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. 2026 Jan 30;24:126. doi: 10.1186/s12916-026-04671-9

Targeting BCL-XL for degradation synergizes with gemcitabine against cholangiocarcinoma

Qinghua Zeng 1,2,#, Yan Zhang 2,#, Yiwen Yang 3,#, Xin Liu 4, Xin Dong 2, Yongzhang Pan 2, Li Hu 2, Ao Zhang 2, Jian Yang 5, Qiuni Luo 2, Xiang Lai 6, Guoping Zhu 1, Xuan Zhang 3,7,✉, Yonghan He 2,7,✉
PMCID: PMC12930748  PMID: 41618329

Abstract

Background

Cholangiocarcinoma (CCA) remains a highly lethal malignancy with a dismal prognosis, primarily driven by therapeutic resistance. A dominant resistance mechanism involves overexpression of anti-apoptotic BCL-2 proteins (BCL-XL, BCL-2, MCL-1). While direct inhibition of these proteins shows efficacy, its clinical utility is frequently limited by dose-dependent hematotoxicity—as exemplified by ABT263, a BCL-XL/BCL-2 dual inhibitor that induces severe thrombocytopenia.

Methods

We performed integrated analyses of BCL-2 family mRNA/protein expression in clinical CCA specimens and preclinical cell lines. Leveraging proteolysis-targeting chimera (PROTAC) technology, we investigated the therapeutic application of BCL-XL-specific degraders, both as monotherapy and in combination with gemcitabine, to selectively target CCA cells while minimizing hematologic toxicity.

Results

Integrated clinical-experimental data identified BCL-XL as a principal determinant of therapeutic sensitivity in CCA. In vitro, the cereblon (CRBN)-based PROTAC XZ739 demonstrated superior efficacy to its von Hippel-Lindau tumor suppressor (VHL)-based counterpart DT2216, reducing CCA cell viability via apoptosis induction. In vivo, XZ739 synergized with gemcitabine to suppress tumor growth in a CCA xenograft model, achieving robust efficacy without significant thrombocytopenia—a critical advance over conventional BCL-XL inhibitors.

Conclusions

These findings establish XZ739 as a promising therapeutic candidate for BCL-XL-dependent CCA, highlighting its translational potential for rational combination with chemotherapy to overcome resistance while mitigating hematologic toxicity.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12916-026-04671-9.

Keywords: BCL-XL, Chemotherapy, Cholangiocarcinoma, PROTAC

Background

Cholangiocarcinoma (CCA) is the second most common primary hepatic malignancy after hepatocellular carcinoma and the leading biliary tract malignancy [1]. Globally, its incidence has risen steadily over decades, with notable geographical variation: from < 6/100,000 in most Western nations to > 80/100,000 in parts of Southeast Asia, primarily reflecting endemic risks like liver fluke infection and chronic biliary inflammation [2, 3]. CCA constitutes ~ 15% of primary liver cancers, with 5-year survival < 10% in most cohorts [2, 4]. CCA originates from bile duct epithelial cells and is classified into intrahepatic, perihilar, and distal subtypes according to anatomical location [5, 6]. It is characterized by aggressive tumor biology, late diagnosis, and limited treatment options, leading to high mortality [6]. Surgery is curative but feasible only in early stages; most patients present with advanced disease. For unresectable cases, standard chemotherapy (gemcitabine-cisplatin, or fluorouracil-oxaliplatin) is used [7], yet outcomes remain poor due to intrinsic chemoresistance, desmoplastic stroma, and genetic heterogeneity [5]. This underscores the urgent need for improved therapies.

A key resistance mechanism involves apoptotic pathway dysregulation. The BCL-2 protein family balances pro-apoptotic (BAX, BAK, PUMA, NOXA) and anti-apoptotic (BCL-2, BCL-XL, MCL-1) members [8, 9]. Tumor overexpression of anti-apoptotic proteins promotes survival and therapy resistance [10]. In CCA, transcriptomics shows upregulated anti-apoptotic BCL-2 family members, with BCL-2 and MCL-1 implicated in tumor growth and resistance [2, 11, 12]. Functional studies confirm differential dependence on these proteins, highlighting BCL-2 family targeting as a viable strategy [2, 12].

However, clinical use of small-molecule BCL-XL inhibitors is limited by dose-dependent thrombocytopenia, as platelets rely on BCL-XL [13–15]. For example, ABT263 (navitoclax), a dual BCL-2/BCL-XL inhibitor, showed preclinical/early clinical efficacy but was discontinued due to severe thrombocytopenia [16, 17]. To address this, we and others have developed various PROTACs, with representative examples such as the VHL-based DT2216 and the CRBN-based XZ739 that selectively degrade BCL-XL. PROTACs recruit E3 ligases (VHL/CRBN) to ubiquitinate and degrade target proteins via proteasomes [18, 19]. Notably, these ligases are less expressed in platelets but abundant in cancer cells, reducing hematologic toxicity [19, 20]. Among reported BCL-XL degraders, DT2216 demonstrated efficacy in hematologic malignancies (e.g., acute lymphoblastic leukemia [21], T-cell lymphomas [22]) with minimal thrombocytopenia. XZ739 also showed potent activity in hematologic cancers with low platelet impact, though its CCA efficacy was unexplored [21, 23].

This study investigated BCL-2 family prognostic/therapeutic roles in CCA. Clinical and experimental data indicate that BCL-XL is upregulated in CCA tissues/cells and drives therapeutic resistance. We found CRBN-based PROTAC XZ739 selectively eliminated BCL-XL-dependent CCA cells while sparing platelets, minimizing toxicity. Combined with gemcitabine, XZ739 markedly suppressed tumor growth in a CCA xenograft model. These results establish BCL-XL as a key resistance driver in CCA and support BCL-XL-targeted PROTACs, especially with chemotherapy, as a novel strategy to improve outcomes.

Methods

Gene expression analysis

Transcriptomic data (TPM values) of eight major cancers, including CCA, prostate adenocarcinoma, uterine corpus endometrial carcinoma, lung adenocarcinoma, acute myeloid leukemia, adrenocortical carcinoma, glioblastoma multiforme, and bladder urothelial carcinoma, were obtained from The Cancer Genome Atlas (TCGA) database [24]. Members of the BCL-2 protein family, including BCL2L1 (BCL-XL coding gene), BCL2, MCL1, BAX, BAK1 (BAK coding gene), BCL2L11 (BIM coding gene), PMAIP1 (NOXA coding gene), and BBC3 (PUMA coding gene), were analyzed. Tumor-normal expression differences were assessed via the Wilcoxon rank-sum test (R 4.4.3), visualized with “ggplot2” boxplots (p < 0.05 significant). TPM data for four CCA cell lines (RBE, HuccT1, SNU1079, SNU1196) were retrieved from CCLE (https://www.broadinstitute.org/ccle/datasets) for preliminary BCL-2 family expression profiling.

