Comprehensive molecular phenotyping of ARID1A-deficient gastric cancer reveals pervasive epigenomic reprogramming and therapeutic opportunities.

OBJECTIVE: Gastric cancer (GC) is a leading cause of cancer mortality, with ARID1A being the second most frequently mutated driver gene in GC. We sought to decipher ARID1A-specific GC regulatory networks and examine therapeutic vulnerabilities arising from ARID1A loss. DESIGN: Genomic profiling of GC patients including a Singapore cohort (>200 patients) was performed to derive mutational signatures of ARID1A inactivation across molecular subtypes. Single-cell transcriptomic profiles of ARID1A-mutated GCs were analysed to examine tumour microenvironmental changes arising from ARID1A loss. Genome-wide ARID1A binding and chromatin profiles (H3K27ac, H3K4me3, H3K4me1, ATAC-seq) were generated to identify gastric-specific epigenetic landscapes regulated by ARID1A. Distinct cancer hallmarks of ARID1A-mutated GCs were converged at the genomic, single-cell and epigenomic level, and targeted by pharmacological inhibition. RESULTS: We observed prevalent ARID1A inactivation across GC molecular subtypes, with distinct mutational signatures and linked to a NFKB-driven proinflammatory tumour microenvironment. ARID1A-depletion caused loss of H3K27ac activation signals at ARID1A-occupied distal enhancers, but unexpectedly gain of H3K27ac at ARID1A-occupied promoters in genes such as NFKB1 and NFKB2. Promoter activation in ARID1A-mutated GCs was associated with enhanced gene expression, increased BRD4 binding, and reduced HDAC1 and CTCF occupancy. Combined targeting of promoter activation and tumour inflammation via bromodomain and NFKB inhibitors confirmed therapeutic synergy specific to ARID1A-genomic status. CONCLUSION: Our results suggest a therapeutic strategy for ARID1A-mutated GCs targeting both tumour-intrinsic (BRD4-assocatiated promoter activation) and extrinsic (NFKB immunomodulation) cancer phenotypes.

Loss of ARID1A activates mTOR signaling and SOX9 in gastric adenocarcinoma-rationale for targeting ARID1A deficiency.

BACKGROUND: Gastric adenocarcinoma (GAC) is a lethal disease with limited therapeutic options. Genetic alterations in chromatin remodelling gene AT-rich interactive domain 1A (ARID1A) and mTOR pathway activation occur frequently in GAC. Targeting the mechanistic target of rapamycin (mTOR) pathway in unselected patients has failed to show survival benefit. A deeper understanding of GAC might identify a subset that can benefit from mTOR inhibition. METHODS: Genomic alterations in ARID1A were analysed in GAC. Mouse gastric epithelial cells from CK19-Cre-Arid1Afl/fl and wild-type mice were used to determine the activation of oncogenic genes due to loss of Arid1A. Functional studies were performed to determine the significance of loss of ARID1A and the sensitivity of ARID1A-deficient cancer cells to mTOR inhibition in GAC. RESULTS: More than 30% of GAC cases had alterations (mutations or deletions) of ARID1A and ARID1A expression was negatively associated with phosphorylation of S6 and SOX9 in GAC tissues and patient-derived xenografts (PDXs). Activation of mTOR signalling (increased pS6) and SOX9 nuclear expression were strongly increased in Arid1A-/- mouse gastric tissues which could be curtailed by RAD001, an mTOR inhibitor. Knockdown of ARID1A in GAC cell lines increased pS6 and nuclear SOX9 and increased sensitivity to an mTOR inhibitor which was further amplified by its combination with fluorouracil both in vitro and in vivo in PDXs. CONCLUSIONS: The loss of ARID1A activates pS6 and SOX9 in GAC, which can be effectively targeted by an mTOR inhibitor. Therefore, our studies suggest a new therapeutic strategy of clinically targeting the mTOR pathway in patients with GAC with ARID1A deficiency.

YAP1 mediates gastric adenocarcinoma peritoneal metastases that are attenuated by YAP1 inhibition.

OBJECTIVE: Peritoneal carcinomatosis (PC; malignant ascites or implants) occurs in approximately 45% of advanced gastric adenocarcinoma (GAC) patients and associated with a poor survival. The molecular events leading to PC are unknown. The yes-associated protein 1 (YAP1) oncogene has emerged in many tumour types, but its clinical significance in PC is unclear. Here, we investigated the role of YAP1 in PC and its potential as a therapeutic target. METHODS: Patient-derived PC cells, patient-derived xenograft (PDX) and patient-derived orthotopic (PDO) models were used to study the function of YAP1 in vitro and in vivo. Immunofluorescence and immunohistochemical staining, RNA sequencing (RNA-Seq) and single-cell RNA-Seq (sc-RNA-Seq) were used to elucidate the expression of YAP1 and PC cell heterogeneity. LentiCRISPR/Cas9 knockout of YAP1 and a YAP1 inhibitor were used to dissect its role in PC metastases. RESULTS: YAP1 was highly upregulated in PC tumour cells, conferred cancer stem cell (CSC) properties and appeared to be a metastatic driver. Dual staining of YAP1/EpCAM and sc-RNA-Seq revealed that PC tumour cells were highly heterogeneous, YAP1high PC cells had CSC-like properties and easily formed PDX/PDO tumours but also formed PC in mice, while genetic knockout YAP1 significantly slowed tumour growth and eliminated PC in PDO model. Additionally, pharmacologic inhibition of YAP1 specifically reduced CSC-like properties and suppressed tumour growth in YAP1high PC cells especially in combination with cytotoxics in vivo PDX model. CONCLUSIONS: YAP1 is essential for PC that is attenuated by YAP1 inhibition. Our data provide a strong rationale to target YAP1 in clinic for GAC patients with PC.

Targeting cancer stem cells with a pan-BCL-2 inhibitor in preclinical and clinical settings in patients with gastroesophageal carcinoma.

OBJECTIVE: Gastro-oesophageal cancers (GEC) are resistant to therapy and lead to poor prognosis. The cancer stem cells (CSCs) and antiapoptotic pathways often confer therapy resistance. We sought to elucidate the antitumour action of a BCL-2 inhibitor, AT101 in GEC in vitro, in vivo and in a clinical trial. METHODS: Extensive preclinical studies in vitro and in vivo were carried out to establish the mechanism action of AT101 on targeting CSCs and antiapoptotic proteins. A pilot clinical trial in patients with GEC was completed with AT-101 added to standard chemoradiation. RESULTS: Overexpression of BCL-2 and MCL-1 was noted in gastric cancer tissues (GC). AT-101 induced apoptosis, reduced proliferation and tumour sphere formation in MCL-1/BCL-2 high GC cells. Interestingly, AT101 dramatically downregulated genes (YAP-1/Sox9) that control CSCs in GEC cell lines regardless of BCL-2/MCL-1 expression. Addition of docetaxel to AT-101 amplified its antiproliferation and induced apoptosis effects. In vivo studies confirmed the combination of AT101 and docetaxel demonstrated stronger antitumour activity accompanied with significant decrease of CSCs biomarkers (YAP1/SOX9). In a pilot clinical trial, 13 patients with oesophageal cancer (EC) received AT101 orally concurrently with chemoradiation. We observed dramatic clinical complete responses and encouraging overall survival in these patients. Clinical specimen analyses revealed that AT-101 dramatically reduced the expression of CSCs genes in treated EC specimens indicating antitumour activity of AT101 relies more on its anti-CSCs activity. CONCLUSIONS: Our preclinical and clinical data suggest that AT-101 overcomes resistance by targeting CSCs pathways suggesting a novel mechanism of action of AT101 in patients with GEC.

Integrated genomic profiling and modelling for risk stratification in patients with advanced oesophagogastric adenocarcinoma.

OBJECTIVE: Prognosis of patients with advanced oesophagogastric adenocarcinoma (mEGAC) is poor and molecular determinants of shorter or longer overall survivors are lacking. Our objective was to identify molecular features and develop a prognostic model by profiling the genomic features of patients with mEGAC with widely varying outcomes. DESIGN: We profiled 40 untreated mEGACs (20 shorter survivors <13 months and 20 longer survivors >36 months) with whole-exome sequencing (WES) and RNA sequencing and performed an integrated analysis of exome, transcriptome, immune profile and pathological phenotypes to identify the molecular determinants, developing an integrated model for prognosis and comparison with The Cancer Genome Atlas (TCGA) cohorts. RESULTS: KMT2C alterations were exclusively observed in shorter survivors together with high level of intratumour heterogeneity and complex clonal architectures, whereas the APOBEC mutational signatures were significantly enriched in longer survivors. Notably, the loss of heterozygosity in chromosome 4 (Chr4) was associated with shorter survival and 'cold' immune phenotype characterised by decreased B, CD8, natural killer cells and interferon-gamma responses. Unsupervised transcriptomic clustering revealed a shorter survivor subtype with distinct expression features (eg, upregulated druggable targets JAK2, MAP3K13 and MECOM). An integrated model was then built based on clinical variables and the identified molecular determinants, which significantly segregated shorter and longer survivors. All the above features and the integrated model have been validated independently in multiple TCGA cohorts. CONCLUSION: This study discovered novel molecular features prognosticating overall survival in patients with mEGAC and identified potential novel targets in shorter survivors.

Multiplex profiling of peritoneal metastases from gastric adenocarcinoma identified novel targets and molecular subtypes that predict treatment response.

OBJECTIVE: Peritoneal carcinomatosis (PC) occurs frequently in patients with gastric adenocarcinoma (GAC) and confers a poor prognosis. Multiplex profiling of primary GACs has been insightful but the underpinnings of PC's development/progression remain largely unknown. We characterised exome/transcriptome/immune landscapes of PC cells from patients with GAC aiming to identify novel therapeutic targets. DESIGN: We performed whole-exome sequencing (WES) and whole transcriptome sequencing (RNA-seq) on 44 PC specimens (43 patients with PC) including an integrative analysis of WES, RNA-seq, immune profile, clinical and pathological phenotypes to dissect the molecular pathogenesis, identifying actionable targets and/or biomarkers and comparison with TCGA primary GACs. RESULTS: We identified distinct alterations in PC versus primary GACs, such as more frequent CDH1 and TAF1 mutations, 6q loss and chr19 gain. Alterations associated with aggressive PC phenotypes emerged with increased mutations in TP53, CDH1, TAF1 and KMT2C, higher level of 'clock-like' mutational signature, increase in whole-genome doublings, chromosomal instability (particularly, copy number losses), reprogrammed microenvironment, enriched cell cycle pathways, MYC activation and impaired immune response. Integrated analysis identified two main molecular subtypes: 'mesenchymal-like' and 'epithelial-like' with discriminating response to chemotherapy (31% vs 71%). Patients with the less responsive 'mesenchymal-like' subtype had high expression of immune checkpoint T-Cell Immunoglobulin And Mucin Domain-Containing Protein 3 (TIM-3), its ligand galectin-9, V-domain Ig suppressor of T cell activation (VISTA) and transforming growth factor-ß as potential therapeutic immune targets. CONCLUSIONS: We have uncovered the unique mutational landscape, copy number alteration and gene expression profile of PC cells and defined PC molecular subtypes, which correlated with PC therapy resistance/response. Novel targets and immune checkpoint proteins have been identified with a potential to be translated into clinics.

LncRNA PVT1 up-regulation is a poor prognosticator and serves as a therapeutic target in esophageal adenocarcinoma.

BACKGROUND: PVT1 has emerged as an oncogene in many tumor types. However, its role in Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) is unknown. The aim of this study was to assess the role of PVT1 in BE/EAC progression and uncover its therapeutic value against EAC. METHODS: PVT1 expression was assessed by qPCR in normal, BE, and EAC tissues and statistical analysis was performed to determine the association of PVT1 expression and EAC (stage, metastases, and survival). PVT1 antisense oligonucleotides (ASOs) were tested for their antitumor activity in vitro and in vivo. RESULTS: PVT1 expression was up-regulated in EACs compared with paired BEs, and normal esophageal tissues. High expression of PVT1 was associated with poor differentiation, lymph node metastases, and shorter survival. Effective knockdown of PVT1 in EAC cells using PVT1 ASOs resulted in decreased cell proliferation, invasion, colony formation, tumor sphere formation, and reduced proportion of ALDH1A1+ cells. Mechanistically, we discovered mutual regulation of PVT1 and YAP1 in EAC cells. Inhibition of PVT1 by PVT1 ASOs suppressed YAP1 expression through increased phosphor-LATS1and phosphor-YAP1 while knockout of YAP1 in EAC cells significantly suppressed PVT1 levels indicating a positive regulation of PVT1 by YAP1. Most importantly, we found that targeting both PVT1 and YAP1 using their specific ASOs led to better antitumor activity in vitro and in vivo. CONCLUSIONS: Our results provide strong evidence that PVT1 confers an aggressive phenotype to EAC and is a poor prognosticator. Combined targeting of PVT1 and YAP1 provided the highest therapeutic index and represents a novel therapeutic strategy.

Identification of DNA mutations in gastric washes from gastric adenocarcinoma patients: Possible implications for liquid biopsies and patient follow-up.

Whereas cancer patients have benefited from liquid biopsies, the scenario for gastric adenocarcinoma (GAC) is still dismal. We used next-generation deep sequencing of TP53-a highly mutated and informative gene in GAC-to assess mutations in tumor biopsies, plasma (PL) and stomach fluids (gastric wash-GW). We evaluated their potential to reveal tumor-derived mutations, useful for monitoring mutational dynamics at diagnosis, progression and treatment. Exon-capture libraries were constructed from 46 patients including tumor biopsies, GW and PL pre and post-treatment (196 samples), with high vertical coverage >8,000×. At diagnosis, we detected TP53 mutations in 15/46 biopsies (32.6%), 7/46 GW- (15.2%) and 6/46 PL-samples (13%). Biopsies and GW were concordant in 38/46 cases (82.6%) for the presence/absence of mutations and, furthermore, four GW-exclusive mutations were identified, suggesting tumor heterogeneity. Considering the combined analysis of GW and PL, TP53 mutations found in biopsies were also identified in 9/15 (60%) of cases, the highest detection level reported for GAC. Our study indicates that GW could be useful to track DNA alterations, especially if anchored to a comprehensive gene-panel designed for this malignancy.

A new intronic quantitative PCR method led to the discovery of transformation from human ascites to murine malignancy in a mouse model.

Purpose: To establish a fast and accurate detection method for interspecies contaminations in the patient-derived xenograft (PDX) models and cell lines, and to elucidate possible mechanisms if interspecies oncogenic transformation is detected. Methods: A fast and highly sensitive intronic qPCR method detecting Gapdh intronic genomic copies was developed to quantify if cells were human or murine or a mixture. By this method, we documented that murine stromal cells were abundant in the PDXs; we also authenticated our cell lines to be human or murine. Results: In one mouse model, GA0825-PDX transformed murine stromal cells into a malignant tumorigenic murine P0825 cell line. We traced the timeline of this transformation and discovered three subpopulations descended from the same GA0825-PDX model: epithelium-like human H0825, fibroblast-like murine M0825, and main passaged murine P0825 displayed differences in tumorigenic capability in vivo. P0825 was the most aggressive and H0825 was weakly tumorigenic. Immunofluorescence (IF) staining revealed that P0825 cells highly expressed several oncogenic and cancer stem cell markers. Whole exosome sequencing (WES) analysis revealed that TP53 mutation in the human ascites IP116-generated GA0825-PDX may have played a role in the human-to-murine oncogenic transformation. Conclusion: This intronic qPCR is able to quantify human/mouse genomic copies with high sensitivity and within a time frame of a few hours. We are the first to use intronic genomic qPCR for authentication and quantification of biosamples. Human ascites transformed murine stroma into malignancy in a PDX model.

Proteogenomic landscape of gastric adenocarcinoma peritoneal metastases.

Advanced gastric adenocarcinoma (GAC) often leads to peritoneal carcinomatosis (PC) and is associated with very poor outcome. Here we report the comprehensive proteogenomic study of ascites derived cells from a prospective GAC cohort (n = 26 patients with peritoneal carcinomatosis, PC). A total of 16,449 proteins were detected from whole cell extracts (TCEs). Unsupervised hierarchical clustering resulted in three distinct groups that reflected extent of enrichment in tumor cells. Integrated analysis revealed enriched biological pathways and notably, some druggable targets (cancer-testis antigens, kinases, and receptors) that could be exploited to develop effective therapies and/or tumor stratifications. Systematic comparison of expression levels of proteins and mRNAs revealed special expression patterns of key therapeutics target notably high mRNA and low protein expression of HAVCR2 (TIM-3), and low mRNA but high protein expression of cancer-testis antigens CTAGE1 and CTNNA2. These results inform strategies to target GAC vulnerabilities.

Galectin-3 Cooperates with CD47 to Suppress Phagocytosis and T-cell Immunity in Gastric Cancer Peritoneal Metastases.

The peritoneal cavity is a common site of gastric adenocarcinoma (GAC) metastasis. Peritoneal carcinomatosis (PC) is resistant to current therapies and confers poor prognosis, highlighting the need to identify new therapeutic targets. CD47 conveys a "don't eat me" signal to myeloid cells upon binding its receptor signal regulatory protein alpha (SIRPα), which helps tumor cells circumvent macrophage phagocytosis and evade innate immune responses. Previous studies demonstrated that the blockade of CD47 alone results in limited clinical benefits, suggesting that other target(s) might need to be inhibited simultaneously with CD47 to elicit a strong antitumor response. Here, we found that CD47 was highly expressed on malignant PC cells, and elevated CD47 was associated with poor prognosis. Galectin-3 (Gal3) expression correlated with CD47 expression, and coexpression of Gal3 and CD47 was significantly associated with diffuse type, poor differentiation, and tumor relapse. Depletion of Gal3 reduced expression of CD47 through inhibition of c-Myc binding to the CD47 promoter. Furthermore, injection of Gal3-deficient tumor cells into either wild-type and Lgals3-/- mice led to a reduction in M2 macrophages and increased T-cell responses compared with Gal3 wild-type tumor cells, indicating that tumor cell-derived Gal3 plays a more important role in GAC progression and phagocytosis than host-derived Gal3. Dual blockade of Gal3 and CD47 collaboratively suppressed tumor growth, increased phagocytosis, repolarized macrophages, and boosted T-cell immune responses. These data uncovered that Gal3 functions together with CD47 to suppress phagocytosis and orchestrate immunosuppression in GAC with PC, which supports exploring a novel combination therapy targeting Gal3 and CD47. SIGNIFICANCE: Dual inhibition of CD47 and Gal3 enhances tumor cell phagocytosis and reprograms macrophages to overcome the immunosuppressive microenvironment and suppress tumor growth in peritoneal metastasis of gastric adenocarcinoma.

Evolution of immune and stromal cell states and ecotypes during gastric adenocarcinoma progression.

