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Background In esophageal cancer (EC), there is a paucity of knowledge regarding the interplay between the tumor immune microenvironment and response to neoadjuvant treatment and, therefore, which factors may influence outcomes. Thus, our goal was to investigate the changes in the immune microenvironment with neoadjuvant treatment in EC by assessing the expression of immune related genes and their association with prognosis. Methods We examined the transcriptome of paired pre- and post-neoadjuvant treated EC specimens. Based on these findings, we validated the presence of tumor-infiltrating neutrophils using CD15+ immunohistochemistry in a discovery cohort of patients with residual pathologic disease. We developed a nomogram as a predictor of progression-free survival (PFS) incorporating the variables CD15+ cell count, tumor regression grade, and tumor grade. Results After neoadjuvant treatment, there was an increase in genes related to myeloid cell differentiation and a poor prognosis associated with high neutrophil (CD15+) counts. Our nomogram incorporating CD15+ cell count was predictive of PFS with a C-index of 0.80 (95% confidence interval [CI] 0.68–0.9) and a concordance probability estimate (CPE) of 0.77 (95% CI 0.69–0.86), which indicates high prognostic ability. The C-index and CPE of the validation cohort were 0.81 (95% CI 0.69–0.91) and 0.78 (95% CI 0.7–0.86), respectively. Conclusions Our nomogram incorporating CD15+ cell count can potentially be used to identify patients at high risk of recurrent disease and thus stratify patients who will benefit most from adjuvant treatment.

Gene List from siRNA screen with APR-246 growth inhibition in H1299 p53-null (Sheet 1) and p53-R273H (Sheet 2)

Figure S5 shows MYC directly regulates SLC7A11 levels and APR-246 sensitivity.

Supplemental Table 1, 2, 3. Supplemental Materials and Methods.

Epigenetics; Tumour-suppressor proteins Epigenética; Proteínas supresoras de tumores Epigenètica; Proteïnes supresores de tumors Colorectal cancers (CRCs) often display histological features indicative of aberrant differentiation but the molecular underpinnings of this trait and whether it directly drives disease progression is unclear. Here, we identify co-ordinate epigenetic inactivation of two epithelial-specific transcription factors, EHF and CDX1, as a mechanism driving differentiation loss in CRCs. Re-expression of EHF and CDX1 in poorly-differentiated CRC cells induced extensive chromatin remodelling, transcriptional re-programming, and differentiation along the enterocytic lineage, leading to reduced growth and metastasis. Strikingly, EHF and CDX1 were also able to reprogramme non-colonic epithelial cells to express colonic differentiation markers. By contrast, inactivation of EHF and CDX1 in well-differentiated CRC cells triggered tumour de-differentiation. Mechanistically, we demonstrate that EHF physically interacts with CDX1 via its PNT domain, and that these transcription factors co-operatively drive transcription of the colonic differentiation marker, VIL1. Compound genetic deletion of Ehf and Cdx1 in the mouse colon disrupted normal colonic differentiation and significantly enhanced colorectal tumour progression. These findings thus reveal a novel mechanism driving epithelial de-differentiation and tumour progression in CRC. This project was supported by NHMRC project grant (1107831), a Cancer Council Victoria Grant (1164674) and the Operational Infrastructure Support Programme, Victorian Government, Australia. JMM was supported by a NHMRC Senior Research Fellowship (1046092). IYL was supported by F J Fletcher Research Scholarship and Randal and Louisa Alcock Scholarship from the University of Melbourne. LJJ was supported by La Trobe University Australian Postgraduate Awards. IN was supported by La Trobe University Postgraduate Research Scholarship. JWTT was supported by the University of Melbourne Australian Postgraduate Awards. OMS is a National Health and Medical Research Council (NHMRC) Senior Research Fellow (APP1136119). Open Access funding enabled and organized by CAUL and its Member Institutions.

APR-246 (eprenetapopt) is in clinical development with a focus on hematologic malignancies and is promoted as a mutant-p53 reactivation therapy. Currently, the detection of at least one TP53 mutation is an inclusion criterion for patient selection into most APR-246 clinical trials. Preliminary results from our phase Ib/II clinical trial investigating APR-246 combined with doublet chemotherapy [cisplatin and 5-fluorouracil (5-FU)] in metastatic esophageal cancer, together with previous preclinical studies, indicate that TP53 mutation status alone may not be a sufficient biomarker for APR-246 response. This study aims to identify a robust biomarker for response to APR-246. Correlation analysis of the PRIMA-1 activity (lead compound to APR-246) with mutational status, gene expression, protein expression, and metabolite abundance across over 700 cancer cell lines (CCL) was performed. Functional validation and a boutique siRNA screen of over 850 redox-related genes were also conducted. TP53 mutation status was not consistently predictive of response to APR-246. The expression of SLC7A11, the cystine/glutamate transporter, was identified as a superior determinant of response to APR-246. Genetic regulators of SLC7A11, including ATF4, MDM2, wild-type p53, and c-Myc, were confirmed to also regulate cancer-cell sensitivity to APR-246. In conclusion, SLC7A11 expression is a broadly applicable determinant of sensitivity to APR-246 across cancer and should be utilized as the key predictive biomarker to stratify patients for future clinical investigation of APR-246.

To explore the clinical utility of circulating tumor DNA (ctDNA) in esophageal adenocarcinoma (EAC) by developing a cost-effective and rapid technique utilising targeted amplicon sequencing.Emerging evidence suggests that levels of ctDNA in the blood can be used to monitor treatment response and in the detection of disease recurrence in various cancer types. Current staging modalities for EAC such as computerised tomography of the chest/abdomen/pelvis (CT) and positron emission tomography (PET) do not reliably detect occult micro-metastatic disease, the presence of which signifies a poor prognosis. After curative-intent treatment, some patients are still at high risk of recurrent disease, and there is no widely accepted optimal surveillance tool for patients with EAC.Sixty-two patients with EAC were investigated for the presence of ctDNA using a tumor-informed approach. We designed a custom targeted amplicon sequencing panel of target specific primers covering mutational foci in 9 of the most commonly mutated genes in EAC. Serial blood samples were taken before and after neoadjuvant treatment (NAT), and during surveillance.Somatic mutations were detected in pre-treatment biopsy samples of 55 out of 62 (89%) EAC patients. Mutations in TP53 (80%) were the most common. Out of these 55 patients, 20 (36%) had detectable ctDNA at baseline. The majority (90%) of patients with detectable ctDNA had either locally advanced tumors, nodal involvement or metastatic disease. In patients with locally advanced tumors, disease free survival (DFS) was more accurately stratified using pre-treatment ctDNA status [HR 4.34 (95% CI 0.93-20.21); P = 0.05] compared to nodal status on PET-CT. In an exploratory subgroup analysis, patients who are node negative but ctDNA positive have inferior DFS [HR 11.71 (95% CI 1.16-118.80) P = 0.04]. In blood samples taken before and following NAT, clearance of ctDNA after NAT was associated with a favourable response to treatment. Furthermore, patients who are ctDNA positive during post-treatment surveillance are at high risk of relapse.Our study shows that ctDNA has potential to provide additional prognostication over conventional staging investigation such as CT and PET. It may also have clinical utility in the assessment of response to NAT and as a biomarker for the surveillance of recurrent disease.

Esophageal adenocarcinoma is of increasing global concern due to increasing incidence, a lack of effective treatments, and poor prognosis. Therapeutic target discovery and clinical trials have been hindered by the heterogeneity of the disease, the lack of "druggable" driver mutations, and the dominance of large-scale genomic rearrangements. We have previously undertaken a comprehensive small-molecule phenotypic screen using the high-content Cell Painting assay to quantify the morphological response to a total of 19,555 small molecules across a panel of genetically distinct human esophageal cell lines to identify new therapeutic targets and small molecules for the treatment of esophageal adenocarcinoma. In this current study, we report for the first time the dose-response validation studies for the 72 screening hits from the target-annotated LOPAC and Prestwick FDA-approved compound libraries and the full list of 51 validated esophageal adenocarcinoma-selective small molecules (71% validation rate). We then focus on the most potent and selective hit molecules, elesclomol, disulfiram, and ammonium pyrrolidinedithiocarbamate. Using a multipronged, multitechnology approach, we uncover a unified mechanism of action and a vulnerability in esophageal adenocarcinoma toward copper-dependent cell death that could be targeted in the future.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that contributes to protein biogenesis as a subunit of the TIM22 complex at the inner mitochondrial membrane. Mutations in AGK cause Sengers syndrome, an autosomal recessive condition characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis. We mapped the proteomic changes in Sengers patient fibroblasts and AGKKO cell lines to understand the effects of AGK dysfunction on mitochondria. This uncovered down-regulation of a number of proteins at the inner mitochondrial membrane, including many SLC25 carrier family proteins, which are predicted substrates of the complex. We also observed down-regulation of SFXN proteins, which contain five transmembrane domains, and show that they represent a novel class of TIM22 complex substrate. Perturbed biogenesis of SFXN proteins in cells lacking AGK reduces the proliferative capabilities of these cells in the absence of exogenous serine, suggesting that dysregulation of one-carbon metabolism is a molecular feature in the biology of Sengers syndrome.

