NAR Cancer最新文献

The DNA damage response (DDR) entails reorganization of proteins and protein complexes involved in DNA repair. The coordinated regulation of these proteomic changes maintains genome stability. Traditionally, regulators and mediators of DDR have been investigated individually. However, recent advances in mass spectrometry (MS)-based proteomics enable us to globally quantify changes in protein abundance, post-translational modifications (PTMs), protein localization, and protein-protein interactions (PPIs) in cells. Furthermore, structural proteomics approaches, such as crosslinking MS (XL-MS), hydrogen/deuterium exchange MS (H/DX-MS), Native MS (nMS), provide large structural information of proteins and protein complexes, complementary to the data collected from conventional methods, and promote integrated structural modeling. In this review, we will overview the current cutting-edge functional and structural proteomics techniques that are being actively utilized and developed to help interrogate proteomic changes that regulate the DDR.

Maintenance of genomic methylation patterns at DNA replication forks by DNMT1 is the key to faithful mitotic inheritance. DNMT1 is often overexpressed in cancer cells and the DNA hypomethylating agents azacytidine and decitabine are currently used in the treatment of hematologic malignancies. However, the toxicity of these cytidine analogs and their ineffectiveness in treating solid tumors have limited wider clinical use. GSK-3484862 is a newly-developed, dicyanopyridine containing, non-nucleoside DNMT1-selective inhibitor with low cellular toxicity. Here, we show that GSK-3484862 targets DNMT1 for protein degradation in both cancer cell lines and murine embryonic stem cells (mESCs). DNMT1 depletion was rapid, taking effect within hours following GSK-3484862 treatment, leading to global hypomethylation. Inhibitor-induced DNMT1 degradation was proteasome-dependent, with no discernible loss of DNMT1 mRNA. In mESCs, GSK-3484862-induced Dnmt1 degradation requires the Dnmt1 accessory factor Uhrf1 and its E3 ubiquitin ligase activity. We also show that Dnmt1 depletion and DNA hypomethylation induced by the compound are reversible after its removal. Together, these results indicate that this DNMT1-selective degrader/inhibitor will be a valuable tool for dissecting coordinated events linking DNA methylation to gene expression and identifying downstream effectors that ultimately regulate cellular response to altered DNA methylation patterns in a tissue/cell-specific manner.

Centromeres play a crucial role in DNA segregation by mediating the cohesion and separation of sister chromatids during cell division. Centromere dysfunction, breakage or compromised centromeric integrity can generate aneuploidies and chromosomal instability, which are cellular features associated with cancer initiation and progression. Maintaining centromere integrity is thus essential for genome stability. However, the centromere itself is prone to DNA breaks, likely due to its intrinsically fragile nature. Centromeres are complex genomic loci that are composed of highly repetitive DNA sequences and secondary structures and require the recruitment and homeostasis of a centromere-associated protein network. The molecular mechanisms engaged to preserve centromere inherent structure and respond to centromeric damage are not fully understood and remain a subject of ongoing research. In this article, we provide a review of the currently known factors that contribute to centromeric dysfunction and the molecular mechanisms that mitigate the impact of centromere damage on genome stability. Finally, we discuss the potential therapeutic strategies that could arise from a deeper understanding of the mechanisms preserving centromere integrity.

RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2 (∼25% of breast and ovarian cancers). Emerging structure activity relationships for RAD52 are complex, making it challenging to transform previously identified disruptors of the RAD52-ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. All six were RAD52 inhibitors (IC₅₀ ∼23-1200 μM) with two of the compounds (Z56 and Z99) selectively killing BRCA-mutant cells and inhibiting cellular activities of RAD52 at micromolar inhibitor concentrations. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA-complemented cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC₅₀ ∼1.3-8 μM), which were only toxic to BRCA-mutant cells. RAD52 complexation by Z56, Z99 and its more specific derivatives provide a roadmap for next generation of cancer therapeutics.

Stromal cells promote extensive fibrosis in pancreatic ductal adenocarcinoma (PDAC), which is associated with poor prognosis and therapeutic resistance. We report here for the first time that loss of the RNA-binding protein human antigen R (HuR, ELAVL1) in PDAC cells leads to reprogramming of the tumor microenvironment. In multiple in vivo models, CRISPR deletion of ELAVL1 in PDAC cells resulted in a decrease of collagen deposition, accompanied by a decrease of stromal markers (i.e. podoplanin, α-smooth muscle actin, desmin). RNA-sequencing data showed that HuR plays a role in cell-cell communication. Accordingly, cytokine arrays identified that HuR regulates the secretion of signaling molecules involved in stromal activation and extracellular matrix organization [i.e. platelet-derived growth factor AA (PDGFAA) and pentraxin 3]. Ribonucleoprotein immunoprecipitation analysis and transcription inhibition studies validated PDGFA mRNA as a novel HuR target. These data suggest that tumor-intrinsic HuR supports extrinsic activation of the stroma to produce collagen and desmoplasia through regulating signaling molecules (e.g. PDGFAA). HuR-deficient PDAC in vivo tumors with an altered tumor microenvironment are more sensitive to the standard of care gemcitabine, as compared to HuR-proficient tumors. Taken together, we identified a novel role of tumor-intrinsic HuR in its ability to modify the surrounding tumor microenvironment and regulate PDGFAA.

