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BIRC2 blockade facilitates immunotherapy of hepatocellular carcinoma

Molecular Cancer volume 24, Article number: 113 (2025) Cite this article

Abstract

Background

The effectiveness of immunotherapy in hepatocellular carcinoma (HCC) is limited, however, the molecular mechanism remains unclear. In this study, we identified baculoviral IAP repeat-containing protein 2 (BIRC2) as a key regulator involved in immune evasion of HCC.

Methods

Genome-wide CRISPR/Cas9 screening was conducted to identify tumor-intrinsic genes pivotal for immune escape. In vitro and in vivo models demonstrated the role of BIRC2 in protecting HCC cells from immune killing. Then the function and relevant signaling pathways of BIRC2 were explored. The therapeutic efficacy of BIRC2 inhibitor was examined in different in situ and xenograft HCC models.

Results

Elevated expression of BIRC2 correlated with adverse prognosis and resistance to immunotherapy in HCC patients. Mechanistically, BIRC2 interacted with and promoted the ubiquitination-dependent degradation of NFκB-inducing kinase (NIK), leading to the inactivation of the non-canonical NFκB signaling pathway. This resulted in the decrease of major histocompatibility complex class I (MHC-I) expression, thereby protecting HCC cells from T cell-mediated cytotoxicity. Silencing BIRC2 using shRNA or inhibiting it with small molecules increased the sensitivity of HCC cells to immune killing. Meanwhile, BIRC2 blockade improved the function of T cells both in vitro and in vivo. Targeting BIRC2 significantly inhibited tumor growth, and enhanced the efficacy of anti-programmed death protein 1 (PD-1) therapy.

Conclusions

Our findings suggested that BIRC2 blockade facilitated immunotherapy of HCC by simultaneously sensitizing tumor cells to immune attack and boosting the anti-tumor immune response of T cells.

Introduction

HCC constitutes approximately 90% of all primary liver tumors and is a prominent contributor to cancer-related mortality on a global scale [1]. Over the past decade, the foremost therapeutic options for HCC have been multi-kinase inhibitors. However, concerns regarding drug resistance and enduring toxicity persisted [2]. Simultaneously, more than ten other pharmaceutical agents have failed to meet clinical endpoints across various phase 3 clinical trials [3].

The noteworthy success of immunotherapy, using immune checkpoint blockade (ICB) primarily encompassing anti-PD-1, anti-programmed death-ligand 1 (PD-L1), and anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) therapies, has laid the groundwork for curative approaches to a multitude of malignancies [4]. The application of PD-1/PD-L1 blockade also displayed encouraging therapeutic potential as a second-line intervention for HCC in phase 1/2 clinical trials [5, 6]. Nevertheless, in phase 3 studies spanning first- and second-line settings, single-agent anti-PD-1 antibodies have fallen short in extending the survival period, with a modest response rate of approximately 15% [7, 8]. In the pursuit of combination therapy, the IMbrave150 study (anti-PD-L1 plus anti-VEGF therapy) marked the pioneering randomized phase 3 clinical trial to unveil a significant enhancement in the overall survival (OS) of patients afflicted with systemic treatment-naive unresectable HCC compared with the conventional first-line multi-kinase inhibitor Sorafenib [9,10,11]. Similarly, anti-PD-1 combined with anti-VEGF therapy has also exhibited favorable tumor response in first-line scenarios (ORIENT-32, CARES-310) [12, 13]. Furthermore, the HIMALAYA study corroborated the survival benefits associated with dual immunotherapy in patients with HCC [14]. However, the overall response rate was still limited in the above combination therapies, with up to 70% of the patients exhibiting drug resistance. It is necessary to unveil the underlying mechanisms of resistance to immunotherapy, and improve the therapeutic efficacy through other combination strategies.

In most cases, HCC arises in chronic inflammatory liver stemming from viral hepatitis or non-alcoholic steatohepatitis (NASH) [15]. Immune dysregulation is a pervasive companion throughout the spectrum of inflammation-associated liver disease and neoplasm [16]. In established HCC cases, impaired secretion of interferon (IFN), tumor necrosis factor (TNF), and granzyme by cytotoxic T cells (CTL) has been linked to an exhausted effector-cell phenotype, ultimately fostering tumor progression [17]. In contrast to CTL which exert anti-tumor functions, other immune cells such as T regulatory cells (Treg), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) contribute to the formation of an immunosuppressive microenvironment [18]. However, investigations into tumor-intrinsic mechanism of resistance to immunotherapy in HCC remain scarce.

