Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma
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Curated by eLife
eLife Assessment:
This manuscript sheds light on the biology of embryonal rhabdomyosarcoma, a common pediatric muscle tumor, by exploiting an established zebrafish model. Specifically, new knowledge is revealed of how the p53 tumor suppressor contributes to progression and extent of disease. This paper will be of interest not only to pediatric oncologists but also the broader cancer research community given the frequency of TP53 mutations as secondary lesions in human cancer.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
In embryonal rhabdomyosarcoma (ERMS) and generally in sarcomas, the role of wild-type and loss- or gain-of-function TP53 mutations remains largely undefined. Eliminating mutant or restoring wild-type p53 is challenging; nevertheless, understanding p53 variant effects on tumorigenesis remains central to realizing better treatment outcomes. In ERMS, >70% of patients retain wild-type TP53 , yet mutations when present are associated with worse prognosis. Employing a kRAS G12D -driven ERMS tumor model and tp53 null (tp53 -/- ) zebrafish, we define wild-type and patient-specific TP53 mutant effects on tumorigenesis. We demonstrate that tp53 is a major suppressor of tumorigenesis, where tp53 loss expands tumor initiation from <35% to >97% of animals. Characterizing three patient-specific alleles reveals that TP53 C176F partially retains wild-type p53 apoptotic activity that can be exploited, whereas TP53 P153Δ and TP53 Y220C encode two structurally related proteins with gain-of-function effects that predispose to head musculature ERMS. TP53 P153Δ unexpectedly also predisposes to hedgehog-expressing medulloblastomas in the kRAS G12D -driven ERMS-model.
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eLife Assessment:
This manuscript sheds light on the biology of embryonal rhabdomyosarcoma, a common pediatric muscle tumor, by exploiting an established zebrafish model. Specifically, new knowledge is revealed of how the p53 tumor suppressor contributes to progression and extent of disease. This paper will be of interest not only to pediatric oncologists but also the broader cancer research community given the frequency of TP53 mutations as secondary lesions in human cancer.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript by Chen et al., describes an in vivo zebrafish assay to test the impact of human tp53 variants on rhabdomyosarcoma tumor pathogenesis. There are a number of novel observations in this study. First, tumor onset is significantly enhanced when the zebrafish rhabdomyosarcoma tumor model is in a tp53 null background, leading to >97% of animals developing tumors, most within the first 20 days of life. Second, introduction of wildtype human tp53 cDNA suppresses tumor initiation, establishing a rapid and effective in vivo assay to examine the activity of tp53 variants on tumor induction and growth. Three tp53 variants identified in human rhabdomyosarcoma patients were tested in standard in vitro assays, but none of the variants demonstrated wild type tp53 activity such as suppression of tumor cell …
Reviewer #1 (Public Review):
The manuscript by Chen et al., describes an in vivo zebrafish assay to test the impact of human tp53 variants on rhabdomyosarcoma tumor pathogenesis. There are a number of novel observations in this study. First, tumor onset is significantly enhanced when the zebrafish rhabdomyosarcoma tumor model is in a tp53 null background, leading to >97% of animals developing tumors, most within the first 20 days of life. Second, introduction of wildtype human tp53 cDNA suppresses tumor initiation, establishing a rapid and effective in vivo assay to examine the activity of tp53 variants on tumor induction and growth. Three tp53 variants identified in human rhabdomyosarcoma patients were tested in standard in vitro assays, but none of the variants demonstrated wild type tp53 activity such as suppression of tumor cell growth or activation of gene expression. In contrast, all three variants were able to suppress tumor onset and initiation in the in vivo zebrafish rhabdomyosarcoma model. The ability of the variants to suppress tumor initiation was attributed to retention of some level of activity, or novel activity, in vivo. Additional molecular data would strengthen the conclusion that the different tp53 variants function as hypomorph or gain of function alleles.
