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FRONTIERS IN CANCER SCIENCE 2020

2-6 NovemberAbstract Booklet

Genome Instituteof Singapore

CHIEF JUDGE:

Ramanuj DasGupta Genome Institute of Singapore,

A*STAR Singapore

POSTER JUDGES:

Jason ChanNational Cancer Centre Singapore

Qingfeng ChenInstitute of Molecular and Cell Biology,

A*STAR Singapore

Jit Kong CheongYong Loo Lin School of Medicine,

National University of Singapore, Singapore

Guo-Liang ChewCancer Science Institute of Singapore

Vaidehi KrishnanDuke-NUS Medical School, Singapore

FCS 20 2002

P001YAP and Tissue Rigidity in the Premalignant Stage of Mouse Lung Squamous Cell Carcinoma (SCC) Model

Siti Fathiah Masre1*, Kiew May Chee1, Muhammad Asyaari Zakaria1, Nor Fadilah Rajab2, Chua Eng Wee3

1Centre for Toxicology and Health Risk Studies, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Kuala Lumpur, 2Centre for Healthy Ageing and Wellness, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Kuala Lumpur, 3Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Kuala Lumpur

Lung squamous cell carcinoma (LSCC), one of the subtypes of non-small cell lung cancer, has caused a large number of deaths worldwide. Yes-Associated-Protein (YAP) is a downstream effector of the hippo signalling pathway that has been found to play an essential role in cancer development. Activation of YAP is suggested to be connected with tissue rigidity via positive feedback loop leading to cancer growth. Also, the role of YAP in the early stage of lung SCC remains to be elucidated. Thus, this study aimed to investigate YAP expression and tissue rigidity in the premalignant LSCC stage. Balb/C mice (n=16) were randomly divided into vehicle control group and N-nitroso-tris-chloroethylurea (NTCU)-induced premalignant group. At week 15, all mice were sacrificed and the lung was biopsied for analysis of histopathology, YAP gene expression, tissue stiffness,

and pYAP protein expression. Histopathological analysis revealed hyperplasia formation with a substantial increase (p<0.05) in the epidermal thickness of the bronchial epithelial cells in the premalignant group as compared to the control group. The expression of YAP gene showed that the premalignant group exhibited higher fold changes than the vehicle group. Picrosirius red staining showed a significant increase (p<0.05) in the collagen expression in the premalignant group compared to the control group which indicate an increased in tissue rigidity. In the case of immunohistochemistry (IHC) analysis, there was a significant increase (p<0.05) in cytoplasmic and nuclear pYAP expression in the premalignant group compared to the control group. In conclusion, this study indicates that YAP could play an important role in inducing tissue rigidity in the early development of pulmonary SCC.

P002DDX3X Loss is Associated with Aggressive Phenotypes in non-Hodgkin’s Lymphomas

Atish Kizhakeyil1, Nurmahirah binte Mohammed Zaini2, Zhi Sheng Poh3, Xinpeng Loh3, Aik Seng Ng1, Zun Siong Low1, Praseetha Prasannan1, Brandon Wong1, Chun Gong4, Michelle GK Tan5, Chandramouli Nagarajan2, Dachuan Huang6, Lim Jing Quan6, Choon-Kiat Ong6,7, Sin Tiong Ong7, Soon-Thye Lim6, Wee-Joo Chng8,9, George Follows10, Daniel Hodson4, Ming-Qing Du11, Yeow Tee Goh2, Suat Hoon Tan12, Nicholas Francis Grigoropoulos2,7,*, Navin Kumar Verma1,13,*

1Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore 2Department of Haematology, Singapore General Hospital, Singapore 3School of Biological Sciences, Nanyang Technological University Singapore, Singapore 4Wellcome MRC Cambridge Stem Cell Institute, Cambridge, UK 5Clinical Translational Sciences, Singapore General Hospital, Singapore 6Lymphoma Genomic Translational Research Laboratory, Division of Medical Oncology, National Cancer Centre Singapore, Singapore 7Duke-NUS Medical School, Singapore 8National University Cancer Institute, Singapore 9Cancer Science Institute of Singapore, National University of Singapore, Singapore 10Addenbrooke’s Hospital NHS Foundation Trust, Cambridge, UK 11Department of Pathology, University of Cambridge, Cambridge, UK 12National Skin Centre Singapore, Singapore 13Skin Research Institute of Singapore, Singapore

Non-Hodgkin’s lymphomas (NHL) is a diverse group of cancer, encompassing the most common diffuse large B-cell lymphoma (DLBCL) to the rarer T cell lymphoma. DDX3X is an RNA helicase and, depending on tumour types, plays tumour suppressive or oncogenic role in cancer. However, whether and how DDX3X is involved in NHLs are not clear. We performed targeted sequencing of DDX3X hotspots on exons 8-15 was performed in duplicate on a validation cohort of 158 unselected cases of DLBCL (Leeds cohort) and DDX3X mutations were found in 5 cases. Using whole exome sequencing, we identified DDX3X mutations in 4 out of 9 (44%) patients with relapsed/refractory DLBCL (R/R-DLBCL) treated with R-CHOP. Kaplan-Meier analysis of the 223 DLBCL cases recorded on cbioportal revealed that patients with DDX3X mutations have worse 5-year overall survival

(22%) compared to patients with wild-type DDX3X (72%, p=0.0217). Using DLBCL patient-derived cell lines, we observed that expression of mutant variant R475C or knockdown of DDX3X in these cells caused significant up-regulation of cytokine signalling pathways (IL-2 and IL-10), and increased phosphorylation of STAT3 and p42/44. Inactivation or DDX3X loss increased resistance to doxorubicin and histone deacetylase targeting drugs, including romidepsin and vorinostat, in DLBCL and CTCL cells, respectively. Importantly, both B- and T-cell lineage DDX3X-depleted cells remained sensitive to pharmacological STAT3 inhibition. These findings provide new insight into the molecular mechanisms of chemoresistance in NHLs and identify DDX3X mutations as a biomarker for both resistance to standard chemotherapy as well as sensitivity to STAT3 inhibition.

FCS 20 2003

P003Rhoj is a Novel Target for Progression and Invasion of Glioblastoma by Impairing Cytoskeleton Dynamics

Mei Wang1-3, #, Xiaochun Jiang 2, #, Hongjin Chen3, Chengfei Zhang3, Haojun Xu3, Chengyun Yao4 *, Hongping Xia1-4* 1 Department of Microbiology and Immunology, Medical School of Southeast University, China. 2 Department of Neurosurgery, Yijishan Hospital of Wannan Medical College, China 3 Department of Pathology, School of Basic Medical Sciences & Sir Run Run Hospital & State Key Laboratory of Reproductive Medicine & Key Laboratory of Antibody Technique of National Health Commission, Nanjing Medical University, China 4 Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, China

Rho GTPases family members were identified as a critical regulator of cell morphology, actin cytoskeleton organization, cell movement, cell cycle, and also contributed to tumor progression, which have been implicated in many cancers’ metastasis and progression. Here, we firstly reported the dysregulation of Rhoj in glioblastoma multiforme (GBM) and aimed to investigate the role and mechanism of Rhoj in GBM. We analyzed the expression of 21 Rho GTPases family members and validated the expression of Rhoj in GBM by immunohistochemistry. We further investigated the role and mechanism of Rhoj in GBM both in vitro and in vivo. We observed that Rhoj is significantly overexpressed in GBM and associated with patients’ survival. However,

the role and underlying molecular mechanism of Rhoj in GBM are still unclear. We demonstrated that transcription factor c-Jun regulated the expression of Rhoj, and Rhoj interacted with moesin to promote GBM cell proliferation and migration by potentiating the activation of Rac1/PAK pathway and cytoskeletal dynamics. Rhoj may promote migration and invasion of GBM cells by regulating epithelial-mesenchymal transition (EMT)-like process. In conclusion, the Rhoj/ Rac1/PAK signaling mediates invasion and progression of GBM and is a potential therapeutic target for GBM treatment. Rhoj may also be a promising biomarker for GBM diagnosis and prognosis.

Dachuan Huang1,2, Jing Quan Lim1,2, Daryl Ming Zhe Cheah1, Yurike Laurensia1, Jane Wan Lu Pang1, Esther Kam Yin Wong1, Burton Kuan Hui Chia1, Jasmine Goh3, Jason Yongsheng Chan5, Edward Kai-Hua Chow3,, Soo Yong Tan4, Soon Thye Lim2,5,6,, Choon Kiat Ong1,2,7

1Lymphoma Genomic Translational Research Laboratory, Division of Medical Oncology, National Cancer Centre, 11 Hospital Drive, Singapore169610 2DUKE-NUS Medical School, 8 College Road, Singapore 169857 3Cancer Science Institute of Singapore, National University of Singapore, Singapore 119074 4Department of Pathology, National University Health System, Singapore 119074 5Division of Medical Oncology, National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610 6Singhealth-DukeNUS Blood Cancer Centre, 11 Hospital Drive, Singapore 169610 7 Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672

P004Whole Genome Sequencing Reveals Potent Therapeutic Strategy for Monomorphic Epitheliotropic Intestinal T-Cell Lymphoma

Introduction Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is a rare aggressive malignancy accounting for majority of intestinal T-cell lymphoma in Asia. There is an urgent need to establish a well-characterized and clinical relevant MEITL cellular and in vivo model to decipher the biological meaning of genetic alterations, and to explore the potential therapeutic targets.

Methods Whole-genome sequencing (WGS) was performed on four MEITL tumors and their corresponding blood samples. Simultaneously, a subcutaneous and orthotropic patient derived xenograft (PDX) model was established, and characterized histologically and genetically. The cytotoxic/cytostatic effect of drugs targeting frequent METIL mutations was systematically

evaluated using Quadratic Phenotypic Optimization Platform (QPOP) and verified in vivo.

Results WGS data yielded an average of 1.52 somatic short-variants per Mb. Recurrent non-silent mutations were found in CREBBP, STAT5B, SETD2, GNAI2 and JAK3 genes. Sanger sequencing, WES and immunohistochemical analysis demonstrated that the PDX tumors are genetically and immunomorphologically stable compared to the primary tumours. The in vitro and in vivo experiments demonstrated a synergistic effect between Rompidpsin and Pimozide.

Conclusions These results show that the key driver mutations in MEITL are druggable and combined targeted therapy might achieve great efficacy for MEITL.

FCS 20 2004

P005Collagen Differences Analysis in a Mouse Model of NTCU-Induced Lung SCC

Muhammad Asyaari Zakaria1, Siti Fathiah Masre1, Nor Fadilah Rajab2, Eng Wee Chua3, Gayathri Thevi Selvarajah4.1Biomedical Science Programme, Center for Toxicology and Health Risk Studies, Faculty of Health Sciences, Universiti Kebangsaan Malaysia (UKM), Malaysia, 2Center for Healthy Ageing and Wellness, Faculty of Health Sciences, Universiti Kebangsaan Malaysia (UKM), Malaysia, 3Faculty of Pharmacy, Universiti Kebangsaan Malaysia (UKM), Malaysia, 4Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia (UPM), Malaysia.

Introduction: Collagen, the main structural protein in the extracellular matrix (ECM) is dysregulated and overproduced in cancer. Collagen able to activate integrin and other receptors essential in promoting cancer cell proliferation, migration, and invasion. It was believed that smoking-associated lung squamous cell carcinoma (SCC) is characterized by collagen alterations that jeopardize lung function. However, the evidence is still elusive. Therefore, we aimed to investigate the content and expression of collagen in lung SCC using a mouse model. Method: Lung SCC was induced by topical application of a nicotine-derived compound; N-nitroso-tris-chloroethyl urea (NTCU). Female BALB/c mice were divided into two groups (n = 6): (1) cancer group (25 µL of 0.04 M NTCU) and (2) control group (25 µL of 70 % acetone). Treatment was given twice a week for 30 weeks on the dorsal of the mouse’s shaved skin. After lung extraction, hematoxylin and eosin (H&E), picro sirius red (PSR) staining and

RNA sequencing were performed. P-value of ≤ 0.05 was assigned as statistical significance.Results: We have successfully developed SCC subtype of lung cancer in Balb/C mice and have found significantly higher levels of collagen content in the cancer group. Significant upregulation of collagen (col) type IV, V, VI, VII, XVII, and XXIV genes were detected and involved in the ECM-receptor interaction pathway through DESeq2 and KEGG analysis. Reactome analysis revealed that these collagens play important role in the ECM organization, collagen formation, assembly of collagen fibrils, and multimeric structure. Interestingly, these genes also involved in collagen degradation which may aid in cancer cell invasion during metastasis. Conclusion: This study suggests that collagen is dysregulated and overproduced in NTCU induced lung SCC. Thus, restoring normal collagen content in the lung may be promising as an alternative cancer therapy in the future.

cell surface expression of memory T cell markers CD27, CCR7 and CD62L. Genes associated with TCR signaling and interferon response were found to be expressed across all cell clusters, yet with elevated levels in D-CT and LA-CT. Furthermore, cell surface expression of different NK receptors including NKG2D, DNAM1 and NKp30 are more enriched in LA-CT compared to the other 2 subsets, suggesting the acquisition of additional NK receptor related functions in this group of cells. In line with this, blocking of TCRγδ and various NK receptors individually with antibodies failed to significantly suppress the cytotoxic activities of these cells. Taken together, our data suggests that as CB-gdT are likely to adopt dynamic memory and signal -specific functional programs in culture. These unique characteristics of our CB-gdT can overcome the challenges of tumor heterogeneity and cell persistence, with the potential of improving outcomes in cell immunotherapy.

P006Single Cell Transcriptome Analysis Revealed Complex Activation Profile Governing the Cytotoxic Activity of In-Vitro Expanded Cord Blood Derived Gamma Delta T Cells

Kar Wai Tan1, Zhihui Li2, Joey Lai3, Dianyan Guo1, Yeh Ching Linn3, William YK Hwang3,4, Shang Li5, Yeow Tee Goh3, and Alice Cheung3

1Department of Clinical Translational Research, Singapore General Hospital, 2Genome Institute of Singapore, 3Department of Haematology, Singapore General Hospital, 4National Cancer Centre of Singapore, 5Cancer and Stem Cell Biology, Duke-NUS Medical School of Singapore

We had previously explored the potential of expanding cord blood (CB) derived γδ T cells (CB-gdT) as well as their corresponding ability to target primary acute myeloid leukemia (AML) cells. Using a feeder cell line-based in vitro expansion protocol, we achieved a clinically relevant scale expansion of γδ T cells exhibiting specific and potent cytotoxicity against a range of human AML cell lines and primary patient samples. Single cell transcriptome analysis showed that key cytotoxic genes including GZMB, GZMA and NKG7 were all highly expressed across all clusters, indicating that the expanded cells were indeed functionally cytotoxic. Comparing against multiple curated gene sets, we have identified 3 main subsets of γδ T cells: Proliferative, Cytotoxic γδ T cells (P-CT), Differentiated Cytotoxic γδ T cells (D-CT) and Late Activated Cytotoxic γδ T cells (LA-CT). P-CT shows an expression profile positively associated with cell proliferation as well as increased

FCS 20 2005

Anh Tuan Nguyen1, Keit Min Tham1 and Frederic Bard1,2

1Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673 2Department of Biochemistry, National University of Singapore, 21 Lower Kent Ridge Road, Singapore 119077

cell stage. Surprisingly, inhibition of GALA in all liver cells does not rescue tumor growth. We next find that when surrounding healthy cells have higher GALA levels than tumor cells, they prevent tumor progression. Cell competition is evident in in vitro co-culture of GALA-high cells versus GALA-low cells. Winner GALA-high cells induce apoptosis in neighboring loser cells through cell-cell contact. Interestingly, about a third of human patients display high levels of GALA in non-cancerous adjacent tissues and have increased survival. By contrast, tumors with high GALA surrounded by low GALA tissues tend to correlate with poor outcome. Together, our results indicate that GALA promotes a competitive growth process in solid cancers based on cell killing.

P007The GALA Pathway Drives Cell Competition between Normal and Tumor Cells in Liver Cancer

Liver cancer represents a major public health problem. Solid tumors grow within a normal tissue, eliminating normal cells in the process. The cell competition between healthy and cancerous cells was first described in Drosophila and is known to limit oncogenic growth and maximize tissue fitness during development. It is unknown whether cell competition occurs during liver cancer. Previous evidences indicate that GalNAc O-glycosylation activation (GALA) pathway is markedly increased and drives tumor malignancy and invasiveness. Here, we establish transgenic mouse models to test the role of GALA in liver cancer cells and healthy surrounding cells. We find that inhibition of GALA specifically in transformed cells induce a growth block at the one-

P008Analyses of Mutational and Structural Variation in a Cohort of Anaplastic Astrocytoma Tumours

Anjali Kannangath1, Ellora Chua Hui Zhen1, Liisi Laaniste2, Sarah Langley3, Kevin O’Neil4 and Federico Roncaroli5, Matthew Williams6, Michael Johnson2, Nathan Harmston1,7

1Science Division, Yale-NUS, Singapore, 2Department of Brain Sciences, Imperial College Faculty of Medicine, London, UK, 3 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore, 4Department of Neurosurgery, Imperial College Healthcare NHS Trust, London, UK, 5Division of Neuroscience and Experimental Psychology, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK, 6Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, UK, 7Cancer and Stem Cell Biology, Duke-NUS, Singapore

Anaplastic Astrocytoma is a diffusely infiltrating, malignant, primary brain tumour (typically classified as a Grade 3 glioma), with lower grade astrocytomas often progressing to this more aggressive form. Despite numerous studies on the mutational landscape of astrocytoma, our understanding of the molecular mechanisms involved in the formation and progression of these tumours is far from complete. Here, we use whole genome sequencing to investigate eight anaplastic astrocytoma (AA) tumours - which exhibited both Grade II and Grade III pathology - and investigated the mutational and structural variant landscape of the Grade III component. This revealed widespread heterogeneity in the location, size and number of

structural variants in Grade 3 AA tumours. Excluding one sample which appears to have undergone a whole-genome doubling event, copy number alterations affecting the key driver genes IDH1/2 and P53 were not observed. However, we did observe heterozygous loss of ATRX in multiple samples, and gain of EGFR in 3/8 tumours. In addition, we show differences in mutational signatures between samples in the cancer cell fraction of specific events, and in the overlap with specific genomic features. These findings highlight the need for further studies investigating the structural variant landscape of gliomas to help understand the mechanisms involved in tumourigenesis and to support the development of individualized therapies.

FCS 20 2006

P009Hepatic SIRT6 Deficit Promotes Liver Tumorigenesis in the Mice Models

Mei Wang 1,2,#, Shan Jiang3,#, Linhua Lan4,#, Xiao Mo1, Ping Xu1, Haojun Xu1, Chengfei Zhang1, Fan Yang3,*, Hongping Xia 1,2*, Jinglin Xia4,*

1Department of Pathology, School of Basic Medical Sciences & Sir Run Run Hospital & State Key Laboratory of Reproductive Medicine & Key Laboratory of Antibody Technique of National Health Commission, Nanjing Medical University, Nanjing, China, 2Department of Immunology, Medical School of Southeast University, Nanjing, China. 3The First Affiliated Hospital of Chongqing Medical University, Chongqing, China 4The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Background SIRT6 belongs to class III sirtuin family with NAD+ -dependent histone deacetylase activities and controls multiple processes including aging, metabolism and inflammation. In recent years, increasing studies showed tumor suppressor role of SIRT6 in HCC development. Methods We established a two-stage DEN followed CCl4 induced liver carcinogenesis in the hepatic-specific SIRT6 HKO mice models. The HCC cells were transfected with SIRT6 shRNA or pcDNA3.1-SIRT6 for knockdown or overexpression of SIRT6. The tumor formation

was examined by the xenograft mice experiments. Results We found that hepatic SIRT6 deficit significantly promotes liver injury and liver cancer through inhibition of the ERK1/2 pathway. SIRT6 was compensatory upregulated in mice tumor tissues and human HCC cells and overexpressed SIRT6 inhibits tumor growth both in vitro and in vivo. Conclusions Taken together, we provide a useful mouse model for delineating the molecular pathways involved in chronic liver diseases and primary liver cancer and suggest that SIRT6 can be a promising target for HCC therapies.

Mai Trinh Nguyen1, Hui Yee Yong1,2,3, Boya Peng5,6, Jie Zheng4, Victor Chin Yong Ho2,5,, Katja Fink1,2,5, Patrick R. Griffin4, Minh Nguyet Le Thi 5,6 and Dahai Luo1,3

1Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore City, Singapore, 2School of Biological Sciences, Nanyang Technological University, Singapore City, Singapore, 3NTU Institute of Structural Biology, Nanyang Technological University, Singapore City, Singapore, 4Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA 5 Singapore Immunology Network, Agency for Science, Technology and Research, Singapore City, Singapore, 5Department of Biomedical Sciences, College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon, Hong Kong, 6Yoon Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore

P010Structure-Guided Design of Immunomodulatory RNAs Specifically Targets the Cytoplasmic Viral RNA Sensor RIG-I and Promising Anticancer

Antiviral immunity responses in human involves cellular innate immunity and host adaptive immunity response. Antiviral cellular innate immunity is primarily mediated by intracellular dsRNA binding protein RIG-I. Upon viral infection, virus intermediates generate dsRNA that such as pathogen associated molecular patterns that bind to pattern recognition receptors such as RIG-I receptor which essentially leads to the activation of cellular innate immunity responses and the activation of antiviral state in the infected cell. Activation of RLR using immunomodulatory RNA (immRNA) could be a promising antiviral strategy. Here, using structure-based selection, we characterized that 3p10LA9 and 3p10LG9, an artificial 25-mer immRNA derived from 3p10L, could robustly

activate RIG-I. We found that the purine/pyrimidine base insertion at position 9 could lead to greater potency than 3p10L. Mechanistically, 3p10LG9 binds to the helicase C-terminus domain (CTD) RNA sensing module of RIG-I, interfering with CTD autoinhibition on N-terminus caspase activation and recruitment domains. In human monocytic and lung fibroblastic cells lines, transfection of 3p10LG9 but not 3p10LG9 lacking 5’ phosphorylation, was able to elicit interferon response in a dose-dependent manner. 3p10LG9 was able to prevent dengue virus infection to a significantly and greater extent than 3p10L. Importantly, 3p10LG9/ 3p10LA9 could act as therapeutic against cancer in mouse model.

FCS 20 2007

P011Identification of Mechanisms in Azacitidine-Venetoclax-Resistant Acute Myeloid Leukemia using Genome-Wide CRISPR/Cas9 Library Screening

Acute myeloid leukemia (AML) is the most common myeloid malignancy with 10-year survival rate < 30%. Currently, a combination therapy of venetoclax, a Bcl-2 inhibitor and azacytidine is a treatment of choice for patients not eligible for conventional chemotherapy. However, majority of patients rapidly develop resistance and suffer disease relapse. Little is known on the underlying mechanism of resistance. To identify the genetic factors that contribute to resistance and druggable targets, we performed CRISPR/Cas9 library screening using de-novo azacytidine-venetoclax resistant AML cells.

We firstly determined the IC20, IC50 and IC80 of a combination of azacitidine and venetoclax on 16 AML cell lines that represent different disease stages, cellular origins and genetic profiles. Two cell lines, SKNO-1 and

Wei Xiang1, Gao Bin Chen2, Zehui Kok1, Yihui Lam1, Charles Chuah1,2, Shang Li2

1Department of Haematology, Singapore General Hospital; 2Cancer and Stem Cell Biology Program, Duke-NUS Medical School

HEL-1, were identified to be de-novo resistant lines with > 1000-fold difference on IC50 compared to sensitive lines. We next performed CRISPR/Cas9 library screening on SKNO-1 and HEL-1 and ranked synthetic lethal genes using the MAGECK algorithm analysis. We identified several gene candidates which might be critically involved in AML de-novo resistance to azacytidine-venetoclax. Some of the identified genes are known to play essential roles in cancer cell growth and survival. Validation will be performed using functional analysis. Our findings will be useful in 1) understanding the mechanisms of azacytidine-venetoclax resistance in AML; 2) identifying therapeutic strategy to overcome resistance; 3) revealing prognostic biomarkers to define risk stratifications and response to venetoclax and azacytidine for AML patients.

P012Computational Modeling of Evolution in Tumor Cell Populations Shows Differences in the Symmetry of Resistance After Treatment with Synergistic or Antagonistic Drug Combinations

E C Saputra1,2, Lisa Tucker-Kellogg1

1Cancer and Stem Cell Biology (CSCB) and Centre for Computational Biology, Duke-NUS Medical, Singapore. 2Current address: Carnegie Mellon School of Computer Science, Pittsburgh, PA, USA.

Introduction: Evolution is easy to observe in individual cases but difficult to generalize, especially when attempting to study evolution of cancer drug-resistance under treatment with multiple drugs. Computational simulations of evolution offer the opportunity to articulate trends and compare relative probabilities. In this work, we use simplified mathematical models of cancer to make predictions about how the synergism of a two-drug treatment (the non-additivity) can bias the probability of whether the most likely drug-resistance to evolve in the future will be against one drug at a time, or against both at once.

Methods: Suppose drug-1 and drug-2 are given simultaneously to a population of tumor cells. If there is any subsequent expansion or treatment failure, we can define the “symmetry of resistance” as the extent to which the post-relapse cells are equally resistant against both drugs (e.g., 44% resistant to drugA and 44% resistant to drugB) versus not equally resistant (e.g., 88% resistant to one drug and fully sensitive to the other).

We studied the symmetry of resistance during simulations of cancer evolution using a computational model that tracks the number of cells and the degree of drug-resistance of each cell over time.

Results: Virtual tumor cells were simulated to evolve under selective pressure from two-drug combinations that were additive, synergistic (more efficacious than additive) or antagonistic (less efficacious than additive). When resistance arose against treatment with synergistic combinations, it more asymmetric (stronger resistance to one drug than then other), whereas when cells escaped antagonistic combinations, the resistance was more likely symmetric (e.g., moderately resistant to both drugs).

Conclusion: Our theoretical model offers a testable prediction, that at relapse after a synergistic treatment, the tumor would be more likely to retain sensitivity to one of the drugs, compared with tumors that develop equally severe relapse after a non-synergistic treatment.

FCS 20 2008

P013Surface Functionalized Extracellular Vesicles for Safe and Targeted Delivery of Anti-Cancer Therapeutics

With the advent of novel drugs in the fight against cancer, there is also a need for a safe, efficient and scalable drug delivery vector, capable of bypassing cellular barriers and delivering therapeutics to target cells. In recent years, naturally derived extracellular vesicles (EVs) have emerged as a safe biocompatible alternative to existing vectors such as viruses and liposomes. However, there are several obstacles such as scalability and high levels of non-specific uptake that need to be overcome before EVs can be successfully translated for clinical use.

Here, we describe a novel enzyme-mediated method for generating target-specific EVs capable of delivering therapeutic cargo to target cells without compromising the endogenous advantageous properties of EVs. EVs engineered using this method were shown to be stably conjugated with a range of targeting moieties at high copy number. The resulting EVs were shown to exhibit antigen-specific targeting capabilities, showing preferential accumulation in target cells both in vitro and

in vivo. In combination with our red blood cell-derived EV (RBCEV) platform, we have developed a biocompatible, scalable and efficient drug delivery system, capable of delivering a range of therapeutic molecules including chemotherapeutic agents and RNA payloads specifically to target cells. The functionality of RBCEVs engineered using this approach was demonstrated by the ability of targeting RBCEVs to specifically deliver encapsulated chemotherapeutics to lung tumor xenografts, resulting in improved treatment outcomes using significantly lower doses of (Paclitaxel) PTX than used clinically. We also demonstrate the ability to induce enhanced gene expression or knockdown in target cells via the encapsulation of nucleic acid payloads in targeting RBCEVs. This simple and biocompatible method for the generation of target-specific EVs addresses much of the short-comings of current EV-based vectors and provides a safe and biocompatible alternative for the delivery of a range of anti-cancer therapeutics.

Jayasinghe Migara Kavishka1,2†, Pham Chanh Tin1,2†, Pham Tuan Thach1,2†, Yang Yuqi2†, Tan Melissa1, Peng Boya1,2, Shi Jiahai2*, Le TN Minh1,2*

1Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 2Department of Biomedical Science, City University of Hong Kong, Hong Kong, *Corresponding Author, † Equal Contribution

P014DNMT3A Loss Leads to Perturbed 3D Genome Organization and Altered Epigenetic Landscapes in Myeloid Leukemia

Deepak Babu*1, Lingshi Kong*1,2, Ruchi Choudhary1,2, Shao Xiaoman1, Qiling Zhou1, Winnie Fam1, Jia Qi Tng1, Daniel Tenen1, Melissa Jane Fullwood1,2,3

1Cancer Science Institute of Singapore, National University of Singapore, 2School of Biological Sciences, Nanyang Technological University, 3Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, *denotes equal contribution

DNA methyltransferases 3A (DNMT3A), is one of the major enzymes responsible for de-novo DNA methylation and is one of the most commonly mutated genes in AML. We asked as to how does loss of function of DNMT3A underlie the pathogenesis of AML. We hypothesized that loss of DNMT3A would lead to reduced genome-wide methylation and aberrant binding of CTCF to DNA. This causes disruption of 3D genome architecture, in particular at the topologically associating domains (TADs). Due to altered TADs, chromatin interactions around key cancer-related genes might be disrupted. As a result we expect to observe a downstream alteration in transcriptional profiles, eventually driving and/or maintaining leukemia pathogenesis.

To test the hypothesis, we carried out an integrated analysis by comparing the differential methylation

(bisulphite sequencing), TADs (HiC) and loops, transcriptional profiles (RNA-seq and qPCR) and histone modification profiles (CTCF, H3K27ac, H3K27me3 and H3K4me3 ChIP-seq) in a myeloid leukemia cell line model with wild-type and loss of DNMT3A.

Bisulphite sequencing revealed an overall reduction in genome-wide methylation upon loss of DNMT3A in the knockout cell line. Comparison of Hi-C data revealed disruption in TAD architecture upon loss of DNMT3A. We observed altered epigenetic landscapes (as revealed by ChIP-seq) around candidate genes whose transcriptional profiles were altered as seen by RNA-seq and validated by qPCR. Interestingly, we noticed the upregulation of several non-coding RNA with potential roles in cancer. Our data revealed a previously unknown, important role for DNMT3A in the 3D genome organization.

FCS 20 2009

P015Design, Investigation and Delivery of Advanced trans-Splicing RNA for Suicide Gene Therapy

Pei She Loh1 and Volker Patzel1.1Department of Microbiology and Immunology, National University of Singapore, Singapore

Spliceosome-mediated RNA trans-splicing represents a technology that allows endogenous labelling of target pre-mRNAs with an artificial trans-splicing RNA triggering the formation of chimeric RNAs and proteins. Hence, the RNA trans-splicing technology enables genetic repair on the pre-mRNA level by exon replacement, labelling of nascent transcripts with fluorophores for diagnostic imaging, and specific reprogramming of cellular functions based on the expression of distinct pre-mRNA biomarkers. We developed a suicide gene therapy approach in which trans-splicing RNA is used to label disease-specific pre-mRNA biomarkers with a death signal leading to selective killing of diseased cells without affecting healthy normal cells. As gene-directed enzyme prodrug therapy system we selected the Herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system. This therapeutic approach is not limited to cancer treatment and is also being explored for targeting of cells that are irreversibly transduced by integrating viruses. Artificial trans-splicing RNAs were featured with binding domains (BD) targeting the pre-mRNA of the

α-fetoprotein, a biomarker of hepatocellular carcinoma (HCC), or alternatively transcripts derived from the human papillomavirus type 16. Rationally designed tsRNAs greatly improved on-target activity and on-target specificity. These suicide vectors triggered selective death of liver cancer cells or HPV-16 transduced cells. Our latest generation of multi-targeting constructs comprising recognition domains for up to 5 different hepatocellular carcinoma (HCC) pre-mRNA biomarkers exhibited the highest cell death activity even at 330-fold reduced GCV concentrations. Currently we explore non-viral dumbbell-shaped DNA minimal vectors for delivery. These are tested in patient-derived xenograft (PDX) 3D cell culture models and will be tested in both immune-deficient and humanized mouse models. For targeted delivery in vivo, HCC-targeting vectors will be furnished with tri-antennary N-acetylgalactosamine (GalNAc3) residues. So far, our observations suggest that the amalgamation of the trans-splicing-based suicide technology with dumbbell vectors provides a powerful tool for specific destruction of aberrant target cells.

P016MYC Overexpression Leads to Gain of c-Myc Binding Sites at Superenhancers and Pre-Existing Chromatin Loops

Yi Xiang SEE 1,2, Kaijing CHEN2, Ömer AN2, Henry YANG2, Melissa J. FULLWOOD1,2,1School of Biological Sciences, Nanyang Technological University, Singapore, 2Cancer Science Institute of Singapore, National University of Singapore, Singapore

The MYC oncogene encodes for the c-Myc protein and is frequently dysregulated across multiple cancer cell types, making it an attractive target for cancer therapy. However, there have been many difficulties in targeting c-Myc, due to its complex network of regulators and the unstructured nature of its protein. For that reason, we are interested in looking at the downstream functions of c-Myc specific to cancer. Overexpression of MYC leads to its binding at active enhancers, resulting in a global transcriptional amplification of active genes. However, the mechanism underlying this c-Myc enhancer invasion has not been well studied.

To that end, we performed ChIP-seq, RNA-seq, 4C-seq and Hi-C on the U2OS Tet-inducible MYC osteosarcoma cell line. MYC overexpression in U2OS cells led to increased c-Myc binding at promoters, typical enhancers and superenhancers, as well as associated changes

in gene expression. Semi-quantitative Hi-C analysis revealed an increase in global chromatin contact frequency, although the increase was not significant in 3C-PCR analysis of selected loci. There was a preferential enrichment of c-Myc at chromatin loop anchors, resulting in upregulation of genes associated with these loops.

Our results suggest that chromatin interactions mediate c-Myc enhancer invasion by facilitating spatial proximity between canonical binding sites at promoters and lower affinity binding sites at enhancers. The accumulation of c-Myc at cancer-type specific superenhancers may then drive the expression of interacting oncogenes that each cancer is highly reliant on. By elucidating the chromatin landscape of c-Myc driven cancers, we can potentially target these superenhancers or their associated oncogenes for cancer therapy, without affecting physiological c-Myc signalling.

FCS 20 2010

P017A Comprehensive Mapping of the 3D Genome Organization in Nasopharyngeal Cancer

Sambhavi Animesh 1*, Ruchi Choudhari 2*, Ng Xin Yi 3, Goh Boon Cher 1, 3,4, Melissa Jane Fullwood 1, 2

1 Cancer Science Institute of Singapore, Centre for Translational Medicine, National University of Singapore, Singapore- 117599, 2Department of Biological Science, Nanyang Technological University, Singapore- 639798, 3Department of Haematology-Oncology, National University Cancer Institute of Singapore, National University Health System, Singapore-119228, 4Department of Pharmacology, Yong Loo Lin School of Medicine, National University Health System, Singapore, Singapore.*Co-first authors

The three-dimensional (3D) organisation of chromatin in the nucleus plays a vital role in the transcriptional regulation by facilitating interactions between gene promoters and distal regulatory elements. Any variations in these tightly regulated features of the 3D genome architecture have a significant impact on gene expression and increase disease risk in several diseases, including cancer. Generally, a high-throughput chromosome conformation capture technique such as Hi-C is used to investigate three-dimensional (3D) chromatin conformation at a very high spatial resolution. Till now, no such studies have been conducted to study the chromatin interactome and 3D genome organization in nasopharyngeal cancer.

Here, we present BiopC, a modified in situ Hi-C method that allows the generation of high-quality genome-wide chromatin conformation maps using solid tumor biopsy samples. BiopC can be performed on a very tiny tissue sample obtained from the needle biopsy. The library

can be prepared in a single microtube from the sample collection to the Hi-C library preparation and thus prevents the sample loss. We employed this method on three nasopharyngeal tumor biopsy sample viz S009, S010, and S012 and nasopharyngeal cancer cell line HK1. We can successfully detect various conformational features such as topologically associating domains (TADs), loops, Frequently interacting regions (FIREs), and stripes. We also profiled the super-enhancer (SE) landscape in three NPC cell lines HK1, C666-1, and HNE1 using the H3k27ac ChIP sequencing data (Yuan et al. Cancer Res; 77(23), 2017). We observed 298 SEs were present in all the three cell lines and are associate with oncogenes via chromatin interactions. The BiopC method discussed here can be employed to study the 3D genome organization in any solid tumor biopsy samples. Such studies will be critical to understand the molecular mechanism of oncogenesis fully and to guide the personalized therapeutic strategies eventually.

P018Aihistonote: Digital Pathology Annotation Platform for Smart Clinical Assessment

Kok Haur ONG1, Xinmi HUO1, Malay SINGH1, Longjie LI 1, Hao HAN 1, Laurent GOLE1, David M YOUNG1, Susan Swee Shan HUE2, Kah Weng LAU2, Soo Yong TAN2*, Weimiao YU1*

1Computational & Molecular Pathology Lab (CMPL), Institute of Molecular and Cell Biology, Singapore, 2Department of Pathology, National University Hospital, Singapore.

AI-driven computational pathology diagnosis is an emerging but rapid growing field. It applies the computational algorithms such as AI and machine learning solutions to classify cancers and other diseases from digital pathological images. Labelling digital pathological images, known as annotation, requires experienced pathologists with proper pathological training. High quality annotated image database is the foundation for AI-based solution development since most of the successful models are supervised learning. It is essential to mark and annotate specific areas/structures to describe the disease and then build the learning models. However, the resource of the pathologists is limited. Therefore, it is difficult and expensive to obtain an extensive, systematic and structured annotation

set of digital pathological images to develop accurate supervised learning models. To facilitate advances in computational pathology, AiHistoNote, a cloud-based structural annotation platform, was developed to enable pathologists to annotate digital pathological images. Based on this cloud-based solution, we are building an annotation dataset of prostate cancer and link annotations to ontology information for accurate AI-based pathological diagnosis. As a result, clinical validation and decision-making by pathologists can be faster, easier and more accurate. Here we demonstrate how we apply this annotation platform to facility the prostate cancer diagnosis using H&E images. In the future, we will extend this platform to other cancer types.

FCS 20 2011

P019Antisense RNA-Mediated Promoter-Specific Regulation Influences Gene Expression in Cancer

Fernando Bellido Molias1, Andre Sim2, Ka Wai Leong1, Omer An1, Yangyang Song1, Vanessa Hui En Ng1, Chen Ying2, Jonathan Göke2, *, Leilei Chen1,3, *

1Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599, Singapore, 2Computational & Systems Biology, Genome Institute of Singapore, Singapore 138672, Singapore, 3Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Natural antisense transcripts (NATs) are a type of RNAs that are transcribed from the same locus, but in the opposite direction to the sense gene transcript. Regardless some non-coding NATs (ncNATs) have been found to regulate key biological events such as cell differentiation and carcinogenesis, the sequence and function of most of them remains completely unexplored. Considering this, and because transcriptional aberrations are a common event during oncogenesis -and are even considered as non-genetic drivers of cancer-, we thoughtfully analyzed the TCGA and GTEx hepatocellular carcinoma (HCC) RNA expression datasets, and we managed to identify a list of 85 ncNATs differentially expressed in HCC. Among them, we characterized the previously undescribed HNF4A-

AS1L and we found out that it specifically upregulates the Promoter P1-driven isoforms of its sense gene HNF4A, which is a well-known liver cancer driver. This highly promoter-specific mechanism facilitates an independent control of the oncogenic and tumor suppressive HNF4A isoforms, eventually promoting HCC development. Our further analysis of the TCGA/GTEx RNA-Seq data from 23 cancer types identified approximately a hundred ncNATs whose expression show correlation with the activity of their associated sense genes at a promoter-specific level -similar to HNF4A-AS1L and HNF4A P1-. This suggests that promoter selection might be a general mechanism employed by ncNATs for a context-specific regulation of alternative isoform expression in cancer.

P020Highly Multiplexed Immuno-Fluorescence Images Data Analysis for Prostate Cancer

Malay SINGH1, Laurent GOLE1, Kok Haur ONG1, Hao HAN1, David M YOUNG1, Susan Swee Shan HUE2, Soo Yong TAN2*, Weimiao YU1*

1Computational & Molecular Pathology Lab, Institute of Molecular and Cell Biology, Singapore,2Department of Pathology, National University Hospital, Singapore.

Prostate Cancer (PCa) is estimated to cause at least 10% of all cancer deaths in US. Virtual microscopy techniques have been developed to aid PCa diagnosis and prognosis. Immune and tumor cell distributions and interaction (inter- and intra-) have been known to provide insights in tumor microenvironment. Multiplexed immune-fluorescence imaging allows the study of various immune and tumor cells in tissue simultaneously. We have developed an automated image processing pipeline to detect and quantify multiple immuno-phenotypes in prostate tissues. Our pipeline incorporates robust image processing and machine learning methods to analyse histopathological tissue whole slide images (WSI).

We have stratified our patient cohort (N=156) according to their Gleason Score (GS, Primary and secondary Gleason grade) annotated by the pathologist. Each patient tissue sample was sliced into 4 consecutive slices. First slice was stained by H&E such that they can be assessed by pathologist. The subsequent 3 slices were stained using UltiMapper I/O PD-L1, UltiMapper I/O

PD-1, and UltiMapper I/O T-act kits respectively. All the 4 slices along with pathologist annotations are Z-stacked. We quantified the immuno-phenotypes along the Z-stack as cell density (#cells/mm2) using our pipeline. We then analysed these immuno-phenotypes across patient groups for statistical significance. We achieved significant results with multiple immuno-phenotypes with p<0.05 for various pairs of patient groups.

Patients graded GS 4+3 (N=55) have higher panCK+ cell density when compared to patients graded GS 3+4 (N=64) with p=0.0234. This result aligns well with various major studies indicating worse pathological stage and higher chance of metastases for men with GS 4+3 when compared to men with GS 3+4. Our results indicate that immuno-phenotype quantification can aid in PCa patient diagnosis and prognosis. Our results incorporating basic cell density paves the path for more sophisticated pipelines quantifying cell-cell interactions, cell distributions which can be used in clinical settings.

FCS 20 2012

P021Identification of a Novel Active KRAS-MTORC2-AKT-ROS Drug-Induced Mitochondrial Morphology Changes and Execution of Mutant Kras Driven Cancer Cells

Kartini Iskandar1, Chuan Han Jonathan Foo2, Liew Qiu Xia Angeline2, Deepika Raman1, Shazib Pervaiz1,2,3

1Department of Physiology, YLL School of Medicine, National University of Singapore, 2NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 3National University Cancer Institute, NUHS, Singapore

RAS is amongst the most commonly mutated oncogenes and known to be involved in the regulation of multiple cancer hallmarks. Interestingly activation of RAS is a potent signal for intracellular ROS generation, which was exploited in our recent work using an experimental small molecule compound (merodantion; C1). These data provided evidence to implicate Akt/PKB upstream of autophagy-associated execution of cancer cells harboring mutant KRAS. Notably, the specific targeting of mutant KRAS expressing cancer cells was a function of KRAS-dependent mitochondrial redox catastrophe. Intrigued by these findings, we further elucidated the signaling network(s) involved in this novel-active KRAS-mediated-therapeutic strategy against mutant KRAS driven cancer cells. We report here that drug-induced activation of mutant KRAS triggers mitochondrial morphology changes, consistent with the activation of fission and/or disruption of the fusion machinery. Using tools like mitochondrial network analysis (MiNA) developed by Valente et al, we observed mitochondria fragmentation indicated by an increase in individual mitochondria and networks as well as decreased branching and branch-length, compared to the untreated cells. These effects could be rescued upon the transient knockdown of KRAS. Notably, active KRAS and Akt/PKB appear to be involved in the phospho-

activation of DRP1 (ser616)-associated with mitochondrial fission-as well as cleavage of the fusion-associated OPA1, thus corroborating the induction of mitochondrial fission. Interestingly, accumulation of the voltage dependent anion channel 1 (VDAC1) was observed in cells following exposure to the small molecule compound. Furthermore, transient silencing of VDAC1 abolished drug-induced mitophagy as evidenced by the absence of LC3 II accumulation in the mitochondrial fraction as well as total cells lysates. Interestingly, knockdown of either KRAS or AKT rescued VDAC1 accumulation and stability. A similar effect was observed upon silencing of DRP1, thus suggesting an axis of mutant active KRAS-Akt-DRP1-VDAC1 in mitochondrial morphology changes and cancer cell execution. Lastly, to unravel the mechanism of AKT activation in this model, we identified mTORC2 as an important upstream mediator. Pharmacological inhibition of mTORC2 via TORIN1 or genetic knockdown of Rictor, inhibited AKT phosphorylation, reduced mitophagy induction, decreased ROS generation and rescued colony forming ability. Taken together, we demonstrate a novel mechanism wherein drug-induced activation of KRAS triggers mTORC2-dependent AKT activation resulting in an increase in ROS and mitofission and subsequent cell death.

P022The MAP/ERK Pathway May Be the Wrong Target to Repress KRAS Signaling in Sporadic Colorectal Carcinomas

Kuen Kuen Lam1, Choong Leong Tang1, Emile Tan1, Siew Heng Wong2, Peh Yean Cheah1,3,4

1Department of Colorectal Surgery, Singapore General Hospital; 2JW Bioscience Pte Ltd; 3Saw Swee Hock School of Public Health; 4Duke-NUS Medical School, National University of Singapore

Colorectal Cancer (CRC) is a major cause of cancer death worldwide. In most CRC, the mitogenic epidermal growth factor receptor (EGFR) pathway is activated, thus anti-EGFR therapy are sometimes prescribed in addition to standard chemotherapy. Nevertheless, anti-EGFR therapy is only beneficial to patients with wild-type KRAS, and even so, these patients quickly develop resistance due to therapy-induced KRAS mutation. KRAS, a small GTPase protein downstream of EGFR, is a central activator of the EGFR signaling pathway. KRAS activates cell proliferation and anti-apoptotic pathways, with the MAPK/ERK and PI3K/Akt pathways being most commonly reported. Up to 40% of CRC harbors KRAS somatic mutations which leads to its constitutive activation even in the absence of EGFR signaling. However, direct inhibition of KRAS is challenging as its protein structure is inadmissible to inhibitor docking. Hence, there have been extensive development of inhibitors targeting the MAPK/ERK pathway believed

to be the main KRAS downstream pathway in several cancers. Phosphorylated ERK1/2 is reported to be the main effectors of this pathway. We surveyed phospho-ERK1/2 normalized to total-ERK1/2 (pERK/tERK) protein levels in 28 pairs of CRC tumor and matched mucosa samples, inclusive of tumors with wild-type KRAS and p.Gly12Asp, p.Gly12Val and p.Gly13Asp activating mutations common in CRC using LICOR Western Blot analysis. Surprisingly, in 20/28 (~71%) of the samples, the tumor has lower or equivalent pERK/tERK compared to its matched mucosa 10 cm or more away. Notably, pERK/tERK is not significantly higher in KRAS-activated vs KRAS-wildtype tumors. Our results suggest that MAPK/ERK is unlikely to be the major KRAS downstream pathway activated in CRC. These results are consistent with the analysis of several published datasets on human CRC tissues. We therefore propose that MAPK/ERK is not a good drug target for sporadic CRC and efforts should instead be diverted to other potential targets.

FCS 20 2013

P023Retention of Runx1-Insufficient EPCR+CD41+ Hematopoietic Stem Cells by Nras Mutation Facilitates Myeloid Leukemogenesis

Michelle Meng Huang Mok1, Ng King Pan1, Chelsia Wang Qiuxia1, Melvin Dai2, Zhang Xiaoming2, Ichiro Taniuchi3, Xin-Yuan Fu1, Yoshiaki Ito1, Lih-Weng Deng2, Motomi Osato1

1Cancer Science Institute of Singapore, National University of Singapore, Singapore, 2Department of Biochemistry, National University of Singapore, Singapore, 3RIKEN Center for Integrative Medical Sciences, Japan

The RUNX1 gene is critical for the generation and maintenance of hematopoietic stem cells (HSCs), and among the most frequently mutated genes in human myeloid leukemia. RUNX1 alterations per se, though being leukemia-prone, are insufficient to cause overt leukemia due to a niche-detachment defect. Additional genetic changes in NRAS and MLL5 have been observed in RUNX1 leukemia, yet the molecular basis for this synergism remains elusive. Here we report that restoration of niche interaction of HSCs is facilitated by such additional genetic changes in myeloid leukemogenesis. Non-myeloablative bone marrow transplantation of wild type cells, the most rigorous method to interrogate niche interaction defects, revealed engraftment competency in the mice carrying Runx1

insufficiency. This result suggests that Runx1 mutant HSCs are outcompeted by transplanted wild type HSCs. Notably, this chimerism was reduced or completely abrogated in mice harboring an additional genetic hit such as Nras or Mll5 mutation. Flow cytometry analysis demonstrated that concurrent genetic alterations lead to the restoration of HSCs expressing endothelial protein C receptor (EPCR, a.k.a. CD201) and CD41, which may serve as a cell-of-origin for myeloid leukemia. Such retention of the specific subset of HSCs in the niche may allow for an increased window of opportunity to acquire subsequent mutations for the full-blown disease. Further elucidation on the niche-related mechanism would provide deeper insights into myeloid leukemogenesis and may lead to novel therapeutics.

P024Glioblastoma Oncolytic Virotherapy using Live-Attenuated Zika Virus (ZIKV) Vaccines

Carla Bianca Luena Victorio1, Wisna Novera1, Alfred Suyang Sun2,3, Eng Eong Ooi4, Ann-Marie Chacko1

1Cancer and Stem Cell Biology Programme, Duke-NUS Medical School, 8 College Rd 169857 Singapore, 2Genome Institute Singapore, 60 Biopolis Street 138672 Singapore, 3National Neuroscience Institute Singapore, 11 Jalan Tan Tock Seng 308433 Singapore, 4Emerging Infectious Diseases Programme, Duke-NUS Medical School, 8 College Rd 169857 Singapore

Glioblastoma multiforme (GBM) is the most common primary malignancy in the central nervous system and among the deadliest of human cancers. Despite aggressive multi-modality treatment—including surgery, radiation, and temozolimide (TMZ) chemotherapy—most patients die from recurring therapy-resistant disease, which underlines the desperate unmet medical need for effective new therapies for GBM patients. Our solution is to develop GBM-targeted oncolytic viruses (OVs)—i.e. cancer-killing viruses— based on live-attenuated Zika Virus (ZIKV) vaccine strains. ZIKV is an RNA virus

previously described to naturally infect glioma stem cells. In vitro, these ZIKV vaccine strains exhibit GBM cell killing, elicit antiviral immune response in infected cells, and inhibit tumour clonogenicity. Compared to the wild-type clinical isolate, the ZIKV vaccine strains also exhibit minimal infectivity in normal non-cancer cells, such as primary endothelia and terminally differentiated neurons derived from human pluripotent stem cells. The increased safety margin and oncolytic activity of ZIKV vaccine strains relative to the clinical isolate suggests better potential as a GBM therapeutic.

FCS 20 2014

MYC is frequently highly expressed in gastrointestinal cancers through unknown mechanisms and remains a major focus in cancer therapeutic research. MicroRNAs are potent post-transcriptional regulators of gene expression and studies have suggested their clinical potential in cancer treatment. In this study, we focus on the MYC coding sequence (CDS) due to the extensive shortening of its 3′ untranslated region observed in colorectal cancer (CRC) patient samples which could abrogate microRNA-mediated regulation. Using unbiased computational and experimental approaches,

Triple-negative breast cancer (TNBC) is the most aggressive and invasive subtype of breast cancer that remains clinically challenging. Despite its higher recurrence and poor prognosis, advances in therapeutic interventions for TNBC have remained limited due to lack of defined targets, and to date, adjuvant chemotherapy continues to be the mainstay treatment for TNBC patients. One of the major impeding factors to the development of effective therapeutics is the intrinsic high heterogeneity of TNBC that convolutes elucidation of its molecular mechanisms and aberrations. Despite gene-based expression studies reporting several subclasses within TNBC, in-depth mechanistic insights on distinct pathways or plausible targets for these subclasses is currently lacking. In this work, using a panel of breast cancer cell lines, we explored subtype-specific proteome changes to unveil pathway aberrations and molecular vulnerabilities within different breast cancer subtypes, more specifically within the TNBC group. We found that expression of specific kinases and proteases underlie

P025Suppression of MYC Expression by MiR-138 Inhibits Cancer Development

Ng Desi1,2†, Velda Teh1†, Chun You Lim1, Xiao Hong Chew1, Avencia Sanchez-Mejias1, ‡, Priyankaa Pitcheshwar1, Shi Wang3, Bei-En Siew4, Kuok-Chung Lee5, Choon-Seng Chong5, Ker-Kan Tan4,5, Yvonne Tay1,2

1Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599, Singapore, 2Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore, 3Department of Pathology, National University Health System, 4Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 5Division of Colorectal Surgery, University Surgical Cluster, National University Health System, ‡ Present address: Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona 08003, Spain, †Contributed equally

we identify and validate microRNA-138 (miR-138) as a MYC CDS-targeting microRNA. We show that miR-138 inhibits MYC expression to suppress CRC and hepatocellular carcinoma (HCC) tumour growth. Critically, the intravenous administration of miR-138 mimics significantly impedes MYC-driven HCC growth in vivo. Overall, we discover miR-138 as a robust suppressor of CRC and HCC progression through its negative regulation of MYC, highlighting the therapeutic potential of miR-138 in MYC-driven cancers.

P026Unraveling Subclass-Specific Molecular Vulnerabilities in Triple-Negative Breast Cancer

Asfa Alli-Shaik1, Max Kosok1,2, Boon Huat Bay2, Jayantha Gunaratne1,2

1Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, 2Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

unique functional differences between the two TNBC subclasses, basal A and basal B, and through systemic investigation of modulated protein interaction networks and assessment of protein co-regulations, we revealed several protein associations involving these functional proteins to be consistently dysregulated in a subclass-specific manner within TNBC. Our analysis highlighted that the basal B TNBC subclass, representing the more aggressive cell line group, displayed the highest number of perturbed associations with particular molecular inclination towards aggressive cancer hallmarks including EMT, and identified several druggable targets including kinases AXL, PEAK1 and TGFBR2, and proteases FAP,UCHL1 and MMP2/14, as specific targets for basal B subclass. Validation experiments further confirmed basal B-specific roles of kinase AXL and protease FAP in mediating aggressive phenotypes, thus emphasizing on the importance of exploiting these novel kinase and protease targets in a subclass-specific manner for improved and tailored cancer treatment.

FCS 20 2015

Background: KIF11 is needed for formation of bipolar spindle in metaphase. KIF11 suppression leads to prolonged mitotic arrest, subsequent cell death in mitosis. We established the efficacy of KIF11 inhibition on tumor proliferation in embryonal brain tumors. Medulloblastoma (MB) is a type of embryonal brain tumor and the most common malignant brain tumor in children. p53 mutation defines a high-risk group in MB patients. Aims: (1) Evaluate in-vitro effects of KIF11 inhibition on tumor proliferation in MB, (2) Compare cell death mechanisms in p53-Wild Type (p53-WT) versus p53-Mutant cells, (3) Investigate in-vivo efficacy of KIF11 inhibition in patient-derived orthotopic xenograft (PDOX) mouse models of MB. Results: KIF11 inhibition effectively suppressed tumor cell proliferation in 4 MB cell lines. KIF11 inhibition resulted in G2M arrest and decrease in G1 phase in both p53-WT and p53-Mutant cells. Only p53-Mutant cells undergo massive late apoptotic/necrotic cell death after KIF11 inhibition while p53-WT cells did not. p53-WT

P028Dysregulated 3’UTR Splicing Promotes Oncogene Expression and Cancer Progression

P027Cell Fates of Medulloblastoma from Anti-Mitotic effects of KIF11 Suppression Differ with p53 Mutant Status

Qian-Wei Lim1,2, Sekar Karthik1, Qi Lin6,7, YuChen Du6,7, Ching C Lau3-5, Adesina Adekunle3,4,7, Jack Su Meng Fen3-5, Angela Major8, M. Tarek Elghetany3,4,8, Kam-Man Hui1,9,11, Xiaonan Li3-6, Wan-Yee Teo1-3,5,9-11

1Humphrey Oei Institute of Cancer Research, National Cancer Center Singapore, 2Pediatric Brain Tumor Research Office, SingHealth-Duke-NUS Academic Medical Center, Singapore, 3Baylor College of Medicine, Houston, TX, USA, 4Dan L. Duncan Cancer Center, Houston, TX, USA, 5Department of Pediatrics, Division of Hematology-Oncology, Texas Children’s Cancer Center, 6Northwestern University Feinberg School of Medicine, IL, USA, 7Department of Molecular Pathology, Texas Children’s Hospital, Houston, TX, USA, 8Department of Pathology, Texas Children’s Hospital, Houston, TX, USA, 9Institute of Molecular and Cell Biology, A*STAR, Singapore,10KK Women’s & Children’s Hospital, Singapore, 11Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore

cells demonstrated increase in pH2AX and c-caspase 3 indicative of DNA-damage and apoptosis after KIF11 inhibition. This phenomenon was not observed in p53-mutant cells. Mutant-p53 protein decreases while WT-p53 protein increases after KIF11 inhibition. c-PARP was elevated after KIF11 inhibition in both p53-WT and p53-Mutant cells, indicating apoptosis. KIF11 expression was enriched among patient MB tumors. In-vivo, PDOX mice bearing p53-WT MB tumor cells demonstrated significant improvement in overall survival with KIF11 inhibition (p=0.0003, Log-rank test). Conclusions: Our results suggest (1) cell death mechanisms with KIF11 inhibition differ with p53 mutant status, (2) WT-p53 may be implicated in DNA-damage repair when pH2AX increased. This p53 function may be aberrated in mutant-p53 as the phenomenon was observed only in WT-p53 and not mutant-p53 cells, (3) Different treatment response with KIF11 inhibition for MBs harboring p53-mutation.

Jia Jia Chan1, Bin Zhang1, Zhi Hao Kwok1, Xiao Hong Chew1, Chun You Lim1, Avencia Sanchez-Mejias1, Henry Yang1, Xavier Roca2, Leilei Chen1,3, Yvonne Tay1,4

1Cancer Science Institute of Singapore, 2School of Biological Sciences, Nanyang Technological University, 3Department of Anatomy, National University of Singapore, 4Department of Biochemistry, National University of Singapore

The majority of mammalian genes generate mRNAs with different 3’ untranslated regions (3’UTRs). Alternative 3’UTRs are produced via multiple mechanisms, and function as important post-transcriptional regulators. They are widespread and have been implicated in disease development through their differential modulation of gene expression and function. In cancer, most known alternative 3’UTRs are derived from alternative polyadenylation, whereas 3’UTR splicing remains poorly understood as most splicing research has focused on changes in protein-coding potential. Through a pan-cancer, transcriptome-wide analysis of 3’UTR splicing, we identify the oncogene CTNNB1 as the top candidate with the most highly dysregulated spliced 3’UTR (3’SP) in

40% of the tumor samples analyzed. The CTNNB1 3’SP is significantly upregulated in hepatocellular carcinoma (HCC), one of the most prevalent and molecularly heterogenous cancers. Furthermore, high levels of its 3’UTR splicing are correlated with poorer survival for HCC patients. 3’UTR splicing of CTNNB1 promotes CTNNB1 expression, cell proliferation and migration. Critically, blocking CTNNB1 3’UTR splicing and the inhibition of its 3’SP variant abrogate its oncogenic phenotype. Overall, our findings highlight the significance of 3’UTR splicing in cancer and suggest that its detection and targeting may represent new avenues for the development of more robust diagnostics and therapeutics.

FCS 20 2016

P029Induction of Metastatic Cancer in the Mouse Oxyntic Stomach Through Isthmal Stem/Progenitor Cells.

Background & Aims: Metaplasia and gastric cancer are reportedly derived from dedifferentiation of gastric chief cells. However, the canonical gastric lineage relationships during adult homeostasis are hotly debated, and the cellular origin of cancer in the gastric corpus remains uncertain. We, therefore, investigated whether chief cells represent the cellular origin of gastric cancer using a novel pepsinogen C specific CreERT2 recombinase mouse model.

Methods: We generated a pepsinogen C-CreERT2 (Pgc-CreERT2) knock-in mouse model and crossed Pgc-CreERT2/+ mice with R26-EYFP mice to generate Pgc-CreERT2/R26-EYFP (Pgc/YFP) mice to investigate the physiological roles of pepsinogen C expressing cells in maintaining epithelial homeostasis. Gastric tissues were collected, followed by lineage-tracing experiments, and histology and immunofluorescence were examined. We further established Pgc-CreERT2;KrasG12D/+ mice and investigated whether gastric chief cells are responsible for metaplasia and the precancerous state in gastric glands. Furthermore, to investigate cancer development

Daisuke Douchi, Akihiro Yamamura, Junichi Matsuo, Mitsuhiro Shimura, Sabirah Chen, Napat Nuttonmanit, Pang ShuChin, Linda Shyue Huey Chuang, and Yoshiaki Ito

Cancer Science Institute of Singapore, National University of Singapore

from chief cells, we crossed Pgc-CreERT2/+ mice with conditional KrasG12D, Apcflox, Trp53flox mice, and then activated the Kras, and inactivated the Wnt/β-catenin, and Trp53 signaling pathways.

Results: In the Pgc/YFP mouse model, YFP labeled chief cells accurately as expected. However, pepsinogen C mRNA was detected throughout chief cell, neck cell and isthmal stem/progenitor regions, although at lower levels. In Pgc-CreERT2;KrasG12D/+ mice, Pgc-expressing cells with a KrasG12D/+ mutation gave rise to pseudopyloric metaplasia, to which we have evidence to be generated from isthmal stem/progenitor cells. Furthermore, Pgc-CreERT2;KrasG12D/+;Apcflox/flox mice presented intramucosal gastric cancer, while Pgc-CreERT2;KrasG12D/+;Apcflox/flox;Trp53flox/flox mice presented invasive and metastatic cancer.

Conclusions: We identified most likely cellular origin of metaplasia and gastric cancer, unexpectedly, to be isthmal stem/progenitor cells by utilizing a Pgc-CreERT2 knock-in mouse model.

P030Widespread Repression of Gene Expression in Cancer by a Wnt/β-catenin/MAPK Pathway

Nathan Harmston1,2, Jun Yi Stanley Lim1, Enrico Petretto3, David M. Virshup1,4 and Babita Madan1

1Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore 169857, 2Science Division, Yale-NUS College, 16 College Avenue, Singapore, Singapore 138527, 3Center for Computational Biology and Program in Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, Singapore, Singapore 169857, 4Department of Pediatrics, Duke University School of Medicine, Durham, NC, USA 27710

Aberrant Wnt signaling drives a number of cancers through regulation of diverse downstream pathways. Wnt/β-catenin signaling achieves this in part by increasing the expression of oncogenes such as MYC and cyclins. In this study, global assessment of the Wnt-regulated transcriptome in vivo in genetically distinct cancers demonstrates that Wnt signaling suppresses the expression of as many genes as it activates. We examined this large set of genes that are upregulated upon inhibition of Wnt signaling in Wnt-addicted pancreatic and colorectal cancer models.

We find that inhibiting Wnt signaling leads to this marked increase in gene expression by activating ERK and JNK. These gene expression changes can be mitigated in part by concurrent inhibition of MEK. Our findings suggest that increased Wnt signaling in cancer may be an essential modulator of oncogenic MAPK signaling to prevent RAS-mediated senescence. These results shift the paradigm from “Wnt/β-catenin turns on genes” to a more nuanced view where Wnt/β-catenin signaling drives both widespread gene repression and activation.

FCS 20 2017

P031Molecular Mimicry of Niche-Interacting Status by ITGA9 Overexpression Underlies the Extramedullary Maintenance of Leukemia Stem Cells in AML.

Akiko Niibori-Nambu1, Chelsia Qiuxia Wang1,2, Desmond Wai Loon Chin1, Jing Yuan Chooi1, Hiroki Hosoi1, Giselle Sek Suan Nah1, Branko Cirovic, Darren Qiancheng Tan1, Peter J.M. Valk3, Toshio Suda1,4, Wee Joo Chng1, Henry Hang1, Vinay Tergaonkar2, Ichiro Taniuchi5 and Motomi Osato1,4, 6,7*

1Cancer Science Institute of Singapore, National University of Singapore, Singapore, 2Institute of Molecular and Cell Biology, A*STAR, Singapore, 3Department of Hamatology, Erasmus University Medical Center, Rotterdam, The Netherlands, 4International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan, 5Laboratory for Transcriptional Regulation, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan, 6Institute of Bioengineering and Nanotechnology, A*STAR, Singapore, 7Department of Pediatrics, National University of Singapore, Singapore

Leukemia stem cells (LSCs), being dormant at steady state, are widely believed to reside in the well-characterized bone marrow niches; however, the capacity of the BM niches which accommodate LSCs is limited, and most LSCs are being maintained outside the BM, namely the extramedullary spaces. The molecular basis for this long-standing paradox remains elusive. Here, we show that integrin a9 overexpression (ITGA9 OE) plays a pivotal role in the extramedullary maintenance of LSCs by molecularly mimicking the niche-interacting status, through the binding with its soluble-form ligand, osteopontin (Opn). Retroviral insertional mutagenesis conducted on a leukemia-prone Runx-deficient mice identified Itga9 OE as a novel leukemogenic event. ITGA9 OE activates p38MAPK and AKT signaling pathways, and facilitates the formation of a super-enhancer for c-MYC.

The elevated c-MYC expression subsequently enhances ribosomal biogenesis to overcome the cell integrity defect caused by the preexisting Runx alteration. The ITGA9-Myc axis, originally found in mice, was further confirmed to be active in various types of human acute myeloid leukemias (AMLs) besides RUNX leukemias. In addition, ITGA9 was experimentally shown to be a functional LSC marker in AML. Notably, the binding of ITGA9 with soluble Opn, a known negative regulator against HSC activation, induced LSC dormancy, while the disruption of ITGA9-soluble Opn interaction caused rapid cell propagation. These findings suggest that the ITGA9 OE leads to both active leukemia cell proliferation and increase of dormant LSCs in a well-balanced manner, thereby maintaining LSCs extramedullarly. The ITGA9-Opn axis would serve as a novel therapeutic target in AML.

P032Investigating Synergistic Drug Combinations with PRMT5 Inhibition for Anti-Leukemia Therapy

Luca Pignata1,2, , Brenda Y. Ha1,Jia Yi Fong1,3, Denis Torre4, Ernesto Guccione1,2,4,5

1Institute of Molecular and Cell Biology, Singapore, 2Department of Biochemistry, National University of Singapore, Singapore, 3NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, 4Department of Oncological Sciences and Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, 5Department of Pharmacological Sciences and Mount Sinai Center for Therapeutics Discovery, Icahn School of Medicine at Mount Sinai,New York

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are hematopoietic malignancies characterized by abnormal proliferation and differentiation of myeloid stem cells. Currently, there is no effective treatment for these diseases. Recent studies have shown that hematopoietic malignancies are susceptible to PRMT5 inhibition as a single agent or in combination with other drugs. In order to identify new and more effective drugs that can synergize with PRMT5 inhibitors, we performed a drug screen using a panel of inhibitors against candidate targets in human AML cell line (MV4-11) treated with different concentrations of PRMT5 inhibitor. Candidate targets for screening were selected based on their established clinical relevance in AML. We found that GSK-LSD1, an inhibitor of the lysine

demethylase LSD1, showed the strongest synergistic effect on viability in MV4-11. Moreover, this anti-proliferative activity correlated with a strong induction of apoptosis, suggesting that the explanation behind the synergism was the activation of the apoptotic pathway. Furthermore, we observed the same phenotype in different human leukemia cell lines (U937, K562 and Molm13). An analogous synergistic effect was seen in vivo where the simultaneous inhibition of PRMT5 and LSD1 resulted in a significant decrease of tumor growth in our mouse xenograft model MV4-11. In summary, our findings reveal a novel synergism from combined inhibition of PRMT5 and LSD1 in AML cell lines, and may offer an improved combination drug therapy approach for the treatment of leukemia.

FCS 20 2018

P033Three-dimensional Genome Organization Maps in Normal Haematopoietic Stem Cells and Acute Myeloid Leukemia

*Co-first authors ^Corresponding author

Acute Myeloid Leukemia (AML) is one type of the blood cancer that is also a highly lethal disease associated with genetic and epigenetic alterations. Oncogenes such as MEIS1 and HOXA9 which regulate stemness-related transcriptional programs may cause AML by aberrant expression associated with dysregulated chromatin interactions. Here we obtained and analyzed the 3D genome organization maps in both AML and

Benny Wang1,2*, Lingshi Kong1,2*, Deepak Babu2*, Ruchi Choudhary1,2, Winnie Fam2, Jia Qi Tng2, Yufen Goh2, Xin Liu3, Fang Fang Song3, Priscella Chia3, Ming Chun Chan4, Omer An2, Cheng Yong Tham2, Touati Benoukraf2,5, Henry Yang2, Wilson Wang4, Wee Joo Chng2,3,6, Daniel Tenen2, Melissa Jane Fullwood1,2,7^1School of Biological Sciences, Nanyang Technological University, 2Cancer Science Institute of Singapore, National University of Singapore, 3National University Cancer Institute, National University Health System, 4Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 5Memorial University of Newfoundland, 6Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, 7Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore.

normal CD34+ clinical haematopoietic stem cells. The Frequently Interacting Region (FIRE) in MEIS1 region were found to be absent in 2 AML samples which shows low MEIS1 levels compare with normal samples and AML samples with the FIRE. The CRISPR excision in this FIRE led to the MEIS1 expression loss. This progress gave us a potential therapeutic way of AML that suggest a new candidate of target epigenetic inhibitors. This process of 3D genomic analysis can make us notice more on FIRE influences in cancer researches.

P034Role of Long Non-Coding RNA ELF3-AS in Hepatocellular Cacinoma

Fernando Bellido Molias1,2 Lim Wei Jie Max1,2 Jasmin Teo Xin Jia1,2

1Cancer Science Institute of Singapore, 2Department of Medicine, National University of Singapore, Singapore

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the fourth most frequent cause of cancer-related deaths in the world. Despite advancing healthcare, 5-year survival of HCC is still very poor. Non-coding antisense RNA (ncNAT) is a relatively new but growing topic in the field of cancer molecular research. Some functions linked to ncNATs include effects on transcription initiation of other genes via promoter competition, direct transcriptional effects, etc.

Initial screening of 6 patient samples using stand-specific RNA sequencing analysis showed an upregulation of ELF3-AS1 in HCC. In addition, data from TCGA indicates that ELF3-AS is a strong biomarker of poor survival. Besides, its counterpart sense gene, ELF3, which encodes a relevant transcription factor in liver, also shows upregulation in HCC. This positive correlation

suggested a potential regulative role of ELF3-AS1 on ELF3. Using HCC cell lines as a model, we explored the functional role of ELF3-AS1 in liver cancer. Silencing of ELF3-AS1 using Antisense LNA GapmeRs and shRNAs in Huh7 and PLC/PRF/5 cells, which abundantly express this ncNAT, revealed a clear decrease in cell proliferation and survival. This effect was confirmed using the cell proliferation kit XTT and colony formation assays. Moreover, overexpression of ELF3-AS1 in these two cell lines and in SNU398, which basal ELF3-AS1 levels are very low, consistently showed an increase in cell growth. Additionally, silencing of ELF3-AS1 showed a specific decrease in ELF3 expression, especially of the ELF3-201 isoform which is, as ELF3-AS1, overly overexpressed in HCC. Luciferase results supported a potential participation of ELF3-AS1 in the promoter activation of ELF3. Further work is ongoing to continue exploring the impact of ELF3-AS1 in HCC development.

FCS 20 2019

P035Benchmarking Joint Multi-Omics Dimensionality Reduction Approaches for Cancer Study

High-dimensional multi-omics data are now standard in cancer research. They can greatly enhance our understanding of cancer onset and progression when effectively integrated. To achieve proper integration, joint Dimensionality Reduction (jDR) methods are among the most efficient approaches. However, several jDR methods are available, urging the need for a comprehensive benchmark with practical guidelines.

We performed a systematic evaluation of nine representative jDR methods using three complementary benchmarks. First, we evaluated their performances in retrieving ground-truth sample clustering from simulated multi-omics datasets. Second, we used TCGA cancer data to assess their strengths in predicting survival, clinical annotations and known pathways/biological processes. Finally, we assessed their classification of multi-omics single-cell data profiled from cancer cell lines.

When performing clustering on simulated multi-omics datasets, intNMF, intrinsically designed as a clustering algorithm, displayed the most promising performance. In the cancer data sub-benchmark, when we evaluated

Laura Cantini1, Pooya Zakeri2, Celine Hernandez1, Aurelien Naldi1, Denis Thieffry1,3, Elisabeth Remy4, Anaïs Baudot2,5

1Computational Systems Biology Team, Institut de Biologie de l’Ecole Normale Supérieure, CNRS, INSERM, Ecole Normale Supérieure, Université PSL, Paris, France, 2Aix Marseille Univ, INSERM, Marseille Medical Genetics, CNRS, Turing Center for Living Systems, Marseille, France, 3 Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore,4Aix Marseille University, CNRS, Centrale Marseille, I2M, Turing Center for Living Systems, Marseille, France, 5Barcelona Supercomputing Center (BSC), Barcelona, Spain.

the associations of the factors with survival or clinical annotations, MCIA, JIVE, MOFA, and RGCCA were the most efficient methods. When assessing the associations of the metagenes with biological annotations, MCIA and tICA were the most efficient. Finally, in the last sub-benchmark, when clustering single-cell multi-omics data, MSFA and tICA, as well as MCIA and intNMF, outperformed other approaches.

Overall, our benchmark suggests that intNMF should be prioritized by researchers focusing on clustering samples. However, intNMF is not effective when assessing the quality of individual factors and metagenes. Researchers interested in exploring factor-level information, such as associations with clinical annotation or survival, should rather consider MCIA, JIVE, MOFA and RGGCA. When focusing on the underlying biology of the metagenes, tICA and MCIA should be prioritized. Finally, our study highlights the versatility of MCIA. The full code developed for of this benchmark study is implemented in a Jupyter notebook (momix) to foster reproducibility, and support users and future developers.

P036CSI NGS Portal: An Online Platform for Automated NGS Data Analysis and Sharing

Ömer An, Kar-Tong Tan, Ying Li, Jia Li, Chan-Shuo Wu, Bin Zhang, Leilei Chen, Henry Yang

Cancer Science Institute of Singapore, National University of Singapore, Singapore

Background: Next-generation sequencing (NGS) has been a widely-used technology in biomedical research for understanding the role of molecular genetics of cells in health and disease. A variety of computational tools have been developed to analyse the vastly growing NGS data, which often require bioinformatics skills, tedious work and significant amount of time.

Methods: To facilitate data processing steps minding the gap between biologists and bioinformaticians, we developed CSI NGS Portal, an online platform which gathers established bioinformatics pipelines to provide fully automated NGS data analysis and sharing in a user-friendly website.

Results: The portal currently provides 17 standard pipelines for analysing data from DNA, RNA, smallRNA, ChIP, RIP, 4C, SHAPE, circRNA, eCLIP, Bisulfite and scRNA sequencing, and is flexible to expand with new pipelines. The users can upload raw data in fastq format and submit jobs in a few clicks, and the results will be self-accessible via the portal to view/download/share in real-time. The output can be readily used as the final report or as input for other tools depending on the pipeline.

Conclusions: Overall, CSI NGS Portal helps researchers rapidly analyse their NGS data and share results with colleagues without the aid of a bioinformatician. The portal is freely available at: https://csibioinfo.nus.edu.sg/csingsportal

FCS 20 2020

P037Single Cell Atlas of Human Hepatocellular Carcinoma

Ankur Sharma1,*, Justine Jia Wen Seow1,9, Charles-Antoine Dutertre2,3,9, Rhea Pai1, Archita Mishra2, Gurmeet Singh2, Shabnam Khalilnezhad2, Regina Men Men Wong1, Ahad Khalilnezhad2, Samydurai Sudhagar1, Hui Min Teo1, Tony Kiat Hon Lim4, Brian K P Goh5, Jerry Chan6, Pierce KH Chow7,*, Florent Ginhoux2,8,*, Ramanuj DasGupta1,10,*

1Genome Institute of Singapore, A*STAR, 60 Biopolis Street, Genome, #02-01, Singapore 138672, Singapore, 2Singapore Immunology Network (SIgN), A*STAR, 8A Biomedical Grove, Immunos Building, Level 3 and 4, Singapore 138648, Singapore, 3Program in Emerging Infectious Disease, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore, 4Department of Pathology, Singapore General Hospital, Singapore 169608, Singapore, 5Department of Hepato-Pancreato-Biliary and Transplant Surgery, Singapore General Hospital, Singapore 169608, Singapore, 6Department of Reproductive Medicine, KK Women’s and Children’s Hospital, Singapore 229899, Singapore, 7Division of Surgical Oncology, National Cancer Centre, Singapore 169610, Singapore,8Shanghai Institute of Immunology, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China, 9These authors contributed equally, 10Lead Contact

P038ZRSR1 Cooperates with ZRSR2 in Regulating Splicing of U12-type Introns in Murine Hematopoietic Cells

Tumors reside and evolve in a complex ecosystem of immune and non-immune stromal cells. We are employing multi-sectoral single-cell RNA-seq to catalogue intra-tumor heterogeneity in human hepatocellular carcinoma (HCC). We have generated single-cell atlas form >100,000 cells from fifteen individual samples, each consisting of 2-5 tumor and matched adjacent normal sectors. In total, we profiled 57 individual tumor and normal sectors consisting of HBV+ and HBV- HCC. We also compared various cryopreservation technologies to understand the impact on cellular recovery. In total we identified >70

distinct cells-states in HCC including novel, previously uncharacterised subpopulations. Most importantly, we consistently observed a marked heterogeneity in tumor infiltrating immune cells suggesting a dynamic tumor-TME interactions. Taken together this resource provides unprecedented insights into biology of liver cancer and paves the way for patient stratification.

Keywords hepatocellular carcinoma, tumor microenvironment, myeloid cells, reprogramming, sc-RNA-seq

Zeya Cao1,2,7, Vikas Madan1,7, Weoi Woon Teoh1, Pushkar Dakle1, Lin Han1,2, Pavithra Shyamsunder1,3, Siqin Zhou1, Jia Li1, Hazimah Binte Mohd Nordin1, Shi Jizhong1, Yu Shuizhou1, Henry Yang1, Md Zakir Hossain1, Wee Joo Chng1,2,4, and H. Phillip Koeffler1,5,6

1Cancer Science Institute of Singapore, 2Department of Medicine, National University of Singapore, Singapore, 3Programme in Cancer and Stem Cell Biology, Duke–NUS Medical School, Singapore, 4Hematology-Oncology, National University Cancer Institute, NUHS, Singapore, 5Cedars-Sinai Medical Center, Division of Hematology/Oncology, UCLA School of Medicine, Los Angeles, USA, 6National University Cancer Institute, National University Hospital Singapore, Singapore, 7These authors contributed equally to this work.

Mutations of the spliceosome genes constitute most frequent genetic alterations in myelodysplastic syndromes (MDS). Somatic inactivating alterations of splicing factor, ZRSR2, occur in ~10% of MDS patients. Mutations/loss of ZRSR2 in human myeloid cells causes aberrant splicing, primarily affecting splicing of the U12-type introns. To investigate further the function of ZRSR2 in hematopoietic development and splicing, we generated Zrsr2 knockout (KO) mice. Our comprehensive analyses of hematopoietic compartment in ZRSR2 KO male mice indicated that ZRSR2 was dispensable for hematopoietic development. Moreover, mis-splicing of U12-type introns in ZRSR2 KO murine myeloid precursors was modest compared to the ZRSR2 mutant MDS bone marrow and ZRSR2 knockdown human AML cells. To decipher the unexpected observation that deletion of Zrsr2 in mice did not impact hematopoietic development and affected modestly splicing of U12-type introns, we investigated the role of a closely-related homolog, ZRSR1. We utilized shRNAs to silence

ZRSR1 in murine myeloid precursors and assessed splicing using RNA-Sequencing. Wildtype myeloid cells expressing Zrsr1 shRNA did not display significant mis-splicing of the U12-type introns. However, knockdown of Zrsr1 in ZRSR2-deficient myeloid cells exacerbated mis-splicing of the U12-introns. This suggested that ZRSR1 contributes to regulation of U12-spliceosome in murine hematopoietic cells. Increased mis-splicing of U12-type introns in ZRSR1/ZRSR2-deficient cells was validated using quantitative RT-PCR. 32D cells lacking either one or both ZRSR proteins also verified the compensatory role of murine ZRSR1 in splicing of U12-type introns. We also demonstrated that aberrant retention of U12-type introns of MAPK9 and MAPK14 caused by deficiency of ZRSR proteins leads to their reduced protein expression in both human and murine myeloid cells. Overall, our study highlights that both ZRSR1 and ZRSR2 are functional components of the murine U12-spliceosome, and depletion of both proteins is required to model accurately ZRSR2-mutant MDS in mice.

FCS 20 2021

P039Ultra-Deep Sequencing Of Blood And Urine To Test For Exposure To Aristolochic Acids

Arnoud Boot1, Po-Hung Lin2, Willie Yu1, Fang Yin Lo3, Jesse Salk3, Clint Valentine3, See Tong Pang2, Steven George Rozen1

1Centre for Computational Biology and program for Cancer Stem Cell Biology, Duke-NUS Medical School, Singapore, 2Division of Urooncology, Department of Urology, Chang Gung University and Memorial Hospital, Linkou, Taiwan, 3TwinStrand Biosciences, Seattle, USA

Aristolochic acids (AAs) are a class of highly potent mutagens and nephrotoxins present in some herbal remedies. AAs contribute to large proportions of urinary tract and liver cancers in Asia. Currently the only available test for AA exposure is DNA-sequencing of tumor tissues. Although this helps attribute a cancer’s etiology, it is less useful for prevention efforts. A non-invasive test for AA-exposure in healthy individuals would make it possible to identify exposure clusters where source control efforts would be most effective, as well as high-risk individuals who could benefit from intensified cancer screening.

Therefore, we used duplex sequencing to detect the AA mutational signature (SBS22) in tissues from 12 upper tract urothelial cancer patients and in blood and urine from a subset of these patients. We also performed whole-genome sequencing (WGS) of all resected tissues.

For patients whose tumors showed SBS22 by WGS, most normal tissues did not show SBS22 by WGS. Conversely, duplexsequencing did not detect SBS22 in most of these tumor tissues, but did detect it in most normal tissues. Thus, clonal expansion enables WGS detection but obstructs duplex sequencing detection of AA exposure. Importantly, however, cellular DNA from urine and blood consistently showed SBS22 in patients whose tumors showed AA-exposure by WGS.

In conclusion; urine and blood samples are ideal substrates for assessing exposure to mutagens such as AAs by duplexsequencing. Furthermore, identification of SBS22 in blood suggests more widespread systemic mutagenesis than previously thought.

P041HSP90 Inhibitor, Geldanamycin, Synergizes Cisplatin Sensitivity in Cancer Stem Cells of Oral Squamous Cell Carcinoma

Amnani Aminuddin1, Eng Wee Chua1, Chee-Onn Leong2,3 & Pei Yuen Ng1

1Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Malaysia, 2School of Pharmacy, International Medical University, Malaysia, 3Centre for Cancer and Stem Cell Research, Institute for Research, Development and Innovation, International Medical University, Malaysia

Cisplatin is the first-line chemotherapeutic agent for the treatment of oral squamous cell carcinoma (OSCC). Nevertheless, the complexity of cisplatin sensitivity and resistance remains an obstacle to its chemotherapeutic efficacy. Current chemotherapy effectively removes the bulk of tumour cells, while a therapy-insensitive subpopulation in a heterogeneous tumour, known as cancer stem cells (CSCs) could not be eliminated, leading to cancer recurrence. Herein, this present work aimed to rapidly identify candidate compounds that inhibit cell viability of CSCs derived from OSCC through a high-throughput screening (HTS). Firstly, the stem cell-like tumour spheres from OSCC SAS cell line were derived using a sphere-forming assay to represent an in vitro model of CSCs. A combination of cisplatin sensitivity testing, flow cytometry, microarray, qPCR and Western blotting analyses demonstrated that the tumour spheres exhibited enhanced cisplatin resistance and marked stemness features, including increased expression

levels of stemness genes and proteins, common CSCs surface markers, and genes involved in glucose and lipid metabolism. The tumour spheres were then treated with a collection of compounds consisted of bioactive compounds and clinical approved drugs for 72 hours prior to read-out of the cell viability using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS). From the primary screening, 174 out of 1463 compounds significantly inhibited the cell viability of the tumour spheres. Further validation screening showed that geldanamycin, an inhibitor of heat shock protein 90 (HSP90), exerted a highly inhibitory effect on the viability of the tumour spheres. Moreover, the compound also sensitized the tumour spheres to cisplatin in the drug combination testing. Altogether, this work highlighted the potential use of geldanamycin in combination therapy with a standard chemotherapeutic agent, cisplatin, against the enhanced cisplatin-resistant CSCs for improving the treatment outcome in OSCC.

FCS 20 2022

P042Oncohistone-like Endogenous Short H2A Histone Variants are Reactivated in Many Cancers

Jing Quan Lim1,2,3*, Dachuan Huang1,2*, Tiffany Tang4*, Daryl Tan5,6*, Jabed Iqbal7, Cedric Chuan Young Ng8,9, Benjamin Mow10, Qi-Chun Cai11, Li-Mei Poon12, Yok-Lam Kwong13, Won Seog Kim14, Tongyu Lin3,15+, Jin-Xin Bei3+, Choon Kiat Ong1,16,17+, Soon Thye Lim18,19+

1Lymphoma Genomic Translational Research Laboratory, Cellular and Molecular Research, National Cancer Centre Singapore, 11 Hospital Drive, 169610, Singapore, 2ONCO-ACP, Duke-NUS Medical School, 8 College Road, 169857, Singapore, 3State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou 510060, China, 4Division of Medical Oncology, National Cancer Centre Singapore, 11 Hospital Drive, 169610, Singapore, 5Raffles Cancer centre, Raffles Hospital, 585 North Bridge Road #10-00, Singapore 188770, Singapore, 6Department of Haematology, Singapore General Hospital, Outram Road, Singapore 169608, 7Department of Pathology, Singapore General Hospital, 20 College Road, Academia, 169856, Singapore, 8Laboratory of Cancer Epigenome, Division of Medical Sciences, National Cancer Centre Singapore, 11 Hospital Drive, 169610, Singapore, 9Division of Cancer and Stem Cell Biology, Duke-NUS Medical School, 8 College Road, 169857, Singapore, 10Mount Elizabeth Medical Centre, 11Guangdong Provincial People???s Hospital, Guangdong Academy of Medical Sciences, 12Department of Haematology-Oncology, National University Cancer Institute of Singapore, National University Health System, Singapore, 13Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, 14Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 15Department of Medical Oncology, Sun Yat-sen University Cancer Center, Guangzhou 510060, China, 16Genome Institute of Singapore, 60 Biopolis Street Genome, 138672, Singapore, 17Duke-NUS Graduate Medical School, 8 College Road, 169857, Singapore, 18Director’s office, National Cancer Centre Singapore, 19Office of Education, Duke-NUS Medical School, Singapore,* Co-first authors,+ Co-senior authors

Guo-Liang Chew1, Marie Bleakley2,3, Robert K. Bradley2, Harmit S. Malik2,4, Steven Henikoff2,4, Antoine Molaro5* & Jay Sarthy2*1Cancer Science Institute of Singapore, National University of Singapore, Singapore, 2Division of Basics Sciences, 3Division of Clinical Research & 4Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 5GReD, Université Clermont Auvergne, CNRS, INSERM, Clermont–Ferrand, France, *Co-corresponding authors

Short H2A (sH2A) histone variants are primarily expressed in testis in placental mammals. Their incorporation into chromatin is associated with nucleosome destabilization and modulation of alternate splicing. Here, we show that sH2As innately possess features like oncohistone mutations associated with nucleosome instability. Through analyses of existing cancer genomics datasets,

we find aberrant sH2A reactivation in a broad array of cancers, which manifest splicing patterns consistent with global nucleosome destabilization. We posit that short H2As are a novel class of “readymade” oncohistones, whose inappropriate expression contributes to chromatin dysfunction in cancer.

P043Whole-Genome Sequencing Identifies Biomarker of Response to Pembrolizumab in Relapsed/Refractory Natural Killer/T Cell Lymphoma

Background Natural killer/T-cell lymphoma (NKTCL) is an aggressive malignancy. Currently, there is no effective salvage therapy for patients with disseminated or relapsed NKTCL and their prognosis remain dismal. The off-label use of programmed death-1 antibody on patients with relapsed/refractory (RR) NKTCL has seen competent clinical response rates in recent times, but not all patients will experience clinical benefit. This indicates the importance of a definitive biomarker that could predict response for patients with RR-NKTCL. Methods Comparative analyses were done on clinical, histological and genetic features to identify potential biomarkers for response. Two-tailed tests were performed whenever the tests were allowed to do so. We then conducted a retrospective and prospective analysis on 19 and two relapse/refractory (RR) Natural killer/T-cell lymphoma (NKTCL) cases, respectively, when in the absence of other viable therapeutic options, were treated with pembrolizumab. Findings The sequencing data from pre-pembrolizumab tumors revealed frequent (21%, 4/19) somatic programmed

death-ligand 1 (PD-L1) structural rearrangements (PD-L1MUT), which was significantly enriched within the responders to pembrolizumab (P=0.03; 4 PD-L1MUT in 9 responders versus 0 PD-L1MUT in 10 non-responders). Analysis of traditional clinical predictors of response to standard chemotherapy and histological PD-L1 expression revealed no significant association with response to pembrolizumab. Using PD-L1MUT as a predictor for response, statistical performance measures such as sensitivity and specificity of 44% (4/9) and 100% (4/4) were achieved, respectively. When treated with pembrolizumb, patients with tumors harboring PD-L1MUT have significant better overall survival (Hazard ratio=2.97e-09, mean=5.55, 95% C.I. [3.84, 7.26] years, P=0.0279, log-rank test) than those with PD-L1WT (mean=2.59, 95% C.I. [1.25, 3.93] years). Finally, we prospectively identified a patient with RR-NKTCL tumor harboring PD-L1MUT, who achieved complete response to pembrolizumab. Conclusions The observed efficacy of pembrolizmab on patients with PD-L1MUT NKTCL highlights PD-L1MUT as a potential biomarker of response to pembrolizumab.

FCS 20 2023

P044Functional Characterisation Guides Classification of Novel BAP1 Germline Variants

P045Recurrent Tumor-Specific Intron Retention Transcripts in AML Serve as Effective Targets for Neoantigen Immunotherapy and Vaccination

Jing Han Hong 1,2, Siao Ting Chong 3, Po-Hsien Lee 4,5, Sock Hoai Chan 3, Jing Tan 6,7, Hong Lee Heng 1,7, Nur Diana Binte Ishak 3, Bin Tean Teh 1,2,4,5,7,8, *, Joanne Ngeow2,3,9,10, *

1Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore 169857, Singapore, 2Institute of Molecular and Cellular Biology, Agency for Science, Technology and Research, Singapore 138673, Singapore, 3Cancer Genetics Service, Division of Medical Oncology, National Cancer Center, Singapore 169610, Singapore, 4Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599, Singapore, 5Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore 138672, Singapore, 6Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, 510060, Guangzhou, Guangdong, China, 7Laboratory of Cancer Epigenome, Division of Medical Sciences, National Cancer Centre Singapore, Singapore 169610, Singapore, 8SingHealth/Duke-NUS Institute of Precision Medicine, National Heart Centre Singapore, Singapore, 9Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 308232, Singapore, 10Oncology Academic Clinical Program, Duke-NUS Medical School, Singapore 169857, Singapore

BAP1 is a tumour suppressor that regulates protein deubiquitination. Cancers with BAP1 inactivating mutations tend to be more aggressive and with poor clinical outcomes. Individuals with hereditary BAP1 germline variants are at significant risks of developing many cancers including clear cell renal cell carcinoma, mesothelioma and uveal melanoma at a younger age. Therapeutic options are limited, suggesting an urgent need to discover and characterise new pathogenic BAP1 germline variants, which is instrumental to identify at risk individuals and their family members, to allow for early detection and therapeutic intervention strategies. In this study, we aim to functionally characterise known BAP1 pathogenic and likely benign germline variants to aid in the evaluation and classification of BAP1 germline variants of unknown significance (VUS). We found that

patient-derived lymphoblastoid cell lines with pathogenic BAP1 germline variants showed reduced expression of epithelial-mesenchymal transition (EMT) markers. Pathogenic variants also tend to lead to the truncation of BAP1. In addition, pathogenic variants are observed to have decreased deubiquitination capabilities and aberrant cellular localisation. Our study summarises a set of functional assays that can be used as a first step to curate novel BAP1 germline variants against known pathogenic and likely benign variants. We show that these functional assays are useful for BAP1 variant curation and may be added in the American College of Medical Genetics and Genomics (ACMG) criteria for BAP1 variant classification. This may help with clinical interpretation with respect to patient phenotype.

Li Jia1,3, Ren Xi1,2,3, Li Ying1, Omer An1, Zhang Bin1 and Henry Yang1,2

1Cancer Science Institute of Singapore, National University of Singapore, 2Department of Biochemistry, National University of Singapore, 3Contributed equally to this work, *Corresponding author/Speaker

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy. On the one hand, a number of mutations have been discovered over the last decades. Although some of them were introduced into diagnostic tests, effective treatments without frequent relapse have not been achieved so far. On the other hand, transcriptome studies remain poorly translated into clinics, although genome-wide expression profiling has significantly contributed to the elucidation of AML pathogenesis. In this work, we have studied splicing of AML transcriptome and found that intron retention widely existed in AML patients. By comparing ~1000 AML RNA-seq samples with 10+k RNA-seq samples of different normal tissues, we have discovered a large amount of AML tumor-specific

intron retention events. Importantly some tumor-specific retained introns could be recurrently identified in 50+% AML patients. Such recurrence was not biased towards primary or relapsed samples, treatment responsive or non-responsive patients, leukemia stem cell enriched or depleted samples, young or elderly patients, and various karyotypes. Furthermore, we showed that those recurrent intron transcripts could be potentially translated to a common set of AML-specific neoantigens. This means that a potential common neoantigen immunotherapy could be developed for a large fraction of AML patients, regardless of karyotyping, treatment outcome, and relapse status. Moreover, potentially effective AML vaccinations could be feasible for a wide range of population with a hope of effectively treating/eliminating an age-related disease.

FCS 20 2024

P046c-Met Activation Leads to the Establishment of a TGFβ-receptor Regulatory Network Required for Bladder Cancer Invasion

P047TIP60-ESRP2 Axis Regulates Alternative Splicing of Integrin Subunit Alpha 6

Pieter Eichhorn1, Wen Jing Sim2, 3, Prasanna Iyengar2,3, Jean Paul Thierry2

1Curtin University, WA, Australia; 2Institute of Molecular and Cellular Biology, Singapore; 3Cancer Science Institute of Singapore, Singapore.

Treatment of muscle-invasive bladder cancer remains a major clinical challenge. Aberrant HGF/c-MET upregulation and activation is frequently observed in bladder cancer correlating with cancer progression and invasion. However, the precise mechanisms underlying HGF/c-MET mediated invasion in bladder cancer remains unknown. As part of a negative feedback loop SMAD7 binds to the E3 ligase SMURF2 targeting the TGFβ receptor for degradation. Under these conditions SMAD7 acts as an agonist disrupting the intermolecular interactions within SMURF2, permitting SMURF2 activation. We demonstrate that HGF stimulates TGFβ signalling by inducing c-SRC mediated phosphorylation

of SMURF2 at two tyrosine residues impeding SMAD7 binding and enhancing SMURF2 C2-HECT domain interaction, resulting in SMURF2 inhibition and TGFβ receptor stabilization. This upregulation of the TGFβ pathway by HGF leads to TGFβ-mediated EMT and invasion. Using clinically relevant orthotopic mouse models we show that inhibition of TGFβ signalling completely prevents HGF induced bladder cancer invasion. Furthermore, we make a rationale for the use of TGFβ receptor and MEK inhibitors in the treatment of high grade non-muscle-invasive bladder cancers or early stage muscle invasive bladder cancers.

Shreshtha Sailesh Bhatia1, Roberto Tirado-Magallanes1, Cheng Yong Tham1, H. Phillip Koeffler1,3,4, Ernesto Guccione6,7, Touati Benoukraf1,5 and Sudhakar Jha1,2,*

1Cancer Science Institute of Singapore, National University of Singapore, Singapore, 2Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 3Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 4National University Cancer Institute, National University Hospital Singapore, Singapore, 5Discipline of Genetics, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, NL, Canada, 6Institute of Molecular and Cell Biology (IMCB), A*STAR, Singapore, 7Tisch Cancer Institute, Department of Oncological Sciences and Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, United States of America

Alternative splicing of RNA transcripts can result in a multitude of variations from the genome. Since splicing can occur co-transcriptionally, chromatin structure has been reported to affect the choice of a splice site. TIP60 is a haploinsufficient tumor suppressor gene that is frequently downregulated in cancers. Since TIP60 is a writer of histone and non-histone modifications, we investigated TIP60’s role in modulating alternative splicing.

RNA sequencing was performed in TIP60-depleted MCF10A cells and with MCF10A cells stably expressing wild-type or catalytically inactive TIP60. Interestingly, when TIP60 was depleted, the majority of the differential splicing events were skipped exon events, including increased exon 25 skipping event of ITGA6. The increase in ITGA6 exon 25 skipping with TIP60 depletion

was rescued by overexpression of wild-type but not catalytically inactive TIP60, suggesting the involvement of TIP60’s catalytic activity. TIP60 depletion resulted in an ITGA6 isoform that has a shorter cytoplasmic domain and has been implicated to promote tumor initiation in breast cancer.

To identify the splicing factor that regulates this event, a siRNA screen of 75 splicing factors was employed. The siRNA screen identified ESRP2 to regulate splicing of ITGA6 exon 25. Mechanistically, TIP60 depletion resulted in decreased ESRP2 protein and this regulation was dependent on TIP60’s catalytic activity.

This study suggests that alternative splicing of ITGA6 exon 25 is regulated by the TIP60-ESRP2 axis and its biological consequence will be discussed at the meeting.

FCS 20 2025

P048PRAMEF2, a Cancer Testis Antigen, Inhibits LATS1 Mediated Tumor Suppression

PRAMEF2, member of the PRAME multigene family, is a Cancer Testis Antigen. All cancer testis antigens are known to be selectively expressed in tumor tissues and adult testicular germ cells, thus making them promising targets in cancer immunotherapy. However, the physiological function and regulation of PRAMEF2 in tumorigenesis remains unexplored. Hence, the initial part of our study reports that PRAMEF2 is downregulated upon metabolic stress in a p53 dependent manner. This established PRAMEF2 as a novel target in the p53 transcriptional regulation repertoire. Next to delineate the function of PRAMEF2 in the cell, we performed a proteomic screen which helped us identify PRAMEF2 as a BC-box containing substrate recognition subunit of a Cullin 2 based E3 ubiquitin ligase complex. This screen also revealed LATS1 to be a novel interacting partner of PRAMEF2. LATS1 is a Ser/Thr Kinase, belonging to the core kinase cascade of the Hippo Pathway. LATS1 negatively regulates YAP, the transcriptional regulator, through its phosphorylation dependent cytoplasmic

Madhurima Ghosh1 and Sanjeev Das1

1National Institute of Immunology, New Delhi, India

retention thereby realising its tumor suppressive function. Here we report that PRAMEF2, under unstressed conditions, ubiquitylates LATS1 resulting in its proteasomal degradation. We mapped the site for ubiquitylation to the conserved Lys860 residue in LATS1. This ubiquitylation mediated degradation of LATS1 reversed its negative effect on YAP leading to its enhanced nuclear accumulation. ChIP and qPCR analysis confirmed that YAP nuclear accumulation increased the expression of its target metastatic and proliferative genes. Thus, PRAMEF2, under unstressed conditions, abrogates the tumor suppressive role of LATS1. Furthermore, studies in mice tumor models confirmed that depletion of PRAMEF2 reduces YAP nuclear accumulation and facilitates LATS1 mediated tumor suppression. Additionally, increased PRAMEF2 levels correlate with decreased LATS1 levels in increasing grades of breast carcinoma. Thus these findings highlight the pivotal role of PRAMEF2 in tumorigenesis and provide a novel mechanistic insight into LATS1 regulation.

P049PRMT5 is a Therapeutic Target in Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Malini RETHNAM1, Darren Qiancheng TAN1, Shi Hao TAN1, Li YING1, Henry YANG1 and Toshio SUDA1

1Cancer Science Institute of Singapore

BACKGROUND Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic cancer with unmet clinical need. Other than Tagraxofusp-erzs, treatment options for BPDCN remain limited. A recurrent feature of BPDCN is dysregulated MYC expression, which is associated with dependency on Protein arginine methyltransferase 5 (PRMT5). Interestingly, it was recently shown that BPDCN was enriched for a PRMT5 gene signature. However, the role of elevated PRMT5 in BPDCN remains unexplored. We hypothesise that pharmacological inhibition of PRMT5 might serve as a potential therapeutic strategy in BPDCN.

METHODS To elucidate the role of PRMT5 in BPDCN, we functionally and transcriptionally profiled the response of patient-derived BPDCN cell line CAL-1 to the PRMT5 inhibitor GSK3326595 (GSK595) in vitro, and in vivo using a CAL-1 xenograft model.

RESULTS PRMT5 is upregulated in human BPDCN cells, relative to healthy plasmacytoid dendritic cells. In vitro, PRMT5 inhibition decreased CAL-1 growth and induced

apoptosis in a dose-dependent manner. Correspondingly, bi-daily oral administration of GSK595 significantly mitigated growth of tumours in CAL-1 xenografts. These illustrate that PRMT5 is essential for BPDCN progression. Transcriptome analysis of GSK595-treated CAL-1 cells revealed upregulation of intron retention events, which were overrepresented for RNA methylation genes; including METTL3 - an RNA methyltransferase writer that catalyses m6A modification on mRNAs. PRMT5 inhibition decreased METTL3 expression, along with its targets - MYC and TAZ. Importantly, a dose-dependent reduction in m6A modification was observed in GSK595-treated CAL-1 cells. Overall, our data implicates METTL3 as a downstream effector of PRMT5 in BPDCN. Current work is ongoing to elucidate the functional importance of PRMT5-mediated METTL3 expression in BPDCN.

CONCLUSIONS In summary, our findings identify a novel link between RNA methylation and PRMT5 in BPDCN. We show that elevated PRMT5 activity is essential in BPDCN and targeting it is a viable therapeutic strategy.

FCS 20 2026

P050Unravelling the Role of TRIP12 in Epithelial-Mesenchymal Transition (EMT) and Cancer Progression

Kwok Kin Lee1, Deepa Rajagopalan1,2, Shreshtha Sailesh Bhatia1, Wee Joo Chng1,3,4, and Sudhakar Jha1,2

1Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599, 2Department of Biochemistry 3Department of Medicine Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, 4Department of Haematology-Oncology, National University Cancer Institute of Singapore, National University Health System

Metastasis remains the leading cause of breast cancer death worldwide. Thyroid hormone receptor interactor 12 (TRIP12) is a potential oncogenic E3 ligase most notably involved in the degradation of tumor suppressor p14ARF. Despite this, publicly available patient datasets show an opposite trend, where high TRIP12 is positively correlated with breast cancer distant-metastasis free survival. Epithelial-Mesenchymal Transition (EMT) is an important step in metastasis where relatively polarized, immotile epithelial cells transit to a relatively motile, aggressive mesenchymal-like state. Our investigation

reveals that TRIP12 inhibits EMT and regulates metastasis-related processes (single cell dissemination and anoikis), delineating a cancer progression inhibitory role for TRIP12. Mechanistically, we find that TRIP12 suppresses EMT through ZEB1 and ZEB2 gene expression. In conclusion, we have identified a novel role of TRIP12 in inhibiting EMT and metastasis-related processes, contrary to its oncogenic role in cancer initiation. This uncovers an added complexity of cancer development regulated by TRIP12.

P051Investigation into the Origins of an Ancient BRCA1 Founder Mutation Identified Among Chinese Families in Singapore

Tarryn Shaw#1, Sock Hoai Chan#1, Jing Xian Teo2, Siao Ting Chong1, Shao-Tzu Li1, Eliza Courtney1, Diana Ishak1, Haresh Sankar3, Zoe Li Ting Ang1, Jianbang Chiang1, Marie Loh3, Li Zhou3, Soo Chin Lee4, Hui-Yuan Yeh3, Arun Mouli Kolinjivadi3, Weng Khong Lim2, Joanne Ngeow1,3

#joint first authors1Cancer Genetics Service, National Cancer Centre Singapore, 2SingHealth Duke-NUS Institute of Precision Medicine, 3Lee Kong Chian School of Medicine, Nanyang Technological University, 4Department of Haematology-Oncology, National University Cancer Institute Singapore

Identification of ancestry-specific pathogenic variants is imperative for diagnostic, treatment, management and prevention strategies, and to understand penetrance/modifiers on risk. Our study aimed to determine the clinical significance of a recurrent BRCA1 c.442-22_442-13del variant of unknown significance identified among 13 carriers from six Chinese families, all with a significant history of breast and/or ovarian cancer. We further aimed to establish whether this was due to a founder effect and explore its origins. Haplotype analysis, using nine microsatellite markers encompassing 2.5 megabase pairs around the BRCA1 locus, identified a common haploblock specific to the variant carriers, confirming a founder effect. Variant age was estimated to date back 77.9 generations to 69 BC using the Gamma approach.

On principal component analysis using single nucleotide polymorphisms merged with 1000Genomes dataset, variant carriers were observed to overlap predominantly with the southern Han Chinese population. To determine pathogenicity of the variant, we assessed the functional effect on RAD51 foci formation as well as replication fork stability upon induction of DNA damage and observed an impaired DNA repair response associated with the variant. In summary, we identified an ancient Chinese founder mutation dating back 77.9 generations, possibly common among individuals of southern Han Chinese descent. Using evidence from phenotypic/family history studies, segregation analysis and functional characterization, the BRCA1 variant was reclassified from uncertain significance to pathogenic.

FCS 20 2027

Hereditary cancer syndrome refers to an increased potential of tumor development due to an inherited mutation, with the possible occurrence at younger ages. Although, hereditary cancer syndromes constitute approximately 5% of all diagnosed malignancies, identification of patients is critical as they demand specific, lifelong attention. Most hereditary breast and ovarian cancers (HBOC) are attributed by germline mutations in the genes BRCA1 and BRCA2. From a functional perspective, most of the predisposing genes such as BRCA1, BRCA2, PALB2, RAD51C and RAD51D are involved in homologous recombination (HR), the major pathway for repair of DNA double-strand breaks in mammalian cells. BRCA1/2 mutated tumors deficient in HR repair benefit from PARP inhibitor which represents an anti-cancer strategy by inducing lethality in cancer cells. Combining immune checkpoint blockade with PARP inhibition has emerged as a field of great interest in clinical practice. However, the data is still scanty especially in Asian population in term of the immune signature of HBOC. In this cohort of clinically

P052Deep Profiling of Immune Landscape as well as Immune Microenvironment of Asian Hereditary Ovarian and Breast Cancers

Li Wen Justina Nadia Lee1*, Siao Ting Chong2*, Arun Kumar Kolinjivadi Chandra Mouli3, Nur Diana Binte Ishak2, Hamed Bashiri1, Tracy Zhijun Tien1, Joe Poh Sheng Yeong1,4,5,6†, Joanne Yuen Yie Ngeow 1,2,3,8†

1Institute of Molecular and Cell Biology, Agency of Science, Technology and Research (A*STAR), Singapore, 2Cancer Genetics Service, Division of Medical Oncology, National Cancer Centre, Singapore, 3Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, 4Cancer Science Institute of Singapore, Yong Loo Lin School of Medicine, Singapore, 5Department of Anatomical Pathology, Singapore General Hospital, Singapore, 6Singapore Immunology Network (SIgN), Agency of Science, Technology and Research (A*STAR), Singapore, 8Oncology Academic Clinical Program, Duke-NUS Medical School Singapore, Singapore, *Contributed equally, †Corresponding authors

genetic tested HBOC, we evaluated formalin-fixed paraffin-embedded (FFPE) archival tissues from 60 hereditary breast cancer and 42 hereditary ovarian cancer utilizing the cutting-edge automated multiplex Immunohistochemistry (m-IHC), followed by precise quantification with pathological image analysis software. Ultimately, the data was used to correlate with the clinicopathological parameter and clinical outcome.The results showed that PD-L1 is significantly higher in ovarian tumors with pathogenic germline BRCA1 and BRCA2. Interestingly, for breast tumors, the similar trend is only observed for BRCA1 but not BRCA2. Further studies are on-going to interrogate the peripheral blood immune cells and other DNA damage relevant markers using techniques such as multi-coloured flow cytometry and multiplex cytokine profiling. We envision a strategic integration of liquid and tissue biopsies, providing a revolutionary strategy to empower novel diagnostic, prognostic and therapeutic strategies of Asian Hereditary Ovarian and Breast cancers.

P053Incidental Finding of Colorectal Cancer in a COVID-19 Patient, followed by Deep Profiling of SARS-CoV-2-associated Immune Landscape and Tumour Microenvironment

Xinru Lim1*, Lawrence Chun Chau Cheung2,3*, Denise Goh1*, Tracy Zhijun Tien1, Jeffrey Chun Tatt Lim1, Jia Lin Ng4, Jenny Guek-Hong Low3,5, Joe Poh Sheng Yeong1,2,6,7†, Tony Kiat Hon Lim2†

1Institute of Molecular and Cell Biology (IMCB), Agency of Science, Technology and Research (A*STAR), Singapore, 2Department of Anatomical Pathology, Singapore General Hospital, Singapore, 3Duke-NUS Medical School, Singapore, 4Department of Colorectal Surgery, Singapore General Hospital, Singapore, 5Department of Infectious Diseases, Singapore General Hospital, Singapore, 6Singapore Immunology Network (SIgN), Agency of Science, Technology and Research (A*STAR), Singapore, 7Cancer Science Institute of Singapore, National University of Singapore, Singapore, *Contributed equally

Background Reports have shown that cancer patients are more vulnerable to COVID-19, with increased disease severity and higher mortality rate. The causes of this vulnerability are likely multifactorial. As little is known about the interplay between COVID-19, cancer and the immune system, it is important to understand the immunopathological processes underlying COVID-19 in cancer patients to further optimise their management.

Methods Hereby, we reported a 45-year-old Indian male with COVID-19 who was incidentally found to have caecal adenocarcinoma. His blood, lymph nodes, normal tissue and tumour samples were obtained 10 days after recovery from COVID-19 for further analysis. Multiplex immunohistochemistry was performed to understand COVID-19-associated tumour immune microenvironment. Moreover, to simulate ex vivo COVID-19 infection, dissociated cells from collected samples were incubated with SARS-CoV-2 peptides or control for 16 hours, followed by 25-colour flow cytometry analysis, to study COVID-19-elicited immune response.

Results Multiplex immunohistochemistry demonstrated upregulated expression of ACE2 in the tumour as compared to adjacent normal tissue, whilst SARS-CoV-2 was detected only in adjacent normal tissue but not within the tumour. We also observed SARS-CoV-2 in other organs such as appendix and lymph nodes, and the presence of tertiary lymphoid structure, abundant T and NK cells were detected within the proximity of the tumour. Additionally, upon stimulation with SARS-CoV-2 peptides, we successfully elicited SARS-CoV-2-specific CD4+ T cells which expressed immune markers such as granzyme B, TNF-a and IFN-g. Deep profiling of the samples is on-going with single-cell sequencing and digital spatial profiling.

Conclusion We believe this is the first report of immune profiling of in situ tumour microenvironment in cancer patient with COVID-19. Our findings showed that, albeit after viral clearance, SARS-CoV-2 viral proteins were still detectable in various tissues, and that SARS-CoV-2-specific T cell responses could also be triggered. Our study highlights the complex interactions between cancer, COVID-19 and the immune system.

FCS 20 2028

P054Mitochondrial MDM2 and TFAM Mediate Metabolic Reprogramming Towards OXPHOS in TKI-Resistant Oncogene-Addicted Cancer

Jie Qing Eu1,2, Li Ren Kong2,3, Jayshree Hirpara1, Boon Cher Goh1,4,5,6, Andrea L Wong1,5,7,8, Shazib Pervaiz2,4,9

1Cancer Science Institute, Singapore, 2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 3Medical Research Council Cancer Unit, University of Cambridge, CB2, 0XZ UK, 4National University Cancer Institute, National University Health System, Singapore, 5Department of Haematology-Oncology, National University Health System, Singapore, 6Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 7Haematology-Oncology Research Group, National University Cancer Institute, Singapore, 8Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 9NUS Graduate School for Integrative Sciences and Engineering, SingaporeThe Warburg effect has been the basic principle of cancer metabolism research in the past decades. However, many studies have disputed the theory of cancer cells switching to aerobic glycolysis for survival due to mitochondria dysfunction. Intriguingly, in the recent years, there are increasing amount of evidences showing an up-regulation of oxidative phosphorylation (OXPHOS) in certain types of cancer. Our group has shown that in oncogene-addicted cancer, prolonged treatment with tyrosine kinase inhibitors (TKI) lead to emergence of resistance, a primary cause of failure in the targeted therapy. Interestingly, when oncogene-addicted cancer cells develop TKI resistance, we observed an increase in OXPHOS activity and mitochondria biogenesis markers, suggesting that a metabolic switch to OXPHOS could be a mechanism of TKI resistance. Additionally, by targeting TKI-resistant cancer with an OXPHOS inhibitor, the resistant cells could be re-sensitized to their respective TKI. Nevertheless, despite the growing interest in mitochondria biogenesis in cancer, there is a lack of inhibitors that are designed to target

proteins regulating mitochondria biogenesis including key regulators PGC1a and TFAM and no clear biomarkers of mitochondria biogenesis and OXPHOS inhibition used in the clinical setting.

Our preliminary results now suggest that an OXPHOS inhibitor currently in a Phase IIa clinical trial, OPC-317, has demonstrated inhibition of mitochondria biogenesis through the downregulation of TFAM and a decrease in mtDNA copy number. In a bid to understand the mechanism, we observed in our model that one of the negative regulators of OXPHOS, mitochondrial MDM2, translocate to the cytosol upon acquiring TKI resistance leading to increase in OXPHOS as well as transcription of mtDNA. Intriguingly, our findings suggest that mitochondrial MDM2 competitively inhibits TFAM translocation to the mitochondria, resulting in inhibition of OXPHOS and mitochondria biogenesis. This project therefore presents a strategy in targeting mitochondria biogenesis in the treatment of TKI-resistant cancer.

P0553-Dimensional Ex Vivo Histology of Excised Basal Cell Carcinoma Enables Easy Detection of Cancer Margins

Yingrou Tan1,2, Yuning Zhang3, Ng Lai Guan2, Tey Hong Liang1,4,5 1Department of Research, National Skin Centre, National Healthcare Group, Singapore. 2Singapore Immunology Network (SIgN), Agency for Science, Technology and Research, A*STAR, Singapor,.3Faculty of Science, National University of Singapore, Singapore, 4Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 5Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore

BackgroundSkin cancer is the 5th most common cancer, with 1.04 million non-melanoma skin cancer cases worldwide in 2018. Skin cancers are managed either through conventional surgical excision or Mohs micrographic surgery (MMS). Margin assessment in conventional excision is achieved through sampling surgical margins through examining 12-16 two-dimensional (2D) sections, but only 0.1-0.2% of a 3cm specimen is examined, making it likely to miss tumour involvement in some margins. This leads to high false negative rates and a higher recurrence with a 5-year recurrence rate of 4.1-10.1% for basal cell carcinoma (BCC). MMS is the gold standard for managing skin cancer, but has a higher cost involved due to the need for specially trained surgeons to perform the procedure.Rapid development of tissue optical clearing techniques in the past 10 years, together with the use of light sheet microscopy has enabled the three-dimensional (3D) visualization of murine and more recently, human tissues.

We propose to leverage on these tissue optical clearing and 3D volumetric imaging techniques to visualize basal cell carcinoma margins in clinical specimens, improving existing false negative rates and to disrupt the current 2D histo(pathological) approach.ResultsWe demonstrate that cancer margins in excised BCC tumours can be easily visualized in 3D using a solvent-based optical clearing method. BCC-positive regions are marked out by staining with epithelial cell adhesion molecule (EpCAM) or a nuclear stain, propidium iodide. Based on these 3D images, it is a relatively simple process to determine if the cancer margins of a sample are clear by mapping the BCC-positive regions onto a tumour map that can be attached to a typical pathology report. This entire workflow can potentially be applied within a clinical histopathology lab as a new way to manage skin cancer, and to reduce the costs associated with MMS for the patient.

FCS 20 2029

P056Keeping the Unimpeded Flow of QI and Blood in Cancer Prevention

Dr Rey

Tiquia Traditional Chinese Medicine Practitioner Philosopher of Science Melbourne, Australia

Seeing ‘qi as matter’ and ‘matter as qi’ and translating Qi, Yin and Yang and the Five elements wu xing as space-time-matter-in-motion, I discuss the balancing attributes of medicinal matter yaowu vis-a-vis cancer as the existential threat to our yinyang existence and its etiology in traditional Chinese medicine. I present a case study of a lung cancer patient whose condition was stabilized by chronoacupuncture and materia medica herbal therapy

i.e. his anti-pathogenic Yang qi [(zheng qi which is the body’s capacity to resist all disease causing agents) and his ‘pathogenic yin qi’ (xie qi which refers to all disease causing factors including cancer as kesou cough )] evolving in a harmonious state of yin and yang balance. Finally, I translated and outlined TCM oncologist, Dr. Jia Kun’s TCM (traditional Chinese medicine) proposals on how to prevent being afflicted by cancer.

P057Intracellular Redox Milieu Regulates Mcl-1 Levels Through Phosphorylation of Mcl-1

Jolin Xiao Hui Lai1, Shazib Pervaiz1,2,3

1 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 2NUS Graduate School for Integrative Sciences and Engineering, Singapore, 3National University Cancer Institute, National University Health System, Singapore

Mcl-1, a member of the Bcl-2 family of anti-apoptotic proteins is highly amplified in malignant cancers as well as implicated in chemotherapy resistance. Due to the short half-life of Mcl-1, the stability of Mcl-1 is regulated largely by post-translational modifications (PTMs) such as phosphorylation and ubiquitination. Where phosphorylation at its PEST domain region at Thr163 residue by kinases result in its stability and extended protein half-life, phosphorylation of Ser159 Mcl-1 results in a shorter half-life due to increased degradation. Given the importance of PTMs in regulating Mcl-1, identification of signalling pathways that could modulate PTMs of Mcl-1 to thereby sensitize tumors to chemotherapy have been explored extensively.

With interest in PTMs, the intracellular redox milieu has been widely reported to modulate protein-protein signalling as well as inducing PTMs, where different ROS species have also been shown to mediate differential effects. For example, enhanced production of O2

- (by either inhibition or downregulation of SOD1/SOD2)

could mediate tumorigenicity and chemoresistance. To that end, our group and others have also previously reported the oncogenicity of superoxide (O2

-), where an elevated O2

- level mediates enhanced anti-apoptotic activity against chemotherapeutic agents through sustained Bcl-2 phosphorylation and the inhibition of O2

-

production conversely sensitized cells to chemotherapy. Nonetheless, the exact mechanisms of how O2

- exerts its pro-survival effects remain elusive.

Our preliminary findings now suggest that elevated intracellular O2

- levels induces Mcl-1 stability in cancer cells through PTMs as a potential mechanism of chemotherapy resistance. Increased level of O2

- induced Mcl-1 was due to the phosphorylation of Thr163 residue of Mcl-1 at the PEST domain which in turn reduced ubiquitination of Mcl-1. An increased O2

- level also mediated resistance to cell death induced by Mcl-1 BH3 specific mimetic, S63845 hence highlighting one of the potential roles O2

- could mediate chemoresistance.

FCS 20 2030

P058Upregulation Of miR-146a During Senescence Promotes Senescence Evasion By Modulating ROS Levels

Liew Qiu Xia Angeline1,2, Shazib Pervaiz1,2

1NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, 2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Cellular senescence is conventionally defined as the permanent state of growth arrest. Traditionally, chemotherapies induce high levels of DNA damage in tumor cells to promote cell death or cellular senescence1. A subpopulation of cells that enters senescence can evade senescence to proliferate, thereby resulting in tumor regrowth which contributes to chemoresistance and tumor aggressiveness1. In fact, a persistent clinical challenge in the management of cancers is tumor recurrence. Multiple studies have demonstrated senescence evasion to be a novel mechanism in which breast cancer cells might recur and/or persist after chemotherapy1-3. However, there is still a lack of understanding of the molecular events that govern the emergence of this tumor regrowth. Notably, Achuthan, et al.2 have reported that these senescence-escaped cells harbor low levels of reactive oxygen species (ROS), unlike senescent cells. As such, it is of high importance to elucidate the regulatory mechanisms that downregulate ROS in senescent cells to understand the mechanisms promoting senescence escape and consequently, the development of chemoresistance

Our preliminary results demonstrate that Doxorubicin can induce senescence in MDA-MB-231 breast carcinoma cells. After an initial phase of apoptotic cell death, these senescent cells can escape growth arrest to proliferate. miR-146a, a biomarker of senescence and a miRNA of interest in our project, was upregulated during senescence. We further observed that these senescence-escaped cells have lower ROS levels compared to senescent cells and this correlates with the upregulation of MnSOD, an antioxidant enzyme. These findings provide evidence that miR-146a, which has been previously shown to downregulate ROS, could be involved in modulating the oxidative stress response in our senescence model thus promoting senescence evasion. Understanding the mechanism behind this conversion from high to low ROS would be beneficial for developing novel strategies to combat chemoresistance by preventing senescence escape.

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