FOXO transcription factors are critical regulators of cell homeostasis and steer cell death, differentiation and longevity in mammalian cells. By combined pharmacophore-modeling-based in silico and fluorescence polarization-based screening we identified small molecules that physically interact with the DNA-binding domain (DBD) of FOXO3 and modulate the FOXO3 transcriptional program in human cells. The mode of interaction between compounds and the FOXO3-DBD was assessed via NMR spectroscopy and docking studies. We demonstrate that compounds S9 and its oxalate salt S9OX interfere with FOXO3 target promoter binding, gene transcription and modulate the physiologic program activated by FOXO3 in cancer cells. These small molecules prove the druggability of the FOXO-DBD and provide a structural basis for modulating these important homeostasis regulators in normal and malignant cells.

• Klíčová slova
• FOXO transcription factors, biochemistry, cancer biology, chemical biology, docking, drug targeting, human, molecular biophysics, pharmacophore modelling, small compounds, structural biology,
• MeSH
• DNA chemie   genetika   metabolismus   MeSH
• genetická transkripce účinky léků   MeSH
• genový knockdown MeSH
• HEK293 buňky MeSH
• knihovny malých molekul chemie   metabolismus   farmakologie   MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární modely MeSH
• nádorové buněčné linie MeSH
• promotorové oblasti (genetika) genetika   MeSH
• protein FOXO3 chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• simulace molekulového dockingu MeSH
• stanovení celkové genové exprese metody   MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• FOXO3 protein, human MeSH Prohlížeč
• knihovny malých molekul MeSH
• protein FOXO3 MeSH

The patients with mantle cell lymphoma (MCL) have translocation t(11;14) associated with cyclin D1 overexpression. We observed that iron (an essential cofactor of dioxygenases including prolyl hydroxylases [PHDs]) depletion by deferoxamine blocked MCL cells' proliferation, increased expression of DNA damage marker γH2AX, induced cell cycle arrest and decreased cyclin D1 level. Treatment of MCL cell lines with dimethyloxalylglycine, which blocks dioxygenases involving PHDs by competing with their substrate 2-oxoglutarate, leads to their decreased proliferation and the decrease of cyclin D1 level. We then postulated that loss of EGLN2/PHD1 in MCL cells may lead to down-regulation of cyclin D1 by blocking the degradation of FOXO3A, a cyclin D1 suppressor. However, the CRISPR/Cas9-based loss-of-function of EGLN2/PHD1 did not affect cyclin D1 expression and the loss of FOXO3A did not restore cyclin D1 levels after iron chelation. These data suggest that expression of cyclin D1 in MCL is not controlled by ENGL2/PHD1-FOXO3A pathway and that chelation- and 2-oxoglutarate competition-mediated down-regulation of cyclin D1 in MCL cells is driven by yet unknown mechanism involving iron- and 2-oxoglutarate-dependent dioxygenases other than PHD1. These data support further exploration of the use of iron chelation and 2-oxoglutarate-dependent dioxygenase inhibitors as a novel therapy of MCL.

• Klíčová slova
• 2-oxoglutarate-dependent enzymes, DNA damage, cell cycle, iron, mantle cell lymphoma, prolyl hydroxylases (EGLN/PHDs),
• MeSH
• aminokyseliny dikarboxylové farmakologie   MeSH
• chelátory železa farmakologie   MeSH
• cyklin D1 metabolismus   MeSH
• deferoxamin farmakologie   MeSH
• deficit železa MeSH
• dioxygenasy antagonisté a inhibitory   metabolismus   MeSH
• down regulace účinky léků   MeSH
• hydroxylace MeSH
• hypoxie buňky účinky léků   MeSH
• inhibitory enzymů farmakologie   MeSH
• kyseliny ketoglutarové farmakologie   MeSH
• lidé MeSH
• lymfom z plášťových buněk enzymologie   MeSH
• messenger RNA genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• poškození DNA MeSH
• prolyl-4-hydroxylasy HIF metabolismus   MeSH
• protein FOXO3 genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny dikarboxylové MeSH
• chelátory železa MeSH
• cyklin D1 MeSH
• deferoxamin MeSH
• dioxygenasy MeSH
• EGLN2 protein, human MeSH Prohlížeč
• FOXO3 protein, human MeSH Prohlížeč
• inhibitory enzymů MeSH
• kyseliny ketoglutarové MeSH
• messenger RNA MeSH
• oxalylglycine MeSH Prohlížeč
• prolyl-4-hydroxylasy HIF MeSH
• protein FOXO3 MeSH

