DNA Checkpoint Research Articles

Abstract 1051: Targeting replication stress pathway provides an avenue for novel combination therapy options including TTfields plus chemo agents which increase replication stress

Abstract Tumor Treating Fields (TTFields) is a new physical therapeutic modality, which has been FDA approved for the treatment of recurrent glioblastoma (GBM) as monotherapy, for newly diagnosed GBM in combination with temozolomide, and for unresectable locally advanced or metastatic malignant pleural mesothelioma (MPM) in combination with pemetrexed and a platinum-based chemotherapy. Clinical trials are ongoing for other cancers, including lung, pancreatic, and ovarian cancers. TTFields are low-intensity, intermediate frequency, alternating electric fields which are loco-regionally applied to tumor sites using non-invasive arrays. The initial mechanism by which TTFields was thought to kill tumor cells was the disruption of mitosis. Using gene expression analysis and functional characterization studies, we discovered a novel role of TTFields in DNA damage repair and replication stress pathways. TTFields treatment decreases Fanconi Anemia (FA) pathway signaling proteins thereby impairs ionizing radiation (IR)-induced DNA damage repair processes. The length of newly replicated DNA slowed as a function of TTFields exposure time and that TTFields increased R-loop formation, which indicates that TTFields induced replication stress. Hence, we hypothesized TTFields increase sensitivity of chemotherapy agents that target and increase replication stress in novel combination therapy options. PARP1 protects DNA breaks, by recruiting DNA repair and checkpoint proteins to the sites of damage and recruiting MRE11 for DNA end processing (required for replication restart and enhanced Chk1 activation). Indeed, TTFields treatment concomitant with the PARP1 inhibitor olaparib followed by IR was found to be synergistic compared to IR or olaparib alone or in combination. However, the degree of sensitization and synergy varied across non-small cell lung cancer cell lines. TTFields increased cell killing efficacy of etoposide synergistically, which forms a ternary complex with topoisomerase II and prevents re-ligation of DNA strands to elicit DNA strand breaks and induce replication stress. The advantage of combining TTFields with AZD6738 (an ataxia telangiectasia and Rad3-related protein (ATR) inhibitor) was tested. ATR is an essential kinase regulator of the replication checkpoint that plays a critical role in safeguarding genome integrity from replication stress. Another novel combination of TTFields together with irinotecan, which traps topoisomerase I- DNA in ternary cleavage complex and inhibits initial cleavage reaction and re-ligation steps to increase replication stress, is also tested. Taken together, our results suggest that different chemo agents that target replication stress pathways can be used in combination with TTFields for cancer therapy, which need to be tested in in-vivo studies and clinical trials. Citation Format: Narasimha Kumar Karanam, Michael Dean Story. Targeting replication stress pathway provides an avenue for novel combination therapy options including TTfields plus chemo agents which increase replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1051.

LncRNA DINOR is a virulence factor and global regulator of stress responses in Candida auris.

The emergent fungal pathogen Candida auris exhibits high resistance to antifungal drugs and environmental stresses, impeding treatment and decontamination1-3. The fungal factors mediating this stress tolerance are largely unknown. In the present study, we performed piggyBac, transposon-mediated, genome-wide mutagenesis and genetic screening in C. auris, and identified a mutant that grew constitutively in the filamentous form. Mapping the transposon insertion site revealed the disruption of a long non-coding RNA, named DINOR for DNA damage-inducible non-coding RNA. Deletion of DINOR caused DNA damage and an upregulation of genes involved in morphogenesis, DNA damage and DNA replication. The DNA checkpoint kinase Rad53 was hyperphosphorylated in dinorΔ mutants, and deletion of RAD53 abolished DNA damage-induced filamentation. DNA-alkylating agents, which cause similar filamentous growth, induced DINOR expression, suggesting a role for DINOR in maintaining genome integrity. Upregulation of DINOR also occurred during exposure to the antifungal drugs caspofungin and amphotericin B, macrophages, H2O2 and sodium dodecylsulfate, indicating that DINOR orchestrates multiple stress responses. Consistently, dinorΔ mutants displayed increased sensitivity to these stresses and were attenuated for virulence in mice. Moreover, genome-wide genetic interaction studies revealed links between the function of DINOR and TOR signalling, an evolutionarily conserved pathway that regulates the stress response. Identification of the mechanism(s) by which DINOR regulates stress responses in C. auris may provide future opportunities for the development of therapeutics.

