Evaluation of the combined epigenetic targeting of LSD1 and HDAC for cell death induction in rhabdomyosarcoma cells

  • Rhabdomyosarcoma is the most common paediatric soft-tissue sarcoma, and for tumour recurrence, the prognosis is still unfavourable. The current standard therapy consisting of surgery, radiation and combined chemotherapy does not consider the specific biology of this tumour. Histone deacetylases (HDACs) and the Lysine-specific demethylase-1 (LSD1) are two epigenetic modifiers which are both part of repressor complexes leading to transcriptional silencing of target genes. Whereas HDACs lead to deacetylation of several lysine-residues within the histone tail, LSD1 is specific for demethylation of H3K4me2 and H3K4me1, as well as in a different context for H3K9me2. Rhabdomyosarcoma is reported to harbour high levels of LSD1, but the functional relevance is yet unclear. HDAC inhibition proved to be effective as single agent treatment, however, the proximity of HDAC1/2 and LSD1 in repressor complexes at the DNA implies a suitable rationale for a combination therapy potentially leading to cooperative effects on target gene transcription. In this study, we aimed to evaluate the potential of a combined LSD1 and HDAC inhibition for cell death induction in rhabdomyosarcoma cell lines. Whereas LSD1 inhibitors failed to induce cell death on their own, the combined inhibition of HDACs and LSD1 resulted in highly synergistic cell death induction. This effect extended to several combinations of LSD1 and HDAC inhibitors as well as to four different rhabdomyosarcoma cell lines, two of embryonal and two of alveolar histology. With the use of the HDAC inhibitor JNJ-26481585 and the reversible LSD1 inhibitor GSK690, we demonstrated that the cell death induced by the combination matches with the details of intrinsic mitochondrial apoptosis. JNJ-26481585/GSK690-induced cell death is partially caspase-dependent and leads to caspase cleavage, followed by substrate cleavage as shown for PARP, as well as loss of the mitochondrial membrane potential. Furthermore, JNJ-26481585 and GSK690 acted together to transcriptionally upregulate the proapoptotic proteins NOXA, BIM and BMF, which resulted in respective changes on protein level for both cell lines. However, the antiapoptotic BCL-2 family proteins BCL-2, MCL-1 and BCL-xL displayed only minor changes in protein levels upon treatment with GSK690 and JNJ-26481585, which did not rely on transcriptional activity. Therefore, the increase in proapoptotic proteins induces a shift towards proapoptotic signalling at the mitochondrial membrane. This shift is functionally relevant since knockdown of a proapoptotic protein or overexpression of one of the antiapoptotic proteins BCL-2 and MCL-1, as well as a stabilized mutant MCL-1, can significantly protect from GSK690/JNJ-26481585-induced cell death. Knockdown of the mitochondrial membrane protein BAK, which is directly guarding the mitochondrial membrane integrity, potently protected from GSK690/JNJ-26481585- induced cell death, directly linking the shift in the BCL-2 family proteins to the observed loss of mitochondrial membrane potential and the further downstream activation of caspases. Furthermore, treatment with JNJ-26481585 and GSK690 resulted in a cell cycle arrest in G2/M phase, indicating additional effects on the tumour cells beside apoptosis induction. Taken together, the combined inhibition of LSD1 and HDACs is a promising strategy for rhabdomyosarcoma treatment.
  • Das Rhabdomyosarkom ist der häufigste pädiatrische Weichteiltumor. Es wird prinzipiell in zwei große Subgruppen unterschieden, die des histologisch embryonalen Phänotyps und die des histologisch alveolären Phänotyps. Allgemein haben die embryonalen Tumore (genannt eRMS) eine deutlich bessere Prognose als die als aggressiv geltenden alveolären Tumore (genannt aRMS), sofern sie nicht metastasiert sind. Beide Tumorsubtypen präsentieren sich mit unterschiedlichen genetischen Auffälligkeiten. So haben die meisten aRMS ein Fusionsprotein aus PAX7 und FOXO1 oder PAX3 und FOXO1. Dies entsteht durch eine chromosomale Translokation zwischen dem Chromosom 2 und 3, bzw. 1 und 3. Die Tumore des embryonalen Phänotyps weisen meist Genmutationen auf, die wichtige regulatorische Proteine wie RAS oder p53 betreffen. Weiterhin tritt auch eine Deletion auf Chromosom 11 häufiger auf. Aktivierende RAS-Mutationen fördern das Zellwachstum und inaktivierende Mutationen von p53 verhindern die natürliche Kontrolle, die bei unreguliertem Wachstum programmierten Zelltod auslösen würde, wodurch diese Mutationen zur Tumorigenität beitragen können. Die momentane Standardtherapie für Rhabdomyosarkompatienten besteht aus chirugischen Eingriffen, Bestrahlung und einer kombinierten Chemotherapie mit den Medikamenten Vincristin, Actinomycin D und Cyclophosphamide. In weiter fortgeschrittenen Tumorstadien finden auch Ifosfamid und Etoposid Anwendung. Trotz dieser vielseitigen und aggressiven Therapie, bleibt die Prognose mit fortschreitendem Tumorwachstum schlecht. ...

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Metadaten
Author:Tinka Haydn
URN:urn:nbn:de:hebis:30:3-476465
Place of publication:Frankfurt am Main
Referee:Volker DötschORCiDGND, Simone FuldaORCiDGND
Document Type:Doctoral Thesis
Language:English
Year of Completion:2017
Year of first Publication:2017
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Granting Institution:Johann Wolfgang Goethe-Universität
Date of final exam:2018/01/12
Release Date:2018/11/22
Page Number:108, xvi
HeBIS-PPN:43914311X
Institutes:Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Sammlungen:Universitätspublikationen
Sammlung Biologie / Biologische Hochschulschriften (Goethe-Universität)
Licence (German):License LogoArchivex. zur Lesesaalplatznutzung § 52b UrhG