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Major risk factors of head and neck squamous cell carcinoma (HNSCC) are tobacco use and human papillomavirus (HPV). HPV E6 oncoprotein leads to p53 degradation, whereas HPV-negative cancers are frequently associated with TP53 mutations. Peposertib is a potent and selective, orally administered small-molecule inhibitor of the catalytic subunit of the DNA-dependent kinase (DNA-PKcs), a key regulator of non-homologous end joining (NHEJ). NHEJ inhibition along with irradiation (IR)-induced DNA double-strand breaks has the potential to increase antitumor treatment efficacy. Here, we investigated the responses of a panel of HNSCC models with distinct HPV and p53 status to treatments with IR, DNA-PKcs inhibition, and their combination in-vitro and in-vivo. IR-induced DNA damage combined with peposertib administration shortly before IR results in decreased cell viability and proliferation and causes DNA repair delay in all studied HNSCC cell lines. However, our data confirm that the actual cell fate upon this treatment is determined by cellular p53 and/or HPV status. Cells lacking functional p53 due to its degradation by HPV or due to a loss-of-function mutation are arrested in the G2/M phase of the cell cycle and eliminated by apoptosis whereas p53-proficient HNSCC cell lines preferentially undergo senescence. This is also recapitulated in-vivo, where HPV+ UD-SCC-2 xenografts display stronger and more durable responses to the combined treatment as compared to p53 wild-type UM-SCC-74A tumors. In conclusion, DNA-PKcs inhibitor peposertib should be further studied as a potential radiosensitizer for HNSCCs, taking into consideration the genetic background and the HPV status of a particular tumor.

Various lines of investigation support a signaling interphase shared by receptor tyrosine kinases and the DNA damage response. However, the underlying network nodes and their contribution to the maintenance of DNA integrity remain unknown. We explored MET-related metabolic pathways in which interruption compromises proper resolution of DNA damage. Discovery metabolomics combined with transcriptomics identified changes in pathways relevant to DNA repair following MET inhibition (METi). METi by tepotinib was associated with the formation of γH2AX foci and with significant alterations in major metabolic circuits such as glycolysis, gluconeogenesis, and purine, pyrimidine, amino acid, and lipid metabolism. 5'-Phosphoribosyl-N-formylglycinamide, a de novo purine synthesis pathway metabolite, was consistently decreased in in vitro and in vivo MET-dependent models, and METi-related depletion of dNTPs was observed. METi instigated the downregulation of critical purine synthesis enzymes including phosphoribosylglycinamide formyltransferase, which catalyzes 5'-phosphoribosyl-N-formylglycinamide synthesis. Genes encoding these enzymes are regulated through E2F1, whose levels decrease upon METi in MET-driven cells and xenografts. Transient E2F1 overexpression prevented dNTP depletion and the concomitant METi-associated DNA damage in MET-driven cells. We conclude that DNA damage following METi results from dNTP reduction via downregulation of E2F1 and a consequent decline of de novo purine synthesis., Significance: Maintenance of genome stability prevents disease and affiliates with growth factor receptor tyrosine kinases. We identified de novo purine synthesis as a pathway in which key enzymatic players are regulated through MET receptor and whose depletion via MET targeting explains MET inhibition-associated formation of DNA double-strand breaks. The mechanistic importance of MET inhibition-dependent E2F1 downregulation for interference with DNA integrity has translational implications for MET-targeting-based treatment of malignancies., (©2024 The Authors; Published by the American Association for Cancer Research.)

The DNA damage response (DDR) is intertwined with signaling pathways downstream of oncogenic receptor tyrosine kinases (RTKs). To drive research into the application of targeted therapies as radiosensitizers, a better understanding of this molecular crosstalk is necessary. We present here the characterization of a previously unreported MET RTK phosphosite, Serine 1016 (S1016) that represents a potential DDR-MET interface. MET S1016 phosphorylation increases in response to irradiation and is mainly targeted by DNA-dependent protein kinase (DNA-PK). Phosphoproteomics unveils an impact of the S1016A substitution on the overall long-term cell cycle regulation following DNA damage. Accordingly, the abrogation of this phosphosite strongly perturbs the phosphorylation of proteins involved in the cell cycle and formation of the mitotic spindle, enabling cells to bypass a G2 arrest upon irradiation and leading to the entry into mitosis despite compromised genome integrity. This results in the formation of abnormal mitotic spindles and a lower proliferation rate. Altogether, the current data uncover a novel signaling mechanism through which the DDR uses a growth factor receptor system for regulating and maintaining genome stability., (© 2023. The Author(s).)

