Function and Regulation of Equilibrative Nucleobase Transporter 1 in Acute Lymphoblastic Leukemia

• Author / Creator

  Ruel, Nicholas M

• Background: 6-Mercaptopurine (6-MP) is a purine nucleobase analog used for the treatment of acute lymphoblastic leukemia (ALL), inflammatory bowel disease, and other disorders. 6-MP is primarily used in ALL, the most common childhood cancer. Enzymes involved in 6-MP metabolism have been shown to cause variability in 6-MP efficacy and toxicity in ALL patients. However, they only partially explain some of the variability seen. A major understudied factor was that the transport pathway for 6-MP into cells remained unknown, a requirement for 6-MPs mechanism of action. In 2015, it was discovered that the SLC43A3 gene encoded for the Equilibrative Nucleobase Transporter 1 (ENBT1). ENBT1 exists as two separate isoforms, differing by 13 amino acids in the first extracellular loop. Notably, it was determined to be potentially responsible for the uptake of nucleobase analogs, like 6-MP, into cells. If ENBT1 is responsible for the uptake of 6-MP into leukemic cells, then it could potentially modify its toxicity and overall efficacy, providing a biomarker for the success of 6-MP therapy in patients.

  Hypothesis: It is hypothesized that ENBT1 is responsible for the transport of 6-MP into leukemia cells and that the variability of the cellular toxicity of 6-MP is due to variation in the expression of ENBT1 in leukemia cells.

  Methods: ENBT1 will be characterized in HEK293 cells, which are innately deficient in ENBT1, with recombinantly expressed ENBT1. Endogenously expressed ENBT1 will also be investigated, specifically in leukemia cells. Furthermore, the induction of oxidative stress and activation of protein kinase C (PKC) will be assessed in leukemia cells, and the impact of this on the ENBT1-mediated 6-MP uptake.

  Results: HEK293 cells were stably transfected with both isoforms of recombinant SLC43A3 and characterized. SLC43A3-encoded ENBT1 was confirmed to be a purine-selective nucleobase transporter that mediated the bidirectional transport of 6-MP. Notably, SLC43A3-transfected HEK293 cells exhibited 10-fold greater sensitivity to 6-MP than empty-vector transfected cells, showing that ENBT1 is critical for 6-MP activity. In a panel of leukemia cells, endogenous SLC43A3 expression varies and is correlated to the overall uptake of 6-MP into the cells. However, SLC43A3 expression did not correlate to the efficacy of 6-MP across the panel of cells. However, knockdown of SLC43A3 in RS4;11 leukemia cells showed a reduction in the uptake of 6-MP and a decreased sensitivity to 6-MP, showing that the endogenous regulation and plasma membrane expression of ENBT1 is important to 6-MP activity. When looking at the regulation of ENBT1, ENBT1-mediated uptake of 6-MP is decreased following induction of oxidative stress by menadione, which can be reversed by the antioxidant TEMPOL. Finally, activation of PKC by the phorbol ester, phorbol 12-myristate 13-acetate, decreases ENBT1-mediated 6-MP uptake via the phosphorylation of threonine 231 on the largest predicted intracellular loop of ENBT1.

  Conclusion: SLC43A3-encoded ENBT1 is integral to the cellular transport of 6-MP and significant changes in expression can significantly impact the efficacy of 6-MP in leukemia cells. Induction of oxidative stress and activation of PKC decreases ENBT1 functional uptake of 6-MP, providing a potential therapeutic target for 6-MP in ALL patients. Taken together, SLC43A3/ENBT1 is a significant determinant in 6-MP efficacy and sensitivity in leukemia cells and should be investigated further in patients to serve as a potential biomarker for 6-MP therapy.

• Subjects / Keywords

  • Nucleobases
  • 6-Mercaptopurine
  • Equilibrative Nucleobase Transporter 1
  • Acute Lymphoblastic Leukemia

• Graduation date

  Fall 2024

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-68xt-9069

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.