Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - ONCOMECHAML (Common Oncogenic Mechanisms in Multi-Partner Translocation Families in Acute Myeloid Leukemia)

Teaser

Acute Myeloid Leukemia (AML) is a frequent cancer of the blood system, which more than 80% of patients do not survive for more than 5 years after diagnosis. Straightforward clinical decisions are complicated by the immense genetic complexity of AML and by the lack of...

Summary

Acute Myeloid Leukemia (AML) is a frequent cancer of the blood system, which more than 80% of patients do not survive for more than 5 years after diagnosis. Straightforward clinical decisions are complicated by the immense genetic complexity of AML and by the lack of personalized treatment options. Many patients exhibit chromosomal aberrations giving rise to fusion proteins, which act as strong driver oncogenes. The chromosomal rearrangements are grouped into “Multi-Partner Translocation” (MPT) families, with one specific gene fused to a variety of recipients. This modular architecture makes MPT families interesting subjects for comparative studies of oncogenic mechanisms. The three most common MPT families in adult and pediatric AML represent translocations of the MLL, RUNX1 and NUP98 genes, all featuring more than 20 members. Despite their high clinical significance, the molecular mechanisms that underlie oncogenic transformation remain unknown for the majority of fusion proteins. Furthermore, it is unclear if transforming mechanisms are conserved within and across different MPT families.
In this project, we aim to delineate critical common components of oncogenic mechanisms in AML driven by MPT families through a comparative analysis of 20 MLL-, RUNX1- and NUP98-fusion proteins. First, we will describe how fusion proteins interact with their partner proteins and how they regulate the expression of target genes. Second, we will identify critical components within the fusion-protein-dependent regulatory landscapes by systematic loss-of-function screening. High-confidence candidates will be further analysed in detail in AML cells via different molecular approaches.
This project will contribute to the clarification of molecular mechanisms underlying fusion-protein-dependent oncogenic transformation. We are convinced that knowledge about the oncogenic mechanisms can be translated into clinical approaches for better patient management strategies for AML patients.

Work performed

We have established a unique experimental pipeline that combines a thorough characterization of cellular effects with functional annotation of novel critical effectors of AML fusion proteins. We have generated a number of advanced cell line and mouse models, which allow for tunable expression of representative fusion proteins of three MPT families. We have used these systems to perform detailed studies of interaction partners of fusion proteins within cells using affinity purification coupled to mass spectrometry (AP-MS) to reveal protein-interactomes of AML fusion proteins. In parallel, we have studied global changes in gene expression by RNA-seq upon fusion protein withdrawal in mouse models of oncogene-dependent leukemia to uncover novel target genes of AML fusion proteins. Analysis of the genomic distribution of fusion proteins by chromatin immunoprecipitation followed by sequencing (ChIP-seq) is currently ongoing. Novel bioinformatic tools were generated and used to intersect these datasets to identify common cellular denominators of MPT families.
The functional relevance of protein interactors and/or target genes is systematically interrogated using functional genomics approaches in clinically relevant model systems. While we initially started to use RNAi loss-of-function screening to validate small lists of candidate proteins, we have recently begun to leverage the CRISPR/Cas9 technology to identify critical effectors of MPT families at a genomic scale. First genome-wide CRISPR/Cas9 screens in fusion-protein-dependent cellular models are currently being analysed. High-confidence hits are subsequently validated using a variety of experimental approaches.
We have recently completed a first study to apply this experimental pipeline to conserved interactors of seven MLL fusion proteins. Functional investigation of 128 conserved MLL-fusion-interactors identified a specific role for the lysine methyltransferase SETD2 in MLL-leukemia. SETD2 loss caused growth arrest and differentiation of AML cells, and led to increased DNA damage. These results uncover a dependency for SETD2 during MLL-leukemogenesis, revealing a novel actionable vulnerability in this disease.

Final results

This project will generate large informative datasets and novel experimental systems that are of relevance for basic and clinical cancer research. In contrast to studies of isolated fusion proteins, our approach will identify novel conserved effectors of AML oncoproteins and thus may contribute to an improved understanding of oncogenic mechanisms of the MLL-, RUNX1 and NUP98 MPT families. We expect to provide datasets that comprehensively describe interaction partners and target genes for the three largest MPT families in AML to the biomedical research community. This will be complemented by robust datasets describing global genetic requirements for proliferation and survival of AML cells with MLL-, RUNX1 and NUP98-fusion proteins. Intersection of data from these orthogonal approaches will reveal novel entry points into molecular mechanisms of fusion protein-mediated leukemogenesis at unprecedented depth. As our selection of candidates will be guided by clinical relevance, the results of this study may directly impact on diagnostic and therapeutic strategies in the management of AML.