Lmp1 enhances aerobic glycolysis in natural killer/t cell lymphoma

ABSTRACT Natural killer/T cell lymphoma (NKTCL) exhibits highly aggressive clinical behavior, and the outcomes for relapsed/refractory patients are still poor. Recently, the mechanism

underlying the effect of Epstein-Barr virus (EBV) infection, which has not been fully defined in NKTCL, has attracted great attention. We explored how LMP1 promoted aerobic glycolysis via

metabolic sequencing combined with mRNA sequencing and immunoprecipitation coupled to mass spectrometry. Experimental assays were used to determine the effects of LMP1 and its downstream

pathway on the function and glucose metabolism of NKTCL cells. The correlations between LMP1 expression in patients and their clinical features, treatment response, and prognosis were

analyzed. Results show that LMP1 enhances NKTCL cell proliferation in vitro and in vivo, inhibits apoptosis, and decreases gemcitabine sensitivity. In addition, LMP1 also enhances aerobic

glycolysis in NKTCL cells, as indicated by increases in glucose uptake, lactate production, and extracellular acidification rate. Clinically, LMP1 expression is correlated with risk

stratification, treatment response, and prognosis, and higher LMP1 expression indicates greater SUVmax for NKTCL patients. Mechanistically, LMP1 competitively binds to TRAF3 to promote cell

proliferation and aerobic glycolysis by regulating the noncanonical NF-κB pathway. The application of an NF-κB pathway inhibitor or reactivation of the NF-κB pathway affects aerobic

glycolysis and the biological function of NKTCL cells. In summary, this study is the first to describe and define in detail how LMP1 affects glucose metabolism in NKTCL and might provide a

novel perspective for further treatment. SIMILAR CONTENT BEING VIEWED BY OTHERS DEPENDENCE ON MITOCHONDRIAL RESPIRATION OF MALIGNANT T CELLS REVEALS A NEW THERAPEUTIC TARGET FOR

ANGIOIMMUNOBLASTIC T-CELL LYMPHOMA Article Open access 19 June 2024 MULTI-OMICS ANALYSES REVEAL THAT HIV-1 ALTERS CD4+ T CELL IMMUNOMETABOLISM TO FUEL VIRUS REPLICATION Article 25 March 2021

A FUNCTIONAL SINGLE-CELL METABOLIC SURVEY IDENTIFIES _ELOVL1_ AS A TARGET TO ENHANCE CD8+ T CELL FITNESS IN SOLID TUMOURS Article Open access 10 March 2025 INTRODUCTION Natural killer/T

cell lymphoma (NKTCL), a subtype of T cell and NK-cell lymphoid proliferations and lymphomas classified by the latest World Health Organization (WHO) [1], presents highly aggressive clinical

behavior and is particularly prevalent in Asian and South American populations [2]. In recent decades, therapeutic regimens containing L-asparaginase, including DDGP [3], modified SMILE

[4], and P-GemOx [5], have been recommended by the National Comprehensive Cancer Network guidelines based on improved clinical prognosis [6]. However, the outcomes for relapsed and

refractory patients are still suboptimal [7]. Considering the close association of NKTCL with Epstein-Barr virus (EBV) infection [8], targeting EBV provides a promising strategy for future

treatment. Latent membrane protein 1 (LMP1), encoded by EBV, promotes oncogenesis to facilitate malignant transformation in EBV-associated malignancies [9], and has been reported to play

fundamental roles in nasopharyngeal carcinoma (NPC) [10, 11]. Thus, studies on the therapeutic potential of LMP1 for the treatment of NKTCL are urgently needed. The reprogramming of

metabolic pathways, regarded as a crucial hallmark of tumors, is associated with both tumorigenesis and disease progression [12]. Among them, the metabolism of glucose for aerobic glycolysis

rather than oxidative phosphorylation, known as the Warburg effect, is an important metabolic feature of malignant lymphomas [13, 14], and can support infinite replication, self-sufficiency

in growth signals, resistance to antigrowth signals, tumor invasion and metastasis, angiogenesis, and disturbance of the microenvironment [15]. It has been widely reported that viral

infection is related to tumor pathogenesis and development, and the mechanism underlying the effect of viral infection on tumor metabolism has attracted great attention in recent years.

