Abstract
Cohesin is a multiprotein complex essential for cell division and three-dimensional genome organisation. Mutations in genes encoding the cohesin-subunits, particularly STAG2, are found in ~13% of acute myeloid leukaemia (AML) cases. This frequency is as high as ~50% in the Down's syndrome-associated AML subtype (DS-AML). AML is an aggressive form of cancer of the bone marrow, with an overall survival rate of less than 30%, with especially poor outcomes for older patients. Previous research has shown that cohesin mutations dysregulate expression of the leukaemia-associated gene RUNX1. In AML, cohesin mutations co-occur with the RUNX1:RUNX1T1 translocation: t(8;21) and with other alterations to
the RUNX1 gene. Wnt signalling also enhances the spatial proximity between RUNX1 and RUNX1T1.
A recent drug screen showed that cohesin-mutant cells were synthetic lethal with Wnt agonism (Chin et al., 2020). The increased sensitivity to Wnt was likely due to enhanced β- catenin stabilisation. Wnt-responsive genes were more sensitized in DS-AML CMK cells engineered to contain a patient-specific mutation in cohesin subunit STAG2. This suggests that that cohesin mutations could progress oncogenesis by enhancing Wnt signalling, but the precise mechanism of this is not known. We hypothesise that Wnt signalling and cohesin mutations cooperate to dysregulate Wnt target genes, and possibly increase the frequency of the AML translocation: t(8;21).
In this project, we aimed to determine whether cohesin mutations alter β-catenin accumulation and/or histone modifications at gene regulatory sites in DS-AML cells. These experiments used isogenic DS-AML CMK cells that are unmodified, or that contain a CRISPR-generated null mutation in the gene encoding the cohesin subunit STAG2. First, we wanted to confirm that CMK cells had stabilised β-catenin in response to Wnt agonism.
Immunofluorescence and cell fractionation followed by immunoblotting showed that the Wnt-agonist CHIR99021 enhances β-catenin nuclear accumulation in STAG2 mutant cells. Experiments in a second STAG2-edited cell line, MCF10A, confirmed that β-catenin accumulation is conserved in STAG2 mutants.
We then used the chromatin immunoprecipitation method CUT&RUN to assess the global binding profile of β-catenin in the parental and STAG2 mutant CMK cells stimulated with CHIR99021. CUT&RUN with antibodies detecting the histone modifications H3K27ac and H3K4me3 was performed to assess whether the chromatin landscape is altered
in STAG2 mutants at key regulatory sites for RUNX1/RUNX1T1 or other Wnt targets. We found increased active histone marks; H3K4me3 and H3K27ac at the P2 promoter
of RUNX1 and increased H3K27ac at regulatory elements of RUNX1 in STAG2 mutant cells upon CHIR99021 treatment. In STAG2 mutants, β-catenin binding to TCF-7 sites was enhanced in the inflammatory-response related IL-10 gene which enhances B cell survival, proliferation, and antibody production, and the KLHL12 gene, an E3 ubiquitin ligase complex that acts as a negative regulator of the Wnt signalling pathway.
We’ve therefore found that STAG2 mutation in addition to Wnt agonism leads to activation of enhancers and increased expression of RUNX1 and RUNX1T1 and an alteration of the chromatin landscape at each of these genes.
These findings are important, as treatments for AML have not altered significantly in more than 30 years and new therapies are urgently needed. Understanding how cohesin mutations co-operate with the Wnt pathway can lead to the development of new AML-therapeutics by contributing new information on the aetiology of AML and identifying new targetable pathways.