Modelling Crebbp loss in BCL2 driven non-Hodgkin’s lymphoma

Abstract

Non-Hodgkin’s lymphomas (NHL) are a spectrum of hematopoietic cancer accounting for 4% of new cancer diagnoses each year. Approximately 95% of all NHL are of B-cell origin; diffuse large B-cell lymphomas (DLBCL) and follicular lymphoma (FL) accounting for 30-40% and 20% of B-NHL respectively. Recent human mutation profiling and resequencing studies have shown that CREBBP and BCL2 are frequently mutated, early events in B-NHL that are often are concurrent. This thesis presents a study of their role in oncogenesis by generating a novel model of B-NHL overexpressing BCL2 in the haematopoetic compartment combined with the conditional heterozygous loss of Crebbp in the germinal centre B-cells. Loss of Crebbp significantly accelerated BCL2 driven lymphomagenesis. Characterisation of these lymphomas by flow cytometry demonstrated that they were phenotypically similar to mature human B-cell lymphomas of germinal centre origin. Additionally an insertional mutagenesis screen was performed in mice sensitised to B-NHL. Somatic MoMuLV caused mutations interact with the sensitising mutations, facilitating tumourigenesis and recapitulating the stepwise and accumulative progression of human disease. Provirus insertions were detected using a novel method of insertion site cloning called UMI-LM-PCR which amplifies the region flanking the provirus. Insertions were then mapped to the mouse genome flagging genes with a putative role in tumourigenesis. This method represents the highest throughput method to date and its applicability to Illumina sequencing permits the most comprehensive quantitative survey of subclonal mutations. This work confirms Crebbp is a tumour suppressor gene and that its loss cooperates with overexpression of BCL2 to accelerate lymphomagenesis. Through insertional mutagenesis screening of this model hundreds of putative cancer genes have been identified including Pou2f2 and Tfrc which have a propensity for mutation specifically in B-cell lymphomas deficient in Crebbp and overexpressing BCL2. These candidates now need to be validated to better characterise their role in B-cell lymphoma