Targeting Nucleotides Dependency Via SIRT3 Inhibition to Overcome Metabolic Plasticity and Flexibility of Dlbcls

Metabolic adaptations are critical for cancer cells to survive in hostile environment such as low nutrient and drug treatment conditions. DLBCLs (Diffuse large B cell lymphoma) originate from a metabolic competitive/challenging environment of germinal center (GC). However, it is unknown whether and how metabolic flexibility (the ability to use different nutrients) and plasticity (the ability to process the same nutrient differently) contribute to survival of DLBCLs. Our prior study identified SIRT3, the only mitochondrial protein deacetylase, as a master regulator of DLBCL metabolism. We developed the first mitochondrial targeting SIRT3 inhibitor (SIRT3-i, called YC8-02) which kills DLBCLs regardless of somatic mutations and cell of origin, suggesting a common metabolic dependency in DLBCLs. Mechanistically, loss or inhibition of SIRT3 reduced TCA cycle metabolites derived from glutamine, which triggered autophagy and disrupted the sensing of amino acids and usage of glucose in DLBCLs. Therefore, we questioned if the metabolic adaptation provides survival advantage and underlies another critical metabolic need in DLBCLs under the metabolic stress induced by SIRT3 inhibition.

Here, we found that DLBCLs specifically gained survival advantage in high glucose conditions (15mM) under SIRT3-i treatment. Glucose serves as an alternative nutrient source to compensate the TCA cycle in SIRT3 deficient cells. The combinatory treatment of SIRT3-i and glucose metabolism inhibitors caused synergistic killing effects to DLBCLs. However, glucose is a common substrate of multiple downstream products, and DLBCLs utilized glucose flexibly to the TCA cycle but exposed defects in de novo biosynthesis of nucleotides. The SIRT3-i treatment led to reductions of nucleotides in PDXs tumors visualized by metabolic imaging (MALDI) in vivo. Importantly, SIRT3-i only induced metabolic changes in tumor tissues without affect normal organs, such as kidneys with high concentration drugs. Thus, we hypothesized that lymphoma cells maintains a cancer specific metabolic program in contrast to normal B cells. Indeed, we observed that glucose and glutamine usage are greatly enhanced in lymphoma cells (vavP-bcl2) than normal B cells in vivo. Lymphoma B cells maintain higher levels of nucleotides than normal B cells, as glucose is actively directed to nucleotide biosynthesis in lymphoma cells in vivo. The glucose tracing experiments showed that glucose-serine-glycine-one carbon pathway was most affected route by SIRT3-i in nucleotides synthesis pathway. Serine tracing experiments validated this finding and high dose of serine protected DLBCLs from SIRT3 treatment in normal glucose (5mM) conditions. One carbon pathway is highly activated to provide substrates for de novo nucleotide synthesis downstream of mitochondrial enzymes including SHMT2 and MTHFD2. Inhibitors of SHMT2 or MTHFD2 effectively kill DLBCLs but can be rescued by supplement of precursors of nucleosides (adenosine, guanosine, hypoxanthine, uridine, cytosine and thymine, AGHUCT). Consistently, both overexpression of SHMT2 or MTHFD2 and AGHUCT rescued DLBCLs from SIRT3-i. These findings indicated that DLBCLs may flexibly use different nutrients from environment to overcome drug induced metabolic stress, which inspired us designing a combinatory treatment of ENT (equilibrative nucleoside transporters) inhibitors with SIRT3-i to block nucleosides freely shared in tumor environments. Under such combo treatment, nether glucose or nucleosides can protect DLBCLs from SIRT3 inhibition. Finally, we confirmed the effectiveness of this new regimen in aggressive lymphoma that can kill recipient C57BL/6 mice in two weeks with systemic disease. The combo treatment eradicated lymphoma cells from the recipient mice in 1.5 weeks with the cure rate at 75%.

Our data revealed that DLBCLs have active metabolic flexibility and plasticity by turning different nutrients, like glucose, serine, and nucleosides, into resources for survival and proliferation. Our evidence shows that nucleotides are the ultimate metabolic needs of DLBCLs to sustain their uncontrolled proliferation. Targeting SIRT3 and ENTs together can restrain nucleotide availability by blocking both de novo biosynthesis and salvage pathway which provided a promising therapy by preventing metabolic flexibility and plasticity of DLBCLs.