The Development of p53-Targeted Therapies for Human Cancers

Round 1

Reviewer 1 Report

This review article provides a list of strategies and agents to increase WT p53 activity in cancers with wt p53 or mutant p53. Status of clinical trials is in many cases included in the description of the various drugs and agents. The article should be useful to those in the p53 field aimed at developing p53 targeting drugs. There are a number of things that should be addressed?

Line 35: The tumor suppressor p53 is critical for normal cell growth…  Is this true? P53 ko mice and cells are viable. I believe it is inaccurate to say it is critical for normal cell growth.

Lin 39: The types of p53 mutations in human cancers include mostly missense mutations (Add % frequency here).

Fig 1 needs a more detailed legend. Also, should indicate if this is a comprehensive or partial list of drugs in Fig 1.

Lin 64: Thirdly, MDM2 promotes the excretion of  p53 from the nucleus and thus inhibits the activities of p53 as a transcriptional factor (should add a reference here).

Line 67: In addition, MDMX can stabilize MDM2, which has a very short half-life, and ultimately promotes p53 degradation.  (should add reference here).

Line 73: (Nutlin; Roche) was the first small  molecule compound identified to specifically disrupt the p53-MDM2 interface and thus  increase p53 stability and activity, leading to cell cycle arrest, apoptosis, senescence and differentiation of cancer cells [10]. (did they show differentiation of cancer cells in ref. 10?)

Line 83: A Phase I study of RG7112 (NCT00623870) in patients with hematologic malignancies has shown that RG7112 can inhibit p53 degradation, but with an increase in non-transcriptional levels, thereby activating p53 target genes and promote (can the authors clarify what “but with an increase in non-transcriptional levels” means?).

On line 46 authors say mutant p53s can still form tetramers with wt p53 in the heterozygous state. On line 184 authors say most p53 mutations inhibit DNA binding  and disrupt tetramerization. Can the authors reconcile these different statements about tetramerization?

Line 224 paragraph: need more discussion here about Zn chelators. If Zn is required for p53 binding and increasing Zn can promote mutant p53 folding into a WT conformation, then Zn chelators might be expected to prevent folding into a WT conformation. Authors should indicate more about how the Zn chelator promotes folding into WT conformation.  Also need more discussion in the following paragraph about COTI-2. Is it  a zinc chelator? How does it work to activate mutant p53?

Line 264 paragraph about RETRA and Prodigiosin talks about these drugs activating p53 family members. This paragraph is under the heading of drugs that cause dispersion of p53 aggregates. Do RETRA and Prodigiosin disrupt mutant p53 aggregates? Is this how they activate p73? If not, this paragraph seems out of place.

Minor editing of English is suggested

Author Response

Point-to-point responses to reviewers:

Reviewer #1 (Comments to the Author): 

This review article provides a list of strategies and agents to increase WT p53 activity in cancers with wtp53 or mutant p53. Status of clinical trials is in many cases included in the description of the various drugs and agents. The article should be useful to those in the p53 field aimed at developing p53 targeting drugs. There are a number of things that should be addressed?

• Line 35: The tumor suppressor p53 is critical for normal cell growth… Is this true? P53 ko mice and cells are viable. I believe it is inaccurate to say it is critical for normal cell growth.

Response: We deleted the “critical for normal cell growth” from the sentence.

• Lin 39: The types of p53 mutations in human cancers include mostly missense mutations (Add % frequency here).

Response: We added the percentage (70-80%) as required.

• Fig 1 needs a more detailed legend. Also, should indicate if this is a comprehensive or partial list of drugs in Fig 1.

Response: We provided detailed legend for Fig 1. 

• Lin 64: Thirdly, MDM2 promotes the excretion of p53 from the nucleus and thus inhibits the activities of p53 as a transcriptional factor (should add a reference here).

Response: We sincerely appreciate the valuable comments. We have checked the literature carefully and added related reference in the revised manuscript.

• Line 67: In addition, MDMX can stabilize MDM2, which has a very short half-life, and ultimately promotes p53 degradation. (should add reference here).

Response: We added the corresponding references.

• Line 73: (Nutlin; Roche) was the first small molecule compound identified to specifically disrupt the p53-MDM2 interface and thus increase p53 stability and activity, leading to cell cycle arrest, apoptosis, senescence and differentiation of cancer cells [10]. (did they show differentiation of cancer cells in ref. 10?)

  Response: We added a reference to support the statement.

• Line 83: A Phase I study of RG7112 (NCT00623870) in patients with hematologic malignancies has shown that RG7112 can inhibit p53 degradation, but with an increase in non-transcriptional levels, thereby activating p53 target genes and promote (can the authors clarify what “but with an increase in non-transcriptional levels” means?).

