Preclinical activity of brincidofovir in Peripheral T-cell and NK/T-cell Lymphoma

Preclinical activity of brincidofovir in Peripheral T-cell and NK/T-cell Lymphoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Preclinical activity of brincidofovir in Peripheral T-cell and NK/T-cell Lymphoma Jason Yongsheng Chan, Elizabeth Chun Yong Lee, Kelila Chai, Boon Yee Lim, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6768176/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Brincidofovir (BCV) is a novel nucleoside phosphonate analogue with unique dual anti-viral and anti-tumor properties. Methods: Activity of BCV was evaluated in forty-four cell-line models, including T/NK-cell non-Hodgkin lymphoma (T/NK-NHL, n=25) and B-cell lymphoma (BCL, n=19), as well as their respective NOD/SCID mice xenograft models (NK/T-cell lymphoma, peripheral T-cell lymphoma, not otherwise specified, TP63 -rearranged anaplastic large cell lymphoma, and MYC / BCL2 -rearranged diffuse large B-cell lymphoma). Potential in vivo immunogenic effects were examined in a syngeneic EL4-C57BL/6 murine lymphoma model. Results: BCV demonstrated potent anti-tumor activity in vitro across the majority of cell-lines regardless of EBV positivity, with IC50 values within clinically-achievable human plasma concentrations (2 µg/ml) in 17/25 (68.0%) T/NK-NHL and in 13/19 (68.4%) BCL. In vivo treatment via intraperitoneal BCV (40mg/kg, 2X per week) significantly inhibited tumor growth in all xenograft models when compared to vehicle control. Notably, RNAseq analysis demonstrated BCV downregulated MYC and MYC-target pathways in T/NK-NHL models. Further mechanistic studies showed that BCV evoked S-phase cell cycle arrest, replication stress, DNA damage and apoptosis, while also triggering STING pathway-mediated interferon responses, PD-L1 expression and immunogenic cell death. In the syngeneic EL4-C57BL/6 model, BCV in combination with anti-PD1 significantly inhibited tumor growth and triggered an immune reaction characterized by highest scores for adaptive immune response, cytokines/chemokines & receptors, cytotoxic cells, dendritic cells, NK CD56dim cells and neutrophils (NanoString Immunology Panel). Conclusions: Taken together, these results demonstrate a novel role of BCV in the treatment of lymphoma, and suggest potential for combination with checkpoint immunotherapy. Biological sciences/Cancer/Cancer therapy/Drug development Health sciences/Health care/Therapeutics/Drug therapy/Molecularly targeted therapy NK/T cell lymphoma nucleoside analogue replication stress immunotherapy PD-L1 immunogenic cell death Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key Points Treatment with brincidofovir induces replication stress, DNA damage and immunogenic cell death in PTCL and NKTCL A phase Ib/II clinical trial is underway to evaluate safety and efficacy in patients with non-Hodgkin lymphoma (NCT06761677) INTRODUCTION The peripheral T-cell lymphomas (PTCL) and natural killer/T-cell lymphomas (NKTCL) represent a group of rare aggressive non-Hodgkin lymphomas with poor prognosis. Collectively, they are more prevalent in certain ethnogeographic regions including across East Asia and represent a major healthcare need. Overall survival outcomes have not significantly improved save for the introduction of brentuximab vedotin for the subset of CD30-positive PTCL in the frontline setting, and with L-asparaginase-based regimens for NKTCL, as compared with historically-adopted anthracycline-based “CHOP”-like regimens. 1 In the salvage setting for relapsed/refractory disease, novel agents such as epigenetic therapies have been approved for PTCL 2 albeit with limited efficacy, while no standard effective treatment currently exists for NKTCL beyond conventional chemotherapy. 3 Previously, certain nucleoside analogues have been suggested to elicit viral-independent anti-tumor activity, in addition to their known anti-viral effects. In particular, cidofovir (CDV), an acyclic nucleoside phosphonate with a broad target range of viral species including the Epstein-Barr virus (EBV), was initially shown to be effective against EBV-related malignancies such as Burkitt lymphoma and nasopharyngeal carcinoma in cell-line and xenograft models. 4 , 5 This anti-tumor effect was mediated through apoptosis, while accompanied by downregulation of EBV-related oncoproteins LMP1 and EBNA2A. 5 Subsequent studies have further demonstrated promising efficacy and safety of topical CDV injection in two patients with locally-recurrent nasopharyngeal carcinoma, 6 as well as intracavitatory administration of CDV with herpesvirus-associated primary effusion lymphoma. 7 More recently, CDV was further demonstrated to evoke anti-cancer activity in viral-negative tumors, through the induction of DNA damage as a mechanism of action 8 – 10 – this led us to a working hypothesis that CDV may represent a unique agent with dual anti-viral and anti-tumor properties, while also being potentially immune-activating as a consequence of DNA damage. 11 However, as its toxicity profile for systemic administration at doses achieving therapeutically-tractable drug concentrations would preclude feasibility, an alternative strategy would be required for clinical translation. Brincidofovir (BCV) is a novel lipid conjugate of CDV with improved intracellular delivery, higher potency and a favourable toxicity profile compared to CDV, carrying significantly reduced risks of nephrotoxicity and myelosuppression. BCV is known to convert to CDV intracellularly after cleavage of its lipid moiety, which then undergoes di-phosphorylation into its active form. While the orally-active formulation is currently approved as a countermeasure against smallpox, the intravenous formulation has been undergoing development for the treatment of adenovirus infection in allogeneic stem cell transplant recipients, given its more favourable gastrointestinal toxicity profile. 12 , 13 Unlike CDV however, the role of BCV in cancer treatment has not been evaluated. Therefore, in this study, we comprehensively investigated the efficacy of BCV in a panel of non-Hodgkin lymphoma cell-line and xenograft models, as well as the mechanisms underlying its anti-tumor activity, paving the way for an early phase clinical trial in patients with lymphoma. MATERIALS AND METHODS Patient data and biospecimen collection Clinical information and archival formalin-fixed paraffin-embedded (FFPE) tissue samples of patients who were diagnosed with NKTCL were obtained from the National Cancer Centre Singapore (Supplementary Table S3). All cases were reviewed by expert hematopathologists at the Singapore General Hospital. Tissue collection and consent protocols were under ethics approval from the SingHealth Centralized Institution Review Board (CIRB 2018/3084). Written informed consent from patients for use of clinical data and biospecimens was obtained in accordance with the Declaration of Helsinki. Cell lines, reagents and quantification of viability Forty-five cell-lines were included in our study, including NKTCL (n = 11), B-cell lymphoma (n = 19), T-cell lymphoma (n = 14) and chronic myeloid leukemia (n = 1). The source and cell culture conditions are summarized in Supplementary Table S16. BCV was obtained from AdooQ (#A13326, Irvine, CA, USA) and prepared based on the manufacturer’s recommendations. Aciclovir, ganciclovir, adefovir, foscarnet, penciclovir, CDV were obtained from Selleck Chemicals (Houston, TX, USA). Cell viability was quantified via Promega CellTiter-Glo® 2.0 Cell Viability Assay (Promega, Madison, WI, USA), as per manufacturer’s protocol. Briefly, cells were seeded into 96-well plates at a concentration of 2 × 10 3 cells in 100 µl media per well. After 96 h, Promega CellTiter-Glo® 2.0 Cell Viability Assay reagent was added to each well and incubated for 10 min at room temperature before measuring for absorbance at 480 nm using Tecan M200 Infinite 96-well plate reader with IControl Software 1.6 (Tecan, Männedorf, Switzerland). Cell viability was assessed as the percentage of mock-treated control absorbance. The growth inhibitory effects were analyzed by generating dose response curves as a plot of the percentage surviving cells versus drug concentration and their IC50s were estimated using GraphPad Prism version 8.0.2 (GraphPad Software, Boston, MA, USA). All reactions were performed in triplicate. Xenograft experiments and in vivo drug treatment For in vivo drug treatment, lymphoma cell lines (NK-S1, PTCL-S1, PTCL-S2 and OCI-LY18) were subcutaneously implanted (0.5 x 10 6 cells) onto the flanks of six-week-old female NSG mice. Similarly, murine EL4 cell lines were implanted into the flanks of six-week-old female C57BL6N mice. BCV (SymBio Pharmaceuticals, Tokyo, Japan) was intraperitoneally administered at a dosage of 40 mg/kg or vehicle control (5% dextrose in water) twice per week, starting after the tumors reach approximately 100 mm 3 in size. The respective treatment subgroups received anti-PD-1 (200 µg twice per week) or isotype vehicle controls (#BE0146 and #BE0089, InVivoMab, Lebanon, NH, USA). All animal studies were conducted in compliance with protocols approved by the SingHealth Institutional Animal Care and Use Committee (IACUC). Tumor measurements were recorded repeatedly until the vehicle control tumor sizes reached approximately 2000 mm 3 , when the mice were euthanized following IACUC guidelines. Tumor sizes in experimental and control groups were averaged at each time point and compared statistically. RNA isolation, whole transcriptome sequencing and gene set enrichment analysis Total RNA was extracted from tumor and cell lines using the AllPrep DNA/RNA FFPE Kit and RNeasy Mini Kit, according to the manufacturer’s protocol (Qiagen, Valencia, CA, USA). The integrity of RNA was determined by electrophoresis using the 2100 Bioanalyzer and/or the 4200 TapeStation (Agilent Technologies, CA, USA). Whole transcriptome sequencing of cell lines was performed on Illumina platforms (NovogeneAIT Genomics, Singapore) using the standard Illumina RNA-seq protocol or on the MGI DNBSEQ-G400 platform using their standard MGI RNA-seq protocol (MGI Tech, China). Transcriptomic profiling of FFPE tumor tissue was performed using the Magnis SureSelect XT HS2 RNA Reagent Kit on the Magnis NGS Prep System (Agilent Technologies, CA, USA) followed by sequencing on the Illumina platform (NovogeneAIT Genomics, Singapore). Read alignment, transcript abundance estimation, identification of differentially-expressed genes and gene set enrichment analysis (GSEA) were performed as per previously described. 