LINC-AC092535.5 regulates MICAL2 mRNA level to inhibit p53-mediated ferroptosis in nasopharyngeal carcinoma.

BackgroundCentromere protein N (CENP-N) has been reported to be highly expressed in malignancies, but its role and mechanism in nasopharyngeal carcinoma (NPC) are unknown.MethodsAbnormal CENP-N expression from NPC microarrays of GEO database was analyzed. CENP-N expression level was confirmed in NPC tissues and cell lines. Stable CENP-N knockdown and overexpression NPC cell lines were established, and transcriptome sequencing after CENP-N knockdown was performed. In vitro and in vivo experiments were performed to test the impact of CENP-N knockdown in NPC cells. ChIP and dual luciferase reporter assays were used to verify the combination of IRF2 and CENP-N. Western blot analysis, cellular immunofluorescence, immunoprecipitation and GST pulldown assays were used to verify the combination of CENP-N and AKT.ResultsCENP-N was confirmed to be aberrantly highly expressed in NPC tissues and cell lines and to be associated with high 18F-FDG uptake in cancer nests and poor patient prognosis. Transcriptome sequencing after CENP-N knockdown revealed that genes with altered expression were enriched in pathways related to glucose metabolism, cell cycle regulation. CENP-N knockdown inhibited glucose metabolism, cell proliferation, cell cycling and promoted apoptosis. IRF2 is a transcription factor for CENP-N and directly promotes CENP-N expression in NPC cells. CENP-N affects the glucose metabolism, proliferation, cell cycling and apoptosis of NPC cells in vitro and in vivo through the AKT pathway. CENP-N formed a complex with AKT in NPC cells. Both an AKT inhibitor (MK-2206) and a LDHA inhibitor (GSK2837808A) blocked the effect of CENP-N overexpression on NPC cells by promoting aerobic glycolysis, proliferation, cell cycling and apoptosis resistance.ConclusionsThe IRF2/CENP-N/AKT axis promotes malignant biological behaviors in NPC cells by increasing aerobic glycolysis, and the IRF2/CENP-N/AKT signaling axis is expected to be a new target for NPC therapy.

NOLC1, an Enhancer of Nasopharyngeal Carcinoma Progression, Is Essential for TP53 to Regulate MDM2 Expression

Serum amyloid A1 (SAA1) was previously identified as a tumor suppressor gene with anti-angiogenic activities in nasopharyngeal carcinoma (NPC). Three SAA1 isoforms (SAA1.1, 1.3, and 1.5) were observed with disproportionate frequencies among the NPC patients and healthy people. SAA1.1 and 1.3 are the functional isoforms to inhibit angiogenesis whereas SAA1.5 was the defective gene. Our immunohistochemical results showed that the loss of SAA1 staining in the metastatic NPC tissues was significantly associated with tumor progression. We aim to investigate the functional roles of the three SAA1 isoforms in NPC metastasis in the present study. We want to investigate whether the functional SAA1.1 and 1.3 isoforms can suppress tumor metastasis by antagonizing the integrin-FAK signaling pathway in the NPC tumor cells. The focal adhesion assay was performed by seeding the tumor cells with or without the integrin alphaVbeta3/beta5 ligand vitronectin. Both the vector-alone control and the SAA1.5-expressing NPC cells began to spread out and adhered to the bottom of the culture dish in the presence of vitronectin from 7 to 9 hours, whereas the SAA1.1 and SAA1.3-cells remained in the round-up morphology with minimal attachment. Interestingly, after 48 hours the SAA1.1 and SAA1.3-expressing cells formed adherents junction among the cells. In order to study the viability of the effects of the three SAA1 isoforms on NPC cells, MTT viability assay was performed. The results showed that the viability of the SAA1.1 and SAA1.3-expressing cells were around 50 % lower than both the vector-alone and the SAA1.5-expressing cells. It is likely that the loss of focal adhesion after seeding of the SAA1.1 and SAA1.3-expressing cells will affect the survival of NPC cells. Furthermore, we found that the presence of the recombinant SAA1.1 and SAA 1.3 proteins could reduce the number of viable NPC cells compared with the solvent control and the SAA1.5 protein. We previously reported that the SAA1 proteins can physically interact with the integrin alphaVbeta3. Taken together, we suggested that the secreted SAA1 proteins from the NPC cells could directly affect the NPC focal adhesion as well as the cell viability by blocking the integrin on the NPC cell surface. We acknowledge the financial support of the General Research Fund (grant number HKBU17115114 to HLL) of the Research Grants Council of the Hong Kong Special Administrative Region. Citation Format: On Ying Man, Hong Lok Lung. The functional role(s) of serum amyloid A1 (SAA1) polymorphisms in integrin-mediated cell adhesion in nasopharyngeal carcinoma (NPC) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5877. doi:10.1158/1538-7445.AM2017-5877

