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Chromatin architecture is a fundamental mediator of genome function. Fasting is a major environmental cue across the animal kingdom, yet how it impacts three-dimensional (3D) genome organization is unknown. Here we show that fasting induces an intestine-specific, reversible and large-scale spatial reorganization of chromatin in Caenorhabditis elegans. This fasting-induced 3D genome reorganization requires inhibition of the nutrient-sensing mTOR pathway, acting through the regulation of RNA Pol I, but not Pol II nor Pol III, and is accompanied by remodelling of the nucleolus. By uncoupling the 3D genome configuration from the animal's nutritional status, we find that the expression of metabolic and stress-related genes increases when the spatial reorganization of chromatin occurs, showing that the 3D genome might support the transcriptional response in fasted animals. Our work documents a large-scale chromatin reorganization triggered by fasting and reveals that mTOR and RNA Pol I shape genome architecture in response to nutrients., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Environment-epigenome interactions are emerging as contributors to disease risk and health outcomes. In fact, organisms outside of the laboratory are constantly exposed to environmental changes that can influence chromatin regulation at multiple levels, potentially impacting on genome function. In this review, we will summarize recent findings on how major external cues impact on 3D chromatin organization in different experimental systems. We will describe environment-induced 3D genome alterations ranging from chromatin accessibility to the spatial distribution of the genome and discuss their role in regulating gene expression., Competing Interests: Declaration of Competing Interest Lorenz Pudelko and Daphne S. Cabianca, authors of the mini-review entitled “The influencers' era: how the environment shapes chromatin in 3D”, declare that they have no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Methionine aminopeptidase 2 (MetAP2) is essential to endothelial cell growth and proliferation during tumor angiogenesis. M8891 is a novel orally bioavailable, potent, selective, reversible MetAP2 inhibitor with antiangiogenic and antitumor activity in preclinical studies. The safety, tolerability, pharmacokinetics, and pharmacodynamics of M8891 monotherapy were assessed in a phase I, first-in-human, multicenter, open-label, single-arm, dose-escalation study (NCT03138538). Patients with advanced solid tumors received 7-80 mg M8891 once daily in 21-day cycles. The primary endpoint was dose-limiting toxicity (DLT) during cycle 1, with the aim to determine the maximum tolerated dose (MTD). Twenty-seven patients were enrolled across six dose levels. Two DLTs (platelet count decrease) were reported, one each at 60 and 80 mg/once daily M8891, resolving after treatment discontinuation. MTD was not determined. The most common treatment-emergent adverse event was platelet count decrease. M8891 plasma concentration showed dose-linear increase up to 35 mg and low-to-moderate variability; dose-dependent tumor accumulation of methionylated elongation factor 1α, a MetAP2 substrate, was observed, demonstrating MetAP2 inhibition. Pharmacokinetic/pharmacodynamic response data showed that preclinically defined target levels required for in vivo efficacy were achieved at safe, tolerated doses. Seven patients (25.9%) had stable disease for 42-123 days. We conclude that M8891 demonstrates a manageable safety profile, with dose-proportional exposure and low-to-moderate interpatient variability at target pharmacokinetic/pharmacodynamic levels at ≤35 mg M8891 once daily. On the basis of the data, 35 mg M8891 once daily is the recommended phase II dose for M8891 monotherapy. This study forms the basis for future development of M8891 in monotherapy and combination studies., Significance: M8891 represents a novel class of reversible MetAP2 inhibitors and has demonstrated preclinical antitumor activity. This dose-escalation study assessed M8891 treatment for patients with advanced solid tumors. M8891 demonstrated favorable pharmacokinetics, tumoral target engagement, and a manageable safety profile, and thus represents a novel antitumor strategy warranting further clinical studies., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Background & Aims: Epigenetic processes regulating gene expression contribute markedly to epithelial cell plasticity in colorectal carcinogenesis. The lysine methyltransferase SUV420H2 comprises an important regulator of epithelial plasticity and is primarily responsible for trimethylation of H4K20 (H4K20me3). Loss of H4K20me3 has been suggested as a hallmark of human cancer due to its interaction with DNMT1. However, the role of Suv4-20h2 in colorectal cancer is unknown., Methods: We examined the alterations in histone modifications in patient-derived colorectal cancer organoids. Patient-derived colorectal cancer organoids and mouse intestinal organoids were genetically manipulated for functional studies in patient-derived xenograft and orthotopic transplantation. Gene expression profiling, micrococcal nuclease assay, and chromatin immunoprecipitation were performed to understand epigenetic regulation of chromatin states and gene expression in patient-derived and mouse intestinal organoids., Results: We found that reduced H4K20me3 levels occurred predominantly in right-sided patient-derived colorectal cancer organoids, which were associated with increased chromatin accessibility. Re-compaction of chromatin by methylstat, a histone demethylase inhibitor, resulted in reduced growth selectively in subcutaneously grown tumors derived from right-sided cancers. Using mouse intestinal organoids, we confirmed that Suv4-20h2-mediated H4K20me3 is required for maintaining heterochromatin compaction and to prevent R-loop formation. Cross-species comparison of Suv4-20h2-depleted murine organoids with right-sided colorectal cancer organoids revealed a large overlap of gene signatures involved in chromatin silencing, DNA methylation, and stemness/Wnt signaling., Conclusions: Loss of Suv4-20h2-mediated H4K20me3 drives right-sided colorectal tumorigenesis through an epigenetically controlled mechanism of chromatin compaction. Our findings unravel a conceptually novel approach for subtype-specific therapy of this aggressive form of colorectal cancer., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Redox regulation of specific cysteines via oxidoreductases of the thioredoxin family is increasingly being recognized as an important signaling pathway. Here, we demonstrate that the cytosolic isoform of the vertebrate-specific oxidoreductase Glutaredoxin 2 (Grx2c) regulates the redox state of the transcription factor SP-1 and thereby its binding affinity to both the promoter and an enhancer region of the CSPG4 gene encoding chondroitin sulfate proteoglycan nerve/glial antigen 2 (NG2). This leads to an increased number of NG2 glia during in vitro oligodendroglial differentiation and promotes migration of these wound healing cells. On the other hand, we found that the same mechanism also leads to increased invasion of glioma tumor cells. Using in vitro (human cell lines), ex vivo (mouse primary cells), and in vivo models (zebrafish), as well as glioblastoma patient tissue samples we provide experimental data highlighting the Yin and Yang of redox signaling in the central nervous system and the enzymatic Taoism of Grx2c., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2022. The Author(s).)

Tepotinib is an oral MET inhibitor approved for metastatic non-small cell lung cancer (NSCLC) harboring MET exon 14 (METex14) skipping mutations. Examining treatment-naive or tepotinib-resistant cells with MET amplification or METex14 skipping mutations identifies other receptor tyrosine kinases (RTKs) that co-exist in cells prior to tepotinib exposure and become more prominent upon tepotinib resistance. In a small cohort of patients with lung cancer with MET genetic alterations treated with tepotinib, gene copy number gains of other RTKs were found at baseline and affected treatment outcome. An Src homology 2 domain-containing phosphatase 2 (SHP2) inhibitor delayed the emergence of tepotinib resistance and synergized with tepotinib in treatment-naive and tepotinib-resistant cells as well as in xenograft models. Alternative signaling pathways potentially diminish the effect of tepotinib monotherapy, and the combination of tepotinib with an SHP2 inhibitor enables the control of tumor growth in cells with MET genetic alterations., Competing Interests: L.P., F.J., C.R., C.S., N.L., M.S., D.M., M.K., C.E., A.B., K.M.S., and N.K. are employees of Merck KGaA. A.B. and M.S. hold stocks in Merck KGaA. No other potential conflict of interest relevant to this article was reported., (© 2020 The Author(s).)

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 avenues.

Cutaneous malignant melanoma (CMM) is the deadliest form of skin cancer and clinically challenging due to its propensity to develop therapy resistance. Reactive oxygen species (ROS) can induce DNA damage and play a significant role in CMM. MTH1 protein protects from ROS damage and is often overexpressed in different cancer types including CMM. Herein, we report that MTH1 inhibitor TH1579 induced ROS levels, increased DNA damage responses, caused mitotic arrest and suppressed CMM proliferation leading to cell death both in vitro and in an in vivo xenograft CMM zebrafish disease model. TH1579 was more potent in abrogating cell proliferation and inducing cell death in a heterogeneous co-culture setting when compared with CMM standard treatments, vemurafenib or trametinib, showing its broad anticancer activity. Silencing MTH1 alone exhibited similar cytotoxic effects with concomitant induction of mitotic arrest and ROS induction culminating in cell death in most CMM cell lines tested, further emphasizing the importance of MTH1 in CMM cells. Furthermore, overexpression of receptor tyrosine kinase AXL, previously demonstrated to contribute to BRAF inhibitor resistance, sensitized BRAF mutant and BRAF/NRAS wildtype CMM cells to TH1579. AXL overexpression culminated in increased ROS levels in CMM cells. Moreover, silencing of a protein that has shown opposing effects on cell proliferation, CAV-1, decreased sensitivity to TH1579 in a BRAF inhibitor resistant cell line. AXL-MTH1 and CAV-1-MTH1 mRNA expressions were correlated as seen in CMM clinical samples. Finally, TH1579 in combination with BRAF inhibitor exhibited a more potent cell killing effect in BRAF mutant cells both in vitro and in vivo. In summary, we show that TH1579-mediated efficacy is independent of BRAF/NRAS mutational status but dependent on the expression of AXL and CAV-1.

Background: Glioblastoma (GBM) is an aggressive form of brain cancer with poor prognosis. Although murine animal models have given valuable insights into the GBM disease biology, they cannot be used in high-throughput screens to identify and profile novel therapies. The only vertebrate model suitable for large-scale screens, the zebrafish, has proven to faithfully recapitulate biology and pathology of human malignancies, and clinically relevant orthotopic zebrafish models have been developed. However, currently available GBM orthotopic zebrafish models do not support high-throughput drug discovery screens., Methods: We transplanted both GBM cell lines as well as patient-derived material into zebrafish blastulas. We followed the behavior of the transplants with time-lapse microscopy and real-time in vivo light-sheet microscopy., Results: We found that GBM material transplanted into zebrafish blastomeres robustly migrated into the developing nervous system, establishing an orthotopic intracranial tumor already 24 hours after transplantation. Detailed analysis revealed that our model faithfully recapitulates the human disease., Conclusion: We have developed a robust, fast, and automatable transplantation assay to establish orthotopic GBM tumors in zebrafish. In contrast to currently available orthotopic zebrafish models, our approach does not require technically challenging intracranial transplantation of single embryos. Our improved zebrafish model enables transplantation of thousands of embryos per hour, thus providing an orthotopic vertebrate GBM model for direct application in drug discovery screens.

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells in vitro . We used an orthotopic zebrafish model to confirm our results in vivo and light-sheet microscopy to follow the effect of MTH1 inhibition in GBM in real time. In conclusion, MTH1 represents a promising target for GBM therapy and MTH1 inhibitors may also be effective in patients that suffer from recurring disease., Competing Interests: CONFLICTS OF INTEREST MTH1 inhibitors are developed in the laboratory of TH for the treatment of cancer.

Cancer cells are commonly in a state of redox imbalance that drives their growth and survival. To compensate for oxidative stress induced by the tumor redox environment, cancer cells upregulate specific nononcogenic addiction enzymes, such as MTH1 (NUDT1), which detoxifies oxidized nucleotides. Here, we show that increasing oxidative stress in nonmalignant cells induced their sensitization to the effects of MTH1 inhibition, whereas decreasing oxidative pressure in cancer cells protected against inhibition. Furthermore, we purified zebrafish MTH1 and solved the crystal structure of MTH1 bound to its inhibitor, highlighting the zebrafish as a relevant tool to study MTH1 biology. Delivery of 8-oxo-dGTP and 2-OH-dATP to zebrafish embryos was highly toxic in the absence of MTH1 activity. Moreover, chemically or genetically mimicking activated hypoxia signaling in zebrafish revealed that pathologic upregulation of the HIF1α response, often observed in cancer and linked to poor prognosis, sensitized embryos to MTH1 inhibition. Using a transgenic zebrafish line, in which the cellular redox status can be monitored in vivo, we detected an increase in oxidative pressure upon activation of hypoxic signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine protected embryos with activated hypoxia signaling against MTH1 inhibition, suggesting that the aberrant redox environment likely causes sensitization. In summary, MTH1 inhibition may offer a general approach to treat cancers characterized by deregulated hypoxia signaling or redox imbalance. Cancer Res; 76(8); 2366-75. ©2016 AACR., (©2016 American Association for Cancer Research.)