In the present study, we employed a comprehensive loss-of-function genetic screening approach in a tumor cell/T cell co-culture model to identify genes that modulate sensitivity to immune attack. BIRC2 emerged as a pivotal player in tumor immune evasion. BIRC2 belongs to the inhibitor of apoptosis (IAP) family, characterized by the presence of the baculovirus IAP repeat (BIR) domain, which is essential for its anti-apoptotic potential [19]. BIRC2 also harbors a carboxy-terminal RING finger domain, conferring ubiquitin ligase (E3) activity [20]. The binding of BIRC2 inhibitors to BIRC2 protein enhances its E3 ligase activity, thereby promoting self-ubiquitination and degradation [21].

We discovered that elevated BIRC2 expression correlated with adverse prognosis and resistance to ICB in HCC patients. Then we systematically explored the multifaceted role of BIRC2 in both tumor cells and T cells. BIRC2 blockade significantly facilitated immunotherapy of HCC by simultaneously sensitizing tumor cells to immune attack and boosting the anti-tumor immune response of T cells. These findings underscore the rationale for considering BIRC2 as a novel target in the immunotherapy of HCC and highlight the potential for a promising combination strategy involving ICB and BIRC2 inhibitors.

Materials and methods

Materials and methods are included in the online supplementary material.

Results

BIRC2 is a tumor-intrinsic immune evasion gene

To elucidate tumor-intrinsic genes pivotal for immune evasion in HCC, we introduced Hepa1-6-OVA cells to a genome-scale CRISPR/Cas9 knockout library. This comprehensive library comprises 94,528 sgRNAs targeting 19,463 protein-coding genes, with an average coverage of five sgRNAs per gene. Subsequently, these cells were exposed to activated OT-I T cells (Fig. 1A). Deep sequencing unveiled a total of 291 genes characterized by sgRNA depletion and 282 genes marked by sgRNA enrichment. Among these, we identified well-documented tumor-intrinsic genes associated with immune evasion which were frequently observed in patients resistant to ICB, such as JAK2, TAP2, and B2M [22]. The comprehensive list of differentially expressed genes (DEGs) identified through our analysis was displayed in Table S1. Importantly, BIRC2 emerged as the top-ranking candidate (Fig. 1B).

Fig. 1
figure 1

BIRC2 is a tumor-intrinsic immune evasion gene. A. Schematic representation of the CRISPR/Cas9 screening process. B. Volcano plot illustrating the normalized fold change of sgRNA in Hepa1-6-OVA cells challenged with OT-I T cells compared to control CD8+ T cells, with selected top candidates highlighted. C. Schematic representation of the competitive assay. D. Apoptosis of Hepa1-6-OVA cells infected with sgBIRC2 after challenging with OT-I T cells. E. Clone formation of Hepa1-6-OVA cells infected with sgBIRC2 after challenging with OT-I T cells. Representative of three independent experiments. Statistical analysis was performed on biological replicates (n = 3); each value represents mean ± SD; two-sided Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. F. Tumor growth of Hepa1-6-OVA cells in NSG and C57BL/6J mice. Representative of two independent experiments (n = 6–9 mice/group); each value represents mean ± SEM. Survival analysis performed using the log-rank (Mantel-Cox) test. *, P < 0.05; **, P < 0.01; ***, P < 0.001

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We then conducted competitive assays to validate the impact of BIRC2 perturbation on HCC cells. This entailed the application of CRISPR/Cas9-mediated single-gene knockout (KO) using two distinct sgRNAs (Figure S1A). BIRC2 knockout (KO) did not exert any discernible influence on the viability of tumor cells (Figure S1B). However, in the co-culture system featuring tumor-specific OT-I T cells targeting Hepa1-6-OVA cells (Fig. 1C), we observed a substantial increase in apoptosis (Fig. 1D) and a reduction in clone formation (Fig. 1E) of BIRC2-KO cells.

Next, we implanted BIRC2-KO Hepa1-6-OVA cells into immune-competent C57BL/6J mice, which were known to generate an adaptive immune response capable of exerting selective pressure on tumors. In parallel, tumor cells were transplanted into immune-deficient NSG mice as a control. Consistent with our in vitro findings, BIRC2-KO demonstrated no impact on the growth of subcutaneous tumors in NSG mice, whereas delayed tumor formation was observed in C57BL/6J mice (Fig. 1F).

We performed immunohistochemistry (IHC) staining in tumors harvested from C57BL/6J mice to analyze the potential changes in innate and adaptive immunity. MDSCs (Figure S1C) and TAMs (Figure S1D) showed no statistically significant differences between the BIRC2-KO and control groups. Similarly, cancer-associated fibroblast (CAFs) exhibited comparable stromal distribution (Figure S1E). BIRC2-KO tumors demonstrated a significant elevation in the infiltration of both CD8+ and CD4+ T cells, as well as the emergence of tertiary lymphoid structures at the invasive margins (Figure S1F-G). A modest increase in B cell density was also noted (Figure S1H). These findings collectively suggested that BIRC2 depletion fostered an immunostimulatory tumor microenvironment by improving adaptive immunity.

