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Abstract The mitotic MTH1 inhibitor TH1579 is a dual inhibitor that inhibits mitosis and incorporation of oxidative DNA damage and leads to cancer-specific cell death. The response to immune checkpoint inhibitor (ICI) treatment is often augmented by DNA damaging agents through the cGAS-STING pathway. This study investigates whether TH1579 can improve the efficacy of immune checkpoint blockades through its immunomodulatory properties. Various human and murine cancer cell lines were treated with mitotic MTH1i TH1579, and the expression of PD-L1 and T-cell infiltration-related chemokines was analysed by flow cytometry and real-time qPCR. Syngeneic mouse models were established to examine the combined effect of TH1579 and PD-L1 blockade. In our investigation, we found that TH1579 upregulates PD-L1 expression at both the protein and mRNA levels in human cancer cell lines. However, in murine cell lines, the increase was less pronounced. An in vivo experiment in a syngeneic mouse melanoma model showed that TH1579 treatment significantly increased the efficacy of atezolizumab, an anti-PD-L1 antibody, compared to vehicle or atezolizumab monotherapy. Furthermore, TH1579 exhibited immune-modulatory properties, elevating cytokines such as IFN-β and chemokines including CCL5 and CXCL10, in a cGAS-STING pathway-dependent manner. In conclusion, TH1579 has the potential to improve ICI treatment by modulating immune checkpoint-related proteins and pathways.

Abstract Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we perform multi-region, single-cell DNA sequencing to characterize the SCNA landscape across tumor-rich and normal tissue in two male patients with localized prostate cancer. We identify two distinct karyotypes: ‘pseudo-diploid’ cells harboring few SCNAs and highly aneuploid cells. Pseudo-diploid cells form numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones. In contrast, aneuploid cells do not form subclones and are detected throughout the prostate, including normal tissue regions. Highly localized pseudo-diploid subclones are confined within tumor-rich regions and carry deletions in multiple tumor-suppressor genes. Our study reveals that SCNAs are widespread in normal and tumor regions across the prostate in localized prostate cancer patients and suggests that a subset of pseudo-diploid cells drive tumorigenesis in the aging prostate.

Abstract Complex DNA damage (CDD), containing two or more DNA lesions within one or two DNA helical turns, is a signature of ionising radiation (IR) and contributes significantly to the therapeutic effect through cell killing. The levels and complexity of CDD increases with linear energy transfer (LET), however, the specific cellular response to this type of DNA damage and the critical proteins essential for repair of CDD is currently unclear. We performed an siRNA screen of ~240 DNA damage response proteins to identify those specifically involved in controlling cell survival in response to high-LET protons at the Bragg peak, compared to low-LET entrance dose protons which differ in the amount of CDD produced. From this, we subsequently validated that depletion of 8-oxoguanine DNA glycosylase (OGG1) and poly(ADP-ribose) glycohydrolase (PARG) in HeLa and head and neck cancer cells leads to significantly increased cellular radiosensitivity specifically following high-LET protons, whilst no effect was observed after low-LET protons and X-rays. We subsequently confirmed that OGG1 and PARG are both required for efficient CDD repair post-irradiation with high-LET protons. Importantly, these results were also recapitulated using specific inhibitors for OGG1 (TH5487) and PARG (PDD00017273). Our results suggest OGG1 and PARG play a fundamental role in the cellular response to CDD and indicate that targeting these enzymes could represent a promising therapeutic strategy for the treatment of head and neck cancers following high-LET radiation.

With diminishing returns and high clinical failure rates from traditional preclinical and animal-based drug discovery strategies, more emphasis is being placed on alternative drug discovery platforms. Ex vivo approaches represent a departure from both more traditional preclinical animal-based models and clinical-based strategies and aim to address intra-tumoural and inter-patient variability at an earlier stage of drug discovery. Additionally, these approaches could also offer precise treatment stratification for patients within a week of tumour resection in order to direct tailored therapy. One tumour group that could significantly benefit from such ex vivo approaches are high-grade gliomas, which exhibit extensive heterogeneity, cellular plasticity and therapy-resistant glioma stem cell (GSC) niches. Historic use of murine-based preclinical models for these tumours has largely failed to generate new therapies, resulting in relatively stagnant and unacceptable survival rates of around 12-15 months post-diagnosis over the last 50 years. The near universal use of DNA damaging chemoradiotherapy after surgical resection within standard-of-care (SoC) therapy regimens provides an opportunity to improve current treatments if we can identify efficient drug combinations in preclinical models that better reflect the complex inter-/intra-tumour heterogeneity, GSC plasticity and inherent DNA damage resistance mechanisms. We have therefore developed and optimised a high-throughput ex vivo drug screening platform; GliExP, which maintains GSC populations using immediately dissociated fresh surgical tissue. As a proof-of-concept for GliExP, we have optimised SoC therapy responses and screened 30+ small molecule therapeutics and preclinical compounds against tumours from 18 different patients, including multi-region spatial heterogeneity sampling from several individual tumours. Our data therefore provides a strong basis to build upon GliExP to incorporate combination-based oncology therapeutics in tandem with SoC therapies as an important preclinical alternative to murine models (reduction and replacement) to triage experimental therapeutics for clinical translation and deliver rapid identification of effective treatment strategies for individual gliomas.