Survival analysis

Disease-free survival (DFS) analysis of BCL2L1, BCL2, and MCL1 in CCA was performed using the GEPIA2 online tool (http://gepia2.cancer-pku.cn) [25]. Patients were stratified into high- and low-expression groups based on the median expression cutoff. The survival plots were generated with the following parameters: hazard ratio (HR) calculation enabled, 95% confidence interval not displayed, and axis units set to months.

Receiver operating characteristic (ROC) curve analysis

The expression levels of BCL2L1, BCL2, and MCL1 were used to construct ROC curves for evaluating their diagnostic efficiency in distinguishing CCA from normal tissues. Tumor and normal samples were labeled according to their tissue type in the dataset (tumor = 1, normal = 0). ROC analysis was conducted in R software using the “pROC” package, and ROC curves were visualized with the ggroc function. The area under the curve (AUC) was calculated, with values > 0.9 considered to indicate excellent diagnostic performance.

Compounds

DT2216 and XZ739 were synthesized at the Shanghai Institute of Materia Medica following previously reported protocols [21, 26]. XZ739 negative compound (XZ739-Neg) was synthesized according to the experimental procedure (Additional file 1). A1331852 (HY-19741), S63845 (HY-100741), ABT199 (HY-15531), QVD (HY-12305), pomalidomide (HY-10984), MG132 (HY-13259), doxorubicin (HY-15142), and gemcitabine (HY-17026) were obtained from MedChemExpress (Shanghai, China). Cisplatin (S1166) was purchased from Selleck Chemicals (Shanghai, China), and ABT263 (GC12405) from GLPBIO (Shanghai, China).

Cell culture

The CCA cell lines SNU1196, SNU1079, RBE, and HuccT1 were cultured in RPMI-1640 medium (C11875500BT, Gibco, MD, USA) supplemented with 10% fetal bovine serum (RY-F22, Royacel Biotechnology Co., Ltd., Lanzhou, China) and 1% penicillin–streptomycin (15140–122, Gibco, MD, USA). Cells were maintained at 37 °C in a humidified incubator with 5% CO2. The human intrahepatic biliary epithelial cell line (HIBEC) was cultured in DMEM medium (C11995500BT, Gibco, MD, USA) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin under the same conditions. SNU1196 and SNU1079 cells were purchased from Huiying Biotechnology (Shanghai, China), RBE and HuccT1 were obtained from the Kunming Cell Bank of the Chinese Academy of Sciences (Kunming, China), and HIBEC was obtained from Zeye Biotechnology (Shanghai, China).

Cell viability assay

Cells were seeded in 96-well plates at a density of 3.5 × 103 in 100 µL complete medium and incubated for 24 h. The medium was replaced with 200 µL of fresh medium containing the indicated compounds at the specified concentrations, followed by incubation for 72 h at 37 °C. Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay (G1111, Promega, Madison, WI, USA) according to the manufacturer’s instructions. Dose–response curves were generated, and IC50 values were calculated using GraphPad Prism version 9.5 (San Diego, CA, USA). Combination index (CI) values were calculated using the CompuSyn software (http://www.combosyn.com).

Western blot

Cells were washed with PBS and lysed on ice with RIPA buffer (BP-115DG, Boston BioProducts) supplemented with protease inhibitors for 30 min, followed by centrifugation at 13,000 × g, 20 min, 4 °C. Protein concentrations were determined using a bicinchoninic acid (BCA) Protein Assay Kit (P0010, Beyotime, Shanghai, China). Equal amounts of protein were denatured in 4 × Laemmli sample buffer (1610747, Bio-Rad, Hercules, CA, USA) containing 5% β-mercaptoethanol at 95 °C for 8-10 min, separated by SDS-PAGE on 12% gels and transferred to 0.22 μm PVDF membranes (Millipore, Bedford, MA, USA) using a wet transfer system (Bio-Rad, Richmond, CA, USA). Membranes were blocked with 5% (w/v) skim milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. After washing, membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. Signals were visualized using a SCG-W3000 Plus imaging system (Servicebio, Wuhan, China) and quantified with ImageJ software (NIH, Bethesda, MD, USA). The antibodies used in this study are listed in Additional file 2: Table S1.

Transfection of small interfering RNA

BCL-XL small interfering RNA (siRNA) and control siRNA were obtained from Sangon Biotech (Shanghai, China) (Additional file 3: Table S2). siRNAs were transfected into SNU1079 cells using Lipofectamine 2000 Transfection Reagent (11668019, Thermo Fisher Scientific, USA) according to the manufacturer’s optimized protocol. Cells were harvested for downstream experiments 72 h post-transfection. Knockdown efficiency was confirmed by Western blot.

Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from cells using TRIzol reagent (15596018, Invitrogen, USA) according to the manufacturer’s instructions. RNA concentration and purity were determined spectrophotometrically, and equal amounts of RNA were reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific, USA). Quantitative PCR amplification was performed with 2 × Universal SYBR qPCR Master Mix (QP101A, YoungGen, Kunming, China) on a CFX-Connect Real-Time PCR System (Bio-Rad, USA), using GAPDH as the endogenous control. Relative gene expression levels were calculated using the 2−ΔΔCt method. Primer sequences are listed in Additional file 3: Table S2.

Colony-forming assay

SNU1079 cells were seeded in 6-well plates at a density of 550 cells per well. After 3 days of incubation, cells were treated with various concentrations of XZ739 for 72 h. The medium was then replaced with fresh complete medium, and cells were further cultured for approximately 2 weeks until individual colonies reached approximately 50 cells. Colonies were fixed with methanol for 20 min, stained with 0.1% crystal violet (110703010, BKMAM, Hunan, China) for 15 min, and subsequently imaged. Colony numbers were quantified using ImageJ software.

Cell scratch test

SNU1079 cells were seeded in 6-well plates and grown to full confluence. A single linear scratch was made in the cell monolayer using a sterile 200 μL pipette tip. Cells were then treated with 0.1 μM XZ739 and incubated for 48 h. Scratch images were captured at 0 and 48 h using a stereomicroscope (SMZ-171-BLED, Motic, Xiamen, China) at 10 × magnification. The wound width was measured and quantified using ImageJ software.

Cell apoptosis assay

SNU1079 cells (1 × 106 per well) were treated with varying concentrations of the indicated compounds. Cells were harvested, washed with PBS, and resuspended in binding buffer. Apoptosis Detection Kit (C1062L, Beyotime) according to the manufacturer’s protocol. Samples were analyzed using an LSR Fortessa flow cytometer (LSR Fortessa, Becton Dickinson, CA, USA), and data were processed with FlowJo software (Tree Star, Ashland, OR, USA).

Cell cycle assay

SNU1079 cells were seeded in 10-cm dishes at a density of 1 × 106 cells per dish and treated with 0.3 μM XZ739 for 24 h. Cells were harvested, washed with PBS, and fixed in 70% ethanol overnight at 4 °C. On the following day, cells were washed with PBS and stained with propidium iodide (PI) from the Cell Cycle and Apoptosis Detection Kit (C1052, Beyotime) for 30 min at room temperature. Cell cycle distribution was analyzed on an LSR Fortessa flow cytometer (LSR Fortessa, Becton Dickinson).

Platelet toxicity assay

Sixteen C57BL/6J mice (7 weeks old, ~ 25 g, equal numbers of males and females) were randomly divided into 4 groups (n = 4 per group). XZ739 (3 mpk and 6 mpk) and ABT263 (6 mpk) were formulated in an injectable vehicle (VEH) consisting of 5% DMSO, 40% PEG300, 5% Tween-80, and 50% saline. Mice in the treatment groups received intraperitoneal injections of 100 μL of the respective formulations, while control mice were injected with an equal volume of saline. Blood samples were collected via facial vein bleeding at 24 and 72 h after injection and analyzed using an automated veterinary hematology analyzer (BC-30 Vet, Mindray, Shenzhen, China). Since XZ739 caused notable platelet toxicity at these doses, subsequent experiments were performed with reduced doses of XZ739 following the same protocol.

In vivo tumor inhibition experiments

Male BALB/c nude mice (approximately 5 weeks old) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and maintained under specific pathogen-free (SPF) conditions at the Kunming Institute of Zoology, Chinese Academy of Sciences. After one week of acclimation, SNU1079 cells (1.5 × 106 cells/mouse in the first experiment; 1.8 × 106 cells/mouse in the second experiment) suspended in 100 μL of a 1:1 mixture of RPMI-1640 medium and Matrigel (356234, Corning, USA) were subcutaneously inoculated into the right flank to establish xenograft tumors. Mice were randomized into groups when tumor volumes reached ~ 100 mm3. Tumor volume was calculated as V = (length × width2)/2. Body weight was measured using a digital balance.

In the first experiment, mice were divided into four groups: VEH, XZ739 (1.5 mpk), XZ739 (2.5 mpk), and ABT263 (40 mpk). XZ739 and ABT263 were formulated in an injectable vehicle (5% DMSO, 40% PEG300, 5% Tween-80, and 50% saline). Initially, all groups received intraperitoneal injections every three days. Due to suboptimal efficacy, the XZ739 (1.5 mpk) dose was escalated to 5 mpk from the fifth injection onward, and the XZ739 (2.5 mpk) group was dosed every two days. Body weight and tumor volume were monitored in parallel with treatment.

In the second mouse experiment, mice were allocated to six experimental groups: VEH, XZ739 (2.5 mpk), gemcitabine (50 mpk), XZ739 + gemcitabine combination, cisplatin (2.5 mpk), and XZ739 + cisplatin combination. XZ739 and gemcitabine were formulated using the same vehicle composition as described above, whereas cisplatin was dissolved in saline. All agents were administered intraperitoneally every three days. For the XZ739 + gemcitabine group, mice received separate administrations of XZ739 (2.5 mpk) together with gemcitabine (50 mpk) for the first three injections. Due to significant drug-induced toxicity manifesting as substantial body weight loss, the gemcitabine dose was reduced to 25 mpk for subsequent administrations, while the XZ739 (2.5 mpk) remained consistent throughout the study. In the XZ739 + cisplatin group, mice received separate administrations of XZ739 at 2.5 mpk together with cisplatin at 2.5 mpk at each injection.

When the mean tumor volume in the control group reached ~ 900 mm3, a final injection was administered. Mice were euthanized 24 h later, and blood and tissue samples were collected for further analysis. All animal experiments were approved by the Animal Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences (IACUC-RE-2025-03-006) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals.

Immunohistochemistry (IHC)

Paraffin-embedded human CCA tissue sections (4 μm) were obtained from Yunnan Cancer Hospital. Sections were deparaffinized in xylene and rehydrated through graded ethanol solutions. Antigen retrieval was performed by heating in citrate buffer (pH 6.0) at 100 °C for 10 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide. Sections were then blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature and incubated with primary antibodies overnight at 4 °C. After washing, sections were incubated with the appropriate secondary antibodies, followed by chromogenic detection, microscopic examination, and image acquisition. The following primary antibodies were used: BCL-XL (1:500, #2764, Cell Signaling Technology, USA), MCL-1 (1:200, #39224, Cell Signaling Technology), and BCL-2 (1:200, MAB-0711, Fuzhou Maixin Biotech, China).

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). Comparisons between two groups were performed using an unpaired two-tailed Student’s t-test; Welch’s correction was applied when the assumption of equal variance was not met. For comparisons among more than two groups, one-way analysis of variance (ANOVA) was conducted. Statistical significance was set at p < 0.05 (* for p < 0.05, ** for p < 0.01, and *** for p < 0.001; ns, not significant).

Results

BCL-XL is a prognostic factor and therapeutic target for CCA

The BCL-2 protein family comprises pro-apoptotic (e.g., BAX, BAK, PUMA, NOXA) and anti-apoptotic (e.g., BCL-2, BCL-XL, MCL-1) members, which regulate cancer cell apoptosis, patient survival, and drug resistance across malignancies. We analyzed BCL-2 family gene expression in eight major cancers (CCA, prostate adenocarcinoma, uterine corpus endometrial carcinoma, lung adenocarcinoma, acute myeloid leukemia, adrenocortical carcinoma, glioblastoma multiforme, and bladder urothelial carcinoma) from TCGA. In most solid cancer types, anti-apoptotic genes showed no significant change or were downregulated in cancerous tissues compared to normal tissues, while pro-apoptotic genes displayed variable expression (Additional file 4: Fig. S1A-C; Additional file 4: Fig. S2A-C). Acute myeloid leukemia showed upregulated BCL2 and MCL1 (Additional file 4: Fig. S2D). Crucially, CCA exhibited concurrent upregulation of both anti-apoptotic (BCL2L1, BCL2, MCL1) and pro-apoptotic (BBC3, BAK1, PMAIP1) genes (Fig. 1A).

Fig. 1.

Fig. 1

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BCL-XL is a prognostic factor and a potential therapeutic target for CCA. A Expression of BCL-2 family genes in CCA. Data are from TCGA. B, C Immunohistochemical analysis of BCL-XL, BCL-2 and MCL-1 in human CCA and adjacent non-tumor tissues, with representative images and quantification of positively stained cells. D-F ROC curve analysis of the diagnostic performance of BCL2L1, BCL2, and MCL1 in CCA based on TCGA data. G-I Progression-free survival analysis of CCA patients stratified by high versus low expression of BCL2L1, BCL2, and MCL1

Focusing on anti-apoptotic proteins for their survival-promoting role in malignancies, we validated BCL-XL, BCL-2, and MCL-1 protein levels via immunohistochemistry in six clinical CCA specimens. All three proteins were elevated in tumor tissues versus adjacent non-tumor tissues, with BCL-XL showing the highest expression (40-fold increase), followed by BCL-2 and MCL-1 (Fig. 1B-C). Diagnostic performance analysis revealed BCL2L1 (AUC = 0.987) and BCL2 (AUC = 0.949) as superior CCA predictors, while MCL1 had moderate performance (AUC = 0.797) (Fig. 1D-F). Survival analysis indicated that higher BCL2L1 expression correlated with shorter patient survival, though statistical significance was not achieved, while BCL2 and MCL1 showed no such association (Fig. 1G-I). These findings establish BCL-XL as both a robust prognostic biomarker and a promising therapeutic target for CCA.

BCL-XL inhibition, not BCL-2 or MCL-1 targeting, reduces CCA cell viability

To investigate the functional roles of anti-apoptotic proteins in CCA, we analyzed four established CCA cell lines (SNU1079, RBE, HuccT1, SNU1196) and one normal biliary epithelial line (HIBEC). Among these, SNU1079, RBE, and HuccT1 represent intrahepatic CCA (ICC), while SNU1196 is an extrahepatic CCA (ECC) subtype. CCLE database analysis revealed consistent high expression of anti-apoptotic gene BCL2L1 and pro-apoptotic genes (BAX, BAK1, PMAIP1) across all CCA lines, whereas MCL1 and BCL2 showed relatively lower expression (Additional file 4: Fig. S3A). Protein profiling confirmed transcriptional findings: BCL-XL was uniformly elevated in all four CCA lines compared to HIBEC (Fig. 2A-B). BCL-2 exhibited comparable levels between malignant and normal cells, except in HuccT1, where it was reduced. MCL-1 levels varied: elevated in SNU1079/RBE but lower in SNU1196/HuccT1 versus HIBEC. Notably, pro-apoptotic proteins BAX, BIM, and NOXA were highly expressed in at least three CCA lines (Fig. 2A-B). These patterns position BCL-XL as a potential therapeutic target.

Fig. 2.

Fig. 2

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BCL-XL is essential for CCA cell survival and CRBN-based PROTAC targeting BCL-XL reduces cell viability. A Representative immunoblot of BCL-2 family proteins in HIBEC and four CCA cell lines. B Quantification of protein levels in (A). C Cell viability of HIBEC and CCA cells after 72 h treatment with BCL-2 family inhibitors (A-1331852, ABT199, S63845, and ABT263) assessed by MTS assay. IC50: half-maximal inhibitory concentration. D Chemical structures of DT2216 and XZ739. E, F Cell viability of HIBEC and SNU1079 cells after 72 h treatment with DT2216 (E) or XZ739 (F), determined by MTS assay. G Representative immunoblot of CRBN and VHL expression in HIBEC and four CCA cell lines. H Quantification of CRBN and VHL levels in (G). Data are presented as mean ± SEM, and the experiments were performed at least three times. Statistical analysis was performed using unpaired two-tailed Student’s t-test or one-way ANOVA, as appropriate. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant

To validate functional dependencies, cells were treated with pathway-specific inhibitors: A1331852 (BCL-XL-selective), ABT199 (BCL-2-selective), S63845 (MCL-1-selective), and ABT263 (BCL-XL/BCL-2 dual inhibitor). Normal HIBEC cells remained viable post-treatment (Additional file 4: Fig. S3B), indicating therapeutic selectivity. Crucially, BCL-XL inhibition with A1331852 markedly reduced viability in SNU1079 (IC50 = 0.2 μM) and SNU1196 (IC50 = 0.07 μM) cells (Fig. 2C; Additional file 4: Fig. S3C-D). ABT263 similarly suppressed these lines (IC50 = 3.5 and 2.32 μM, respectively), confirming BCL-XL sensitivity. Small interfering RNA (siRNA)-mediated knockdown of BCL-XL significantly reduced SNU1079 cell viability via apoptosis induction (Additional file 4: Fig. S3E-G), thereby confirming the functional dependence of the cells on BCL-XL for survival. By contrast, BCL-2 or MCL-1-specific inhibitors showed no efficacy (Fig. 2C; Additional file 4: Fig. S3C-D; Fig. 3H-I). Collectively, protein expression profiling and inhibitor sensitivity assays establish BCL-XL as a critical survival determinant in CCA cells, distinct from BCL-2/MCL-1 which lack functional impact in this context.

Fig. 3.

Fig. 3

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XZ739 induces apoptosis and inhibits clonogenicity and migration by selectively degrading BCL-XL in SNU1079 cells. A XZ739-induced apoptosis is caspase-dependent. SNU1079 cells were pretreated with QVD (10 μM, 2 h) followed by XZ739 (0.3 μM, 48 h). Representative flow cytometry plots are shown. B Quantification of apoptotic cells from (A). C XZ739 inhibited colony formation of SNU1079 cells after 72 h of treatment. D XZ739 (0.1 μM) suppressed SNU1079 cell migration after 48 h of treatment. E, F Quantification of colonies (C) and migration rates (D). G XZ739 induced dose-dependent degradation of BCL-XL and upregulated cPARP expression after 24 h treatment. H DT2216 induced time-dependent degradation of BCL-XL and PARP cleavage. I BCL-XL degradation persisted up to 96 h after XZ739 removal, following 24 h treatment. J ABT263, pomalidomide (POMA), or their combination did not affect BCL-XL levels in SNU1079 cells. K Pretreatment with ABT263 blocked BCL-XL degradation by XZ739. L Pretreatment with POMA, a CRBN ligand, blocked BCL-XL degradation by XZ739. M MG132 (proteasome inhibitor) blocked BCL-XL degradation by XZ739. Data are presented as mean ± SEM, and the experiments were performed at least three times. Statistical analysis was performed using unpaired two-tailed Student’s t-test or one-way ANOVA, as appropriate. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant

Targeting BCL-XL with PROTAC technology reduces CCA cell viability

While BCL-XL-selective inhibitors (e.g., A1331852) and dual BCL-XL/BCL-2 inhibitors (e.g., ABT263) demonstrate safety in normal biliary epithelial cells (Fig. 2C; Additional file 4: Fig. S3B), their clinical utility is severely limited by dose-dependent thrombocytopenia. This toxicity arises because platelets express high BCL-XL levels and depend on it for survival [27–29]. Conventional medicinal chemistry cannot overcome this on-target toxicity, prompting development of PROTACs to mitigate platelet toxicity while maintaining efficacy [22, 26]. PROTACs are heterobifunctional molecules that hijack the ubiquitin-proteasome system (UPS) to degrade target proteins [30, 31].