14 A gene set is significantly enriched if its False Discovery Rate (FDR) q-value is below 0.05. Single cell RNA sequencing Single-cell RNA-seq (scRNAseq) libraries were prepared from NK-S1 and KAI-3 cell lines with (1 µg/ml for 48h, 1 µg/ml for 72h) or without treatment with BCV. Each cell was captured and uniquely-barcoded using the 10X Chromium Next GEM Single Cell 3' Kit v3 (PN1000268) and 10X Chromium Controller according to manufacturer’s protocol (10X Genomics, CA, USA). Briefly, an estimate of 16,000 cells were loaded at a concentration of 1200 cells/µl in an attempt to recover 10,000 cells. Following Gel Beads-in-emulsion (GEMs) generation, cell lysis and dissolution of the Gel Bead within each reaction vesicle enabled reverse transcription of polyadenylated mRNA, producing cDNA tagged with both a universal cell barcode and unique molecular index (UMI). The generated cDNA from each sample was used for 3’ gene expression (3'GEX) scRNA-seq library generation. Enzymatic fragmentation of the cDNA transcripts was carried out, followed by End-Repair and A-tailing, adaptor ligation and final library amplification PCR with unique sample indices for each sample. Final library quality was determined using the Agilent Bioanalyzer High Sensitivity DNA Kit and sequenced on the Illumina platform (NovogeneAIT, Singapore). Analysis of single-cell sequencing data The reads were demultiplexed and aligned against the GRCh38 reference genome using 10X Cell Ranger v9.0.0 (10X Genomics, CA, USA). The single-cell data were loaded into count matrices, and samples were merged based on their cell line identity, KAI-3 and NK-S1, using Seurat v5.2.1. 15 A total of 17051 and 9196 cells were retained for KAI-3 and NK-S1 respectively, after filtering for cells with less than 20% mitochondrial genes and expressed at least 450 genes. These cells were then subjected to scaling, log-normalization of gene expression measurements, principal component analysis with the top 2,000 genes selected by variance stabilizing transformation approach, construction of the nearest-neighbor graph using the first 15 principal components, and clustering of cells at 0.2 resolution with the Louvain algorithm as implemented by Seurat. UMAP was used to visualize the distribution of cells in the projection of the significant principal components. Differentially expressed genes were identified using a reference cluster and subsequently ranked based on the signed fold change and p-adjusted values. Significantly enriched pathways were determined using Fast Gene Set Enrichment Analysis, v1.30.0. Statistical analysis Statistical analysis of mean values was performed through t-tests/one-way ANOVA. Median values were compared using Mann-Whitney U tests. High and low TLE1 scores were dichotomized using the median split method. Progression-free survival (PFS) was defined as the time from diagnosis until progression or death from any cause. Overall survival (OS) was measured from diagnosis until the date of death from any cause or censored at last follow-up for survivors. Survival analyses were conducted using the Kaplan-Meier method and log-rank tests. All statistical tests assumed a 2-sided test with a significance level of 0.05 unless otherwise stated and performed using MedCalc for Windows version 19.0.7 (MedCalc Software, Ostend, Belgium). Data availability Gene expression data and single cell transcriptomic data have been deposited in Gene Expression Omnibus (accession number GSE293367). RESULTS In vitro and in vivo activity of BCV in NKTCL BCV, a novel lipid-conjugated nucleoside phosphonate analogue of CDV (Fig. 1 A), was evaluated for its preclinical anti-lymphoma activity in a panel of forty-four lymphoma cell-line models (NKTCL, n = 11; T-cell lymphoma, n = 14; B-cell lymphoma n = 19) (Fig. 1 B). BCV inhibited viability in all NKTCL cell-lines in a dose- and time-dependent manner, with IC50 values within clinically-achievable human plasma concentrations (2 µg/ml). Highest sensitivity (IC50 < 1 µg/ml) was demonstrated in four cell-lines KAI-3, NK-S1, NK-92 and KHYG-1 (IC50 36.0 to 303.6 ng/ml) (Figs. 1 C-D; Figure S1 ), accompanied by increase in sub-G1 fraction and S-phase arrest on cell cycle analyses (Figure S1 C). In contrast, CDV and other selected anti-viral agents (acyclovir, ganciclovir, adefovir, foscarnet, and penciclovir) did not show potent anti-lymphoma activity (Figure S2 ). Of note, the activity of BCV appeared to be independent of EBV positivity and was potent against the EBV-negative KHYH-1 cell-line, though BCV was able to downregulate both EBNA1 and LMP1 protein expression levels in the EBV-positive KAI-3 and NK-S1 cell-lines (Figures S3A-B). Intraperitoneal BCV (40 mg/kg, 2X per week) inhibited tumor growth in NOD/SCID mice NK-S1 xenografts, compared with vehicle alone (tumor volume: p = 0.0005; tumor weight: p = 0.0006) (Figs. 1 E-F). No significant toxicity or body weight reduction was observed (Fig. 1 G). RNAseq of all NKTCL cell-lines and Hallmark gene set enrichment analysis (GSEA) demonstrated that MYC target pathways and cell cycle-related pathways (E2F targets, G2M checkpoint) were prominently upregulated in the four sensitive cell-lines compared to the rest (FDR q < 0.01) (Fig. 2 A and Tables S1-2). Notably, TLE1 , a known transcriptional repressor of the MYC oncogene, was the topmost downregulated gene (log2FoldChange − 7.39, adjusted p < 0.0001) in the four BCV-sensitive cell-lines. This finding was verified on qPCR ( p = 0.0061) and Western blot (Figs. 2 B-D). Interestingly, patients with TLE1 -low NKTCL had worse progression-free survival (HR 6.10, 95% CI 2.04 to 18.2, p = 0.0012) and worse overall survival (HR 3.12, 95% CI 1.06 to 9.23, p = 0.0394) (Figs. 2 E-G). In keeping with results from cell-lines, RNAseq on NKTCL patient samples (n = 53) showed that TLE1 -low tumors were similarly enriched for genes involved in MYC target and cell cycle pathways (Fig. 2 H-I and Tables S3-5). Molecular pathways regulated by BCV in NKTCL In order to elucidate the molecular mechanisms underlying the anti-lymphoma effect of BCV, RNAseq and GSEA was performed on BCV-treated cell-lines (KAI-3 and NK-S1). Among common downregulated genes in both cell lines treated with increasing doses of BCV (0.1 µg/ml and 1 µg/ml) revealed common downregulated (n = 51) genes, including MYC , as well as genes involved in chromatin remodeling. Common upregulated (n = 103) genes included those involved in various signaling pathways, such as interferon alpha and gamma response, TNFA signaling, inflammatory response, p53 pathway, and apoptosis. Notably, the top downregulated pathways on the KAI-3 and NK-S1 cell-lines were both MYC Targets V1 (normalized enrichment scores [NES] -2.56 and − 2.12, respectively; both FDR q-value < 0.001) (Figs. 3 A-B and Tables S6-9). A dose-dependent downregulation of MYC expression by BCV was confirmed on qPCR and Western blot. BCV induced cyclin E and phospho-P53 (serine 20) expression, cleavage of PARP and caspase-3, indicating S-phase arrest and programmed cell death by apoptosis. Similar results were observed in NK-92 and KHYG-1 cell-lines (Figs. 3 C-D and S3C). In keeping with these results, results from RNAseq and GSEA showed that immune-related, DNA replication and repair pathways were upregulated in KAI-3 and NK-S1 post-BCV treatment (Fig. 3 E). Additionally, we investigated the effects of BCV on the JAK-STAT pathway, a key signaling pathway in NKTCL. 3 BCV treatment led to a dose and time-dependent decrease in total and phospho-STAT1, STAT3, and STAT5 protein expression in NK-S1 and KAI-3 cell lines (Figure S3D). Single cell RNA sequencing (scRNAseq) revealed distinct temporal cell states evoked by BCV treatment (1 µg/ml) for 48h and 72h, in both KAI-3 and NK-S1 cell-lines (Figure S4). In both cell-lines, a significant shift in transcriptomic cell states occurred upon BCV treatment at 48h and 72h, with emergence of different cell clusters marked by perturbation of MYC and mTORC signalling, DNA repair, cell cycle and apoptosis pathways, as well as immune-mediated signals, amongst others (Table S10). Immunogenic cell death response triggered by BCV To further investigate the cell death response evoked by BCV, we performed confocal microscopy and showed that BCV triggered micronuclei formation and DNA fragmentation in both KAI-3 and NK-S1 cell-lines (Fig. 4 A). Western blot demonstrated increase in replication stress markers (p-CHK1 S317 and S345, RRM2, p-RPA2 S33), p-H2AX (DNA double strand break) and p-TBK1 (STING pathway activation) (Fig. 4 B). Gene expression levels of type I ( IFNA , IFNB1 ) and II ( IFNY ) interferons and cytokines ( CCL5 , CXCL10 ) were upregulated following BCV treatment, as did the proportion of surface calreticulin-expressing cells and amount of extracellular HMGB1 released, indicative of immunogenic cell death (IMCD) (Figs. 4 C-E). Notably, the immune checkpoint PD-L1 gene ( CD274 ) and protein expression were markedly increased following BCV treatment (Figs. 4 F-G), the latter including both nuclear and membranous protein compartments (Figs. 4 H-I). There was a significant increase in cytoplasmic histone H3 protein expression following BCV treatment, indicative of cytoplasmic DNA release (Fig. 4 I). The in vivo response to BCV treatment on NK-S1 xenograft tumors similarly showed increased protein expression of PD-L1 in BCV-treated mice compared with vehicle-treated controls, with a 1.9-fold higher median H-score ( p = 0.0007) (Figs. 4 J-K). In NK-92 and KHYG-1 cell-lines, total and surface PD-L1 protein expression, as well as surface calreticulin expression were similarly increased following BCV treatment (Figures S3E-G). In vitro and in vivo activity of BCV in PTCL Next, BCV was investigated in a cohort of T-cell lymphoma cell-lines (anaplastic large cell lymphoma [ALCL], n = 9; PTCL not otherwise specified [PTCL-NOS], n = 2; primary cutaneous T-cell lymphoma [CTCL], n = 1). BCV inhibited viability across all T-cell lymphoma cell-lines in a dose-dependent manner (median IC50 = 593 ng/ml; range, 60.2 to 2785 ng/ml). Marked sensitivity (IC50 < 1000 ng/ml) was demonstrated in most cell-lines (seven of 11), including our in-house PTCL-S1 ( TP63 -rearranged ALCL) and PTCL-S2 (PTCL-NOS) cell-line models (IC50 = 177 ng/ml and 1664 ng/ml, respectively) (Figs. 5 A and S5). 