The emerging concept of generating cancer stem cells from epithelial-mesenchymal transition has attracted great interest; however, the factors and molecular mechanisms that govern this putative tumor-initiating process remain largely elusive. We report here that miR-200a not only regulates epithelial-mesenchymal transition but also stem-like transition in nasopharyngeal carcinoma cells. We first showed that stable knockdown of miR-200a promotes the transition of epithelium-like CNE-1 cells to the mesenchymal phenotype. More importantly, it also induced several stem cell-like traits, including CD133(+) side population, sphere formation capacity, in vivo tumorigenicity in nude mice, and stem cell marker expression. Consistently, stable overexpression of miR-200a switched mesenchyme-like C666-1 cells to the epithelial state, accompanied by a significant reduction of stem-like cell features. Furthermore, in vitro differentiation of the C666-1 tumor sphere resulted in diminished stem-like cell population and miR-200a induction. To investigate the molecular mechanism, we demonstrated that miR-200a controls epithelial-mesenchymal transition by targeting ZEB2, although it regulates the stem-like transition differentially and specifically by β-catenin signaling. Our findings reveal for the first time the function of miR-200a in shifting nasopharyngeal carcinoma cell states via a reversible process coined as epithelial-mesenchymal to stem-like transition through differential and specific mechanisms.

Nasopharyngeal carcinoma (NPC) is one common head and neck malignancy with leading cause of cancer-related death. Long noncoding RNAs (lncRNAs) have been reported to play essential roles in progression, prognosis and treatment of NPC. However, the exact role of lncRNA zinc finger antisense 1 (ZFAS1) in NPC progression and its potential mechanism remain largely unknown.The expressions of ZFAS1 and microRNA-135a (miR-135a) were measured in NPC tissues or cells by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between ZFAS1 and miR-135a was explored by luciferase reporter assay and RNA immunoprecipitation (RIP). Cell proliferation, apoptosis, migration and invasion were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5 -diphenyl-2-H-tetrazolium bromide (MTT) assay, flow cytometry or trans-well assay, respectively. Our data showed the expression of ZFAS1 was up-regulated and miR-135a was down-regulated in NPC tissues and cells. miR-135a was bound to ZFAS1 in NPC cells. Moreover, knockdown of ZFAS1 or addition of miR-135a inhibited cell proliferation, migration and invasion but promoted apoptosis in NPC cells. Besides, down-regulation of miR-135a reversed abrogation of ZFAS1-mediated inhibition of proliferation, migration and invasion and increase of apoptosis in NPC cells. Our data suggested Inhibition of ZFAS1 protected against proliferation, migration and invasion but contributed to apoptosis by sponging miR-135a in NPC cells, providing a novel avenue for NPC treatment.

BackgroundRadioresistance greatly hinders the treatment of nasopharyngeal carcinoma (NPC). Long noncoding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) has been corroborated to participate in diverse cancers, including NPC. Our aim was to investigate the underlying molecular mechanism of PVT1 in NPC radioresistance.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was utilized to measure the expression levels of PVT1, microRNA (miR)-515-5p and phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) in NPC tissues and cells. Cell counting kit-8 (CCK8) assay, colony formation assay and flow cytometry assay were employed to detect cell proliferation, radiosensitivity and apoptosis, respectively. The protein levels of Cyclin D1, B-cell lymphoma 2 associated X (Bax), Cleaved-caspase-3, PIK3CA, protein kinase B (AKT) and phosphorylated AKT (p-AKT) in samples were measured by Western blot. The starBase was used to predict the binding sites between miR-515-5p and PVT1 or PIK3CA. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the interaction. Xenograft tumor model was established to investigate the biological role of PVT1 in vivo.ResultsThe levels of PVT1 and PIK3CA were upregulated in NPC tissues and cells, opposite to the expression of miR-515-5p. Knockdown of PVT1 inhibited cell proliferation, radioresistance and promoted cell apoptosis in NPC cells. Meanwhile, PVT1 silencing downregulated Cyclin D1, and upregulated Bax and Cleaved-casp-3 in NPC cells after radiotherapy. Besides, miR-515-5p interacted with PVT1 and targeted PIK3CA in NPC cells. Further studies indicated that PVT1 regulated radioresistance via miR-515-5p/PIK3CA axis and modulated the AKT pathway by interacting with miR-515-5p. Moreover, knockdown of PVT1 suppressed tumor growth in vivo.ConclusionDownregulation of PVT1 inhibited proliferation, radioresistance and promoted apoptosis by downregulating PIK3CA via sponging miR-515-5p in NPC cells.

