Our initial studies on p15Ink4b provided evidence that the gene is a tumor suppressor for myeloid leukemia in mice using p15Ink4b-deficient mice. 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 Ink4b 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 Cre-loxP system. Mice, homozygous for the conditional allele, have been generated and shown to express p15Ink4b at normal levels. These mice have been 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 have been crossed with LysMCre mice for specific deletion in the myeloid lineage. Mice are currently being analyzed for susceptibility to retrovirus-induced leukemia. The loss of Ink4b expression in a high proportion of myeloid based diseases and increased AML susceptibility in Ink4b-deficient mice implies an important function for this CDKI in the maintenance of normal myelopoiesis. Despite this, little is known about how loss of Ink4b contributes to myelopoietic diseases or of the potential role of Ink4b in myeloid cell formation.We have recently investigated the role of Ink4b in hematopoiesis using Ink4b knock-out mice (Ink4b-/-). Bone marrow (BM) from Ink4b-/- mice show no changes in the frequency of hematopoietic stem cells but contained greater numbers of bi-potent granulocyte-macrophage progenitors and this was found to be intrinsic to the p15Ink4b cells. Interestingly, Ink4b-/- progenitors did not cycle more frequently than wild-type progenitors and showed no differences in apoptotic potential. However, loss of Ink4b was shown to affect differentiation of the common myeloid progenitors (CMP) resulting in an imbalance between erythroid and myeloid potential. This work demonstrates a novel role for Ink4b during differentiation. In another area of research our laboratory has examined human AML subtypes for hypermethylation of the INK4b locus. Inhibitors of DNA methylation are promising options for the treatment of human AML and MDS. So far, it was not investigated whether or not INK4b is hypermethylated in all cytogenetic subtypes of AML. A comparison of levels of INK4b methylation in AML with the three most common cytogenetic alterations, inv(16), t(8;21) and t(15;17), revealed a strikingly low level of methylation in all leukemias with inv(16) compared to the other types. Surprisingly, the expression level of INK4b in inv(16)+ AML samples was low and comparable to that of the other subtypes. An investigation into an alternative mechanism of INK4b silencing determined that the loss of INK4b expression was caused by inv(16)-encoded CBFa-SMMHC (a subunit of the core binding factor fused in frame to the smooth muscle myosin heavy chain). The silencing was manifested in an inability to activate the normal expression of INK4b RNA. CBFa-SMMHC was shown to displace RUNX1 from a newly determined core binding factor (CBF) site in the promoter of INK4b.