16 In vivo , intraperitoneal BCV (40mg/kg, 2X per week) inhibited tumor growth in both NOD/SCID mice PTCL-S1 and PTCL-S2 xenografts, compared with vehicle alone. For PTCL-S1, at 21 days post-treatment with BCV, tumor volume was significantly reduced (81.9 mm 3 vs 1207.1 mm 3 , p < 0.0001), as was tumor weight (0.021 g vs vehicle: 0.812 g, p < 0.0001) (Figs. 5 B-C). For PTCL-S2, both tumor volume ( p = 0.0113) and tumor weight ( p = 0.0039) were both significantly reduced with BCV treatment as compared to vehicle control (Figs. 6SA-B). Like in NKTCL, RNAseq and GSEA showed that MYC target and cell cycle pathways were significantly downregulated in PTCL-S1 cell-line post-BCV treatment. Upregulation of interferon ( IFNB1 , IFNY ), cytokine ( CCL5 , CXCL10 ), and CD274 gene expression was observed. Western blot analysis showed downregulation of MYC protein and upregulation of apoptosis markers (cleaved PARP, cleaved caspase-3), phospho-H2AX, phospho-CHK1 (Ser317/Ser345), p-RPA2 (Ser33) and PDL1 in BCV-treated PTCL-S1 and PTCL-S2 cell-lines (Figs. 5 D-E and S6C; Tables S11-12). BCV treatment on PTCL-S1 xenograft tumors similarly showed increased protein expression of PD-L1 in BCV-treated mice compared with vehicle-treated controls ( p = 0.0212) (Figs. 5 F-G). Immune response activation by BCV and anti-PD1 immune checkpoint blockade In order to investigate the in vivo immune response further, we first examined the effect of BCV in murine lymphoma cell-lines EL-4 and TK-1, demonstrating a dose-dependent inhibition on cell viability in both models (IC50 516 ng/ml and 444 ng/ml, respectively), as well as increased surface expression of PD-L1 (Figs. 6 A-C). Deploying a syngeneic EL4-C57BL/6 model, BCV alone or in combination with anti-PD1 (intraperitoneal 200 µg 2X per week) significantly inhibited tumor growth at 8 days post-treatment, compared to isotype or anti-PD1 treatment alone. However, no significant difference in tumor volume or weight was observed with the combination of BCV and anti-PD1, compared with BCV alone (Figs. 6 D-F). Intriguingly, at this early time point, examination on H&E staining showed a significant immune response in BCV-treated groups, which appeared particularly brisk with the addition of anti-PD1 (Fig. 6 G). NanoString profiling showed a significantly heavier immune infiltration in the BCV + anti-PD1 combination group, as corroborated by the highest scores for adaptive immune response, cytokines/chemokines & receptors, cytotoxic cells, dendritic cells, NK CD56 dim cells and neutrophils. Gene expression of CCL2 , CCL12 , CXCL9 , GZMB , CHIL3 and CTLA4 were also highest in the combination treatment group (Figs. 6 H-I and S7-9; Tables S13-14). Extending preclinical activity of BCV to B-cell lymphoma models Finally, BCV was investigated in a cohort of B-cell lymphoma cell-lines. BCV inhibited viability in all B-cell lymphoma cell-lines in a dose-dependent manner (IC50 79.8 ng/ml to 8414 ng/ml). Marked sensitivity (IC50 < 1 µg/ml) was demonstrated in nine cell-lines, including the MYC / BCL2 -rearranged double-hit DLBCL cell-line OCI-LY18 (IC50 500 ng/ml) (Figs. 7 A and S10). Intraperitoneal BCV (40 mg/kg, 2X per week) inhibited tumor growth in NOD/SCID mice OCI-LY18 xenografts, compared with vehicle alone (tumor volume: p < 0.0001; tumor weight: p = 0.0317) (Figs. 7 B-C). Interestingly, in contrast to T and NK/T cell-lines, RNAseq showed that the transcriptional repressor TLE1 was instead amongst the top upregulated genes in the BCV-sensitive cell-lines (IC50 < 1 µg/ml) (log2foldchange = 8.12, adjusted p = 0.0004), which was validated on qPCR ( p = 0.0350) (Figs. 7 D-E and Table S15). DLBCL patient samples from two independent datasets showed that TLE1 -high tumors conferred worse overall survival (GSE11318, n = 200: HR 2.77, 95%CI 1.78 to 4.30, p < 0.0001; GSE10846, n = 414: HR 1.94, 95%CI 1.35 to 2.80, p = 0.0004). DISCUSSION In this study, we investigated the preclinical therapeutic activity of the novel nucleoside analogue BCV in a spectrum of non-Hodgkin lymphoma cell-lines. Originally intended as an anti-viral agent against smallpox and other viruses, our observations in vitro and in vivo support its potential role as an anti-cancer agent, regardless of the presence of any known associated viruses. Furthermore, by examining transcriptomic profiles of the cell-line models, we identified potential signaling pathways modulating the response to BCV, including STING pathway activation and PD-L1 expression, implying prospective synergies with checkpoint immunotherapy. Previous studies on CDV have demonstrated growth inhibitory effects in various cancer types, including viral-negative tumors such as melanoma, 17 glioblastoma, 18 as well as squamous carcinoma of the head and neck (HNSCC) and cervix. 9 , 10 In a vascular tumor model induced by basic fibroblast growth factor (FGF2)-overexpressing endothelial cells (FGF2-T-MAE) in mice, CDV evoked S-phase cell cycle arrest and apoptosis. 19 In glioblastoma, the cytotoxic effect of CDV was mediated through apoptosis, regardless of the presence of associated cytomegalovirus infection. In this study, CDV was shown to incorporate into tumor cell DNA, thereby promoting replication fork stalling and initiating the formation of DNA double-strand breaks. 18 Similarly findings were also observed in HNSCC and cervical carcinoma. 9 , 10 In keeping with these earlier studies, our current results support a similar mechanism of action for BCV in PTCL and NKTCL, in which BCV triggers replication stress and cell cycle arrest, as well as DNA damage and apoptosis. The cyclic GMP–AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a key mediator of the inflammatory response to DNA damage, including that induced by some exogenous anti-tumor agents. 11 BCV exposure leads to accumulation of cytosolic DNA, cGAS/STING pathway activation, triggering an interferon response and upregulation of the PD-L1 immune checkpoint protein. In addition, BCV evokes an immunogenic cell death (ICD), as evidenced by increased cell membranous expression of calreticulin, a danger-associated molecular pattern (DAMP) molecule, where it acts as an “eat me” signal for immature dendritic cells. 20 An increased in secreted high mobility group box 1 (HMGB1) protein levels was also observed, responsible for the optimal release and presentation of tumor antigens to dendritic cells, dendritic cell maturation, and synthesis of proinflammatory molecules. 21 , 22 Indeed, our results using a syngeneic EL4-C57BL/6 lymphoma model further suggests a robust local inflammatory response in response to addition of PD1 blockade to BCV treatment, including activation of adaptive immune responses, cytokines/chemokines & receptors, cytotoxic cells, dendritic cells, NK CD56 dim cells and neutrophils. The potential for combination with immune checkpoint blockade, as well as with other DNA damaging agents or radiation therapy deserves to be evaluated in further studies. TLE1 functions as a transcriptional co-repressor that can antagonize the functions of a number of transcription factors involved in key signals mediating oncogenesis and inflammation, including Notch, Wnt, and NF-κB pathways. 23 In a prior study by Fraga et al., TLE1 was found to be epigenetically inactivated in various hematologic malignancies through CpG island promoter hypermethylation. Reintroduction of TLE1 caused growth inhibition, whereas depletion resulted in growth enhancement, supporting its role as a tumor suppressor. 24 Our results showed that in NKTCL cell-lines, TLE1 gene expression was inversely correlated with sensitivity to BCV. TLE1 -low tumors were enriched for MYC target pathways, which was prominently downregulated by BCV treatment. In patients with NKTCL, we further demonstrated that low tumor levels of TLE1 gene expression was associated with worse survival outcomes. Previously, in patients with T-cell acute lymphoblastic leukemia (T-ALL), low TLE1 expression was similarly associated with higher relapse rate and shorter survival. 25 , 26 Taken together, this implies that TLE1 gene expression may be both a potential predictive and prognostic biomarker in NKTCL, and that patients with low tumor TLE1 expression might derive greater benefit to BCV therapy. Interestingly however, our results demonstrated the converse instead for B-cell lymphoma – TLE1 was significantly upregulated in the cell-lines which were more sensitive to BCV, and high TLE1 gene expression instead conferred poor survival in patients with DLBCL. The specific mechanisms of TLE1 function might be contextual upon the hematopoietic lineage and will require further elucidation in future studies. Our work demonstrates the therapeutic potential of BCV in non-Hodgkin lymphoma, regardless of the presence of EBV. While our study remains limited as a preclinical investigation of BCV, remarkable in vivo anti-tumor activity was demonstrated in classically aggressive forms of lymphoma including NKTCL, ALK-negative ALCL, and double-hit DLBCL xenograft models. We have also gained insight into potential mechanisms of anti-tumor action of BCV, including its ability to evoke immunogenic cell death. In conclusion, we propose that BCV is an attractive agent for the treatment of non-Hodgkin lymphoma, particularly PTCL and NKTCL. A clinical trial (NCT06761677) is underway to confirm its safety and efficacy in patients with relapsed and/or refractory non-Hodgkin lymphoma. Further translational studies will be required to validate predictive biomarkers of response. Declarations Funding This work was supported by the Tanoto Foundation Professorship in Medical Oncology, New Century Foundation Limited, Ling Foundation, Singapore Ministry of Health’s National Medical Research Council Research Transition Award (TA21jun-0005), Large Collaborative Grant (OFLCG18May-0028 and OFLCG23May-0039), TETRAD II Collaborative Centre Grant (CG21APR2002), SingHealth Duke-NUS AM/ACP-Designated Philanthropic Fund Grant Award (08/FY2023/EX/27-A65), as well as the Khoo Bridge Funding Award (Duke-NUS-KBrFA/2025/0090) provided by Duke-NUS Medical School and the “Estate of Tan Sri Khoo Teck Puat”. Study drug and funding were also provided by SymBio Pharmaceuticals Limited. Authorship Contributions J.Y.C. analyzed the data and prepared the first draft the manuscript; S.T.L. provided clinical information and samples; H.Y.T., J.Q.L. performed the bioinformatic analyses; E.C.Y.L., K.X.Y.C., J.Y.L., B.K., B.Y.L., Z.L., T.K.K., J.S.K., K.S.L., N.A.B.M.T., D.H. processed tissue and performed experiments; T.B.T., M.H., K.F., contributed to data interpretation; J.Y.C. and C.K.O. conceptualized the study, interpreted the results, had unrestricted access to all data, and revised the manuscript. All authors agreed to submit the manuscript, read and approved the final draft, and take full responsibility for its content. Disclosure of Conflicts of Interests This work is supported in part by funding from SymBio Pharmaceuticals Limited to J.Y.C. and C.K.O.; M.H. and K.F. are employees of SymBio Pharmaceuticals Limited. The remaining authors declare no competing financial interests. References O'Connor OA, Ma H, Chan JYS, Kim SJ, Yoon SE, Kim WS. 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Nat Med. 2007;13(1):54–61. doi: 10.1038/nm1523 . Epub 2006 Dec 24. PMID: 17187072. Semino C, Angelini G, Poggi A, Rubartelli A. NK/iDC interaction results in IL-18 secretion by DCs at the synaptic cleft followed by NK cell activation and release of the DC maturation factor HMGB1. Blood. 2005;106(2):609–16. doi: 10.1182/blood-2004-10-3906 . Epub 2005 Mar 31. PMID: 15802534. Kazama H, Ricci JE, Herndon JM, Hoppe G, Green DR, Ferguson TA. Induction of immunological tolerance by apoptotic cells requires caspase-dependent oxidation of high-mobility group box-1 protein. Immunity. 