Publications by authors named "Wai Leong Tam"

48 Publications

Association of Rare CYP39A1 Variants With Exfoliation Syndrome Involving the Anterior Chamber of the Eye.

Authors:
Zheng Li Zhenxun Wang Mei Chin Lee Matthias Zenkel Esther Peh Mineo Ozaki Fotis Topouzis Satoko Nakano Anita Chan Shuwen Chen Susan E I Williams Andrew Orr Masakazu Nakano Nino Kobakhidze Tomasz Zarnowski Alina Popa-Cherecheanu Takanori Mizoguchi Shin-Ichi Manabe Ken Hayashi Shigeyasu Kazama Kenji Inoue Yosai Mori Kazunori Miyata Kazuhisa Sugiyama Tomomi Higashide Etsuo Chihara Ryuichi Ideta Satoshi Ishiko Akitoshi Yoshida Kana Tokumo Yoshiaki Kiuchi Tsutomu Ohashi Toshiya Sakurai Takako Sugimoto Hideki Chuman Makoto Aihara Masaru Inatani Kazuhiko Mori Yoko Ikeda Morio Ueno Daniel Gaston Paul Rafuse Lesya Shuba Joseph Saunders Marcelo Nicolela George Chichua Sergo Tabagari Panayiota Founti Kar Seng Sim Wee Yang Meah Hui Meng Soo Xiao Yin Chen Anthi Chatzikyriakidou Christina Keskini Theofanis Pappas Eleftherios Anastasopoulos Alexandros Lambropoulos Evangelia S Panagiotou Dimitrios G Mikropoulos Ewa Kosior-Jarecka Augustine Cheong Yuanhan Li Urszula Lukasik Monisha E Nongpiur Rahat Husain Shamira A Perera Lydia Álvarez Montserrat García Héctor González-Iglesias Andrés F V Cueto Luis F V Cueto Federico Martinón-Torres Antonio Salas Çilingir Oguz Nevbahar Tamcelik Eray Atalay Bilge Batu Murat Irkec Dilek Aktas Burcu Kasim Yury S Astakhov Sergei Y Astakhov Eugeny L Akopov Andreas Giessl Christian Mardin Claus Hellerbrand Jessica N Cooke Bailey Robert P Igo Jonathan L Haines Deepak P Edward Steffen Heegaard Sonia Davila Patrick Tan Jae H Kang Louis R Pasquale Friedrich E Kruse André Reis Trevor R Carmichael Michael Hauser Michele Ramsay Georg Mossböck Nilgun Yildirim Kei Tashiro Anastasios G P Konstas Miguel Coca-Prados Jia Nee Foo Shigeru Kinoshita Chie Sotozono Toshiaki Kubota Michael Dubina Robert Ritch Janey L Wiggs Francesca Pasutto Ursula Schlötzer-Schrehardt Ying Swan Ho Tin Aung Wai Leong Tam Chiea Chuen Khor

JAMA 2021 02;325(8):753-764

Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore.

Importance: Exfoliation syndrome is a systemic disorder characterized by progressive accumulation of abnormal fibrillar protein aggregates manifesting clinically in the anterior chamber of the eye. This disorder is the most commonly known cause of glaucoma and a major cause of irreversible blindness.

Objective: To determine if exfoliation syndrome is associated with rare, protein-changing variants predicted to impair protein function.

Design, Setting, And Participants: A 2-stage, case-control, whole-exome sequencing association study with a discovery cohort and 2 independently ascertained validation cohorts. Study participants from 14 countries were enrolled between February 1999 and December 2019. The date of last clinical follow-up was December 2019. Affected individuals had exfoliation material on anterior segment structures of at least 1 eye as visualized by slit lamp examination. Unaffected individuals had no signs of exfoliation syndrome.

Exposures: Rare, coding-sequence genetic variants predicted to be damaging by bioinformatic algorithms trained to recognize alterations that impair protein function.

Main Outcomes And Measures: The primary outcome was the presence of exfoliation syndrome. Exome-wide significance for detected variants was defined as P < 2.5 × 10-6. The secondary outcomes included biochemical enzymatic assays and gene expression analyses.

Results: The discovery cohort included 4028 participants with exfoliation syndrome (median age, 78 years [interquartile range, 73-83 years]; 2377 [59.0%] women) and 5638 participants without exfoliation syndrome (median age, 72 years [interquartile range, 65-78 years]; 3159 [56.0%] women). In the discovery cohort, persons with exfoliation syndrome, compared with those without exfoliation syndrome, were significantly more likely to carry damaging CYP39A1 variants (1.3% vs 0.30%, respectively; odds ratio, 3.55 [95% CI, 2.07-6.10]; P = 6.1 × 10-7). This outcome was validated in 2 independent cohorts. The first validation cohort included 2337 individuals with exfoliation syndrome (median age, 74 years; 1132 women; n = 1934 with demographic data) and 2813 individuals without exfoliation syndrome (median age, 72 years; 1287 women; n = 2421 with demographic data). The second validation cohort included 1663 individuals with exfoliation syndrome (median age, 75 years; 587 women; n = 1064 with demographic data) and 3962 individuals without exfoliation syndrome (median age, 74 years; 951 women; n = 1555 with demographic data). Of the individuals from both validation cohorts, 5.2% with exfoliation syndrome carried CYP39A1 damaging alleles vs 3.1% without exfoliation syndrome (odds ratio, 1.82 [95% CI, 1.47-2.26]; P < .001). Biochemical assays classified 34 of 42 damaging CYP39A1 alleles as functionally deficient (median reduction in enzymatic activity compared with wild-type CYP39A1, 94.4% [interquartile range, 78.7%-98.2%] for the 34 deficient variants). CYP39A1 transcript expression was 47% lower (95% CI, 30%-64% lower; P < .001) in ciliary body tissues from individuals with exfoliation syndrome compared with individuals without exfoliation syndrome.

Conclusions And Relevance: In this whole-exome sequencing case-control study, presence of exfoliation syndrome was significantly associated with carriage of functionally deficient CYP39A1 sequence variants. Further research is needed to understand the clinical implications of these findings.
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http://dx.doi.org/10.1001/jama.2021.0507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903258PMC
February 2021

Recent advances in proteome-wide label-free target deconvolution for bioactive small molecules.

Med Res Rev 2021 Feb 3. Epub 2021 Feb 3.

Department of Urology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen Urology Minimally Invasive Engineering Center, Shenzhen, Guangdong, China.

Small-molecule drugs modulate biological processes and disease states through engagement of target proteins in cells. Assessing drug-target engagement on a proteome-wide scale is of utmost importance in better understanding the molecular mechanisms of action of observed beneficial and adverse effects, as well as in developing next generation tool compounds and drugs with better efficacies and specificities. However, systematic assessment of drug-target engagement has been an arduous task. With the continuous development of mass spectrometry-based proteomics instruments and techniques, various chemical proteomics approaches for drug target deconvolution (i.e., the identification of molecular target for drugs) have emerged. Among these, the label-free target deconvolution approaches that do not involve the chemical modification of compounds of interest, have gained increased attention in the community. Here we provide an overview of the basic principles and recent biological applications of the most important label-free methods including the cellular thermal shift assay, pulse proteolysis, chemical denaturant and protein precipitation, stability of proteins from rates of oxidation, drug affinity responsive target stability, limited proteolysis, and solvent-induced protein precipitation. The state-of-the-art technical implications and future outlook for the label-free approaches are also discussed.
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http://dx.doi.org/10.1002/med.21788DOI Listing
February 2021

Intratumoural immune heterogeneity as a hallmark of tumour evolution and progression in hepatocellular carcinoma.

Nat Commun 2021 01 11;12(1):227. Epub 2021 Jan 11.

Translational Immunology Institute (TII), SingHealth-DukeNUS Academic Medical Centre, Singapore, Singapore.

The clinical relevance of immune landscape intratumoural heterogeneity (immune-ITH) and its role in tumour evolution remain largely unexplored. Here, we uncover significant spatial and phenotypic immune-ITH from multiple tumour sectors and decipher its relationship with tumour evolution and disease progression in hepatocellular carcinomas (HCC). Immune-ITH is associated with tumour transcriptomic-ITH, mutational burden and distinct immune microenvironments. Tumours with low immune-ITH experience higher immunoselective pressure and escape via loss of heterozygosity in human leukocyte antigens and immunoediting. Instead, the tumours with high immune-ITH evolve to a more immunosuppressive/exhausted microenvironment. This gradient of immune pressure along with immune-ITH represents a hallmark of tumour evolution, which is closely linked to the transcriptome-immune networks contributing to disease progression and immune inactivation. Remarkably, high immune-ITH and its transcriptomic signature are predictive for worse clinical outcome in HCC patients. This in-depth investigation of ITH provides evidence on tumour-immune co-evolution along HCC progression.
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http://dx.doi.org/10.1038/s41467-020-20171-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801667PMC
January 2021

UniPath: a uniform approach for pathway and gene-set based analysis of heterogeneity in single-cell epigenome and transcriptome profiles.

Nucleic Acids Res 2021 02;49(3):e13

Department for Computational Biology, Indraprastha Institute of Information Technology, Delhi 110020, India.

Recent advances in single-cell open-chromatin and transcriptome profiling have created a challenge of exploring novel applications with a meaningful transformation of read-counts, which often have high variability in noise and drop-out among cells. Here, we introduce UniPath, for representing single-cells using pathway and gene-set enrichment scores by a transformation of their open-chromatin or gene-expression profiles. The robust statistical approach of UniPath provides high accuracy, consistency and scalability in estimating gene-set enrichment scores for every cell. Its framework provides an easy solution for handling variability in drop-out rate, which can sometimes create artefact due to systematic patterns. UniPath provides an alternative approach of dimension reduction of single-cell open-chromatin profiles. UniPath's approach of predicting temporal-order of single-cells using their pathway enrichment scores enables suppression of covariates to achieve correct order of cells. Analysis of mouse cell atlas using our approach yielded surprising, albeit biologically-meaningful co-clustering of cell-types from distant organs. By enabling an unconventional method of exploiting pathway co-occurrence to compare two groups of cells, our approach also proves to be useful in inferring context-specific regulations in cancer cells. Available at https://reggenlab.github.io/UniPathWeb/.
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http://dx.doi.org/10.1093/nar/gkaa1138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897496PMC
February 2021

Novel carfilzomib-based combinations as potential therapeutic strategies for liposarcomas.

Cell Mol Life Sci 2021 Feb 26;78(4):1837-1851. Epub 2020 Aug 26.

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

Proteasome inhibitors, such as bortezomib and carfilzomib, have shown efficacy in anti-cancer therapy in hematological diseases but not in solid cancers. Here, we found that liposarcomas (LPS) are susceptible to proteasome inhibition, and identified drugs that synergize with carfilzomib, such as selinexor, an inhibitor of XPO1-mediated nuclear export. Through quantitative nuclear protein profiling and phospho-kinase arrays, we identified potential mode of actions of this combination, including interference with ribosome biogenesis and inhibition of pro-survival kinase PRAS40. Furthermore, by assessing global protein levels changes, FADS2, a key enzyme regulating fatty acids synthesis, was found down-regulated after proteasome inhibition. Interestingly, SC26196, an inhibitor of FADS2, synergized with carfilzomib. Finally, to identify further combinational options, we performed high-throughput drug screening and uncovered novel drug interactions with carfilzomib. For instance, cyclosporin A, a known immunosuppressive agent, enhanced carfilzomib's efficacy in vitro and in vivo. Altogether, these results demonstrate that carfilzomib and its combinations could be repurposed for LPS clinical management.
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http://dx.doi.org/10.1007/s00018-020-03620-wDOI Listing
February 2021

Epigenomes of Human Hearts Reveal New Genetic Variants Relevant for Cardiac Disease and Phenotype.

Circ Res 2020 Aug 12;127(6):761-777. Epub 2020 Jun 12.

From the Cardiovascular Research Institute, National University Health System, Singapore (W.L.W.T., C.G.A.-N., E.W., C.J.M.L., H.S.T., A.P., Z.W., B.P., M.I.A., R.S.Y.F.).

Rationale: Identifying genetic markers for heterogeneous complex diseases such as heart failure is challenging and requires prohibitively large cohort sizes in genome-wide association studies to meet the stringent threshold of genome-wide statistical significance. On the other hand, chromatin quantitative trait loci, elucidated by direct epigenetic profiling of specific human tissues, may contribute toward prioritizing subthreshold variants for disease association.

Objective: Here, we captured noncoding genetic variants by performing epigenetic profiling for enhancer H3K27ac chromatin immunoprecipitation followed by sequencing in 70 human control and end-stage failing hearts.

Methods And Results: We have mapped a comprehensive catalog of 47 321 putative human heart enhancers and promoters. Three thousand eight hundred ninety-seven differential acetylation peaks (FDR [false discovery rate], 5%) pointed to pathways altered in heart failure. To identify cardiac histone acetylation quantitative trait loci (haQTLs), we regressed out confounding factors including heart failure disease status and used the G-SCI (Genotype-independent Signal Correlation and Imbalance) test to call out 1680 haQTLs (FDR, 10%). RNA sequencing performed on the same heart samples proved a subset of haQTLs to have significant association also to gene expression (expression quantitative trait loci), either in (180) or through long-range interactions (81), identified by Hi-C (high-throughput chromatin conformation assay) and HiChIP (high-throughput protein centric chromatin) performed on a subset of hearts. Furthermore, a concordant relationship between the gain or disruption of TF (transcription factor)-binding motifs, inferred from alternative alleles at the haQTLs, implied a surprising direct association between these specific TF and local histone acetylation in human hearts. Finally, 62 unique loci were identified by colocalization of haQTLs with the subthreshold loci of heart-related genome-wide association studies datasets.

Conclusions: Disease and phenotype association for 62 unique loci are now implicated. These loci may indeed mediate their effect through modification of enhancer H3K27 acetylation enrichment and their corresponding gene expression differences (bioRxiv: https://doi.org/10.1101/536763). Graphical Abstract: A graphical abstract is available for this article.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.317254DOI Listing
August 2020

Controls the Dopaminergic/Oligodendroglial Fate through Wnt/β-catenin Signaling Regulation.

Cells 2020 03 13;9(3). Epub 2020 Mar 13.

Department of Biology, University of Padova, 35131 Padova, Italy.

During the development of the central nervous system, the proliferation of neural progenitors and differentiation of neurons and glia are tightly regulated by different transcription factors and signaling cascades, such as the Wnt and Shh pathways. This process takes place in cooperation with several microRNAs, some of which evolutionarily conserved in vertebrates, from teleosts to mammals. We focused our attention on , as its role in the regulation of cell signaling during neural development is still unclear. Specifically, we used human stem cell cultures and whole zebrafish embryos to study, in vitro and in vivo, the role of in the development of dopaminergic (DA) neurons, a cell type primarily affected in Parkinson's disease. We demonstrated that the zebrafish homologue of () is expressed in the forebrain during the development of DA neurons. Moreover, we identified 143 target genes downregulated by , including the neural fate markers TCF4 and TCF12, as well as the Wnt pathway effector TCF7L2. We then demonstrated that negatively regulates the proliferation of DA-progenitors by inhibiting Wnt/β-catenin signaling in zebrafish embryos. In parallel, positively regulates Shh signaling, thus controlling the balance between oligodendroglial and DA neuronal cell fates. In summary, this study identifies a new molecular cross-talk between Wnt and Shh signaling pathways during the development of DA-neurons. Being mediated by a microRNA, this mechanism represents a promising target in cell differentiation therapies for Parkinson's disease.
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http://dx.doi.org/10.3390/cells9030711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140713PMC
March 2020

Genomic landscape of lung adenocarcinoma in East Asians.

Nat Genet 2020 02 3;52(2):177-186. Epub 2020 Feb 3.

Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

Lung cancer is the world's leading cause of cancer death and shows strong ancestry disparities. By sequencing and assembling a large genomic and transcriptomic dataset of lung adenocarcinoma (LUAD) in individuals of East Asian ancestry (EAS; n = 305), we found that East Asian LUADs had more stable genomes characterized by fewer mutations and fewer copy number alterations than LUADs from individuals of European ancestry. This difference is much stronger in smokers as compared to nonsmokers. Transcriptomic clustering identified a new EAS-specific LUAD subgroup with a less complex genomic profile and upregulated immune-related genes, allowing the possibility of immunotherapy-based approaches. Integrative analysis across clinical and molecular features showed the importance of molecular phenotypes in patient prognostic stratification. EAS LUADs had better prediction accuracy than those of European ancestry, potentially due to their less complex genomic architecture. This study elucidated a comprehensive genomic landscape of EAS LUADs and highlighted important ancestry differences between the two cohorts.
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http://dx.doi.org/10.1038/s41588-019-0569-6DOI Listing
February 2020

New High-Throughput Screening Identifies Compounds That Reduce Viability Specifically in Liver Cancer Cells That Express High Levels of SALL4 by Inhibiting Oxidative Phosphorylation.

Gastroenterology 2019 12 22;157(6):1615-1629.e17. Epub 2019 Aug 22.

Department of Pathology, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts.

Background & Aims: Some oncogenes encode transcription factors, but few drugs have been successfully developed to block their activity specifically in cancer cells. The transcription factor SALL4 is aberrantly expressed in solid tumor and leukemia cells. We developed a screen to identify compounds that reduce the viability of liver cancer cells that express high levels of SALL4, and we investigated their mechanisms.

Methods: We developed a stringent high-throughput screening platform comprising unmodified SNU-387 and SNU-398 liver cancer cell lines and SNU-387 cell lines engineered to express low and high levels of SALL4. We screened 1597 pharmacologically active small molecules and 21,575 natural product extracts from plant, bacteria, and fungal sources for those that selectively reduce the viability of cells with high levels of SALL4 (SALL4 cells). We compared gene expression patterns of SALL4 cells vs SALL4-knockdown cells using RNA sequencing and real-time polymerase chain reaction analyses. Xenograft tumors were grown in NOD/SCID gamma mice from SALL4 SNU-398 or HCC26.1 cells or from SALL4 patient-derived xenograft (PDX) cells; mice were given injections of identified compounds or sorafenib, and the effects on tumor growth were measured.

Results: Our screening identified 1 small molecule (PI-103) and 4 natural compound analogues (oligomycin, efrapeptin, antimycin, and leucinostatin) that selectively reduced viability of SALL4 cells. We performed validation studies, and 4 of these compounds were found to inhibit oxidative phosphorylation. The adenosine triphosphate (ATP) synthase inhibitor oligomycin reduced the viability of SALL4 hepatocellular carcinoma and non-small-cell lung cancer cell lines with minimal effects on SALL4 cells. Oligomycin also reduced the growth of xenograft tumors grown from SALL4 SNU-398 or HCC26.1 cells to a greater extent than sorafenib, but oligomycin had little effect on tumors grown from SALL4 PDX cells. Oligomycin was not toxic to mice. Analyses of chromatin immunoprecipitation sequencing data showed that SALL4 binds approximately 50% of mitochondrial genes, including many oxidative phosphorylation genes, to activate their transcription. In comparing SALL4 and SALL4-knockdown cells, we found SALL4 to increase oxidative phosphorylation, oxygen consumption rate, mitochondrial membrane potential, and use of oxidative phosphorylation-related metabolites to generate ATP.

Conclusions: In a screening for compounds that reduce the viability of cells that express high levels of the transcription factor SALL4, we identified inhibitors of oxidative phosphorylation, which slowed the growth of xenograft tumors from SALL4 cells in mice. SALL4 activates the transcription of genes that regulate oxidative phosphorylation to increase oxygen consumption, mitochondrial membrane potential, and ATP generation in cancer cells. Inhibitors of oxidative phosphorylation might be used for the treatment of liver tumors with high levels of SALL4.
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http://dx.doi.org/10.1053/j.gastro.2019.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309153PMC
December 2019

A Near-Infrared Probe Tracks and Treats Lung Tumor Initiating Cells by Targeting HMOX2.

J Am Chem Soc 2019 09 4;141(37):14673-14686. Epub 2019 Sep 4.

Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, Agency for Science , Technology and Research (A*STAR) , Singapore 138667 , Singapore.

Tumor initiating cells (TIC) are resistant to conventional anticancer therapy and associated with metastasis and relapse in cancer. Although various TIC markers and their antibodies have been proposed, it is limited to the use of antibodies for in vivo imaging or treatment of TIC. In this study, we discovered heme oxygenase 2 (HMOX2) as a novel biomarker for TIC and developed a selective small molecule probe TiNIR (tumor initiating cell probe with near infrared). TiNIR detects and enriches the functionally active TIC in human lung tumors, and through the photoacoustic property, TiNIR also visualizes lung TIC in the patient-derived xenograft (PDX) model. Furthermore, we demonstrate that TiNIR inhibits tumor growth by blocking the function of HMOX2, resulting in significantly increased survival rates of the cancer model mice. The novel therapeutic target HMOX2 and its fluorescent ligand TiNIR will open a new path for the molecular level of lung TIC diagnosis and treatment.
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http://dx.doi.org/10.1021/jacs.9b06068DOI Listing
September 2019

Cancer Stem Cells: Concepts, Challenges, and Opportunities for Cancer Therapy.

Methods Mol Biol 2019 ;2005:43-66

Genome Institute of Singapore, Singapore, Singapore.

Cancer stem cells (CSCs) are a subpopulation of cancer cells with self-renewal capacity, that fuel tumor growth and contribute to the heterogeneous nature of tumors. First identified in hematological malignancies, CSC populations have to date been proposed in solid tumors in various organs. In vitro and in vivo assays, mouse genetic models, and more recently single-cell sequencing technologies and other '-omics' methodologies have not only facilitated the identification of novel CSC populations but also revealed and clarified novel properties of CSCs. Increasingly, both cell-autonomous and CSC niche factors are recognized as important contributors of CSC properties. The deepened understanding of CSC properties and characteristics would enable and facilitate the rational design of CSC-specific therapeutics that would, ideally, have high selectivity for cancer cells, eliminate tumor bulk, and prevent tumor recurrence. Addressing these issues would form some of the key challenges of the CSC research field in the coming years.
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http://dx.doi.org/10.1007/978-1-4939-9524-0_4DOI Listing
March 2020

Publisher Correction: Methionine is a metabolic dependency of tumor-initiating cells.

Nat Med 2019 Jun;25(6):1022

Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

In the version of this article originally published, there is an error in Fig. 5a. Originally, 'MAT2A' appeared between 'Methionine' and 'Homocysteine'. 'MAT2A' should have been 'MTR'. The error has been corrected in the PDF and HTML versions of this article.
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http://dx.doi.org/10.1038/s41591-019-0486-3DOI Listing
June 2019

Methionine is a metabolic dependency of tumor-initiating cells.

Nat Med 2019 05 6;25(5):825-837. Epub 2019 May 6.

Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

Understanding cellular metabolism holds immense potential for developing new classes of therapeutics that target metabolic pathways in cancer. Metabolic pathways are altered in bulk neoplastic cells in comparison to normal tissues. However, carcinoma cells within tumors are heterogeneous, and tumor-initiating cells (TICs) are important therapeutic targets that have remained metabolically uncharacterized. To understand their metabolic alterations, we performed metabolomics and metabolite tracing analyses, which revealed that TICs have highly elevated methionine cycle activity and transmethylation rates that are driven by MAT2A. High methionine cycle activity causes methionine consumption to far outstrip its regeneration, leading to addiction to exogenous methionine. Pharmacological inhibition of the methionine cycle, even transiently, is sufficient to cripple the tumor-initiating capability of these cells. Methionine cycle flux specifically influences the epigenetic state of cancer cells and drives tumor initiation. Methionine cycle enzymes are also enriched in other tumor types, and MAT2A expression impinges upon the sensitivity of certain cancer cells to therapeutic inhibition.
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http://dx.doi.org/10.1038/s41591-019-0423-5DOI Listing
May 2019

Clonal MET Amplification as a Determinant of Tyrosine Kinase Inhibitor Resistance in Epidermal Growth Factor Receptor-Mutant Non-Small-Cell Lung Cancer.

J Clin Oncol 2019 04 24;37(11):876-884. Epub 2019 Jan 24.

1 National Cancer Centre Singapore, Singapore.

Purpose: Mesenchymal epithelial transition factor ( MET) activation has been implicated as an oncogenic driver in epidermal growth factor receptor ( EGFR)-mutant non-small-cell lung cancer (NSCLC) and can mediate primary and secondary resistance to EGFR tyrosine kinase inhibitors (TKI). High copy number thresholds have been suggested to enrich for response to MET inhibitors. We examined the clinical relevance of MET copy number gain (CNG) in the setting of treatment-naive metastatic EGFR-mutant-positive NSCLC.

Patients And Methods: MET fluorescence in situ hybridization was performed in 200 consecutive patients identified as metastatic treatment-naïve EGFR-mutant-positive. We defined MET-high as CNG greater than or equal to 5, with an additional criterion of MET/centromeric portion of chromosome 7 ratiο greater than or equal to 2 for amplification. Time-to-treatment failure (TTF) to EGFR TKI in patients identified as MET-high and -low was estimated by Kaplan-Meier method and compared using log-rank test. Multiregion single-nucleotide polymorphism array analysis was performed on 13 early-stage resected EGFR-mutant-positive NSCLC across 59 sectors to investigate intratumoral heterogeneity of MET CNG.

Results: Fifty-two (26%) of 200 patients in the metastatic cohort were MET-high at diagnosis; 46 (23%) had polysomy and six (3%) had amplification. Median TTF was 12.2 months (95% CI, 5.7 to 22.6 months) versus 13.1 months (95% CI, 10.6 to 15.0 months) for MET-high and -low, respectively ( P = .566), with no significant difference in response rate regardless of copy number thresholds. Loss of MET was observed in three of six patients identified as MET-high who underwent postprogression biopsies, which is consistent with marked intratumoral heterogeneity in MET CNG observed in early-stage tumors. Suboptimal response (TTF, 1.0 to 6.4 months) to EGFR TKI was observed in patients with coexisting MET amplification (five [3.2%] of 154).

Conclusion: Although up to 26% of TKI-naïve EGFR-mutant-positive NSCLC harbor high MET CNG by fluorescence in situ hybridization, this did not significantly affect response to TKI, except in patients identified as MET-amplified. Our data underscore the limitations of adopting arbitrary copy number thresholds and the need for cross-assay validation to define therapeutically tractable MET pathway dysregulation in EGFR-mutant-positive NSCLC.
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http://dx.doi.org/10.1200/JCO.18.00177DOI Listing
April 2019

Phenotypic and molecular features underlying neurodegeneration of motor neurons derived from spinal and bulbar muscular atrophy patients.

Neurobiol Dis 2019 04 1;124:1-13. Epub 2018 Nov 1.

Stem Cell and Regenerative Biology, Genome Institute of Singapore, Singapore 138672, Singapore. Electronic address:

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of polyglutamine region in the androgen receptor. To gain insights into mechanisms of SBMA, four wild-type and five SBMA iPSC lines were differentiated to spinal motor neurons (sMNs) with high efficiency. SBMA sMNs showed neurite defects, reduced sMN survival and decreased protein synthesis levels. Microarray analysis revealed a dysregulation in various neuronal-related signalling pathways in SBMA sMNs. Strikingly, FAM135B a novel gene of unknown function, was found drastically downregulated in SBMA sMNs. Knockdown of FAM135B in wild-type sMNs reduced their survival and contributed to neurite defects, similar to SBMA sMNs, suggesting a functional role of FAM135B in SBMA. The degenerative phenotypes and dysregulated genes revealed could be potential therapeutic targets for SBMA.
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http://dx.doi.org/10.1016/j.nbd.2018.10.019DOI Listing
April 2019

Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies.

J Exp Med 2018 07 7;215(7):1913-1928. Epub 2018 Jun 7.

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

RAF kinase inhibitors are clinically active in patients with BRAF (V600E) mutant melanoma. However, rarely do tumors regress completely, with the majority of responses being short-lived. This is partially mediated through the loss of negative feedback loops after MAPK inhibition and reactivation of upstream signaling. Here, we demonstrate that the deubiquitinating enzyme USP28 functions through a feedback loop to destabilize RAF family members. Loss of USP28 stabilizes BRAF enhancing downstream MAPK activation and promotes resistance to RAF inhibitor therapy in culture and in vivo models. Importantly, we demonstrate that USP28 is deleted in a proportion of melanoma patients and may act as a biomarker for response to BRAF inhibitor therapy in patients. Furthermore, we identify Rigosertib as a possible therapeutic strategy for USP28-depleted tumors. Our results show that loss of USP28 enhances MAPK activity through the stabilization of RAF family members and is a key factor in BRAF inhibitor resistance.
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http://dx.doi.org/10.1084/jem.20171960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028519PMC
July 2018

Identification of Tumor Initiating Cells with a Small-Molecule Fluorescent Probe by Using Vimentin as a Biomarker.

Angew Chem Int Ed Engl 2018 03 14;57(11):2851-2854. Epub 2018 Feb 14.

Singapore Bioimaging Consortium, Agency for Science Technology and Research (A*STAR), Singapore, 138667, Singapore.

Tumor initiating cells (TICs) have been implicated in clinical relapse and metastasis of a variety of epithelial cancers, including lung cancer. While efforts toward the development of specific probes for TIC detection and targeting are ongoing, a universal TIC probe has yet to be developed. We report the first TIC-specific fluorescent chemical probe, TiY, with identification of the molecular target as vimentin, a marker for epithelial-to-mesenchymal transition (EMT). TiY selectively stains TICs over differentiated tumor cells or normal cells, and facilitates the visualization and enrichment of functionally active TICs from patient tumors. At high concentration, TiY also shows anti-TIC activity with low toxicity to non-TICs. With the unexplored target vimentin, TiY shows potential as a first universal probe for TIC detection in different cancers.
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http://dx.doi.org/10.1002/anie.201712920DOI Listing
March 2018

Elucidating the genomic architecture of Asian EGFR-mutant lung adenocarcinoma through multi-region exome sequencing.

Nat Commun 2018 01 15;9(1):216. Epub 2018 Jan 15.

Cancer Stem Cell Biology, Genome Institute of Singapore, Singapore, 138672, Singapore.

EGFR-mutant lung adenocarcinomas (LUAD) display diverse clinical trajectories and are characterized by rapid but short-lived responses to EGFR tyrosine kinase inhibitors (TKIs). Through sequencing of 79 spatially distinct regions from 16 early stage tumors, we show that despite low mutation burdens, EGFR-mutant Asian LUADs unexpectedly exhibit a complex genomic landscape with frequent and early whole-genome doubling, aneuploidy, and high clonal diversity. Multiple truncal alterations, including TP53 mutations and loss of CDKN2A and RB1, converge on cell cycle dysregulation, with late sector-specific high-amplitude amplifications and deletions that potentially beget drug resistant clones. We highlight the association between genomic architecture and clinical phenotypes, such as co-occurring truncal drivers and primary TKI resistance. Through comparative analysis with published smoking-related LUAD, we postulate that the high intra-tumor heterogeneity observed in Asian EGFR-mutant LUAD may be contributed by an early dominant driver, genomic instability, and low background mutation rates.
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http://dx.doi.org/10.1038/s41467-017-02584-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768770PMC
January 2018

Chromosome 1q21.3 amplification is a trackable biomarker and actionable target for breast cancer recurrence.

Nat Med 2017 Nov 25;23(11):1319-1330. Epub 2017 Sep 25.

Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Biopolis, Singapore.

Tumor recurrence remains the main reason for breast cancer-associated mortality, and there are unmet clinical demands for the discovery of new biomarkers and development of treatment solutions to benefit patients with breast cancer at high risk of recurrence. Here we report the identification of chromosomal copy-number amplification at 1q21.3 that is enriched in subpopulations of breast cancer cells bearing characteristics of tumor-initiating cells (TICs) and that strongly associates with breast cancer recurrence. Amplification is present in ∼10-30% of primary tumors but in more than 70% of recurrent tumors, regardless of breast cancer subtype. Detection of amplification in cell-free DNA (cfDNA) from blood is strongly associated with early relapse in patients with breast cancer and could also be used to track the emergence of tumor resistance to chemotherapy. We further show that 1q21.3-encoded S100 calcium-binding protein (S100A) family members, mainly S100A7, S100A8, and S100A9 (S100A7/8/9), and IL-1 receptor-associated kinase 1 (IRAK1) establish a reciprocal feedback loop driving tumorsphere growth. Notably, this functional circuitry can be disrupted by the small-molecule kinase inhibitor pacritinib, leading to preferential impairment of the growth of 1q21.3-amplified breast tumors. Our study uncovers the 1q21.3-directed S100A7/8/9-IRAK1 feedback loop as a crucial component of breast cancer recurrence, serving as both a trackable biomarker and an actionable therapeutic target for breast cancer.
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http://dx.doi.org/10.1038/nm.4405DOI Listing
November 2017

Novel therapeutic targets on the horizon for lung cancer.

Lancet Oncol 2016 08;17(8):e347-e362

Division of Medical Oncology, National Cancer Centre Singapore, Singapore; Cancer Therapeutics Research Laboratory, National Cancer Centre Singapore, Singapore; Genome Institute of Singapore, A*STAR, Singapore. Electronic address:

Lung cancer is a leading cause of cancer-related mortality worldwide, and is classically divided into two major histological subtypes: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). Although NSCLC and SCLC are considered distinct entities with different genomic landscapes, emerging evidence highlights a convergence in therapeutically relevant targets for both histologies. In adenocarcinomas with defined alterations such as EGFR mutations and ALK translocations, targeted therapies are now first-line standard of care. By contrast, many experimental and targeted agents remain largely unsuccessful for SCLC. Intense preclinical research and clinical trials are underway to exploit unique traits of lung cancer, such as oncogene dependency, DNA damage response, angiogenesis, and cellular plasticity arising from presence of cancer stem cell lineages. In addition, the promising clinical activity observed in NSCLC in response to immune checkpoint blockade has spurred great interest in the field of immunooncology, with the scope to develop a diverse repertoire of synergistic and personalised immunotherapeutics. In this Review, we discuss novel therapeutic agents for lung cancer that are in early-stage development, and how prospective clinical trials and drug development may be shaped by a deeper understanding of this heterogeneous disease.
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http://dx.doi.org/10.1016/S1470-2045(16)30123-1DOI Listing
August 2016

Tumour-initiating cell-specific miR-1246 and miR-1290 expression converge to promote non-small cell lung cancer progression.

Nat Commun 2016 06 21;7:11702. Epub 2016 Jun 21.

Genome Institute of Singapore, Singapore 138672, Singapore.

The tumour-initiating cell (TIC) model accounts for phenotypic and functional heterogeneity among tumour cells. MicroRNAs (miRNAs) are regulatory molecules frequently aberrantly expressed in cancers, and may contribute towards tumour heterogeneity and TIC behaviour. More recent efforts have focused on miRNAs as diagnostic or therapeutic targets. Here, we identified the TIC-specific miRNAs, miR-1246 and miR-1290, as crucial drivers for tumour initiation and cancer progression in human non-small cell lung cancer. The loss of either miRNA impacted the tumour-initiating potential of TICs and their ability to metastasize. Longitudinal analyses of serum miR-1246 and miR-1290 levels across time correlate their circulating levels to the clinical response of lung cancer patients who were receiving ongoing anti-neoplastic therapies. Functionally, direct inhibition of either miRNA with locked nucleic acid administered systemically, can arrest the growth of established patient-derived xenograft tumours, thus indicating that these miRNAs are clinically useful as biomarkers for tracking disease progression and as therapeutic targets.
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http://dx.doi.org/10.1038/ncomms11702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4919505PMC
June 2016

Activation of PKA leads to mesenchymal-to-epithelial transition and loss of tumor-initiating ability.

Science 2016 Mar;351(6277):aad3680

Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Ludwig Center for Molecular Oncology at MIT, Cambridge, MA 02142, USA.

The epithelial-to-mesenchymal transition enables carcinoma cells to acquire malignancy-associated traits and the properties of tumor-initiating cells (TICs). TICs have emerged in recent years as important targets for cancer therapy, owing to their ability to drive clinical relapse and enable metastasis. Here, we propose a strategy to eliminate mesenchymal TICs by inducing their conversion to more epithelial counterparts that have lost tumor-initiating ability. We report that increases in intracellular levels of the second messenger, adenosine 3',5'-monophosphate, and the subsequent activation of protein kinase A (PKA) induce a mesenchymal-to-epithelial transition (MET) in mesenchymal human mammary epithelial cells. PKA activation triggers epigenetic reprogramming of TICs by the histone demethylase PHF2, which promotes their differentiation and loss of tumor-initiating ability. This study provides proof-of-principle for inducing an MET as differentiation therapy for TICs and uncovers a role for PKA in enforcing and maintaining the epithelial state.
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http://dx.doi.org/10.1126/science.aad3680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131720PMC
March 2016

IRAK1 is a therapeutic target that drives breast cancer metastasis and resistance to paclitaxel.

Nat Commun 2015 Oct 27;6:8746. Epub 2015 Oct 27.

Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, A*STAR (Agency for Science, Technology and Research), 60 Biopolis Street, 02-01, Biopolis 138672, Singapore.

Metastatic tumour recurrence due to failed treatments remains a major challenge of breast cancer clinical management. Here we report that interleukin-1 receptor-associated kinase 1 (IRAK1) is overexpressed in a subset of breast cancers, in particular triple-negative breast cancer (TNBC), where it acts to drive aggressive growth, metastasis and acquired resistance to paclitaxel treatment. We show that IRAK1 overexpression confers TNBC growth advantage through NF-κB-related cytokine secretion and metastatic TNBC cells exhibit gain of IRAK1 dependency, resulting in high susceptibility to genetic and pharmacologic inhibition of IRAK1. Importantly, paclitaxel treatment induces strong IRAK1 phosphorylation, an increase in inflammatory cytokine expression, enrichment of cancer stem cells and acquired resistance to paclitaxel treatment. Pharmacologic inhibition of IRAK1 is able to reverse paclitaxel resistance by triggering massive apoptosis at least in part through inhibiting p38-MCL1 pro-survival pathway. Our study thus demonstrates IRAK1 as a promising therapeutic target for TNBC metastasis and paclitaxel resistance.
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http://dx.doi.org/10.1038/ncomms9746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4640083PMC
October 2015

Distinct EMT programs control normal mammary stem cells and tumour-initiating cells.

Nature 2015 Sep 2;525(7568):256-60. Epub 2015 Sep 2.

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.

Tumour-initiating cells (TICs) are responsible for metastatic dissemination and clinical relapse in a variety of cancers. Analogies between TICs and normal tissue stem cells have led to the proposal that activation of the normal stem-cell program within a tissue serves as the major mechanism for generating TICs. Supporting this notion, we and others previously established that the Slug epithelial-to-mesenchymal transition-inducing transcription factor (EMT-TF), a member of the Snail family, serves as a master regulator of the gland-reconstituting activity of normal mammary stem cells, and that forced expression of Slug in collaboration with Sox9 in breast cancer cells can efficiently induce entrance into the TIC state. However, these earlier studies focused on xenograft models with cultured cell lines and involved ectopic expression of EMT-TFs, often at non-physiological levels. Using genetically engineered knock-in reporter mouse lines, here we show that normal gland-reconstituting mammary stem cells residing in the basal layer of the mammary epithelium and breast TICs originating in the luminal layer exploit the paralogous EMT-TFs Slug and Snail, respectively, which induce distinct EMT programs. Broadly, our findings suggest that the seemingly similar stem-cell programs operating in TICs and normal stem cells of the corresponding normal tissue are likely to differ significantly in their details.
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http://dx.doi.org/10.1038/nature14897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764075PMC
September 2015

Musashi proteins are post-transcriptional regulators of the epithelial-luminal cell state.

Elife 2014 Nov 7;3:e03915. Epub 2014 Nov 7.

Department of Biology, Massachusetts Institute of Technology, Cambridge, United States.

The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis, we found that Msi proteins regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT), and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited the translation of Jagged1, a factor required for EMT, and repressed EMT in cell culture and in mammary gland in vivo. Knockdown of Msis in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types.
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http://dx.doi.org/10.7554/eLife.03915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381951PMC
November 2014

A breast cancer stem cell niche supported by juxtacrine signalling from monocytes and macrophages.

Nat Cell Biol 2014 Nov 28;16(11):1105-17. Epub 2014 Sep 28.

1] Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA [2] MIT Ludwig Center for Molecular Oncology, Cambridge, Massachusetts 02139, USA [3] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.

The cell-biological program termed the epithelial-mesenchymal transition (EMT) confers on cancer cells mesenchymal traits and an ability to enter the cancer stem cell (CSC) state. However, the interactions between CSCs and their surrounding microenvironment are poorly understood. Here we show that tumour-associated monocytes and macrophages (TAMs) create a CSC niche through juxtacrine signalling with CSCs. We performed quantitative proteomic profiling and found that the EMT program upregulates the expression of CD90, also known as Thy1, and EphA4, which mediate the physical interactions of CSCs with TAMs by directly binding with their respective counter-receptors on these cells. In response, the EphA4 receptor on the carcinoma cells activates Src and NF-κB. In turn, NF-κB in the CSCs induces the secretion of a variety of cytokines that serve to sustain the stem cell state. Indeed, admixed macrophages enhance the CSC activities of carcinoma cells. These findings underscore the significance of TAMs as important components of the CSC niche.
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http://dx.doi.org/10.1038/ncb3041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4296514PMC
November 2014