Melatonin inhibits EMT and PD‐L1 expression through the ERK1/2/FOSL1 pathway and regulates anti‐tumor immunity in HNSCC

Melatonin is an endogenous hormone with various biological functions and possesses anti‐tumor properties in multiple malignancies. Immune evasion is one of the most important hallmarks of head and neck squamous cell carcinoma (HNSCC) and is closely related to tumor progression. However, as an immune modulator under physiological conditions, the roles of melatonin in tumor immunity in HNSCC remains unclear. In this study, we found that the endogenous melatonin levels in patients with HNSCC were lower than those in patients with benign tumors in head and neck. Importantly, lower melatonin levels were related to lymph node metastasis among patients with HNSCC. Moreover, melatonin significantly suppressed programmed death‐ligand 1 (PD‐L1) expression and inhibited epithelial–mesenchymal transition (EMT) of HNSCC through the ERK1/2/FOSL1 pathway in vitro and in vivo. In SCC7/C3H syngeneic mouse models, anti‐programmed death‐1 (PD‐1) antibody combined with melatonin significantly inhibited tumor growth and modulated anti‐tumor immunity by increasing CD8⁺ T cell infiltration and decreasing the regulatory T cell (Treg) proportion in the tumor microenvironment. Taken together, melatonin inhibited EMT and downregulated PD‐L1 expression in HNSCC through the ERK1/2/FOSL1 pathway and exerted synergistic effects with anti‐PD‐1 antibody in vivo, which could provide promising strategies for HNSCC treatment.     

Circulating naïve and effector memory T cells correlate with prognosis in head and neck squamous cell carcinoma

T‐cell memory is an important mechanism for long‐term protection against diverse pathogens. Generation and persistence of memory T cells are vital components of anti‐tumor immunity, given their ability to persist for prolonged durations, as well as activate and migrate rapidly. In the present study, we investigated the clinical and prognostic significance of T‐cell subsets in the peripheral circulation of patients with head and neck squamous cell carcinoma (HNSCC). Moreover, we calculated the enrichment scores of T‐cell subsets in primary tumor tissues and compared their clinical characteristics using a public database. Multivariate survival analyses of circulating T‐cell parameters revealed that clinical parameters, except M factor, were not independent prognostic factors, whereas proportions of CD8⁺ T cells, naïve T cells (T_Ns), effector memory T cells (T_EMs), and CD38⁺CD8⁺ T cells were independent prognostic factors, suggesting the importance of these peripheral T‐cell parameters as independent prognostic biomarkers. Consistent with these results, the T‐cell enrichment analysis indicated that enrichment of CD8⁺ T_Ns in the tumor microenvironment was an independent prognostic factor. Moreover, an ex vivo experiment demonstrated significantly less cytotoxic activity in CD38⁺ T cells than in CD38⁻ T cells. These findings suggest that T‐cell memory‐related parameters in both systemic immunity and the tumor microenvironment could be used as prognostic biomarkers regardless of clinical characteristics. Further characterization of circulating T cells would lead to the development of novel biomarkers for patients with HNSCC.     

Targeting EZH2 regulates tumor growth and apoptosis through modulating mitochondria dependent cell-death pathway in HNSCC

EZH2 is a negative prognostic factor and is overexpressed or activated in most human cancers including head and neck squamous cell carcinoma (HNSCC). Analysis of The Cancer Genome Atlas (TCGA) HNSCC data indicated that EZH2 over-expression was associated with high tumor grade and conferred poor prognosis. EZH2 inhibition triggered cell apoptosis, cell cycle arrest and decreased cell growth in vitro. MICU1 (mitochondrial calcium uptake1) was shown to be down regulated when EZH2 expression was inhibited in HNSCC. When the EZH2 and MICU1 were inhibited, HNSCC cells became susceptible to cell cycle arrest and apoptosis. Mitochondrial membrane potential and cytosolic Ca²⁺ concentration analysis suggested that EZH2 and MICU1 were required to maintain mitochondrial membrane potential stability. A xenograft tumor model was used to confirm that EZH2 depletion inhibited HNSCC cell growth and induced tumor cell apoptosis. In summary, EZH2 is a potential anti-tumor target in HNSCC.     

HPV+ HNSCC‐derived exosomal miR‐9‐5p inhibits TGF‐β signaling‐mediated fibroblast phenotypic transformation through NOX4

Human papillomavirus (HPV) is a significant risk factor for head and neck squamous cell carcinoma (HNSCC). HPV⁺ HNSCC patients have a higher survival rate, which may be related to its unique tumor microenvironment. Exosomes are emerging as a communication tool between tumor cells and the tumor microenvironment, including cancer‐associated fibroblasts (CAFs). In this study, 111 clinical samples tissues and public sequencing data were analyzed. Our study found fewer CAFs infiltrated in HPV⁺ HNSCC, and poor CAF infiltration level was associated with a good prognosis. HPV⁺ HNSCC cell‐derived exosomes can significantly reduce the phenotypic transformation of fibroblasts. miR‐9‐5p, as a miRNA enriched in HPV⁺ HNSCC cell‐derived exosomes, can be transferred to fibroblasts. miR‐9‐5p mimic transfection decreased the expression of NOX4 and the level of intracellular reactive oxygen species (ROS), which inhibited the transforming growth factor beta 1(TGF‐β1)‐induced increase of αSMA levels. Therefore, these results indicated that HPV⁺ HNSCC‐derived exosomal miR‐9‐5p inhibits TGF‐β signaling‐mediated fibroblast phenotypic transformation through NOX4, which is related to the excellent prognosis of HPV patients.     

Inhibition of growth and survival of human head and neck squamous cell carcinoma cells by curcumin via modulation of nuclear factor-kappaB signaling

Increased expression of proinflammatory and proangiogenic factors are associated with aggressive tumor growth and decreased survival of patients with head and neck squamous cell carcinoma (HNSCC). In as much as genes that are regulated by nuclear factor NF-kappaB suppress apoptosis, induce proliferation, and mediate inflammation, angiogenesis and tumor metastasis, agents that suppress NF-kappaB activation have potential as treatment for various cancers including HNSCC. We demonstrate that all HNSCC cell lines expressed constitutively active NF-kappaB and IkappaBalpha kinase (IKK), which is needed for NF-kappaB activation. Treatment of MDA 686LN cells with curcumin (diferuloylmethane), a pharmacologically safe chemopreventive agent, inhibited NF-kappaB activation through abrogation of IKK. As a result expression of various cell survival and cell proliferative genes including Bcl-2, cyclin D1, IL-6, COX-2 and MMP-9 was suppressed. This, in turn, inhibits proliferation of all HNSCC cell lines, arrests cell cycle in G1/S phase (MDA 686LN) and induces apoptosis as indicated by upstream and downstream caspase activation, PARP cleavage, annexin V staining in MDA 686LN cells. Suppression of NF-kappaB by cell-permeable p65-based peptide and NBD peptide also inhibited the proliferation and induced apoptosis in these cells. Our results indicate that curcumin is a potent inhibitor of cell proliferation and an inducer of apoptosis in HNSCC through suppression of IKK-mediated NF-kappaB activation and of NF-kappaB-regulated gene expression.     

Genetic analysis of single disseminated tumor cells in the lymph nodes and bone marrow of patients with head and neck squamous cell carcinoma

Considering the limited information on the biology and molecular characteristics of disseminated tumor cells (DTCs) in head and neck squamous cell carcinoma (HNSCC), we examined the genomic alterations in DTCs from HNSCCs and their potential clinical relevance. To analyze both the lymphatic and hematogenous routes of tumor cell dissemination, we investigated samples from lymph nodes (LNs) and bone marrow (BM) of 49 patients using immunofluorescence double staining for epithelial cells expressing cytokeratin 18 (KRT18) and/or epithelial cell adhesion molecules (EpCAM, CD326). The identified marker‐positive cells were isolated by micromanipulation followed by single‐cell whole‐genome amplification and metaphase‐based comparative genomic hybridization (mCGH) to determine genome‐wide copy number alterations. The findings were correlated with clinical parameters and follow‐up data. We detected chromosomal aberrations in KRT18‐ and EpCAM‐positive cells from both compartments; BM‐derived cells showed a significantly higher percentage of aberrant genome (PAG) per cell than cells detected in LNs. No significant association was found between DTC data and clinical follow‐up. Genomic profiling of BM‐DTCs revealed genomic alterations typical for HNSCC, suggesting hematogenous dissemination of subclones around the time of surgery. In contrast, DTC data in LNs revealed that several marker‐positive cells were not of malignant origin, indicating the presence of epithelial glandular inclusions in parts of the processed neck LN samples. Therefore, DTC detection of LNs in the neck based only on epithelial markers is not advisable and requires detection of chromosomal instability (CIN), gene mutations, or additional markers, which have yet to be identified. Nevertheless, our investigation paves the way for larger studies to focus on HNSCC BM‐DTCs with high‐resolution methods to gain deeper insights into the biology of hematogenous metastasis in this cancer.     

Digital scoring of EpCAM and slug expression as prognostic markers in head and neck squamous cell carcinomas

Head and neck squamous cell carcinomas (HNSCCs) have poor clinical outcome owing to therapy resistance and frequent recurrences that are among others attributable to tumor cells in partial epithelial‐to‐mesenchymal transition (pEMT). We compared side‐by‐side software‐based and visual quantification of immunohistochemistry (IHC) staining of epithelial marker EpCAM and EMT regulator Slug in n = 102 primary HNSCC to assess optimal analysis protocols. IHC scores incorporated expression levels and percentages of positive cells. Digital and visual evaluation of membrane‐associated EpCAM yielded correlating scorings, whereas visual evaluation of nuclear Slug resulted in significantly higher overall scores. Multivariable Cox proportional hazard analysis defined the median EpCAM expression levels resulting from visual quantification as an independent prognostic factor of overall survival. Slug expression levels resulting from digital quantification were an independent prognostic factor of recurrence‐free survival, locoregional recurrence‐free survival, and disease‐specific survival. Hence, we propose to use visual assessment for the membrane‐associated EpCAM protein, whereas nuclear protein Slug assessment was more accurate following digital measurement.     

Loss of TGF-β signaling and PTEN promotes head and neck squamous cell carcinoma through cellular senescence evasion and cancer-related inflammation

The molecular mechanisms that contribute to the initiation and progression of head and neck squamous cell carcinoma (HNSCC) have not been completely delineated. Our observations indicate that defects in the transforming growth factor-β and PI3K/Akt signaling pathways are common in human HNSCCs. Conditional activation of the PI3K/Akt pathway due to Pten deletion in the mouse head and neck epithelia gives rise to hyperproliferation, but only a few lesions progress to HNSCC. However, Pten-deficient mice developed full-penetrance HNSCC in combination with type I TGF-β receptor (Tgfbr1) deletion. Molecular analysis revealed enhanced cell proliferation, decreased apoptosis, and increased expression of CCND1 in the basal layer of the head and neck epithelia, as well as in the tumors of Tgfbr1/Pten double conditional knockout (2cKO) mice. Furthermore, neoplastic transformation involves senescence evasion, and is associated with an increased number of putative cancer stem cells. In addition, the nuclear factor-κB pathway activation, myeloid-derived suppressor cell infiltration, angiogenesis and immune suppression in the tumor microenvironment, all of which are characteristics of human HNSCCs, contribute significantly to head and neck carcinogenesis in 2cKO mice. These tumors display pathology and multiple molecular alterations resembling human HNSCCs. This suggests that the Tgfbr1/Pten 2cKO mouse model is suitable for preclinical intervention, and that it has significant implications in the development of diagnostic cancer biomarkers and effective strategies for prevention and treatment of HNSCCs.     

DNA double strand break repair defect and sensitivity to poly ADP-ribose polymerase (PARP) inhibition in human papillomavirus 16-positive head and neck squamous cell carcinoma

Patients with human papillomavirus-positive (HPV+) head and neck squamous cell carcinomas (HNSCCs) have increased response to radio- and chemotherapy and improved overall survival, possibly due to an impaired DNA damage response. Here, we investigated the correlation between HPV status and repair of DNA damage in HNSCC cell lines. We also assessed in vitro and in vivo sensitivity to the PARP inhibitor veliparib (ABT-888) in HNSCC cell lines and an HPV+ patient xenograft. Repair of DNA double strand breaks (DSBs) was significantly delayed in HPV+ compared to HPV− HNSCCs, resulting in persistence of γH2AX foci. Although DNA repair activators 53BP1 and BRCA1 were functional in all HNSCCs, HPV+ cells showed downstream defects in both non-homologous end joining and homologous recombination repair. Specifically, HPV+ cells were deficient in protein recruitment and protein expression of DNA-Pk and BRCA2, key factors for non-homologous end joining and homologous recombination respectively. Importantly, the apparent DNA repair defect in HPV+ HNSCCs was associated with increased sensitivity to the PARP inhibitor veliparib, resulting in decreased cell survival in vitro and a 10–14 day tumor growth delay in vivo. These results support the testing of PARP inhibition in combination with DNA damaging agents as a novel therapeutic strategy for HPV+ HNSCC.     

Role of JNK activation in paclitaxel-induced apoptosis in human head and neck squamous cell carcinoma

It has been reported that paclitaxel activates cell cycle arrest and increases caspase protein expression to induce apoptosis in head and neck squamous cell carcinoma (HNSCC) cell lines. However, the potential signaling pathway regulating this apoptotic phenomenon remains unclear. The present study used OEC-M1 cells to investigate the underlying molecular mechanism of paclitaxel-induced apoptosis. Following treatment with paclitaxel, cell viability was assessed via the MTT assay. Necrosis, apoptosis, cell cycle and mitochondrial membrane potential (∆Ψm) were analyzed via flow cytometric analyses, respectively. Western blot analysis was performed to detect the expression levels of proteins associated with the MAPK and caspase signaling pathways. The results demonstrated that low-dose paclitaxel (50 nM) induced apoptosis but not necrosis in HNSCC cells. In addition, paclitaxel activated the c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38 mitogen-activated protein kinase. The paclitaxel-activated JNK contributed to paclitaxel-induced apoptosis, activation of caspase-3, −6, −7, −8 and −9, and reduction of ∆Ψm. In addition, caspase-8 and −9 inhibitors, respectively, significantly decreased paclitaxel-induced apoptosis. Notably, Bid was truncated following treatment with paclitaxel. Taken together, the results of the present study suggest that paclitaxel-activated JNK is required for caspase activation and loss of ∆Ψm, which results in apoptosis of HNSCC cells. These results may provide mechanistic basis for designing more effective paclitaxel-combining regimens to treat HNSCC.     

A combination of a ribonucleotide reductase inhibitor and histone deacetylase inhibitors downregulates EGFR and triggers BIM-dependent apoptosis in head and neck cancer

Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common malignant neoplasm and more than 50% of patients succumb to this disease. HNSCCs are characterized by therapy resistance, which relies on the overexpression of anti-apoptotic proteins and on the aberrant regulation of the epidermal growth factor receptor (EGFR). As inherent and acquired resistance to therapy counteracts improvement of long-term survival, novel multi-targeting strategies triggering cancer cell death are urgently required. We investigated how induction of replicational stress by the ribonucleotide reductase inhibitor hydroxyurea (HU) combined with histone deacetylase inhibitors (HDACi) exerts anti-tumor activity. We treated HNSCC cell lines and freshly isolated tumor cells with HDACi, such as the clinically approved anti-epileptic drug valproic acid (VPA), in combination with HU. Our data demonstrate that at clinically achievable levels VPA/HU combinations efficiently block proliferation as well as clonogenic survival, and trigger apoptosis of HNSCC cells. In the presence of VPA/HU, such tumor cells increase expression of the pro-apoptotic BCL-2 family protein BIM, independent of wild-type p53 signaling and in the absence of increased expression of the p53 targets PUMA and BAX. The pro-apoptotic activity of BIM in HNSCCs was found critical for tumor cell death; ectopic overexpression of BIM induced HNSCC apoptosis and RNAi-mediated depletion of BIM protected HNSCC cells from VPA/HU. Also, significantly elevated BIM levels (p less than 0.01) were detectable in the apoptotic tumor centers versus proliferating tumor margins in HNSCC patients (n=31), underlining BIM's clinical relevance. Importantly, VPA/HU treatment additionally reduces expression and cell surface localization of EGFR. Accordingly, in a xenograft mouse model, VPA/HU efficiently blocked tumor growth (P less than 0.001) correlating with BIM induction and EGFR downregulation. We provide a molecular rationale for the potent anti-cancer activities of this drug combination. Our data suggest its exploitation as a potential strategy for the treatment of HNSCC and other tumor entities characterized by therapy resistance linked to dysregulated EGFR activation.     

UBE2T promotes β‐catenin nuclear translocation in hepatocellular carcinoma through MAPK/ERK‐dependent activation

Ubiquitin‐conjugating enzyme E2T (UBE2T) has been implicated in many types of cancer including hepatocellular carcinoma (HCC). Epithelial–mesenchymal transition (EMT) process plays a fundamental role during tumor metastasis and progression. However, the molecular mechanisms underlying EMT in HCC in accordance with UBE2T still remain unknown. In this study, we showed that UBE2T overexpression augmented the oncogenic properties and specifically EMT in HCC cell lines, while its silencing attenuated them. UBE2T affected the activation of EMT‐associated signaling pathways: MAPK/ERK, AKT/mTOR, and Wnt/β‐catenin. In addition, we revealed that the epithelial protein complex of E‐cadherin/β‐catenin, a vital regulator of signal transduction in tumor initiation and progression, was totally disrupted at the cell membrane. In particular, we observed that UBE2T overexpression led to E‐cadherin loss accompanied by a simultaneous elevation of both cytoplasmic and nuclear β‐catenin, while its silencing resulted in a strong E‐cadherin turnover at the cell membrane. Interestingly, chemical inhibition of the MAPK/ERK, AKT/mTOR, and Wnt/β‐catenin signaling pathways demonstrated that the nuclear translocation of β‐catenin and subsequent EMT was enhanced mainly by MAPK/ERK. Collectively, our findings demonstrate the UBE2T/MAPK‐ERK/β‐catenin axis as a critical regulator of cell state transition and EMT in HCC.     

Heat shock protein 47 confers chemoresistance on pancreatic cancer cells by interacting with calreticulin and IRE1α

