Regulation von Apoptose, Autophagie und DNA-Integrität in Leukämiezellen durch replikativen Stress und pharmakologische Hemmung der Histondeacetylasen

Die chronische myeloische Leukämie (CML) ist eine Krebserkrankung, die durch das onkogene BCR/ABL Fusionsprotein charakterisiert ist. Die zielgerichtete Therapie mit Tyrosinkinase-Inhibitoren ermöglicht in bis zu 80% der Fälle eine normale Lebenserwartung. Allerdings bleiben leukämische Stammzellen meist erhalten und können zu einer Neuerkrankung führen. Derzeit werden Histondeacetylase-Inhibitoren (HDACi) entwickelt, die als vielversprechende Krebsmedikamente in der Therapie von hämatologischen Erkrankungen eingesetzt werden. Dabei sind Kombinationstherapien mit Chemotherapeutika im Fokus, da sie effizienter als Monotherapien sind. Hydroxyurea wird aktuell als stabiles Medikament gegen Sichelzellanämie sowie gegen chronische myeloproliferative Erkrankungen eingesetzt. Aufgrund der Funktion als Ribonukleotidreduktase-Inhibitor wird Hydroxyurea in Laboren oft dazu verwendet, gezielt replikativen Stress durch Depletion von dNTPs zu induzieren. Replikativer Stress setzt in den Zellen eine komplexe Zellantwort in Gang. Dabei spielen Prozesse wie die DNA-Schadensantwort, Apoptose und Autophagie eine wichtige Rolle. In dieser Arbeit wurde untersucht, ob der Klasse I HDACi MS 275 CML Zellen gegenüber Hydroxyurea sensitiviert. Die Daten zeigen, dass MS 275 die Expression des CDKi p21 und einen G1 Arrest der Zellen auslöst. Hydroxyurea hingegen arretiert die Zellen in der S Phase und fördert die Autophagie. Die kombinierte Gabe von MS 275 und Hydroxyurea führt zu einer signifikanten und synergistischen Zunahme der Apoptose, sowie zu einer Caspase abhängigen Proteolyse von Autophagieregulatoren. Als kritischen Faktor für eine von dem Tumorsuppressor p53-unabhängige Apoptoseinduktion wurde das pro apoptotische Protein NOXA ermittelt. Experimente mit einer genetischen Eliminierung von NOXA sowie Versuche mit NOXA-defizienten MEFs belegen eine unabdingbare Rolle von NOXA in der Aktivierung der Apoptosekaskade nach Kombinationsbehandlung in CML Zellen. Als weitere wichtige Faktoren des Apoptosewegs wurden c-ABL und p73 als vorgeschaltete Regulatoren von NOXA ausfindig gemacht. Die Untersuchung der DNA Schadensantwort ergab, dass MS 275 die von Hydroxyurea induzierte Aktivierung von Checkpoint-Kinasen, sowie insbesondere die Mengen an DNA Strangbrüchen deutlich erhöht. Die pharmakologische Hemmung von ATR und CHK1 hat eine zusätzliche Zunahme an DNA Schäden in MS-275/Hydroxyurea behandelten CML Zellen zur Folge. Die Inaktivierung der ATR-CHK1 Achse führt zu einer signifikanten Reduktion der durch MS-275/Hydroxyurea induzierten Apoptose. Diese Daten legen eine bisher unbekannte pro-apoptotische Rolle von ATR und CHK1 durch eine transkriptionelle Regulation von NOXA offen. Zudem wird dargelegt, dass Klasse I HDACs unerlässlich für die DNA-Reparatur, die Hemmung der Apoptose sowie für den Erhalt der Autophagie in CML Zellen nach replikativem Stress sind. Der aufgedeckte Mechanismus kann als generelles Konzept für die Wirkungsweise von Hydroxyurea und HDACi, insbesondere im Kontext der CML verstanden werden. Da die Behandlungsstrategie mit Hydroxyurea und HDACi vielversprechende Daten liefert, ermutigt dies zu Analysen der Kombinationstherapie in klinischen Studien.Chronic myeloid leukemia (CML) is a hematological disorder characterized by the presence of the oncogenic fusion protein BCR/ABL. The targeted therapy with tyrosine kinase inhibitors allows in up to 80% of all CML cases an ordinary life expectancy. However, remaining leukemic stem cells can lead to new cases. At present, histone deacetylase inhibitors (HDACi) are newly developed and used as promising cancer drugs in therapy of leukemia. Combination therapies of chemotherapeutics are gaining in importance more and more, as they show higher efficacies than monotherapies. Hydroxyurea is currently the stable drug for the treatment of sickle cell anemia and chronic myeloproliferative disorders. As hydroxyurea acts as a ribonucleotide reductase inhibitor, it is commonly used in laboratories for generating replicative stress through depletion of the dNTP pool. Replicative Stress induces a complex cellular program involving DNA damage response, apoptosis and autophagy. In this thesis, I analysed if the class I HDACi MS-275 sensitizes CML cells towards hydroxyurea. Here I show that MS-275 induces the expression of the CDKi p21 and stalls the cells in G1 phase. Whereas hydroxyurea arrests the cells in S phase and triggers autophagy. The additional application of MS-275 leads to a significant and synergistic increase in apoptosis as well as to a caspase-dependent cleavage of autophagic regulators. As critical factor in inducing p53-independent apoptosis the pro-apoptotic protein NOXA was determined. Genetic elimination of NOXA as well as experiments with NOXA deficient MEFs confirmed the important role of this protein in activation of the apoptotic cascade upon MS-275/Hydroxyurea treatment in CML cells. As further critical factors in the apoptotic pathway c-ABL and p73 were found as upstream regulators of NOXA. The investigation of the DNA damage response revealed that the class I HDACi MS 275 enhances the hydroxyurea-induced activation of checkpoint kinases as well as the amount of DNA strand breaks, notably. The pharmacological inhibition of ATR and CHK1 results in a distinct accumulation of DNA damage in MS-275/Hydroxyurea-treated CML cells. The inactivation of the ATR-CHK1 axis evokes a significant reduction of the MS 275/Hydroxyurea induced apoptosis. My data reveal a previously unidentified pro-apoptotic role of ATR and CHK1 through a transcriptional control of NOXA. Furthermore, I demonstrate that class I HDACs are necessary to initiate DNA repair, to prevent apoptosis and to sustain autophagy during replicative stress in CML cells. The disclosed mechanism is to be taken as a concept for the efficacy of hydroxyurea and HDACi especially in the context of CML. As the therapeutic strategy of hydroxyurea and HDACi show promising results, this encourages one to introduce the combinatorial therapy in clinical trials