We further explored the effects of BIRC2 knockdown (KD) in human HCC cells. Immunoblotting revealed the presence of BIRC2 protein in the majority of the examined HCC cell lines (Figure S2A). PLC/PRF/5 cells, characterized by the highest BIRC2 expression, were selected for subsequent experiments. We successfully generated stable cell lines with BIRC2-KD using shRNAs (Figure S2B). As illustrated in Figure S2C-D, BIRC2-KD induced a modest inhibition of cell proliferation in vitro, while with no discernible impact on tumor growth in NSG mice in vivo. When co-cultured with activated peripheral blood mononuclear cells (PBMC) (Figure S2E), BIRC2-KD significantly augmented apoptosis (Figure S2F) and suppressed clone formation (Figure S2G) of HCC cells.

BIRC2 correlates with poor survival and resistance to ICB in HCC patients

We examined the expression of BIRC2 in HCC tissues. Elevated BIRC2 protein (Figure S3A) and mRNA (Figure S3B) expression was observed in tumor tissues compared with that in adjacent non-tumorous liver tissues. IHC staining highlighted an increased BIRC2 protein level in tumor tissues (Figure S3C).

To assess BIRC2 expression in a broader cohort, we employed IHC analysis on an HCC microarray comprising 765 patients. Three independent pathologists scored the level of BIRC2 protein as 0, 1, 2 or 3 (Fig. 2A). The association between BIRC2 expression and patients’ clinicopathological characteristics was summarized in Table 1. Univariate and multivariate Cox regression analyses of survival predictors were summarized in Table 2. No significant difference of BIRC2 expression was observed across different histological subtypes, including trabecular, pseudo-glandular, solid, macrotrabecular-massive (MTM), fibrolamellar, steatohepatitis, clear cell and other types (Figure S3D).

Fig. 2
figure 2

BIRC2 expression correlates with poor survival and resistance to ICB in HCC patients. A. Representative HE and IHC images of BIRC2 in HCC tissues. B. Representative IHC images of CD8 and CD4 in high- and low-BIRC2 groups. C. The number of CD8+ and CD4+ T cells in high- and low-BIRC2 groups. D. Representative images of tumor-infiltrating lymphocytes in high- and low-BIRC2 groups. E. Kaplan-Meier analysis of the OS comparing high and low BIRC2 expression in HCC patients (n = 765). F. Kaplan-Meier analysis of the DFS comparing high and low BIRC2 expression in HCC patients (n = 765). G. Kaplan-Meier analysis of OS comparing high and low BIRC2 expression in HCC patients who received combined immunotherapy (n = 144). H. Kaplan-Meier analysis of PFS comparing high and low BIRC2 expression in HCC patients who were treated with anti-PD-1 plus Lenvatinib (n = 47). Representative of three independent experiments. Statistical analysis was performed on biological replicates (n = 3); each value represents mean ± SD; two-sided Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001

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Table 1 Association of BIRC2 expression with clinicopathological features in HCC patients
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Table 2 Cox proportional hazards model analysis of prognostic factors in HCC patients
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As illustrated in Fig. 2B-D, BIRC2 expression exhibited a significant negative correlation with the number of tumor-infiltrating CD8+ and CD4+ T cells, consistent with the data from the GSE10143 (Figure S3E) and GSE10186 (Figure S3F) databases. High BIRC2 levels indicated shorter OS (Fig. 2E) and disease-free survival (DFS) (Fig. 2F) in HCC patients. Furthermore, data from The Cancer Genome Atlas (TCGA) and GSE27150 databases revealed that high BIRC2 expression was associated with poor clinical outcome (Figure S4A). Previous studies have shown that HCC from non-viral etiology were more resistant to ICI therapy [23]. We compared the expression of BIRC2 in HCC with viral (HBV), non-viral (NASH) and other unknown etiologies. No significant difference was observed (Figure S4B). High expression of BIRC2 suggested shorter OS in the HBV as well as the NASH subgroup (Figure S4C).

Among our cohort of 765 patients, 144 individuals underwent combined immunotherapy, and high BIRC2 expression was significantly associated with reduced OS in these patients (Fig. 2G). We further validated BIRC2 expression in another cohort comprising 47 HBV-associated HCC treated with anti-PD-1 in combination with Lenvatinib (Tables 3 and 4). Kaplan-Meier analysis disclosed that patients with high BIRC2 levels exhibited an unfavorable progression-free survival (PFS) (Fig. 2H).

Table 3 Association of BIRC2 expression with clinicopathological features in HCC patients who have received anti-PD-1 plus lenvatinib therapy
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Table 4 Cox proportional hazards model analysis of prognostic factors in HCC patients who have received anti-PD-1 plus lenvatinib therapy
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Analysis using the TIDE tool indicated that high BIRC2 expression was associated with poor prognosis in melanoma (Figure S4D). Furthermore, high BIRC2 expression attenuated or negated the survival benefits associated with CTL in melanoma (Figure S4E) and lung cancer (Figure S4F).