We have previously identified prenylated Rab acceptor 1 (PRA1) as a novel cellular interacting partner for Epstein-Barr virus-encoded oncoprotein, latent membrane protein 1 (LMP1). The intracellular trafficking and full signaling of LMP1 requires its interaction with PRA1. To further explore the role of PRA1 in Epstein-Barr virus-associated nasopharyngeal carcinoma (NPC) cells, we generated several PRA1-knockdown cell clones, which exhibited altered cell morphology and increased cell motility. We identified proteins differentially expressed in the knockdown clones by means of isobaric mass tags labeling coupled with multidimensional liquid chromatography-mass spectrometry. We validated a panel of proteins, which showed consistent up-regulation in PRA1-knockdown clones and participated in regulating lipid homeostasis and cell migration. Immunofluorescence staining further revealed altered localization of these proteins and accumulation of intracellular cholesterol in PRA1-knockdown clones. These effects were phenocopied by treatment with a cholesterol transport inhibitor, U18666A. Moreover, overexpressed PRA1 was able to alleviate the dysregulation of these affected proteins either from PRA1 knockdown or U18666A treatment, implying a role for PRA1 in regulating the levels of these affected proteins in response to altered cholesterol homeostasis. We further demonstrated that LMP1 expression caused PRA1 sequestration in NPC cells, leading to a consequence reminiscent of PRA1 knockdown. Finally, the immunohistochemistry showed a physiological relevance of the PRA1-associated proteome-wide changes in NPC biopsy tissues. In sum, our findings delineated novel roles of PRA1 in lipid transport and cell migration, and provided additional insights into the molecular basis of NPC morphogenesis, namely a consequence of LMP1-PRA1 interaction.

IntroductionAntidifferentiation noncoding RNA (ANCR) is a newly identified long noncoding RNA, which is reported to function as an oncogene in multiple human cancers. However, its function in nasopharyngeal carcinoma (NPC) and underlying mechanism are still unclear.Materials and methodsWe explored the expression of ANCR in NPC tissues and cells by real-time PCR and analyzed the relationship between ANCR expression and clinicopathological characteristics of NPC patients by Pearson’s chi-squared test. Then we inhibited ANCR expression in NPC cells using siRNAs and evaluated the effect of ANCR expression on cell proliferation, colony formation, and radiosensitivity by cell counting kit-8 assay and colony formation assay. We used RT-PCR and Western blot analyses to search target genes of ANCR. Also, we used RNA immunoprecipitation (RIP) assay and chromatin immunoprecipitation assay to study the molecular mechanism in this regulation.ResultsWe found that ANCR was upregulated in NPC tissues and cells. ANCR expression was significantly correlated with tumor size and TNM stage. Further, ANCR knockdown inhibited NPC cell growth and radiation resistance. Mechanistically, we found that PTEN was upregulated in ANCR knockdown NPC cells. In addition, RIP assay indicated that EZH2, the oncogenic histone methyltransferase of polycomb repressive complex 2, interacted with ANCR in NPC cells. More importantly, the binding of EZH2 and deposition of relevant negative histone marker H3K27me3 on PTEN promoter depended on ANCR expression.ConclusionANCR expression is upregulated in NPC and promotes NPC growth and radiation resistance through an epigenetic regulation of PTEN expression.

