Cell Cycle Checkpoint Defects Research Articles

Deposition of onco-histone H3.3-G34W leads to DNA repair deficiency and activates cGAS/STING-mediated immune responses.

Mutations in histone H3.3-encoding genes causing mutant histone tails are associated with specific cancers such as pediatric glioblastomas (H3.3-G34R/V) and giant cell tumor of the bone (H3.3-G34W). The mechanisms by which these mutations promote malignancy are not completely understood. Here we show that cells expressing H3.3-G34W exhibit DNA double-strand breaks (DSBs) repair defects and increased cellular sensitivity to ionizing radiation (IR). Mechanistically, H3.3-G34W can be deposited to damaged chromatin, but in contrast to wild-type H3.3, does not interact with non-homologous end-joining (NHEJ) key effectors KU70/80 and XRCC4 leading to NHEJ deficiency. Together with defective cell cycle checkpoints reported previously, this DNA repair deficiency in H3.3-G34W cells led to accumulation of micronuclei and cytosolic DNA following IR, which subsequently led to activation of the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, thereby inducing release of immune-stimulatory cytokines. These findings suggest a potential for radiotherapy for tumors expressing H3.3-G34W, which can be further improved by combination with STING agonists to induce immune-mediated therapeutic efficacy.

CSIG-15. DEFINING THE ROLE OF HISTONE H3 K27 METHYLATION AND H3.3 S31 PHOSPHORYLATION IN H3.3K27M MUTANT DIFFUSE MIDLINE GLIOMAS

Abstract Diffuse midline gliomas (DMG) are aggressive high-grade gliomas that characteristically occurs in children. Heterozygous K27M mutation occurs in histone H3.3 as early events in DMG and are sufficient to drive a global reduction of H3K27 methylation (H3K27me) on all histone H3 variants. Histone H3K27me is responsible for epigenetic gene repression, and loss of K27me is associated with epigenetic dysregulation. However, histone H3.3 has a unique Chk1 phosphorylation site, Serine 31, critical for mitotic checkpoint regulation. Masking of S31 phosphorylation results in the abrogation of cell cycle checkpoints that monitor the alignment and segregation of chromosomes. DMG cells have decreased levels of mitotic S31P and are chromosomally unstable and aneuploid. Genomic-editing of H3.3 to revert the K27M mutation restores mitotic S31P and K27me and significantly reduces the rate of chromosome instability (CIN). Expression of H3.3 K27M or a non-phosphorylatable S31A mutant in normal, diploid cells results in chromosome missegregation and cell cycle checkpoint defects; and cells fail to undergo G1 cycle arrest leading to aneuploidy. H3.3 K27M and S31A inhibit p53 accumulation. In a mouse model of DMG, H3.3K27M and H3.3S31A promoted the development of high-grade gliomas, whereas H3.3WT controls did not. H3.3S31A is WT for K27me, demonstrating that loss of S31P is oncogenic. It remains unclear if it is the H3.3 K27M mutations or the associated loss of H3K27me3 that drives the reduction of S31P. The PRC2 complex is responsible for H3K27me, catalyzed by methyltransferases subunits EZH1 and EZH2. We used genome editing to knockout EZH1 and EZH2 in DMG cells and untransformed human diploid cell lines. Deletion of EZH2 causes a loss in K27 tri-methylation, and EZH1/2 knockouts causes a global loss of K27me. Ongoing studies examine how the loss of K27me affects gene expression, proliferation, H3.3S31P, chromosome missegregation, and aneuploid.

Automated classification of mitotic catastrophe by use of the centromere fragmentation morphology.

Mitotic catastrophe is a common mode of tumor cell death. Cancer cells with a defective cell-cycle checkpoint often enter mitosis with damaged or under replicated chromosomes following genotoxic treatment. Premature condensation of the under-replicated (or damaged) chromosomes results in double-stranded DNA breaks at the centromere (centromere fragmentation). Centromere fragmentation is a morphological marker of mitotic catastrophe and is distinguished by the clustering of centromeres away from the chromosomes. We present an automated 2-step system for segmentation of cells exhibiting centromere fragmentation. The first step segments individual cells from clumps. We added two new terms, weighted local repelling term (WLRt) and weighted gradient term (WGt), in the energy functional of the traditional Chan-Vese based level set method. WLRt was used to generate a repelling force when contours of adjacent cells merged and then penalized the overlap. WGt enhances gradients between overlapping cells. The second step consists of a new algorithm, SBaN (shape-based analysis of each nucleus), which extracts features like circularity, major-axis length, minor-axis length, area, and eccentricity from each chromosome to identify cells with centromere fragmentation. The performance of SBaN algorithm for centromere fragmentation detection was statistically evaluated and the results were robust.

