ARIH1 signaling promotes anti-tumor immunity by targeting PD-L1 for proteasomal degradation

Cancer expression of PD-L1 suppresses anti-tumor immunity. PD-L1 has emerged as a remarkable therapeutic target. However, the regulation of PD-L1 degradation is not understood. Here, we identify several compounds as inducers of PD-L1 degradation using a high-throughput drug screen. We find EGFR inhibitors promote PD-L1 ubiquitination and proteasomal degradation following GSK3α-mediated phosphorylation of Ser279/Ser283. We identify ARIH1 as the E3 ubiquitin ligase responsible for targeting PD-L1 to degradation. Overexpression of ARIH1 suppresses tumor growth and promotes cytotoxic T cell activation in wild-type, but not in immunocompromised mice, highlighting the role of ARIH1 in anti-tumor immunity. Moreover, combining EGFR inhibitor ES-072 with anti-CTLA4 immunotherapy results in an additive effect on both tumor growth and cytotoxic T cell activation. Our results delineate a mechanism of PD-L1 degradation and cancer escape from immunity via EGFR-GSK3α-ARIH1 signaling and suggest GSK3α and ARIH1 might be potential drug targets to boost anti-tumor immunity and enhance immunotherapies.

P rogrammed death ligand-1 (PD-L1) is constitutively expressed on the surface of cancer cells [1][2][3][4] . The interaction between PD-L1 and its receptor, programmed death protein-1 (PD-1), which is mainly expressed on the surface of T cells, results in cancer cell evasion from immune surveillance 5 . PD-1 and PD-L1 have become important immune checkpoint blockade immunotherapy targets for different types of cancers 5,6 . However, resistance to such immunotherapy approaches is prevalent and the mechanisms of resistance are not well understood.
Proteasomal degradation of PD-L1 has been reported to be promoted by cyclin D-CDK4-mediated phosphorylation followed by Cullin 3 SPOP -dependent ubiquitination 7 , as well as glycogen synthase kinase 3-β (GSK3β)-mediated phosphorylation followed by β-TrCP-dependent ubiquitination 8 . However, the dynamic ubiquitin modification of PD-L1 for proteasomal degradation should mostly occur in the intracellular segment of PD-L1, which is not addressed before. Ariadne-1 homolog (ARIH1) is a member of the Ariadne family of E3 ubiquitin ligases with a cognate E2 enzyme UBCH7 [9][10][11] . ARIH1 is known to play a role in protein translation regulation in response to DNA damage and to ubiquitinate EIF4E2 12 . The role of ARIH1 in PD-L1 degradation or anti-tumor immunity is not known.
Epidermal growth factor receptor (EGFR) is a major receptor tyrosine kinase, frequently overactivated in cancers, with established roles in cell growth and survival [13][14][15] . Patients with EGFRmutant-driven tumors develop resistance to EGFR tyrosine kinase inhibitor treatments, which are usually accompanied by the acquisition of EGFR mutations 16 . Clinical retrospectives suggest that EGFR mutations are associated with low response rates to immune therapies in non-small cell lung cancer 17,18 . Importantly, EGFR mutations are associated with increased PD-L1 expression [19][20][21] . EGFR activation promotes PD-L1 expression via Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling pathway 22 and inhibits its degradation via phosphorylation mediated by GSK3β 8 in the extracellular part of PD-L1, contributing to cancer escape from anti-tumor immunity. These findings strongly indicate that EGFR works as a critical regulator of tumor immune surveillance via regulation of PD-L1 expression, but the mechanism of this regulation is not fully understood, especially as the reported phosphorylation sites are in the extracellular part of PD-L1.
In this study, we screened a panel of 2125 Food and Drug Administration (FDA)-approved drugs or drug candidates and found that ES-072, a third-generation EGFR inhibitor, induced a potent degradation of PD-L1. Our mechanistic findings suggest that inhibition of EGFR activates GSK3α by suppressing AKT activity, which subsequently promotes the phosphorylation at the intracellular Ser279 and Ser283 residues of PD-L1, leading to ARIH1-mediated ubiquitination and proteasome-mediated degradation. Cancer-associated mutation of ARIH1 compromises ubiquitination of PD-L1. Together, our results suggest ARIH1 is the E3 ligase for PD-L1, which could lead to the development of therapeutic strategies to overcome immunotherapy resistance in cancers and enhance checkpoint blockade therapy efficacy.

Results
A high-throughput screen of 2125 FDA-approved drugs or drug candidates identifies promoters of PD-L1 degradation. To establish a fluorescence-based high-throughput screening protocol for the membrane levels of endogenous PD-L1, we used a phycoerythrin-conjugated anti-PD-L1 antibody and interferon-γ (IFNγ)-treated U937 cells (histiocytic lymphosarcoma cell line). IFNγ enhances the basal expression levels of PD-L1, allowing for a wider dynamic range and, thus, a better screening system 23,24 .