Summary: SAMHD1 is a dNTP triphosphohydrolase governing nucleotide pool homeostasis and can detoxify chemotherapy metabolites controlling their clinical responses. To understand SAMHD1 biology and investigate the potential of targeting SAMHD1 as neoadjuvant to current chemotherapies, we set out to discover selective small-molecule inhibitors. Here, we report a discovery pipeline encompassing a biochemical screening campaign and a set of complementary biochemical, biophysical, and cell-based readouts for rigorous characterization of the screen output. The identified small molecules, TH6342 and analogs, accompanied by inactive control TH7126, demonstrated specific, low μM potency against both physiological and oncology-drug-derived substrates. By coupling kinetic studies with thermal shift assays, we reveal the inhibitory mechanism of TH6342 and analogs, which engage pre-tetrameric SAMHD1 and deter oligomerization and allosteric activation without occupying nucleotide-binding pockets. Altogether, our study diversifies inhibitory modes against SAMHD1, and the discovery pipeline reported herein represents a thorough framework for future SAMHD1 inhibitor development.

Abstract Background Chemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized. Methods The differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model. Results We provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model. Conclusions Our in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.

With diminishing returns and high clinical failure rates from traditional preclinical and animal-based drug discovery strategies, more emphasis is being placed on alternative drug discovery platforms. Ex vivo approaches represent a departure from both more traditional preclinical animal-based models and clinical-based strategies and aim to address intra-tumoural and inter-patient variability at an earlier stage of drug discovery. Additionally, these approaches could also offer precise treatment stratification for patients within a week of tumour resection in order to direct tailored therapy. One tumour group that could significantly benefit from such ex vivo approaches are high-grade gliomas, which exhibit extensive heterogeneity, cellular plasticity and therapy-resistant glioma stem cell (GSC) niches. Historic use of murine-based preclinical models for these tumours has largely failed to generate new therapies, resulting in relatively stagnant and unacceptable survival rates of around 12-15 months post-diagnosis over the last 50 years. The near universal use of DNA damaging chemoradiotherapy after surgical resection within standard-of-care (SoC) therapy regimens provides an opportunity to improve current treatments if we can identify efficient drug combinations in preclinical models that better reflect the complex inter-/intra-tumour heterogeneity, GSC plasticity and inherent DNA damage resistance mechanisms. We have therefore developed and optimised a high-throughput ex vivo drug screening platform; GliExP, which maintains GSC populations using immediately dissociated fresh surgical tissue. As a proof-of-concept for GliExP, we have optimised SoC therapy responses and screened 30+ small molecule therapeutics and preclinical compounds against tumours from 18 different patients, including multi-region spatial heterogeneity sampling from several individual tumours. Our data therefore provides a strong basis to build upon GliExP to incorporate combination-based oncology therapeutics in tandem with SoC therapies as an important preclinical alternative to murine models (reduction and replacement) to triage experimental therapeutics for clinical translation and deliver rapid identification of effective treatment strategies for individual gliomas.

The exploitation of the DNA damage response and DNA repair proficiency of cancer cells is an important anticancer strategy. The replication and repair of DNA are dependent upon the supply of deoxynucleoside triphosphate (dNTP) building blocks, which are produced and maintained by nucleotide metabolic pathways. Enzymes within these pathways can be promising targets to selectively induce toxic DNA lesions in cancer cells. These same pathways also activate antimetabolites, an important group of chemotherapies that disrupt both nucleotide and DNA metabolism to induce DNA damage in cancer cells. Thus, dNTP metabolic enzymes can also be targeted to refine the use of these chemotherapeutics, many of which remain standard of care in common cancers. In this review article, we will discuss both these approaches exemplified by the enzymes MTH1, MTHFD2 and SAMHD1. © 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Spatial heterogeneity in prostate cancer can contribute to its resistance to androgen deprivation therapy (ADT). Here, the authors analyse prostate cancer samples before and after ADT using Spatial Transcriptomics, and find heterogeneous pre-treatment tumour cell populations and stromal cells that are associated with resistance.

Autoimmune hepatitis (AIH) is an inflammatory liver disease driven by the hyperactivation of various intrahepatic antigen‐specific T cells due to a breach of immune tolerance. Studies in immunometabolism demonstrate that activated T cells harbor increased levels of reactive oxygen species that cause oxidative DNA damage. In this study, we assessed the potential of DNA damage repair enzyme MutT homolog 1 (MTH1) as a therapeutic target in AIH and karonudib as a novel drug for patients with AIH. We report herein that MTH1 expression was significantly increased in liver samples from patients with AIH compared to patients with chronic hepatitis B and nonalcoholic fatty liver disease and from healthy controls. In addition, the expression of MTH1 was positively correlated with AIH disease severity. We further found abundant T cells that expressed MTH1 in AIH. Next, we found that karonudib significantly altered T‐cell receptor signaling in human T cells and robustly inhibited proliferation of human T cells in vitro. Interestingly, our data reflected a preferential inhibition of DNA damage repair in activated T cells by karonudib. Moreover, MTH1 was required to develop liver inflammation and damage because specific deletion of MTH1 in T cells ameliorated liver injury in the concanavalin A (Con A)‐induced hepatitis model by inhibiting T‐cell activation and proliferation. Lastly, we validated the protective effect of karonudib on the Con A‐induced hepatitis model. Conclusion: MTH1 functions as a critical regulator in the development of AIH, and its inhibition in activated T cells reduces liver inflammation and damage.

