Title: Characterisation of altered epigenetic cell states following loss of CREBBP function and their role in the induction and treatment of ALL
Funder: Cancer Research UK

Abstract: Background: B-acute lymphoblastic leukaemia (B-ALL) is an aggressive malignancy of immature B-cell progenitors. It remains the commonest cause of cancer-related death in children and is associated with dismal survival in adults. Mutations in the histone acetyltransferase CREBBP are associated with a number of haematological and solid malignancies and confer a high risk of relapse and chemo-resistance in B-ALL. Our established inducible Crebbp-/- murine lymphoma model exhibits an expanded putative pre-leukaemic IL7R+ progenitor, abnormal DNA damage responses and a marked reduction in the activating histone mark H3K27ac, yet with limited transcriptional impact. We hypothesise that the altered epigenetic programming of these pre-leukaemic cells provides an epigenetically-permissive “cancer cell state” through which subsequent driver oncogenes exert their impact. Targeting this cell state could provide a novel approach to the therapeutic challenges of tumour heterogeneity and epigenetically-mediated chemo-resistance, in B lineage malignancies and perhaps beyond. Aims: 1. We will generate a novel murine model of CREBBP-mutated B-ALL, and integrate detailed bulk and single-cell epigenetic, transcriptional and signalling analyses to network model how Crebbp loss contributes to clonal evolution and chemo-resistance in B-ALL. 2. We will genome-engineer a CREBBP-mutant human B-ALL cell line to aid drug discovery. Methods: 1. We hypothesise that this CREBBP-mutant pre-leukaemic IL7R+ compartment will transform to B-ALL when combined with a potent co-operative second-hit mutation (e.g. K- RAS). We will cross our existing mice to create an inducible Mx1CreCrebbp(fl/fl)K-Ras(G12D) model. Mice developing leukaemia will be analysed by flow cytometry to identify the critical cellular compartments for detailed analysis. These compartments will be analysed by bulk, and where applicable single cell, transcriptional (RNA-Seq), epigenetic (ChIP, ATAC-seq, pCHiC) and signalling (IL7R, pre-B cell receptor (BCR) and BCR) assays. These results will be combined into a detailed network-level model identifying the pathways driving clonal dominance, transformation and chemo-resistance in CREBBP-mutant B-ALL. 2. We will genome engineer the near-haploid human cell line NALM16 to harbour the CREBBP(R1446C) mutation for use in drug testing. We will identify candidate therapeutics by testing hypothesised vulnerabilities, including those identified in 1 and through an agnostic genome-wide CRISPR drop-out screen, which will also validate targets identified in aim 1. We will test leading drug candidates in vivo. Outcomes: We will identify multiple potential therapeutic vulnerabilities for CREBBP-mutant high-risk B-ALL and pre-clinically validate candidate drugs. Understanding how CREBBP loss primes an oncogenically-permissive epigenetic landscape may have therapeutic implications for related B lineage or CREBBP-mutated malignancies, and perhaps beyond.

Lay abstract: Background: B-acute lymphoblastic leukaemia (B-ALL) is an aggressive malignancy of immature B-cell progenitors. It remains the commonest cause of cancer-related death in children and is associated with dismal survival in adults. Mutations in the histone acetyltransferase CREBBP are associated with a number of haematological and solid malignancies and confer a high risk of relapse and chemo-resistance in B-ALL. Our established inducible Crebbp-/- murine lymphoma model exhibits an expanded putative pre-leukaemic IL7R+ progenitor, abnormal DNA damage responses and a marked reduction in the activating histone mark H3K27ac, yet with limited transcriptional impact. We hypothesise that the altered epigenetic programming of these pre-leukaemic cells provides an epigenetically-permissive “cancer cell state” through which subsequent driver oncogenes exert their impact. Targeting this cell state could provide a novel approach to the therapeutic challenges of tumour heterogeneity and epigenetically-mediated chemo-resistance, in B lineage malignancies and perhaps beyond. Aims: 1. We will generate a novel murine model of CREBBP-mutated B-ALL, and integrate detailed bulk and single-cell epigenetic, transcriptional and signalling analyses to network model how Crebbp loss contributes to clonal evolution and chemo-resistance in B-ALL. 2. We will genome-engineer a CREBBP-mutant human B-ALL cell line to aid drug discovery. Methods: 1. We hypothesise that this CREBBP-mutant pre-leukaemic IL7R+ compartment will transform to B-ALL when combined with a potent co-operative second-hit mutation (e.g. K- RAS). We will cross our existing mice to create an inducible Mx1CreCrebbp(fl/fl)K-Ras(G12D) model. Mice developing leukaemia will be analysed by flow cytometry to identify the critical cellular compartments for detailed analysis. These compartments will be analysed by bulk, and where applicable single cell, transcriptional (RNA-Seq), epigenetic (ChIP, ATAC-seq, pCHiC) and signalling (IL7R, pre-B cell receptor (BCR) and BCR) assays. These results will be combined into a detailed network-level model identifying the pathways driving clonal dominance, transformation and chemo-resistance in CREBBP-mutant B-ALL. 2. We will genome engineer the near-haploid human cell line NALM16 to harbour the CREBBP(R1446C) mutation for use in drug testing. We will identify candidate therapeutics by testing hypothesised vulnerabilities, including those identified in 1 and through an agnostic genome-wide CRISPR drop-out screen, which will also validate targets identified in aim 1. We will test leading drug candidates in vivo. Outcomes: We will identify multiple potential therapeutic vulnerabilities for CREBBP-mutant high-risk B-ALL and pre-clinically validate candidate drugs. Understanding how CREBBP loss primes an oncogenically-permissive epigenetic landscape may have therapeutic implications for related B lineage or CREBBP-mutated malignancies, and perhaps beyond.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS