Graduation Date

Fall 12-20-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Cancer Research

First Advisor

Shannon M. Buckley

Second Advisor

Jennifer Black

Third Advisor

Gargi Ghosal

Fourth Advisor

R. Katherine Hyde

Abstract

Hematopoiesis is a critical system that provides blood cells necessary for nutrient and oxygen transfer throughout an organism and for protection from harmful agents. It is maintained throughout life by the hematopoietic stem cells (HSCs) that reside within the bone marrow (BM) and are responsible for providing billions of new cells each day. HSCs maintain a careful balance between a quiescent state, that sustains the integrity of the stem cells, and a proliferative state, that provides new cells to replenish those that undergo apoptosis. Hematopoiesis is regulated by a variety of intrinsic pathways and extrinsic signals such as cytokine signaling, transcription factor expression, and post-translational regulation, including the ubiquitin proteasome system (UPS). Dysregulation of these pathways can lead to hematopoietic failure or malignant transformation. The key components of the UPS are the E3 ubiquitin ligases that determine which proteins are degraded within the cell.

We identified FBXO9, the substrate recognition component of the Skp1-Cul1-F-box E3 ligase complex, as an important regulator of normal and malignant hematopoiesis. To facilitate study of FBXO9 in HSC maintenance and transformation, we engineered the first tissue specific conditional Fbxo9 knockout mouse model. Analysis of BM and blood derived from Fbxo9-/- mice revealed that its loss does not overtly impact HSC function and very few alterations were seen in normal hematopoiesis. A mass spectrometry analysis of Fbxo9-/- BM under conditions of myeloablative stress revealed that knockout of this E3 ligase resulted in the differential expression of proteins involved in metabolism, a process important to maintaining the balance between quiescence and differentiation and in preventing malignant transformation.

When systems important for maintaining HSCs are dysregulated, it often results in the formation of malignancies such as acute myeloid leukemia (AML). AML is the most common form of acute leukemia and second most common form of childhood leukemia. Analysis of adult and pediatric patient datasets revealed low Fbxo9 expression across all AML subtypes when compared to other F-box proteins in the SCF family. Furthermore, patients with inv(16)/t(16;16) have particularly low expression even when compared to other AML subtypes. Utilizing our Fbxo9 knockout mouse model, we observed that loss of Fbxo9 concurrent with expression of the inv(16) fusion protein, CBFB-SMMHC, led to the rapid onset and progression of AML. Alone, CBFB-SMMHC expression results in a block in myeloid differentiation that eventually gives rise to AML but when expression of the fusion protein is combined with loss of Fbxo9, the resulting AML has a more aggressive phenotype with high frequency of immature blasts in the BM and peripheral organs. A mass spectrometry analysis of Fbxo9-/- inv(16) AML tumor cells revealed that loss of Fbxo9 leads to an increase in proteasome component expression and proteasome activity. Furthermore, loss of this E3 ligase resulted in greater sensitivity to proteasome inhibition treatment. Taken together, our studies identify FBXO9 as a novel tumor suppressor of AML and provide evidence that inhibiting the proteasome in patients with low FBXO9 expression could prove more efficacious in treating the disease.

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