The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation.

TIMELESS (TIM) in the fork protection complex acts as a scaffold of the replisome to prevent its uncoupling and ensure efficient DNA replication fork progression. Nevertheless, its underlying basis for coordinating leading and lagging strand synthesis to limit single-stranded DNA (ssDNA) exposure remains elusive. Here, we demonstrate that acute degradation of TIM at ongoing DNA replication forks induces the accumulation of ssDNA gaps stemming from defective Okazaki fragment (OF) processing. Cells devoid of TIM fail to support the poly(ADP-ribosyl)ation necessary for backing up the canonical OF processing mechanism mediated by LIG1 and FEN1. Consequently, recruitment of XRCC1, a known effector of PARP1-dependent single-strand break repair, to post-replicative ssDNA gaps behind replication forks is impaired. Physical disruption of the TIM-PARP1 complex phenocopies the rapid loss of TIM, indicating that the TIM-PARP1 interaction is critical for the activation of this compensatory pathway. Accordingly, combined deficiency of FEN1 and the TIM-PARP1 interaction leads to synergistic DNA damage and cytotoxicity. We propose that TIM is essential for the engagement of PARP1 to the replisome to coordinate lagging strand synthesis with replication fork progression. Our study identifies TIM as a synthetic lethal target of OF processing enzymes that can be exploited for cancer therapy.

Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection.

Poly(ADP-ribosyl)ation (PARylation), catalyzed mainly by poly(ADP-ribose) polymerase (PARP)1, is a key posttranslational modification involved in DNA replication and repair. Here, we report that TIMELESS (TIM), an essential scaffold of the replisome, is PARylated, which is linked to its proteolysis. TIM PARylation requires recognition of auto-modified PARP1 via two poly(ADP-ribose)-binding motifs, which primes TIM for proteasome-dependent degradation. Cells expressing the PARylation-refractory TIM mutant or under PARP inhibition accumulate TIM at DNA replication forks, causing replication stress and hyper-resection of stalled forks. Mechanistically, aberrant engagement of TIM with the replicative helicase impedes RAD51 loading and protection of reversed forks. Accordingly, defective TIM degradation hypersensitizes BRCA2-deficient cells to replication damage. Our study defines TIM as a substrate of PARP1 and elucidates how the control of replisome remodeling by PARylation is linked to stalled fork protection. Therefore, we propose a mechanism of PARP inhibition that impinges on the DNA replication fork instability caused by defective TIM turnover.

The Adaptive Mechanisms and Checkpoint Responses to a Stressed DNA Replication Fork.

DNA replication is a tightly controlled process that ensures the faithful duplication of the genome. However, DNA damage arising from both endogenous and exogenous assaults gives rise to DNA replication stress associated with replication fork slowing or stalling. Therefore, protecting the stressed fork while prompting its recovery to complete DNA replication is critical for safeguarding genomic integrity and cell survival. Specifically, the plasticity of the replication fork in engaging distinct DNA damage tolerance mechanisms, including fork reversal, repriming, and translesion DNA synthesis, enables cells to overcome a variety of replication obstacles. Furthermore, stretches of single-stranded DNA generated upon fork stalling trigger the activation of the ATR kinase, which coordinates the cellular responses to replication stress by stabilizing the replication fork, promoting DNA repair, and controlling cell cycle and replication origin firing. Deregulation of the ATR checkpoint and aberrant levels of chronic replication stress is a common characteristic of cancer and a point of vulnerability being exploited in cancer therapy. Here, we discuss the various adaptive responses of a replication fork to replication stress and the roles of ATR signaling that bring fork stabilization mechanisms together. We also review how this knowledge is being harnessed for the development of checkpoint inhibitors to trigger the replication catastrophe of cancer cells.

Replisome dysfunction upon inducible TIMELESS degradation synergizes with ATR inhibition to trigger replication catastrophe.