FOXO transcription factors regulate cellular homeostasis, longevity and response to stress. FOXO1 (also known as FKHR) is a key regulator of hepatic glucose production and lipid metabolism, and its specific inhibition may have beneficial effects on diabetic hyperglycemia by reducing hepatic glucose production. Moreover, all FOXO proteins are considered potential drug targets for drug resistance prevention in cancer therapy. However, the development of specific FOXO inhibitors requires a detailed understanding of structural differences between individual FOXO DNA-binding domains. The high-resolution structure of the DNA-binding domain of FOXO1 reported in this study and its comparison with structures of other FOXO proteins revealed differences in both their conformation and flexibility. These differences are encoded by variations in protein sequences and account for the distinct functions of FOXO proteins. In particular, the positions of the helices H1, H2 and H3, whose interface form the hydrophobic core of the Forkhead domain, and the interactions between hydrophobic residues located on the interface between the N-terminal segment, the H2-H3 loop, and the recognition helix H3 differ among apo FOXO1, FOXO3 and FOXO4 proteins. Therefore, the availability of apo structures of DNA-binding domains of all three major FOXO proteins will support the development of FOXO-type-specific inhibitors.

• Klíčová slova
• DNA-binding domain, FOXO1, Forkhead domain, nuclear magnetic resonance, structure,
• MeSH
• forkhead box protein O1 chemie   genetika   metabolismus   MeSH
• forkhead transkripční faktory chemie   genetika   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární modely MeSH
• myši MeSH
• protein FOXO3 chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• sekundární struktura proteinů MeSH
• sekvenční analýza proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• forkhead box protein O1 MeSH
• forkhead transkripční faktory MeSH
• protein FOXO3 MeSH

• MeSH
• chromozomální delece * MeSH
• chronická lymfatická leukemie * krev   genetika   mortalita   MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• lidské chromozomy, pár 6 genetika   MeSH
• míra přežití MeSH
• nádorové proteiny * biosyntéza   genetika   MeSH
• přežití bez známek nemoci MeSH
• protein FOXO3 * biosyntéza   genetika   MeSH
• regulace genové exprese u leukemie * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• dopisy MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• FOXO3 protein, human MeSH Prohlížeč
• nádorové proteiny * MeSH
• protein FOXO3 * MeSH

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in virulence of Bordetella pertussis. CyaA penetrates myeloid cells expressing the complement receptor 3 (αM β2 integrin CD11b/CD18) and subverts bactericidal capacities of neutrophils and macrophages by catalysing unregulated conversion of cytosolic ATP to the key signalling molecule adenosine 3',5'-cyclic monophosphate (cAMP). We show that the signalling of CyaA-produced cAMP hijacks, by an as yet unknown mechanism, the activity of the tyrosine phosphatase SHP-1 and activates the pro-apoptotic BimEL-Bax cascade. Mitochondrial hyperpolarization occurred in human THP-1 macrophages within 10 min of exposure to low CyaA concentrations (e.g. 20 ng ml(-1) ) and was accompanied by accumulation of BimEL and association of the pro-apoptotic factor Bax with mitochondria. BimEL accumulation required cAMP/protein kinase A signalling, depended on SHP-1 activity and was selectively inhibited upon small interfering RNA knockdown of SHP-1 but not of the SHP-2 phosphatase. Moreover, signalling of CyaA-produced cAMP inhibited the AKT/protein kinase B pro-survival cascade, enhancing activity of the FoxO3a transcription factor and inducing Bim transcription. Synergy of FoxO3a activation with SHP-1 hijacking thus enables the toxin to rapidly trigger a persistent accumulation of BimEL, thereby activating the pro-apoptotic programme of macrophages and subverting the innate immunity of the host.