Having established BCL-XL as a critical survival determinant in CCA cells, we evaluated two BCL-XL-specific PROTACs: DT2216, a VHL-recruiting degrader derived from ABT263 (Fig. 2D, upper) [26]; another is XZ739, a CRBN-recruiting PROTAC also derived from ABT263 (Fig. 2D, lower) [23]. Viability assays revealed that DT2216 did not affect SNU1079 cells (Fig. 2E), whereas XZ739 potently reduced their viability (Fig. 2F). Both compounds remained non-toxic to normal HIBEC cells (Fig. 2E-F), consistent with prior inhibitor findings (Fig. 2C). The differential efficacy stems from E3 ligase expression patterns: CRBN was highly expressed while VHL was relatively low in CCA cells (Fig. 2G-H). These data demonstrate that CRBN-based PROTAC outperforms VHL-based counterpart in suppressing CCA cell activity, and has a preferable safety profile.

XZ739 induces apoptosis, inhibits clonogenicity, and blocks migration via selective BCL-XL degradation in SNU1079 cells

Flow cytometry confirmed that reduced cell viability with XZ739 primarily resulted from apoptosis (15% apoptotic rate at 0.3 μM/48 h vs. 5% vehicle) (Fig. 3A-B), not cell cycle arrest (Additional file 4: Fig. S4A). This apoptosis was fully blocked by pan-caspase inhibitor QVD (Fig. 3A-B). Additionally, XZ739 significantly suppressed colony formation and migration (Fig. 3C-F). Mechanistically, XZ739 dose- and time-dependently reduced BCL-XL protein levels without affecting BCL-2/MCL-1 (Fig. 3G-H; Additional file 4: Fig. S4B-E), confirming its degradation selectivity. Concomitant increases in cleaved PARP (cPARP) drove apoptotic cell death (Fig. 3G-H). The degradation occurred post-translationally, as XZ739 did not alter anti-apoptotic gene expression (Additional file 4: Fig. S4F-H). To assess the degradation specificity of XZ739, we synthesized a negative compound (XZ739-Neg) lacking CRBN-recruiting capacity and evaluated its degradation efficacy against BCL-XL and its effect on CCA cell viability (Additional file 4: Fig. S4I). As anticipated, XZ739 demonstrated dose-dependent degradation of BCL-XL, whereas XZ739-Neg exhibited no capacity to induce BCL-XL degradation even at elevated concentrations (Additional file 4: Fig. S4J). Consistent with these findings, XZ739 significantly reduced CCA cell viability in a dose-dependent manner, while XZ739-Neg showed no measurable impact (Additional file 4: Fig. S4K). Given that CRBN-based PROTACs may target known CRBN neosubstrates, including IKZF family proteins (IKZF1/IKZF3), CK1α, and GSPT1 [32], we further quantified the protein levels of these targets following XZ739 treatment. As demonstrated in Additional file 4: Fig. S4L, XZ739 exhibited dose-dependent degradation of BCL-XL at 30 and 100 nM concentrations, while sparing IKZF1, IKZF3, CK1α, and GSPT1. These results confirm the selective degradation of BCL-XL by XZ739. Cells treated with 0.1 μM XZ739 for 24 h showed near-complete BCL-XL depletion at 24 h post-washout, persisting for ≥ 96 h (Fig. 3I), demonstrating prolonged degradation activity. This sustained effect positions XZ739 as a long-acting BCL-XL-targeting therapeutic for CCA.

XZ739 degrades BCL-XL via the ubiquitin-proteasomal pathway

We next investigated the mechanism by which XZ739 induces BCL-XL degradation. PROTACs function as bifunctional molecules, linking a target-binding ligand to an E3 ligase-recruiting module [33, 34]. To validate this, we first tested ABT263 (BCL-XL ligand) and pomalidomide (CRBN ligand) in SNU1079 cells. Neither compound alone nor their combination induced BCL-XL degradation (Fig. 3J). However, pre-incubation with excess ABT263 or pomalidomide blocked XZ739-mediated BCL-XL degradation (Fig. 3K-L), confirming ligand competition. Further, the proteasome inhibitor MG-132 prevented BCL-XL degradation (Fig. 3M), directly implicating the ubiquitin-proteasome system. These findings collectively establish that XZ739 functions as a CRBN-dependent PROTAC, requiring both its ligands and proteasomal activity for targeted BCL-XL degradation in CCA cells.

XZ739 monotherapy fails to suppress CCA progression in xenograft models

Based on in vitro data, we evaluated the effect of XZ739 on CCA in vivo. BCL-XL PROTACs mitigate platelet toxicity as platelets express lower CRBN/UBA1/SFT levels—key ubiquitin-proteasome components [35]. To assess the impact of XZ739 on platelet count, C57BL/6 mice received 0.5, 1, 2, 3, and 6 mpk XZ739, or 6 mpk ABT263, with blood counts measured at 24 and 72 h. While 2 mpk XZ739 caused transient platelet reduction (recoverable by 72 h), doses ≥ 3 mpk induced sustained thrombocytopenia (≥ 50% reduction), mirroring ABT263 effects (Fig. 4A; Additional file 4: Fig. S5A-B). Neither compound altered monocyte counts (Additional file 4: Fig. S5C), suggesting < 3 mpk as a safe in vivo dose.

Fig. 4.

Fig. 4

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XZ739 alone does not suppress CCA progression in vivo. A Platelet toxicity assessment showing 2 mpk XZ739 as a safe dose in vivo. C57BL/6 mice were treated with XZ739 (0.5, 1, or 2 mpk), and blood counts were measured at 24 h and 72 h (n = 4 mice per group). B Schematic of the first SNU1079 xenograft study. C, D Body weight (D) and tumor volume (E) changes over time (n = 6 for VEH, n = 7 for other groups). E Photographs of resected tumors. F Tumor weights at sacrifice (day 25 post-engraftment) (n = 6 for VEH, n = 7 for other groups). G Hematological parameters (PLT, WBC, RBC, HGB, LYM) one day after final injection (n = 6 for VEH; n = 7 for other groups). H Immunoblot analysis of BCL-XL, BCL-2, MCL-1 and cPARP in tumor tissues (n = 3 mice per group), with quantification of BCL-XL. Data are presented as mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t-test or one-way ANOVA, as appropriate. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant

We next tested XZ739 in SNU1079 xenograft nude mice (Fig. 4B). Initial 1.5/2.5 mpk XZ739 (q3d) or 40 mpk ABT263 from day 16 showed no tumor growth inhibition by day 28. Increasing XZ739 to 5 mpk (or switching 2.5 mpk to q2d) also failed to suppress tumor growth by day 40 (Fig. 4C-F), indicating that BCL-XL inhibition/degradation alone is insufficient for CCA control.

