NSD2 contributes to oncogenic RAS-driven transcription in lung cancer cells through long-range epigenetic activation

The histone methyltransferase NSD2/WHSC1/MMSET is overexpressed in a number of solid tumors but its contribution to the biology of these tumors is not well understood. Here, we describe that NSD2 contributes to the proliferation of a subset of lung cancer cell lines by supporting oncogenic RAS transcriptional responses. NSD2 knock down combined with MEK or BRD4 inhibitors causes co-operative inhibitory responses on cell growth. However, while MEK and BRD4 inhibitors converge in the downregulation of genes associated with cancer-acquired super-enhancers, NSD2 inhibition affects the expression of clusters of genes embedded in megabase-scale regions marked with H3K36me2 and that contribute to the RAS transcription program. Thus, combinatorial therapies using MEK or BRD4 inhibitors together with NSD2 inhibition are likely to be needed to ensure a more comprehensive inhibition of oncogenic RAS-driven transcription programs in lung cancers with NSD2 overexpression.This work was supported by Eli Lilly and Company. We would like to thank the Genomics and Flow Cytometry units at the CNIO for technical help, C. Pantoja and the CNIO-Lilly Cell Signaling Therapies Laboratory for sharing protocols and reagents.S

Comprehensive Plasma Metabolomic Profile of Patients with Advanced Neuroendocrine Tumors (NETs). Diagnostic and Biological Relevance

Purpose: High-throughput “-omic” technologies have enabled the detailed analysis of metabolic networks in several cancers, but NETs have not been explored to date. We aim to assess the metabolomic profile of NET patients to understand metabolic deregulation in these tumors and identify novel biomarkers with clinical potential. Methods: Plasma samples from 77 NETs and 68 controls were profiled by GC−MS, CE−MS and LC−MS untargeted metabolomics. OPLS-DA was performed to evaluate metabolomic differences. Related pathways were explored using Metaboanalyst 4.0. Finally, ROC and OPLS-DA analyses were performed to select metabolites with biomarker potential. Results: We identified 155 differential compounds between NETs and controls. We have detected an increase of bile acids, sugars, oxidized lipids and oxidized products from arachidonic acid and a decrease of carnitine levels in NETs. MPA/MSEA identified 32 enriched metabolic pathways in NETs related with the TCA cycle and amino acid metabolism. Finally, OPLS-DA and ROC analysis revealed 48 metabolites with diagnostic potential. Conclusions: This study provides, for the first time, a comprehensive metabolic profile of NET patients and identifies a distinctive metabolic signature in plasma of potential clinical use. A reduced set of metabolites of high diagnostic accuracy has been identified. Additionally, new enriched metabolic pathways annotated may open innovative avenues of clinical research

Differential Role of Human Choline Kinase α and β Enzymes in Lipid Metabolism: Implications in Cancer Onset and Treatment

11 pages, 6 figures, 1 table.Background The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of 1phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKα and ChoKβ isoforms, the first one with two different variants of splicing. Recently ChoKα has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKβ in carcinogenesis has been reported. Methodology/Principal Findings Here we compare the in vitro and in vivo properties of ChoKα1 and ChoKβ in lipid metabolism, and their potential role in carcinogenesis. Both ChoKα1 and ChoKβ showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKβ display an ethanolamine kinase role, ChoKα1 present a dual choline/ethanolamine kinase role, suggesting the involvement of each ChoK isoform in distinct biochemical pathways under in vivo conditions. In addition, while overexpression of ChoKα1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKβ overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKα1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKβ mRNA levels were observed. Finally, MN58b, a previously described potent inhibitor of ChoK with in vivo antitumoral activity, shows more than 20-fold higher efficiency towards ChoKα1 than ChoKβ. Conclusion/Significance This study represents the first evidence of the distinct metabolic role of ChoKα and ChoKβ isoforms, suggesting different physiological roles and implications in human carcinogenesis. These findings constitute a step forward in the design of an antitumoral strategy based on ChoK inhibition.This work has been supported by grants to JCL from Comunidad de Madrid (GR-SAL-0821-2004), Ministerio de Ciencia e Innovación (SAF2008-03750, RD06/0020/0016), Fundación Mutua Madrileña, and by a grant to ARM from Fundación Mutua Madrileña.Peer reviewe

Generation and characterization of monoclonal antibodies against choline kinase α and their potential use as diagnostic tools in cancer

6 pages, 3 figures, 1 table.Choline kinase α (ChoKα) is a metabolic enzyme involved in the synthesis of phosphatidylcholine, recently implicated in cancer onset since it is overexpressed in a variety of human cancers such as mammary, lung, colorectal and prostate adenocarcinomas. Furthermore, overexpression of ChoKα in human HEK293T cells confers them oncogenic properties with the induction of tumors after subcutaneous injection in nude mice. ChoKα levels in tumor samples have been analyzed using polyclonal antibodies and Western blotting. These techniques have considerable limitations and do not allow for a precise and efficient evaluation of the real significance of ChoK overexpression in human carcinogenesis. We developed a set of monoclonal antibodies with high specificity and sensitivity against ChoKα, and characterized their properties. We provide evidence that the newly generated MoAbs against ChoKα have potential use in cancer diagnosis by conventional immunohistochemistry techniques.This work was supported by Laboratorios INDAS, and by grants CAM 08.1/0047/2003, FIS C03-08 and FIS C03-10 from MSyC.Peer reviewe

CREBBP/EP300 bromodomains are critical to sustain the GATA1/MYC regulatory axis in proliferation

Abstract Background The reported antitumor activity of the BET family bromodomain inhibitors has prompted the development of inhibitors against other bromodomains. However, the human genome encodes more than 60 different bromodomains and most of them remain unexplored. Results We report that the bromodomains of the histone acetyltransferases CREBBP/EP300 are critical to sustain the proliferation of human leukemia and lymphoma cell lines. EP300 is very abundant at super-enhancers in K562 and is coincident with sites of GATA1 and MYC occupancy. In accordance, CREBBP/EP300 bromodomain inhibitors interfere with GATA1- and MYC-driven transcription, causing the accumulation of cells in the G0/G1 phase of the cell cycle. The CREBBP/CBP30 bromodomain inhibitor CBP30 displaces CREBBP and EP300 from GATA1 and MYC binding sites at enhancers, resulting in a decrease in the levels of histone acetylation at these regulatory regions and consequently reduced gene expression of critical genes controlled by these transcription factors. Conclusions Our data shows that inhibition of CREBBP/EP300 bromodomains can interfere with oncogene-driven transcriptional programs in cancer cells and consequently hold therapeutic potential

Choline kinase is a novel oncogene that potentiates RhoA-induced carcinogenesis

Choline kinase is overexpressed in human breast, lung, colorectal, and prostate tumors, a finding that suggests the involvement of this enzyme in carcinogenesis. Here we show that overexpression of choline kinase induce oncogenic transformation of human embryo kidney fibroblasts and canine epithelial Madin-Darby canine kidney cells. Choline kinase lays downstream of RhoA signaling and is activated through ROCK kinase, one of the best-characterized RhoA effectors. In keeping with this, coexpression of RhoA and choline kinase potentiates both anchorage independent growth and tumorigenesis. Finally, choline kinase–mediated transformation is sensitive to MN58b, a well-characterized specific choline kinase inhibitor. These results provide the definitive evidence that choline kinase has oncogenic properties and that choline kinase inhibition constitutes a novel valid antitumor strategy.SAF2001-2042FIS C03-08FIS C03-107.616 JCR (2005) Q1, 11/123 OncologyUE