Efficacious therapeutic approaches are urgently needed to improve outcomes in patients with oesophageal adenocarcinoma (OAC). However, oncogenic drivers amenable to targeted therapy are limited and their functional characterisation is essential. Among few targeted therapies available, anti‐human epidermal growth factor receptor 2 (HER2) therapy showed only modest benefit for patients with OAC. Herein, we investigated the potential oncogenic role of growth factor receptor bound protein 7 (GRB7), which is reported to be co‐amplified with HER2 (ERBB2) in OAC. GRB7 was highly expressed in 15% of OAC tumours, not all of which could be explained by co‐amplification with HER2, and was associated with a trend for poorer overall survival. Knockdown of GRB7 decreased proliferation and clonogenic survival, and induced apoptosis. Reverse phase protein array (RPPA) analyses revealed a role for PI3K, mammalian target of rapamycin (mTOR), MAPK, and receptor tyrosine kinase signalling in the oncogenic action of GRB7. Furthermore, the GRB7 and HER2 high‐expressing OAC cell line Eso26 showed reduced cell proliferation upon GRB7 knockdown but was insensitive to HER2 inhibition by trastuzumab. Consistent with this, GRB7 knockdown in vivo with an inducible shRNA significantly inhibited tumour growth in cell line xenografts. HER2 expression did not predict sensitivity to trastuzumab, with Eso26 xenografts remaining refractory to trastuzumab treatment. Taken together, our study provides strong evidence for an oncogenic role for GRB7 in OAC and suggests that targeting GRB7 may be a potential therapeutic strategy for this cancer. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Background and AimsOesophageal adenocarcinoma (OAC) is of increasing global concern due to increasing incidence, a lack of effective treatments, and poor prognosis. Therapeutic target discovery and clinical trials have been hindered by the heterogeneity of the disease, lack of driver mutations, and the dominance of large-scale genomic rearrangements. In this work we have characterised three potent and selective hit compounds identified in an innovative high-content phenotypic screening assay. The three hits include two approved drugs; elesclomol and disulfiram, and another small molecule compound, ammonium pyrrolidinedithiocarbamate. We uncover their mechanism of action, discover a targetable vulnerability, and gain insight into drug sensitivity for biomarker-based clinical trials in OAC.MethodsElesclomol, disulfiram, and ammonium pyrrolidinedithiocarbamate were systematically characterised across panels of oesophageal cell lines and patient-derived organoids. Drug treated oesophageal cell lines were morphologically profiled using a high-content, imaging platform. Compounds were assessed for efficacy across patient-derived organoids. Metabolomics and transcriptomics were assessed for the identification of oesophageal-cancer specific drug mechanisms and patient stratification hypotheses.ResultsHigh-content profiling revealed that all three compounds were highly selective for OAC over tissue-matched controls. Comparison of gene expression and morphological signatures unveiled a unified mechanism of action involving the accumulation of copper selectively in cancer cells, leading to dysregulation of proteostasis and cancer cell death. Basal omic analyses revealed proteasome and metabolic markers of drug sensitivity, forming the basis for biomarker-based clinical trials in OAC.ConclusionsIntegrated analysis of high-content imaging, transcriptomic and metabolomic data has revealed a new therapeutic mechanism for the treatment of OAC and represents an alternative target-agnostic drug discovery strategy.

Significant advances have been made in our understanding of the tumor immune microenvironment (TIM) and tumor infiltrating lymphocytes (TILs). Nevertheless, there is little understanding of the changes in the TIM in response to neoadjuvant chemoradiotherapy (CRT). Thus, our aim was to investigate the changes in the TIM with neoadjuvant CRT in EC by assessing the immune cell infiltrate, the expression of immune related genes, and their association with treatment response and prognosis. Methods To decipher the effects of neoadjuvant CRT on the TIM, we obtained 58 paired pre-treatment and post neoadjuvant CRT treated EC specimens. TILs and tumor infiltrating neutrophils (TIN) were quantified in pre-treatment biopsies and surgical resection specimens following neoadjuvant CRT. To evaluate the immune transcriptomics, RNA was extracted from these specimens and gene expression was assessed using the Nanostring Platform based on the PanCancer Immune Profiling Panel. Immunohistochemistry (IHC) was performed to validate findings from the immune transcriptomics. Results TIL counts were not prognostic for disease specific survival (DSS). We observed higher expression of immune-suppressive inflammatory chemokines (TGFß-1 and IL-16) in post-neoadjuvant treated samples compared to pre-treatment biopsies. In samples collected after neoadjuvant CRT, low expression of genes related to anti-tumor T cell cytotoxic activity1 was significantly associated with disease recurrence. In patients with residual disease, multivariate analysis revealed a high neutrophil count, but not TIL count, was significantly associated with inferior DSS (HR 3.8 [1.3– 10.8]; p = 0.01). Conclusion In EC, the tumor microenvironment after neoadjuvant CRT remains largely immune-suppressive. We discovered that the presence of TINs in patients with residual disease post neoadjuvant CRT is an independent adverse prognostic factor. Collectively, our results suggest that an inflammatory pro-tumoral microenvironment associated with TINs may contribute to treatment resistance and progressive disease in EC.

Extensive genomic analysis of patient samples has identified genes whose mutation or loss map malignant progression from Barrett’s metaplasia, through low- (CDKN2A) and high-grade dysplasia (TP53), to invasive adenocarcinoma (SMAD4). Interestingly, loss of SMAD4 has been found to occur exclusively in the invasive disease stage, but the reason for this is unknown. This work aimed to characterise the role of SMAD4 in esophageal adenocarcinoma (EAC) tumorigenesis and identify novel therapeutic targets for SMAD4-deficient EAC. Methods We developed a novel in vivo tumorigenesis model that demonstrates progression of dysplastic Barrett’s esophagus (BE) to invasive EAC upon knockout of SMAD4. We conducted parallel genome-wide CRISPR-Cas9 knockout screens, both in vitro and in vivo, on a background of either wildtype-SMAD4 or SMAD4-knockout dysplastic BE cells to identify co-operative drivers of tumorigenesis in vivo, as well as synthetic lethal interactions to identify potential therapeutic targets in SMAD4-deficient EAC. Functional validation of hits was performed using cell-based assays and drugs targeting candidate molecular targets. Results We identified a synthetic lethal relationship between SMAD4-deficiency and cell cycle checkpoint inhibition, suggesting a role for SMAD4 in maintaining genomic stability and a potential novel therapeutic avenue for SMAD4-deficient EAC. A concurrent in vivo CRISPR-Cas9 tumorigenesis screen produced tumors 4-fold faster than loss of SMAD4 alone and identified regulators of mTOR signalling and SMAD4 as co-operative drivers of tumorigenesis in EAC. Interestingly, these tumorigenic cells exhibited an inherent dependency on specific translation mechanisms downstream of mTOR. Meanwhile, wildtype-SMAD4 BE cells failed to thrive in vivo with mTOR modifications alone, indicating a true co-operative effect at play with SMAD4 loss. Conclusion This study uncovered a potential gatekeeping role of SMAD4 in maintaining genomic stability and inhibiting mTOR-mediated EAC tumorigenesis. In sum, loss of SMAD4 was found to increase genomic instability, thereby rendering EAC cells sensitive to cell cycle checkpoint impediment, whilst simultaneously co-operating with modulated mTOR signalling to promote tumorigenesis in EAC xenograft models.

Chronic inflammation is a known risk factor for gastrointestinal cancer. The evidence that nonsteroidal anti-inflammatory drugs suppress the incidence, growth, and metastasis of gastrointestinal cancer supports the concept that a nonsteroidal anti-inflammatory drug target, cyclooxygenase, and its downstream bioactive lipid products may provide one of the links between inflammation and cancer. Preclinical studies have demonstrated that the cyclooxygenase-2–prostaglandin E2 pathway can promote gastrointestinal cancer development. Although the role of this pathway in cancer has been investigated extensively for 2 decades, only recent studies have described its effects on host defenses against transformed epithelial cells. Overcoming tumor-immune evasion remains one of the major challenges in cancer immunotherapy. This review summarizes the impacts of the cyclooxygenase-2–prostaglandin E2 pathway on gastrointestinal cancer development. Our focus was to highlight recent advances in our understanding of how this pathway induces tumor immune evasion.

BACKGROUND AND AIMS: Understanding the mechanisms by which tumors adapt to therapy is critical for developing effective combination therapeutic approaches to improve clinical outcomes for patients with cancer. METHODS: To identify promising and clinically actionable targets for managing colorectal cancer (CRC), we conducted a patient-centered functional genomics platform that includes approximately 200 genes and paired this with a high-throughput drug screen that includes 262 compounds in four patient-derived xenografts (PDXs) from patients with CRC. RESULTS: Both screening methods identified exportin 1 (XPO1) inhibitors as drivers of DNA damage-induced lethality in CRC. Molecular characterization of the cellular response to XPO1 inhibition uncovered an adaptive mechanism that limited the duration of response in TP53mutated, but not in TP53-wild type CRC models. Comprehensive proteomic and transcriptomic characterization revealed that the ATM/ATR-CHK1/2 axes were selectively engaged in TP53mutant CRC cells upon XPO1 inhibitor treatment, and that this response was required for adapting to therapy and escaping cell death. Administration of KPT-8602, an XPO1 inhibitor, followed by AZD-6738, an ATR inhibitor, resulted in dramatic antitumor effects and prolonged survival in TP53-mutant models of CRC. CONCLUSION: Our findings anticipate tremendous therapeutic benefit and support the further evaluation of XPO1 inhibitors, especially in combination with DNA damage checkpoint inhibitors, to elicit an enduring clinical response in patients with CRC harboring TP53 mutations.

The prevalence and dire implications of mutations in the tumour suppressor, p53, highlight its appeal as a chemotherapeutic target. We recently showed that impairing cellular antioxidant systems via inhibition of SLC7A11, a component of the system xc− cystine-glutamate antiporter, enhances sensitivity to mutant-p53 targeted therapy, APR-246. We investigated whether this synergy extends to other genes, such as those encoding enzymes of the pentose phosphate pathway (PPP). TKT, one of the major enzymes of the PPP, is allegedly regulated by NRF2, which is in turn impaired by accumulated mutant-p53 protein. Therefore, we investigated the relationship between mutant-p53, TKT and sensitivity to APR-246. We found that mutant-p53 does not alter expression of TKT, nor is TKT modulated directly by NRF2, suggesting a more complex mechanism at play. Furthermore, we found that in p53null cells, knockdown of TKT increased sensitivity to APR-246, whilst TKT overexpression conferred resistance to the drug. However, neither permutation elicited any effect on cells overexpressing mutant-p53 protein, despite mediating oxidative stress levels in a similar fashion to that in p53-null cells. In sum, this study has unveiled TKT expression as a determinant for sensitivity to APR-246 in p53-null cells.