Nasopharyngeal carcinoma (NPC) is prevalent in southeastern Asia, and its tumorigenesis is rather complex. The purpose of this research was to identify the pivotal genes that may be altered during the early stage of NPC progression. Eleven genes were selected by comparative microarray analysis of NPC versus normal nasomucosal cells. The expression of SPARC (secreted protein, acidic, cysteine-rich) was statistically significantly downregulated in NPC cells. In exploring the mechanism underlying the decreased transcription of SPARC in NPC cells, we found that the transcription factor SRY (sex-determining region Y)-box 5 (SOX-5) is upregulated in NPC cells. RNA interference of SOX-5 by short hairpin RNA (shRNA) in NPC cells caused a dramatic increase in SPARC and chromosome immunoprecipitation assay showed SOX-5 can bind directly to SPARC promoter, suggesting that SOX-5 acts as a key transcriptional repressor of SPARC. We further demonstrate that shRNA knockdown of SOX-5 suppressed the proliferation of NPC cells, as well as their migratory ability, which was also observed when SPARC was overexpressed in NPC cells. Alternatively, blocking SPARC with an antagonistic antibody reversed the effects of SOX-5 knockdown. In 66 NPC patients’ biopsy specimens, overexpression of SOX-5 in tumor cells correlated clinically with poor survival. Our study suggests that SOX-5 transcriptionally downregulates SPARC expression and plays an important role in regulation of NPC progression. SOX-5 is a potential tumor marker for advanced NPC prognosis.

Nasopharyngeal carcinoma (NPC) is rare worldwide but common in southern China, including Hong Kong. The endemic NPC among southern Chinese is typically non-keratinizing carcinoma, which is almost 100% associated with Epstein-Barr virus (EBV) infection. Patient-derived xenografts (PDXs), given their close resemblance with patient tumors, serve as important models in preclinical evaluation for novel therapeutic drugs. For unclear reasons, it has been difficult to establish NPC PDXs in vivo and cell lines in vitro. Currently, there are few NPC PDXs available for research, including X666, X2117, C15, C17 and C18. However, all of them have been passaged in nude mice for over 25 years and may deviate from their original NPC tumors in patients. In vitro, C666-1 is the only EBV-positive (EBV+ve) NPC cell line which has been used extensively in investigations. C666-1 was established from the NPC xenograft (X666), which had been propagated for a long period of time. Most if not all the other previously reported NPC cell lines have lost their EBV episomes upon in vitro propagation. Furthermore, many of these reported NPC cell lines have been shown with genetic contamination of HeLa cells. Hence, their applications in NPC studies are limited. The lack of representative nasopharyngeal carcinoma (NPC) models has also seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. In this study, we report the successful establishment and comprehensive characterization of four new NPC PDXs (all EBV+ve) and three NPC cell lines (one EBV+ve; two EBV-ve). These newly established EBV+ve NPC PDXs and cell lines significantly recapitulate the mutation profiles of their original NPC tumors, and harbor common genetic alterations reported in NPC, which supports their potential applications in the investigations of NPC pathogenesis. We postulate that lytic EBV reactivation may be an intrinsic barrier to the successful establishment of EBV+ve NPC PDXs and cell lines. The ROCK inhibitor (Y-27632) was shown to be able to suppress epithelial differentiation and lytic reactivation of EBV in infected NPC cells. Establishment of a new EBV-positive NPC cell line, NPC43, was achieved directly from patient NPC tissues by including Y-27632 in culture medium. NPC43 cells exhibited tumorigenicity in immunodeficient mice, and could be induced to undergo EBV lytic reactivation with production of infectious visions. The establishment and characterization of new NPC PDXs and cell lines will provide valuable experimental tools for NPC and EBV research. Funding support: Research Grant Council, Hong Kong: AoE NPC grant (AoE/M-06/08), Theme-based Research Scheme grant (T12-401/13-R) and Collaborative Research Fund (C7027-16G), General Research Fund (106140069, 106160185, 17111516, 17110315) and HMRF grant (04151726, 13142201). Citation Format: Weitao Lin, Yim Ling Yip, Lin Jia, Wen Deng, Kwok Wai Lo, Chin Man Tsang, Maria Li Lung, George Sai Tsao. Establishment and characterization of new tumor xenografts and cancer cell lines from EBV positive nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 56.

Background: Accumulating studies discloses that long non-coding RNAs (lncRNAs) serve important roles in human tumorigenesis, including nasopharyngeal carcinoma (NPC). The purpose of the present study was to determine the role of lncRNA FEZF1-AS1 in NPC.Materials and methods: The expression levels of FEZF1-AS1 in NPC tissues and cell lines were detected by RT-qPCR analysis. MTT assay was performed to investigate the proliferation of NPC cells in vitro, whereas the migration and invasion of NPC cells were determined by wound healing assay and transwell assay. A nude mouse tumor model was established to study the role of FEZF1-AS1 in NPC tumorigenesis in vivo. The expression levels of proteins were detected by Western blot assay.Results: The results showed that FEZF1-AS1 expression was increased in the NPC tissues and cell lines, and the higher expression of FEZF1-AS1 was closely associated with poor prognosis of NPC patients. We further observed that knockdown of FEZF1-AS1 inhibited the proliferation of NPC cells in vitro and suppressed NPC xenograft growth in vivo through inducing G2/M cell cycle arrest. The migratory and invasive abilities of NPC cells were also reduced upon FEZF1-AS1 knockdown. Moreover, we demonstrated that inhibition of FEZF1-AS1 remarkably suppressed epithelial–mesenchymal transition (EMT) and reduced β-catenin accumulation in nucleus in NPC cells.Conclusions: Collectively, we showed that FEZF1-AS1 might be a key regulator of cell cycle, EMT and Wnt/β-catenin signaling in NPC cells, which may be helpful for understanding of pathogenesis of NPC.

BackgroundRecently, long non-coding RNAs (lncRNAs) have been elucidated to play essential roles in cancers, and the recognition of lncRNA expression patterns in nasopharyngeal carcinoma (NPC) may be helpful for indicating novel mechanisms underlying NPC carcinogenesis. Herein, we conducted this study to probe into the function of lncRNA ZNF667-AS1 in NPC progression with the involvement of microRNA-1290 (miR-1290) and actin-binding LIM protein 1 (ABLIM1).Materials and MethodsIn silico analysis screened differentially expressed genes and miRNAs in NPC and predicted potential mechanisms. ZNF667-AS1 expression was detected in NPC tissues and cells. The gain-and-loss function assays were performed to explore the effects of lncRNA ZNF667-AS1 and miR-1290 in NPC cell biological behaviors. In vivo experiments were further conducted to confirm the in vitro results.ResultsIn silico analysis predicted that ZNF667-AS1 was diminished in NPC, which may downregulate ABLIM1 through sponging miR-1290. ZNF667-AS1 was poorly expressed in NPC tissues and cells, and overexpression of ZNF667-AS1 inhibited growth of NPC cells. ZNF667-AS1 competitively bound with miR-1290, thereby upregulating ABLIM1. miR-1290 resulted in the promotion of NPC cell progression by suppressing ABLIM1. Overexpression of ZNF667-AS1 or suppression of miR-1290 inhibited tumorigenicity of NPC cells in vivo.ConclusionThis study highlights that lncRNA ZNF667-AS1 promotes ABLIM1 expression by sponging miR-1290 to suppress NPC cell progression.

Nasopharyngeal carcinoma is a type of otolaryngological malignancy with high incidence. Long non-coding RNAs (lncRNAs) are closely related to nasopharyngeal carcinoma. LncRNA AFAP1-AS1 (AFAP1-AS1) has been found to play important roles in nasopharyngeal carcinoma progression and poor prognosis. However, the mechanism underlying AFAP1-AS1 in regulating nasopharyngeal carcinoma is still unclear. In current study, AFAP1-AS1 was found to be up-regulated in nasopharyngeal carcinoma tissues and cells. AFAP1-AS1 overexpression and knockdown were conducted in nasopharyngeal carcinoma cells. The results proved that AFAP1-AS1 promoted the survival and migration of nasopharyngeal carcinoma cells. Additionally, specificity protein 1 (SP1) was enhanced in nasopharyngeal carcinoma tissues and cells, and induced AFAP1-AS1 expression. The interaction between AFAP1-AS1 and miR-497-5p was confirmed. AFAP1-AS1 was demonstrated to regulate CELF1, a target gene of miR-497-5p. Further functional analysis revealed that AFAP1-AS1 knockdown attenuated SP1-induced nasopharyngeal carcinoma progression. These results indicate that SP1-induced AFAP1-AS1 facilitates nasopharyngeal carcinoma progression by regulating miR-497-5p/CELF1 pathway, which provides a new target for nasopharyngeal carcinoma treatment.

