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. However, Ink4b was shown to affect differentiation of common myeloid progenitor (CMP) cells. The resulting imbalance was shown to favor the production of granulocyte-macrophage progenitors at the expense of erythroid-megakaryocyte progenitor (MEP) cells. This work demonstrates a novel cell cycle-independent role for Ink4b during cell fate at the erythroid/myeloid bifurcation. Since GATA-1 and PU.1 instruct erythroid and myeloid cell fates and inhibit each others transcriptional function we set out to determine if expression of these genes or other genes involved in lineage choice were altered in common myeloid progenitors from In4b-deficient compared to wild-type mice. RT-PCR results showed that PU.1 levels were unaffected while GATA-1, GATA-2, and GATA-3 were down-regulated in common myeloid progenitors. Whether p15Ink4b affects the common myeloid progenitor differentiation through its known capacity to interact with Cdk4/6 or by participating in an unrelated pathway is currently unclear.
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|Bies, Juraj; Sramko, Marek; Fares, Joanna et al. (2010) Myeloid-specific inactivation of p15Ink4b results in monocytosis and predisposition to myeloid leukemia. Blood 116:979-87|