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Access Type

WSU Access

Date of Award

January 2025

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Cancer Biology

First Advisor

Yubin Ge

Abstract

Acute myeloid leukemia (AML) is an aggressive and extremely heterogenous form of acute leukemia that has a sharp rise in incidence with increasing age. Unfortunately, as patients age, they have limited therapeutic options due to the intensive nature of chemotherapeutic agents and decreased resilience. There has been increased focus on development of targeted therapeutics with improved tolerability for these patients. This has led to a new standard of care in the combination of venetoclax (VEN) and azacitidine (AZA) for AML patients who are ≥75 years or unfit for intensive chemotherapy. Though initially promising, resistance to the combination therapy is clinical challenge and VEN+AZA-resistant patients have dismal outcomes. Though these therapies are used in combination, there have yet to be pointed investigations into resistance to both VEN and AZA and there is a need for a model to do so. Another major contributor to poor outcomes in AML is the persistence of leukemia stem cells (LSCs) in patients following treatment. LSCs lead to disease recurrence and resistance to therapy and are associated with worse prognosis in AML patients but are difficult to target therapeutically. Here we seek to develop models to study VEN+AZA resistance in AML and investigate the use of the novel imipridone, ONC213, against both VEN+AZA resistant cells and LSCs. In this dissertation work, we sought to design models to study the resistance to VEN+AZA, investigate mechanisms of resistance and characterize resistant cell lines, and to explore therapeutic options to address resistance through the novel imipridone ONC213. To better understand the mechanisms of resistance, we developed VEN+AZA-resistant AML cell lines, MV4-11/VEN+AZA-R and ML-2/VEN+AZA-R, from previous sensitive MV4-11 (FLT3-ITD) and ML-2 (FLT3 wt) parental cells through step-wise increases of VEN+AZA in the media. These VEN+AZA resistant cell lines show >300-fold persistent resistance compared to the parental lines. We demonstrate that these cells have unique metabolic profiles, including significantly increased changes in nucleotide metabolic pathways, changes in fatty acid and amino acid metabolism and increased utilization and reliance on glycolysis. Changes in fatty acid metabolites are supported by increases in the fatty acid transporter CD36 and changes in deoxynucleotide metabolism are supported by increases in ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2) in the resistant cells compared to the parental cells. VEN+AZA resistant cells demonstrate increased utilization and reliance on glycolysis and inhibition of glycolysis with 2-Deoxy-D-glucose (2-DG) re-sensitized the resistant cells to VEN+AZA therapy. VEN+AZA-R cells also have increased levels of the antiapoptotic protein Mcl-1, which is commonly increased in cells with resistance to VEN. Overexpression of Mcl-1 and knockdown of Bax, respectively, result in resistance to VEN+AZA in parental MV4-11 cells. Our results provide insight into the molecular mechanisms contributing to VEN+AZA resistance and provide a model for further investigations into characterization and development of treatment strategies. To investigate how to overcome VEN+AZA resistance in these models, we utilized ONC213, an imipridone agent being studied in our lab with known mitotoxic properties and antileukemic activity. ONC213 resensitizes VEN+AZA-R cells to VEN therapy in a dose dependent manner and the combination of ONC213+VEN demonstrates synergy and potent antileukemic activity against VEN+AZA-resistant and -sensitive cells and against primary AML patient samples. ONC213+VEN demonstrates efficacy against cell line derived xenograft models. Further ONC213+VEN effectively targets AML progenitor and LSCs both ex vivo and in vivo, extending the activity of the combination to target an additional cause of relapse in AML. The combination of ONC213 and VEN induces a potent integrated stress response (ISR) in AML cells that is associated with reduced mitochondria function and strong reductions in Mcl-1levels. The reduction of Mcl-1 and targeting mitochondrial respiration is important to ONC213+VEN activity as demonstrated in Mcl-1 overexpression (OE) and Bak/Bax knockdown (KD) models. Collectively, the work done in this dissertation study establishes a new model for studying VEN+AZA resistance, examines a new agent, ONC213, to address this resistance, and determines that ONC213+VEN induction of mitochondrial stress in AML cells is a powerful tool to address both VEN+AZA resistance and for targeting LSCs.

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