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Basic tumor immunology
Original research
Tumor cell-intrinsic SETD2 inactivation sensitizes cancer cells to immune checkpoint blockade through the NR2F1-STAT1 pathway
  1. Xufen Zheng1,
  2. Yuxiang Luo1,
  3. Yangjie Xiong1,
  4. Xiaoxiao Liu1,
  5. Chunling Zeng1,
  6. Xiaojing Lu1,
  7. Xiaofang Wang1,
  8. Yumei Cheng1,
  9. Simin Wang1,
  10. Haoqi Lan1,
  11. Kai Wang1,
  12. Zhonghui Weng1,
  13. Wenbo Bi1,
  14. Xinxin Gan2,
  15. Xiaona Jia1,
  16. Linhui Wang2 and
  17. Yuexiang Wang1
  1. 1CAS Key Laboratory of Tissue Microenvironment and Tumor, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China
  2. 2Department of Urology, Changhai Hospital, Naval Medical University, Shanghai, China
  1. Correspondence to Dr Yuexiang Wang; yxwang76{at}sibs.ac.cn; Dr Linhui Wang; wanglinhuicz{at}163.com

  • XZ, YL and YX are joint first authors.

Abstract

Background Cancer immunotherapies can induce durable tumor regression, but most patients do not respond. SETD2 mutation has been linked to the efficacy of immune checkpoint inhibitors (ICIs) immunotherapy. The functional importance of the SETD2 inactivation and how to modulate immunotherapy response remains unclear.

Methods To explore the function of SETD2 in immunotherapy, knockout and subsequent functional experiments were conducted. Bulk RNA-seq, ATAC-seq, Chip-seq and single-cell RNA-seq were performed to dissect the mechanism and explore the immune microenvironment of mouse tumor. Flow cytometry was used to assess cell surface antigen and intratumoral T cell levels.

Results We comprehensively determine the effect of SETD2 inactivation in ICIs therapy and elucidate the mechanistic impact on tumor immunity. Murine syngeneic tumors harboring Setd2 inactivation are sensitive to ICIs. By bulk and single-cell RNA-seq, we further reveal that SETD2 inactivation reprograms intratumoral immune cells and inflames the tumor microenvironment, which is characterized by high infiltration of T cells and enhanced antigen presentation to activate CD8+ T cell-mediated killing. Mechanistically, via an integrated multiomics analysis using ATAC-seq, ChIP-seq and RNA-seq, we demonstrate that SETD2 inactivation reduces NR2F1 transcription by impairing H3K36me3 deposition and chromatin accessibility, which activates the STAT1 signaling pathway to promote chemokines and programmed cell death protein-1 (PD-1) expression and enhance antigen presentation. All these regulatory mechanisms synergistically promote the effects of anti-programmed cell death ligand 1 immunotherapy in Setd2-knockout syngeneic mouse models. The SETD2-NR2F1-STAT1 regulatory axis is conserved in human and murine cancers. Finally, cancer patients harboring SETD2 mutations who received ICIs show increased durable clinical benefits and survival.

Conclusions These findings provide novel insights into the biology of SETD2 inactivation regulation and reveal a new potential therapeutic biomarker for ICIs immunotherapy in various refractory cancers.

  • Biomarkers, Tumor
  • Immune Checkpoint Inhibitors
  • Immunotherapy
  • Tumor Microenvironment

Data availability statement

Data are available in a public, open access repository. All sequencing datasets (scRNA-seq, RNA-seq, ATAC-seq, ChIP-seq and WES) reported in this study have been deposited in the National Omics Data Encyclopedia (https://www.biosino.org/node/) under accession no. OEP003840.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/jitc-2023-007678

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    WHAT IS ALREADY KNOWN ON THIS TOPIC

    • SETD2 has been identified as a tumor suppressor in multiple cancers. Bioinformatic analysis has shown that SETD2 is potentially correlated with the efficacy of immune checkpoint inhibitor (ICI) immunotherapy, but the experimental evidence and the precise molecular mechanism are unclear.

    WHAT THIS STUDY ADDS

    • This study identified SETD2 deficiency as a biomarker for ICIs immunotherapy through comprehensive experiments. We found regulatory axis involving SETD2, NR2F1 and STAT1 in cancer cells as molecular mechanism of tumor immune regulation mediated by SETD2 inactivation.

    HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

    • These findings provide a rationale for immunotherapeutic strategies for the treatment of patients with SETD2 deficiency.

    Background

    Immune checkpoint inhibitors (ICIs) mainly include antibodies that targeting programmed cell death protein-1 (PD-1), programmed cell death ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4).1 ICIs immunotherapies have revolutionized the treatment of patients with a wide range of advanced cancers, including malignant melanoma, renal cell carcinoma, non-small cell lung cancer, Hodgkin’s lymphoma, breast cancer, cervical cancer, gastric carcinoma, and head and neck squamous cell carcinoma.2 However, only a minority of cancer patients benefit from ICIs immunotherapy. Given the potential toxicities and the significant economic cost of these agents, it is of vital significance to identify patients who are more likely to benefit from ICIs.1 3

    SETD2 is the sole histone methyltransferase responsible for the trimethylation of histone H3 at lysine 36 (H3K36me3) and plays a critical role through H3K36me3.4 H3K36me3 is recognized and functions in modulating genomic instability by regulating DNA mismatch repairing, homologous recombination repairing initiated by DNA double-strand breaks and participates in intragenic transcription initiation, transcription elongation and RNA processing in alternative splicing.5–12 Genomic mutations of SETD2 are prevalent in ~5% of human cancers and promote tumor progression and metastasis, including clear cell renal cell carcinoma,13 14 pancreatic cancer,15 colorectal cancer,16 glioma,17 gastrointestinal stromal tumor,18 prostate cancer and breast cancer.19 20 However, no small molecules or antibodies that specifically target SETD2-deficient tumors and show therapeutic potential have been developed. In addition, SETD2 inactivation promotes cancer metastasis through distinct mechanisms in various cancer types.15 19 21 For instance, SETD2 inactivation promotes prostate cancer metastasis by reducing EZH2 methylation at K735 and inhibiting EZH2 degradation.19 In Kras-driven murine pancreatic cancer, SETD2 inactivation enhances metastasis by promoting acinar-to-ductal metaplasia and epithelia-mesenchymal transition.15 Loss of SETD2-mediated H3K36me3 induces a genome-wide increase in chromatin accessibility, which increases MMP1 transcription and accelerates clear cell renal cell carcinoma metastases.21 Therefore, it is challenging to develop patient-oriented therapies based on the pathway downstream of SETD2 in a manner that is effective across multiple cancers.

    Recently, bioinformatic analysis of TCGA datasets has shown that SETD2 mutation is potentially correlated with the efficacy of ICI immunotherapy22; however, the functional importance of the SETD2 inactivation and how to modulate immunotherapy response remains unclear. Although the tumor suppressor function of SETD2 has been well established, the role of tumor cell-intrinsic SETD2 on antitumor immunity has been largely unexplored. Here, using various murine and human cancer cell lines, syngeneic murine models, patient samples, publicly available multiomics and clinical datasets, we present a comprehensive evaluation of the functional implications of SETD2 inactivation, including its role in altering the immune microenvironment and shaping ICIs response. We further demonstrate that the SETD2-NR2F1-STAT1 axis modulates the sensitivity of various lethal cancers to ICIs.

    Methods

    Detailed methods have been described in online supplemental file 1.

    Supplemental material

    Results

    Setd2-knockout sensitizes tumors to ICIs immunotherapy in syngeneic murine models

    SETD2 encodes a histone H3K36 trimethyltransferase that is mutated with high prevalence in human clear cell renal cell carcinoma, pancreatic cancer, melanoma, non-small cell lung cancer, among others (online supplemental figure S1A). SETD2, with 85% amino acid similarity, is highly homologous between human and mouse. To test whether SETD2 inactivation sensitizes tumors to immune checkpoint blockade, we established a syngeneic mouse tumor cell line, namely, KC cells, which were derived from murine spontaneous pancreatic adenocarcinoma samples from Pdx1cre; LSL-KrasG12D C57BL/6 mice (online supplemental figure S1B).23 We selected the pancreatic adenocarcinoma because pancreatic cancer is still one of the most lethal diseases and novel therapeutic strategies are urgently needed in clinical practice.24 According to TCGA dataset, genomic mutations that inactivate SETD2 occur in ~1.4% of pancreatic cancers. Whole-exome sequencing (WES) and RNA-seq confirmed that Setd2 gene was not mutated and that it was normally expressed in KC cells (online supplemental table S1,S2). Setd2-knockout (KO) KC cells were established using CRISPR/Cas9 genome-editing technology (figure 1A). KC-sgSetd2 cells showed similar rates of proliferation in vitro (online supplemental figure S1C) and of tumor growth in vivo in BALB/C immunodeficient mice compared with the Setd2-expressing control cells (online supplemental figure S1D). As Cd274 was upregulated in KC-sgSetd2 cells (online supplemental table S2), anti-PD-L1 antibody was used as the ICI in our study. To determine the effect of Setd2 on the response to immunotherapy, equal numbers of KC-sgSetd2 and control cells were subcutaneously (s.c.) transplanted into C57BL/6 immunocompetent mice, followed by intraperitoneal (i.p.) injection with the anti-PD-L1 antibody. Anti-PD-L1 therapy administration markedly decreased the growth of KC-sgSetd2 tumors compared with the vehicle control but had no effect on KC-control tumors (figure 1B–E). These results demonstrate that Setd2 knockout sensitizes pancreatic adenocarcinoma to anti-PD-L1 immunotherapy.

    Supplemental material

    Supplemental material

    Figure 1
    Figure 1

    Setd2 knockout sensitizes murine tumors to anti-PD-L1 immunotherapy. (A) Western blotting confirmation of Setd2 knockout in KC cells. (B–E) Setd2 knockout enhances sensitivity to anti-PD-L1 therapy in KC cells. KC-ctrl or KC-sgSetd2 tumors treated with anti-PD-L1 or vehicle control are shown (n=5 for each group). A total of 4×106 KC-ctrl or KC-sgSetd2 cells were injected s.c. and anti-PD-L1 antibody or PBS was injected i.p. into C57BL/6 mice at the indicated time points. (B) Schematic of anti-PD-L1 administration; (C, E) growth curves (multiple t-test, n.s. p>0.05, *p<0.05), tumor weight at the end point (unpaired Student’s t-test, n.s. p>0.05, **p<0.01) and (D) representative tumor images are shown. Error bars represent the mean±SEM. (F) Western blotting confirmation of SETD2 expression in B16F10 cells. (G–J) Setd2 knockout improves sensitivity to anti-PD-L1 therapy in B16F10 cells. B16F10-ctrl or B16F10-sgSetd2 tumors treated with anti-PD-L1 or vehicle control are shown (n=5 for each group). A total of 1×106 cells were injected s.c. and anti-PD-L1 antibody or PBS was injected i.p. into C57BL/6 mice at the indicated time points. (G) Schematic of anti-PD-L1 administration; (H, J) growth curves (multiple t-test, n.s. p>0.05, *p<0.05, ****p<0.0001), tumor weight at the end point (unpaired Student’s t-test, n.s. p>0.05, ***p<0.001) and (I) representative tumor images are shown. Error bars represent the mean±SEM.

    To provide additional evidence supporting this, we extended these findings to a second mouse tumor cell line (B16F10, murine melanoma) that expressed Setd2 (figure 1F), and B16F10 cells are completely resistant to immune checkpoint blockade with antibodies targeting the PD-1 and/or CTLA-4 receptors.25 Similar to KC cells, ablation of Setd2 in B16F10 cells did not appreciably decelerate cell proliferation in vitro (online supplemental figure S1E) or in immunodeficient mice (online supplemental figure S1F) but had a profound effect on the efficacy of anti-PD-L1 treatment in immunocompetent mice (figure 1G, H and I). In contrast, anti-PD-L1 treatment was ineffective against control B16F10 tumors (figure 1I, J). Hence, tumor-intrinsic Setd2 inactivation sensitizes both KC pancreatic adenocarcinoma and B16F10 melanoma to immune checkpoint blockade.

