Irreversible JNK blockade overcomes PD-L1-mediated resistance to chemotherapy in colorectal cancer
Lei Sun1,2, Árpád V. Patai1, Tara L. Hogenson3, Martin E. Fernandez-Zapico3, Bo Qin 1 ✉ and Frank A. Sinicrope 1,4,5 ✉
© The Author(s), under exclusive licence to Springer Nature Limited 2021
Colorectal cancer (CRC) cells have low or absent tumor cell PD-L1 expression that we previously demonstrated can confer chemotherapy resistance. Here, we demonstrate that PD-L1 depletion enhances JNK activity resulting in increased BimThr116 phosphorylation and its sequestration by MCL-1 and BCL-2. Activated JNK signaling in PD-L1-depeted cells was due to reduced mRNA stability of the CYLD deubiquitinase. PD-L1 was found to compete with the ribonuclease EXOSC10 for binding to CYLD mRNA. Thus, loss of PD-L1 promoted binding and degradation of CYLD mRNA by EXOSC10 which enhanced JNK activity. An irreversible JNK inhibitor (JNK-IN-8) reduced BimThr116 phosphorylation and unsequestered Bim from MCL-1 and BCL-2 to promote apoptosis. In cells lacking PD-L1, treatment with JNK-IN-8, an MCL-1 antagonist (AZD5991), or their combination promoted apoptosis and reduced long-term clonogenic survival by anticancer drugs. Similar effects of the JNK inhibitor on cell viability were observed in CRC organoids with suppression of PD-L1. These data indicate that JNK inhibition may represent a promising strategy to overcome drug resistance in CRC cells with low or absent PD-L1 expression.

Oncogene; https://doi.org/10.1038/s41388-021-01910-6

INTRODUCTION
Programmed death ligand 1 (PD-L1, also known as CD274 or B7H1), is expressed on multiple tumor cell types including human colorectal cancer (CRC) cells. PD-L1 is an immune checkpoint protein that enables tumor cells to escape immune surveillance through binding its receptor PD-1 [1, 2]. In some tumor types, the level of PD-L1 expression measured in tumor plus immune cells is a predictive biomarker whereby low or absent PD-L1 is associated with poor response to immune checkpoint inhibitors [3]. Data also suggest non-immune, tumor intrinsic roles for PD-L1 in tumor growth and progression [4], and we recently reported that deletion of PD-L1 in human CRC cells can confer resistance to chemotherapy. Specifically, deletion of PD-L1 was shown to suppress the pro-apoptotic BH3-only Bim (BCL-2 interacting mediator of cell death) protein and confer resistance to diverse anticancer drugs [5]. Bim can be regulated by transcription factors (FOXO, AP-1) [6–8] or by modification of Bim phosphorylation at multiple sites by members of the MAP kinase family [9]. Bim phosphorylation has been shown to increase or decrease its proapoptotic activity [9, 10] by influencing its binding to pro- survival (BCL-2, BCL-XL and MCL-1) proteins [11–13]. Development of a strategy aimed at restoring Bim activity may lead to new approaches for the treatment of tumors with low or absent PD-L1 expression.
Bim can be regulated by JNK (c-Jun N-Terminal Kinase) via phosphorylation [10]. JNKs are a family of protein kinases (JNK1, JNK2, and JNK3) that have variable and complex functions in cancer including cell stress signaling, tumor cell apoptosis as well as proliferation and invasion that may depend on cellular
context and/or tumor type [14–16]. These functions of JNK are mediated through phosphorylation of its main substrate and transcription factor c-JUN at Ser63 that leads to its stabilization and activation [17]. Tumor-necrosis factor (TNF) receptor- associated factor 2 (TRAF2) is an E3 ligase and upstream regulator of JNK signaling [18, 19]. K63-linked polyubiquitina- tion of TRAF2 results in its binding to the apoptosis signal- regulating kinase 1 (ASK1) complex that activates mitogen- activated protein kinases 4 and 7 (MKK4/7) to initiate JNK signaling [14–16]. In contrast, K63-linked polyubiquitin chains can be removed from TRAF2 by the deubiquitinase CYLD which can suppress JNK signaling. Inhibitors of JNK have been developed and include the irreversible inhibitor JNK-IN-8 that displays high specificity compared to nonselective, reversible inhibitors such as SP600125 [20].
In this report, we demonstrate that loss of PD-L1 can increase BimThr116 phosphorylation resulting in its sequestration and functional inactivation to confer multiple drug resistance. BimThr116 phosphorylation was mediated by JNK whose kinase activity is enhanced by a reduction in the CYLD deubiquitinase due to its mRNA degradation that can activate JNK signaling in PD-L1 depleted cells. We utilized an irreversible JNK inhibitor (JNK- IN-8) that covalently binds cysteine in the JNK catalytic site, and potently inhibits the phosphorylation of c-JUN ser63 [20]. JNK-IN-8 was shown to inhibit BimThr116 phosphorylation and enable free Bim to promote chemotherapy-induced apoptosis. As an alter- native therapeutic strategy, the MCL-1 inhibitor (AZD5991) was used to unsequester Bim and its combination with JNK-IN-8 was evaluated.

