Comprehensive Functional Studies on Translocation Products in AML
Chromosomal translocations are frequent events in leukemia. Often, this event fuses two genes, thereby creating novel proteins with oncogenic functions. In many cases, the molecular mechanism of cellular transformation of these translocation products is unknown. We have already established a number of cancer cell lines with inducible expression of affinity tagged variants of translocation products for a comprehensive analysis of their oncogenic activities.
We are using affinity purification coupled to mass spectroscopy (AP-MS) analysis to identify the interactomes of selected fusion proteins. Subsequently, we attempt to obtain functional insights into the identified interactors through the use of customized shRNA libraries in cell line models expressing the respective translocation product. Novel hits from shRNA screens will be followed up using cell lines and primary cells in in vitro and in vivo assays.
In parallel to functional studies we will study the changes in gene expression patterns that are induced upon expression of selected translocation products by RNA-seq. If applicable, the genomic distribution of the same translocation products proteins will be analyzed using ChIP-seq. Bioinformatic intersection of ChIP-seq- and gene expression datasets will allow the discrimination of direct transcriptional target genes of translocation proteins.
Identification of Critical Effectors of MLL Fusions in AML
Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene on chromosome 11 are found in 10 % of all human acute leukemias. The MLL gene is fused to a plethora of recipient loci, resulting in the expression of >60 different fusion proteins. In the majority of those, MLL’s N-terminus is retained within the fusion protein, while tis C-terminus is lost, leading to loss of MLL’s H3K4-specific methyltransferase activity. The different fusion partners of MLL, however, often lead to the recruitment of novel enzymatic activities to the MLL N-terminus, thereby creating hybrid proteins with novel cellular activities that contribute to leukemic transformation. While functional analysis of selected MLL fusion proteins has revealed novel molecular mechanisms of transformation as well as novel targeting strategies, it is not clear if these oncogenic mechanisms represent a common vulnerability in the entire MLL-fusion family. We hypothesize that all fusion proteins within the MLL-translocation family share common oncogenic mechanisms. Identification of common critical effectors of MLL fusion proteins will inform novel diagnostic and therapeutic strategies to improve the management of AML (See Image Gallery).
In summary, our data highlight the functional relevance of combined proteomic-genomic cellular screening to identify critical effectors of MLL-FPs. In addition, our study identifies a novel role for MIC1 in the maintenance of genomic integrity during initiation and progression of MLL-rearranged AML and establishes SMIC1 as a therapeutic target in leukemia. We are currently continuing to investigate the roles of MIC1 in AML using diverse approach, incuding ChIP-seq, Whole Exome Sequencing as well as classical molecular biological approaches.
Figure 1: Systematic functional interrogation of conserved interaction partners of MLL fusion proteins. (A) AP-MS-derived protein-protein interaction network of 7 selected MLL fusion proteins (schematic fusion proteins). Shown are top 300 interactors per fusion protein (based on p-values, grey and cyan circles). Cyan circles represent 128 proteins that interact with ≥5 of 7 MLL fusion proteins. (B) Experimental workflow to systematically test a functional contribution of 128 conserved MLL-fusion interaction partners (network, top) using an shRNA strategy in positive- and negative selection readouts.