Transposon mutagenesis reveals cooperation of ETS family transcription factors with signaling pathways in erythro-megakaryocytic leukemia

Abstract

To define genetic lesions driving leukemia, we targeted credependent Sleeping Beauty (SB) transposon mutagenesis to the blood-forming system using a hematopoietic-selective vav 1 oncogene (vav1) promoter. Leukemias of diverse lineages ensued, most commonly lymphoid leukemia and erythroleukemia. The inclusion of a transgenic allele of Janus kinase 2 (JAK2)V617F resulted in acceleration of transposon-driven disease and strong selection for erythroleukemic pathology with transformation of bipotential erythro-megakaryocytic cells. The genes encoding the E-twenty-six (ETS) transcription factors Ets related gene (Erg) and Ets1 were the most common sites for transposon insertion in SB-induced JAK2V617F-positive erythroleukemias, present in 87.5% and 65%, respectively, of independent leukemias examined. The role of activated Erg was validated by reproducing erythroleukemic pathology in mice transplanted with fetal liver cells expressing translocated in liposarcoma (TLS)-ERG, an activated form of ERG found in human leukemia. Via application of SB mutagenesis to TLS-ERG-induced erythroid transformation, we identified multiple loci as likely collaborators with activation of Erg. Jak2 was identified as a common transposon insertion site in TLS-ERG-induced disease, strongly validating the cooperation between JAK2V617F and transposon insertion at the Erg locus in the JAK2V617F-positive leukemias. Moreover, loci expressing other regulators of signal transduction pathways were conspicuous among the common transposon insertion sites in TLS-ERGdriven leukemia, suggesting that a key mechanism in erythroleukemia may be the collaboration of lesions disturbing erythroid maturation, most notably in genes of the ETS family, with mutations that reduce dependence on exogenous signals.T he study of cancer-causing genes using insertional mutagenesis in the mouse (1, 2) provides a powerful complement to human cancer genomics for functional identification and characterization of the genetic lesions driving tumor development. Transposons are DNA elements with the unique capacity to change their genomic position, usually via expression of a transposase, an enzyme that catalyzes excision of the transposon from the genome and facilitates its reintegration elsewhere. Although most mammals lack active endogenous transposons, the Sleeping Beauty (SB) system has been developed recently to adapt the well-studied fish Tc/mariner transposon to allow insertional mutagenesis in the mouse (2). Temporally controlled or tissue-specific SB-mediated genetic screens have been designed by imposing cre-recombinase control over the expression of the transposase (2). Moreover, by activating transposition in mice carrying a predisposing germ-line genetic lesion, insertional mutations that cooperate with that particular lesion in cancer pathogenesis can be isolated specifically.To define genetic lesions driving leukemia, we targeted SB transposon mutagenesis to the blood-forming system by intercrossing mice transgenic for both a transposon array and a cre-inducible ...

Early Lineage Priming by Trisomy of Erg Leads to Myeloproliferation in a Down Syndrome Model

“…Conversely, the numbers of GMPs and bipotential megakaryocyte-erythroid progenitor populations (BEMP, CD150 + CD9 hi , CD150 + FcγR + ) were reduced in Erg +/Mld2 mice haploinsufficient for functional Erg . Trisomic Ts65Dn/Erg +/+/+ mice were noted to have significantly fewer CFU-E, an anomaly that was not evident in Ts65Dn/Erg +/+/Mld2 mice and Erg +/Mld2 mice had an expanded population of these late erythroid progenitors, supporting previous data that suggested Erg normally restrains terminal erythroid differentiation [ 22 , 24 , 25 ]. Consistent with the immunophenotypic analyses, in clonogenic assays, Ts65Dn/Erg +/+/+ bone marrow demonstrated stimuli-specific increases in the numbers of granulocyte, macrophage and megakaryocyte colony-forming units (CFU).…”

“…This finding is consistent with changes in human DS foetal livers [ 15 ] and other independent murine models of Erg overexpression [ 24 , 40 ]. Indeed, aberrant megakaryocyte-erythroid differentiation and erythroid maturation block may be a potential unifying mechanism for Erg in predisposing to acute bipotential megakaryocytic-erythroid leukemia [ 25 ]. The selective expansion of CD150 + CD9 hi cells within bipotential megakaryocyte-erythroid progenitor compartment in Ts(17 16 )65Dn mice is similar to that observed in murine models of thrombopoietin driven myeloproliferation [ 33 , 34 ], providing additional evidence that this progenitor population correlates with the degree of megakaryocytosis in disease models.…”

“…Erg has previously been shown to be essential for normal hematopoietic stem cell function [ 21 – 23 ]. Moreover, Erg deregulation can cause erythro-megakaryocytic leukemia in mice [ 24 , 25 ], and is implicated in acute myeloid and lymphoid malignancy in humans. In t(16;21) AML that carry the ERG/TLS-FUS fusion, complex karyotype AML with amplification of 21q, normal karyotype adult AML, MLL -rearranged paediatric AML and in T-ALL, high levels of ERG expression correlate with poor prognosis [ 26 – 29 ].…”

Analyzing tumor heterogeneity and driver genes in single myeloid leukemia cells with SBCapSeq

“…Another 110 CCGs, including Flt3 and Runx1 , have human orthologs with mutations identified by exome or WGS 19 in at least one AML genome (P = 3.5 x 10 −7 , Bonferroni corrected). Notably, many CCGs overlapped with human AML CCGs that fall within the long-tail regions of nonsynonymous coding mutations identified by the Cancer Genome Atlas for AML 19 and with CCGs identified in two different mouse models of leukemia that also used SB mutagenesis 20,21 (Supplementary Note). We conclude from these findings that SB captures the genes mutated by the diversity of mutational processes in human AML.…”

A computational approach for comparative oncogenomics using mouse models

“…Currently the Sleeping Beauty transposon system (described below in detail) has been used to study a wide range of cancers. This included T-cell acute lymphoblastic lymphoma (T-ALL), colorectal carcinoma, malignant peripheral nerve sheath tumors, hepatocellular carcinoma (HCC), glioblastoma multiforme (GBM), pancreatic cancer, medulloblastoma, B-cell precursor acute lymphoblastic leukemia, chronic lymphocytic leukemia, and erythroleukemia [8,31,[35][36][37][38][39][40][41]. While Sleeping Beauty can be sufficient for tumor formation in some tissues of wild type mice, it is not always sufficient [28].…”