Author response:
The following is the authors’ response to the current reviews.
Reviewer 1:
While BAP1 mutant UM cell lines were included for some of the experiments, it seems the in-vivo data mentioned in the response to the reviewers comment is missing? The authors stated that "MP46 (Supplementary Fig. 3a) is BAP1-null uveal melanoma cell line with no detectable protein expression (Amirouchene-Angelozzi et al., Mol Oncol 2014), and we have observed strong tumor growth inhibition in this CDX model with our BAF ATPase inhibitor." But the CDX model data shown in Figure 4 is from 92.1 cells. If this data is available, then the manuscript would benefit from its addition.
We thank the reviewer for bringing this to our attention. As the reviewer mentioned, we show 92-1 CDX model in our manuscript. Additionally, strong tumor growth inhibition in MP-46 CDX model treated with our BAF ATPase inhibitor can be found in Vaswani et al., 2025 (PMID:39801091, https://pubmed.ncbi.nlm.nih.gov/39801091/).
Reviewer 3:
Supplementary Figure 2C
Is the T910M mutation in the parental MP41 cells heterozygous? If so, the authors should indicate this in the figure legend. If this is a homozygous mutation, the authors should explain how the inhibitors suppress SMARCA4 activity in cells that have a LOF mutation.
We thank the reviewer for bringing this to our attention. We updated the figure legend accordingly to reflect the genotype of the mutations highlighted in the table.
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary:
The presented study by Centore and colleagues investigates the inhibition of BAF chromatin remodeling complexes. The study is well-written, and includes comprehensive datasets, including compound screens, gene expression analysis, epigenetics, as well as animal studies. This is an important piece of work for the uveal melanoma research field, and sheds light on a new inhibitor class, as well as a mechanism that might be exploited to target this deadly cancer for which no good treatment options exist.
Strengths:
This is a comprehensive and well-written study.
Weaknesses:
There are minimal weaknesses.
We thank the reviewer for the positive comments.
Reviewer #2 (Public Review):
Summary:
The authors generate an optimized small molecule inhibitor of SMARCA2/4 and test it in a panel of cell lines. All uveal melanoma (UM) cell lines in the panel are growth-inhibited by the inhibitor making the focus of the paper. This inhibition is correlated with the loss of promoter occupancy of key melanocyte transcription factors e.g. SOX10. SOX10 overexpression and a point mutation in SMARCA4 can rescue growth inhibition exerted by the SMARCA2/4 inhibitor. Treatment of a UM xenograft model results in growth inhibition and regression which correlates with reduced expression of SOX10 but not discernible toxicity in the mice. Collectively the data suggest a novel treatment of uveal melanoma.
Strengths:
There are many strengths of the study including the strong challenge of the on-target effect, the assays used, and the mechanistic data. The results are compelling as are the effects of the inhibitor. The in vivo data is dose-dependent and doses are low enough to be meaningful and associated with evidence of target engagement.
Weaknesses:
The authors introduce the field stating that SMARCA4 inhibitors are more effective in SMARCA2 deficient cancers and the converse. Since the desirable outcome of cancer therapy would be synthetic lethality it is not clear why a dual inhibitor is desirable. Wouldn't this be associated with more side effects? It is not known how the inhibitor developed here impacts normal cells, in particular T cells which are essential for any durable response to cancer therapies in patients. Another weakness is that the UM cell lines used do not molecularly resemble metastatic UM. These UM most frequently have mutations in the BAP1 tumor suppressor gene. It is not clear if the described SMARCA2/4 inhibitor is efficacious in BAP1 mutant UM cell lines in vitro or BAP1 mutant patient-derived xenografts in vivo.
We thank the reviewer for their insightful and constructive comments. As we demonstrate in Fig. 1d, uveal melanoma cells are selectively and deeply sensitive to BAF ATPase inhibition, and provides a therapeutic window. This is confirmed in Fig. 4a-c, as we demonstrated robust tumor growth inhibition, achieved at a dose well-tolerated in xenograft study. FHD-286, a dual BRM/BRG1 inhibitor similar to FHT-1015 with optimized physical properties, has been evaluated in a Phase I trial in patients with metastatic uveal melanoma (NCT04879017) and manuscript describing results of this clinical trial is currently in preparation.
As the reviewer mentioned, BAP1 loss is a signature of metastatic uveal melanoma. MP38 is a BAP1 mutant uveal melanoma cell line, and we demonstrated growth inhibition and robust caspase 3/7 activity in response to FHT-1015 (Supplementary Fig. 3a and 3f). MP46 (Supplementary Fig. 3a) is BAP1-null uveal melanoma cell line with no detectable protein expression (Amirouchene-Angelozzi et al., Mol Oncol 2014), and we have observed strong tumor growth inhibition in this CDX model with our BAF ATPase inhibitor.
Reviewer #3 (Public Review):
Summary:
This manuscript reports the discovery of new compounds that selectively inhibit SMARCA4/SMARCA2 ATPase activity that work through a different mode as previously developed SMARCA4/SMARCA2 inhibitors. They also demonstrate the anti-tumor effects of the compounds on uveal melanoma cell proliferation and tumor growth. The findings indicate that the drugs exert their effects by altering chromatin accessibility at binding sites for lineage-specific transcription factors within gene enhancer regions. In uveal melanoma, altered expression of the transcription factor, SOX10, and SOX10 target gene underlies the anti-proliferative effects of the compounds. This study is significant because the discovery of new SMARCA4/SMARCA2 inhibitory compounds that can abrogate uveal melanoma tumorigenicity has therapeutic value. In addition, the findings provide evidence for the therapeutic use of these compounds in other transcription factor-dependent cancers.
Strengths:
The strengths of this manuscript include biochemical evidence that the new compounds are selective for SMARCA4/SMARCA2 over other ATPases and that the mode of action is distinct from a previously developed compound, BRM014, which binds the RecA lobe of SMARCA2. There is also strong evidence that FHT1015 suppresses uveal melanoma proliferation by inducing apoptosis. The in vivo suppression of tumor growth without toxicity validates the potential therapeutic utility of one of the new drugs. The conclusion that FHT1015 primarily inhibits SMARCA4 activity and thereby suppresses chromatin accessibility at lineage-specific enhancers is substantiated by ATAC-seq and ChIP-seq studies.
Weaknesses:
The weaknesses include a lack of more precise information on which SMARCA4/SMARCA2 residues the drugs bind. Although the I1173M/I1143M mutations are evidence that the critical residues for binding reside outside the RecA lobe, this site is conserved in CHD4, which is not affected by the compounds. Hence, this site may be necessary but not sufficient for drug binding or specifying selectivity. A more precise evaluation of the region specifying the effect of the new compounds would strengthen the evidence that they work through a novel mode and that they are selective. Another concern is that the mechanisms by which FHT1015 promotes apoptosis rather than simply cell cycle arrest are not clear. Does SOX10 or another lineage-specific transcription factor underlie the apoptotic effects of the compounds?
We thank the reviewer for the valuable comments.
We believe that our dual ATPase inhibitor is selective and additional insights into binding specificity and selectivity for earlier stage compounds of this series were recently published in Vaswani et al., 2025 (PMID:39801091, https://pubmed.ncbi.nlm.nih.gov/39801091/).
The reviewer also poses a great question regarding the mechanism of apoptosis. The mechanism of apoptosis is extremely complex, but we observed a decrease in pro-survival BCL-2 protein expression in response to FHT-1015, in the experiment corresponding to Supplementary Fig. 5e. In the experiment described in Fig. 3k, we also monitored caspase 3/7 activity over time, and SOX10 overexpression rescued 92-1 cells from FHT-1015 induced apoptosis. This suggests the role of SOX10 as an important mediator of response to BAF ATPase inhibition, including apoptosis induced by FHT-1015.
Additional Reviews:
The referees would like to draw the authors' attention to the following issues that would best benefit from additional revision.
The clinical relevance of the study would be strengthened by the use of uveal melanoma cell lines with BAP1 mutations that better represent metastatic uveal melanoma. The use of patient-derived xenografts would also be pertinent and would be a useful addition. Similarly, attention to the effects of the inhibitor on non-cancerous proliferative cells such as blood/T/immune cells would also strengthen the manuscript. As the study reports the administration of one of the inhibitors in mice for the xenograft experiments, it would be important to assess any potential effects on blood cell counts and better discuss the eventual toxicity or lack of toxicity and how it was assessed.
The authors should better explain how SOX10 over expression can rescue viability in the presence of the inhibitor. Similarly given the critical roles of BRG1, SOX10, and MITF in cutaneous melanoma some specific discussion on the sensitivity of cutaneous melanoma cells to the inhibitor should be considered, and potential differences with uveal melanoma highlighted.
Aside from these issues, the authors are urged to consider the other points mentioned below.
Reviewer #1 (Recommendations For The Authors):
Figure 1d, as well as the text in the manuscript referring to this figure, would benefit from indicating specific cell lines used for UM. The same for the sentence in line 153.
We thank the reviewer for bringing this to our attention. We have added the cell line names and updated the manuscript accordingly.
For any of the studies conducted, is there any link with the genetics of UM? E.g. BAP1 wildtype/BAP1 mutant?
As addressed above in the public review section, MP38 is a BAP1 mutant uveal melanoma cell line, and we demonstrated growth inhibition and robust caspase 3/7 activity in response to FHT-1015 (Supplementary Fig. 3a and 3f). MP46 (Supplementary Fig. 3a) is BAP1-null uveal melanoma cell line with no detectable protein expression (Amirouchene-Angelozzi et al., Mol Oncol 2014), and we have observed strong tumor growth inhibition in this CDX model with our BAF ATPase inhibitor.
Row 191 - How were peaks classified as enhancer-occupied?
We used annotatePeaks function of HOMER package to annotate genomic locations, as well as H3K27ac ChIP-seq to annotate peaks as enhancer-occupied. We thank the reviewer to pointing it out and have updated the manuscript accordingly to include this information.
Row 259, the two cell lines should be named, also in Figure 3i.
We have added the cell line names and updated the manuscript accordingly.
Reviewer #2 (Recommendations For The Authors):
As a proof of concept, this study is truly excellent and the authors should be commended. However, it is desirable that new knowledge in cancer is translated to the clinic. To this end there are a few things needed to strengthen the study.
I am rephrasing my statements from the public review to say that I would recommend testing the inhibitor in T cells (side effects) and BAP1 mutant cell lines (for clinical relevance).
As addressed in the public review section, MP38 is a BAP1 mutant uveal melanoma cell line, and we demonstrated growth inhibition and robust caspase 3/7 activity in response to FHT-1015 (Supplementary Fig. 3a and 3f). MP46 (Supplementary Fig. 3a) is BAP1-null uveal melanoma cell line with no detectable protein expression (Amirouchene-Angelozzi et al., Mol Oncol 2014), and we have observed strong tumor growth inhibition in this CDX model with our BAF ATPase inhibitor.
Regarding concerns for any potential side effect on T cells, we observed an increase in both CD4 and CD8 T-cell populations in the peripheral blood and the spleen, when naïve, non-tumor bearing CD-1 mice were dosed with SMARCA2/4 dual ATPase inhibitor FHD-286 once daily for 14 days. FHD-286 is a compound similar to FHT-1015 described in Vaswani et al., 2025 (PMID:39801091, https://pubmed.ncbi.nlm.nih.gov/39801091/). In addition, FHD-286 has been tested in tumor bearing syngeneic models. When B16F10 tumor bearing C57BL/6 were dosed with FHD-286 for 10 days, we observed an increase in CD69+ activated CD8 T-cell infiltration in the tumor microenvironment (doi:10.1136/jitc-2022-SITC2022.0888).
Reviewer #3 (Recommendations For The Authors):
(1) Determine drug binding by crystal structure or generate additional SMARCA4 or SMARCA2 mutations in the region near I1173/I1143 that are not conserved in CHD4 and test them in an ATPase assay for effects on drug inhibition. For example, Q1166 in SMARCA4 and Q1136 in SMARCA4 could be changed to Alanine as in CHD4. Would this abrogate drug inhibition?
We believe that our dual ATPase inhibitor is selective and additional insights into binding specificity and selectivity for earlier stage compounds of this series were recently published in Vaswani et al., 2025 (PMID:39801091, https://pubmed.ncbi.nlm.nih.gov/39801091/).
(2) The finding that SOX10 can rescue the antiproliferative effects of FHT1015 suggests that SMARCA4 is primarily needed for SOX10 expression. However, the co-occupancy of SMARCA4 and SOX10 at enhancers suggests that they cooperate to promote chromatin accessibility. It is unclear how over-expression of SOX10 can promote chromatin accessibility in drug-inhibited cells since SOX10 does not have chromatin remodeling activity. ATAC-seq in cells over-expressing SOX10 and treated with the drug could identify SOX10-dependent targets that do not require SMARCA4 activity and clarify the mechanism. It would also be informative to determine if SOX10 over-expression abrogates the effects of FHT1015 on both cell cycle and apoptosis, helping to resolve whether it is a partial or complete rescue of proliferation.
We agree that running ATAC-seq in cells overexpressing SOX10 would clarify this mechanism. However, shifts in corporate strategy deprioritized any further experiments for this project. One potential mechanism that SOX10 overexpression can partially rescue BAF inhibition phenotype is through overexpressed SOX10 localizing to open chromatin regions (mostly promoters) across the genome. We know from our ATAC-seq data (Fig. 2) that BAF inhibition leads to loss of chromatin accessibility at SOX10 enhancer sites, while promoter regions are only partially affected. Therefore, we think that overexpression of SOX10 would allow upregulation of its target genes via binding to the promoter regions. In this model, the enhancer-driven SOX10 target genes are likely to remain silenced.
(3) Although the in vivo studies indicate that the drugs are well-tolerated, additional in vitro studies to determine the effects of the drug on the proliferation/survival of non-cancerous cells would further validate their therapeutic utility.
Author Response: The reviewer raises a critical question. FHD-286, a dual BRM/BRG1 inhibitor similar to FHT-1015 with optimized physical properties, has been evaluated in a Phase I trial in patients with metastatic uveal melanoma (NCT04879017), and it was well tolerated at continuous daily dose of up to 7.5 mg QD and at intermittent dose of up to 17.5 mg QD. Manuscript describing results of this clinical trial is currently in preparation.
