The Hox a Locus Is Strongly Targeted by MMLV in an E2a-PBX1 Induced B-Cell Leukemia Model.

We previously developed a B-ALL model from E2a-PBX1 transgenic mice (Bijl et al. Abstract 469 ASH 2003 and paper in preparation). We now exploit this model to identify oncogenic collaborators to E2a-PBX1. To achieve this, 18 newborn E2a-PBX1 transgenic mice and 23 control littermates were intraperitonally injected with MMLV. The occurrence of B-ALL in E2a-PBX1 transgenic animals was significantly accelerated when compared to control littermates (mean survival 162 ± 31 versus 191 ± 50 days, respectively, P=0.03), suggesting the presence of E2a-PBX1 specific collaborators. Inverse PCR was performed on genomic DNA isolated from seven E2a-PBX1 and six control tumors. Seventy-two different retroviral insertion sites were recovered from the tumors. Six common integration sites (CIS) for MMLV were identified; including two novel CIS, i.e. Pde4d, a phosphodiesterase, and a hypothetical phospholipase A930027K05Rik. Strikingly, the Hox a locus was targeted in six of seven E2a-PBX1 tumors, and only one of six control tumors. MMLV integrants in the Hox a locus were detected in a 19kb region located between Hoxa6/Hoxa7 and Hoxa10. Two tumors were targeted twice in this locus. Q-PCR analysis for expression of all Hox a cluster genes identified a significant increase for the Hoxa3 to Hoxa10 genes in the transgenic tumors when compared to the expression in a control MMLV induced B-cell tumor (see table, below). Hoxa7 and Hoxa6 were the most frequently and strongly activated (6/6 and 5/6 transgenic tumors, respectively), showing values of 5700 and 6600 fold over expression in one particular tumor. Interestingly, a Hox a locus insertion in a control tumor (table: last row) did not have any effect on gene activation. In this study we demonstrate a preference for MMLV to target the Hox a locus in an E2a-PBX1context. The resultant gene activation suggests that several members of the Hox a locus are functional collaborators to E2a-PBX1 in the induction of B-cell leukemia. Fold difference in expression for Hox a cluster genes in Hox a targeted tumors compared to a control tumor. Insertions a3 a4 a5 a6 a7 a9 a10 a9/a10 −1 1 4 3 138 2 4 a3 17 55 1067 5700 6608 171 62 a10 3 1 11 98 38 3 −4 a9/a10 1 −1 2 21 9 2 −2 a9/a10 2x 13 6 19 221 191 85 2 a7/a9 + a10 8 4 11 66 44 9 149 a10 −2 −6 2 2 −2 −8 −5