Proteasome Inhibition Induces IRF1 Downstream Molecules Independently of JAK Activity in Cancers

Abstract

Loss-of-function mutations in Janus kinase 1/2 (JAK1/2) cause low tumor immunogenicity through defects in the induction of the transcription factor interferon regulatory factor 1 (IRF1), resulting in non-responsiveness to cancer immunotherapy reagents. Therefore, the discovery of reagents that increase IRF1 expression independent of JAK activity is clinically important for the success of cancer immunotherapy reagents. We herein demonstrated that proteasome inhibitors activated IRF1 downstream pathways in a JAK-independent manner in various cancer types. Proteasome inhibitors increased IRF1 expression by inhibiting the degradation of IRF1 in melanoma. Furthermore, proteasome inhibitors induced the expression of the IRF1 downstream molecules, programmed death-ligand 1 (PD-L1), PD-L2, and human leukocyte antigen class I molecules. The induction of IRF1 expression by proteasome inhibitors was also detected in cancer types other than melanoma. Moreover, we showed that the induction of IRF1 expression was independent of JAK activity by genetic or chemical inhibition of JAK. Therefore, proteasome inhibitors may serve as adjuvants that potentiate the efficacy of cancer immunotherapy reagents by enhancing cancer immunogenicity.

INTRODUCTION

Recent advances in cancer treatment have been achieved through the development of cancer immunotherapy reagents, such as immune checkpoint inhibitors (ICIs)^1–4); however, drug resistance caused by low cancer immunogenicity remains a significant obstacle. Intrinsic or acquired resistance to ICIs can occur due to the loss of Janus kinase 1/2 (JAK1/2), resulting in lower cancer immunogenicity.^5,6) As well as ICIs, lower cancer immunogenicity may also reduce the efficacy of bispecific antibodies for cancer therapy, including the TCR/anti-CD3 bispecific fusion protein targeting gp100, tebentafusp, which was recently developed and shown to prolong overall survival in uveal melanoma,^4,7) because of the requirement of cancer recognition by TCR. These findings prompted us to speculate that the activation of JAK downstream molecules may be a rational therapeutic strategy to overcome resistance to cancer immunotherapy reagents.

The transcription factor interferon regulatory factor 1 (IRF1), a downstream molecule in the JAK signaling pathway, is considered as a key regulator of cellular immunogenicity, because numerous IRF1 target genes play pivotal roles in determining the immunogenic properties of cells, including genes involved in antigen presentation (such as human leukocyte antigen [HLA] class I, transporter associated with antigen processing, and beta-2 microglobulin)⁸⁾ and ligands for immune inhibitory receptors (programmed death-ligand 1 [PD-L1] and PD-L2).⁹⁾ Although we identified the JAK-independent induction of IRF1 at the transcriptional level in melanoma through the SRY-box transcription factor 10–IRF4 (SOX10–IRF4) axis,¹⁰⁾ this is limited to cancer types with melanin production because the SOX10–IRF4 axis is only present in melanin-producing cells due to the pigmentation-associated enhancer region of the IRF4 gene.^11,12) Therefore, the identification of mechanisms that induce IRF1 in other cancer types may be important for improving the efficacy of cancer immunotherapy reagents.

Instead of the transcriptional regulation of IRF1, the post-transcriptional or post-translational regulation of IRF1 is a potential target for increasing the expression of the IRF1 protein. IRF1 has a short half-life and is degraded through the ubiquitin–proteasome pathway.^13,14) Previous studies reported that IRF1 was phosphorylated by IκB kinase or glycogen synthase kinase-3β (GSK3β), resulting in its ubiquitination and degradation.^15,16) Although the targeting of IκB kinase and GSK3β is still limited to some cancer types, similar to the targeting of SOX10, proteasome inhibitors may be broadly applied to the treatment of cancers. Several proteasome inhibitors, such as bortezomib, carfilzomib, and ixazomib, have already been approved by the U.S. Food and Drug Administration (FDA); however, it currently remains unclear whether proteasome inhibitors are useful in combination with ICIs.

In the present study, we identified the proteasome inhibitors bortezomib and MG132 as chemical inducers of IRF1 expression in cancers. In addition to melanoma, we also showed that MG132 induced IRF1 in other cancer types, including lung cancer, colorectal cancer, and breast cancer. As a result of IRF1 downstream targets, PD-L1, PD-L2, and HLA class I molecules were also induced by proteasome inhibitors. Furthermore, we demonstrated that the induction of IRF1 by proteasome inhibition was independent of JAK activity. These results suggest the potential of proteasome inhibitors to enhance the efficacy of cancer immunotherapy by increasing tumor immunogenicity.

MATERIALS AND METHODS

Reagents and Plasmids

The proteasome inhibitors MG132 and bortezomib were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Recombinant human interferon-γ (IFN-γ) or mouse IFN-γ was purchased from PeptoTech (Cranbury, NJ, U.S.A.). The plasmids used were p3xFLAG-hIRF1 and pcDNA3.1-HA/UBC, which were the subcloned human IRF1 cDNA into the p3xFLAG-CMV7.1 vector (Sigma-Aldrich, St. Louis, MO, U.S.A.) and human UBC cDNA into the pcDNA3.1-HA vector (Thermo Fisher Scientific, Waltham, MA, U.S.A.), respectively. LentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid #52962; http://n2t.net/addgene:52962; RRID:Addgene_52962).¹⁷⁾

Cell Cultures

The human melanoma cell lines UACC257 and Malme-3M were obtained from the NCI (Bethesda, MD, U.S.A.). Other human cell lines were supplied by ATCC (Manassas, VA, U.S.A.). A2058, SK-MEL-28, DLD-1, A549, MDA-MB-231, and B16-F10 cells were cultured in RPMI-1640 medium (Nissui, Tokyo, Japan) containing 10% fetal bovine serum and penicillin/streptomycin/l-glutamine. A2058 ISRE-Luc stable cells were established by the transfection with pGL4.26-ISRE,¹⁸⁾ selected, and maintained with hygromycin.

In small interfering RNA (siRNA) knockdown experiments, siRNA for IRF1 (s7503, Thermo Fisher Scientific) or negative control #1 (Thermo Fisher Scientific) was used for transfection at a final concentration of 12.5 nM in melanoma cells using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific).

Stable A2058 cells expressing spCas9 were established by lentiviral infection with lentiCas9-Blast and referred to as A2058/Cas9. After transfection with guide RNA for JAK1 (CRISPR1089856_SGM, Thermo Fisher Scientific), A2058/JAK1-KO cells were established by single-cell cloning. The frameshift mutation of JAK1 was observed by DNA sequencing.

Western Blotting Analysis

Whole-cell extracts or nuclear extracts were prepared using the method of Schreiber et al.¹⁹⁾ and subjected to Western blotting analysis using an anti-IRF1 antibody (Cell Signaling Technology, Danvers, MA, U.S.A.), anti-β-actin antibody (Santa Cruz, Dallas, TX, U.S.A.), anti-HA antibody (Roche, Basel, Switzerland), anti-FLAG antibody (Merck, Darmstadt, Germany), and anti-histone H3 antibody (Abcam, Cambridge, U.K.). Band intensities were measured by ImageJ and normalized to those of each control lane.

Immunoprecipitation Assay

Cells were lysed in cell lysis buffer (20 mM HEPES, pH 7.6, 1 mM EGTA, 1.5 mM MgCl₂, 0.5 M NaCl, 20% glycerol, 0.1% Triton X-100). Aliquots of lysates containing 500 μg protein were incubated with an anti-IRF1 antibody (Cell Signaling Technology; 1 μL/sample) or anti-rabbit-immunoglobulin G antibody (Cell Signaling Technology) at 4°C for 14 h on a rotator, followed by incubation with 25 μL of Dynabeads™ Protein G (Invitrogen, Waltham, MA, U.S.A.) for 1.5 h at 4°C on a rotator. Immunoprecipitates were washed and denatured in sample buffer at 95°C for 5 min. Lysates and immunoprecipitates were subjected to Western blotting analysis using an anti-HA-biotin antibody (Roche) and anti-FLAG antibody (Merck).

Quantitative RT-PCR (qRT-PCR)

Total RNAs were prepared from human cancer cells using the RNeasy Plus Mini Kit (Thermo Fisher Scientific). qRT-PCR was performed using the One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real-Time) (TaKaRa Bio, Shiga, Japan). The primers used were as follows: 5′-CTT CCA TGG GAT CTG GAA GA-3′ (sense) and 5′-GAC CCT GGC TAG AGA TGC AG-3′ (antisense) for IRF1 mRNA; 5′-CCA GCA CAC TGA GAA TCA ACA-3′ (sense) and 5′-ATT TGG AGG ATG TGC CAG AG-3′ (antisense) for PD-L1 mRNA; and 5′-GCA CAG AGC CTC GCC TT-3′ (sense) and 5′-GTT GTC GAC GAC GAG CG-3′ (antisense) for β-actin mRNA. All reactions were run in triplicate, and mRNA levels were normalized to β-actin mRNA expression.

Flow Cytometry

After 96 h of siRNA transfection, cells were treated with proteasome inhibitors for 8 h or with IFN-γ for 24 h. The cells were stained with a phycoerythrin-conjugated anti-human CD274 antibody (eBioscience, San Diego, CA, U.S.A.), allophycocyanin-conjugated anti-human PD-L2 antibody (R&D Systems, Minneapolis, MN, U.S.A.), fluorescein isothiocyanate (FITC)-conjugated anti-human HLA-ABC antibody (BD Pharmingen, San Diego, CA, U.S.A.), or FITC-conjugated anti-major histocompatibility complex (MHC) class I (H-2Db) antibody (eBioscience). All flow cytometry data were analyzed using FlowJo software (TreeStar Software, Woodburn, OR, U.S.A.).

Statistical Analysis

Statistical significance was assessed using GraphPad Prism (GraphPad Software, San Diego, CA, U.S.A.). For comparisons involving more than 3 groups, one- or two-way ANOVA followed by Bonferroni post hoc tests was performed, while the unpaired Student’s t-test was used for comparisons between 2 groups. A p-value of less than 0.01 or 0.05 was regarded as statistically significant.

RESULTS

Proteasome Inhibitors Increased IRF1 Protein Expression Levels in Melanoma

To establish whether IRF1 is regulated by ubiquitin–proteasome degradation, we initially examined IRF1 expression in A2058 cells treated with the proteasome inhibitors MG132 and bortezomib. As shown in Fig. 1A, IRF1 protein expression was increased by both proteasome inhibitors. Furthermore, IRF1 induction was observed in UACC257, SK-MEL-28, and Malme-3M cells treated with MG132 (Fig. 1B). We also observed the accumulation of ubiquitinated IRF1 in A2058 cells (Fig. 1C), suggesting the degradation of ubiquitinated IRF1 under basal conditions. After MG132 treatment, the induction of IRF1 expression was weaker at the mRNA level than at the protein level (Fig. 1D). These results suggest that proteasome inhibitors increased IRF1 protein expression in melanoma cells.

Fig. 1. Proteasome Inhibitors Increase IRF1 Protein Expression Levels in Melanoma

(A) A2058 cells were treated with 10 μM MG132 (left panel) or 100 nM bortezomib (right panel) for the indicated times, and whole-cell lysates were then subjected to Western blotting. Band intensities were measured by ImageJ and normalized to those of each control lane. (B) Human melanoma cells were treated with MG132 for 8 h and then subjected to Western blotting. Other conditions were similar to those in (A). (C) A2058 cells were transfected with expression vectors for HA-tagged UBC (HA-UBC), FLAG-tagged IRF1 (FLAG-IRF1), and/or an empty vector. At 24 h post-transfection, cells were treated with 10 μM MG132 for 8 h and then subjected to Western blotting. (D) A2058 cells were treated with 10 μM MG132 for 8 h. Total RNA was subjected to qRT-PCR. Results are normalized to IRF1 expression in DMSO-treated cells. Data are presented as the mean ± standard deviation (S.D.) of 3 independent experiments. *p < 0.01 vs. DMSO-treated cells by the Student’s t-test.

Proteasome Inhibitors Activate IRF1 Downstream Pathways in Melanoma

We next investigated the nuclear localization of IRF1 after MG132 treatment. Similar to total IRF1 protein expression in Fig. 1, nuclear IRF1 expression increased after MG132 treatment (Fig. 2A). The expression of PD-L1 mRNA, one of the IRF1 downstream molecules, was also increased by MG132 (Fig. 2B). We also determined the increased transcriptional activity of IRF1 after MG132 treatment by measuring luciferase mRNA expression under the regulation of the IFN-stimulated response element, which is regulated by IRF1 (Fig. 2C). To further examine the effects of IRF1 transcriptional activity on IRF1 downstream molecules other than PD-L1, we assessed the expression of PD-L2 and HLA class I molecules after proteasome inhibition. Consistently, the expression of PD-L2 and HLA class I molecules on the cell surface significantly increased in A2058 cells treated with MG132 (Fig. 2D). These results indicate that the induction of IRF1 by proteasome inhibition activated the IRF1 downstream pathway. To confirm the significance of IRF1 after MG132 treatment, we next checked PD-L1 expression as a marker for melanoma immunogenicity, because the induction of HLA class I molecules was observed in a smaller window compared with that of PD-L1 (Fig. 2D). The knockdown of IRF1 significantly impaired PD-L1 induction by MG132 in A2058 cells (Fig. 2E). Collectively, these results indicate that proteasome inhibitors activated IRF1 downstream pathways in melanoma cells.

Fig. 2. Proteasome Inhibitors Activate IRF1 Downstream Pathways in Melanoma

(A) A2058 cells were treated with 10 μM MG132 (left panel) for 8 h, and nuclear extracts were then subjected to Western blotting. Other conditions were similar to those in Fig. 1A. (B) A2058 cells were treated with 10 μM MG132 for 8 h. Total RNA was subjected to qRT-PCR. Results are normalized to PD-L1 expression in DMSO-treated cells. Other conditions were similar to those in Fig. 1D. (C) A2058 ISRE-Luc cells were treated with 10 μM MG132 for 8 h. Total RNA was subjected to qRT-PCR. Results are normalized to Luc2 expression in DMSO-treated cells. Other conditions were similar to those in Fig. 1D. (D) A2058 cells were treated with 10 μM MG132 (left panel) for 8 h and then subjected to flow cytometry. (E) A2058 cells were transfected with siCNTL or siIRF1 and treated with 10 μM MG132 for 8 h. After 96 h of transfection, cells were subjected to flow cytometry (left) and Western blotting. Mean fluorescent intensities are presented as the mean ± S.D. of at least 3 independent experiments. *p < 0.05 vs. mean fluorescent intensity in siCNTL-transfected cells by two-way ANOVA followed by the Bonferroni post hoc test. Other conditions were similar to those in Fig. 1A or 2B.

Proteasome Inhibitors Activate IRF1 Downstream Pathways in Various Cancer Types

Since we previously reported the transcriptional induction of IRF1 by histone deacetylase (HDAC) inhibitors in melanoma and clear cell sarcoma,^10,20) we speculated whether the post-translational regulation of IRF1 may be conserved in cancer types other than melanoma or clear cell sarcoma. Therefore, we examined IRF1 expression in A549 lung cancer cells, MDA-MB-231 breast cancer cells, and DLD-1 colorectal cancer cells treated with MG132. IRF1 protein expression was increased by MG132 treatment, similar to melanoma cells (Fig. 3A). In addition, MG132 treatment increased PD-L1 mRNA expression in all 3 cell types (Fig. 3B). We then investigated PD-L1 protein expression after MG132 treatment. Due to differences in cytotoxicity among cancer cells, we applied a lower concentration of MG132 to prevent MG132-induced cell death. The results obtained showed an increase in PD-L1 expression by MG132 in DLD-1 and A549 cells, but not in MDA-MB-231 cells (Fig. 3C). These results suggest that, with some exceptions, proteasome inhibitors enhance the immunogenicity of various cancers, including melanoma, lung cancer, and colorectal cancer.

Fig. 3. Proteasome Inhibitors Activate IRF1 Downstream Pathways in Various Cancer Types

(A, B) DLD-1 colorectal cancer cells, A549 lung cancer cells, and MDA-MB-231 breast cancer cells were treated with 10 μM MG132 for 8 h and then subjected to Western blotting (A) or qRT-PCR (B). Results are normalized to PD-L1 expression in DMSO-treated cells. Data are presented as the mean ± S.D. of 3 independent experiments. *p < 0.05 vs. DMSO-treated cells by two-way ANOVA followed by the Bonferroni post hoc test. Other conditions were similar to those in Fig. 1A. (C) DLD-1 colorectal cancer cells, A549 lung cancer cells, and MDA-MB-231 breast cancer cells were treated with 5 μM MG132 for 24 h and then subjected to flow cytometry.

Proteasome Inhibitors Induce IRF1 Expression Independently of JAK Activity

To investigate whether the induction of IRF1 expression by proteasome inhibition was independent of JAK activity, A2058 cells were treated with the JAK inhibitor baricitinib. As shown in Figs. 4A and 4B, the induction of both IRF1 and PD-L1 by MG132 was not impaired by baricitinib treatment. To confirm its independence from JAK activity, we established JAK1-knockout cells (A2058/JAK1-KO). IFN-γ induced IRF1 expression in A2058 cells, but not in A2058/JAK1-KO cells (Fig. 4C). In contrast, MG132 induced IRF1 expression in both A2058 and A2058/JAK1-KO cells. Consistent with the induction of IRF1, the MG132 treatment induced PD-L1 expression, even in A2058/JAK1-KO cells, whereas IFN-γ treatment did not (Fig. 4D). We further confirmed the increased H-2Db expression, which is an MHC class I molecule in mouse, in a JAK-independent manner after MG132 treatment in mouse B16F10 melanoma cells, though IFN-γ-induced H-2Db was completely suppressed by baricitinib (Fig. 4E). These results strongly suggest that proteasome inhibition induces tumor immunogenicity independently of JAK activity.

Fig. 4. Proteasome Inhibitors Induce IRF1 Expression Independently of JAK Activity

(A) A2058 cells were pretreated with 0.5 μM baricitinib for 30 min. After pretreatment, cells were treated with 10 μM MG132 for 8 h and then subjected to Western blotting. (B) After pretreatment with 0.5 μM baricitinib, cells were treated with 10 μM MG132 for 8 h and then subjected to flow cytometry. (C) A2058 or A2058/JAK1-KO cells were treated with 10 μM MG132 for 8 h or 10 ng/mL IFN-γ for 3 h and then subjected to Western blotting. Other conditions were similar to those in Fig. 1A. (D) A2058 or A2058/JAK-KO cells were treated with 10 μM MG132 for 8 h or 10 ng/mL IFN-γ for 24 h and then subjected to flow cytometry. (E) B16F10 cells were treated with 100 nM MG132 for 24 h or 50 IU/mL mouse IFN-γ for 24 h and then subjected to flow cytometry.