Out of 2125 FDA-approved drugs or drug candidates screened, 160 were found to reduce the membranal PD-L1 levels (Supplementary Fig. 1e and Supplementary Table 1). We classified the positive hits according to the signaling pathways they were known to be involved in, which included the JAK/ STAT pathway, the phosphatidylinositol 3-kinase (PI3K)/AKT/ mammalian target of the rapamycin pathway, pathways that regulate the cell cycle, and protein tyrosine kinases (Fig. 1a). The screen confirmed the existing knowledge about PD-L1 regulation, as 13 different JAK inhibitors were found to decrease plasma membrane PD-L1 levels by more than 25%; this also indicated that the screen setup is valid, as JAK was the top drug target inhibition of which resulted in the PD-L1 level decrease.
On the other hand, EGFR has also been strongly linked to PD-L1 protein level regulation. From the EGFR inhibitors that promote PD-L1 level decrease, AZD9291 and ES-072 were selected for our screen follow-up and mechanistic studies to investigate what drives PD-L1 degradation downstream of EGFR inhibition. AZD9291, Osimertinib, is a potent and selective mutant EGFR (L858R/ T790M) inhibitor, which is widely used in clinic 26,27 . ES-072 is a third-generation EGFR inhibitor designed by our team to overcome drug-resistance-induced EGFR mutation L858R/T790M. Confirming the screen findings, both ES-072 and AZD9291 dramatically reduced the IFNγ-induced membranal PD-L1 levels in U937 cells following treatment with the compounds, as judged by western blotting and flow cytometry results ( Fig. 1b-d). The effect of the EGFR inhibitors on PD-L1 levels was time-dependent, reaching a plateau following 12 h of treatment ( Fig. 1e-g). Similar findings were seen in H1975 cells ( Supplementary Fig. 2a-d) and interleukin-4 (IL-4)-treated peritoneal-derived macrophages (PDMs) (Supplementary Fig. 2e-g).
EGFR inhibitors are known to promote PD-L1 degradation via the GSK3α/β-TrCP pathway 8 . Surprisingly, contrary to the previous reports, we found that knockdown of β-TrCP did not fully block the reduction of PD-L1 levels induced following EGFR inhibition (Fig. 1h, i), whereas GSK3α/β inhibitor LY2090314 did block this reduction ( Fig. 1j-l). LY2090314 was found to be highly specific for GSK3α with an half maximal inhibitory concentration (IC50) value of 0.87 ± 0.09 nM (Supplementary Table 2). These results indicated that a β-TrCP-independent, yet GSK3α/βdependent mechanism regulating PD-L1 levels downstream of EGFR should exist.
ES-072 promotes proteasomal PD-L1 degradation via EGFR inhibition. The rest of our studies were continued using ES-072 due to its stability and specificity (Supplementary Table 3). As, upon EGFR inhibition, PD-L1 has been shown to be degraded via the proteasome, we first determined whether ES-072 treatment also results in a proteasome-dependent degradation of PD-L1. Both western blotting and flow cytometry analysis of plasma membrane PD-L1 levels confirmed that EGFR inhibition results in a proteasome-mediated PD-L1 degradation, as the decrease in the PD-L1 levels induced by ES-072 was rescued by the proteasome inhibitor MG132 ( Supplementary Fig. 3a-c).
In accord with previous reports 28,29 , EGF treatment promoted an increase of PD-L1 levels ( Supplementary Fig. 3d-f), whereas small interfering RNA (siRNA)-mediated knockdown of EGFR decreased PD-L1 levels ( Supplementary Fig. 3g). ES-072 was found to be highly specific for EGFR in an in vitro kinase-profiling screen, where it was found to inhibit wild-type, T790M, and T790M/L858R EGFR with IC50 values of 8.9, <0.5, and 1.75 nM, respectively (Supplementary Table 3). Furthermore, the decrease of PD-L1 levels induced by ES-072 is dependent on EGFR ( Supplementary Fig. 3h). These results suggest that the effect of ES-072 on PD-L1 is via EGFR, consistent with the notion that ES-072 treatment targets PD-L1 for proteasomal degradation, whereas EGF treatment decreased the basal level of these ubiquitination events ( Supplementary Fig. 3i). Together, these data indicated that ES-072 induced the decrease of PD-L1 through inhibition of EGFR, induction of K48-linked ubiquitination, and proteasomal degradation.
PD-L1 degradation depends on its phosphorylation at Ser279 and Ser283. To determine the mechanism behind ES-072-induced PD-L1 degradation, we tested whether PD-L1 undergoes any phosphorylation changes following treatment with this EGFR inhibitor. As judged by mass spectrometry, ES-072 induced several PD-L1 phosphorylation events (Fig. 2a-c and Supplementary  Table 4). The top two most robustly induced phosphorylation events on the cytosolic side of PD-L1 were on Ser279 and Ser283 (Fig. 2a, b and Supplementary Table 4). It should be noted that both Ser279 and Ser283 are located in the intracellular segment of PD-L1 (Fig. 2c). Mutating these residues to the phosphorylation-resistant alanine showed that both of the sites play an important role in ES-072-induced PD-L1 degradation, and that dual S279A; S283A (2SA) mutation blocks PD-L1 degradation more potently than either S279A or S283A alone (Fig. 2d). Consistently, chase experiments using protein synthesis inhibitor cycloheximide showed that these U937 cells were incubated with IFNγ (100 ng/mL) for 48 h, treated with the drugs at 10 μM for 12 h. Ruxolitinib (Rux) was used as a positive control. The hit compounds that induced the decrease of PD-L1 levels are shown in blue. The depth of blue represents decreased level of PD-L1. The heatmap represents the targeted pathways obtained from the high-throughput screening, based upon decreased membrane PD-L1 level detected by flow cytometry. b Immunoblotting of PD-L1 in U937 cells treated with ES-072 (ES) or AZD9291 (AZD) at indicated concentrations for 12 h. ES and AZD are EGFR inhibitors. c, d Median fluorescence intensity (MFI) (c) and relative quantification (d) of PD-L1 in U937 cells treated with 10 μM ES-072 or 10 μM AZD9291 for 12 h. Data represent means ± SEM, n = 18, 6 independent repeats, ****P < 0.0001. e-g Immunoblotting (e, f) and flow cytometry (g) analysis of PD-L1 levels in U937 cells treated with 10 μM ES-072 or 10 μM AZD9291 for the indicated times. h, i Immunoblottings (h) of PD-L1 and β-TrCP, relative quantification (i) of PD-L1 MFI in H1975 cells transfected with non-targeting siRNA (si-CTRL) or β-TrCP siRNAs (si-β-TrCP) and treated with or without 10 μM ES-072/AZD9291 for 12 h. Data represent means ± SEM, n = 9, 3 independent repeats, NS: no significant; ****P < 0.0001. j-l Immunoblotting (j) and flow cytometry analysis (k) with relative quantification (l) of PD-L1 in H1975 cells treated with 10 μM ES-072 or AZD9291, and/or 5 μM LY2090314 (LY) for 12 h. LY is a GSK3 inhibitor. Data represent means ± SEM, n = 9, 3 independent repeats, ****P < 0.0001. Source data are provided as a Source Data file. phosphorylation events are critical for PD-L1 turnover, as the 2SA mutation delayed PD-L1 degradation (Fig. 2e).
To confirm that endogenous PD-L1 is phosphorylated following ES-072 treatment, we generated a phospho-specific antibody against PD-L1 Ser279. Consistent with mass spectrometry data, ES-072 treatment induced robust phosphorylation at Ser279 in both endogenous and recombinant PD-L1, which was blocked by the S279A mutation, indicating the specificity of the antibody (Fig. 2f, g). Notably, immunohistochemistry and western blotting analysis of tumor biopsies obtained from alveolar adenocarcinoma patients showed that PD-L1 phosphorylation levels at Ser279 are higher in EGFR-WT tumors compared to EGFR-mutant-driven tumors, and this difference in phosphorylation levels correlated with lower PD-L1 levels in EGFR-WT tumors ( Fig. 2h-k). The EGFR mutations found in these patients were L858R, E542K, P753R, and Δex19, which have been linked to the activation of EGFR and the resistance to EGFR inhibitors in non-small cell lung cancer 30 .
These results indicate that PD-L1 phosphorylation events at Ser279 and Ser283 are regulated downstream of the EGFR signaling pathway and are critical for PD-L1 degradation.
To determine the kinase responsible for the Ser279/283 phosphorylation events that drive PD-L1 degradation following ES-072 treatment, we performed a large-scale PD-L1 immunoprecipitation experiment followed by mass spectrometry-based proteomic analysis, which revealed several interactors of PD-L1 (Fig. 3b). These results were also confirmed by western blotting experiments (Fig. 3c). Notably, the top endogenous kinase, the interaction of which with PD-L1 was enhanced by ES-072 treatment, was found to be GSK3α, but not GSK3β, despite equal expression levels for GSK3α and GSK3β in HEK293T cells (Fig. 3b, c). This result was also confirmed by tunicamycin treatment, a specific N-linked glycosylation inhibitor, using HEK293T cells with exogenous PD-L1 expression (Supplementary Fig. 4a-c).
We further validated these findings by testing for the ability of ES-072 to induce the interaction between PD-L1 and endogenous GSK3α, using proximity ligation assay (PLA), which showed that there is a basal level of GSK3α/PD-L1 interaction, which is enhanced upon ES-072 treatment (Fig. 3d). Interestingly, Ser279 and Ser283 are also a potential match for the consensus motif of Fig. 4d) 32,33 . ES-072 treatment promoted GSK3α activation in an EGFR-and AKTdependent manner, as judged by the decrease of phospho-GSK3α at Ser21, which is a marker of GSK3α inhibition (Fig. 3e). ES-072 showed a higher activation effect on GSK3α when compared to the other two EGFR inhibitors, as judged by loss of the inhibitory phosphorylation of GSK3α ( Supplementary Fig. 4e). These findings suggest that GSK3α could be the kinase that directly phosphorylates PD-L1 at Ser279/283 following EGFR inhibition by ES-072.
