Homologous Recombination-mediated DNA Repair Research Articles

Global crotonylome reveals CDYL-regulated RPA1 crotonylation in homologous recombination-mediated DNA repair.

Previously, we reported that chromodomain Y-like (CDYL) acts as a crotonyl-coenzyme A hydratase and negatively regulates histone crotonylation (Kcr). However, the global CDYL-regulated crotonylome remains unclear. Here, we report a large-scale proteomics analysis for protein Kcr. We identify 14,311 Kcr sites across 3734 proteins in HeLa cells, providing by far the largest crotonylome dataset. We show that depletion of CDYL alters crotonylome landscape affecting diverse cellular pathways. Specifically, CDYL negatively regulated Kcr of RPA1, and mutation of the Kcr sites of RPA1 impaired its interaction with single-stranded DNA and/or with components of resection machinery, supporting a key role of RPA1 Kcr in homologous recombination DNA repair. Together, our study indicates that protein crotonylation has important implication in various pathophysiological processes.

Radiation Responses of 2D and 3D Glioblastoma Cells: A Novel, 3D-specific Radioprotective Role of VEGF/Akt Signaling through Functional Activation of NHEJ.

Glioblastoma is resistant to conventional treatments and has dismal prognosis. Despite promising in vitro data, molecular targeted agents have failed to improve outcomes in patients, indicating that conventional two-dimensional (2D) in vitro models of GBM do not recapitulate the clinical scenario. Responses of primary glioblastoma stem-like cells (GSC) to radiation in combination with EGFR, VEGF, and Akt inhibition were investigated in conventional 2D cultures and a three-dimensional (3D) in vitro model of GBM that recapitulates key GBM clinical features. VEGF deprivation had no effect on radiation responses of 2D GSCs, but enhanced radiosensitivity of GSC cultures in 3D. The opposite effects were observed for EGFR inhibition. Detailed analysis of VEGF and EGF signaling demonstrated a radioprotective role of Akt that correlates with VEGF in 3D and with EGFR in 2D. In all cases, positive correlations were observed between increased radiosensitivity, markers of unrepaired DNA damage and persistent phospho-DNA-PK nuclear foci. Conversely, increased numbers of Rad51 foci were observed in radioresistant populations, indicating a novel role for VEGF/Akt signaling in influencing radiosensitivity by regulating the balance between nonhomologous end-joining and homologous recombination-mediated DNA repair. Differential activation of tyrosine kinase receptors in 2D and 3D models of GBM explains the well documented discrepancy between preclinical and clinical effects of EGFR inhibitors. Data obtained from our 3D model identify novel determinants and mechanisms of DNA repair and radiosensitivity in GBM, and confirm Akt as a promising therapeutic target in this cancer of unmet need.

Abstract C067: Mechanistic study of the superior anti-cancer properties of a first-in-class small molecule targeting PCNA

Abstract Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was predominantly expressed in a broad range of cancer cells and tumor tissues, but not significantly in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interdomain connector loop of PCNA that is known to be a major binding site for many of PCNA’s interacting proteins. A cell permeable peptide harboring the L126-Y133 sequence inhibited PCNA function in cancer cells and selectively kills cancer cells and xenograft tumors. Based on these observations, we sought small molecules targeting this peptide region of PCNA as potential broad-spectrum anti-cancer agents. Our effort led to a drug candidate, AOH1996, which selectively kills a broad range of cancer cells at high nanomolar concentrations, but is not associated with significant toxicity to non-malignant cells. It also works synergistically with DNA damaging chemotherapeutic drugs, such as cisplatin and irinotecan, to selectively kill cancer cells. This compound is orally available to animals and suppresses tumor growth in a dosage form compatible to clinical applications. Importantly, it doesn’t cause significant toxicity at 2.5 times its effective dose. Mechanistically, AOH1996 competes with T3, a known PCNA ligand, for binding to PCNA. However, the mechanism by which AOH1996 exerts its effect on cancer cells may not be identical to what have been reported for the T3 analogs. In particular, we found that AOH1996 interferes with the association of PCNA and its binding proteins, leading to DNA replication stress, blockade of homologous recombination-mediated DNA repair, and induction of apoptosis in cancer cells. These findings demonstrated the potential of this compound as a novel therapeutic agent warranting clinical investigation for cancer treatment. We have started planning a phase 1 clinical study for this compound. Citation Format: Long Gu, Min Li, Robert Lingeman, Robert J Hickey, Yilun Liu, Linda H Malkas. Mechanistic study of the superior anti-cancer properties of a first-in-class small molecule targeting PCNA [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 C067. doi:10.1158/1535-7163.TARG-19-C067