Studies have shown that EBV and its encoded LMP1 can potentially alter glucose metabolism in NPC, through increasing the expression of vascular endothelial growth factor to stimulate

angiogenesis [16], inhibiting glucose deprivation and metabolism to restore sensitivity to apoptosis induction [17], and decreasing monocyte migration and T cell activation to promote immune

escape [18]. Hence, targeting LMP1 may represent a promising approach for treating EBV-positive malignancies by interfering with aerobic glycolysis. However, the effect of LMP1 on glucose

metabolism in EBV-associated NKTCL and the detailed mechanism have not been fully defined. In this study, we explored how LMP1 promoted aerobic glycolysis via metabolic sequencing combined

with mRNA sequencing and immunoprecipitation (IP) coupled to mass spectrometry. Experimental assays determined the effects of LMP1 and its downstream pathway on the function and glucose

metabolism of NKTCL cells. Clinically, the correlations between LMP1 expression in NKTCL patients and their clinical features, treatment response, prognosis, and the baseline maximum

standardized uptake value (SUVmax) before treatment were analyzed. In summary, this study is the first to describe and define in detail the effect of LMP1 on glucose metabolism in NKTCL and

might provide a novel perspective for further treatment. MATERIALS AND METHODS PATIENTS AND CLINICAL DATA A total of 58 formalin-fixed, paraffin-embedded tumor tissues from NKTCL patients

were obtained from the First Affiliated Hospital of Zhengzhou University. All patients were reviewed and interpreted independently by three experienced pathologists, and diagnoses were made

according to the latest WHO classification criteria. After diagnosis, enrolled patients received DDGP regimen, including gemcitabine, dexamethasone, cisplatin, and pegaspargase, as the

first-line treatment. Immunohistochemistry (IHC) of LMP1 was performed according to standard procedures. The LMP1 antibody was listed in Additional File 1: Table S1. Staining was assessed

according to the staining intensity and the positively stained area by a pathologist and verified by two other pathologists without prior knowledge of the patients’ information, and the

specific evaluation criteria and definition of cut-off value were described in Additional File 2. The overall response rate (ORR) was defined as the proportion of patients who achieved a

complete response (CR) or partial response (PR). Efficacy evaluation was conducted every 2 treatment cycles. Overall survival (OS) was defined as the interval from the date of first

treatment to the date of death for any reason. Progression-free survival (PFS) was defined as the interval from the date of first treatment to the date of disease progression or death for

any reason. The SUVmax was collected from the reports on pretreatment 18F-FDG PET-CT examinations. The clinical features of the patients and the correlations of these features with LMP1

expression were summarized in Table 1. CELL LINES AND CULTURE YT and NKYS cell lines were obtained from Dr. Wing C. Chan (City of Hope Medical Center), the SNT16 cell line was a gift from

Guangzhou Bairui Biomedical Technology Company, Ltd. (China), and the SNK6 cell line was kindly provided by Dr. Norio Shimizu and Yu Zhang of Chiba University. The culture conditions were

mentioned in Additional File 2. CONSTRUCTION OF STABLE CELL LINES Sequences for over-expression (OE)-LMP1 and its vector (OE-vector) were designed by Guangzhou Bairui Biomedical Technology

Company, Ltd. (China). Sequences for short hairpin RNA (shRNA) of LMP1 (shLMP1) and negative control (shNC), and OE-RelB and its vector (OE-vector) were designed by Shanghai Genechem

Company, Ltd. (China). Brief procedures were mentioned in Additional File 2. CELL PROLIFERATION ASSAY Cell proliferation assay was conducted with Cell Counting Kit-8 (CCK-8) reagent

(UElandy, China). Brief procedures were mentioned in Additional File 2. Independent experiments were repeated at least three times. CELL APOPTOSIS ASSAY Cell apoptosis assay was conducted

with APC-Annexin V/PI Apoptosis Detection Kit (UElandy, China). Brief procedures were mentioned in Additional File 2. Independent experiments were repeated at least three times. GEMCITABINE

SENSITIVITY ASSAY Gemcitabine sensitivity assay was conducted with CCK-8 reagent (UElandy, China). Brief procedures were mentioned in Additional File 2. Independent experiments were repeated

at least three times. MRNA SEQUENCING AND METABOLIC SEQUENCING ANALYSIS Total RNA extraction, mRNA library construction and sequencing, sample preparation, LC–MS detection, and data

analysis were performed by Suzhou PANOMIX Biomedical Tech Co., LTD. (China). Brief procedures were mentioned in Additional File 2. DROPLET DIGITAL PCR (DDPCR) RNA was extracted using

RNAsimple Total RNA Kit (TIANGEN, China). cDNA was synthesized by UEIris RT mix with DNase (UElandy, China). The primers were synthesized by Hangzhou Shangyasai Biotechnology Co., Ltd.

(China) and Sangon Biotech Co., Ltd. (China), with sequences listed in Additional File 1: Table S2. Brief procedures were mentioned in Additional File 2. Independent experiments were

repeated at least three times. GLUCOSE UPTAKE AND LACTATE PRODUCTION DETECTION ASSAYS Glucose uptake detection assay was conducted with the Glucose Oxidase Method Kit (APPLYGEN, China), and

the lactate production detection assay was conducted with the Lactic Acid Assay kit (Nanjing Jiancheng Bioengineering Institute, China). Brief procedures were mentioned in Additional File 2.

Independent experiments were repeated at least three times. GLYCOLYSIS STRESS ASSAY Extracellular acidification rate (ECAR) of NKTCL cells was conducted with Seahorse XF96 Flux Analyzer

(Agilent, USA) and Glycolysis Stress Test Kit (Agilent, USA). Cell adhesion was described in Additional File 2. Independent experiments were repeated at least three times. MASS SPECTROMETRY

ANALYSIS Total sample preparation, mass spectrometry detection, and data analysis were performed as previously [19]. Brief procedures were mentioned in Additional File 2. The raw sequencing

data was mentioned in Additional File 3. IP, CO-IP, AND SILVER STAINING Silver staining was conducted with Fast Silver Stain Kit (Beyotime, China). Brief procedures were mentioned in

Additional File 2. The antibodies were listed in Additional File 1: Table S1. WESTERN BLOTTING Brief procedures were mentioned in Additional File 2. Primary and secondary antibodies were

listed in Additional File 1: Table S1. XENOGRAFT TUMOR ASSAY BALB/c‐Nu nude mice and NOD-Scid mice were purchased from the GemPharmatech Company (China). Brief procedures were mentioned in

Additional File 2. Animals were randomly selected and at least five animals in each group. The antibodies were listed in Additional File 1: Table S1. STATISTICAL ANALYSIS Statistical

analyses were performed using SPSS software version 25.0 (IBM Corp., USA) and GraphPad Prism version 8.0 (GraphPad Software, Inc., USA). Data was expressed as the mean ± standard deviation

for repeated measurements. Comparisons between groups were performed using Student’s _t_-test and analysis of variance. PFS and OS were analyzed using the Kaplan–Meier method and log-rank

test. The correlation between LMP1 expression with clinical features and treatment response was assessed using the χ2-test. A value of _P_ < 0.05 was considered statistically significant.

