Cellular functions of the protein kinase ATM and their relevance to human disease.

The protein kinase ataxia telangiectasia mutated (ATM) is a master regulator of double-strand DNA break (DSB) signalling and stress responses. For three decades, ATM has been investigated extensively to elucidate its roles in the DNA damage response (DDR) and in the pathogenesis of ataxia telangiectasia (A-T), a human neurodegenerative disease caused by loss of ATM. Although hundreds of proteins have been identified as ATM phosphorylation targets and many important roles for this kinase have been identified, it is still unclear how ATM deficiency leads to the early-onset cerebellar degeneration that is common in all individuals with A-T. Recent studies suggest the existence of links between ATM deficiency and other cerebellum-specific neurological disorders, as well as the existence of broader similarities with more common neurodegenerative disorders. In this Review, we discuss recent structural insights into ATM regulation, and possible aetiologies of A-T phenotypes, including reactive oxygen species, mitochondrial dysfunction, alterations in transcription, R-loop metabolism and alternative splicing, defects in cellular proteostasis and metabolism, and potential pathogenic roles for hyper-poly(ADP-ribosyl)ation.

Poly-ADP-ribosylation drives loss of protein homeostasis in ATM and Mre11 deficiency.

Loss of the ataxia-telangiectasia mutated (ATM) kinase causes cerebellum-specific neurodegeneration in humans. We previously demonstrated that deficiency in ATM activation via oxidative stress generates insoluble protein aggregates in human cells, reminiscent of protein dysfunction in common neurodegenerative disorders. Here, we show that this process is driven by poly-ADP-ribose polymerases (PARPs) and that the insoluble protein species arise from intrinsically disordered proteins associating with PAR-associated genomic sites in ATM-deficient cells. The lesions implicated in this process are single-strand DNA breaks dependent on reactive oxygen species, transcription, and R-loops. Human cells expressing Mre11 A-T-like disorder mutants also show PARP-dependent aggregation identical to ATM deficiency. Lastly, analysis of A-T patient cerebellum samples shows widespread protein aggregation as well as loss of proteins known to be critical in human spinocerebellar ataxias that is not observed in neocortex tissues. These results provide a hypothesis accounting for loss of protein integrity and cerebellum function in A-T.

Response of the microbiome-gut-brain axis in Drosophila to amino acid deficit.

A balanced intake of macronutrients-protein, carbohydrate and fat-is essential for the well-being of organisms. An adequate calorific intake but with insufficient protein consumption can lead to several ailments, including kwashiorkor1. Taste receptors (T1R1-T1R3)2 can detect amino acids in the environment, and cellular sensors (Gcn2 and Tor)3 monitor the levels of amino acids in the cell. When deprived of dietary protein, animals select a food source that contains a greater proportion of protein or essential amino acids (EAAs)4. This suggests that food selection is geared towards achieving the target amount of a particular macronutrient with assistance of the EAA-specific hunger-driven response, which is poorly understood. Here we show in Drosophila that a microbiome-gut-brain axis detects a deficit of EAAs and stimulates a compensatory appetite for EAAs. We found that the neuropeptide CNMamide (CNMa)5 was highly induced in enterocytes of the anterior midgut during protein deprivation. Silencing of the CNMa-CNMa receptor axis blocked the EAA-specific hunger-driven response in deprived flies. Furthermore, gnotobiotic flies bearing an EAA-producing symbiotic microbiome exhibited a reduced appetite for EAAs. By contrast, gnotobiotic flies with a mutant microbiome that did not produce leucine or other EAAs showed higher expression of CNMa and a greater compensatory appetite for EAAs. We propose that gut enterocytes sense the levels of diet- and microbiome-derived EAAs and communicate the EAA-deprived condition to the brain through CNMa.

Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice.

Achieving complete electrooxidation of ethanol by single atomic Rh decoration of Pt nanocubes.

SignificanceDirect ethanol fuel cells are attracting growing attention as portable power sources due to their advantages such as higher mass-energy density than hydrogen and less toxicity than methanol. However, it is challenging to achieve the complete electrooxidation to generate 12 electrons per ethanol, resulting in a low fuel utilization efficiency. This manuscript reports the complete ethanol electrooxidation by engineering efficient catalysts via single-atom modification. The combined electrochemical measurements, in situ characterization, and density functional theory calculations unravel synergistic effects of single Rh atoms and Pt nanocubes and identify reaction pathways leading to the selective C-C bond cleavage to oxidize ethanol to CO2. This study provides a unique single-atom approach to tune the activity and selectivity toward complicated electrocatalytic reactions.

Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress.

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

High tumor infiltrating lymphocytes are significantly associated with pathological complete response in triple negative breast cancer treated with neoadjuvant KEYNOTE-522 chemoimmunotherapy.