1Gastrointestinal Research Unit, Mayo Clinic, Rochester, MN, USA. 2Department of Gastrointestinal Surgery, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China. 3Schulze Center for Novel Therapeutics, Division of Oncology Research, Department of Oncology, Mayo Clinic, Rochester, MN, USA. 4Departments of Medicine
and Oncology, Mayo Clinic, Rochester, MN, USA. 5 Mayo Comprehensive Cancer Center, Rochester, MN, USA. ✉email: [email protected]; [email protected]
Received: 3 December 2020 Revised: 11 June 2021 Accepted: 15 June 2021

MATERIALS & METHODS
Cell culture and reagents
RKO, HCT15 and DLD1 human CRC cell lines were obtained from the ATCC (Manassas, VA). All cell lines were authenticated by short tandem repeat analysis and routinely tested using MycoAlert Mycoplasma detection set (Lonza, Allendale, NJ). CRC cell lines were cultured in RPMI medium (Invitrogen, Carlsbad, CA, catalog no.11875) supplemented with 10% (v/v) fetal bovine serum (FBS). HEK293T were cultured with Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO, catalog no.D5796) supplemented with 10% FBS. Monolayer cultures were established and cells were passaged a few times before treatment(s). Cells were treated with single drug or drug combinations utilizing the following agents: ABT-263 (Selleckchem, Houston, TX, catalog no.S1001), AZD5991(AstraZe- neca, Cambridge, UK, catalog no.HY-101533), cobimetinib (GDC- 0973/XL-518; Active Biochem, Hong Kong, PRC, catalog no.A- 1180), pan-p38 inhibitor Doramapimod (Sellekchem, catalog no. S1574) and JNK-IN-8 (Sellekchem, catalog no.S4901). All drugs were dissolved in dimethyl sulfoxide (DMSO) at specified concentrations and stored at −20 °C. Drugs were diluted in media at the time of cell treatment. For immunoblotting and immuno- precipation experiments, the following antibodies were utilized [all purchased from Cell Signaling, Danvers, MA]: anti-Bcl-2 (catalog no.4223), anti-Mcl-1 (catalog no.94296), anti-cleaved caspase-3 (Asp175) [catalog no.9664], anti-α-tubulin (catalog no.3873), anti-PD-L1 (catalog no.13684), anti-phospho-p44/42 MAPK (Erk1/2) [Thr202/Tyr204] (catalog no.4370), anti-p44/42 MAPK (Erk1/2) (catalog no.4695), anti-phospho-c-Jun (Ser63) (catalog no.91952), anti-c-Jun antibody (60A8) [catalog no.9165], anti-JNK antibody (catalog no.9252), anti-p38 antibody (catalog no.8690), anti-Phospho-p38 MAPK (Thr180/Tyr182) (catalog no.4511) and anti-Bim (catalog no.2933).

Lentiviral CRISPR knockout and lentiviral shRNA knockdown Human PD-L1 (CD274) guide RNA (target sequence TACCGCTG- CATGATCAGCTA) was cloned into lentiviral vector pLentiCRISPRv2 that was purchased from GenScript (Piscataway, NJ). Recombinant lentiviruses were produced in HEK293T cells as previously described [5]. Cells were incubated with lentiviruses in the presence of 8 μg/ml polybrene according to the manufacturer’s instructions. When the cells were about 80% confluent after lentivirus infection, they were cultured in growth media and then selected using puromycin (1:5000 v/v). Knockdown efficiency was verified 48 h after transfection by immunoblotting. Production and transduction of lentivirus into target cells and elimination of non- transduced target cells were performed per standard procedures, as described previously [21].

Apoptosis assay
Apoptosis was analyzed by Annexin V and PI labeling as described previously [5]. In brief, floating and adherent cells were collected following 48 h of drug treatment, transferred to 15 ml tubes and then centrifuged for 5 min at 1500 rpm. Cells were washed by ice cold phosphate-buffered saline (PBS) twice, resuspended in 1 × Annexin V binding solution (BD Biosciences, San Jose, CA, catalog no. 556454) and then stained with Annexin V conjugated with fluorescein isothiocyanate (BD Biosciences, catalog no. 556419) and PI. Apoptotic cells were quantified by flow cytometry and early and late apoptosis were combined. All samples were protected from light and incubated at room temperature for 30 min. Results were analyzed by MATLAB (Mathworks, Natick, MA).

Immunoblotting and immunoprecipitation
Cells were lysed with NETN buffer [20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% Nonidet P-40, 100 mM NaCl], 10 mM NaF, 50 mM
β-glycerophosphate, and 1 mg/ ml each of pepstatin A and aprotinin. To remove cellular debris, lysates were centrifuged at 10,000 rpm for 15 min. After centrifugation, proteins of interest
were immunoprecipitated by incubating lysates with 2 µg of indicated antibody and 20 µl protein A or protein G Sepharose beads (GE Healthcare, Marlborough, MA) overnight at 4 °C. Immunoprecipitates were then centrifuged at 8000 rpm for 1 min, and washed with cold NETN buffer twice. After addition of 40 µl of 1× Laemmli buffer to the immunoprecipitates, samples were boiled for 10 min followed by SDS–PAGE separation. Proteins were then transferred to PVDF membranes using the semi-dry method (Trans-Blot® Turbo™ Transfer System, Bio-Rad, Hercules, CA). The membrane was blocked with 5% milk for 1 h, incubated with the indicated primary antibody overnight, washed with PBS-T buffer three times, and then blotted with goat anti-rabbit HRP or goat anti-mouse HRP secondary antibodies (Jackson Immunor- esearch, West Grove, PA) for 1 h. Membranes were washed with PBS-T buffer three times and then the signal was detected using an Azure Imaging system (Dublin, CA).

RNA immunoprecipitation assay and QRT-PCR
Native RNA immunoprecipitation (RIP) was performed with Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore). Briefly, cells were fixed with 0.3% formaldehyde for 10 min and then incubated with glycine to final concentration at 0.2 M. Next cells were lysed with lysis buffer containing 50 mM Tris HCl (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1% (v/v) TritonX-100, 0.1% (w/v)
sodium deoxycholate and sonicated with sonicator (10% AMP, 5 s sonication, 5 s pause, 10 cycles). The supernatant was incubated with target antibody and protein A/G magnetic beads followed by incubation overnight at 4 °C. Then the beads were washed two times with lysis buffer which is composed of 50 mM Tris HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1% (v/v) TritonX-100, 0.1% (w/v)
sodium deoxycholate), LiCl buffer (10 mM Tris HCl, pH 8, 250 mM LiCl, 0.5% (v/v) NP-40, 0.1% (w/v) sodium deoxycholate, 1 mM EDTA), and washing buffer [100 mM NaCl buffer (10 mM Tris HCl, pH 8, 1 mM EDTA, 100 mM NaCl)]. The RNA was eluted off by elution buffer [100 mM Tris HCl (pH 8), 10 mM EDTA, 1% (w/v) SDS] and extracted with Trizol (Invitrogen).

RNA extraction, reverse transcription and quantitative RT-PCR Quick-RNA miniPrep kit (Zymo, Irvine, CA) was used for extracting RNA. PrimeScript RT-PCR Kit (Takara) was used for reverse transcription. CYLD primer was obtained from Qiagen. SYBR Green PCR Master Mix (Applied Biosystems) was used for Quantitative RT-PCR.

Organoid culture and viability assay
Patient-derived CRC organoids was generated under an IRB- approved protocol that required patient informed consent. CRC organoids were cultured in DMEM with glutamax 1% B27, 50 μg/ mL ascorbic acid, 20 μg/mL insulin, 0.25 μg/mL hydrocortisone, 100 ng/mL FGF2, 100 nM all-trans retinoic acid and 10 μM Y267632, 1% Penicillin-Streptomycin. The organoids were infected with control shRNA or PD-L1 shRNA lentivirus, selected with puromycin and incubated with indicated treatments. Three days later, organoid viability was analyzed with CellTiter-Glo 3D Cell Viability Assay kit (Promega, Madison, WI).

Clonogenic survival assay
A clonogenic survival assay was performed as described previously [22]. Five hundred cells were seeded in six-well plates. After attachment, cells were treated with different drugs at the indicated doses. After 8–10 days in cell culture, cell colonies were visualized by fixation in 10% methanol/10% acetic acid and then stained with 0.5% crystal violet in 10% methanol. Each condition was performed in triplicate.

Statistical analysis
Data derived from the apoptosis assay, organoid viability assay and clonogenic survival assay were presented as mean ± standard

deviation (SD) or standard error of the mean (SEM). All cell culture experiments were performed in triplicate. Student’s t test (two- tailed) and two way ANOVA were performed as indicated, and results were considered statistically significant if *p < 0.05 or **p < 0.01, as shown.

RESULTS
Depletion of PD-L1 enhances Bim phosphorylation to promote its interaction with MCL-1 and BCL-2
Previously, we reported that loss of PD-L1 attenuated DNA damage and apoptosis induced by diverse anticancer drugs that was reversed by restoring wild-type PD-L1 in human CRC cell lines [5]. The effect of PD-L1 on chemosensitivity was confirmed in MC38 murine tumor xenografts generated from PD-L1 knockout vs parental CRC cells. Here, we sought to determine the mechanism by which PD-L1 depletion can regulate chemotherapy-induced apoptosis.
Since Bim can be sequestered by BCL-2 family proteins [23], we determined whether release of Bim from pro-survival BCL-2 family proteins can enhance chemotherapy-induced apoptosis. To address this question, we first examined the interaction between Bim and MCL-1/BCL-2 in PD-L1 knockout and parental cells by immunoprecipitation. We observed increased binding of Bim to MCL-1 and to BCL-2 in PD-L1 knockout RKO cells vs parental cells (Fig. 1A, B). Similar results were observed in DLD1 and HCT15 CRC cell lines (Fig. 1C, D; Supplementary Fig. 1A). Since Bim phosphorylation status can alter its binding to BCL-2 [10], we examined the Thr116 phosphorylation site on Bim that we found to show enhanced phosphorylation in PD-L1 knockout vs vector alone cells (Fig. 1A). Increased sequestration of Bim by MCL-1 and BCL-2 was specific to PD-L1 knockout cells (Fig. 1A). To confirm our result, we incubated cell lysates with lambda phosphatase and observed dephosphorylation of Bim that disrupted its interaction with MCL-1 and BCL-2 in PD-L1 knockout cells (Fig. 1A). We then determined the specificity of the BimThr116 phosphorylation site for promoting its interaction with MCL-1 and BCL-2 in PD-L1 knockout cells. We observed increased Bim phosphorylation at Thr116 shown to be mediated by JNK [24], but not at Ser69 (Fig. 1B) which is mediated by ERK1/2 signaling [25].
In CRC cell lines with mutationally-activated RAF/RAS signaling, we observed that loss of PD-L1 was shown to attenuate apoptosis induced by the MEK inhibitor, cobimetinib [5]. Despite the ability of cobimetinib to induce Bim expression, this effect was insufficient to reverse apoptosis resistance in PD-L1 depleted cells [5]. To address the potential for cross-talk with other signaling molecules including p38, we treated CRC cell lines with cobimetinib, a pan p38 inhibitor (Doramapimod), or vehicle and found that phosphorylation of Bim at Thr116 was not regulated by MEK-ERK signaling or p38 kinase (Supplementary Fig. 1B). Furthermore, the p38 pan-inhibitor did not inhibit JNK activity shown by lack of suppression of c-JUN phosphorylation in three different CRC cell lines (Fig. 1E; Supplementary Fig. 1C, D). We also performed knockdown of both MCL-1 and BCL-2 in CRC cells that did not alter BimThr116 nor JNK activity (Supplementary Fig. 2A-C). Furthermore, MCL-1 and BCL-2 double knockdown markedly enhanced apoptotic signaling independent of PD-L1 status (Supplementary Fig. 2A-C). Therefore, loss of PD-L1 results in enhanced phosphorylation of Bim at Thr116 which can promote its binding to and sequestration by MCL-1 and BCL-2.

