Identification of the MYBL2-mediated tumor suppression pathway and the essential cooperating mutations in myeloid malignancies

• Applicant: Dr. Stefan Heinrichs
• Subject Area: Hematology, Oncology
• Term: from 2017 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 349046532

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders characterized by inefficient hematopoiesis due clonal expansion of mutated cells with acquired stem cell properties and impaired differentiation potential. One heterozygously common deleted region (CDR) is located on the long arm of chromosome 20 and predicted to harbor a tumor suppressor gene. Strikingly, in samples of patients with the 20q CDR, mutations of alleles on the intact chromosome 20 within this CDR had not been identified. Hence, the presence of a classical tumor suppressor requiring biallelic inactivation appeared to be unlikely. Instead, the monoallelic loss by deletion may sufficiently reduce gene expression levels to promote cell transformation.In search of a gene fulfilling these criteria, we identified the transcription factor MYBL2 as gene-dosage dependent tumor suppressor located within the 20q CDR. Surprisingly, two thirds of MDS patients with a normal karyotype are also affected by MYBL2 downregulation indicating a frequent disease mechanism. Indeed, downregulation of Mybl2 in mice by RNAi revealed a strong clonal advantage and expansion of the affected hematopoietic cells. However, this discovery has remained inconsequential for the therapy of MDS due to the lack of knowledge of its mechanistic impact leading to the dominance of the MDS clone.Thus, in this grant proposal, we aim (1) to identify the functional consequences of the downregulation of the transcription factor MYBL2 and (2) to determine the MYBL2-interacting pathways required for the pathogenesis of MDS. A key feature of our published model was the downregulation of Mybl2 using stable RNAi in vivo mimicking the downregulation in patient samples. We have now developed a reversible, doxycycline (dox)-dependent Mybl2 RNAi vector and tested it in vitro. Using this vector, we could immortalized primary murine HSPCs by Mybl2 knockdown. Importantly, these cells cease proliferation upon dox removal. We will use this system to determine, in vitro and in vivo, those Mybl2 target genes that are responsible for this phenotype in immunophenotypically equal cell populations with and without dox. In addition, we will use conditional Mybl2 knockout animals to define all Mybl2 target genes. Direct target genes will be determined by ChIP-Seq studies. In aim 2, we will identify genes that support the development of MDS in cooperation with the downregulation of Mybl2 leading to the development of new MDS mouse models. We will mainly use Crispr/Cas9-mediated genomic engineering, which has been established in our lab, to knockout 8 different MDS tumor suppressor genes. Inactivation of one or two of them will be modeled with the reversible Mybl2 knockdown. The resulting new MDS mouse model will allow to address key questions including the requirement for low Mybl2 levels for the maintenance of the disease. Ultimately, these models will facilitate the identification of novel drug targets.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Peter Horn