Background and aim: Allergic asthma is a complex inflammatory disease involving type 2 innate lymphoid cells, type 2 T helper cells, macrophages, and eosinophils. The disease is characterized by wheezing, dyspnea, coughing, chest tightness and variable airflow limitation for which there is no cure and is symptomatically treated with inhaled corticosteroids and β2-agonists. Molecular mechanisms underlying its complex pathogenesis are not fully understood. However, 8-oxoguanine DNA glycosylase-1 (OGG1), a DNA repair protein may play a central role, as OGG1 deficiency decreases both innate and allergic inflammation.Methods: Using a murine ovalbumin (OVA) model of allergic airway inflammation we assessed the utility of an inhibitor of OGG1 (TH5487) in this disease context. Cytokines and chemokines, promoting immune cell recruitment were measured using a 23-multiplex assay and Western blotting. Additionally, immune cell recruitment to bronchi was measured using flow cytometry. Histological analyses and immunofluorescent staining were used to confirm immune cell influx and goblet cell hyperplasia of the airways. A PCR array was used to assess asthma-related genes in murine lung tissue following TH5487 treatment. Finally, airway hyperresponsiveness was determined using in vivo lung function measurement.Results: In this study, administration of TH5487 to mice with OVA-induced allergic airway inflammation significantly decreased goblet cell hyperplasia and mucus production. TH5487 treatment also decreased levels of activated NF-κB and expression of proinflammatory cytokines and chemokines resulting in significantly lower recruitment of eosinophils and other immune cells to the lungs. Gene expression profiling of asthma and allergy-related proteins after TH5487 treatment revealed differences in several important regulators, including down regulation of Tnfrsf4, Arg1, Ccl12 and Ccl11, and upregulation of the negative regulator of type 2 inflammation, Bcl6. Furthermore, the gene Clca1 was upregulated following TH5487 treatment, which should be explored further due to its ambiguous role in allergic asthma. In addition, the OVA-induced airway hyperresponsiveness was significantly reduced by TH5487 treatment.Conclusion: Taken together, the data presented in this study suggest OGG1 as a clinically relevant pharmacological target for the treatment of allergic inflammation.

BackgroundPARP1 plays a critical role in the base excision repair (BER) pathway, and PARP1 inhibition leads to specific cell death, through a synthetic lethal interaction, in the context of BRCA1/2 deficiency. To date, up to five different PARP inhibitors (PARPi), have been approved, nevertheless, the acquisition of resistance to PARPi is common and there is increasing interest in enhancing responses and expand their use to other tumour types.MethodsWe hypothesized that other BER members could be additional synthetic lethal partners with mutated BRCA genes. To test this, we decided to evaluate the glycosylase OGG1 as a potential candidate, by treating BRCA1 proficient and deficient breast cancer cells with PARPi olaparib and the OGG1 inhibitor TH5478.ResultsKnocking out BRCA1 in triple-negative breast cancer cell lines causes hypersensitivity to the OGG1 inhibitor TH5487. Besides, TH5487 enhances the sensitivity to the PARP inhibitor olaparib, especially in the context of BRCA1 deficiency, reflecting an additive interaction.DiscussionThese results provide the first evidence that OGG1 inhibition is a promising new synthetic lethality strategy in BRCA1-deficient cells, and could lead to a new framework for the treatment of hereditary breast and ovarian cancer.

Abstract Chemo-immunotherapy has improved survival in B-cell lymphoma patients, but refractory/relapsed diseases still represent a major challenge, urging for development of new therapeutics. Karonudib (TH1579) was developed to inhibit MTH1, an enzyme preventing oxidized dNTP-incorporation in DNA. MTH1 is highly upregulated in tumor biopsies from patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma, hence confirming a rationale for targeting MTH1. Here, we tested the efficacy of karonudib in vitro and in preclinical B-cell lymphoma models. Using a range of B-cell lymphoma cell lines, karonudib strongly reduced viability at concentrations well tolerated by activated normal B cells. In B-cell lymphoma cells, karonudib increased incorporation of 8-oxo-dGTP into DNA, and prominently induced prometaphase arrest and apoptosis due to failure in spindle assembly. MTH1 knockout cell lines were less sensitive to karonudib-induced apoptosis, but were displaying cell cycle arrest phenotype similar to the wild type cells, indicating a dual inhibitory role of the drug. Karonudib was highly potent as single agent in two different lymphoma xenograft models, including an ABC DLBCL patient derived xenograft, leading to prolonged survival and fully controlled tumor growth. Together, our preclinical findings provide a rationale for further clinical testing of karonudib in B-cell lymphoma.

