Reactivation Of Mutant P53 Research Articles

Zinc Metallochaperones as Mutant p53 Reactivators: A New Paradigm in Cancer Therapeutics.

Restoration of wild-type structure and function to mutant p53 with a small molecule (hereafter referred to as “reactivating” mutant p53) is one of the holy grails in cancer therapeutics. The majority of TP53 mutations are missense which generate a defective protein that is targetable. We are currently developing a new class of mutant p53 reactivators called zinc metallochaperones (ZMCs) and, here, we review our current understanding of them. The p53 protein requires the binding of a single zinc ion, coordinated by four amino acids in the DNA binding domain, for proper structure and function. Loss of the wild-type structure by impairing zinc binding is a common mechanism of inactivating p53. ZMCs reactivate mutant p53 using a novel two-part mechanism that involves restoring the wild-type structure by reestablishing zinc binding and activating p53 through post-translational modifications induced by cellular reactive oxygen species (ROS). The former causes a wild-type conformation change, the later induces a p53-mediated apoptotic program to kill the cancer cell. ZMCs are small molecule metal ion chelators that bind zinc and other divalent metal ions strong enough to remove zinc from serum albumin, but weak enough to donate it to mutant p53. Recently we have extended our understanding of the mechanism of ZMCs to the role of cells’ response to this zinc surge. We found that cellular zinc homeostatic mechanisms, which normally function to maintain free intracellular zinc levels in the picomolar range, are induced by ZMCs. By normalizing zinc levels, they function as an OFF switch to ZMCs because zinc levels are no longer sufficiently high to maintain a wild-type structure. This on/off switch leads to a transient nature to the mechanism of ZMCs in which mutant p53 activity comes on in a few hours and then is turned off. This finding has important implications for the translation of ZMCs to the clinic because it indicates that ZMC concentrations need not be maintained at high levels for their activity. Indeed, we found that short exposures (as little as 15 min) were adequate to observe the mutant p53 reactivating activity. This switch mechanism imparts an advantage over other targeted therapeutics in that efficacy can be accomplished with minimal exposure which minimizes toxicity and maximizes the therapeutic window. This on/off switch mechanism is unique in targeted cancer therapeutics and will impact the design of human clinical trials.

Aminobenzothiazole derivatives stabilize the thermolabile p53 cancer mutant Y220C and show anticancer activity in p53-Y220C cell lines

Many cancers have the tumor suppressor p53 inactivated by mutation, making reactivation of mutant p53 with small molecules a promising strategy for the development of novel anticancer therapeutics. The oncogenic p53 mutation Y220C, which accounts for approximately 100,000 cancer cases per year, creates an extended surface crevice in the DNA-binding domain, which destabilizes p53 and causes denaturation and aggregation. Here, we describe the structure-guided design of a novel class of small-molecule Y220C stabilizers and the challenging synthetic routes developed in the process. The synthesized chemical probe MB710, an aminobenzothiazole derivative, binds tightly to the Y220C pocket and stabilizes p53-Y220C in vitro. MB725, an ethylamide analogue of MB710, induced selective viability reduction in several p53-Y220C cancer cell lines while being well tolerated in control cell lines. Reduction of viability correlated with increased and selective transcription of p53 target genes such as BTG2, p21, PUMA, FAS, TNF, and TNFRSF10B, which promote apoptosis and cell cycle arrest, suggesting compound-mediated transcriptional activation of the Y220C mutant. Our data provide a framework for the development of a class of potent, non-toxic compounds for reactivating the Y220C mutant in anticancer therapy.

Synergistic Antitumor Effect of BKM120 with Prima-1Met Via Inhibiting PI3K/AKT/mTOR and CPSF4/hTERT Signaling and Reactivating Mutant P53

Background/Aims: PI3KCA and mutant p53 are associated with tumorigenesis and the development of cancers. NVP-BKM120, a selective pan-PI3K inhibitor, exerts the antitumor activity by suppressing the PI3K signaling pathway. Prima-1^Met, a low molecular weight compound, can rescue the gain-of-function of mutant p53 by restoring its transcriptional function. In this study, we investigated whether PI3K inhibition combined with mutant p53 reactivation could enhance the antitumor effect in thyroid cancer cells. Methods: The effects of BKM120 and Prima-1^Met on the proliferation, apoptosis, migration and invasion of thyroid cancer cells were measured by MTT, colony formation, flow cytometry, wound-healing and transwell assays, respectively. Thyroid differentiation was assessed by detecting the expression levels of specific markers using RT-PCR and Western blot. The in vivo antitumor efficacy was analyzed in a mouse xenograft model. Results: The combinational treatment of BKM120 and Prima-1^Met significantly enhanced the inhibitions of cell viability, colony formation, migration and invasion, and the induction of apoptosis in thyroid cell lines, and synergistically suppressed tumor xenograft growth by inhibiting the PI3K/Akt/mTOR and EMT signaling pathways, up-regulating p53 targeted genes, and triggering the release of cytochrome c. Moreover, the combination of BKM120 and Prima-1^Met suppressed the stemlike traits of thyroid cancer cells and promoted their differentiation by upregulating the expression of thyroid-specific differentiation markers and repressing the expression of cancer stem cell markers. Furthermore, the mechanism study demonstrated that the combinational treatment synergistically abrogated the binding of CPSF4 at the promoter of hTERT and thus suppressed hTERT expression. Consistently, overexpression of hTERT rescued the inhibitions of cell viability, invasion and stem-like traits mediated by the combination of BKM120 and Prima-1^Met. Conclusion: Our results showed that the combination of BKM120 with Prima-1^Met synergistically suppressed the growth of thyroid cancer cells and tumor xenografts via inhibiting PI3K/Akt/mTOR and CPSF4/hTERT signaling and reactivating mutant p53.

Abstract B211: Cellular zinc homeostatic mechanisms function as an off switch for zinc metallochaperone-mediated reactivation of mutant p53

Abstract The p53 transcription factor functions as one of cancer’s most potent tumor suppressors and is the most frequently mutated gene in human cancer. The majority of p53 mutations (>70%) are missense that generate a defective protein found at high levels in cells that is targetable. Restoration of wild type structure and function of mutant p53 with a small molecule (so-called reactivation) is a highly sought after goal in anticancer drug development. The p53 protein requires the binding of a single zinc ion to fold properly, and mutations that impair the protein’s ability to bind zinc (and cause it to misfold) are highly prevalent in cancer. We recently discovered a new class of small-molecule zinc chelators named zinc metallochaperones (ZMCs) that reactivate zinc-deficient mutant p53 through a novel mechanism involving both zinc ionophore activity to raise intracellular zinc concentrations and donation to restore zinc binding to mutant p53. This induces a wild type conformation change and a p53-mediated apoptotic program. The lead compound (ZMC1) displays a transient pharmacodynamics (p21 levels) in vitro. We hypothesized that the regulation of these pharmacodynamics is governed by cellular zinc homeostatic mechanisms that function to restore zinc to its physiologic picomolar levels. We examined the entire suite of zinc homeostatic genes in response to ZMC1 and manipulated several metallothionein genes by knockout and knockdown. The net effect of this was to increase the peak and duration of intracellular zinc levels that lead to a more potent and sustained duration of p21 expression. This translated to increased sensitivity to ZMC1. We further postulated that this pharmacodynamics would allow the drug to function with very minimal exposure, and colony formation studies in vitro indicated that a 2-hour exposure was as effective as a 72-hour exposure. We then sought to translate this mechanism in vivo using a genetically engineered murine model of KPC pancreatic cancer (Pdx-1Cre; KrasG12D) that expresses either the p53R172H (zinc deficient) allele or p53R270H (non-zinc deficient). Pharmacokinetic (PK) studies of the drug revealed a short half-life (15 minutes) indicating a minimal exposure. Despite this, daily, intermittent dosing at the maximum tolerated dose resulted in a statistically significant increase in the overall survival of the KPC-p53R172H mice while having no such effect in the KPC-p53R270H. We sought to improve the efficacy of ZMC1 by preloading it with zinc in a 2:1 molar ratio based on the crystal structure. The drug-zinc complex (Zn-1) increased the median survival of KPCp53-R172H mice from 26 days to 35 days (ZMC1 monomer versus Zn-1). These studies indicate that cellular zinc homeostatic mechanisms function as an “off” switch for ZMCs, which has important implications for the translation of ZMCs in humans. Principally, this allows the drug to function with minimal exposure, which minimizes potential zinc toxicity. ZMC1 as monotherapy improves survival in an allele-specific mutant p53 manner. Furthermore, ZMC1 can be optimized by synthesizing it complexed with zinc. Overall, this “off” switch is novel for a targeted molecular therapeutic and represents a significant departure from the traditional paradigm where the goal is to develop a compound that binds the target with a PK profile that provides maximal exposure. Citation Format: Xin Yu, Samuel Kogan, Ying Chen, Ashley T. Tsang, Tracy Withers, Hongxia Lin, Chunxia Chen, Dirk F. Moore, Joseph Bertino, Chang Chan, Darren R. Carpizo. Cellular zinc homeostatic mechanisms function as an off switch for zinc metallochaperone-mediated reactivation of mutant p53 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B211.