2008;29(1):21–32. doi: 10.1016/j.immuni.2008.05.013 . PMID: 18631454; PMCID: PMC2704496. Yu G, Chen Y, Hu Y, Zhou Y, Ding X, Zhou X. Roles of transducin-like enhancer of split (TLE) family proteins in tumorigenesis and immune regulation. Front Cell Dev Biol. 2022;10:1010639. doi: 10.3389/fcell.2022.1010639 . PMID: 36438567; PMCID: PMC9692235. Fraga MF, Berdasco M, Ballestar E, Ropero S, Lopez-Nieva P, Lopez-Serra L, Martín-Subero JI, Calasanz MJ, Lopez de Silanes I, Setien F, Casado S, Fernandez AF, Siebert R, Stifani S, Esteller M. Epigenetic inactivation of the Groucho homologue gene TLE1 in hematologic malignancies. Cancer Res. 2008;68(11):4116-22. doi: 10.1158/0008-5472.CAN-08-0085 . PMID: 18519670. Brassesco MS, Pezuk JA, Cortez MA, Bezerra Salomão K, Scrideli CA, Tone LG. TLE1 as an indicator of adverse prognosis in pediatric acute lymphoblastic leukemia. Leuk Res. 2018;74:42–46. doi: 10.1016/j.leukres.2018.09.010 . Epub 2018 Sep 26. PMID: 30286331. Aref S, El-Ghonemy MS, Atia DM, Elbaiomy MM, Abdelsalam SA, Tawfik A, El-Sebaie A. Prognostic Value of TLE1 Gene Expression in Patients with T-cell Acute Lymphoblastic Leukemia. Asian Pac J Cancer Prev. 2021;22(5):1653–1658. doi: 10.31557/APJCP.2021.22.5.1653. PMID: 34048198; PMCID: PMC8408389. Additional Declarations Yes there is potential conflict of interest. This work is supported in part by funding from SymBio Pharmaceuticals Limited to J.Y.C. and C.K.O.; M.H. and K.F. are employees of SymBio Pharmaceuticals Limited. The remaining authors declare no competing financial interests. Supplementary Files SupplementalTables.xlsx Supplemental Tables SupplementalData.docx Supplemental Data Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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12:40:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6768176/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6768176/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83864287,"identity":"287304d2-a229-444c-a71d-5edfed97b8d0","added_by":"auto","created_at":"2025-06-03 20:58:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3300381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepurposing brincidofovir for treatment of NK/T cell lymphoma.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Chemical structures of antiviral drugs cidofovir and brincidofovir (BCV).\u003c/p\u003e\n\u003cp\u003e(B) Overview of lymphoma cell-line models used in the study.\u003c/p\u003e\n\u003cp\u003e(C) IC50 of BCV across NKTCL cell lines including the EBV-negative KHYG-1 cell line.\u003c/p\u003e\n\u003cp\u003e(D) BCV inhibits cell growth of NKTCL cell lines in a dose-dependent and time-dependent manner. Drug treatments were performed in triplicates, and results are represented by mean values and standard deviations.\u003c/p\u003e\n\u003cp\u003e(E-F) BCV inhibited tumor growth in the NK-S1 xenograft model (tumor volume: \u003cem\u003ep\u003c/em\u003e = 0.0005, two-tailed t-test; tumor weight: \u003cem\u003ep\u003c/em\u003e = 0.0006, Mann-Whitney U test). NSG mice were treated twice per week IP with either vehicle or BCV (40 mg/kg) 7 days after subcutaneous flank inoculation with 0.5 x 10\u003csup\u003e6\u003c/sup\u003e NK-S1 cells (n = 8 per group) (scale bar: 10 mm).\u003c/p\u003e\n\u003cp\u003e(G) No significant effect of BCV on mice body weight was demonstrated.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6768176/v1/5c30f6c062f2d8f571a2d87a.png"},{"id":83864379,"identity":"766e16e6-f349-46e2-a0e5-602c67f2e8c6","added_by":"auto","created_at":"2025-06-03 21:06:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3565855,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential gene expression pathways associated with BCV sensitivity in NK/T cell lymphoma cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Gene set enrichment analysis showed that \u003cem\u003eMYC\u003c/em\u003e target pathways and cell cycle-related pathways were prominently upregulated in the four sensitive cell-lines compared to the rest . ** FDR q-value \u0026lt; 0.001, * FDR q-value \u0026lt; 0.05\u003c/p\u003e\n\u003cp\u003e(B) Volcano plot showing top differentially-expressed genes between top 4 sensitive NKTCL cell lines versus the rest. TLE was the top differentially-expressed gene (log2foldchange -7.39, adj. \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e(C) TLE1 gene expression was significantly higher in the more resistant NKTCL cells lines, validated on qPCR (\u003cem\u003ep\u003c/em\u003e = 0.0061, Mann-Whitney U test).\u003c/p\u003e\n\u003cp\u003e(D) TLE1 protein expression was not detectable in the top 4 sensitive NKTCL cell lines.\u003c/p\u003e\n\u003cp\u003e(E) In NKTCL patients, low TLE1 gene expression (cut-off as determined by ROC analysis), was associated with (F) poorer progression-free survival and (G) overall survival.\u003c/p\u003e\n\u003cp\u003e(H) Volcano plot showing top differentially-expressed genes between TLE1-high and TLE1 -low NKTCL tumors.\u003c/p\u003e\n\u003cp\u003e(I) Gene set enrichment analysis showed that \u003cem\u003eMYC\u003c/em\u003e target pathways and cell cycle-related pathways were prominently upregulated in TLE1-low NKTCL tumor samples . ** FDR q-value \u0026lt; 0.001, * FDR q-value \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6768176/v1/e39b603f90fbd3772b9271ae.png"},{"id":83864288,"identity":"e2c36269-4c22-47e7-ad20-79b44b792fab","added_by":"auto","created_at":"2025-06-03 20:58:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5175571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBrincidofovir inhibits MYC oncogenic signaling while activating DNA repair and immune-related pathways.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Whole transcriptomic sequencing of KAI-3 and NK-S1 cell lines treated with increasing doses of BCV (0.1 µg/ml and 1 µg/ml) revealed common downregulated (n = 51) and upregulated (n = 103) genes involved in various signaling pathways, based on MSigDB over-representation analysis (FDR q \u0026lt; 0.05). Data was obtained from 3 biological replicates.\u003c/p\u003e\n\u003cp\u003e(B) Gene set enrichment analysis showed that MYC targets were the top Hallmark gene sets downregulated in KAI-3 (MYC TARGETS V1: NES -2.56, FDR q \u0026lt; 0.001) and NK-S1 (MYC TARGETS V1: NES = -2.12, FDR q \u0026lt; 0.001) post-BCV treatment.\u003c/p\u003e\n\u003cp\u003e(C) Downregulation of \u003cem\u003eMYC\u003c/em\u003egene expression following BCV treatment in both KAI-3 and NK-S1 cell lines was validated by qPCR. Results shown are mean values and standard deviations from three independent experiments.\u003c/p\u003e\n\u003cp\u003e(D) Western blot demonstrated decreased protein expression of MYC, while phopho-p53, cyclin E, cleaved PARP and cleaved caspase-3 were increased.\u003c/p\u003e\n\u003cp\u003e(E) Gene set enrichment analysis showed that immune-related, DNA replication and repair pathways were upregulated post-BCV treatment. ** FDR q-value \u0026lt; 0.001, * FDR q-value \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6768176/v1/8b42dabc07f65b87f632fa02.png"},{"id":83864293,"identity":"33f36886-0722-40b3-99ce-c8ce3b5e1ff1","added_by":"auto","created_at":"2025-06-03 20:58:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10722019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBCV triggers DNA damage, STING pathway activation, and immune signals in NKTCL cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Micronuclei formation and DNA fragmentation 48h following BCV treatment (scale bar: 10 µm).\u003c/p\u003e\n\u003cp\u003e(B) Western blot demonstrating dose- and time-dependent increase in replication stress markers, including p-CHK1 (S317 and S345), RRM2, and p-RPA2 (S33). Both p-H2AX (indicative of DNA double strand break) and p-TBK1 (indicating STING pathway activation) are also upregulated in the same manner.\u003c/p\u003e\n\u003cp\u003e(C) Upregulation of type I (IFNA, IFNB1) and II (IFNY) interferon and cytokine (CCL5, CXCL10) gene expression (qPCR) following BCV treatment (* \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05; ** \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; one-way ANOVA).\u003c/p\u003e\n\u003cp\u003e(D) Increase in proportion of calreticulin-expressing cells and (E) extracellular release of HMGB1 upon BCV treatment.\u003c/p\u003e\n\u003cp\u003e(F) Upregulation of PD-L1 protein expression on Western blot, (G) CD274 gene expression on qPCR, and (H) PD-L1-expressing cells on flow cytometry. qPCR, flow cytometry, and HMGB1 release assay data shown are mean values and standard deviations from three independent experiments.\u003c/p\u003e\n\u003cp\u003e(I) BCV treatment increased cytoplasmic DNA levels (indicated by histone H3). PD-L1 protein levels were upregulated in both membranous and nuclear fractions.\u003c/p\u003e\n\u003cp\u003e(J) Representative images of PD-L1 IHC on NK-S1 xenograft tumors in vehicle and BCV-treated mice (scale bar: 20 µm).\u003c/p\u003e\n\u003cp\u003e(K) IHC on NK-S1 xenograft tumors showed increased protein expression of PD-L1 in BCV-treated mice compared with vehicle-treated controls (\u003cem\u003ep\u003c/em\u003e = 0.0007, Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6768176/v1/7a3eab2d5935a4581caa754f.png"},{"id":83864292,"identity":"45e37cd0-04b4-44ca-b884-d4bd4a6a83d0","added_by":"auto","created_at":"2025-06-03 20:58:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8146857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePre-clinical efficacy of brincidofovir in peripheral T-cell lymphoma.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) IC50 of BCV across various T-cell lymphoma cell lines.\u003c/p\u003e\n\u003cp\u003e(B) BCV inhibited tumor growth in the PTCL-S1 xenograft model (tumor volume: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, two-tailed t-test; (C) tumor weight: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, Mann-Whitney U test). NSG mice were treated twice per week IP with either vehicle or BCV (40 mg/kg) 7 days after subcutaneous flank inoculation with 0.5 x 10\u003csup\u003e6\u003c/sup\u003e PTCL-S1 cells (n = 10 per group) (scale bar: 10 mm).\u003c/p\u003e\n\u003cp\u003e(D) Gene set enrichment analysis showed that several pathways were deregulated in PTCL-S1 post-BCV treatment, including downregulation of MYC target pathways. ** FDR q-value \u0026lt; 0.001, * FDR q-value \u0026lt; 0.05\u003c/p\u003e\n\u003cp\u003e(E) Western blot demonstrated decrease protein expression of MYC, while cyclin E, cleaved PARP, cleaved caspase-3 were increased. p-H2AX, p-CHK1, p-RPA2, and PD-L1 were similarly increased.\u003c/p\u003e\n\u003cp\u003e(F) Representative images of PD-L1 IHC on PTCL-S1 xenograft tumors in vehicle and BCV-treated mice (scale bar: 50 µm).\u003c/p\u003e\n\u003cp\u003e(G) IHC on PTCL-S1 xenograft tumors showed increased protein expression of PD-L1 in BCV-treated mice compared with vehicle-treated controls (\u003cem\u003ep\u003c/em\u003e = 0.0212, Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6768176/v1/1c005e761d78f5a9782203af.png"},{"id":83864295,"identity":"cf58f20a-26a9-4f74-9604-3c34c075b052","added_by":"auto","created_at":"2025-06-03 20:58:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":14794152,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination of BCV and anti-PD1 immune checkpoint blockade.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) BCV inhibits cell growth of murine T-cell lymphoma cell lines EL-4 and TK-1 in a dose-dependent manner. Drug treatments were performed in triplicate, and results are represented by mean values and standard deviations.\u003c/p\u003e\n\u003cp\u003e(C) BCV increased PD-L1-expression in both murine T-cell lymphoma cell lines on flow cytometry.