Gremlin-1 Overexpression in Mouse Lung Reduces Silica-Induced Lymphocyte Recruitment - A Link to Idiopathic Pulmonary Fibrosis through Negative Correlation with CXCL10 Chemokine

Idiopathic pulmonary fibrosis (IPF) is characterized by activation and injury of epithelial cells, the accumulation of connective tissue and changes in the inflammatory microenvironment. The bone morphogenetic protein (BMP) inhibitor protein gremlin-1 is associated with the progression of fibrosis both in human and mouse lung. We generated a transgenic mouse model expressing gremlin-1 in type II lung epithelial cells using the surfactant protein C (SPC) promoter and the Cre-LoxP system. Gremlin-1 protein expression was detected specifically in the lung after birth and did not result in any signs of respiratory insufficiency. Exposure to silicon dioxide resulted in reduced amounts of lymphocyte aggregates in transgenic lungs while no alteration in the fibrotic response was observed. Microarray gene expression profiling and analyses of bronchoalveolar lavage fluid cytokines indicated a reduced lymphocytic response and a downregulation of interferon-induced gene program. Consistent with reduced Th1 response, there was a downregulation of the mRNA and protein expression of the anti-fibrotic chemokine CXCL10, which has been linked to IPF. In human IPF patient samples we also established a strong negative correlation in the mRNA expression levels of gremlin-1 and CXCL10. Our results suggest that in addition to regulation of epithelial-mesenchymal crosstalk during tissue injury, gremlin-1 modulates inflammatory cell recruitment and anti-fibrotic chemokine production in the lung.Peer reviewe

Ex vivo culture of cells derived from circulating tumour cell xenograft to support small cell lung cancer research and experimental therapeutics

Background and Purpose: Small cell lung cancer (SCLC) is an aggressive disease with median survival of <2 years. Tumour biopsies for research are scarce, especially from extensive-stage patients, with repeat sampling at disease progression rarely performed. We overcame this limitation for relevant preclinical models by developing SCLC circulating tumour cell derived explants (CDX), which mimic the donor tumour pathology and chemotherapy response. To facilitate compound screening and identification of clinically relevant biomarkers, we developed short-term ex vivo cultures of CDX tumour cells. Experimental Approach: CDX tumours were disaggregated, and the human tumour cells derived were cultured for a maximum of 5 weeks. Phenotypic, transcriptomic and pharmacological characterization of these cells was performed. Key Results: CDX cultures maintained a neuroendocrine phenotype, and most changes in the expression of protein-coding genes observed in cultures, for up to 4 weeks, were reversible when the cells were re-implanted in vivo. Moreover, the CDX cultures exhibited a similar sensitivity to chemotherapy compared to the corresponding CDX tumour in vivo and were able to predict in vivo responses to therapeutic candidates. Conclusions and Implications: Short-term cultures of CDX provide a tractable platform to screen new treatments, identify predictive and pharmacodynamic biomarkers and investigate mechanisms of resistance to better understand the progression of this recalcitrant tumour. © 2018 The British Pharmacological Societ

Small-molecule activation of OGG1 increases oxidative DNA damage repair by gaining a new function

Oxidative DNA damage is recognised by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1), initiating repair. Here, we describe a small molecule (TH10785) that interacts with the Phe319 and Gly42 amino acids of OGG1, increases the enzyme activity 10-fold and generates a novel β,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and ageing.European Research Council TAROX-695376Swedish Research Council 2015-00162 and 2018-03406Ministry of Science and Innovation, Spain/State Research Agency, Spain/10.13039/501.100011033European Regional Development Fund (ERDF) BFU2017-83900-PCrafoord Foundation 20190532Alfred Osterlund FoundationSwedish Pain Relief FoundationSwedish Cancer Society CAN 2018/0658 and CAN 2017/716Torsten and Ragnar Soderberg foundationDr. Ake-Olsson Foundation for Hematological Research 2020-00306Thomas Helleday Foundation for medical research postdoctoral stipendsNTNU Enabling Technology Programme on BiotechnologyEMBO Short-Term Fellowship 9005FEBS Short-Term FellowshipScandinavian ExchangeGerman Research Foundation (DFG) 239748522Sonderforschungsbereich (SFB) 1127Leibniz AwardNorwegian Research Council 303369Karolinska Institutet Research Foundation 2020-02186Lars Hiertas Minne StiftelseAsociacion Espanola Contra Cancer grant Postdoctoral AECC 2020 POSTD20042BENIInstituto de Salud Carlos III CP19/00063, PI20/00329 and PI19/00640European Social Fund (ESF)Innovative Medicines Initiative 2 Joint Undertaking (JU) 875510European Union's Horizon 2020 research and innovation program, Marie Sklodowska-Curie 722729Accepte

Development of a chemical probe against NUDT15

The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6 thio d GTP, thereby dictating the clinical response of this standard of care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15 dependent chemotherapeutics. Lead compound TH1760 demonstrated low nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6 thioguanine through enhanced accumulation of 6 thio d GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenue

Validation and development of MTH1 inhibitors for treatment of cancer

Background: Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. Material and methods: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8-oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling, proteomics, cellular thermal shift assays, kinase and CEREP panel were used for target engagement, mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E-mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. Results: Here, we demonstrate that recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579, (Karonudib), an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. Conclusion: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept

Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress

The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors. Helleday and colleagues describe a nanomolar MTHFD2 inhibitor that causes replication stress and DNA damage accumulation in cancer cells via thymidine depletion, demonstrating a potential therapeutic strategy in AML tumors in vivo