Discovering and targeting the wider super-enhancer network of acute lymphoblastic leukaemia

Background B cell acute lymphoblastic leukaemia (B-ALL) is the most common childhood malignancy and despite survival being high (children, ~90%), outcome is worse for adults and those with refractory/relapse or high-risk disease.

As genomic rearrangements are risk-stratifying, research efforts strive to develop approaches specifically targeting these events, to generate kinder treatments with improved outcomes.

Whilst translocations involving inappropriate pairing of super-enhancers and proto-oncogenes are well reported, little is known about the activity of other super-enhancers and their role in leukaemia.

Fusion genes are common in B-ALL (~60%), they drive the leukaemia, define subtypes and risk stratification, but we do not know how they are regulated.

Around 6.2% of the genome is marked by super-enhancers and whilst some are involved in chromosomal rearrangements, the wider impact of their dysregulation in B-ALL is currently understudied.

By controlling cell identity and tumour suppressor genes, any changes in their activity could contribute to the dysregulated proliferation and differentiation observed in B-ALL. Aims: 1. Identify location, activity and 3D connections of super-enhancers in normal immature B-cells 2. Functionally interrogate super-enhancer involvement in fusion gene expression 3.

Characterise the wider involvement of super-enhancers in B-ALL comparing to normal B cells 4.

Identify druggable targets to reverse aberrant super-enhancer activity in B-ALL Methods To understand the wider involvement of super-enhancers, we need to acquire in-depth knowledge of their activity during normal immature B-cell development, and identify whether changes in location, activity and genes they regulate contribute to leukaemia onset and progression.

With the development of low cell input technologies that profile the epigenome (CUT&RUN/Tag, liCHiC, ATAC-seq) we can now map their location across early B cell maturation to understand which genes they regulate (UMI-4C/RNA-seq).

Modelling changes to super-enhancers using CRISPR (aCas9, dCas9) will enable us to understand how they impact fusion gene expression and more widely, inactivate and activate tumour suppressor genes and proto-oncogenes.

Polymer modelling (HiP-HoP) will identify critical and potentially targetable regions of super-enhancers, with proximity labelling techniques (Caspex) highlighting proteins responsible for leukemic gene dysregulation. Chemical perturbation/activation will assess protein-protein interactions as novel targets for therapeutic development.

How the results of this research will be used This research programme will facilitate a step change in the understanding of super-enhancer involvement in B-ALL.

By studying the underlying mechanisms and showing disease-specific effects, we can develop effective translation into patient benefit.

Selectively targeting aberrant, cell-type specific super-enhancer activity provides a novel approach for future treatments.