DNA interaction is a druggable target for

The EMBO Journal Peer Review Process File - EMBO-2014-89213

Manuscript EMBO-2014-89213

Gli1/DNA interaction is a druggable target for Hedgehogdependent tumor Paola Infante, Mattia Mori, Romina Alfonsi, Francesca Ghirga, Federica Aiello, Sara Toscano, Cinzia Ingallina, Mariangela Siler, Danilo Cucchi, Agnese Po, Evelina Miele, Davide D'Amico, Gianluca Canettieri, Enrico De Smaele, Elisabetta Ferretti, Isabella Screpanti, Gloria Uccello Barretta, Maurizio Botta, Bruno Botta, Alberto Gulino and Lucia Di Marcotullio Corresponding author: Lucia Di Marcotullio, La Sapienza University of Rome

Review timeline:

Submission date: Editorial Decision: Revision received: Editorial Decision: Revision received: Accepted:

10 June 2014 16 July 2014 03 October 2014 27 October 2014 04 November 2014 12 November 2014

Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) Editor: Thomas Schwarz-Romond 1st Editorial Decision

16 July 2014

Thank you very much for the opportunity to consider your paper that describes a hypothesis-driven drug development and validation approach in the context of Hh-driven tumorigenesis for publication in The EMBO Journal. The rather consistent comments from two expert scientists reveal significant interest in this dataset. You will also recognize that both demand (i) further-reaching results to corroborate specificity of the compound and (ii) convincing establishment of its biological activity in better suited in-vivo system followed by thorough pathological examination of the endpoint result after treatment. I hope you understand that these requests aim at solidifying your interesting observations and increase translational confidence in the study. Conditioned on such amendments (and I am pleased to see that ref#2 offers a variety of constructive suggestions), I am happy to invite a revised manuscript for continued consideration at The EMBO Journal. Please do not hesitate to get in touch in case I can be of any assistance/you would like to discuss feasibility, amount and timeline of some of the requested experiments.

© European Molecular Biology Organization

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I am happy to engage to facilitate eventual presentation of your results in The EMBO Journal and remain with best regards. -----------------------------------------------REFEREE COMMENTS Referee #1: In the manuscript the authors describe the characteristics of GLI-binding to DNA building on the previously published crystal structure of the Zn-finger region in complex with an oligonucleotide encompassing the specific recognition sequence. Further, a natural small molecule, Glabrescione B (GlaB), that interferes with binding of GLI1 or GLI2 to DNA is identified and is shown to inhibit Hh-pathway activity in vitro and in vivo in murine allograft models of medulloblastoma and BCC. Given the accumulating evidence of Hh-pathway activation at steps downstream of the receptor level (the target of presently available inhibitors) and the evident resistance development in patients treated with receptor level inhibitors there is an important need and interest to delineate alternative ways to block the Hh-signaling pathway. Major points; 1. One concern is the statement in the paper that "there is a lack of information on the structural and functional requirements of GLI1/DNA interaction" in light of the previously published structure of the GLI1 Zn-finger region in complex with DNA. Findings presented in the present ms (e.g. on the role of K350 in DNA binding) are in several cases obvious based on the structure. The MD analysis largely confirms what was already known and it is not apparent what is truly novel. The rationale to limit the examination of the effect on DNA binding to K350 and K340 mutations is not clear given that all basic residues appear to have an effect on DNA binding. How were the results of the gel retardation assay quantified and the bands corresponding to free probe defined? Were all GLI mutant proteins expressed at the same level? 2. Another major concern is the specificity of GlaB for GLI, which is not addressed. Additional evidence to corroborate that the observed effects of GlaB is dependent on GLI inhibition is also needed. For example by showing inhibition of GLI-DNA binding by ChIP in vitro and in vivo, rescue experiments by Gli1 expression and comparison to targeting of Gli1 and Gli2 by siRNA. 3. The rationale for the selected screening criteria is not clear and it would be interesting to know from the virtual screen how often GlaB ends up in the selected position. Moreover, the K350A single mutant, which may be the most interesting mutant is not analyzed. It is also surprising that a higher concentration of GlaB is required for a significant effect in the EMSA assay compared to the cell based assays. What is the Kd for the binding of GlaB to GLI1? Minor points; 4. In the experiments using transfected Gli1 and MEFs comparison to a GlaB related negative control would be valuable. 5. Since different doses of GlaB were used in the in vivo experiments with medulloblastoma and BCC analyzing the dose-response relationship including the correlation between tumor growth inhibition and Gli1 mRNA reduction would be of interest. 6. In some places residue 380 is denoted K and in others R. There are also some problems with the English in the ms.

