Investigation of Ikaros-dependent transcriptional regulation of novel VPAC1 splice variants during epigenetic transformation

Leukemia is caused by uncontrolled proliferation of hematopoietic cells that results in more deaths than any other cancer among children and young adults under the age of 20. The anti-leukemic, transcription factor, Ikaros, is selectively expressed in, and required for proper immune cell development. A reduction in Ikaros DNA binding due to a loss in Ikaros protein, or an overabundance of non-DNA binding, dominant negative Ikaros isoforms, causes leukemogenesis. Ikaros has been shown to regulate transcription of a gene set that controls cellular proliferation, including the neuropeptide receptor vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor-I (VPAC1). Uncovering the mechanisms involved in Ikaros gene regulation is essential to determine how its dysregulation leads to leukemia. Ikaros-mediated transcriptional regulation of VPAC1 is critical to better understand leukemic transformations as the anti-leukemic activities of Ikaros are hypothesized to be driven by VPAC1 signaling. The specific objectives of the research addressed here were to 1.) investigate Ikaros mediated downregulation of VPAC1 using an Ikaros negative cell system, 2.) biochemically characterize how the epigenetic mechanism(s) controlling spontaneous cellular transformation is impinged by Ikaros, and 3.) explain pro- and anti-leukemic activities of VPAC1 by seeking to find novel putative VPAC1 splice variants in human tissue. We successfully established a cell culture based system to study Ikaros downregulation of VPAC1. Additionally, we discovered a ground-breaking observation of a VPAC1 expression spike that occurred during the epigenetic transition to a tumorigenic phenotype. Lastly, we identified 4 putative, novel, splice variants of VPAC1 that were highly expressed in brain tissue and immune cells (T cells). This research provides a superior foundation of knowledge regarding Ikaros regulation of the VPAC1 GPCR gene and provides significant insight into epigenetic cellular transformation leading to leukemogenesis.