\u003c/p\u003e\n\u003cp\u003e(D-F) BCV inhibited tumor growth in the EL-4 xenograft model in C57BL/6J mice (tumor volume: \u003cem\u003ep\u003c/em\u003e = 0.0001, two-tailed t-test; tumor weight: \u003cem\u003ep\u003c/em\u003e = 0.0267, Mann-Whitney U test). BCV alone (IP 40 mg/kg) or in combination with anti-PD1 (IP 200 µg 1X per week) significantly inhibited tumor growth at 8 days post-treatment, compared to isotype or anti-PD1 treatment alone. No significant difference in tumor volume or weight was observed with the combination of BCV and anti-PD1, compared with BCV alone. Subcutaneous flank inoculation with 0.5 x 10\u003csup\u003e6\u003c/sup\u003e EL-4 cells (n = 10 per group) (scale bar: 10 mm).\u003c/p\u003e\n\u003cp\u003e(G) Representative H\u0026amp;E images showing increased apoptosis in BCV-treated tumors and brisk immune infiltration in the BCV + anti-PD1 combination group, relative to non-BCV treated groups (20X magnification; scale bar: 50 µm; inset shows 40X magnification).\u003c/p\u003e\n\u003cp\u003e(H) NanoString Mouse Immunology Panel demonstrated the highest scores for adaptive immune response, apoptosis, basic cell functions, cytokines/chemokines \u0026amp; receptors pathways in the combination group.\u003c/p\u003e\n\u003cp\u003e(I) Cell types significantly enriched in the BCV + anti-PD1 combination group are shown.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6768176/v1/d796b3999177f85da79eafcb.png"},{"id":83864291,"identity":"5897a253-47b5-4a54-8a98-3b22e8202ed4","added_by":"auto","created_at":"2025-06-03 20:58:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3313288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePre-clinical efficacy of brincidofovir in B-cell lymphoma.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) IC50 of BCV across B-cell lymphoma cell lines.\u003c/p\u003e\n\u003cp\u003e(B-C) BCV inhibited tumor growth in the OCI-LY18 xenograft model (tumor volume: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, two-tailed t-test; tumor weight: \u003cem\u003ep\u003c/em\u003e = 0.0317, Mann-Whitney U test). NSG mice were treated twice per week IP with either vehicle or BCV (40 mg/kg) following flank inoculation with 0.5 x 10\u003csup\u003e6\u003c/sup\u003e OCI-LY18 cells (n = 5 per group) (scale bar: 10 mm).\u003c/p\u003e\n\u003cp\u003e(D) Volcano plot showing top differentially-expressed genes between top sensitive B-cell lymphoma cell lines (n = 9) versus the rest. TLE was amongst the top differentially-expressed gene (log2foldchange 8.12, adj. \u003cem\u003ep\u003c/em\u003e = 0.0004).\u003c/p\u003e\n\u003cp\u003e(E) \u003cem\u003eTLE1\u003c/em\u003e gene expression was significantly higher in the more sensitive B-cell lymphoma cells lines, validated on qPCR (\u003cem\u003ep\u003c/em\u003e = 0.0350).\u003c/p\u003e\n\u003cp\u003e(F) In DLBCL patients, high \u003cem\u003eTLE1\u003c/em\u003e gene expression (above median) was associated with poorer progression-free survival and overall survival.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6768176/v1/204a104e524dafa2885ead61.png"},{"id":89283657,"identity":"d35d15ea-5e6d-484a-b256-2b9a08056ae3","added_by":"auto","created_at":"2025-08-18 10:54:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":48346552,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6768176/v1/0584f7ae-5c44-480e-b9f6-6faf009adf3c.pdf"},{"id":83864294,"identity":"c25c72d6-fea3-4113-aeea-75b25a5f4db9","added_by":"auto","created_at":"2025-06-03 20:58:58","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4517992,"visible":true,"origin":"","legend":"Supplemental Tables","description":"","filename":"SupplementalTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6768176/v1/e8de9bf82c4f78f25cf63624.xlsx"},{"id":83864296,"identity":"dfa248ed-b625-440d-92cc-1190d985f412","added_by":"auto","created_at":"2025-06-03 20:58:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10661189,"visible":true,"origin":"","legend":"Supplemental Data","description":"","filename":"SupplementalData.docx","url":"https://assets-eu.researchsquare.com/files/rs-6768176/v1/938adfd25d2027d436498729.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential conflict of interest.\nThis work is supported in part by funding from SymBio Pharmaceuticals Limited to J.Y.C. and C.K.O.; M.H. and K.F. are employees of SymBio Pharmaceuticals Limited. The remaining authors declare no competing financial interests.","formattedTitle":"Preclinical activity of brincidofovir in Peripheral T-cell and NK/T-cell Lymphoma","fulltext":[{"header":"Key Points","content":"\u003cul\u003e\n \u003cli\u003eTreatment with brincidofovir induces replication stress, DNA damage and immunogenic cell death in\u0026nbsp;PTCL and NKTCL\u003c/li\u003e\n \u003cli\u003eA phase Ib/II clinical trial is underway to evaluate safety and efficacy in patients with non-Hodgkin lymphoma (NCT06761677)\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eThe peripheral T-cell lymphomas (PTCL) and natural killer/T-cell lymphomas (NKTCL) represent a group of rare aggressive non-Hodgkin lymphomas with poor prognosis. Collectively, they are more prevalent in certain ethnogeographic regions including across East Asia and represent a major healthcare need. Overall survival outcomes have not significantly improved save for the introduction of brentuximab vedotin for the subset of CD30-positive PTCL in the frontline setting, and with L-asparaginase-based regimens for NKTCL, as compared with historically-adopted anthracycline-based \u0026ldquo;CHOP\u0026rdquo;-like regimens.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e In the salvage setting for relapsed/refractory disease, novel agents such as epigenetic therapies have been approved for PTCL\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e albeit with limited efficacy, while no standard effective treatment currently exists for NKTCL beyond conventional chemotherapy.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003ePreviously, certain nucleoside analogues have been suggested to elicit viral-independent anti-tumor activity, in addition to their known anti-viral effects. In particular, cidofovir (CDV), an acyclic nucleoside phosphonate with a broad target range of viral species including the Epstein-Barr virus (EBV), was initially shown to be effective against EBV-related malignancies such as Burkitt lymphoma and nasopharyngeal carcinoma in cell-line and xenograft models.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e This anti-tumor effect was mediated through apoptosis, while accompanied by downregulation of EBV-related oncoproteins LMP1 and EBNA2A.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Subsequent studies have further demonstrated promising efficacy and safety of topical CDV injection in two patients with locally-recurrent nasopharyngeal carcinoma,\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e as well as intracavitatory administration of CDV with herpesvirus-associated primary effusion lymphoma.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e More recently, CDV was further demonstrated to evoke anti-cancer activity in viral-negative tumors, through the induction of DNA damage as a mechanism of action\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e \u0026ndash; this led us to a working hypothesis that CDV may represent a unique agent with dual anti-viral and anti-tumor properties, while also being potentially immune-activating as a consequence of DNA damage.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e However, as its toxicity profile for systemic administration at doses achieving therapeutically-tractable drug concentrations would preclude feasibility, an alternative strategy would be required for clinical translation.\u003c/p\u003e \u003cp\u003eBrincidofovir (BCV) is a novel lipid conjugate of CDV with improved intracellular delivery, higher potency and a favourable toxicity profile compared to CDV, carrying significantly reduced risks of nephrotoxicity and myelosuppression. BCV is known to convert to CDV intracellularly after cleavage of its lipid moiety, which then undergoes di-phosphorylation into its active form. While the orally-active formulation is currently approved as a countermeasure against smallpox, the intravenous formulation has been undergoing development for the treatment of adenovirus infection in allogeneic stem cell transplant recipients, given its more favourable gastrointestinal toxicity profile.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Unlike CDV however, the role of BCV in cancer treatment has not been evaluated. Therefore, in this study, we comprehensively investigated the efficacy of BCV in a panel of non-Hodgkin lymphoma cell-line and xenograft models, as well as the mechanisms underlying its anti-tumor activity, paving the way for an early phase clinical trial in patients with lymphoma.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient data and biospecimen collection\u003c/h2\u003e \u003cp\u003eClinical information and archival formalin-fixed paraffin-embedded (FFPE) tissue samples of patients who were diagnosed with NKTCL were obtained from the National Cancer Centre Singapore (Supplementary Table S3). All cases were reviewed by expert hematopathologists at the Singapore General Hospital. Tissue collection and consent protocols were under ethics approval from the SingHealth Centralized Institution Review Board (CIRB 2018/3084). Written informed consent from patients for use of clinical data and biospecimens was obtained in accordance with the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell lines, reagents and quantification of viability\u003c/h3\u003e\n\u003cp\u003eForty-five cell-lines were included in our study, including NKTCL (n\u0026thinsp;=\u0026thinsp;11), B-cell lymphoma (n\u0026thinsp;=\u0026thinsp;19), T-cell lymphoma (n\u0026thinsp;=\u0026thinsp;14) and chronic myeloid leukemia (n\u0026thinsp;=\u0026thinsp;1). The source and cell culture conditions are summarized in Supplementary Table S16. BCV was obtained from AdooQ (#A13326, Irvine, CA, USA) and prepared based on the manufacturer\u0026rsquo;s recommendations. Aciclovir, ganciclovir, adefovir, foscarnet, penciclovir, CDV were obtained from Selleck Chemicals (Houston, TX, USA). Cell viability was quantified via Promega CellTiter-Glo\u0026reg; 2.0 Cell Viability Assay (Promega, Madison, WI, USA), as per manufacturer\u0026rsquo;s protocol. Briefly, cells were seeded into 96-well plates at a concentration of 2 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells in 100 \u0026micro;l media per well. After 96 h, Promega CellTiter-Glo\u0026reg; 2.0 Cell Viability Assay reagent was added to each well and incubated for 10 min at room temperature before measuring for absorbance at 480 nm using Tecan M200 Infinite 96-well plate reader with IControl Software 1.6 (Tecan, M\u0026auml;nnedorf, Switzerland). Cell viability was assessed as the percentage of mock-treated control absorbance. The growth inhibitory effects were analyzed by generating dose response curves as a plot of the percentage surviving cells versus drug concentration and their IC50s were estimated using GraphPad Prism version 8.0.2 (GraphPad Software, Boston, MA, USA). All reactions were performed in triplicate.