Endonucleases responsible for DNA repair of helix-distorting DNA lesions in the thermophilic crenarchaeon Sulfolobus acidocaldarius in vivo.

The DNA repair mechanisms of hyperthermophiles can provide important insights for understanding how genetic information is maintained under extreme environments. Recent biochemical studies have identified a novel endonuclease in hyperthermophilic archaea, NucS/EndoMS, that acts on branched DNA substrates and mismatched bases. NucS/EndoMS is thought to participate in the DNA repair of helix-distorting DNA lesions, including UV-induced DNA damage and DNA adducts, and mismatched bases; however, the specific in vivo role of NucS/EndoMS in hyperthermophilic archaeal DNA repair has not been reported. To explore the role of this protein, we knocked out the nucS/endoMS gene of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. While the nucS/endoMS-deleted strain exhibited sensitivity to DNA adducts, it did not have high mutation rates or any sensitivity to UV irradiation. It has been proposed that the XPF endonuclease is involved in homologous recombination-mediated stalled-fork DNA repair. The xpf-deficient strain exhibited sensitivity to helix-distorting DNA lesions, but the sensitivity of the nucS/endoMS and xpf double knockout strain did not increase compared to that of the single knockout strains. We conclude that the endonuclease NucS/EndoMS works with XPF in homologous recombination-mediated stalled-fork DNA repair for the removal of helix-distorting DNA lesions in S. acidocaldarius.

Abstract 3027: Pharmacological regulation of the ABCG2 multidrug transporter in A549 non-small cell lung cancer cells

Abstract The notoriously drug resistant non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths and the ABCG2 multidrug transporter was shown to be involved in NSCLC drug resistance. Direct drug interactions with this transporter have been extensively studied, while a potential modulation of ABCG2 expression by drugs or hormones is less explored. To study the drug-dependent regulation of ABCG2 expression, we used CRISPR-Cas9 and homologous recombination-mediated DNA repair to generate fluorescent lung cancer reporter cells with preserved endogenous regulatory regions. In A549 lung adenocarcinoma cells, which have two wild type copies of the ABCG2 gene, we replaced the initial coding region of ABCG2 with a green fluorescent protein (eGFP)-coding sequence. In addition, since our previous studies demonstrated that an N-terminally eGFP-tagged ABCG2 protein is properly localized and functional, we have generated A549 cells in which the native ABCG2 coding region was modified to express an eGFP-tagged ABCG2 protein. The eGFP knock-in cells sensitively reported ABCG2 transcriptional regulation, while the cells expressing an eGFP-ABCG2 fusion protein allowed the assessment of the full, functional transporter. Using the engineered A549 cells, we found that several drugs, including anti-cancer medications, glucocorticoids, HDAC inhibitors and hypoxia-mimicking agents, caused a significant induction of ABCG2 expression. In these experiments, an intronic regulatory element was found to play a key role in the specific glucocorticoid induction of ABCG2. We suggest that our transgenic reporter cell lines, amenable to high-throughput studies, can be used to investigate the induction of ABCG2-based drug resistance in cancer and predict potential drug-drug interactions. Understanding how the generation of drug-tolerant persister cells occurs via ABCG2 expression may help the selective elimination of these multidrug resistant cancer cells. Supported by the NKFIH-OTKA grant K115375. Citation Format: Daniella Kovacsics, Nikolett Mészáros, Borbála Tihanyi, Zsolt Matula, Adrienn Borsy, Edit Szabó, György Várady, Tamás Orbán, Ágota Apáti, Ábel Fóthi, Anna Brózik, Balázs Sarkadi. Pharmacological regulation of the ABCG2 multidrug transporter in A549 non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3027.

