Regulo-Interactome Modules of Hematopoietic Stem Cells

All mature blood cells arise from a small subset of pluripotent hematopoietic stem cells (HSC). HSCs continuously decide between self renewal as immature cells and differentiation into mature cells of all blood lineages. Although the hierarchical organization of the hematopoietic system has been characterized in detail in the last years, and many gene products involved in this process are known, the make-up and logic of the molecular circuitry responsible for self renewal and pluripotency remain largely unknown. Here we propose the analysis and preliminary validation of regulo-interactome modules of HSCs. The project is made possible by recent technological developments and integral access to a well-organised functional proteomics, genomics and bioinformatic laboratory and proximity to a clinical hemato-oncological department. As a starting point, we will express tagged versions of 8 transcription factors that have been implicated in normal and leukemic stem cell function in murine hematopoietic progenitors. We propose first to characterize HSC- specific protein complexes by affinity purification followed by mass spectrometry (AP-MS). This will assign the individual components to a limited number of protein complexes (the "HSC interactome"). Second, we will use chromatin-immunoprecipitation followed by deep sequencing (ChIP-seq) and mRNA expression profiling to elucidate the genome wide chromatin occupancy and the influence on gene expression of the complexes that were identified by the AP-MS approach (the "HSC regulome"). Using bioinformatic integration, these independent datasets will lead to the establishment of a functional protein interaction network coupled to a gene regulation network (the "regulo-interactome of HSCs"), revealing how coordination of action between the molecular machines involved empowers the transcriptional programs of HSCs. The obtained network will be extended to biochemical and functional testing of the molecular machines, including novel components. Selected protein-protein interactions will be subjected to detailed biochemical characterization. As the potential to undergo self renewal is a major characteristic of normal but also leukemic stem cells, we will test if any of the selected new components has a role in self renewal of HSCs in in vitro assays. Moreover, we will screen primary samples from patients with diverse hematological malignancies for alterations in gene copy numbers of novel components of the HSC-specific transcriptional machinery. Thus, the ultimate goal of this integrated project will be the elucidation of the molecular circuitry required for stem cell properties and the discovery of proteins and mechanisms that can be validated and exploited as diagnostic and/or therapeutic targets in the fight against leukemia.

Acute myeloid leukemia (AML) is an aggressive cancer of the blood system. Over the past years, it has become clear that mutations in distinct genes can be strong drivers of leukemia development. Contrarily, it is often unclear how particular gene mutations alter cellular functions to ultimately induce cancer. Therefore, a better understanding of the molecular mechanisms underlying gene mutations in leukemia is critical to develop better therapeutic strategies to treat patients in a personalized fashion.The transcription factor CCAAT/enhancer binding protein alpha (C/EBPa) is an important regulator of granulocytic differentiation. In 9% of all patients with AML, the gene encoding C/EBPa is mutated. However, these mutations are not causing complete protein loss, but are rather supposed to alter the function of C/EBPa through the expression of a N-terminally truncated isoform of the protein (termed p30). While expression of p30 induces enhanced proliferation and blocks granulocytic differentiation, the molecular mechanisms of p30-dependent functions are incompletely understood.We used an integrated approach consisting of gene expression profiling, affinity purification coupled to mass spectrometry and chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) to show that the leukemic p30 variant - but not wild-type C/EBPa - specifically interacts with the MLL protein complex via the Wdr5 protein. The MLL protein is a histone methyltransferase that acts as a positive regulator of transcriptional processes by marking histones in active promoters with a tri-methylation on their Lys-4 residue (H3K4me3). shRNA-mediated downregulation of Wdr5 reversed the p30-induced differentiation block and interfered with p30-dependent pre-leukemic self renewal of primary cells from p30/p30 mice. ChIP-seq studies showed that the p30 variant showed greater overlap with H3K4me3-decorated genomic regions than the wild-type C/EBPa protein, indicating that p30 may utilize specific ways of transcriptional regulation that are different from its wild-type counterpart. Finally, we show that p30-expressing cells displayed very specific sensitivity to inhibition of the MLL protein complex using a small molecule inhibitor that disrupts the MLL-Menin interaction (MI-2). MI-2 induced growth arrest and terminal myeloid differentiation and global analysis of gene expression showed that p30-dependent target genes are downregulated upon MI-2 treatment.Overall, our results propose a new mechanism of p30-dependent leukemia development and validate the MLL complex as a therapeutic target in AML with mutated C/EBPa. This work may have significant impact on the development of targeted inhibition strategies to inhibit the MLL complex in AML.