DISCUSSION

Although proteasome inhibition impacts many proteins, IRF1 is a critical molecule in the induction of PD-L1 by proteasome inhibitors (Fig. 2E). IRF1 ubiquitination, essential for proteasomal degradation, is regulated by ligases like CHIP and cIAP2.^21,22) Since ubiquitinated IRF1 accumulated after proteasome inhibition (Fig. 1C), those ubiquitin ligases remain functional in melanoma and other cancer types used in this study. This conserved degradation mechanism highlights IRF1 as a potential drug target in various cancers.

Although ICIs are approved for various cancer types, including lung cancer and melanoma, one of the resistance mechanisms to ICIs is JAK mutations, which result in defects in the induction of both IRF1 and its downstream molecules. In addition, these defects may affect the efficacy of current TCR/anti-CD3 bispecific antibodies, such as tebentafusp, because of the loss of antigen presentation in cancers. In this context, we showed the induction of IRF1 by proteasome inhibitors in a JAK-independent manner using both genetic and chemical inhibition of JAK (Fig. 4). Since we previously demonstrated that JAK-independent induction of IRF1 by an HDAC inhibitor increased the efficacy of anti-PD-1 antibody in melanoma,¹⁰⁾ proteasome inhibition in this study may be another strategy for improving the efficacy of not only ICIs but also TCR/anti-CD3 bispecific antibodies in cancer patients with JAK mutations. Given the FDA approval of bortezomib for multiple myeloma and mantle cell lymphoma,^23–25) it may also benefit cancer patients receiving immunotherapy.

Although we demonstrated an increase in immunogenicity by proteasome inhibition, the therapeutic effects of ICIs may also be enhanced through increases in the number of T cells in the tumor microenvironment. In lung cancer and hepatocellular carcinoma, IRF1 has been shown to regulate the synthesis of the chemokine CXCL10.^26,27) Therefore, the increased expression of IRF1 in cancer cells may lead to CXCL10-mediated recruitment of cytotoxic T cells, which may enhance the effects of ICIs. This hypothesis is also supported by the expression level of CXCL10 in the tumor microenvironment being an indicator of the outcomes of patients treated with ICIs.²⁸⁾

Since HDAC inhibitors increase IRF1 expression through the SOX10–IRF4 axis linked to the pigmentation-associated enhancer of the IRF4 region,^10–12) the effects of HDAC inhibitors are limited to melanin-producing cancers, such as melanoma and clear cell sarcoma. In contrast, proteasome inhibition increased IRF1 expression across various cancers. While differences in PD-L1 induction by proteasome inhibition were observed among various cancers (Fig. 3), breast cancers, at least MDA-MB-231 cells, may have other PD-L1 regulatory mechanisms. In this context, considering that IRF1 knockdown did not fully block PD-L1 induction by MG132, the PD-L1 protein itself may be regulated by proteasomal degradation through the E3 ligases ARIH1 and CUL3.^29,30) In addition, we detected the weak induction of IRF1 mRNA expression in A2058 cells treated with MG132, which was not statistically significant (Fig. 1D). Since IRF1 may regulate the activation of STAT1,³¹⁾ it may still be possible to induce IRF1 mRNA after treatment with MG132. Nevertheless, the present results support proteasome inhibition as a strategy to induce IRF1 in some cancer types.

In summary, we herein demonstrated that proteasome inhibitors enhanced JAK-independent immunogenicity against cancers, such as melanoma, lung cancer, and colorectal cancer, by inhibiting the degradation of the transcription factor IRF1. The clinical significance of this study is supported by cancer immunotherapy reagents that require cancer immunogenicity for their effects. Therefore, the induction of IRF1 by proteasome inhibition may provide therapeutic opportunities using cancer immunotherapy reagents for cancer patients with lower cancer immunogenicity.

Acknowledgments

We thank the members of the Sakurai Laboratory for their discussions and suggestions. The present study was supported in part by JSPS KAKENHI Grant Numbers: 24K10355 (S.Y.) and 23K24026 (H.S.), as well as research Grants from Takeda Science Foundation (S.Y.) and the GSK Japan Research Grant 2021 (S.Y.).

Conflict of Interest

The authors declare no conflict of interest.

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