Mechanistic study of the superior anti-cancer properties of a first-in-class small molecule targeting PCNA.

e14636 Background: Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was predominantly expressed in a broad range of cancer cells and tumor tissues, but not significantly in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interdomain connector loop of PCNA that is known to be a major binding site for many of PCNA’s interacting proteins. A cell permeable peptide harboring the L126-Y133 sequence inhibited PCNA function in cancer cells and selectively kills cancer cells and xenograft tumors. Methods: Based on these observations, we sought small molecules targeting this peptide region of PCNA as potential broad-spectrum anti-cancer agents. Results: Our effort led to a drug candidate, AOH1996, which selectively kills a broad range of cancer cells at high nanomolar concentrations, but is not associated with significant toxicity to non-malignant cells. It also works synergistically with DNA damaging chemotherapeutic drugs, such as cisplatin and irinotecan, to selectively kill cancer cells. This compound is orally available to animals and suppresses tumor growth in a dosage form compatible to clinical applications. Importantly, it doesn’t cause significant toxicity at 2.5 times its effective dose. Mechanistically, AOH1996 competes with T3, a known PCNA ligand, for binding to PCNA. However, the mechanism by which AOH1996 exerts its effect on cancer cells may not be identical to what have been reported for the T3 analogs. In particular, we found that AOH1996 interferes with the association of PCNA and MCM7 to euchromatin, leading to DNA replication stress, blockade of homologous recombination-mediated DNA repair, and induction of apoptosis in cancer cells. Conclusions: These findings demonstrated the potential of this compound as a novel therapeutic agent warranting clinical investigation for cancer treatment. We have started planning a phase 1 clinical study for this compound.

Cell-intrinsic programmed death ligand-1 (PD-L1) inhibits cytotoxic chemo, promotes DNA damage repair, and enhances ATM/ATR signaling following exposure to DNA damaging agents in bladder, melanoma, and ovarian cancer cells

Abstract We have shown that cancer cell-intrinsic PD-L1 signals increase mTORC1 and unbundle actin. To test if tumor cell PD-L1 affects the DNA damage response (DDR) we made PD-L1KO mouse ID8agg (ovarian) and B16 (melanoma), and human bladder RT4 cancer lines. The DNA damaging agent gemcitabine (gem) reduced PD-L1KO ID8agg and RT4 viability in vitro vs. control (con) (ID8agg: 29X vs. 1.3X; RT4: 2X vs 1.1X). 4h gem increased phospho (p)-γH2AX, a marker of DNA double strand breaks: 2X in PD-L1KO vs. con RT4. P-γH2AX stayed abnormally high after gem removal, further showing defective DDR in PD-L1KO. 24h gem also induced more p-γH2AX in PD-L1KO vs. con ID8agg, and 48h gem induced more apoptosis in PD-L1KO vs. con RT4. Cisplatin, a DNA damaging agent distinct from gem, reduced PD-L1KO ID8agg viability 9X vs. 2X in con. No viability difference was seen in cisplatin treated PD-L1KO vs. con RT4. Thus, PD-L1 chemo resistance effects are cell type and/or agent specific. To define DDR mechanism(s), we found that 24h gem decreased p-Chk2 (ATM DDR pathway), due to reduced total Chk2, but not p-Chk1 or total Chk1 (ATR DDR pathway) in PD-L1KO vs. con RT4. 20 Gy X-rays (DNA damage distinct vs. gem), reduced p-Chk1 in PD-L1KO but not con B16 and induced less p-RPA32 in PD-L1KO vs. con B16, suggesting defects specifically in homologous recombination mediated DNA repair. Thus, cell-intrinsic PD-L1 DDR control is insult and/or cell type specific. RT4 DDR effects were independent of mTOR or actin. DDR affects immunotherapy so PD-L1 cell-intrinsic DDR control could be exploited in treatment or response biomarkers. DDR inhibitors, chemo or radiation combos could improve anti-PD-L1 efficacy in distinct cancers, subjects of ongoing work, as is testing tumor cell intrinsic PD-1 effects.

