Human endogenous retroviruses as epigenetic therapeutic targets in TP53-mutated diffuse large B-cell lymphoma

TP53 mutation (TP53mut) occurs in 10–20% of diffuse large B-cell lymphoma (DLBCL) cases and serves as an unfavorable biomarker of DLBCL progression. It confers resistance to immunochemotherapy, high-dose chemotherapy, autologous stem cell transplantation, and anti-CD19 chimeric antigen receptor T-cell therapy. Therapeutic targeting of TP53mut remains a significant challenge in DLBCL treatment. Here we assessed TP53mut in 667 patients with newly diagnosed DLBCL, including 576 patients treated with immunochemotherapy rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and 91 patients with decitabine plus R-CHOP (DR-CHOP, NCT02951728 and NCT04025593). TP53mut independently predicted an inferior prognosis in R-CHOP-treated DLBCL, although this could be mitigated by DR-CHOP treatment. In TP53mut patients, multiple viral regulation pathways were repressed, resulting in the inhibition of immune modulation, as revealed by gene set enrichment analysis. TP53mut DLBCL exhibited increased methyltransferase SUV39H1 expression and H3K9 trimethylation (H3K9me3), contributing to repression of endogenous retroviruses (ERVs) and immunosuppressive tumor microenvironment. In TP53mut DLBCL cell lines, decitabine down-regulated SUV39H1, inhibited H3K9me3 occupancy on ERVs, and triggered ERV expression, thereby unleashing interferons program and CD4+T/CD8+T cell activation. Molecular silencing of SUV39H1 significantly abrogated decitabine-induced H3K9me3 inhibition and ERV expression. In TP53mut patient-derived xenograft models and TP53mut patients, the anti-tumor effect was improved upon the use of combined treatment of decitabine and doxorubicin via SUV39H1-H3K9me3-ERVs axis. Collectively, our findings highlight an ERV regulatory circuitry in TP53mut DLBCL and the crucial roles ERVs for epigenetically reprogramming tumor microenvironment for treating TP53mut-driven cancers.


INTRODUCTION
Diffuse large B-cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin lymphoma and TP53 mutation (TP53 mut ) is an important unfavorable genetic alteration, 1 predominantly occurring in the DNA binding domain (DBD). 2 Functionally, TP53 mutants are classified as structural mutants (R175, R249, G245, and Y220) and DNA contact surface mutants (R248 and R273).Structural mutants reduce the thermostability of the protein, leading to improper folding at physiological temperatures and loss of DNA binding activity.DNA contact surface mutants are situated within the DNA core binding region and hinder the DNA-protein binding. 3P53 mut confers resistance not only to conventional immunochemotherapy, 1,4,5 but also to autologous stem-cell transplantation (ASCT), 6,7 and chimeric antigen receptor T-cell (CAR-T) therapy in DLBCL, 8 due to the immunosuppressive tumor microenvironment (TME), repression of interferon (IFN) response and inhibition of T-cell activation. 8,9The IFN family includes two main classes: type I and type II IFNs, 10 both of which suppress tumors by directly acting on tumor cells and by indirectly enhancing anti-tumor immunity. 11,12Type I IFNs (mainly IFN-α and IFN-β) play a pivotal role in host defense against viral infection. 13Type II IFN (IFN-γ) is induced by IFN-α-or IFNβ-mediated activation of STAT4. 14-Aza-2'-deoxycytidine (decitabine) is a hypomethylating agent that exhibits anti-tumor activity by reactivating tumor suppressor genes. 15In myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), the outcome of TP53 mut patients is significantly improved by decitabine treatment. 16,17Recently, we have reported a phase I/II trial of decitabine plus R-CHOP (DR-CHOP, NCT02951728) and found that all five TP53 mut patients achieve durable remission. 18Response to decitabine is related to a significant elevation of serum IFN-γ and increased peripheral blood CD3 + CD4 + T and CD3 + CD8 + T cells. 18However, the role of decitabine in reprogramming TME needs further investigation in TP53 mut DLBCL.
Human endogenous retroviruses (ERVs) are remnants of ancestral viral infections and permanently integrate within the human genome. 19DNA methylation and trimethylation of histone H3 on lysine 9 (H3K9me3) are important epigenetic mechanisms in modulating ERV expression, 20 inhibiting IFN-γ production, and impairing T-cell activation. 21DNA methyltransferase 1 (DNMT1) is the main factor inducing DNA methylation; meanwhile, the depletion of DNMT1 leads to ERV de-repression. 22Decitabine induces DNA demethylation through DNMT1 depletion and restores ERV transcription, provoking the anti-tumor activity. 23UV39 family and PRDM family members function as the main methyltransferases of H3K9. 24SUV39 family members repress ERV transcription through H3K9 methylation. 21,25Whether H3K9 methylation can be targeted by decitabine remains of great interest.The viral mimicry anti-tumor effects exerted by ERVs also command great attention.
Here we first explored the pivotal role of TP53 mut in modulating the ERV expression and the TME reprogramming in a histone methylation-dependent manner.Then we illustrated the underlying mechanism of decitabine on ERV regulatory circuitry and anti-tumor immunity in TP53 mut DLBCL.Our findings thus provide both in vitro and in vivo evidence that ERV functions as a new epigenetic therapeutic target in TP53 mut -driven cancers.
Age, sex, ECOG performance status, Ann Arbor stage, serum LDH level, extranodal involvement, and TP53 mutational status were included in univariate analysis.Variables demonstrating significance with p < 0.200 on univariate analysis were included in the multivariate model. 26Univariate analysis showed that age >60 years, ECOG ≥ 2, Ann Arbor stage III/IV, elevated serum LDH, multiple extranodal involvement, and TP53 mut were unfavorable prognostic indicators for both PFS and OS in the R-CHOP cohort.By multivariate analysis, Ann Arbor stage III/IV, elevated serum LDH, and TP53 mut were independent factors predicting poor PFS and OS (all p ≤ 0.001) (Supplementary Table 2).However, in the DR-CHOP cohort, univariate analysis showed only ECOG ≥ 2 as an adverse prognostic factor for both PFS and OS (p = 0.017 and 0.027, respectively).By multivariate analysis, ECOG ≥ 2 was an independent factor predicting poor PFS and OS (p = 0.037 and 0.013, respectively) (Supplementary Table 3).
TP53 mut is associated with ERV down-regulation and immunosuppressive TME in DLBCL To identify the potential role of TP53 mut on the TME of DLBCL, we performed RNA sequencing on 280 TP53 mut DLBCL (67 with TP53 mut and 213 with TP53 wt ).Interestingly, in TP53 mut patients, in addition to activated pathways involved in TP53-targeted biological functions such as positive regulation of cell cycle and negative regulation of intrinsic apoptosis (Supplementary Fig. 2a), multiple viral regulation pathways (regulation of viral genome replication, response to virus, and defense response to virus) were shown to be repressed, accompanied by the inhibition of immune modulation (response to IFN-gamma, response to type I IFN, and T cell activation), as revealed by gene set enrichment analysis (GSEA) (Fig. 2a).
To further determine the potential virus involved in TP53 mut DLBCL, viral sequences were assessed and quantified by the Kraken 2 27 and ERVs were assessed and quantified by Repeat-Masker (hg19) based on the database downloaded from Repeating Elements.Among the main virus genera detected, only ERVs were remarkably down-regulated in TP53 mut DLBCL (Supplementary Table 4).The volcano plot showed significantly lower expression levels of multiple ERVs in TP53 mut than in TP53 wt tumors (Fig. 2b).ERVL-E, MER21C, HERV16, and HERVK11 were the most significantly down-regulated ERVs (Fig. 2c).Meanwhile, tracking tumor immunophenotype (TIP) analysis indicated that the recruitment activities of T cells (p = 0.031), CD4 + T (p = 0.002), and CD8 + T cells (p = 0.019) were significantly decreased in TP53 mut DLBCL, as compared to the TP53 wt DLBCL (Fig. 2d).Consistent with the lower infiltration rate of T cells, the enriched genes involved in MHC-I and MHC-II protein complex, as well as IFN-stimulated genes, were significantly repressed in TP53 mut DLBCL (Supplementary Fig. 2b).
To explore the underlying mechanisms of TP53 mut on ERV expressions, we established TP53 mut (p.R175H, p.R248Q, p.R273C) cell lines in both GCB (SU-DHL-4) and ABC (OCI-LY-3) subtypes.Representative structural mutant R175H, as well as DNA contact surface mutants R248Q and R273C, were used in our experimental models to illustrate the aberrant function of mutant TP53.In both SU-DHL-4 (Fig. 3f) and OCI-LY-3 cells (Supplementary Fig. 4a), expression of SUV39H1 was higher in TP53 mut cells than in TP53 wt cells.Comparable to the gene expression, the high protein expression of SUV39H1 was confirmed by western blot (Fig. 3g and Supplementary Fig. 4b) and immunofluorescence staining (Supplementary Fig. 4c).As per the literature review, the expression of E2F1 is up-regulated in TP53-knockdown cells. 29In our study, E2F1 expression levels were significantly higher in TP53 mut DLBCL patients (Supplementary Fig. 4d) and TP53 mut cell lines (Supplementary Fig. 4e and 4f) than TP53 wt counterparts.According to the human transcription factor target database, 30 E2F1 could bind to the promoter of SUV39H1.Luciferase-reporter assay was then performed, and the results confirmed that E2F1 could directly activate SUV39H1 through transcriptional regulation (Supplementary Fig. 4g).As the results of elevated expression of SUV39H1, H3K9me3 levels were significantly increased in TP53 mut cells by western blot (Fig. 3g and Supplementary Fig. 4b) and immunofluorescence staining (Fig. 3h).
Molecular silencing of SUV39H1 was induced by transfecting with SUV39H1 short hairpin RNA (shRNA) in TP53 mut SU-DHL-4 and OCI-LY-3 cell lines, and vector, as well as non-targeting shRNA, were used as control (Supplementary Fig. 5a, b).Lack of SUV39H1 expression resulted in a significant decrease of H3K9me3 (Supplementary Fig. 5b) and an increase in ERV expression (Supplementary Fig. 5c), indicating that ERV repression was dependent on SUV39H1 expression in TP53 mut DLBCL.As a consequence of ERV activation, remarkable up-regulation of IFN (Supplementary Fig. 5d) and increased CD4 + T, CD8 + T cells (Supplementary Fig. 5e) were observed in SUV39H1-shRNA cells, as compared to control cells.
ChIP-seq was further performed to investigate the genomic distribution of H3K9me3 and revealed a significant enrichment of H3K9me3 on the whole genome in TP53 mut cells, as compared to TP53 wt cells (Fig. 3i and Supplementary Fig. 6a).Of note, H3K9me3 was significantly enriched on the sequence of ERVL-E, MER21C, HERV16, and HERVK1 (Fig. 3j), resulting in repression of these ERVs in TP53 mut SU-DHL-4 and OCI-LY-3 cells (Fig. 3k and Supplementary Fig. 6b).Consequently, concentrations of IFN [IFN-α, (p = 0.043), IFN-β (p = 0.015), and IFN-γ (p < 0.001)] were lower in the supernatants of TP53 mut cells than in TP53 wt cells (Supplementary Fig. 7a).To validate the influence of TP53 mut on TME, TP53 mut cells were co-cultured with peripheral blood mononuclear cells (PBMCs).TP53 mut cells inhibited the growth of CD4 + T and CD8 + T cells, as compared to TP53 wt cells (Supplementary Fig. 7b).Since DNMT1 was reported to play a crucial role in ERV repression, we evaluated the expression of DNMT1.However, no significant difference was observed between TP53 mut and TP53 wt DLBCL patients (Supplementary Fig. 8a) and TP53 wt cell lines (Supplementary Fig. 8b-8d), suggesting DNMT1 may not be involved in regulating ERVs in TP53 mut DLBCL.

DISCUSSION
7][8] As an alternative mechanism of lymphoma progression, TP53 mut alters host innate and adaptive immune responses, 9 thus contributing to immunosuppressive TME, with decreasing CD4 + T and CD8 + T cell components. 8,9To our knowledge, this is the first report to identify ERVs as TP53 mut -downstream targets in DLBCL, whose inhibition depended on a histone-methylation manner, resulting from increased SUV39H1 expression and H3K9 methylation.Consistently, molecular silencing of TP53 in solid tumor cells increases SUV39H1 expression and induces H3K9me3, leading to cell cycle progression and chemoresistance. 29,31,32Of note, as revealed by Chip-seq, TP53 mut mediated enrichment of H3K9me3 for whole genome and H3K9me3 deposition sites on ERVs are at least part of the H3K9 epigenetic program to regulate H3K9me3-target genes.Therefore, the genome-wide epigenetic downregulation by TP53 mut was associated with ERV repression and consequent IFN inhibition, contributing to impaired anti-tumor immunity in TP53 mut DLBCL.Our study not only proposed a functional role of TP53 mut on TME within DLBCL, but also provided a potential rationale to overcome the adverse effect of TP53 mut in DLBCL progression.
Activation of TP53 induces viral mimicry response, unleashes the interferon program, and increases CD4 + T and CD8 + T cell proportion, raising possibilities of targeting ERVs as an anti-tumor approach since directly targeting TP53 mut is challenging. 33Moreover, we were inspired by our previous study, in which DR-CHOPresponding TP53 mut DLBCL patients have elevated serum IFN concentrations and increased proportions of peripheral blood CD3 + CD4 + T and CD3 + CD8 + T cells. 18In this study, we confirmed our previous results that DR-CHOP improved the outcome of TP53 mut DLBCL patients and further elucidated the underlying mechanism of DR-CHOP on epigenetic regulation through modulating ERV expression, IFN production, and T cell activation in TP53 mut DLBCL.Mutant TP53 shapes the immune landscape of the TME by down-regulating MHC-1/II expression and T cell infiltration, 9 decitabine treatment increased ERV expression, IFN production, and mediated viral mimicry anti-tumor immunity, which is why, TP53 mut DLBCL, resistant to R-CHOP, could be eliminated by decitabine combined with chemotherapy (DR-CHOP).Consistently, in MDS and AML, decitabine induces higher IFN production 34 and improves responses in TP53 mut patients. 35,36These observations demonstrated that reprogramming TME was essentially involved in decitabine-treated TP53 mut hematological malignancies through fostering IFN production and T cell activation.
Axis of virus-IFN-T-cell activation is a well-known mechanism in anti-virus treatment.Growing preclinical and clinical evidence suggests that hypomethylating agents can induce DNA demethylation and ERV reactivation, resulting in a viral mimicry state to generate anti-tumor immune responses. 18,37However, the expression of DNMT1 showed no significant difference between TP53 mut and TP53 wt DLBCL patients and cell lines.Furthermore, the bisulfite-PCR analysis showed that the sequences of ERVL-E, MER21C, HERV16, and HERVK11 were mainly unmethylated, and decitabine failed to change the unmethylated status of these ERVs, indicating that DNA methylation was less involved in TP53 mut DLBCL.Consistent with our results, in mouse embryonic stem cells, 69 ERV subfamilies were de-repressed in the SETDB1 knockout cells, and only 5 were de-repressed in the DNMT1/DNMT3A/DNMT3B triple knockout cells, indicating that the majority of ERVs were controlled by histone methylation rather than DNA methylation. 38ndeed, in TP53 mut DLBCL cell lines and PDX murine models, we illustrated that decitabine induced ERV expression through the down-regulation of SUV39H1 and inhibition of H3K9me3.Furthermore, molecular silencing of SUV39H1 abrogated the effects of decitabine and doxorubicin on H3K9me3 and ERV expressions, underlining the pivotal role of SUV39H1 in epigenetic reprogramming upon decitabine treatment.This is consistent with solid tumors in which decitabine impairs SUV39H1 recruitment and inhibits H3K9me3, thereby blocking tumor cell growth and metastasis. 39t is also worth pointing out that upon DR-CHOP treatment, TP53 mut DLBCL obtained 3-year PFS and 3-year OS with 72.1% and 79.1%, respectively, similar to TP53 wt DLBCL that received R-CHOP. 7Collectively, our findings highlighted a TP53/SUV39H1/ H3K9me3 regulatory circuitry and assigned ERVs as promising therapeutic targets in cancer treatment.Future studies should focus on better understanding the role of ERVs as possible tumor antigens and the underlying mechanism for epigenetically modulating TME for immunotherapy. 37n conclusion, decitabine sensitized chemotherapy and orchestrated with R-CHOP in TP53 mut DLBCL via SUV39H1-H3K9me3-ERVs axis, underlines a new anti-tumor mechanism of decitabine through modulating histone methylation.These findings further defined the clinical rationale for ERVs as potential epigenetic therapeutic targets to reprogram TME in treating TP53 mut -driven cancers.