Consistent with this hypothesis, purified GSK3α robustly phosphorylated wild-type, but not 2SA synthetic peptides in vitro, at Ser279, which we used to represent the intracellular region of PD-L1 (Fig. 3f). Furthermore, purified GSK3α also robustly phosphorylated purified PD-L1 in vitro, at Ser279 (Fig. 3g). Consistently, knockdown of GSK3α, but not GSK3β, blocked the phosphorylation of PD-L1 at Ser279 (Fig. 3h and Supplementary Fig. 4f). These results indicate that GSK3α directly phosphorylates PD-L1 at Ser279. In agreement with the data showing that Ser279/Ser283 phosphorylation is important for ES-072-induced PD-L1 degradation, and that these sites are phosphorylated by GSK3α, knockdown of GSK3α in U937 and H1975 cells partially rescued PD-L1 degradation induced by ES-072 ( Fig. 3i and Supplementary Fig. 4g). Similar results were seen when GSK3α was knocked down in HEK293T cells overexpressed with Flag-PD-L1 (Fig. 3j).
Overall, these findings showed that ES-072 treatment results in inhibition of EGFR and activation of GSK3α, which phosphorylates PD-L1 at its cytosolic Ser279 and Ser283 residues to target PD-L1 for proteasomal degradation. Our results also indicate that this mechanism is distinct from the previously reported phosphorylation of PD-L1 by GSK3β at T180 and S184, at the extracellular region of PD-L1 8 .
E3 ubiquitin ligase ARIH1 directly ubiquitinates PD-L1 and targets it for proteasomal degradation, following EGFR inhibition. To determine which E3 ubiquitin ligase promotes PD-L1 ubiquitination and proteasomal degradation following ES-072 treatment, we analyzed our mass spectrometry immunoprecipitation data for the presence of known mediators of ubiquitination. In addition to cullin ligases, which are known to mediate PD-L1 degradation 7,8 , we found that ES-072 promoted PD-L1 interaction with E3 ubiquitin ligases called ARIH1 and ring finger protein 25 (RNF25) 34 (Fig. 4a).
These results indicate that ARIH1 directly ubiquitinates PD-L1 and targets it for proteasomal degradation, following ES-072induced inhibition of EGFR.
Phosphorylation of PD-L1 at Ser279/283 mediated by GSK3α promotes PD-L1/ARIH1 interaction and subsequent PD-L1 ubiquitination and degradation. We next tested whether GSK3α-mediated PD-L1 phosphorylation at Ser279/283 is the mechanism of recruitment of ARIH1 to PD-L1. HEK293T cells stably expressing ARIH1-HA were transfected with wild-type PD-L1 or the phosphorylation-resistant S279A, S283A, and 2SA mutants. Remarkably, PD-L1 ubiquitination induced by ARIH1 overexpression was partially reduced by the S279A and S283A mutations, and strongly inhibited by the 2SA double mutation of these GSK3α phosphorylation sites (Fig. 5a). In accord with this, ARIH1 interaction with PD-L1 was strongly reduced by the 2SA mutation (Fig. 5a, HA blot in the IP-Flag-PD-L1 panel).
These findings suggest that PD-L1 degradation is mediated by its GSK3α-driven phosphorylation at Ser279/283 and subsequent recruitment of ARIH1 to ubiquitinate PD-L1 via K48-linked ubiquitin chains that target it for proteasomal degradation. ARIH1 promotes anti-tumor immunity via PD-L1 degradation. Immunohistochemistry analysis of biopsies obtained from healthy volunteer lung tissues (control) and lung cancer patients showed that, although protein levels of PD-L1 and phospho-GSK3α (i.e., inhibited GSK3α) were strongly elevated in tumor tissues, ARIH1 protein levels were higher in control samples ( Supplementary Fig. 7). This finding is consistent with our discovery that ARIH1 promotes PD-L1 degradation and suggest that loss of ARIH1 expression in cancer is a mechanism of PD-L1 accumulation that drives escape from anti-tumor immunity. The mechanisms that result in decreased ARIH1 expression levels in cancer remain to be elucidated.
Notably, ARIH1 mutation found in large cell lung carcinoma (Y392C) 35 blocked ARIH1-induced PD-L1 ubiquitination and degradation (Fig. 6a, b), indicating that mutational inactivation of ARIH1 in cancer could lead to accumulation of PD-L1, to promote escape from anti-tumor immunity. This observation suggests a potential mechanism of why this mutation occurs in clinical patients with large cell lung carcinoma.