KEOPS complex promotes homologous recombination via DNA resection.

KEOPS complex is one of the most conserved protein complexes in eukaryotes. It plays important roles in both telomere uncapping and tRNA N6-threonylcarbamoyladenosine (t6A) modification in budding yeast. But whether KEOPS complex plays any roles in DNA repair remains unknown. Here, we show that KEOPS complex plays positive roles in both DNA damage response and homologous recombination-mediated DNA repair independently of its t6A synthesis function. Additionally, KEOPS displays DNA binding activity in vitro, and is recruited to the chromatin at DNA breaks in vivo, suggesting a direct role of KEOPS in DSB repair. Mechanistically, KEOPS complex appears to promote DNA end resection through facilitating the association of Exo1 and Dna2 with DNA breaks. Interestingly, inactivation of both KEOPS and Mre11/Rad50/Xrs2 (MRX) complexes results in synergistic defect in DNA resection, revealing that KEOPS and MRX have some redundant functions in DNA resection. Thus we uncover a t6A-independent role of KEOPS complex in DNA resection, and propose that KEOPS might be a DSB sensor to assist cells in maintaining chromosome stability.

UBQLN4 Regulates DNA Repair Pathway Choice.

The proteasomal shuttle factor UBQLN4 represses homologous recombination-mediated DNA repair (HRR).

Discovery of peptide ligands targeting a specific ubiquitin-like domain–binding site in the deubiquitinase USP11

Ubiquitin-specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes. Defined noncatalytic domains in USP4 and USP15 are known to interact with E3 ligases and substrate recruitment factors. No such interactions have been reported for these domains in the paralog USP11, a key regulator of DNA double-strand break repair by homologous recombination. We hypothesized that USP11 domains adjacent to its protease domain harbor unique peptide-binding sites. Here, using a next-generation phage display (NGPD) strategy, combining phage display library screening with next-generation sequencing, we discovered unique USP11-interacting peptide motifs. Isothermal titration calorimetry disclosed that the highest affinity peptides (KD of ∼10 μm) exhibit exclusive selectivity for USP11 over USP4 and USP15 in vitro. Furthermore, a crystal structure of a USP11–peptide complex revealed a previously unknown binding site in USP11's noncatalytic ubiquitin-like (UBL) region. This site interacted with a helical motif and is absent in USP4 and USP15. Reporter assays using USP11-WT versus a binding pocket–deficient double mutant disclosed that this binding site modulates USP11's function in homologous recombination–mediated DNA repair. The highest affinity USP11 peptide binder fused to a cellular delivery sequence induced significant nuclear localization and cell cycle arrest in S phase, affecting the viability of different mammalian cell lines. The USP11 peptide ligands and the paralog-specific functional site in USP11 identified here provide a framework for the development of new biochemical tools and therapeutic agents. We propose that an NGPD-based strategy for identifying interacting peptides may be applied also to other cellular targets.

BET proteins regulate homologous recombination-mediated DNA repair: BRCAness and implications for cancer therapy.

