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The lack of predictive preclinical models is a fundamental barrier to translating knowledge about the molecular pathogenesis of cancer into improved therapies. Insertional mutagenesis (IM) in mice is a robust strategy for generating malignancies that recapitulate the extensive inter- and intra-tumoral genetic heterogeneity found in advanced human cancers. While the central role of "driver" viral insertions in IM models that aberrantly increase the expression of proto-oncogenes or disrupt tumor suppressors has been appreciated for many years, the contributions of cooperating somatic mutations and large chromosomal alterations to tumorigenesis are largely unknown. Integrated genomic studies of T lineage acute lymphoblastic leukemias (T-ALLs) generated by IM in wild-type (WT) and Kras mutant mice reveal frequent point mutations and other recurrent non-insertional genetic alterations that also occur in human T-ALL. These somatic mutations are sensitive and specific markers for defining clonal dynamics and identifying candidate resistance mechanisms in leukemias that relapse after an initial therapeutic response. Primary cancers initiated by IM and resistant clones that emerge during in vivo treatment close key gaps in existing preclinical models, and are robust platforms for investigating the efficacy of new therapies and for elucidating how drug exposure shapes tumor evolution and patterns of resistance., Author summary A lack of predictive cancer models is a major bottleneck for prioritizing new anti-cancer drugs for clinical trials. We comprehensively profiled a panel of primary mouse T lineage leukemias initiated by insertional mutagenesis and found remarkable similarities with human T-ALL in regard to overall mutational burden, the occurrence of specific somatic mutations and large chromosomal alterations, and concordant gene expression signatures. We observed frequent duplication of the Kras oncogene with loss of the normal allele, which has potential therapeutic implications that merit further investigation in human leukemia and in other preclinical models. Mutations identified in mouse leukemias that relapsed after in vivo treatment with signal transduction inhibitors were also observed in relapsed human T-ALL, indicating that this model system can be utilized to investigate strategies for overcoming intrinsic and acquired drug resistance. Finally, preclinical models similar to the one described here that are characterized by a normal endogenous tumor microenvironment and intact immune system will become increasingly important for testing immunotherapy approaches for human cancer.

Somatic mutations affecting ETV6 often occur in acute lymphoblastic leukemia (ALL), the most common childhood malignancy. The genetic factors that predispose to ALL remain poorly understood. Here we identify a novel germline ETV6 p. L349P mutation in a kindred affected by thrombocytopenia and ALL. A second ETV6 p. N385fs mutation was identified in an unrelated kindred characterized by thrombocytopenia, ALL and secondary myelodysplasia/acute myeloid leukemia. Leukemic cells from the proband in the second kindred showed deletion of wild type ETV6 with retention of the ETV6 p. N385fs. Enforced expression of the ETV6 mutants revealed normal transcript and protein levels, but impaired nuclear localization. Accordingly, these mutants exhibited significantly reduced ability to regulate the transcription of ETV6 target genes. Our findings highlight a novel role for ETV6 in leukemia predisposition., Author Summary Inherited mutations of transcription factors have recently been associated with susceptibility to acute leukemia. Here we report two unrelated kindreds with inherited mutations in ETV6, the gene encoding the transcription factor ETS variant 6. These families were characterized by a low platelet count (thrombocytopenia) and acute lymphoblastic leukemia (ALL). Sequencing a panel of genes identified germline ETV6 mutations associated with leukemia and thrombocytopenia in multiple individuals tested. In one family, there was a substitution within the DNA binding domain of ETV6, termed L349P, and in the second there were five base pairs missing in ETV6 (N385fs), causing an abnormally truncated protein. We overexpressed the ETV6 mutants in the HeLa cell line and measured protein levels and localization within the cells. Instead of localizing to the nucleus, as expected for a transcription factor, the mutant proteins were found in the cytoplasm. The mutant proteins also showed decreased ability to regulate the expression of other genes typically suppressed by ETV6. These findings suggest that germline ETV6 mutations cause a new type of heritable leukemia. This discovery makes possible the pre-symptomatic diagnosis of leukemia susceptibility in families with germline ETV6 mutations, and also provides new information on the causes of leukemia.

The LMO2 oncogene is deregulated in the majority of human T-cell leukemia cases and in most gene therapy-induced T-cell leukemias. We made transgenic mice with enforced expression of Lmo2 in T-cells by the CD2 promoter/enhancer. These transgenic mice developed highly penetrant T-ALL by two distinct patterns of gene expression: one in which there was concordant activation of Lyl1, Hhex, and Mycn or alternatively, with Notch1 target gene activation. Most strikingly, this gene expression clustering was conserved in human Early T-cell Precursor ALL (ETP-ALL), where LMO2, HHEX, LYL1, and MYCN were most highly expressed. We discovered that HHEX is a direct transcriptional target of LMO2 consistent with its concordant gene expression. Furthermore, conditional inactivation of Hhex in CD2-Lmo2 transgenic mice markedly attenuated T-ALL development, demonstrating that Hhex is a crucial mediator of Lmo2's oncogenic function. The CD2-Lmo2 transgenic mice offer mechanistic insight into concordant oncogene expression and provide a model for the highly treatment-resistant ETP-ALL subtype.

Five X-linked severe combined immunodeficiency patients (SCID-X1) successfully treated with autologous bone marrow stem cells infected ex vivo with an IL2RG-containing retrovirus subsequently developed T-cell leukemia and four contained insertional mutations at LMO2. Genetic evidence also suggests a role for IL2RG in tumor formation, although this remains controversial. Here, we show that the genes and signaling pathways deregulated in murine leukemias with retroviral insertions at Lmo2 are similar to those deregulated in human leukemias with high LMO2 expression and are highly predictive of the leukemias induced in SCID-X1 patients. We also provide additional evidence supporting the notion that IL2RG and LMO2 cooperate in leukemia induction but are not sufficient and require additional cooperating mutations. The highly concordant nature of the genetic events giving rise to mouse and human leukemias with mutations at Lmo2 are an encouraging sign to those wanting to use mice to model human cancer and may help in designing safer methods for retroviral gene therapy., Author Summary Twenty patients with X-linked severe combined immunodeficiency (SCID-X1) have been successfully treated by gene therapy. Unfortunately, five of these patients have developed T-cell leukemia two or more years after receiving the therapeutic gene IL2RG on a retroviral vector. The leukemias developed because the vector inserted itself near cancer-causing genes and disrupted their normal regulation. Remarkably, in four patients, the vector inserted near a known T-cell oncogene, LMO2. We have found that in mice, similar retroviruses cause T-cell leukemias by inserting near Lmo2. We have found two leukemias that have retroviral insertions near Lmo2 and Il2rg in the same cell. The probability of these insertions happening by chance is exceedingly small and these results imply that these two genes are deregulated together to induce leukemia. Our data show that Lmo2 and Il2rg cooperate but may not be sufficient for leukemia development and additional mutations contribute to leukemia development. We have also found cooperating retroviral insertions in genes that are abnormally expressed in human T-cell leukemias. The mouse models provide unique insight into the pathogenesis of T-cell leukemia, and they are highly predictive of the leukemias caused by SCID-X1 gene therapy.

The degree to which gene expression covaries between different primary tissues within an individual is not well defined. We hypothesized that expression that is concordant across tissues is more likely influenced by genetic variability than gene expression which is discordant between tissues. We quantified expression of 11,873 genes in paired samples of primary leukemia cells and normal leukocytes from 92 patients with acute lymphoblastic leukemia (ALL). Genetic variation at >500,000 single nucleotide polymorphisms (SNPs) was also assessed. The expression of only 176/11,783 (1.5%) genes was correlated (p