Understanding tumor microenvironment (TME) reprogramming in gastric adenocarcinoma (GAC) progression may uncover novel therapeutic targets. Here, we performed single-cell profiling of precancerous lesions, localized and metastatic GACs, identifying alterations in TME cell states and compositions as GAC progresses. Abundant IgA+ plasma cells exist in the premalignant microenvironment, whereas immunosuppressive myeloid and stromal subsets dominate late-stage GACs. We identified six TME ecotypes (EC1-6). EC1 is exclusive to blood, while EC4, EC5, and EC2 are highly enriched in uninvolved tissues, premalignant lesions, and metastases, respectively. EC3 and EC6, two distinct ecotypes in primary GACs, associate with histopathological and genomic characteristics, and survival outcomes. Extensive stromal remodeling occurs in GAC progression. High SDC2 expression in cancer-associated fibroblasts (CAFs) is linked to aggressive phenotypes and poor survival, and SDC2 overexpression in CAFs contributes to tumor growth. Our study provides a high-resolution GAC TME atlas and underscores potential targets for further investigation.

Patient-derived cell lines and orthotopic mouse model of peritoneal carcinomatosis recapitulate molecular and phenotypic features of human gastric adenocarcinoma.

BACKGROUND: Gastric adenocarcinoma with peritoneal carcinomatosis (PC) is therapy resistant and leads to poor survival. To study PC in depth, there is an urgent need to develop representative PC-derived cell lines and metastatic models to study molecular mechanisms of PC and for preclinical screening of new therapies. METHODS: PC cell lines were developed from patient-derived PC cells. The tumorigenicity and metastatic potential were investigated by subcutaneously (PDXs) and orthotopically. Karyotyping, whole-exome sequencing, RNA-sequencing, and functional studies were performed to molecularly define the cell lines and compare genomic and phenotypic features of PDX and donor PC cells. RESULTS: We established three PC cell lines (GA0518, GA0804, and GA0825) and characterized them in vitro. The doubling times were 22, 39, and 37 h for GA0518, GA0804, and GA0825, respectively. Expression of cancer stem cell markers (CD44, ALDH1, CD133 and YAP1) and activation of oncogenes varied among the cell lines. All three PC cell lines formed PDXs. Interestingly, all three PC cell lines formed tumors in the patient derived orthotopic (PDO) model and GA0518 cell line consistently produced PC in mice. Moreover, PDXs recapitulated transcriptomic and phenotypic features of the donor PC cells. Finally, these cell lines were suitable for preclinical testing of chemotherapy and target agents in vitro and in vivo. CONCLUSION: We successfully established three patient-derived PC cell lines and an improved PDO model with high incidence of PC associated with malignant ascites. Thus, these cell lines and metastatic PDO model represent excellent resources for exploring metastatic mechanisms of PC in depth and for target drug screening and validation by interrogating GAC for translational studies.

An improved strategy for CRISPR/Cas9 gene knockout and subsequent wildtype and mutant gene rescue.

A fluorescence marker mOrange was inserted to the popular pLentiCrispr-V2 to create pLentiCrispr-V2-mOrange (V2mO) that contained both a puromycin selection and a fluorescent marker, making viral production and target transduction visible. Lentiviruses packaged with this plasmid and appropriate guide RNAs (gRNAs) successfully knocked out the genes RhoA, Gli1, and Gal3 in human gastric cancer cell lines. Cas9-gRNA editing efficiency could be estimated directly from Sanger electropherograms of short polymerase chain reaction products around the gRNA regions in Cas9-gRNA transduced cells. Single cloning of transduced target cell pools must be performed to establish stable knockout clones. Rescue of wildtype (RhoA and Gal3) and mutant (RhoA.Y42C) genes into knockout cells was successful only when cDNAs, where gRNAs bind, were modified by three nucleotides while the amino acid sequences remained unchanged. Stringent on-target CRISPR/Cas9 editing was observed in Gal3 gene, but not in RhoA gene since RhoA.Y42C already presented a nucleotide change in gRNA5 binding site. In summary, our improved strategy added these advantages: adding visual marker to the popular lentiviral system, monitoring lentiviral production and transduction efficiencies, cell-sorting Cas9+ cells in target cells by fluorescence-activated cell sorting, direct estimation of gene editing efficiency of target cell pools by short PCR electropherograms around gRNA binding sites, and successful rescue of wildtype and mutant genes in knockout cells, overcoming Cas9 editing by modifying cDNAs.