Bromodomain and Extra-Terminal (BET) proteins are historically involved in regulating gene expression and BRD4 was recently found to be involved in DNA damage regulation. Aims of our study were to assess BRD4 regulation in homologous recombination-mediated DNA repair and to explore novel clinical strategies through the combinations of the pharmacological induction of epigenetic BRCAness in BRCA1 wild-type triple negative breast cancer (TNBC) cells by means of BET inhibitors and compounds already available in clinic. Performing a dual approach (chromatin immunoprecipitation and RNA interference), the direct relationship between BRD4 and BRCA1/RAD51 expression was confirmed in TNBC cells. Moreover, BRD4 pharmacological inhibition using two BET inhibitors (JQ1 and GSK525762A) induced a dose-dependent reduction in BRCA1 and RAD51 levels and is able to hinder homologous recombination-mediated DNA damage repair, generating a BRCAness phenotype in TNBC cells. Furthermore, BET inhibition impaired the ability of TNBC cells to overcome the increase in DNA damage after platinum salts (i.e., CDDP) exposure, leading to massive cell death, and triggered synthetic lethality when combined with PARP inhibitors (i.e., AZD2281). Altogether, the present study confirms that BET proteins directly regulate the homologous recombination pathway and their inhibition induced a BRCAness phenotype in BRCA1 wild-type TNBC cells. Noteworthy, being this strategy based on drugs already available for human use, it is rapidly transferable and could potentially enable clinicians to exploit platinum salts and PARP inhibitors-based treatments in a wider population of TNBC patients and not just in a specific subgroup, after validating clinical trials.

The Anticancer Activity of a First-in-class Small-molecule Targeting PCNA.

Proliferating cell nuclear antigen (PCNA) plays an essential role in regulating DNA synthesis and repair and is indispensable to cancer cell growth and survival. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was ubiquitously expressed in a broad range of cancer cells and tumor tissues, but not significantly in nonmalignant cells. We found the L126-Y133 region of caPCNA is structurally altered and more accessible to protein-protein interaction. A cell-permeable peptide harboring the L126-Y133 sequence blocked PCNA interaction in cancer cells and selectively kills cancer cells and xenograft tumors. On the basis of these findings, we sought small molecules targeting this peptide region as potential broad-spectrum anticancer agents. By computer modeling and medicinal chemistry targeting a surface pocket partly delineated by the L126-Y133 region of PCNA, we identified a potent PCNA inhibitor (AOH1160) and characterized its therapeutic properties and potential toxicity. AOH1160 selectively kills many types of cancer cells at below micromolar concentrations without causing significant toxicity to a broad range of nonmalignant cells. Mechanistically, AOH1160 interferes with DNA replication, blocks homologous recombination-mediated DNA repair, and causes cell-cycle arrest. It induces apoptosis in cancer cells and sensitizes them to cisplatin treatment. AOH1160 is orally available to animals and suppresses tumor growth in a dosage form compatible to clinical applications. Importantly, it does not cause significant toxicity at 2.5 times of an effective dose. These results demonstrated the favorable therapeutic properties and the potential of AOH1160 as a broad-spectrum therapeutic agent for cancer treatment.

Resistance to UV Irradiation Caused by Inactivation of nurA and herA Genes in Thermus thermophilus.

NurA and HerA are thought to be essential proteins for DNA end resection in archaeal homologous recombination systems. Thermus thermophilus, an extremely thermophilic eubacterium, has proteins that exhibit significant sequence similarity to archaeal NurA and HerA. To unveil the cellular function of NurA and HerA in T. thermophilus, we performed phenotypic analysis of disruptant mutants of nurA and herA with or without DNA-damaging agents. The nurA and herA genes were not essential for survival, and their deletion had no effect on cell growth and genome integrity. Unexpectedly, these disruptants of T. thermophilus showed increased resistance to UV irradiation and mitomycin C treatment. Further, these disruptants and the wild type displayed no difference in sensitivity to oxidative stress and a DNA replication inhibitor. T. thermophilus NurA had nuclease activity, and HerA had ATPase. The overexpression of loss-of-function mutants of nurA and herA in the respective disruptants showed no complementation, suggesting their enzymatic activities were involved in the UV sensitivity. In addition, T. thermophilus NurA and HerA interacted with each other in vitro and in vivo, forming a complex with 2:6 stoichiometry. These results suggest that the NurA-HerA complex has an architecture similar to that of archaeal counterparts but that it impairs, rather than promotes, the repair of photoproducts and DNA cross-links in T. thermophilus cells. This cellular function is distinctly different from that of archaeal NurA and HerA.IMPORTANCE Many nucleases and helicases are engaged in homologous recombination-mediated DNA repair. Previous in vitro analyses in archaea indicated that NurA and HerA are the recombination-related nuclease and helicase. However, their cellular function had not been fully understood, especially in bacterial cells. In this study, we performed in vivo analyses to address the cellular function of nurA and herA in an extremely thermophilic bacterium, Thermus thermophilus As a result, T. thermophilus NurA and HerA exhibited an interfering effect on the repair of several instances of DNA damage in the cell, which is in contrast to the results in archaea. This finding will facilitate our understanding of the diverse cellular functions of the recombination-related nucleases and helicases.

Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling.

The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we used a combined genetic-phosphoproteomic approach to manipulate Mec1 activation and globally monitor Mec1 signaling, allowing us to delineate distinct checkpoint-independent modes of Mec1 action. Using cells in which endogenous Mec1 activators were genetically ablated, we found that expression of "free" Mec1 activation domains (MADs) can robustly activate Mec1 and rescue the severe DNA replication and growth defects of these cells back to wild-type levels. However, unlike the activation mediated by endogenous activator proteins, "free" MADs are unable to stimulate Mec1-mediated suppression of gross chromosomal rearrangements (GCRs), revealing that Mec1's role in genome maintenance is separable from a previously unappreciated proreplicative function. Both Mec1's functions in promoting replication and suppressing GCRs are independent of the downstream checkpoint kinases. Additionally, Mec1-dependent GCR suppression seems to require localized Mec1 action at DNA lesions, which correlates with the phosphorylation of activator-proximal substrates involved in homologous recombination-mediated DNA repair. These findings establish that Mec1 initiates checkpoint signaling, promotes DNA replication, and maintains genetic stability through distinct modes of action.

BRCA1 or CDK12 loss sensitizes cells to CHK1 inhibitors.

A broad spectrum of tumors develop resistance to classic chemotherapy, necessitating the discovery of new therapies. One successful strategy exploits the synthetic lethality between poly(ADP-ribose) polymerase 1/2 proteins and DNA damage response genes, including BRCA1, a factor involved in homologous recombination-mediated DNA repair, and CDK12, a transcriptional kinase known to regulate the expression of DDR genes. CHK1 inhibitors have been shown to enhance the anti-cancer effect of DNA-damaging compounds. Since loss of BRCA1 increases replication stress and leads to DNA damage, we tested a hypothesis that CDK12- or BRCA1-depleted cells rely extensively on S-phase-related CHK1 functions for survival. The silencing of BRCA1 or CDK12 sensitized tumor cells to CHK1 inhibitors in vitro and in vivo. BRCA1 downregulation combined with CHK1 inhibition induced excessive amounts of DNA damage, resulting in an inability to complete the S-phase. Therefore, we suggest CHK1 inhibition as a strategy for targeting BRCA1- or CDK12-deficient tumors.

Abstract 5883: CHD1 as regulator of cell adhesion in Ets fusion negative prostate cancer

