Abstract
Non-essential amino acids are becoming increasingly important in cancer metabolism because of their roles beyond protein translation. These include providing precursors for macromolecule biosynthesis, serving as substrates for epigenetic modifications, regulating redox states, and modulating cell signaling. Acute lymphoblastic leukemia (ALL) is one such example where asparagine is shown to be conditionally essential for cancer growth and proliferation. Exploiting this metabolic vulnerability, L-Asparaginase (ASNase) has been used in the clinic to successfully treat ALL patients through depleting the circulating asparagine.
Our investigation into the mechanism of resistance to ASNase in ALL cells yielded a striking correlation between asparagine bioavailability and c-MYC protein levels. c-MYC is a key transcription factor and has been shown to regulate the transcription of more than 10% of genes, including many metabolic genes, to meet the demands of tumor growth and progression. In addition, it has a well-documented role in normal hematopoiesis and hematological malignancies, including ALL, where high c-MYC levels have been shown to be a marker for leukemic initiation and growth. Using asparagine auxotrophic ALL cells that do not express asparagine synthetase (ASNS), the asparagine biosynthesis enzyme, we found that c-MYC protein levels were acutely depleted when asparagine was withdrawn from the tissue culture medium. However, c-MYC protein levels were sustained when ASNS cDNA was re-introduced into these cells. These results indicate the possibility of a reciprocal association between metabolism and a critical oncogene MYC.
We also discovered that asparagine depletion inhibited the translation of c-MYC without changing its mRNA expression or altering the rate of protein degradation. Mechanistic Target of Rapamycin Complex 1 (mTORC1) and General Controlled Nonderepressible 2 (GCN2) are the two central molecular components involved in amino acid sensing and translation control. Asparagine starvation notably caused a decrease in global protein synthesis, mediated by suppression of mTORC1 and simultaneous activation of GCN2. Our studies using small molecule inhibitors and polysome profiling showed that repression of c-MYC mRNA translation is not due to the activation of the GCN2 pathway or a consequence of global translation suppression. In addition, both the '5' and '3' untranslated regions (UTRs) were not required for this inhibitory effect on c-MYC mRNA translation during asparagine depletion. Our results suggest that while asparagine depletion causes a defect in global translation at the initiation step, its effect on MYC mRNA translation is possibly due to elongation stalling.
While ASNase can efficiently deplete circulating asparagine, its clinical applications are limited to ALL due to its low-level expression of ASNS. However, aspartate, a critical substrate for asparagine biosynthesis, can be pharmacologically targeted using electron transport chain (ETC) inhibitors. Our studies in ASNase-resistant ALL and lymphomas showed that while asparagine depletion alone did not affect c-MYC protein expression, co-treatment with ETC inhibitors (phenformin) showed a significant decrease in c-MYC protein levels. We extended this observation to test the synergy of ASNase and phenformin to treat an orthotopic MYC-driven B-cell lymphoma model. While either of the single treatments only modestly reduced the tumor burden, the double treatment showed the most significant reduction in the tumor burden, which correlated with a significant reduction of c-MYC protein expression. These results suggest that perturbing the asparagine biosynthesis pathway can be clinically employed to inhibit c-MYC protein expression and improve therapeutic outcomes.
Disclosures
Wek:HiberCell: Membership on an entity's Board of Directors or advisory committees.