Cellworks Omics Biology Model (CBM) to predict therapy response and identify novel biomarkers for 5FU-based combination therapy in gastric cancer patients.

e16091 Background: Using a 5FU backbone, a variety of combination chemotherapy regimens have been adopted for gastric cancer. However, the genetic heterogeneity of the disease suggests certain drugs would lead to better outcomes for specific patients. In principle, the incorporation of molecular profiling information into treatment selection can generate superior survival for patients. Therefore, we conducted a pilot study using the CBM to identify novel genomic biomarkers of response and resistance to several 5FU-based regimens. Methods: 12 gastric cancer patients treated with 5FU-cisplatin (N = 4), 5FU-VP16 (N = 5) and 5FU-cisplatin-docetaxel (N = 3) were selected from the TCGA database. Mutation and copy number aberrations from each case served as input for CBM to generate patient-specific protein network maps generated from PubMed and other resources. Disease-biomarkers unique to each patient were identified within protein network maps. Drug impact on the disease network was digitally simulated to determine treatment efficacy by measuring effect of chemotherapy on the cell growth score, i.e., a composite of cell proliferation, viability, apoptosis, metastasis, DNA damage and other cancer hallmarks. Effectively, the mechanism of action of each drug was mapped to each patient’s genome and the biological consequences of genomic abnormalities were correlated with response. Results: Of the 12 patients in the study, 10 patients responded to 5FU-based regimens and 2 were non-responders. CBM correctly predicted the therapy response in each therapy segment. Key response criteria were: 5FU-cisplatin - ARID1A (LOF), RAD51 (LOF), MBD1 (LOF), DUT (LOF). 5FU-VP16 - FBXW7 (DEL), RAD51D (DEL), RAD51C (DEL), XRCC2 (DEL), XRCC3 (DEL). 5FU-cisplatin-docetaxel - ARID1A (DEL), CHEK1 (DEL), REV3L (DEL), XRCC3(DEL) and TUBB (AMP). Resistance biomarkers were also identified for these drugs. FBXW7 loss caused cisplatin resistance, but also predicted sensitivity to VP16. Interestingly, all five 5FU-VP16 responders harbored FBXW7 loss. Similarly, in two 5FU-cisplatin-docetaxel non-responders, KIFC1 (GOF), YBX1(AMP), CDC20 (AMP) were key biomarkers for docetaxel resistance. In one of these patients, ATM (DEL), RBBP8 (DEL) and GSTP1 (DEL) predicted response to VP16. Conclusions: The efficacy of individual cytotoxic agents varies in the population. Mutations and copy number changes in DNA checkpoint, DNA repair, and tumor suppressor genes impact responsive to chemotherapy and should be routinely assessed. Notably, 5FU-VP16 combination could have delivered better antitumor efficacy for several non-responders or 25% of the patients in this small cohort. CBM is able to identify both the most active as well inefficacious components of the treatment upfront. We suggest that this approach should be prospectively evaluated in a larger cohort.

Synergy of Immunostimulatory Genetherapy with Immune Checkpoint Blockade Motivates Immune Response to Eliminate Cancer

AbstractNormalizing the tumor‐induced immune deficiency in the immunosuppressive tumor microenvironment (TME) through increasing the efficient infiltration and activation of antitumoral immunity in TME is the core of promising immunotherapy. Herein, a Cyclo(Arg‐Gly‐Asp‐d‐Phe‐Lys) (RGD) peptides‐modified combinatorial immunotherapy system based on the self‐assembly of the nanoparticles named RGD‐DMA composed of RGD‐PEG‐PLA, methoxy poly(ethylene glycol)‐poly(lactide) (MPEG‐PLA) and 1,2‐Dioleoyl‐3‐trimethylammonium‐propane (DOTAP) is used to codeliver the immunostimulatory chemokine CCL19‐encoding plasmid DNA (CCL19 pDNA) and immune checkpoint ligand PD‐L1 inhibitor (BMS‐1). The RGD‐DMA/pCCL19‐BMS‐1 system not only exhibited significant inhibition of tumor progression but also induced locally high concentrations of immunostimulatory cytokines at tumor sites without causing an obviously systemic inflammatory response. The immunosuppressive TME is efficaciously reshaped by the coadministration of RGD‐DMA/pCCL19 and BMS‐1, as indicated by the activated T lymphocytes, increased intratumoral‐infiltration of mature dendritic cells (DCs), and the repolarization of macrophages from pro‐tumoral M2‐phenotype toward tumoricidal M1‐phenotype. The upregulated PD‐L1 expression at tumor sites caused by the increased IFN‐γ levels after immunostimulatory gene therapy further demonstrated the synergistic effects of BMS‐1 in counteracting the inhibitory role of PD‐L1 expression in antitumor immunity. Therefore, the combination of immunostimulating therapy and immune checkpoint inhibitor that synergistically target multiple immune regulatory pathways demonstrates significant potential as a novel immunotherapy approach.

ZmRAD17 Is Required for Accurate Double-Strand Break Repair During Maize Male Meiosis.

RAD17, a replication factor C (RFC)-like DNA damage sensor protein, is involved in DNA checkpoint control and required for both meiosis and mitosis in yeast and mammals. In plant, the meiotic function of RAD17 was only reported in rice so far. Here, we identified and characterized the RAD17 homolog in maize. The Zmrad17 mutants exhibited normal vegetative growth but male was partially sterile. In Zmrad17 pollen mother cells, non-homologous chromosome entanglement and chromosome fragmentation were frequently observed. Immunofluorescence analysis manifested that DSB formation occurred as normal and the loading pattern of RAD51 signals was similar to wild-type at the early stage of prophase I in the mutants. The localization of the axial element ASY1 was normal, while the assembly of the central element ZYP1 was severely disrupted in Zmrad17 meiocytes. Surprisingly, no obvious defect in female sterility was observed in Zmrad17 mutants. Taken together, our results suggest that ZmRAD17 is involved in DSB repair likely by promoting synaptonemal complex assembly in maize male meiosis. These phenomena highlight a high extent of divergence from its counterpart in rice, indicating that the RAD17 dysfunction can result in a drastic dissimilarity in meiotic outcome in different plant species.

Monitoring Schizosaccharomyces pombe genome stress by visualizing end-binding protein Ku.

ABSTRACTStudies of genome stability have exploited visualization of fluorescently tagged proteins in live cells to characterize DNA damage, checkpoint, and repair responses. In this report, we describe a new tool for fission yeast, a tagged version of the end-binding protein Pku70 which is part of the KU protein complex. We compare Pku70 localization to other markers upon treatment to various genotoxins, and identify a unique pattern of distribution. Pku70 provides a new tool to define and characterize DNA lesions and the repair response.

Chromothripsis, DNA repair and checkpoints defects

Chromothripsis is a unique form of genome instability characterized by tens to hundreds of DNA double-strand breaks on one or very few chromosomes, followed by error-prone repair. The derivative chromosome(s) display massive rearrangements, which lead to the loss of tumor suppressor function and to the activation of oncogenes. Chromothripsis plays a major role in cancer as well as in other conditions, such as congenital diseases. In this review, we discuss the repair processes involved in the rejoining of the chromosome fragments, the role of DNA repair and checkpoint defects as a cause for chromothripsis as well as DNA repair defects resulting from chromothripsis. Finally, we consider clinical implications and potential therapeutic vulnerabilities that could be utilized to eliminate tumor cells with chromothripsis.

