ARID2 deficiency promotes tumor progression and is associated with higher sensitivity to chemotherapy in lung cancer

The survival rate in lung cancer remains stubbornly low and there is an urgent need for the identification of new therapeutic targets. In the last decade, several members of the SWI/SNF chromatin remodeling complexes have been described altered in different tumor types. Nevertheless, the precise mechanisms of their impact on cancer progression, as well as the application of this knowledge to cancer patient management are largely unknown. In this study, we performed targeted sequencing of a cohort of lung cancer patients on genes involved in chromatin structure. In addition, we studied at the protein level the expression of these genes in cancer samples and performed functional experiments to identify the molecular mechanisms linking alterations of chromatin remodeling genes and tumor development. Remarkably, we found that 20% of lung cancer patients show ARID2 protein loss, partially explained by the presence of ARID2 mutations. In addition, we showed that ARID2 deficiency provokes profound chromatin structural changes altering cell transcriptional programs, which bolsters the proliferative and metastatic potential of the cells both in vitro and in vivo. Moreover, we demonstrated that ARID2 deficiency impairs DNA repair, enhancing the sensitivity of the cells to DNA-damaging agents. Our findings support that ARID2 is a bona fide tumor suppressor gene in lung cancer that may be exploited therapeutically.Financial Support: I. V. is supported by SAF2012-31627 and SAF2016-76758-R grants from the Spanish Ministerio de Economía y Competitividad (MINECO), by a Fundación Ramón Areces grant and by ERC2014-StG637904 grant from the European Research Council. I. V has been awardee of the Programa Ramón y Cajal (MINECO, Spain). T. M has been awardee of the Ayudas para la contratación de investigadores predoctorales (MINECO, Spain). B. M is awardee of the Ayudas para la formación de profesorado universitario (FPU, Ministerio de Educación y Formación Profesional, Spain). PC laboratory is supported by grant SAF-2015-63638R (MINECO/FEDER, UE); by Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) and by Asociación Española Contra el Cáncer (AECC), grant GCB141423113. BC has been supported by a Retos Jóvenes Investigadores grant SAF2015-73364-JIN (AEI/FEDER, UE) and a grant from Fundación Francisco Cobos. P. S. is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001152), the UK Medical Research Council (FC001152). HUCA/IUOPA which is jointly financed by Servicio de Salud del Principado de Asturias, Instituto de Salud Carlos III and Fundación Bancaria Cajastur. This research was funded in part by the Wellcome Trust [FC001152]

JAK2 V617F hematopoietic clones are present several years prior to MPN diagnosis and follow different expansion kinetics.

TO THE EDITOR: The JAK2 V617F mutation is the most common somatic mutation in the classical myeloproliferative neoplasms (MPNs), present in >95% of cases of polycythemia vera (PV) and ∼50% of essential thrombocythemia (ET) and myelofibrosis (MF).1⇓⇓-4 It is usually the sole identifiable driver mutation in MPNs5 and was recently also identified as a driver of age-related clonal hemopoiesis in healthy individuals.6⇓⇓-9 In order to investigate the preclinical clonal evolution of MPNs, we identified 12 individuals with a JAK2 V617F mutant MPN, who 4.6 to 15.2 years previously (median 10.2 years) had also donated blood to register with the Cyprus Bone Marrow Donor Registry at the Karaiskakio FoundationThis work was supported by the Wellcome Trust Sanger Institute (WT098051). T. McKerrell is funded by a Wellcome Trust Clinician Scientist Fellowship (100678/Z/12/Z). G.S.V. is funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA), and work in his laboratory is also funded by Cancer Research UK, Bloodwise, the Kay Kendall Leukaemia Fund, and Celgene. I.V. is supported by the Spanish Ministerio de Economía y Competitividad, Programa Ramón y Cajal

Colorectal adenomas contain multiple somatic mutations that do not coincide with synchronous adenocarcinoma specimens

We have performed a comparative ultrasequencing study of multiple colorectal lesions obtained simultaneously from four patients. Our data show that benign lesions (adenomatous or hyperplastic polyps) contain a high mutational load. Additionally multiple synchronous colorectal lesions show non overlapping mutational signatures highlighting the degree of heterogeneity between multiple specimens in the same patient. Observations in these cases imply that considering not only the number of mutations but an effective oncogenic combination of mutations can determine the malignant progression of colorectal lesions

Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies.

