Phosphoglycerate Dehydrogenase Is Required for Germinal Center Formation and Is a Therapeutic Target in MYC-driven Lymphoma

The fields of cancer- and immuno-metabolism have re-emerged as areas of significant translational potential. Even though the upregulation of glycolysis by proliferating lymphocytes is the basis for widely used clinical tests such as FDG-PET, little is known about which metabolic pathways are involved in the utilization of glucose to support B-cell proliferation. The synthesis of serine from glucose has been demonstrated to be a key metabolic pathway supporting cellular proliferation in some healthy and malignant cell types. Importantly, this pathway is regulated by MYC, which is known to be essential for germinal centre formation and is commonly dysregulated in lymphoma. Despite this, the role that the serine synthesis pathway (SSP) plays in germinal center biology and pathology has not been previously investigated. We performed a comprehensive characterization of the role of the SSP in germinal center B cells and lymphomas derived from these cells. We demonstrate that upregulation of a functional SSP is a metabolic hallmark of B-cell activation and the germinal center reaction. We show that both human and murine resting naïve B cells lack expression of the first two enzymes in this pathway, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) enzymes. However, B-cell activation, predominantly through the B-cell receptor, robustly induces the expression of these enzymes in vitro, resulting in an acquired ability to synthesize serine, glycine and the purine nucleotides adenosine and guanosine from glucose. This is reflected in striking expression of PHGDH and PSAT1 within germinal centers but not in marginal zones confirming that this upregulation is occurring in germinal B cells activated in vivo. We then proceeded to investigate the impact of inhibiting PHGDH on germinal center formation and high-affinity antibody production in vivo. This was done both genetically, using a conditional B-cell knockout mouse model, and pharmacologically using a specific inhibitor of PHGDH, PH-755. Importantly, we show that PHGDH inhibition impairs germinal center formation with a resultant reduction in high-affinity antibody production. Mechanistic experiments demonstrate that PHGDH inhibition effectively blocks cells from synthesising serine and glycine from glucose, making them unable to proliferate in environments that lack these amino acids. We then investigated role of PHDGH and PSAT1 in Burkitt Lymphoma (BL), Diffuse Large B Cell Lymphoma (DLBCL) and Chronic Lymphocytic Leukemia (CLL). Notably, very high expression of these two proteins was observed in BL, with intermediate-to-high expression in DLBCL and relatively low expression in CLL, where expression was restricted to proliferation centers. Given the heterogeneity of expression in DLBCL patients, we next interrogated a published GSE database (Lenz et al. NEJM 2008) to investigate the impact on outcome. Notably high expression of PSAT1 was significantly associated with a poorer overall survival rate in DLBCL. We then investigated whether the SSP could be a therapeutic target in lymphoma. We demonstrate that PHGDH inhibition effectively inhibits de novo serine, glycine and purine nucleotide synthesis from glucose, resulting in impaired proliferation and increased apoptosis in a panel of human BL cell lines in vitro. We then analyzed the impact of PHGDH inhibition on lymphoma development in vivo using Eµ-myc mice, which harbour Myc coupled to the IgH enhancer characteristic of BL. Importantly we confirm the role of MYC by demonstrating that Eµ-Myc B-cells show significantly higher expression of PHGDH and PSAT1 expression resulting in increased serine and glycine synthesis when compared to control cells. We demonstrate that pharmacological inhibition of PHGDH using PH-755 impairs lymphoma progression in this model. We confirm the importance of PHGDH by showing that genetic ablation of Phgdh in Eµ-myc cells in a tamoxifen inducible system (using Eµ-myc/+;Rosa26-CreER T2/+;Phgdh fl/fl mice) also results in a significant reduction in lymphoma progression. Taken together, this work represents the first report of the role of the SSP in the biology of the germinal centre response and lymphomas derived from these cells. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in MYC-driven lymphoma, which is an area of significant unmet need. Disclosures Gribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria. Calado: Myricx Pharma: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties: Cancer Treatments. WO patent WO 2020/128475 A1 (2020).

Brg1 Supports B Cell Proliferation and Germinal Center Formation Through Enhancer Activation

Activation and differentiation of B cells depend on extensive rewiring of gene expression networks through changes in chromatin structure and accessibility. The chromatin remodeling complex BAF with its catalytic subunit Brg1 was previously identified as an essential regulator of early B cell development, however, how Brg1 orchestrates gene expression during mature B cell activation is less clear. Here, we find that Brg1 is required for B cell proliferation and germinal center formation through selective interactions with enhancers. Brg1 recruitment to enhancers following B cell activation was associated with increased chromatin accessibility and transcriptional activation of their coupled promoters, thereby regulating the expression of cell cycle-associated genes. Accordingly, Brg1-deficient B cells were unable to mount germinal center reactions and support the formation of class-switched plasma cells. Our findings show that changes in B cell transcriptomes that support B cell proliferation and GC formation depend on enhancer activation by Brg1. Thus, the BAF complex plays a critical role during the onset of the humoral immune response.

Rap1 Is Essential for B-Cell Locomotion, Germinal Center Formation and Normal B-1a Cell Population

Integrin regulation by Rap1 is indispensable for lymphocyte recirculation. In mice having B-cell-specific Rap1a/b double knockouts (DKO), the number of B cells in lymph nodes decreased to approximately 4% of that of control mice, and B cells were present in the spleen and blood. Upon the immunization with NP-CGG, DKO mice demonstrated the defective GC formation in the spleen, and the reduced NP-specific antibody production. In vitro, Rap1 deficiency impaired the movement of activated B cells along the gradients of chemoattractants known to be critical for their localization in the follicles. Furthermore, B-1a cells were almost completely absent in the peritoneal cavity, spleen and blood of adult DKO mice, and the number of B-cell progenitor/precursor (B-p) were reduced in neonatal and fetal livers. However, DKO B-ps normally proliferated, and differentiated into IgM+ cells in the presence of IL-7. CXCL12-dependent migration of B-ps on the VCAM-1 was severely impaired by Rap1 deficiency. Immunostaining study of fetal livers revealed defects in the co-localization of DKO B-ps and IL-7-producing stromal cells. This study proposes that the profound effects of Rap1-deficiency on humoral responses and B-1a cell generation may be due to or in part caused by impairments of the chemoattractant-dependent positioning and the contact with stromal cells.

TFH cells depend on Tcf1-intrinsic HDAC activity to suppress CTLA4 and guard B-cell help function

Precise regulation of coinhibitory receptors is essential for maintaining immune tolerance without interfering with protective immunity, yet the mechanism underlying such a balanced act remains poorly understood. In response to protein immunization, T follicular helper (TFH) cells lacking Tcf1 and Lef1 transcription factors were phenotypically normal but failed to promote germinal center formation and antibody production. Transcriptomic profiling revealed that Tcf1/Lef1-deficient TFH cells aberrantly up-regulated CTLA4 and LAG3 expression, and treatment with anti-CTLA4 alone or combined with anti-LAG3 substantially rectified B-cell help defects by Tcf1/Lef1-deficient TFH cells. Mechanistically, Tcf1 and Lef1 restrain chromatin accessibility at the Ctla4 and Lag3 loci. Groucho/Tle corepressors, which are known to cooperate with Tcf/Lef factors, were essential for TFH cell expansion but dispensable for repressing coinhibitory receptors. In contrast, mutating key amino acids in histone deacetylase (HDAC) domain in Tcf1 resulted in CTLA4 derepression in TFH cells. These findings demonstrate that Tcf1-instrinsic HDAC activity is necessary for preventing excessive CTLA4 induction in protein immunization–elicited TFH cells and hence guarding their B-cell help function.