Chromosome 16 inversion, inv(16), is one of the most common chromosome abnormalities in human acute myeloid leukemia (AML). A fusion gene between the core binding factor B (CBFB) gene and the myosin heavy chain 11 (MYH11) gene is generated by this inversion. CBFB encodes transcription factor CBFbeta while MYH11 encodes the smooth muscle form of myosin heavy chain (SMMHC). CBFbeta is the obligate partner of RUNX proteins, a group of related transcription factors playing critical roles in development and diseases. Using a mouse knock-in strategy, we showed that the fusion gene Cbfb-MYH11 dominantly represses Runx/Cbfb function, since embryos heterozygous for the knocked-in fusion gene had similar phenotypes as the Runx1 or Cbfb null embryos (block of hematopoiesis and lethality due to vascular defect). In order to study the function of Cbfb gene in T cell development in adult mice, we used a mouse line with floxed exons 5 and 6 of Cbfb inserted 5? to the Cbfb-MYH11 fusion cassette, which produced pseudo-normal mice (loxKI). By crossing the loxKI mice with mice expressing the Cre gene under the control of the T cell-specific Lck promoter (LckCre), we generated LckCre-loxKI double positive mice, in which the floxed exon 5 and 6 were deleted and Cbfb-MYH11 re-expressed only in the thymus when Lck started to express. T cell development in the Tg(Lck-Cre)/Cbfb+/loxKI mice was severely impaired. The thymus of the mutant was about half the size of their littermate controls. The total number of thymocytes was reduced by 10 to 20-fold. Furthermore, the size of the mature T cell population in the spleen was smaller. Tunnel assay and annexin V staining showed increased apoptosis of the mutant thymocytes. FACS analysis of thymocytes from 4 to 12 week old mice showed a developmental blockage at double negative T cell stage 3. TCRbeta signaling and TCR?? cell development were found to be normal, suggesting that the defect from Cbfb-MYH11 is downstream of TCRbeta signaling. In vitro reporter assay showed that Cbfb-MYH11 was able to de-repress CD4 silencer function, consistent with its dominant-negative effect on Runx functions. Such de-repression would have resulted in increased CD4/CD8 ratio in vivo, as reported previously for the Runx1 or Runx3 deficiencies. However, the CD4 /CD8 ratio decreased unexpectedly in the LckCre-loxKI double positive mice. We will cross the LckCre-loxKI double positive mice with mice carrying mutations of the Runx-binding sites in the CD4 silencer, to see if Cbfb-MYH11 regulates CD4 expression through mechanisms other than affecting the silencer. Our data suggested that Cbfb plays an important role in T cell development. It is likely that the phenotype reflects the combined effect of missing all three Runx genes, since the phenotype described here is more severe than either Runx1 or Runx3 null alone. A manuscript reporting these findings is in preparation. Molecularly the fusion protein CBFbeta-SMMHC is able to interact with Runx1, homo-dimerize and multimerize. CBFbeta-SMMHC can sequester Runx1 in the cytoplasm. It has higher affinity for Runx1 and protects Runx1 from ubiquitin degradation better than the wild type Cbfbeta does. In addition, certain region of SMMHC may serve as a transcription repressor. However, the in vivo functional importance of these mechanisms is not clear. We intend to determine the relative importance of CBFbeta-SMMHC functional domains through the knock-in approach. Five targeting constructs with three C-terminal and two internal deletions have been generated, deleting the domains for multimerization, transcriptional repression, Runx1 sequestration domain, Runx1 stabilization, and high affinity Runx1 binding, respectively. ES cell lines for two of the constructs have been generated. Targeted ES cells will be used to generate chimeric mice, which will be bred to wild type female mice to assess the ability of the truncated Cbfb-MYH11 genes to block embryonic hematopoiesis. Either the chimeras or the F1 heterozygous mice (if they are viable) will then be treated with ENU (to induce leukemia) and monitored for leukemia development. Results from these knock-in mice will help us to determine which functional domains are important for leukemogenesis. Such knowledge may enhance our understanding of the leukemogenic process and provide more specific targets for developing therapeutic chemicals for leukemia treatment. Chromosomal rearrangements affecting RUNX1 and CBFB are common in acute leukemias. These mutations result in the expression of fusion proteins that act in a dominant negative manner to suppress the normal function of the RUNX1/CBFbeta complex. In addition, loss-of-function mutations in RUNX1 have been identified in sporadic cases of AML and in association with familial platelet disorder with propensity to develop AML. We recently showed that Runx1 deficient chimeric mice were indeed more prone to leukemogenesis. Therefore, we hypothesize blockage of RUNX1 function is an important step in leukemogenesis for CBFbeta-SMMHC. In collaboration with Drs. John Bushweller and Milton Brown in University of Virginia, we have initiated a project to develop small chemicals specifically inhibiting the interaction between CBFbeta-SMMHC and RUNX1, which therefore are expected to release the blockage of RUNX1 function. This is a project funded by the Leukemia and Lymphoma Society and will be a teamwork. Dr. Bushweller will identify lead compounds in silico through computer modeling. Dr. Brown will synthesize the chemicals and test their binding capability by NMR and FRET assays. We are using a cell-based assay to assess if the chemical inhibitors can block the interaction between CBFbeta-SMMHC and Runx1, and will test their ability to block leukemia development in our mouse models. Such chemicals may become the next generation of leukemia drugs, which are highly specific and less toxic.
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