BIRC2 regulates IFNγ response in HCC cells

To decipher the relevant signaling pathways of BIRC2, we conducted RNA sequencing (RNA-seq). The results from the KEGG (Fig. 3A) and GSEA (Figure S5A) analysis consistently indicated IFNγ response as the most prominently affected biological process.

Fig. 3
figure 3

BIRC2 suppresses the expression of HLA-ABC and PD-L1 induced by IFNγ. A. KEGG analysis of significant pathways up-regulated in PLC cells infected with shBIRC2. B. Expression of HLA-ABC and PD-L1 in PLC cells infected with shBIRC2 and treated with IFNγ. C. Expression of HLA-ABC and PD-L1 in PLC cells infected with shBIRC2 and cultured in conditioned medium. D. Membrane expression of HLA-ABC and PD-L1. E. Expression and subcellular localization of HLA-ABC and PD-L1. Representative of three independent experiments. Statistical analysis was performed on biological replicates (n = 3); each value represents mean ± SD; two-sided Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001

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IFNγ exerts anti-proliferative and pro-apoptotic effects in tumor cells [24]. It has also been reported to up-regulate the expression of MHC-I, known as human leukocyte antigen-ABC (HLA-ABC) in humans and H2Kb in mice, as well as PD-L1 [25]. We then proceeded to validate the impact of BIRC2 perturbation on IFNγ response in HCC cells. BIRC2-KD enhanced IFNγ-triggered growth inhibition (Figure S5B) and apoptosis (Figure S5C). This was corroborated by an increase in the cleavage of caspase and PARP proteins (Figure S5D). The levels of HLA-ABC and PD-L1 were markedly elevated in response to IFNγ in the BIRC2-KD group (Fig. 3B). To ascertain whether HLA-ABC and PD-L1 were regulated by tumor cell-extrinsic autocrine factors, we compared the protein levels in tumor cells cultured in conditioned medium derived from BIRC2-KD cells. Notably, no significant differences were observed between different medium conditions (Fig. 3C). Furthermore, flow cytometry (Fig. 3D) and immunofluorescence (IF) (Fig. 3E) analyses revealed a remarkable increase in the expression of HLA-ABC and PD-L1 on the cell membrane induced by IFNγ in BIRC2-deficient cells.

In parallel, we assessed the expression of H2Kb and PD-L1 in Hepa1-6-OVA cells. Consistent with the findings in PLC cells, BIRC2-KO enhanced the expression of H2Kb and PD-L1 in response to IFNγ (Figure S5E). More importantly, BIRC2-KO immediately facilitated tumor antigen presentation in HCC cells (Figure S5F).

BIRC2 inactivates the non-canonical NFκB pathway in HCC cells

IFNγ activates several transcriptional activators of MHC-I and PD-L1. Within the context of MHC-I promoter regulation, the interferon-sensitive response element (ISRE) binds to interferon regulatory factor (IRF) family members, while NFκB interacts with the NFκB-binding sites of enhancer A [26]. We first sought to elucidate whether BIRC2 plays a role in the regulation of the JAK/STAT/IRF pathway. As illustrated in Fig. 4A, BIRC2-KD did not influence the expression of IFNγ receptor 1 (IFNGR1) or the phosphorylation of Janus kinase (JAK) or signal transducer and activator of transcription (STAT) proteins, all of which were induced by IFNγ. Our subsequent investigation focused on the impact of BIRC2 on the NFκB pathway.

Fig. 4
figure 4

BIRC2 inactivates the non-canonical NFκB pathway in HCC cells. A. Expression of IFNGR1 and phosphorylation of JAK/STAT family proteins. B. Phosphorylation of NFκB p65 and processing of NFκB p105/p50. C. Processing of NFκB p100/p52. D. Nuclear accumulation of IRF1. E. Nuclear accumulation of NFκB p65, p50, and p52. F. Nuclear translocation of NFκB p65 and p52

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The canonical NFκB pathway predominantly involves RELA (p65), NFκB1 (p105/p50) and cREL, and the non-canonical NFκB pathway involves NFκB2 (p100/p52) and RELB [27]. As shown in Fig. 4B-C, BIRC2-KD did not modulate the canonical NFκB pathway, but expedited the activation of the non-canonical NFκB pathway. Separation of cytoplasmic and nuclear proteins revealed that BIRC2-KD did not activate IRF1 (Fig. 4D), while facilitating the nuclear accumulation of NFκB p52 (Fig. 4E). Translocation of NFκB p52, as opposed to p65, from the cytoplasm to the nucleus was substantiated by IF (Fig. 4F).