JNK activation mediates Bim phosphorylation status in CRC cell lines
Phosphorylation of Bim has been reported to occur by JNK, a family of protein kinases that play an essential role in cell stress signaling pathways and in the regulation of tumor growth and cell death [24]. We, therefore, tested the hypothesis that inhibition of JNK kinase can suppress binding of Bim to MCL-1/BCL-2. Treatment of PD-L1 knockout and parental cells with cobimetinib
alone or combined with the irreversible JNK inhibitor (JNK-IN-8) was shown to potently suppress Bim phosphorylation at Thr116 shown to be responsible for its interaction with MCL-1 and BCL-2 in CRC cell lines (Fig. 1F). This finding supports the role of JNK as a mediator of Bim phosphorylation shown here to promote interaction with BCL-2 family proteins in PD-L1 depleted cells.

PD-L1 depletion promotes degradation of CYLD mRNA that can enhance JNK activity
To explore the mechanism of upregulated JNK activity in PD-L1 depleted cells, we probed upstream regulators of JNK signaling. We detected decreased expression of the CYLD deubiquitinase in PD-L1 knockout cells, but not other upstream regulators of JNK signaling such as MKK4, MKK7, TRAF2, or ASK1 (Fig. 2A). As a deubiquitinase, CYLD removes K63 polyubiquitin chains from its substrate TRAF2 that results in inhibition of TRAF2 activity [26, 27]. In PD-L1 knockout CRC cell lines with reduced CYLD expression, we consequently observed increased K63 -linked ubiquitination of TRAF2 (Fig. 2B). The reduction of CYLD in cells with PD-L1 deletion occurred independent of the proteasome since the proteasome inhibitor MG132 failed to restore CYLD expression (Fig. 2C). Importantly, reconstitution of PD-L1 in knockout cells was shown to restore CYLD protein expression (Fig. 2A). To elucidate the mechanism of decreased CYLD expression, we examined its mRNA level which was decreased in PD-L1 knockout vs parental cells (Fig. 2D) as was the half-life of CYLD mRNA (Fig. 2E). In our previous report, PD-L1 was shown to act as an RNA-binding protein to regulate mRNA stability of DNA damage-related genes [28]. Through competition with the RNA exosome, intracellular PD-L1 protected targeted RNAs from degradation, thereby increasing cellular resistance to DNA damage [28]. Based on our previous finding that PD-L1 can compete with the ribonuclease EXOSC10 to stabilize the targeted mRNA [28], we hypothesized that EXOSC10 can degrade CYLD mRNA. To test this hypothesis, we performed an RNA immunoprecipitation (RIP) assay that detected an interaction between the PD-L1 protein and CYLD mRNA (Fig. 2F). In cells with depletion of PD-L1, we observed increased binding of EXOSC10 to CYLD mRNA (Fig. 2G). In addition, knockdown of EXOSC10 was shown to restore CYLD mRNA expression (Fig. 2H). Transfection of EXOSC10 siRNA suppressed JNK activity indicated by significantly reduced c-JUN Ser63 phosphorylation in RKO cells (Fig. 2I), and consistent results were observed in the HCT15 and DLD1 CRC cell lines (Supplementary Fig. 3A, B). Together, these findings demonstrate that loss of PD-L1 increases the interaction of EXOSC10 with CYLD mRNA resulting in its ribonuclease-mediated degradation and downstream activation of JNK signaling.

Targeting JNK kinase reverses drug resistance mediated by
PD-L1 depletion
To determine the translational value of the aforementioned findings, we determined whether inhibition of JNK1-3 kinases using an irreversible covalent inhibitor, i.e., JNK-IN-8, can suppress phosphorylation of BimThr116 and disrupt its interaction with MCL- 1/BCL-2 and thereby, overcome drug resistance. Treatment of CRC cell lines with JNK-IN-8 was shown to potently suppress its known downstream target of c-JUN phosphorylation at Ser63 [29] (Fig. 3A), and effectively inhibited Bim phosphorylation at Thr116. Cobimetinib suppressed Bim phosphorylation at Ser69, but not Thr116. The combination of JNK-IN-8 and cobimetinib enhanced caspase-3 cleavage in PD-L1 knockout cells but not in parental cells (Fig. 3A). Moreover, JNK-IN-8 was shown to overcome resistance to both oxaliplatin and CPT-11 in PD-L1-depleted cells (Fig. 3B, C), as shown by its ability to promote apoptosis measured by annexin V and PI labeling and suppression of long-term clonogenic survival (Fig. 3D-G; Supplementary Fig. 4). Treatment with the JNK-IN-8 inhibitor did not alter either ERK nor p38 activity in any of the three CRC cell lines (Fig. 3A; Supplementary Fig. 5A,

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Fig. 1 Irreversible inhibition of JNK using JNK-IN-8 suppresses the interaction of Bim with MCL-1/BCL-2 in PD-L1 knockout cells. A, B Parental and PD-L1 knockout RKO cells were lysed directly or in the presence of lambda (λ) phosphatase, and incubated with antibodies against Bim. Immunoblotting was performed with the indicated antibodies. C, D Control and PD-L1 shRNA knockdown DLD1 and HCT15 cells were lysed and incubated with anti-Bim antibody. Immunoblotting was performed with the indicated antibodies. E Parental and PD-L1 knockout RKO cells were treated with the p38 pan-inhibitor, doramapimod. F Immunoprecipation was performed in PD-L1 parental and knockout RKO cells treated with JNK-IN-8 (10 µM), cobimetinib (5 µM), or their combination for 16 h, then incubated with an anti-Bim antibody. Immunoprecipitates were probed with the indicated antibodies.

D). Importantly, the combination of JNK-IN-8 with a cytotoxic drug restored apoptosis in PD-L1 knockout cell to a similar extent as seen in parental CRC cells (Fig. 3A-C).
Next, we evaluated the combination of JNK-IN-8 with che- motherapy in two additional CRC cell lines, DLD1 and. HCT15. JNK- IN-8 was shown to enhance apoptotic signaling indicated by caspase-3 cleavage in PD-L1 knockdown vs parental cell lines treated with cobimetinib, CPT-11 or oxaliplatin (Supplementary Fig. 5A-F). JNK-IN-8 was also shown suppress long-term colony formation of DLD1 and HCT15 cells treated with cobimetinib, CPT- 11 or oxaliplatin (Supplementary Fig. 5G-L). These findings are entirely consistent with those generated in the RKO cell line (Fig. 3A-G). To further confirm our results, we utilized a patient-derived CRC organoid model. We generated CRC organoid cells with
knockdown of PD-L1 which displayed resistance to oxaliplatin, CPT-11 and cobimetinib as demonstrated in an ATP-based organoid cell viability assay (Fig. 4A-C). Furthermore, treatment with JNK-IN-8 sensitized PD-L1 knockdown organoids to antic- ancer drug-induced apoptosis (Fig. 4D-F). Using a second CRC organoid line, we observed similar results (Fig. 4G-I). These data demonstrate that JNK inhibition can overcome apoptosis resis- tance conferred by low or absent PD-L1 in human CRC cells and as confirmed in a human CRC organoid model.

BH3-only mimetic drugs release Bim from its sequestration by MCL-1 or BCL-2 to enhance apoptosis
We hypothesized that targeting MCL-1 and/or BCL-2 can unsequester Bim and, thereby, sensitize PD-L1-depleted cells to

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Fig. 2 Loss of PD-L1 destabilizes CYLD mRNA and activates JNK signaling in RKO cells. A Parental cells, PD-L1 knockout cells alone or reconstituted with HA-PD-L1 were lysed and immunoblotted with indicated antibodies. B PD-L1 parental (+/+) and knockout (−/−) cells were treated with a proteasome inhibitor (MG132) and lysates were incubated with an anti-TRAF2 antibody. Immunoblotting was performed with the indicated antibodies. C PD-L1 parental (+/+) and knockout (−/−) cells were treated with DMSO or MG132. Cells were then lysed and immunoblotted as shown. D qRT-PCR analysis of CYLD mRNA expression in PD-L1 +/+ and −/− cells. E PD-L1 + / + and −/− cells were treated with actinomycin D (5 µg/ml) and CYLD mRNA stability was evaluated. F An RNA immunoprecipitation assay (RIP) was performed using an anti-PD-L1 antibody and control IgG. G RIP assay was also performed with an anti-EXOSC10 antibody and IgG. H qRT-PCR analysis of CYLD mRNA expression shown with different treatments. I Cells with the same treatment as in (H) were lysed and immunoblotted with the indicated antibodies. Analysis of the ratio of p-c-JUN to total c-JUN was performed using densitometry. Data are presented as mean ± SD, n = 3; **p < 0.01.

chemotherapy-induced apoptosis. We utilized a small molecule, potent and selective inhibitor of MCL-1, AZD5991, which has entered human clinical trials [30, 31]. AZD5991 alone or combined with cobimetinib was shown to disrupt the interaction between Bim and MCL-1 in PD-L1 knockout and parental CRC cells (Fig. 5A). Similarly, the BCL-2 inhibitor ABT-263 was also shown to dissociate Bim from its sequestration by BCL-2 (Fig. 5B). Using CRC cell lines with activation of RAF/RAS signaling, we found that AZD5991 can
sensitize PD-L1 knockout and parental cells to apoptosis induction by cobimetinib (Supplementary Fig. 6A), oxaliplatin, or CPT-11 (Supplementary Fig. 6C, D), as indicated by cleavage of caspase-3 and induction of Annexin V labeling. Drug treatment induced apoptosis to a similar extent in both PD-L1 knockout and parental RKO cells (Supplementary Fig. 6A-D). Results for the combination of AZD5991 and anticancer drugs were similar in HCT15 and in DLD1 CRC cell lines with knockdown or intact PD-L1

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Fig. 3 The JNK inhibitor JNK-IN-8 sensitizes PD-L1 knockout cells to multiple anticancer drugs. A–C PD-L1 parental (+/+) and knockout (−/−) RKO cells were treated with JNK-IN-8 (400 nM) or cobimetinib (5 µM) (A) alone or in combination, and with oxaliplatin (20 µM) (B) or CPT- 11 (10 µM) (C) for 48 h. Cells were then subjected to immunoblotting, including analysis of cleaved caspase-3 (CC3). D PD-L1 parental and knockout cells were treated as shown, and apoptosis was analyzed by Annexin V labeling. Data are presented as mean ± SD, n = 3; **p < 0.01. E–G PD-L1 parental and knockout RKO cells were each treated with JNK-IN-8 (400 nM) or cobimetinib (5 µM) alone or combined with (E) oxaliplatin (20 µM) (F) or CPT-11 (10 µM) (G). Cells were analyzed in assays for colony formation. Data are presented as mean ± SD, n = 3; **p < 0.01.

(Supplementary Fig. 7A-F). The ability of AZD5991 to enhance cell death by anticancer drugs was further shown in a colony formation assay indicating a sustained effect on cell viability compared to vehicle (Supplementary Figs. 6E-G and 7G-L). Together, these results indicate that MCL-1 antagonism impairs
the neutralization of Bim by MCL-1 to effectively reverse drug resistance due to PD-L1 depletion.
Given that a BH3 mimetic or a JNK1-3 inhibitor can each overcome drug resistance mediated by loss of PD-L1, we examined the combination of cobimetinib with AZD5991 or JNK-IN-8, as well

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Organoid viability (%)
B

DMSO
JNK-IN-8
JNK-IN-8
+CPT
E
CPT
Organoid 1
PD-L1
shRNA - + - + - + - +
Tubulin

CC3 PD-L1

Organoid viability (%)
C
1.4
1.2
1
0.8
0.6
0.4
0.2
0
DMSO CPT-11 JNK-IN-8

Organoid 1

**
JNK-IN-8+ CPT-11

Ctrl shRNA PD-L1 shRNA

F

DMSO
combimetinib
JNK-IN-8
JNK-IN-8
+combimetinib
Organoid 1
PD-L1
shRNA - + - + - + - +
Tubulin

CC3

DMSO cobimetinib JNK-IN-8 cobimetinib
+JNK-IN-8
PD-L1

Tubulin

Organoid viability (%)
G
1.2

1

0.8

0.6

0.4

0.2
Organoid 2

*

Ctrl shRNA PD-L1 shRNA
H
Organoid viability (%)
1.4
1.2
1
0.8
0.6
0.4
0.2

Organoid 2
*

Ctrl shRNA PD-L1 shRNA

0
DMSO oxaliplatin JNK-IN-8 JNK-IN-8+
oxaliplatin
0
DMSO CPT-11 JNK-IN-8 JNK-IN-8+
CPT-11

Organoid viability (%)
I 1.2
1

0.8

0.6

0.4

0.2
Organoid 2

**

Ctrl shRNA PD-L1 shRNA

Control shRNA

PD-L1 shRNA
PD-L1

0
DMSO cobimetinib JNK-IN-8 cobimetinib
+JNK-IN-8
Tubulin

Fig. 4 Patient-derived CRC organoids with PD-L1 knockdown were sensitized to chemotherapy by JNK inhibition. A–C Parental and PD-L1 knockdown CRC organoids cells were treated with JNK-IN-8 (400 nM) alone or in combination with oxaliplatin (20 µM) (A), CPT-11 (10 µM) (B) or cobimetinib (5 µM) (C). Cells were then analyzed in a 3D cell culture viability assay with data presented as mean ± SEM, n = 3; **p < 0.01. D–F Parental and PD-L1 knockdown CRC organoids cells were treated with oxaliplatin (20 µM) (D), CPT-11 (10 µM) (E) or cobimetinib (5 µM) (F) alone or combined with JNK-IN-8 (400 nM) for 72 h. Cells were subjected to immunoblotting including analysis of cleaved caspase-3 (CC3). G–I The experiment outlined in (A–C) was then repeated in a second organoid (Organoid 2) with analysis again in a 3D cell viability assay. Data are presented as mean ± SEM, n = 3; **p < 0.01, *p < 0.05.

as all three drugs in PD-L1 knockout and parental cells. The drug triplet induced apoptosis to a significantly greater extent than did cobimetinib combined with low dose AZD5991 or JNK-IN-8 in PD- L1 knockout cells (Fig. 6A). We then examined the effect of the drug triplet on the interaction of Bim with MCL-1 in an immunoprecipitation assay. Treatment with JNK-IN-8 or AZD5991

inhibited BimThr116 phosphorylation and reduced the interaction between Bim and MCL-1 as did the triplet combination with cobimetinib (Fig. 6B), and potently reversed drug resistance in cells with PD-L1 loss. Moreover, low dose triple drug treatment markedly suppressed the long-term viability of PD-L1 knockout cells to a greater extent than did a drug doublet or monotherapy (Fig. 6C, D).

A
Cobimetinib
IgG MCL-1

cobimetinib, or a pan-p38 inhibitor. To exclude potential cross-talk among the three major MAPKs, we found that cobimetinib did not alter p38 or JNK activity, the p38 inhibitor did not alter JNK activity, and treatment with JNK-IN-8 did not alter ERK nor p38 activity.
We observed for the first time that BimThr116 phosphorylation was mediated by elevated JNK activity that was a consequence of loss of PD-L1. Analysis of the mechanism by which JNK activation

DMSO
DMSO
AZD5991
AZD5991+
Cobimetinib

IP
PD-L1 +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/-

Input
B
can enhance BimThr116 phosphorylation revealed that it is due to suppression of the CYLD deubiquitination enzyme that can cleave lysine 63-linked polyubiquitin chains from target proteins that regulate cell survival or cell proliferation [34]. We observed a reduced level of CYLD mRNA expression that was due to its accelerated degradation by the ribonuclease EXOSC10 in PD-L1 knockout cells. Downregulation of CYLD at the RNA level has been observed in different cancer types where it can serve as a tumor suppressor [35]. The intracellular exosome complex (EXOSC), of which EXOSC10 is one of the subunits of the RNA exosome core, is responsible for degrading and processing protein-coding and non-coding transcripts [36, 37]. The ability of EXOSC10 to degrade CYLD was confirmed by knockdown of EXOSC10, which also suppressed JNK activity.
In this report, we describe a novel strategy to restore Bim- mediated, drug-induced apoptosis in PD-L1 depleted CRC cells and in human organoids. In an effort to circumvent drug resistance in PD-L1 depleted cells due to increased JNK activity, we targeted c-JUN/JNK signaling by upstream inhibition of the activator enzymes JNK1-3 using JNK-IN-8. Covalent inhibitors such as JNK-IN-8 are attractive drugs and experimental tools since they

IP
DMSO
DMSO
ABT-263
Cobimetinib
ABT-263+
Cobimetinib
result in rapid and irreversible inhibition of JNK with high

Input
in
Fig. 5 BH3 mimetics inhibit the interaction of Bim with MCL-1/ BCL-2 in PD-L1 knockout cells. A, B PD-L1 parental (+/+) and knockout (−/−) RKO cells were treated with cobimetinib (5 µM) alone or combined with small molecule inhibitors of BCL-2 (ABT-263 [2 µM]) (A) or MCL-1 (AZD5991 [10 µM]) (B) for 16 h. Cells were lysed and immunoprecipitated with an anti-MCL-1 antibody and then probed with the indicated antibodies.
DISCUSSION
The role of tumor cell PD-L1 in responsiveness to cytotoxic chemotherapy is unknown. The ability to address this question in the clinic is complicated by the routine assessment of PD-L1 in tumor tissue by the Combined Positive Score, which considers the aggregate number of PD-L1 staining cells in tumor as well as lymphocytes and macrophages [32]. In this report, we found that suppression of PD-L1 confers resistance to diverse anticancer drugs in multiple CRC cell lines and in two patient-derived colon cancer organoids. Chemoresistance in PD-L1 depleted cells was a consequence of functional inactivation of Bim proteins due to their sequestration by MCL-1 and BCL-2 proteins. Specifically, loss of tumor cell PD-L1 promoted phosphorylation of Bim at Thr116 leading to its functional inactivation due to enhanced interaction with MCL-1/BCL-2 proteins. Multi-site phosphorylation of Bim is a regulatory mechanism known to control its apoptotic activity [10, 33], and occurs by multiple mechanisms including JNK signaling [34]. In our CRC cells with constitutive MEK-ERK activation, other MAP kinases or p38 did not affect phosphoryla- tion of Bim at Thr116 as shown by use of the MEK-ERK inhibitor,
selectivity [20]. We report the first data using JNK-IN-8 in CRC cells where we observed potent suppression of c-JUN phosphor- ylation at Ser63. Since c-JUN is a downstream target of JNK [38], this finding confirms enhanced JNK activity in our PD-L1 knockout cells. Whereas JNK1 and JNK2 are ubiquitiously expressed in multiple tissue types, JNK3 is mostly localized to brain and to a lesser extent in the heart and testis [39]. JNK-IN-8 has high affinity for all three JNK isoforms and forms a covalent bond with Cys116 in the catalytic sites of both JNK1 and JNK2. JNK-IN-8 is far more selective for JNK than are reversible inhibitors such as SP600125 that has been frequently used to study JNK functions but inhibits a wide range of kinases, including p38, CDK1, and MEK/ERK [40]. Treatment with JNK-IN-8 has been shown to overcome resistance to a BRAF inhibitor in human melanoma cells [41, 42], and was also able to sensitize pancreatic cancer cells to 5-fluorouracil [43]. Furthermore, JNK-IN-8 was synergistic with the HER2 inhibitor lapatinib in inducing cell death in triple negative breast cancer cells [44]. Importantly, treatment of CRC cells with JNK-IN-8 inhibited Bim phosphorylation at Thr116, but not Ser69, which was shown to unsequester Bim from MCL-1 or BCL-2 and, thereby, enable its proapoptotic activity. BimSer69 is regulated by MEK/ERK
[25] and prevents the proteasomal degradation of Bim [10]. In CRC cells with RAF/RAS activation, the MEK1/2 inhibitor cobimetinib suppressed Bim phosphorylation at Ser69, but not Thr116. BimThr116 and BimSer69 phosphorylation sites are equivalent to the mouse BimThr112 and BimSer65 sites, respectively [10]. The importance of Bim phosphorylation by JNK has been shown in MEF cells treated with UV radiation whereby phosphorylation at Thr116 occurred in wild-type MEF, but not JNK-deficient fibroblasts [10]. Of note, JNK is not the only kinase that can phosphorylate Bim at this specific site [10]. We found that the combination of JNK-IN-8 with CPT-11, oxaliplatin or cobimetinib can cooperatively enhance apoptosis and, thereby, overcome limitations of anticancer drugs in PD-L1 deficient CRC cells. The ability of JNK-IN-8 to enhance apoptosis to these three anticancer drugs was also shown in an organoid model. The JNK pathway was shown to mediate cell survival following treatment with inhibitors of BRAF and MEK in BRAFV600E metastatic melanoma

Cobimetinib
AZD5991
JNK-IN-8
Cobimetinib+ AZD5991
Cobimetinib+
+JNK-IN-8
AZD5991+ JNK-IN-8
AZD5991+
Cobimetinib+ JNK-IN-8
A B IgG BIM
DMSO
Cobimetinib
Cobimetinib
AZD5991+ JNK-IN-8

Cobimetinib+
+JNK-IN-8
Cobimetinib+ AZD5991

AZD5991+
Cobimetinib+ JNK-IN-8

+/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/-
PD-L1

C 120

100

Cell Survival (%)
80

60

40

PD-L1+/+_DMSO PD-L1-/-_DMSO PD-L1+/+_JNK-IN-8 PD-L1-/-_JNK-IN-8
PD-L1+/+_AZD5991
**
PD-L1-/-_AZD5991
PD-L1+/+_JNK-IN-8+AZD5991 PD-L1-/-_JNK-IN-8+AZD5991
pBim T116 MCL1
Bim

20

0
0 0.002 0.004 0.006 0.008 0.01 0.012
D Cobimetinib (μM)

120

100

Cell Survival (%)
80

60

40

PD-L1 +/+_DMSO
PD-L1 -/-_DMSO
**
PD-L1 +/+_JNK-IN-8 PD-L1 -/-_JNK-IN-8 PD-L1 +/+_AZD5991 PD-L1 -/-_AZD5991
PD-L1 +/+_JNK-IN-8+AZD5991
PD-L1 -/-_JNK-IN-8+AZD5991

20

0 0 0.2 0.4 0.6 0.8 1 1.2
CPT-11 (μM)

Fig. 6 Combination of inhibitors of JNK (JNK-IN-8), MCL-1 (AZD5991), and MEK1/2 (cobimetinib) overcomes drug resistance in PD-L1 knockout cells. A PD-L1 parental (+/+) and knockout (−/−) RKO cells were treated with JNK-IN-8 (400 nM), AZD5991 (6 nM) or cobimetinib (2.5 µM) alone or in various combinations for 48 h. Cells were probed with the indicated antibodies and subjected to immunoblotting. B Immunoprecipitation was performed using an anti-Bim antibody in these same drug-treated cells, as shown in (A), for 16 h. Immunoprecipitates were probed with the indicated antibodies and blotted. C–D PD-L1 parental and knockout RKO cells were seeded into six-well plates and treated with indicated concentrations of cobimetinib (C) or CPT-11 (D) combined with AZD5991 (6 nM) or JNK-IN-8 (400 nM). After 14 days, colonies were fixed and stained using Giemsa. Data are presented as mean ± SD, n = 3; **p < 0.01.

[41, 42]. Our data underscore the important role of JNK as a regulator of apoptotic signaling in CRC cells where it can serve as a druggable therapeutic target. Selective inhibitors of JNK1, JNK2, or JNK3 isoforms are emerging that may improve efficacy, as shown by the selective targeting of JNK with the peptide D-JNKI-1 (XG-102, AM-111) [45].
Since targeting the BH3 domain-binding groove of MCL-1 with BH3 mimetic agents is an effective strategy to promote apoptosis [46], we utilized a novel and highly selective small molecule antagonist of MCL-1 (AZD5991) in combination with cobimetinib. While cobimeti- nib is known to induce Bim [5], its induction was insufficient to induce

apoptosis in PD-L1 depleted CRC cells. However, the combination of cobimetinib with AZD5991 or JNK-IN-8 released Bim from its sequestration by MCL-1 and BCL-2 to promote apoptosis which was markedly enhanced for the drug triplet in PD-L1 deficient cells. This combinatorial strategy allowed use of lower doses of each drug which can avoid or reduce the potential for in vivo toxicities. Combining antagonists of MCL-1/BCL-2 and MEK may be an effective therapy for patients whose tumors have low or absent tumor cell PD-L1 expression, which is commonly found in human CRCs. In human CRCs, low or absent PD-L1 was associated with poorer patient survival at both the level of mRNA [5] and protein [47, 48].

In summary, depletion of PD-L1 accelerates degradation of CYLD mRNA by EXOSC10, resulting in enhanced JNK signaling activation. Loss of tumor cell PD-L1 creates a dependency on Bim whose physiologically relevant interaction with anti-apoptotic MCL-1 or BCL-2 proteins is regulated by JNK-mediated Bim phosphorylation at Thr116 in CRC cells. We exploited the vulnerability of these cells to disruption of this protein-protein interaction to unsequester Bim from MCL-1/BCL-2 using a novel JNK inhibitor that prevented JNK-mediated phosphorylation of BimThr116shown to neutralize its proapoptotic activity or alterna- tively, BH3 mimetics (shown schematically in Supplementary Fig. 8). Future directions include confirmation of our study findings in a murine tumor xenograft model generated from CRC cells with manipulated PD-L1 expression. In addition, further study in human CRC PDX models are warranted to determine whether the combination of a JNK inhibitor with an anticancer drug can overcome drug resistance in human CRC cells that typically have low or absent PD-L1 expression. Taken together, these findings provide a mechanism for the reduced apoptotic activity of Bim in CRC cells with loss of PD-L1, and suggest novel therapeutic strategies to reverse drug resistance aimed at unsequestering Bim from MCL-1/BCL-2 using an irreversible JNK inhibitor in CRCs with low or absent tumor cell PD-L1 expression.

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ACKNOWLEDGEMENTS
This study was supported, by NCI R01 CA210509-01A1 and Mayo Clinic Center for Biomedical Discovery Pilot Grant Program (both to FAS). LS is supported by the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China. AP is supported by the Rosztoczy Foundation Hungarian Scholarship Program.
AUTHOR CONTRIBUTIONS
FS and BQ designed the experiments, BQ and LS performed the experiments, TLH and MEF provided the organoid model, LS, BQ, and FS analyzed and interpreted the study data. BQ and FS wrote the paper that was reviewed by all authors.

COMPETING INTERESTS
The authors declare no competing interests.

ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41388-021-01910-6.

Correspondence and requests for materials should be addressed to B.Q. or F.A.S.

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