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Molecular Genetics of Myeloid Malignancy Predisposition: Insights into Pathogenesis and Therapeutic Translation
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| 자료유형 | 학위논문 |
|---|---|
| 서명/저자사항 | Molecular Genetics of Myeloid Malignancy Predisposition: Insights into Pathogenesis and Therapeutic Translation. |
| 개인저자 | Krutein, Michelle C. |
| 단체저자명 | University of Washington. Pathology. |
| 발행사항 | [S.l.]: University of Washington., 2019. |
| 발행사항 | Ann Arbor: ProQuest Dissertations & Theses, 2019. |
| 형태사항 | 116 p. |
| 기본자료 저록 | Dissertations Abstracts International 81-04B. Dissertation Abstract International |
| ISBN | 9781687959355 |
| 학위논문주기 | Thesis (Ph.D.)--University of Washington, 2019. |
| 일반주기 |
Source: Dissertations Abstracts International, Volume: 81-04, Section: B.
Advisor: Horwitz, Marshall S. |
| 이용제한사항 | This item must not be sold to any third party vendors.This item must not be added to any third party search indexes. |
| 요약 | Preleukemic diseases are highly informed by genetic predisposition and require appropriate models for studying pathogenesis and the progression to hematological malignancy. Although rare, familial platelet disorder (FPD) and severe congenital neutropenia (SCN) have few or no therapies available to patients and also possess high rates of leukemic transformation to myeloid malignancy. With these points in mind, my graduate work aimed to elucidate the molecular mechanisms of ELANE-associated severe congenital neutropenia, identify new genetic mutations causing preleukemic diseases, and evaluate novel therapies for platelet disorder with predisposition to myeloid malignancy. Familial platelet disorder with predisposition to acute myelogenous leukemia is an autosomal dominant disorder caused by monoallelic mutation of RUNX1, initially resulting in half-normal levels of RUNX1 protein. Patients develop leukemia only after a protracted prodrome consisting of thrombocytopenia and bleeding diathesis relating to functional platelet granule deficiency, suggesting that early intervention affords an opportunity for preventing malignant transformation. We hypothesize that pharmacological inhibition of RUNX1 protein degradation may normalize RUNX1 protein levels and restore platelet numbers and function. RUNX1 is rapidly degraded through the ubiquitin-proteasome pathway. Moreover, RUNX1 auto-regulates its own expression. A predicted kinetic property of auto-regulatory circuits is that transient perturbations of steady-state levels result in continued maintenance of expression at adjusted levels, even after inhibitors of degradation or inducers of transcription are withdrawn, suggesting that transient inhibition of RUNX1 degradation may have lasting effects. Here we evaluate cell lines, FPD/AML patient derived induced pluripotent stem cells (iPSC), and FPD/AML primary bone marrow cells and show that, in some circumstances, transient expression of exogenous RUNX1 or inhibition of steps leading to RUNX1 ubiquitylation and proteasomal degradation restore RUNX1 levels, thereby advancing megakaryocytic differentiation in vitro. Thus, drugs retarding RUNX1 proteolytic degradation may represent a therapeutic avenue for treating bleeding complications and preventing leukemia in FPD/AML.Heterozygous mutations in ELANE, encoding the potent serine protease, neutrophil elastase (NE), cause cyclic neutropenia (CyN) and are the most common cause of severe congenital neutropenia (SCN). Patient presentation is marked by profoundly low neutrophil counts accompanied by predisposition to myelodysplasia (MDS) and acute myeloid leukemia (AML). There is no unified theory of SCN or CyN pathogenesis. However, out of the >100 mutations recorded in SCN and CyN, none of these mutations have been found to encompass the three catalytic residues necessary for NE proteolysis, which may suggest retention of these residues is important for SCN and CyN pathology. To address this question, I developed novel iPSC models of EA-associated severe congenital neutropenia via genome editing strategies using CRISPR-Cas9 targeting with homology directed integration of synthetically designed non-viral vectors. These vectors either harbored an aggressive SCN mutation or a single residue substitution of the catalytic serine of neutrophil elastase. Additional work must be performed to generate iPSC lines possessing both the SCN and catalytic inactivation mutation in cis as well as a wild type ELANE control iPSC line. All vectors contained a green fluorescent protein (GFP) gene trap whereby mutant NE expression is announced through GFP reporting.These cell lines allow us the opportunity to investigate how catalytic activity of neutrophil elastase influences neutrophil development and SCN pathology, both questions that are unanswered or under scrutiny in the field. It is also critical to mention that our method of genome editing creates models whereby expression of the mutant protein is detectable through a reporter. This characteristic makes our models superior to other existing iPSC models that have not been able to achieve mutant protein reporting due to direct reprogramming of primary SCN samples. Additionally, these integration vectors can be easily adapted to harbor any desired changes in exon 4 or 5 of ELANE. Lastly, although neither homozygous or S202A mutations in ELANE have been observed in normal, SCN, or CyN individuals, these novel cell models will provide us with the tools to determine the interaction between NE proteolysis and granulopoiesis in both normal and diseased states.Congenital neutropenia is a genetically heterogeneous disease whereby our lab has contributed to this growing list of genetic factors found causative of neutropenia. In order to expand the current knowledge of neutrophil development and biology it is critical for us to continue the search for novel genes or mutations that produce a neutropenic phenotype. Genetic screening of neutropenic children in two unrelated families revealed the same T679I variant of unknown significance in the gene SUZ12. A critical transcription factor governing stem cell differentiation, SUZ12 protein normally facilitates epigenetic remodeling through global H3K27me3 yet has not been reported as having a role in neutrophil development specifically. I reprogrammed primary patient samples to SUZ12-iPSCs and subsequently subjected them to hematopoietic stem cell (HSC) and neutrophil differentiation which recapitulated phenotypes observed in patients. Epigenetic landscape evaluation of SUZ12-iPSCs via western blot and chromatin immunoprecipitation sequencing (ChIPseq) revealed reduced H3K27me3 repressive genome markers, elevated H3K4me3 activation markers, and some differences in SUZ12 binding. These studies reflect the first report of mutations in epigenetic proteins, more specifically SUZ12, as being causative of SCN. |
| 일반주제명 | Pathology. |
| 언어 | 영어 |
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