Targeting mutant p53 for efficient cancer therapy.

The tumour suppressor gene TP53 is the most frequently mutated gene in cancer. Wild-type p53 can suppress tumour development by multiple pathways. However, mutation of TP53 and the resultant inactivation of p53 allow evasion of tumour cell death and rapid tumour progression. The high frequency of TP53 mutation in tumours has prompted efforts to restore normal function of mutant p53 and thereby trigger tumour cell death and tumour elimination. Small molecules that can reactivate missense-mutant p53 protein have been identified by different strategies, and two compounds are being tested in clinical trials. Novel approaches for targeting TP53 nonsense mutations are also underway. This Review discusses recent progress in pharmacological reactivation of mutant p53 and highlights problems and promises with these strategies.

Abstract 2570: Induction of mild oxidative stress as a strategy for reactivation of mutant p53 proteins: KSS72, a small molecule derived from ethacrynic acid restores the biological functions of R248W/Q mutant

Abstract p53 gene mutations in 50% of human cancers drive the emergence of oncogenic genomes and confer resistance to anticancer drugs & apoptosis. Therefore, a pharmacological reactivation of p53 mutant proteins to their wild-type forms has emerged as an intense research area. We have shown that induction of oxidative protein modifications primarily underlie the mechanistic conversion of mutant to wt or wt-like p53 forms (Eur J Medchem 107:233, 2016; Int J Oncol 48:1426, 2016). R248W/Q DNA contact mutations in the p53 core are highly frequent and cause functional inactivation. No small molecules reactivating this p53 mutation are available. Here, we describe the design and synthesis of a small molecule KSS72 using a cell-based screening and its potency in reactivating the R248 p53 mutants. KSS72 is a non-diuretic hydrophobic analog [1-(2,3-dichloro-4-methoxyphenyl)-2-methylenebutan-1-one] of ethacrynic acid (Edecrin) obtained by removing the carboxylate side chain. KSS-72 retained the ability to bind with GSH, inhibit GSTs and induce marked redox-imbalance in tumor cells. KSS72 displayed preferential cytotoxicity (3-4 fold more cell killing) against cancer cells expressing the allele specific R248W mutation (MiaPaca-2) in comparison with the wt p53 or p53-null cancer cell lines. Further, we developed isogenic cell lines expressing the R248W and R248Q mutants in the p53-null H1299 lung cancer cells and evaluated their sensitivity towards KSS72; these models again verified the greater drug efficacy. To prove the hypothesis the higher efficacy of KSS72 stems from the reactivation of mutant p53, the conformation-specific antibodies (Pab1620 for wt and Pab420 for mutant) were employed. Immunoprecipitation and immunostaining using confocal microscopy showed that KSS72 treatment of R248W or R248Q p53-cells results in a marked reciprocal loss of mutant protein and gradual increase of wt-like protein. EMSA revealed a time-dependent restoration of DNA-binding of the mutant p53, which was accompanied by a distinct upregulation of pro-apoptotic signaling pathways mediated by PUMA, Bax, p21cip1 and MDM2 proteins. A strong G2/M arrest and significant apoptosis were observed. Other data showed that KSS72 restores the wt structure and function by covalently binding the p53R248 protein. Pharmacokinetics showed that KSS72 has excellent oral bioavailability with a half-life of 3.5 h. In MiaPaca-2 xenografts developed in nude mice, there was a marked tumor growth suppression with KSS72 as a single drug. In conclusion, KSS72 is an attractive lead compound for further development because p53 reactivation occurred without discernible organ toxicities after oral doses and a wide therapeutic window. The redox-sensitive cysteines involved in the p53 reactivation are being identified by mass spectrometry (supported by CPRIT grants RP130266 & RP170207 to KSS). Citation Format: Surendra R. Punganuru, Hanumantha Rao Madala, Kalkunte S. Srivenugopal. Induction of mild oxidative stress as a strategy for reactivation of mutant p53 proteins: KSS72, a small molecule derived from ethacrynic acid restores the biological functions of R248W/Q mutant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2570. doi:10.1158/1538-7445.AM2017-2570

Mutant p53 targeting by the low molecular weight compound STIMA-1.

Reactivation of mutant p53 in human tumor cells should induce cell death by apoptosis and thus eliminate the tumor. Several small molecules that reactivate mutant p53 have been identified. Here we show that STIMA-1, a low molecular weight compound with some structural similarities to the previously identified molecule CP-31398, can stimulate mutant p53 DNA binding in vitro and induce expression of p53 target proteins and trigger apoptosis in mutant p53-expressing human tumor cells. Human diploid fibroblasts are significantly more resistant to STIMA-1 than mutant or wild type p53-carrying tumor cells. STIMA-1 may provide new insights into possible mechanisms of mutant p53 reactivation and thus facilitate the development of novel anticancer drugs that target mutant p53-carrying tumors.

