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Most lncRNAs display species-specific expression patterns suggesting that animal models of cancer may only incompletely recapitulate the regulatory crosstalk between lncRNAs and oncogenic pathways in humans. Among these pathways, Sonic Hedgehog (SHH) signaling is aberrantly activated in several human cancer entities. We unravel that aberrant expression of the primate-specific lncRNA HedgeHog Interacting Protein-AntiSense 1 (HHIP-AS1) is a hallmark of SHH-driven tumors including medulloblastoma and atypical teratoid/rhabdoid tumors. HHIP-AS1 is actively transcribed from a bidirectional promoter shared with SHH regulator HHIP. Knockdown of HHIP-AS1 induces mitotic spindle deregulation impairing tumorigenicity in vitro and in vivo. Mechanistically, HHIP-AS1 binds directly to the mRNA of cytoplasmic dynein 1 intermediate chain 2 (DYNC1I2) and attenuates its degradation by hsa-miR-425-5p. We uncover that neither HHIP-AS1 nor the corresponding regulatory element in DYNC1I2 are evolutionary conserved in mice. Taken together, we discover an lncRNA-mediated mechanism that enables the pro-mitotic effects of SHH pathway activation in human tumors.

The utility of patient-derived tumor cell lines as experimental models for glioblastoma has been challenged by limited representation of the in vivo tumor biology and low clinical translatability. Here, we report on longitudinal epigenetic and transcriptional profiling of seven glioblastoma spheroid cell line models cultured over an extended period. Molecular profiles were associated with drug response data obtained for 231 clinically used drugs. We show that the glioblastoma spheroid models remained molecularly stable and displayed reproducible drug responses over prolonged culture times of 30 in vitro passages. Integration of gene expression and drug response data identified predictive gene signatures linked to sensitivity to specific drugs, indicating the potential of gene expression-based prediction of glioblastoma therapy response. Our data thus empowers glioblastoma spheroid disease modeling as a useful preclinical assay that may uncover novel therapeutic vulnerabilities and associated molecular alterations.

Background: Glioblastoma is the most common and most lethal primary brain cancer. CBF1 (also known as Recombination signal Binding Protein for immunoglobulin kappa J, RBPJ) is the cardinal transcriptional regulator of the Notch signalling network and has been shown to promote cancer stem-like cells (CSCs) in glioblastoma. Recent studies suggest that some of the malignant properties of CSCs are mediated through the activation of pro-invasive programme of epithelial-to-mesenchymal transition (EMT). Little is known whether CBF1 is involved in the EMT-like phenotype of glioma cells. Methods: In a collection of GBM neurosphere lines, we genetically inhibited CBF1 and investigated the consequences on EMT-related properties, including in vitro invasiveness by Boyden chambers assay, chemoresistance using a clinical drug library screen and glycolytic metabolism assessing live-cell extracellular acidification rate. We also compared CBF1 expression in cells exposed to low and high oxygen tension. In silico analysis in large-scale Western and Eastern patient cohorts investigated the clinical prognostic value of CBF1 expression in low- and high-grade glioma as well as medulloblastoma. Results: Mean CBF1 expression is significantly increased in isocitrate dehydrogenase 1 (IDH1) R132H mutant glioblastoma and serves as prognostic marker for prolonged overall survival in brain tumours, particularly after therapy with temozolomide. Hypoxic regions of glioblastoma have higher CBF1 activation and exposure to low oxygen can induce its expression in glioma cells in vitro. CBF1 inhibition blocks EMT activators such as zinc finger E-box-binding homeobox 1 (ZEB1) and significantly reduces cellular invasion and resistance to clinically approved anticancer drugs. Moreover, we indicate that CBF1 inhibition can impede cellular glycolysis. Conclusions: Mean CBF1 activation in bulk tumour samples serves as a clinical predictive biomarker in brain cancers but its intratumoral and intertumoral expression is highly heterogeneous. Microenvironmental changes such as hypoxia can stimulate the activation of CBF1 in glioblastoma. CBF1 blockade can suppress glioblastoma invasion in vitro in particular in cells undergone EMT such as those found in the hypoxic niche. Targeting CBF1 can be an effective anti-EMT therapy to impede invasive properties and chemosensitivity in those cells.

Pilocytic astrocytomas as well as other pediatric low-grade gliomas (pLGG) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are