INTRODUCTION: For patients with locally advanced triple negative breast cancer (TNBC), the standard of care is to administer the KEYNOTE-522 (K522) regimen, including chemotherapy and immunotherapy (pembrolizumab) given in the neoadjuvant setting. Pathological complete response (pCR) is more likely in patients who receive the K522 regimen than in patients who receive standard chemotherapy. Studies have shown that pCR is a strong predictor of long-term disease-free survival. However, factors predicting pCR to K522 are not well understood and require further study in real-world populations. METHODS: We evaluated 76 patients who were treated with the K522 regimen at our institution. Twenty-nine pre-treatment biopsy slides were available for pathology review. Nuclear grade, Nottingham histologic grade, Ki-67, lymphovascular invasion, and tumor infiltrating lymphocytes (TIL) were evaluated in these 29 cases. For the cases that did not have available slides for review from pre-treatment biopsies, these variables were retrieved from available pathology reports. In addition, clinical staging, race, and BMI at the time of biopsy were retrieved from all 76 patients' charts. Binary logistic regression models were used to correlate these variables with pCR. RESULTS: At the current time, 64 of 76 patients have undergone surgery at our institution following completion of K522 and 31 (48.4%) of these achieved pCR. In univariate analysis, only TIL was significantly associated with pCR (p = 0.014) and this finding was also confirmed in multivariate analysis, whereas other variables including age, race, nuclear grade, Nottingham grade, Ki-67, lymphovascular invasion, BMI, pre-treatment tumor size, and lymph node status were not associated with pCR (p > 0.1). CONCLUSION: Our real-world data demonstrates high TIL is significantly associated with pCR rate in the K522 regimen and may potentially serve as a biomarker to select optimal treatment. The pCR rate of 48.4% in our study is lower than that reported in K522, potentially due to the smaller size of our study; however, this may also indicate differences between real-world data and clinical trial results. Larger studies are warranted to further investigate the role of immune cells in TNBC response to K522 and other treatment regimens.

Correlation of HER2 Protein Level With mRNA Level Quantified by RNAscope in Breast Cancer.

Trastuzumab deruxtecan (T-DXd) has been approved by the US Food and Drug Administration (FDA) to treat patients with metastatic HER2-positive and HER2-low breast cancer, and clinical trials are examining its efficacy against early-stage breast cancer. Current HER2 immunohistochemical (IHC) assays are suboptimal in evaluating HER2-low breast cancers and identifying which patients would benefit from T-DXd. HER2 expression in 526 breast cancer tissue microarray (TMA) cores was measured using the FDA-approved PATHWAY and HercepTest IHC assays, and the corresponding RNA levels were evaluated by RNAscope. HER2 protein levels by regression analysis using a quantitative immunofluorescence score against cell line arrays with known HER2 protein levels determined by mass spectrometry were available in 48 of the cores. RNAscope was also performed in 32 metastatic biopsies from 23 patients who were subsequently treated with T-DXd, and the results were correlated with response rate. HER2 RNA levels by RNAscope strongly correlated with HER2 protein levels (P < .0001) and with HER2 IHC H-scores from the PATHWAY and HercepTest assays (P < .0001). However, neither protein levels nor RNA levels significantly differed between cases scored 0, ultralow, and 1+ by PATHWAY and HercepTest. The RNA levels were significantly higher (P = .030) in responders (6.4 ± 8.2 dots/cell, n = 12) than those in nonresponders (2.6 ± 2.2, n = 20) to T-DXd. RNAscope is a simple assay that can be objectively quantified and is a promising alternative to current IHC assays in evaluating HER2 expression in breast cancers, especially HER2-low cases, and may identify patients who would benefit from T-DXd.

The residue of salinomycin in the muscles of olive flounder (Paralichthys olivaceus) and black rockfish (Sebastes Schlegeli) after oral administration analyzed by LC-Tandem-MS.

BACKGROUND: Salinomycin, an antibiotic, have potential as a veterinary drug for fish due to its anti-parasitic activity against several fish parasites. Thus the residual levels of salinomycin in muscles of two significant aquaculture species in Korea, olive flounder and black rockfish, were analyzed using HPLC-MS-MS. RESULTS: The proper method to analyze the residual salinomycin in fish muscles using LC-MS-MS was settled and the method was validated according to CODEX guidelines. The residues in three distinct groups for two fish species were analyzed using the matrix match calibration curves at points of five different times following oral administration. After oral administration, salinomycin rapidly breaks down in both olive flounder and black rockfish. After 7th days, the average residue in all groups of two fish spp. decreased below limit of quantitation (LOQ). CONCLUSION: Due to low residue levels in fish muscles, salinomycin may therefore be a treatment that is safe for both fish and humans. This result could contribute to establishment of MRL (minimal residual limit) for approval of salinomycin for use in aquaculture.

Nbs1 Converts the Human Mre11/Rad50 Nuclease Complex into an Endo/Exonuclease Machine Specific for Protein-DNA Adducts.

The human Mre11/Rad50/Nbs1 (hMRN) complex is critical for the sensing, processing, and signaling of DNA double-strand breaks. The nuclease activity of Mre11 is essential for mammalian development and cell viability, although the regulation and substrate specificity of Mre11 have been difficult to define. Here we show that hMRN catalyzes sequential endonucleolytic and exonucleolytic activities on both 5' and 3' strands of DNA ends containing protein adducts, and that Nbs1, ATP, and adducts are essential for this function. In contrast, Nbs1 inhibits Mre11/Rad50-catalyzed 3'-to-5' exonucleolytic degradation of clean DNA ends. The hMRN endonucleolytic cleavage events are further stimulated by the phosphorylated form of the human C-terminal binding protein-interacting protein (CtIP) DNA repair enzyme, establishing a role for CtIP in regulating hMRN activity. These results illuminate the important role of Nbs1 and CtIP in determining the substrates and consequences of human Mre11/Rad50 nuclease activities on protein-DNA lesions.