BIRC2 interacts with and mediates ubiquitination-dependent degradation of NIK

To unveil the potential interactors with BIRC2, we conducted co-immunoprecipitation (co-IP) coupled with liquid chromatography tandem mass spectrometry (LC/MS) in PLC cells. In alignment with the data predicted by online databases (Figure S6A), LC/MS analyses revealed a direct physical interaction between BIRC2 and NIK, the essential upstream kinase of the non-canonical NFκB pathway (Figure S6B). This interaction was further corroborated by IF, which demonstrated that BIRC2 exhibited diffuse cytoplasmic co-localization with NIK in PLC cells (Fig. 5A). Anti-NIK antibody effectively precipitated BIRC2 protein, and reciprocal immunoprecipitation using anti-BIRC2 antibody confirmed the direct mutual interaction between the two proteins in both PLC cells and HEK293T cells stably transfected with BIRC2- and NIK-overexpression plasmids (Fig. 5B). We also confirmed the BIRC2-NIK interaction in both Hepa1-6 cells and HEK293T cells stably transfected with mouse BIRC2- and NIK-overexpression plasmids (Figure S6C).

Fig. 5
figure 5

BIRC2 interacts with and mediates ubiquitination-dependent degradation of NIK. A. Co-localization of BIRC2 and NIK. B. Co-IP assay of BIRC2 and NIK in PLC and HEK293T cells. C. Co-IP assay of ubiquitination in PLC cells infected with shBIRC2. D. Expression of HLA-ABC and PD-L1 in PLC cells infected with shBIRC2 and treated with IFNγ and MG132. E. Expression of HLA-ABC and PD-L1 on the membrane of PLC cells infected with shBIRC2 and treated with IFNγ and B022. Representative of three independent experiments. Statistical analysis was performed on biological replicates (n = 3); each value represents mean ± SD; two-sided Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001

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Given BIRC2’s established role as an E3 ligase, we investigated ubiquitination of NIK. As shown in Fig. 5C, BIRC2 significantly enhanced ubiquitination-dependent degradation of NIK. In the presence of the proteasome inhibitor MG132, a substantial increase in the levels of HLA-ABC and PD-L1 was detected (Fig. 5D). Furthermore, the selective NIK inhibitor, B022, reversed the inhibitory effect of BIRC2 on the expression of HLA-ABC and PD-L1 induced by IFNγ (Fig. 5E). These results collectively suggested that BIRC2 restrained the IFNγ response in HCC cells through interaction with and regulation of ubiquitination-dependent degradation of NIK.

TNFα transcriptionally induces the expression of BIRC2 via NFκB p65

As detailed earlier, BIRC2 functioned as a tumor-intrinsic CTL-evasion gene, suggesting its potential regulation in the context of immune killing. To elucidate which cytokines induced the expression of BIRC2, we exposed PLC cells to PBMC supernatant and a selection of commonly encountered effector cytokines, including IL2, IFNα, IFNβ, IFNγ, and TNFα. Our observations revealed that TNFα substantially elevated the expression of BIRC2 at both mRNA (Figure S7A) and protein (Figure S7B) levels.

We then analyzed the promoter sequence of the human BIRC2 gene and identified two potential binding sites for NFκB p65, a transcription factor downstream of the TNFα signaling cascade (Figure S7C). Through dual luciferase reporter assay, we confirmed that the transcriptional activity of the BIRC2 promoter relied on the binding sites for NFκB p65, evident in both HEK293T (Figure S7D) and PLC cells (Figure S7E). ChIP analysis further validated that TNFα significantly increased the recruitment of NFκB p65 to the BIRC2 promoter (Figure S7F).

BIRC2 blockade inhibits tumor growth in in situ HCC models

BIRC2 is known to express in various cell types, including T cells. Originally, BIRC2 antagonists were used in cancer therapy aimed to induce apoptosis by relieving caspase inhibition [28]. Therefore, an ideal drug should exert minimal adverse effects on T cells. Among several candidates, we selected LCL161, a monovalent BIRC2 inhibitor, for our subsequent experiments (Figure S8A). Consistent with shRNA knockdown results, LCL161 significantly enhanced IFNγ-induced apoptosis (Figure S8B) and the expression of HLA-ABC and PD-L1 in PLC cells (Figure S8C).

Next, we assessed the therapeutic efficacy of LCL161 in different in situ HCC models. LCL161 was administered into DEN/CCl4 model either from the 22nd or 24th weeks, representing early- or late-stage tumors, respectively (Figure S9A). LCL161 treatment resulted in a notable decrease in the number and size of both early- and late-stage tumors (Figure S9B). HE staining indicated that LCL161 facilitated the infiltration of lymphocytes into the tumor microenvironment (Figure S9C). IHC staining confirmed that LCL161 significantly increased the number of both CD8+ and CD4+ T cells (Figure S9D). Moreover, LCL161 also reduced the level of BIRC2 protein in tumor tissues in vivo (Figure S9E).