The diagnosis of hematologic malignancies relies on multidisciplinary workflows involving morphology, flow cytometry, cytogenetic, and molecular genetic analyses. Advances in cancer genomics have identified numerous recurrent mutations with clear prognostic and/or therapeutic significance to different cancers. In myeloid malignancies, there is a clinical imperative to test for such mutations in mainstream diagnosis; however, progress toward this has been slow and piecemeal. Here we describe Karyogene, an integrated targeted resequencing/analytical platform that detects nucleotide substitutions, insertions/deletions, chromosomal translocations, copy number abnormalities, and zygosity changes in a single assay. We validate the approach against 62 acute myeloid leukemia, 50 myelodysplastic syndrome, and 40 blood DNA samples from individuals without evidence of clonal blood disorders. We demonstrate robust detection of sequence changes in 49 genes, including difficult-to-detect mutations such as FLT3 internal-tandem and mixed-lineage leukemia (MLL) partial-tandem duplications, and clinically significant chromosomal rearrangements including MLL translocations to known and unknown partners, identifying the novel fusion gene MLL-DIAPH2 in the process. Additionally, we identify most significant chromosomal gains and losses, and several copy neutral loss-of-heterozygosity mutations at a genome-wide level, including previously unreported changes such as homozygosity for DNMT3A R882 mutations. Karyogene represents a dependable genomic diagnosis platform for translational research and for the clinical management of myeloid malignancies, which can be readily adapted for use in other cancers

Leukemia-associated somatic mutations drive distinct patterns of age-related clonal hemopoiesis.

Clonal hemopoiesis driven by leukemia-associated gene mutations can occur without evidence of a blood disorder. To investigate this phenomenon, we interrogated 15 mutation hot spots in blood DNA from 4,219 individuals using ultra-deep sequencing. Using only the hot spots studied, we identified clonal hemopoiesis in 0.8% of individuals under 60, rising to 19.5% of those ≥90 years, thus predicting that clonal hemopoiesis is much more prevalent than previously realized. DNMT3A-R882 mutations were most common and, although their prevalence increased with age, were found in individuals as young as 25 years. By contrast, mutations affecting spliceosome genes SF3B1 and SRSF2, closely associated with the myelodysplastic syndromes, were identified only in those aged >70 years, with several individuals harboring more than one such mutation. This indicates that spliceosome gene mutations drive clonal expansion under selection pressures particular to the aging hemopoietic system and explains the high incidence of clonal disorders associated with these mutations in advanced old age

The 5-Hydroxymethylcytosine Landscape of Prostate Cancer

Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease. SIGNIFICANCE: In prostate cancer, 5-hydroxymethylcytosine delineates oncogene activation and stage-specific cell states and can be analyzed in liquid biopsies to detect cancer phenotypes. See related article by Wu and Attard, p. 3880.publishedVersionPeer reviewe

Caracterización molecular del papel de los complejos remodeladores de la cromatina en la progresión tumoral

ABSTRACT: The mutational landscape of cancer has been robustly analyzed in the last decades using next-generation sequencing (NGS) allowing the identification of somatic mutations in several SWI/SNF chromatin remodeling complex subunits such as SMARCA4, SMARCA2, ARID1A, ARID1B, ARID2 and PBRM1 in different tumor types. Several studies propose the modulation of specific transcriptional programs and defects in DNA repair mechanisms as potential explanations of the role of SWI/SNF complex alterations in the pathogenesis of cancer. The primary aim of this thesis is to determine which chromatin remodeling genes/complexes play an important role in tumor development, as well as to identify the molecular mechanisms and, finally, to determine if the alterations in these complexes can be used as diagnostic, prognostic or therapeutic tools to improve the management of cancer patients. We have performed target sequencing of 732 samples (479 tumors and 257 matched normal and 45 cell lines) from several human cancer types, using custom probes designed against the coding sequence of 250 genes including chromatin regulators as well as recurrently mutated cancer genes. Validation of the potential mutations found in the screening was assessed using high-coverage PCR-based ultrasequencing. Finally, proliferation, migration and invasion studies in vitro and in vivo were performed to validate the functional impact of some of the alterations in cancer cell lines. This comprehensive genomic analysis identified 4920 somatic mutations among all the samples studied. More than 800 of these mutations are located in the genes of the components of the four main ATP-dependent chromatin remodeling complexes (SWI/SNF, ISWI, CHD, and INO80). Interestingly, genomic alterations in chromatin remodelers (mainly SWI/SNF) appear to be mutually exclusive, indicating a functional redundancy for some of these genes. In addition, we identify new candidate genes who had not previously de-scribed as driver genes in cancer or have not been described in the tumor type in which they have been found in this study. These genes include ARID2, SRCAP, SMARCA5, CTCF, CHD4, SSX1, JHDM1D or SMAD4. We have tested the effect of ARID2 silencing on proliferation, migration and invasion studies in vitro and in vivo and we have identified the transcriptional alterations produced by this silencing. Furthermore, we have checked the effect of ARID2-deficiency on response to DNA damaging treatments like cisplatin or etoposide. We saw that ARID2-deficiency increased cell sensitivity to both cisplatin and etoposide evidenced by a lower IC50. Our results are in agreement with the notion that chromatin structure plays an important role in tumor progression and that some genes coding for components of ATP-dependent chromatin remodeling complexes are bona fide cancer driver genes essential for tumor development. We found evidence that alterations in these genes might be used as prognostic markers as well as biomarkers for patient stratification in the study of response to specific cancer treatments as is the case of ARID2 in lung cancer. ARID2 deficiency seems to exert a tumor suppressor role by two complementary mechanisms, firstly, generating a pro-tumoral transcriptional program in the cell and, subsequently, affecting the DNA repair mechanisms generating genetic instability. This dual role of ARID2 deficiency in cancer probably extends to other SWI/SNF subunits that could be independent of the function of the canonical SWI/SNF complexes.RESUMEN: En las últimas décadas, se ha analizado los patrones mutacionales del cáncer de manera robusta utilizando secuenciación de nueva generación (NGS). Esto ha permitido la identificación de mutaciones somáticas en varias subunidades del complejo SWI/SNF en diferentes tipos tumorales como SMARCA4, SMARCA2, ARID1A, ARID1B, ARID2 y PBRM1. Varios estudios proponen que la modulación de programas transcripcionales específicos así como defectos en los mecanismos de reparación del ADN pueden explicar el papel de las alteraciones del complejo SWI/SNF en la progresión del cáncer. El objetivo principal de esta tesis es determinar qué genes/complejos juegan un papel importante en el desarrollo tumoral, así como identificar los mecanismos moleculares por los cuales los defectos en estos genes juegan un papel importante en el desarrollo tumoral y si, finalmente, estas alteraciones se pueden utilizar con valor diagnóstico, pronóstico o terapéutico para mejorar el manejo de los pacientes de cáncer. Hemos llevado a cabo la secuenciación dirigida de 732 muestras (479 tumores y 257 normales y 45 líneas celulares) de varios tipos de cáncer humano, utilizando sondas diseñadas específicamente contra la secuencia codificante de 250 genes que incluyen regula-dores de cromatina además de genes recurrentemente mutados en cáncer. La validación de algunas de las mutaciones encontradas se evaluó mediante PCR acoplada a secuenciación a alta cobertura. Finalmente, se realizaron estudios de proliferación, migración e invasión in vitro e in vivo para validar el impacto funcional de algunas de las alteraciones en líneas celulares tumorales. Este análisis genómico integral identificó 4920 mutaciones somáticas entre todas las muestras estudiadas. Más de 800 de estas mutaciones se encuentran en los genes de los componentes de los cuatro principales complejos de remodelación de cromatina dependientes de ATP (SWI/SNF, ISWI, CHD e INO80). Curiosamente, las alteraciones genómicas en los remodeladores de cromatina (principalmente SWI/SNF) parecen ser mutua-mente excluyentes, lo que indica una redundancia funcional en algunos de estos genes. Además, identificamos nuevos genes candidatos que no se habían postulado anterior-mente como implicados en cáncer o no han sido descritos como importantes en los subtipos en los que han sido encontrados en nuestro estudio. Estos genes incluyen ARID2, SRCAP, SMARCA5, CTCF, CHD4, SSX1, JHDM1D o SMAD4. Hemos comprobado el efecto del silenciamiento de ARID2 en proliferación, migración e invasión in vitro e in vivo. También hemos identificado las alteraciones transcripcionales resultado de la deficiencia en ARID2. Por último, hemos verificado el efecto de la deficiencia de ARID2 en la res-puesta a los tratamientos que dañan el ADN como el cisplatino o el etopósido observando que esta deficiencia aumenta la sensibilidad celular a ambos compuestos evidenciado por una IC50 más baja. Nuestros resultados concuerdan con la idea de que la estructura de la cromatina desempeña un papel importante en la progresión tumoral y que algunos genes que codifican componentes de los complejos remodeladores de cromatina dependientes de ATP son genes esenciales para el desarrollo de tumores. Encontramos evidencia de que alteraciones en estos genes podrían usarse como marcadores de pronóstico y biomarcadores para la estratificación de pacientes en el estudio de la respuesta a tratamientos específicos contra el cáncer, como es el caso de ARID2 en el cáncer de pulmón. La deficiencia de ARID2 parece ejercer un papel de supresor tumoral mediante dos mecanismos complementarios, en primer lugar provocando un programa transcripcional pro-tumoral en la célula y en segundo lugar afectando a la reparación del ADN provocando inestabilidad genética. Este doble papel que parece jugar ARID2 en cáncer de pulmón probablemente sea extensivo a otras subunidades del complejo SWI/SNF y a su vez podría ser independientes de la función canónica de los complejos SWI/SNF.Esta tesis ha sido realizada en el Instituto de Biomedicina y Biotecnología de Cantabria (Santander) y una parte en el Francis Crick Institute (London). La financiación necesaria para la realización de esta Tesis doctoral ha sido aportada por: MInisterio de Economía y Competitividad a traves de los Proyectos del Plan Nacional: SAF2012-31627 y SAF2016-76758-R. La Fundacion Ramon Areces a traves del Proyecto: Proyecto de investigación de ciencias de la vida 2015: Caracterización molecular del papel de la disfunción mitocondrial en el desarrollo tumoral. European Research Council a través del proyecto ERC-2014-STG-637904 INTRAHETEROSEQ. El autor de esta tesis ha disfrutado de una Ayuda para contratos predoctorales para la formación de doctores 2013, referencia BES-2013-062983, concedida por el MInisterio de Economía y Competitividad, al igual que una Ayuda a la movilidad predoctoral para la realización de estancias breves en centro de I+D españoles y extranjeros de la convocatoria 2015, referencia EEBB-I-16-11749

AARDVARK: an automated reversion detector for variants affecting resistance kinetics.

SUMMARY: Resistance to two classes of FDA-approved therapies that target DNA repair-deficient tumors is caused by mutations that restore the tumor cells DNA repair function. Identifying these reversion mutations currently requires manual annotation of patient tumor sequence data. Here we present AARDVARK, an R package that automatically identifies reversion mutations from DNA sequence data. AVAILABILITY AND IMPLEMENTATION: AARDVARK is implemented in R (≥3.5). It is available on GitHub at https://github.com/davidquigley/aardvark. It is licensed under the MIT license