Nasopharyngeal carcinoma (NPC) is a kind of head-neck malignant tumor. lncRNA-PVT1 can promote the proliferation of carcinoma cells, and induce cells to have stem cell-like potentials. However, the function of PVT1 in NPC cells is not clear. The expressions of lncRNA-PVT1 and the expressions of the stem cell markers in NPC tissues or cell lines were investigated by qRT-PCR or western blot. The cell proliferation, and the ability of NPC cells to form spherical, clonal colonies were investigated by MTT assay, colony formation assay, and tumor-sphere formation assay. Cancer stem cells surface markers were detected by flow cytometry and western blot. PI3K/AKT signal activation in NPC cells was determined by western blot. PVT1 was significantly up-regulated in both NPC tissues and cell lines and associated with poor prognosis. PVT1 knockdown reduced NPC cells viability, clonogenicity, the cell surface CD44+/CD24- stem phenotype, and the expressions of the stem cell markers in NPC cells, including Oct4, c-Myc, SOX2, and ALDH. Furthermore, PVT1 negatively regulates the expression levels of miR-1207 in NPC cells and spheres cells, which is critical for NPC stemness. Knockdown of miR-1207 promoted stem phenotype and the expressions of the stem cell markers in NPC cells. Moreover, phosphor-PI3K (p-PI3K) and phosphor-AKT (p-AKT) were found to be down-regulated after PVT1 siRNAs transfection in NPC cells. And miR-1207 inhibitor transfection reversed the all the effects brought by PVT1 knockdown. Pvt1 promotes cancer stem cell-like properties in NPC cells via inhibiting miR-1207 and activating the PI3K/AKT signal pathway.

Patients diagnosed with Nasopharyngeal carcinoma (NPC) initially respond well to chemo- and radiotherapy but eventually develop resistance to these treatments. Hence, novel and improved treatment strategies are needed for NPC. The BCL-2 family proteins are critical regulators of the intrinsic apoptosis pathway. These proteins are up-regulated in many cancers hence they have become attractive therapeutic targets. Given that different cell population rely on different anti-apoptotic proteins for survival, it is crucial to determine which proteins are important for NPC survival. A human apoptosis RT2 Profiler PCR Array was first employed to profile the anti-apoptotic gene expressions in both the NPC cell lines. The HK1 cells expressed all the anti-apoptotic genes (MCL-1, BFL-1, BCL-2, BCL-XL, and BCL-w). On the other hand, C666-1 expressed all except for BFL-1 (undetectable level). Given that there are no specific BFL-1 inhibitors, the role of BFL-1 in NPC cell survival was determined by deleting the gene using the CRISPR/Cas9 technique. The BFL-1 single guide RNAs were cloned into the PX459 plasmid (pSpCas9(BB)-2A-Puro) and transfected into the NPC cells to establish BFL-1 knock-out cells. Parallel to this experiment, the contributions of the other anti-apoptotic proteins in NPC survival was determined by treating NPC cell lines HK1, C666-1 and C17 with BH3 mimetics ABT-199, A-1331852 and S63845 which inhibit BCL-2, BCL-XL and MCL-1, respectively, alone and in combination, in both 2D and 3D cell culture models. The HK1 and C666-1 cells were resistant to single agent treatment of all three drugs, suggesting that none of these anti-apoptotic proteins singly mediate survival of the cells. The NPC C17 cells however were sensitive to single agent treatment of S63845 indicating that these cells rely on MCL-1 for survival. Co-inhibition of BCL-XL and MCL-1 with A1331852 and S63845 significantly inhibited cell proliferation in all three cell lines. Similar data were obtained with 3-dimensional spheroid cell culture models. Treatment with a combination of A1331852 and S63845 resulted in inhibition of growth and invasion of the 3D spheroids generated from the HK1 cells. Co-inhibition of BCL-2 and MCL-1 with ABT-199 and S63845, also inhibited cell proliferation of the NPC cell lines. However, the effect of the combination was not as pronounced as combination of A1331852 and S63845. Collectively, our data demonstrate that BCL-XL and MCL-1 are crucial for NPC survival and targeting these proteins with selective inhibitors may be potentially useful as treatment strategies for the management of NPC. Citation Format: Siti Fairus Abdul Rahman, Kalaivani Muniandy, Ghows Azzam, Nethia Mohana Kumaran. Anti-apoptotic proteins BCL-XL and MCL-1 are crucial for nasopharyngeal carcinoma (NPC) cell survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 65.