Inhibiting the system xC\u2212/glutathione axis selectively targets cancers with mutant-p53 accumulation

TP53, a critical tumour suppressor gene, is mutated in over half of all cancers resulting in mutant-p53 protein accumulation and poor patient survival. Therapeutic strategies to target mutant-p53 cancers are urgently needed. We show that accumulated mutant-p53 protein suppresses the expression of SLC7A11, a component of the cystine/glutamate antiporter, system xC−, through binding to the master antioxidant transcription factor NRF2. This diminishes glutathione synthesis, rendering mutant-p53 tumours susceptible to oxidative damage. System xC− inhibitors specifically exploit this vulnerability to preferentially kill cancer cells with stabilized mutant-p53 protein. Moreover, we demonstrate that SLC7A11 expression is a novel and robust predictive biomarker for APR-246, a first-in-class mutant-p53 reactivator that also binds and depletes glutathione in tumours, triggering lipid peroxidative cell death. Importantly, system xC− antagonism strongly synergizes with APR-246 to induce apoptosis in mutant-p53 tumours. We propose a new paradigm for targeting cancers that accumulate mutant-p53 protein by inhibiting the SLC7A11–glutathione axis.

Thiosemicarbazones Functioning as Zinc Metallochaperones to Reactivate Mutant p53.

Small-molecule restoration of wild-type structure and function to mutant p53 (so-called mutant reactivation) is a highly sought-after goal in cancer drug development. We previously discovered that small-molecule zinc chelators called zinc metallochaperones (ZMCs) reactivate mutant p53 by restoring zinc binding to zinc-deficient p53 mutants. The lead compound identified from the NCI-60 human tumor cell lines screen, NSC319726 (ZMC1), belongs to the thiosemicarbazone (TSC) class of metal ion chelators that bind iron, copper, magnesium, zinc, and other transition metals. Here, we have investigated the other TSCs, NSC319725 and NSC328784, identified in the same screen, as well as the more well studied TSC, 3-AP (Triapine), to determine whether they function as ZMCs. We measured the zinc Kd zinc ionophore activity, ability to restore zinc to purified p53 DNA binding domain (DBD), and ability to restore site-specific DNA binding to purified R175H-DBD in vitro. We tested all four TSCs in a number of cell-based assays to examine mutant p53 reactivation and the generation of reactive oxygen species (ROS). We found that NSC319725 and NSC328784 behave similarly to ZMC1 in both biophysical and cell-based assays and are heretofore named ZMC2 (NSC319725) and ZMC3 (NSC328784). 3-AP generates a ROS signal similar to ZMC1-3, but it fails to function as a ZMC both in vitro and in cells and ultimately does not reactivate p53. These findings indicate that not all TSCs function as ZMCs, and much of their activity can be predicted by their affinity for zinc.

Full-length p53 tetramer bound to DNA and its quaternary dynamics.

P53 is a major tumor suppressor that is mutated and inactivated in ~50% of all human cancers. Thus, reactivation of mutant p53 using small molecules has been a long sought-after anticancer therapeutic strategy. Full structural characterization of the full-length oligomeric p53 is challenging because of its complex architecture and multiple highly flexible regions. To explore p53 structural dynamics, here we developed a series of atomistic integrative models with available crystal structures of the full-length p53 (fl-p53) tetramer bound to three different DNA sequences: a p21 response element, a puma response element and a nonspecific DNA sequence. Explicitly solvated, all-atom molecular dynamics simulations of the three complexes (totaling nearly 1 μs of aggregate simulation time) yield final structures consistent with electron microscopy maps and, for the first time, show the direct interactions of the p53 C-terminal with DNA. Through a collective principal component analysis, we identify sequence-dependent differential quaternary binding modes of the p53 tetramer interfacing with DNA. Additionally, L1 loop dynamics of fl-p53 in the presence of DNA is revealed, and druggable pockets of p53 are identified via solvent mapping to aid future drug discovery studies.

Reactivation of mutant p53 by capsaicin, the major constituent of peppers.

