P18.17.A FROM STERILITY TO SELECTIVITY: A C. ELEGANS SYNTHETIC LETHALITY SCREEN REVEALS NON-CONSERVED VULNERABILITIES IN ATRX-DEFICIENT GLIOMA

The overall prognosis for pancreatic cancer remains dismal and potent chemotherapeutic agents that selectively target this cancer are critically needed. Elevated expression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is frequent in pancreatic cancer, and it offers promising tumor-selective targeting. Recently, KP372-1 was identified as a novel NQO1 redox cycling agent that induces cytotoxicity in cancer cells by creating redox imbalance; however, the mechanistic basis of KP372-1-induced cytotoxicity remains elusive. Here, we show that KP372-1 sensitizes NQO1-expressing pancreatic cancer cells and spares immortalized normal pancreatic duct cells, hTERT-HPNE. Notably, we found that KP372-1 is ~ 10- to 20-fold more potent than β-lapachone, another NQO1 substrate, against pancreatic cancer cells. Mechanistically, our data strongly suggest that reactive oxygen species produced by NQO1-dependent redox cycling of KP372-1 cause robust DNA damage, including DNA breaks. Furthermore, we found that KP372-1-induced DNA damage hyperactivates the central DNA damage sensor protein poly(ADP-ribose) polymerase 1 (PARP1) and activates caspase-3 to initiate cell death. Our data also show that the combination of KP372-1 with PARP inhibition creates enhanced cytotoxicity in pancreatic cancer cells. Collectively, our study provides mechanistic insights into the cytotoxicity instigated by KP372-1 and lays an essential foundation to establish it as a promising chemotherapeutic agent against cancer.

Ageing is defined as the gradual decline of normal physiological functions in a time-dependent manner. Significant progress has been made in characterising the regulatory processes involved in the mechanisms of ageing which would have been hindered without the use of model organisms. Use of alternative model organisms greatly diversifies our understanding of different factors underpinning the ageing process and the potential translation for human application. Unique characteristics make Daphnia an attractive model organism for research into mechanisms underlying ageing, such as transparent body, short generation time, well-characterised methylome, regenerative capabilities and available naturally occurring ecotypes. Most interestingly, genetically identical female and male Daphnia have evolved different average lifespans, providing a unique opportunity for understanding the underlying mechanisms of ageing and regulation of lifespan. Investigating sex differences in longevity could provide insight into principal mechanisms of ageing and lifespan regulation. In this study we provide evidence in support of establishing genetically identical female and male Daphnia as unique and valuable resources for research into mechanisms of ageing and begin to delineate the mechanisms involved in sex differences in lifespan. We identify significant differences between genders in physiological markers such as lifespan, growth rate, heart rate and swimming speed in addition to molecular markers such as lipid peroxidation product accumulation, thiol content decline and age-dependent decline in DNA damage repair efficiency. Overall, our data indicates that investigating sex differences in longevity in the clonal organism Daphnia under controlled laboratory conditions can provide insight into principal mechanisms of ageing and lifespan regulation.

Posttranslational modifications (PTMs) provide dynamic regulation of the cellular proteome, which is critical for both normal cell growth and for orchestrating rapid responses to environmental stresses, e.g. genotoxins. Key PTMs include ubiquitin, the Small Ubiquitin-like MOdifier SUMO, and phosphorylation. Recently, SUMO-targeted ubiquitin ligases (STUbLs) were found to integrate signaling through the SUMO and ubiquitin pathways. In general, STUbLs are recruited to target proteins decorated with poly-SUMO chains to ubiquitinate them and drive either their extraction from protein complexes, and/or their degradation at the proteasome. In fission yeast, reducing or preventing the formation of SUMO chains can circumvent the essential and DNA damage response functions of STUbL. This result indicates that whilst some STUbL "targets" have been identified, the crucial function of STUbL is to antagonize SUMO chain formation. Herein, by screening for additional STUbL suppressors, we reveal crosstalk between the serine/threonine phosphatase PP2A-Pab1B55 and the SUMO pathway. A hypomorphic Pab1B55 mutant not only suppresses STUbL dysfunction, but also mitigates the phenotypes associated with deletion of the SUMO protease Ulp2, or mutation of the STUbL cofactor Rad60. Together, our results reveal a novel role for PP2A-Pab1B55 in modulating SUMO pathway output, acting in parallel to known critical regulators of SUMOylation homeostasis. Given the broad evolutionary functional conservation of the PP2A and SUMO pathways, our results could be relevant to the ongoing attempts to therapeutically target these factors.

