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Arsenic and an Old Place: Rescuing p53 Mutants in Cancer

2021; Cell Press; Volume: 39; Issue: 2 Linguagem: Inglês

10.1016/j.ccell.2021.01.012

1878-3686

Stewart N. Loh,

Advanced biosensing and bioanalysis techniques

Tumor suppressor p53 lacks conventional drug binding pockets that would facilitate rescue of cancer-driving mutations. In this issue, Chen et al. discover a new role for an old drug, arsenic trioxide, in binding and stabilizing p53. The arsenic atom binds in a conserved, cryptic site and reactivates multiple p53 mutants. Tumor suppressor p53 lacks conventional drug binding pockets that would facilitate rescue of cancer-driving mutations. In this issue, Chen et al. discover a new role for an old drug, arsenic trioxide, in binding and stabilizing p53. The arsenic atom binds in a conserved, cryptic site and reactivates multiple p53 mutants. Reactivating mutant p53 has the potential to treat many tumor types, yet not a single drug has been approved to restore mutated p53 tumor-suppressing activity. In this issue, Chen et al., 2020Chen S. Wu J.-L. Liang Y. Tang Y.-G. Song H.-X. Wu L.-L. Xing Y.-F. Yan N. Li Y.-T. Wang Z.-Y. et al.Arsenic Trioxide Rescues Structural p53 Mutations through a Cryptic Allosteric Site.Cancer Cell. 2020; (S1535-6108(20)30605-X. Published online December 24, 2020)https://doi.org/10.1016/j.ccell.2020.11.013Abstract Full Text Full Text PDF Scopus (25) Google Scholar report rescue of p53 mutants by arsenic trioxide, an FDA-approved drug for the treatment of leukemia. By doing so, they address this gap which has been a long-standing priority in cancer treatment. p53's undruggable reputation originates from the broad spectrum of mutations found in cancer patients. An ideal chemical would rescue a number of these mutations despite the different mechanisms by which they inactivate p53. About 90% of tumorigenic p53 alterations encode for missense mutations in its DNA binding domain (DBD) and are spread out at almost every codon (Baugh et al., 2018Baugh E.H. Ke H. Levine A.J. Bonneau R.A. Chan C.S. Why are there hotspot mutations in the TP53 gene in human cancers?.Cell Death Differ. 2018; 25: 154-160https://doi.org/10.1038/cdd.2017.180Crossref PubMed Scopus (155) Google Scholar). The class of "hotspot" mutations which knocks out DNA binding by removing key DNA contact residues are likely to be the most challenging ones to be rescued by small molecules. Other hotspot mutations lie outside the DNA binding groove. They impair DNA binding by indirect mechanisms such as reducing DBD's thermodynamic stability and/or affinity for its single essential zinc ion or distorting the DNA binding pocket without changing the identities of DNA contact residues (Blanden et al., 2020Blanden A.R. Yu X. Blayney A.J. Demas C. Ha J.-H. Liu Y. Withers T. Carpizo D.R. Loh S.N. Zinc shapes the folding landscape of p53 and establishes a pathway for reactivating structurally diverse cancer mutants.eLife. 2020; 9: e61487https://doi.org/10.7554/eLife.61487Crossref Scopus (6) Google Scholar). Some success has been achieved in restoring DNA binding function to these mutants; for example, PhiKan and related molecules fill the hydrophobic cavity in DBD left by the Tyr220→Cys (Y220C) substitution, restoring lost side chain interactions and partially reinstating thermodynamic stability (Boeckler et al., 2008Boeckler F.M. Joerger A.C. Jaggi G. Rutherford T.J. Veprintsev D.B. Fersht A.R. Targeted rescue of a destabilized mutant of p53 by an in silico screened drug.Proc. Natl. Acad. Sci. USA. 2008; 105: 10360-10365Crossref PubMed Scopus (248) Google Scholar). Their action is specific to the Y220C variant. DBD coordinates Zn2+ via three Cys and one His residue. Zinc metallochaperones (e.g., ZMC1/NSC319726) elevate intracellular zinc levels, thereby increasing zinc binding and thermodynamic stability of DBD (Yu et al., 2014Yu X. Blanden A.R. Narayanan S. Jayakumar L. Lubin D. Augeri D. Kimball S.D. Loh S.N. Carpizo D.R. Small molecule restoration of wildtype structure and function of mutant p53 using a novel zinc-metallochaperone based mechanism.Oncotarget. 