Referee #3: The authors set out to identify novel drugs to inhibit binding of the transcription factor to Gli target sequences, aiming at developing a therapy of SHH driven tumors. To this end, they started with a bio-informatics / structural biology approach to first identify potential binding/ interaction sites. They identified a number of putative sites of interaction in the zinc finger domains of Gli , namely ZF4 and ZF5. A number of putative sides were identified as amenable by mutagenesis, and were


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validated by site-directed mutagenesis. Mutants of ZF4 and ZF5 were generated and the impact of individual mutations tested by a Gli transactivation (luciferase) assay. The authors have subsequently selected a medium (ZF4; K340A) and a highly effective (ZF4; K350A) mutation. Subsequent experiments aim at identifying compounds that would interfere with these sites in vitro. The compound GlaB was identified as an effective inhibitor of Gli. The following experiments then validate the effects of GlaB. These experiments comprise cell based assays by inhibiting growth, importantly in the absence of significant cytotoxicity. A set of experiments was designed to inform about in vivo applicability and effectiveness, addressed in allografting experiments of PTCH-/- cell preparation injected heterotopcially into the flank of nude mice. In vivo administration of GlaB into grafted mice resulted in a reduction of tumors size and preliminary histological examination suggests reduction of proliferating cells. This is an elegant and well-designed study, demonstrating a hypothesis-driven drug development and validation. The strengths of the study are the detailed functional analysis of the binding sites and the interaction of the inhibitory drug with the target site. There are a number of shortcomings, though, which will need to be addressed to demonstrate the in vivo effects more convincingly, supported by more relevant in vivo experiments and higher endpoint quality data. Also there are controls which I would like to see included. The authors may already have generated these but may not have included them. Major points. In vivo validation of drug effectiveness. The authors have used PTCH-/- cell lines derived from cerebellar tumors arising in PTCH-/- mice. These cells were then heterotopcially allografted into the flank of (genetically) immunosuppressed mice. There are technical and biological concerns about this approach: (1) technical: (a) The measurement of the size of these tumors: no indication is given how the size and growth dynamics were monitored. State of the art determination of the size and the grown dynamics involve transplantation of cells carrying a luminescent (or otherwise labelled) reporter which can be monitored in vivo (For example, see PMID: 24982206; 24913958; 18843112). Other in vivo imaging methods, such as MRI may be utilised, but it is acknowledged that small animal MRI with sufficient resolution, e.g. 8-10T field strength may not be easily available. (b) The analysis of the resulting tumors is superficial and substandard. The histological image (Fig 6C) of both untreated and treated tumour shows non-specific background only and no proliferating cells, and is of inacceptable quality. The authors do not describe the potentially very important changes occurring in the tumors: Do tumours simply stop growing, do they undergo apoptosis, or do they undergo neuronal differentiation? What is the nature of the pink hypocellular tissue in Fig 6C, GalB treated ? Connective tissue, differentiated tumor, neural tissue, or simply host connective tissue? (2) Biological: (a) It is well known that CNS tumors can considerable change morphology and growth dynamics when transplanted outside the CNS. It would be important to address this (at least) by orthotopic transplantation into the CNS, ideally into the cerebellum but if not technically possible at least into the forebrain. Adequate pathological characterisation using a panel of biologically relevant markers is essential to show the effects of the drug on the tumor cells. Ideally the tumor cells should be labelled with a tracer (beta galactosidase for example) to track their fate. (b) The authors have not considered testing a true in vivo effect, for example by treating P4-P8 mice with a proliferating external granular layer (EGL) with GlaB, and analyse the effect on the EGL: What will happen to the EGL in vivo, will there be a reduction of cell proliferation, lesser migration towards the Internal granular layer, or premature differentiation and developmental reduction of the cerebellar size? These data will be essential to draw conclusions about the biological effects in the drug in this translational setting. (c) It needs to be tested, using a cohort of PTCH-/- mice (or an alternative, transgenic model with an SHH dependent pathway alteration and a higher medulloblastoma incidence): and treat one cohort with GLAB. Again, an adequate histological endpoint analysis including state of the art quantitative histological imaging is essential. (3) What is the reason of a reduction of cell growth and NS formation, apoptosis, or less proliferation? This needs to be addressed. Minor points:


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Fig 2D: The authors need to include the absence of an effect on non -target genes, e.g. from the wnt pathway or at least housekeeping genes, to demonstrate the selective action of GlaB. Fig 4e; Fig E7: The authors may consider adapting the scale a maximum of 30 or 40% to visualise the effects (or absence of) GlaB on cell viability. ---------------------------------------------Comments regarding this referee's expertise (self-assessment) 1) It is important to note that my scientific expertise does not cover an in-depth understanding of structural biology. Therefore is it essential that another reviewer with specific expertise assesses this aspect of the study. Comments regarding the experiments 2) I have made a number of suggestions regarding the assessment of in vivo effect of GlaB. I gave a number of options and recommendations [2a,. b, c], but I feel it would be unreasonable to ask to carry out all these experiments. It would be important that the authors address at least ONE, ideally perhaps two of the points. ((i) Postnatal developmental effects of GlaB on the cerebellar EGL, (ii) orthotopic allografting into the brain, (iii) using a transgenic brain tumor model (PTCH-/- mice for example) rather than injected cells; (iv) bioluminescence or similar imaging of the growth progression or reduction). However, no matter which one or two of the suggested approaches the authors decide on, the referee insists on a state of the art pathological analysis of the resulting tumors to be carried out and that these data are presented adequately.

1st Revision - authors' response

03 October 2014

Point-by-point replies to Reviewers’ comments. Reviewer #1 Reviewer: 1. One concern is the statement in the paper that "there is a lack of information on the structural and functional requirements of GLI1/DNA interaction" in light of the previously published structure of the GLI1 Zn-finger region in complex with DNA. Findings presented in the present ms (e.g. on the role of K350 in DNA binding) are in several cases obvious based on the structure. The MD analysis largely confirms what was already known and it is not apparent what is truly novel. - The rationale to limit the examination of the effect on DNA binding to K350 and K340 mutations is not clear given that all basic residues appear to have an effect on DNA binding. - How were the results of the gel retardation assay quantified and the bands corresponding to free probe defined? - Were all GLI mutant proteins expressed at the same level? Authors: We agree with the reviewer that a crystal structure describing the Gli1ZF/DNA complex is available and represents a profitable starting point for further elucidation. However, it is widely accepted that crystal structures represent static snapshots of dynamic systems in non-physiological conditions (i.e. solid state, presence of crystallization enhancers, crystal packing effects) becoming good tools for further in depth analysis, such as molecular dynamics simulations. In contrast, using X-ray structures without computational refinement may led to uncertainty during the ligand design step and computational studies may underline protein motions that are not described by the crystal structure. (Relevant papers in the field: Marco E, Gago F. ChemMedChem 2007, 2, 1388-1401; Phillips GN. Biology Reports 2009, 1:38, Schwartz W, Sakmar TP. Nature 477, 540–541). Finally, protein conformations solved by X-ray crystallography may be biased by unpredictable, and often neglected, crystal packing effects. In the specific case of the Gli1ZF/DNA complex, the crystal structure was solved at medium to low resolution (2.6 Å), using cobalt ions instead of the physiological zinc within the Zn-coordination system of each zinc finger. Since Zn-coordination is crucial for Gli1 activity, we expect that the replacement with cobalt may have determined slight conformational changes with respect to the native form.


