Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch.

During tumor development, promoter CpG islands (CGIs) that are normally silenced by Polycomb repressive complexes (PRCs) become DNA hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) catalyze CpG methylation at PRC-regulated regions remains unclear. Here we report a cryo-EM structure of the DNMT3A long isoform (DNMT3A1) N-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine 119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 N-terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Furthermore, aberrant redistribution of DNMT3A1 to Polycomb target genes inhibits their transcriptional activation during cell differentiation and recapitulates the cancer-associated DNA hypermethylation signature. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for countering promoter CGI DNA hypermethylation, a major molecular hallmark of cancer.

Nucleosome conformation dictates the histone code.

Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has been proposed that these PTMs form localized 'codes' that are read by specialized regions (reader domains) in chromatin-associated proteins (CAPs) to regulate downstream function. Substantial effort has been made to define [CAP: histone PTM] specificities, and thus decipher the histone code and guide epigenetic therapies. However, this has largely been done using the reductive approach of isolated reader domains and histone peptides, which cannot account for any higher-order factors. Here, we show that the [BPTF PHD finger and bromodomain: histone PTM] interaction is dependent on nucleosome context. The tandem reader selectively associates with nucleosomal H3K4me3 and H3K14ac or H3K18ac, a combinatorial engagement that despite being in cis is not predicted by peptides. This in vitro specificity of the BPTF tandem reader for PTM-defined nucleosomes is recapitulated in a cellular context. We propose that regulatable histone tail accessibility and its impact on the binding potential of reader domains necessitates we refine the 'histone code' concept and interrogate it at the nucleosome level.

Editor's Choice - Hybrid vs. Open Surgical Reconstruction for Iliofemoral Occlusive Disease: A Prospective Randomised Trial.

OBJECTIVE: The aim of this non-inferiority randomised trial was to compare the short and midterm safety and efficacy of hybrid repair (HR) and open reconstruction (OR) for patients with co-existing iliac and common femoral artery (CFA) occlusive disease. METHODS: The study was registered on the ClinicalTrials.gov register (identifier: NCT02580084). From 2015 to 2017, eligible patients presenting with combined iliac and CFA occlusive disease were randomised to either HR or OR. HR group patients underwent recanalisation and stenting of iliac arteries combined with CFA endarterectomy and patch angioplasty. The OR group underwent aortofemoral bypass with simultaneous CFA endarterectomy. Short (30 day) and midterm (36 month) outcomes including morbidity, mortality, and patency rates were compared between groups. RESULTS: Of 427 patients assessed, 202 were randomised (102 HR and 100 OR). The average hospital length of stay was shorter in the HR group (8.2 ± 4.2 days HR group vs. 15.7 ± 6.9 days OR group, p < .001); the 30 day peri-operative morbidity rate was 8.8% in the HR group vs. 21% in the OR group (p = .030). There was no significant difference in the 36 month mortality rate (p = .16). The cumulative primary patency rates were 93% (HR) vs. 93% (OR) at 12 months and 91% (HR) vs. 89% (OR) at 36 months (p = .38). The limb salvage rates were 99% (HR) vs. 99% (OR) at 12 months and 98% (HR) vs. 97% (OR) at 36 months (p = .49). CONCLUSION: The results of this first non-inferiority randomised study support the safety and midterm efficacy of hybrid procedures for patients with iliofemoral peripheral arterial disease. HR patients had a shorter length of stay with reduced peri-operative morbidity and similar medium term patency rates.

The dCypher Approach to Interrogate Chromatin Reader Activity Against Posttranslational Modification-Defined Histone Peptides and Nucleosomes.

Bulk chromatin encompasses complex sets of histone posttranslational modifications (PTMs) that recruit (or repel) the diverse reader domains of Chromatin-Associated Proteins (CAPs) to regulate genome processes (e.g., gene expression, DNA repair, mitotic transmission). The binding preference of reader domains for their PTMs mediates localization and functional output, and are often dysregulated in disease. As such, understanding chromatin interactions may lead to novel therapeutic strategies, However the immense chemical diversity of histone PTMs, combined with low-throughput, variable, and nonquantitative methods, has defied accurate CAP characterization. This chapter provides a detailed protocol for dCypher, a novel approach for the rapid, quantitative interrogation of CAPs (as mono- or multivalent Queries) against large panels (10s to 100s) of PTM-defined histone peptide and semisynthetic nucleosomes (the potential Targets). We describe key optimization steps and controls to generate robust binding data. Further, we compare the utility of histone peptide and nucleosome substrates in CAP studies, outlining important considerations in experimental design and data interpretation.