We established a 4T1 cell line with stable ARIH1-OE to evaluate the role of ARIH1 in PD-L1 degradation and tumorigenesis. In accord with our previous findings, ARIH1-OE reduced the protein levels of PD-L1 (Fig. 6c). ARIH1 overexpression had no effect on cell proliferation in vitro (Supplementary Fig. 8a) and on tumor growth in immunodeficient nude mice ( Supplementary Fig. 8b-d). However, we observed a dramatically suppressed tumor growth in the ARIH1-OE group in immunocompetent BALB/c mice, the majority of which exhibited a complete tumor regression (Fig. 6d-f). The levels of total and activated CD8 + cytotoxic T cells (GzmB + ) that infiltrated the tumor microenvironment were significantly increased in the ARIH1-OE group (Fig. 6g, h and Supplementary  Fig. 9). Consistently, ARIH1 overexpression resulted in an increased expression of inflammatory cytokines, including IFNγ, tumor necrosis factor-α, and T-cell chemokines C-C motif chemokine ligand 5 (CCL-5) and C-X-C motif chemokine ligand 10 (CXCL-10), as judged by quantitative reverse transcription   Fig. 3b, except ubiquitination-related proteins are shown in the heatmap. b Co-IP analysis for the interaction of ARIH1 and endogenous PD-L1 in HEK293T cells transfected with HA-ARIH1; HA-tagged empty vector (HA-EV) was transfected as a negative control. c Recombinant PD-L1 was purified using a GST pull-down assay and incubated with HEK293T lysates, which were transfected with HA-ARIH1. The interaction between ARIH1 and PD-L1 was detected by Immunoblot assay. d Co-IP analysis for the interaction of ARIH1 and PD-L1 in HEK293T cells transfected with HA-ARIH1 and Flag-PD-L1, treated with or without 10 μM ES-072 for 24 h; Flag-tagged empty vector (Flag-EV) was transfected as a negative control. e, f PD-L1 level in 20 μg/mL cycloheximide (CHX)-treated HEK293T cells transfected with or without HA-ARIH1 (e) and ARIH1-siRNA (f). g, h Immunoblots of PD-L1 and ARIH1 (HA) in H1975 cells transfected with ARIH1-HA (g) or ARIH1-siRNAs (h). i HEK293T cells were transfected with Flag-PD-L1. Co-IP analysis for the interaction of K48-ubiquitin and PD-L1 in HEK293T cells transfected with ARIH1-siRNAs or HA-ARIH1 and treated with MG132 (10 μM, a proteasome inhibitor) for 6 h. j Immunoblots of PD-L1 and ARIH1 in U937 cells transfected with ARIH1-siRNAs, following treatment with 10 μM ES-072 for 24 h. k HEK293T cells were transfected with HA-K48-ubiquitin and Flag-PD-L1. Co-IP analysis for the interaction of K48-ubiquitin and PD-L1 in HEK293T cells transfected with ARIH1-siRNAs and treated with or without 10 μM ES-072 for 24 h. l Recombinant PD-L1 and ARIH1 were purified in transfected HEK293T cells, respectively. An in vitro ubiquitination assay of PD-L1 was performed with purified GST-UBA1 (E1), His-UBCH7 (E2) in the presence or absence of ubiquitin or HA-ARIH1 (E3). Source data are provided as a Source Data file.  Fig. 10a-d).

PCR (qRT-PCR) of the whole tumor mRNA (Supplementary
Recent studies have explored to intercept two immune checkpoint pathways, PD-L1 and CTLA4, collectively, to improve the efficacy of immunotherapy 36 . Next, we used the 4T1 tumor xenograft model and performed an anti-CTLA4 immune checkpoint blockade treatment in the presence/absence of ES-072. Tumor growth was significantly decreased both by the ES-072 treatment and the anti-CTLA4 treatment. Notably, a further decrease of tumor growth and even complete regression was observed when ES-072 and anti-CTLA4 treatments were combined (Fig. 6i-k). The levels of total and activated CD8 + cytotoxic T cells (GzmB + ) in the tumors were also significantly increased upon the ES-072/anti-CTLA4 combination treatment when compared to either of the single treatments (Fig. 6l, m). Furthermore, western blotting analysis of the tumor lysates showed that ES-072 treatment decreased PD-L1 levels, while increasing its phosphorylation and ubiquitylation levels (Fig. 6n).
Taken together, our data indicate that ARIH1 plays a role in promoting anti-tumor immunity, and that ES-072 is a promising agent to boost anti-tumor immunity and anti-CTLA4 immune checkpoint blockade immunotherapies.

Discussion
Our study identified that ES-072, an EGFR inhibitor, is an anti-PD-L1 agent that induces a signaling cascade downstream of GSK3α activation. Following ES-072 treatment, GSK3α phosphorylates PD-L1 at Ser279 and Ser283, which promotes recruitment of the E3 ubiquitin ligase ARIH1 that marks PD-L1 for proteasomal degradation (Fig. 7). Our tumor xenograft experiments revealed an important function of ARIH1 in promoting anti-tumor immunity. This finding indicates that various cancer therapies, including immune checkpoint blockade and cell-based immunotherapies, can be enhanced by supplementation of this inhibitor into the treatment regimens, as not only will it drive EGFR inhibition, decreasing cancer growth, but it will also drive degradation of PD-L1, enhancing anti-tumor immunity.