Abstract The most common oxidative DNA lesion is 8-oxoguanine which is mainly recognized and excised by the 8-oxoG DNA glycosylase 1 (OGG1), initiating the base excision repair (BER) pathway. Telomeres are particularly sensitive to oxidative stress (OS) which disrupts telomere homeostasis triggering genome instability. In the present study, we have investigated the effects of inactivating BER in OS conditions, by using a specific inhibitor of OGG1 (TH5487). We have found that in OS conditions, TH5487 blocks BER initiation at telomeres causing an accumulation of oxidized bases, that is correlated with telomere losses, micronuclei formation and mild proliferation defects. Moreover, the antimetabolite methotrexate synergizes with TH5487 through induction of intracellular reactive oxygen species (ROS) formation, which potentiates TH5487-mediated telomere and genome instability. Our findings demonstrate that OGG1 is required to protect telomeres from OS and present OGG1 inhibitors as a tool to induce oxidative DNA damage at telomeres, with the potential for developing new combination therapies for cancer treatment.

Epithelial–myoepithelial carcinoma (EMC) is a rare subtype of salivary gland neoplasms. Since the initial description of the cancer, just over 300 cases have been reported. EMCs occupy a biphasic cellular differentiation-state defined by the constitution of two cell types representing epithelial and myoepithelial lineages, yet the functional consequence of the differentiation-state heterogeneity with respect to therapy resistance of the tumors remains unclear. The reported local recurrence rate of the cases is approximately 30%, and while distant metastases are rare, a significant fraction of these cases are reported to receive no survival benefit from radio- or chemotherapy given in addition to surgery. Moreover, no targeted therapies have been reported for these neoplasms. We report here the first use and application of ex vivo drug screening together with next generation sequencing to assess targeted treatment strategies for a rare metastatic epithelial–myoepithelial carcinoma. Results of the ex vivo drug screen demonstrate significant differential therapeutic sensitivity between the epithelial and myoepithelial intra-tumor cell lineages suggesting that differentiation-state heterogeneity within epithelial–myoepithelial carcinomas may present an outlet to partial therapeutic responses to targeted therapies including MEK and mTOR inhibitors. These results suggest that the intra-tumor lineage composition of EMC could be an important factor to be assessed when novel treatments are being evaluated for management of metastatic EMC.

Abstract Background The majority of copy number callers requires high read coverage data that is often achieved with elevated material input, which increases the heterogeneity of tissue samples. However, to gain insights into smaller areas within a tissue sample, e.g. a cancerous area in a heterogeneous tissue sample, less material is used for sequencing, which results in lower read coverage. Therefore, more focus needs to be put on copy number calling that is sensitive enough for low coverage data. Results We present MetaCNV, a copy number caller that infers reliable copy numbers for human genomes with a consensus approach. MetaCNV specializes in low coverage data, but also performs well on normal and high coverage data. MetaCNV integrates the results of multiple copy number callers and infers absolute and unbiased copy numbers for the entire genome. MetaCNV is based on a meta-model that bypasses the weaknesses of current calling models while combining the strengths of existing approaches. Here we apply MetaCNV based on ReadDepth, SVDetect, and CNVnator to real and simulated datasets in order to demonstrate how the approach improves copy number calling. Conclusions MetaCNV, available at https://bitbucket.org/sonnhammergroup/metacnv , provides accurate copy number prediction on low coverage data and performs well on high coverage data.

Abstract Background Fanconi anemia is a rare disease clinically characterized by malformations, bone marrow failure and an increased risk of solid tumors and hematologic malignancies. The only therapies available are hematopoietic stem cell transplantation for bone marrow failure or leukemia, and surgical resection for solid tumors. Therefore, there is still an urgent need for new therapeutic options. With this aim, we developed a novel high-content cell-based screening assay to identify drugs with therapeutic potential in FA. Results A TALEN-mediated FANCA-deficient U2OS cell line was stably transfected with YFP-FANCD2 fusion protein. These cells were unable to form fluorescent foci or to monoubiquitinate endogenous or exogenous FANCD2 upon DNA damage and were more sensitive to mitomycin C when compared to the parental wild type counterpart. FANCA correction by retroviral infection restored the cell line’s ability to form FANCD2 foci and ubiquitinate FANCD2. The feasibility of this cell-based system was interrogated in a high content screening of 3802 compounds, including a Prestwick library of 1200 FDA-approved drugs. The potential hits identified were then individually tested for their ability to rescue FANCD2 foci and monoubiquitination, and chromosomal stability in the absence of FANCA. Conclusions While, unfortunately, none of the compounds tested were able to restore cellular FANCA-deficiency, our study shows the potential capacity to screen large compound libraries in the context of Fanconi anemia therapeutics in an optimized and cost-effective platform.

To understand complex diseases, high-throughput data are generated at large and multiple levels. However, extracting meaningful information from large datasets for comprehensive understanding of cell phenotypes and disease pathophysiology remains a major challenge. Despite tremendous advances in understanding molecular mechanisms of cancer and its progression, current knowledge appears discrete and fragmented. In order to render this wealth of data more integrated and thus informative, we have developed a GECIP toolbox to investigate the crosstalk and the responsible genes’/proteins’ connectivity of enriched pathways from gene expression data. To implement this toolbox, we used mainly gene expression datasets of prostate cancer, and the three datasets were GSE17951, GSE8218, and GSE1431. The raw samples were processed for normalization, prediction of differentially expressed genes, and the prediction of enriched pathways for the differentially expressed genes. The enriched pathways have been processed for crosstalk degree calculations for which number connections per gene, the frequency of genes in the pathways, sharing frequency, and the connectivity have been used. For network prediction, protein–protein interaction network database FunCoup2.0 was used, and cytoscape software was used for the network visualization. In our results, we found that there were enriched pathways 27, 45, and 22 for GSE17951, GSE8218, and GSE1431, respectively, and 11 pathways in common between all of them. From the crosstalk results, we observe that focal adhesion and PI3K pathways, both experimentally proven central for cellular output upon perturbation of numerous individual/distinct signaling pathways, displayed highest crosstalk degree. Moreover, we also observe that there were more critical pathways which appear to be highly significant, and these pathways are HIF1a, hippo, AMPK, and Ras. In terms of the pathways’ components, GSK3B, YWHAE, HIF1A, ATP1A3, and PRKCA are shared between the aforementioned pathways and have higher connectivity with the pathways and the other pathway components. Finally, we conclude that the focal adhesion and PI3K pathways are the most critical pathways, and since for many other pathways, high-rank enrichment did not translate to high crosstalk degree, the global impact of one pathway on others appears distinct from enrichment.

Previously, we reported that MTH1 inhibitors TH588 and TH1579 selectively induce oxidative damage and kill Ras-expressing or -transforming cancer cells, as compared to non-transforming immortalized or primary cells. While this explains the impressive anti-cancer properties of the compounds, the molecular mechanism remains elusive. Several oncogenes induce replication stress, resulting in under replicated DNA and replication continuing into mitosis, where TH588 and TH1579 treatment causes toxicity and incorporation of oxidative damage. Hence, we hypothesized that oncogene-induced replication stress explains the cancer selectivity. To test this, we overexpressed c-Myc in human epithelial kidney cells (HA1EB), resulting in increased proliferation, polyploidy and replication stress. TH588 and TH1579 selectively kill c-Myc overexpressing clones, enforcing the cancer cell selective killing of these compounds. Moreover, the toxicity of TH588 and TH1579 in c-Myc overexpressing cells is rescued by transcription, proteasome or CDK1 inhibitors, but not by nucleoside supplementation. We conclude that the molecular toxicological mechanisms of how TH588 and TH1579 kill c-Myc overexpressing cells have several components and involve MTH1-independent proteasomal degradation of c-Myc itself, c-Myc-driven transcription and CDK activation.

Computational chemistry has now been widely accepted as a useful tool for shortening lead times in early drug discovery. When selecting new potential drug targets, it is important to assess the likelihood of finding suitable starting points for lead generation before pursuing costly high-throughput screening campaigns. By exploiting available high-resolution crystal structures, an in silico druggability assessment can facilitate the decision of whether, and in cases where several protein family members exist, which of these to pursue experimentally. Many of the algorithms and software suites commonly applied for in silico druggability assessment are complex, technically challenging and not always user-friendly. Here we applied the intuitive open access servers of DoGSite, FTMap and CryptoSite to comprehensively predict ligand binding pockets, druggability scores and conformationally active regions of the NUDIX protein family. In parallel we analyzed potential ligand binding sites, their druggability and pocket parameter using Schrödinger's SiteMap. Then an in silico docking cascade of a subset of the ZINC FragNow library using the Glide docking program was performed to assess identified pockets for large-scale small-molecule binding. Subsequently, this initial dual ranking of druggable sites within the NUDIX protein family was benchmarked against experimental hit rates obtained both in-house and by others from traditional biochemical and fragment screening campaigns. The observed correlation suggests that the presented user-friendly workflow of a dual parallel in silico druggability assessment is applicable as a standalone method for decision on target prioritization and exclusion in future screening campaigns.

Abstract The deoxycytidine analogue cytarabine (ara‐C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara‐C efficacy by hydrolysing the active triphosphate metabolite ara‐CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1‐mediated barrier to ara‐C efficacy in primary blasts and mouse models of AML, displaying SAMHD1‐dependent synergy with ara‐C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara‐CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara‐C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML.

