Acute myeloid leukaemia (AML) results from an uncontrolled proliferation of immature myeloid progenitor cells accompanied by a block in differentiation. Whole genome sequencing efforts have contributed to the ever-expanding genetic classification of AML. The presence of specific co-operating mutations is required for malignant transformation. While there is good evidence that certain mutation combinations lead to AML transformation, little is known about the mechanisms by which they cooperate.
Cohesin is a multiprotein complex made up of four major subunits – SMC1A, SMC3, RAD21 and STAG1/2. Mutations in cohesin genes occur in 12-20% of AML and myelodysplastic syndrome (MDS) cases1. Cohesin is an essential protein complex for sister chromatid cohesion, and is also involved in transcriptional regulation via 3D-genome organization. Genes that are frequently co-mutated with cohesin in AML and MDS include TET2, DNMT3A, RUNX1 and NPM12.
Zebrafish are a tractable model system with a haematopoietic system that is conserved among vertebrates. We are using CRISPR-Cas9 to generate zebrafish with combinatorial mutations that are characteristic of AML. Gene-specific mutations in the early exons of the four stag paralogs, namely stag1a, stag1b, stag2a and stag2b have been generated. CRISPR-injected and stable F1 embryos mutant for stag2a and stag2b exhibited severe abnormal developmental phenotypes such as perturbation of the dorso-ventral axis. Preliminary analysis of the kidney marrow of an existing rad21 mutant revealed a significant increase in precursor populations compared to wildtype. Zebrafish carrying mutations in the frequently co-occurring epigenetic modifier tet2 have also been generated. Frameshift mutations in the functional oxygenase domain of tet2 correlated with a ubiquitous downregulation of tet2 expression by whole mount in situ hybridization. Future work will focus on the generation and characterization of the combinatorial mutant models to provide insight into mechanisms of cooperativity.