MOLECULAR PATHOGENESIS OF CHROMOSOME 16 INVERSION INHUMA

16号染色体反转INHUMA的分子发病机制

• 批准号: 7315950
• 负责人: PU PAUL LIU
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7315950
• 关键词: MOLECULAR; PATHOGENESIS; CHROMOSOME; 16; INVERSION

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 in T cell development, we analyzed mice compound-heterozygous for a null and a hypomorphic GFP knockin alleles (Cbfb-/GFP), and with a conditional, dominant-negative MYH11 knockin allele (Cbfb+/56M). CD4 expression was derepressed and thymocyte development was blocked at DN1 and DN2 stages in E17.5 Cbfb -/GFP thymus, with a 20-fold reduction of total thymocyte numbers. Although Cbfb-MYH11 derepressed CD4 expression in in vitro reporter assays, such derepression was not observed in the Cbfb-MYH11 thymus. However, in adult thymus, Cbfb-MYH11 expression led to a 10-fold reduction in thymocyte numbers, resulting from both impaired survival of CD4+CD8+ thymocytes and a differentiation block at DN3 stage. Our data suggest that Cbfb is critical for several stages of T cell development and help to explain why CBFB-MYH11+ cells cannot be detected in the T cell lineage in AML patients with this fusion gene. A manuscript reporting these findings is submitted. 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 four 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. So far, chimeras have been generated with ES cells containing two internal deletions of CBFbeta-SMMHC. 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 team effort. 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. In humans, heterozygous mutations of RUNX2 are associated with Cleidocranial Dysplasia (CCD). Runx2+/- mice show a similar phenotype to CCD. We showed previously that the Runx-binding partner Cbfbeta is also important for skeletal formation; mice homozygous for a hypomorphic allele of Cbfb have a phenotype similar to Runx2+/- mice. In addition, we have investigated whether Cbfbeta plays a role is skeletal development in humans. We have identified a patient with CBFB haploinsufficiency but no mutations in RUNX2, who has delayed cranial ossification similar to that seen in CCD patients. This supports our data from mouse that Cbfbeta and Runx2 play a similar role in bone formation. To further understand the role of Cbfbeta during skeletogenesis, we used the Cre-lox system to express the dominant negative fusion gene, Cbfb-MYH11, in chondrocytes or osteoblasts. Expression of Cbfb-MYH11 in osteoblasts had no effect on survival or skeletal development, while expression of Cbfb-MYH11 in chondrocytes resulted in decreased ossification and perinatal leathality. This suggests that chondrocyte is the Cbfbeta target in skeletogenesis. To address whether Cbfb and Runx2 cooperate in skeletogenesis, we crossed Runx2+/- mice and mice carrying mutations in Cbfb. We found that Runx2+/- mice also defective in Cbfb show increased severity of the skeletal phenotype. The increase in severity inversely correlates with Cbfb dosage indicating that Runx2 and Cbfb cooperate during skeletogenesis. We are interested in identifying other factors that cooperate with Runx2 and have started a modifier screen by analyzing skeletal development in newborns from crosses between Runx2+/- and ENU-treated BalbC/J mice. To date, we?ve found a naturally occurring mutation in the BalbC/J background that partially rescues the Runx2+/- phenotype and an ENU induced mutation that enhances the Runx2+/- phenotype. We are in the process of mapping these mutations and screening for others.