Dissertation
Using Retroviral Vector Genotoxicity To Study Hematopoietic Stem Cell Diseases
Doctor of Philosophy (PhD), Washington State University
01/2015
Handle:
https://hdl.handle.net/2376/118398
Abstract
Myelodysplastic syndromes (MDS) are a group of hematopoietic stem-cell disorders marked by dysplasia that often progresses to acute myeloid leukemia (AML). The molecular analysis and treatment of MDS/AML has been impeded by the heterogeneity of the disease. Our goal was to identify the collaborating gene alterations implicated in MDS and the progression to AML to further define the disease heterogeneity for patient prognosis. Mutations in the transcription factor RUNX1 have been identified in 15-40% of MDS patients that progressed to AML. Here we performed a forward retroviral mutagenesis screens in mouse CD105+, Sca-1+ bone marrow cells, transduced with the RUNX1 D171N mutant, to identify potential collaborating gene mutations using retroviral mediated genotoxicity.
Interestingly, among integration sites identified using our novel shuttle vector rescue technique, inositol-trisphosphate 3-kinase B (ITPKB) was recovered. We identified ITPKB as an independent prognostic factor in AML patients. ITPKB is a known master regulator of hematopoietic stem cells, and may be a novel therapeutic target for AML patients with alterations.
To increase the sensitivity of our retroviral mutagenesis screen, we adapted the modified genomic sequencing PCR protocol for use on the Illumina MiSeq paired end platform. Using this sensitive protocol, over 400 unique retroviral integration sites were recovered. Analysis of the top gene candidates recovered from mutagenized cultures identified a new gene signature in AML patient samples. The “GIST” signature is comprised of gene alterations in GRK5, ITPKB, SERINC3, and TUBB3 genes. Patients harboring alterations in these genes had greatly reduced survival times, higher rates of relapse, and differential responses to induction therapies. These findings highlight the power of our approach to identify potential biomarkers for AML diagnosis and treatment.
Using retroviral mutagenesis in an in vivo bone marrow transplantation assay, we identified retroviral integration sites near genes that promoted long term survival of RUNX1 D171N mutant cells, and induced MDS. This highlights the heterogeneous molecular features of MDS/AML and provides crucial insights for the understanding of tumorigenic pathways. Thus, we have developed a useful retroviral mutagenesis screen for MDS/AML that should help in the understanding of the molecular basis of MDS and the progression of AML.
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Details
- Title
- Using Retroviral Vector Genotoxicity To Study Hematopoietic Stem Cell Diseases
- Creators
- Dustin Thomas Rae
- Contributors
- Grant D Trobridge (Advisor)Gary G Meadows (Committee Member)Buel D Rodgers (Committee Member)David X Liu (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Pharmacy and Pharmaceutical Sciences, College of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 133
- Identifiers
- 99900581525801842
- Language
- English
- Resource Type
- Dissertation