Patients and study design
This study enrolled a total of 667 newly diagnosed DLBCL patients, with 576 in the R-CHOP cohort and the remaining patients in the DR-CHOP cohort (n = 91).Our previous phase I/II trial  40 excluding mediastinal large B-cell lymphoma or primary central nervous system DLBCL.Treatment response was evaluated according to the International Workshop Criteria. 41The Hospital Review Board approved the study with informed consent obtained following the Declaration of Helsinki.
In vitro co-culture system PBMCs were obtained from healthy donors through ficoll density gradient centrifugation, yielding a heterogeneous population consisting of myeloid and lymphoid cells, including ~15% B cells, 70% T cells, 5% monocytes, and 10% natural killer (NK) cells.The effector (E) to target (T) ratio refers to the proportion of PBMCs to lymphoma cells.In this study, the E:T ratio was set at 5:1, as recommended in previous research. 43PBMCs were grown in RPMI-1640 medium with 10% heat-inactivated fetal bovine serum (FBS) added in a humidified atmosphere containing 5% CO 2 at 37 °C.

DNA sequencing and viral genome detection
Genomic DNA was isolated from frozen tumor tissue using a Wizard® Genomic DNA Purification Kit (Promega, Wisconsin-Madison, USA) and from formalin-fixed paraffin-embedded (FFPE) tumor tissue using a GeneRead DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.For 128 patients, whole genome sequencing (WGS) was performed on frozen tumor tissue.For 153 patients, whole exome sequencing (WES) was performed on frozen tumor tissue (n = 125) and FFPE tumor tissue quality-controlled by agarose gel electrophoresis (n = 28).Targeted sequencing was performed on formalin-fixed paraffin-embedded tumor samples of 386 patients with genomic DNA extracted using GeneRead DNA formalin-fixed paraffinembedded Tissue Kit (Qiagen, Hilden, Germany).A targeted sequencing panel was assessed using a custom Sure Select library (Agilent Technologies), including 55 genes related to the pathogenesis of DLBCL according to literature, or associated with significant alternations in gene expression, as revealed by RNA sequencing.
RNA sequencing RNA sequencing was performed on frozen tumor samples of 280 patients with RNA extracted using the RNeasy MinElute Cleanup Kit (Qiagen, Dusseldorf, German).RNA-seq protocols are optimized for 1 μg of total RNA.RNA integrity was assessed using RNA 6000 Nano Kit (Agilent, California, USA) on Agilent 2100 Bioanalyzer (Agilent, California, USA) with an RNA Integrity Number (RIN) value ≥ 8. High-quality RNA showed a 28S rRNA band at 4.5 kb at twice the intensity of the 18S rRNA band at 1.9 kb.Then RNA-seq was performed using Illumina HiSeq 2000 (Illumina, California, USA) at 40-60 million 150 bp paired-end reads per sample depth.
Viral sequences were assessed and quantified by the Kraken 2. 27 ERVs were assessed and quantified by RepeatMasker (hg19) based on a database downloaded from Repeating Elements.The ERVs' expression was further assessed based on the mean expression of the top 4 ERVs.

Bioinformatic analyses
Bioinformatic analyses were performed by r 4.0.3, using R package "sva" to remove the batch effect.Raw reads were normalized, and differentially expressed genes (DEGs) were obtained with R package "limma" (v3.38.3).The gene set enrichment analysis was performed using the R package 'clusterProfiler' (v3.10.1).GO enrichment analysis and data visualization.The methylation score was assessed by single-sample gene set enrichment analysis (ssGSEA) in the GSVA R package (v1.45.0) based on hallmark genes for methylation processes downloaded from the Molecular Signatures Database (MSigDB). 28terferon assessment Serum specimens of DLBCL patients were collected before (day 0) and after one cycle of DR-CHOP (day 21).Culture supernatants of cell lines and lysed PDX samples were collected before and after decitabine and doxorubicin exposition.Concentrations of interferons in serum and culture supernatant were measured using interferon-α ELISA kit (cat.no.70-EK199-96, MultiSciences Biotechnology), interferon-β ELISA kit (cat.no.70-EK1236-96, MultiSciences Biotechnology) and interferon-γ ELISA kit (cat.no.70-EK180-96, MultiSciences Biotechnology) according to manufacturer's protocols.Cytokine concentrations were determined by Fig. 5 Decitabine combined with doxorubicin on ERV activation, IFN production, and anti-tumor effect in TP53 mut PDX models.a Decitabine and doxorubicin treatment significantly retarded tumor growth of TP53 mut PDX models, as compared to untreated and single treatment groups.b Decitabine and doxorubicin down-regulated SUV39H1 expression and H3K9me3 score in TP53 mut tumors.c Decreased H3K9me3 expression revealed by immunohistochemistry staining on TP53 mut tumors.Left panel, representative immunohistochemistry staining images for H3K9me3.Right panel, proportion of H3K9me3 expression.Scale bars, 50 μm.Expression levels were assessed according to the percentage of positive cells: + denoted <25%; ++ denoted 25-49%; +++ denoted 50-74%; ++++ denoted 75-100%.d High expression of ERVs in lysed TP53 mut PDX tumors treated with decitabine and doxorubicin, as compared to untreated and single treatment groups.e Up-regulated gene ontology (GO) terms in TP53 mut tumors treated with decitabine combined with doxorubicin, as compared to single doxorubicin treatment.f Significantly activated signaling pathways identified by RNA-seq in TP53 mut tumors treated with decitabine combined with doxorubicin, as compared to single doxorubicin treatment.Enrichment plots of response to virus signaling pathway, type I/type II interferon signaling pathway, and T cell activation involved in immune response pathway by GSEA analysis.g The concentrations of IFN-α, IFN-β, and IFN-γ in TP53 mut tumors treated with decitabine and doxorubicin, as compared to untreated and single treatment groups

Lentivirus packaging and transfection
The coding sequence of the human E2F1 gene (GenBank Accession: NM_005225.3)with C-terminal 3×flag was synthesized and cloned into a pCMV vector between HindIII and BamHI sites.The resulting plasmid was confirmed by Sanger sequencing.To overexpress E2F1 in SU-DHL-4 and OCI-LY-3 cells, purified plasmid E2F1-over or vector were transfected into HEK-293T using lipofectamine 2000 (Invitrogen, Cambridge, MA, USA).The HEK-293T cell culture supernatant was condensed into a viral concentration of ~3 × 10 8 transducing units/mL.The lentiviral particles were incubated with SU-DHL-4 and OCI-LY-3 cells, respectively, for 72 h.Stably transfected cells were generated through the introduction of GFP.
Methylation-specific PCR Cells that underwent treatment with decitabine and/or doxorubicin were collected for DNA isolation utilizing a genomic DNA kit as per the instruction provided by the manufacturer (A1120, Promega, Wisconsin-Madison, USA), and then the DNA samples were treated using DNA Bisulfite Conversion Kit (DP215-02, Tiangen, China) and methylation-specific PCR Kit (EM101, Tiangen, China).The methylation and unmethylation primers utilized in this study are detailed in the Supplementary Table 5.The methylation-specific PCR products (200 ng) were electrophoresed on 2% agarose gels and visualized through ethidium bromide staining.
Patient-derived tumor xenografted model Four-week-old female NSG and NOD-SCID mice were obtained from Shanghai Laboratory Animal Center (Shanghai, China) to establish and maintain PDX models.Heterotopic PDX models were generated as previously described. 49Decitabine and doxorubicin, either alone or in combination, were applied to PDX models with a low passage number (P3-P5) to preserve the genetic integrity of the parental tumors.Tumor volumes were calculated as 0.5 × a × b 2 , where 'a' is the length and 'b' is the width.Treatments were started after the tumor became about 0.5 × 0.5 cm on the surface (day 0).The dose and administration schedule were as follows: decitabine pretreatment (10 mg/m 2 , 5 days), followed by doxorubicin (0.6 mg/kg every other day) four times, while the control group was untreated.Tumor-bearing mice were then euthanized by CO 2 asphyxiation.Animals were used according to the ARRIVE guidelines and the protocols approved by Shanghai Rui Jin Hospital Animal Care and Use Committee.

Statistics
All statistical analyses were performed in GraphPad Prism software (GraphPad Software, San Diego, CA, version 7.0), SPSS v23.0, and R v3.6.1 Survival Estimates were calculated using the Kaplan-Meier method, and survival curves were compared by the log-rank test.Univariate hazard estimate was generated with Cox proportional hazards models.Variables with a p-value <0.2 were included in the multivariate logistic regression analysis. 26Unpaired t-test with or without Welch's correction was used to compare different groups, and Pearson rank correlation was used to calculate the correlation between the two groups.Fisher's exact tests were applied to compare non-ordinal categorical variables.A two-sided p-value of <0.05 was considered statistically significant.