Summary Background Clinical services for Barrett’s esophagus have been rising worldwide including Australia, but little is known of the long-term outcomes of such patients. Retrospective studies using data at baseline are prone to both selection and misclassification bias. We investigated the clinical characteristics and outcomes of Barrett’s esophagus patients in a prospective cohort. Methods We recruited patients diagnosed with Barrett’s esophagus in tertiary settings across Australia between 2008 and 2016. We compared baseline and follow-up epidemiological and clinical data between Barrett’s patients with and without dysplasia. We calculated age-adjusted incidence rates and estimated minimally and fully adjusted hazard ratios (HR) to identify those clinical factors related to disease progression. Results The cohort comprised 268 patients with Barrett’s esophagus (median follow-up 5 years). At recruitment, 224 (84%) had no dysplasia, 44 (16%) had low-grade or indefinite dysplasia (LGD/IND). The age-adjusted incidence of esophageal adenocarcinoma (EAC) was 0.5% per year in LGD/IND compared with 0.1% per year in those with no dysplasia. Risk of progression to high-grade dysplasia/EAC was associated with prior LGD/IND (fully adjusted HR 6.55, 95% confidence interval [CI] 1.96–21.8) but not long-segment disease (HR 1.03, 95%CI 0.29–3.58). Conclusions These prospective data suggest presence of dysplasia is a stronger predictor of progression to cancer than segment length in patients with Barrett’s esophagus.

The mechanisms by which cells respond and adapt to oxidative stress are largely unknown but are key to developing a rationale for cancer therapies that target antioxidant pathways. APR-246 is a mutant-p53 targeted therapeutic currently under clinical investigation in myeloid dysplastic syndrome (MDS) and acute myeloid leukemia1. Whilst the mechanism of action of APR-246 is thought to be reactivation of wild-type p53 activity through covalent modification of cysteine residues in the core domain of mutant-p53 protein2,3, here we report that the anti-neoplastic capacity of APR-246 lies predominantly in the conjugation of free cysteine. Genome-wide CRISPR perturbation screening, metabolite profiling and proteomics in response to APR-246 treatment in mutant-p53 cancer cells highlighted the role of GSH and mitochondrial metabolism in determining APR-246 efficacy. APR-246 sensitivity was increased through loss of key enzymes in mitochondrial one-carbon metabolism,SHMT2andMTHFD1L, due to diminished glycine supply forde novoGSH synthesis. Critically, we show that APR-246 induces iron-dependent, apoptotic machinery-independent cell death, ferroptosis. Whole-cell proteomics analyses indicated an upregulation of proteins involved in iron-sulfur cluster biogenesis (eg. FDX1). GSH, acetyl-CoA and NADH levels were also depleted in APR-246 treated cells. Importantly, we found that APR-246 inhibits iron-sulfur cluster biogenesis in the mitochondria of cancer cells through cysteine conjugation. This work not only details novel determinants of APR-246 activity in cancer cells, but also provides a clinical roadmap for targeting antioxidant pathways in tumours - beyond targeting mutant-p53 tumours.

PurposeAPR-246 (Eprenetapopt) is in clinical development with a focus on haematological malignancies and is marketed as a mutant-p53 reactivation therapy. Currently, the detection of at least oneTP53mutation is an inclusion criterion for patient selection into most clinical trials. Preliminary results from our phase Ib/II clinical trial investigating APR-246 combined with combination chemotherapy (cisplatin and 5-Fluorouracil) in metastatic oesophageal cancer, together with previous pre-clinical studies, indicate thatTP53mutation status alone may not be a sufficient biomarker for response to APR-246. This study aimed to identify a robust biomarker for response to APR-246.MethodsCorrelation analysis of the PRIMA-1 activity (lead compound to APR-246) with mutational status, gene expression, protein expression and metabolite abundance across over 800 cancer cell lines was performed. Functional validation and a boutique siRNA screen of over 750 redox-related genes were also conducted.ResultsTP53mutation status was not predictive of response to APR-246. The expression of SLC7A11, the cystine/glutamate transporter, was identified as a superior determinant of response to APR-246. Genetic regulators of SLC7A11, including ATF4, MDM2, wild-type p53 and c-Myc were confirmed to also regulate cancer cell sensitivity to APR-246.ConclusionsSLC7A11 expression is the major determinant of sensitivity to APR-246 and should be utilised as a predictive biomarker in future clinical investigation of APR-246.

Background & Aims Esophageal adenocarcinoma (EAC) develops from its precursor Barrett’s esophagus through intermediate stages of low- and high-grade dysplasia. However, knowledge of genetic drivers and molecular mechanisms implicated in disease progression is limited. Herein, we investigated the effect of Mothers against decapentaplegic homolog 4 (SMAD4) loss on transforming growth factor β (TGF-β) signaling functionality and in vivo tumorigenicity in high-grade dysplastic Barrett’s cells. Methods An in vivo xenograft model was used to test tumorigenicity of SMAD4 knockdown or knockout in CP-B high-grade dysplastic Barrett’s cells. RT2 polymerase chain reaction arrays were used to analyze TGF-β signaling functionality, and low-coverage whole-genome sequencing was performed to detect copy number alterations upon SMAD4 loss. Results We found that SMAD4 knockout significantly alters the TGF-β pathway target gene expression profile. SMAD4 knockout positively regulates potential oncogenes such as CRYAB, ACTA2, and CDC6, whereas the CDKN2A/B tumor-suppressor locus was regulated negatively. We verified that SMAD4 in combination with CDC6-CDKN2A/B or CRYAB genetic alterations in patient tumors have significant predictive value for poor prognosis. Importantly, we investigated the effect of SMAD4 inactivation in Barrett’s tumorigenesis. We found that genetic knockdown or knockout of SMAD4 was sufficient to promote tumorigenesis in dysplastic Barrett’s esophagus cells in vivo. Progression to invasive EAC was accompanied by distinctive and consistent copy number alterations in SMAD4 knockdown or knockout xenografts. Conclusions Altogether, up-regulation of oncogenes, down-regulation of tumor-suppressor genes, and chromosomal instability within the tumors after SMAD4 loss implicates SMAD4 as a protector of genome integrity in EAC development and progression. Foremost, SMAD4 loss promotes tumorigenesis from dysplastic Barrett’s toward EAC., Graphical abstract

The Acylglycerol Kinase (AGK) is a mitochondrial lipid kinase that contributes to protein biogenesis as a subunit of the TIM22 complex at the inner mitochondrial membrane. Mutations in AGK cause Sengers syndrome, an autosomal recessive condition characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy and lactic acidosis. We undertook proteomic profiling of Sengers patient fibroblasts and an AGKKO cell line to map the proteomic changes that ensue upon AGK dysfunction. This uncovered extensive remodelling of mitochondrial one-carbon metabolism enzymes and showed that inner membrane serine transporters, Sideroflexins (SFXNs), are novel substrates of the TIM22 complex. Deletion of SFXN1 recapitulates the remodelling of one-carbon metabolism observed in Sengers patient cells. Proliferation of cells lacking AGK is perturbed in the absence of exogenous serine and rescuable through addition of formate, highlighting the dysregulation of one carbon metabolism as a key molecular feature in the biology of Sengers syndrome.

Background: Circulating tumor DNA (ctDNA) has clinical utility in monitoring treatment response and in the detection of disease recurrence in breast and colorectal cancer1,2. The aim of this study was to explore the role of ctDNA in the management of patients with esophageal cancer (EC). Methods:Blood samples and tumor biopsies were collected from 79 patients after diagnosis of EC. In patients planned for surgery, blood samples were taken before and after neoadjuvant treatment, and during the surveillance period. Blood and biopsy tissue samples were analysed for mutations using a custom targeted amplicon-based approach to cover mutational foci across 9 of the most commonly mutated genes in EC. Results:Somatic mutations in treatment-naïve EC tumor biopsies were detected in 71 out of 79 (90%) patients. Out of these 71 cases, 23 (32%) had detectable tumor-informed ctDNA in their plasma. The majority (90%) of patients who were ctDNA positive had either locally advanced or metastatic disease. For node negative locally advanced patients treated with curative intent, positive ctDNA status at diagnosis is a poor prognostic marker (HR 11.71; 1.16 - 118.80; p=0.037). In blood samples taken before and following neoadjuvant therapy (NAT), reversal of a ctDNA positive status after NAT was associated with an excellent response to treatment. In patients who had serial plasma samples taken during surveillance, detection of ctDNA was associated with inferior disease specific survival (HR 4.36; 1.07 - 17.88; p = 0.04). Discussion:This study demonstrates that ctDNA may have clinical utility in the management of patients with EC by providing additional prognostic information at pre-treatment staging. In the absence of a widely accepted paradigm for surveillance after curative-intent treatment, assessment of ctDNA in post treatment blood samples may lead to the detection of early recurrent disease. 1. Dawson, S.-J. et al. Analysis of Circulating Tumor DNA to Monitor Metastatic Breast Cancer. New Engl J Medicine 368, 1199-1209 (2013).2. Tie, J. et al. Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer. Sci Transl Med 8, 346ra92 (2016). Citation Format: Carlos Suhady Cabalag, Michael Yates, Mariana B. Corrales, Paul Yeh, Stephen Q. Wong, Bonnie Zhang, Kenji M. Fujihara, Lynn Chong, Michael Hii, Sarah-Jane Dawson, Wayne A. Phillips, Cuong P. Duong, Nicholas J. Clemons. Utility of circulating tumor DNA in esophageal cancer: A potential prognostic biomarker in tumor staging, monitoring of treatment response and detection of recurrent disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 539.

Esophageal cancer is the 8th most common cancer worldwide and has the 6th highest mortality rate of all cancers. The 5-year survival rate following esophageal adenocarcinoma (EAC) diagnosis is dismal at less than 15 percent, indicating a dire need for improved therapeutic strategies and early detection. EAC develops stepwise following exposure to chronic gastric reflux: From pre-malignant Barrett's metaplasia, through stages of low- and high-grade dysplasia until developing into invasive cancer. Mutation or loss of common tumor suppressor genes TP53 and SMAD4 act as markers for cancer progression, occurring in high-grade dysplastic tissue and invasive EAC, respectively. Our novel in vivo tumorigenesis model demonstrates progression of Barrett's metaplasia to EAC, in which SMAD4-deficient Barrett's metaplasia cells form tumors in immunodeficient mice after a period of latency and in a dose-dependent manner. This delayed tumor growth onset suggests further drivers are required for oncogenesis, and these SMAD4-deficient cells and tumors display a greater degree of genomic instability than wildtype-SMAD4 controls. A genome-wide CRISPR-Cas9 knockout screen unveiled a synthetic lethal relationship between SMAD4-deficiency and cell cycle checkpoint inhibition, suggesting a role for SMAD4 in maintaining genomic stability and a potential novel therapeutic avenue for SMAD4-deficient EAC. Additionally, a concurrent in vivo CRISPR-Cas9 tumorigenesis screen produced tumors 4-fold faster than the previous model and identified regulators of mTOR signaling as co-operative drivers of tumorigenesis in EAC. Wildtype-SMAD4 cells failed to generate tumors despite undergoing the same genetic perturbations, indicating a potential gatekeeping effect of SMAD4 in mTOR-mediated EAC tumorigenesis. In sum, loss of SMAD4 acts as a double-edged sword, increasing genomic instability and thereby rendering EAC cells sensitive to cell cycle checkpoint inhibition, whilst simultaneously co-operating with modulated mTOR signaling to promote tumorigenesis in EAC xenograft models. Citation Format: Julia V. Milne, Jovana R. Gotovac, Kenji M. Fujihara, Cuong P. Duong, Wayne A. Phillips, Nicholas J. Clemons. SMAD4 as a potential gatekeeper for genomic instability and mTOR-mediated tumorigenesis in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2671.

Human Tim8a and Tim8b are members of an intermembrane space chaperone network, known as the small TIM family. Mutations in TIMM8A cause a neurodegenerative disease, Mohr-Tranebjærg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells or how mutation of this protein leads to a neurodegenerative disease. We show that hTim8a is required for the assembly of Complex IV in neurons, which is mediated through a transient interaction with Complex IV assembly factors, in particular the copper chaperone COX17. Complex IV assembly defects resulting from loss of hTim8a leads to oxidative stress and changes to key apoptotic regulators, including cytochrome c, which primes cells for death. Alleviation of oxidative stress with Vitamin E treatment rescues cells from apoptotic vulnerability. We hypothesise that enhanced sensitivity of neuronal cells to apoptosis is the underlying mechanism of MTS.

Human Tim8a is a member of an intermembrane space chaperone network, known as the small TIM family, which transport hydrophobic membrane proteins through this compartment. Mutations in TIMM8A cause a neurodegenerative disease, Mohr-Tranebjærg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells and consequently how lack of hTim8a leads to a neurodegenerative disease. We identified a novel cell-specific function of hTim8a in the assembly of Complex IV, which is mediated through a transient interaction with the copper chaperone COX17. Complex IV assembly defects in cells lacking hTim8a leads to oxidative stress and changes to key apoptotic regulators, including cytochrome c and Bax, which primes cells for cell death. Alleviation of oxidative stress using Vitamin E rescues cells from apoptotic vulnerability. We hypothesis that enhanced sensitivity of neuronal cells to apoptosis is the underlying mechanism of MTS.

The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. The compound APR‐246 (PRIMA‐1Met/Eprenetapopt) is converted to methylene quinuclidinone (MQ) that targets mutant p53 protein and perturbs cellular antioxidant balance. APR‐246 is currently tested in a phase III clinical trial in myelodysplastic syndrome (MDS). By in vitro, ex vivo, and in vivo models, we show that combined treatment with APR‐246 and inhibitors of efflux pump MRP1/ABCC1 results in synergistic tumor cell death, which is more pronounced in TP53 mutant cells. This is associated with altered cellular thiol status and increased intracellular glutathione‐conjugated MQ (GS‐MQ). Due to the reversibility of MQ conjugation, GS‐MQ forms an intracellular drug reservoir that increases availability of MQ for targeting mutant p53. Our study shows that redox homeostasis is a critical determinant of the response to mutant p53‐targeted cancer therapy., Tumor suppressor TP53 is mutated in a large fraction of tumors. APR‐246/Eprenetapopt is the most clinically advanced mutant p53‐targeting drug candidate (Phase III). Besides restoring wild type p53 activity, the active product MQ also disrupts the redox balance, resulting in cancer cell death.