ABSTRACT: Background: Osteosarcoma (OS) is the most common primary malignant bone tumour. Unfortunately, no new treatments are approved and over the last 30 years the survival rate remains only 30% at 5 years for poor responders justifying an urgent need of new therapies. The Mutt homolog 1 (MTH1) enzyme prevents incorporation of oxidized nucleotides into DNA and recently developed MTH1 inhibitors may offer therapeutic potential as MTH1 is overexpressed in various cancers. Methods: The aim of this study was to evaluate the therapeutic benefits of targeting MTH1 with two chemical inhibitors, TH588 and TH1579 on human osteosarcoma cells. Preclinical efficacy of TH1579 was assessed in human osteosarcoma xenograft model on tumour growth and development of pulmonary metastases. Findings: MTH1 is overexpressed in OS patients and tumour cell lines, compared to mesenchymal stem cells. In vitro, chemical inhibition of MTH1 by TH588 and TH1579 decreases OS cells viability, impairs their cell cycle and increases apoptosis in OS cells. TH1579 was confirmed to bind MTH1 by CETSA in OS model. Moreover, 90 mg/kg of TH1579 reduces in vivo tumour growth by 80.5% compared to non-treated group at day 48. This result was associated with the increase in 8-oxo-dG integration into tumour cells DNA and the increase of apoptosis. Additionally, TH1579 also reduces the number of pulmonary metastases. Interpretation: All these results strongly provide a pre-clinical proof-of-principle that TH1579 could be a therapeutic option for patients with osteosarcoma. Funding: This study was supported by La Ligue Contre le Cancer, la SFCE and Enfants Cancers Santé. Key words: Osteosarcoma, Bone tumours, MTH1, ROS, DNA damage

Targeting the glycolytic PFKFB3 enzyme is being studied as a therapeutic strategy against cancer. Here the authors identify PFKFB3 as being involved in homologous recombination (HR) repair of DNA double strand breaks (DSBs) and present a PFKFB3 inhibitor.

Heterogeneity within tumors presents a challenge to cancer treatment. Here, the authors investigate transcriptional heterogeneity in prostate cancer, examining expression profiles of different tissue components and highlighting expression gradients in the tumor microenvironment.

NUDIX hydrolases are an important family of nucleotide-metabolizing enzymes. Here, the authors identify potent, small molecule inhibitors of NUDT5, which is implicated in ADP-ribose and 8-oxo-guanine metabolism, and confirm its role in gene regulation and proliferation in breast cancer cells.

Tumor hypoxia and hypoxic adaptation of cancer cells represent major barriers to successful cancer treatment. We revealed that improved antioxidant capacity contributes to increased radioresistance of cancer cells with tolerance to chronic-cycling severe hypoxia/reoxygenation stress. We hypothesized, that the improved tolerance to oxidative stress will increase the ability of cancer cells to cope with ROS-induced damage to free deoxy-nucleotides (dNTPs) required for DNA replication and may thus contribute to acquired resistance of cancer cells in advanced tumors to antineoplastic agents inhibiting the nucleotide-sanitizing enzyme MutT Homologue-1 (MTH1), ionizing radiation (IR) or both. Therefore, we aimed to explore potential differences in the sensitivity of cancer cells exposed to acute and chronic-cycling hypoxia/reoxygenation stress to the clinically relevant MTH1-inhibitor TH1579 (Karonudib) and to test whether a multi-targeting approach combining the glutathione withdrawer piperlongumine (PLN) and TH1579 may be suited to increase cancer cell sensitivity to TH1579 alone and in combination with IR. Combination of TH1579 treatment with radiotherapy (RT) led to radiosensitization but was not able to counteract increased radioresistance induced by adaptation to chronic-cycling hypoxia/reoxygenation stress. Disruption of redox homeostasis using PLN sensitized anoxia-tolerant cancer cells to MTH1 inhibition by TH1579 under both normoxic and acute hypoxic treatment conditions. Thus, we uncover a glutathione-driven compensatory resistance mechanism towards MTH1-inhibition in form of increased antioxidant capacity as a consequence of microenvironmental or therapeutic stress.

The NUDIX hydrolases are known to be involved in several cellular processes and diseases, such as cancer, but remain poorly characterized as a family. Here, the authors provide a comprehensive analysis of the structural, biochemical, and expression properties of 18 human NUDIX proteins, and begin to address their functional inter-relationships.

DNA damage caused by reactive oxygen species may result in genetic mutations or cell death. Base excision repair (BER) is the major pathway that repairs DNA oxidative damage in order to maintain genomic integrity. In mammals, eleven DNA glycosylases have been reported to initiate BER, where each recognizes a few related DNA substrate lesions with some degree of overlapping specificity. 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most abundant DNA oxidative lesions, is recognized and excised mainly by 8-oxoguanine DNA glycosylase 1 (OGG1). Further oxidation of 8-oxoG generates hydantoin lesions, which are recognized by NEIL glycosylases. Here, we demonstrate that NEIL1, and to a lesser extent NEIL2, can potentially function as backup BER enzymes for OGG1 upon pharmacological inhibition or depletion of OGG1. NEIL1 recruitment kinetics and chromatin binding after DNA damage induction increase in cells treated with OGG1 inhibitor TH5487 in a dose-dependent manner, whereas NEIL2 accumulation at DNA damage sites is prolonged following OGG1 inhibition. Furthermore, depletion of OGG1 results in increased retention of NEIL1 and NEIL2 at damaged chromatin. Importantly, oxidatively stressed NEIL1- or NEIL2-depleted cells show excessive genomic 8-oxoG lesions accumulation upon OGG1 inhibition, suggesting a prospective compensatory role for NEIL1 and NEIL2. Our study thus exemplifies possible backup mechanisms within the base excision repair pathway.

Tumours with homologous recombination (HR) defects become sensitive to PARPi. Here, the authors show that androgen receptor (AR) regulates HR and AR inhibition activates the PARP pathway in vivo, thus inhibition of both AR and PARP is required for effective treatment of high risk prostate cancer.