• Klíčová slova
• bordetella, cell injury/sublethal injury, microbial-cell interaction, toxins, virulence,
• MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• AMP cyklický metabolismus   MeSH
• apoptóza fyziologie   MeSH
• Bordetella pertussis metabolismus   patogenita   MeSH
• fagocyty metabolismus   mikrobiologie   MeSH
• forkhead transkripční faktory genetika   metabolismus   MeSH
• interakce hostitele a patogenu fyziologie   MeSH
• lidé MeSH
• makrofágy metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• protein BCL2L11 MeSH
• protein FOXO3 MeSH
• protein X asociovaný s bcl-2 metabolismus   MeSH
• proteiny regulující apoptózu metabolismus   MeSH
• protoonkogenní proteiny metabolismus   MeSH
• signální transdukce MeSH
• tyrosinfosfatasa nereceptorového typu 6 metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• AMP cyklický MeSH
• BAX protein, human MeSH Prohlížeč
• forkhead transkripční faktory MeSH
• FOXO3 protein, human MeSH Prohlížeč
• membránové proteiny MeSH
• protein BCL2L11 MeSH
• protein FOXO3 MeSH
• protein X asociovaný s bcl-2 MeSH
• proteiny regulující apoptózu MeSH
• protoonkogenní proteiny MeSH
• PTPN6 protein, human MeSH Prohlížeč
• tyrosinfosfatasa nereceptorového typu 6 MeSH

Acute leukemia is considered to be a two- or multiple-step process. Although there is a considerable knowledge regarding the character of the "first hit," the nature of the "second hit" remains unanswered in most of the cases including leukemias with MLL gene rearrangement. We demonstrate here a striking sequence of events, which include a covert, protracted preleukemic phase characterized by a dominant MLL/FOXO3A clone with intact myeloid differentiation and the subsequent acquisition of a secondary genetic abnormality, leading to overt lymphoblastic leukemia. Backtracking of the secondary acute lymphoblastic leukemia (sALL) with the MLL rearrangement showed no blasts in the bone marrow (BM) during the protracted preleukemic phase. However, at the same time (more than 1 year before the sALL diagnosis) the MLL/FOXO3A was present in up to 90% of BM cells including myeloid lineage, suggesting that the fusion arose in a multipotent progenitor. To identify potential "second hit" precipitating sALL we compared DNA in preleukemic versus fully leukemic samples. The analysis revealed a 10 Mb gain on 19q13.32 in the sALL, absent in the preleukemic specimen. These data provide insight into the dynamics of leukemogenesis in secondary leukemia with MLL rearrangement.

• MeSH
• akutní promyelocytární leukemie genetika   metabolismus   patologie   terapie   MeSH
• cytogenetické vyšetření MeSH
• forkhead transkripční faktory genetika   metabolismus   MeSH
• fúze genů * MeSH
• genová přestavba MeSH
• histonlysin-N-methyltransferasa MeSH
• jednonukleotidový polymorfismus MeSH
• lidé MeSH
• lidské chromozomy, pár 19 genetika   MeSH
• mladiství MeSH
• myeloidní buňky metabolismus   MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• nádorové kmenové buňky metabolismus   patologie   MeSH
• pre-B-buněčná leukemie genetika   metabolismus   patologie   terapie   MeSH
• preleukemie genetika   MeSH
• protein FOXO3 MeSH
• protoonkogenní protein MLL genetika   metabolismus   MeSH
• regulace genové exprese u leukemie MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• forkhead transkripční faktory MeSH
• FOXO3 protein, human MeSH Prohlížeč
• histonlysin-N-methyltransferasa MeSH
• KMT2A protein, human MeSH Prohlížeč
• nádorové biomarkery MeSH
• protein FOXO3 MeSH
• protoonkogenní protein MLL MeSH