Histone deacetylase inhibitors dysregulate DNA repair proteins and antagonize metastasis-associated processes

Purpose!#!We set out to determine whether clinically tested epigenetic drugs against class I histone deacetylases (HDACs) affect hallmarks of the metastatic process.!##!Methods!#!We treated permanent and primary renal, lung, and breast cancer cells with the class I histone deacetylase inhibitors (HDACi) entinostat (MS-275) and valproic acid (VPA), the replicative stress inducer hydroxyurea (HU), the DNA-damaging agent cis-platinum (L-OHP), and the cytokine transforming growth factor-β (TGFβ). We used proteomics, quantitative PCR, immunoblot, single cell DNA damage assays, and flow cytometry to analyze cell fate after drug exposure.!##!Results!#!We show that HDACi interfere with DNA repair protein expression and trigger DNA damage and apoptosis alone and in combination with established chemotherapeutics. Furthermore, HDACi disrupt the balance of cell adhesion protein expression and abrogate TGFβ-induced cellular plasticity of transformed cells.!##!Conclusion!#!HDACi suppress the epithelial-mesenchymal transition (EMT) and compromise the DNA integrity of cancer cells. These data encourage further testing of HDACi against tumor cells

Oncogenic Kinase Cascades Induce Molecular Mechanisms That Protect Leukemic Cell Models from Lethal Effects of De Novo dNTP Synthesis Inhibition

The ribonucleotide reductase inhibitor hydroxyurea suppresses de novo dNTP synthesis and attenuates the hyperproliferation of leukemic blasts. Mechanisms that determine whether cells undergo apoptosis in response to hydroxyurea are ill-defined. We used unbiased proteomics to uncover which pathways control the transition of the hydroxyurea-induced replication stress into an apoptotic program in chronic and acute myeloid leukemia cells. We noted a decrease in the serine/threonine kinase RAF1/c-RAF in cells that undergo apoptosis in response to clinically relevant doses of hydroxyurea. Using the RAF inhibitor LY3009120, we show that RAF activity determines the sensitivity of leukemic cells toward hydroxyurea. We further disclose that pharmacological inhibition of the RAF downstream target BCL-XL with the drug navitoclax and RNAi combine favorably with hydroxyurea against leukemic cells. BCR-ABL1 and hyperactive FLT3 are tyrosine kinases that causally contribute to the development of leukemia and induce RAF1 and BCL-XL. Accordingly, the ABL inhibitor imatinib and the FLT3 inhibitor quizartinib sensitize leukemic cells to pro-apoptotic effects of hydroxyurea. Moreover, hydroxyurea and navitoclax kill leukemic cells with mutant FLT3 that are resistant to quizartinib. These data reveal cellular susceptibility factors toward hydroxyurea and how they can be exploited to eliminate difficult-to-treat leukemic cells with clinically relevant drug combinations.Pharmaceutical Sciences, Faculty ofNon UBCReviewedFacultyResearche