BackgroundMutations in the p53 oncosuppressor gene are highly frequent in human cancers. These alterations are mainly point mutations in the DNA binding domain of p53 and disable p53 from transactivating target genes devoted to anticancer activity. Mutant p53 proteins are usually more stable than wild-type p53 and may not only impair wild-type p53 activity but also acquire pro-oncogenic functions. Therefore, targeting mutant p53 to clear the hyperstable proteins or change p53 conformation to reactivate wild-type p53 protein functions is a powerful anticancer strategy. Several small molecules have been tested for p53 reactivation in mutant p53-carrying cells while studies exploiting the effect of natural compounds are limited. Capsaicin (CPS) is the major constituent of peppers and show antitumor activity by targeting several molecular pathway, however, its effect on mutant p53 reactivation has not been assessed yet. In this study we aimed at investigating whether mutant p53 could be a new target of capsaicin-induced cell death and the underlying mechanisms.Methodsp53 levels were analysed by western blot upon capsaicin treatment in the presence of the autophagy inhibitor chloroquine. The mutant p53 reactivation was evaluated by chromatin-immunoprecipitation (ChIP) assay and semi-quantitative RT-PCR analyses of wild-type p53 target genes. The specific wild-type p53 activation was determined by using the inhibitor of p53 transactivation function, pifithrin-α and siRNA for p53.ResultsHere, we show that capsaicin induced autophagy that was, at least in part, responsible of mutant p53 protein degradation. Abrogation of mutant p53 by capsaicin restored wild-type p53 activities over mutant p53 functions, contributing to cancer cell death. Similar effects were confirmed in cancer cells bearing tumor-associated p53 mutations and in H1299 (p53 null) with overexpressed p53R175H and p53R273H mutant proteins.ConclusionThese findings demonstrate for the first time that capsaicin may reduce mutant p53 levels and reactivate wild-type p53 protein in mutant p53-carrying cells and the p53 reactivation contributes to capsaicin-induced cell death.

Reactivation of mutant p53: Constraints on mechanism highlighted by principal component analysis of the DNA binding domain.

Most p53 mutations associated with cancer are located in its DNA binding domain (DBD). Many structures (X-ray and NMR) of this domain are available in the protein data bank (PDB) and a vast conformational heterogeneity characterizes the various free and complexed states. The major difference between the apo and the holo-complexed states appears to lie in the L1 loop. In particular, the conformations of this loop appear to depend intimately on the sequence of DNA to which it binds. This conclusion builds upon recent observations that implicate the tetramerization and the C-terminal domains (respectively TD and Cter) in DNA binding specificity. Detailed PCA analysis of the most recent collection of DBD structures from the PDB have been carried out. In contrast to recommendations that small molecules/drugs stabilize the flexible L1 loop to rescue mutant p53, our study highlights a need to retain the flexibility of the p53 DNA binding surface (DBS). It is the adaptability of this region that enables p53 to engage in the diverse interactions responsible for its functionality. Proteins 2016; 84:1443-1461. © 2016 Wiley Periodicals, Inc.

Abstract 3833: Restoration of wildtype structure and function of mutant p53 by thiosemicarbozones using a novel zinc metallochaperone based mechanism

Abstract NSC319726 (ZMC1) is a small molecule that reactivates mutant p53 by restoration of WT structure and function to the most common p53 missense mutant (p53-R175H). We identified that ZMC1 functions as a zinc-metallochaperone, providing an optimal concentration of zinc to facilitate proper folding of p53 protein, and increasing cellular reactive oxygen species to transactivate the newly conformed p53-R175H (via post-translational modifications). ZMC1 was identified from an in silico screen of the NCI anti-cancer drug screen along with two other thiosemicarbazones (TSCs), NSC319725 and NSC328784. We investigated these TSCs to determine if they could reactivate mutant p53 using a zinc metallochaperone mechanism. We found that indeed these compounds could reactivate mutant p53 by functioning as zinc metallochaperones. In distinction, Triapine the only TSC in clinical development, does not function as a zinc metallochaperone and is not a mutant p53 reactivator. Citation Format: Xin Yu, Adam R. Blanden, Ashley T. Tsang, Saif Zaman, John Gelleran, David Augeri, S. David Kimball, Stewart N. Loh, Darren R. Carpizo. Restoration of wildtype structure and function of mutant p53 by thiosemicarbozones using a novel zinc metallochaperone based mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3833.

Abstract 2097: Translating a mutant p53 reactivating drug (ZMC1) in murine pancreatic cancer models

