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40 Sustained MYB Activity Is Necessary for Oncogenic Transcription in KMT2A-Rearranged Acute Lymphoblastic Leukemia through Enhancer-Promoter Interactions and Epigenetic Modifications at Enhancers

Program: Oral and Poster Abstracts
Type: Oral
Session: 603. Lymphoid Oncogenesis: Basic: Mechanisms of Acute Lymphoblastic Leukemia Development and Progression
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research, Biological Processes, molecular biology
Saturday, December 9, 2023: 10:15 AM

I-Jun Lau, DPhil, BMBCh1*, Joe R Harman, PhD2*, Nicole E Jackson2*, Nicholas Denny, BA, BMBS, MRCP2*, Alastair L Smith2*, Paresh Vyas, FMedSci, DPhil, FRCPath2,3*, James Oliver Davies, FRCPath, MRCP2*, Jim Hughes, BSc, PhD4*, Nicholas Crump, PhD5* and Thomas Arthur Milne2

1MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Abingdon, United Kingdom
2MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom
3Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom
4Centre for Computational Biology, Medical Research Council (MRC) Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom
5Hugh & Josseline Langmuir Centre for Myeloma Research Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom

Aberrant enhancer activity is a hallmark of many cancers including KMT2A-rearranged acute lymphoblastic leukemia (ALL), an aggressive leukemia subtype with an event-free survival of 19–45% in infant-ALL (<1 year of age) and 50–59% in childhood-ALL cases. Targeting enhancer activity may provide a novel therapeutic strategy for this hard-to-treat disease. Transcription factors are key effectors of enhancer activity. They recognise and bind to specific DNA sequences at enhancers, driving the expression of target genes from these distal loci. MYB is an important hematopoietic transcription factor that is commonly overexpressed in KMT2A-rearranged leukemias and is required for leukemia survival, where loss of MYB has been shown to abrogate the leukemia phenotype. However, the precise mechanism by which MYB contributes to enhancer function, and the stages of enhancer activation at which it acts are unknown.

We employed a well-characterized chromatin anchoring system to interrogate MYB function and determine whether it can recruit specific enhancer activities de novo. By fusing the MYB transactivation domain (MYBTA) to the Tet repressor (TetR) DNA binding domain, MYBTA is directed to an array of Tet operator (TetO) sequences incorporated into a neutral genomic region devoid of native gene regulatory elements. We found that MYBTA alone is sufficient to recruit key enhancer-associated proteins, including P300, Brd4 and Mediator to the TetO locus. We observed MYBTA-dependent deposition of H3K27ac, increased chromatin accessibility and transcription not just at the TetO locus, but remarkably, also at regions more than 50kb away. Using the chromosome conformation capture (3C) technique NG Capture-C, we observed MYBTA-induced DNA interactions between TetO and the distal sites of transcription, as well as more long-range interactions up to 400kb distal to where MYBTA is bound. Together, these observations suggest that MYBTA binding alone is sufficient to establish an enhancer-like regulatory element, which activates distant cryptic promoters. This activity is dependent on the continued presence of MYBTA, as disruption of MYBTA binding results in a rapid loss of transcription and acquired chromatin features, implicating MYB in enhancer maintenance as well as initiation.

To confirm our observations in a disease context, we tested the requirement for MYB at endogenous enhancers in SEM cells, a model of KMT2A-rearranged ALL. By stably introducing a degradation tag (FKBP12F36V) to the endogenous MYB gene, we were able to rapidly induce protein degradation and observe the immediate effects on chromatin features. Using transient transcriptome sequencing, we noted significant transcriptional downregulation of known MYB target genes, including BCL2, MYC, LMO4 and CDK6. We also observed decreases in enhancer H3K27ac levels and eRNA transcription, most marked at MYB-bound loci. To investigate the requirement for MYB in maintaining physical proximity between enhancer and promoter, we performed the base pair-resolution 3C technique Micro-Capture-C (MCC). MCC revealed highly punctate interactions between enhancer elements and their target promoters. At BCL2 and MYC, MYB degradation results in a reduction in interactions with the promoter at discrete regions within the enhancers. This argues for highly localised MYB-dependent activity at enhancers, driving interaction with and activation of distal target promoters.

Overall, our data argue for a role for MYB in establishing enhancers de novo, indicating that MYB overexpression may directly result in onco-enhancer activity at key genes in vivo. At a subset of MYB-bound enhancers, the continued presence of MYB is absolutely required to maintain enhancer activity and oncogene upregulation. Further exploration of associated co-factors that mediate MYB-dependent enhancer activity may identify novel targets for therapeutic exploitation.

Disclosures: Harman: Dark Blue Therapeutics: Current Employment. Vyas: Jazz: Honoraria; Astellas: Honoraria; Abbvie: Consultancy, Honoraria; Pfizer: Honoraria; Gilead: Honoraria; BMS: Research Funding; Auron Therapeutics: Current holder of stock options in a privately-held company. Milne: Dark Blue Therapeutics: Consultancy, Current holder of stock options in a privately-held company.

*signifies non-member of ASH