Epstein-Barr virus (EBV) is associated with multiple types of cancers, many of which express the viral oncoprotein Latent Membrane Protein 1 (LMP1). LMP1 contributes to both epithelial and B-cell transformation. Although metabolism reprogramming is a cancer hallmark, much remains to be learned about how LMP1 alters lymphocyte oncometabolism. To gain insights into key B-cell metabolic pathways subverted by LMP1, we performed systematic metabolomic analyses on B cells with conditional LMP1 expression. This approach highlighted that LMP highly induces de novo purine biosynthesis, with xanthosine-5-P (XMP) as one of the most highly LMP1-upregulated metabolites. Consequently, IMPDH inhibition by mycophenolic acid (MPA) triggered death of LMP1-expressing EBV-transformed lymphoblastoid cell lines (LCL), a key model for EBV-driven immunoblastic lymphomas. Whereas MPA instead caused growth arrest of Burkitt lymphoma cells with the EBV latency I program, conditional LMP1 expression triggered their death, and this phenotype was rescuable by guanosine triphosphate (GTP) supplementation, implicating LMP1 as a key driver of B-cell GTP biosynthesis. Although both IMPDH isozymes are expressed in LCLs, only IMPDH2 was critical for LCL survival, whereas both contributed to proliferation of Burkitt cells with the EBV latency I program. Both LMP1 C-terminal cytoplasmic tail domains critical for primary human B-cell transformation were important for XMP production, and each contributed to LMP1-driven Burkitt cell sensitivity to MPA. Metabolomic analyses further highlighted roles of NF-kB, mitogen activated kinase and protein kinase C downstream of LMP1 in support of XMP abundance. Of these, only protein kinase C activity was important for supporting GTP levels in LMP1 expressing Burkitt cells. MPA also de-repressed EBV lytic antigens, including LMP1 itself in latency I Burkitt cells, highlighting crosstalk between the purine biosynthesis pathway and the EBV epigenome. These results suggest novel oncometabolism-based therapeutic approaches to LMP1-driven lymphomas.
Copyright: © 2025 Burton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cytotoxic lymphocytes are crucial to our immune system, primarily eliminating virus-infected or cancerous cells via perforin/granzyme killing. Perforin forms transmembrane pores in the plasma membrane, allowing granzymes to enter the target cell cytosol and trigger apoptosis. The prowess of cytotoxic lymphocytes to efficiently eradicate target cells has been widely harnessed in immunotherapies against haematological cancers. Despite efforts to achieve a similar outcome against solid tumours, the immunosuppressive and acidic tumour microenvironment poses a persistent obstacle. Using different types of effector cells, including therapeutically relevant anti-CD19 CAR T cells, we demonstrate that the acidic pH typically found in solid tumours hinders the efficacy of immune therapies by impeding perforin pore formation within the immunological synapse. A nanometre-scale study of purified recombinant perforin undergoing oligomerization reveals that pore formation is inhibited specifically by preventing the formation of a transmembrane β-barrel. The absence of perforin pore formation directly prevents target cell death. This finding uncovers a novel layer of immune effector inhibition that must be considered in the development of effective immunotherapies for solid tumours.
© 2025. The Author(s).

Our investigation uncovers that nanomolar concentrations of salinomycin, monensin, nigericin, and narasin (a group of potassium/sodium cation carriers) robustly enhance surface expression of CD20 antigen in B-cell-derived tumor cells, including primary malignant cells of chronic lymphocytic leukemia and diffuse large B-cell lymphoma. Experiments in vitro, ex vivo, and animal model reveal a novel approach of combining salinomycin or monensin with therapeutic anti-CD20 monoclonal antibodies or anti-CD20 CAR-T cells, significantly improving non- Hodgkin lymphoma (NHL) therapy. The results of RNA-seq, genetic editing, and chemical inhibition delineate the molecular mechanism of CD20 upregulation, at least partially, to the downregulation of MYC, the transcriptional repressor of the MS4A1 gene encoding CD20. Our findings propose the cation carriers as compounds targeting MYC oncogene, which can be combined with anti-CD20 antibodies or adoptive cellular therapies to treat NHL and mitigate resistance, which frequently depends on the CD20 antigen loss, offering new solutions to improve patient outcomes.

