Authors:	Switzeny, Olivier
Title:	Role of epigenetic biomarkers and IDH1 mutation in anticancer drug resistance of high‐grade gliomas
Online publication date:	15-Dec-2017
Year of first publication:	2017
Language:	english
Abstract:	Both genetic and epigenetic changes contribute to the development of human cancer. Malignant brain tumors (WHO grade III - IV) account for about 60 % of all gliomas. Me-dian survival is 12-20 months from the time of diagnosis. Patients undergo surgical re-section and concomitant treatment with radiotherapy plus temozolomide (TMZ). Two molecular markers have been found to increase the progression free survival (PFS) and the overall survival (OS) of patients. The first marker is the promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT), a well-established predictive marker. The current state of the art for determining the MGMT promoter methylation status is methylation specific PCR (MSP), which has limits in specificity and sensitivity. We developed a high-resolution melt (HRM) analysis assay and compared it with MSP and pyrosequencing regarding its predictive value. The second molecular marker is a somatic mutation in the isocitrate dehydrogenase 1 (IDH1) gene, an important prognos-tic marker. It is a gain of function mutation that produces the oncometabolite 2-hydroxyglutarate (2-HG) by NADPH-dependent reduction of alpha-ketoglutarate (α-KG). 2-HG is a competitive inhibitor of α-KG dependent enzymes (ten-eleven translocation methylcytosine dioxygenases (TETs), histone lysine demethylases (KDMs), AlkB homologs (ALKBHs), e.g.). This is reflected in a general DNA and histone hypermethylation in mu-tated IDH1 tumor cells. The impairment of the DNA repair enzymes belonging to the ALKBH family has not been studied so far and could open the door to new tailor-made therapies, by exploiting this altered DNA repair pathway. To determine whether the IDH1 mutation can be exploited for targeted therapy, a cell line based approach was chosen. Artesunate (ART) causes lipid peroxidation and generates intracellular ROS that gives rise to the formation of 1, N6-ethenoadenine which is a substrate for ALKBH2. We tested whether the impairment of ALKBH2 in IDH1 mutated cells leads to an increased sensitivity to ART. Further, ALKBH2 knock-out cells were generated to test if the cytotox-icity of ART is depended on the ALKBH2 functionality. The MGMT promoter of 14 glio-blastoma cell lines with known MGMT activity and 83 formalin-fixed samples from high-grade glioma patients treated with radiation and TMZ was analyzed by HRM, MSP, and pyrosequencing. The data were compared as to PFS and OS of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level rele-vant for PFS and OS was determined. In a multivariate Cox regression model, methyla-tion of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan–Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when HRM was used instead of MSP. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommend-ed being used routinely for determination of the MGMT status of gliomas. To determine whether the IDH1 mutation can be exploited for targeted therapy, a doxycycline induci-ble TET-on system in LN319 glioma cells was chosen. Upon doxycycline treatment, either IDH1 mt or IDH1 wt were overexpressed. Cells expressing the mutated IDH1 enzyme were more sensitive to ART compared to cells expressing the wild-type IDH1 enzyme, which was reflected in higher apoptosis levels. To test if the oncometabolite 2-HG is the key metabolite responsible for the observed phenotype in IDH1 mutated cells, LN18 and T98G glioma cell lines were exposed to exogenous 2-HG together with increasing doses of ART. 2-HG was able to sensitize both cell lines dose dependently towards ART treat-ment. To confirm that ALKBH2 is involved in the counteraction and repair of ART in-duced cytotoxicity and DNA damage, CRISPR/Cas9 ALKBH2 knock-out clones of LN18 and T98G glioma cell lines were generated, and their sensitivity to ART was compared to their parental counterparts. ALKBH2 knock-out cells showed increased levels of apopto-sis compared to the wild-type counterparts. The increased sensitivity of IDH1 mt cells to artesunate