Recent studies show that PD-L1 expression and degradation levels are regulated by several protein tyrosine kinases, which seem to converge on the PI3K-AKT-GSK3 pathway, yet the mechanisms of PD-L1 degradation regulation were not fully clear [37][38][39] . Our findings suggest that the EGFR-AKT-GSK3α-ARIH1 axis is critical for the regulation of PD-L1 degradation.
These results also provide a broad insight into how cancer cells with RTK-activating mutations that drive inhibition of GSK3 may promote escape from anti-tumor immunity by preventing PD-L1 degradation and place RTK inhibitors that result in activation of GSK3 as putative enhancers of the PD-1/PD-L1 immune checkpoint blockade immunotherapies. Our work may pave the way for the augmentation of current immunotherapies that involve targeting PD-1/PD-L1 and help to overcome resistance to such therapies.
GSK3α and GSK3β share 97% amino acid similarity in their kinase domains 40 . The isoforms have both unique and overlapping functions, and one isoform cannot completely compensate for the loss function of another one. The most significant evidence for this is that GSK3α-deficient mice show increased insulin sensitivity 41 , whereas GSK3β deletion in mice results in late embryonic death 42 . Growth factors, such as EGF, result in inactivation of GSK3 through Ser9 phosphorylation of GSK3α or Ser21 phosphorylation of GSK3β by AKT 43,44 .
GSK3β has been reported to promote degradation of PD-L1 via phosphorylation at extracellular T180 and S184, contributing to anti-tumor immunity 8 . These two phosphorylation sites are located in the extracellular region of PD-L1, and their control by GSK3β is not fully understood. In this study, we found that GSK3α, but not GSK3β, co-immunoprecipitated with glycosylated PD-L1 following EGFR inhibition by ES-072 (Supplementary Fig. 4a-c). Our mechanistic studies showed that GSK3α phosphorylates PD-L1 at Ser279/283, at the cytosolic region of the receptor (Fig. 2c). The dual-mode of PD-L1 regulation by GSK3α via its cytosolic region and GSK3β via its extracellular region highlights the importance of oncogenic signaling pathways that converge on the EGFR-AKT-GSK3α/β axis in suppressing PD-L1 degradation and therefore promoting cancer escape from anti-tumor immunity [45][46][47] . Whether GSK3α or GSK3β on PD-L1 regulation are the only critical factors remains to be determined.
GSK3 is a tumor suppressor, with important roles in both solid tumors and blood tumors [48][49][50][51] , where it regulates cancer cell viability and proliferation 52,53 . Aberrant expression of GSK3α in lung cancer has prognostic significance for clinical treatment 54 . In light of these reports and findings of our study, GSK3-activating agents are likely to promote a strong PD-L1 degradation phenotype and inhibit cancer escape from anti-tumor immunity, thereby enhancing various cancer therapies, including immunotherapies.
In addition to ARIH1, PD-L1 is also targeted for degradation by Cul3 SPOP and β-TrCP 7,8 E3 ubiquitin ligases. Together, these findings suggest that combination treatments that target more than one of these E3 ubiquitin ligases may have potentially additive/synergistic effects on PD-L1 degradation in cancer, allowing a more potent stimulation of anti-tumor immunity.
In summary, our study sheds light on the mechanisms of cancer escape from anti-tumor immunity via increased PD-L1 protein levels downstream of EGFR overexpression and overactivation in cancer. Our work suggests that GSK3α-and ARIH1activating agents, as well as EGFR inhibitors, are likely to stimulate anti-tumor immunity and therefore enhance existing cancer therapies by triggering the PD-L1 degradation pathway this work delineates. PDMs were obtained from male BALB/c mice. PDMs, U937, and H1975 cells were cultured in RPMI-1640 (Hyclone, with L-glutamine); HEK293T cells were cultured in Dulbecco's modified Eagle's medium (Hyclone, with L-glutamine, with 4.5 g/L glucose, without pyruvate). These media were supplemented with 10% heatinactivated fetal calf serum (Gibco) and 1% Penicillin/Streptomycin (Gibco). All cells were cultured at 37°C with 5% CO 2 . Cells were transiently transfected with DNA using Lipofectamine 2000 or Lipofectamine 3000.

Methods
Plasmids construction and RNA interference. Flag-PD-L1 (WT and mutants), GSK3α-HA, and ARIH1-HA (WT and mutants) were amplified by PCR and fused the fragments into pCMV3 via seamless cloning. The constructs and siRNAs were transfected into cell lines (HEK293T, H1975, and U937) with Lipofectamine 2000 or Lipofectamine 3000, according to the the manufacturer's protocol. siRNAs used in this study are provided in Supplementary Table 5.
