Nathalene Truffaux

Reduced H3K27me3 and DNA Hypomethylation Are Major Drivers of Gene Expression in K27M Mutant Pediatric High-Grade Gliomas

Two recurrent mutations, K27M and G34R/V, within histone variant H3.3 were recently identified in ∼50% of pHGGs. Both mutations define clinically and biologically distinct subgroups of pHGGs. Here, we provide further insight about the dominant-negative effect of K27M mutant H3.3, leading to a global reduction of the repressive histone mark H3K27me3. We demonstrate that this is caused by aberrant recruitment of the PRC2 complex to K27M mutant H3.3 and enzymatic inhibition of the H3K27me3-establishing methyltransferase EZH2. By performing chromatin immunoprecipitation followed by next-generation sequencing and whole-genome bisulfite sequencing in primary pHGGs, we show that reduced H3K27me3 levels and DNA hypomethylation act in concert to activate gene expression in K27M mutant pHGGs.

Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine gliomas (DIPGs) are highly infiltrative malignant glial neoplasms of the ventral pons that, due to their location within the brain, are unsuitable for surgical resection and consequently have a universally dismal clinical outcome. The median survival time is 9–12 months, with neither chemotherapeutic nor targeted agents showing substantial survival benefit in clinical trials in children with these tumors. We report the identification of recurrent activating mutations in the ACVR1 gene, which encodes a type I activin receptor serine/threonine kinase, in 21% of DIPG samples. Strikingly, these somatic mutations (encoding p.Arg206His, p.Arg258Gly, p.Gly328Glu, p.Gly328Val, p.Gly328Trp and p.Gly356Asp substitutions) have not been reported previously in cancer but are identical to mutations found in the germ line of individuals with the congenital childhood developmental disorder fibrodysplasia ossificans progressiva (FOP) and have been shown to constitutively activate the BMP–TGF-β signaling pathway. These mutations represent new targets for therapeutic intervention in this otherwise incurable disease.

Functionally defined therapeutic targets in diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNA-seq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi–histone deacetylase inhibitor panobinostat demonstrated therapeutic efficacy both in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4 revealed that the two had synergistic effects. Together, these data suggest a promising therapeutic strategy for DIPG.

Recurrent MET fusion genes represent a drug target in pediatric glioblastoma

Pediatric glioblastoma is one of the most common and most deadly brain tumors in childhood. Using an integrative genetic analysis of 53 pediatric glioblastomas and five in vitro model systems, we identified previously unidentified gene fusions involving the MET oncogene in ∼10% of cases. These MET fusions activated mitogen-activated protein kinase (MAPK) signaling and, in cooperation with lesions compromising cell cycle regulation, induced aggressive glial tumors in vivo. MET inhibitors suppressed MET tumor growth in xenograft models. Finally, we treated a pediatric patient bearing a MET-fusion-expressing glioblastoma with the targeted inhibitor crizotinib. This therapy led to substantial tumor shrinkage and associated relief of symptoms, but new treatment-resistant lesions appeared, indicating that combination therapies are likely necessary to achieve a durable clinical response.

Erratum: Functionally defined therapeutic targets in diffuse intrinsic pontine glioma(Nature Medicine (2015) 21 (555-559) DOI: 10.1038/nm.3855)

Catherine S Grasso, Yujie Tang, Nathalene Truffaux, Noah E Berlow, Lining Liu, Marie-Anne Debily, Michael J Quist, Lara E Davis, Elaine C Huang, Pamelyn J Woo, Anitha Ponnuswami, Spenser Chen, Tessa B Johung, Wenchao Sun, Mari Kogiso, Yuchen Du, Lin Qi, Yulun Huang, Marianne Hütt-Cabezas, Katherine E Warren, Ludivine Le Dret, Paul S Meltzer, Hua Mao, Martha Quezado, Dannis G van Vuurden, Jinu Abraham, Maryam Fouladi, Matthew N Svalina, Nicholas Wang, Cynthia Hawkins, Javad Nazarian, Marta M Alonso, Eric H Raabe, Esther Hulleman, Paul T Spellman, Xiao-Nan Li, Charles Keller, Ranadip Pal, Jacques Grill & Michelle Monje Nat. Med. 21, 555–559 (2015); doi:10.1038/nm.3855; published online 4 May 2015; corrected after print 15 June 2015

Preclinical evaluation of dasatinib alone and in combination with cabozantinib for the treatment of diffuse intrinsic pontine glioma.

BACKGROUND Platelet-derived growth factor receptor A is altered by amplification and/or mutation in diffuse intrinsic pontine glioma (DIPG). We explored in vitro on new DIPG models the efficacy of dasatinib, a multi-tyrosine kinase inhibitor targeting this receptor. METHODS Gene expression profiles were generated from 41 DIPGs biopsied at diagnosis and compared with the signature associated with sensitivity/resistance to dasatinib. A panel of 12 new DIPG cell lines were established from biopsy at diagnosis, serially passaged, and characterized by gene expression analyses. Effects of dasatinib (1-10 μM) on proliferation, invasion, and cytotoxicity were determined on 4 of these cell lines using live-cell imaging and flow cytometry assays. Downstream signaling and receptor tyrosine kinases (RTKs) were assessed by western blot and phospho-RTK array. The effect of the combination with the c-Met inhibitor cabozantinib was studied on cellular growth and invasion analyzed by the Chou-Talaly method. RESULTS DIPG primary tumors and cell lines exhibited the gene expression signature of sensitivity to dasatinib. Dasatinib reduced proliferation (half-maximal inhibitory concentration = 10-100 nM) and invasion (30%-60% reduction) at 100 nM in 4/4 cultures and induced apoptosis in 1 of 4 DIPG cell lines. Activity of downstream effectors of dasatinib targets including activin receptor 1 was strongly reduced. Since multiple RTKs were activated simultaneously in DIPG cell lines, including c-Met, which can be also amplified in DIPG, the benefit of the combination of dasatinib with cabozantinib was explored for its synergistic effects on proliferation and migration/invasion in these cell lines. CONCLUSION Dasatinib exhibits antitumor effects in vitro that could be increased by the combination with another RTK inhibitor targeting c-Met.

Abstract 2763: Activity of dasatinib and potential escape mechanisms in diffuse intrinsic pontine glioma (DIPG) models.

Background Diffuse intrinsic pontine glioma (DIPG) have a dismal prognosis and their biology is not yet well explored. Using an integrated molecular profiling approach we revealed PDGFRA gene amplification and mutations in the extracellular domain as a characteristic of the oligodendroglial subgroup in newly diagnosed pediatric DIPG. Therefore we explored the therapeutic potential of dasatinib, an inhibitor of the PDGFR, Src, and Ephrin tyrosine-kinases in newly developed DIPG models. Materials and Methods A unique panel of 12 DIPG cell lines has been established from fresh tumor material biopsies in newly diagnosed children with DIPG with a 100% success rate. Four were oligoastrocytomas Grade 2 and 3, five astrocytomas Grade 2, 1 GBM and 2 non further specified; two had a gain of the PDGFRA gene. Effects of dasatinib on proliferation, invasion and cytotoxicity in vitro were determined at concentrations of 0.001-1 μM using the Live-cell imaging assay of the IncuCyte system. Cell growth and cell invasion were monitored by confluence algorithm, and cytotoxicity was determined by loss of membrane integrity generating nuclear staining by a fluorescent live-cell imaging reagent. Tyrosine-kinase receptors (RTK) and downstream signaling expression was assessed by phospho-RTK array and Western blot analysis. Results Anti-proliferative effects of dasatinib were observed in all 6 cell lines tested with IC50s between 0.02-0.07 μM. This compared favorably with the sensitive adult glioma T98G cell line and the pediatric supratentorial glioma SF188 cell line (0.15 μM and 0.3 μM, respectively). Significant dose-dependent cytotoxic effects were observed in two DIPG cell lines (NEM145 and NEM 168) and anti-invasive effects were seen in all cells at 0.1 μM. The two most sensitive cell lines exhibited PDGFRA activation and their primary tumors had a gain of PDGFRA. While Src was activated in all cell lines, we noticed that the less sensitive cell lines had MET overexpression. Conclusion Primary DIPG cell lines are highly sensitive in vitro to dasatinib suggesting its exploration is children with DIPG. Sensitivity may be linked not exclusively to PDGFRA activation but also to Src activation. The role of MET activation in escape mechanisms is currently explored. Citation Format: Nathalene Truffaux, Ludivine Le Dret, Stephanie Puget, Gilles Vassal, Birgit Geoerger, Jacques Grill. Activity of dasatinib and potential escape mechanisms in diffuse intrinsic pontine glioma (DIPG) models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2763. doi:10.1158/1538-7445.AM2013-2763