Poly-ADP-ribosylation, that is, adding ADP-ribose moieties to a protein, is a unique type of protein post-translational modification that regulates various cellular processes such as DNA repair, mitosis, transcription, and cell growth. Small-molecule inhibitors of poly-ADP-ribose polymerase 1 (PARP1) have been developed as anticancer agents because inhibition of PARP enzymes may be a synthetic lethal strategy for cancers with or BRCA2 mutations. However, there are still questions surrounding PARP inhibitors. Data were collected from Pubmed, Medline, through searching of these keywords: "PARP", "BRCA", "Synthetic lethal" and "Tankyrase inhibitors". We describe the current knowledge of PARP inhibition and its effects on DNA damage; mechanisms of resistance to PARP inhibitors; the evolution of PARP inhibitors; and the potential use of PARP5a/b (tankyrases) inhibitors in cancer treatment. PARP inhibitors are already showing promise as therapeutic tools, especially in the management of BRCA-mutated breast and ovarian cancers but also in tumors with dysfunctional BRCA genes. Small-molecule tankyrase inhibitors are important for increasing our understanding of tankyrase biology.

SUMOylation has been implicated in many cellular processes that are important for cancer cell survival, including cell cycle, chromosome structure and segregation, nuclear and subnuclear organization, transcription and DNA damage repair. However, a potent and selective inhibitor to target the SUMO pathway has been lacking. The SUMO-activating enzyme (SAE) is an essential enzyme in the pathway that initiates the SUMOylation process. Here we report the identification of the first mechanism-based SAE inhibitors with nanomolar potency in cellular assays. These inhibitors selectively block SAE enzyme activity and total SUMOylation in cells, which leads to reduced cancer cell proliferation. Moreover, SAE inhibition resulted in disruption of PML nuclear bodies and redistribution of DAXX. In vivo administration of SAE inhibitor into tumor bearing mice results in modulation of several biomarkers including a significant reduction in SUMO-conjugates and E2 Ubc9 thioesters, demonstrating SUMO pathway inhibition. Our results demonstrate the feasibility of inhibiting the SUMO pathway with small molecule inhibitors and provide tools to study the SUMO biology in cancer. Citation Format: Sai M Pulukuri, Xingyue He, Stephen Grossman, Jessica Riceberg, Erik Koenig, Teresa Soucy, Keli Song, Anna Kreshock, Dylan England, Hirotake Mizutani, Larry Dick, James Brownell, John Newcomb, Steve Langston, Eric Lightcap, Katherine Galvin. Probing the roles of SUMOylation in cancer cell biology using a selective SUMO activating enzyme inhibitor [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 1147. doi:10.1158/1538-7445.AM2017-1147

Decision letter: SET-9 and SET-26 are H3K4me3 readers and play critical roles in germline development and longevity

Histone post-translational modifications have been shown to contribute to DNA damage repair. Prior studies have suggested that specific H3K79 methylation states play distinct roles in the response to UV-induced DNA damage. To evaluate these observations, we examined the effect of altered H3K79 methylation patterns on UV-induced G1/S checkpoint response and sister chromatid exchange (SCE). We found that the di- and trimethylated states both contribute to activation of the G1/S checkpoint to varying degrees, depending on the synchronization method, although methylation is not required for checkpoint in response to high levels of UV damage. In contrast, UV-induced SCE is largely a product of the trimethylated state, which influences the usage of gene conversion versus popout mechanisms. Regulation of H3K79 methylation by H2BK123 ubiquitylation is important for both checkpoint function and SCE. H3K79 methylation is not required for the repair of double-stranded breaks caused by transient HO endonuclease expression, but does play a modest role in survival from continuous exposure. The overall results provide evidence for the participation of H3K79 methylation in UV-induced recombination repair and checkpoint activation, and further indicate that the di- and trimethylation states play distinct roles in these DNA damage response pathways.