In vitro high-throughput drug screens. U937 cells (2 × 10 5 ) were preincubated with 100 μg/L IFNγ for 48 h and plated in 96-well plates per well (Corning). Then 10 μM FDA-approved drugs or drug candidates were added and incubated for 12 h. All compounds were commercially purchased. Treatments were performed twice; each plate contained a negative control (dimethyl sulfoxide) and a positive control (Ruxolitinib). U937 cells were centrifuged for 5 min at 1000 × g and the supernatant discarded. The collected cells were washed with phosphate-buffered saline (PBS) twice and incubated with PE anti-human CD274 at 4°C for 30 min. Then the incubated U937 cells were washed and resuspended with PBS, and the protein level of membrane PD-L1 reflected by PD-L1-PE median fluorescence intensity (MFI) was determined using flow cytometry analysis. The hit compounds were picked and classified according to the PD-L1-PE-MFI and the targeted pathways.
Western blot analysis. For western blot analysis, cells were collected and washed with PBS, then lysed in radioimmunoprecipitation assay (RIPA) buffer (1% Triton X-100, 100 mM Tris-HCl pH 8.8, 100 mM NaCl, 0.5 mM EDTA). After incubation on ice for 30 min, the lysates were centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was collected and the protein concentration was measured by bicinchoninic acid reaction. Protein samples were added with 2× loading buffer and heated at 100°C for 10 min, separated with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride membranes, and blocked with 5% non-fat milk in Phosphate Buffered Saline with 0.1% Tween-20 (PBST) for 1 h at room temperature. The membranes were probed with the corresponding primary antibodies at 4°C overnight and horseradish peroxidaseconjugated secondary antibodies at room temperature for 1 h. Signals were detected using chemiluminescence reagents (#4AW001-500, 4A649 Biotech, Co.).
Immunoprecipitation. For immunoprecipitation between PD-L1 and GSK3α/ ARIH1/K48, cells were lysed in RIPA buffer (1% Triton X-100, 100 mM Tris-HCl pH 8.8, 100 mM NaCl, 0.5 mM EDTA) supplemented with a complete protease inhibitor cocktail (Bimake, added fresh), and mixed with antibodies at 4°C for 4 h; protein A/G agarose beads were added and incubated at 4°C overnight. Beads were washed three times with RIPA buffer and subjected to western blotting.
Mass spectrometry and data analysis. Flag-tagged PD-L1 was overexpressed in HEK293T cells and were trypsin-digested. PD-L1 was immunoprecipitated with beads following immunoprecipitation. The resulting peptides were subjected to the phosphopeptide enrichment using TiO 2 beads. The enriched phospho-peptides were analyzed on the Q Exactive™ HF mass spectrometer (Thermo Fisher Scientific). The identification and quantification of phosphorylated peptides were done by MaxQuant. The tandem mass spectra were searched against the UniProt human protein database together with a set of commonly observed contaminants. The precursor mass tolerance was set as 20 p.p.m. and the fragment mass tolerance was set as 0.1 Da. The 33 cysteine carbamide methylation was set as a static modification and the methionine oxidation, as well as serine, threonine, and tyrosine phosphorylation, were set as variable modifications. The false discovery rate (FDR) at peptide spectrum match level was controlled below 1%.
Duolink® PLA fluorescence analysis. For the interaction between PD-L1 and GSK3α/GSK3β, the samples were pre-treated with respect to fixation, retrieval, and/or permeabilization. Then the samples were incubated with Duolink® Blocking Solution in a heated humidity chamber for 60 min at 37°C. The samples were incubated with diluted primary antibody in a humidity chamber overnight at 4°C. The samples were washed with 1× Wash Buffer A at room temperature for 5 min twice after the primary antibody solution was moved then incubated with diluted PLUS, and MINUS PLA probes (1 : 5) in a pre-heated humidity chamber for 1 h at 37°C. The samples were washed with 1× Wash Buffer A at room temperature for 5 min twice after the PLA probes were moved, then incubated with ligation solution in a pre-heated humidity chamber for 30 min at 37°C. The samples were washed with 1× Wash Buffer A at room temperature for 5 min twice after the ligation solution was moved then incubated with amplification solution in a pre-heated humidity chamber for 100 min at 37°C. The samples were washed with 1× Wash Buffer B for 10 min twice and 0.01x Wash Buffer B for 1 min at room temperature after the amplification solution was moved. The slides were mounted with a coverslip using a minimal volume of Duolink® PLA Mounting Medium with 4′,6diamidino-2-phenylindole analyzed with confocal microscope. The images were collected using Cytation 3 and were analyzed using Gen5 2.0.
Flow cytometry analysis of membrane PD-L1. For flow cytometric analysis for membrane PD-L1, H1975 or HEK293T cells were collected by centrifugation at 1000 × g for 5 min, incubated with PBS (0.5% bovine serum albumin) for 10 min at room temperature. The cells were probed with PE-conjugated PD-L1 antibody (#329706, Biolegend) and a matched isotype control at 4°C for 30 min in the dark. After washing three times with PBS, the cells were analyzed using flow cytometry (Beckman Coulter Cytoflex) and data were analyzed using CytExpert V2.3 and and FlowJo X software.
GST-pull down. GST-tagged PD-L1 was expressed in Escherichia coli BL21 and purified. HA-tagged ARIH1 was expressed in HEK293T cells and purified using magnetic HA beads. For reaction, purified GST-PD-L1 was first incubated with glutathione-Sepharose 4B beads at 4°C for 1 h. Then, the HA-tagged protein was added and incubated at 4°C for 2 h. The mixture was boiling with 1× loading buffer for 10 min, then subjected to western blotting.
In vitro ubiquitination assays. Plasmids GST-UBA1 (E1), Flag-PD-L1, HA-ARIH1 (E3), and ARIH1 inactive mutant were transfected into HEK293T cells. Post transfection, cells were collected and lysed in RIPA Lysis Buffer at 4°C for 1 h. Then, the lysates were incubated with indicated beads at 4°C overnight. His-tagged protein UBCH7 (E2) ubiquitin was purified by E. coli BL21 expression. Reactions were performed in a 30 μL reaction mixture at 37°C for 2 h in the presence of His-Ub, E1, E2, E3, Flag-PD-L1, ATP regeneration solution (Enzo Life Sciences) and Ubiquitin Reaction Buffer (Enzo Life Sciences). All reactions were terminated by boiling 10 min with SDS sample buffer and then subjected to western blotting.
Immunohistochemistry. EGFR-WT tumors and EGFR-mutant-driven tumors from primary lung adenocarcinoma tissues were obtained from 8 patients (4 cases each group, median age: 60 years old, range from 47 to 81) at the Sir Run Run Shaw Hospital, Zhejiang University. The four EGFR mutations are L858R point mutation of exon 21; E542K point mutation of exon 10 and deletion of exon 19 (ex19del 745-750); P753R point mutation of exon 19; L858R point mutation of exon 21. All samples were collected with signed informed consent according to the internal review and ethics boards of Sir Run Run Shaw Hospital. These tissues were rapidly excised, fixed with 4% paraformaldehyde, and embedded in paraffin for tissue sections (5 μm thick) and immunohistochemical staining. The primary antibodies used are anti-PD-L1 (1 : 200), anti-p-PD-L1 (Ser279) (1 : 200), anti-p-GSK3α (1 : 200), and anti-ARIH1 (1 : 200). Visualization of cell nuclei was performed with hematoxylin and analysis was done using the Olympus BX61 light microscope.
Generation of ARIH1-OE stable 4T1 cell lines. Briefly, HEK293T cells were transfected with PCDH-CTRL and PCDH-mouse ARIH1 with packaging plasmids. Medium with secreted viruses was collected at 48, 72, and 96 h, and was filtered through 0.45 μm filters. Twenty-four hours post infection, the medium was replaced with fresh medium and the infected 4T1 cells were selected with 4 μg/mL puromycin for 3 days.
Tumor xenograft experiments. Female BALB/c mice or nude mice (aged 8-10 weeks) were purchased from Shanghai SLAC Laboratory Animal, Co., Ltd (Shanghai, China). All the animal experiments were strictly conducted in accordance with the protocols approved by the Ethics Committee for Animal Studies at Zhejiang University, China.
For xenograft model with control or ARIH1-stable 4T1 cells, 5 × 10 5 control or ARIH1-stable 4T1 cells suspended in 50 μL PBS and Matrigel (1 : 1 v/v) were injected into the fourth breast fat pad. On days 3-5 after injection, tumor size was measured and calculated by using the formula 1/2 × length × width 2 . Tumor weight was recorded on the day of killing.
Immunoprecipitation assay with mouse tumor tissues. For immunoprecipitation between PD-L1 and ubiquitin, fresh xenograft tissues were lysed in RIPA buffer (1% Triton X-100, 100 mM Tris-HCl pH 8.8, 100 mM NaCl, 0.5 mM EDTA) supplemented with a complete protease inhibitor cocktail (Bimake, added fresh) and mixed with anti-PD-L1 at 4°C for 4 h; protein A/G agarose beads were added and incubated at 4°C overnight. Beads were washed three times with RIPA buffer and subjected to western blotting.
qRT-PCR analysis for tumor cytokines. Fresh tumor tissues were lysed and total RNA was extracted using TRIzol™ Plus RNA Purification Kit (Invitrogen). cDNA was synthesized from purified RNA using the PrimeScript RT reagent Kit (TAKARA, RR047A) according to the manufacturer's instructions. Quantitative PCR was performed using a StepOnePlus Real-Time PCR Systems (ABI). The comparative Ct method was used for the data analysis and mouse β-actin mRNA was used as an internal control. The sequences of primers used for qRT-PCR are provided in Supplementary Table 6.
Statistics and reproducibility. Numerical data are presented as means ± SEM; all data analyses were performed using GraphPad Prism (version 7.0, GraphPad Software, Inc.). Unpaired Student's t-test was used to analyze the flow cytometry data. One-way analysis of variance was used to analyze the statistical differences among the groups with P-values indicated in the related graphs. The level of statistical significance was set at a p < 0.05. All assays were carried out at least three independent times with the same results.