Manel Esteller Research Articles

Abstract 534: Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2

Abstract Background: Copanlisib is a PI3K inhibitor with selectivity against PI3Kδ and PI3Kα and is approved for treating patients with follicular lymphoma and marginal zone lymphoma (MZL). Preclinical studies have demonstrated strong synergism of copanlisib and BCL2 inhibitor venetoclax in different lymphoma models, and the combination is under clinical evaluation. Understanding the resistance mechanisms could help the design of improved therapies. Hence, we generated MZL cell lines resistant to the copanlisib/venetoclax combination. Methods: All cells were tested for cell identity and Mycoplasma status. Resistance was confirmed stable after 3-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out by confirming sensitivity to vincristine. Targeted agents already in the clinics were combined with copanlisib/venetoclax. Cells underwent transcriptome and methylation profiling, targeted DNA sequencing, and immunophenotypic and immunoblotting analyses. Results: We developed SSK41-derived models with resistance to copanlisib (n.=1) or venetoclax (n.=1) as single agents and to copanlisib/venetoclax (n.=2) by long IC50 or by upfront IC90 exposure. The single-drug-resistant cells showed only partial resistance to copanlisib/venetoclax. In contrast, the cells that developed exposing cells to copanlisib/venetoclax IC90 exhibited a more substantial decrease in sensitivity to the combination. Copanlisib/venetoclax induced apoptosis and cell cycle arrest in PAR but not in resistant cells. Sensitivity to other PI3K, BCL2, and BTK inhibitors was decreased in all resistant lines. The response to MCL1-i, mTOR-i, and epigenetic agents (decitabine, HDAC, BET, and EZH2 inhibitors) was maintained. Triple combinations of copanlisib/venetoclax with the AURKA inhibitor alisertib, PLK inhibitor rigosertib, or the multikinase inhibitor amcasertib were active in both parental and resistant cells. No acquired mutations were observed via targeted DNA sequencing designed to cover various coding genomic regions known to be recurrently mutated in mature B-cell neoplasms, including BCL2, nor via RNA-Seq for PIK3CA/B/G/D genes. Integration of RNA and methylation profiles showed deregulation of genes involved in type-I IFN, chemokines (CCL25, CXCL13), apoptosis regulation, and JAK-STAT signaling. Resistant cells upregulated CD19 expression compared to parental cells. Immunoblotting showed the inability of copanlisib/venetoclax to block BCR signaling in resistant cells. Conclusions: We created four novel MZL-derived models of secondary resistance to PI3K and BCL2 inhibitors, which will help clarify possible modalities to overcome resistance. Novel potential targets were already identified, such as AURKA or PLK, to be further exploited. Citation Format: Alberto J. Arribas, Luciano Cascione, Eleonora Cannas, Helen Bellerjeau, Federica Fuzio, Aleix Noguera, Andrea Rinaldi, Manel Esteller, Emanuele Zucca, Andrea Alimonti, Davide Rossi, Anastasios Stathis, Francesco Bertoni. Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 534.

Abstract 394: ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms

Abstract Background: Marginal zone lymphoma (MZL) is an indolent yet incurable B cell malignancy. Two BTK inhibitors, ibrutinib and zanubrutinib, are FDA approved for relapsed/refractory MZL patients. PI3K inhibitors have also shown clinical activity. The identification of the mechanisms of resistance can provide useful information to optimize the use of the agents. We previously reported an IL6 driven MZL model of PI3K inhibitors resistance developed by prolonged exposure to the PI3Kδ inhibitor idelalisib (Arribas, Haematologica 2022). Here, we present the detailed characterization of a second model with resistance to both BTK and PI3K inhibitors. Methods: MTT assay. RNA-Seq, whole exome sequencing, miRNA and methylation profiling. FACS and ELISA analyses. Results: Resistant cells, developed by continuous exposure of the cell line Karpas1718 to idelalisib, showed resistance to various inhibitors of BTK (ibrutinib, zanubrutinib, acalabrutinib and pirtobrutinib) and PI3K (idelalisib, duvelisib, copanlisib and umbralisib). No mutations affecting BTK, PLCG2 or CXCR4 were identified in resistant cells, which had higher expression of genes involved in ERBB signaling (HBEGF, NRG2, ERBB4), cell proliferation (PBK, MKI67, TCL1A) and DNA recombination (RAG1, RAG2) than parental cells. We confirmed cell surface ERBB4 up-regulation, and the cytoplasmatic expression and secretion of its ligand HBEGF in resistant cells, which led to increased levels of p-AKT and p-ERK. The miRNAs miR-29c and let-7c, known negative regulators of the HBEGF-ERBB axis, were fully methylated and down-regulated in resistant compared to parental cells. ERBB4 genetic silencing improved sensitivity to PI3Kδ inhibitor, and exposure to let-7c or miR-29c mimics decreased secreted HBEGF and recovered sensitivity to PI3K inhibitors in resistant cells. Addition of recombinant HBEGF (rHBEGF) induced resistance to BTK and to PI3K inhibitors in parental cells and in other lymphoma models including mantle cell lymphomas and diffuse large B cell lymphomas (DLBCL). The rHBEGF induced resistance was reverted adding the ERBB inhibitor lapatinib. To extend our findings to the clinical context, using two MZL and one DLBCL expression datasets, we showed HBEGF and ERBB4 expression in clinical specimens. Finally, HBEGF levels appeared elevated in the serum of CLL patients with primary or acquired resistance to PI3Kδ or to BTK inhibitors, compared to patients responding to the drugs and paired for similar clinical features. Conclusions: We characterized a novel B cell lymphoma model of secondary resistance to BTK and PI3K inhibitors. Our results indicate that epigenetic plasticity led to the activation of HBEGF-ERBB signaling sustaining resistance to BTK/PI3K inhibitors, which can be overcome using epigenetic agents and ERBB inhibitors. These therapeutics approaches could be tested in novel clinical trials. AJA, SN: equally contributed. Citation Format: Alberto J. Arribas, Sara Napoli, Luciano Cascione, Laura Barnabei, Giulio Sartori, Eleonora Cannas, Eugenio Gaudio, Chiara Tarantelli, Afua A. Mensah, Filippo Spriano, Antonella Zucchetto, Francesca M. Rossi, Andrea Rinaldi, Manuel Castro de Moura, Anastasios Stathis, Georg Stussi, Valter Gattei, Jennifer R. Brown, Manel Esteller, Emanuele Zucca, Davide Rossi, Francesco Bertoni. ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 394.