\u003c/p\u003e\n\u003ch3\u003eXenograft experiments and in vivo drug treatment\u003c/h3\u003e\n\u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e drug treatment, lymphoma cell lines (NK-S1, PTCL-S1, PTCL-S2 and OCI-LY18) were subcutaneously implanted (0.5 x 10\u003csup\u003e6\u003c/sup\u003e cells) onto the flanks of six-week-old female NSG mice. Similarly, murine EL4 cell lines were implanted into the flanks of six-week-old female C57BL6N mice. BCV (SymBio Pharmaceuticals, Tokyo, Japan) was intraperitoneally administered at a dosage of 40 mg/kg or vehicle control (5% dextrose in water) twice per week, starting after the tumors reach approximately 100 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e in size. The respective treatment subgroups received anti-PD-1 (200 \u0026micro;g twice per week) or isotype vehicle controls (#BE0146 and #BE0089, InVivoMab, Lebanon, NH, USA). All animal studies were conducted in compliance with protocols approved by the SingHealth Institutional Animal Care and Use Committee (IACUC). Tumor measurements were recorded repeatedly until the vehicle control tumor sizes reached approximately 2000 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, when the mice were euthanized following IACUC guidelines. Tumor sizes in experimental and control groups were averaged at each time point and compared statistically.\u003c/p\u003e\n\u003ch3\u003eRNA isolation, whole transcriptome sequencing and gene set enrichment analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from tumor and cell lines using the AllPrep DNA/RNA FFPE Kit and RNeasy Mini Kit, according to the manufacturer\u0026rsquo;s protocol (Qiagen, Valencia, CA, USA). The integrity of RNA was determined by electrophoresis using the 2100 Bioanalyzer and/or the 4200 TapeStation (Agilent Technologies, CA, USA). Whole transcriptome sequencing of cell lines was performed on Illumina platforms (NovogeneAIT Genomics, Singapore) using the standard Illumina RNA-seq protocol or on the MGI DNBSEQ-G400 platform using their standard MGI RNA-seq protocol (MGI Tech, China). Transcriptomic profiling of FFPE tumor tissue was performed using the Magnis SureSelect XT HS2 RNA Reagent Kit on the Magnis NGS Prep System (Agilent Technologies, CA, USA) followed by sequencing on the Illumina platform (NovogeneAIT Genomics, Singapore). Read alignment, transcript abundance estimation, identification of differentially-expressed genes and gene set enrichment analysis (GSEA) were performed as per previously described.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e A gene set is significantly enriched if its False Discovery Rate (FDR) q-value is below 0.05.\u003c/p\u003e\n\u003ch3\u003eSingle cell RNA sequencing\u003c/h3\u003e\n\u003cp\u003eSingle-cell RNA-seq (scRNAseq) libraries were prepared from NK-S1 and KAI-3 cell lines with (1 \u0026micro;g/ml for 48h, 1 \u0026micro;g/ml for 72h) or without treatment with BCV. Each cell was captured and uniquely-barcoded using the 10X Chromium Next GEM Single Cell 3' Kit v3 (PN1000268) and 10X Chromium Controller according to manufacturer\u0026rsquo;s protocol (10X Genomics, CA, USA). Briefly, an estimate of 16,000 cells were loaded at a concentration of 1200 cells/\u0026micro;l in an attempt to recover 10,000 cells. Following Gel Beads-in-emulsion (GEMs) generation, cell lysis and dissolution of the Gel Bead within each reaction vesicle enabled reverse transcription of polyadenylated mRNA, producing cDNA tagged with both a universal cell barcode and unique molecular index (UMI). The generated cDNA from each sample was used for 3\u0026rsquo; gene expression (3'GEX) scRNA-seq library generation. Enzymatic fragmentation of the cDNA transcripts was carried out, followed by End-Repair and A-tailing, adaptor ligation and final library amplification PCR with unique sample indices for each sample. Final library quality was determined using the Agilent Bioanalyzer High Sensitivity DNA Kit and sequenced on the Illumina platform (NovogeneAIT, Singapore).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of single-cell sequencing data\u003c/h2\u003e \u003cp\u003eThe reads were demultiplexed and aligned against the GRCh38 reference genome using 10X Cell Ranger v9.0.0 (10X Genomics, CA, USA). The single-cell data were loaded into count matrices, and samples were merged based on their cell line identity, KAI-3 and NK-S1, using Seurat v5.2.1.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e A total of 17051 and 9196 cells were retained for KAI-3 and NK-S1 respectively, after filtering for cells with less than 20% mitochondrial genes and expressed at least 450 genes. These cells were then subjected to scaling, log-normalization of gene expression measurements, principal component analysis with the top 2,000 genes selected by variance stabilizing transformation approach, construction of the nearest-neighbor graph using the first 15 principal components, and clustering of cells at 0.2 resolution with the Louvain algorithm as implemented by Seurat. UMAP was used to visualize the distribution of cells in the projection of the significant principal components. Differentially expressed genes were identified using a reference cluster and subsequently ranked based on the signed fold change and p-adjusted values. Significantly enriched pathways were determined using Fast Gene Set Enrichment Analysis, v1.30.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of mean values was performed through t-tests/one-way ANOVA. Median values were compared using Mann-Whitney U tests. High and low TLE1 scores were dichotomized using the median split method. Progression-free survival (PFS) was defined as the time from diagnosis until progression or death from any cause. Overall survival (OS) was measured from diagnosis until the date of death from any cause or censored at last follow-up for survivors. Survival analyses were conducted using the Kaplan-Meier method and log-rank tests. All statistical tests assumed a 2-sided test with a significance level of 0.05 unless otherwise stated and performed using MedCalc for Windows version 19.0.7 (MedCalc Software, Ostend, Belgium).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eData availability\u003c/h3\u003e\n\u003cp\u003eGene expression data and single cell transcriptomic data have been deposited in Gene Expression Omnibus (accession number GSE293367).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro and in vivo activity of BCV in NKTCL\u003c/h2\u003e \u003cp\u003eBCV, a novel lipid-conjugated nucleoside phosphonate analogue of CDV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), was evaluated for its preclinical anti-lymphoma activity in a panel of forty-four lymphoma cell-line models (NKTCL, n\u0026thinsp;=\u0026thinsp;11; T-cell lymphoma, n\u0026thinsp;=\u0026thinsp;14; B-cell lymphoma n\u0026thinsp;=\u0026thinsp;19) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). BCV inhibited viability in all NKTCL cell-lines in a dose- and time-dependent manner, with IC50 values within clinically-achievable human plasma concentrations (2 \u0026micro;g/ml). Highest sensitivity (IC50\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026micro;g/ml) was demonstrated in four cell-lines KAI-3, NK-S1, NK-92 and KHYG-1 (IC50 36.0 to 303.6 ng/ml) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), accompanied by increase in sub-G1 fraction and S-phase arrest on cell cycle analyses (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). In contrast, CDV and other selected anti-viral agents (acyclovir, ganciclovir, adefovir, foscarnet, and penciclovir) did not show potent anti-lymphoma activity (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Of note, the activity of BCV appeared to be independent of EBV positivity and was potent against the EBV-negative KHYH-1 cell-line, though BCV was able to downregulate both EBNA1 and LMP1 protein expression levels in the EBV-positive KAI-3 and NK-S1 cell-lines (Figures S3A-B). Intraperitoneal BCV (40 mg/kg, 2X per week) inhibited tumor growth in NOD/SCID mice NK-S1 xenografts, compared with vehicle alone (tumor volume: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0005; tumor weight: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0006) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). No significant toxicity or body weight reduction was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRNAseq of all NKTCL cell-lines and Hallmark gene set enrichment analysis (GSEA) demonstrated that \u003cem\u003eMYC\u003c/em\u003e target pathways and cell cycle-related pathways (E2F targets, G2M checkpoint) were prominently upregulated in the four sensitive cell-lines compared to the rest (FDR q\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Tables S1-2). Notably, \u003cem\u003eTLE1\u003c/em\u003e, a known transcriptional repressor of the \u003cem\u003eMYC\u003c/em\u003e oncogene, was the topmost downregulated gene (log2FoldChange \u0026minus;\u0026thinsp;7.39, adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the four BCV-sensitive cell-lines. This finding was verified on qPCR (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0061) and Western blot (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). Interestingly, patients with \u003cem\u003eTLE1\u003c/em\u003e-low NKTCL had worse progression-free survival (HR 6.10, 95% CI 2.04 to 18.2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0012) and worse overall survival (HR 3.12, 95% CI 1.06 to 9.23, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0394) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G). In keeping with results from cell-lines, RNAseq on NKTCL patient samples (n\u0026thinsp;=\u0026thinsp;53) showed that \u003cem\u003eTLE1\u003c/em\u003e-low tumors were similarly enriched for genes involved in \u003cem\u003eMYC\u003c/em\u003e target and cell cycle pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I and Tables S3-5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMolecular pathways regulated by BCV in NKTCL\u003c/h2\u003e \u003cp\u003eIn order to elucidate the molecular mechanisms underlying the anti-lymphoma effect of BCV, RNAseq and GSEA was performed on BCV-treated cell-lines (KAI-3 and NK-S1). Among common downregulated genes in both cell lines treated with increasing doses of BCV (0.1 \u0026micro;g/ml and 1 \u0026micro;g/ml) revealed common downregulated (n\u0026thinsp;=\u0026thinsp;51) genes, including \u003cem\u003eMYC\u003c/em\u003e, as well as genes involved in chromatin remodeling. Common upregulated (n\u0026thinsp;=\u0026thinsp;103) genes included those involved in various signaling pathways, such as interferon alpha and gamma response, TNFA signaling, inflammatory response, p53 pathway, and apoptosis. Notably, the top downregulated pathways on the KAI-3 and NK-S1 cell-lines were both MYC Targets V1 (normalized enrichment scores [NES] -2.56 and \u0026minus;\u0026thinsp;2.12, respectively; both FDR q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B and Tables S6-9). A dose-dependent downregulation of MYC expression by BCV was confirmed on qPCR and Western blot. BCV induced cyclin E and phospho-P53 (serine 20) expression, cleavage of PARP and caspase-3, indicating S-phase arrest and programmed cell death by apoptosis. Similar results were observed in NK-92 and KHYG-1 cell-lines (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D and S3C). In keeping with these results, results from RNAseq and GSEA showed that immune-related, DNA replication and repair pathways were upregulated in KAI-3 and NK-S1 post-BCV treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Additionally, we investigated the effects of BCV on the JAK-STAT pathway, a key signaling pathway in NKTCL.