could be rescued by exogenous α-KG. IDH1 mutated cells and cells with de-fective ALKBH2 were both characterized by an increased DNA damage response and increased S/G2 cell cycle fraction compared to the appropriate controls when treated with ART. This work has shown that ALKBH2 plays a major role in the DNA repair of DNA damage induced by artesunate. Strong evidence has been found to claim that the IDH1 mutation leads to an impairment the functionality of ALKBH2 by the production of 2-HG. This metabolic attenuation of ALKBH2 in cancer cells can be exploited to increase apop-tosis by drugs, which induce DNA lesions being substrates of ALKBH2. Für die Entwicklung hochgradig maligner Gliome sind keine exogenen Risikofaktoren bekannt, je-doch treten sie am häufigsten im fortgeschrittenen Erwachsenenalter auf. Die Transformation von Gliazellen zu stammzellähnlichen malignen Gliomzellen ist eng an genetische und epigenetische Veränderungen der DNA geknüpft. Insbesondere sind DNA-Reparaturenzyme, die am Erhalt der genomischen und epigenetischen Integrität beteiligt sind, in malignen Gliomen dereguliert. Zwei Biomarker beeinflussen wesentlich das Überleben von Gliompatienten in positiver Weise. Diese sind auf epigenetischer Ebene die Promoter-Hypermethylierung der O6-Methylguanin-DNA-Methyltransferase (MGMT) und, auf genetischer Ebene, somatische Mutationen der Isocitrat-Dehydrogenase 1 (IDH1). Tumorbiopsien von Patienten werden routinemäßig auf diese zwei Mar-ker untersucht und klassifiziert. Gängigen Methoden zur Quantifizierung der Promotermethylie-rung mangelt es an Spezifität und Sensitivität sowie eines klaren Schwellenwertes, ab wann es zu einer Stilllegung des MGMT-Gens kommt. In dieser Arbeit wurde die High-resolution melt (HRM) Analyse als alternative Methode zur methylierungsspezifischen PCR (MSP) zum Zweck der Quanti-fizierung der Promotormethylierung von 83 hochgradigen Gliomen unter Verwendung paraffinier-ten Tumormaterials angewendet. Die Prädiktion der Überlebenszeit sowie das progressionsfreie Intervall wurde mit den Ergebnissen von zwei anderen Methoden verglichen: MSP und Pyrose-quenzierung (PSQ). Die HRM Methode erwies sich als günstige, schnelle und, im Vergleich zu PSQ, gleichwertige Methode zur Quantifizierung der MGMT- Promotormethylierung im Tumorgewebe. Ein Methylierungs-Schwellenwert, ab dem es zu einem verbesserten Ansprechen auf die applizier-te Chemotherapie kommt, konnte definiert werden. Die HRM Methode ermöglicht es, Therapie-resistente von Therapie-sensitiven Tumorentitäten zu unterscheiden sowie unwirksame Therapie-ansätze im Vorhinein auszuschließen und damit die Lebensqualität von Gliompatienten zu verbes-sern. Die Ursachen für das mehr als dreifach längere Überleben von Patienten, die an einem IDH1 mutierten Gliom leiden, sind noch nicht hinlänglich bekannt. IDH1 - Mutationen sind meist „gain-of function“ Mutationen, die zur Produktion von 2-Hydroxyglutarsäure (2-HG) statt α-Ketoglutarsäure (α-KG) führen. 2-HG inhibiert α-KG abhängige Enzyme, zu welchen u.a. auch das AlkB-homologe Protein ALKBH2 gehört. ALKBH2 ist ein DNA-Reparaturprotein, das über oxidative Demethylierung DNA-Schäden repariert. Die reparierten Läsionen sind u.a. Etheno-DNA-Basenaddukte, die muta-gen sind und die DNA-Replikation behindern. Artesunate ist ein TCM-Präparat, das in der Malaria-therapie verwendet wird. Es induziert über die Lipidperoxidation Etheno-DNA-Basenaddukte, die ein Substrat für ALKBH2 darstellen. In dieser Arbeit konnte gezeigt werden, dass verstärkte Apoptose nach Applikation von Artesunate in IDH1 mutierten Zellen, in ALKBH2 knock-out Gliom-zellen sowie in 2-HG vorbehandelten Zellen stattfindet. Die Sensibilisierung gegenüber Artesunate ist 2-HG abhängig. Diese Arbeiten haben Grundlagen für eine verbesserte Therapie von IDH-1 mu-tierten Gliomen geschaffen.
DDC:	570 Biowissenschaften 570 Life sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 04 Medizin
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-1389
URN:	urn:nbn:de:hebis:77-diss-1000016966
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	https://rightsstatements.org/vocab/InC/1.0/
Extent:	177 Seiten
Appears in collections:	JGU-Publikationen