Abstract Prostate cancer (PCa) develops into lethal disease in about 10% of men diagnosed with this malignancy. Common genomic changes are seen in PCa such as alterations in CDKN1B, RB1, TP53, PTEN, NKX3-1, MYC and androgen receptor. Oncogenic fusions involving the Ets family are seen in 50-70% PCa patients, the most common of which is TMPRSS2-ERG. TMPRSS2-ERG influences cell migration, particularly when PTEN is co-deleted or PIK3CA activating mutations are present. However, in the absence of ERG fusions, molecular drivers of the PCa phenotype are less clear but include alterations in SPOP (6-15% of PCa), SPINK1 (10%), MAP3K7 (18-38%) and CHD1 (15-27%). CHD1 (Chromo-domain Helicase DNA Binding Protein-1) is a chromatin remodeling factor with many roles including homologous recombination mediated DNA repair of double strand breaks and maintenance of genomic stability and therefore it is hypothesized to cause the predominant intrachromosomal rearrangements observed in Ets fusion negative tumors. This study was undertaken to evaluate the cellular effects that follow CHD1 deletion that could aid PCa initiation and progression. CHD1 was knocked out in the non tumorigenic, HPV16 immortalised prostate epithelial cell line, RWPE-1, using CRISPR/Cas9. Compared to parental RWPE-1 and non target cells (NT2), the CHD1 KO cells had a smaller and rounder morphology and were less adherent to tissue culture plates. To study expression of extracellular matrix and adhesion molecules, RT profiler assay was performed using RWPE-1 parental and CHD1 KO cells. CHD1 KO cells showed a decrease in the expression of SPARC, MMP2, ITGA2, ITGA5, ITGA6, FN1, LAMB3, collagen, tenascin and vitronectin as compared to parentals and NT2. Reduction in extracellular matrix and adhesion molecules and altered morphology of CHD1 KO cells as compared to parentals and NT2 cells suggest that loss of CHD1 reduces cell-cell and cell-matrix adhesion. These results suggest that in the absence of PTEN loss in Ets negative tumors, deletion of CHD1 gene could change cell adhesion dynamics ascribing a new role for CHD1 in the development and progression of PCa. Citation Format: Aparna Kareddula, Whitney Petrosky, Irina Tereshchenko, Daniel Medina, Hana Aviv, Eric Singer, Robert S. DiPaola, Kim M. Hirshfield. CHD1 as regulator of cell adhesion in Ets fusion negative prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5883. doi:10.1158/1538-7445.AM2017-5883

Shutting down the power supply for DNA repair in cancer cells.

Phosphoglycerate mutase 1 (PGAM1) functions in glycolysis. In this issue, Qu et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201607008) show that PGAM1 inactivation leads to nucleotide depletion, which causes defective homologous recombination-mediated DNA repair, suggesting that targeting metabolic enzymes increases cancer cell susceptibility to DNA damaging agents.

Small Molecule Inhibition of the Ubiquitin-specific Protease USP2 Accelerates cyclin D1 Degradation and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell Lymphoma Models

Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 μm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.

Turning the concept of synthetic lethality on its head

One of the most significant discoveries in cancer treatment over the past 15 years has been the advent of poly(ADP-ribose) polymerase (PARP) inhibitors, and their exploitation of the concept of synthetic lethality in the treatment of BRCA1/2 mutant cancers (1,2). In synthetic lethality, the loss of each gene or protein by itself does not affect cell viability, but the loss of both results in cell death. The goal of this approach is to optimise targeted tumor cell death, while sparing normal tissue (the “magic bullet” first proposed by Paul Ehlrich in the late 19th century). PARP inhibitors in BRCA1/2 mutant tumors have been the classical example of this, where the loss of homologous recombination-mediated DNA repair through BRCA1/2 mutation renders cells sensitive to inhibition or loss of PARP1.

Incision of damaged DNA in the presence of an impaired Smc5/6 complex imperils genome stability

The Smc5/6 complex is implicated in homologous recombination-mediated DNA repair during DNA damage or replication stress. Here, we analysed genome-wide replication dynamics in a hypomorphic budding yeast mutant, smc6-P4. The overall replication dynamics in the smc6 mutant is similar to that in the wild-type cells. However, we captured a difference in the replication profile of an early S phase sample in the mutant, prompting the hypothesis that the mutant incorporates ribonucleotides and/or accumulates single-stranded DNA gaps during replication. We tested if inhibiting the ribonucleotide excision repair pathway would exacerbate the smc6 mutant in response to DNA replication stress. Contrary to our expectation, impairment of ribonucleotide excision repair, as well as virtually all other DNA repair pathways, alleviated smc6 mutant's hypersensitivity to induced replication stress. We propose that nucleotide incision in the absence of a functional Smc5/6 complex has more disastrous outcomes than the damage per se. Our study provides novel perspectives for the role of the Smc5/6 complex during DNA replication.