA damage checkpoint responses

TopBP1 has been identified as a direct activator of ATR and interacts with the Nbs1 subunit of the Mre11-Rad50-Nbs1 (MRN) complex in egg extracts in a checkpoint-regulated manner. In this study, we show that Mdc1 associates with both TopBP1 and Nbs1 in egg extracts and human cells. We cloned a cDNA encoding the full-length version of Xenopus Mdc1. The association between Mdc1 and TopBP1 involves the first pair of BRCT repeats in TopBP1. The N-terminal region (161–230) of Mdc1 is required for this binding. The interaction between Mdc1 and Nbs1 involves the two tandem BRCT repeats of Nbs1. Functional studies with mutated forms of Mdc1, TopBP1, and Nbs1 indicate that the BRCT-dependent association of these proteins is critical for a normal checkpoint response to DSBs. TopBP1 cannot interact with Nbs1 in Mdc1-depleted egg extracts, suggesting that Mdc1 connects TopBP1 and Nbs1 together. These findings suggest that Mdc1 is a crucial mediator of the DNA damage checkpoint response.

Abstract 2729: Estrogen receptor-negative breast cancer cell lines exhibit hypersensitivity to the CHK1 inhibitor LY2606368

Abstract Checkpoint kinase 1 (CHK1) inhibitors are currently under investigation as chemopotentiating agents due to the role of CHK1 in establishing DNA damage checkpoints in the cell cycle. A novel CHK1/CHK2 inhibitor, LY2606368, as a single agent causes replication catastrophe, DNA double strand breaks and apoptosis (King C et al. Mol Cancer Ther. 2015). Accordingly, LY2606368 is currently in clinical development as a single agent and in combination with both cytotoxic and targeted agents. As subsets of breast cancers exhibit genomic instability and DNA repair deficiencies, we assessed the effect of LY2606368 as a single agent on breast cancer cell lines. We characterized the IC50's for growth inhibition by LY2606368 of a large panel of cell lines assembled on the basis of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression status. Triple negative breast cancer (TNBC) is a subtype of breast cancer that is pathologically negative for expression of ER/PR and HER2 protein. As previously reported for other CHK1 inhibitors (Bryant C et al. BMC Cancer. 2014; Albiges L et al. Breast. 2014), TNBC cell lines exhibited high sensitivity to LY2606368. Interestingly, hypersensitivity to LY2606368 was observed in ER-negative cells, regardless of HER2 status. In addition, ER-positive cells were comparatively resistant suggesting that high sensitivity to LY2606368 occurs in the absence of ER and is not restricted to TNBC. No correlation was found between TP53 mutational status and sensitivity to LY2606368. Consistent with the observed hypersensitivity, ER-negative cell lines exposed to LY2606368 exhibited high levels of γH2AX and phospho-Ser1981-ATM demonstrating appearance of DNA double strand breaks. The concomitant appearance of phospho-Ser345-Chk1 marked aborted checkpoint activation by upstream detector/effector kinases (e.g. ATR, ATM). ER-positive cells did not engage in initiation of the DNA damage response or significant checkpoint activation when exposed to comparable doses of LY2606368. Collectively, these results suggest that the CHK1 inhibitor LY2606368 is likely to be more cytotoxic in ER-negative breast cancer types. Homologous recombination and other DNA repair deficiencies can explain the need for CHK1-dependent checkpoint activation during each replication cycle. On the other hand, ER-positive breast cancers might be less sensitive to monotherapy because of the absence of unresolved DNA damaging events that create checkpoint dependency. In summary, triple negative status does not directly determine the sensitivity of breast cancer cell lines to the CHK1 inhibitor LY2606368, although TNBC cells are in general hypersensitive. The absence of ER might be a more promising marker of sensitivity to LY2606368 as a monotherapy in breast cancer. Citation Format: Jean-Bernard Lazaro, Kalindi Parmar, Geoffrey I. Shapiro, Alan D. D’Andrea. Estrogen receptor-negative breast cancer cell lines exhibit hypersensitivity to the CHK1 inhibitor LY2606368. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2729.