The structure of DNA replication forks is preserved by TIMELESS (TIM) in the fork protection complex (FPC) to support seamless fork progression. While the scaffolding role of the FPC to couple the replisome activity is much appreciated, the detailed mechanism whereby inherent replication fork damage is sensed and counteracted during DNA replication remains largely elusive. Here, we implemented an auxin-based degron system that rapidly triggers inducible proteolysis of TIM as a source of endogenous DNA replication stress and replisome dysfunction to dissect the signaling events that unfold at stalled forks. We demonstrate that acute TIM degradation activates the ATR-CHK1 checkpoint, whose inhibition culminates in replication catastrophe by single-stranded DNA accumulation and RPA exhaustion. Mechanistically, unrestrained replisome uncoupling, excessive origin firing, and aberrant reversed fork processing account for the synergistic fork instability. Simultaneous TIM loss and ATR inactivation triggers DNA-PK-dependent CHK1 activation, which is unexpectedly necessary for promoting fork breakage by MRE11 and catastrophic cell death. We propose that acute replisome dysfunction results in a hyper-dependency on ATR to activate local and global fork stabilization mechanisms to counteract irreversible fork collapse. Our study identifies TIM as a point of replication vulnerability in cancer that can be exploited with ATR inhibitors.

The TIMELESS effort for timely DNA replication and protection.

Accurate replication of the genome is fundamental to cellular survival and tumor prevention. The DNA replication fork is vulnerable to DNA lesions and damages that impair replisome progression, and improper control over DNA replication stress inevitably causes fork stalling and collapse, a major source of genome instability that fuels tumorigenesis. The integrity of the DNA replication fork is maintained by the fork protection complex (FPC), in which TIMELESS (TIM) constitutes a key scaffold that couples the CMG helicase and replicative polymerase activities, in conjunction with its interaction with other proteins associated with the replication machinery. Loss of TIM or the FPC in general results in impaired fork progression, elevated fork stalling and breakage, and a defect in replication checkpoint activation, thus underscoring its pivotal role in protecting the integrity of both active and stalled replication forks. TIM is upregulated in multiple cancers, which may represent a replication vulnerability of cancer cells that could be exploited for new therapies. Here, we discuss recent advances on our understanding of the multifaceted roles of TIM in DNA replication and stalled fork protection, and how its complex functions are engaged in collaboration with other genome surveillance and maintenance factors.

Hepatic Abscess as Early Presentation of Perforated Diverticulitis.

A hepatic abscess is a rare condition which can have multiple etiologies, including biliary disease, intra-abdominal collections (appendicitis, diverticulitis, and inflammatory bowel disease), abdominal surgery, liver transplantation, and liver trauma. The diagnosis is primarily based on imaging findings of ultrasound and computed tomography scan. Management includes antibiotics and percutaneous drainage or surgical drainage of the abscess. Here, we present a case of a 43-year-old Hispanic female who initially presented with left lower quadrant and right upper quadrant abdominal pain for 1 week. She was found to have a hepatic abscess and pelvic abscess likely secondary to perforated diverticulum.

Validating TDP1 as an Inhibition Target for the Development of Chemosensitizers for Camptothecin-Based Chemotherapy Drugs.

Cancer chemotherapy sensitizers hold the key to maximizing the potential of standard anticancer treatments. We have a long-standing interest in developing and validating inhibitors of the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) as chemosensitizers for topoisomerase I poisons such as topotecan. Herein, by using thieno[2,3-b]pyridines, a class of TDP1 inhibitors, we showed that the inhibition of TDP1 can restore sensitivity to topotecan, results that are supported by TDP1 knockout cell experiments using CRISPR/Cas9. However, we also found that the restored sensitivity towards topoisomerase I inhibitors is likely regulated by multiple complementary DNA repair pathways. Our results showed that one of these pathways is likely modulated by PARP1, although it is also possible that other redundant and partially overlapping pathways may be involved in the DNA repair process. Our work thus raises the prospect of targeting multiple DNA repair pathways to increase the sensitivity to topoisomerase I inhibitors.

