Mutant Isocitrate Dehydrogenase 1 Expression Enhances Response of Gliomas to the Histone Deacetylase Inhibitor Belinostat

Round 1

Reviewer 1 Report

This paper effectively demonstrates in in vitro experiments that the expression of a mutant form of IDH1 in glioma cells results in reduced survival (Fig. 3) and increased apoptosis (Fig. 4) when exposed to the DHAC inhibitor belinostat. They go on to show that in a phase 1 trial in which belinostat was added to standard-of-care RT/TMZ therapy for patients with newly-diagnosed GBMs that in the one patient who had an IDH mutant tumor that there was a more robust response assessed by MRI scan than in other patients.  Taken together, these data suggest that IDH1 mutation status may be a suitable biomarker for response to HDAC inhibition. 

A few minor comments:

1)    Author line:

Superscript 1 should be 3:Department of Radiation Oncology, Johns Hopkin University, Baltimore, MD

Superscript 2 should be 4: Department of Radiology and Imaging Sciences, Emory University, Atlanta, GA

2)    Fig. 3 – some sort of statistic to show that the difference between control and mtIDH1 is stat. significant

3)    Fig. 4 – some sort of statistic to show that the difference between control and mtIDH1 is stat. significant

Author Response

We thank reviewer #1 for reviewing our manuscript.  For comment 1), we made the correction to the superscripts.  For comments 2) and 3), we performed student’s t-test for comparisons and have modified Figures 3 & 4 and their corresponding legend to show the significant p-values and descriptions.

Reviewer 2 Report

The aim of the paper was to investigate the response of gliomas to the histone deacetylase inhibitor belinostat. For glioma tumors it is highly significant to search for novelty treatment regimens and diagnostic means in order to effectively treat both IDH1 mutation and wild type IDH glioblastomas. The authors showed high potency of belinostat – new generation histone deacetylase inhibitor – in treatment of glioblastoma. Its highly enhanced response was proved in IDH1 R132 H mutation glioblastoma, which makes it specifically promising for this group of patients and may potentially improve their clinical outcomes.

The authors also understand and make it clear that further search for alternative metabolites to be applied in MR spectroscopy may prove to be better for tracking tumors harboring IDH mutations. Additional sophisticated MR spectroscopy techniques may as well allow better assessment of those metabolites.

The manuscript is clear, relevant for the filed and presented in a well-structured manner. The figures and table properly show the data and are easy to interpret. The cited references are relevant. The conclusions in the Discussion part are coherent and well supported by the listed citations. Although it was not a part of this study, the authors included some other important findings of their research presented in other papers (lines 453-465).

My specific comments:

The belinostat-containing treatment regimen was applied only in one case of IDH1 R132H mutation glioblastoma. Further investigation for a larger group of such patients would be advised.

Author Response

We also thank reviewer #2 for evaluating our manuscript.  In response to this reviewer’s specific comments, we agree that our study is the limited by having only one case of an IDH1 mutant GBM treated with the belinostat-containing regimen however striking the response was for this patient. Since we are not able to treat additional patients in this manner at this time, we have added a paragraph discussing limitations of our study with discussion of this particular point (p. 11 lines  491-495).

Reviewer 3 Report

In the manuscript titled "Mutant isocitrate dehydrogenase 1 expression enhances response of gliomas to the histone deacetylase inhibitor belinostat," prepared by Chang et al., The author tested the tumor-suppressing effect of HDAC inhibitor belinostat on IDH1 mutant glioma. They performed in vitro analysis and reported the MRI data from a clinical study. 

The author used NHA and LN229 for IDH mutant transduction. These are established cell lines that are driven by oncogenes irrelevant to IDH mutant glioma in humans. The author should consider using patient-derived glioma cells with intrinsic IDH mutation.

The waterfall plot shows the responses of patients receiving RT/TMZ/Bel treatment. There is only one case with IDH mutant glioma. Without proper control settings, it is hard to determine whether RT/TMZ/Bel is more effective than RT/TMZ. Due to the lack of sufficient case numbers, it is impossible to tell whether there is statistical significance between IDH WT GBM and IDH mutant GBM.

Author Response

Finally, we would like to also thank reviewer #3 for evaluating our manuscript.  In response to this reviewer’s comments on the problems with using established cell lines, we agree that this is a potential limitation and that assessment of patient-derived IDH mutant glioma cells could further support our findings.  We are interested in pursuing this extension of our study but feel that it is beyond the scope of this current study.  We have now discussed this potential limitation in the Discussion (p. 11, lines 483-491).  Similar to reviewer #2, this reviewer felt that it is hard to draw firm conclusions on the basis of results in one patient.  Similar to our response above, since we are not going to be able to get further data on additional IDH mutant GBM patients treated with the belinostat-containing regimen at this time, we have further discussed this limitation in the manuscript (p. 11 lines  491-495).  There was also a mention of need to improve the English in this manuscript.  Despite the fact that the author (HKS) that was responsible for the majority of the writing is a native English speaker, we did have the manuscript extensively proofread again to correct any remaining grammatical errors and improve the stylistic expression. 

Round 2

Reviewer 3 Report

I don't think the author addressed my concerns.

Author response: He/she asked us to do more experiments using patient-derived IDH mutant lines. Given that we were given just a couple of weeks to respond to the reviewer comments, we responded by further discussing the limitations of our study brought up by this reviewer since we clearly could not have done the experiments asked by this reviewer within this time (could take upwards to several months to even a year to obtain grow and generate the needed IDH-mutant cell lines). This reviewer felt that our system where we introduced expression of mutant IDH1 in normal human
astrocytes immortalized by standard means was artificial in nature but I would argue that since this was a normal astrocyte line that we used, there is a very low likelihood that additional mutations that may be affecting response to an HDACi is present. In fact, their suggestion of using a patient-derived IDH mutant line where we would then need to knock out IDH1 mutant expression would be similarly artificial (if not more so) especially since using something like shRNA to knockout mutant IDH1 expression may actually affect the expression of wild type IDH1 expression in that system which would lead to additional confounding issues with the suggested experiment(s).