PROJECT SUMMARY/ABSTRACT Deletions of all or part of chromosome 7 [-7/del(7q)] are among the most common karyotypic abnormalities in myeloid diseases, particularly high-risk myeloid diseases. Myelodysplastic syndrome (MDS) is a series of clonal disorders characterized by ineffective hematopoiesis, leading to peripheral cytopenias and dysplasia in one or more blood lineages with risk of transformation to acute leukemia. -7/del(7q) is found in 10% of adult MDS cases and, strikingly, in up to 50% of pediatric MDS cases. The presence of -7/del(7q) is associated with a poor karyotype and higher risk MDS, and carries a worse prognosis than cases with diploid chromosome 7. -7/del(7q) is often the only cytogenetic finding, and in a subset of pediatric MDS cases is the sole detectable molecular abnormality, strongly suggesting a driving role for chromosome 7 deletions in disease pathogenesis. There have been no new therapies for MDS in over a decade, highlighting an urgent need to better understand the recurrent genetic features of MDS that may lead to new treatment options. The lack of synteny between human and mouse chromosome 7 is a major barrier in the development of animal models of -7/del(7q). In a breakthrough in the field, our lab identified CUX1, a homeobox transcription factor shown to regulate cell proliferation and apoptosis, as a haploinsufficient myeloid tumor suppressor gene located in a commonly deleted region of 7q. Our lab engineered a doxycycline-inducible shRNA CUX1- knockdown mouse, and mice deficient in Cux1 develop a myeloid disease with trilineage dysplasia and lethal anemia, hallmarks of MDS. These data strongly support a role for this 7q-encoded gene in MDS etiology. However, chromosome 7 deletions are often large and span additional genes that alter hematopoiesis or lead to myeloid disease when deleted in mouse models. These data suggest 7q may be a contiguous gene syndrome region, in which loss of multiple neighboring genes en bloc contributes to disease development. Our preliminary data show CUX1 loss in human K562 leukemia cells decreases the repressive epigenetic histone mark H3K27me3. We further show that combined loss of Cux1 and the 7q gene Ezh2, an H3K27 methyltransferase, in murine hematopoietic progenitors synergistically increases myeloid cell expansion in vitro, compared to either gene alone. These data provide support for the hypothesis that 7q is a contiguous gene syndrome region. This proposal aims to: 1) leverage CRISPR-Cas9 gene editing to identify combinatorial 7q gene deletions that cooperate with Cux1 loss to drive del(7q) pathogenesis; and 2) define mechanisms by which loss of 7q genes drives del(7q) pathogenesis by characterizing the epigenetic and transcriptional landscape of cells deficient in Cux1 alone or with combined Ezh2 loss. This proposal will advance our understanding of chromosome 7 deletions and provide me with a specialized skill set in myeloid neoplasia and genomics that will propel me towar...