Nathalie Legrave

The synthesis of serine from glucose is a key metabolic pathway supporting cellular proliferation in healthy and malignant cells. Despite this, the role that this aspect of metabolism plays in germinal center biology and pathology is not known. Here, we performed a comprehensive characterization of the role of the serine synthesis pathway in germinal center B cells and lymphomas derived from these cells. We demonstrate that upregulation of a functional serine synthesis pathway is a metabolic hallmark of B cell activation and the germinal center reaction. Inhibition of phosphoglycerate dehydrogenase (PHGDH), the first and rate-limiting enzyme in this pathway, led to defective germinal formation and impaired high-affinity antibody production. In addition, overexpression of enzymes involved in serine synthesis was a characteristic of germinal center B cell–derived lymphomas, with high levels of expression being predictive of reduced overall survival in diffuse large B cell lymphoma. Inhibition of PHGDH induced apoptosis in lymphoma cells, reducing disease progression. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in lymphoma.

Summary: Plasticity of cancer metabolism can be a major obstacle for efficient targeting of tumour-specific metabolic vulnerabilities. Here, we identify and quantify the compensatory mechanisms following the inhibition of major pathways of central carbon metabolism in c-MYC-induced liver tumours. We find that glutaminase isoform Gls2, expressed in normal liver, compensates for the deletion of Gls1 isoform expressed in tumours. Inhibiting both glutaminases significantly delays tumourigenesis but does not completely block glutamine catabolism through the Krebs cycle. We reveal that glutamine catabolism is then driven by amidotransferases. Consistently, the synergistic effect of glutaminase and amidotransferase inhibitors on proliferation of mouse and human tumour cells is observedin vitroandin vivo. Furthermore, when Gls1 is deleted the Krebs cycle activity and tumour formation can also be significantly affected if glycolysis is co-inhibited (Gls1^KO/Hk2^KO). Finally, the inhibition of either serine (Psat1^KO) or fatty acid (Fasn^KO) biosynthesis can be compensated by uptake of circulating nutrients. Thus, removing these nutrients from the diet produces synergistic effects on suppression of tumourigenesis. These results highlight the high flexibility of tumour metabolism and demonstrate how targeting compensatory mechanisms can improve a therapeutic outcome.

Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. The calcium-dependent protein kinase 3 ( Tg CDPK3) was previously identified as a key mediator in this process, as Tg CDPK3 knockout (Δ cdpk3 ) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with Δ cdpk3 parasites revealed changes in the Tg CDPK3-dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that Tg CDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of Tg CDPK3, a putative transporter of the major facilitator superfamily (MFS) and show that it is rapidly phosphorylated after induction of calcium signalling. Conditional knockout (KO) of the transporter reveals an essential role in the lytic cycle during intracellular growth with a transcriptome signature of amino acid-starved parasites. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import. Complementation with phosphorylation site mutants shows that phosphorylation of serine 56 (S56) by Tg CDPK3 gives the parasites a growth benefit in competition assays. Collectively, these findings validate an important, albeit non-essential role for Tg CDPK3 in the regulation of metabolic processes, in addition to motility. Author summary: Toxoplasma gondii is an obligate intracellular parasite. To survive and spread throughout the host it must repeatedly infect, replicate within and exit, host cells. These recurring cycles of infection and egress rely on signalling pathways that allow the parasites to sense and respond rapidly to their environment. While some key kinases and secondary messengers within these pathways have been identified, functional analysis of non-kinases has been very limited. This is especially true for candidates that are not predicted to play a role in active motility or are not known to function in established signalling pathways. Here we have followed up on an unexpected target of the T. gondii calcium-dependent kinase 3 ( Tg CDPK3), a plant-like calcium dependent kinase, that was previously shown to play an important role in calcium-mediated exit from the host cell. We show that, in addition to controlling motility of the parasite (as previously shown), Tg CDPK3 phosphorylates an essential tyrosine transporter in the plasma membrane. Mutational analysis of the phosphorylation sites demonstrates an important role in maintaining parasite fitness, thus demonstrating that Tg CDPK3 plays a pleiotropic role in controlling both egress and metabolism.