2014; 5: 8879-8892Crossref PubMed Scopus (58) Google Scholar). Alkylating agents such as PRIMA-1/APR-246 covalently modify thiolate groups and other nucleophiles via hetero-Michael addition, and they appear to stabilize p53 by forming adducts to about half of DBD's ten Cys residues (Zhang et al., 2018Zhang Q. Bykov V.J.N. Wiman K.G. Zawacka-Pankau J. APR-246 reactivates mutant p53 by targeting cysteines 124 and 277.Cell Death Dis. 2018; 9: 439https://doi.org/10.1038/s41419-018-0463-7Crossref PubMed Scopus (95) Google Scholar). Michael addition is promiscuous, however, and raising intracellular zinc increases availability of the metal to all proteins. APR-246 and ZMC1 consequently modify proteins other than p53 and cause cell death by p53-independent pathways as well as on-target mechanisms. When Chen et al., 2020Chen S. Wu J.-L. Liang Y. Tang Y.-G. Song H.-X. Wu L.-L. Xing Y.-F. Yan N. Li Y.-T. Wang Z.-Y. et al.Arsenic Trioxide Rescues Structural p53 Mutations through a Cryptic Allosteric Site.Cancer Cell. 2020; (S1535-6108(20)30605-X. Published online December 24, 2020)https://doi.org/10.1016/j.ccell.2020.11.013Abstract Full Text Full Text PDF Scopus (25) Google Scholar set out to discover drugs that rescue mutant p53, they focus on compounds that would likely bind to at least two cysteine residues. This strategy has the strength of exploiting either the Cys-modifying or zinc-binding modalities described above. When they find that one of the lead molecules was As2O3 (arsenic trioxide, or ATO) and that it rescued p53-R175H, a known zinc-binding mutant, they anticipate that arsenic would act as a "super zinc," replacing the endogenous metal with one that bound more tightly to its coordinating Cys residues (Cys176, Cys238, Cys242). In fact, displacement of Zn(II) by As(III) in the RING domain of the promyelotic leukemia-retinoic acid receptor alpha fusion protein (PML-RARα) is the mechanism by which ATO exerts its therapeutic effects against acute promyelotic leukemia (Kaiming et al., 2018Kaiming C. Sheng Y. Zheng S. Yuan S. Huang G. Liu Y. Arsenic trioxide preferentially binds to the ring finger protein PML: understanding target selection of the drug.Metallomics. 2018; 10: 1564-1569https://doi.org/10.1039/C8MT00202ACrossref Google Scholar). Unexpectedly, native mass spectrometry indicates that ATO-treated DBD bound arsenic and zinc simultaneously. Only the zinc ion was lost in denaturing conditions, suggesting that As binds DBD covalently. The X-ray crystal structures of arsenic complexed to two DBD mutants (G245S and R249S) reveal that the arsenic binding pocket (ABP) is composed of the sulfurs of three non-zinc coordinating Cys residues (Cys124, Cys135, Cys141) and that of Met133 (Figure 1A). The ABP was verified by mutating each of the three Cys residues to Ala and observing loss of ATO's ability to induce binding by an antibody (PAb1620) that recognizes the native conformation of DBD. Unlike the zinc binding site, which is solvent accessible, the ABP is obscured by the side chain of Cys124. The ABP buttresses the DNA binding groove and is 15.4 Å from the zinc ion (Figure 1B), making it well situated to function as potential allosteric regulatory site. One of ATO's most striking properties is that it refolds mutant p53 more efficiently than any of the nine other reported p53-reactivating molecules that were tested in this study. Conformation-specific antibody is one of the primary means to assess p53 (mis)folding in cells. Like most p53 mutants, p53-R175H is overexpressed in cancer cells and is recognized by PAb240 antibody (specific to misfolded/unfolded p53) but not by PAb1620 . In p53-null H1299 cells transfected with p53-R175H, ATO increased the PAb1620 epitope by 74-fold compared to 2.6-fold for the zinc metallochaperone ZMC1 and 1.9-fold for the Michael-addition compound PRIMA-1, the next most effective agents. ATO treatment caused corresponding loss of the PAb240 epitope. Refolding of p53 mutants is likely due to enhanced thermodynamic stability: ATO increased the apparent melting temperature of DBD-R175H by up to 6.5°C (20 μM ATO). How well does ATO perform against other p53 mutants, in cells and in vivo? Chen et al., 2020Chen S. Wu J.-L. Liang Y. Tang Y.-G. Song H.-X. Wu L.-L. Xing Y.-F. Yan N. Li Y.-T. Wang Z.-Y. et al.Arsenic Trioxide Rescues Structural p53 Mutations through a Cryptic Allosteric Site.Cancer Cell. 2020; (S1535-6108(20)30605-X. Published online December 24, 2020)https://doi.org/10.1016/j.ccell.2020.11.013Abstract Full Text Full Text PDF Scopus (25) Google Scholar examined a panel of 25 common mutants and found that ATO induced most of them to refold as judged by PAb1620 binding. The p53 transcriptional function rescue results vary among mutants. As expected, ATO treatment of cultured cells did not trigger transcription of p53 target genes (PUMA or CDKN1A) by DNA contact mutants, which serves as the negative control p53 group. Some of the other mutants for which ATO increased PAb1620 binding, unfortunately, also showed a similar negative response to ATO treatment in cell-based luciferase/PUMA transcriptional assays. The authors speculate that As-mediated stabilization of DBD's global fold plays an essential role in mutant p53 reactivation but maintaining the local structure of the DNA binding groove is also important. For example, arsenic binding refolds R249S and G245S but only modestly enhances their transcriptional activities, which may be explained by the subtle alterations they observed in the L3 loop that contains both DNA- and zinc-binding residues (Figure 1B). Nevertheless, ATO treatment rescued a significant number of mutants to varying degrees in cells, and mice bearing cell line-derived xenografts (expressing p53-R175H under an inducible promoter) responded robustly to ATO, with tumor sizes held to 10%–20% that of the untreated group after 48 days. The ABP triad of Cys124, Cys135, and Cys141 is highly conserved among vertebrate p53s. Curiously, homologs such as p63 and p73 and some invertebrate p53 orthologs contain Trp and Leu at the equivalent of positions 124 and 135, and these substitutions may stabilize the proteins by filling the ABP with bulky hydrophobic side chains. Indeed, when Chen et al., 2020Chen S. Wu J.-L. Liang Y. Tang Y.-G. Song H.-X. Wu L.-L. Xing Y.-F. Yan N. Li Y.-T. Wang Z.-Y. et al.Arsenic Trioxide Rescues Structural p53 Mutations through a Cryptic Allosteric Site.Cancer Cell. 2020; (S1535-6108(20)30605-X. Published online December 24, 2020)https://doi.org/10.1016/j.ccell.2020.11.013Abstract Full Text Full Text PDF Scopus (25) Google Scholar individually replaced Cys124, Cys135, and Cys141 with Val, they observed an increase in PAb1620 epitope in the absence of ATO. Why this energetically unfavorable arrangement of Cys residues has persisted through evolution is unclear, but it may play a role in maintaining p53's innate instability (Blanden et al., 2020Blanden A.R. Yu X. Blayney A.J. Demas C. Ha J.-H. Liu Y. Withers T. Carpizo D.R. Loh S.N. Zinc shapes the folding landscape of p53 and establishes a pathway for reactivating structurally diverse cancer mutants.eLife. 2020; 9: e61487https://doi.org/10.7554/eLife.61487Crossref Scopus (6) Google Scholar). Arsenic has a high affinity for Cys sulfhydryl groups, especially in clusters of two or three, and arsenic inactivates up to 200 transcription factors and enzymes (Shen et al., 2013Shen S. Li X.-F. Cullen W.R. Weinfeld M. Le X.C. Arsenic binding to proteins.Chem. Rev. 2013; 113: 7769-7792https://doi.org/10.1021/cr300015cCrossref PubMed Scopus (417) Google Scholar). Protein inactivation is the source of arsenic toxicity although it can be lifesaving in the case of PML-RARα. Chen et al., 2020Chen S. Wu J.-L. Liang Y. Tang Y.-G. Song H.-X. Wu L.-L. Xing Y.-F. Yan N. Li Y.-T. Wang Z.-Y. et al.Arsenic Trioxide Rescues Structural p53 Mutations through a Cryptic Allosteric Site.Cancer Cell. 2020; (S1535-6108(20)30605-X. Published online December 24, 2020)https://doi.org/10.1016/j.ccell.2020.11.013Abstract Full Text Full Text PDF Scopus (25) Google Scholar demonstrate that arsenic can also restore protein function, which is exactly what is needed in the fight against mutant p53-driven cancers. Whether ABPs exist in other proteins, and whether arsenic binding can stabilize their structures while preserving their functions, remain tantalizing questions. This work was supported by NIH grant R01 CA200800 . The author declares no competing interests. Chromaffin Cells with Sympathoblast Signature: Too Similar to Keep Apart?Bedoya-Reina et al.Cancer CellDecember 31, 2020In BriefDong et al. (2020) built an atlas of developing neural crest cells and their derivatives in humans and used it to trace the origins of neuroblastoma. We compared these datasets with the developing murine adrenal anlagen and found that clusters designated as human chromaffin cells resemble the developing sympathoblast in mouse. 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