A chemical probe targeting the PWWP domain alters NSD2 nucleolar localization.

Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.

A trivalent nucleosome interaction by PHIP/BRWD2 is disrupted in neurodevelopmental disorders and cancer.

Mutations in the PHIP/BRWD2 chromatin regulator cause the human neurodevelopmental disorder Chung-Jansen syndrome, while alterations in PHIP expression are linked to cancer. Precisely how PHIP functions in these contexts is not fully understood. Here we demonstrate that PHIP is a chromatin-associated CRL4 ubiquitin ligase substrate receptor and is required for CRL4 recruitment to chromatin. PHIP binds to chromatin through a trivalent reader domain consisting of a H3K4-methyl binding Tudor domain and two bromodomains (BD1 and BD2). Using semisynthetic nucleosomes with defined histone post-translational modifications, we characterize PHIPs BD1 and BD2 as respective readers of H3K14ac and H4K12ac, and identify human disease-associated mutations in each domain and the intervening linker region that likely disrupt chromatin binding. These findings provide new insight into the biological function of this enigmatic chromatin protein and set the stage for the identification of both upstream chromatin modifiers and downstream targets of PHIP in human disease.

Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies.

Proteases are involved in the control of numerous physiological processes, and their dysregulation has been identified in a wide range of pathologies, including cancer. Protease activity is normally tightly regulated post-translationally and therefore cannot be accurately estimated based on mRNA or protein expression alone. While several types of zymography approaches to estimate protease activity exist, there remains a need for a robust and reliable technique to measure protease activity in biological tissues. We present a novel quantitative ex vivo zymography (QZ) technology based on Probody® therapeutics (Pb-Tx), a novel class of protease-activated cancer therapeutics that contain a substrate linker cleavable by tumor-associated proteases. This approach enables the measurement and comparison of protease activity in biological tissues via the detection of Pb-Tx activation. By exploiting substrate specificity and selectivity, cataloguing and differentiating protease activities is possible, with further refinement achieved using protease-specific inhibitors. Using the QZ assay and human tumor xenografts, patient tumor tissues, and patient plasma, we characterized protease activity in preclinical and clinical samples. The QZ assay offers the potential to increase our understanding of protease activity in tissues and inform diagnostic and therapeutic development for diseases, such as cancer, that are characterized by dysregulated proteolysis.

Aß initiates brain hypometabolism, network dysfunction and behavioral abnormalities via NOX2-induced oxidative stress in mice.

A predominant trigger and driver of sporadic Alzheimer's disease (AD) is the synergy of brain oxidative stress and glucose hypometabolism starting at early preclinical stages. Oxidative stress damages macromolecules, while glucose hypometabolism impairs cellular energy supply and antioxidant defense. However, the exact cause of AD-associated glucose hypometabolism and its network consequences have remained unknown. Here we report NADPH oxidase 2 (NOX2) activation as the main initiating mechanism behind Aß1-42-related glucose hypometabolism and network dysfunction. We utilize a combination of electrophysiology with real-time recordings of metabolic transients both ex- and in-vivo to show that Aß1-42 induces oxidative stress and acutely reduces cellular glucose consumption followed by long-lasting network hyperactivity and abnormalities in the animal behavioral profile. Critically, all of these pathological changes were prevented by the novel bioavailable NOX2 antagonist GSK2795039. Our data provide direct experimental evidence for causes and consequences of AD-related brain glucose hypometabolism, and suggest that targeting NOX2-mediated oxidative stress is a promising approach to both the prevention and treatment of AD.

Two competing mechanisms of DNMT3A recruitment regulate the dynamics of de novo DNA methylation at PRC1-targeted CpG islands.

Precise deposition of CpG methylation is critical for mammalian development and tissue homeostasis and is often dysregulated in human diseases. The localization of de novo DNA methyltransferase DNMT3A is facilitated by its PWWP domain recognizing histone H3 lysine 36 (H3K36) methylation1,2 and is normally depleted at CpG islands (CGIs)3. However, methylation of CGIs regulated by Polycomb repressive complexes (PRCs) has also been observed4-8. Here, we report that DNMT3A PWWP domain mutations identified in paragangliomas9 and microcephalic dwarfism10 promote aberrant localization of DNMT3A to CGIs in a PRC1-dependent manner. DNMT3A PWWP mutants accumulate at regions containing PRC1-mediated formation of monoubiquitylated histone H2A lysine 119 (H2AK119ub), irrespective of the amounts of PRC2-catalyzed formation of trimethylated histone H3 lysine 27 (H3K27me3). DNMT3A interacts with H2AK119ub-modified nucleosomes through a putative amino-terminal ubiquitin-dependent recruitment region, providing an alternative form of DNMT3A genomic targeting that is augmented by the loss of PWWP reader function. Ablation of PRC1 abrogates localization of DNMT3A PWWP mutants to CGIs and prevents aberrant DNA hypermethylation. Our study implies that a balance between DNMT3A recruitment by distinct reader domains guides de novo CpG methylation and may underlie the abnormal DNA methylation landscapes observed in select human cancer subtypes and developmental disorders.