Choline kinase is a novel oncogene that potentiates RhoA-induced carcinogenesis

Choline kinase is overexpressed in human breast, lung, colorectal, and prostate tumors, a finding that suggests the involvement of this enzyme in carcinogenesis. Here we show that overexpression of choline kinase induces oncogenic transformation of human embryo kidney fibroblasts and canine epithelial Madin-Darby canine kidney cells. Choline kinase lays downstream of RhoA signaling and is activated through ROCK kinase, one of the best-characterized RhoA effectors. In keeping with this, coexpression of RhoA and choline kinase potentiates both anchorage independent growth and tumorigenesis. Finally, choline kinase-mediated transformation is sensitive to MN58b, a well-characterized specific choline kinase inhibitor. These results provide the definitive evidence that choline kinase has oncogenic properties and that choline kinase inhibition constitutes a novel valid antitumor strategy. ©2005 American Association for Cancer Research.Grant support: McyT grant SAF2001-2042 and MSyC grants FIS C03-08 and FIS C03-10.Peer Reviewe

MOESM1 of CREBBP/EP300 bromodomains are critical to sustain the GATA1/MYC regulatory axis in proliferation

Additional file 1: Table S1. gRNAs used for gene editing. Fig. S1. Statistical analysis of the CRISPR-Cas9 growth competition experiments. Tukey Kramer analysis of the adjusted percentages of growth inhibition caused by gRNAs targeting different regions of CREBBP (A) or EP300 (B). 5′ coding region (5′), non-conserved aminoacids of the bromodomain (ncBD), conserved aminoacids of the bromodomain (cBD) and non-target (NT). Fig. S2. Enrichment of gene expression changes after treatment with CBP30 and I-CBP112. (A) p-values for enrichment of SE-associated genes (SE) and genes with top levels of EP300 (EP300) in genes upregulated and downregulated by CBP30 and I-CBP112 treatments. (B) GSEA analysis of changes in gene expression caused by the indicated treatments and gene sets. Fig. S3. GATA1 mRNA expression in cancer cell lines and patients. (A) mRNA levels of GATA1 determined by microarray in CCLE lines grouped by cancer type. (B) GATA1 mRNA levels determined by RNAseq in cancer patients according to TCGA. Fig. S4. Expression of GATA1 splicing variants in K562 (A) Three variants are expressed in K562 according to the analysis of the RNA-seq experiment (B) Graph shows the levels of expression of the different variants in K562 cells treated with vehicle or two concentrations of CBP30. P-values for significant changes (p ≤ 0.05) are shown. Fig. S5. Human myeloma cell lines with MYC amplifications or translocations are sensitive to CBP30. (A) IC50s of growth inhibition in KMS11 or MM1S cells treated with JQ1, C646 and CBP30 for 7 days. (B) mRNA (upper panel) and protein (lower panel) levels of MYC in KMS11 or MM1S cells treated with 2 µM CBP30, 10 µM C646 and 150 nM JQ1 for 48 hours

OncoChok. Validación de nuevos factores pronóstico e identificación de nuevas dianas terapéuticas en carcinogénesis humana

El consorcio OncoChok está integrado por el Laboratorio de Oncología Traslacional liderado por el Dr. Juan Carlos Lacal en el Instituto de Investigaciones Biomédicas de Madrid "Alberto Sols" (IIBM) del Consejo Superior de Investigaciones Cientificas (CSIC), y tres de los hospitales universitarios más importantes de la Comunidad de Madrid: Hospital Clínico, Hospital Doce de Octubre y Hospital La Paz y la empresa Translational Cancer Drugs Pharma (TCD Pharma).-- Más información sobre el programa en: http://www.oncochok.com/.El Programa OncoChok investiga la implicación del enzima colino quinasa alfa (ChoKa) como marcador de diagnóstico, pronóstico o predictor de respuesta al tratamiento antitumoral, así como su utilidad como una nueva diana terapéutica en distintos tipos de cáncer (pulmón, mama, páncreas, colorrectal y vejiga). También propone identificar sus posibles reguladores y efectores, su participación en el proceso de carcinogénesis y dilucidar el mecanismo de acción de sus inhibidores. El estudio se completa con la posible implicación del enzima ChoKa y sus reguladores en el desarrollo y progresión del cáncer y el análisis de su posible relación con parámetros clínico-patológicos.OncoChok es un proyecto de investigación subvencionado por la Comunidad de Madrid (CAM).Peer reviewe