ObjectiveTo clarify the prognostic role of tumour protein 53 (TP53) mutations in patients with oesophageal adenocarcinoma (OAC) as there is a need for biomarkers that assist in guiding management for patients with OAC.DesignA systematic review was conducted using MEDLINE, Embase, PubMed and Current Contents Connect to identify studies published between January 1990 and February 2015 of oesophageal cancer populations (with OAC diagnoses >50% of cases) that measured tumoural TP53 status and reported hazard ratios (HR), or adequate data for estimation of HR for survival for TP53-defined subgroups. Risk of bias for HR estimates was assessed using prespecified criteria for the appraisal of relevant domains as defined by the Cochrane Prognosis Methods Group including adherence to Grading of Recommendations, Assessment, Development and Evaluation and REporting recommendations for tumor MARKer prognostic studies guidelines, as well as assay method used (direct TP53 mutation assessment vs immunohistochemistry) and adjustment for standard prognostic factors. A pooled HR and 95% CI were calculated using a random-effects model.ResultsSixteen eligible studies (11 with OAC only and 5 mixed histology cohorts) including 888 patients were identified. TP53 mutations were associated with reduced survival (HR 1.48, 95% CI 1.16 to 1.90, I2=33%). A greater prognostic effect was observed in a sensitivity analysis of those studies that reported survival for OAC-only cohorts and were assessed at low risk of bias (HR 2.11, 95% CI 1.35 to 3.31, I2=0%).ConclusionsPatients with OAC and TP53 gene mutations have reduced overall survival compared with patients without these mutations, and this effect is independent of tumour stage.

Barrett's esophagus (BE) is clinically significant, as it is the only known precursor lesion for esophageal adenocarcinoma. To develop improved therapies for the treatment of BE, a greater understanding of the disease process at the molecular genetic level is needed. However, achieving a greater understanding will require improved preclinical models so that the disease process can be more closely studied and novel therapies can be tested. Our concise review highlights progress in the development of preclinical models for the study of BE and identifies the most suitable model in which to test novel therapies.

Objectives p53 is a critical tumour suppressor and is mutated in 70% of oesophageal adenocarcinomas (OACs), resulting in chemoresistance and poor survival. APR-246 is a first-in-class reactivator of mutant p53 and is currently in clinical trials. In this study, we characterised the activity of APR-246 and its effect on p53 signalling in a large panel of cell line xenograft (CLX) and patient-derived xenograft (PDX) models of OAC. Design In vitro response to APR-246 was assessed using clonogenic survival, cell cycle and apoptosis assays. Ectopic expression, gene knockdown and CRISPR/Cas9-mediated knockout studies of mutant p53 were performed to investigate p53-dependent drug effects. p53 signalling was examined using quantitative RT-PCR and western blot. Synergistic interactions between APR-246 and conventional chemotherapies were evaluated in vitro and in vivo using CLX and PDX models. Results APR-246 upregulated p53 target genes, inhibited clonogenic survival and induced cell cycle arrest as well as apoptosis in OAC cells harbouring p53 mutations. Sensitivity to APR-246 correlated with cellular levels of mutant p53 protein. Ectopic expression of mutant p53 sensitised p53-null cells to APR-246, while p53 gene knockdown and knockout diminished drug activity. Importantly, APR-246 synergistically enhanced the inhibitory effects of cisplatin and 5-fluorouracil through p53 accumulation. Finally, APR-246 demonstrated potent antitumour activity in CLX and PDX models, and restored chemosensitivity to a cisplatin/5-fluorouracil-resistant xenograft model. Conclusions APR-246 has significant antitumour activity in OAC. Given that APR-246 is safe at therapeutic levels our study strongly suggests that APR-246 can be translated into improving the clinical outcomes for OAC patients.

Interactions between cancer cells and cancer-associated fibroblasts (CAF) play an important role in tumour development and progression. In this study we investigated the functional role of CAF in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of EAC patients (183 patients) for CAF markers related to disease mortality. We characterized CAF and normal oesophageal fibroblasts (NOF) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3-D organotypic culture and xenograft models were used to examine effects on EAC cell function, and dissect molecular mechanisms regulating invasion. Most EAC (93%) contained CAF with a myofibroblastic (?-SMA-positive) phenotype, which correlated significantly with poor survival (p?=?0.016; HR 7. 1 (1.7-29.4). Primary CAF, isolated from EAC, have a contractile, myofibroblastic phenotype, and promote EAC cell invasion in vitro (Transwell assays, p?=?

The advent of multi-omic profiling of tumors, together with the increasing affordability of high-throughput drug screening programs, has helped usher in a new era of molecular targeted therapies for many solid malignancies. However, there has been limited success in esophageal adenocarcinoma. Until recently, preclinical drug development for this cancer has been largely limited to a small number of cell line models. Now, the increasing availability of patient-derived xenografts and novel metastatic models are helping to bridge the gap between scientific discovery and patient care. These platforms are valuable adjuncts for drug testing. Nevertheless, further work is required to develop systems that model the tumor microenvironment, including cancer cell interactions with the immune system, which will be particularly relevant for the translation of novel immunotherapies for esophageal adenocarcinoma.