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2779 The Transcriptional Landscape of Ph+B-ALL Is Orchestrated By Long-Range Enhancer-Promoter Interactions and the Coordinated Action of Phosphorylation-Dependent and Phosphorylation-Independent Transcription Factors

Program: Oral and Poster Abstracts
Session: 603. Lymphoid Oncogenesis: Basic: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Lymphoid Leukemias, ALL, genomics, hematopoiesis, Diseases, Lymphoid Malignancies, Biological Processes, Technology and Procedures, omics technologies
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Han Leng Ng, PhD1*, Mark E Robinson, PhD2*, Valeria Malysheva, PhD3*, Ozgen Deniz, MSc, BSc, PhD4*, Nicholas Crump, PhD5*, Kadriye Nehir Cosgun, PhD2*, Kaiyue Helian, MSc6*, Mikhail Spivakov, PhD7*, Markus Müschen, MD8 and Niklas Feldhahn, PhD9*

1Immunology & Inflammation, Centre for Haematology, Imperial College London, London, United Kingdom
2Center of Molecular and Cellular Oncology, Yale University, New Haven, CT
3Computational Neurobiology, Department of Biomedical Sciences, VIB Center for Molecular Neurology, University of Antwerp, Antwerp, Belgium
4Barts Cancer Institute, Queen Mary University of London, LONDON, United Kingdom
5Hugh & Josseline Langmuir Centre for Myeloma Research Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom
6Department for Immunology & Inflammation, Imperial College London, London, United Kingdom
7London Institute for Medical Sciences, London, United Kingdom
8Center of Molecular and Cellular Oncology, Yale Cancer Center, Yale School of Medicine, New Haven, CT
9Immunology&Inflammation, Centre for Haematology, Imperial College London, London, ENG, United Kingdom

Background and Hypothesis: B-cell precursor leukaemia (B-ALL) subtypes can be distinguished by their initiating genetic lesions. Likewise, each subtype exhibits a unique transcriptional program that drives the malignancy and defines the subtype. This often relates to the initiating genetic alteration that affects genes encoding transcription factors (TFs). However, for B-ALLs not driven by TF alterations it is less clear how the transcriptional program is established. Ph+B-ALL is a historically poor prognosis B-ALL driven by the BCR::ABL1 oncogene, a constitutive tyrosine kinase that activates its targets via phosphorylation. We hypothesised that BCR::ABL1 initiates the transcriptional signature of Ph+B-ALL cells by a two-step mechanism involving phosphorylation-activatable TFs, which then induce the expression of additional TFs that consecutively establish the Ph+B-ALL-defining transcriptome. As gene expression is often modulated by cis-regulatory elements such as enhancers, we further speculated that BCR::ABL1 reprograms the enhancer landscape to promote the transcriptional changes that define Ph+B-ALL.

Results: Using an in vitro model of BCR::ABL1-induced transformation of murine B-cell precursors, we show that transcriptional deregulation by BCR::ABL1 is a continuous process evolving until full transformation is established. Only a minority of induced transcriptional changes occur immediately upon BCR::ABL1 expression (3 days), while most affected genes require an extended period of BCR::ABL1 to become deregulated. Furthermore, less than half of deregulated genes in murine Ph+B-ALL and less than 10% of human Ph+B-ALL-defining genes were sensitive to 24h BCR::ABL1 inhibition, suggesting an involvement of phosphorylation-dependent and phosphorylation-independent TFs. TF motif enrichment analysis of active chromatin in human and mouse Ph+B-ALL cells indicated the phosphorylation-dependent TF STAT5 and the phosphorylation-independent TF ETV5 as main mediators. Degron-induced rapid degradation of each TF in Ph+B-ALL cells confirmed their unique and overlapping roles and coordinated action. In parallel to gene expression changes, Ph+B-ALL cells further acquire changes in their enhancer signatures defined by H3K27ac+ at non-promoter regions, and these enhancer signatures can be efficiently used to distinguish Ph+B-ALL from other B-ALL subtypes. Analysis of chromatin interactions of these active enhancers with respective active promoters by Promoter-Capture Hi-C (PCHI-C) in combination with H3K27ac-ChIP-Seq showed that most active genes in Ph+B-ALL cells physically interact with an enhancer, including many genes described to be essential for Ph+B-ALL cells, such as CCND2, BCL2, BCL2L1, and ETV5. Using PCHI-C and H3K27ac-HiChIP we further show that, like gene expression and enhancer signatures, physical contacts of promoters and enhancers can be efficiently used to identify Ph+B-ALL cells and distinguish them from other B-ALLs such as KMT2A-rearranged B-ALL. Subtype-specific chromatin interactions that allow this differentiation directly relate to the transcriptional programs that define the respective B-ALL subtype.

Conclusion: We describe here the molecular details of how signals from an oncogenic tyrosine kinase become converted into changes in gene expression that define the B-lineage leukaemia subtype Ph+B-ALL. Our work highlights the complexity of these changes by acknowledging the role of gene regulatory elements/enhancers in this process and further describe for the first time that B-ALL subtypes can be distinguished by their enhancer usage and promoter-enhancer interactions. Uncovering the precise enhancer regions for the genes deregulated in Ph+B-ALL and the TFs recruited to them may open new windows for therapeutic intervention.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH