PF524 EVALUATION OF SERINE BIOSYNTHESIS PATHWAY AS A POTENTIAL THERAPEUTIC TARGET IN BURKITT LYMPHOMA
Authors: M. Noyszewska-Kania, E. Bialopiotrowicz, M. Cybulska, A. Grochowska, M. Kopczynski, M. Mikula, N. Kachamakova-Trojanowska, A. Loboda, M. Prochorec-Sobieszek, A. Szumera-Ciećkiewicz, M. Firczuk, A. Graczyk-Jarzynka, R. Zagozdzon, A. Zabek, P. Mlynarz, P. Gorniak, M. Szydlowski, E. Jablonska, A. Polak, P. Kowalczyk, K. Piechna, T. Rzymski, K. Brzozka, P. Juszczynski
Journal: HemaSphere (2019)

Background: Burkitt lymphoma (BL) is a highly proliferative tumor characterized by high anabolic requirements. Short BL doubling time is promoted by overexpression of cMYC, which controls genes involved in cell cycle progression and cellular metabolism, including glycolysis. Glycolytic intermediates in cancer can be diverted to other metabolic pathways, such as serine biosynthesis pathway. Serine supports one carbon metabolism, a metabolic network that couples glycolysis to folate and methionine cycle. Aims: As these metabolic circuits are essential for proliferating cells, we assessed the biological consequences of knockdown of two genes involved in serine synthesis, PHGDH and PSAT1 in BL cells, and evaluated serine synthesis pathway targeting as a potential therapeutic strategy in BL. Methods: PSAT1 and PHGDH mRNA and protein expression in BL were assessed in publicly available gene expression datasets and by immunohistochemistry. PSAT1 and PHGDH gene knockouts were achieved by CRISPR/Cas9 editing. Commercially available PHGDH inhibitor NCT-503 was used for in vitro and in vivo studies. Proliferation and clonogenicity were assessed using MTS and methylcellulose assays, respectively. Reactive oxygen species (ROS) and glutathione (GSH) levels were analyzed with CM-H2DCFDA and GSH/GSSG-Glo tests. Global DNA methylation was investigated by flow cytometry and 5’-methylcytosine staining. Gene expression was analyzed with quantitative RT-PCR. For in vivo experiments, NSG/J mice were subcutaneously inoculated with either wild-type Namalwa/Raji cells or cells with PHGDH/PSAT1 knockdown. NCT-503 or its inactive enantiomer were administered ip (40 mg/kg). Metabolic profiles of xenografts were analyzed using magnetic resonance NMR spectroscopy. Results: PSAT1 and PHGDH protein levels were significantly higher in BL than DLBCL patients both at transcript and protein levels (p = 0.0016 and p = 0.0001, respectively). Expression of both genes was regulated by cMYC via ATF4 transcription factor: MYC knockdown decreased ATF4 transcription factor expression and, as a consequence, downregulated PSAT1 and PHGDH expression. Genetic disruption of PHGDH/PSAT1 significantly decreased BL cells proliferation and clonogenicity. Similar results were observed for BL cell lines incubated with NCT-503. Incubation of BL cells with NCT-503 promoted ROS production and decreased glutathione (GSH) level. Prolonged incubation with NCT-503 induced apoptosis of BL cells. As serine biosynthesis pathway is linked to methionine cycle responsible for production of methyl group donors, we assessed global DNA and histone methylation following PHGDH inhibition. NCT-503 decreased DNA and histones methylation and led to re-expression of ID4, KLF4, CDKN2B and TXNIP genes. Finally, we assessed the effect of PHGDH/PSAT1 inhibition in vivo. Surprisingly, neither PSAT1 knockdown nor PHGDH inhibition delayed BL-xenograft growth in mice. Explanted tumors (PSAT1 knockout versus controls) exhibited similar metabolomic profiles, indicating the capacity of BL cells to compensate loss of serine biosynthesis pathway in vivo. Summary/Conclusion: Altogether, our experiments demonstrate that serine biosynthesis pathway is not an effective therapeutic target in BL. Although inhibition of serine synthesis enzymes inhibits BL cell proliferation in vitro, these effects are not recapitulated in vivo. Comparison of metaboliomic profiles of xenografts with active and disrupted serine pathway suggests that deficiency of serine biosynthesis can by effectively compensated.