RESULTS LMP1 INFLUENCES THE AGGRESSIVE BIOLOGICAL BEHAVIORS OF NKTCL CELLS AND IS RELATED TO THE CLINICAL CHARACTERISTICS OF PATIENTS To determine the role of LMP1 in the tumorigenesis and

development of NKTCL, we examined LMP1 expression in 6 NKTCL cell lines (Fig. 1A). From this, we selected YT and SNT16 to generate OE-LMP1 cells (Additional File 4: Fig. S1A), and NKYS and

SNK6 to generate shLMP1 cells (Additional File 4: Fig. S1B). Subsequently, cell proliferation, apoptosis resistance, and gemcitabine sensitivity were assessed in vitro. As expected, LMP1

overexpressing NKTCL cells, including YTOE-LMP1, SNT16OE-LMP1 cells, NKYSshNC, and SNK6shNC cells, exhibited significantly enhanced cell proliferation (Fig. 1B), inhibition of the

starvation-induced apoptosis (Fig. 1C) and reduced gemcitabine sensitivity (Fig. 1D). The role of LMP1 in tumorigenesis and tumor growth in vivo was also assessed in NKTCL xenograft mouse

models (Additional File 4: Fig. S1C). We found that the mice injected with YTOE-LMP1 cells exhibited a greater tumor burden than the mice injected with YTOE-vector cells (Fig. 1E, F) and

that the mice injected with NKYSshLMP1 cells exhibited a lower tumor burden (Fig. 1G, H). In addition, the expression of Ki67 was related to the expression of the related protein LMP1 in

mouse tissues (Fig. 1I). Furthermore, LMP1 expression was analyzed and scored by IHC staining in samples from 58 patients with pathologically verified NKTCL from the First Affiliated

Hospital of Zhengzhou University (Fig. 1J). Interestingly, we found that LMP1 expression was significantly related to the Prognostic Index for Natural Killer cell lymphoma-Epstein-Barr virus

(PINK-E) score of NKTCL patients (Table 1). Moreover, the results demonstrated that patients with low LMP1 expression were more likely to achieve a CR or a PR after treatment (Table 2), and

patients with aberrantly high LMP1 expression had a worse prognosis, indicated by shorter OS and PFS (Fig. 1K). Taken together, these findings indicate that LMP1 supports NKTCL cell

biological functions both in vitro and in vivo and is associated with the risk stratification, treatment response, and prognosis of NKTCL patients. LMP1 INFLUENCES THE AEROBIC GLYCOLYSIS IN

NKTCL CELLS AND IS RELATED TO SUVMAX OF PATIENTS To explore the functional role of metabolism in LMP1-enhanced NKTCL cell proliferation, untargeted metabolomics sequencing was conducted, and

778 differentially expressed metabolites between NKYSshNC and NKYSshLMP1 cells were identified (Additional File 4: Fig. S2A–D). KEGG enrichment analysis revealed that the main

differentially expressed metabolic pathways were glucose-related metabolic pathways (Fig. 2A), and the function of LMP1 in aerobic glycolysis in NPC has been confirmed [20]. Therefore, we

assessed glucose uptake and lactate production in the 6 NKTCL cell lines. Interestingly, the LMP1-positive cell lines SNK6, KAI3, and NKYS took up more glucose and produced more lactate than

did the LMP1-negative cell lines KHYG1, SNT16 and YT (Fig. 2B). OE-LMP1 and shLMP1 had the same effects on YT and SNT16 cells (Fig. 2C), and on NKYS and SNK6 cells (Fig. 2D). Moreover, the

changes in the ECAR induced by LMP1 were measured using a glycolysis stress assay. As expected, the OE-LMP1 cells had an increased ECAR, while the shLMP1 cells had a significantly decreased

ECAR (Fig. 2E, F). In addition, the expression of glycolysis-related genes and proteins was also investigated and recapitulated (Fig. 2G, H). Subsequently, 18F-FDG uptake was analyzed in 42

NKTCL patients with LMP1 expression scored who underwent PET-CT examination before treatment. These findings suggested that aberrantly high LMP1 expression was accompanied by a greater

SUVmax than low LMP1 expression (Fig. 2I, J). In summary, these results demonstrate that LMP1 strengthens NKTCL cell biological function by contributing to increased aerobic glycolysis. LMP1

REGULATES AEROBIC GLYCOLYSIS VIA COMPETITIVE BINDING WITH TRAF3 TO ACTIVATE THE NONCANONICAL NF-ΚB PATHWAY The underlying mechanism by which LMP1 regulates aerobic glycolysis in NKTCL

remains unclear, and to address this problem, we performed RNA sequencing using RNA extracted from NKYSshNC and NKYSshLMP1 cells, and KEGG enrichment analysis indicated that the NF-κB

signaling pathway was significantly enriched in the shNC group (Fig. 3A). Furthermore, we identified one LMP1 binding partner, TRAF3, from the SNK6 cell line using IP coupled to mass

spectrometry according to Significance Analysis of the INTeractome (SAINT) score [21, 22] (Fig. 3B, Additional File 3). It has been previously reported that LMP1 sequesters and binds TRAF3

more effectively than CD40 to regulate the NF-κB pathway in B-cell lymphoma cells [23]. Accordingly, a CO-IP assay was implemented to verify the competitive binding of LMP1 and CD40 with

TRAF3 in NKTCL cells. The results showed that NKYSshNC and SNK6shNC cells bound more TRAF3, and NKYSshLMP1 and SNK6shLMP1 cells bound more CD40 (Fig. 3C). Depending on the dysregulated

function of TRAF3 in the noncanonical NF-κB pathway, degradation of TRAF3 after competitive binding to LMP1 with increased affinity also promoted the accumulation of NIK, which activated the

downstream noncanonical NF-κB signaling pathway, as confirmed by CO-IP assay (Fig. 3C). The expression of noncanonical NF-κB pathway-related genes and proteins was tested by ddPCR (Fig. 3D)

and western blotting in NKTCL cells (Fig. 3E), and by IHC staining in NKTCL xenograft tumor tissues (Fig. 3F), and these results collectively suggested that shLMP1 inhibited the activation

of the downstream noncanonical NF-κB pathway. In brief, LMP1 competes with CD40 for TRAF3 binding and subsequently activates the noncanonical NF-κB signaling pathway in NKTCL cells to

promote aerobic glycolysis (Fig. 4). ACTIVATION OF THE NONCANONICAL NF-ΚB PATHWAY AFFECTS AEROBIC GLYCOLYSIS IN NKTCL CELLS To confirm the effect of the noncanonical NF-κB pathway

reactivation on aerobic glycolysis in NKTCL, NKYSshLMP1 and SNK6shLMP1 cells with OE-RelB were generated (Additional File 4: Fig. S3). In addition, the NF-κB pathway inhibitor BAY 11-7082

was applied to the YT and SNT16 cells with OE-LMP1. BAY 11-7082 greatly reduced glucose uptake and lactate production compared with those in OE-LMP1 cells (Fig. 5A, B), and

NKYSshLMP1+OE-RelB and SNK6shLMP1+OE-RelB cells took up more glucose and produced more lactate than NKYSshLMP1+OE-vector and SNK6shLMP1+OE-vector cells (Fig. 5C, D). Moreover, the glycolysis

stress assay indicated that the ECAR was significantly decreased by the application of BAY 11-7082 in YT and SNT16 cells (Fig. 5E, F) and increased by OE-RelB in NKYS and SNK6 cells (Fig.

