Mechanism of leukemogenesis by the inv(16) chimeric gene CBFB/PEBP2B-MHY11 Katsuya Shigesada* ,1 , Bart van de Sluis 2 and P Paul Liu* ,2 1 Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan; 2 Oncogenesis and Development Section, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA Inv(16)(p13q22) is associated with acute myeloid leukemia subtype M4Eo that is characterized by the presence of myelomonocytic blasts and atypical eosinophils. This chromosomal rearrangement results in the fusion of CBFB and MYH11 genes. CBFb normally interacts with RUNX1 to form a transcriptionally active nuclear complex. The MYH11 gene encodes the smooth muscle myosin heavy chain. The CBFb-SMMHC fusion protein is capable of binding to RUNX1 and form dimers and multimers through its myosin tail. Previous results from transgenic mouse models show that Cbfb-MYH11 is able to inhibit dominantly Runx1 function in hematopoiesis, and is a key player in the pathogenesis of leukemia. In recent years, molecular and cellular biological studies have led to the proposal of several models to explain the function of CBFb-SMMHC. In this review, we will first focus our attention on the molecular mechanisms proposed in the recent publications. We will next examine recent gene expression profiling studies on inv(16) leukemia cells. Finally, we will describe a recent study from one of our labs on the identification of cooperating genes for leukemogenesis with CBFB-MYH11. Oncogene (2004) 23, 4297–4307. doi:10.1038/sj.onc.1207748 Keywords: CBFb-MYH11; CBFb-SMMHC; inv(16); leukemia; hematopoiesis Molecular basis for the dominant inhibition of RUNX1-dependent transcription by CBFb-SMMHC Identification of dual functional domains in CBFb-SMMHC It has been shown that the phenotype of heterozygous Cbfb-MYH11 knockin mice is very similar to that of Runx1 knockout mice (Castilla et al., 1996). The results imply that CBFb-SMMHC inactivates the function of Runx1 nearly completely despite the presence of a residual normal Cbfb/PEBP2b. In order for this to occur, CBFb-SMMHC would have to outcompete Cbfb at the step of heterodimerization with Runx1 first of all, subsequent to which a certain mechanism(s) should act to bring Runx1 into a functionally incompetent state in the end (repression). If one of these steps were lacking or impaired, CBFb-SMMHC would fail to elicit any substantial dominant-negative effect regardless of how well the other step might work. Until recently, however, most functional studies of CBFb-SMMHC have centered around the mechanism of repression, paying relatively little attention to the first heterodimerization step. Nevertheless, evidence sugges- tive of its enhanced ability for heterodimerization (hyper-heterodimerization) has come from our previous finding that CBFb-SMMHC is predominantly localized in the cytoplasm in association with the actin cytoske- leton, and simultaneously capable of sequestering RUNX proteins to the cytoplasm in a manner over- riding the intrinsic or artificially modulated ability of Runx to localize in the nucleus (Lu et al., 1995; Adya etal., 1998; Kanno etal., 1998). In sharp contrast, Cbfb tends to localize in the cytoplasm in a diffuse pattern by itself, and can be partly, although not completely, translocated into the nucleus only through heterodimer- ization with Runx protein. Furthermore, CBFb- SMMHC can stabilize RUNX1 against intracellular proteasome-mediated degradation much more strongly than Cbfb (Huang et al., 2001a). To characterize the hyper-heterodimerization activity of CBFb-SMMHC more directly, we conducted ex- tensive functional analyses using a series of CBFb- SMMHC deletions as truncated exon by exon either C- terminally or internally (Huang etal., 2003). Through in vitro binding experiments by means of co-immunopre- cipitation and GST-pulldown assays as well as an intracellular hyperprotection assay, it was confirmed that CBFb-SMMHC can heterodimerize with RUNX1 at an affinity higher than that of CBFb by one order of magnitude or more. Parallel analyses with RUNX1 deletions revealed that the region of RUNX1 required for hyper-heterodimerization is the Runt domain. Further, a minimum region of the myosin tail respon- sible for hyper-heterodimerization was further mapped to exons 33–36 (residues 166–363). The myosin tail alone, of course, did not show any detectable binding to the Runt domain. However, appreciable hetrodimeriza- tion activities became detectable when the myosin tail was fused to PEBP2b proteins made heterodimerization- defective due to double point mutations (residues 64 and *Correspondence: K Shigesada; E-mail: kshigesa@virus.kyoto-u.ac.jp; P Liu; E-mail: pliu@mail.nih.gov Oncogene (2004) 23, 4297–4307 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc