Reactivation of Mutant p53 and Induction of Apoptosis in Human Tumor Cells by Maleimide Analogs

Reactivation of mutant p53 is likely to provide important benefits for treatment of chemotherapy- and radiotherapy-resistant tumors. We demonstrate here that the maleimide-derived molecule MIRA-1 can reactivate DNA binding and preserve the active conformation of mutant p53 protein in vitro and restore transcriptional transactivation to mutant p53 in living cells. MIRA-1 induced mutant p53-dependent cell death in different human tumor cells carrying tetracycline-regulated mutant p53. The structural analog MIRA-3 showed antitumor activity in vivo against human mutant p53-carrying tumor xenografts in SCID mice. The MIRA scaffold is a novel lead for the development of anticancer drugs specifically targeting mutant p53. Reactivation of mutant p53 is likely to provide important benefits for treatment of chemotherapy- and radiotherapy-resistant tumors. We demonstrate here that the maleimide-derived molecule MIRA-1 can reactivate DNA binding and preserve the active conformation of mutant p53 protein in vitro and restore transcriptional transactivation to mutant p53 in living cells. MIRA-1 induced mutant p53-dependent cell death in different human tumor cells carrying tetracycline-regulated mutant p53. The structural analog MIRA-3 showed antitumor activity in vivo against human mutant p53-carrying tumor xenografts in SCID mice. The MIRA scaffold is a novel lead for the development of anticancer drugs specifically targeting mutant p53. The p53 tumor suppressor responds to various types of cellular stress, e.g. DNA damage, hypoxia, and oncogenic signaling, and triggers cell cycle arrest, apoptosis, or senescence (1Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2279) Google Scholar). This is achieved through transcriptional transactivation of specific target genes carrying p53 DNA binding motifs (1Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2279) Google Scholar, 2Sherr C.J. Harvey Lect. 2000; 96: 73-92PubMed Google Scholar). p53 is frequently mutated in tumors, indicating a strong selection for inactivation of the p53 tumor suppressor pathway during tumor development (3Hollstein M. Sidransky D. Vogelstein B. Harris C.C. Science. 1991; 253: 49-53Crossref PubMed Scopus (7400) Google Scholar). Most p53 mutations are missense point mutations clustering in the core domain (residues 94–292) that harbors the p53-specific DNA binding activity (4Beroud C. Soussi T. Nucleic Acids Res. 1998; 26: 200-204Crossref PubMed Scopus (166) Google Scholar). Amino acid substitutions in the core domain result in loss of specific DNA binding, either because of substitution of residues that contact DNA (so-called DNA contact mutants) or substitution of residues that are critical for the structural integrity of the DNA binding interface of the core (so-called structural mutants) (5Bullock A.N. Fersht A.R. Nat. Rev. Cancer. 2001; 1: 68-76Crossref PubMed Scopus (477) Google Scholar). As a result, mutant p53 proteins fail to activate transcription of p53 target genes and, hence, do not trigger a p53-dependent biological response. Inability to activate expression of the target genes appears to have consequences for mutant p53 expression levels in tumor cells. Several negative regulators of p53, including the ubiquitin ligase MDM2, which targets p53 for proteasome-mediated degradation, and the more recently discovered Pirh2 (6Leng R.P. Lin Y. Ma W. Wu H. Lemmers B. Chung S. Parant J.M. Lozano G. Hakem R. Benchimol S. Cell. 2003; 112: 779-791Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar) and COP1 (7Dornan D. Wertz I. Shimizu H. Arnott D. Frantz G.D. Dowd P. O'Rourke K. Koeppen H. Dixit V.M. Nature. 2004; 429: 86-92Crossref PubMed Scopus (583) Google Scholar) are themselves p53 target genes. Thus, loss of p53-dependent transcription will cause impaired p53 degradation. This may at least partially explain the fact that mutant p53 is expressed at high levels in many tumors (8Bartek J. Bartkova J. Vojtesek B. Staskova Z. Lukas J. Rejthar A. Kovarik J. Midgley C.A. Gannon J.V. Lane D.P. Oncogene. 1991; 6: 1699-1703PubMed Google Scholar). Radiotherapy and chemotherapy, which are currently used, are thought to eliminate tumors through induction of p53-dependent apoptosis (9Lowe S.W. Bodis S. McClatchey A. Remington L. Ruley H.E. Fisher D.E. Housman D.E. Jacks T. Science. 1994; 266: 807-810Crossref PubMed Scopus (1537) Google Scholar). Consistent with this notion, tumors harboring p53 mutations are generally more resistant to chemotherapy than wild-type p53-carrying tumors (10Greenblatt M.S. Bennett W.P. Hollstein M. Harris C.C. Cancer Res. 1994; 54: 4855-4878PubMed Google Scholar, 11Campling B.G. el-Deiry W.S. Methods Mol. Med. 2003; 75: 53-77PubMed Google Scholar, 12Geisler S. Borresen-Dale A.L. Johnsen H. Aas T. Geisler J. Akslen L.A. Anker G. Lonning P.E. Clin. Cancer Res. 2003; 9: 5582-5588PubMed Google Scholar, 13Gadducci A. Cosio S. Muraca S. Genazzani A.R. Eur. J. Gynaecol. Oncol. 2002; 23: 390-396PubMed Google Scholar). Accordingly, novel and more efficient strategies for treatment of mutant p53-carrying tumors should greatly improve clinical outcome for many tumor types. Several studies have demonstrated ways to restore normal function to mutant p53 (14Bykov V.J. Selivanova G. Wiman K.G. Eur. J. Cancer. 2003; 39: 1828-1834Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). A synthetic peptide derived from the p53 C terminus was shown to induce mutant p53-dependent cell death (15Selivanova G. Iotsova V. Okan I. Fritsche M. Strom M. Groner B. Grafstrom R.C. Wiman K.G. Nat. Med. 1997; 3: 632-638Crossref PubMed Scopus (311) Google Scholar). Introduction of second site suppressor mutations in p53 stabilized folding of the mutant protein and restored specific DNA binding (16Nikolova P.V. Wong K.B. DeDecker B. Henckel J. Fersht A.R. EMBO J. 2000; 19: 370-378Crossref PubMed Scopus (138) Google Scholar). More recently, attempts have been made to identify small molecules that reactivate mutant p53. Rastinejad and co-workers (17Foster B.A. Coffey H.A. Morin M.J. Rastinejad F. Science. 1999; 286: 2507-2510Crossref PubMed Scopus (679) Google Scholar) screened a chemical library using a protein assay and identified molecules (CP31398 and CP257042) that can preserve wild-type conformation of p53, rescue its transactivation capacity and restore its antitumor activity in vivo. We have previously screened a chemical library from the National Cancer Institute and identified PRIMA-1, a compound that induces mutant p53-dependent apoptosis and restores native conformation, DNA binding, and transcriptional transactivation to mutant p53, and inhibits tumor growth in vivo (18Bykov V.J. Issaeva N. Shilov A. Hultcrantz M. Pugacheva E. Chumakov P. Bergman J. Wiman K.G. Selivanova G. Nat. Med. 2002; 8: 282-288Crossref PubMed Scopus (848) Google Scholar). Despite the successful identification of the mutant p53-reactivating molecules described above, further screening for additional mutant p53-reactivating molecules remains a highly important task. Compounds identified so far may not be useful in the clinic for various reasons, for example unfavorable pharmacokinetics or toxicity profiles. Development of novel mutant p53-targeting drugs with different modes of action should increase the likelihood of achieving clinical success. Here we describe a novel class of low molecular weight compounds that are structurally different from PRIMA-1 but show similar capacity to restore wild-type conformation and function to mutant p53, and induce tumor cell death in a mutant p53-dependent manner. Cells, Kits, and Antibodies—The human Saos-2 osteosarcoma and H1299 lung adenocarcinoma cell lines are p53-null. The sublines Saos-2-His273 and H1299-His175 carry the indicated tetracycline-regulated mutant p53 expression constructs (Tet-off). Human HCT116 colon carcinoma cells carry wild-type p53 (p53+/+), and the isogenic HCT116 (p53-/-) cells are p53-null. SW480 colon carcinoma cells express the endogenous His273/Ser309 mutant p53. CaspaTag TM Pan fluorescein caspase (VAD) activity kit was from Intergen (Oxford, UK), carboxyfluorescein (FAM)-labeled caspase inhibitor FAM-DEVD-FMK 1The abbreviations used are: FMK, fluoromethylketone; Z, benzyloxycarbonyl; GST, glutathione S-transferase; FACS, fluorescence-activated cell sorter; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; EGFP, enhanced green fluorescent protein; MIRA, mutant p53-dependent induction of rapid apoptosis; SCID, severe combined immunodeficiency. was obtained from Immunohistochemistry Technologies (Bloomington, MN), polyclonal rabbit anti-p53 and anti-Bax antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), and fluorescein isothiocyanate-conjugated anti-rabbit Ig was from Vector Laboratories (Burlingame, CA). Plasmids—The plasmids encoding the GST-human wild-type p53 fusion protein and the GST-human mutant p53 protein His175 were described earlier (36Selivanova G. Iotsova V. Kiseleva E. Strom M. Bakalkin G. Grafstrom R.C. Wiman K.G. Nucleic Acids Res. 1996; 24: 3560-3567Crossref PubMed Scopus (65) Google Scholar). Chemical Library—A library of low molecular weight compounds was obtained from the NCI, National Institutes of Health, Bethesda, MD. For more information, see web site dtp.nci.nih.gov. Chemical Library Screening and Growth Suppression Assays— Saos-2 cells carrying tetracycline-regulated His273 mutant p53 were used for screening of the Diversity Set from NCI, National Institutes of Health (15Selivanova G. Iotsova V. Okan I. Fritsche M. Strom M. Groner B. Grafstrom R.C. Wiman K.G. Nat. Med. 1997; 3: 632-638Crossref PubMed Scopus (311) Google Scholar). Mutant p53 expression was shut off by incubation of cells with doxycycline for 1 week (5 μg/ml). Cells were grown in 96-well plates at a density of 3,000 cells per well with or without doxycycline and treated with 25 μm of the compounds from the NCI, National Institutes of Health Diversity Set of low molecular weight compounds. After 48 h of incubation, the reagent WST-1 (Roche Applied Science) was added. The degree of WST-1 reduction, which reflects cell viability, was measured in a microplate reader at λ490 nm according to the manufacturer's instructions (Roche Applied Science). were and treated with compounds. After 48 h of incubation, cells were by with treated with A and with were a were by the were treated with MIRA-1 p53-dependent induction of rapid for and in plates at cells per were with and DNA proteins were described (15Selivanova G. Iotsova V. Okan I. Fritsche M. Strom M. Groner B. Grafstrom R.C. Wiman K.G. Nat. Med. 1997; 3: 632-638Crossref PubMed Scopus (311) Google Scholar). were in binding 1 and 1 in G. Iotsova V. Kiseleva E. Strom M. Bakalkin G. Grafstrom R.C. Wiman K.G. Nucleic Acids Res. 1996; 24: 3560-3567Crossref PubMed Scopus (65) Google of and were at for or The was with or without compounds. for was described by B.A. Coffey H.A. Morin M.J. Rastinejad F. Science. 1999; 286: 2507-2510Crossref PubMed Scopus (679) Google the treatment were with 25 with The was to plates and at for The were with and by in at for 1 were with by of antibodies or in were at for were with After a with was with at for plates were with PBS, and a was added. at nm was by the in vivo studies were by the in and was in with For treatment with SCID were was with H1299-His175 cells (5 in were to for to the of tumors. of for of or 1 of MIRA-3 for or Mutant p53-dependent Growth Suppression by previously screened the Diversity of low molecular weight compounds from NCI, National Institutes of Health using Saos-2 osteosarcoma cells carrying tetracycline-regulated His273 mutant p53 and the WST-1 cell This to the identification of PRIMA-1, a that inhibits growth of human tumor cells in a mutant p53-dependent in vitro and in vivo (18Bykov V.J. Issaeva N. Shilov A. Hultcrantz M. Pugacheva E. Chumakov P. Bergman J. Wiman K.G. Selivanova G. Nat. Med. 2002; 8: 282-288Crossref PubMed Scopus (848) Google Scholar). We identified low molecular weight compound in this This compound growth of Saos-2-His273 cells mutant p53 but not growth of the cells in the of doxycycline that off mutant p53 expression was Several structural of MIRA-1 from the were for mutant p53-dependent growth of showed mutant p53-dependent activity similar to that of The of and are shown in Most including and not have the growth of Saos-2-His273 cells at to 25 the active a in the of the MIRA which is in the we MIRA-1 induce mutant p53-dependent growth in human carcinoma or H1299 lung adenocarcinoma cells carrying His175 or His273 mutant p53 of the The are in and in I. cell lines mutant p53 of the we mutant p53-dependent growth similar to that in Saos-2-His273 cells. The the mutant and the cells was The MIRA-1 and MIRA-3 showed similar activity MIRA-1 of normal human treatment with 25 μm MIRA-1 was inhibits cell growth in a mutant p53-dependent with 25 of doxycycline in a The of MIRA-1 to growth in a mutant p53-dependent was further using a Saos-2 or Saos-2-His273 cells were treated with different of MIRA-1 and in The cells were with and for the of with μm MIRA-1 the of by Saos-2 cells of but was efficient in Saos-2 cells of MIRA the to Mutant MIRA-1 was to induce mutant p53-dependent apoptosis, we and DNA of Saos-2 and Saos-2-His273 cells treated with μm MIRA-1 for 48 MIRA-1 a increase in the of cells with a DNA in the of mutant p53, indicating DNA and cell of the of cells 48 h of treatment of Saos-2 and Saos-2-His273 cells with We MIRA-3 treatment caspase activity using the CaspaTag As shown in MIRA-3 induced caspase in the mutant Saos-2 cells but not in the indicating induction of mutant p53-dependent that MIRA-1 restore the function to mutant p53, we the of caspase growth Saos-2-His273 cells were treated with MIRA-1 in the or of the caspase and of cell death was by and and expressed of cells with DNA cell death induced by MIRA-1 by and cell death induced by MIRA-1 by not Thus, we that MIRA-1 and its active induce mutant p53-dependent cell death by the of MIRA-1 its through transcriptional transactivation and protein we the of growth of cells with to of MIRA-1 in a increase in cell not the of cells carrying the p53 which transactivation domain because of additional mutations at and was at least than that of cells MIRA-1 treatment not the that transcriptional transactivation by p53 is critical for cell of p53 by the molecular of of mutant p53, we the of the compound p53 A of the acid substitutions in p53 that in tumors the native conformation of the core in loss of the for the and of by the Y. S. Science. 1994; PubMed Scopus Google Scholar). of the wild-type p53 a similar we MIRA-1 the native conformation of p53 We that 25 μm MIRA-1 the in wild-type and mutant p53 proteins for at MIRA-1 partially of p53 protein shown by of at The by was not by incubation with MIRA-1 not We MIRA was of the of p53 protein with the during incubation at of p53 protein with 25 μm MIRA-1 for h at in increase of the and MIRA-3 induced the MIRA that were in WST-1 cellular assay were to induce the Thus, MIRA of wild-type and mutant p53 at but restore native conformation to wild-type p53 at of DNA of Mutant p53 specific DNA binding is critical for p53 tumor suppressor we MIRA this activity using a of p53 at for DNA binding in the of incubation at this in the of MIRA-1 DNA binding of p53 in a We MIRA the specific DNA binding activity of mutant p53 proteins in from human tumor cells. MIRA-1 enhanced DNA binding of the mutant p53 in from cells and His175 mutant p53 in from cells or antibodies were to the to further of a of mutant p53 proteins using of human tumor cell lines carrying different p53 mutations that MIRA-1 DNA binding of of mutant p53 proteins the DNA binding of mutant DNA DNA of DNA of DNA of DNA of DNA of DNA DNA of DNA binding in a of the of Mutant p53 in that MIRA-1 can the specific DNA binding of mutant p53 in we MIRA-1 restore transcriptional transactivation to mutant p53 in living cells. We treated cells with a with that treatment with μm MIRA-1 for h induced expression in cells. induction of and in cells treated with μm MIRA-1 for h p53 levels treatment with MIRA-1 As further of the of MIRA-1 to rescue transcriptional transactivation of mutant p53, we MIRA was to induce p53 target genes in a mutant p53-dependent manner. of H1299-His175 cells mutant p53 with MIRA-1 in a induction of and treatment of the cells in the of mutant p53 expression and treatment of H1299 cells not cause induction of MDM2, MIRA-1 induced and in SW480 colon carcinoma cells carrying endogenous His273/Ser309 mutant p53 but not in HCT116 colon carcinoma cells that carry wild-type p53. We not of wild-type p53 in cells treatment with We the of MIRA-3 treatment the p53 target in Saos-2-His273 cells. As shown in MIRA-3 induced in Saos-2-His273 cells in a manner. expression was in the Saos-2 cells. provide for of p53-dependent transcriptional transactivation by of in the in the in of the activity of many compounds in the Diversity Set a of human tumor cell the of of tumor cells to a compound with mutant p53 expression and a compound growth of tumor cells mutant p53 or wild-type p53. of the activity of MIRA-1 the of human tumor cell lines a for MIRA-1 and mutant p53 levels the of cell lines carrying mutant p53 according to V.J. Issaeva N. Selivanova G. Wiman K.G. 2002; 23: PubMed Scopus Google Scholar). we not tumor cell lines and mutant p53. of MIRA-3 in the antitumor of the MIRA of compounds in we SCID with H1299-His175 cells and treated the with of MIRA-3 tumors treated with tumors with of with MIRA-3 at a of 1 in tumor of with MIRA-3 at this not result in weight loss or during of treatment with and and weight loss that of the Thus, MIRA-3 antitumor in vivo but at The high of p53 mutations in a of human tumors and the mutant p53 and to and chemotherapy in many clinical studies the to novel efficient strategies for targeting mutant p53 in tumors. We previously screened the Diversity Set of low molecular weight compounds from the NCI, National Institutes of Health and identified low molecular weight compounds that showed of mutant tumor cells. of PRIMA-1, been described (18Bykov V.J. Issaeva N. Shilov A. Hultcrantz M. Pugacheva E. Chumakov P. Bergman J. Wiman K.G. Selivanova G. Nat. Med. 2002; 8: 282-288Crossref PubMed Scopus (848) Google Scholar). Here we MIRA-1 and its active a structurally of compounds that targets mutant p53. MIRA-1 induces cell death in a mutant p53-dependent with a that is than that of The for MIRA-1 in Saos-2 His273 cells was μm the for PRIMA-1 is μm the (18Bykov V.J. Issaeva N. Shilov A. Hultcrantz M. Pugacheva E. Chumakov P. Bergman J. Wiman K.G. Selivanova G. Nat. Med. 2002; 8: 282-288Crossref PubMed Scopus (848) Google Scholar). The of MIRA-1 for mutant human tumor cells carrying mutant p53 is similar to that of We a of at least for the mutant the cells and Thus, MIRA-1 mutant p53-dependent cell PRIMA-1, cell death DNA and caspase indicating cell death by This is with indicating that MIRA the p53 is to that MIRA-1 and PRIMA-1 with to the of cell death MIRA-1 induces cell death h not PRIMA-1 cells h (18Bykov V.J. Issaeva N. Shilov A. Hultcrantz M. Pugacheva E. Chumakov P. Bergman J. Wiman K.G. Selivanova G. Nat. Med. 2002; 8: 282-288Crossref PubMed Scopus (848) Google Scholar). This may a different molecular of action or different of cellular degradation. We have shown that MIRA-1 mutant p53 by MIRA-1 was to preserve native conformation of wild-type and mutant p53 demonstrated by with the antibodies and treatment with MIRA-1 DNA binding of wild-type p53 and enhanced the DNA binding of mutant of p53, and DNA contact MIRA-1 treatment of a p53-dependent in mutant cells and MIRA-1 its analog MIRA-3 induced the p53 target genes and in a mutant p53-dependent manner. a mutant of p53 carrying substitution at residues and in the transactivation domain and the His175 substitution in the core domain was resistant to by This that MIRA-1 induces apoptosis of p53-dependent transcriptional that the of MIRA-1 that protein is for that MIRA-1 and of its structural by the the native and conformation of p53 the native conformation, to of transactivation of target genes and induction of p53-dependent DNA binding demonstrated that MIRA-1 DNA binding of but not mutant of p53. MIRA-1 DNA binding of the mutant in cells but not in and the His273 mutant in cells was not by MIRA-1 in the DNA binding assay but MIRA-1 to Saos-2 or cells in WST-1 are with the of cellular in the of MIRA-1 to reactivate mutant p53. MIRA compounds tumor growth in vivo we treated SCID carrying human tumor xenografts with This MIRA-1 analog was in tumor cell growth and apoptosis by WST-1 assay and We a antitumor activity of MIRA-3 in vivo. the compound was at the used, indicating a of toxicity is a in further lead of MIRA of compounds. MIRA-1 is structurally from the previously described mutant p53-targeting compounds PRIMA-1 (18Bykov V.J. Issaeva N. Shilov A. Hultcrantz M. Pugacheva E. Chumakov P. Bergman J. Wiman K.G. Selivanova G. Nat. Med. 2002; 8: 282-288Crossref PubMed Scopus (848) Google Scholar) and and (17Foster B.A. Coffey H.A. Morin M.J. Rastinejad F. Science. 1999; 286: 2507-2510Crossref PubMed Scopus (679) Google and a novel class of mutant p53-reactivating we have shown that MIRA-1 restores native conformation and transcriptional transactivation to mutant p53 and induce tumor cell apoptosis in a mutant p53-dependent its molecular of action remains to be The MIRA compounds a which may with and in proteins of structural of MIRA-1 demonstrated that a of compounds were to reactivate mutant p53. We that the of the in the is critical for the mutant p53-dependent activity this were in cell and to preserve native conformation of wild-type or mutant p53 proteins The of the is important for the of compounds to with and This the that of residues in p53 by active MIRA compounds a in the native of the of or is to have p53 conformation and The p53 core domain conformation is for specific DNA binding residues Y. S. Science. 1994; PubMed Scopus Google Scholar) that are targets for studies have indicated that p53 activity is to in cells S. H. H. 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For induces of the cellular to the of cells by 2004; 6: PubMed Scopus Google Scholar, A. T. A. S. I. M. S. K. J. Oncol. 2000; Google Scholar). have been shown to have anticancer activity in vitro and in vivo Chung 1997; PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar). we that MIRA-1 and its active are of mutant p53 and trigger mutant p53-dependent The MIRA compounds described here for the development of novel anticancer that rescue wild-type conformation and function of mutant p53. of compounds should at and and further molecular of The targeting of mutant p53 by small molecules MIRA will more efficient and clinical in the We for HCT116 Institute of for and and the of Cancer and NCI, National Institutes of Health for the library of low molecular weight compounds. Reactivation of mutant p53 and induction of apoptosis in human tumor cells by of PDF