Mechanisms and Treatment of Hajdu Cheney Syndrome

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PROJECT SUMMARY/ABSTRACT Notch receptors play a critical role in cell fate decisions and in the regulation of bone remodeling, either directly or through the induction of their target genes, namely Hairy Enhancer of Split (Hes) and Hes-related with YRPW motif (Hey). Hajdu Cheney Syndrome (HCS) is a devastating disease characterized by developmental abnormalities, acroosteolysis and bone loss with fractures. HCS is associated with mutations in exon 34 of NOTCH2 upstream of the PEST domain leading to NOTCH2 stabilization and gain-of-function. We created a mouse model of HCS (Notch2tm1.1Ecan) that presents with osteopenia due to enhanced osteoclastogenesis and bone resorption. These events are secondary to an increase in receptor activator of nuclear factor Kappa B ligand (RANKL) by cells of the osteoblast lineage, and to direct effects of NOTCH2 in cells of the myeloid lineage. In this lineage, the expression of HES1 is induced by NOTCH2 and the inactivation of Hes1 in the osteoclast lineage reverses the in vitro and in vivo phenotype of HCS mutants. Moreover, HES1 induces osteoclastogenesis directly and as a result causes osteopenia in vivo. This reveals a previously unrecognized function of HES1 in osteoclast differentiation and function that will be explored as part of the proposed research. An additional goal of the proposed work is to develop ways to correct the skeletal manifestations of HCS by targeting the mutation with Notch2 antisense oligonucleotides (ASO), a strategy that would be applicable to other genetic disorders of the skeleton. Our specific aims are: Aim 1) To determine the role of HES1 in osteoclastogenesis. Our goals are to induce and inactivate Hes1 specifically in cells of the osteoclast lineage to determine its contribution to osteoclast differentiation and bone remodeling as determined by microcomputed tomography and histomorphometry; Aim 2) To establish that the Notch2tm1.1Ecan mutation can be targeted. We will determine whether the Notch2tm1.1Ecan mutation can be downregulated specifically and the Notch2tm1.1Ecan skeletal phenotype ameliorated by the administration of antisense oligonucleotides targeting the Notch26955C>T mutation; and Aim 3) To validate the mechanisms of the HCS phenotype and ASO approach in NOTCH2 mutant-induced pluripotent (iPS) cells. To this end, we created NOTCH2HCS mutant iPS cell lines to study the impact of the mutation on osteoclastogenesis and the efficacy of ASOs in downregulating NOTCH2 mutant alleles. The goals of the proposed work are to understand the mechanisms and develop specific antisense technology to treat the skeletal manifestations of a devastating NOTCH2-associated disease.