Response: We clarified the statement as “RG7112 does not affect p53 transcription but can inhibit p53 degradation, thereby activating p53 target genes and promote cell cycle arrest and apoptosis in leukemia cells.”

• On line 46 authors say mutant p53s can still form tetramers with wtp53 in the heterozygous state. On line 184 authors say most p53 mutations inhibit DNA binding and disrupt tetramerization. Can the authors reconcile these different statements about tetramerization?

Response: We revised the statement as “Most p53 mutations can affect the transcriptional activities of wtp53, inducing the loss of DNA-binding activity directly or indirectly by the disruption of normal tetramerization of wtp53.”

• Line 224 paragraph: need more discussion here about Zn chelators. If Zn is required for p53 binding and increasing Zn can promote mutant p53 folding into a WT conformation, then Zn chelators might be expected to prevent folding into a WT conformation. Authors should indicate more about how the Zn chelator promotes folding into WT conformation. Also need more discussion in the following paragraph about COTI-2. Is it a zinc chelator? How does it work to activate mutant p53?

Response: We provided more information on Zn binding chemicals and COT1-2 in the revised manuscript.

• Line 264 paragraph about RETRA and Prodigiosin talks about these drugs activating p53 family members. This paragraph is under the heading of drugs that cause dispersion of p53 aggregates. Do RETRA and Prodigiosin disrupt mutant p53 aggregates? Is this how they activate p73? If not, this paragraph seems out of place.

Response: Mutant p53 can aggregate with transcriptional regulators p63 and p73, preventing their entry into the nucleus. RETRA and Prodigiosin can dismantle mutant p53-p63/p73 aggregates, releasing p73 and activating genes shared with wild-type p53.  We clarified these points in the revised manuscript.

Reviewer 2 Report

nuscript by Lu Y et al review the strategies to develop p53 targeting therapies. It is well written and up-to date. Although it is not entirely comprehensive ( a number of compounds have not been mentioned – but there are so many of them now…), this review will be appreciated by a broad audience.

 

 However, this manuscript should be corrected, since there are a number of errors, especially in references.

 

Here are my comments:

 

Line 11: epigenetic silencing of wild type p53 is not a major mechanism of p53 disfunction, as it stated in Summary. In addition, this mechanism has not been mentioned in the text, and there is no reference.

 

Line 37: Ref #1 is not relevant to what is stated in this sentence

 

Line 64: Ref #9 is not a proper reference for p53 N-terminus binding to MDM2

 

Lines 75-76: in the cited paper there was no induction of differentiation or senescence by nutlin

 

Line 134: no reference about NVP-CGM097 clinical trial

 

Line 137: wrong ref! ref #20 is about the discovery of NVP-CGM097, but not about MK-8242

 

Line 186: It is not explained, which mutations lead to the disruption of tetramerization?  

 

Line 197: wrong ref #31

 

Line 203: wrong ref #37- the cited paper is about restoring the mutant p63 function, nothing about mutant p53 aggregation in this paper

 

Line 208: wrong ref #33,34

 

Line 229. Not an original paper is cited, should be PMID: 25294809

 

Line 242: nonsense-mediated mRNA decay degrades mRNA, not the protein, as it is written

 

Line 258: it would be good to mention first that mutp53 forms aggregates

 

Line 268: ref to RETRA discovery is missing

 

Line 336: cited is the paper reporting Phase I trial, not Phase III, as written

 

Line 348: the concept of synthetic lethality is not adequately explained 

Text editing is needed, for example, line 102, should be: …clinical trials have NOT yet been published…; line 225, should be: …p53 to unfolded and aggregated STATE…; etc

Author Response

Reviewer #2 (Comments to the Author):

This manuscript by Lu Y et al review the strategies to develop p53 targeting therapies. It is well written and up-to date. Although it is not entirely comprehensive ( a number of compounds have not been mentioned – but there are so many of them now…), this review will be appreciated by a broad audience. However, this manuscript should be corrected, since there are a number of errors, especially in references.

• Line 11: epigenetic silencing of wild type p53 is not a major mechanism of p53 disfunction, as it stated in Summary. In addition, this mechanism has not been mentioned in the text, and there is no reference.

Response: We delete the statement on epigenetic silencing of p53 from the text.

• Line 37: Ref #1 is not relevant to what is stated in this sentence

Response: We corrected reference 1 with a proper citation.  

• Line 64: Ref #9 is not a proper reference for p53 N-terminus binding to MDM2

Response: We corrected reference 9 with an appropriate citation.   

• Lines 75-76: in the cited paper there was no induction of differentiation or senescence by nutlin

Response: As suggested, a reference has been added to support the statement. This article shows SK-N-SH cells surviving nutlin treatment adopted a senescence-like phenotype, whereas CLB-GA and NGP cells underwent neuronal differentiation.

• Line 134: no reference about NVP-CGM097 clinical trial

Response: We added the reference.

• Line 137: wrong ref! ref #20 is about the discovery of NVP-CGM097, but not about MK-8242

Response: We corrected reference 20 with an appropriate one. 

• Line 186: It is not explained, which mutations lead to the disruption of tetramerization?

Response: We added the p53 mutations that affect tetramerization in the following paragraph: Mutp53 tends to form aggregates due to the exposure of adhesion sequences originally wrapped inside its hydrophobic core, so structural mutp53, such as R110P, R175H, R248Q, R249S, and R282W, can form amyloid aggregates.

• Line 197: wrong ref #31

Response: We corrected reference 31 with an appropriate one. 

• Line 203: wrong ref #37- the cited paper is about restoring the mutant p63 function, nothing about mutant p53 aggregation in this paper

Response: We corrected reference 37 with an appropriate one. 

• Line 208: wrong ref #33,34

Response: We corrected these references with appropriate ones. 

• Line 229. Not an original paper is cited, should be PMID: 25294809

Response: We corrected the reference with the appropriate one. 

• Line 242: nonsense-mediated mRNA decay degrades mRNA, not the protein, as it is written

Response: We fixed the typo.

• Line 258: it would be good to mention first that mutp53 forms aggregates

Response: We added the statement in the revised manuscript.

• Line 268: ref to RETRA discovery is missing

Response: We provided the citation on RETRA discovery.

• Line 336: cited is the paper reporting Phase I trial, not Phase III, as written

Response: We corrected the typo as suggested.

• Line 348: the concept of synthetic lethality is not adequately explained

Response: We explained the concept of synthetic lethality.

Reviewer 3 Report

This is a well-organized review article. It is relatively short but summarizes the essence of the fields. Specific suggestions are as follows.

 

Where Y220C is described on page 8, the authors should refer to Guiley & Shokat, Cancer Discov 2023; 13: 56-69.

 

Where the authors mention stapled peptides on page 4, they should refer to the work by David Lane, a p53 pioneer, for example,

Mortensen ACL et al., Front Oncol 2019; 9: 923

Lundsten S et al., Nanomaterials (Basel) 2020; 10: 783

 

Page 7, section 2.2.2.: HSP40/DNAJA1 inhibitors and statins should be mentioned with the following references:

Parrales A et al., Nat Cell Biol 2016; 18: 1233-1243

Nishikawa S et al., Cell Death Discov 2022; 8: 437

Alalem M et al., Cancers (Basel) 2022; 14: 4187

 

By referring to Malekzadeh P et al., J Clin Invest 2019; 129: 1109-1114, the authors should state that p53 hot spot mutations can be processed and presented on the surface of tumor cells as HLA-restricted neoantigens, which can be targeted by T cell-based cancer immunotherapy.

 

The authors may want to mention a possible conflict between p53-mediated tumor suppression and accelerated aging phenotypes. An interesting commentary on this point is: Horikawa I, Cancer Res 2020; 80: 5164-5165.

Author Response

Reviewer #3 (Comments to the Author):

This is a well-organized review article. It is relatively short but summarizes the essence of the fields. Specific suggestions are as follows.

• Where Y220C is described on page 8, the authors should refer to Guiley & Shokat, Cancer Discov 2023; 13: 56-69.

Response: We corrected the citation.

• Where the authors mention stapled peptides on page 4, they should refer to the work by David Lane, a p53 pioneer, for example, Mortensen ACL et al., Front Oncol 2019; 9: 923 Lundsten S et al., Nanomaterials (Basel) 2020; 10: 783

      Response: We corrected the citations as suggested.

• Page 7, section 2.2.2.: HSP40/DNAJA1 inhibitors and statins should be mentioned with the following references: Parrales A et al., Nat Cell Biol 2016; 18: 1233-1243 Nishikawa S et al.,Cell Death Discov 2022; 8: 437 Alalem M et al., Cancers (Basel) 2022; 14: 4187

Response: We added the suggested citations.

• By referring to Malekzadeh P et al., J Clin Invest 2019; 129: 1109-1114, the authors should state that p53 hot spot mutations can be processed and presented on the surface of tumor cells as HLA-restricted neoantigens, which can be targeted by T cell-based cancer immunotherapy.

Response: Thank you for the suggestion and we have added the part “2.4 Cancer immunotherapy for mutp53” mentioned with the work by Malekzadeh P et al.

• The authors may want to mention a possible conflict between p53-mediated tumor suppression and accelerated aging phenotypes. An interesting commentary on this point is: Horikawa I, Cancer Res 2020; 80: 5164-5165.

Response: Thanks for the suggestion, we discussed this issue in the conclusion of the revised manuscript.

Round 2

Reviewer 2 Report

mo further comments

no further comments