CREATION OF ISOGENIC CELL LINE MODELS OF 17P DELETION TO STUDY CLONAL EVOLUTION AND MECHANISMS OF THERAPY RESISTANCE IN CLL AND DLBCL

Abstract

Introduction: A deletion of chromosome 17p (del17p), on which the tumour suppressor gene TP53 is located, is a prevalent genetic aberration in many cancer types. In chronic lymphocytic leukaemia (CLL), diffuse large B-cell lymphoma (DLBCL), and other B cell malignancies this chromosomal mutation is associated with high fitness advantage in the setting of therapy, and the most adverse clinical outcome. Whilst resistance to the chemoimmunotherapies often used in the treatment of CLL and DLBCL can be explained by the functional loss of p53, such deactivation does not explain the increase in aggressive behaviour of the malignant cells. To study this, we aimed to develop in-vitro models of 17p deletion in CLL and DLBCL by creating TP53 (15.6 kbp) and 17p (20.5 Mbp) deletions by means of CRISPR-Cas9 genome editing. Methods: The efficiency of the gene editing machineries were firstly assessed in the adherent human colorectal cancer cell line HCT116 prior to their application on the B-cell lines, which are less amenable to genetic manipulation. TP53 gene removal (15.6 kbp) was performed by transient transfection of a vector encoding Cas9 as well as single guide (sg) RNAs targeting upstream and downstream of the TP53 coding region. For deletion of 17p (20.5 Mbp) sgRNAs targeting exon 2 of GEMIN4 (telomeric) and intron 5 of MAP2K3 (centromeric) were designed. PCR was used to confirm deletions. Results: Isogenic HCT116 cell lines with hemizygous and homozygous TP53 deletion, as well as hemizygous 17p deletion were obtained (Figure 1). HG-3 cells, a CLL cell line, were successfully transduced with inducible-Cas9, and subsequently with the sgRNAs for removal of TP53 and 17p. Keyword: Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.