Abstract 1374: Preclinical investigation of ATR inhibition alone and in combination with PARP inhibition in high risk neuroblastoma

Abstract Background:Neuroblastoma (NB) is the commonest extra-cranial malignant solid tumour of childhood and one of the most difficult to cure. DNA damage response (DDR) defects are frequently observed in high risk NB including allelic loss and loss of function mutations in key DDR genes, oncogene induced replication stress (RS) and cell cycle checkpoint dysfunction. Cancer cells with defective cell cycle checkpoint signalling and/or increased oncogene-driven RS are acutely dependent on the DNA damage sensor kinase ATR. This study aims to identify features of NB cell lines leading to ATR inhibitor sensitivity. As PARP inhibition causes RS through unrepaired single strand DNA breaks progressing to replication, we hypothesise that ATR inhibition will increase PARP inhibitor cytotoxicity. Aims: 1) To determine which molecular features lead to sensitivity to VE-821 (ATR inhibitor) in cell lines derived from high-risk NB tumours. 2) To assess synergism between PARP inhibition with olaparib and ATR inhibition in high risk NB cell lines and to measure RS. Materials and Methods: Cell proliferation in response to 72 hours treatment with VE-821 was assessed by XTT assay (Roche) in a panel of 11 NB cell lines and the effect of ATR inhibition on olaparib growth inhibition in 4 cell lines: SHSY5Y, SKNAS, NGP and N20_R1. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS respectively. RS was also measured by γH2AX foci formation using immunofluorescent microscopy. Results: VE-821 caused significantly more growth inhibition in MYCN amplified cell lines and cell lines with low ATM protein expression by XTT assay (p<0.05 Mann-Whitney U test). Olaparib (5 µM) treatment increased CHK1S345 and H2AXS129 phosphorylation after 24 hours treatment in all cell lines. H2AXS129 phosphorylation and foci number was further increased with the addition of VE-821 (1 µM). ATR inhibition prevented CHK1S345 phosphorylation. In cell proliferation assays, combination index analysis (Calcusyn) showed that ATR inhibition by VE-821 is synergistic with olaparib at sub lethal concentrations (<1 µM) (CI value 0.04-0.89). Conclusion: MCYN amplification and low ATM protein expression are determinants of ATRi sensitivity in NB cell lines. ATR inhibition by VE-821 is synergistic with olaparib at sub lethal concentrations (<1 µM) and further increases the replication stress caused by PARP inhibition. Citation Format: Harriet E. Southgate, Lindi Chen, Nicola J. Curtin, Deborah A. Tweddle. Preclinical investigation of ATR inhibition alone and in combination with PARP inhibition in high risk neuroblastoma [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 1374.

Synthetic lethality strategies: Beyond BRCA1/2 mutations in pancreatic cancer.

Cancer cells are often characterized by abnormalities in DNA damage response including defects in cell cycle checkpoints and/or DNA repair. Synthetic lethality between DNA damage repair (DDR) pathways has provided a paradigm for cancer therapy by targeting DDR. The successful example is that cancer cells with BRCA1/2 mutations are sensitized to poly(adenosine diphosphate [ADP]‐ribose)polymerase (PARP) inhibitors. Beyond the narrow scope of defects in the BRCA pathway, “BRCAness” provides more opportunities for synthetic lethality strategy. In human pancreatic cancer, frequent mutations were found in cell cycle and DDR genes, including P16, P73, APC, MLH1, ATM, PALB2, and MGMT. Combined DDR inhibitors and chemotherapeutic agents are under preclinical or clinical trials. Promoter region methylation was found frequently in cell cycle and DDR genes. Epigenetics joins the Knudson's “hit” theory and “BRCAness.” Aberrant epigenetic changes in cell cycle or DDR regulators may serve as a new avenue for synthetic lethality strategy in pancreatic cancer.

53BP1 loss rescues embryonic lethality but not genomic instability of BRCA1 total knockout mice

BRCA1 is critical for DNA double-strand break (DSB) repair by homologous recombination (HR). BRCA1 deficient mice are embryonic lethal. Previous studies have shown that 53BP1 knockout (KO) rescues embryonic lethality of BRCA1 hypomorphic mutant mice by restoring HR. Here, we show that 53BP1 KO can partially rescue embryonic lethality of BRCA1 total KO mice, but HR is not restored in BRCA1-53BP1 double knockout (DKO) mice. As a result, BRCA1-53BP1 DKO cells are extremely sensitive to PARP inhibitors (PARPi). In addition to HR deficiency, BRCA1-53BP1 DKO cells have elevated microhomology-mediated end joining (MMEJ) activity and G2/M cell cycle checkpoint defects, causing severe genomic instability in these cells. Interestingly, BRCA1-53BP1 DKO mice rapidly develop thymic lymphoma that is 100% penetrant, which is not observed in any BRCA1 mutant mice rescued by 53BP1 KO. Taken together, our study reveals that 53BP1 KO can partially rescue embryonic lethality caused by complete BRCA1 loss without rescuing HR-related defects. This finding suggests that loss of 53BP1 can support the development of cancers with silenced BRCA1 expression without causing PARPi resistance.

Abstract P5-04-26: Identification of preclinical mechanisms driving acquired resistance to endocrine therapy in estrogen-receptor positive (ER+) breast cancer cells

Abstract Estrogen Receptor positive (ER+) breast cancer accounts for the majority of breast cancer cases and standard of care for these tumors is treatment with endocrine therapy, including the blockade of estrogen production (i.e. aromatase inhibitors; AIs) as well as the use of antagonists of ER function, i.e. selective estrogen receptor modulators (SERMs, i.e. tamoxifen) and selective estrogen receptor degraders (SERDs, i.e. fulvestrant). Despite the initial dependency of ER+ breast tumors on estrogen and ER for their survival and proliferation, treatment in the metastatic setting invariably leads to therapeutic resistance. While mechanisms of resistance to AIs include mutations in the estrogen receptor gene ESR1, less is known about mechanisms of resistance to SERMs and SERDs, thus it is essential to further investigate the latter, in order to successfully treat relapsed patients. To pre-clinically model cell-autonomous acquired resistance to these agents, we used T47D, an ER+ and p53- estrogen-responsive cell line treated with increasing concentrations of the SERM/SERD hybrid (SSH) ER-targeting agent GDC-0810 over the period of several months during which individual clones with acquired resistance to GDC-0810 were selected. GDC-0810-resistant clones were cross-resistant to other endocrine agents, including SERMs (tamoxifen) and SERDs (fulvestrant), consistent with general loss of dependency on ER. Surprisingly, the cells also lost sensitivity to palbociclib, the latter likely linked to their loss of one copy of the retinoblastoma (Rb) tumor suppressor gene. Comprehensive genetic and phenotypic characterization of the resistant clones relative to the parental cells revealed multiple mutations and deletions in DNA repair and cell cycle genes, and associated defects in DNA repair and cell cycle checkpoints. Cell cycle, proteomic, and mRNA expression analysis of parental versus resistant clones at baseline and upon DNA damage, identified a distinct cell cycle profile in the GDC-0810-resistant clones, characterized by accumulation of cells in the mitotic phase. A broad chemical screen identified pharmacologic inhibitors of cell cycle regulators and chemotherapeutic drug classes that preferentially target the ER-independent, GDC-0810 resistant clones compared to the parental cells. Our work provides novel insights into mechanisms and biomarkers of acquired resistant to estrogen therapies in ER+ breast cancer and reveals the acquisition of actionable dependencies that may potentially be exploited in resistant tumors. Furthermore, our studies provide rationale for testing specific chemotherapy regimens upon endocrine resistance accompanied by cell cycle and DNA repair checkpoint dysfunction in ER+ breast cancer. Citation Format: Ong C, Daemen A, Merrick K, O'Brien T, Friedman L, Hatzivassiliou G. Identification of preclinical mechanisms driving acquired resistance to endocrine therapy in estrogen-receptor positive (ER+) breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-26.

The Superior Efficacy of Anti-PD-1/PD-L1 Immunotherapy in KRAS-Mutant Non-Small Cell Lung Cancer that Correlates with an Inflammatory Phenotype and Increased Immunogenicity

