Methylation-independent silencing of the tumor suppressor INK4b (p15) by CBFbeta-SMMHC in acute myelogenous leukemia with inv(16).

The tumor suppressor gene INK4b (p15) is silenced by CpG island hypermethylation in most acute myelogenous leukemias (AML), and this epigenetic phenomenon can be reversed by treatment with hypomethylating agents. Thus far, it was not investigated whether 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 with the other types. Surprisingly, the expression level of INK4b in inv(16)+ AML samples was low and comparable with 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 core binding factor beta-smooth muscle myosin heavy chain (CBFbeta-SMMHC). The silencing was manifested in an inability to activate the normal expression of INK4b RNA as shown in vitamin D3-treated U937 cells expressing CBFbeta-SMMHC. CBFbeta-SMMHC was shown to displace RUNX1 from a newly determined CBF site in the promoter of INK4b. Importantly, this study (a) establishes that the gene encoding the tumor suppressor p15(INK4b) is a target of CBFbeta-SMMHC, a finding relevant to the leukemogenesis process, and (b) indicates that, in patients with inv(16)-containing AML, reexpression from the INK4b locus in the leukemia would not be predicted to occur using hypomethylating drugs.

A novel retrovirus provides the cooperating oncogenic event(s) required to demonstrate the tumor suppressor activity of p15Ink4b in myeloid cells in vivo.

Cancer is a multistep process resulting from an accumulation of several genetic changes. The determination of cooperating events in experimental models can help scientists decipher specific neoplastic pathways and place genes with similar functions in complementation groups. In leukemia models, retrovirus tagging is a powerful approach to determine genes that cooperate with oncogenic transgenes or tumor suppressors that have undergone targeted deletion. Experimental models for B and T cell leukemias involving transgenic c-myc were the first to show the utility of retroviral tagging. Here we review these experiments and present examples of new models of myeloid leukemia where retroviruses have collaborated with a transgene [Cbfbeta-MYH111 from Inv(16)] and with loss of a tumor suppressor (Ink4b) mice to induce disease.

Hypermethylation of the Ink4b locus in murine myeloid leukemia and increased susceptibility to leukemia in p15(Ink4b)-deficient mice.

The Ink4b gene (Cdkn2b) encodes p15(Ink4b), a cyclin-dependent kinase inhibitor. It has been implicated in playing a role in the development of acute myeloid leukemia (AML) in man, since it is hypermethylated with high frequency. We provide evidence that the gene is a tumor suppressor for myeloid leukemia in mice. The evidence is twofold: (1) retrovirus-induced myeloid leukemias of the myelomonocytic phenotype were found to have hypermethylation of the 5' CpG island of the Ink4b gene, and this could be correlated with reduced mRNA expression, as demonstrated by TaqMan real-time PCR. p15(Ink4b) mRNA expression in a leukemia cell line, with hypermethylation at the locus, was induced following treatment with 5-aza-2'-deoxycytidine. (2) Targeted deletion of one allele in mice by removal of exon 2 increases their susceptibility to retrovirus-induced myeloid leukemia. Mice deficient in both alleles were not more susceptible to myeloid disease than those deficient in one allele, raising the possibility that there are opposing forces related to the development of myeloid leukemia in Ink4b null mice.