Using p15Ink4b embryonal knock-out mice, our initial studies of p15Ink4b provided preliminary evidence that the gene is a tumor suppressor for myeloid leukemia. Subsequently, we developed an improved model that more closely resembles the disease in man by deleting the gene specifically in myeloid lineage cells. The new model employing the Cre-loxP system allows conditional deletion of the gene. We successfully accomplished the myeloid tissue specific deletion by crossing INK4b(exon 2) floxed mice with LysMCre mice and showed by PCR technology that Ink4b was deleted in common myeloid progenitors. The p15Ink4bfl/fl-LysMcre mice develop progressive monocytosis in the peripheral blood accompanied by increased numbers of myeloid and monocytic cells in the bone marrow. Morphological characteristics of p15Ink4bfl/fl-LysMcre mice most closely resemble a mild myeloproliferative form of chronic myelomonocytic leukemia (CMML). In accord with our findings, hypermethylation of p15INK4b was reported previously in approximately 60% of CMML cases. In the p15Ink4bfl/fl-LysMcre mice spontaneous progression from chronic disease to acute leukemia was not observed. Nevertheless, MOL4070LTR retrovirus integrations provided cooperative genetic mutations resulting in a high frequency of myeloid leukemia in knockout mice. Two common retrovirus insertion sites near c-myb and Sox4 genes were identified and their transcript upregulated in leukemia, suggesting a collaborative role of their protein products with p15Ink4b-deficiency in promoting malignant disease. This animal model demonstrates that p15Ink4b plays an active role in the establishment of preleukemic conditions. In addition, this is the first demonstration that p15Ink4b can act as a tumor suppressor in the presence of a fully functional p16Ink4a locus. Previously, tumor suppressor activity of p15Ink4b was demonstrated by others only in an animal model where both p16INK4a and p15INK4b genes were simultaneously inactivated and the animals developed a wide spectrum of tumors with prevalence of skin tumors and soft tissue sarcomas, but not of myeloid origin.p15INK4b is a member of the INK4 family member of cyclin-dependent kinase inhibitors. However, since INK4b is the only member that is inactivated in acute myeloid leukemia, we have been interested in determining if there is any other function specific to the myeloid lineage that can be assigned to this gene. For these experiments we used the two knock-out models described above. Initial investigations of hematopoiesis in Ink4b- deficient mice showed that they have greater numbers of bi-potent granulocyte-macrophage progenitors (GMP) and this was found to be intrinsic to the cells. Interestingly, Ink4b-deficient granulocyte-macrophage progenitors did not cycle more frequently than wild-type progenitors and showed no differences in apoptotic potential or self-renewal potential. However, Ink4b was shown to affect differentiation of common myeloid progenitor (CMP) cells, resulting in an imbalance of down-stream progenitors. In vitro analysis of progenitor cells from knock-out mice demonstrated that loss of Ink4b causes an increase in GMP and decrease in MEP (megakaryocyte-erythroid progenitor) potential. Based upon the data obtained from the knockout mice, we hypothesized that p15Ink4b is required for efficient erythropoiesis. We have now shown that p15Ink4b promotes erythroid differentiation and suppresses myeloid differentiation of hematopoietic progenitors under normal and stress conditions. It was found that p15Ink4b is expressed more highly in committed megakaryocyte-erythroid progenitors (MEP) than granulocyte-macrophage progenitors (GMP). More importantly, mice lacking p15Ink4b have lower numbers of primitive red cell progenitors and a severely impaired response to 5-fluorouracil and phenylhydrazine induced hematopoietic stress. Introduction of p15Ink4b in multi-potential progenitor cells produced changes at the molecular level,including activation of MEK/ERK signaling, increase GATA-1, EpoR, decrease Pu1 and GATA-2 expression. These changes rendered cells more permissive to erythroid commitment and less permissive to myeloid commitment, as demonstrated by an increase in early BFU-E formation with a concomitant decrease in myeloid progenitors. Our results indicate that p15Ink4b functions in hematopoiesis, by maintaining proper lineage commitment of progenitors and assisting in rapid red blood cell replenishment following stress.Recently, we found that expression of p15Ink4b is strongly induced in a biphasic manner during development and activation of mouse bone marrow-derived DCs (BM-DCs), suggesting an important role for p15Ink4b in DC maturation. Interestingly, myeloid-specific deletion of p15Ink4b in mice resulted in significantly fewer and less mature CD8- conventional DCs (cDCs) as compared to wild type mice. Consistent with this data, ex-vivo generated bone marrow-derived DCs (BM-DCs) from the knockout mice have markedly lower levels of expression of the antigen presenting (MHCII) and the co-stimulatory molecules (CD80, CD86) than wild-type controls. Accordingly, these cells show a reduced ability to uptake antigen and to stimulate allogeneic T-cell responses. The re-expression of p15Ink4b in progenitors results in a restoration of MHCII expression, as well as co-stimulatory molecules, confirming a positive role for p15Ink4b in cDCs development. Furthermore, p15Ink4b expression increases phosphorylation of Erk1/Erk2 protein kinases that leads to an increased transactivation activity of the PU.1 transcription factor, an important regulator of DC development. Taken together, our results indicate a novel role for p15Ink4b in mDC development, and suggest that frequent inactivation of the gene in myeloid malignancies could lead to an inefficient anti-leukemic immune response during leukemogenesis. Our data also have an important translational significance. AML blasts isolated from patients, and differentiated ex-vivo into DCs, represent a powerful immunotherapy tool. However, AML-DCs have reportedly a partially impaired maturation process as compared to DCs from healthy donors. We propose that re-expression of p15INK4b in AML-DCs may overcome some of the limitations of a DC-based immunotherapy for AML patients.
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