The Transforming Potential of the Homeobox Gene Cdx2 Is Depending on Its N-Terminal Transactivation Domain In Vivo and Can Be Antagonized by the MEK1/2 Inhibitor in a Mouse Model of t(12;13) Positive AML.

In AML the translocation t(12;13)(p13;q12) results in the ectopic expression of the homeobox gene Cdx2 and the expression of the ETV6-CDX2 fusion. We have shown that the ectopic expression of the proto-oncogene Cdx2 and not the expression of the ETV6-CDX2 fusion is the key event in initiating myeloid leukemogenesis in a murine model of t(12;13) AML (PNAS, Rawat et. al. 2004). We now analyzed the functional relevance of the different Cdx2 domains and explored the potential of kinase inhibitors to antagonize Cdx2 induced leukemia. For this we generated different mutants, inactivating the PBX1-interacting motif (W167A-Cdx2), or deleting the N-terminal transactivation domain (Ndel-Cdx2). Expression of Cdx2 and the different mutants was induced in primary murine BM cells by retroviral gene transfer. Target genes were identified by cDNA microarray analysis. Mice transplanted with BM cells expressing Cdx2 and its W167A-Cdx2 mutant developed transplantable AML (n=14, avg. latency 90 days). In contrast, mice transplanted with the NDel-Cdx2 mutant did not show any leukemic phenotype in vivo (n=13). In order to identify gene expression signatures associated with Cdx2 induced transformation, we performed microarray analysis on highly purified normal Sca1+/lin− HSC and Sca1+/lin+ progenitor cells transduced with the leukemogenic Cdx2 compared to the non-leukemogenic NDel-Cdx2 mutant and the GFP control vector after 72h of retrovirally induced expression of the different constructs. Compared to the NDel-Cdx2 mutant and the GFP control Cdx2 up regulated genes, which are associated with self-renewal (Wint2, Hoxb3, Etv6, Abcg2,), leukemogenesis (Lmo2, Pim-2, Hoxa9) and in signal transduction pathways (e.g. MAPK family). In addition, Cdx2 transduced BM cells showed an activated Erk1/2 pathway on the protein level. Based on these results we tested whether inhibition of the MAPK pathway would impair the leukemogenic potential of Cdx2. When Cdx2 transduced BM cells were incubated with the MEK1/2 inhibitor PD98059, a 78% reduction of viable cells (n=3, p<0.03) and of the proportion of blast-like Sca1+ positive cells were observed compared to untreated cells (n=3, p<0.005) in liquid culture after 7 days. Furthermore, incubation with the MEK1/2 inhibitor PD98059 decreased the activity of Cdx2 at the level of the short-term repopulating stem cell 8-fold as assessed in the ΔCFU-S after 7 days in vitro culture (n=7, p<0.001). In contrast, incubation with the p38 specific inhibitor SB 28049 did not show any decrease in Cdx2 activity in ΔCFU-S assay, indicating that the transforming potential of Cdx2 depends on the MEK1/2 pathway, but not on the p38 pathway. These data demonstrate that the leukemogenic potential of the homeobox gene Cdx2 depends on the N-terminal activation domain. Furthermore, our data link the oncogenic capacity of the transcription factor Cdx2 to MAPK signaling, opening the possibility to counteract homeobox-associated leukemogenesis by kinase inhibitors.