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Reviewer #2 (Public Review):
In this paper, the authors exploit an established zebrafish model of embryonal rhabdomyosarcoma driven by a mutation in the kRAS oncogene to understand the contribution of secondary mutations to the presentation and behavior of the disease. Specifically, they focus on mutations in TP53, a potent tumor suppressor and the most commonly mutated gene in human cancer. Using a zebrafish mutant line in which tp53 has been deleted, they demonstrate that loss of p53 greatly accelerates tumor onset, frequency, size and number of lesions. This aggressive behavior is driven by hyperproliferation rather than reduced apoptosis, an outcome process that can be prevented by co-expression of wild type human or zebrafish p53. Subsequently, two patient-specific TP53 lesions are expressed in human cancer cells and in the …
Reviewer #2 (Public Review):
In this paper, the authors exploit an established zebrafish model of embryonal rhabdomyosarcoma driven by a mutation in the kRAS oncogene to understand the contribution of secondary mutations to the presentation and behavior of the disease. Specifically, they focus on mutations in TP53, a potent tumor suppressor and the most commonly mutated gene in human cancer. Using a zebrafish mutant line in which tp53 has been deleted, they demonstrate that loss of p53 greatly accelerates tumor onset, frequency, size and number of lesions. This aggressive behavior is driven by hyperproliferation rather than reduced apoptosis, an outcome process that can be prevented by co-expression of wild type human or zebrafish p53. Subsequently, two patient-specific TP53 lesions are expressed in human cancer cells and in the zebrafish rhabdomyosarcoma model lacking p53 and distinct tumor patterns are observed. The protein structural consequences of these specific p53 abnormalities are determined, providing insights into their observed functional features and how these could be manipulated as a therapeutic strategy. These studies position the zebrafish as a robust animal model for determining the contributions of specific secondary tp53 mutations to rhabdomyosarcoma, an approach that could be applied more broadly to secondary tp53 mutations in other cancers.
Strengths
The manuscript is well-written and experiments logically flow from one to the other. The studies exploit unique tools at the authors' disposal and highlight how the conserved genetics and optical clarity of the zebrafish model can provide insight into nuanced genotype-phenotype correlations of specific p53 lesions. The figures are nicely displayed and the data demonstrating the different tumor progression patterns are compelling. This approach can be applied to better understand the natural history of patient specific TP53 variants in the context of embryonal rhabdomyosarcoma and other tumors for which a zebrafish model is available.
Weaknesses
While zebrafish expressing the KRASG12D mutation develop embryonal rhabdomyosarcoma, this mutation is infrequent in human tumors. The explanations of transgenic approaches are very limited. The order in which these lesions occur may also be significant, and this is not addressed by the authors. In Li Fraumeni syndrome, the TP53 mutations occur first and are germline and modifier mutations occur secondarily and are somatic. Comparison of the corresponding zebrafish and human mutant p53 cDNAs is not described. Exogenously-provided p53 cDNAs expressed under the rag2 promoter may not function the same as if the endogenous tp53 genes were mutated using genomic engineering.
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Reviewer #3 (Public Review):
Using a robust transient transgenic approach in the zebrafish embryonal rhabdomyosarcoma (ERM) model, Chen et. al. identified diverse activities of several disease-relevant TP53 variants in ERM pathogenesis. The useful tools established in this study would allow rapid in vivo assessment of the effect of newly identified TP53 mutations on ERM tumorigenesis.
Strengths:
• It's the first time to dissect the activities of several rare patient-specific TP53 mutations in ERM tumor initiation and progression in vivo.• This study demonstrates the robustness of transient co-injection transgenic approach for rapid structure function analyses of disease-relevant variants in vivo.
• This study also suggests distinct activities of different TP53 structure variants, such as their potential functions as a hypomorphic …
Reviewer #3 (Public Review):
Using a robust transient transgenic approach in the zebrafish embryonal rhabdomyosarcoma (ERM) model, Chen et. al. identified diverse activities of several disease-relevant TP53 variants in ERM pathogenesis. The useful tools established in this study would allow rapid in vivo assessment of the effect of newly identified TP53 mutations on ERM tumorigenesis.
Strengths:
• It's the first time to dissect the activities of several rare patient-specific TP53 mutations in ERM tumor initiation and progression in vivo.• This study demonstrates the robustness of transient co-injection transgenic approach for rapid structure function analyses of disease-relevant variants in vivo.
• This study also suggests distinct activities of different TP53 structure variants, such as their potential functions as a hypomorphic allele, a gain-of-function mutation, or a predisposition mutant for the head musculature ERMs.
Weaknesses:
• The role of tp53 loss in promoting the initiation of kRASG12D-driven ERM has been demonstrated previously using a similar strategy by coauthors (Ignatius, M. S., eLife, 2018; Langenau, D. M., Genes Dev, 2007).• The data from TP53-null SaOS2 osteosarcoma cell line did not consistently support the findings from in vivo zebrafish studies, which is confusing and would need to be addressed.
• It is not clear how overexpression of the TP53P153△ or TP53Y220C mutant induced different effects on the tumor initiation and cell survival of kRASG12D-driven ERM but led to similarly enhanced head ERM development.
• This study mainly applied the overexpression approach to understand the function of TP53 mutants in ERM pathogenesis and demonstrated the distinct effects of their overexpression on kRASG12D-driven tumor initiation, cell survival and proliferation. However, these mutations are not gained or amplified in human ERMs. Hence, overexpression approach could provide some insights of their function, but cannot faithfully mimic the ERM disease situation to uncover the real function of these mutants in ERM pathogenesis.
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