The Leukemogenic Potential of Cdx2 Is Dependent on Its N-Terminal Serine Sites and Can Be Counteracted by MAPK Inhibitors

The homeobox transcription factor Cdx2 is one of the most frequent ectopically expressed proto-oncogenes in human AML and when retrovirally expressed causes AML in mice (Rawat et. al. PNAS 2004 and Blood 2008). As the leukemogenic potential of Cdx2 was dependent on its N-terminal transactivation domain, we now extended structure-function analyses by inactivating the evolutionarily conserved MAPK phosphorylation sites (S60, S99, S108, S156, S60-S99-108 (3S), and S60-S99-108-156 (4S)) in Cdx2. Peripheral blood analysis of the mice, transplanted with bone marrow cells retrovirally transduced with different Cdx2 serine mutants (n=5) after six weeks, revealed that all the Cdx2 serine mutants showed significant growth advantage over non-transduced carrier cells. However, assessment of engraftment after 16 weeks showed that mice transplanted with BM expressing the S99, the S108 mutants, the 3S and 4S failed to develop leukemic engraftment in contrast to wild type Cdx2, indicating that the S99 and S108 serine sites are critical for leukemic transformation. Furthermore, mice transplanted with BM expressing Cdx2 wild type (n=24), the S60 (n=3) or S156 mutant (n=5) developed AML after median latency of 120, 90 and 167 days post transplantation, respectively. In contrast S99, S108 and 3S mice developed AML after very long latency of 328 (5/5 mice), 341(3/5 mice) and 336 (3/5 mice) days post transplantation, respectively. Interestingly, 4S mice (n=5), did not develop any disease up to an observation time of 400 days, indicating that the transforming potential of Cdx2 was dependent on multiple N-terminal serines. As it was shown that the transcriptional activity of Cdx2 is dependent on the phosphorylation status of N-terminal serines in non-hematopoietic tissue we tested the phosphorylation status in hematopoietic cells: murine cells retrovirally transduced with Cdx2 and human AML cell lines positive for CDX2 expression showed a phosphorylated form of CDX2 and an activated Erk1/2 pathway, in contrast to AML cell lines negative for CDX2 expression. Based on this we tested whether inhibition of the MAPK pathway would impair the transforming potential of Cdx2. When Cdx2 transduced BM cells were incubated with MEK1/2 inhibitors (PD98059, U0126) for 14 days in liquid culture, viability of the cells was reduced by 78% (n=3, p<0.03). Incubation with the inhibitors decreased the activity of Cdx2 at the level of the short-term repopulating stem cell 8-fold as assessed in the rCFU-S after 7 days in vitro culture (n=7, p<0.001). In contrast, Mek1/2 inhibitors did not change the yield of day 12 CFU-S for AML1-ETO expressing progenitor cells in this assay. These data demonstrate that the leukemogenic potential of the homeobox gene Cdx2 depends on N-terminal serines. Furthermore, our data link the oncogenic capacity of the transcription factor Cdx2 to the MAPK signaling, opening the possibility to counteract homeobox-associated leukemogenesis by kinase inhibitors.