Fig. 1
Fig. 1 Mutational profile and survival analysis of patients with TP53 mut DLBCL.a Mutational profile of TP53 mut DLBCL patients.b, c PFS and OS of patients upon R-CHOP (b) or DR-CHOP (c) treatment according to TP53 mut status

Fig. 2
Fig. 2 ERV repression and immunosuppressive TME in TP53 mut DLBCL.a Down-regulated gene ontology (GO) terms in TP53 mut DLBCL patients, as compared to TP53 wt DLBCL patients.The color of points indicates the -log (p-value) of dysregulated pathways in two groups.The size of points indicates the number of genes included in each gene set.b Volcano plot image of the ERV expression in TP53 mut DLBCL and TP53 wt DLBCL.The gray dashed line corresponds to p = 0.05.The significantly depressed ERVs in TP53 mut DLBCL are annotated.c ERV expression in DLBCL according to TP53 mut status.d Immunity activity scores of indicated immune cells in TP53 mut DLBCL and TP53 wt DLBCL.P-values comparing different scores in two groups.TP53 mut , mutant TP53; TP53 wt , wild-type TP53

Fig. 3
Fig. 3 H3K9me3 enrichment and ERV expression in TP53 mut DLBCL.a Correlations of the H3K9me3 score with ERV expression.b H3K9me3 score in DLBCL patients according to TP53 mut status.c Immunohistochemistry staining of H3K9me3 in DLBCL according to TP53 mut status.Left panel, representative immunohistochemistry staining for H3K9me3.Right panel, proportion of H3K9me3 expression.Scale bars, 100 μm.Expression levels were assessed according to the percentage of positive cells: + denoted <25%; ++ denoted 25-49%; +++ denoted 50-74%; ++++ denoted 75-100%.d Correlations of SUV39H1 expression with the H3K9me3 score.e SUV39H1 expression according to RNAsequencing data of lymphoma samples from TP53 mut DLBCL and TP53 wt DLBCL.f SUV39H1 expression in TP53 mut or TP53 wt SU-DHL-4 cell line.g H3K9me3 expression in TP53 mut or TP53 wt SU-DHL-4 cell line by western blot.h H3K9me3 expression in TP53 mut or TP53 wt SU-DHL-4 cell line by immunofluorescence staining.Scale bars, 40 μm.i Distribution of H3K9me3 on the whole genome in TP53 mut p.R175H or TP53 wt SU-DHL-4 cell line.j H3K9me3 signal profiles in the genomic regions of ERVs in TP53 mut or TP53 wt SU-DHL-4 cell line.Upper, relative enrichment of H3K9me3 domains at the indicated ERVs; lower, the genome browser view of H3K9me3 signals on the indicated ERVs.Signals represent ChIPseq RPM.k Expression of ERVs in TP53 mut or TP53 wt SU-DHL-4 cell line

Fig. 4
Fig. 4 Decitabine combined with doxorubicin on ERV activation in TP53 mut SU-DHL-4 cells.a SUV39H1 expression detected by qRT-PCR in TP53 mut SU-DHL-4 cell line treated with decitabine, doxorubicin, alone or in combination.b The expression of SUV39H1 and H3K9me3 detected by western blot in TP53 mut SU-DHL-4 cell line upon indicated treatment group.c-e ERV expression (c), IFN production (d), as well as percentages of CD4 + T and CD8 + T cells (e) upon indicated treatment group.f Expression of SUV39H1 and H3K9me3 in SUV39H1 knock-down SU-DHL-4 cell line upon indicated treatment group.g ERV expression in SUV39H1 knock-down SU-DHL-4 cell line upon indicated treatment group

Fig. 6
Fig. 6 Induction of ERV expression and IFN production upon DR-CHOP or R-CHOP treatment in DLBCL patients.a-c Increased ERV expression (a), IFN production (b), as well as percentages of CD3 + CD4 + T and CD3 + CD8 + T cells (c) in TP53 mut patients after one cycle of DR-CHOP.d-f ERV expression (d), IFN production (e), as well as percentages of CD3 + CD4 + T and CD3 + CD8 + T cells (f) in TP53 mut patients after one cycle of R-CHOP