Background: Mutations in KRAS are a common oncogene driver detected in approximately 30% of non-squamous non-small cell lung cancer (NSCLC) cases, conferring a poor prognosis without effective targeted therapy. Immune checkpoint inhibitors against PD-1/ PD-L1 yield improved survival rates of KRAS-mutant NSCLC patients while changing the paradigm of cancer therapy. Yet, the underlying association between KRAS mutations and immune responses remains unclear. Methods: We performed an integrated analysis of the data from publicly available repositories and from clinical center cohorts to explore the association between KRAS mutation status and tumor immunity-associated features, including PD-L1 expression, CD8+ tumor-infiltrating lymphocytes (TILs) and tumor mutational burden (TMB). Three-pool analysis was performed to assess the efficacies of different PD-1/PD-L1 inhibitors regarding objective response rates (ORR), durable clinical benefit, and overall survival (OS) in NSCLC patients with or without KRAS mutations. In addition, a KRAS-mutant lung adenocarcinoma mouse model was established to estimate the relative efficacy of anti-PD-L1 monoclonal antibody (mAb) monotherapy or combination treatment with docetaxel versus docetaxel alone. Findings: Pool analysis of 23 public studies suggested that patients with KRAS mutations had increased PD-L1 expression (OR=1.87; 95% CI: 1.34-2.61; P=0.0002). The combined analysis of PD-L1 and CD8+ TIL in the immunostained group showed increased dual-positive (PD-L1+/TIL+) cells in the KRAS-mutant group (P=0.008), suggesting KRAS mutations induce an inflammatory phenotype. Additional data verified a prominently increased KRAS mutant-associated TMB (TCGA: P=0.0155; Broad database: P=0.0001), indicating KRAS mutations promote tumor immunogenicity in NSCLC. Further molecular analyses revealed that KRAS mutations caused various defects in cell cycle checkpoints and DNA damage response pathways. Two-pool analysis confirmed that PD-1/PD-L1 inhibitors resulted in better ORR (OR=1.51; 95% CI: 1.17-1.96; P=0.002) and improved OS compared with docetaxel chemotherapy (HR=0.64; 95% CI: 0.43-0.95; P=0.03) KRAS-mutant NSCLC patients. Finally, anti-PD-L1 mAb significantly reduced tumor growth compared with docetaxel in KRASG12C lung adenocarcinoma mice (P<0.001), while PD-L1 mAb combined with docetaxel did not promote an anti-tumor response. Interpretation: KRAS-mutant NSCLC patients show remarkable clinical benefit from anti-PD-1/PD-L1 immunotherapy. We also uncovered a complex relationship among KRAS mutations, inflammatory phenotypes, and tumor immunogenicity. Importantly, our findings demonstrate that PD-1/PD-L1 inhibitor-based monotherapy may be more effective in KRAS-mutant NSCLC patients. Funding: This work was mainly supported by CIFMS. Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: This study was approved by the Ethics Committee of CICAMS, and all participants provided written informed consent.

Cell cycle checkpoint defects in gastrointestinal malignancies.

680 Background: Cyclin Dependent Kinases (CDKs) play a significant role in cell cycle regulation. Aberrations involving the cell cycle pathway genes can lead to uncontrolled cell proliferation and genomic instability. These could potentially be targeted with CDK4/6 inhibitors. The frequency and type of these alterations in GI tumors is largely unknown. Methods: We analyzed the frequency of abnormalities in cell cycle genes in patients with diverse GI malignancies (colorectal, liver, pancreas, gastroesophageal, anal, appendix) that underwent next generation sequencing from January 2013 to August 2017. Results: Aberrations in the cell cycle pathway were identified in 33 of 299 (11%) of cancers. The frequency of aberrations was as follows: CDKN2A/B in 10 (30.3%), CCND1 in 7 patients (pts) (21.2%), CCND2 in 2 pts (6%), CEBPA in 2 pts (6%), CDK6 in 2 pts (6%), CDK8 in 2 pts (6%) and CDK2 in 1 (3%). Alteration involving multiple genes of cell cycle noted in 7 patients (21.2%) with combination of CCND1 and CDKN2A being most common combination. The cell cycle checkpoint defects were most frequently seen in 9 pts with colon (27%), 8 pts with hepatobiliary (27%), 8 pts with pancreatic (24%), 7 pts with esophageal (21%), and less commonly in small bowel (6%) and GIST (6%). Conclusions: The alterations in the cell cycle pathway are most common in certain GI tumors mainly colon, pancreatic, hepatobiliary and esophageal tumors. Future clinical trials exploring the potential role of targeted agents such as CDK4/6 inhibitors alone or in combination with other targeted agents such as MEK inhibitors requires further exploration in these tumors.

Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies.

Radiotherapy has been accounted as the most comprehensive cancer treatment modality over the past few decades. However, failure of this treatment modality occurs in several malignancies due to the resistance of cancer cells to radiation. It was previously reported by the present authors that defective cell cycle checkpoints could be used as biomarkers for predicting the responsiveness to radiation in individual patients with cholangiocarcinoma (CCA). However, identification of functional defective cell cycle checkpoints from cells from a patient's tissues is cumbersome and not applicable in the clinic. The present study evaluated the radiosensitization potential of etoposide in p53-defective CCA KKU-M055 and KKU-M214 cell lines. Treatment with etoposide enhanced the responsiveness of two p53-defective CCA cell lines to radiation independent of G2 checkpoint function. In addition, etoposide treatment increased radiation-induced cell death without altering the dominant mode of cell death of the two cell lines. These findings indicate that etoposide could be used as a radiation sensitizer for p53-defective tumors, independent of the function of G2 checkpoint.

