Student Theses and Dissertations

Date of Award

2021

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Birsoy Laboratory

Abstract

Tumor environment influences the response to anti-cancer therapy, but which extracellular nutrients impact drug sensitivity is largely unknown. In this work, we used functional genomics to identify metabolic modifiers of the response to L-asparaginase (ASNase), a therapy that depletes plasma asparagine and targets leukemic cells with insufficient asparagine synthesis. Our approach revealed thiamine pyrophosphate kinase 1 (TPK1), which converts vitamin B1 (thiamine) into the cofactor thiamine pyrophosphate (TPP), as a metabolic dependency under ASNase treatment. In glutamine-anaplerotic leukemia cells, we found that TPP availability enables asparagine synthesis from extracellular glutamine. Mechanistically, TPP is critical for the activity of alpha-ketoglutarate dehydrogenase (AKGDH), a TCA cycle enzyme that catalyzes a step in the overall conversion of glutamine to asparagine. When TPP availability is limiting for cell proliferation of TPK1 KOs, ASNase sensitivity is significantly increased. Standard cell culture media formulations provide thiamine at a concentration that is ~100-fold higher than that observed in human plasma. While thiamine is generally not limiting for cell proliferation under standard culture conditions, a DNA-barcode competition assay identified a subset of leukemia cell lines that grow sub-optimally under lower, more physiological thiamine levels. These cell lines are characterized by low expression of SLC19A2, a high affinity thiamine transporter. Intriguingly, SLC19A2 expression was necessary for not only optimal growth, but also for maintaining ASNase resistance, when standard media thiamine was lowered to the concentration of human plasma. Importantly, analyzing RNAseq data of pediatric acute lymphoblastic leukemia (ALL) tumor samples revealed that SLC19A2 is the primary thiamine transporter expressed in these cancers, and that SLC19A2-low tumors exist among patients. To model such tumors, we used a SLC19A2-low cell line to generate orthotopic tumors in NSG mice. Remarkably, humanizing blood thiamine content of mice through diet sensitized these leukemia cells to ASNase in vivo. Altogether, our work reveals that utilization of thiamine is a determinant of ASNase response for some cancer cells, and that over-supplying vitamins may impact therapeutic response in leukemia. Additionally, our work adds to the recent literature that demonstrates how physiological levels of certain nutrients in cell culture can affect therapy. Specifically, our work provides the first proof of principle that humanizing the vitamin levels of both in vitro and in vivo models can affect drug sensitivity. This has broad implications for the screening and validation of new therapeutic candidates.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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