Insertional Mutagenesis and Deep Profiling Reveals Gene Hierarchies and a Myc/p53-Dependent Bottleneck in Lymphomagenesis

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.

From cytopenia to leukemia: the role of Gfi1 and Gfi1b in blood formation

“…More specifically, this study provided evidence that Gfi1 is an "oncorequisite" factor required for T-cell lymphomagenesis by limiting the ability of p53 to induce apoptosis during malignant transformation; that is, loss of Gfi1 led to reactivation of p53 in T-cell tumor cells and induced cell death. Recent work from Huser and colleagues 73 also suggested that data from retrovirally induced cancer models have wide implications for the genetics of human lymphomas. They conclude that a wide range of genes can accomplish the final step in this type of lymphomagenesis with the common end point of growth factor-independent proliferation.…”

“…They conclude that a wide range of genes can accomplish the final step in this type of lymphomagenesis with the common end point of growth factor-independent proliferation. 73 They also suggest that the action of a small network of genes that includes Gfi1 is sufficient to overcome mechanisms such p53 activation that would otherwise inhibit malignant transformation. 73 …”

“…73 They also suggest that the action of a small network of genes that includes Gfi1 is sufficient to overcome mechanisms such p53 activation that would otherwise inhibit malignant transformation. 73 …”

Somatic mutations in murine models of leukemia and lymphoma: Disease specificity and clinical relevance

“…In these experiments, it is anticipated that the mutation(s) engineered into the germline have disrupted one or more critical pathways required for malignant transformation, and that complementary pathways will become activated with time, possibly because of spontaneous somatic mutations. The fact that additional mutations can collaborate with transgenes to induce malignant transformation has been shown by acceleration of disease onset in transgenic mice through the use of mutagens such as N ‐ethyl‐ N ‐nitrosourea (ENU) or retroviral insertions …”

“…The fact that additional mutations can collaborate with transgenes to induce malignant transformation has been shown by acceleration of disease onset in transgenic mice through the use of mutagens such as N-ethyl-N-nitrosourea (ENU) or retroviral insertions. 4,5 Investigators have traditionally used two approaches to identify genes and/or pathways that can complement a genetically engineered mutation. A targeted or candidate gene approach studies a limited number of genes that are known or suspected to be involved in malignant transformation, using PCR amplification and Sanger sequencing of target genes.…”