Abstract 153: Epigenetic loss of NSUN5 in glioma drives a global protein synthesis depletion along with the emergence of a stress adaptive translational program

Abstract One of the known hallmarks of cancer is an aberrant RNA transcriptome, and yet, it cannot always be fully due to genetic or epigenetic alterations at the transcriptional start sites of the genes. Indeed, recent evidences point out to new malignant alterations at the RNA level itself. RNA molecules have been found to show more than 100 differently chemically modified nucleotides, and such post-transcriptional modifications play a role in controlling RNA homeostasis, being all grouped under the term ‘epitranscriptome'. In our study, we reported that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate 5-methylcytosine RNA methyltransferase. As NSUN5 activity has not been formally described in human cells yet, we studied its potential targets in the human transcriptome, demonstrating its role in the methylation of the cytosine 3782 in the 28S ribosomal RNA (rRNA). Logically, we found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA, and this depletion drives an overall depletion of protein synthesis under stress conditions, analyzed by O-propargyl-puromycin (OP-Puro) and [3H] leucine incorporation into nascent proteins. However, together with the depletion of global protein synthesis, we observed the emergence of an adaptive translational program for survival under conditions of cellular stress, assessed by combining three NGS-experiments: RNA-seq, Ribo-seq and SILAC. Moreover, as one of the genes increasing its expression upon NSUN5 silencing is the stress-related enzyme NQO1, we found out that NSUN5-depleted gliomas are sensitive to bioactivatable substrates of the NQO1 enzyme. Most importantly, we observed that NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival, conferring it value as a predictor of better patient outcome for this otherwise devastating disease. Citation Format: Vanessa Ortiz Barahona, Maxime Janin, Manel Esteller. Epigenetic loss of NSUN5 in glioma drives a global protein synthesis depletion along with the emergence of a stress adaptive translational program [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 153.

Abstract 5416: Positioning the NQO1-bioactivatable drug isobutyl-deoxynyboquinone in diffuse intrinsic pontine glioma (DIPG): an exceptional therapeutic opportunity in pediatric brain tumor

Abstract Purpose: Diffuse Intrinsic Pontine Glioma (DIPG), is a very aggressive pediatric cancer with poor overall survival and no effective treatment. Despite numerous clinical trials, the overall survival remains at 9 months after diagnosis and only radiotherapy has shown a relative efficacy. Therefore, it is primordial to increase our understanding of the biology of DIPG tumors, as well as finding new healthcare strategies to tackle this pediatric disease. Recently, we discovered that NQO1, a stress-related protein, was overexpressed in 3 out of 6 DIPG patient-derived cell lines, as well as in half of the primary tissues. Interestingly, NQO1 overexpression can be targeted by substrates that induce an excessive oxidative stress into the cells that finally push them to apoptosis, while normal surrounding tissues with basal expression of NQO1 would be keep safe. Experimental procedure: This discovery prompted us to test in in vitro and in vivo assays a promising drug named isobutyl-deoxynyboquinone (IB-DNQ): a NQO1 bioactivatable substrate. We first tested the drug response of the 6 cell lines before testing it on orthotopic xenograft mouse models. We also investigated by which mechanism NQO1 is regulated in DIPG. Finally, we performed experiments in order to decipher by which mechanism NQO1 is regulated in DIPG. Results: We saw that NQO1 overexpressing cells are very sensitive to the drug, compared to the cell lines with normal expression. Moreover, we started to validate the use of IB-DNQ in vivo using a DIPG cell line treated or not (mock) with IB-DNQ and also a NQO1-knockdown model in orthotopic xenograft mice models, confirming that IB-DNQ crosses the blood brain barrier and increases the overall survival. NQO1 increased expression in DIPG is surprisingly not due to a NRF2-mediated transcriptional regulation as we did not observe a correlation between NQO1 transcript abundance and protein expression in DIPG cell lines, indicating that NRF2 pathway could not be implicated, but translational or post-translational regulation could be operating. We performed a polysome profiling of 4 patient-derived cell lines, a protein stability assay and a large scale proteome for the 6 cell lines (pending results). Conclusion: Our preliminary results, including drug efficacy, are very encouraging for the development of this new therapeutic in DIPG. This discovery represents a promising opportunity to tackle this devastating disease and a new hope for the patients and their families. Citation Format: Maxime Henri Janin, Vanessa Ortiz Barahona, Pere Llinas Arias, Carolina De La Torre, Angel Montero Carcaboso, Andres Morales La Madrid, Paul Hergenrother, Manel Esteller. Positioning the NQO1-bioactivatable drug isobutyl-deoxynyboquinone in diffuse intrinsic pontine glioma (DIPG): an exceptional therapeutic opportunity in pediatric brain tumor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5416.

Abstract 1422: Clinical association of miR-155-5p with breast cancer and its relevance for treatment with PARP inhibitors

Abstract INTRODUCTION. miR-155-5p is a well-known oncogenic microRNA frequently overexpressed in human cancers, including breast cancer (BC), but its putative role as biomarker remains controversial. Thus, we aimed at evaluating miR-155-5p levels in a large BC cohort, and its association with clinical parameters. Then, since miR-155-5p was found to be epigenetically controlled by BRCA1, and to increase the tumor mutation load, we analysed miR-155-5p correlation with defects in Homologous Recombination (HR) genes (i.e. HRD), and its capability to alter the response to the first-in-class PARP (Poly-[ADP-ribose]-polymerase-1 (PARP-1)) inhibitor Olaparib (AZD2281). METHODS. We measured miR-155-5p expression in a retrospective cohort of breast cancer patients (CSS cohort, n=283; median follow-up=81 months) by RT-qPCR. Then, we used the TCGA Breast Cancer (TCGA-BC) dataset (n=1095) to validate our results, and evaluate the association between miR-155-5p and mutations in HR genes. Last, we ectopically modulated miR-155-5p in three wtBRCA1/2 cell lines (i.e. MDA-MB-231, MDA-MB-468, MDA-MB-453) and one mutBRCA1 cell line (MDA-MB-436) by using mirVana miR-155-5p mimic or inhibitor, and we measured cell viability before and after treatment with Olaparib or vehicle by PrestoBlue viability reagent. RESULTS. High miR-155-5p levels were associated with unfavourable prognostic indicators including high tumor grade (overall P=0.0007; G2 vs G3 P=0.0053), reduced expression of ER (r= -0.243; P=0.0002) and PgR (r= -0.240; P<0.0001), and high Ki-67 expression (r=0.215; P=0.0005). Consistently, miR-155-5p was differentially expressed across the BC subgroups identified by the surrogate molecular classification in the CSS-cohort (P=0.0008) and by the PAM50 panel in the TCGA-BC dataset (P=0.0001), with the Triple Negative and HER2-amplified tumors showing the highest levels. The analysis of the TCGA BRCA dataset also found higher miR-155 levels in the patient subgroup carrying somatic mutations in HR genes compared to the subgroup without somatic mutations in HR genes (P<0.0001). Last, the in vitro drug testing confirmed that mutBRCA1 MDA-MB-436 cells, exhibiting miR-155-5p endogenous levels 1000-fold higher than the other TNBC cell lines, had a higher sensitivity to a short-term (72hrs) treatment with Olaparib alone (IC50 25.80, 95%CI 24.52-27.15) compared to the other cell lines. Importantly, ectopic overexpression of miR-155-5p in wtBRCA1/2 cell lines, followed by Olaparib exposure, resulted in a significant reduction in viability (p<0.001) compared to Olaparib alone in all cell lines. However, miR-155-5p inhibition in BRCA1mut MDA-MB-436 cell line did not affect the higher sensitivity to PARP inhibition, suggesting that sensitivity to PARPi is not limited to HRD. CONCLUSIONS. Overall, our data point to a role of miR-155-5p in Homologous Recombination Deficiency (HRD), and suggest a putative novel mechanism of synthetic lethality mediated by PARP inhibition and miR-155-5p to promote cancer cell death. Citation Format: Barbara Pasculli, Raffaela Barbano, Andrea Fontana, Tommaso Biagini, Maria Pia Di Viesti, Michelina Rendina, Vanna Maria Valori, Maria Morritti, Sara Bravaccini, Sara Ravaioli, Evaristo Maiello, Paolo Graziano, Roberto Murgo, Massimiliano Copetti, Tommaso Mazza, Vito Michele Fazio, Manel Esteller, Paola Parrella. Clinical association of miR-155-5p with breast cancer and its relevance for treatment with PARP inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1422.

