Date of Award

Winter 2022

Project Type

Dissertation

Program or Major

Molecular and Evolutionary Systems Biology

Degree Name

Doctor of Philosophy

First Advisor

Brian Barth

Second Advisor

Feixia Chu

Third Advisor

Don Wojchowski

Abstract

Acute myeloid leukemia (AML) is a clonal hematopoietic malignancy with highly heterogeneous molecular and clinical profiles. It is one of the deadliest blood cancers, resulting in approximately 10,000 deaths each year. Impaired hematopoiesis is one of AML’s hallmarks. This entails defective erythropoiesis and proliferation of undifferentiated myeloblasts. AML is also characterized by chemoresistance and high relapse rates. Despite significant advances in treatments, there is no cure for AML. Therefore, there is an emerging and unmet need to shift away from traditional chemotherapeutics and focus on targeted therapies with curative potential. Growth/differentiation factor 1 (GDF1) is a member of the TGFβ superfamily with noted roles in embryonic development. GDF1 is produced from the bicistronic Cers1-Gdf1 gene, which also encodes for ceramide synthase 1 (Cers1). Gdf1 is either downregulated or not expressed at all in AML patients. Our research group has further shown that Gdf1 is downregulated in the bone marrow of transgenic AML mice. Additionally, we have shown that recombinant GDF1 exerts anti-AML efficacy by downregulating pathways of ceramide neutralization. Ceramide is a pro-apoptotic lipid that serves at the center of sphingolipid metabolism. The sphingolipid metabolic pathway is finely tuned, and its dysregulation has implications in cancer. Therefore, sphingolipids and their enzymes may be useful targets for therapeutic discovery. Historically, natural products have played a significant role in the discovery and development of novel drugs. This is attributed to the unique chemical structures and properties of their bioactive compounds. Devil’s club (Oplopnax horridus) is a medicinal plant native to Alaska and the Pacific Northwest. It has been an essential cultural element of indigenous Alaskan people for many centuries. Moreover, different parts of Devil’s club have been utilized to treat a plethora of pathologies including cancer. Recent studies have further demonstrated that Devil’s club extracts have an anti-proliferative effect against colorectal, ovarian, breast, and leukemia cell lines. This study sought to first investigate the role of GDF1 as a regulator of hematopoiesis in AML. Treatment of transgenic AML mice demonstrated that GDF1 was effective in restoring impaired hematopoiesis by i) stimulating the expansion of erythroid progenitors, and ii)decreasing the population of immature myeloid cells. These findings helped to establish an anti-AML role for GDF1. Next, Devil’s club was utilized as an ethnobotanical source for the identification of novel anti-AML agents. This helped to understand the impact of Devil’s club extract and fractions on Gdf1 expression as well as more directly on sphingolipid metabolic targets on sphingolipid metabolism. Initially, an extract and multiple fractions of Devil’s club were screened for an ability to increase the expression of Gdf1. Both an organic phase of an ethanolic extract, as well as fractions derived by separating this extract by silica flash column chromatography, were effective in upregulating Gdf1 expression in AML cell lines. Additionally, Devil’s club upregulated the bicistronic transcript Cers1-Gdf1, but downregulated the expression of the monocistronic Cers1 in a human AML cell line. This is interesting because it may suggest that a bioactive component of this Devil’s club extract regulates the process of alternative mRNA splicing. Further investigation showed that Devil’s club fractions downregulated the expression of Asah1 and Ugcg, which encode for acid ceramidase and glucosylceramide synthase, respectively. Therefore, Devil’s club bioactives also directly regulate aspects of sphingolipid metabolism. Altogether, the present study revealed the ability of GDF1 to modulate hematopoiesis in AML. More so, and for the first time, this study highlighted a unique role for Devil’s club bioactives as regulators of GDF1 and sphingolipid metabolism. In the future, these bioactive compounds found in Devil’s club could serve as leads for development of novel anti-AML therapeutics.

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