    SETD2 inactivation activates the IFNγ pathway by upregulating Stat1

    To determine the global gene expression patterns regulated by Setd2 loss, we performed RNA-seq on paired KC-KO/KC-WT C57BL/6 allograft tumors in vivo and KC-sgSetd2/KC-control cells in vitro (online supplemental table S2). GSEA revealed that gene sets that regulate the immune response, cytokine production, interferon-γ (IFNγ) response, antigen processing and presentation were significantly upregulated in both KC-KO tumors and KC-sgSetd2 cells (online supplemental figure S2A, figure 2A). The IFNγ pathway plays a critical role in the tumor response to immunotherapy.26 Notably, STAT1, a key transcription factor in the IFNγ pathway,27 was significantly upregulated and activated in KC-KO tumors and KC-sgSetd2 cells (figure 2B, C, online supplemental figure S2B,C). SETD2-STAT1 regulation was further demonstrated by western blotting and RT-qPCR in B16F10-sgSetd2 murine melanoma cells (figure 2D, E). Furthermore, SETD2 inactivation upregulated the IFNγ-induced chemokine genes Cxcl9, Cxcl10, and Cxcl11 in vitro (figure 2F, G, online supplemental figure S2D) and in vivo (figure 2H, online supplemental figure S2E,F). The STAT1-IFNγ regulating axis has been reported to promote antitumor immunity by recruiting CXCR3+ CD8+ T cells and CXCR3+ NK cells.28 Taken together, these results demonstrate that Setd2 inactivation increases STAT1 expression and activation, and then activates the IFNγ pathway, which promotes immune-related genes expression and immune-related pathway activation.

    Figure 2
    Figure 2

    Setd2 inactivation induces STAT1 expression and activation. (A) Leading-edge plots from the GSEA highlighting the enriched pathways associated with STAT1 activation in KC-sgSetd2 cells. (B–E) Loss of Setd2 promotes STAT1 expression and activation in (B) KC cells and (D) B16F10 cells. Setd2 inactivation increases the mRNA levels of Stat1 in (C) KC cells and (E) B16F10 cells. *p<0.05, ****p<0.0001 by unpaired Student’s t-test. Error bars represent the mean±SEM of three replicates. (F, G) Setd2 inactivation increases the expression of STAT1-activated chemokine genes in (F) KC cells and (G) B16F10 cells. Control or Setd2-KO cells were treated with or without 50 ng/mL IFNγ for 20 hours and total RNA was extracted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by unpaired Student’s t-test. Error bars represent the mean±SEM of three replicates. (H)Setd2 inactivation in allograft tumors increases Cxcl9, Cxcl10 and Stat1 expression. Total mRNA was extracted from KC-ctrl or KC-sgSetd2 tumors from C56BL/6 mice (n=5 for each group). *p<0.05 by unpaired Student’s t-test. Error bars represent the mean±SEM. GSEA, gene set enrichment analysis.

    Loss of SETD2 promotes PD-L1 expression and antigen presentation

    It is well known that the IFNγ-JAK-STAT1 axis is the prominent pathway that contributes to PD-L1 expression and MHC-I antigen processing and presentation.29 30 In addition to chemokine genes, we found that PD-L1 expression and antigen presentation were enhanced on IFNγ-STAT1 pathway activation in our Setd2-deficient models. The upregulation of Pdl1 was confirmed at the transcriptional level by RT-qPCR in Setd2-knockout cells and allograft tumors (figure 3A, B, online supplemental figure S3A,B). PD-L1 protein expression on the cell membrane was increased, as shown by flow cytometry analysis (figure 3C, D, online supplemental figure S3C). It has been reported that upregulation of PD-L1 results in potent immune suppression and tumor immune escape, which explains the potential cause of the immunosuppression in Setd2-deficient tumors.31

    Figure 3
    Figure 3

    Setd2 inactivation promotes IFNγ-STAT1-induced PD-L1 expression and MHC-I antigen presentation. (A)Setd2 knockout promotes Pdl1 expression in KC cells and B16F10 cells. Setd2- knockout or control cells were treated with or without 50 ng/mL IFNγ for 20 hours and total RNA was extracted. **p<0.01 by unpaired Student’s t-test. Error bars represent the mean±SEM of three replicates. (B) Setd2 inactivation increases Pdl1 and H2-K1 expression in allograft tumors. Total mRNA was extracted from KC-ctrl or KC-sgSetd2 tumors from C57BL/6 mice (n=5 for each group). *p<0.05 by unpaired Student’s t-test. Error bars represent the mean±SEM. (C, D)Setd2 knockout promotes PD-L1 expression on the cell surface of (C) KC cells and (D) B16F10 cells. Setd2-knockout or control cells were treated with or without 50 ng/mL IFNγ for 12 hours and PD-L1 expression was detected by flow cytometry. Iso, isotype. MFI, mean fluorescence intensity. **p<0.01, ****p<0.0001 by unpaired Student’s t-test. Error bars represent the mean±SEM of three replicates. (E) Setd2 knockout promotes H2-K1 mRNA expression in KC cells. Cells were treated with or without 50 ng/mL IFNγ for 20 hours and total RNA was extracted. ****p<0.0001 by unpaired Student’s t-test. Error bars represent the mean±SEM of three replicates. (F) Setd2 knockout promotes H-2Kb/Db expression on the cell surface of KC cells. Cells were treated with or without 50 ng/mL IFNγ for 12 hours and H-2Kb/Db expression was assessed by flow cytometry. ***p<0.001, ****p<0.0001 by unpaired Student’s t-test. Error bars represent the mean±SEM of three replicates. (G)Setd2 knockout promotes SIINFEKL-H-2Kb expression on the cell surface in KC cells. Cells were transduced with OVA lentivirus and treated with or without 50 ng/mL IFNγ for 12 hours and SIINFEKL-H-2Kb expression was assessed by flow cytometry. ***p<0.001 by unpaired Student’s t-test. Error bars represent the mean±SEM of three replicates. (H) Setd2 deficiency sensitizes KC cells to effector CD8+ T cell-mediated killing. Apoptosis was analyzed in CD45− tumor cells at the indicated time points. Error bars represent±SEM of three replicates. ****p<0.0001 by unpaired Student’s t-test. T, tumor cell. E, effector CD8+ T cell. VNA, viable non-apoptotic cell. VA, viable apoptotic cell. NVA, non-viable apoptotic cell. NVNA, non-viable non-apoptotic cell.

    The upregulation of the MHC-I complex core gene H2-K1 in Setd2-knockout cells was also shown at the transcriptional level by RT-qPCR (figure 3E, online supplemental figure S3D), and the H-2Kb/Db protein expression level on the cell surface was consistently increased (figure 3F, online supplemental figure S3E). The upregulation of H2-K1 was further demonstrated in vivo (figure 3B, online supplemental figure S3F). Taking the GSEA results together, we show that Setd2 loss promotes MHC-I antigen-processing and presentation.

    Furthermore, we constitutively expressed neoantigen ovalbumin (OVA) in Setd2-KO KC cells, confirmed the upregulation of SIINFEKL-H-2Kb levels on the surface of sgSetd2-OVA cells (figure 3G, online supplemental figure S3G), and then cocultured with OT-1 effector CD8+ T cells or injected subcutaneously into OT-1 mice. Both in vitro and in vivo assays showed that Setd2 deficiency sensitized the KC cells to effector CD8+ OT-1 cell-mediated killing (figure 3H, online supplemental figure S3H,I). Setd2 knockout also sensitized B16F10 cells to effector CD8+ T cell cytotoxicity (online supplemental figureS3J). Activation of the PD-L1/PD1 axis inhibits T cell proliferation, activation and survival mainly by attenuating the T cell receptor (TCR) and CD28 signaling pathways.31 We found that IFNγ-stimulated PD-L1 expression was unable to inhibit the cytotoxicity of OT-1 effector CD8+ T cells activated by specific peptides in vitro, and this results might occur because the activation signaling due to the interaction of highly expressed SIINFEKL-MHC-I and high-affinity OT-1-TCR was stronger than the suppression signaling of the PD-L1/PD1 interaction.32

    In addition, Setd2 deficiency elevated the tumor mutation burden (TMB), as determined by WES (online supplemental figure S3K), (online supplemental table S1), which was validated in multiple human cancers (online supplemental figure S3L).The result is consistent with a previous report that SETD2 functions in DNA double-strand break repair and the accumulation of genomic mutations in Setd2 deficient tumors.9 Taken together, our data indicate that Setd2 deficiency activates the IFNγ pathway by upregulating Stat1 and further increases Pdl1 expression, IFNγ-induced chemokine gene expression and MHC-I antigen presentation, which synergistically improving the effects of anti-PD-L1 immunotherapy.

    SETD2 inactivation promotes Stat1 expression by downregulating Nr2f1

    As the sole enzyme responsible for the trimethylation of lysine 36 on histone 3 (H3K36me3), a histone mark related to actively transcribed regions, most functions of SETD2 are mediated by H3K36me3.33 Chromatin immunoprecipitation sequencing (ChIP-seq) was performed to map the distribution of H3K36me3 in KC-KO and KC-WT cells. Consistent with the previous reports, H3K36me3 was widely distributed in whole genomic regions, including gene bodies, promoters, and intergenic zones (online supplemental figure S4A, online supplemental table S3).15 SETD2 loss induced alterations in chromatin accessibility and transcriptional output via H3K36me3 loss.21 Assay for transposase-accessible chromatin using sequencing (ATAT-seq) was also carried out in KC-ctrl and KC-sgSetd2 cells to assess the impact of chromatin accessibility due to Setd2 loss on Stat1 expression. Loss of SETD2 in KC cells led to genome-wide alterations in chromatin accessibility, including introns, promoters and intergenic regions (online supplemental figure S4B, online supplemental table S4).

    Supplemental material

    Supplemental material

    ChIP-seq and ATAC-seq analyses did not show significant differences in H3K36me3 deposition and chromatin accessibility for Stat1, suggesting that Stat1 expression was indirectly regulated by Setd2 loss. The reduction in Stat1 at the transcriptional level led us to determine that Setd2 deficiency may regulate the expression of potential transcription factors that increase the transcription of Stat1. Nr2f1 was the most significantly downregulated transcription factor among the differentially expressed genes and showed the consistent decreasing trends in H3K36me3 enrichment of ChIP-seq and chromatin accessibility of ATAC-seq in Setd2-inactivated cells (figure 4A, B, online supplemental figure S4C,D), (online supplemental table S5). NR2F1 is an orphan nuclear receptor belonging to the steroid/thyroid hormone receptor superfamily and functions as a transcriptional regulator that can both activate and repress target gene expression.34 We identified the NR2F1 binding motif ‘AGGTCA’in the Stat1 promoter region through a bioinformatics approach (online supplemental figure S4E).34 To experimentally investigate whether Stat1 transcription was regulated by NR2F1, Nr2f1 lentivirus was transduced into Setd2-knockout and control cells (figure 4C, online supplemental figure S4F) and the reduction in Stat1 expression was confirmed at both the mRNA level by RT-qPCR and the protein level by western blotting (figure 4D, online supplemental figure S4G), followed by the downregulation of a series of IFNγ-induced genes (figure 4E, online supplemental figure S4H). Furthermore, to assess whether NR2F1 directly binds to the Stat1 promoter, we constructed a Flag-Nr2f1 lentiviral vector and determined its ability to inhibit STAT1 expression and activation (online supplemental figure S4I). Direct binding was confirmed by ChIP‒qPCR assay (figure 4F). In addition, the GFP reporter assay showed that NR2F1 significantly inhibited GFP expression driven by the Stat1 promoter (figure 4G, H). These results indicated that Setd2 loss impaired the transcription of Nr2f1 by decreasing H3K36me3 deposition on the Nr2f1 gene body and reducing chromatin accessibility proximal to the Nr2f1 promoter region, which further increased the expression and activation of STAT1 in a negative feedback.

    Supplemental material

    Figure 4
    Figure 4

    Setd2 inactivation induces STAT1 expression and activation by downregulating Nr2f1 expression. (A) Setd2 loss suppresses Nr2f1 expression in KC cells. ***p<0.001 by unpaired Student’s t-test. Error bars represent±SEM of three replicates. (B) ChIP-seq, ATAC-seq and RNA-seq tracks of the Nr2f1 gene locus in the indicated cells. (C, D) NR2F1 overexpression inhibits STAT1 expression and activation. KC-ctrl or KC-sgSetd2 cells were transduced with Nr2f1 or control lentivirus. EV, empty vector. (C) Nr2f1 mRNA levels and (D) STAT1 expression and activation were shown. **p<0.01, ***p<0.001, ****p<0.0001, n.s. p>0.05 by unpaired Student’s t-test. Error bars represent the mean±SEM of three replicates. (E) NR2F1 overexpression impairs the expression of Stat1 downstream genes in KC-sgSetd2 cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by unpaired Student’s t-test. Error bars represent±SEM of three replicates. (F) NR2F1 directly binds to the Stat1 promoter region in KC cells, as determined by ChIP‒qPCR assay. KC-sgSetd2 cells were transduced with Flag-Nr2f1 and control lentivirus. ChIP assays were performed with anti-Flag and isotype IgG. **p<0.01, n.s. p>0.05 by unpaired Student’s t-test. Error bars represent±SEM of three replicates. (G, H) NR2F1 suppresses GFP expression driven by the Stat1 promoter in HEK293T cells. MFI of GFP and mCherry was assessed by flow cytometry. SP, Stat1 promoter. ****p<0.0001, n.s. p>0.05 by unpaired Student’s t-test. Error bars represent±SEM of three replicates.

    SETD2 deficiency reprograms the tumor immune microenvironment

    To systemically elucidate the regulation of SETD2-NR2F1-STAT1 axis to the tumor immune microenvironment, we established Setd2-knockout (KC-KO) and Setd2-wildtype (KC-WT) KC monoclonal cell lines (online supplemental figure S5A) and performed RNA-seq assays in C57BL/6 allograft tumors (online supplemental table S2). Immune cell infiltration was significantly higher in KC-KO tumors than that in KC-WT tumors by single sample gene set enrichment analysis (ssGSEA) and ESTIMATE analysis (figure 5A, online supplemental figure S5B).35 Many immune-related genes, including the immune inhibitory genes Cd274, Ctla4, Lag3 and Ido1, were upregulated in KO tumors. In addition, the expression of some immune stimulatory genes and chemokine genes, such as Ccl5 and Cxcl10, was also upregulated in KO tumors (figure 5B).36 37

    Figure 5
    Figure 5

    Bulk RNA-seq and single cell RNA-seq analysis for intratumoral immune infiltration. (A) Heatmap showing the profiles of intratumoral immune cells in KC-WT or KC-KO allograft tumors through bulk RNA-seq, n=3. The relative infiltration of each cell type is normalized into a z-score. (B) Heatmap showing the relative expression of immune-related genes in KC-WT or KC-KO allograft tumors, n=3. (C, D) Setd2 loss alters the intratumoral infiltration of myeloid cells and lymphocytes. (C) Clusters of intratumoral immune cell populations and (D) statistics of individual cell populations identified from single-cell transcriptomic data. Individual clusters are indicated by colors. (E, F)Setd2 loss reprograms macrophage compartments. (E) t-SNE plots showing secondary clusters of macrophages and (F) statistics of M1- and M2-like macrophages. (G, H) Setd2 loss reprograms T lymphocyte subgroups. (G) t-SNE plots showing secondary clusters of T lymphocytes and (H) population statistics of individual cell types. (I, J) Cytotoxic CD8+ T cells are significantly increased through anti-PD-L1 treatment in KC-sgSetd2 tumors. (I) Trajectory analysis showing CD8+ T cell status in different groups and (J) statistics of the indicated cell states. (K, L) Intratumoral T cells infiltrations was increased in (K) KC-sgSetd2 and (L) B16F10-sgSetd2 tumors treated with anti-PD-L1 by flow cytometry analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by unpaired Student’s t-test. Error bars represent the mean±SEM.

    Single-cell RNA-seq (scRNA-seq) analysis of sorted CD45+ immune cells of Setd2-deficient and control KC tumors that were isolated from PBS-treated C57BL/6 immunocompetent mice showed that the proportions of infiltrating T cells, neutrophils, natural killer (NK) cells, and dendritic cells were significantly increased, while the proportion of infiltrating macrophages was significantly reduced in Setd2-deficient tumors compared with control KC tumors (figure 5C, D, online supplemental figure S5C,D). Given the dual function of tumor-associated macrophages (TAMs),38 we reclustered macrophages into M1-like and M2-like macrophages and found a decrease in both types of macrophage proportions, while a dramatic decrease in M2-like macrophages in Setd2-deficient tumors was shown (figure 5E, F, online supplemental figure S5E,F). In view of the crucial role of T cells in antitumor immunity,39 we annotated T cell subsets according to their function and marker genes (figure 5G, online supplemental figure S5G,J). Infiltration of naïve and effector T cell subsets, including naïve CD4+ T cells, regulatory CD4+ T cells (Tregs), CD4+ helper T cells, naïve CD8+ T cells and effector CD8+ T cells, was significantly enhanced in Setd2-knockout allografts, mainly manifesting as an increase in effector CD8+ T cells and Tregs (figure 5H). These results indicate that Setd2 loss inflames the tumor microenvironment (TME) by reducing M2 macrophage infiltration and increasing effector T cell infiltration.