The breast and ovarian predisposition protein BRCA1 is a required component of the mammalian response to double-stranded DNA damage. Its conserved BRCT domains are required for BRCA1 accumulation to sites of repair, while the conserved N-terminal RING domain is able to catalyse the conjugation of ubiquitin and act as an E3 ubiquitin ligase. Disruption of either of these domains by missense mutation is associated with disease development. The SUMO conjugation pathway has been implicated in DNA damage response in model organisms, and in Caenorhabditis elegans the Brac1 binding partner Bard1 associates with the SUMO E2 conjugating enzyme Ubc9. In mammalian cells, BRCA1 has been found to be associated with free SUMO-1 resulting in altered transcription. We undertook to examine the potential influence of the SUMO pathway on BRCA1 response to genotoxic stress. Using a range of biochemical and cell-biology techniques, we have shown that BRCA1 is modified by SUMO in response to genotoxic stress, and co-localises at sites of DNA damage with SUMO1, SUMO2/3 and the SUMO conjugating enzyme Ubc9. PIAS SUMO E3 ligases co-localise with and modulate SUMO modification of BRCA1, and are required for BRCA1 ubiquitin ligase activity in cells. In vitro SUMO modification of the BRCA1:BARD1 heterodimer greatly increases its ligase activity, identifying it as a SUMO regulated ubiquitin ligase. Further, PIAS SUMO ligases are required for complete accumulation of double-strand DNA damage repair proteins subsequent to RNF8 accrual, and for proficient double-strand break repair. Because the two features of BRCA1 activity regulated by the SUMO pathway, ubiquitin ligase activity and accumulation at sites of DNA damage, are also inhibited by some BRCA1 mutations that predispose to breast cancer and ovarian cancer, it seems highly likely that the SUMO pathway will be of relevance to cancer predisposition and development.

Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 microm circle plasmid (2 microm). Here we show that accumulation of 2 microm in the SUMO pathway mutants siz1Delta siz2Delta, slx5Delta, and slx8Delta is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 microm. Characterization of this species from siz1Delta siz2Delta showed that it contains tandem copies of the 2 mum sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 microm-encoded Flp recombinase and the cellular homologous recombination repair (HRR) pathway. Importantly, reduced SUMO attachment to Flp is sufficient to induce formation of this species. Our data suggest a model in which Flp that cannot be sumoylated causes DNA damage, whose repair via HRR produces an intermediate that generates tandem copies of the 2 microm sequence. This intermediate may be a rolling circle formed via break-induced replication (BIR), because mutants defective in BIR contain reduced levels of the HMW form. This work also illustrates the importance of using cir(o) strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 microm amplification.

Rad53 is an essential protein kinase governing DNA damage and replication stress checkpoints in budding yeast. It also appears to be involved in cellular morphogenesis processes. Mass spectrometry analyses revealed that Rad53 is phosphorylated at multiple SQ/TQ and at SP/TP residues, which are typical consensus sites for phosphatidylinositol 3-kinase-related kinases and CDKs, respectively. Here we show that Clb-CDK1 phosphorylates Rad53 at Ser(774) in metaphase. This phosphorylation event does not influence the DNA damage and replication checkpoint roles of Rad53, and it is independent of the spindle assembly checkpoint network. Moreover, the Ser-to-Asp mutation, mimicking a constitutive phosphorylation state at site 774, causes sensitivity to calcofluor, supporting a functional linkage between Rad53 and cellular morphogenesis.

DNA is vulnerable to damage resulting from endogenous metabolites, environmental and dietary carcinogens, some anti-inflammatory drugs, and genotoxic cancer therapeutics. Cells respond to DNA damage by activating complex signalling networks that decide cell fate, promoting not only DNA repair and survival but also cell death. The decision between cell survival and death following DNA damage rests on factors that are involved in DNA damage recognition, and DNA repair and damage tolerance, as well as on factors involved in the activation of apoptosis, necrosis, autophagy and senescence. The pathways that dictate cell fate are entwined and have key roles in cancer initiation and progression. Furthermore, they determine the outcome of cancer therapy with genotoxic drugs. Understanding the molecular basis of these pathways is important not only for gaining insight into carcinogenesis, but also in promoting successful cancer therapy. In this Review, we describe key decision-making nodes in the complex interplay between cell survival and death following DNA damage.