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e BCV treatment led to a dose and time-dependent decrease in total and phospho-STAT1, STAT3, and STAT5 protein expression in NK-S1 and KAI-3 cell lines (Figure S3D). Single cell RNA sequencing (scRNAseq) revealed distinct temporal cell states evoked by BCV treatment (1 \u0026micro;g/ml) for 48h and 72h, in both KAI-3 and NK-S1 cell-lines (Figure S4). In both cell-lines, a significant shift in transcriptomic cell states occurred upon BCV treatment at 48h and 72h, with emergence of different cell clusters marked by perturbation of MYC and mTORC signalling, DNA repair, cell cycle and apoptosis pathways, as well as immune-mediated signals, amongst others (Table S10).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunogenic cell death response triggered by BCV\u003c/h2\u003e \u003cp\u003eTo further investigate the cell death response evoked by BCV, we performed confocal microscopy and showed that BCV triggered micronuclei formation and DNA fragmentation in both KAI-3 and NK-S1 cell-lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Western blot demonstrated increase in replication stress markers (p-CHK1 S317 and S345, RRM2, p-RPA2 S33), p-H2AX (DNA double strand break) and p-TBK1 (STING pathway activation) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Gene expression levels of type I (\u003cem\u003eIFNA\u003c/em\u003e, \u003cem\u003eIFNB1\u003c/em\u003e) and II (\u003cem\u003eIFNY\u003c/em\u003e) interferons and cytokines (\u003cem\u003eCCL5\u003c/em\u003e, \u003cem\u003eCXCL10\u003c/em\u003e) were upregulated following BCV treatment, as did the proportion of surface calreticulin-expressing cells and amount of extracellular HMGB1 released, indicative of immunogenic cell death (IMCD) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E). Notably, the immune checkpoint PD-L1 gene (\u003cem\u003eCD274\u003c/em\u003e) and protein expression were markedly increased following BCV treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-G), the latter including both nuclear and membranous protein compartments (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-I). There was a significant increase in cytoplasmic histone H3 protein expression following BCV treatment, indicative of cytoplasmic DNA release (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). The \u003cem\u003ein vivo\u003c/em\u003e response to BCV treatment on NK-S1 xenograft tumors similarly showed increased protein expression of PD-L1 in BCV-treated mice compared with vehicle-treated controls, with a 1.9-fold higher median H-score (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0007) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-K). In NK-92 and KHYG-1 cell-lines, total and surface PD-L1 protein expression, as well as surface calreticulin expression were similarly increased following BCV treatment (Figures S3E-G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro and in vivo activity of BCV in PTCL\u003c/h2\u003e \u003cp\u003eNext, BCV was investigated in a cohort of T-cell lymphoma cell-lines (anaplastic large cell lymphoma [ALCL], n\u0026thinsp;=\u0026thinsp;9; PTCL not otherwise specified [PTCL-NOS], n\u0026thinsp;=\u0026thinsp;2; primary cutaneous T-cell lymphoma [CTCL], n\u0026thinsp;=\u0026thinsp;1). BCV inhibited viability across all T-cell lymphoma cell-lines in a dose-dependent manner (median IC50\u0026thinsp;=\u0026thinsp;593 ng/ml; range, 60.2 to 2785 ng/ml). Marked sensitivity (IC50\u0026thinsp;\u0026lt;\u0026thinsp;1000 ng/ml) was demonstrated in most cell-lines (seven of 11), including our in-house PTCL-S1 (\u003cem\u003eTP63\u003c/em\u003e-rearranged ALCL) and PTCL-S2 (PTCL-NOS) cell-line models (IC50\u0026thinsp;=\u0026thinsp;177 ng/ml and 1664 ng/ml, respectively) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and S5).\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eIn vivo\u003c/em\u003e, intraperitoneal BCV (40mg/kg, 2X per week) inhibited tumor growth in both NOD/SCID mice PTCL-S1 and PTCL-S2 xenografts, compared with vehicle alone. For PTCL-S1, at 21 days post-treatment with BCV, tumor volume was significantly reduced (81.9 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e vs 1207.1 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), as was tumor weight (0.021 g vs vehicle: 0.812 g, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). For PTCL-S2, both tumor volume (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0113) and tumor weight (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0039) were both significantly reduced with BCV treatment as compared to vehicle control (Figs.\u0026nbsp;6SA-B). Like in NKTCL, RNAseq and GSEA showed that MYC target and cell cycle pathways were significantly downregulated in PTCL-S1 cell-line post-BCV treatment. Upregulation of interferon (\u003cem\u003eIFNB1\u003c/em\u003e, \u003cem\u003eIFNY\u003c/em\u003e), cytokine (\u003cem\u003eCCL5\u003c/em\u003e, \u003cem\u003eCXCL10\u003c/em\u003e), and \u003cem\u003eCD274\u003c/em\u003e gene expression was observed. Western blot analysis showed downregulation of MYC protein and upregulation of apoptosis markers (cleaved PARP, cleaved caspase-3), phospho-H2AX, phospho-CHK1 (Ser317/Ser345), p-RPA2 (Ser33) and PDL1 in BCV-treated PTCL-S1 and PTCL-S2 cell-lines (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E and S6C; Tables S11-12). BCV treatment on PTCL-S1 xenograft tumors similarly showed increased protein expression of PD-L1 in BCV-treated mice compared with vehicle-treated controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0212) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmune response activation by BCV and anti-PD1 immune checkpoint blockade\u003c/h2\u003e \u003cp\u003eIn order to investigate the \u003cem\u003ein vivo\u003c/em\u003e immune response further, we first examined the effect of BCV in murine lymphoma cell-lines EL-4 and TK-1, demonstrating a dose-dependent inhibition on cell viability in both models (IC50 516 ng/ml and 444 ng/ml, respectively), as well as increased surface expression of PD-L1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). Deploying a syngeneic EL4-C57BL/6 model, BCV alone or in combination with anti-PD1 (intraperitoneal 200 \u0026micro;g 2X per week) significantly inhibited tumor growth at 8 days post-treatment, compared to isotype or anti-PD1 treatment alone. However, no significant difference in tumor volume or weight was observed with the combination of BCV and anti-PD1, compared with BCV alone (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F). Intriguingly, at this early time point, examination on H\u0026amp;E staining showed a significant immune response in BCV-treated groups, which appeared particularly brisk with the addition of anti-PD1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). NanoString profiling showed a significantly heavier immune infiltration in the BCV\u0026thinsp;+\u0026thinsp;anti-PD1 combination group, as corroborated by the highest scores for adaptive immune response, cytokines/chemokines \u0026amp; receptors, cytotoxic cells, dendritic cells, NK CD56\u003csup\u003edim\u003c/sup\u003e cells and neutrophils. Gene expression of \u003cem\u003eCCL2\u003c/em\u003e, \u003cem\u003eCCL12\u003c/em\u003e, \u003cem\u003eCXCL9\u003c/em\u003e, \u003cem\u003eGZMB\u003c/em\u003e, \u003cem\u003eCHIL3\u003c/em\u003e and \u003cem\u003eCTLA4\u003c/em\u003e were also highest in the combination treatment group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-I and S7-9; Tables S13-14).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eExtending preclinical activity of BCV to B-cell lymphoma models\u003c/h2\u003e \u003cp\u003eFinally, BCV was investigated in a cohort of B-cell lymphoma cell-lines. BCV inhibited viability in all B-cell lymphoma cell-lines in a dose-dependent manner (IC50 79.8 ng/ml to 8414 ng/ml). Marked sensitivity (IC50\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026micro;g/ml) was demonstrated in nine cell-lines, including the \u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e-rearranged double-hit DLBCL cell-line OCI-LY18 (IC50 500 ng/ml) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and S10). Intraperitoneal BCV (40 mg/kg, 2X per week) inhibited tumor growth in NOD/SCID mice OCI-LY18 xenografts, compared with vehicle alone (tumor volume: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; tumor weight: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0317) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C). Interestingly, in contrast to T and NK/T cell-lines, RNAseq showed that the transcriptional repressor \u003cem\u003eTLE1\u003c/em\u003e was instead amongst the top upregulated genes in the BCV-sensitive cell-lines (IC50\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026micro;g/ml) (log2foldchange\u0026thinsp;=\u0026thinsp;8.12, adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004), which was validated on qPCR (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0350) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-E and Table S15). DLBCL patient samples from two independent datasets showed that \u003cem\u003eTLE1\u003c/em\u003e-high tumors conferred worse overall survival (GSE11318, n\u0026thinsp;=\u0026thinsp;200: HR 2.77, 95%CI 1.78 to 4.30, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; GSE10846, n\u0026thinsp;=\u0026thinsp;414: HR 1.94, 95%CI 1.35 to 2.80, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we investigated the preclinical therapeutic activity of the novel nucleoside analogue BCV in a spectrum of non-Hodgkin lymphoma cell-lines. Originally intended as an anti-viral agent against smallpox and other viruses, our observations \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e support its potential role as an anti-cancer agent, regardless of the presence of any known associated viruses. Furthermore, by examining transcriptomic profiles of the cell-line models, we identified potential signaling pathways modulating the response to BCV, including STING pathway activation and PD-L1 expression, implying prospective synergies with checkpoint immunotherapy.\u003c/p\u003e \u003cp\u003ePrevious studies on CDV have demonstrated growth inhibitory effects in various cancer types, including viral-negative tumors such as melanoma,\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e glioblastoma,\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e as well as squamous carcinoma of the head and neck (HNSCC) and cervix.