    The relative ratio of effector CD8+ T cells and Tregs indicated that effector CD8+ T cells exerted a major antitumor effect with anti-PD-L1 treatment (online supplemental figure S5K). To further explore the effect of CD8+ T cells in the context of anti-PD-L1 administration, we assessed the status of CD8+ T cells. Trajectory analysis demonstrated that there was a significant increase in the proportion of cytotoxic CD8+ T cells in KC-sgSetd2 tumors treated with anti-PD-L1 immunotherapy (figure 5I, J, online supplemental figure S5L), and the anti-PD-L1 immunotherapy regulated the differentiation of CD8+ T cells, turning them from the exhausted to the cytotoxic state in both Setd2-expressing and Setd2-deficient tumors (online supplemental figure S5M). Flow cytometry analysis further validated that anti-PD-L1 treatment significantly increased the infiltration of CD3+ T cells, CD4+ T cells and CD8+ T cells in Setd2-knockout subcutaneous allografts (figure 5K, L, online supplemental figure S5N). Compared with KC-ctrl group, Setd2 knockout significantly increased the infiltration of CD45+ immune cells and anti-PD-L1 therapy further increased the infiltration of CD3+ T cells in orthotopic allografts (online supplemental figure S5O). The findings were validated in human TCGA clear cell renal cell carcinoma (ccRCC) cohort. The immune cell infiltration was significantly higher in SETD2-deleted tumors than that in high-expressed tumors (online supplemental figure S5P,Q), which was consistent with the findings in mouse pancreatic orthotopic tumors (online supplemental figure S5O). These results demonstrated that the inflamed TME due to Setd2 loss contributed to anti-PD-L1 treatment and the main role played by cytotoxic CD8+ T cells in immunotherapy administration. All the results show that Setd2 deficiency has a complicated interaction with the TME. On the one hand, Setd2 deficiency promotes chemokines production to inflame immune microenvironment with increased infiltration of T cells and enhances antigen presentation to activate CD8+ T cell-mediated killing. On the other hand, activation of STAT1 promotes the expression of immune inhibitory checkpoint molecules such as PD-L1, which rivalries with antitumor immunity. All the above mechanisms facilitate the benefit of ICIs immunotherapy.

    SETD2-NR2F1-STAT1 axis in human cancers

    To confirm the findings in murine tumors that SETD2 inactivation inhibits STAT1 expression and activation in a NR2F1-dependent manner in human cancers, we knocked out SETD2 with individual SETD2 sgRNA in the human pancreatic ductal adenocarcinoma cell line YAPC, which normally expresses SETD2 and is widely used in pancreatic cancer research (figure 6A).40 The upregulation of STAT1 was confirmed at both the mRNA and the protein levels, and the upregulation of CXCL10 and PDL1 mediated by STAT1 activation was also validated in YAPC-sgSETD2 cells (figure 6A, B). Next, we performed GSEA analysis to obtain a global view of transcriptome differences in SETD2-knockout and control YAPC cells. Several pathways were enriched in SETD2-knockout YAPC cells, similar to the gene signatures in KC-sgSetd2 cells (figure 6C), and the expression of immune-related genes also increased as that in KC-KO cells (figure 6D).

    Figure 6
    Figure 6

    SETD2-NR2F1-STAT1 axis in human ccRCC and pancreatic cancer. (A) SETD2 knockout promotes STAT1 expression and activation in YAPC cells. (B) SETD2 knockout increases the mRNA levels of STAT1, PDL1 and CXCL10 in YAPC cells. ***p<0.001, ****p<0.0001 by unpaired Student’s t-test. Error bars represent the mean±SEM of three replicates. (C) Leading-edge plots from the GSEA highlight the enriched pathways associated with STAT1 activation in YAPC-sgSETD2 cells. (D) Column charts showing the mRNA levels of the indicated genes in YAPC-ctrl or YAPC-sgSETD2 cells according to RNA-seq. (E) Spearman correlation of expression levels between SETD2 and STAT1 in human pancreatic cancer41 cohort, tumors with unknown-significance of SETD2 mutations were excluded, n=89). (F) Spearman correlation of SETD2 and NR2F1 mRNA levels in human pancreatic cancer (ICGC PACA-CA cohort, n=234). (G) Correlations between NR2F1 and STAT1, SETD2 and STAT1 expression in human pancreatic cancer41 cohort, tumors with unknown-significance of SETD2 mutations were excluded, n=89). SETD2-high represents the tumors of the top one-third and SETD2-low represents the tumors of the bottom one-third based on the SETD2 expression level. *p<0.05 by unpaired Student’s t-test. Error bars represent±SEM. (H, I) Correlation between SETD2 and NR2F1 expression levels in the Renji pancreatic cancer TMA cohort assessed by (H) immunohistochemistry staining and (I) representative pictures of expression levels (n=75). IHC, immunohistochemistry. High represents expression higher than or equal to tumor adjacent normal tissues. Low represents expression lower than tumor adjacent normal tissues. ***p<0.001 by Fisher’s exact test. (J–L) SETD2-NR2F1-STAT1 axis in human ccRCC (TCGA renal cell carcinoma cohort). Spearman correlations of (J) NR2F1 and STAT1 and (K) SETD2 and NR2F1 mRNA levels in human ccRCC cohort, n=569. (L) Correlation between SETD2 and NR2F1 expression. SETD2-high represents the top 50 tumors, and SETD2-low represents the bottom 50 tumors based on SETD2 mRNA levels in the SETD2 WT cohort. SETD2-loss represents 29 tumors with SETD2 inactivation mutations. ****p<0.0001, n.s. p>0.05 by unpaired Student’s t-test. Error bars represent±SEM.

    Analysis of public datasets on the Nature, 2016 and PACA-CA (ICGC) pancreatic cancer cohorts revealed the inverse correlations between SETD2 and STAT1 expression, NR2F1 and STAT1 expression, and SETD2 and PDL1 expression (figure 6E, F, online supplemental figure S6A).41 We further refined the cohort based on the expression levels of SETD2 and NR2F1. Unpaired comparison revealed that the expression of STAT1 and PDL1 was lower in tumors with high SETD2 expression (figure 6G, online supplemental figure S6B) and that STAT1 expression was significantly increased in NR2F1-low tumors (figure 6G), which was consistent with the findings in murine tumors. Next, GSEA of differentially expressed genes in this human pancreatic cancer cohort based on the SETD2 expression level also demonstrated the same enriched gene signatures identified in SETD2-knockout YAPC cells (online supplemental figure S6C). Finally, we performed immunohistochemistry (IHC) staining with validated antibodies against SETD2 and NR2F1 on a human tissue microarray consisting of 75 patients with pancreatic ductal adenocarcinoma and demonstrated a significantly positive correlation of SETD2 and NR2F1 expression (figure 6H, I).

    According to TCGA datasets, SETD2 is mutated with high prevalence (16%) in ccRCC and the majority of these mutations are inactivating mutations and lead to loss of intact protein expression.42 Genomic profiling of ccRCC tumors in TCGA and RECA-EU (ICGC) cohorts revealed negative correlations between the expression of NR2F1 and STAT1, SETD2 and the immune inhibited genes CTLA-4, IDO1, HAVCR2, and LAG3 (figure 6J, online supplemental figure S6D), and a positive correlation between the expression of SETD2 and NR2F1 (figure 6K, online supplemental figure S6E). The mRNA level of NR2F1 was significantly higher in SETD2-high tumors than that in SETD2-low and SETD2-loss tumors (figure 6L). GSEA of differentially expressed genes (SETD2-loss/SETD2-WT) also showed significantly enriched pathways that were functionally similar to human pancreatic cancer (online supplemental figure S6F). Overall, these results demonstrate that the mechanism of SETD2-NR2F1-STAT1 regulation is highly conserved in human cancer cell lines and patient samples, highlighting a role for SETD2 deficiency in the efficacy of ICIs immunotherapy in human cancers.

    Cancer patients harboring SETD2 mutations benefit from ICIs immunotherapy

    The genomic and clinical outcome data of 1662 advanced cancer patients who were treated with ICIs (anti-PD-1/PD-L1/CTLA-4) in MSK-IMPACT were retrieved and analyzed.43 44 Among the 476 cancer genes, 127 genes showed correlations with beneficial effects of immunotherapy (p<0.05). The top genes included known predictive biomarkers for ICIs therapy, including TP53, PAK7, TET1, PTPRD/PTPRT and STK11.45–49 SETD2 ranked among the top three hits of the 476 genes. Patients with inactivating mutations in SETD2 had more favorable outcomes from immunotherapy (p<0.001, rank=3, figure 7A, B), which is consistent with previous report.22 This result was externally validated with additional melanoma (figure 7C, D) and non-small cell lung cancer cohorts (online supplemental figure S7A,B).50 51

    Figure 7
    Figure 7

    Patients with cancer SETD2 mutations benefit from immune checkpoint inhibitors (ICIs) immunotherapy. (A) Correlation between gene mutations and immunotherapy effects (MSK-IMPACT with anti-PD-1/PD-L1/CTLA-4, pan-cancer, including hepatobiliary cancer, colorectal cancer, thyroid cancer, renal non-clear cell carcinoma, renal clear cell carcinoma, glioma, glioblastoma, non-small cell lung cancer, bladder cancer, leukemia, prostate cancer, endometrial cancer, esophagogastric cancer, melanoma, cholangiocarcinoma, breast cancer, head and neck cancer, cervical cancer, ovarian epithelial tumor, pleural mesothelioma, ocular melanoma, sarcoma, adrenocortical carcinoma). P values of overall survival from Kaplan-Meier plotter were assessed by a two-sided log-rank test. Genes are ordered from the largest to the smallest significant difference along the x-axis, with the logarithm of the p value on the y-axis. (B) Kaplan-Meier analysis of the SETD2 MUT and SETD2 WT groups in the ICIs cohorts (MSK-IMPACT with anti-PD-1/PD-L1/CTLA-4, pan-cancer). MUT represents SETD2 loss-of-function mutations, including nonsense, frameshift, splice mutations and missense mutations predicted to be damaging by PPH2 and SIFT. (C, D)SETD2 mutations and ICIs immunotherapy response in melanoma cohorts (Science. 2015, cohort with anti-CTLA-4 therapy). (C) Kaplan-Meier analysis of the SETD2 MUT and SETD2 WT groups and (D) correlation of immunotherapy response between SETD2 MUT and SETD2 WT groups. MUT (n=10). WT (n=100). NR, no response; R, response; WT, wild type.

    Discussion

    Immune checkpoint blockade induces durable tumor regression in only a minority of patients with advanced cancers. Understanding the mechanisms that determine tumor sensitivity to these drugs could potentially expand the number of patients who benefit to ICIs. Our comprehensive study shows that SETD2 inactivation is sufficient to induce antitumor immunity and impart sensitivity to ICI therapy with rigorous experimental evidence in both human and mouse. Mechanistically, via an integrated multiomics analysis using ATAC-seq, ChIP-seq and RNA-seq, we show that tumor cell-intrinsic SETD2 inactivation inhibits NR2F1 expression through reducing H3K36me3 deposition in the NR2F1 gene body and chromatin accessibility in the proximal NR2F1 promoter region, which promotes STAT1 expression and activation, consequently upregulating the expression of PD-L1 and chemokines, and enhancing antigen presentation (online supplemental figure S7C). Hence, our results also provide a mechanistic basis for SETD2 inactivation-mediated sensitivity to ICIs therapy.

    In contrast to the study by Chen et al showing that SETD2 directly mediates STAT1 methylation via its methyltransferase activity, which enhances IFN-activated STAT1 phosphorylation,52 our study demonstrates that NR2F1 functions as a transcription factor to repress STAT1 transcription and that SETD2 inactivation promotes STAT1 expression and phosphorylation by inhibiting NR2F1 expression. Meanwhile, unlike in lung adenocarcinoma, where SETD2 deficiency participates in STAT1-mediated IL8 upregulation through epigenetic modification,53 causing tumor immune invasion, we did not detect IL8 expression through RNA sequencing. Nevertheless, these results suggest that SETD2 may exert different molecular mechanisms under specific conditions.

    Previous research has demonstrated the function of SETD2-mediated H3K36me3 modification in DNA mismatch repair,9 indicating its role in the accumulation of mutational burden across various tumor types, which has been confirmed in our study (online supplemental file 1). Furthermore, besides the established mechanism that sensitizes to ICI, we have discovered a regulatory axis involving SETD2, NR2F1 and STAT1 in PDAC cells. In our experimental models, SETD2 knockout cell lines were subjected to further analysis without undergoing passage, thereby preventing substantial accumulation of TMB within the short duration. Notably, changes in the immune pathway were detected shortly after SETD2 knock out in vitro. Hence, the NR2F1-STAT1 molecular mechanism holds greater significance in the immune regulatory process of SETD2 compared with the alteration of mutation burden.