Depsipeptide FR901228 (FK228) is a new kind of histone deacetylase inhibitors (HDACi) that induces growth arrest and cell death in a variety of tumor cells. Though its effects on oncogene expression and degradation have been documented, the detailed mechanism of FK228-induced cytotoxicity is still undefined. In this study, a differential proteomic analysis was performed to identify proteins associated with FK228-induced cytotoxicity in human lung cancer cells. Two-dimensional gel electrophoresis (2-DE) revealed a distinct protein profile of H322 cells in response to FK228 treatment, and 45 protein spots with significant alteration were screened. In total, 27 proteins were identified by mass spectrometry and involved in signal transduction, transcriptional regulation, metabolism, cytoskeletal organization, and protein folding, synthesis and degradation, consistent with multiple effects of FK228 on tumor cells. Notably, a novel target protein, thioredoxin reductase (TrxR), was identified, which is downregulated in FK228-sensitive cancer cells, but upregulated in resistant cells. The expression level of TrxR was negatively correlated with ROS accumulation, DNA damage and apoptosis, implicating TrxR in FK228-induced apoptosis and HDACi sensitivity in cancer cells. Thus, proteomic analysis provides new information about target proteins important for FK228-induced cytotoxicity in cancer cells.

Eric J. Hall Amato J. Giaccia Radiobiology for the Radiologist, 6th Edition 2006 Lippincott Williams & Wilkins Philadelphia 656 pp. $99.00 hardcover ISBN: 0-7817-4151-3

We identified YDR499W as a Saccharomyces cerevisiae open reading frame with homology to several checkpoint proteins, including S. cerevisiae Rfc5p and Schizosaccharomyces pombe Rad26. Disruption of YDR499W (termed LCD1) results in lethality that is rescued by increasing cellular deoxyribonucleotide levels. Cells lacking LCD1 are very sensitive to a range of DNA-damaging agents, including UV irradiation, and to the inhibition of DNA replication. LCD1 is necessary for the phosphorylation and activation of Rad53p in response to DNA damage or DNA replication blocks, and for Chk1p activation in response to DNA damage. LCD1 is also required for efficient DNA damage-induced phosphorylation of Rad9p and for the association of Rad9p with the FHA2 domain of Rad53p after DNA damage. In addition, cells lacking LCD1 are completely defective in the G(1)/S and G(2)/M DNA damage checkpoints. Finally, we reveal that endogenous Mec1p co-immunoprecipitates with Lcd1p both before and after treatment with DNA-damaging agents. These results indicate that Lcd1p is a pivotal checkpoint regulator, involved in both the essential and checkpoint functions of the Mec1p pathway.

Genetic information is under constant attack from endogenous and exogenous sources, and the use of model organisms has provided important frameworks to understand how genome stability is maintained and how various DNA lesions are repaired. The advance of high throughput next generation sequencing (NGS) provides new inroads for investigating mechanisms needed for genome maintenance. These emerging studies, which aim to link genetic toxicology and mechanistic analyses of DNA repair processes in vivo, rely on defining mutational signatures caused by faulty replication, endogenous DNA damaging metabolites, or exogenously applied genotoxins; the analysis of their nature, their frequency and distribution. In contrast to classical studies, where DNA repair deficiency is assessed by reduced cellular survival, the localization of DNA repair factors and their interdependence as well as limited analysis of single locus reporter assays, NGS based approaches reveal the direct, quantal imprint of mutagenesis genome-wide, at the DNA sequence level. As we will show, such investigations require the analysis of DNA derived from single genotoxin treated cells, or DNA from cell populations regularly passaged through single cell bottlenecks when naturally occurring mutation accumulation is investigated. We will argue that the life cycle of the nematode Caenorhabditis elegans, its genetic malleability combined with whole genome sequencing provides an exciting model system to conduct such analysis.