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Additionally, and surely more relevant to our case, the crystal structure does not provide by itself information on the energy of the system as well as on the individual contribution of Gli1 residues to the binding affinity to DNA. For these reasons, we decided to investigate the conformational dynamics of the system (Gli1 is intrinsically highly flexible indeed) and to monitor the role of some lysine residues in DNA binding. As a proof, whether the crystal structure may suggest only a qualitative role for all these lysine residues, we found a significant quantitative discrimination of their role by MD simulations, further confirmed by in vitro mutagenesis studies (see revised Figures 1B and 1C, residues have different energy contribution in DNA binding). By looking at the X-ray structure without performing such studies, it was truly difficult, if not impossible, to discriminate quantitatively the contribution of DNA-interacting residues and to identify the putative ligand binding site. All this matter has been addressed and clarified in revised Results, page 6. Overall, we feel that our computational analysis provided novel insight into the Gli1ZF/DNA interaction. Finally, we have clarified that radiolabeled bands were directly quantitated by routine autoradiographic methods and assessed by the ratio of probe bound to GliZF versus free probe using Molecular Dynamics PhosphorImager (IMAGE QUANT software) (revised Materials and Methods). The same amounts of recombinant proteins were used, as shown in revised Expanded Figure E2. Reviewer: 2. Another major concern is the specificity of GlaB for GLI, which is not addressed. Additional evidence to corroborate that the observed effects of GlaB is dependent on GLI inhibition is also needed. For example by showing inhibition of GLI-DNA binding by ChIP, rescue experiments by Gli1 expression and comparison to targeting of Gli1 and Gli2 by siRNA. Authors: We have addressed the specificity of GlaB for Gli by showing the GlaB-induced inhibition of GLI-DNA binding to specific Ptch1 promoter by ChIP (new data in revised Figure 2G). We also observed that knock down of Gli1 and Gli2 by siRNA abrogates the response to GlaB, indicating that these proteins are required for the drug effect (new data in revised Figure 2H). We have further addressed the GlaB specificity issue by investigating the ability of the compound to modulate the enzymatic activity of a set of proteins involving some potential post-synthetic regulator of Gli function. New data shown in revised Expanded Figure E11 show that GlaB was inactive in those enzymatic assays, ruling out a possible indirect action upon Gli. Reviewer: It is also surprising that a higher concentration of GlaB is required for a significant effect in the EMSA assay compared to the cell based assays. What is the Kd for the binding of GlaB to GLI1? Authors: We believe that the higher GlaB concentrations observed in EMSA are not surprising because of the use of recombinant ZF region alone in in vitro assays compared to cell based assays using endogenous proteins. The pKd for GlaB binding to Gli1ZF was estimated in silico by means of XSCORE, a tool based on three different empirical scoring functions, which predicts with high accuracy the binding free energy of a ligand to any given target receptor (Wang R et al, . Comput.-Aided Mol. Des. 2002, 16, 11-26; Wang R. et al, . Med. Chem. 2003, 46, 2287-2303). Based on this simulation, the pKd of GlaB binding to Gli1ZF was estimated to 5.35, which corresponds to a low micromolar affinity in good agreement with biological activity data. In a rescue experiment in which we monitored in silico the GlaB binding to the Gli1ZF-K340A mutant, the estimated pKd was 5.20, showing that GlaB has a lower affinity for the K340A Gli1ZF mutant as observed via NMR and in vitro. These pKd values has been added to the revised Figure 3B). Reviewer: The rationale for the selected screening criteria is not clear and it would be interesting to know from the virtual screen how often GlaB ends up in the selected position. Moreover, the K350A single mutant, which may be the most interesting mutant is not analyzed Authors: According with reviewer suggestions, we have added the analysis of molecular docking simulations performed within the framework of the virtual screening. In particular, the 10 lowest energy clusters of GlaB have been showed, as well as GlaB poses within the lowest-energy cluster, which has been selected for further discussions. These data show that lowest-energy clusters of GlaB are located within ZF4 and ZF5, in correspondence of K340 and K350. Notably, the GlaB isoflavone core keeps the same orientation within this putative binding site, whereas prenyl groups


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may occupy three different position within the N-terminal part of ZF5 (revised Expanded Figure E12). Moreover, predicted binding modes of GlaB to K340A, K350 and the double K340A/K350A Gli1ZF mutants, as well as the relative delta-energy of binding have been monitored in silico. Results clearly show that K350 is the major anchor point for GlaB binding to Gli1. Indeed, in Gli1ZF-K340A, GlaB is still able to H-bond to K350 within the putative binding site at the interface of ZF4 and ZF5, although showing a lower theoretical affinity for Gli1ZF. When K350 is replaced by alanine (i.e. Gli1ZF-K350A and Gli1ZF-K340A/K350A), GlaB does not bind anymore within the putative ligand binding site, but interacts non-specifically within ZF2 and ZF3 (revised Figure 3B). Notably, the estimated binding affinity for ZF2 and ZF3 is significantly lower than for ZF4 and ZF5, in agreement with NMR analysis. Overall, this data reinforces the evidence that K350 is the most relevant anchor point for GlaB binding to Gli1 as well as that GlaB is not able to bind within the Gli1ZF when K350 is mutated to alanine. Minor points; Reviewer: In the experiments using transfected Gli1 and MEFs comparison to a GlaB related negative control would be valuable. Authors: A negative control has been added in revised Figure 2D and 2F. Reviewer: Since different doses of GlaB were used in the in vivo experiments with medulloblastoma and BCC analyzing the dose-response relationship including the correlation between tumor growth inhibition and Gli1 mRNA reduction would be of interest. In some places residue 380 is denoted K and in others R. There are also some problems with the English in the ms. Authors: No significant differences were observed in GlaB effects in MB vs BCC: indeed, we actually used a slight reduction of dose of GlaB in MB compared to BCC in vivo experiments (75 mmol/Kg and 100 mmol/Kg, respectively). Typos and english have been carefully revised.

