Research Focus

Thesis Project Title: Modeling Severe Congenital Neutropenia with iPSCs
Neutrophils are a critical component of blood, functioning as the first line of defense against infection. Neutrophil homeostasis requires constant production and is vital for a proper immune response to pathogens. Neutropenia is characterized by an abnormally low number of neutrophils in the blood, as a result either of diminished production or increased cell death. Congenital neutropenia is identified as either cyclic neutropenia (CN) where neutrophil levels drastically dip and rise over a 21-day period, or as severe congenital neutropenia (SCN), in which counts are chronically low due to an arrest in neutrophil precursor differentiation at the promyelocyte stage. Those affected by neutropenia are significantly more susceptible to bacterial and fungal insults, and such infections can rapidly result in sepsis. Importantly, SCN can progress to myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML).
Although genetically heterogeneous, CN and SCN are most frequently attributable to heterozygous mutations in the gene ELANE, which encodes the neutrophil granule serine protease, neutrophil elastase (NE). The role of NE in regulating granulopoiesis remains unclear, although biochemical and cell biological abnormalities including premature catalytic activation of the enzyme, subcellular mislocalization, and induction of the unfolded protein response (UPR) and ER stress response have been described. However, ELANE expression is brief, and is restricted to promyelocytes. Therefore, it has been difficult to determine how expression of wild type or mutant ELANE contributes to promyelocytic maturation.
Mouse knock-in models of human ELANE mutations fail to recapitulate neutropenia. For this reason, the Horwitz lab and collaborators generated patient-derived iPSCs from individuals with a variety of ELANE mutations that fully recapitulate disease features, including promyelocytic maturation arrest, neutropenia, monocytosis, subcellular mislocalization, and induction of UPR and ER stress responses. An immediate goal is to define the exact point during induced iPSC differentiation when ELANE is expressed and determine its consequent downstream effects. By using single cell RNA-seq analysis, we hope to capture the brief burst during which ELANE is transcribed and examine how its finite expression modulates granulopoiesis in iPSCs derived from both patients and healthy controls in order to shed light upon how ELANE facilitates neutrophil maturation and its mutations contribute to characteristic cellular phenotypes.

Awards
Poster session winner, Annual Pathology Department Retreat 2015 University of Washington Cardiovascular Pathology Training Grant recipient 2015

Publications

A high-throughput-compatible FRET-based platform for identification and characterization of botulinum neurotoxin light chain modulators.
Caglič D, Bompiani KM, Krutein MC, Čapek P, Dickerson TJ.
J Vis Exp. 2013 Dec 27;(82):50908. doi: 10.3791/50908.
Identification of clinically viable quinolinol inhibitors of botulinum neurotoxin A light chain. Caglič D, Krutein MC, Bompiani KM, Barlow DJ, Benoni G, Pelletier JC, Reitz AB, Lairson LL, Houseknecht KL, Smith GR, Dickerson TJ