Pheochromocytoma Presenting as Testicular Pain: An Unusual Case Report.

Pheochromocytoma (PCC) is a rare catecholamine-secreting tumor that arises from chromaffin cells of the adrenal medulla which are derived from the neural crest. This report illustrates a 51-year-old Caucasian male with a history of hypertension diagnosed two years ago who presented to the hospital due to acute onset of right testicular pain of 3-day duration. Laboratory results and imaging revealed a presumptive diagnosis of PCC. The patient had undergone robot-assisted laparoscopic right adrenalectomy 14 days after being diagnosed with PCC due to perioperative management with phenoxybenzamine. The final pathology report revealed a PCC. At follow-up two weeks after discharge, the patient reported complete resolution of his testicular pain.

Gastric Cancer Presenting as Persistent Pneumonia: An Unusual Case Report.

Worldwide, gastric cancer is the fifth most common cancer and the third leading cause of cancer deaths, which carries a poor prognosis as only 28.3% are expected to survive after five years. The incidence varies depending on the geographical locations and dietary patterns. Here, we present a case of a 59-year-old Hispanic male with a 10-month history of recurrent bilateral pneumonia and dysphagia. Diagnostic workup revealed metastatic gastric adenocarcinoma.

Cryptogenic Organizing Pneumonia Secondary to Mycoplasma pneumoniae Infection: A Case Report.

Cryptogenic organizing pneumonia (COP), previously known as bronchiolitis obliterans organizing pneumonia (BOOP), is a serious lung disease that can cause significant and long-term damage to the distal respiratory tract. Here we describe an unusual case in which an adult patient with recent mycoplasma pneumonia developed worsening lower airway disease despite initiation of multiple antibiotic therapies, illustrating the immunological effects related to Mycoplasma infections. It is critical that clinicians learn how to recognize early signs of COP in order to tailor appropriate therapy, reduce morbidity, and improve quality of life.

Hyperammonemic Encephalopathy: A Complication of Gastric Bypass Surgery.

Hyperammonemia is a metabolic abnormality characterized by elevated levels of ammonia in the blood. This case report illustrates a 72-year-old Caucasian female with a history of prior gastric bypass surgery done 15 years ago, who was admitted multiple times for acute encephalopathy over the course of a few months. The patient was found to have a gastro-gastric fistula seen on a CT scan of the abdomen, which was the culprit of her acute encephalopathy. The patient underwent fistula closure via esophagogastroduodenoscopy.

Cannabis Butane Hash Oil Dabbing Induced Lung Injury Mimicking Atypical Pneumonia.

"Dabbing" is the inhalation of concentrated marijuana, usually in butane solvent. This case report illustrates a previously healthy 25-year-old caucasian male with a 10-year history of cannabis butane hash oil (BHO) use. The patient presented with dyspnea and cough. The evaluation included a chest x-ray, basic laboratory investigations, computerized tomography angiogram of the chest and echocardiogram. Patient was diagnosed with acute lung injury mimicking atypical pneumonia. He was treated with steroids and had clinically improved and advised to stop dabbing. Further studies are needed to elucidate the full spectrum of the adverse effects of dabbing.

Promising New Inhibitors of Tyrosyl-DNA Phosphodiesterase I (Tdp 1) Combining 4-Arylcoumarin and Monoterpenoid Moieties as Components of Complex Antitumor Therapy.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme in humans, and a current and promising inhibition target for the development of new chemosensitizing agents due to its ability to remove DNA damage caused by topoisomerase 1 (Top1) poisons such as topotecan and irinotecan. Herein, we report our work on the synthesis and characterization of new Tdp1 inhibitors that combine the arylcoumarin (neoflavonoid) and monoterpenoid moieties. Our results showed that they are potent Tdp1 inhibitors with IC50 values in the submicromolar range. In vivo experiments with mice revealed that compound 3ba (IC50 0.62 µM) induced a significant increase in the antitumor effect of topotecan on the Krebs-2 ascites tumor model. Our results further strengthen the argument that Tdp1 is a druggable target with the potential to be developed into a clinically-potent adjunct therapy in conjunction with Top1 poisons.

