USP28 deletion and small-molecule inhibition destabilizes c-MYC and elicits regression of squamous cell lung carcinoma
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Summary:
This paper is of general interest to cancer biologists focusing on identifying new targets for cancer therapy particularly in the context of squamous cell lung carcinoma. The authors demonstrate that genetic ablation of the deubiquitinase USP28 reduces the growth of lung squamous cell carcinomas but not lung adenocarcinomas in a mouse model of lung cancer, and that that this restriction of growth is accompanied by loss of expression of several USP28 targets. They also describe activity of a new small molecule compound in controlling the growth of lung squamous cell carcinomas in mouse genetic and xenograft models, and reducing expression of USP28 targets. They demonstrate that USP28 is one target of the newly identified compound, but they do not establish whether it is the only and biologically relevant target of this compound.
Reviewer #3 opted to reveal their name to the authors in the decision letter after review.
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Abstract
Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient’s 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28 -induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.
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Reviewer #3:
The prevalent treatment options for LSCC are limited in efficacy. Through genetic inactivation of Usp28 in a novel lung cancer mouse model, and chemical inhibition of Usp28 in induced LSCC in mice and human LSCC xenograft tumors, the authors demonstrated the specific dependency of LSCC (but not LADC) on the protein deubiquitinase Usp28. The authors also showed that loss of Usp28 by either means leads to depletion of the oncoproteins c-Myc, p63 and c-Jun in LSCC. Finally, the authors described a novel small molecule that is specific for Usp25/28 among a group of assessed deubiquitinases. Based on these results, the authors suggested chemically targeting USP28 as a potential therapeutic option for human LSCC patients.
Strengths: The presentation of the work is clear, concise and easily readable. The data presented largely …
Reviewer #3:
The prevalent treatment options for LSCC are limited in efficacy. Through genetic inactivation of Usp28 in a novel lung cancer mouse model, and chemical inhibition of Usp28 in induced LSCC in mice and human LSCC xenograft tumors, the authors demonstrated the specific dependency of LSCC (but not LADC) on the protein deubiquitinase Usp28. The authors also showed that loss of Usp28 by either means leads to depletion of the oncoproteins c-Myc, p63 and c-Jun in LSCC. Finally, the authors described a novel small molecule that is specific for Usp25/28 among a group of assessed deubiquitinases. Based on these results, the authors suggested chemically targeting USP28 as a potential therapeutic option for human LSCC patients.
Strengths: The presentation of the work is clear, concise and easily readable. The data presented largely supports the authors' conclusions on the role of USP28 in LSCC tumorigenesis and that inhibition of USP28 is a viable therapeutic option for LSCC treatment. The generation of the KFCU mice model that can give rise to both LADC and LSCC concurrently is interesting and presents a valuable tool for the wider cancer community.
Weakness: The manuscript can benefit from a deeper analysis of the relationship between FBW7 and USP28 in patient cohorts. A comparison of the activity/efficacy of FT206 to existing USP28 inhibitors will also be helpful.
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Reviewer #2:
In this work Ruiz et al, use a couple of elegant mouse genetic models - KFCU (Fbxw7 deletion and mutant Ras over-expression) and KPCU (p53 deletion and mutant Ras over-expression) - to generate both LADC and LSCC tumors. Using this system, the authors show that deletion of USP28 resulted in less LSCC but not LADC tumor formation. However, both tumor types showed an overall decrease in tumor size (in KFCU; data are not shown in KPCU). These results are the genetic proof of concept that USP28 inhibition will be particularly detrimental in the context of LSCC tumors. They further test a compound (FT206) that was previously found to target USP28 and show that indeed this compound is specific for USP28 binding among USPs and can reduce the tumor numbers and size only in LSCC tumors and not LADC in the KF model and in three …
Reviewer #2:
In this work Ruiz et al, use a couple of elegant mouse genetic models - KFCU (Fbxw7 deletion and mutant Ras over-expression) and KPCU (p53 deletion and mutant Ras over-expression) - to generate both LADC and LSCC tumors. Using this system, the authors show that deletion of USP28 resulted in less LSCC but not LADC tumor formation. However, both tumor types showed an overall decrease in tumor size (in KFCU; data are not shown in KPCU). These results are the genetic proof of concept that USP28 inhibition will be particularly detrimental in the context of LSCC tumors. They further test a compound (FT206) that was previously found to target USP28 and show that indeed this compound is specific for USP28 binding among USPs and can reduce the tumor numbers and size only in LSCC tumors and not LADC in the KF model and in three separate LSCC cell line xenograft models. Altogether, they make the argument that targeting LSCC tumors with chemical inhibitors of USP28 is a promising clinical strategy for LSCC cancers. Overall this paper is interesting and the results provided in vivo are strong and nicely demonstrate an on-target effect of FT206 and its specificity in LSCC tumors. The work is very similar to a recent publication of (Prieto-Garcia EMBO Mol Med 2020) describing very similar results for USP28 dependency in LSCC tumors and previous findings regarding the chemical matter used in this paper (FT206).
The major strengths of this paper is that the authors use several very elegant mouse models to establish that Usp28 is a good candidate target for potential therapeutic development designated for LSCC patients. They also show the proof of concept using a compound that is described as a Usp28 inhibitor (FT206). It should be noted that much of the genetic data, showing the importance of Usp28 in LSCC was previously described (Prieto-Garcia EMBO Mol Med 2020) including the potential benefit of chemical inhibition of USP28 . A potential weakness is that there is no rigorous characterizing of Usp28 substrate ubiquitination and degradation following FT206 treatment. This work will likely motivate the development of the USP28 inhibitor(s) for further preclinical assessment in Usp28 dependent tumors such as LSCC.
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Reviewer #1:
The authors investigate a role for a candidate new inhibitor of USP28 in destabilizing c-MYC to reduce the growth of lung squamous carcinomas. They demonstrate that c-MYC levels are higher in lung squamous cell carcinomas (LSCC) versus lung adenocarcinomas (LADC), and depletion of c-MYC reduces LSCC cell growth. The deubiquitinase USP28 is known to stabilize c-MYC; the authors show that depletion of USP28 also decreases c-MYC protein levels. USP28 action opposes that of a ubiquitin complex targeted by the FBXW7 tumor suppressor. The authors create a new mouse model in which FLP recombinase initially causes deletion of FBXW7 and activation of KRAS to cause tumorigenesis with LSCC and LADC, followed by tamoxifen-dependent CRE recombinase deletion of USP28. Loss of USP28 in this model reduced numbers of LSCC but not LADC, and …
Reviewer #1:
The authors investigate a role for a candidate new inhibitor of USP28 in destabilizing c-MYC to reduce the growth of lung squamous carcinomas. They demonstrate that c-MYC levels are higher in lung squamous cell carcinomas (LSCC) versus lung adenocarcinomas (LADC), and depletion of c-MYC reduces LSCC cell growth. The deubiquitinase USP28 is known to stabilize c-MYC; the authors show that depletion of USP28 also decreases c-MYC protein levels. USP28 action opposes that of a ubiquitin complex targeted by the FBXW7 tumor suppressor. The authors create a new mouse model in which FLP recombinase initially causes deletion of FBXW7 and activation of KRAS to cause tumorigenesis with LSCC and LADC, followed by tamoxifen-dependent CRE recombinase deletion of USP28. Loss of USP28 in this model reduced numbers of LSCC but not LADC, and led to decreased expression of c-MYC and other short-lived proteins such as c-JUN and deltap63. A limitation of the data shown is that tumor number calculations are shown for a relatively small number of mice. Deletion of USP28 also did not restrict LADC growth in a second mouse model, with tumors forming based on activation of KRAS and loss of TP53. The authors then describe a compound, FT206, which they show is a specific inhibitor of USP28 among other ubiquitinases. They demonstrate that this compound reduces expression of c-MYC, c-JUN, and deltap63, but do not demonstrate this effect is directly mediated through USP28. They also show FT206 reduces growth of LSCC but not LADC in the KRAS/FBXW7 tumor model, and in human LSCC xenografts. These latter data suggest the compound FT206 may be useful as a lead compound. However, the current data are not sufficient to demonstrate FT206 binding and biological effect is specific for USP28, as the compound may also bind and regulate other non-deubiquitinase proteins.
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Summary:
This paper is of general interest to cancer biologists focusing on identifying new targets for cancer therapy particularly in the context of squamous cell lung carcinoma. The authors demonstrate that genetic ablation of the deubiquitinase USP28 reduces the growth of lung squamous cell carcinomas but not lung adenocarcinomas in a mouse model of lung cancer, and that that this restriction of growth is accompanied by loss of expression of several USP28 targets. They also describe activity of a new small molecule compound in controlling the growth of lung squamous cell carcinomas in mouse genetic and xenograft models, and reducing expression of USP28 targets. They demonstrate that USP28 is one target of the newly identified compound, but they do not establish whether it is the only and biologically relevant target of this compound.
Summary:
This paper is of general interest to cancer biologists focusing on identifying new targets for cancer therapy particularly in the context of squamous cell lung carcinoma. The authors demonstrate that genetic ablation of the deubiquitinase USP28 reduces the growth of lung squamous cell carcinomas but not lung adenocarcinomas in a mouse model of lung cancer, and that that this restriction of growth is accompanied by loss of expression of several USP28 targets. They also describe activity of a new small molecule compound in controlling the growth of lung squamous cell carcinomas in mouse genetic and xenograft models, and reducing expression of USP28 targets. They demonstrate that USP28 is one target of the newly identified compound, but they do not establish whether it is the only and biologically relevant target of this compound.
Reviewer #3 opted to reveal their name to the authors in the decision letter after review.
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