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PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple-negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single-guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR, and MAPK signaling. BRAF V600E mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf inhibitors, PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments. Keywords: human PTEN protein, CRISPR/Cas systems, tumor suppressor genes, melanoma, breast neoplasms

Background: Gastric cancer (GC) is the leading cause of cancer death in Chile, claiming >3,000 lives per year. On April 2021, the US Food and Drug Administration approved the combined use of Nivolumab (immunotherapy) plus chemotherapy as frontline treatment for advanced GC. Unfortunately, as occurs with any therapy, a proportion of treated patients are either unresponsive or display poor responses. Our study sought to obtain a profile of GC patients categorized as responders or non-responders to immunotherapy in order to identify better predictors of response and novel targets for treatment refractory patients. Methods: A total of 50 GC patients were included into our study. Demographic and basic information was obtained from medical records. Additionally, we retrieved information on response to treatment, histological type, PDL1 and HER2 status. Nucleic acid samples were collected from a subset of 45 patients (responders and non-responders) and analyzed by massive analysis of cDNA ends (MACE-seq) assessing 3’ mRNA sequencing. Results: Patients were predominantly male (76%) and advanced stage (III/IV; 98%). Within this subset, 26 were responders and 24 were non-responders. Among clinical variables, body mass index was significantly higher on responders versus non-responders (p=0.026). Among responders, tumors were predominantly located on the gastro-esophagic junction. As expected, responders also displayed a higher prevalence of HER2- and PDL1+. Our 3’ MACE analysis confirmed distinct profiles on responders compared to non-responders. Conclusions: Our findings confirm distinctive profiles among Chilean GC patients categorized as responders or non-responders to immunotherapy. These results could deliver novel markers that may serve as predictors of response to immunotherapy (as an alternative to PDL1) or to identify patients more likely to be refractory to treatment. Future studies should further confirm and validate these findings in a larger sample. Citation Format: Mauricio P. Pinto, Matias Muñoz Medel, Miguel Córdova Delgado, Ignacio N. Retamal, Patricio Manque, Alejandro Berkovits, Maria Paz Rodriguez, Juvenal A. Rios, Benjamin Garcia-Bloj, Gareth Owen, Marcelo Garrido. Distinctive profiles on immunotherapy responders versus non responders in Chilean gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1969.

// Benjamin Garcia-Bloj 1, 2, 4 , Colette Moses 1, 2 , Agustin Sgro 1 , Janice Plani-Lam 1 , Mahira Arooj 1, 5 , Ciara Duffy 1, 2 , Shreyas Thiruvengadam 1 , Anabel Sorolla 1 , Rabab Rashwan 1 , Ricardo L. Mancera 5 , Andrea Leisewitz 4 , Theresa Swift-Scanlan 6 , Alejandro H. Corvalan 3, 4 , Pilar Blancafort 1, 2 1 Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia 2 School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, WA, 6009, Australia 3 Advanced Center for Chronic Diseases (ACCDiS), Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile 4 Faculty of Medicine, School of Medicine, Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile 5 School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth WA 6845, Australia 6 School of Nursing, Virginia Commonwealth University, Richmond, Virginia, 23298, USA Correspondence to: Pilar Blancafort, email: pilar.blancafort@uwa.edu.au Keywords: CRISPR/dCas9, ZF, TALE, tumor suppressor genes, gene reactivation Received: June 01, 2016 Accepted: July 19, 2016 Published: August 09, 2016 ABSTRACT The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO , in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.

Tools for editing the genome and epigenome have revolutionised the field of molecular biology and represent a new frontier in targeted therapeutic intervention., Tools for editing the genome and epigenome have revolutionised the field of molecular biology and represent a new frontier in targeted therapeutic intervention. Although efficiencies and specificities of genome editing technologies have improved with the development of TALEs and CRISPR platforms, intracellular delivery of these larger constructs still remains a challenge using existing delivery agents. Viral vectors, including lentiviruses and adeno-associated viruses, as well as some non-viral strategies, such as cationic polymers and liposomes, are limited by packaging capacity, poor delivery, toxicity, and immunogenicity. We report a highly controlled synthetic strategy to engineer a flexible dendritic polymer using click chemistry to overcome the aforementioned delivery challenges associated with genome engineering technologies. Using a systematic approach, we demonstrate that high transfection efficiencies and packaging capacity can be achieved using this non-viral delivery methodology to deliver zinc fingers, TALEs and CRISPR/dCas9 platforms.

High expression of PTPN21 in B-cell non-Hodgkin’s gastric lymphoma, a positive mediator of STAT5 activity

BACKGROUND AND AIM : Several studies have indicated that continuous exposure to high concentrations of Nitric Oxide (NO), produced by inducibile-NO synthase (iNOS), promote neoplastic transformation within many human cancers, including colon cancer (CC). Recently, it has also been suggested that high NO synthesis is a distinctive feature of "cancer stem cells" (CSC), a tumor subpopulation with self-renewal capacity, that may be identified by the expression of the CD133 surface marker. Aims of this study were to explore the contribution of NO in the definition of colon CSC features and evaluate potential strategies to treat CC by modulating NO production. MATERIAL AND METHODS : By immunohistochemistry analysis we evaluated iNOS and CD133 expression in 20 samples of human CC. Using the DAF-2DA detection system, we assayed the production of intracellular NO in 5 colon CSC lines obtained from human CC tissues. By FACS sorter, we purified the NO high andNO low fractions from all colon CSC lines and we compared the tumorigenic potential of both cell fractions by in vitro and in vivo assays. To tested the potential antitumor effects of iNOS modulation, we treated colon CSCs with the selective iNOS inhibitor 1400W or we stably transfected these cells with two distinct iNOS-directed short-harpin RNA(shRNAs). RESULTS : Immunohistochemistry analysis revealed that, irrespective of grade, higher iNOS expression was associated with increased CD133 levels in all CC samples. Moreover, we observed that NOhigh cells display higher expression levels of stem cell markers and increased tumorigenic properties in vitro and in vivo than NOlow fraction. Both iNOSdirected shRNAs and iNOS inhibitor treatment decreased the rate of cell-cycle transit in colon CSCs and reduced tumorigenic capacity in vitro and in vivo, confirming an integral role for NO and endogenous iNOS activity in the biology of colon CSCs. Interestingly, the knockdown of iNOS expression was associated with a significant down regulation of stem cell markers, such as CD133, BMI and b-Catenin. CONCLUSIONS : This study identifies for the first time a cell-autonomous colon CSC specific source of NO, and it highlights the biological effects of the iNOS within colon CSCs. In the long run, these data could serve as an impetus for evaluation of iNOS-directed therapies as a component of multimodal treatment regimens for human CC.