Background: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and is generally caused by viral infections or consumption of mutagens, such as alcohol. While liver transplantation and hepatectomy is curative for some patients, many relapse into disease with few treatment options such as tyrosine kinase inhibitors, for example, sorafenib or lenvatinib. The need for novel systemic treatment approaches is urgent. Methods: MTH1 expression profile was first analyzed in a HCC database and MTH1 mRNA/protein level was determined in resected HCC and paired paracancerous tissues with polymerase chain reaction (PCR) and immunohistochemistry. HCC cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA. 8-oxoG was measured by the modified comet assay. The effect of MTH1 inhibition on tumor growth was explored in HCC xenograft in vivo models. Results: MTH1 protein level is elevated in HCC tissue compared with paracancerous liver tissue and indicates poor prognosis. The MTH1 inhibitor Karonudib (TH1579) and siRNA effectively introduce toxic oxidized nucleotides into DNA, 8-oxoG, and kill HCC cell lines in vitro . Furthermore, we demonstrate that HCC growth in a xenograft mouse model in vivo is efficiently suppressed by Karonudib. Conclusion: Altogether, these data suggest HCC relies on MTH1 for survival, which can be targeted and may open up a novel treatment option for HCC in the future.

Recent RNA virus outbreaks such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV) have caused worldwide health emergencies highlighting the urgent need for new antiviral strategies. Targeting host cell pathways supporting viral replication is an attractive approach for development of antiviral compounds, especially with new, unexplored viruses where knowledge of virus biology is limited. Here, we present a strategy to identify host-targeted small molecule inhibitors using an image-based phenotypic antiviral screening assay followed by extensive target identification efforts revealing altered cellular pathways upon antiviral compound treatment. The newly discovered antiviral compounds showed broad-range antiviral activity against pathogenic RNA viruses such as SARS-CoV-2, EBOV and Crimean-Congo hemorrhagic fever virus (CCHFV). Target identification of the antiviral compounds by thermal protein profiling revealed major effects on proteostasis pathways and disturbance in interactions between cellular HSP70 complex and viral proteins, illustrating the supportive role of HSP70 on many RNA viruses across virus families. Collectively, this strategy identifies new small molecule inhibitors with broad antiviral activity against pathogenic RNA viruses, but also uncovers novel virus biology urgently needed for design of new antiviral therapies.

RNA viruses have gained plenty of attention during recent outbreaks of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Zika virus (ZIKV), and Ebola virus. ZIKV is a vector borne Flavivirus that is spread by mosquitoes and it mainly infects neuronal progenitor cells. One hallmark of congenital ZIKV disease is a reduced brain size in fetuses, leading to severe neurological defects. The World Health Organization (WHO) is urging the development of new antiviral treatments against ZIKV, as there are no efficient countermeasures against ZIKV disease. Previously, we presented a new class of host-targeting antivirals active against a number of pathogenic RNA viruses, such as SARS-CoV-2. Here, we show the transfer of the image-based phenotypic antiviral assay to ZIKV-infected brain cells, followed by mechanism-of-action studies and a proof-of-concept study in a three-dimensional (3D) organoid model. The novel antiviral compounds showed a therapeutic window against ZIKV in several cell models and rescued ZIKV-induced neurotoxicity in brain organoids. The compound’s mechanism-of-action was pinpointed to late steps in the virus life cycle, impairing the formation of new virus particles. Collectively, in this study, we expand the antiviral activity of new small molecule inhibitors to a new virus class of Flaviviruses, but also uncover compounds’ mechanism of action, which are important for the further development of antivirals.

8-oxoguanine DNA glycosylase (OGG1) is the main DNA glycosylase responsible for the excision of 7,8-dihydro-8-oxoguanine (8-oxoG) from duplex DNA to initiate base excision repair. This glycosylase activity is relevant in many pathological conditions including cancer, inflammation, and neurodegenerative diseases. To have a better understanding of the role of OGG1, we previously reported TH5487, a potent active site inhibitor of OGG1. Here, we further investigate the consequences of inhibiting OGG1 with TH5487. TH5487 treatment induces accumulation of genomic 8-oxoG lesions. Furthermore, it impairs the chromatin binding of OGG1 and results in lower recruitment of OGG1 to regions of DNA damage. Inhibiting OGG1 with TH5487 interferes with OGG1′s incision activity, resulting in fewer DNA double-strand breaks in cells exposed to oxidative stress. This study validates TH5487 as a potent OGG1 inhibitor that prevents the repair of 8-oxoG and alters OGG1–chromatin dynamics and OGG1′s recruitment kinetics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Citrullinated histone H3 (H3Cit) is a central player in the neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs). NETs have been shown to elicit harmful effects on the host, and were recently proposed to promote tumor progression and spread. Here we report significant elevations of plasma H3Cit in patients with advanced cancer compared with age-matched healthy individuals. These elevations were specific to cancer patients as no increase was observed in severely ill and hospitalized patients with a higher non-malignant comorbidity. The analysis of neutrophils from cancer patients showed a higher proportion of neutrophils positive for intracellular H3Cit compared to severely ill patients. Moreover, the presence of plasma H3Cit in cancer patients strongly correlated with neutrophil activation markers neutrophil elastase (NE) and myeloperoxidase (MPO), and the inflammatory cytokines interleukin-6 and -8, known to induce NETosis. In addition, we show that high levels of circulating H3Cit strongly predicted poor clinical outcome in our cohort of cancer patients with a 2-fold increased risk for short-term mortality. Our results also corroborate the association of NE, interleukin-6 and -8 with poor clinical outcome. Taken together, our results are the first to unveil H3Cit as a potential diagnostic and prognostic blood marker associated with an exacerbated inflammatory response in patients with advanced cancer.

