Veterinärmedizinische Universität Wien Forschungsinformationssystem VetDoc

Grafischer Link zur Startseite der Vetmeduni Vienna

Investigation of molecular mechanisms underlying the functional cooperation between CEBPA and TET2 mutations in Acute Myeloid Leukemia

Abstract
Despite some improvements over the last decade, Acute Myeloid Leukemia (AML) has a dismal overall survival (OS). One of the main reasons for the few treatment options in AML is the extensive heterogeneity of the mutational landscape and the resulting genomic and epigenomic changes. To develop new treatment strategies it is crucial to understand the mechanisms underlying the different subtypes and mutational changes at a molecular level.In this project we focus specifically on the CEBPA mutations in combination with TET2 mutations, which is one of the most frequently co-mutated genes with CEBPA. CEBPA encodes a transcription factor and is expressed as the full-length protein, named p42, or a shorter isoform p30 due to the usage of two distinct translation initiation codons. Mutations in CEBPA are identified in 10-15% of AML patients and are most frequently N-terminal frameshift mutations that ablate the expression of p42 and lead to overexpression of p30. Approximately 90% of AML patients with CEBPA mutations harbor co-occurring mutations and TET2 mutations are observed in 33-45% of CEBPA-mutated AML.We have previously observed that patients with CEBPA-TET2 co-mutations have a significantly worse OS compared to CEBPA-mutated TET2 wildtype patients. Analysis of RNA-sequencing (RNA-seq) data from CEBPA-TET2 mutated patients revealed a distinct gene expression profile of this subgroup. To model this mutational combination, we used CRISPR/Cas9 technology to introduce Tet2 mutations into previously established Cebpa-mutated cell lines. The observed proliferative advantage of the co-mutated cells recapitulated the aggressive phenotype observed in the clinics. In AML mouse models for CEBPA and TET2 mutations, we observed a significantly shorter disease latency in the mutational combination. Integration of RNA-seq data from our models with patient gene expression data revealed a list of target genes that could be co-regulated by C/EBPα p30 and mutation of TET2. In line with the function of TET2 in demethylating the DNA, we found the majority of target genes down-regulated upon TET2 loss.We next aim to determine the mechanism, by which loss of TET2 functionally cooperates with mutated CEBPA. We hypothesize that C/EBPα p30 binding is a pre-requisite for TET2 binding at specific genomic regions and that TET2-recruitment to these regions results in expression of specific genes. These C/EBPα-TET2 co-regulated genes might include potential tumor suppressors that could moderate the effect of CEBPA mutations. Therefore, loss of TET2 and subsequent loss of gene expression of these genes could lead to a more aggressive phenotype in CEBPA-mutated AML patients. To study this, we first aim to show the interaction between C/EBPα p30 and TET2 via co-immunoprecipitation (Co-IP). Next, we will determine the p30-dependent TET2 distribution in the genome via TET2 chromatin immunoprecipitation sequencing (ChIP-seq) in Cebpa-mutated cell lines upon Cebpa knockdown. Combinatorial data analysis including previously performed C/EBPα ChIP-seq and RNA-seq in the same cell lines together with RNA-seq from the mouse models will reveal valuable insights into the interaction dynamics between C/EBPα and TET2. Integration of appropriate patient data will increase the human relevance of this study. Therefore, while this project will significantly contribute to basic research, the findings will also be applicable for the development of novel patient management strategies.
Kurzbezeichnung
CaTAML
Projektleitung
Heyes Elizabeth Claire
Laufzeit
01.09.21-01.09.22
Art der Forschung
Grundlagenforschung
Beteiligte Vetmed-Organisationseinheiten
Institut für Medizinische Biochemie
Gefördert durch
Fellinger Krebsforschung - Gemeinnütziger Verein zur Förderung der Krebsforschung, Montleartstrasse 37, 1160 Wien, Österreich
© Veterinärmedizinische Universität Wien Hilfe und DownloadsErklärung zur Barrierefreiheit