Heterogeneous Nuclear Ribonucleoprotein K (Hnrnp K) Overexpression and Its Interaction With Runx1 Rna In Acute Myeloid Leukemia

<p>Acute myeloid leukemia (AML) is an often devastating hematologic malignancy with 5-year overall survival lingering near 20%. Acquiring a deeper understanding of molecular underpinnings of leukemogenesis will provide a basis for developing more effective therapeutic strategies for patients with AML.</p> <p>Here, we identified overexpression of hnRNP K as a recurrent abnormality in a subset (~20%) of AML patients. High levels of this RNA-binding protein associated with inferior clinical outcomes in <em>de novo </em>AML. Thus, to evaluate its putative oncogenic capacity in myeloid disease, we overexpressed hnRNP K in murine hematopoietic stem and progenitor cells isolated from fetal liver cells (FLCs). We revealed that hnRNP K-overexpression alters self-renewal capacity and differentiation potential of these cells <em>in vitro. </em>Such findings were recapitulated <em>in vivo, </em>as murine recipients of hnRNP K-overexpressing FLCs developed fatal myeloproliferative phenotypes.</p> <p>To elucidate mechanisms by which hnRNP K overexpression causes myeloid neoplasia, we took an unbiased approach utilizing RNA-immunoprecipitation sequencing (fRIP-Seq). Among RNA transcripts interacting with hnRNP K was <em>RUNX1</em>—a pivotal transcriptional regulator of definitive hematopoiesis commonly mutated or translocated in AML. Consensus hnRNP K binding sites were identified in the 5’ UTR and near the 3’ splice site in intron 5-6 of <em>RUNX1. </em>Fluorescence anisotropy studies confirmed these interactions were direct, and abrogated when hnRNP K binding sites within <em>RUNX1 </em>were mutated. Manipulating hnRNP K expression in human cell lines and murine FLCs substantially altered <em>RUNX1 </em>splicing surrounding exon 6. RNA-sequencing of FLCs confirmed these data, exposing <em>RUNX1 </em>as a significantly differentially spliced entity in the context of hnRNP K overexpression. Importantly, the protein product of this spliced product (RUNX1ΔEx6) exhibited disparate transcriptional activity in reporter assays compared to full-length RUNX1. Furthermore, we identified KH3 as the hnRNP K domain most critical for these splicing alterations; deletion of KH3 markedly abrogated hnRNP K overexpression phenotypes <em>in vitro. </em></p> <p>In sum, we established hnRNP K as an oncogene in myeloid leukemia that binds <em>RUNX1 </em>RNA, altering its splicing and subsequent transcriptional activity. These findings shed light on a mechanism of myeloid leukemogenesis, paving the way for drug discovery efforts to improve outcomes for patients with this disease.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2020

Authors dc:creator

• Aitken, Marisa
• <p>https://orcid.org/0000-0002-7937-531X</p>

Contributors dc:contributor

• Sean Post, Ph.D.
• Scott Evans, M.D.
• Marina Konopleva, M.D., Ph.D.

• hnRNP K
• RNA binding proteins
• RNA splicing
• AML
• myeloproliferative disease
• mouse model
• Animal Experimentation and Research
• Cancer Biology
• Cell Biology
• Genetics
• Hemic and Lymphatic Diseases
• Immune System Diseases
• Immunopathology
• Medicine and Health Sciences

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/994

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-2043