Similarly, in the c-Myc model, LCL161 substantially delayed tumor development (Figure S10A) and increased lymphocyte infiltration (Figure S10B-C).

BIRC2 blockade promotes the anti-tumor function of T cells

We also assessed the effect of BIRC2 blockade on T cells. As illustrated in Figure S11A, LCL161 significantly enhanced the function of T cells, manifested by increased production of effector cytokines IFNγ and TNFα. We then performed RNA-seq on T cells treated with LCL161. KEGG analysis highlighted the inflammatory response as the most prominent biological process affected (Figure S11B). Western blot analysis suggested that BIRC2 blockade activated the non-canonical NFκB pathway in T cells, consistent with the observations in tumor cells, as opposed to the canonical NFκB pathway (Figure S11C).

Furthermore, we evaluated the anti-tumor function of T cells in vivo. Hepa1-6-OVA cells were subcutaneously transplanted into C57BL/6J mice, then the xenografts were extracted, and function of tumor-infiltrating lymphocytes was assessed via flow cytometry (FCM). As shown in Figure S11D, both CD8+ and CD4+ T cells in the tumor microenvironment exhibited an effector phenotype, characterized by augmented production of IFNγ and TNFα in the LCL161-treated group.

LCL161 had no significant impact on the activation (Figure S12A) or proliferation (Figure S12B) of CD8+ and CD4+ T cells. There were no significant alterations in the levels of phosphorylated ERK1/2 proteins, indicating that BIRC2 did not modulate downstream signaling of T cell receptor (TCR) (Figure S12C).

To further investigate whether BIRC2 overexpression affects T cell exhaustion, we transfected PBMC with BIRC2-overexpression plasmids. An upregulation of inhibitory costimulatory molecules LAG-3 and TIM-3 on the membrane of both CD8+ and CD4+ T cells was observed, which suggested that BIRC2 promoted T cell exhaustion (Figure S12D).

BIRC2 blockade enhances the efficacy of anti-PD-1 therapy

Mice bearing Hepa1-6-OVA tumors were intravenously injected with OT-I T cells as adoptive T cell transfer (ACT) therapy, followed by treatment with LCL161, anti-PD-1 antibody, or a combination of both, respectively. As shown in Fig. 6A-C, LCL161 treatment alone demonstrated some degree of anti-tumor efficacy, albeit not as robust as the anti-PD-1 antibody. The most favorable therapeutic outcomes, characterized by a sustained response, were observed in the combination therapy group. Furthermore, combination therapy significantly extended the survival time.

Fig. 6
figure 6

BIRC2 blockade enhances the efficacy of anti-PD-1 therapy. A. Representative images of tumors in the Hepa1-6-OVA/OT-I model treated with LCL 161, anti-PD-1 antibody, or combination therapy. B. Tumor growth curve of the Hepa1-6-OVA/OT-I model. C. Survival curve of the Hepa1-6-OVA/OT-I model. D. Production of IFNγ and TNFα in OT-I T cells. E. IFNγ and TNFα levels in the serum of the mice. F. Representative images of TUNEL staining. Representative of two independent experiments (n = 7 mice/group); each value represents mean ± SEM. Survival analysis performed using the log-rank (Mantel-Cox) test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. G. Representative images of tumors in the DEN/CCl4 model treated with LCL 161, anti-PD-1 antibody, or combination therapy. H. Schematic model of this study

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Tumor-specific OT-I T cells exhibited effector characteristics, marked by enhanced production of anti-tumor cytokines IFNγ and TNFα in the single-drug groups, although the magnitude of this effect was surpassed by combination therapy (Fig. 6D). Meanwhile, IFNγ and TNFα levels in the serum of the treated mice were also elevated (Fig. 6E). Both LCL161 and anti-PD-1 antibody alone induced apoptosis in tumor cells, whereas combination therapy demonstrated a synergistic enhancement of antitumor effect (Fig. 6F). Moreover, combination therapy also demonstrated promising therapeutic efficacy in the DEN/CCl4 model (Fig. 6G).

NIK and HLA-ABC expression correlates with favorable survival in HCC patients

Lastly, we assessed the expression of NIK and HLA-ABC in serial sections of HCC tumor microarray. As shown in Figure S13A-C, elevated levels of NIK were associated with a favorable OS and DFS in HCC patients. In the cohort comprising 47 patients treated with anti-PD-1 in combination with Lenvatinib, high NIK levels were similarly correlated with extended PFS (Figure S13D). Spearman’s correlation analysis revealed a negative association between the expression of BIRC2 and NIK (Figure S13E).

