Using p15Ink4b-deficient mice, our initial studies of p15Ink4b provided preliminary evidence that the gene is a tumor suppressor for myeloid leukemia. Recently 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. 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 new 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. Morphological characteristics of p15Ink4bfl/fl-LysMcre mice most closely resemble the myeloproliferative form of chronic myelomonocytic leukemia (CMML). In with accord with our finding, hypermethylation of p15INK4b was reported previously in approximately 60% of CMML cases. The majority of patients in a study of CMML were diagnosed with class I CMML with less than 10% of blast in the BM. However, even within this category, a higher proportion of methylation was detected in a more aggressive form of CMML-1 with 5-10% of blast as compared to patients with less than 5% of blasts in the BM. These results argue that myeloid-specific inactivation of p15Ink4b which mimics suppression of its expression in CMML is not just a passive result of a global change in DNA methylation frequently observed in precancerous lesions, but instead it may be of functional significance in the establishment of these preleukemic conditions. Overall, this work correlates with the frequent and specific suppression of p15INK4b expression, via an epigenetic mechanism, in human myeloid diseases. 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. In addition, Ink4b-null mice show defects in MEP/BFU-E formation and expansion of GMP/CFU-GM during recovery from PHZ induced anemic stress. 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. Quantification of p15Ink4b expression in FACS purified mouse hematopoietic progenitors revealed that increased or decreased expression of this message was concomitant with differentiation of common myeloid progenitors into megakaryocyte-erythroid progenitor and granulocyte-macrophage progenitor cells, respectively. Using methylcellulose based media optimized for detection of primitive burst-forming unit erythroid colonies we found that the bone marrow of p15Ink4b knock-out animals had significantly fewer primitive BFU-E colonies. Expression of the protein in p15Ink4b null linage negative bone marrow cells, from the inducible pLVX-pTuner-Green lentiviral vector, restored the erythroid differentiation to the level seen in wild type. Similar results were observed in mouse hematopoietic progenitor cell line (EML) engineered to express p15Ink4b using the inducible pTuner system. Notably, induction of p15Ink4b protein for 24 hours was sufficient to increase commitment of the EML cell line to erythroid lineage, assayed by primitive BFU-E colony formation and increased expression of erythroid lineage specific genes. Of interest, increased expression of p15 was associated with increased expression of EpoR and Gata1 mRNA and protein, hypophosphorylated Rb protein, and decreased expression of myeloid lineage specific transcription factor PU.1 protein. Furthermore, p15 null mice show delayed recovery from chemically induced anemia assayed by colony formation in methylcellulose based media and flow-cytometric analysis of hematopoietic progenitors frequency. In summary, our results implicated a novel function of p15Ink4b in the bifurcation of erythroid and myeloid differentiation, thereby contributing to the development of MDS and AML. 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 p15 in DC maturation. Interestingly, myeloid-specific deletion of p15Ink4b in mice resulted in significantly fewer and less mature myeloid DCs (mDCs) as compared to wild type mice. Consistent with this data, BM cells from knockout mice cultured in vitro generated fewer numbers of BM-DCs that express lower levels of the antigen presenting (MHCII) and the co-stimulatory (CD80, CD86) molecules. Re-expression of p15Ink4b resulted in an increase in the expression of both co-stimulatory molecules confirming a role for p15Ink4b in the regulation of the maturation process of DCs. Additionally, the incomplete maturation of BM-DCs correlated with a reduced ability to activate T cells in a MHCII-mismatched mixed leukocyte reaction suggesting that loss of p15Ink4b affects the function of BM-DCs. 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 p15 in AML-DCs may overcome some of the limitations of a DC-based immunotherapy for AML patients.
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