Reviewer #3 Reviewer: This is an elegant and well-designed study, demonstrating a hypothesis-driven drug development and validation. The strengths of the study are the detailed functional analysis of the binding sites and the interaction of the inhibitory drug with the target site. There are a number of shortcomings, though, which will need to be addressed to demonstrate the in vivo effects more convincingly, supported by more relevant in vivo experiments and higher endpoint quality data. Also there are controls which I would like to see included. The authors may already have generated these but may not have included them. Major points. In vivo validation of drug effectiveness. The authors have used PTCH-/- cell lines derived from cerebellar tumors arising in PTCH-/- mice. These cells were then heterotopically allografted into the flank of (genetically) immunosuppressed mice. There are technical and biological concerns about this approach: (1) technical: (a) The measurement of the size of these tumors: no indication is given how the size and growth dynamics were monitored. State of the art determination of the size and the grown dynamics involve transplantation of cells carrying a luminescent (or otherwise labelled) reporter which can be monitored in vivo (For example, see PMID: 24982206; 24913958; 18843112). Other in vivo imaging methods, such as MRI may be utilised, but it is acknowledged that small animal MRI with sufficient resolution, e.g. 8-10T field strength may not be easily available. (b) The analysis of the resulting tumors is superficial and substandard. The histological image (Fig 6C) of both untreated and treated tumour shows non-specific background only and no proliferating cells, and is of inacceptable quality. The authors do not describe the potentially very important changes occurring in the tumors: Do tumours simply stop growing, do they undergo apoptosis, or