New Hydrazinothiazole Derivatives of Usnic Acid as Potent Tdp1 Inhibitors.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising therapeutic target in cancer therapy. Combination chemotherapy using Tdp1 inhibitors as a component can potentially improve therapeutic response to many chemotherapeutic regimes. A new set of usnic acid derivatives with hydrazonothiazole pharmacophore moieties were synthesized and evaluated as Tdp1 inhibitors. Most of these compounds were found to be potent inhibitors with IC50 values in the low nanomolar range. The activity of the compounds was verified by binding experiments and supported by molecular modeling. The ability of the most effective inhibitors, used at non-toxic concentrations, to sensitize tumors to the anticancer drug topotecan was also demonstrated. The order of administration of the inhibitor and topotecan on their synergistic effect was studied, suggesting that prior or simultaneous introduction of the inhibitor with topotecan is the most effective.

Novel Inhibitors of DNA Repair Enzyme TDP1 Combining Monoterpenoid and Adamantane Fragments.

BACKGROUND AND OBJECTIVE: The DNA repair enzyme tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a current inhibition target to improve the efficacy of cancer chemotherapy. Previous studies showed that compounds combining adamantane and monoterpenoid fragments are active against TDP1 enzyme. This investigation is focused on the synthesis of monoterpenoid derived esters of 1-adamantane carboxylic acid as TDP1 inhibitors. METHODS: New esters were synthesized by the interaction between 1-adamantane carboxylic acid chloride and monoterpenoid alcohols. The esters were tested against TDP1 and its binding to the enzyme was modeling. RESULTS: 13 Novel ester-based TDP1 inhibitors were synthesized with yields of 21-94%; of these, nine esters had not been previously described. A number of the esters were found to inhibit TDP1, with IC50 values ranging from 0.86-4.08 µM. Molecular modelling against the TDP1 crystal structure showed a good fit of the active esters in the catalytic pocket, explaining their potency. A non-toxic dose of ester, containing a 3,7- dimethyloctanol fragment, was found to enhance the cytotoxic effect of topotecan, a clinically used anti-cancer drug, against the human lung adenocarcinoma cell line A549. CONCLUSION: The esters synthesized were found to be active against TDP1 in the lower micromolar concentration range, with these findings being corroborated by molecular modeling. Simultaneous action of the ester synthesized from 3,7-dimethyloctanol-1 and topotecan revealed a synergistic effect.

A Novel Class of Tyrosyl-DNA Phosphodiesterase 1 Inhibitors That Contains the Octahydro-2H-chromen-4-ol Scaffold.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that mends topoisomerase 1-mediated DNA damage. Tdp1 is a current inhibition target for the development of improved anticancer treatments, as its inhibition may enhance the therapeutic effect of topoisomerase 1 poisons. Here, we report a study on the development of a novel class of Tdp1 inhibitors that is based on the octahydro-2H-chromene scaffold. Inhibition and binding assays revealed that these compounds are potent inhibitors of Tdp1, with IC50 and KD values in the low micromolar concentration range. Molecular modelling predicted plausible conformations of the active ligands, blocking access to the enzymatic machinery of Tdp1. Our results thus help establish a structural-activity relationship for octahydro-2H-chromene-based Tdp1 inhibitors, which will be useful for future Tdp1 inhibitor development work.

A Library of Phosphoproteomic and Chromatin Signatures for Characterizing Cellular Responses to Drug Perturbations.