Leishmania major and Trypanosoma cruzi present distinct DNA damage responses

Leishmania major and Trypanosoma cruzi are medically relevant parasites and interesting model organisms, as they present unique biological processes. Despite increasing data regarding the mechanisms of gene expression regulation, there is little information on how the DNA damage response (DDR) occurs in trypanosomatids. We found that L. major presented a higher radiosensitivity than T. cruzi. L. major showed G1 arrest and displayed high mortality in response to ionizing radiation as a result of the inefficient repair of double-strand breaks (DSBs). Conversely, T. cruzi exhibited arrest in the S/G2 cell cycle phase, was able to efficiently repair DSBs and did not display high rates of cell death after exposure to gamma irradiation. L. major showed higher resistance to alkylating DNA damage, and only L. major was able to promote DNA repair and growth recovery in the presence of MMS. ASF1c overexpression did not interfere with the efficiency of DNA repair in either of the parasites but did accentuate the DNA damage checkpoint response, thereby delaying cell fate after damage. The observed differences in the DNA damage responses of T. cruzi and L. major may originate from the distinct preferred routes of genetic plasticity of the two parasites, i.e., DNA recombination versus amplification.

The Deubiquitinase USP9X Maintains DNA Replication Fork Stability and DNA Damage Checkpoint Responses by Regulating CLASPIN during S-Phase.

Coordination of the multiple processes underlying DNA replication is key for maintaining genome stability and preventing tumorigenesis. CLASPIN, a critical player in replication fork stabilization and checkpoint responses, must be tightly regulated during the cell cycle to prevent the accumulation of DNA damage. In this study, we used a quantitative proteomics approach and identified USP9X as a novel CLASPIN-interacting protein. USP9X is a deubiquitinase involved in multiple signaling and survival pathways whose tumor suppressor or oncogenic activity is highly context dependent. We found that USP9X regulated the expression and stability of CLASPIN in an S-phase-specific manner. USP9X depletion profoundly impairs the progression of DNA replication forks, causing unscheduled termination events with a frequency similar to CLASPIN depletion, resulting in excessive endogenous DNA damage. Importantly, restoration of CLASPIN expression in USP9X-depleted cells partially suppressed the accumulation of DNA damage. Furthermore, USP9X depletion compromised CHK1 activation in response to hydroxyurea and UV, thus promoting hypersensitivity to drug-induced replication stress. Taken together, our results reveal a novel role for USP9X in the maintenance of genomic stability during DNA replication and provide potential mechanistic insights into its tumor suppressor role in certain malignancies. Cancer Res; 76(8); 2384-93. ©2016 AACR.

How DNA‐Damage Machinery Protects the Genomes of Mouse Oocytes

During meiosis hundreds of endogenously induced DNA double stranded breaks (DSB) are made by the SPO11 endonuclease in order to assure homologous chromosome pairing (synapsis) and their proper segregation. Because DSBs are potentially mutagenic, quality control mechanisms are in place to guarantee that they are all repaired, preferentially by interhomolog recombination, otherwise apoptosis is induced. It is currently accepted that this “pachytene checkpoint” senses DSB repair and synapsis by independent mechanisms. However, we have experimental evidence that oocytes defective for either DNA repair or synapsis are both eliminated by a canonical DNA damage response checkpoint pathway (dependent upon signaling of the CHK2 kinase to downstream effectors p53 and p63). During the first meiotic division, mechanisms are in place to inhibit the DNA‐repair machinery from using the sister chromatid as recombination repair donors, making the homologous chromosome the substrate of choice. Whereas this block to sister chromatid repair of DSBs assures interhomolog (IH) recombination, it also prevents the chromosomes that fail to undergo IH repair from repairing residual DSBs. Here, we present evidence consistent with a model whereby asynaptic oocytes accumulate SPO11‐independent DSBs, and are eliminated via CHK2 activation.

WWOX modulates the ATR-mediated DNA damage checkpoint response.

For many decades genomic instability is considered one of the hallmarks of cancer. Role of the tumor suppressor WWOX (WW domain-containing oxidoreductase) in DNA damage response upon double strand breaks has been recently revealed. Here we demonstrate unforeseen functions for WWOX upon DNA single strand breaks (SSBs) checkpoint activation. We found that WWOX levels are induced following SSBs and accumulate in the nucleus. WWOX deficiency is associated with reduced activation of ataxia telangiectasia and Rad3-related protein (ATR) checkpoint proteins and increased chromosomal breaks. At the molecular level, we show that upon SSBs WWOX is modified at lysine 274 by ubiquitination mediated by the ubiquitin E3 ligase ITCH and interacts with ataxia telangiectasia-mutated (ATM). Interestingly, ATM inhibition was associated with reduced activation of ATR checkpoint proteins suggesting that WWOX manipulation of ATR checkpoint proteins is ATM-dependent. Taken together, the present findings indicate that WWOX plays a key role in ATR checkpoint activation, while its absence might facilitate genomic instability.

