High incidence of proviral integrations in the Hoxa locus in a new model of E2a–PBX1-induced B-cell leukemia

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Abstract
Relevant mouse models of E2a–PBX1-induced pre-B cell leukemia are still elusive. We now report the generation of a pre-B leukemia model using E2a–PBX1 transgenic mice, which lack mature and precursor T-cells as a result of engineered loss of CD3ϵ expression (CD3ϵ–/–). Using insertional mutagenesis and inverse-PCR, we show that B-cell leukemia development in the E2a–PBX1 × CD3ϵ–/– compound transgenic animals is significantly accelerated when compared to control littermates, and document several known and novel integrations in these tumors. Of all common integration sites, a small region of 19 kb in the Hoxa gene locus, mostly between Hoxa6 and Hoxa10, represented 18% of all integrations in the E2a–PBX1 B-cell leukemia and was targeted in 86% of these leukemias compared to 17% in control tumors. Q-PCR assessment of expression levels for most Hoxa cluster genes in these tumors revealed an unprecedented impact of the proviral integrations on Hoxa gene expression, with tumors having one to seven different Hoxa genes overexpressed at levels up to 6600-fold above control values. Together our studies set the stage for modeling E2a–PBX1-induced B-cell leukemia and shed new light on the complexity pertaining to Hox gene regulation. In addition, our results show that the Hoxa gene cluster is preferentially targeted in E2a–PBX1-induced tumors, thus suggesting functional collaboration between these oncogenes in pre-B-cell tumors.