Activation of Signal Transducer and Activator of Transcription 3 (STAT3) has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z' = 0,8). This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set). Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents.

The risks of non-cancerous diseases associated with exposure to low doses of radiation are at present not validated by epidemiological data, and pose a great challenge to the scientific community of radiation protection research. Here, we show that premature senescence is induced in human fibroblasts when exposed to chronic low dose rate (LDR) exposure (5 or 15 mGy/h) of gamma rays from a 137Cs source. Using a proteomic approach we determined differentially expressed proteins in cells after chronic LDR radiation compared to control cells. We identified numerous proteins involved in protection against oxidative stress, suggesting that these pathways protect against premature senescence. In order to further study the role of oxidative stress for radiation induced premature senescence, we also used human fibroblasts, isolated from a patient with a congenital deficiency in glutathione synthetase (GS). We found that these GS deficient cells entered premature senescence after a significantly shorter time of chronic LDR exposure as compared to the GS proficient cells. In conclusion, we show that chronic LDR exposure induces premature senescence in human fibroblasts, and propose that a stress induced increase in reactive oxygen species (ROS) is mechanistically involved.

Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR) repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB) sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP) and promotes DSB resection, allowing Replication Protein A (RPA) and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis.

Treatments with Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors have offered patients carrying cancers with mutated BRCA1 or BRCA2 genes a new and in many cases effective option for disease control. There is potentially a large patient population that may also benefit from PARP inhibitor treatment, either in monotherapy or in combination with chemotherapy. Here, we describe the multifaceted role of PARP inhibitors and discuss which treatment options could potentially be useful to gain disease control without potentiating side effects.

Abstract Poly (ADP‐ribose) polymerase (PARP) inhibitors effectively kill tumours defective in the BRCA1 or BRCA2 genes through the concept of synthetic lethality. It is suggested that PARP inhibitors cause an increase in DNA single‐strand breaks (SSBs), which are converted during replication to irreparable toxic DNA double‐strand breaks (DSBs) in BRCA1/2 defective cells. There are a number of recent reports challenging this model. Here, alternative models that are not mutually exclusive are presented to explain the synthetic lethality between BRCA1/2 and PARP inhibitors. One such model proposes that PARP inhibition causes PARP‐1 to be trapped onto DNA repair intermediates, especially during base excision repair. This may in turn cause obstruction to replication forks, which require BRCA‐dependent homologous recombination to be resolved. In another model, PARP is directly involved in catalysing replication repair in a distinct pathway from homologous recombination. Experimental evidence supporting these novel models to explain the PARP‐BRCA synthetic lethality are discussed.

Accumulation of somatic mutations is critical for the transition of a normal cell to become cancerous. Mutations cause amino acid substitutions that change properties of proteins. However, it has not been studied as to what extent the composition and accordingly chemical properties of the cell proteome is altered as a result of the increased mutation load in cancer. Here, we analyzed data on amino acid substitutions caused by mutations in about 2000 protein coding genes from the Cancer Cell Line Encyclopedia that contains information on nucleotide and amino acid alterations in 782 cancer cell lines, and validated the analysis with information on amino acid substitutions for the same set of proteins in the Catalogue of Somatic Mutations in Cancer (COSMIC; v78) in circa 18,000 tumor samples. We found that nonsynonymous single nucleotide substitutions in the analyzed proteome subset ultimately result in a net gain of cysteine, histidine, and tryptophan at the expense of a net loss of arginine. The extraordinary loss of arginine may be attributed to some extent to composition of its codons as well as to the importance of arginine in the functioning of prominent tumor suppressor proteins like p53.

Homologous recombination (HR) is intricately associated with replication, transcription and DNA repair in all organisms studied. However, the interplay between all these processes occurring simultaneously on the same DNA molecule is still poorly understood. Here, we study the interplay between transcription and HR during ultraviolet light (UV)-induced DNA damage in mammalian cells. Our results show that inhibition of transcription with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) increases the number of UV-induced DNA lesions (γH2AX, 53BP1 foci formation), which correlates with a decrease in the survival of wild type or nucleotide excision repair defective cells. Furthermore, we observe an increase in RAD51 foci formation, suggesting HR is triggered in response to an increase in UV-induced DSBs, while inhibiting transcription. Unexpectedly, we observe that DRB fails to sensitise HR defective cells to UV treatment. Thus, increased RAD51 foci formation correlates with increased cell death, suggesting the existence of a futile HR repair of UV-induced DSBs which is linked to transcription inhibition.

Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG(-/-) and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ.