Metabolic Profiling Identifies De Novo Nucleotide Synthesis As a Potential Metabolic Vulnerability for Targeted Therapy Against Mantle Cell Lymphoma

Introduction: Cancer cells exhibit dramatic alterations in cellular metabolism, such as enhanced de novo nucleotide synthesis, to support cell growth, proliferation and survival. The abundance of the nucleotide pool as well as the level and activity of different rate-limiting enzymes belonging to the nucleotide synthetic pathway limit the maximal proliferative capacity of cells. Maintenance of an adequate pool of deoxyribonucleotide triphosphates is essential for DNA replication and DNA repair, and consequently, the genetic integrity of nuclear and mitochondrial genomes. We and others have demonstrated that mantle cell lymphoma (MCL) undergoes metabolic reprogramming to progress and develop resistance to targeted therapy; however, the contribution of de novo nucleotide synthesis to the development and progression of MCL remains poorly understood. In contrast, oncogenic Myc is demonstrated to be highly upregulated in a subset of MCL. In addition to its pro-glycolysis, pro-biogenesis and pro-tumor growth functions, oncogenic levels of Myc induce the expression of multiple genes involved in the nucleotide biosynthetic pathway (e.g., IMPDH2, CTPS1, and CAD). Myc-induced glutamine metabolism also increases the abundance and activity of different rate-limiting enzymes that produce the molecular precursors required for de novo nucleotide synthesis. The _γ-nitrogen amide group of glutamine is an indispensable donor of nitrogen for de novo synthesis of both nucleobases purine and pyrimidine. Here, we hypothesize that a subset of MCL depends on de novo nucleotide synthesis for anabolic cell growth and cancer progression due to aberrant Myc expression and Myc-induced glutaminolysis.

Methods: Primary MCL biopsy, apheresis, and blood specimens as well as MCL cell lines were utilized for metabolic and functional analyses. Liquid Chromatography Mass Spectrometry (LC-MS) metabolomics was employed to measure the steady-state level of metabolites. Western-blotting and real-time qPCR were utilized determine protein and gene expression levels. BrdU incorporation and the Cell-Trace Violet Cell Proliferation Assay were employed to assess DNA synthesis and cell proliferation. Cell viability was measured with the Cell Titer-Glo Cell Viability Assay. Pharmacological agents were employed to inhibit either de novo nucleotide synthesis or glutaminolysis.

Results: Metabolomics profiling of steady-state levels of intracellular metabolites showed significant increases in N-carbamoyl aspartate/dihydroorotate and 5-phosphoribosyl-1-pyrophosphate (PRPP), which are critical intermediates in de novo pyrimidine and purine synthesis, respectively, as well as CTP, dUTP, dCTP, in a subset of MCL, indicating remarkably upregulated de novo nucleotide synthesis. The protein and mRNA levels of c-Myc and its target genes involved in the metabolism of nucleotides (IMPDH2, CTPS1, CAD) were significantly increased. Inhibition of pyrimidine synthesis with DON (6-diazo-5-oxo-L-norleucine), a CTPS1 inhibitor, dramatically reduced the pool of pyridine nucleotides, leading to remarkable apoptosis and halted cell proliferation of a subset of MCL cell lines. Consistent with c-Myc overexpression, increased glutamine uptake was also observed in a subset of MCL cell lines. Glutamine deprivation or pharmacological inhibition of glutamine metabolism showed a similar effect on the inhibition of pyrimidine synthesis as DON (6-diazo-5-oxo-L-norleucine), manifested by a significant reduction of pyrimidine nucleoside triphosphate levels, a dramatic increase in apoptosis, and retarded cell proliferation of a subset of MCL cell lines.

Conclusions: Our preliminary results indicate that de novo nucleotide synthesis is upregulated in a subset of MCL with aberrant c-Myc expression. The expression of genes involved in nucleotide metabolism as well as glutaminolysis is also elevated in these cancer cells. Disruption of de novo nucleotide synthesis or glutaminolysis induces apoptosis and suppresses proliferation of a subset of MCL. Myc does not possess enzymatic activity and is considered "undruggable"; therefore, the inhibition of Myc target genes such as those involved in de novo nucleotide synthesis and glutaminolysis presents a promising alternative approach. Taken together, MCL dependency on de novo nucleotide synthesis may represent a metabolic vulnerability for targeted therapy for MCL.