5G, H). Furthermore, the expression of glycolysis-related proteins was also confirmed by western blotting (Fig. 5I, J). In summary, the inhibition or reactivation of the NF-κB pathway

affects aerobic glycolysis in NKTCL cells. ACTIVATION OF THE NONCANONICAL NF-ΚB PATHWAY AND AEROBIC GLYCOLYSIS AFFECT THE AGGRESSIVE BIOLOGICAL FUNCTION OF NKTCL CELLS Whether the activation

of the noncanonical NF-κB pathway and aerobic glycolysis affect the tumorigenesis and development of NKTCL cells remains to be studied. Thus, cell proliferation, apoptosis resistance, and

gemcitabine sensitivity were assessed in vitro. As expected, inhibition of the NF-κB pathway significantly suppressed the proliferation of NKTCL cells (Fig. 6A), and interestingly, OE-RelB

attenuated the decrease in NKTCL cell proliferation caused by shLMP1, which was also inhibited by 2-DG, an aerobic glycolysis inhibitor (Fig. 6B). Consistently, in the apoptosis resistance

and gemcitabine sensitivity experiments, BAY 11-7082 increased starvation-induced cell apoptosis (Fig. 6C), and OE-RelB decreased this effect, and the effect of OE-RelB was inhibited by

treatment with 2-DG (Fig. 6D). Moreover, BAY 11-7082 reduced gemcitabine sensitivity in NKTCL cells (Fig. 6E) and OE-RelB promoted gemcitabine sensitivity, which was inhibited by 2-DG

treatment (Fig. 6F). In summary, the activation of the noncanonical NF-κB pathway and aerobic glycolysis affect the proliferation and development of NKTCL cells modulated by LMP1. DISCUSSION

Previous studies have comprehensively illuminated the phenotypic effects and mechanism of LMP1 on NPC. LMP1 plays an important role in the tumorigenesis and development of NPC through

activating multiple signaling pathways, including cell proliferation and survival, angiogenesis, and invasion pathways [11, 24, 25]. In addition, LMP1 can affect cell-cell interactions,

antigen presentation, cytokine and chemokine production, and modulation of the tumor microenvironment [10, 26, 27]. LMP1 is also reported to enhance cell proliferation in EBV-driven

malignancies and serves as a prognostic marker for NKTCL patients [28, 29]. Therefore, we detected LMP1 expression in 6 NKTCL cell lines and established stable OE-LMP1 and shLMP1 cells. Our

results showed that high LMP1 expression promoted NKTCL cell proliferation in vitro and in vivo, and was positively correlated with Ki67 expression in xenograft mouse tissues. In addition,

experimental assays demonstrated that LMP1 inhibited starvation-induced apoptosis and reduced the gemcitabine sensitivity of NKTCL cells. Clinically, LMP1 expression was scored in 58 NKTCL

patient tissues, and its correlations with clinical features, treatment response, and prognosis were analyzed. These findings suggest that LMP1 affects the tumorigenesis and development of

NKTCL cells and serves as an indicator of the risk stratification, treatment response, and prognosis of NKTCL patients. In recent years, studies on metabolic reprogramming, especially

glucose metabolism, have attracted increased attention. Based on the results of untargeted metabolic sequencing, we found that compared with LMP1-negative cells, LMP1-positive NKTCL cells

exhibited greater glucose uptake and lactate production. Consistent results were obtained for stable OE-LMP1 and shLMP1 cells. Moreover, the glycolysis stress assay suggested that the ECAR

was affected by LMP1 expression. The expression of glycolysis-related genes and proteins also recapitulated this finding. In addition, we analyzed the available 18F-FDG uptake of NKTCL

patients who underwent PET-CT examination before treatment, and the correlation between the SUVmax and LMP1 expression showed that aberrantly high LMP1 expression was accompanied by a

greater SUVmax in NKTCL patients. Mechanistically, some studies have indicated that LMP1 promotes aerobic glycolysis by regulating oncogenic signaling pathways [17, 30], involving in

epigenetic processes [31, 32], and activating glucose metabolic enzymes in NPC [20]. To date, few investigations have focused on the mechanism by which LMP1 regulates aerobic glycolysis in