// Florian Selt 1, 2 , Juliane Hohloch 1 , Thomas Hielscher 3 , Felix Sahm 4, 5 , David Capper 4, 5 , Andrey Korshunov 4, 5 Diren Usta 1 , Sebastian Brabetz 6 , Johannes Ridinger 1 , Jonas Ecker 1, 2 , Ina Oehme 1 , Jan Gronych 7, 8 , Viktoria Marquardt 9 , David Pauck 9 , Heidi Bachli 10 , Charles D. Stiles 11 , Andreas von Deimling 4, 5 , Marc Remke 9 , Martin U. Schuhmann 12 , Stefan M. Pfister 2, 6 , Tilman Brummer 13 , David T.W. Jones 6 , Olaf Witt 1, 2, * , Till Milde 1, 2, * 1 Clinical Cooperation Unit Pediatric Oncology (G340), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany 2 Center for Individualized Pediatric Oncology (ZIPO) and Section of Pediatric Brain Tumors, Department of Pediatric Oncology, Hematology and Immunology, University Hospital Heidelberg, Heidelberg, Germany 3 Division of Biostatistics (C060), German Cancer Research Center (DKFZ), Heidelberg, Germany 4 Department of Neuropathology, University Hospital Heidelberg, Heidelberg, Germany 5 Clinical Cooperation Unit Neuropathology (G380), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany 6 Division of Pediatric Neurooncology (B062), German Cancer Research Center (DKFZ), Heidelberg, Germany, and German Cancer Consortium (DKTK), Heidelberg, Germany 7 Division of Molecular Genetics (B060), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany 8 AbbVie Deutschland GmbH & Co. KG, Medical Immunology, Wiesbaden, Germany (current affiliation) 9 Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Dusseldorf, Germany, and Department of Pediatric Neuro-Oncogenomics, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany 10 Department of Neurosurgery, University Hospital Heidelberg, Heidelberg, Germany 11 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA 12 Department of Neurosurgery, University Hospital Tubingen, Tubingen, Germany 13 Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University and University Medical Centre, Freiburg, Germany * These authors have contributed equally to this work Correspondence to: Florian Selt, email: f. selt@dkfz.de Keywords: pediatric low grade glioma, pilocytic astrocytoma, KIAA1549:BRAF-fusion, oncogene-induced senescence (OIS), MAPK-inhibitors Received: November 18, 2016 Accepted: November 23, 2016 Published: December 17, 2016 ABSTRACT Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. Activation of the MAPK pathway is well established as the oncogenic driver of the disease. It is most frequently caused by KIAA1549:BRAF fusions, and leads to oncogene induced senescence (OIS). OIS is thought to be a major reason for growth arrest of PA cells in vitro and in vivo , preventing establishment of PA cultures. Hence, valid preclinical models are currently very limited, but preclinical testing of new compounds is urgently needed. We transduced the PA short-term culture DKFZ-BT66 derived from the PA of a 2-year old patient with a doxycycline-inducible system coding for Simian Vacuolating Virus 40 Large T Antigen (SV40-TAg). SV40-TAg inhibits TP53/CDKN1A and CDKN2A/RB1, two pathways critical for OIS induction and maintenance. DNA methylation array and KIAA1549:BRAF fusion analysis confirmed pilocytic astrocytoma identity of DKFZ-BT66 cells after establishment. Readouts were analyzed in proliferating as well as senescent states, including cell counts, viability, cell cycle analysis, expression of SV40-Tag, CDKN2A (p16), CDKN1A (p21), and TP53 (p53) protein, and gene-expression profiling. Selected MAPK inhibitors (MAPKi) including clinically available MEK inhibitors (MEKi) were tested in vitro . Expression of SV40-TAg enabled the cells to bypass OIS and to resume proliferation with a mean doubling time of 45h allowing for propagation and long-term culture. Withdrawal of doxycycline led to an immediate decrease of SV40-TAg expression, appearance of senescent morphology, upregulation of CDKI proteins and a subsequent G1 growth arrest in line with the re-induction of senescence. DKFZ-BT66 cells still underwent replicative senescence that was overcome by TERT expression. Testing of a set of MAPKi revealed differential responses in DKFZ-BT66. MEKi efficiently inhibited MAPK signaling at clinically achievable concentrations, while BRAF V600E- and RAF Type II inhibitors showed paradoxical activation. Taken together, we have established the first patient-derived long term expandable PA cell line expressing the KIAA1549:BRAF-fusion suitable for preclinical drug testing.

Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38−) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)–resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.

The synthesis and biological evaluation of potent hydroxamate-based dual HDAC1/6 inhibitors with modest HDAC6 preference and a novel alkoxyurea connecting unit linker region are described. The biological studies included the evaluation of antiproliferative effects and HDAC inhibitory activity in the human ovarian cancer cell line A2780, the human squamous carcinoma cell line Cal27, and their cisplatin resistant sublines A2780CisR and Cal27CisR. The three most potent compounds 1g-i showed IC50 values in the low muM and sub-muM range. 1g-i revealed low nM IC50 values for HDAC6 with up to 15-fold preference over HDAC1, >3500-fold selectivity over HDAC4, and >100-fold selectivity over HDAC8. Furthermore, their ability to enhance cisplatin sensitivity was analyzed in Cal27 and Cal27CisR cells. Notably, a 48 h preincubation of 1g-i significantly enhanced the antiproliferative effects of cisplatin in Cal27 and Cal27CisR. 1g-i interacted synergistically with cisplatin. These effects were more pronounced for the cisplatin resistant subline Cal27CisR.

Targeting TGFβ/Smad signaling is an attractive strategy for several therapeutic applications given its role as a key player in many pathologies, including cancer, autoimmune diseases and fibrosis. The class of b-annelated 1,4-dihydropyridines (DHPs) represents promising novel pharmacological tools as they interfere with this pathway in a novel fashion, i.e. through induction of TGFβ receptor type II degradation. In the present work, >40 rationally designed, novel DHPs were synthesized and evaluated for TGFβ inhibition, substantially expanding the current understanding of the SAR profile. Key findings include that the 2-position tolerates a wide variety of polar functionalities, suggesting that this region could possibly be solvent-exposed within the (thus far) unknown cellular target. A structural explanation for pathway selectivity is provided based on a diverse series of 4″-substituted DHPs, including molecular electrostatic potential (MEP) calculations. Moreover, the absolute configuration for the chiral 4-position was determined by X-ray crystal analysis and revealed that the bioactive (+)-enantiomers are (R)-configured. Another key objective was to establish a 3D-QSAR model which turned out to be robust (r2 = 0.93) with a good predictive power (r2pred = 0.69). This data further reinforces the hypothesis that this type of DHPs exerts its novel TGFβ inhibitory mode of action through binding a distinct target and that unspecific activities that would derive from intrinsic properties of the ligands (e.g., lipophilicity) play a negligible role. Therefore, the present study provides a solid basis for further ligand-based design of additional analogs or DHP scaffold-derived compounds for hit-to-lead optimization, required for more comprehensive pharmacological studies in vivo.