Similarly, high expression of HLA-ABC suggested a favorable OS and DFS (Figure S14A-C). High HLA-ABC levels indicated a better PFS in HCC patients who received combination immunotherapy (Figure S14D). The level of HLA-ABC was negatively associated with BIRC2 (Figure S14E).

Furthermore, we assessed the expression of PD-L1 in HCC tissues. Unfortunately, PD-L1 levels did not correlate with survival outcomes (Figure S15A). Although the expression of BIRC2 and PD-L1 also displayed a negative correlation, it was not statistically significant (Figure S15B).

Collectively, we have provided a line of comprehensive evidence that BIRC2 promoted immune evasion of HCC via ubiquitination-dependent degradation of NIK and inhibition of MHC-I expression, thus inhibiting tumor antigen presentation. BIRC2 blockade facilitated immunotherapy of HCC by simultaneously sensitizing tumor cells to immune attack and boosting the anti-tumor immune response of T cells. (Fig. 6H).

Discussion

Immunotherapy has significantly increased the long-term survival of HCC patients. However, the overall response rate is still limited. Current immunotherapy approaches, primarily involving ICB, ACT, and personalized cancer vaccines, all aim to invigorate the immune system [29]. There is an urgent need to elucidate the tumor-intrinsic mechanisms of resistance to immunotherapy, and develop more effective therapeutic strategies for HCC.

Tumor cells employ diverse mechanisms to adapt to immune pressure, such as loss of antigenicity and reduction of immunogenicity. Both these factors contribute to the establishment of an immunosuppressive microenvironment, ultimately facilitating evasion from T cell-mediated cytolysis [30]. In this study, we identified BIRC2 as a key regulator of immune escape in HCC cells. We further demonstrated that elevated BIRC2 expression correlated with adverse prognosis and resistance to immunotherapy in HCC patients.

BIRC2 has been confirmed to play an important role in resistance to apoptosis in tumor cells [31, 32]. Recent in vitro and in vivo screenings suggested that loss of BIRC2 may enhance the sensitivity of tumor cells to immune attack in various malignancies, including renal cell carcinoma, melanoma, colorectal cancer, and breast cancer [33,34,35,36]. Whereas these studies did not delve into the precise mechanisms of BIRC2. It was reported that knockdown of BIRC2 upregulated CXCL9 expression and recruited inflammatory cells into tumor microenvironment in melanoma and breast cancer [37]. BIRC2 limited antigen presentation and T cell recognition of tumor cells by suppressing IRF1 activity in melanoma [38]. However, studies regarding BIRC2 and immunotherapy of HCC remained scarce. Here, we elucidated a novel signaling cascade in HCC in which BIRC2 inactivated the non-canonical NFκB pathway by interacting with and regulating the ubiquitination-dependent degradation of NIK. This finding aligned with a previous study which demonstrated that BIRC2 depletion in HIV-induced NIK-dependent activation of the NFκB signaling [39]. By inhibiting the processing of NFκB p100/p52, BIRC2 suppressed the downstream expression of MHC-I, which played a crucial role in antigen presentation. Loss of heterozygosity in MHC-I has been correlated with acquired resistance to ICB [40, 41]. Several investigations have sought to identify regulators of MHC-I and enhance the efficacy of immunotherapy [42, 43]. In our study, we observed no significant change in innate immunity, while BIRC-KO remarkably enhanced adaptive immunity, which suggested that BIRC2 fostered an immunosuppressive tumor microenvironment by downregulation of MHC-I expression and inhibition of antigen presentation. Thus, targeting BIRC2 may represent a promising strategy for promoting tumor antigen presentation in cancer immunotherapy.

Given the shared regulatory pathways of MHC-I and PD-L1, an increase in MHC-I levels often coincides with an elevated PD-L1 expression. Overexpression of PD-L1 enables tumor cells to evade immune attack and has been associated with post-operative recurrence in patients with HCC [44]. On October 21, 2022, the FDA approved anti-PD-L1 (Durvalumab) in combination with anti-CTLA-4 (Tremelimumab) therapy for adult patients with unresectable HCC based on the results from the HIMALAYA study [45]. However, our results did not reveal any significant correlation between the expression of PD-L1, patient survival, or the level of BIRC2. The impact of BIRC2 blockade on PD-L1-low HCC should be further investigated.

The BIRC2 antagonist used in this study, LCL161, demonstrated promising therapeutic effects in HCC. While some phase 1 clinical trials using LCL161 as monotherapy or in combination with chemotherapy drugs such as Topotecan in patients with advanced solid tumors reported failure [46, 47], LCL161 was found to elicit durable anti-tumor responses in patients with multiple myeloma (MM) or myelofibrosis in phase 2 clinical trials [48, 49]. LCL161 is administered orally and is well-tolerated in vivo, making it a promising candidate for clinical application.