Abstract Pancreatic cancer therapy suffers from a lack of effective chemotherapy. TP53 is second only to KRAS as the most commonly mutated gene in pancreatic cancer with point mutations occurring in 75% of patients. We identified ZMC1 as an allele specific mutant p53 reactivator and lead compound for mutant p53 targeted drug development. ZMC1 restores wildtype structure and function by functioning as a zinc metallochaperone to restore zinc binding to mutant p53 proteins with impaired zinc binding. Our aim was to translate this novel mechanism in vivo using murine pancreatic cancer models. We investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of ZMC1 and performed efficacy studies for allele specificity in nude mice with subcutaneous tumors from murine pancreatic cancer cell lines derived from a genetically engineered mouse model (KPC) expressing mutant KrasG12D and different alleles of TP53 (WT, null, p53R172H, p53R270H). Tumor growth inhibition became apparent only in KPCp53-R172H xenografts but not in KPCp53-R270H. We then performed efficacy studies in the autochthonous KPC model and found that, ZMC1 extended the median survival from 15 to 26 days in the KPCp53-R172H mice (p = 0.05) but not in the KPCp53-R270H mice. We sought to improve the efficacy of ZMC1 by synthesizing it complexed with zinc (Zn-1) in a 2:1 molar ratio. Zn-1 significanlty increased the median survival of KPCp53-R172H mice from 15 to 35 days (p = 0.0042). The apoptosis rate in the tumors (by Immunohistochemistry staining with Cleaved Caspase 3) was also increased by treatment of ZMC1 and Zn-1. These studies indicate that ZMC1 can function in vivo as a mutant p53 targeted anti-cancer drug at doses that are well tolerated. Furthermore, ZMC1 can be optimized by synthesizing it complexed with zinc. Citation Format: Xin Yu, Ashley T. Tsang, Tracy Withers, John Gelleran, David Augeri, S. David Kimball, Darren R. Carpizo. Translating a mutant p53 reactivating drug (ZMC1) in murine pancreatic cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2097.

Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth.

Mutations in the p53 tumor-suppressor gene are prevalent in human cancers. The majority of p53 mutations are missense, which can be classified into contact mutations (that directly disrupts the DNA-binding activity of p53) and structural mutations (that disrupts the conformation of p53). Both of the mutations can disable the normal wild-type (WT) p53 activities. Nevertheless, it has been amply documented that small molecules can rescue activity from mutant p53 by restoring WT tumor-suppressive functions. These compounds hold promise for cancer therapy and have now entered clinical trials. In this study, we show that cruciferous-vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutant under in vitro and in vivo conditions, revealing a new mechanism of action for a dietary-related compound. PEITC exhibits growth-inhibitory activity in cells expressing p53 mutants with preferential activity toward p53R175, one of the most frequent ‘hotspot' mutations within the p53 sequence. Mechanistic studies revealed that PEITC induces apoptosis in a p53R175 mutant-dependent manner by restoring p53 WT conformation and transactivation functions. Accordingly, in PEITC-treated cells the reactivated p53R175 mutant induces apoptosis by activating canonical WT p53 targets, inducing a delay in S and G2/M phase, and by phosphorylating ATM/CHK2. Interestingly, the growth-inhibitory effects of PEITC depend on the redox state of the cell. Further, PEITC treatments render the p53R175 mutant sensitive to degradation by the proteasome and autophagy in a concentration-dependent manner. PEITC-induced reactivation of p53R175 and its subsequent sensitivity to the degradation pathways likely contribute to its anticancer activities. We further show that dietary supplementation of PEITC is able to reactivate WT activity in vivo as well, inhibiting tumor growth in xenograft mouse model. These findings provide the first example of mutant p53 reactivation by a dietary compound and have important implications for cancer prevention and therapy.

Targeting of Mutant p53 and the Cellular Redox Balance by APR-246 as a Strategy for Efficient Cancer Therapy.

TP53 is the most frequently mutated gene in cancer. The p53 protein activates transcription of genes that promote cell cycle arrest or apoptosis, or regulate cell metabolism, and other processes. Missense mutations in TP53 abolish specific DNA binding of p53 and allow evasion of apoptosis and accelerated tumor progression. Mutant p53 often accumulates at high levels in tumor cells. Pharmacological reactivation of mutant p53 has emerged as a promising strategy for improved cancer therapy. Small molecules that restore wild type activity of mutant p53 have been identified using various approaches. One of these molecules, APR-246, is a prodrug that is converted to the Michael acceptor methylene quinuclidinone (MQ) that binds covalently to cysteines in p53, leading to refolding and restoration of wild type p53 function. MQ also targets the cellular redox balance by inhibiting thioredoxin reductase (TrxR1) and depleting glutathione. This dual mechanism of action may account for the striking synergy between APR-246 and platinum compounds. APR-246 is the only mutant p53-targeting compound in clinical development. A phase I/IIa clinical trial in hematological malignancies and prostate cancer showed good safety profile and clinical effects in some patients. APR-246 is currently tested in a phase Ib/II trial in patients with high-grade serous ovarian cancer.

Abstract PR12: Mutant p53 drives early events in fallopian tube tumorigenesis through mesenchyme-associated autocrine production of matrix that supports survival and mesothelial intercalation.