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e In a vascular tumor model induced by basic fibroblast growth factor (FGF2)-overexpressing endothelial cells (FGF2-T-MAE) in mice, CDV evoked S-phase cell cycle arrest and apoptosis.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e In glioblastoma, the cytotoxic effect of CDV was mediated through apoptosis, regardless of the presence of associated cytomegalovirus infection. In this study, CDV was shown to incorporate into tumor cell DNA, thereby promoting replication fork stalling and initiating the formation of DNA double-strand breaks.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Similarly findings were also observed in HNSCC and cervical carcinoma.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e In keeping with these earlier studies, our current results support a similar mechanism of action for BCV in PTCL and NKTCL, in which BCV triggers replication stress and cell cycle arrest, as well as DNA damage and apoptosis.\u003c/p\u003e \u003cp\u003eThe cyclic GMP\u0026ndash;AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a key mediator of the inflammatory response to DNA damage, including that induced by some exogenous anti-tumor agents.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e BCV exposure leads to accumulation of cytosolic DNA, cGAS/STING pathway activation, triggering an interferon response and upregulation of the PD-L1 immune checkpoint protein. In addition, BCV evokes an immunogenic cell death (ICD), as evidenced by increased cell membranous expression of calreticulin, a danger-associated molecular pattern (DAMP) molecule, where it acts as an \u0026ldquo;eat me\u0026rdquo; signal for immature dendritic cells.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e An increased in secreted high mobility group box 1 (HMGB1) protein levels was also observed, responsible for the optimal release and presentation of tumor antigens to dendritic cells, dendritic cell maturation, and synthesis of proinflammatory molecules.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Indeed, our results using a syngeneic EL4-C57BL/6 lymphoma model further suggests a robust local inflammatory response in response to addition of PD1 blockade to BCV treatment, including activation of adaptive immune responses, cytokines/chemokines \u0026amp; receptors, cytotoxic cells, dendritic cells, NK CD56\u003csup\u003edim\u003c/sup\u003e cells and neutrophils. The potential for combination with immune checkpoint blockade, as well as with other DNA damaging agents or radiation therapy deserves to be evaluated in further studies.\u003c/p\u003e \u003cp\u003eTLE1 functions as a transcriptional co-repressor that can antagonize the functions of a number of transcription factors involved in key signals mediating oncogenesis and inflammation, including Notch, Wnt, and NF-κB pathways.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e In a prior study by Fraga et al., TLE1 was found to be epigenetically inactivated in various hematologic malignancies through CpG island promoter hypermethylation. Reintroduction of TLE1 caused growth inhibition, whereas depletion resulted in growth enhancement, supporting its role as a tumor suppressor.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Our results showed that in NKTCL cell-lines, \u003cem\u003eTLE1\u003c/em\u003e gene expression was inversely correlated with sensitivity to BCV. \u003cem\u003eTLE1\u003c/em\u003e-low tumors were enriched for \u003cem\u003eMYC\u003c/em\u003e target pathways, which was prominently downregulated by BCV treatment. In patients with NKTCL, we further demonstrated that low tumor levels of \u003cem\u003eTLE1\u003c/em\u003e gene expression was associated with worse survival outcomes. Previously, in patients with T-cell acute lymphoblastic leukemia (T-ALL), low \u003cem\u003eTLE1\u003c/em\u003e expression was similarly associated with higher relapse rate and shorter survival.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Taken together, this implies that \u003cem\u003eTLE1\u003c/em\u003e gene expression may be both a potential predictive and prognostic biomarker in NKTCL, and that patients with low tumor \u003cem\u003eTLE1\u003c/em\u003e expression might derive greater benefit to BCV therapy. Interestingly however, our results demonstrated the converse instead for B-cell lymphoma \u0026ndash; \u003cem\u003eTLE1\u003c/em\u003e was significantly upregulated in the cell-lines which were more sensitive to BCV, and high \u003cem\u003eTLE1\u003c/em\u003e gene expression instead conferred poor survival in patients with DLBCL. The specific mechanisms of TLE1 function might be contextual upon the hematopoietic lineage and will require further elucidation in future studies.\u003c/p\u003e \u003cp\u003eOur work demonstrates the therapeutic potential of BCV in non-Hodgkin lymphoma, regardless of the presence of EBV. While our study remains limited as a preclinical investigation of BCV, remarkable \u003cem\u003ein vivo\u003c/em\u003e anti-tumor activity was demonstrated in classically aggressive forms of lymphoma including NKTCL, ALK-negative ALCL, and double-hit DLBCL xenograft models. We have also gained insight into potential mechanisms of anti-tumor action of BCV, including its ability to evoke immunogenic cell death.\u003c/p\u003e \u003cp\u003eIn conclusion, we propose that BCV is an attractive agent for the treatment of non-Hodgkin lymphoma, particularly PTCL and NKTCL. A clinical trial (NCT06761677) is underway to confirm its safety and efficacy in patients with relapsed and/or refractory non-Hodgkin lymphoma. Further translational studies will be required to validate predictive biomarkers of response.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Tanoto Foundation Professorship in Medical Oncology, New Century Foundation Limited, Ling Foundation, Singapore Ministry of Health\u0026rsquo;s National Medical Research Council Research Transition Award (TA21jun-0005), Large Collaborative Grant (OFLCG18May-0028 and OFLCG23May-0039), TETRAD II Collaborative Centre Grant (CG21APR2002),\u0026nbsp;SingHealth Duke-NUS AM/ACP-Designated Philanthropic Fund Grant Award (08/FY2023/EX/27-A65), as well as the Khoo Bridge Funding Award (Duke-NUS-KBrFA/2025/0090) provided by Duke-NUS Medical School and the \u0026ldquo;Estate of Tan Sri Khoo Teck Puat\u0026rdquo;.\u0026nbsp;Study drug and funding were also provided by SymBio Pharmaceuticals Limited.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthorship Contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.Y.C. analyzed the data and prepared the first draft the manuscript; S.T.L. provided clinical information and samples; H.Y.T., J.Q.L. performed the bioinformatic analyses; E.C.Y.L., K.X.Y.C., J.Y.L., B.K., B.Y.L., Z.L., T.K.K., J.S.K., K.S.L., N.A.B.M.T., D.H. processed tissue and performed experiments; T.B.T., M.H., K.F., contributed to data interpretation; J.Y.C. and C.K.O. conceptualized the study, interpreted the results, had unrestricted access to all data, and revised the manuscript. All authors agreed to submit the manuscript, read and approved the final draft, and take full responsibility for its content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDisclosure of Conflicts of Interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported in part by funding from SymBio Pharmaceuticals Limited to J.Y.C. and C.K.O.; M.H. and K.F. are employees of SymBio Pharmaceuticals Limited. The remaining authors declare no competing financial interests.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eO'Connor OA, Ma H, Chan JYS, Kim SJ, Yoon SE, Kim WS. Peripheral T-cell lymphoma: From biology to practice to the future. 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PMID: 34048198; PMCID: PMC8408389.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NK/T cell lymphoma, nucleoside analogue, replication stress, immunotherapy, PD-L1, immunogenic cell death","lastPublishedDoi":"10.21203/rs.3.rs-6768176/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6768176/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e Brincidofovir (BCV) is a novel nucleoside phosphonate analogue with unique dual anti-viral and anti-tumor properties.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Activity of BCV was evaluated in forty-four cell-line models, including T/NK-cell non-Hodgkin lymphoma (T/NK-NHL, n=25) and B-cell lymphoma (BCL, n=19), as well as their respective NOD/SCID mice xenograft models (NK/T-cell lymphoma, peripheral T-cell lymphoma, not otherwise specified, \u003cem\u003eTP63\u003c/em\u003e-rearranged anaplastic large cell lymphoma, and \u003cem\u003eMYC\u003c/em\u003e/\u003cem\u003eBCL2\u003c/em\u003e-rearranged diffuse large B-cell lymphoma). Potential \u003cem\u003ein vivo\u003c/em\u003eimmunogenic effects were examined in a syngeneic EL4-C57BL/6 murine lymphoma model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e BCV demonstrated potent anti-tumor activity \u003cem\u003ein vitro\u003c/em\u003e across the majority of cell-lines regardless of EBV positivity, with IC50 values within clinically-achievable human plasma concentrations (2 µg/ml) in 17/25 (68.0%) T/NK-NHL and in 13/19 (68.4%) BCL. \u003cem\u003eIn vivo\u003c/em\u003e treatment via intraperitoneal BCV (40mg/kg, 2X per week) significantly inhibited tumor growth in all xenograft models when compared to vehicle control. Notably, RNAseq analysis demonstrated BCV downregulated \u003cem\u003eMYC\u003c/em\u003e and MYC-target pathways in T/NK-NHL models. Further mechanistic studies showed that BCV evoked S-phase cell cycle arrest, replication stress, DNA damage and apoptosis, while also triggering STING pathway-mediated interferon responses, PD-L1 expression and immunogenic cell death. In the syngeneic EL4-C57BL/6 model, BCV in combination with anti-PD1 significantly inhibited tumor growth and triggered an immune reaction characterized by highest scores for adaptive immune response, cytokines/chemokines \u0026amp; receptors, cytotoxic cells, dendritic cells, NK CD56dim cells and neutrophils (NanoString Immunology Panel).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Taken together, these results demonstrate a novel role of BCV in the treatment of lymphoma, and suggest potential for combination with checkpoint immunotherapy.\u003c/p\u003e","manuscriptTitle":"Preclinical activity of brincidofovir in Peripheral T-cell and NK/T-cell Lymphoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 20:58:53","doi":"10.21203/rs.3.rs-6768176/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0d06a3a9-a6bc-4517-aabd-04b464fade62","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49235965,"name":"Biological sciences/Cancer/Cancer therapy/Drug development"},{"id":49235966,"name":"Health sciences/Health care/Therapeutics/Drug therapy/Molecularly targeted therapy"}],"tags":[],"updatedAt":"2025-08-18T10:45:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-03 20:58:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6768176","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6768176","identity":"rs-6768176","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}