Recently we have been focusing on the role of tumor suppressors in models of myeloid leukemia developed in our laboratory. Cyclin dependent kinase (cdk) inhibitors are tumor suppresses that play an important role in cell cycle control and have been inactivated in human leukemias either by deletion, promoter hypermethylation or by mutation. In particular, in a high proportion of human AML, MDS and ALL the promoter region of the gene encoding the cyclin dependent kinase (cdk) inhibitor p15INK4b has been found to be hypermethylated. In the mouse, the gene encoding the cdk inhibitor p15INK4b (Cdkn2b) is located on Chr 4 proximal to Cdkn2a which encodes the cdk inhibitor p16INK4a. Additionally, Cdkn2a encodes, through alternative splicing, p19ARF, a positive regulator of the p53 tumor suppressor pathway. p15INK4b is particularly interesting because it is upregulated at the transcription level in myeloid cells by differentiation and growth-inhibiting cytokines and is highly expressed in mature cells of the monocytic lineage. In one area of research we have completed an examination of myeloid leukemias induced by c-Myb and c-Myc for alterations in INK4 gene. The neoplasms induced by endogenous c-myb, through retroviral insertional mutagenesis generally do not express p15Ink4b RNA. A lack of expression in the Myb tumors is not due to deletion or methylation. Further studies in vitro have indicate that the c-Myb itself can repress the expression of p15INK4b. The Myc tumors that were induced by a retrovirus carrying the c-myc gene, in contrast to the Myb tumors general do express p15Ink4b RNA and protein, suggesting that c-Myc can bypass the effects of these tumor suppressor. Two Myc tumors had aberrant INK4 transcripts that were determined to be fusions of p15Ex1 with p16Ex2 or 3 and this was due to deletion of a region encompassing Ex1? for p19Arf. Interestingly, most of the Myc tumors have deleted p19Arf exons even in some cases where p16Ink4a is not deleted. Therefore, our data would suggest that alterations in the Cdkn4a locus in the Myc induced tumors may primarily be due to attempts by the cell to inactivate p19ARF and, therefore, the p53 tumor suppressor pathway. The laboratory has now embarked on a systematic analysis of p15Ink4b. We recently created a new recombinant retrovirus, called MOL4070LTR, that is unique in its ability to infect all strains of mice and to induce myeloid and lymphoid disease in approximately equal proportions. Infection with this virus, which is proving to be extremely useful as a model for induction of myeloid leukemias, induces leukemias with hypermethylation of the p15Ink4b gene, thus mimicking the situation in man. The lab is in the process of determining the common initiation sites for methylation and analyzing the spread of methylation through the extended promoter region of the gene in these leukemias. In addition, we are correlating specific methylation patterns with transcription during progression from the primary tumor stage to transplantation and tissue culture establishment. For the purpose of demonstrating a tumor suppressor function for p15Ink4b in mice, we have developed a mouse with targeted deletion of this gene. The resulting mouse has been used in combination with inoculation of the MOL4070LTR leukemogenic retrovirus. Initial experiments are providing the preliminary data to suggest that p15Ink4b is a suppressor of myeloid leukemogenesis, because the incidence of leukemia is higher in mice with the targeted deletion than it is in wild-type mice.
