Our laboratory has embarked on a systematic analysis of p15INK4b, which in human acute myeloid leukemia (AML) is inactivated more frequently than any other tumor suppressor gene with cell cycle regulatory properties. p15Ink4b is a cyclin-dependent kinase inhibitor and like other Ink4 family members binds to early G1 cyclin-dependent kinases 4 and 6, thereby preventing their interaction with D cyclins. We have previously provided evidence that the gene is a tumor suppressor for myeloid leukemia in mice. First, retrovirus-induced myeloid leukemias of the myelomonocytic phenotype were found to have hypermethylation within the gene's promoter region. In some cases where leukemias did not have hypermethylation in the primary state, the neoplastic cells developed methylation following transplantation in syngeneic mice. A second type of study carried out in p15Ink4b-deficient mice provided the most convincing data regarding the tumor suppressor role of Ink4b. Use of the mice in a retrovirus-induced leukemogenesis protocol showed that mice heterozygous for Ink4b deficiency had increased susceptibility to myeloid leukemia. Mice were inoculated with retrovirus MOL4070LTR, a unique myeloid disease-inducing virus, which was recently constructed and produced in our laboratory. The retrovirus provided the cooperating events through insertional mutagenesis that allowed the demonstration of the susceptibility to leukemia provided by loss of one allele. Mice deficient in both alleles were not more susceptible than those deficient in one allele, raising the possibility that there are opposing forces to the development of myeloid leukemia in Ink4b null mice. To assist in our understanding of why Ink4b-/- mice are not more susceptible to leukemia than Ink4b-/+ mice we have developed a conditional knockout of Ink4b based on the loxP-Cre system. Mice, homozygous for the conditional allele, have been generated and shown to express p15Ink4b at normal levels. These mice are now being crossed with Mx1Cre mice which will allow deletion of exon 2 of Ink4b following treatment of mice with pI-pC. In addition, mice with the conditional knockout allele are being crossed with LysMCre mice for specific deletion in the myeloid lineage. Mice will be subsequently analyzed for susceptibility to retrovirus-induced leukemia.In leukemia, the normal balance between the proliferation and differentiation of hematopoietic stem and progenitor cells can be disrupted. To determine if p15Ink4b plays a role in regulating the levels hematopoietic precursors, we examined the hematopoietic progenitor pool in Ink4b-/- mice. In myeloid progenitor colony assays, bone marrow from Ink4b-/- mice were found to contain a 1.4 fold greater number of progenitors committed to the formation of granulocytes and macrophages (CFU-GM). This in vitro data was supported by flow cytometric analysis which determined that Ink4b-/- BM contained a 3-fold greater proportion of granulocyte-macrophage progenitors (GMP) (11+/-1.2% vs 34+/-9%) concomitant with a 2-fold decrease in common myeloid progenitors (CMP) (56+/-2 % vs 34+/-7 %). GMP isolated from Ink4b-/- bone marrow also demonstrated a 3-fold greater propensity to form CFU-GM. Despite these differences, the proportion of cycling GMP or CMP in Ink4b-/- and wt mice were identical as determined by propidium iodide and hoecsht dye DNA stains as well as in vivo BrdU incorporation. However, in response to specific cytokines, Ink4b-/- CMP showed an increased potential for GMP differentiation and a decreased capacity to form megakaryocyte-erythroid progenitors (MEP). Our work demonstrates that p15Ink4b functions in maintaining normal levels of CFU-GM and may regulate CMP differentiation in response to specific factors. In another area of research our laboratory has examined human AML subtypes for hypermethylation of the INK4B locus.
