CRISPR-Cas9: A Revolutionary Tool for Cancer Modelling

The cancer-modelling field is now experiencing a conversion with the recent emergence of the RNA-programmable CRISPR-Cas9 system, a flexible methodology to produce essentially any desired modification in the genome. Cancer is a multistep process that involves many genetic mutations and other genome rearrangements. Despite their importance, it is difficult to recapitulate the degree of genetic complexity found in patient tumors. The CRISPR-Cas9 system for genome editing has been proven as a robust technology that makes it possible to generate cellular and animal models that recapitulate those cooperative alterations rapidly and at low cost. In this review, we will discuss the innovative applications of the CRISPR-Cas9 system to generate new models, providing a new way to interrogate the development and progression of cancers.This work was supported by FIS project no PI14/01884 to Sandra Rodriguez-Perales from the Spanish plan nacional I+d+, Instituto de Salud Carlos III FEDER. The funders had no role in the study design, data collection and analysis, decision to publish nor preparation of the manuscript.S

A faecal microbiota signature with high specificity for pancreatic cancer

Cancer prevention; Intestinal microbiology; Pancreatic tumoursPrevenció del càncer; Microbiologia intestinal; Tumors pancreàticsPrevención de cáncer; Microbiología intestinal; Tumores pancreáticosBackground Recent evidence suggests a role for the microbiome in pancreatic ductal adenocarcinoma (PDAC) aetiology and progression. Objective To explore the faecal and salivary microbiota as potential diagnostic biomarkers. Methods We applied shotgun metagenomic and 16S rRNA amplicon sequencing to samples from a Spanish case–control study (n=136), including 57 cases, 50 controls, and 29 patients with chronic pancreatitis in the discovery phase, and from a German case–control study (n=76), in the validation phase. Results Faecal metagenomic classifiers performed much better than saliva-based classifiers and identified patients with PDAC with an accuracy of up to 0.84 area under the receiver operating characteristic curve (AUROC) based on a set of 27 microbial species, with consistent accuracy across early and late disease stages. Performance further improved to up to 0.94 AUROC when we combined our microbiome-based predictions with serum levels of carbohydrate antigen (CA) 19–9, the only current non-invasive, Food and Drug Administration approved, low specificity PDAC diagnostic biomarker. Furthermore, a microbiota-based classification model confined to PDAC-enriched species was highly disease-specific when validated against 25 publicly available metagenomic study populations for various health conditions (n=5792). Both microbiome-based models had a high prediction accuracy on a German validation population (n=76). Several faecal PDAC marker species were detectable in pancreatic tumour and non-tumour tissue using 16S rRNA sequencing and fluorescence in situ hybridisation. Conclusion Taken together, our results indicate that non-invasive, robust and specific faecal microbiota-based screening for the early detection of PDAC is feasible.We acknowledge funding from EMBL, CNIO, World Cancer Research (#15–0391), the European Research Council (ERC-AdG-669830 MicrobioS), the BMBF-funded Heidelberg CenterCentre for Human Bioinformatics (HD-HuB) within the German Network for Bioinformatics Infrastructure (de.NBI #031A537B), Fondo de Investigaciones Sanitarias (FIS), Instituto de Salud Carlos III-FEDER, Spain (grant numbers PI15/01573, PI18/01347, FIS PI17/02303); Red Temática de Investigación Cooperativa en Cáncer, Spain (grant numbers RD12/0036/0034, RD12/0036/0050, RD12/0036/0073); III beca Carmen Delgado/Miguel Pérez-Mateo de AESPANC-ACANPAN; EU-6FP Integrated Project (#018771-MOLDIAG-PACA); EU-FP7-HEALTH (#259737-CANCERALIA). Funders had no involvement in the study design, patient enrolment, analysis, manuscript writing or reviewing

Playing “hide and seek” with Texas tortoises: value of a detector dog

Texas tortoises (Gopherus berlandieri) were once considered common and abundant throughout southern Texas with densities as high as 16 tortoises per hectare. Today, density estimates are 0.25 tortoises per hectare, which constitutes about a 98% population decline. Because of their low numbers and elusive behavior, Texas tortoises can be difficult to find. We demonstrate the value of using a detector dog as a time saving method in locating Texas tortoises. We glued VHF radio transmitters onto 9 adult tortoises and released them in a 5-ha plowed and short-grass pasture that contained mesquite (Prosopsis glandulosa) mottes, habitat conducive for Texas tortoise habitat selection. We calculated the Detectability Index (DI) as the detection rate (# tortoises found/minute) × percent tortoises from the known population found within 60 minutes. We compared DIs via telemetry, detector dog, and “cold” (no equipment or knowledge) human searches. We used the time required to find all tortoises when a searcher had knowledge of locations as the baseline. Our baseline DI was 0.79, followed by telemetry (0.13) and detector dogs (0.11), while “cold” searches was 0.02. Telemetry, detector dog, and cold searches were 6-fold, 7-fold, and nearly 40-fold slower, respectively, than having knowledge of tortoise locations. However, the combination of using detector dogs with telemetry resulted in a 50% time savings than single methods. Telemetry was useful in locating a generalized area with a tortoise but a detector dog was 2X faster in visually locating the tortoise once the area was identified. Therefore, we recommend the use of detector dogs as a time-saving method when conducting research on Texas tortoises

Somatic genome editing with the RCAS-TVA-CRISPR-Cas9 system for precision tumor modeling

To accurately recapitulate the heterogeneity of human diseases, animal models require to recreate multiple complex genetic alterations. Here, we combine the RCAS-TVA system with the CRISPR-Cas9 genome editing tools for precise modeling of human tumors. We show that somatic deletion in neural stem cells of a variety of known tumor suppressor genes (Trp53, Cdkn2a, and Pten) leads to high-grade glioma formation. Moreover, by simultaneous delivery of pairs of guide RNAs we generate different gene fusions with oncogenic potential, either by chromosomal deletion (Bcan-Ntrk1) or by chromosomal translocation (Myb-Qk). Lastly, using homology-directed-repair, we also produce tumors carrying the homologous mutation to human BRAF V600E, frequently identified in a variety of tumors, including different types of gliomas. In summary, we have developed an extremely versatile mouse model for in vivo somatic genome editing, that will elicit the generation of more accurate cancer models particularly appropriate for pre-clinical testing.A.C.-G is recipient of a Severo-Ochoa PhD fellowship. C.M. and V.M. are recipients of a "La Caixa "PhD fellowship. We thank A.J. Schuhmacher for the initial assistance with the intracranial injections in adult mice and C.S. Clemente-Troncone for the technical support. We thank Carmen Blanco, David Olmeda, and Marisol Soengas for sharing reagents and Orlando Dominguez for the help with the design of the BRAF high- throughput sequencing. We sincerely thank Dr. José Luis Rodríguez Peralto (Hospital U. 12 de Octubre Madrid) for the BRAF V600 IHCs staining. This research was supported by funds from the Acción Estratégica en Salud Spanish National Research and Development Plan, Instituto de Salud Carlos III (ISCIII), cofounder by FEDER (ERDF) (PI14/01884) to S.R.-P., by a 017 Leonardo Grant for Researchers and Cultural Creators from the BBVA Foundation and a grant from the Seve Ballesteros Foundation to M.S.S

The insecticides permethrin and chlorpyrifos show limited genotoxicity and no leukemogenic potential in human and murine hematopoietic stem progenitor cells

Altres ajuts: European Food and Safety Authority, EFSA.PRAS.2018.04-CT1; Consejo Nacional de Ciencia y Tecnología, CB-2012-01-183467; Centro de Investigación Biomédica en Red en Cáncer, PID2019-104695RBI00; Asociación Española Contra el Cáncer, AECC INVES211226MOLI

A human genome editing-based MLL::AF4 B-cell ALL model recapitulates key cellular and molecular leukemogenic features

The cellular ontogeny and location of the MLL-breakpoint influence the capacity of MLL-edited CD34+ HSPCs to initiate pro-B-ALL, and recapitulate the molecular features of MLL-AF4+ infant B-ALL patients. We provide key insights into the cellular-molecular leukemogenic determinants of MLL-AF4+ infant B-ALL

MGMT genomic rearrangements contribute to chemotherapy resistance in gliomas.

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ

DNA methylation profiles and their relationship with cytogenetic status in adult acute myeloid leukemia

Background: Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required. Methodology/Principal Findings: We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients. Conclusions/Significance: Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature

DNA Methylation Profiles and Their Relationship with Cytogenetic Status in Adult Acute Myeloid Leukemia

Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required.We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients.Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature

Comprehensive characterization of a novel, oncogenic and targetable SEPTIN6::ABL2 fusion in T-ALL

S