Notably, 2.5 mpk XZ739 spared platelets, while 5 mpk induced thrombocytopenia (Fig. 4G). Organ index analyses revealed no effects on lung/kidney/heart but mild spleen/liver increases (Additional file 4: Fig. S5D-H). Western blot confirmed XZ739 reached tumors and reduced BCL-XL but did not activate PARP (Fig. 4H and Additional file 4: Fig. S5I-K), suggesting inadequate apoptotic triggering despite target engagement. These findings suggest that XZ739, at an appropriate dose (2.5 mpk), effectively degrades BCL-XL while minimizing platelet toxicity in vivo—a major obstacle in targeting BCL-XL. However, the limited efficacy of XZ739 in CCA suggests that cholangiocarcinoma progression may rely on multiple survival mechanisms.

Synergistic suppression of CCA progression by XZ739-chemotherapy combinations

XZ739 is capable of degrading BCL-XL; however, it fails to trigger cell apoptosis in vivo. This prompts us to speculate that BCL-XL in CCA tissue might function as a sensitizer for apoptosis in vivo. Subsequently, we examined the response of CCA cells to classical chemotherapeutic agents, namely gemcitabine, cisplatin, and doxorubicin. The former two are the most commonly employed chemotherapeutics for patients with advanced CCA [36]. Gemcitabine alone demonstrated excellent activity against RBE, HuccT1, and SNU1079 cells, with IC50 values lower than 1 μM (Fig. 5A-B; Additional file 4: Fig. S6A). Doxorubicin was highly effective in eliminating all 4 CCA cell lines (Additional file 4: Fig. S6A). SNU1196 cells were sensitive to doxorubicin but resistant to the other two compounds (Additional file 4: Fig. S6A). Despite the potency of these chemotherapeutic agents against CCA cell lines, they also pose a significant toxicity to normal biliary epithelial cells (Fig. 5A-B; Additional file 4: Fig. S6A), which raises safety concerns.

Fig. 5.

Fig. 5

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XZ739 synergizes with chemotherapeutic agents to suppress CCA in vitro. A, B Viability of HIBEC and CCA cells after 72 h treatment with gemcitabine (A) or cisplatin (B), assessed by MTS assay. C-E Synergistic effects of gemcitabine and XZ739 (1:3 ratio, 72 h) in SNU1079 cells. Dose-response curves, CI plots and CI values were generated by CompuSyn. F-H Synergistic effects of cisplatin and XZ739 (1:3 ratio, 72 h) in SNU1079 cells. CI analyses were generated by CompuSyn. I, J Apoptosis analysis showing synergy between gemcitabine and XZ739, but not cisplatin and XZ739. Cells were treated with 0.3 μM XZ739, 0.1 μM gemcitabine or cisplatin, or their combinations for 48 h. Representative flow cytometry plots (I) and quantification (J) are shown. K, L Immunoblot analysis of BCL-XL, PARP, and cPARP in SNU1079 cells treated with XZ739 (30 nM), gemcitabine (10 nM), cisplatin (10 nM), or their combinations for 24 h. Data are presented as mean ± SEM, and the experiments were performed at least three times. Statistical analysis was performed using unpaired two-tailed Student’s t-test or one-way ANOVA, as appropriate. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant

Next, we investigated whether XZ739 has a synergistic effect when combined with chemotherapeutic drugs. If so, the combined use of XZ739 and these drugs might enhance the ability to kill CCA cells while simultaneously reducing the toxicity of chemotherapeutic drugs to normal cells. As shown in Fig. 5C-H and Additional file 4: Fig. S6B-G, all the tested chemotherapeutic drugs exhibited a potent synergistic effect when combined with XZ739. Among them, the combination of gemcitabine and XZ739 performed the best, with all combination index (CI) values less than 0.35 (Fig. 5C-E). The synergistic effect of XZ739 in combination with gemcitabine was further validated by flow cytometry and Western blot assays, which showed an increased number of apoptotic cells and higher levels of the apoptotic marker cPARP (Fig. 5I-K). In contrast, the combination of XZ739 with cisplatin demonstrated a weaker or no synergistic effect in these assays (Fig. 5I and L).

Furthermore, we conducted a second animal experiment to evaluate the combination role of XZ739 with chemotherapeutic agents in CCA. A safe dose of XZ739 (2.5 mpk), the reported dose of gemcitabine (50 mpk), and cisplatin (2.5 mpk), as well as their combinations, were utilized in the treatment (Fig. 6A). Treatment with XZ739 at 2.5 mpk alone did not affect body weight (Fig. 6B), and this dose also did not induce platelet toxicity (Fig. 4G), indicating that XZ739 is well tolerated in mice. Consistent with the data obtained from the first batch of animal experiments, XZ739 alone had no impact on CCA growth (Fig. 6B-E). Although gemcitabine and cisplatin alone could suppress cancer growth, they also led to a reduction in the body weight of mice (Fig. 6B), suggesting their potential toxicity as observed in vitro experiments (Fig. 5A-B; Additional file 4: Fig. S6A). Notably, the combination of 50 mpk gemcitabine with 2.5 mpk XZ739 significantly decreased body weight (Fig. 6B). To determine whether the combination of gemcitabine with XZ739 could maintain the inhibitory effect on CCA while reducing toxicity caused by gemcitabine, we decreased the dose of gemcitabine from 50 to 25 mpk starting from day 16. As anticipated, the new administration strategy quickly restored the body weight of the mice while sustaining the suppressive effect on tumor growth (Fig. 6B-E), thereby demonstrating a favorable synergistic effect. In contrast, cisplatin alone or in combination with XZ739 exhibited a good suppressive effect but did not display any synergistic effect, which was in line with the in vitro results (Fig. 5I and L). The suppressive effect of XZ739 in combination with chemotherapeutic agents was associated with its degradation activity on BCL-XL, as indicated by Western blot (Fig. 6F-I). Collectively, these results suggest that the combination of XZ739 with gemcitabine can synergistically suppress CCA progression and improve the safety window in vivo.

Fig. 6.

Fig. 6

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XZ739 synergizes with chemotherapeutic agents to suppress CCA in vivo. A Schematic of the second SNU1079 xenograft study. B Body weight changes in tumor-bearing mice (n = 6 mice per group at treatment start). C-E Combination of XZ739 and gemcitabine suppressed tumor growth. Representative images of resected tumors (C), tumor growth curves (D) and tumor weights at sacrifice (E) are shown (n = 6 mice per group at treatment start). F, G Immunoblot analysis of BCL-XL, BCL-2, and MCL-1 in tumor tissues from VEH, XZ739, gemcitabine (Gem), and XZ739 + Gem groups (F), or VEH, XZ739, cisplatin (Cis), and XZ739 + Cis groups (G) (n = 3 mice per group). H, I Quantification of BCL-XL, BCL-2, and MCL-1 protein levels in (H) and (I). (n = 3 mice per group). Data are presented as mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t-test or one-way ANOVA, as appropriate. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant

Discussion

CCA remains an aggressive malignancy with limited therapeutic options and dismal clinical outcomes, primarily attributable to intrinsic chemoresistance and the scarcity of effective targeted therapies. Dysregulation of the BCL-2 family of proteins represents a pivotal mechanism through which tumor cells evade apoptosis, with BCL-XL emerging as a particularly critical determinant of survival. In this study, we establish BCL-XL as the major survival dependency in CCA and demonstrate that its selective degradation via the CRBN-based PROTAC XZ739 effectively sensitizes tumors to chemotherapy while mitigating platelet toxicity—a major limitation of conventional BCL-XL inhibitors.