Identification of novel, in vivo active Chk1 inhibitors utilizing structure guided drug design.

Chk1 kinase is a critical component of the DNA damage response checkpoint especially in cancer cells and targeting Chk1 is a potential therapeutic opportunity for potentiating the anti-tumor activity of DNA damaging chemotherapy drugs. Fragment elaboration by structure guided design was utilized to identify and develop a novel series of Chk1 inhibitors culminating in the identification of V158411, a potent ATP-competitive inhibitor of the Chk1 and Chk2 kinases. V158411 abrogated gemcitabine and camptothecin induced cell cycle checkpoints, resulting in the expected modulation of cell cycle proteins and increased cell death in cancer cells. V158411 potentiated the cytotoxicity of gemcitabine, cisplatin, SN38 and camptothecin in a variety of p53 deficient human tumor cell lines in vitro, p53 proficient cells were unaffected. In nude mice, V158411 showed minimal toxicity as a single agent and in combination with irinotecan. In tumor bearing animals, V158411 was detected at high levels in the tumor with a long elimination half-life; no pharmacologically significant in vivo drug-drug interactions with irinotecan were identified through analysis of the pharmacokinetic profiles. V158411 potentiated the anti-tumor activity of irinotecan in a variety of human colon tumor xenograft models without additional systemic toxicity. These results demonstrate the opportunity for combining V158411 with standard of care chemotherapeutic agents to potentiate the therapeutic efficacy of these agents without increasing their toxicity to normal cells. Thus, V158411 would warrant further clinical evaluation.

BMI1, ATM and DDR.

BMI1, ATM and DDR.

Centrosome aberrations in human mammary epithelial cells driven by cooperative interactions between p16INK4a deficiency and telomere-dependent genotoxic stress.

Virtually all human cancers display chromosome instability (CIN), a condition in which chromosomes are gained or lost at a high rate. CIN occurs early in cancer development where it may undermine the advance of the neoplastic disease. With the aim of establishing the mechanisms underlying CIN in cancer, we investigated possible links between telomere-dysfunction and centrosome defects, which were seen to coincide in early in breast carcinogenesis using human mammary epithelial cells (HMECs). In this study, we show that TP53 proficient vHMECs cells develop centrosome aberrations when telomere-dysfunction genotoxic stress is produced in the presence of a defective p16INK4a setting and in parallel with an activation of the DNA damage checkpoint response. These aberrations consist of the accumulation of centrosomes in polyploid vHMECs, plus centriole overduplication in both diploid and polyploid cells, thus reflecting that distinct mechanisms underlie the generation of centrosome aberrations in vHMECs. Transduction of vHMEC with hTERT, which rescued the telomere dysfunction phenotype and consequently reduced DNA damage checkpoint activation, led to a progressive reduction of centrosome aberrations with cell culture, both in diploid and in polyploid vHMECs. Radiation-induced DNA damage also raised centrosome aberrations in vHMEC-hTERT. Collectively, our results, using vHMECs define a model where p16INK4a deficiency along with short dysfunctional telomeres cooperatively engenders centrosome abnormalities before p53 function is compromised.

Chronic Replication Problems Impact Cell Morphology and Adhesion of DNA Ligase I Defective Cells.

Moderate DNA damage resulting from metabolic activities or sub-lethal doses of exogenous insults may eventually lead to cancer onset. Human 46BR.1G1 cells bear a mutation in replicative DNA ligase I (LigI) which results in low levels of replication-dependent DNA damage. This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence. Stable transfection of wild type LigI, as in 7A3 cells, prevents DNA damage and ATM activation. Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration. Comparison of gene expression profiles in the two cell lines detects Bio-Functional categories consistent with the morphological and migration properties of LigI deficient cells. Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors. These observations extend the influence of the DNA damage response checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.

Utilizing murine inducible telomerase alleles in the studies of tissue degeneration/regeneration and cancer.

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.