Abstract 466: A novel DNA methylation-based approach for molecular subtyping and improved prognostication of colorectal cancer using formalin-fixed and paraffin-embedded tissue

Abstract Background: Colorectal cancer (CRC) is characterized by marked inter-tumor heterogeneity, both molecularly and clinically. Patient stratification using transcriptional subtyping show promise to resolve the heterogeneity and guide precision medicine. This requires high quality RNA, which can be purified from fresh frozen (FF) tissue, but not from clinically collected formalin-fixed paraffin-embedded (FFPE) tissue. Consequently, transcriptional subtyping is not applicable to most CRC patients. Aim: To establish a DNA methylation-based approach for molecular characterization and subtyping, which is compatible with both FF and FFPE tissue, and use this to improve patient prognostication as compared to histopathological TNM staging. Methods: Using paired RNA expression and DNA methylation profiles (450K array) from 394 CRCs, we identified 200 CpG sites genome-wide, whose methylation levels correlated best with expression for each gene. With this information, we 1) devised an approach, methCORR, that can infer RNA expression in both FF and FFPE samples using only DNA methylation profiles, 2) established a methCORR network that clusters genes according to overlap in the CpG sites associated with their expression level and 3) used the network to determine and characterize biological traits associated with tumor aggressiveness. Results: The methCORR-inferred RNA expression profiles in FF tissue consistently exhibited high correlation to matched RNA-seq. profiles (median Pearson r=0.97; range 0.91-0.98). We found a higher correlation between FF RNA-seq. and FFPE inferred RNA profiles (median r= 0.97; range 0.96-0.98) than between FF and FFPE RNA-seq. profiles (median r= 0.78; range 0.51-0.92). Gene expression profiles were inferred in two FF (n=231, n=203), and two FFPE cohorts (n=113, n=56). Clustering of these profiles identified the same two CRC subtypes independently in all cohorts. Characterization using the methCORR network showed that the two subtypes resembled conventional and serrated CRC. Moreover, methCORR network analysis identified subtype-specific traits that associated strongly with tumor aggressiveness, such as T-cell, fibroblast, and epithelial-mesenchymal transition activity, and thus allowed identification of subtype-specific prognostic biomarkers. These better predicted relapse-free survival (HR=3.22, 95%CI 2.00-5.17) than TNM staging (HR=1.99, 95%CI 1.28-3.08). Finally, we derived four simple and clinically applicable DNA methylation-specific PCR assays for subtyping and prognostication of CRC FFPE samples. Conclusion: We have developed a novel method for characterization, subtyping, and prognostication of CRC, which is compatible with FF and FFPE samples. We envision that application of the methCORR approach to other cancer types will generate similar fruitful results. Citation Format: Trine B. Mattesen, Mads H. Rasmussen, Juan Sandoval, Halit Ongen, Sigrid S. Árnadóttir, Anders H. Madsen, Søren Laurberg, Emmanouil T. Dermitzakis, Manel Esteller, Claus L. Andersen, Jesper B. Bramsen. A novel DNA methylation-based approach for molecular subtyping and improved prognostication of colorectal cancer using formalin-fixed and paraffin-embedded tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 466.

Abstract 1225: Aberrant SMAD3 signaling in tumor-associated fibroblasts modulates fibrosis and response to the anti-fibrotic drug nintedanib in non-small cell lung cancer

Abstract A hallmark of non-small cell lung cancer (NSCLC) is a fibrotic/desmoplastic stroma rich in activated fibroblasts, which are critical regulators of cancer progression, response to therapies and radiotherapy resistance. Paradoxically, we recently reported that the important pro-fibrotic TGF-β transcription factor SMAD3 was epigenetically down-regulated through promoter hypermethylation in tumor associated fibroblasts (TAFs) from NSCLC patients compared to patient-matched control fibroblasts. In addition, we reported that the clinically approved antifibrotic drug nintedanib elicited a stronger inhibition of both the fibrotic phenotype and its associated tumor-promoting effects in TAFs from adenocarcinoma (ADC) patients compared to squamous cell carcinoma (SCC) patients upon TGF-β1 stimulation in vitro, which was consistent with the selective therapeutic response to nintedanib observed in a clinical trial in ADC (but not SCC) patients. Altogether, these previous results suggest that TGF-β1 signaling may be altered in lung TAFs, and that such alteration may depend on their histologic subtype. In this study we addressed these questions by determining the expression and activity of SMAD3 and its closely related homologue SMAD2 in patient-derived TAFs and paired control fibroblasts, and by dissecting their potential contribution to the differential therapeutic responses to nintedanib observed in ADC and SCC. In vitro studies revealed a marked SMAD3 epigenetic repression through promoter hypermethylation, a low pSMAD3/pSMAD2 ratio and a limited fibrotic phenotype selectively in SCC-TAFs. In contrast, ADC-TAFs overexpressed a panel of fibrotic markers upon TGF-β1 stimulation concomitantly with a high pSMAD3/pSMAD2 ratio and a limited SMAD3 promoter methylation. Histologic analysis of a large patient cohort (112 ADC, 96 SCC) confirmed that the extent of fibrosis is larger in ADC than SCC patients. In addition, knocking-down SMAD3 in ADC-TAFs was sufficient to reduce the antifibrotic and antigrowth effects of nintedanib in vitro and in tumor xenografts in vivo. On the other hand, long-term exposure of pulmonary fibroblasts to cigarette smoke condensate was sufficient to hypermethylate the SMAD3 promoter. Since SCC and ADC tumors typically arise in the upper airways and distal pulmonary sites, respectively, it is conceivable that fibroblasts might be more exposed to the smoking epigenetic effects on SMAD3 in SCC. In summary, we report for the first time that tumor fibrosis is higher in ADC than SCC patients, in association with a selective therapeutic response to the antifibrotic drug nintedanib in the former, and identify the subtype-specific extent of SMAD3 epigenetic repression in TAFs and the subsequent aberrant SMAD3/SMAD2 imbalance as major regulatory mechanisms of tumor fibrosis and response to nintedanib in NSCLC. Citation Format: Rafael Ikemori, Marta Gabasa, Miguel Vizoso, Paula Duch, Sebastian Moran, Sabrina Gea-Sorli, Paloma Bragado, Toni Jauset, Manel Esteller, Laura Soucek, Eduard Monsó, Víctor Peinado, Cristina Fillat, Frank Hilberg, Noemí Reguart, Jordi Alcaraz. Aberrant SMAD3 signaling in tumor-associated fibroblasts modulates fibrosis and response to the anti-fibrotic drug nintedanib in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1225.

Abstract 1819: Non-coding RNAs as key players for understanding oncogenic pathways in ovarian cancer: Studying new therapeutic strategies

Abstract The largest part of the human genome is transcribed into non-coding RNAs (ncRNAs) which are functional transcripts displaying relevant roles in many biological processes, and their dysregulation has been shown to be involved in the development of diseases like cancer. The group of long non-coding RNAs (lncRNAs) has been elucidated to be key regulators of gene expression by acting at different levels, as regulators of epigenetic modifications and chromatin remodelling by acting as structural scaffolds, decoys or guides in the nucleus; as well as post-transcriptional regulators of messenger RNA stability and translation in the cytoplasm. Hence, the knowledge about their mechanisms of action may provide a better understanding of cell biology and likewise, lncRNAs may be used as biomarkers or therapeutical tools. The aim of the current project has been to describe the mechanism of action of a lncRNA called RPSAP52 (Ribosomal Protein SA Pseudogene 52) in ovarian cancer, as well as to study the potential use of RPSAP52 as a tool for therapeutical strategies. Our previous results indicate that RPSAP52, which is an antisense transcript to the well described oncogene HMGA2 (High Mobility Group A2), positively regulates HMGA2 expression through the formation of an R loop structure, and it has been shown that RPSAP52 enhances the oncofetal pathway HMGA2-IGF2BP2-RAS axis through LIN28B-dependent and independent inhibition of let-7 miRNAs in breast cancer and sarcomas. Since the dysregulation of this pathway has been described to be involved in the development of ovarian cancer; which is among the most common types of cancer with poor prognosis, the goal of this project has been to reveal the relevance of RPSAP52 in this system. The oncogenic potential of RPSAP52 has been confirmed by the analysis of a battery of xenograft samples from ovarian cancer patients, in which significant higher levels of RPSAP52 in those samples versus normal ovary have been observed, and it is positively correlated with higher levels of the sense gene HMGA2. Preliminary in vitro studies have shown that the depletion of RPSAP52 with shRNAs is related with a down-regulation of the oncogenic protein LIN28A in SKOV3 cells. Our findings suggest that RPSAP52 may be involved in the pathogenesis of ovarian cancer, and a further analysis of the link between RPSAP52 and LIN28A needs to be performed, together with the study of other candidate protein partners of RPSAP52 revealed by RNA pull down and RAP-MS techniques. Interestingly, the use of the LNA Gapmers as a strategy for the depletion of RPSAP52 also allows us to use them in vivo to confirm in vitro results as well as to study its potential role as a therapeutic target in ovarian cancer. Citation Format: Anaís Sánchez Castillo, Cristina Oliveira Mateos, Marta Soler Riera, Manel Esteller, Sonia Guil. Non-coding RNAs as key players for understanding oncogenic pathways in ovarian cancer: Studying new therapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1819.

Abstract 474: Genome-wide DNA methylation profiling reveals novel candidate epigenetic gatekeepers in hepatocarcinogenesis

Abstract Epigenetic deregulation is a critical event in human malignancies. Besides mutations in TERT promoter, found in 20% of dysplastic nodules, little is known about the key molecular alterations driving early hepatocarcinogenesis. The aims of this study were: 1) to analyze DNA methylation changes during the transition from preneoplastic lesions to early hepatocellular carcinoma (HCC), and 2) to identify candidate epigenetic gatekeepers in the transition between dysplasia and early tumors. Methylome profiling was done with Illumina HumanMethylation450 (485,000 CpG, 96% of known CpG islands). We evaluated differentially methylated CpG sites between groups using an F-test. To detect novel epigenetic gatekeepers we defined hypermethylation as a B value higher than 0.5. We profiled 144 fresh-frozen tissues from resection or liver transplant specimens and integrated these data with our previously report on DNA methylation in HCC (Villanueva et al. Hepatology 2015) resulting in: 16 normal liver, 139 cirrhosis, 8 dysplastic nodules and 227 HCC samples. Correlation between methylation and RNA expression (n= 361) was quantified with the Pearson’s coefficient. Patients were mostly male (76%), with a median age of 66, and with underlying liver disease mainly due to hepatitis C (43%) and B (23%). All tumors were early stage according to BCLC classification, including 43 cases below 2 cm in diameter. A phylo-epigenetic tree derived from the euclidean distances between differentially DNA methylated sites (n=421,997) reveals a gradient of methylation changes that spans normal, cirrhotic, dysplastic nodules and HCC. Epigenetic analysis confirmed closer proximity of dysplasia to HCC than to cirrhotic tissue. We validated aberrant methylation of previously reported HCC epidrivers within the unpublished dataset (e.g. EFBN2, TBX15) (Villanueva et al. Hepatology 2015). Focusing on CpG sites located in promoter regions (i.e., TSS200, TSS1500, 5’UTR, and 1st exon), we selected candidates hypermethylated in less than 1% of normal and cirrhotic tissue, in all high grade dysplastic nodules, and in more than 50% of small HCC nodules (<2 cm). When we integrated DNA methylation and gene expression we found a significant (all P<0.001) inverse correlation in TSPYL5 (r=-0.31), KCNA3 (r=-0.33), LDBH (r=-0.46) and SPINT2 (r=-0.43). Whole-genome DNA methylation profiles accurately discriminate between different histological lesions along the human hepatocarcinogenesis spectrum. We report novel epigenetic gatekeepers in the transition between dysplastic nodules and early HCC. Citation Format: Gabriela Hernandez Meza, Johann von Felden, Amanda Craig, Sergi Sayols, Anna Portela, Manel Esteller, Myron Schwartz, Vincenzo Mazzaferro, Josep Llovet, Augusto Villanueva. Genome-wide DNA methylation profiling reveals novel candidate epigenetic gatekeepers in hepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 474.

Abstract 4725: Efficacy of a new small-molecule inhibitor of histone deacetylase 6 (HDAC6) in preclinical models of B-cell lymphoma and acute myeloid leukemia

Abstract Histone deacetylase 6 (HDAC6) is a protein modifier that is an increasingly attractive pharmacological target. Interestingly, the observation that the HDAC6 knock-out mouse is not lethal, in contrast to those undergoing complete loss of class I, II and III HDACs, suggests that specific HDAC6 inhibitors may be better tolerated than pan-HDAC inhibitors or drugs that target the other HDAC classes. In this regard, the compound ACY-1215 (Rocilinostat), the described selective HDAC6 inhibitor, is undergoing clinical trials for the treatment of multiple myeloma. Taking into account the previous information about HDAC6 inhibitor structures, the structural differences between HDAC6 and other HDAC isoforms and also the structural information of other developed HDAC inhibitors, we have previously designed and synthesized a new potential HDAC6 selective inhibitor, QTX125 with growth inhibitory effects in mantle cell lymphoma (MCL) cell lines, mouse models and ex vivo treatment of primary samples obtained from patients with MCL. Herein, we have extended these findings to show that the newly identified HDAC6 inhibitor QTX125 is also able to inhibit the growth of preclinical models of other B-cell lymphomas such as follicular lymphoma and Burkitt’s cell lymphoma, but also of acute acute myeloid leukemia. In addition beyond a-tubulin, a well known HDAC6 target, we have developed a pharmacological and proteomic screening to identify other proteins modified by HDAC6 that can contribute to the described lymphoma and leukemia phenotypes. Citation Format: Lorea Villanueva, Montserrat Perez-Salvia, Eneko Aldaba, Yosu Vara, Myriam Fabre, Cristina Ferrer, Carme Masdeu, Aizpea Zubia, Eider San Sebastian, Dorleta Otaegui, Pere Llinàs-Arias, Margalida Rosselló-Tortella, María Berdasco, Fernando Setien, Catia Moutinho, Alberto Villanueva, Eva González-Barca, Josep Muncunill, José Tomás Navarro, Miguel Ángel Piris, Fernando Cossio, Manel Esteller. Efficacy of a new small-molecule inhibitor of histone deacetylase 6 (HDAC6) in preclinical models of B-cell lymphoma and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4725.

Abstract 4904: High levels of microRNA-210-3p are associated with increased risk of disease progression in breast cancer patients treated with docetaxel

Abstract MiR-210-3p is the most hypoxia-sensitive miRNA showing overexpression in different human cancers, but its role as biomarker in breast cancer (BC) is still controversial. In our study, we performed the expression analysis of miR-210-3p in a retrospective cohort of breast cancer patients with a median follow-up of 91 months enrolled according to the REMARK guidelines (n=284). As controls, 13 normal breast tissues (NBTs) from reductive mammoplasty were also profiled. By this analysis, we found that miR-210-3p levels were significantly increased in tumours as compared with NBTs (P=0.021). According to the surrogate molecular classification, the Triple Negative subgroup showed the highest levels of miR-210, followed by Luminal B tumours, whereas Luminal A, and HER2-amplified tumours showed the lowest expression levels (P=0.0137). The evaluation of the association of miR-210-3p with time-to-event outcomes in patients without synchronous metastases (n=268) demonstrated an association between risk of disease progression (HR 2.13, 95%CI 0.33-3.39, P=0.002) and higher levels of miR-210-3p in the subgroup treated with docetaxel in sequential therapy with anthracyclines (ECD). Moreover, a cut-off value of 20.966 as established by ROC curve analysis allowed to discriminate patients who developed distant metastases with an accuracy of 85% at 3-years (AUC 0.87, CI 0.69-1) and 83% at 5-years follow up (AUC 0.832, CI 0.656-1). Instead, the accuracy in discriminating patients who died for the disease was of 79.6% at both 5- (AUC 0.804, CI 0.517-1) and 10-years (AUC 0.804 CI 0.517-1) follow up. Overall, our data support the putative involvement of miR-210-3p in the response to treatments including docetaxel and suggest its eligibility to become a biomarker of early metastases development in ECD-treated breast cancer patients. Citation Format: BARBARA PASCULLI, RAFFAELA BARBANO, MICHELINA RENDINA, ANDREA FONTANA, MASSIMILIANO COPETTI, VANNA MARIA VALORI, MARIA MORRITTI, EVARISTO MAIELLO, VITO MICHELE FAZIO, MANEL ESTELLER, PAOLA PARRELLA. High levels of microRNA-210-3p are associated with increased risk of disease progression in breast cancer patients treated with docetaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4904.

Abstract 5394: Initial results from TRANSCAN ERA-NET BREMIR project: MicroRNAs expression profiling for identification of breast cancer patients at high risk to develop distant metastases

Abstract The mortality of breast cancer (BC) is primarily caused by metastatic spread to distant organs, rendering the ability to predict, detect and eliminate metastases one of the most important challenges in patients' management. This study is aimed to evaluate the potential role of several candidate miRNAs as predictive markers of the metastatic processes. The TRANSCAN ERA-NET BREMIR project has been designed to identify miRNA biomarkers able to predict distant metastases development. The correlation of miRNAs expression with distant metastasis free survival is evaluated in a large retrospective cohort (n=302). The best candidate markers will be validated in the independent prospective cohorts which currently include 505 patients. Additionally, miRNA potentialities as circulating biomarkers for treatment monitoring and early metastases detection will be evaluated in plasma samples. The analysis of the retrospective cohort has identified miR-10b-5p, miR-10a-5p, miR-210-3p, miR-30a-5p, miR200a-3p,miR200b-3p,miR200c-3p, miR141-3p, miR-429, miR-155-5p, miR-9-3p and miR-9-5p as crucially involved in BC progression. For miR-9-5p and miR-9-3p we also found a strong inverse correlation with ER and PgR expression, suggesting that they may be directly involved in hormone regulated pathways. To identify novel miRNAs associated with tumour metastases potentials, we also executed a global miRNA profiling on a selected number of breast cancer cases by using the Affymetrix GeneChip® miRNA 4.0. In particular, we analysed 34 BC cases characterized by different outcomes: 17 non metastatic after 10-years follow-up and 17 who developed metastatic disease within 5-years follow-up. As controls, 3 Normal Breast Tissues from reductive mammoplasty, 3 in situ carcinomas and 3 breast cancer cases with synchronous metastases were also evaluated. This analysis identified 8 miRNAs differentially expressed between metastatic and non metastatic cases. The association with study endpoints is currently under evaluation on the entire retrospective cohort. Our hope is that this study will lead to the identification of novel biomarkers allowing a better management of breast cancer patients. Citation Format: Paola Parrella, Raffaela Barbano, Ella Evron, Barbara Pasculli, Orazio Palumbo, Michelina Rendina, Raffaella Stallone, Michela Coco, Andrea Fontana, Tommaso Mazza, Massimiliano Copetti, Vanna Maria Valori, Maria Morritti, Paolo Graziano, Roberto Murgo, Evaristo Maiello, Massimo Carella, Vito Michele Fazio, Manel Esteller. Initial results from TRANSCAN ERA-NET BREMIR project: MicroRNAs expression profiling for identification of breast cancer patients at high risk to develop distant metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5394.

Abstract 612: Methylation changes in DNA of CD8 T cells following CD137 costimulation

Abstract Introduction: CD137 ligation imprints long term changes in the behavior of costimulated T-cells (1). There is not a satisfactory mechanistic explanation yet. Experimental procedures: to determine the specific DNA methylation changes occurring upon CD137 costimulation, purified human CD8+ T cells from three healthy donors were activated in vitro for 5 days with anti-CD3 monoclonal antibody and either with an anti-CD137 monoclonal antibody or its corresponding isotype (huIgG4). Activated lymphocytes were left 5 days in culture with huIL-7 without further stimulation. Such back-to-resting CD8+ lymphocytes were reestimulated with anti-CD3 for 12, 24 and 36h to validate the expression of the genes differentially methylated upon primary stimulation at mRNA and protein levels. DNA methylation profiles of both activated and resting cell subsets were characterized with Infinium 450K DNA methylation array (Illumina). To further confirm our observations, identical experimental procedure was performed with a different anti-CD137 agonist antibody (mIgG1 isotype) in a CD8+ T-cell donor. Differentially methylated genes between anti-CD3+anti-CD137 versus their corresponding control counterparts were validated by pyrosequencing on activated and resting CD8+ from independent group of healthy donors (n=8 for hIgG anti-CD137 and n= 11 for mIgG anti-CD137). Expression changes were confirmed by qRT-PCR and flow cytometry in activated, rested and reestimulated CD8+ lymphocytes. Results: 853 genes were differentially methylated in hIgG4 anti-CD137-treated CD8+ T cells as compared with their controls, 52 of which were shared with mIgG1 anti-CD137-costimulated CD8+ T lymphocytes. A number of differentially methylated genes are involved in i) T cell migration, ii) T cell activation, survival and homeostasis and iii) regulation of gene expression including key T-cell transcription factors. Conclusions: CD137 costimulation induces CD8+ T lymphocytes that are poised to respond more effectively to a second antigen exposure. These acquired functions are imprinted in the genomic DNA of the CD8+ T cells by DNA methylation changes upon CD137 co-stimulation, and involve key genes for CD8+ T cells.

Abstract 3483: Genome-wide analysis of the antisense transcriptome in cancer

Abstract Natural antisense transcripts (NATs) are the most abundant class of long noncoding RNAs (lncRNAs). Importantly, NATs are one of the most unknown ncRNAs. However, due to their highly gene locus-specific effects, NATs may provide a unique entry point for therapeutic intervention in targeted gene regulation. For this reason we are interested in studying the regulation of sense/antisense transcription in cancer related locus. In our previous work we found that, at the important cancer-related locus vimentin (VIM) there is a previously uncharacterized antisense transcript (VIM-AS1), which is transcribed head-to-head with VIM gene. This antisense transcript enhances R loop formation; it allows the binding of transcription factors as NF-kB, low nucleosome occupancy and the transcription of VIM gene. Our results are the first example of R loop mediated enhancement of gene expression involving head-to-head antisense transcription at cancer related locus. We also observed that in colon and breast cancer there was a positive expression correlation between VIM and VIM-AS1 and we have observed that hypermethylation silences both transcripts in colon primary tumors. In the present work we have seen that this positive correlation between sense and antisense transcripts is present in most of the tumor types from patients that we have obtained from Genomic Data Commons Data Portal (GDC Portal). Importantly this positive correlation correlates better in the cases in which the antisense transcript is downregulated or maintains the expression than in tumors where VIM and VIM-AS1 are up-regulated. Furthermore, we wanted to see if the cellular function is also coordinated. VIM is a component of the intermediate filament family of proteins that has an important role in epithelial mesenchymal transition. We found that specific depletion of VIM-AS1 results in a decrease of VIM expression, and now we have found that this depletion produces low migration rate and more invasion capability, as expected. On the other hand, the mechanisms used by antisense transcripts to regulate their corresponding sense mRNAs are not fully understood and in view of these results we are investigating from a genome-wide perspective the regulation of sense/antisense transcription, the involvement of R loop structures and DNA methylation in cancer. For this, we have applied a number of techniques: RNAseq strand specific, Illumina’s HumanMethylation450 Bead Chip array, RNAseq strand specific with overexpression of RNaseH (which specifically degrades the RNA in RNA/DNA hybrids), and DRIPseq (which immunoprecipitates the hybrids DNA-RNAs). The analysis of these data sheds light on the study of the role of sense/antisense transcripts in cancer-related locus. Citation Format: Raquel Boque-Sastre, Manuel Castro de Moura, Antonio Gomez, Sònia Guil, Manel Esteller. Genome-wide analysis of the antisense transcriptome in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3483. doi:10.1158/1538-7445.AM2017-3483

Abstract 2936: Molecular characterization of the immune subclass of hepatocellualr carcinoma

Abstract Background: Immune checkpoint inhibitors have emerged as a promising therapeutic approach in different solid tumors, including hepatocellular carcinoma (HCC). Nonetheless, little is known about the immune-component of HCC or potential biomarkers of response to these therapies. Aims: To perform comprehensive characterization of the HCC immunological profile and to identify biomarkers to select immunotherapy candidates. Methods: We performed gene expression array deconvolution through non-negative matrix factorization in 228 resected HCCs. Characterization of the transcriptional landscape was conducted using >1,000 signatures representing distinct immune cells by gene set enrichment and nearest template prediction analyses. Presence of immune infiltration, tertiary lymphoid structure (TLS), PD-1 and PD-L1 immunostainings was investigated using immunohistochemistry. DNA methylation profile of 450K CpG sites was analyzed to identify those with significant differences for each group. Extensive validation of the immune classifier was performed in 728 independent HCC samples. Results: Overall, an immune-related subclass of HCC was identified in ~27% of patients. The immune subclass was characterized by gene signatures identifying immune cells (i.e. T cells, TLS, cytotox, p<0.001), signatures of response to immune checkpoint therapy (p<0.001), presence of high immune infiltration (p=0.01), TLS (≥5 foci, 19/51 vs 33/168, p=0.01) and PD-1 and PD-L1 protein expression (p<0.05). The methylation levels of 363 CpG sites in 192 immune response gene promoters were able to capture the Immune class (ANOVA, p<0.05, Δβ>0.2 Tukey test). Integration with HCC molecular classifications revealed significant enrichment of the Immune subclass with IFN and S1 (p<0.001) and exclusion of the CTNNB1 and S2 (p<0.001) subclasses. The immune class contains two distinct microenvironment-based types: A) Exhausted immune response type (~35%) characterized by stromal activation, T cell exhaustion signatures, and presence of immunosuppressive components such as TGFB, LGALS1, M2 macrophages and pathways able to recruit myeloid-derived-suppressor cells (FDR<0.01); and B) Active immune response type (~65%) characterized by overexpression of adaptive immune response genes and IFN signaling (p<0.001). Tumors within the active immune response type showed a trend towards better survival vs rest (p=0.07). Conclusions: Around 27% of HCC patients belong to the Immune class, characterized by activation of immune cells and signatures of response to immunotherapies. Within this subclass, two distinct types have been characterized by presenting active or exhausted immune responses, a feature that provides the rationale for precision medicine-based therapies. Note: This abstract was not presented at the meeting. Citation Format: Daniela Sia, Yang Jiao, Iris Martinez, Olga Kuchuk, Carlos Villacorta Martin, Manuel Castro de Moura, Juan Putra, Genis Camprecios, Swan Thung, Samuel Waxman, Vincenzo Mazzaferro, Manel Esteller, Augusto Villanueva, Josep Maria Llovet. Molecular characterization of the immune subclass of hepatocellualr carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2936. doi:10.1158/1538-7445.AM2017-2936

Abstract 4734: miR-9-5p expression in breast cancer correlates with hormone receptor status and affects patients survival

Abstract miR-9 (hsa-miR-9-5p, miR-9-5p) is a highly conserved microRNA (miRNA) primarily expressed in the central nervous system. The three promoters of miR-9 (miR-9-1 at 1q22, miR-9-2 at 5q14.3 and miR-9-3 at 15q26.1) are embedded within CpG islands, and hypermethylation in at least one of these regions has been reported in several tumor types, thus miR-9-5p has been designated as an epigenetically regulated miRNA. However, only a partial or none correlation between methylation status of miR-9 loci and miR-9-5p expression has been demonstrated so far. Furthermore, the expression levels of miR-9-5p resulted highly variable across different carcinomas and conflicting evidences also exist about its functional role within the tumor context. In light of this, we took the effort of clarifying the role of miR-9-5p in primary breast cancer tissues and of evaluating its potential as clinical relevant prognostic biomarker. We analysed miR-9-5p expression and miR-9 promoters methylation status in 129 breast cancer cases and 12 normal breast tissues (NBTs), by qRT-PCR and MSP respectively. Overall, miR-9-5p was increased in tumors compared to NBTs (P<0.001). No significant correlation between promoter methylation and miR-9-5p expression was found either in tumors or in NBTs. Interestingly, miR-9-5p expression was inversely correlated with ER and PgR positivity (P=0.004 and P=0.003, respectively). Consistently, triple negative breast cancer (TNBC) showed the highest miR-9-5p levels and luminal subtype the lowest (P=0.04). The analysis of the TCGA Breast Cancer dataset (n=256) confirmed our results and further demonstrated that miR-9-5p was differentially expressed between the two luminal subtypes (P=0.009) and HER2-amplified compared to TNBCs (P<0.0001). In Univariable Cox regression analysis miR-9-5p expression was significantly associated with higher risk of death (P=0.023). Ingenuity Pathway Analysis exploring the putative interactions among miR-9-5p, ER and PgR upstream and downstream regulators suggested a regulatory loop by which miR-9-5p is induced by steroid hormone receptor and acts within hormone-receptor regulated pathways. Our data suggest miR-9-5p may help refine the molecular classification of breast cancer subtypes and provide additional prognostic information. Citation Format: Raffaela Barbano, Barbara Pasculli, Michelina Rendina, Andrea Fontana, Caterina Fusilli, Massimiliano Copetti, Stefamo Castellana, Vanna Maria Valori, Maria Morritti, Paolo Graziano, Luigi Ciuffreda, Michelina Coco, Francesco Picardo, Tommaso Mazza, Ella Evron, Roberto Murgo, Evaristo Maiello, Manel Esteller, Vito Michele Fazio, Paola Parrella. miR-9-5p expression in breast cancer correlates with hormone receptor status and affects patients survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4734. doi:10.1158/1538-7445.AM2017-4734

Abstract 484: Epigenetic regulation of hsa-miR-3663 in colon cancer

Abstract Colon Cancer is the third most common cancer in the world and a major cause of morbidity and mortality. Molecular mechanisms of colon carcinoma have been deeply studied and current evidences indicate that microRNAs play a pivotal role in its tumorigenesis and progression. MicroRNAs (miRNAs) are small 19 to 22 nucleotides of RNA classified as non-coding RNAs that negatively regulate gene expression at the post-translational level controlling numerous biological processes including development, cell proliferation, apoptosis, differentiation and cell migration. Aberrant expression of miRNAs due to epigenetic alterations has been associated with carcinogenesis. Aimed to identify epigenetically regulated miRNAs involved in colon cancer, we analyzed DNA methylation profiles available in The Cancer Genome Atlas. One of the main hits identified was hsa-miR-3663, hypermethylated in 26% of colon cancer patients (n=286, p< 0.001) in comparison with complete lack of methylation in normal tissues (n=38), suggesting its potential function as a putative tumor suppressor. In order to study the role of DNA methylation controlling hsa-miR-3663, HumanMethylation450K methylation profiles were generated for a panel of colon cancer cell lines (n=10), including the HCT116-DNA methyltransferase knock-out model (DKO, double knockout of DNMT1 and DNMT3B). We found a significant correlation between methylation status of hsa-miR-3663 and expression in colon cancer cell lines. Furthermore, epigenetic regulation of hsa-miR-3663 was confirmed in HCT116 methylated cell line by restored expression upon treatment with DNA demethylating agent 5-aza-2′-deoxycytidine and in the HCT116-DKO model. In order to elucidate the function of hsa-miR-3663, we stably expressed hsa-miR-3663 in a panel of five methylated colorectal cancer cell lines (HCT116, RKO, DLD1, SW48 and SW480). Our initial functional assays revealed that hsa-miR-3663 is not involved in cell proliferation or migration. We are now performing additional assays to further study hsa-miR-3663 and its potential role in colon cancer. Note: This abstract was not presented at the meeting. Citation Format: Laia Pique, Humberto Jorge Ferrerira, Manel Esteller. Epigenetic regulation of hsa-miR-3663 in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 484. doi:10.1158/1538-7445.AM2017-484

Abstract 2130A: Epigenetic silencing of TGFβ1, BCL6, KILLIN and CTSZ is related to trastuzumab resistance in HER2-positive breast cancer models

Abstract Introduction: The major clinical problem for HER2 breast cancer target therapies is the acquisition of resistance. Trastuzumab has been used for the treatment of HER2-positive early stage and metastatic breast cancers. Notoriously, up to 62% patients that respond to the initial treatment develop resistance within a year. Different cellular and molecular alterations may contribute to the development of Trastuzumab resistance, including epigenetic changes. The aim of our study has been the evaluation of epigenetic differences between Trastuzumab-sensitive and -resistant breast cancer cell lines to determine whether they might be used as potential biomarkers to help in the treatment assessment and monitoring. Experimental procedures: To understand the epigenetic changes that may be associated with Trastuzumab resistance we compared the DNA methylation status of ∼450000 CpG sites from a DNA methylation microarray performed in Trastuzumab-sensitive and resistant cells. To identify the differentially methylated CpGs we considered differences between methylation groups of ≥60%. The functions associated to genes were analysed using the Gene Ontology and were used to select a small number of candidate genes. Their differences in promoter methylation status were validated by expression techniques (qPCR) and subsequently confirmed by demethylation agent (5-aza-DC) treatment followed by qPCR. The Trastuzumab resistant HER2-positive cell line (SKTR) had been previously developed in our laboratory by exposing SKBr3 cells to Trastuzumab, starting with 1μM for three months of exposure and increasing up to 2 μM for a 12 months period. Summary of the new, unpublished data: The DNA methylation microarray data showed that different 5’-CpG island sites had altered methylation profile when comparing Trastuzumab-resistant (SKTR) and -sensible SKBr3 cells. We selected 4 candidate genes with a hypermethylation profile in -resistant vs. -sensible cells: TGFβ1 (Transforming Growth Factor Beta 1), BCL6 (B-Cell CLL/Lymphoma 6), KILLIN (P53-Regulated DNA Replication Inhibitor) and CTSZ (Cathepsin Z). The hypermethylation of these four candidate genes in SKTR was in agreement with their expression downregulation observed by PCR analysis. SKTR treatment with the demethylation agent 5-aza-2’-deoxycytidine restored the expression levels of all four genes to the levels of Trastuzumab-sensitive cells. Although previously related to breast cancer, TGFβ1, BCL6, KILLIN and CTSZ have not earlier been associated with the development of Trastuzumab resistance in breast cancer or in other cancer types and may be further studied as potential biomarkers. Conclusions: These results provide a basis for future studies to validate the hypermethylation status of these genes as a predictive or monitoring biomarker of Trastuzumab resistance in HER2+ breast cancer patients. Citation Format: Sonia Palomeras, Angel Diaz-Lagares, Sarrats Ariadna, Crujeiras Ana Belen, Giro-Perafita Ariadna, Juan Sandoval, Marc Rabionet, Manel Esteller, Teresa Puig. Epigenetic silencing of TGFβ1, BCL6, KILLIN and CTSZ is related to trastuzumab resistance in HER2-positive breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2130A.

Abstract 2770: DERL3 hypermethylation alters rhabdomyosarcoma cell metabolism

Abstract Physiological cellular activity requires a precise control of the proteome. However, cancer cells have a distorted protein profile. It is likely that intrinsic defects in protein homeostasis participate in human tumorigenesis, such as alterations in de novo protein synthesis, or defects in protein degradation. In this last case, the ubiquitin (Ub) proteasome system (UPS) targets a variety of proteins, including functional proteins that are no longer needed. Without appropriate protein homeostasis maintained by the UPS, healthy cells can undergo malignant transformation, and this observation has been therapeutically exploited by the development of proteasome inhibitors as anticancer agents. Many of the cytoplasm UPS-degraded proteins are retrotranslocated from the endoplasmic reticulum (ER). In this regard, ER possesses a quality control mechanism, termed the ER-associated degradation mechanism (ERAD), that is induced in response to ER stress by a transcriptional program, known as the unfolded protein response (UPR), which leads to the accelerated degradation of unfolded proteins. Within the ERAD pathway, the Derlin proteins play a critical role. It has been proposed that Derlins form an export channel in the membrane of the ER through which the ERAD substrates pass to reach the proteasome. A previous study in our lab has shown that DERL3 silencing by DNA methylation deregulate SLC2A1 (GLUT1) degradation, promoting the Warburg effect. SLC2A1 is a key glucose receptor up-regulated in most solid tumors, allowing cancer cells to get the glucose they need for tumor progression. In this study we use an embryonic rhabdomyosarcoma cell line as a model to find out the role of DERL3 in these tumors, as the percentage of promoter hypermethylation is these tumors is approximately 60%. Rhabdomyosarcoma (RMS) is one of the leading causes of cancer related deaths among children, being the histological variant of embryonic rhabdomyosarcoma the most common soft tissue sarcoma of childhood and adolescence, with 350 cases per year in USA. The restoration of DERL3 expression in vitro promotes GLUT1 degradation and GLUT4 up-regulation, becoming cells sensitive to insulin. Moreover, it has been shown that DERL3 confers sensitivity against an ERAD related drug, Eeyarestatin I, opening a new approach for RMS treatment. All together, these initial findings highlight the role of DERL3 in RMS metabolism. Citation Format: Pere Llinas, Paula Lopez, Manel Esteller. DERL3 hypermethylation alters rhabdomyosarcoma cell metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2770.

Abstract 2763: DNA methylation profiling unveils TGF-β hyperresponse in tumor associated fibroblasts from lung cancer patients

Abstract There is growing interest in defining the aberrant molecular differences between normal and tumor-associated fibroblasts (TAFs) that support tumor progression. For this purpose, we recently conducted a genome-wide DNA methylation profiling of TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer (NSCLC) patients, and reported a widespread hypomethylation concomitantly with focal gain of DNA methylation; in addition, we found evidence that a fraction of lung TAFs are fibrocytes in origin. Of note, the aberrant epigenome of lung TAFs had a global impact in gene expression and a selective impact on the TGF-β pathway. To get insights on the functional implications of the latter impact, we analyzed the response of lung TAFs to exogenous TGF-β1 in terms of activation and contractility. We found a larger expression of a panel of activation markers including α-SMA and collagen-I in TAFs compared to control fibroblasts. Likewise, TGF-β1 elicited a larger contractility in TAFs than in CFs as assessed by traction force microscopy. These findings reveal that lung TAFs are hyperresponsive to TGF-β1, which may underlie the expansion and/or maintenance of the tumor-promoting desmoplastic stroma in lung cancer. Citation Format: Miguel Vizoso, Marta Puig, F. Javier Carmona, María Maqueda, Antonio Gomez, Anna Labernardie, Marta Gabasa, Saioa Mendizuri, Rafael Ikemori, Xavier Trepat, Sebastian Moran, Enrique Vidal, Noemí Reguart, Alexandre Perera, Manel Esteller, Jordi Alcaraz. DNA methylation profiling unveils TGF-β hyperresponse in tumor associated fibroblasts from lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2763.