Overall survival in patients with metastatic renal cell carcinoma in Russia, Kazakhstan, and Belarus: a report from the RENSUR3 registry.

BACKGROUND: Real-world data describing outcomes of treatment among metastatic renal cell carcinoma (mRCC) patients are limited and heterogeneous. AIM: RENSUR3 registry study assessed real-world data on the use of therapies in mRCC and overall survival (OS) in Russia, Kazakhstan, and Belarus. METHODS: Patients were included in the retrospective multicenter registry study. To be eligible, patients were required to have mRCC diagnosed from January 2015 to January 2016. Anonymized data were collected through an online registry. The outcomes of interest were patient characteristics, treatment patterns, and OS. RESULTS: 1094 mRCC patients were identified. Mean age was 62.3 (SD, 11.2) years. Four hundred and forty-four (41%) patients were 65 years and older. Primary tumor has not been removed in 503 (46%) patients. Subtype of RCC based on WHO classification (clear-cell or other) has been reported in 402 (37%) patients. In total, 595 (54.4%) patients received systemic therapy for metastatic disease. 58% of elderly patients (≥65) were not treated compared to 37% of younger patients. Cytokines and targeted therapy were used in 298 (50.1%) and 297 (49.9%) of 595 treated patients, respectively. Median OS was 11.9 months (95% CI 10.9-12.9). The 1- and 3-year OS rates were 49.6% and 19.3%. CONCLUSIONS: Half of patients received no systemic therapy or had only cytokines for mRCC in Russia, Kazakhstan, and Belarus, which doubtless negatively affected OS in this population. Novel therapies should be considered as life prolonging and a priority.

Size of canine hepatocellular carcinoma as an adverse prognostic factor for surgery.

OBJECTIVE: Liver neoplasms are problematic among small domestic animals. The etiological cause of hepatocellular carcinomas in domestic animals is still unknown although it is believed that chronic infections and toxic substances can affect the development of this type of tumor. This study aimed to analyze the clinical and morphological characteristics of canine hepatocellular carcinoma. MATERIALS AND METHODS: In total, 6,958 cancer operations were performed in the clinic. Liver tumors were detected in 123 dogs in vivo and 375 dogs postmortem. All animals with suspected liver neoplasm were assessed, including history, clinical examination, complete blood count, biochemical blood tests, radiographic examination, and ultrasound with a biopsy for performing cytological and histological analyses. RESULTS: Hepatocellular carcinomas have nonspecific clinical manifestations, also a characteristic aspect of other tumors of the hepatobiliary system. The hematological changes have an impact on the prognosis, and biochemical abnormalities reflect the changes in liver activity. The cytological diagnosis of hepatocellular tumors is difficult because of hepatocyte atypia in highly differentiated carcinomas. Finally, a histological examination was performed in all the dogs diagnosed with hepatocellular carcinoma. CONCLUSION: Hematological changes in dogs with hepatocellular carcinoma affect their prognosis. Biochemical abnormalities of this pathology reflect the changes in liver activity, not indicating a specific pathology. However, an increase in the activity of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase is an unfavorable prognostic sign. In this study, five of seven dogs with a tumor size of more than 5.0 cm had a life expectancy of 30, 51, and 91 days, suggesting that the size of the tumor is an adverse prognostic factor.

Characterization of the plant homeodomain (PHD) reader family for their histone tail interactions.

BACKGROUND: Plant homeodomain (PHD) fingers are central "readers" of histone post-translational modifications (PTMs) with > 100 PHD finger-containing proteins encoded by the human genome. Many of the PHDs studied to date bind to unmodified or methylated states of histone H3 lysine 4 (H3K4). Additionally, many of these domains, and the proteins they are contained in, have crucial roles in the regulation of gene expression and cancer development. Despite this, the majority of PHD fingers have gone uncharacterized; thus, our understanding of how these domains contribute to chromatin biology remains incomplete. RESULTS: We expressed and screened 123 of the annotated human PHD fingers for their histone binding preferences using reader domain microarrays. A subset (31) of these domains showed strong preference for the H3 N-terminal tail either unmodified or methylated at H3K4. These H3 readers were further characterized by histone peptide microarrays and/or AlphaScreen to comprehensively define their H3 preferences and PTM cross-talk. CONCLUSIONS: The high-throughput approaches utilized in this study establish a compendium of binding information for the PHD reader family with regard to how they engage histone PTMs and uncover several novel reader domain-histone PTM interactions (i.e., PHRF1 and TRIM66). This study highlights the usefulness of high-throughput analyses of histone reader proteins as a means of understanding how chromatin engagement occurs biochemically.