Inactivation of JNK2 as carcinogenic factor in colitis-associated and sporadic colorectal carcinogenesis.

We recently reported that dysregulated c-Jun N-terminal kinases (JNK) activity causes defective cell cycle checkpoint control, inducing neoplastic transformation in a cellular ulcerative colitis (UC) model. In the quiescent chronic phase of UC, p-p54 JNK was down-regulated and p-p46 JNK was up-regulated. Both were up-regulated in the acute phase. Consequently, increased p21WAF1 and γ-H2AX, two JNK-regulated proteins, induced cell cycle arrest. Their down-regulation led to checkpoint override, causing increased proliferation and undetected DNA damage in quiescent chronic phase, all characteristics of tumorigenesis. We investigated expression of p-JNK2, p-JNK1-3, p21WAF1, γ-H2AX and Ki67 by immunohistochemistry in cases of quiescent UC (QUC), active UC (AUC), UC-dysplasia and UC-related colorectal carcinoma (UC-CRC). Comparison was made to normal healthy colorectal mucosa, sporadic adenoma and colorectal carcinoma (CRC), diverticulitis and Crohns disease (CD). We found p-JNK2 up-regulation in AUC and its early down-regulation in UC-CRC and CRC carcinogenesis. With down-regulated p-JNK2, p21WAF1 was also decreased. Ki67 was inversely expressed, showing increased proliferation early in UC-CRC and CRC carcinogenesis. p-JNK1-3 was increased in AUC and QUC. Less increased γ-H2AX in UC-CRC compared to CRC gave evidence that colitis-triggered inflammation masks DNA damage, thus contributing to neoplastic transformation. We hypothesize that JNK-dependent cell cycle arrest is important in AUC, while chronic inflammation causes dysregulated JNK activity in quiescent phase that may contribute to checkpoint override, promoting UC carcinogenesis. We suggest restoring p-JNK2 expression as a novel therapeutic strategy to early prevent the development of UC-related cancer.

Abstract 2748: Targeting DNA repair deficient cancers with the cell-penetrating autoantibody 3E10

Abstract There is growing evidence that many solid tumors have defective DNA repair pathways and cell cycle checkpoints. This has spurred development of therapeutics to specifically target cancer cells with defective DNA repair, but few have been successful outright due to problems such as general toxicity. One novel anti-cancer agent of interest to us is 3E10, a unique cell-penetrating, anti-DNA autoantibody. This molecule is non-toxic to normal cells on its own but has deleterious effects on cells with DNA repair deficiencies. Specifically, 3E10 has been shown to be synthetically lethal in BRCA2-deficient human cancer cells, to be synergistic with radiation and other commonly used DNA damaging therapies, and to reduce the efficiency of Rad51 mediated strand exchange and thus homologous recombination (HR). The goal of this project is to better understand how 3E10 can be used as a new paradigm for cancer treatment. New evidence suggests that 3E10 inhibits Exo1 exonuclease activity. Additionally, preliminary data shows that 3E10 physically interacts with Rad51, ultimately inhibiting Rad51 nuclear localization and foci formation. Additionally, a point mutation in 3E10 that confers increased DNA binding mediates increased cell death in BRCA2-deficient human cancer cells. Furthermore, studies investigating the potential therapeutic effect of 3E10 in patient derived primary melanoma cell lines show that 3E10 is synthetically lethal with PTEN deficiency. This data suggests that 3E10 inhibits HR by binding to DNA at double strand breaks and occupying sites normally processed by core repair machinery, as well as by physically interacting with Rad51. Overall, 3E10 holds great promise as a novel therapeutic agent for various cancers, including PTEN deficient melanomas. Reference: Hansen, J. et al. Targeting cancer with a lupus autoantibody. Science Translational Medicine 4, (2012) Citation Format: Audrey L. Turchick, Peter M. Glazer. Targeting DNA repair deficient cancers with the cell-penetrating autoantibody 3E10. [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 2748.

ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective chronic lymphocytic leukemia cells

AbstractTP53 and ataxia telangiectasia mutated (ATM) defects are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukemia (CLL). Currently, therapies capable of providing durable remissions in relapsed/refractory TP53- or ATM-defective CLL are lacking. Ataxia telangiectasia and Rad3-related (ATR) mediates response to replication stress, the absence of which leads to collapse of stalled replication forks into chromatid fragments that require resolution through the ATM/p53 pathway. Here, using AZD6738, a novel ATR kinase inhibitor, we investigated ATR inhibition as a synthetically lethal strategy to target CLL cells with TP53 or ATM defects. Irrespective of TP53 or ATM status, induction of CLL cell proliferation upregulated ATR protein, which then became activated in response to replication stress. In TP53- or ATM-defective CLL cells, inhibition of ATR signaling by AZD6738 led to an accumulation of unrepaired DNA damage, which was carried through into mitosis because of defective cell cycle checkpoints, resulting in cell death by mitotic catastrophe. Consequently, AZD6738 was selectively cytotoxic to both TP53- and ATM-defective CLL cell lines and primary cells. This was confirmed in vivo using primary xenograft models of TP53- or ATM-defective CLL, where treatment with AZD6738 resulted in decreased tumor load and reduction in the proportion of CLL cells with such defects. Moreover, AZD6738 sensitized TP53- or ATM-defective primary CLL cells to chemotherapy and ibrutinib. Our findings suggest that ATR is a promising therapeutic target for TP53- or ATM-defective CLL that warrants clinical investigation.

Development of genetically flexible mouse models of sarcoma using RCAS-TVA mediated gene delivery.

Sarcomas are a heterogeneous group of mesenchymal malignancies and unfortunately there are limited functional genomics platforms to assess the molecular pathways contributing to sarcomagenesis. Thus, novel model systems are needed to validate which genes should be targeted for therapeutic intervention. We hypothesized that delivery of oncogenes into mouse skeletal muscle using a retroviral (RCAS-TVA) system would result in sarcomagenesis. We also sought to determine if the cell type transformed (mesenchymal progenitors vs. terminally differentiated tissues) would influence sarcoma biology. Cells transduced with RCAS vectors directing the expression of oncoproteins KrasG12D, c-Myc and/or Igf2 were injected into the hindlimbs of mice that expressed the retroviral TVA receptor in neural/mesenchymal progenitors, skeletal/cardiac muscle or ubiquitously (N-tva, AKE and BKE strains respectively). Disrupting the G1 checkpoint CDKN2 (p16/p19−/−) resulted in sarcoma in 30% of p16/p19−/−xN-tva mice with a median latency of 23 weeks (range 8–40 weeks). A similar incidence occurred in p16/p19−/−xBKE mice (32%), however, a shorter median latency (10.4 weeks) was observed. p16/p19−/−xAKE mice also developed sarcomas (24% incidence; median 9 weeks) yet 31% of mice also developed lung sarcomas. Gene-anchored PCR demonstrated retroviral DNA integration in 86% of N-tva, 93% of BKE and 88% of AKE tumors. KrasG12D was the most frequent oncogene isolated. Oncogene delivery by the RCAS-TVA system can generate sarcomas in mice with a defective cell cycle checkpoint. Sarcoma biology differed between the different RCAS models we created, likely due to the cell population being transformed. This genetically flexible system will be a valuable tool for sarcoma research.

Unexpected Function of the Glucanosyltransferase Gas1 in the DNA Damage Response Linked to Histone H3 Acetyltransferases in Saccharomyces cerevisiae

Chromatin organization and structure are crucial for transcriptional regulation, DNA replication, and damage repair. Although initially characterized in remodeling cell wall glucans, the β-1,3-glucanosyltransferase Gas1 was recently discovered to regulate transcriptional silencing in a manner separable from its activity at the cell wall. However, the function of Gas1 in modulating chromatin remains largely unexplored. Our genetic characterization revealed that GAS1 had critical interactions with genes encoding the histone H3 lysine acetyltransferases Gcn5 and Sas3. Specifically, whereas the gas1 gcn5 double mutant was synthetically lethal, deletion of both GAS1 and SAS3 restored silencing in Saccharomyces cerevisiae. The loss of GAS1 also led to broad DNA damage sensitivity with reduced Rad53 phosphorylation and defective cell cycle checkpoint activation following exposure to select genotoxins. Deletion of SAS3 in the gas1 background restored both Rad53 phosphorylation and checkpoint activation following exposure to genotoxins that trigger the DNA replication checkpoint. Our analysis thus uncovers previously unsuspected functions for both Gas1 and Sas3 in DNA damage response and cell cycle regulation.

