Utilization of Cancer Cell Line Screening to Elucidate the Anticancer Activity and Biological Pathways Related to the Ruthenium-Based Therapeutic BOLD-100

Round 1

Reviewer 1 Report

 

In this manuscript, the authors explore the sensitivity of a large panel of cancer cell lines to BOLD-100, an anticancer compound based on rutenium. The aim of the study was to identify those tumors with higher sensitivity to this methal-based anticancer drug, and also to deep into its mechanims of action. Overall, the work is well planned and well written. However, I don’t think it represents a significant advance of the type that would warrant publication in Cancers for the following reasons:

-       Using medians for BOLD-100 IC50s, the authors claim increased activity in cell lines from esophageal cancer, blood cancers, and bladder cancer. However, in blood cancers, in bladder cancer and in all tissues types in general it can be observed variations in sensitivity that seems to depend on cell lines rather than on tissues types. These results suggest that sensitivity could be related with molecular alterations or some specifyc characteristic present in hypersensitive cell lines. Moreover, the authors show that sensitivity also depend on growth media, indicating that differences in sensitivity would require in vivo data.

-       Utilizing the cancer cell line genomic data from CCLE, the authors performed a statistical modeling analysis in order to identify  candidate genes that associate with BOLD-100 susceptibility. They found that expression levels of genes associated with ribosome biogenesis were inversely related to the susceptibility to BOLD-100. This result seems to be in agreement with a previous publication showing that BOLD-100 interacts with ribosomal proteins and its accompanied by ER stress-induction and modulation of GRP78 in cancer cells (38). To increase the impact of the work the authors should carry out experiments that validate their bioinformatinc findings, they could analyze transcription rates, the induction of the UPR in different cell lines by BOLD-100, and also analyze the RP-MDM2-P53 axis, as indicated in the discussion.

 

 

Author Response

Thank you for your review. We agree that within tissue type variation in sensitivity varies between different cancer types. For example, multiple myeloma cell lines had limited variability while breast cancer cell lines had large variability. As suggested by the reviewer, this variability may be due to unidentified characteristics within the hypersensitive cell lines. In certain cancer types we attempted to address this by analyzing known subgroups within each tissue. For example, in breast cancer cell lines we separated the lines based on their Her2, ER, and PR status (see Figure 1C). Additionally, we also completed a regression analysis to identify molecular characteristics within each tissue type. Unfortunately, due to the small number of cell lines within each tissue type, this analysis was not powered sufficiently to identify significant characteristics and therefore was not included in the manuscript. Finally, in the pan-cancer molecular marker analysis, we took into consideration both tissue type and growth media in our multiple regression model. We have altered the text in both the results and discussion to indicate that variation in tissue type may be driven by specific changes in hypersensitive cell lines. 

We also agree that further downstream studies are required to validate and expand on our current analysis. We believe that the scope of our current work generates hypotheses on BOLD-100 and its anticancer properties, which provides a foundation for an in-depth investigation into the role of ribosomal processes in metal-based cancer therapeutics. Though the implementation of these studies is outside the scope of our current work, we have addressed the potential for future research in the revised manuscript.

Reviewer 2 Report

The article is well designed and written and this approach could provide a platform for translationar research, not just in BOLD-100, to expand the findings reported in this manuscript. However, as the authors themselves have discussed, larger cell screens should be performed to identify significant gene expression associations. Authors should discuss this point further.

Author Response

Thank you for your review. We agree that larger screens would likely identify significantly more gene expression associations. Unfortunately, in this study we already utilized the maximum number of cancer cell lines (319) available to us within our resources. As suggested by the reviewer, we have discussed this point further in the revised manuscript.

Reviewer 3 Report

The article "Utilization of Cancer Cell Screening and Bioinformatics Analy-2 sis to Elucidate Key Biological Pathways Critical to the Activity 3 of the Ruthenium-Based Anticancer Drug BOLD-100" written by Brian J. Park, Paromita Raha, Jim Pankovich, Mark Bazett, presents the results after investigating BOLD-100 (sodium trans-[tetrachlorobis(1H indazole)ruthenate(III)]), on 319 cancer cell line panel spanning 24 tissues.  It includes various type of cancer cell lines, the methods are well presented, the conclusions are apropiate with the results, and these findings could lead to understanding of  mechanistic activity of the molecule. 

In my opionion this article is valuable and I consider it should be published afte a minor recheck of the manuscript.

Author Response

Thank you for your review.

Reviewer 4 Report

In manuscript, Park et al firstly performed in-vitro killing assay on 319 cancer cell lines with BOLD-100 treatment. Then, based on the experimental results they utilized bioinformatics to analyze BOLD-100’s sensitivity profile and compared function of BOLD-100 to cisplatin as well as other anticancer compounds in cancer cell treatment. Also, they demonstrated the potential of genes related to ribosomal processes that function in BOLD-100 anti-cancer activity and try to use machine learning to predict BOLD-100 sensitivity in different cancers. The manuscript provides us some information as to the promising function of BOLD-100 in cancer treatment. However, most of the results were based on experiments or databases from cancer cell lines and the mechanism of BOLD-100 function is still unclear. Although the authors mentioned the function of BOLD-100 might be ribosomal processes related, they do not show any experimental results for validation. The following are some specific points.

 

Major points:

1.     Since the BOLD-100 has already been used in clinical trial, in 319 cancer cell lines are there any cell lines that is the same tissue lineage as the clinical one? If there are, could the authors please specifically compare those to the clinical used ones?

2.     All the data used for analysis in the manuscript are from cancer cell lines. Are there any clinical data or results from patient derived in-vitro model that could be used for validation? Cancer cell line in vitro is quite an artificial system and I am not sure how significant it is when making conclusions only based on this.

3.     If the authors claim ribosomal process is important in BOLD-100 mediated anti-tumor activity, could the authors please provide further evidence to prove this point? For example, to show if suppression or activation of ribosomal process will disturb BOLD-100 mediated cancer killing activity.

 

Author Response

Thank you for your review. BOLD-100 is currently being tested in a Phase 1b/2 clinical trial in combination with FOLFOX for the treatment of patients with advanced gastric, colon, bile duct and pancreatic cancer (NCT04421820). All four of these cancer types were represented within the 319 cancer cell lines. Interestingly, pancreatic cancer cell lines were the fourth most responsive tissue type by median value, while bile duct and colon cancer cell lines were both more responsive to BOLD-100 then their relative response to cisplatin (see FIgure 1D). This trial has not published clinical efficacy results yet; however at ASCO 2022 early preliminary results were shown in a poster (DOI: 10.1200/JCO.2022.40.16_suppl.3031) suggesting an improvement in progression-free survival in the metastatic colorectal cancer patients. As the full data is not available at this time, we are unable to do a specific comparison between this in vitro work and the clinical results. We have updated the discussion to discuss this concept in more detail. 

 

We agree that two-dimensional cell models have limitations in representing the complex molecular landscape of cancer and future work using more advanced systems such as organoids, animal models, and patient populations are required. We do not have access to clinical results related to BOLD-100 at this time, as the current clinical trial is not expected to be completed until later in 2023. Unfortunately, although we agree it would strengthen the work, there are no patient derived samples collected in patients treated with BOLD-100. That said, we believe that our current work provides a platform for subsequent translational research and our presented findings generated hypotheses on the bioactivity and efficacy of BOLD-100. We have updated the discussion to highlight this limitation. 

 

In regards to our bioinformatics findings on BOLD-100’s potential relationship with ribosomal processes, we agree that downstream studies are required to validate our findings. There is prior evidence that BOLD-100 interacts with select ribosomal proteins [Reference 38] and our current work provides support for this. We have addressed the need for validation assays in the revision; however, the execution of these studies is outside the scope of our current work.

Reviewer 5 Report

The manuscript under evolution is an important presentation of how bioinformatic analysis can be utilized to understand underlying mechanism of action for novel drug modalities. It can be accepted in its current form.

Author Response

Thank you for your review.

Round 2

Reviewer 1 Report

 

The authors agree with all my comments; first, that variations in sensitivity  seems to depend on cell lines rather than on tissues types. I suggested that it would be appropiate to get in vivo data, but the authors did not provide new data in the revised version. Second, I suggested that to increase the impact of the work the authors should carry out experiments that validate their bioinformatic findings. The authors agree, but consider that these studies are outside the scope of their current work.

Therefore, I still consider that although the work is well planned and well written it does not represent a significant advance to be publish in Cancers, a journal with a high impact factor, it might be more appropiate for other journals.

 

 

Author Response

Thank you for your review and feedback. Although we attempted to address these concerns in our initial revisions by emphasizing the limitations of the current methodology and the importance of broadening our scope in future work, we respect your concerns and conclusions.

Reviewer 4 Report

Thanks the authors for the response. One last thing I want to discuss is about the title. The current title is focusing on using the indicating methods to identify novel pathway. In that case, the manuscript should emphasize more on the novel pathways they identified and show more evidence to prove them.  If it is not what the authors want to highlight, I suggest to reconsider the title currently used.

Author Response

Thank you for your review and support in improving the quality of manuscript. We agree that the initial title of our manuscript was ambitious. In order to better reflect our findings, we have modified our manuscript title to be less definitive and to emphasize more the nature of the study. The new title is “Utilization of Cancer Cell Line Screening to Elucidate the Anticancer Activity and Biological Pathways Related to the Ruthenium-Based Therapeutic BOLD-100”