Pancreatic ductal adenocarcinoma (PDAC) is one of the most chemoresistant cancers. An understanding of the molecular mechanism by which PDAC cells have a high chemoresistant potential is important for improvement of the poor prognosis of patients with PDAC. Here we show for the first time that disruption of heat shock protein 47 (HSP47) enhances the efficacy of the therapeutic agent gemcitabine for PDAC cells and that the efficacy is suppressed by reconstituting HSP47 expression. HSP47 interacts with calreticulin (CALR) and the unfolded protein response transducer IRE1α in PDAC cells. Ablation of HSP47 promotes both the interaction of CALR with sarcoplasmic/endoplasmic reticulum Ca²⁺‐ATPase 2 and interaction of IRE1α with inositol 1,4,5‐triphosphate receptor, which generates a condition in which an increase in intracellular Ca²⁺ level is prone to be induced by oxidative stimuli. Disruption of HSP47 enhances NADPH oxidase‐induced generation of intracellular reactive oxygen species (ROS) and subsequent increase in intracellular Ca²⁺ level in PDAC cells after treatment with gemcitabine, resulting in the death of PDAC cells by activation of the Ca²⁺/caspases axis. Ablation of HSP47 promotes gemcitabine‐induced suppression of tumor growth in PDAC cell‐bearing mice. Overall, these results indicated that HSP47 confers chemoresistance on PDAC cells and suggested that disruption of HSP47 may improve the efficacy of chemotherapy for patients with PDAC.     

MK‐6, a novel not‐α IL‐2, elicits a potent antitumor activity by improving the effector to regulatory T cell balance

IL‐2 is a pleiotropic cytokine that regulates immune cell homeostasis. Its immunomodulatory function has been used clinically as an active immunotherapy agent for metastatic cancers. However, severe adverse effects, including the vascular leak syndrome and the preferential stimulation of anti‐immunogenic Treg rather than effector T cells, have been obstacles. We newly designed a mutein IL‐2, Mutakine‐6 (MK‐6), with reduced IL‐2Rα–binding capability. MK‐6 induced comparable cell growth potential toward IL‐2Rβγ–positive T cells but was far less efficient in in vitro Treg proliferation and STAT5 activation. Unlike IL‐2, in vivo administration of MK‐6 produced minimal adverse effects. Using CT26 and B16F10‐syngeneic tumor models, we found MK‐6 was highly efficacious on tumor regression. Serum albumin conjugation to MK‐6 prolonged in vivo half‐life and accumulated in CT26 tumors, showing enhanced antitumor effect. Tumor‐infiltrating leukocytes analysis revealed that albumin‐fused MK‐6 increased the ratio of effector CD8⁺ T cells to CD4⁺ Treg cells. These results demonstrated that MK‐6 is an efficient immunomodulator potentially used for improved immunotherapy with decreased adverse effects and attenuated Treg stimulation.     

PD‐1‐induced T cell exhaustion is controlled by a Drp1‐dependent mechanism

Programmed cell death‐1 (PD‐1) signaling downregulates the T‐cell response, promoting an exhausted state in tumor‐infiltrating T cells, through mostly unveiled molecular mechanisms. Dynamin‐related protein‐1 (Drp1)‐dependent mitochondrial fission plays a crucial role in sustaining T‐cell motility, proliferation, survival, and glycolytic engagement. Interestingly, such processes are exactly those inhibited by PD‐1 in tumor‐infiltrating T cells. Here, we show that PD‐1^pos CD8⁺ T cells infiltrating an MC38 (murine adenocarcinoma)‐derived murine tumor mass have a downregulated Drp1 activity and more elongated mitochondria compared with PD‐1^neg counterparts. Also, PD‐1^pos lymphocytic elements infiltrating a human colon cancer rarely express active Drp1. Mechanistically, PD‐1 signaling directly prevents mitochondrial fragmentation following T‐cell stimulation by downregulating Drp1 phosphorylation on Ser616, via regulation of the ERK1/2 and mTOR pathways. In addition, downregulation of Drp1 activity in tumor‐infiltrating PD‐1^pos CD8⁺ T cells seems to be a mechanism exploited by PD‐1 signaling to reduce motility and proliferation of these cells. Overall, our data indicate that the modulation of Drp1 activity in tumor‐infiltrating T cells may become a valuable target to ameliorate the anticancer immune response in future immunotherapy approaches.     

LS‐106, a novel EGFR inhibitor targeting C797S,exhibits antitumor activities both in vitro and in vivo

With the wide clinical use of the third‐generation epidermal growth factor receptor (EGFR) inhibitor osimertinib for the treatment of EGFR‐mutated non–small cell lung cancer (NSCLC), acquired resistance caused by EGFR C797S tertiary mutation has become a concern. Therefore, fourth‐generation EGFR inhibitors that could overcome this mutation have gained increasing attention in recent years. Here, we identified LS‐106 as a novel EGFR inhibitor against C797S mutation and evaluated its antitumor activity both in vitro and in vivo. In cell‐free assay, LS‐106 potently inhibited the kinase activities of EGFR^{19del/T790M/C797S} and EGFR^{L858R/T790M/C797S} with IC₅₀ values of 2.4 nmol/L and 3.1 nmol/L, respectively, which was more potent than osimertinib. Meanwhile, LS‐106 exhibited comparable kinase inhibitory effect to osimertinib on EGFR^L858R/T790M and wild‐type EGFR. Results from cellular experiments demonstrated that LS‐106 potently blocked the phosphorylation of EGFR C797S triple mutations in the constructed BaF3 cells that highly expressed EGFR^{19del/T790M/C797S} or EGFR^{L858R/T790M/C797S}, and thus inhibited the proliferation of these cells. We also constructed tumor cells harboring EGFR^{19del/T790M/C797S} (named PC‐9‐OR cells) using the CRISPR/Cas9 system and found that LS‐106 markedly suppressed the activation of EGFR^{19del/T790M/C797S} and the proliferation of PC‐9‐OR cells. Moreover, cells harboring EGFR^{19del/T790M/C797S} underwent remarkable apoptosis upon LS‐106 treatment. In vivo experiments further demonstrated that oral administration of LS‐106 caused significant tumor regression in a PC‐9‐OR xenograft model, with a tumor growth inhibition rate (TGI) of 83.5% and 136.6% at doses of 30 and 60 mg/kg, respectively. Taken together, we identified LS‐106 as a novel fourth‐generation EGFR inhibitor against C797S mutation and confirmed its preclinical antitumor effects in C797S–triple‐mutant tumor models.     

Synergistic induction of apoptosis by the Bcl-2 inhibitor ABT-737 and imatinib mesylate in gastrointestinal stromal tumor cells

Background: Although imatinib mesylate has revolutionized the management of patients with gastrointestinal stromal tumor (GIST), resistance and progression almost inevitably develop with long‐term monotherapy. To enhance imatinib‐induced cytotoxicity and overcome imatinib‐resistance in GIST cells, we examined the antitumor effects of the pro‐apoptotic Bcl‐2/Bcl‐xL inhibitor ABT‐737, alone and in combination with imatinib.Methods: We treated imatinib‐sensitive, GIST‐T1 and GIST882, and imatinib‐resistant cells with ABT‐737 alone and with imatinib. We determined the anti‐proliferative and apoptotic effects by cell viability assay, flow cytometric apoptosis and cell cycle analysis, immunoblotting, and nuclear morphology. Synergism was determined by isobologram analysis.Results: The IC50 of single‐agent ABT‐737 at 72 h was 10 μM in imatinib‐sensitive GIST‐T1 and GIST882 cells, and 1 μM in imatinib‐resistant GIST48IM cells. ABT‐737 and imatinib combined synergistically in a time‐ and dose‐dependent manner to inhibit the proliferation and induce apoptosis of all GIST cells, as evidenced by cell viability and apoptosis assays, caspase activation, PARP cleavage, and morphologic changes. Isobologram analyses revealed strongly synergistic drug interactions, with combination indices <0.5 for most ABT‐737/imatinib combinations. Thus, clinically relevant in vitro concentrations of ABT‐737 have single‐agent antitumor activity and are synergistic in combination with imatinib.Conclusion: We provide the first preclinical evidence that Bcl‐2/Bcl‐xL inhibition with ABT‐737 synergistically enhances imatinib‐induced cytotoxicity via apoptosis, and that direct engagement of apoptotic cell death may be an effective approach to circumvent imatinib‐resistance in GIST.