Although the value of proteomics has been demonstrated, cost and scale are typically prohibitive, and gene expression profiling remains dominant for characterizing cellular responses to perturbations. However, high-throughput sentinel assays provide an opportunity for proteomics to contribute at a meaningful scale. We present a systematic library resource (90 drugs × 6 cell lines) of proteomic signatures that measure changes in the reduced-representation phosphoproteome (P100) and changes in epigenetic marks on histones (GCP). A majority of these drugs elicited reproducible signatures, but notable cell line- and assay-specific differences were observed. Using the "connectivity" framework, we compared signatures across cell types and integrated data across assays, including a transcriptional assay (L1000). Consistent connectivity among cell types revealed cellular responses that transcended lineage, and consistent connectivity among assays revealed unexpected associations between drugs. We further leveraged the resource against public data to formulate hypotheses for treatment of multiple myeloma and acute lymphocytic leukemia. This resource is publicly available at https://clue.io/proteomics.

Rectal Cancer in the Eye: A Case Report of Orbital Metastasis.

Orbital metastasis from colorectal cancer is extremely rare. In this case report, we describe a 48-year-old woman who presented with recurrent severe headaches and new onset constipation with no known history of cancer. After vigilant workup, imaging, and biopsies, she was diagnosed with orbital metastasis from a primary rectal carcinoma. She was started on chemotherapy and radiation therapy. Her chemotherapy regimen consisted of FLOX (leucovorin + fluorouracil + oxaliplatin), along with panitumumab, which she tolerated well. She received chemotherapy for seven months before she lost her battle with cancer.

Castration Resistance in Prostate Cancer Is Mediated by the Kinase NEK6.

In prostate cancer, the development of castration resistance is pivotal in progression to aggressive disease. However, understanding of the pathways involved remains incomplete. In this study, we performed a high-throughput genetic screen to identify kinases that enable tumor formation by androgen-dependent prostate epithelial (LHSR-AR) cells under androgen-deprived conditions. In addition to the identification of known mediators of castration resistance, which served to validate the screen, we identified a mitotic-related serine/threonine kinase, NEK6, as a mediator of androgen-independent tumor growth. NEK6 was overexpressed in a subset of human prostate cancers. Silencing NEK6 in castration-resistant cancer cells was sufficient to restore sensitivity to castration in a mouse xenograft model system. Tumors in which castration resistance was conferred by NEK6 were predominantly squamous in histology with no evidence of AR signaling. Gene expression profiling suggested that NEK6 overexpression stimulated cytoskeletal, differentiation, and immune signaling pathways and maintained gene expression patterns normally decreased by castration. Phosphoproteome profiling revealed the transcription factor FOXJ2 as a novel NEK6 substrate, with FOXJ2 phosphorylation associated with increased expression of newly identified NEK6 transcriptional targets. Overall, our studies establish NEK6 signaling as a central mechanism mediating castration-resistant prostate cancer. Cancer Res; 77(3); 753-65. ©2016 AACR.

Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes.

Profiling post-translational modifications represents an alternative dimension to gene expression data in characterizing cellular processes. Many cellular responses to drugs are mediated by changes in cellular phosphosignaling. We sought to develop a common platform on which phosphosignaling responses could be profiled across thousands of samples, and created a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of responses to chemical perturbagens.To develop the assay, we investigated the coordinate regulation of phosphosites in samples derived from three cell lines treated with 26 different bioactive small molecules. Phosphopeptide analytes were selected from these discovery studies by clustering and picking 1 to 2 proxy members from each cluster. A quantitative, targeted parallel reaction monitoring assay was developed to directly measure 96 reduced-representation probes. Sample processing for proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment have been fully automated, making possible the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12%. This highly reproducible process allowed ∼95% of the reduced-representation phosphopeptide probes to be detected in ∼200 samples.The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and timepoints to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of biological conditions, suitable for profiling thousands of samples. We believe the assay will prove highly useful for classification of known and novel drug and genetic mechanisms through comparison of phosphoproteomic signatures.