Targeting Pediatric Glioblastomas by Combining OLIG2 Inhibitor CT-179 with Fractionated Radiation in a Panel of Patient-Derived Orthotopic Xenograft Mouse Models

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The research by Holly Lindsay et al. evaluated the therapeutic efficacy of the OLIG2 inhibitor CT-179 in pHGG, measuring OLIG2 mRNA expression levels in 10 patient-derived orthotopic xenograft models. They also analyzed the in vitro activity of CT-179 alone or in combination with radiotherapy in monolayer and neurosphere cells and assessed its brain penetration in tumor-bearing PDOX mice.  Ultimately, they found that orally administered CT-179 can cross the blood-brain barrier and, when combined with radiotherapy, has the potential to inhibit pGBM growth.

However, I think the authors should address the following issues before considering accepting the paper:

1. Once an academic term has been introduced with its full name at the outset of the article, it is unnecessary to repeat the expansion in subsequent references. Please revise.
2. Several of the figures are very minimalistic and data-light. For example, Figure 2. In vitro anti-tumor activities of CT-179. Figures 2A and 2C are too blurry. It is recommended to adjust the image layout to make them clearer.
3. The quality of the manuscript would be improved if a dedicated section on statistical analysis were added to the “Materials and Methods” section. Clearly describing statistical methods is standard practice in pharmaceutical and analytical chemistry research, especially when presenting quantitative data and validating parameters.
4. Words like “in vitro” and “in vivo” appear in both regular and italicized fonts in the text; this needs to be corrected for consistency.
5. A recent Nature Communications article has already discussed how “transcription factors ASCL1 and OLIG2 drive glioblastoma development and jointly regulate tumor cell type and migration.” The authors then suggest that suppressing OLIG2 doesn’t fully demonstrate the novelty of their work, appearing more like a supplement to previous research.  Furthermore, a recent Nature Communications article has already discussed “Suppressing recurrence in Sonic Hedgehog subgroup medulloblastoma using the OLIG2 inhibitor CT-179.” The author did not clearly present the main innovative aspects of this article; these need to be added or improved upon.
6. What is the toxicity of the inhibitor CT-179 to normal cells, and what are its side effects? What is its oral bioavailability?
7. What is the sensitization mechanism of the combined treatment of inhibitors and radiotherapy proposed by the authors? Are there any changes in DNA damage repair markers, such as γH2AX, after the combined therapy?
8. Has the author considered using “induced relapse” PDOX models, i.e., testing the effects of the treatment regimen on tumors that have relapsed after standard treatment?
9. Regarding the experiments with PDOX tumors, we suggest the authors include a simple flowchart to provide further explanation.
10. Why are there no error bars or statistical significance analysis in Figure 1B?

Author Response

Comments and Suggestions for Authors

The research by Holly Lindsay et al. evaluated the therapeutic efficacy of the OLIG2 inhibitor CT-179 in pHGG, measuring OLIG2 mRNA expression levels in 10 patient-derived orthotopic xenograft models. They also analyzed the in vitro activity of CT-179 alone or in combination with radiotherapy in monolayer and neurosphere cells and assessed its brain penetration in tumor-bearing PDOX mice.  Ultimately, they found that orally administered CT-179 can cross the blood-brain barrier and, when combined with radiotherapy, has the potential to inhibit pGBM growth.

However, I think the authors should address the following issues before considering accepting the paper:

1. Once an academic term has been introduced with its full name at the outset of the article, it is unnecessary to repeat the expansion in subsequent references. Please revise.

Response: Yes, revised as suggested.

1. Several of the figures are very minimalistic and data-light. For example, Figure 2. In vitro anti-tumor activities of CT-179. Figures 2A and 2C are too blurry. It is recommended to adjust the image layout to make them clearer.

Response: The resolution on our submission was set as very high. The blurry may have been caused the conversion of the manuscript from word to PDF.  We do recognize that the font size were pretty small and have increased them to improve the visibility. The revised figure 2 is now attached on page 5, lines 143-149.

1. The quality of the manuscript would be improved if a dedicated section on statistical analysis were added to the “Materials and Methods” section. Clearly describing statistical methods is standard practice in pharmaceutical and analytical chemistry research, especially when presenting quantitative data and validating parameters.

Response: This is a very good point. The original section was somehow lost during the copy and paste fitting into the journal designated format. It is now inserted back on page 13, lines 502-508

Statistical Analysis

Values were presented as mean ± standard deviation (SD).  The effect of treatment on cell proliferation was analyzed with two-way analysis of variance (ANOVA). Changes of animal survival times were analyzed with Kaplan-Meier method followed by pair-wise comparisons with using the Gehan-Wilcoxon test. No new code was developed. All statistical analyses were performed by using SigmaPlot 14.0 (Systat Software, Inc., San Jose, CA).  P<0.05 was considered statistically significant.

 

1. Words like “in vitro” and “in vivo” appear in both regular and italicized fonts in the text; this needs to be corrected for consistency.

Response: Yes, corrected throughout the manuscript.

1. A recent Nature Communications article has already discussed how “transcription factors ASCL1 and OLIG2 drive glioblastoma development and jointly regulate tumor cell type and migration.” The authors then suggest that suppressing OLIG2 doesn’t fully demonstrate the novelty of their work, appearing more like a supplement to previous research.  Furthermore, a recent Nature Communications article has already discussed “Suppressing recurrence in Sonic Hedgehog subgroup medulloblastoma using the OLIG2 inhibitor CT-179.” The author did not clearly present the main innovative aspects of this article; these need to be added or improved upon.

Response: We appreciate the reviewer of highlighting these recent publications. Similar to most studies on specific gene(s) or pathways, no single study or a few studies can fully expose the biological activities or clinical implications. While we do recognize the contributions of these two studies (reference #34 and #28 in our original submission) that were published recently (2025 and 2024), the primary goal or the innovation of our current study is to demonstrate the preclinical therapeutic efficacy in pediatric high-grade glioma to facilitate the translation into clinical trials. Our use of a relatively large panel of clinical relevant PDOX model and the combination with a standard therapy (radiation) provides an additional layer of significance. We also wish to point out that while our studies were conducted, these two papers have not published yet. It is because of the time-consuming nature of detailed in vivo therapeutic efficacy (i.e., the extension of animal survival times) that held our team back a little bit.

1. What is the toxicity of the inhibitor CT-179 to normal cells, and what are its side effects? What is its oral bioavailability?

Response: As we reported in the methods, we used body weight changes as a surrogate marker of drug toxicity.  As we All the animals treated with CT-179 maintained relatively stable body weight and no animal died sooner than the untreated control. These data indicated that the overall toxicity to normal cells is minimum.  We have therefore added this data to the

As for the oral bioavailability, we described in the result section 2.4 on page 6, lines 176 -193. We added “oral bioavailability” on the section title and in the text to highlight this point.   

1. What is the sensitization mechanism of the combined treatment of inhibitors and radiotherapy proposed by the authors? Are there any changes in DNA damage repair markers, such as γH2AX, after the combined therapy?

Response: This again is a very good question. The primary reason of combining CT-179 with radiation is to aim for clinical trials as it remains a challenge, if not impossible, to test a single agent in relatively rare pediatric high-grade gliomas (compare with adult cancers). Combination with existing standard therapy is clinically a highly desired strategy even for phase I clinical trials.  We have add the following sentence in the manuscript (lines196-198 on page 6):   Demonstrating an additive or synergistic anti-tumor activity by combining CT-179 with XRT would facilitate the initiation of clinical trials.

As for the mechanism, we have provided some thoughts in the Discussion section (line 369-373).   As for the DNA damage, the reviewer has raised a very good point. We have added the following in the manuscript (on page 11, line 391-398): 

Indeed, DNA damage has long been recognized as one of the primary mechanisms of XRT induced tumor cell killing. Our previous study has shown that 2 Gy/day for 5 days alone caused massive (nearly 100%) expression of γH2X in the PDOX models of pediatric GBM and, at the same time, triggered the exit of putative cancer stem cells from quiescent G₀ phase into active proliferation [37]. Since OLIG2 expression is closely related to cancer stem cells, suppression of this critical gene by CT-179 may have contributed to the increased anti-tumor activities of XRT in the current studies. 

1. Has the author considered using “induced relapse” PDOX models, i.e., testing the effects of the treatment regimen on tumors that have relapsed after standard treatment?

Response: This is a good point. As shown in Table 1, we have 6 PDOX models derived from recurrent gliomas. One of them, IC-1621GBM and IC-3752GBM was subjected to in vivo efficacy analysis.  We have added the following to highlight this point on Page 11, Lines 357-358: Although these PDOX models were highly malignant and difficult-to-treat tumors, including models derived from recurrent tumors (IC-1621GBM and IC-3752GBM),  imcomplete inhibition of OLIG2 in by CT-179 may have limited the overall efficacy.

1. Regarding the experiments with PDOX tumors, we suggest the authors include a simple flowchart to provide further explanation.

Response: The overall experimental design of this study is pretty straightforward. We tried but the flowchart looks overly simple.

1. Why are there no error bars or statistical significance analysis in Figure 1B?

Response: Those data were extracted from a gene expression profiling. It was mentioned in the Result section on page 4 line 111-112. But we agree with the reviewer that it will help the readers by adding following description in the figure legend on page 8 line 135: Data were extracted from gene expression profiling.

Reviewer 2 Report

Comments and Suggestions for Authors

1. It seems authors does not discussed about previous reported works?
2. Better to highlight the key point of this research and how merit over other reported work related to this.
3. Line 53: Mentioned children? age factor of childrens should be mention.
4. In your research Fig 1A. Patient MRI mentioned this belongs to children MRI or belongs to major human? 
5. pediatric 490 high-grade glioma in this research have you done previously. if so mention some discussion about past and current also compare the results

Comments on the Quality of English Language

need to minor revision

Author Response

Comments and Suggestions for Authors

1. It seems authors does not discussed about previous reported works?

Response: We have included multiple publications include the two papers mentioned by the first reviewer. There is always

1. Better to highlight the key point of this research and how merit over other reported work related to this.

Response: Although we summarized our findings at the end of the discussion on page 11 line 408-411, we agree with the reviewer that provide a few works to highlight the key points should help the readers. It is now revised on page 11 line 397-398:  These data provided key points supporting additional in vivo studies to optimize the dosing, frequency, and length of CT-179 treatment to further improve its therapeutic efficacy to provide strong pre-clinical rationale for the initiation of clinical trials for patients with pGBM.

We have also included a section of Conclusion at the very end of the manuscript (after Materials and Methods) following the journal’s manuscript template. It once again highlighted our key and novel findings.

 

1. Line 53: Mentioned children? age factor of childrens should be mention.

Response: Yes, added. “younger than 21 year old”.

1. In your research Fig 1A. Patient MRI mentioned this belongs to children MRI or belongs to major human? 

Response: It is the real MRI image of patient 3752GBM from which the PDOX model IC- 3752GBM were established. We revised the figure legend on page 4 line 131: A. Representative MRI and whole mouse brain section from pGBM 3752 from which the animal model IC-3752GBM were established.

 

1. pediatric 490 high-grade glioma in this research have you done previously. if so mention some discussion about past and current also compare the results.

Response: Something might have been cut off from the comments, making it difficult to address at this moment.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript presents important preclinical data on a novel therapeutic strategy for pHGG and merits publication after major revisions. This study addresses a pressing issue in the field of pediatric neuro-oncology and provides valuable preclinical research insights. However, some content still requires revision to enhance clarity, rigor, and impact.

• Abbreviations should not be used in the title.
• Line 46, when "BBB" is first mentioned, the full name should be used.
• The title of Figure 2 should be placed immediately after the figure.
• I didn't find Table 1 in the manuscript.
• Line 257, the author did not provide the supplementary table 1.
• Lines 168, The title here lacks a serial number. We hope the author can review the entire text.
• The author should explain the methods of data processing and statistics in the fourth part.
• Lines 92-106, The author directly jumps from the research background to the research method section. What is CT-179 mentioned here? The research background does not mention this. The author's writing method needs to be strengthened.
• In the conclusion section, it is suggested that the author include some additional forward-looking content.

Author Response

Comments and Suggestions for Authors

This manuscript presents important preclinical data on a novel therapeutic strategy for pHGG and merits publication after major revisions. This study addresses a pressing issue in the field of pediatric neuro-oncology and provides valuable preclinical research insights. However, some content still requires revision to enhance clarity, rigor, and impact.

• Abbreviations should not be used in the title.

Response: Yes, corrected.

• Line 46, when "BBB" is first mentioned, the full name should be used.

Response: Yes, corrected in the abstract on page 2 line 46. While in the main text, it was already spelled out on page 6 line 176.

• The title of Figure 2 should be placed immediately after the figure.

Response:  Yes, corrected.

• I didn't find Table 1 in the manuscript.

This is a very good catch, thank you. Table 1 is now added back.

• Line 257, the author did not provide the supplementary table 1.

Response: Yes, corrected. It is now Table 2.

• Lines 168, The title here lacks a serial number. We hope the author can review the entire text.

Response: Yes, corrected.

• The author should explain the methods of data processing and statistics in the fourth part.

Response: Yes, added on page 14, line 502-508.

• Lines 92-106, The author directly jumps from the research background to the research method section. What is CT-179 mentioned here? The research background does not mention this. The author's writing method needs to be strengthened.

Response: We don’t quite get the point of this question. This paragraph from lines 92-106 is an extended description of know molecular regulations on OLIG2 to justify it as a good therapeutic target. It is still the background information, not the research method.

• In the conclusion section, it is suggested that the author include some additional forward-looking content.

Response: This is a good suggestion, but we have included such forward-looking content in the main text at the end of the discussion on page 14, Lines 510-522. To keep a smooth flow of information, we respectively request to keep it as is.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed most of my previous concerns, and I have a little suggestion:

For the toxicity of the inhibitor CT-179 to normal cells, the authors could cite other literature to support their finding that the overall toxicity to normal cells is minimal, as no tests have been conducted on other normal cell types.

Author Response

Comments: For the toxicity of the inhibitor CT-179 to normal cells, the authors could cite other literature to support their finding that the overall toxicity to normal cells is minimal, as no tests have been conducted on other normal cell types.

Response: Yes, indeed. A previous study has revealed low toxicity in norma cells. Our revision is therefore made on Page 11, Lines 348-355:

One additionally encouraging aspect of CT-179 is that the CT-179 exhibited a safety profile in vivo in our animal models. All the mice tolerated the treatment very well no significant loss of body weights during the therapies acting alone or in combinations. This finding is further supported by a recent study in which Li et al has demonstrated minimum off-target effects of CT-179 in 12 normal human primary cell lines at biologically relevant concentrations using the BioMAP® Diversity PLUS® platform, a cell-based screen used to assess the safety of pre-clinical experimental therapies on normal healthy tissues [27].

 

Reviewer 3 Report

Comments and Suggestions for Authors

My concerns are well addressed.

Author Response

Thanks a lot. 

 

Xiao-Nan