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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., Accurate recapitulation of human disease in animal models requires generation of complex and heterogeneous genetic variation. Here the authors combine RCAS-TVA with CRISPR-Cas9 to generate mouse models of cancer.

Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin β family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway., The epidermal growth factor receptor (EGFR) signalling is regulated at multiple levels. Here the authors show that the importin RanBP6 acts as a tumor suppressor in Glioblastoma and regulates EGFR signalling through promoting translocation of STAT3 to the nuclei and repressing EGFR transcription.

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells in vitro. We used an orthotopic zebrafish model to confirm our results in vivo and light-sheet microscopy to follow the effect of MTH1 inhibition in GBM in real time. In conclusion, MTH1 represents a promising target for GBM therapy and MTH1 inhibitors may also be effective in patients that suffer from recurring disease. We thank the staff of the zebrafish core facility for their excellent service, K.Edfelt and C.Sjögren for administrative help, S.Eriksson and F.Pineiro for lab support, and J.Carreras-Puigvert for help with the cell profiler software. We acknowledge M.Scobie, K.Vallin, T.Koolmeister, M.Henriksson, O.Wallner and S.Jacques for synthesis of MTH1 inhibitors and M.Guo and M.Nister for providing the GBM cultures (all Karolinska Institutet). Sí

Background A critical challenge in the management of Glioblastoma Multiforme (GBM) tumors is the accurate diagnosis and assessment of tumor progression in a noninvasive manner. We have identified Membrane-type 1 matrix metalloproteinase (MT1-MMP) as an attractive biomarker for GBM imaging since this protein is actively involved in tumor growth and progression, correlates with tumor grade and is closely associated with poor prognosis in GBM patients. Here, we report the development of an immunoPET tracer for effective detection of MT1-MMP in GBM models. Methods An anti-human MT1-MMP monoclonal antibody (mAb), LEM2/15, was conjugated to p-isothiocyanatobenzyl-desferrioxamine (DFO-NCS) for Zr-89 labeling. Biodistribution and PET imaging studies were performed in xenograft mice bearing human GBM cells (U251) expressing MT1-MMP and non-expressing breast carcinoma cells (MCF-7) as negative control. Two orthotopic brain GBM models, patient-derived neurospheres (TS543) and U251 cells, with different degrees of blood-brain barrier (BBB) disruption were also used for PET imaging experiments. Results Zr-89 labeling of DFO-LEM2/ 15 was achieved with high yield (>90\%) and specific activity (78.5 MBq/mg). Biodistribution experiments indicated that Zr-89-DFO-LEM2/15 showed excellent potential as a radiotracer for detection of MT1-MMP positive GBM tumors. PET imaging also indicated a specific and prominent Zr-89-DFO-LEM2/15 uptake in MT1-MMP+ U251 GBM tumors compared to MT1-MMP-MCF-7 breast tumors. Results obtained in orthotopic brain GBM models revealed a high dependence of a disrupted BBB for tracer penetrance into tumors. Zr-89-DFO-LEM2/15 showed much higher accumulation in TS543 tumors with a highly disrupted BBB than in U251 orthotopic model in which the BBB permeability was only partially increased. Histological analysis confirmed the specificity of the immunoconjugate in all GBM models. Conclusion A new anti MT1-MMP-mAb tracer, Zr-89-DFO-LEM2/15, was synthesized efficiently. In vivo validation showed high-specific-contrast imaging of MT1-MMP positive GBM tumors and provided strong evidence for utility of MT1-MMP-targeted immunoPET as an alternate to nonspecific imaging of GBM. MAM JLMT AJS FMA: Crowdfunding platform ``Precipita´´ FECYT (Spanish Foundation for Science and Technology) [http://www.precipita.es/precipitado/deteccion-y-seguimiento-del-cance r-cerebral-mas-comun-y-danino.html]. AGA JLMT: Regional Government of Madrid Angiobodies Programme S2010/BMD-2312 [http://www.madrid.org/cs/Satellite?buscador=true\&c=CM\_ConvocaPresta c\_FA\&cid=1142619643182\&language=es\&pagename=ComunidadMadrid\%2FEstru ctura]. AJS: BBVA Foundation grant for Research, Innovation and Cultural Creation and M+Vision Advanced fellowship. MS: Marie Curie CIG grant \& Seve Ballesteros Foundation grant. Sí