Epstein-Barr virus (EBV) is associated with multiple types of cancers, many of which express the key viral oncoprotein Latent Membrane Protein 1 (LMP1). LMP1 is the only EBV-encoded protein whose expression is sufficient to transform both epithelial and B-cells. Although metabolism reprogramming is a cancer hallmark, much remains to be learned about how LMP1 alters lymphocyte oncometabolism. To gain insights into key B-cell metabolic pathways subverted by LMP1, we performed systematic metabolomic analyses on B cells with conditional LMP1 expression. This approach highlighted that LMP highly induces de novo purine biosynthesis, with xanthosine-5-P (XMP) as one of the most highly LMP1-upregulated metabolites. Consequently, IMPDH inhibition by mycophenolic acid (MPA) triggered apoptosis of LMP1-expressing EBV-transformed lymphoblastoid cell lines (LCL), a key model for EBV-driven immunoblastic lymphomas. Whereas MPA instead caused growth arrest of Burkitt lymphoma cells with the EBV latency I program, conditional LMP1 expression triggered their apoptosis. Although both IMPDH isozymes are expressed in LCLs, only IMPDH2 was critical for LCL survival, whereas both contributed to proliferation of Burkitt cells with the EBV latency I program. Both LMP1 C-terminal cytoplasmic tail domains critical for primary human B-cell transformation were important for XMP production, and each contributed to LMP1-driven Burkitt cell sensitivity to MPA. MPA also de-repressed EBV lytic antigens including LMP1 in latency I Burkitt cells, highlighting crosstalk between the purine biosynthesis pathway and the EBV epigenome. These results suggest novel oncometabolism-based therapeutic approaches to LMP1-driven lymphomas. IMPORTANCE Altered metabolism is a hallmark of cancer, yet much remains to be learned about how EBV rewires host cell metabolism to support multiple malignancies. While the oncogene LMP1 is the only EBV-encoded gene that is sufficient to transform murine B-cells and rodent fibroblasts, knowledge has remained incomplete about how LMP1 alters host cell oncometabolism to aberrantly drive infected B-cell growth and survival. Likewise, it has remained unknown whether LMP1 expression creates metabolic vulnerabilities that can be targeted by small molecule approaches to trigger EBV-transformed B-cell programmed cell death. We therefore used metabolomic profiling to define how LMP1 signaling remodels the B-cell metabolome. We found that LMP1 upregulated purine nucleotide biosynthesis, likely to meet increased demand. Consequently, LMP1 expression sensitized Burkitt B-cells to growth arrest upon inosine monophosphate dehydrogenase blockade. Thus, while LMP1 itself may not be a therapeutic target, its signaling induces dependence on downstream druggable host cell nucleotide metabolism enzymes, suggesting rational therapeutic approaches.

Stem cell therapy holds significant potential for skeletal muscle repair, with in vitro-generated human muscle reserve cells (MuRCs) emerging as a source of quiescent myogenic stem cells that can be injected to enhance muscle regeneration. However, the clinical translation of such therapies is hampered by the need for fetal bovine serum (FBS) during the in vitro generation of human MuRCs. This study aimed to determine whether fresh allogeneic human platelet-rich plasma (PRP) combined or not with hyaluronic acid (PRP-HA) could effectively replace xenogeneic FBS for the ex vivo expansion and differentiation of human primary myoblasts. Cells were cultured in media supplemented with either PRP or PRP-HA and their proliferation rate, cytotoxicity and myogenic differentiation potential were compared with those cultured in media supplemented with FBS. The results showed similar proliferation rates among human myoblasts cultured in PRP, PRP-HA or FBS supplemented media, with no cytotoxic effects. Human myoblasts cultured in PRP or PRP-HA showed reduced fusion ability upon differentiation. Nevertheless, we also observed that human MuRCs generated from PRP or PRP-HA myogenic cultures, exhibited increased Pax7 expression and delayed re-entry into the cell cycle upon reactivation, indicating a deeper quiescent state of human MuRCs. These results suggest that allogeneic human PRP effectively replaces FBS for the ex vivo expansion and differentiation of human myoblasts and favors the in vitro generation of Pax7High human MuRCs, with important implications for the advancement of stem cell-based muscle repair strategies.
© 2024. The Author(s).