HSP90 is necessary for the ACK1-dependent phosphorylation of STAT1 and STAT3

Signal transducers and activators of transcription (STATs) are latent, cytoplasmic transcription factors. Janus kinases (JAKs) and activated CDC42-associated kinase-1 (ACK1/TNK2) catalyse the phosphorylation of STAT1 and the expression of its target genes. Here we demonstrate that catalytically active ACK1 promotes the phosphorylation and nuclear accumulation of STAT1 in transformed kidney cells. These processes are associated with STAT1-dependent gene expression and an interaction between endogenous STAT? and ACK1. Moreover, the E3 ubiquitin ligase seven-in-absentia homolog-2 (SIAH2), which targets ACK1 through valine-909 for proteasomal degradation, attenuates the ACK1-STAT1 signalling node. We further show that ACK1 promotes the phosphorylation and nuclear accumulation of STAT3 in cultured cells and that the levels of ACK1 correlate positively with the levels of tyrosine phosphorylated STAT3 in primary lung adenocarcinoma (ADC) cells. Global analysis of ACK1 interaction partners validated the interaction of ACK1 with heat shock protein 90 (HSP90 alpha/beta). Inhibition of this chaperone with the novel drug Onalespib (AT13387) demonstrates that HSP90 is an upstream regulator of the ACK1-dependent phosphorylation of STAT? and STAT3. In addition to these molecular insights, our data offer a pharmacological strategy to control the ACK1-STAT signalling axis

Analysis of the interplay between all-trans retinoic acid and histone deacetylase inhibitors in leukemic cells

The treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) induces granulocytic differentiation. This process renders APL cells resistant to cytotoxic chemotherapies. Epigenetic regulators of the histone deacetylases (HDACs) family, which comprise four classes (I-IV), critically control the development and progression of APL. We set out to clarify the parameters that determine the interaction between ATRA and histone deacetylase inhibitors (HDACi). Our assays included drugs against class I HDACs (MS-275, VPA, and FK228), panHDACi (LBH589, SAHA), and the novel HDAC6-selective compound Marbostat-100. We demonstrate that ATRA protects APL cells from cytotoxic effects of SAHA, MS-275, and Marbostat-100. However, LBH589 and FK228, which have a superior substrate-inhibitor dissociation constant (Ki) for the class I deacetylases HDAC1, 2, 3, are resistant against ATRA-dependent cytoprotective effects. We further show that HDACi evoke DNA damage, measured as induction of phosphorylated histone H2AX and by the comet assay. The ability of ATRA to protect APL cells from the induction of p-H2AX by HDACi is a readout for the cytoprotective effects of ATRA. Moreover, ATRA increases the fraction of cells in the G1 phase, together with an accumulation of the cyclin-dependent kinase inhibitor p21 and a reduced expression of thymidylate synthase (TdS). In contrast, the ATRA-dependent activation of the transcription factors STAT1, NF-kappa B, and C/EBP hardly influences the responses of APL cells to HDACi. We conclude that the affinity of HDACi for class I HDACs determines whether such drugs can kill naive and maturated APL cells

Class I histone deacetylases regulate p53/NF-κB crosstalk in cancer cells

The transcription factors NF-kappa B and p53 as well as their crosstalk determine the fate of tumor cells upon therapeutic interventions. Replicative stress and cytolcines promote signaling cascades that lead to the co-regulation of p53 and NF-kappa B. Consequently, nuclear p53/NF-kappa B signaling complexes activate NF-kappa B-dependent survival genes. The 18 histone deacetylases (HDACs) are epigenetic modulators that fall into four classes (I-IV). Inhibitors of histone deacetylases (HDACi) become increasingly appreciated as anti-cancer agents. Based on their effects on p53 and NF-kappa B, we addressed whether clinically relevant HDACi affect the NF-kappa B/p53 crosstalk. The chemotherapeutics hydroxyurea, etoposide, and fludarabine halt cell cycle progression, induce DNA damage, and lead to DNA fragmentation. These agents co-induce p53 and NF-kappa B-dependent gene expression in cell lines from breast and colon cancer and in primary chronic lymphatic leukemia (CLL) cells. Using specific HDACi, we find that the class I subgroup of HDACs, but not the class lib deacetylase HDAC6, are required for the hydroxyurea-induced crosstalk between p53 and NF-kappa B. HDACi decrease the basal and stress-induced expression of p53 and block NF-kappa B-regulated gene expression. We further show that class I HDACi induce senescence in pancreatic cancer cells with mutant p53. (C) 2016 Elsevier Inc. All rights reserved