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do they undergo neuronal differentiation? What is the nature of the pink hypocellular tissue in Fig 6C, GalB treated? Connective tissue, differentiated tumor, neural tissue, or simply host connective tissue? (2) Biological: (a) It is well known that CNS tumors can considerable change morphology and growth dynamics when transplanted outside the CNS. It would be important to address this (at least) by orthotopic transplantation into the CNS, ideally into the cerebellum but if not technically possible at least into the forebrain. Adequate pathological characterisation using a panel of biologically relevant markers is essential to show the effects of the drug on the tumor cells. Ideally the tumor cells should be labelled with a tracer (beta galactosidase for example) to track their fate. (b) The authors have not considered testing a true in vivo effect, for example by treating P4-P8 mice with a proliferating external granular layer (EGL) with GlaB, and analyse the effect on the EGL: What will happen to the EGL in vivo, will there be a reduction of cell proliferation, lesser migration towards the Internal granular layer, or premature differentiation and developmental reduction of the cerebellar size? These data will be essential to draw conclusions about the biological effects in the drug in this translational setting. (c) It needs to be tested, using a cohort of PTCH-/- mice (or an alternative, transgenic model with an Shh dependent pathway alteration and a higher medulloblastoma incidence): and treat one cohort with GLAB. Again, an adequate histological endpoint analysis including state of the art quantitative histological imaging is essential. (3) What is the reason of a reduction of cell growth and NS formation, apoptosis, or less proliferation? This needs to be addressed. Comments regarding the experiments I have made a number of suggestions regarding the assessment of in vivo effect of GlaB. I gave a number of options and recommendations [2a, b, c], but I feel it would be unreasonable to ask to carry out all these experiments. It would be important that the authors address at least ONE, ideally perhaps two of the points. (i) Postnatal developmental effects of GlaB on the cerebellar EGL (ii) orthotopic allografting into the brain (iii) using a transgenic brain tumor model (PTCH-/- mice for example) rather than injected cells (iv) bioluminescence or similar imaging of the growth progression or reduction). However, no matter which one or two of the suggested approaches the authors decide on, the referee insists on a state of the art pathological analysis of the resulting tumors to be carried out and that these data are presented adequately. Authors: Since the Referee is offering us to revise the manuscript addressing only one of a variety of constructive suggestions, we have followed his/her advice. We decided to address two suggestions: in vivo short term treatment of 6-days old neonatal cerebella (option [i]) and orthotopic allografting into the brain (option [ii]), since the additional option (a transgenic brain tumor model (PTCH-/mice for example) requires too long term experiments to generate and treat the transgenic mice, which would not be compatible with a timely communication to the scientific audience of this pharmacologically competitive data). More specifically: 1) We decided to include the biological activity in better suited in vivo system by addressing in vivo treatment of 6-days old neonatal cerebella where Hedgehog promotes proliferation of granule progenitors residing in EGL (External Granular Layer) as well as of ex vivo isolated granule progenitors after in vivo treatment. GlaB-mediated targeting of Gli antagonizes all these effects (as evaluated by EGL morphometry, immunohistochemical staining, qRT-PCR and western blotting of several proliferative markers (Ki67, PCNA, Cyclins D1 and D2, NMyc, etc) (revised Figure 4 new panels B, C, D, E and F). 2) We transplanted the human MB Daoy cells (belonging to the Shh-MB subgroup) into the cerebellum of NOD/SCID mice. Tumor volume (calculated along serial histologic brain sections as described in revised Materials and Methods) displayed a significant reduction of the tumor mass formed by Daoy cells in GlaB-treated mice compared to the control (new revised Figure 7). Such a reduced tumor mass was likely caused by inhibition of tumor cell proliferation, since the percentages of Ki67 was decreased in GlaB-treated tumors (new revised Figure 7). 3) We have included in the revised manuscript a thorough pathological examination of the tumor allografts after in vivo treatment with GlaB, by quantifying immunohistochemical effects through cell imaging software and biochemical analysis (western blotting and qRT-PCR mRNA levels) of


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proliferating (Cyclins D1 and D2, Ki67, PCNA, NMyc, etc) or apoptotic (TUNEL assays and cleaved Caspase-3 protein levels). Interestingly, GlaB was also able to reduce the expression of Nanog gene, previously reported to be a direct transcriptional target of Hh/Gli and to be responsible for the enhancement of stemness features of Mb cells (Po et al, EMBO J. 2010; Garg et al, EMBO J., 2013) (revised Figure 6 new panel E). 4) We have clarified that the reduction of cell growth and medulloblastoma neurospheres formation is due to less proliferation (as evaluated by BrdU uptake) followed by apoptosis (evaluated by TUNEL assay), in agreement with the previously reported prosurvival function of Gli and proapoptotic activity of Hh/Gli antagonists (Mazzà et al, 2013; Zaidi et al, 2013; Hou etal, 2014; Pan et al, 2012). Indeed, exposure of dissociated neurosphere cells to GlaB for 24 or 48h impairs their ability to be labeled by BrdU, while apoptosis (TUNEL staining and overexpression of cleaved Caspase-3) was observed only after 48h of treatment. These events would then result in inhibition of their clonogenic ability (number and size of secondary neurospheres) (revised Figure 5 new panels C, D, E, F and G). Minor points: Reviewer: Fig 2D: The authors need to include the absence of an effect on non -target genes, e.g. from the wnt pathway or at least housekeeping genes, to demonstrate the selective action of GlaB. Fig 4e; Fig E7: The authors may consider adapting the scale a maximum of 30 or 40% to visualise the effects (or absence of) GlaB on cell viability. Authors: The selective action of GlaB in Figure 2D has been confirmed by the lack of activity on an unrelated housekeeping Pfkfb3 gene. Furthermore, all qRT-PCR data throughout the manuscript have been normalized to HPRT, b2-microglobulin and GAPDH housekeeping genes. The original Figure addressed by the reviewer has been replaced by revised Figure 5G in the revised manuscrpt: a more careful analysis of cell death in medulloblastoma stem cells (TUNEL staining and western blotting of cleaved Caspase-3) revealed a proapoptotic effect of GlaB, although only after 48h.

2nd Editorial Decision

27 October 2014

The original referees commented on your revised manuscript. Both suggest minor amendments before ultimate acceptance of your study. Please check their comments carefully (particularly the details requested from ref#2; I would also encourage a more unbiased presentation of your results on GlaB-specificity, noticing that significant experimental revisions aimed at addressing this point). I hope that this message will be received as good news and would be grateful for your timely attention and response as to facilitate rapid production/publication. I take the liberty to congratulate already at this stage to a nice paper and look forward receiving the minor final amendments shortly! -----------------------------------------------Referee #1: In the revised version the authors have clarified several issues resulting in an improved manuscript. The structural data add to the existing knowledge from the published crystal structure that provides the basis for the understanding of GLI1/DNA interaction. It would be important to provide a more thorough and unbiased analysis of the specificity of GlaB. Referee #2:


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The revision of the manuscript has addresses most all my concerns regarding the quality of the histology, and the appropriateness of the interpretation. The implementation of the additional experiments has significantly improved the quality and feel that my comments have been satisfactorily addressed. Note: I have only checked that the authors have addressed the points I made. A few minor technical details need to be corrected or completed: Figure 8. Inhibition of Gli-dependent BCC tumor cell growth: Scale bars in figure 8 F: "represent 50 µm for H&E, 7.5 µm for Gli1, 15 µm for Ki67, 25 µm for TUNEL stainings". Please check the 7.5 um scale bar. It looks more like 25um (in fact all these images may have an incorrect value for the scale bar). There may be a systematic miscalculation of the scale bar! (a single erythrocyte has a diameter of 7.5um!) 2) "To study the GlaB effect in vivo, we turned to an allograft model of MB cells. Nude mice were grafted with spontaneous primary MB from Ptch1+/- mice"; and "Spontaneous MB from Ptch1+/mice were isolated, minced, pipetted to obtain a single-cell suspension and grafted s.c. at the posterior flank of female BALB/c nude mice" : how many cells were injected? (the authors have for example given the cell number for the BCC graft experiment)

2nd Revision - authors' response

04 November 2014

Replies to Reviewers Reviewer #1 Reviewer: In the revised version the authors have clarified several issues resulting in an improved manuscript. The structural data add to the existing knowledge from the published crystal structure that provides the basis for the understanding of GLI1/DNA interaction. It would be important to provide a more thorough and unbiased analysis of the specificity of GlaB. Authors: We thank the Reviewer for the positive comment on our revised manuscript. According to the reviewer’s comment and the editor’s suggestion, we have amended the manuscript and provided a more unbiased analysis of the specificity of GlaB (see: page 5, lane 27; page 9, lanes 14 and 18; page 10, lane 9 and 13; page 16, lane 18).

Reviewer #2 Reviewer: The revision of the manuscript has addresses most all my concerns regarding the quality of the histology, and the appropriateness of the interpretation. The implementation of the additional experiments has significantly improved the quality and feel that my comments have been satisfactorily addressed. Note: I have only checked that the authors have addressed the points I made. Authors: We are grateful to the reviewer for his nice comment and for helping us improving the overall quality of our work. Reviewer: A few minor technical details need to be corrected or completed: Figure 8. Inhibition of Gli-dependent BCC tumor cell growth: Scale bars in figure 8 F: "represent 50 µm for H&E, 7.5 µm for Gli1, 15 µm for Ki67, 25 µm for TUNEL stainings".


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Please check the 7.5 um scale bar. It looks more like 25um (in fact all these images may have an incorrect value for the scale bar). There may be a systematic miscalculation of the scale bar! (a single erythrocyte has a diameter of 7.5um!) Authors: We apologize for the mistake. We have modified the scale bar for all images presented in Figure 8F and reported the correct values where necessary (page 36, line 24). Reviewer: "To study the GlaB effect in vivo, we turned to an allograft model of MB cells. Nude mice were grafted with spontaneous primary MB from Ptch1+/- mice"; and "Spontaneous MB from Ptch1+/- mice were isolated, minced, pipetted to obtain a single-cell suspension and grafted s.c. at the posterior flank of female BALB/c nude mice": how many cells were injected? (the authors have for example given the cell number for the BCC graft experiment) Authors: We have included this information in the revised Materials and Methods, according to the Reviewer suggestion (page 21, line 5).


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