Purpose: Oncogene addiction provides important therapeutic opportunities for precision oncology treatment strategies. To date the cellular circuitries associated with driving oncoproteins, which eventually establish the phenotypic manifestation of oncogene addiction, remain largely unexplored. Data suggest the DNA damage response (DDR) as a central signaling network that intersects with pathways associated with deregulated addicting oncoproteins with kinase activity in cancer cells., Experimental: DESIGN: We employed a targeted mass spectrometry approach to systematically explore alterations in 116 phosphosites related to oncogene signaling and its intersection with the DDR following inhibition of the addicting oncogene alone or in combination with irradiation in MET-, EGFR-, ALK- or BRAF (V600)-positive cancer models. An NSCLC tissue pipeline combining patient-derived xenografts (PDXs) and ex vivo patient organotypic cultures has been established for treatment responsiveness assessment., Results: We identified an 'oncogene addiction phosphorylation signature' (OAPS) consisting of 8 protein phosphorylations (ACLY S455, IF4B S422, IF4G1 S1231, LIMA1 S490, MYCN S62, NCBP1 S22, P3C2A S259 and TERF2 S365) that are significantly suppressed upon targeted oncogene inhibition solely in addicted cell line models and patient tissues. We show that the OAPS is present in patient tissues and the OAPS-derived score strongly correlates with the ex vivo responses to targeted treatments., Conclusions: We propose a score derived from OAPS as a quantitative measure to evaluate oncogene addiction of cancer cell samples. This work underlines the importance of protein phosphorylation assessment for patient stratification in precision oncology and corresponding identification of tumor subtypes sensitive to inhibition of a particular oncogene., (© 2022. The Author(s).)

CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-an early step in NHEJ-yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion., (© 2021 Bosch-Guiteras et al.; Published by Cold Spring Harbor Laboratory Press.)

Increasing evidence suggests that interference with growth factor receptor tyrosine kinase (RTK) signaling can affect DNA damage response (DDR) networks, with a consequent impact on cellular responses to DNA-damaging agents widely used in cancer treatment. In that respect, the MET RTK is deregulated in abundance and/or activity in a variety of human tumors. Using two proteomic techniques, we explored how disrupting MET signaling modulates global cellular phosphorylation response to ionizing radiation (IR). Following an immunoaffinity-based phosphoproteomic discovery survey, we selected candidate phosphorylation sites for extensive characterization by targeted proteomics focusing on phosphorylation sites in both signaling networks. Several substrates of the DDR were confirmed to be modulated by sequential MET inhibition and IR, or MET inhibition alone. Upon combined treatment, for two substrates, NUMA1 S395 and CHEK1 S345, the gain and loss of phosphorylation, respectively, were recapitulated using invivo tumor models by immunohistochemistry, with possible utility in future translational research. Overall, we have corroborated phosphorylation sites at the intersection between MET and the DDR signaling networks, and suggest that these represent a class of proteins at the interface between oncogene-driven proliferation and genomic stability., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Background: To evaluate the effects of a protocol designed to optimize the use of preoperative chest X-rays (CXRs) in the evaluation of patients undergoing anesthesia for elective surgery., Design: Observational prospective study., Setting: General 350 bedded hospital, with main surgical branches., Methods: a), Protocol: routine CXRs can be avoided in patients aged less than 60 years, nonsmokers, without acute-chronic respiratory, cardiovascular symptoms, free from neoplastic diseases, not candidates to major vascular, abdominal or thoracic surgery, not treated with immunosuppressive therapy, nor immigrants from areas of endemic TB. b) All out patient subjects admitted to anesthesiology service for evaluation prior to elective surgery., Results: Out of 5198 patients, 3795 were enrolled in the protocol; in 152 cases, preoperative CXRs were performed, 3456 patients (57.2% ASA 1; 42% ASA 2; 0.8% ASA 3) underwent surgery without CXRs. Thirty-four percent of patients had general anesthesia, 54.5% regional anesthesia, 20.6% regional-peripheral anesthesia with/without MAC. No critical events nor major complications were observed in the perioperative period in these subjects. Preoperative CXRs (performed in 152 cases) yelded useful informations with effect on the clinical management in 20 instances., Conclusions: In a context of adequate preoperative anesthesiologic evaluation, this protocol proved to be effective in reducing the number of routine preoperative CXRs in patients undergoing elective surgery. This resulted in a substantial reduction of radiation exposure both to the subject and to the general population, and costs saving, without evident negative side-effects.

The anaesthetic technique chosen for a laparohysterectomy in a woman affected by Apert's acrocephalosyndactilia is described. Difficulties in performing tracheal intubation were overcome by mean of loco-regional anesthesia (LRA). In order to minimize the anaesthetic risk, a standardised preoperative evaluation and assessment integrating the usual investigations and the possibility of employing intubation techniques as alternative to direct laryngoscopy are suggested.

Immigrants find it hard--although not all in the same way--to adapt to the language, the social structure and the way of life of their host country. Different factors overlap: the foreigners' social and cultural background, their education and their adaptability to a foreign environment, their plans for the future, the political and legal situation in the host country as well as the local population's acceptance of and tolerance and respect for a foreign culture. The fact that 75% of all foreigners in Switzerland possess a residence permit can be considered as rather positive; however, this does not necessarily mean that these people are familiar with the local language and way of life. Isolation and a continuously postponed return to the home country are conditions well known even among resident foreigners. Seasonal workers, on the other hand, feel great uncertainty and concern. In critical life situations, for instance caused by illness, accidents or unemployment, the process of rehabilitation can strongly be influenced by earlier living conditions. The Swiss legislation on foreigners as well as the legislation on social security can often bring about fundamental fateful questions. In order to find efficient solutions to the wide range of possible difficulties, a whole-hearted interdisciplinary cooperation is extremely important.

In this paper the Authors describe the anesthetic techniques used for abdominal surgery in a 19 year-old woman with "central-core disease". This uncommon congenital neuromuscular disorder is frequently related to malignant hyperthermia syndrome. Total intravenous anesthesia with the association propofol-fentanyl was well tolerated by the patient. There was no evidence of malignant hyperthermia during the procedure and cardiovascular stability was excellent.