NKTCL, which is the novel aspect of our study. To further explain the role of LMP1 in aerobic glycolysis in NKTCL, RNA sequencing was conducted and KEGG enrichment analysis revealed that the

NF-κB signaling pathway was significantly enriched in the high LMP1 expression group. Subsequently, via IP coupled to mass spectrometry, we identified one LMP1-interacting protein, TRAF3,

which plays a negative role in regulating the NF-κB signaling pathway [33]. Previous studies demonstrated that LMP1 led to TRAF3 sequestration in B-lymphoma cells, which inhibited the

negative regulation of pro-survival membrane, cytoplasmic, and nuclear signaling events by TRAF3 [23]. Therefore, the interactions between LMP1 and TRAF3 and between TRAF3 and CD40 upon LMP1

availability were examined in our research. The results showed that cells with high LMP1 expression exhibited greater binding of LMP1 and TRAF3, and shLMP1 cells exhibited greater binding

of LMP1 and CD40. Because of its competitive binding to LMP1 with greater affinity, the subsequent degradation of TRAF3 also protected NIK from self-degradation and subsequently caused its

accumulation, which activated the downstream noncanonical NF-κB signaling pathway. The expression of noncanonical NF-κB pathway-related genes and proteins also confirmed this phenomenon.

Several studies have shown that the NF-κB pathway governs glycolysis via direct engagement of the cellular networks, with profound implications on inflammation, metabolic diseases, and

tumorigenesis [34, 35]. A study on diffuse large B-cell lymphoma (DLBCL) showed that TP53 mutations cooperated with c-Rel to promote NF-κB functions and led to enhanced invasion and

metastasis in malignant cells [36]. Moreover, the activation of the NF-κB signaling pathway increased glucose uptake by inducing the plasma membrane localization of GLUT1, blocking

apoptosis, and promoting B-cell lymphoma growth [37]. However, this effect on NKTCL is not fully understood, and whether the activation of the noncanonical NF-κB signaling pathway affects

aerobic glycolysis and the biological function of NKTCL cells remains to be explored. Thus, in our study, an NF-κB pathway inhibitor was applied to YTOE-LMP1 and SNT16 OE-LMP1 cells, and

OE-RelB cells were established from NKYSshLMP1 and SNK6shLMP1 cells. The results showed that with the inhibition of the NF-κB pathway, NKTCL cells exhibited suppression of aerobic glycolysis

including decreased glucose uptake, lactate production, and ECAR, while OE-RelB cells showed promotion of aerobic glycolysis. In addition, glycolysis-related genes and proteins were also

examined. Further experiments confirmed that inhibition of the NF-κB pathway could suppress the aggressive behavior of NKTCL cells and that of OE-RelB cells could restore these behaviors.

Moreover, the addition of 2-DG, an inhibitor of glycolysis, significantly inhibited biological cellular functions. Recently, accumulating evidence has also revealed the immune-related

effects of LMP1 in NKTCL. Li et al. highlighted the crucial role of malignant NK cells with LMP1 expression in reshaping the cellular landscape and fostering an immunosuppressive

microenvironment [38], and two other studies suggested that LMP1 expression is positively correlated with PD-L1 expression in NKTCL [8, 39]. In addition, LMP1 also serves as a promising

target for novel adaptive T cell immunotherapy for the treatment of NKTCL [40]. Moreover, glycolysis has been implicated in the regulation of the tumor microenvironment and development by

inhibiting monocyte migration, suppressing T cell activation, and promoting the release of cytokines in DLBCL [41]. Thus, whether enhancement of aerobic glycolysis is involved in the

regulation of the NKTCL microenvironment and whether immunotherapy can attenuate abnormal glycolysis in NKTCL cells need to be elucidated in further studies. In summary, the present study is

the first to reveal the role and detailed mechanism of LMP1 in promoting aerobic glycolysis and aggressive biological functions in NKTCL. Our results elucidate the effects of viral

infection on abnormal metabolism in NKTCL patients, which expands the understanding of the pathogenesis and progression of this disease and might provide a promising perspective for the

treatment of NKTCL. DATA AVAILABILITY Raw untargeted metabolic sequencing data are available in the Metabolights under the accession number MTBLS9482. Raw mRNA sequencing data are deposited