Consistent with prior reports, our transcriptomic and immunohistochemical analyses confirm widespread upregulation of BCL-2 family proteins in CCA tissues, with BCL-XL exhibiting the most pronounced elevation and correlating with adverse patient outcomes [37, 38]. Similar associations have been documented in hepatocellular, pancreatic, and colorectal cancers, where BCL2L1 overexpression confers chemoresistance by suppressing intrinsic apoptosis [39–41]. These findings underscore the prognostic and therapeutic relevance of BCL-XL across diverse malignancies. Therapeutic targeting of BCL-XL has historically been constrained by on-target thrombocytopenia, as platelets rely on BCL-XL for survival [14]. For instance, ABT263 was initially developed as an anticancer agent but was later repurposed as a senolytic due to dose-limiting platelet toxicity [42, 43]. To circumvent this challenge, we leveraged PROTAC technology, which exploits E3 ligase-mediated ubiquitination and degradation of target proteins [44]. Notably, direct comparative analysis revealed that CRBN-based XZ739 reduced CCA cell viability more potently than the VHL-based DT2216, an effect correlated with higher CRBN expression in our models. This suggests that CRBN recruitment may confer particular advantages in CCA. Crucially, XZ739 demonstrates selective induction of BCL-XL degradation in CCA cells while sparing platelets, a phenomenon attributed to differential CRBN expression—abundant in CCA cell lines but markedly reduced in platelets [35, 45]. Additionally, our previous work revealed that CRBN-associated ubiquitin-activating (E1, UBA1) and ubiquitin-conjugating (E2, SFT) enzymes exhibit deficient expression in platelets [35], which may functionally impair BCL-XL degradation by disrupting the ubiquitin transfer required for proteasomal targeting. Therefore, the disfavored degradation in platelets could provide a therapeutic window for CCA treatment by minimizing hematopoietic toxicity while preserving anti-tumor efficacy.

Mechanistically, XZ739 operates through a durable, catalytic mode of action distinct from occupancy-driven inhibitors. Unlike ABT263 or A1331852, which require continuous binding to neutralize pro-apoptotic proteins like BIM [17, 46], PROTACs induce repeated ubiquitination and proteasomal degradation of their targets. Once BCL-XL is degraded, the PROTAC molecule can be recycled to eliminate residual protein, thereby sustaining suppression even after drug withdrawal [47]. This explains the prolonged efficacy of XZ739 observed in our in vitro models.

In vivo, however, XZ739 monotherapy achieved efficient BCL-XL degradation in xenograft tumors but failed to significantly suppress growth. This discrepancy likely stems from multiple factors, including adaptive resistance mechanisms (e.g., metabolic reprogramming or activation of parallel pro-survival pathways) [48, 49], pharmacokinetic limitations (e.g., insufficient intratumoral drug exposure), target saturation, or compensatory feedback mechanisms. Our data did not support compensatory MCL-1 upregulation; instead, MCL-1 levels decreased following XZ739 treatment, suggesting that alternative mechanisms drive in vivo resistance. We observed that a higher dose of XZ739 induced less pronounced BCL-XL degradation compared to a lower dose in XZ739 monotherapy. As a bifunctional molecule, XZ739 mediates its pharmacological activity through simultaneous binding to both CRBN and BCL-XL. However, at excessively high concentrations, XZ739 may preferentially form binary complexes (CRBN-XZ739 or BCL-XL-XZ739) rather than the desired ternary complex (CRBN-XZ739-BCL-XL), a phenomenon known as the “hook effect”. This dose-dependent paradoxical reduction in target engagement can compromise degradation efficiency [50]. Optimal structural modification of the BCL-XL PROTAC may reduce the hook effect and enable adequate exposure of the CAA tissue to the BCL-XL degrader, facilitating complete degradation of BCL-XL.

These observations highlight that while BCL-XL is a promising target, its inhibition is most effective within rational combination strategies rather than as monotherapy. To explore such strategies, we systematically screened chemotherapeutic agents in combination with XZ739. Gemcitabine and cisplatin are the most commonly used chemotherapeutic agents for patients with advanced CCA [36]. However, treatment with gemcitabine alone yields a median survival of only about 8 months, and even in combination with cisplatin, the median survival extends to merely around 1 year [51]. This limited therapeutic benefit is largely attributed to the development of chemoresistance [52]. In our study, we identified BCL-XL as a major factor for chemoresistance in CCA. In vitro and in vivo studies further demonstrate that BCL-XL degrader XZ739, combined with gemcitabine, synergistically and effectively suppresses CCA. This combination can significantly reduce the systemic toxicity of gemcitabine. Mechanistically, this synergy may arise from complementary actions on cell fate: gemcitabine induces S-phase arrest, whereas BCL-XL degradation relieves the sequestration of pro-apoptotic proteins, thereby enhancing their activity [53, 54]. Pro-apoptotic factors such as BAX, BAK, PUMA, and NOXA are frequently upregulated in CCA [55], and prior studies indicate that the balance between pro- and anti-apoptotic proteins strongly influences drug sensitivity [8, 10]. By releasing these pro-apoptotic factors, XZ739 lowers the apoptotic threshold, rendering tumor cells more vulnerable to gemcitabine-induced DNA damage. This integrated model provides a more comprehensive explanation for the observed synergy beyond cell cycle regulation alone and aligns with prior reports in other malignancies [56, 57]. Therefore, the combination of a CRBN-based degrader and gemcitabine, particularly in its potential to overcome chemoresistance and minimize hematopoietic toxicity, may enhance the response rate in clinical treatment and/or reduce systemic toxicity.

Several limitations warrant consideration. First, XZ739 activity was evident only in a subset of CCA cell lines, reflecting tumor heterogeneity and underscoring the need for predictive biomarkers to guide patient selection. Second, although XZ739 exhibited strong efficacy against CCA cells in vitro, it failed to suppress CCA progression in vivo, indicating that the underlying mechanisms warrant further investigation. Third, as most experiments were conducted in CCA cell lines and immunodeficient mouse models, future studies employing patient-derived xenograft and organoid models will be essential to validate these findings and enhance their translational relevance to human CCA therapy.

Conclusions

In conclusion, this study identifies BCL-XL as a clinically relevant therapeutic target in CCA and demonstrates that CRBN-based PROTAC degradation offers a viable strategy to overcome the limitations of conventional inhibitors. The robust synergy between XZ739 and gemcitabine provides a mechanistic rationale for combination regimens, highlighting the translational potential of PROTAC-mediated BCL-XL targeting in CCA. Future investigations integrating biomarker-guided patient stratification, comprehensive safety assessment, and exploration of additional combinatorial approaches will be essential to translate these preclinical findings into clinical benefit.

Supplementary Information

12916_2026_4671_MOESM1_ESM.docx (52.6KB, docx)

Additional file 1. General methods for chemical synthesis.

12916_2026_4671_MOESM2_ESM.docx (19.2KB, docx)

Additional file 2: Table S1. List of antibodies used in this study.

12916_2026_4671_MOESM3_ESM.docx (18.9KB, docx)

Additional file 3: Table S2. List of siRNA sequences and primers used in this study.

12916_2026_4671_MOESM4_ESM.docx (2.8MB, docx)

Additional file 4: Fig. S1. Expression of BCL-2 family genes in prostate adenocarcinoma, uterine corpus endometrial carcinoma, and lung adenocarcinoma. Fig. S2. Expression of BCL-2 family genes in adrenocortical carcinoma, glioblastoma multiforme, bladder urothelial carcinoma, and acute myeloid leukemia. Fig. S3. Expression and functional assessment of BCL-2 family members in CCA cell lines. Fig. S4. Effect of XZ739 on cell cycle and BCL-2 family gene expression in SNU1079 cells. Fig. S5. In vivo safety evaluation of XZ739. Fig. S6. Synergistic effects of XZ739 and chemotherapeutic drugs in vitro. Fig. S7. In vivo combination therapy with XZ739 and chemotherapeutic drugs.

Acknowledgements

We would like to thank Yusong Meng from the Core Technology Facility of Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS), for providing us with valuable technical assistance in flow cytometry assays.

Abbreviations

CCA

Cholangiocarcinoma

PROTAC

Proteolysis-targeting chimera

VHL

Von Hippel-Lindau

CRBN

Cereblon

DFS

Disease-free survival

AUC

Area under the curve

CI

Combination index

ICC

Intrahepatic CCA

ECC

Extrahepatic CCA

UPS

Ubiquitin-proteasome system

cPARP

Cleaved PARP

mpk

Mg/kg

Authors’ contributions

QHZ, YZ, and YWY contributed equally to this work. QHZ and YZ were responsible for methodology, writing – original draft, and writing – review & editing. YWY carried out the synthesis of the compound. XL1 (Xin Liu) performed the methodology and resources. XD, YZP, LH, AZ, QNL, and XL2 (Xiang Lai) performed methodology. JY and GPZ provided resources. YHH was responsible for writing – review & editing, writing – original draft, supervision, funding acquisition, and conceptualization. XZ performed writing – review & editing, supervision, funding acquisition, and conceptualization. All authors read and approved the final manuscript.

Funding

This work was supported by the National Key R&D Program of China (2023YFC3603300 to YHH), Yunnan Fundamental Research Projects (202305AH340006 to YHH), National Natural Science Foundation of China (82471599 to YHH, 22277131 to XZ), and CAS “Light of West China” Program (xbzg-zdsys-202312 to YHH). Both YHH and XZ are supported by the Pioneer Hundred Talents Program of the Chinese Academy of Sciences. In addition, YHH is supported by the Yunnan Revitalization Talent Support Program Young Talent Project. GPZ is supported by the Outstanding Innovative Research Team for Molecular Enzymology and Detection in Anhui Provincial Universities (2022AH010012).

Data availability

All datasets analyzed in this study are publicly available. Transcriptomic data were obtained from The Cancer Genome Atlas (TCGA) database ([https://www.cancer.gov/ccg/research/genome‐sequencing/tcga](https:/www.cancer.gov/ccg/research/genome%E2%80%90sequencing/tcga)) [24]. Gene expression data for cholangiocarcinoma cell lines were retrieved from the Cancer Cell Line Encyclopedia (CCLE) database ([https://www.sites.broadinstitute.org/ccle/datasets](https:/www.sites.broadinstitute.org/ccle/datasets)). Disease-free survival analyses were performed using the GEPIA2 web tool ([http://gepia2.cancer-pku.cn](http:/gepia2.cancer-pku.cn)) [25].

Declarations

Ethics approval and consent to participate

Human samples were collected in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital, and the Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences (Approval No. KIZRKX-2025-SQ-046). Written informed consent was obtained from all participants.

All animal experiments were approved by the Animal Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences (IACUC-RE-2025-03-006) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals. During the experiment, the maximum tumor volume in nude mice did not exceed the ethically permitted limit of 1500 mm3.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Qinghua Zeng, Yan Zhang and Yiwen Yang contributed equally to this work.

Contributor Information

Xuan Zhang, Email: zhangxuan@simm.ac.cn.

Yonghan He, Email: heyonghan@mail.kiz.ac.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12916_2026_4671_MOESM1_ESM.docx (52.6KB, docx)

Additional file 1. General methods for chemical synthesis.

12916_2026_4671_MOESM2_ESM.docx (19.2KB, docx)

Additional file 2: Table S1. List of antibodies used in this study.

12916_2026_4671_MOESM3_ESM.docx (18.9KB, docx)

Additional file 3: Table S2. List of siRNA sequences and primers used in this study.

12916_2026_4671_MOESM4_ESM.docx (2.8MB, docx)

Additional file 4: Fig. S1. Expression of BCL-2 family genes in prostate adenocarcinoma, uterine corpus endometrial carcinoma, and lung adenocarcinoma. Fig. S2. Expression of BCL-2 family genes in adrenocortical carcinoma, glioblastoma multiforme, bladder urothelial carcinoma, and acute myeloid leukemia. Fig. S3. Expression and functional assessment of BCL-2 family members in CCA cell lines. Fig. S4. Effect of XZ739 on cell cycle and BCL-2 family gene expression in SNU1079 cells. Fig. S5. In vivo safety evaluation of XZ739. Fig. S6. Synergistic effects of XZ739 and chemotherapeutic drugs in vitro. Fig. S7. In vivo combination therapy with XZ739 and chemotherapeutic drugs.

Data Availability Statement

All datasets analyzed in this study are publicly available. Transcriptomic data were obtained from The Cancer Genome Atlas (TCGA) database ([https://www.cancer.gov/ccg/research/genome‐sequencing/tcga](https:/www.cancer.gov/ccg/research/genome%E2%80%90sequencing/tcga)) [24]. Gene expression data for cholangiocarcinoma cell lines were retrieved from the Cancer Cell Line Encyclopedia (CCLE) database ([https://www.sites.broadinstitute.org/ccle/datasets](https:/www.sites.broadinstitute.org/ccle/datasets)). Disease-free survival analyses were performed using the GEPIA2 web tool ([http://gepia2.cancer-pku.cn](http:/gepia2.cancer-pku.cn)) [25].

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