Date of Award

Spring 2021

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

W. K Thomas

Abstract

Acute myeloid leukemia (AML) is cancer of the myeloid lineage of blood cells. In AML, hematopoietic precursors acquire mutations or chromosomal changes that cause differentiation arrest. This results in an uncontrolled proliferation of these malignant cells in the bone marrow that can interfere with normal blood cell production. AML progresses rapidly and can be fatal within weeks if left untreated. The only current curative treatment is bone marrow transplant, which has potentially life-threatening side effects. Thus, a better understanding of the underlying biology of AML is needed to enable the development of better therapeutic modalities.Ceramides are a family of wax-like lipids in the broader category of sphingolipids. Ceramide is comprised of a sphingoid base linked to a fatty acid through an amide group. Recently, ceramides have been appreciated for their bioactive functions including as regulators of apoptosis. This is significant because ceramide metabolic routes persist in AML that may be exploited for therapeutic development. Recently, it was shown that nanoliposomal C6-ceramide (Lip-C6) exerts unique therapeutic efficacy towards AML with myelodysplastic syndrome-related changes (AML-MRC). In contrast, other forms of AML were resistant to Lip-C6 due to enhanced ceramide metabolism. Interestingly, in these Lip-C6-resistant AMLs the gene encoding for growth/differentiation factor 1 (GDF1) is downregulated. GDF1 is encoded from a rare bicistronic gene that also encodes for ceramide synthase 1. GDF1 is presently only appreciated to have roles in embryonic and cardiac development. However, its genetic link to a ceramide biosynthesizing enzyme as well as its inverse relation to Lip-C6-resistant AML suggest that it may also exert a role in ceramide metabolism. This dissertation research first explored a hypothesis that GDF1 regulates ceramide detoxification in AML. GDF1 expression was variably expressed across AML subtypes but was mostly downregulated in Lip-C6-resistant AML. Moreover, there was an inverse relationship between GDF1 expression and genes encoding for ceramide neutralizing enzymes. This inverse relationship was validated as treatment of AML cell lines with recombinant GDF1 downregulated the expression of these same genes. Interestingly, recombinant GDF1 was also able to uniquely promote SMAD2/3 phosphorylation while concurrently downregulating STAT2 tyrosine phosphorylation in a transforming growth factor beta receptor 1 (TGFR1)-dependent manner. Next, this dissertation research evaluated the ability of GDF1 to regulate hematopoiesis and exert an anti-AML therapeutic effect. Recombinant GDF1 restored hematopoiesis and promote erythropoiesis (red blood cell development) both in vitro and in vivo. GDF1 was unable to impact hematopoiesis in normal bone marrow, suggesting that its hematopoietic-regulatory therapeutic effect was specific to abnormal and malignant situations. Furthermore, GDF1 treatment exerted combinatorial anti-AML efficacy with cytarabine and extended the overall survival of mice engrafted with a highly aggressive AML. The final aspect of this dissertation studied a novel transgenic obese AML mouse model to evaluate links between obesity and AML. Obesity provokes profound changes in lipid homeostasis including by upregulating sphingolipid biosynthesis. Transgenic obese AML mice developed a robust leukemia burden compared with non-obese counterparts. More so, genes responsible for regulation of the ceramide-mediated NADPH oxidase 2 were upregulated in these transgenic obese AML mice. This demonstrated a further sphingolipid-mediated dysfunction that can contribute to the development and progression of AML. Overall, this dissertation research has uncovered important sphingolipid metabolic biology that underlies the development and progression of AML. This has revealed pathways associated with resistance to ceramide-elevating therapeutics such as Lip-C6. Most noteworthy, this research has identified for the first time an ability for GDF1 to regulate ceramide metabolism, hematopoiesis, and erythropoiesis. It has also shown that GDF1 can exert effects through a unique TGFR1-dependent mechanism regulating both SMAD2/3 and STAT3 signaling. Collectively, this has revealed an anti-AML therapeutic ability for GDF1 linked to the regulation of ceramide metabolism.

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