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prognosis and tumor immune microenvironment. Front Cell Dev Biol. 2023;11:1070777. Article  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS YT and NKYS cell

lines were obtained from Dr. Wing C. Chan (City of Hope Medical Center), SNT16 cell line was a gift from Guangzhou Bairui Biomedical Technology Company, Ltd. (China), and SNK6 cell line was

kindly provided by Dr. Norio Shimizu and Yu Zhang of Chiba University. I would like to show great gratitude to them all. FUNDING This work was supported by the National Natural Science

Foundation of China (81970184; 82170183; 82070209; U1904139), Funding for Scientific Research and Innovation Team of The First Affiliated Hospital of Zhengzhou University (QNCXTD2023012),

Henan Province Youth Health Science and Technology Innovation Project (LJRC2023014), Joint Construction Project of Medical Science and Technology of Henan Province of China (LHGJ20220386),

and Henan Province Health Commission Co-Construction Project (SB201901044). The work was also supported by the Oncology Department and State Key Laboratory of Esophageal Cancer Prevention

& Treatment and Henan Key Laboratory for Esophageal Cancer Research of the First Affiliated Hospital of Zhengzhou University. AUTHOR INFORMATION Author notes * These authors contributed

equally: Wenting Song, Yuyang Gao, Jiazhuo Wu. AUTHORS AND AFFILIATIONS * Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China Wenting Song,

 Yuyang Gao, Jiazhuo Wu, Hongwen Li, Zhuangzhuang Shi, Chen Gong, Zihe Zhang, Zhaoming Li & Mingzhi Zhang * State Key Laboratory of Esophageal Cancer Prevention & Treatment and Henan

Key Laboratory for Esophageal Cancer Research, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China Wenting Song, Yuyang Gao & Jiazhuo Wu Authors * Wenting

Song View author publications You can also search for this author inPubMed Google Scholar * Yuyang Gao View author publications You can also search for this author inPubMed Google Scholar *

Jiazhuo Wu View author publications You can also search for this author inPubMed Google Scholar * Hongwen Li View author publications You can also search for this author inPubMed Google

Scholar * Zhuangzhuang Shi View author publications You can also search for this author inPubMed Google Scholar * Chen Gong View author publications You can also search for this author

inPubMed Google Scholar * Zihe Zhang View author publications You can also search for this author inPubMed Google Scholar * Zhaoming Li View author publications You can also search for this

author inPubMed Google Scholar * Mingzhi Zhang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Wenting Song: Investigation, Project

administration, Formal analysis, Data curation, Visualization, Writing-Original draft; Yuyang Gao: Investigation, Formal analysis, Validation; Jiazhuo Wu: Investigation, Formal analysis,

Validation; Hongwen Li: Formal analysis, Project administration; Zhuangzhuang Shi: Formal analysis; Chen Gong: Formal analysis; Zihe Zhang: Formal analysis; Zhaoming Li: Funding acquisition,

Resources, Supervision, Writing-review & editing; Mingzhi Zhang: Conceptualization, Funding acquisition, Resources, Supervision, Writing-review & editing. CORRESPONDING AUTHOR

Correspondence to Mingzhi Zhang. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ETHICS APPROVAL This study was conducted in accordance with the

declaration of Helsinki and approved by the Ethics Committee for Scientific Research and Clinical Trials of the First Affiliated Hospital of Zhengzhou University (approval number:

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aerobic glycolysis in natural killer/T cell lymphoma. _Cell Death Dis_ 15, 604 (2024). https://doi.org/10.1038/s41419-024-06999-7 Download citation * Received: 11 March 2024 * Revised: 09

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