Arid4b alters cell cycle and cell death dynamics during mouse embryonic stem cell differentiation.

Cell division and death play an important role in embryonic development. Cell specialization is accompanied with slow proliferation and quiescence. Cell death is important for morphogenesis. Gene expression changes during differentiation is coordinated by lineage-specific transcription factors and chromatin factors. It is not yet fully understood how alterations in gene expression and cell cycle/death mechanisms are connected. We previously identified a chromatin protein Arid4b as a critical factor for meso/endoderm differentiation of mouse embryonic stem cells (mESCs). The differentiation defect of Arid4b-deficient mESCs might be due to misregulation of cell proliferation or death. Here, we identified a role for Arid4b in cell cycle rewiring at the onset of differentiation. Arid4b-deficient differentiating cells have less proliferative capacity and their cell cycle profile is more similar to mESC stage than the differentiating wild-type cells. We found no evidence of increased DNA damage or checkpoint activation. Our investigation of cell death mechanisms found no contribution from autophagy but revealed a slight increase in Caspase-3 activation implying early apoptosis in Arid4b-deficient differentiating cells. Taken together, our data suggest Arid4b regulates cell cycle alterations during exit from pluripotency. Future studies will be instrumental in understanding whether these changes directly contribute to Arid4b-dependent differentiation control.

DNA repair, recombination, and damage signaling.

DNA must be accurately copied and propagated from one cell division to the next, and from one generation to the next. To ensure the faithful transmission of the genome, a plethora of distinct as well as overlapping DNA repair and recombination pathways have evolved. These pathways repair a large variety of lesions, including alterations to single nucleotides and DNA single and double-strand breaks, that are generated as a consequence of normal cellular function or by external DNA damaging agents. In addition to the proteins that mediate DNA repair, checkpoint pathways have also evolved to monitor the genome and coordinate the action of various repair pathways. Checkpoints facilitate repair by mediating a transient cell cycle arrest, or through initiation of cell suicide if DNA damage has overwhelmed repair capacity. In this chapter, we describe the attributes of Caenorhabditis elegans that facilitate analyses of DNA repair, recombination, and checkpoint signaling in the context of a whole animal. We review the current knowledge of C. elegans DNA repair, recombination, and DNA damage response pathways, and their role during development, growth, and in the germ line. We also discuss how the analysis of mutational signatures in C. elegans is helping to inform cancer mutational signatures in humans.

Kinetochore-independent mechanisms of sister chromosome separation.

Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.

Targeting DNA Repair and Chromatin Crosstalk in Cancer Therapy.

Simple SummaryTargeting aberrant DNA repair in cancers in addition to transcription and replication is an area of interest for cancer researchers. Inhibition of DNA repair selectively in cancer cells leads to cytotoxic or cytostatic effects and overcomes survival advantages imparted by chromosomal translocations or mutations. In this review, we highlight the relevance of DNA repair-linked events in developmental diseases and cancers and also discuss mechanisms to overcome these events that participate in different cellular processes.Aberrant DNA repair pathways that underlie developmental diseases and cancers are potential targets for therapeutic intervention. Targeting DNA repair signal effectors, modulators and checkpoint proteins, and utilizing the synthetic lethality phenomena has led to seminal discoveries. Efforts to efficiently translate the basic findings to the clinic are currently underway. Chromatin modulation is an integral part of DNA repair cascades and an emerging field of investigation. Here, we discuss some of the key advancements made in DNA repair-based therapeutics and what is known regarding crosstalk between chromatin and repair pathways during various cellular processes, with an emphasis on cancer.

Mechanism of intrinsic resistance of lung squamous cell carcinoma to epithelial growth factor receptor-tyrosine kinase inhibitors revealed by high-throughput RNA interference screening.

Although targeted therapy has achieved a great breakthrough in the treatment of lung adenocarcinoma, there are still no effective targeted drugs for lung squamous cell carcinoma (SqCC). In addition, as immunotherapy can only prolong the overall survival (OS) of lung SqCC by ≤5 months, chemotherapy and radiotherapy are still the main types of therapy for advanced SqCC. The expression level of epithelial growth factor receptor (EGFR) in patients with lung SqCC is higher compared with those with adenocarcinoma, but the former group is intrinsically resistant to EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Therefore, if the drug resistance in patients with lung SqCC could be reversed, the majority of patients may benefit from EGFR-TKIs. In the present study, the high-throughput RNA interference technology was used to screen the genes involved in the EGFR-TKI erlotinib resistance of lung SqCCs, and integrin-linked kinase (ILK) was identified to be the most effective. The role of ILK in erlotinib resistance was further studied in cell lines, and the expression of ILK was analyzed in patients with SqCC and adenocarcinoma. Finally, the mechanism of ILK in EGFR-TKIs resistance was analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) and ingenuity pathway analysis (IPA). The results demonstrated that the ILK gene knockdown could overcome erlotinib resistance by inhibiting cell proliferation, inducing apoptosis and blocking the cell cycle at the G2/M phase. The expression of ILK in patients with SqCC was significantly higher compared with those with adenocarcinoma with sensitizing EGFR mutations. In addition, the cell cycle pathway ‘G2/M DNA damage and checkpoint regulation’ was identified to be significantly inhibited by ILK knockdown in IPA, KEGG and GO analysis. The results of the present study may improve the understanding of EGFR-TKI resistance in lung SqCCs, thus promoting the development of potential targeted therapies for lung SqCCs.

Functional characterization of Candida glabrata ORF, CAGL0M02233g for its role in stress tolerance and virulence

Present investigation is aimed to analyze the role of an uncharacterized ORF of Candida glabrata (CBS138), CAGL0M02233g (an ortholog of RAD53, a key DNA checkpoint effector in Saccharomyces cerevisiae) in tolerance of various stresses and in biofilm formation. The CAGL0M02233g was cloned in p416TEF shuttle vector for constitutive expression under TEF1 promoter in BG14 strain (ura3 auxotrophic C. glabrata), and upregulated expression of the cloned ORF was confirmed by immunoblotting. The constitutive expression of CAGL0M02233g rendered cells resistant to the DNA damage stressor (MMS), replication stressor (HU) and hypoxia mimetic (CoCl2) in plate spot and growth curve assays. Hypoxia (a low oxygen condition) is an imperative host factor that influences Candida pathogenesis. Biofilm formation by the BG14 cells transformed with p416TEF-CAGL0M02233g (REX cell) was reduced to approximately 50% under hypoxia. It is notable that biofilm formation by the REX cells was significantly lower than that of BG14 cells transformed with p416TEF vector (VC cell) under hypoxia. The biofilm of the REX cells has shown higher susceptibility to fluconazole than that of VC cells under hypoxia and REX cells at normoxia. This is the first report on the function of CAGL0M02233g in tolerance of various stressors and in modulation of the biofilm under hypoxia.

Abstract 2379: Elaborating the mechanism of PARP synthetic lethality in ATM loss

Abstract PARP inhibitors have transformed the treatment for ovarian cancer, which is known to have high incidence of homologous recombination (HR) deficiency through the loss of BRCA1 or BRCA2 genes. 30-35% of ovarian cancer patients have defects in BRCA1/2 genes caused by germline, somatic or epigenetic alterations. PARP inhibitors induce cell death in HR-deficient cells through the mechanism of synthetic lethality where cancer cells cannot tolerate the loss of both SSB and DSB repair machinery. Although genetic or epigenetic alterations in other pathways such as Fanconi anemia, or in non-authentic homologous recombination repair (HRR) genes such as PTEN, IDH, and CDK12 have been reported to contribute to HR deficiency, mechanistic understanding of HR loss remains to be determined. Another DNA damage repair (DDR) protein, ATM, a kinase that phosphorylates and activates major DNA damage checkpoints, i.e. CHK2 & BRCA1, has also being increasingly recognized recently as a synthetic lethal partner with PARP inhibitors in multiple tumor indications such as prostate, lung and colorectal cancers. ATM functions in both cell cycle arrest and DNA repair in response to DSBs, however how cells respond to inhibition of PARP in the absence of ATM is not yet fully understood. In this study, we demonstrated that loss of ATM function, i.e. via generating an ATM KO by CRISPR/Cas9 in two genetic backgrounds, i.e. in DLD-1 and HeLa cell lines, induced HR-deficiency indicated by the impairment of Rad51-foci formation and increased phosphorylation of histone H2AX in ATM KO (-/-) cell lines. Furthermore DLD-1 ATM KO (-/-) cell line demonstrated increased sensitivity to niraparib (i.e. 45-fold difference in sensitivity compared to the DLD-1 parental, HR-proficient cell line) in the 14day colony formation assay. In comparison, BRCA2 (-/-) KO in DLD-1 gave a 125-fold increase compared to parental cell line. In DLD-1 ATM KO (-/-) cell line, we have also detected that loss of ATM was associated with low BRCA1 and BRCA2 protein expression suggestive of downstream effect on impairment of HR-mediated DNA checkpoint signaling in ATM loss. Thus, PARPi synthetic lethality phenotype observed in tumors with ATM loss could be regulated through the combination effect coming from the loss of both of ATM & BRCA1/2 and the subsequent effect on both HR gene functions. Our findings highlight that ATM has a critical role in DNA repair for the synergism with PARP inhibitors. Citation Format: Asli Muvaffak, Kevin G. Coleman. Elaborating the mechanism of PARP synthetic lethality in ATM loss [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 2379.

Active transcription and Orc1 drive chromatin association of the AAA+ ATPase Pch2 during meiotic G2/prophase.

Pch2 is an AAA+ protein that controls DNA break formation, recombination and checkpoint signaling during meiotic G2/prophase. Chromosomal association of Pch2 is linked to these processes, and several factors influence the association of Pch2 to euchromatin and the specialized chromatin of the ribosomal (r)DNA array of budding yeast. Here, we describe a comprehensive mapping of Pch2 localization across the budding yeast genome during meiotic G2/prophase. Within non-rDNA chromatin, Pch2 associates with a subset of actively RNA Polymerase II (RNAPII)-dependent transcribed genes. Chromatin immunoprecipitation (ChIP)- and microscopy-based analysis reveals that active transcription is required for chromosomal recruitment of Pch2. Similar to what was previously established for association of Pch2 with rDNA chromatin, we find that Orc1, a component of the Origin Recognition Complex (ORC), is required for the association of Pch2 to these euchromatic, transcribed regions, revealing a broad connection between chromosomal association of Pch2 and Orc1/ORC function. Ectopic mitotic expression is insufficient to drive recruitment of Pch2, despite the presence of active transcription and Orc1/ORC in mitotic cells. This suggests meiosis-specific 'licensing' of Pch2 recruitment to sites of transcription, and accordingly, we find that the synaptonemal complex (SC) component Zip1 is required for the recruitment of Pch2 to transcription-associated binding regions. Interestingly, Pch2 binding patterns are distinct from meiotic axis enrichment sites (as defined by Red1, Hop1, and Rec8). Inactivating RNAPII-dependent transcription/Orc1 does not lead to effects on the chromosomal abundance of Hop1, a known chromosomal client of Pch2, suggesting a complex relationship between SC formation, Pch2 recruitment and Hop1 chromosomal association. We thus report characteristics and dependencies for Pch2 recruitment to meiotic chromosomes, and reveal an unexpected link between Pch2, SC formation, chromatin and active transcription.

Fractionation-Dependent Radiosensitization by Molecular Targeting of Nek1.