DNA-methylating environmental carcinogens such as N-nitrosodimethylamine (NDMA) and certain alkylators used in chemotherapy form O ⁶-methylguanine (m6G) as a functionally critical intermediate. NDMA is a multi-organ carcinogen found in contaminated water, polluted air, preserved foods, tobacco products, and many pharmaceuticals. Only ten weeks after exposure to NDMA, neonatally-treated mice experienced elevated mutation frequencies in liver, lung and kidney of ∼35-fold, 4-fold and 2-fold, respectively. High-resolution mutational spectra (HRMS) of liver and lung revealed distinctive patterns dominated by GC→AT mutations in 5'-Pu-G-3' contexts, very similar to human COSMIC mutational signature SBS11. Commonly associated with alkylation damage, SBS11 appears in cancers treated with the DNA alkylator temozolomide (TMZ). When cells derived from the mice were treated with TMZ, N-methyl-N-nitrosourea, and streptozotocin (two other therapeutic methylating agents), all displayed NDMA-like HRMS, indicating mechanistically convergent mutational processes. The role of m6G in shaping the mutational spectrum of NDMA was probed by removing MGMT, the main cellular defense against m6G. MGMT-deficient mice displayed a strikingly enhanced mutant frequency, but identical HRMS, indicating that the mutational properties of these alkylators is likely owed to sequence-specific DNA binding. In sum, the HRMS of m6G-forming agents constitute an early-onset biomarker of exposure to DNA methylating carcinogens and drugs.

The exonuclease domain of DNA polymerases epsilon's catalytic subunit (POLE) removes misincorporated nucleotides, called proofreading. POLE-exonuclease mutations cause colorectal- and endometrial cancers with an extreme burden of single nucleotide substitutions. We recently reported that particularly the hereditary POLE exonuclease mutation N363K predisposes in addition to aggressive giant cell glioblastomas. We knocked-in this mutation homozygously into human cell lines and compared its properties to knock-ins of the likewise hereditary POLE L424V mutation and to a complete proofreading-inactivating mutation (exo-null). We found that N363K cells have higher mutation rates as both L424V- or exo-null mutant cells. In contrast to L424V cells, N363K cells expose a growth defect, replication stress and DNA damage. In non-transformed cells, these burdens lead to aneuploidy but macroscopically normal nuclei. In contrast, transformed N363K cells phenocopy the enlarged and disorganized nuclei of giant cell glioblastomas. Taken together, our data characterize a POLE exonuclease domain mutant that not only causes single nucleotide hypermutation, but in addition DNA damage and chromosome instability, leading to an extended tumor spectrum. Our results expand the understanding of the polymerase exonuclease domain and suggest that an assessment of both the mutational potential and the genetic instability might refine classification and treatment of POLE-mutated tumors.

Immune checkpoint inhibitors (ICIs) have led to durable responses in cancer patients, yet their efficacy varies significantly across cancer types and patients. To stratify patients based on their potential clinical benefits, there have been substantial research efforts in identifying biomarkers and computational models that can predict the efficacy of ICIs, and it has become difficult to keep track of all of them. It is also difficult to compare findings of different studies since they involve different cancer types, ICIs, and various other details. To make it easy to access the latest information about ICI efficacy, we have developed a knowledgebase and a corresponding web-based portal (https://iciefficacy.org/). Our knowledgebase systematically records information about latest publications related to ICI efficacy, predictors proposed, and datasets used to test them. All information recorded is checked carefully by a manual curation process. The web-based portal provides functions to browse, search, filter, and sort the information. Digests of method details are provided based on the original descriptions in the publications. Evaluation results of the effectiveness of the predictors reported in the publications are summarized for quick overviews. Overall, our resource provides centralized access to the burst of information produced by the vibrant research on ICI efficacy.

Telomerase is a specialized reverse transcriptase that synthesizes telomeric repeats at the ends of linear chromosomes. Telomerase is transiently expressed in germ and stem cells, but nearly all somatic cells silence it after differentiating. However, the vast majority of cancer cells reactivate and constitutively express telomerase to maintain replicative immortality. Because of this, telomerase has remained a promising broad-spectrum chemotherapeutic target for over 30 years. However, various challenges associated with obtaining high-resolution structural data for telomerase have limited the development of rationally designed structure-based therapeutics. Various techniques and model systems have been utilized to advance our understanding of the structural biology of telomerase. In particular, multiple high-resolution cryogenic electron microscopy (cryo-EM) structures published within the past few years have revealed new components of the telomerase complex with near atomic resolution structural models. Additionally, these structures have provided details for how telomerase is recruited to telomeres and its mechanism of telomere synthesis. With these new pieces of evidence, and the promising outlook for future refinements to our current models, the possibility of telomerase specific chemotherapeutics is becoming more tangible than ever. This review summarizes these recent advancements and outlines outstanding questions in the field.

R-loops are abundant and dynamic structures ubiquitously present in human cells both in the nuclear and mitochondrial genomes. They form in cis in the wake of transcription complexes and in trans apart from transcription complexes. In this review, we focus on the relationship between R-loops and topoisomerases, and cancer genomics and therapies. We summarize the topological parameters associated with the formation and resolution of R-loops, which absorb and release high levels of genomic negative supercoiling (Sc-). We review the deleterious consequences of excessive R-loops and rationalize how human type IA (TOP3B) and type IB (TOP1) topoisomerases regulate and resolve R-loops in coordination with helicase and RNase H enzymes. We also review the drugs (topoisomerase inhibitors, splicing inhibitors, G4 stabilizing ligands) and cancer predisposing genes (BRCA1/2, transcription, and splicing genes) known to induce R-loops, and whether stabilizing R-loops and thereby inducing genomic damage can be viewed as a strategy for cancer treatment.