LncRNA PVT1 Is a Poor Prognosticator and Can Be Targeted by PVT1 Antisense Oligos in Gastric Adenocarcinoma.

Gastric adenocarcinoma (GAC) is inherently resistant or becomes resistant to therapy, leading to a poor prognosis. Mounting evidence suggests that lncRNAs can be used as predictive markers and therapeutic targets in the right context. In this study, we determined the role of lncRNA-PVT1 in GAC along with the value of inhibition of PVT1 using antisense oligos (ASOs). RNA scope in situ hybridization was used to analyze PVT1 expression in tumor tissue microarrays (TMAs) of GAC and paired normal tissues from 792 patients. Functional experiments, including colony formation and invasion assays, were performed to evaluate the effects of PVT1 ASO inhibition of PVT1 in vitro; patient-derived xenograft models were used to evaluate the anti-tumor effects of PVT1 ASOs in vivo. LncRNA-PVT1 was upregulated in GACs compared to the matched adjacent normal tissues in the TMA. LncRNA PVT1 expression was positively correlated with larger tumor size, deeper wall invasion, lymph node metastases, and short survival duration. Inhibition of PVT1 using PVT1 ASOs significantly suppressed tumor cell growth and invasion in vitro and in vivo. PVT1 expression was highly associated with poor prognosis in GAC patients and targeting PVT1 using PVT1 ASOs was effective at curtailing tumor cell growth in vitro and in vivo. Thus, PVT1 is a poor prognosticator as well as therapeutic target. Targeting PVT1 using PVT1 ASOs provides a novel therapeutic strategy for GAC.

YAP1-Mediated CDK6 Activation Confers Radiation Resistance in Esophageal Cancer - Rationale for the Combination of YAP1 and CDK4/6 Inhibitors in Esophageal Cancer.

PURPOSE: Esophageal cancer is a lethal disease that is often resistant to therapy. Alterations of YAP1 and CDK6 are frequent in esophageal cancer. Deregulation of both molecules may be responsible for therapy resistance. EXPERIMENTAL DESIGN: Expressions of YAP1 and CDK6 were examined in esophageal cancer cells and tissues using immunoblotting and immunohistochemistry. YAP1 expression was induced in esophageal cancer cells to examine YAP1-mediated CDK6 activation and its association with radiation resistance. Pharmacologic and genetic inhibitions of YAP1 and CDK6 were performed to dissect the mechanisms and assess the antitumor effects in vitro and in vivo. RESULTS: YAP1 expression was positively associated with CDK6 expression in resistant esophageal cancer tissues and cell lines. YAP1 overexpression upregulated CDK6 expression and transcription, and promoted radiation resistance, whereas treatment with the YAP1 inhibitor, CA3, strongly suppressed YAP1 and CDK6 overexpression, reduced Rb phosphorylation, as well as sensitized radiation-resistant/YAP1high esophageal cancer cells to irradiation. CDK4/6 inhibitor, LEE011, and knock down of CDK6 dramatically inhibited expression of YAP1 and sensitized resistant esophageal cancer cells to irradiation indicating a positive feed-forward regulation of YAP1 by CDK6. In addition, suppression of both the YAP1 and CDK6 pathways by the combination of CA3 and LEE011 significantly reduced esophageal cancer cell growth and cancer stem cell population (ALDH1 + and CD133 + ), sensitized cells to irradiation, and showed a strong antitumor effect in vivo against radiation-resistant esophageal cancer cells. CONCLUSIONS: Our results document that a positive crosstalk between the YAP1 and CDK6 pathways plays an important role in conferring radiation resistance to esophageal cancer cells. Targeting both YAP1 and CDK6 pathways could be a novel therapeutic strategy to overcome resistance in esophageal cancer.