Our study has some limitations. First, findings in cells and mouse models may not comprehensively represent the complexity of the tumor microenvironment in patients. The validation of these data in animal models with larger sample sizes or, ideally, in clinical settings, is essential to ensure their relevance in the treatment of HCC. Second, HCC is known for its heterogeneity, and the effectiveness of BIRC2 blockade may vary among patients. In our study, only approximately 30% of patients exhibited high expression of BIRC2. Further exploration is needed to determine which patients would benefit from BIRC2 antagonist therapy. Third, the newly updated American Association for the Study of Liver Disease (AASLD) guideline advocates that all HCC should be biopsied not only for diagnosis but also for targeted therapy. However, acquisition of tumor tissues via biopsy remains challenging. We have tried to test serum BIRC2 level using ELISA kits as a surrogate method, but failed. Fourth, HBV-associated HCC accounted for more than 90% of the cases in our cohort. The cell line PLC/PRF/5 used in mechanism experiments has been proved to secrete HBsAg. We focused on this subtype in the present study. Although the prevalence of HBV remains the major cause of HCC in the Asia-Pacific region, especially in China, NASH and alcohol-associated liver disease are on the rise [50]. The efficacy of BIRC2 blockade in HCC with non-viral etiologies needs to be further explored. Additionally, it is crucial to investigate the long-term and potential side effects of anti-BIRC2 therapy in HCC patients.

In conclusion, our study suggests that targeting BIRC2 represents a unique approach to facilitate the immunotherapy of HCC by simultaneously sensitizing tumor cells to immune killing and boosting the anti-tumor immune response of T cells. Future elucidation of the underlying mechanisms may pave the way for novel therapeutic strategies for HCC.

Data availability

All data were available in the main text or the supplementary materials. The data could be provided by Jingping Yun pending scientific review and a completed material transfer agreement. Raw sequencing data files were deposited in the Genome Sequence Archive for Human (GSA-Human) database (https://bigd.big.ac.cn/gsa-human/browse/HRA008191). Other raw data files were deposited in the Research Data Deposit (https://www.researchdata.org.cn/Search.aspx?k=RDDB2023991582).

Abbreviations

BIRC2:

Baculoviral IAP repeat containing protein 2

HCC:

Hepatocellular carcinoma

ICB:

Immune checkpoint blockade

NIK:

NFκB-inducing kinase

OS:

Overall survival

OVA:

Ovalbumin

PD1:

Programmed death protein 1

PFS:

Progression free survival

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Funding

This study was supported by grants from the National Natural Science Foundation of China (82203530, 82372978, 82072611, 82273046, 82103220, 82203002), the Guangdong Fundamental and Applied Fundamental Research Fund (2021A1515110959, 2023A1515010158) and the Cancer Innovative Research Program of Sun Yat-sen University Cancer Center (CIRP-SYSUCC-0042).

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  1. Lingyi Fu, Shuo Li and Jie Mei contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Oncology in South China, Guangdong Provincial Clinical Research Center for Cancer, Sun Yat-sen University Cancer Center, Guangzhou, China

    Lingyi Fu, Shuo Li, Jie Mei, Ziteng Li, Xia Yang, Chengyou Zheng, Nai Li, Yansong Lin, Chao Cao, Lixuan Liu, Liyun Huang, Xiujiao Shen, Yuhua Huang & Jingping Yun

  2. Department of Pathology, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, 510060, China

    Lingyi Fu, Shuo Li, Ziteng Li, Xia Yang, Chengyou Zheng, Nai Li, Yansong Lin, Chao Cao, Lixuan Liu, Liyun Huang, Xiujiao Shen, Yuhua Huang & Jingping Yun

  3. Department of Liver Surgery, Sun Yat-Sen University Cancer Center, Guangzhou, China

    Jie Mei

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Contributions

Conceptualization: Lingyi Fu, Jingping Yun. Methodology: Lingyi Fu, Shuo Li, Jie Mei, Ziteng Li, Xiujiao Shen, Xia Yang, Chengyou Zheng, Liyun Huang, Nai Li, Yansong Lin, Chao Cao, Lixuan Liu. Investigation and visualization: Lingyi Fu, Shuo Li, Jie Mei. Funding acquisition: Lingyi Fu, Yuhua Huang, Jingping Yun. Supervision: Lingyi Fu, Yuhua Huang, Jingping Yun. Writing & editing: Lingyi Fu, Yuhua Huang, Jingping Yun.

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Correspondence to Jingping Yun.

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Fu, L., Li, S., Mei, J. et al. BIRC2 blockade facilitates immunotherapy of hepatocellular carcinoma. Mol Cancer 24, 113 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12943-025-02319-5

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Keywords

Molecular Cancer

ISSN: 1476-4598