    Recently, the development and application of single-cell RNA sequencing has facilitated delineation of the landscape of immune cells within the TME at the single-cell level.54 Tumor-antagonizing immune cells mainly include effector T cells, NK cells, dendritic cells, M1 macrophages and N1 neutrophils, while tumor-promoting immune cells largely include Tregs, myeloid-derived suppressor cells (MDSCs), M2 macrophages and N2 neutrophils.55 Several studies have confirmed that the response to ICIs therapy is related to tumor-infiltrating lymphocytes and other naïve immune cells in the TME.56 Our study demonstrates how SETD2 inactivation reprograms the TME to an immune inflamed state. Through single-cell transcriptome sequencing of intratumoral immune cells, we show that Setd2 deficiency increases the proportion of infiltrating T lymphocytes, including Tregs, CD4+ helper T cells and effector CD8+ T cells, and reduces the infiltration of M2-like macrophages. This immune microenvironment was in balance with the high expression of PD-L1 in tumor cells and more susceptible to ICIs immunotherapy. With anti-PD-L1 administration, the proportions of Tregs and M2-like macrophages were slightly decreased, while cytotoxic CD8+ T cells, the central effectors of antitumor immunity,39 were robustly enhanced in Setd2-deficient allografts. Taken together, all these cellular mechanisms synergistically enhance the benefit of anti-PD-L1 immunotherapy. A recent study showed that Setd2-deficient pancreatic tumor cells enhance neutrophil recruitment in mice,57 which was also observed in our single-cell RNA sequencing, but due to the limited number of neutrophils, further sequencing and analysis is required to determine whether the neutrophils are polarized toward an immunosuppressive phenotype.

    SETD2 functions as a tumor suppressor, and its mutations account for 5% of all cancers in TCGA cohort. Most of these mutations are inactivating mutations and lead to loss of intact protein expression, which makes SETD2 a poor therapeutic target in cancers. Our comprehensive study reveals the link between SETD2 inactivation and sensitivity to ICIs therapy, providing a rationale for immunotherapeutic strategies for the treatment of patients with SETD2 deficiency, despite the naturally aggressive tumor biological behavior and unfavorable prognosis. This study has clinical implications. The results presented here identify a novel biomarker for improving immunotherapy. Despite the extraordinary success, immunotherapy with ICIs has demonstrated objective responses only in a minority of advanced patients, emphasizing the critical need to identify biomarkers to select the appropriate patients for treatment, to identify the cause of resistance, and to develop strategies for treating non-responders. The current predictive biomarkers of response to ICIs include TMB, PD-L1 expression, neoantigen load, T cell infiltration and others.58–64 But the durable benefit of ICIs is limited to a minority of patients with these predictors. Recent study showed that TMB cannot be applied generically across melanoma subtypes as a predictive biomarker for ICIs therapy.65 In addition, the mutational number defining TMB-high appears to vary across cancer types.43 Although the greatest survival benefit was observed in patients with PD-L1-strong positive lung cancer, ICIs therapy also significantly improved the survival of PD-L1-negative patients with lung cancer.66 These findings indicated that more accurate biomarkers are needed.67 68 The clinical response to ICIs therapy remains incompletely characterized.69 70 The mechanistic basis for the variation in response patterns or long-term clinical benefits remains poorly explained. Indeed, ICIs-resistant tumors became responsive to immunotherapy when SETD2 was inactivated in both pancreatic cancer and melanoma syngeneic mouse models in our study. Through eligible clinical data, we validated our findings in melanoma and pan-cancer cohorts, it is regret that the clinical data of immunotherapy in pancreatic cancers is limited. Most of the clinical data lack detailed gene expression or genome information. Our findings warrant confirmation in subsequent randomized trials, especially in pancreatic cancer.

    Conclusion

    Collectively, our data derived from cell lines, syngeneic mouse models, and patient specimens revealed the prominent significance of SETD2 deficiency in augmenting tumor immunogenicity and activating tumor immune microenvironment through the NR2F1-STAT1 signaling pathway. This work not only provides insights into SETD2 tumor suppressor biology, but also provides evidence that SETD2-mutant cancers could benefit from ICIs immunotherapy, highlighting the predictive value of SETD2 mutation status in guiding immunotherapy.

    Supplemental material

    Data availability statement

    Data are available in a public, open access repository. All sequencing datasets (scRNA-seq, RNA-seq, ATAC-seq, ChIP-seq and WES) reported in this study have been deposited in the National Omics Data Encyclopedia (https://www.biosino.org/node/) under accession no. OEP003840.

    Ethics statements

    Patient consent for publication

    Ethics approval

    This study involves human participants and the tissue microarray (pancreatic ductal adenocarcinoma and matched normal samples) was from Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital (Shanghai, China) and with institutional review board approval (KY2020-116). Participants gave informed consent to participate in the study before taking part.

    Acknowledgments

    We thank Dr Jonathan Fletcher at Brigham and Women’s Hospital/Harvard Medical School for critical and constructive comments.

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    Supplementary materials

    Footnotes

    • XZ, YL and YX contributed equally.

    • Contributors YW and LW are the guarantors of the study. XZ, LW and YW conceived and designed the research. XZ, YL, YX, XLiu, XW, CZ, XLu, SW, HL and YW performed experiments, bioinformatics investigation and analyzed the data. XZ, YL, XG and LW reviewed the histopathologic diagnoses. WB, XJ, ZW and KW provided scientific advice and helpful comments into the project. XZ, YL, YX and YW prepared figures and tables. XZ, YL, YX and YW wrote the manuscript. All authors read and approved the final manuscript.

    • Funding This work was supported by grants from the Basic Research Project of Shanghai Science and Technology Commission (20JC1419200); the Innovation Program of Shanghai Science and technology committee (20Z11900300); the National Natural Science Foundation of China (82072974, 82120108020); the funds by the Chinese Academy of Sciences.

    • Competing interests No, there are no competing interests.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.