Radiological Imaging in Ataxia Telangiectasia: a Review

The human genetic disorder ataxia telangiectasia (A-T) is characterised by neurodegeneration, immunodeficiency, radiosensitivity, cell cycle checkpoint defects, genomic instability and cancer predisposition. Progressive cerebellar ataxia represents the most debilitating aspect of this disorder. At present, there is no therapy available to cure or prevent the progressive symptoms of A-T. While it is possible to alleviate some of the symptoms associated with immunodeficiency and deficient lung function, neither the predisposition to cancer nor the progressive neurodegeneration can be prevented. Significant effort has focused on improving our understanding of various clinical, genetic and immunological aspects of A-T; however, little attention has been directed towards identifying altered brain structure and function using MRI. To date, most imaging studies have reported radiological anomalies in A-T. This review outlines the clinical and biological features of A-T along with known radiological imaging anomalies. In addition, we briefly discuss the advent of high-resolution MRI in conjunction with diffusion-weighted imaging, which enables improved investigation of the microstructural tissue environment, giving insight into the loss in integrity of motor networks due to abnormal neurodevelopmental or progressive neurodegenerative processes. Such imaging approaches have yet to be applied in the study of A-T and could provide important new information regarding the relationship between mutation of the ataxia telangiectasia mutated (ATM) gene and the integrity of motor circuitry.

Cyclin D1 Maintains Mantle Cell Lymphoma Through CDK4-Independent Regulation Of DNA Replicative Checkpoints

Mantle cell lymphoma (MCL) is rarely curable and therapy resistance often leaves few viable treatment options for patients. Previous studies have identified the importance of cyclin D1 (CCND1) translocation and overexpression in MCL pathogenesis, which leads to increased cyclin-dependent kinase 4 (CDK4) activity and accelerated cell cycle progression. However, targeting this abnormal cell cycle control, mainly through CDK4 inhibition causes only G1-phase growth arrest without significant cell death (Marzec et al. 2006). In contrast, prolonged inhibition of CCND1 with RNA interference induces apoptosis in MCL cell lines (Weinstein et al. 2012), suggesting an essential function of CCND1 independent of CDK4 activity. The mechanism of this non-catalytic role of CCND1 in maintaining MCL cell survival is largely unknown.To clarify the cell cycle role of CCND1 in addition to its CDK4-dependent function, we compared the effects of CCND1 and CDK4 silencing on MCL cell survival. MCL cell lines co-expressing GFP and doxycycline-inducible shRNA targeting CCND1 or CDK4 were generated. Cells with similar GFP expression levels were FACS sorted to normalize for shRNA expression. Both CCND1 and CDK4 silencing resulted in G1-phase arrest, but only CCND1-silenced cells demonstrated a marked increase in apoptosis. Investigation of the potential cause of apoptosis revealed significant accumulation of DNA double-strand breaks following CCND1 ablation, as measured by nuclear gamma-H2AX focus formation. Interestingly, CCND1-silenced cells exhibited a significant increase in 53BP1+ nuclear bodies in G1-phase, reminiscent of 53BP1 foci observed by Lukas and colleagues in cells undergoing aphidicolin-induced replication stress (Lukas et al. 2011). Analysis of replication fork movement in CCND1-depleted cells showed substantially reduced fork speed and increased frequency of origin firing, both of which are indicative of replication stress. In contrast, knockdown of CDK4 did not result in slower forks or increase in the frequency of origin firing. Genomic instability associated with replication stress was also apparent in CCND1-silenced cells, including increased micronucleus formation and recurrent chromatid gaps or breaks detected by cytokinesis-block assay and karyotyping, respectively.Analysis of DNA replicative and damage checkpoints revealed that both ATR-CHEK1 and ATM-CHEK2 pathways were activated by phosphorylation following CCND1 silencing in MCL cell lines, a xenograft animal model, and primary tumor samples, but not in non-MCL tumors. Interestingly, this activation (with the exception of ATM phosphorylation) was unsustainable over time and did not cause down-regulation of the downstream targets CDC25 and CDK1/2 but, instead, we observed an increase in CDC25A/B protein levels and CDK1/2 activity, indicating defective cell cycle checkpoints. Exposing CCND1-silenced cells to replication stress-inducing or DNA-damaging agents such hydroxyurea, aphidicolin, etoposide or ionizing radiation further amplified the checkpoint defects seen in unperturbed cells. We did not observe any significant difference in this checkpoint signaling in control and CDK4 knockdown cells under these conditions. Furthermore, CCND1-deficient cells were more sensitive to pharmacological inhibition of ATR and CHEK1 but not ATM, confirming a constitutive role of CCND1 in the ATR-CHEK1 pathway.In conclusion, these studies revealed an unexpected CDK4-independent role of CCND1 in maintaining DNA replicative checkpoints to prevent replication stress and genome instability in MCL cells. As most cancer treatments rely on agents that create DNA replication stress, targeting this function of CCND1 could provide a rational approach to overcome resistance to conventional therapies in MCL. Disclosures:No relevant conflicts of interest to declare.

