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Summary of Patients and Clinical Data.

Supplementary Figure 1. Unsupervised Clustering on CCA samples. Supplementary Figure 2. Alterations Found in CCA Clusters. Supplementary Figure 3. MAP2K4 Homozygous Deletions and ERBB2 Amplifications. Supplementary Figure 4. Alterations Related to Structural Variations Found in CCAs. Supplementary Figure 5. FIREFLY Analysis of Pathways Systematically Dysregulated by Somatic Promoter Mutations that Alter Transcription Factor Binding. Supplementary Figure 6. Epigenetic Clusters and Mutation Signatures.

Structural Variations across 71 WGS CCAs.

Summary of CCA Alterations.

Further details of methods, in addition to the supplementary figure and table legends.

Alterations in CCA Clusters.

Coverage Statistics and List of Genes for Targeted Sequencing.

Point mutations in the ligand binding domain of retinoic acid receptor alpha (RARα) have been implicated in breast fibroepithelial tumors development. However, their role in the tumorigenesis of solid tumors is currently unknown. In this study, using a combination of biochemical and cellular assays, we evaluated the functional consequences of known tumor associated RARα mutations on retinoic acid signaling. All of the clinically associated mutants tested showed diminished transcriptional activities compared to wild type RARα. These mutants also exhibited a dominant negative effect, an activity which has previously been linked to developmental defects and tumor formation in mice. X-ray crystallography showed that mutants remain relatively intact structurally and the loss of transcriptional activity is due to altered co-activator recruitment. In agreement with our biochemical analyses, transcriptomics and cell growth analysis showed that the mutant RARα proteins confer resistance to growth inhibition in the presence of its ligand in phyllodes tumor cells. Although the mutations impair the receptor responses to retinoic acid, certain mutant RARα are partially reactivatable with alternative synthetic agonists. Our data provide insights into the mechanisms by which RARα mutations impact tumorigenesis.

Aldehyde dehydrogenases (ALDH) are overexpressed in various types of cancers. One of the ALDH family genes, ALDH1A2, is aberrantly expressed in more than 50% of cases of T-cell acute lymphoblastic leukemia (T-ALL). However, its molecular function and role in the pathogenesis of T-ALL are largely unknown. Chromatin immunoprecipitation-sequencing and RNA-sequencing analyses showed that the oncogenic transcription factor TAL1 and its regulatory partners bind to the intronic regulatory element of the ALDH1A2 gene, directly inducing a T-ALL-specific isoform with enzymatic activity. ALDH1A2 was preferentially expressed in the TAL1-positive T-ALL subgroup. In TALL cell lines, depletion of ALDH1A2 inhibited cell viability and induced apoptosis. Interestingly, gene expression and metabolomic profiling revealed that ALDH1A2 supported glycolysis and the tricarboxylic acid cycle, accompanied by NADH production, by affecting multiple metabolic enzymes to promote ATP production. Depletion of ALDH1A2 increased the levels of reactive oxygen species, while the levels were reduced by ALDH1A2 overexpression both in vitro and in vivo. Overexpression of ALDH1A2 accelerated tumor onset and increased tumor penetrance in a zebrafish model of T-ALL. Taken together, our results indicate that ALDH1A2 protects against intracellular stress and promotes T-ALL cell metabolism and survival. ALDH1A2 overexpression enables leukemic clones to sustain a hyper-proliferative state driven by oncogenes.

Supplemental material, Supplemental_Material_for_TSA_CRAFT_by_Lee_et_al_(2) for TSA-CRAFT: A Free Software for Automatic and Robust Thermal Shift Assay Data Analysis by Po-Hsien Lee, Xi Xiao Huang, Bin Tean Teh and Ley-Moy Ng in SLAS Discovery

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.

Aldehyde dehydrogenases (ALDHs) have been implicated as a stem cell marker both in normal and malignant cells. Elevated ALDH activity is associated with the stemness capability as well as the drug resistance in various cancers. As a notable example, ALDH1A2, one of ALDH family genes, is ectopically overexpressed in T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological disease that results from malignant transformation of T-cell progenitors. However, its roles and molecular functions in T-ALL pathogenesis are poorly understood. In our study, we demonstrated that the oncogenic transcription factor TAL1 directly induces an expression of ALDH1A2 in T-ALL cells, while normal T-cells do not express this gene. The TAL1 transcriptional complex binds to an intragenic regulatory element of ALDH1A2 and aberrantly activates the alternative promoter of the short isoform, which is specific to T-ALL cells. Using in vitro enzymatic assay, we found that the short isoform still retain enzymatic activity in catalyzing the oxidation of aldehydes. Importantly, ALDH1A2 promotes the viability and survival of T-ALL cells. Unbiased gene expression and metabolome profiling demonstrated that ALDH1A2 promotes glycolysis and mitochondria respiration, thereby supporting energy production. Additionally, expression of the short ALDH1A2 attenuated the amount of reactive oxygen species (ROS) both in vitro and in vivo. Furthermore, forced expression of short ALDH1A2 in T-cells increased overall penetrance of T-cell malignancy induced by the AKT2 oncogene in a zebrafish model. Taken together, our data suggests that ALDH1A2 is crucial and advantageous for T-ALL cell viability and survival by mediating energy production and by protecting cancer cells from oxidative stresses, thus serving as a requisite to maintain the hyperproliferative state of T-ALL cells (“onco-requisite”). Citation Format: CHUJING ZHANG, Stella Amanda, Cheng Wang, Tze King Tan, Zulfaqar Ali Muhammad, Wei Zhong Leong, Ley Moy Ng, Shojiro Kitajima, Zhenhua Li, Allen Eng Juh Yeoh, Shi Hao Tan, Takaomi Sanda. Oncorequisite role of an aldehyde dehydrogenase in the pathogenesis of T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-301.

Breast fibroepithelial tumors comprise a heterogeneous spectrum of pathological entities, from benign fibroadenomas to malignant phyllodes tumors. Although MED12 mutations have been frequently found in fibroadenomas and phyllodes tumors, the landscapes of genetic alterations across the fibroepithelial tumor spectrum remain unclear. Here, by performing exome sequencing of 22 phyllodes tumors followed by targeted sequencing of 100 breast fibroepithelial tumors, we observed three distinct somatic mutation patterns. First, we frequently observed MED12 and RARA mutations in both fibroadenomas and phyllodes tumors, emphasizing the importance of these mutations in fibroepithelial tumorigenesis. Second, phyllodes tumors exhibited mutations in FLNA, SETD2 and KMT2D, suggesting a role in driving phyllodes tumor development. Third, borderline and malignant phyllodes tumors harbored additional mutations in cancer-associated genes. RARA mutations exhibited clustering in the portion of the gene encoding the ligand-binding domain, functionally suppressed RARA-mediated transcriptional activation and enhanced RARA interactions with transcriptional co-repressors. This study provides insights into the molecular pathogenesis of breast fibroepithelial tumors, with potential clinical implications.

We have completed the genetic characterization of all eight gene segments for four low pathogenic avian influenza (LPAI) viruses. The objective of this study was to detect the presence of novel signatures that may serve as early warning indicators of the conversion of LPAI viruses to high pathogenic avian influenza (HPAI) viruses. This study included three H5N2 and one H5N3 viruses that were isolated from live poultry imported into Singapore as part of the national avian influenza virus (AIV) surveillance program. Based on the molecular criterion of the World Organisation for Animal Health (OIE), sequence analysis with the translated amino acid (aa) sequence of the hemagglutinin (HA) gene revealed the absence of multibasic aa at the HA cleavage site, identifying all four virus isolates as LPAI. Detailed phylogenetic tree analyses using the HA and neuraminidase (NA) genes clustered these isolates in the Eurasian H5 lineage, but away from the HPAI H5 subtypes. This analysis further revealed that the internal genes clustered to different avian and swine subtypes, suggesting that the four isolates may possibly share their ancestry with these different influenza subtypes. Our results suggest that the four LPAI isolates in this study contained mainly avian signatures, and the phylogenetic tree for the internal genes further suggests the potential for reassortment with other different circulating avian subtypes. This is the first comprehensive report on the genetic characterization of LPAI H5N2/3 viruses isolated in South-East Asia.

Musical Chairs The plant hormone abscisic acid (ABA) helps plants to respond to changes in the environment, such as drought. Physiological responses are initiated when ABA binds to its receptor. In the absence of ABA, downstream kinases are held inactive by phosphatases. Soon et al. (p. 85 , published online 24 November; see the Perspective by Leung ) now show that both the hormone-receptor complex and the downstream kinase bind to the same site on the phosphatase. Thus, in the presence of hormone, the phosphatase is occupied and unable to interfere with downstream kinase activity.

Four hundred specimens were collected from pediatric patients hospitalized in Singapore; 21 of these specimens tested positive for human metapneumovirus (HMPV), with the A2 genotype predominating. A 5% infection rate was estimated, suggesting that HMPV is a significant cause of morbidity among the pediatric population of Singapore.