Probody Therapeutic Design of 89Zr-CX-072 Promotes Accumulation in PD-L1-Expressing Tumors Compared to Normal Murine Lymphoid Tissue.

PURPOSE: Probody therapeutic CX-072 is a protease-activatable antibody that is cross-reactive with murine and human programmed death-ligand 1 (PD-L1). CX-072 can be activated in vivo by proteases present in the tumor microenvironment, thereby potentially reducing peripheral, anti-PD-L1-mediated toxicities. To study its targeting of PD-L1-expressing tissues, we radiolabeled CX-072 with the PET isotope zirconium-89 (89Zr). EXPERIMENTAL DESIGN: 89Zr-labeled CX-072, nonspecific Probody control molecule (PbCtrl) and CX-072 parental antibody (CX-075) were injected in BALB/c nude mice bearing human MDA-MB-231 tumors or C57BL/6J mice bearing syngeneic MC38 tumors. Mice underwent serial PET imaging 1, 3, and 6 days after intravenous injection (pi), followed by ex vivo biodistribution. Intratumoral 89Zr-CX-072 distribution was studied by autoradiography on tumor tissue sections, which were subsequently stained for PD-L1 by IHC. Activated CX-072 species in tissue lysates were detected by Western capillary electrophoresis. RESULTS: PET imaging revealed 89Zr-CX-072 accumulation in MDA-MB-231 tumors with 2.1-fold higher tumor-to-blood ratios at 6 days pi compared with 89Zr-PbCtrl. Tumor tissue autoradiography showed high 89Zr-CX-072 uptake in high PD-L1-expressing regions. Activated CX-072 species were detected in these tumors, with 5.3-fold lower levels found in the spleen. Furthermore, 89Zr-CX-072 uptake by lymphoid tissues of immune-competent mice bearing MC38 tumors was low compared with 89Zr-CX-075, which lacks the Probody design. CONCLUSIONS: 89Zr-CX-072 accumulates specifically in PD-L1-expressing tumors with limited uptake in murine peripheral lymphoid tissues. Our data may enable clinical evaluation of 89Zr-CX-072 whole-body distribution as a tool to support CX-072 drug development (NCT03013491).

Main dependencies in reduction of radiation exposure to the population of the Southern Urals.

Dynamics of soil contamination, food chain, and radiation doses to population at the East-Urals Radioactive Trace (EURT) and the Karachay Radioactive Trace (KRT) are reviewed. Gamma spectrometric analysis of samples was performed according to standard methodology; 90Sr was determined by the extraction method. Over 80% of radionuclides in soil are contained in the upper 20-cm layer. Biologically available forms of Sr and insoluble forms of 137Cs are predominant. The main reasons for reduction in milk contamination are radioactive decay and sequestration of radionuclides in soil. Current annual intake of radionuclides with food: EURT--310 Bq (90Sr), KRT--324 Bq (90Sr) and 732 Bq (137Cs).

Spontaneous epileptic manifestations in a DCX knockdown model of human double cortex.

Previous reports indicate that in utero knockdown of doublecortin (DCX) results in the genesis of a subcortical heterotopia reminiscent of the doublecortex observed in female patients with DCX mutations. It has also been shown that these rats display an increased susceptibility to convulsant agents and increased cortical neurons excitability; but it is presently unknown whether they display spontaneous seizures. Furthermore, the link between the size of heterotopia and the clinical manifestation remained to be elucidated. Using video-electrocorticogram recordings, we now report that DCX knockdown induces frequent spontaneous seizures commonly associated with myoclonic jerks in adult rats. Surprisingly, epilepsy occurred even in rats with very small subcortical heterotopias, as revealed by histological analysis of recorded animals. Moreover, the severity of the epileptic manifestations was positively correlated with both the size of the subcortical heterotopia and the age of recorded animals; thus, epileptic features were not detected in immature affected rats. In conclusion, our data demonstrate for the first time that subtle alterations can yield epilepsy and reveal a strong correlation between thicknesses of subcortical heterotopia, age of affected individuals and clinical impairment.