53BP1 Is Limiting for NHEJ Repair in ATM-deficient Model Systems That Are Subjected to Oncogenic Stress or Radiation

The DNA damage response (DDR) factors ataxia telangiectasia mutated (ATM) and p53 binding protein 1 (53BP1) function as tumor suppressors in humans and mice, but the significance of their mutual interaction to the suppression of oncogenic translocations in vivo has not been investigated. To address this question, the phenotypes of compound mutant mice lacking 53BP1 and ATM (Trp53bp1(-/-)/Atm(-/-)), relative to single mutants, were examined. These analyses revealed that loss of 53BP1 markedly decreased the latency of T-lineage lymphomas driven by RAG-dependent oncogenic translocations in Atm(-/-) mice (average survival, 14 and 23 weeks for Trp53bp1(-/-)/Atm(-/-) and Atm(-/-) mice, respectively). Mechanistically, 53BP1 deficiency aggravated the deleterious effect of ATM deficiency on nonhomologous end-joining (NHEJ)-mediated double-strand break repair. Analysis of V(D)J recombinase-mediated coding joints and signal joints in Trp53bp1(-/-)/Atm(-/-) primary thymocytes is, however, consistent with canonical NHEJ-mediated repair. Together, these findings indicate that the greater NHEJ defect in the double mutant mice resulted from decreased efficiency of rejoining rather than switching to an alternative NHEJ-mediated repair mechanism. Complementary analyses of irradiated primary cells indicated that defects in cell-cycle checkpoints subsequently function to amplify the NHEJ defect, resulting in more frequent chromosomal breaks and translocations in double mutant cells throughout the cell cycle. Finally, it was determined that 53BP1 is dispensable for the formation of RAG-mediated hybrid joints in Atm(-/-) thymocytes but is required to suppress large deletions in a subset of hybrid joints. The current study uncovers novel ATM-independent functions for 53BP1 in the suppression of oncogenic translocations and in radioprotection.

DNA repair and cell cycle checkpoint defects as drivers and therapeutic targets in melanoma

The ultraviolet radiation (UVR) component of sunlight is the major environmental risk factor for melanoma, producing DNA lesions that can be mutagenic if not repaired. The high level of mutations in melanomas that have the signature of UVR-induced damage indicates that the normal mechanisms that detect and repair this damage must be defective in this system. With the exception of melanoma-prone heritable syndromes which have mutations of repair genes, there is little evidence for somatic mutation of known repair genes. Cell cycle checkpoint controls are tightly associated with repair mechanisms, arresting cells to allow for repair before continuing through the cell cycle. Checkpoint signaling components also regulate the repair mechanisms. Defects in checkpoint mechanisms have been identified in melanomas and are likely to be responsible for increased mutation load in melanoma. Loss of the checkpoint responses may also provide an opportunity to target melanomas using a synthetic lethal approach to identify and inhibit mechanisms that compensate for the defective checkpoints.

Targeting DNA double-strand break signalling and repair: recent advances in cancer therapy

Genomic instability, a hallmark of almost all human cancers, drives both carcinogenesis and resistance to therapeutic interventions. Pivotal to the ability of a cell to maintain genome integrity are mechanisms that signal and repair deoxyribonucleic acid (DNA) double-strand breaks (DSBs), one of the most deleterious lesions induced by ionising radiation and various DNA-damaging chemicals. On the other hand, many current therapeutic regimens that effectively kill cancer cells are based on the induction of excessive DSBs. However, these drugs often lack selectivity for tumour cells, which results in severe side effects for the patients, thus compromising their therapeutic potential. Therefore, the development of novel tumour-specific treatment strategies is required. Unlike normal cells, however, cancer cells are often characterised by abnormalities in the DNA damage response including defects in cell cycle checkpoints and/or DNA repair, rendering them particularly sensitive to the induction of DSBs. Therefore, new anticancer agents designed to exploit these vulnerabilities are becoming promising drugs for enhancing the specificity and efficacy of future cancer therapies. Here, we summarise the latest preclinical and clinical developments in cancer therapy based on the current knowledge of DSB signalling and repair, with a special focus on the combination of small molecule inhibitors with synthetic lethality approaches.