Abstract
Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC, -Runx2) develop rapid onset tumours that can be accelerated and rendered polyclonal by neonatal Moloney murine leukaemia virus (MoMLV) infection. RIM/DS analysis of 28 polyclonal lymphomas identified 771 common insertion sites (CISs) defining a ‘progression network’ that encompassed a remarkably large fraction of known MoMLV target genes, with further strong indications of oncogenic selection above the background of MoMLV integration preference. Progression driven by RIM was characterised as a Darwinian process of clonal competition engaging proliferation control networks downstream of cytokine and T-cell receptor signalling. Enhancer mode activation accounted for the most efficiently selected CIS target genes, including Ccr7 as the most prominent of a set of chemokine receptors driving paracrine growth stimulation and lymphoma dissemination. Another large target gene subset including candidate tumour suppressors was disrupted by intragenic insertions. A second RIM/DS screen comparing lymphomas of wild-type and parental transgenics showed that CD2-MYC tumours are virtually dependent on activation of Runx family genes in strong preference to other potent Myc collaborating genes (Gfi1, Notch1). Ikzf1 was identified as a novel collaborating gene for Runx2 and illustrated the interface between integration preference and oncogenic selection. Lymphoma target genes for MoMLV can be classified into (a) a small set of master regulators that confer self-renewal; overcoming p53 and other failsafe pathways and (b) a large group of progression genes that control autonomous proliferation in transformed cells. These findings provide insights into retroviral biology, human cancer genetics and the safety of vector-mediated gene therapy.
Highlights
The oncogenic potential of murine c-retroviruses (MLVs) stems from proviral integration into host DNA, a mutagenic process which can result in activation or disruption of critical host cell genes [1]
Cancers are known to arise by a series of mutational and non-mutational events but the advent of cancer genome sequencing highlights the growing challenge of separating important from irrelevant mutations
In this study we combined retroviral tagging with generation sequencing to achieve a comprehensive description of lymphoma development and progression in transgenic mouse model systems
Summary
The oncogenic potential of murine c-retroviruses (MLVs) stems from proviral integration into host DNA, a mutagenic process which can result in activation or disruption of critical host cell genes [1]. By sequential integrations in the nascent tumour cell, MLVs can drive multiple steps in the oncogenic process. These features have led to the use of MLVs as screening tools for genes relevant to cancer, haematopoietic malignancies. The reach of this approach has grown considerably with the development of high throughput methods for cloning and sequencing analysis of host-virus junctions at insertion sites, facilitating screens of large tumour panels and identifying hundreds of genes of potential relevance to cancer. Target gene interactions can be explored functionally when combined with manipulation of the mouse genome and mice with an activated oncogene or mutant tumour suppressor gene in the germ-line
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