NIMA (never-in-mitosis gene A)-related kinase 1 (Nek1) is shown to impact on different cellular pathways such as DNA repair, checkpoint activation, and apoptosis. Its role as a molecular target for radiation sensitization of malignant cells, however, remains elusive. Stably transduced doxycycline (Dox)-inducible Nek1 shRNA HeLa cervix and siRNA-transfected HCT-15 colorectal carcinoma cells were irradiated in vitro and 3D clonogenic radiation survival, residual DNA damage, cell cycle distribution, and apoptosis were analyzed. Nek1 knockdown (KD) sensitized both cell lines to ionizing radiation following a single dose irradiation and more pronounced in combination with a 6 h fractionation (3 × 2 Gy) regime. For preclinical analyses we focused on cervical cancer. Nek1 shRNA HeLa cells were grafted into NOD/SCID/IL-2Rγc−/− (NSG) mice and Nek1 KD was induced by Dox-infused drinking water resulting in a significant cytostatic effect if combined with a 6 h fractionation (3 × 2 Gy) regime. In addition, we correlated Nek1 expression in biopsies of patients with cervical cancer with histopathological parameters and clinical follow-up. Our results indicate that elevated levels of Nek1 were associated with an increased rate of local or distant failure, as well as with impaired cancer-specific and overall survival in univariate analyses and for most endpoints in multivariable analyses. Finally, findings from The Cancer Genome Atlas (TCGA) validation cohort confirmed a significant association of high Nek1 expression with a reduced disease-free survival. In conclusion, we consider Nek1 to represent a novel biomarker and potential therapeutic target for drug development in the context of optimized fractionation intervals.

The Anticancer Drug 3-Bromopyruvate Induces DNA Damage Potentially Through Reactive Oxygen Species in Yeast and in Human Cancer Cells.

3-bromopyruvate (3-BP) is a small molecule with anticancer and antimicrobial activities. 3-BP is taken up selectively by cancer cells’ mono-carboxylate transporters (MCTs), which are highly overexpressed by many cancers. When 3-BP enters cancer cells it inactivates several glycolytic and mitochondrial enzymes, leading to ATP depletion and the generation of reactive oxygen species. While mechanisms of 3-BP uptake and its influence on cell metabolism are well understood, the impact of 3-BP at certain concentrations on DNA integrity has never been investigated in detail. Here we have collected several lines of evidence suggesting that 3-BP induces DNA damage probably as a result of ROS generation, in both yeast and human cancer cells, when its concentration is sufficiently low and most cells are still viable. We also demonstrate that in yeast 3-BP treatment leads to generation of DNA double-strand breaks only in S-phase of the cell cycle, possibly as a result of oxidative DNA damage. This leads to DNA damage, checkpoint activation and focal accumulation of the DNA response proteins. Interestingly, in human cancer cells exposure to 3-BP also induces DNA breaks that trigger H2A.X phosphorylation. Our current data shed new light on the mechanisms by which a sufficiently low concentration of 3-BP can induce cytotoxicity at the DNA level, a finding that might be important for the future design of anticancer therapies.

Characterisation of unessential genes required for survival under conditions of DNA stress

BackgroundGenomic instability is a hallmark of cancer. Cancer progression depends on the development and amplification of mutations that alter the cellular response to threats to the genome. This can lead to DNA replication stress and the potential loss of genetic integrity of the newly formed cells. This study utilised fission yeast to map the interactions occurring in some of the most crucial pathways in both DNA replication and checkpoint monitoring involving Rad4, the Schizosaccharomyces pombe (S. pombe) TopBP1 homologue. We have modelled conditions of replication stress in the genetically tractable fission yeast, S. pombe using the hypomorphic rad4-116 allele. Synthetic genetic analysis was used to identify processes required for cell survival under conditions of DNA replication stress. With the aim of mapping the genetic interactions of rad4 and its mutant allele, rad4-116, several genes that could have an interaction with rad4 during replication stress have emerged as attractive. ResultsInteractions with genes involved in chromatin remodelling, such as hip1, and replication fork stalling resolution, such as mrc1, swi1 and swi3 were explored and confirmed. The interactions of Rad4 with each of the genes provided separate and distinct tumour formation pathways, as evident in the synthetically lethal interactions. Even within the same complex, rad4-116 double mutants behaved differently proving that Rad4 interacts at different levels and functions with the same proteins. ConclusionResults from this study provide a novel view of the rad4 interactions, the association of Rad4 with the replisome. The study also provides the groundwork on a theoretical and practical level for the exploration and separation of interactions of TopBP1 with the histone chaperone family and the replisome.

Abstract A091: Targeting homologous recombination repair defects in lung cancer

Abstract Non-Small Cell Lung Cancer (NSCLC) is the leading cause of cancer related mortality worldwide, and accounts for 80-85% of all lung cancer diagnoses. While targeted therapies against epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) fusions have proven to be effective for the treatment of NSCLC, only a fraction of NSCLC patients (i.e. <20%) benefit from these targeted agents. Although immunotherapy approaches, specifically PD-1:PD-L1 blockade, appear to be more broadly efficacious, there are still a substantial proportion of patients who may not benefit from these treatments. To investigate the potential of targeting the DNA Damage Response (DDR) pathway in lung cancer, as an alternative therapeutic approach for these patients we sought to identify whether functionally relevant HRR (Homologous Recombination Repair)-defects could be synthetically lethal with niraparib monotherapy in NSCLC PDX models. Approximately 3% of lung cancers harbor mutations in ATM (i.e. 3.5% in lung adenocarcinomas & 1.4% in lung squamous cell carcinoma), which is involved in HRR as well as multiple other DNA repair and checkpoint functions, but the functional consequences of this alteration remain to be established. Studies in the past few years have demonstrated the potential of targeting the DDR pathway as a promising therapeutic strategy for lung cancer, i.e. in small cell lung cancer (SCLC) where overall response rates to immune checkpoint blockade remain poor. In addition, inhibitors targeting DDR proteins have shown promise in preclinical models of lung cancer, and are under clinical investigation as single agents and in combination with other targeted and immunotherapy agents. In this study, we have evaluated niraparib sensitivity in 57 NSCLC PDX models containing both BRCA and non-BRCA HRR mutations (n=17) as well as HRR WT models (n=40). This analysis demonstrated that niraparib sensitive models include both HRR mutant and HRR WT lung tumors (TGI > 70%). Amongst the PDX models containing a biallelic HRR mutation (ATM, BAP1, RAD51D & XRCC2), only ATM biallelic mutant models were sensitive to niraparib (2 out of 6). Furthermore, none of the of nine monoallelic mutant models (ATM, ATR, BRCA1/2, RAD50 & RAD52) were sensitive to niraparib. Surprisingly, 7.5% (3 out of 40) of the HRR WT PDX models were sensitive to niraparib. Further analysis is required to elucidate the molecular mechanisms driving niraparib sensitivity in HRR WT NSCLC. Citation Format: Asli Muvaffak, Yinghui Zhou, Bin Feng, Sarah Wang, Jing Ju Wang, Sridhar Ramaswamy, Kevin G Coleman. Targeting homologous recombination repair defects in lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A091. doi:10.1158/1535-7163.TARG-19-A091

Functional analysis of genetic variants in the high-risk breast cancer susceptibility gene PALB2

Heterozygous carriers of germ-line loss-of-function variants in the DNA repair gene PALB2 are at a highly increased lifetime risk for developing breast cancer. While truncating variants in PALB2 are known to increase cancer risk, the interpretation of missense variants of uncertain significance (VUS) is in its infancy. Here we describe the development of a relatively fast and easy cDNA-based system for the semi high-throughput functional analysis of 48 VUS in human PALB2. By assessing the ability of PALB2 VUS to rescue the DNA repair and checkpoint defects in Palb2 knockout mouse embryonic stem (mES) cells, we identify various VUS in PALB2 that impair its function. Three VUS in the coiled-coil domain of PALB2 abrogate the interaction with BRCA1, whereas several VUS in the WD40 domain dramatically reduce protein stability. Thus, our functional assays identify damaging VUS in PALB2 that may increase cancer risk.