BACKGROUND: Autophagy is a crucial factor contributing to radioresistance during radiotherapy. Although Lys05 has proven its ability to improve the results of radiotherapy through the inhibition of autophagy, molecular mechanisms of this inhibition remain elusive. We aimed to describe the molecular mechanisms involved in Lys05-induced inhibition of autophagy. MATERIALS AND METHODS: Radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative) and methods of quantitative phosphoproteomics were employed to define the molecular mechanisms involved in Lys05-induced inhibition of autophagy. RESULTS: We confirmed that at an early stage after irradiation, autophagy was induced, whereas at a later stage after irradiation, it was inhibited. The early-stage induction of autophagy was characterized mainly by the activation of biosynthetic and metabolic processes through up- or down-regulation of the critical autophagic regulatory proteins Sequestosome-1 (SQSTM1) and proline-rich AKT1 substrate 1 (AKT1S1). The late-stage inhibition of autophagy was attributed mainly to down-regulation of Unc-51 like autophagy-activating kinase 1 (ULK1) through phosphorylation at Ser638. CONCLUSION: This work contributes to emerging phosphoproteomic insights into autophagy-mediated global signaling in lung cancer cells, which might consequently facilitate the development of precision medicine therapeutics.

• Klíčová slova
• Lys05, Phosphoproteomics, autophagy, inhibitor, lung cancer,
• MeSH
• aminochinoliny farmakologie   MeSH
• autofagie * MeSH
• fosfoproteiny analýza   metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• nádory plic farmakoterapie   metabolismus   patologie   radioterapie   MeSH
• nemalobuněčný karcinom plic farmakoterapie   metabolismus   patologie   radioterapie   MeSH
• polyaminy farmakologie   MeSH
• proteom analýza   metabolismus   MeSH
• radiosenzibilizující látky farmakologie   MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminochinoliny MeSH
• fosfoproteiny MeSH
• Lys01 MeSH Prohlížeč
• polyaminy MeSH
• proteom MeSH
• radiosenzibilizující látky MeSH

Current anti-cancer strategy takes advantage of tumour specific abnormalities in DNA damage response to radio- or chemo-therapy. Inhibition of the ATR/Chk1 pathway has been shown to be synthetically lethal in cells with high levels of oncogene-induced replication stress and in p53- or ATM- deficient cells. In the presented study, we aimed to elucidate molecular mechanisms underlying radiosensitization of T-lymphocyte leukemic MOLT-4 cells by VE-821, a higly potent and specific inhibitor of ATR. We combined multiple approaches: cell biology techniques to reveal the inhibitor-induced phenotypes, and quantitative proteomics, phosphoproteomics, and metabolomics to comprehensively describe drug-induced changes in irradiated cells. VE-821 radiosensitized MOLT-4 cells, and furthermore 10 μM VE-821 significantly affected proliferation of sham-irradiated MOLT-4 cells. We detected 623 differentially regulated phosphorylation sites. We revealed changes not only in DDR-related pathways and kinases, but also in pathways and kinases involved in maintaining cellular metabolism. Notably, we found downregulation of mTOR, the main regulator of cellular metabolism, which was most likely caused by an off-target effect of the inhibitor, and we propose that mTOR inhibition could be one of the factors contributing to the phenotype observed after treating MOLT-4 cells with 10 μM VE-821. In the metabolomic analysis, 206 intermediary metabolites were detected. The data indicated that VE-821 potentiated metabolic disruption induced by irradiation and affected the response to irradiation-induced oxidative stress. Upon irradiation, recovery of damaged deoxynucleotides might be affected by VE-821, hampering DNA repair by their deficiency. Taken together, this is the first study describing a complex scenario of cellular events that might be ATR-dependent or triggered by ATR inhibition in irradiated MOLT-4 cells. Data are available via ProteomeXchange with identifier PXD008925.

• MeSH
• aminokyselinové motivy MeSH
• ATM protein antagonisté a inhibitory   MeSH
• biologické markery MeSH
• fosfoproteiny * chemie   metabolismus   MeSH
• fosforylace MeSH
• genová ontologie MeSH
• inhibitory proteinkinas farmakologie   MeSH
• kontrolní body buněčného cyklu účinky léků   účinky záření   MeSH
• lidé MeSH
• metabolom * MeSH
• metabolomika metody   MeSH
• nádorové buněčné linie MeSH
• proteom * MeSH
• proteomika metody   MeSH
• pyraziny farmakologie   MeSH
• radiosenzibilizující látky farmakologie   MeSH
• signální transdukce MeSH
• sulfony farmakologie   MeSH
• tolerance záření účinky léků   MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• výpočetní biologie metody   MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide MeSH Prohlížeč
• ATM protein MeSH
• ATR protein, human MeSH Prohlížeč
• biologické markery MeSH
• fosfoproteiny * MeSH
• inhibitory proteinkinas MeSH
• MTOR protein, human MeSH Prohlížeč
• proteom * MeSH
• pyraziny MeSH
• radiosenzibilizující látky MeSH
• sulfony MeSH
• TOR serin-threoninkinasy MeSH

Cancer treatments such as radiotherapy and most of the chemotherapies act by damaging DNA of cancer cells. Upon DNA damage, cells stop proliferation at cell cycle checkpoints, which provides them time for DNA repair. Inhibiting the checkpoint allows entry to mitosis despite the presence of DNA damage and can lead to cell death. Importantly, as cancer cells exhibit increased levels of endogenous DNA damage due to an excessive replication stress, inhibiting the checkpoint kinases alone could act as a directed anti-cancer therapy. Here, we review the current status of inhibitors targeted towards the checkpoint effectors and discuss mechanisms of their actions in killing of cancer cells.

• Klíčová slova
• ATM, ATR, Chk1, DNA damage response, Wee1, cancer, checkpoint, inhibitor, p53, replication stress,
• MeSH
• buněčná smrt účinky léků   MeSH
• cílená molekulární terapie metody   MeSH
• inhibitory proteinkinas farmakologie   terapeutické užití   MeSH
• kontrolní body buněčného cyklu účinky léků   MeSH
• lidé MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• nádory farmakoterapie   enzymologie   metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• inhibitory proteinkinas MeSH
• nádorový supresorový protein p53 MeSH