Abstract High-grade serous ovarian cancer (HGS-OvCa) harbor p53 mutations and predominantly originates from the secretory epithelial cell compartment of the fallopian tube fimbriae. From this site, tumor cells detach, survive in the peritoneal cavity, and form cellular clusters that clear the mesothelium and expand to form peritoneal masses. To examine the contribution of mutant p53 to any of these phenotypic alterations associated with HGS-OvCA tumor progression, we developed live-cell microscopy assays that recapitulate these phenotypes and examined dynamic processes regulated by depletion of p53 or gain-of-function (GOF) mutations in p53 in cultured secretory fallopian tube epithelial cells. Expression of GOF mutant variants of p53, but not depletion of endogenous wild-type p53, promoted survival and cell-cell aggregation under conditions of cell detachment, leading to the formation of spheroids with mesothelium-clearing capacity. These phenotypes were blocked by mutant p53 reactivation and depended on the expression of the mesenchyme transcription factor TWIST1, inhibition of p63 and de novo formation of a unique cell-matrix adhesion niche supported by autocrine fibronectin production and activation of integrins in detached cells. Inhibition of fibronectin or integrin α5 in suspension blocked cell-to-cell adhesion leading to HGS-OvCa spheroid disintegration and ultimately anoikis. These results indicate the detached FTE cells-expressing GOF mutants of p53 undergo a mesenchymal transition under conditions of matrix detachment leading to autocrine production of matrix and anchorage-independent survival and proliferation, as well as mesothelial clearance activity. These findings provide important new insights into activities of mutant p53 that may play a critical role in early events in tumorigenesis of high-grade ovarian serous tumors and provide a rationale for the development of therapeutics that target ovarian cancer cells transiting through the peritoneal cavity. This abstract is also presented as Poster B03. Citation Format: Marcin P. Iwanicki, Hsing-Yu Chen, Ioannis Zervantonakis, Marian Novak, Taru Muranen, Tan A. Ince, Ronny Drapkin, Joan S. Brugge. Mutant p53 drives early events in fallopian tube tumorigenesis through mesenchyme-associated autocrine production of matrix that supports survival and mesothelial intercalation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR12.

Reactivation of wild-type and mutant p53 by tryptophanolderived oxazoloisoindolinone SLMP53-1, a novel anticancer small-molecule.

Restoration of the p53 pathway, namely by reactivation of mutant (mut) p53, represents a valuable anticancer strategy. Herein, we report the identification of the enantiopure tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a novel reactivator of wild-type (wt) and mut p53, using a yeast-based screening strategy. SLMP53-1 has a p53-dependent anti-proliferative activity in human wt and mut p53R280K-expressing tumor cells. Additionally, SLMP53-1 enhances p53 transcriptional activity and restores wt-like DNA binding ability to mut p53R280K. In wt/mut p53-expressing tumor cells, SLMP53-1 triggers p53 transcription-dependent and mitochondrial apoptotic pathways involving BAX, and wt/mut p53 mitochondrial translocation. SLMP53-1 inhibits the migration of wt/mut p53-expressing tumor cells, and it shows promising p53-dependent synergistic effects with conventional chemotherapeutics. In xenograft mice models, SLMP53-1 inhibits the growth of wt/mut p53-expressing tumors, but not of p53-null tumors, without apparent toxicity. Collectively, besides the potential use of SLMP53-1 as anticancer drug, the tryptophanol-derived oxazoloisoindolinone scaffold represents a promissing starting point for the development of effective p53-reactivating drugs.

Amiloride induces potent antitumoral activity in multiple myeloma through the reactivation of mutant p53

PO-302 Amiloride induces potent antitumoral activity in multiple myeloma through the reactivation of mutant p53 E. Rojas, I. Misiewicz-Krzeminska, S.H. Garcia, T. Paino, L.A. Corchete, I. Isidro, M.V. Mateos, E. Ocio, N.C. Gutierrez Hematology Departament, University Hospital, IBSAL IBMCC (USAL-CSIC) Funding: “Instituto de Salud Carlos III” (PI13/00111), “Gerencia Regional de Salud” (BIO/SA57/13), “Asociacion Espanola contra el cancer” (AECC: GCB120981SAN). Background: The antihypertensive agent, amiloride, has been demonstrated to inhibit tumor cell proliferation in different experimental models. Here, we investigate the antimyeloma effect of amiloride in myeloma cell lines and primary samples, and the potential mechanisms involved. Methods: Seven human myeloma cell lines (NCI-H929, JJN3, KMS12-BM, KMS12-PE, U266, MM1S and RPMI-8226) and 6 bone marrow (BM) samples from MM patients were used for the experiments. Cell viability, apoptosis and cell cycle analyses were carried. The measuring of mitochondrial membrane potential (m) was evaluated by flow cytometry analyses. The gene expression was quantified by Taqman assay qRT-PCR. Synergy with other antimyeloma agents was calculated with the “CalcuSyn” software program. Results: To test the antiproliferative effect of amiloride, myeloma cells were treated with increasing concentrations of the compound (0.1mM-1mM) for 24, 48 and 72 hours. Amiloride displayed potent antimyeloma activity in the panel of 7 cell lines. A substantial induction of apoptotic cell death (greater than 40% at 24 h and 60% at 48 h) and cell cycle blockade was observed in all MM cell lines. The apoptosis induced by amiloride was not related to the TP53 mutational status, since amiloride was still able to induce cell death in p53 mutated cell lines, such as U-266, KMS12-BM and JJN3. Moreover, this drug caused a notable decrease of m. Mechanistic studies showed that apoptosis triggered by amiloride was associated with the overexpression of BAX, BAK1, BBC3, TNFRSF10B, FAS, CDKN1B and CDKN1A, even in those MM cell lines in which p53 gene was mutated. The cell death was reduced in MM cell lines under the treatments with p53 inhibitors (pifithrin, PFT and PFT), even in p53 mutated cell lines. These results suggest the reactivation of mutant p53 in amiloride-induced apoptosis in myeloma cells. The double combinations of amiloride with bortezomib, dexamethasone and melphalan were synergistic in NCI-H929, JJN3, RMPI and MM1S cell lines. In vitro studies confirmed the antimyeloma efficacy of amiloride in patient samples and revealed significant lower cytotoxicity in the remaining cell subpopulations compared to tumor plasma cells. Conclusions: Our results demonstrate a potent and selective antimyeloma effect of amiloride independent of p53 status. The present data support the investigation of amiloride as a treatment option for MM patients, either alone or in combination.

Cdc37 suppressing in multiple myeloma blocks proliferation but induces bortezomib resistance

PO-302 Amiloride induces potent antitumoral activity in multiple myeloma through the reactivation of mutant p53 E. Rojas, I. Misiewicz-Krzeminska, S.H. Garcia, T. Paino, L.A. Corchete, I. Isidro, M.V. Mateos, E. Ocio, N.C. Gutierrez Hematology Departament, University Hospital, IBSAL IBMCC (USAL-CSIC) Funding: “Instituto de Salud Carlos III” (PI13/00111), “Gerencia Regional de Salud” (BIO/SA57/13), “Asociacion Espanola contra el cancer” (AECC: GCB120981SAN). Background: The antihypertensive agent, amiloride, has been demonstrated to inhibit tumor cell proliferation in different experimental models. Here, we investigate the antimyeloma effect of amiloride in myeloma cell lines and primary samples, and the potential mechanisms involved. Methods: Seven human myeloma cell lines (NCI-H929, JJN3, KMS12-BM, KMS12-PE, U266, MM1S and RPMI-8226) and 6 bone marrow (BM) samples from MM patients were used for the experiments. Cell viability, apoptosis and cell cycle analyses were carried. The measuring of mitochondrial membrane potential (m) was evaluated by flow cytometry analyses. The gene expression was quantified by Taqman assay qRT-PCR. Synergy with other antimyeloma agents was calculated with the “CalcuSyn” software program. Results: To test the antiproliferative effect of amiloride, myeloma cells were treated with increasing concentrations of the compound (0.1mM-1mM) for 24, 48 and 72 hours. Amiloride displayed potent antimyeloma activity in the panel of 7 cell lines. A substantial induction of apoptotic cell death (greater than 40% at 24 h and 60% at 48 h) and cell cycle blockade was observed in all MM cell lines. The apoptosis induced by amiloride was not related to the TP53 mutational status, since amiloride was still able to induce cell death in p53 mutated cell lines, such as U-266, KMS12-BM and JJN3. Moreover, this drug caused a notable decrease of m. Mechanistic studies showed that apoptosis triggered by amiloride was associated with the overexpression of BAX, BAK1, BBC3, TNFRSF10B, FAS, CDKN1B and CDKN1A, even in those MM cell lines in which p53 gene was mutated. The cell death was reduced in MM cell lines under the treatments with p53 inhibitors (pifithrin, PFT and PFT), even in p53 mutated cell lines. These results suggest the reactivation of mutant p53 in amiloride-induced apoptosis in myeloma cells. The double combinations of amiloride with bortezomib, dexamethasone and melphalan were synergistic in NCI-H929, JJN3, RMPI and MM1S cell lines. In vitro studies confirmed the antimyeloma efficacy of amiloride in patient samples and revealed significant lower cytotoxicity in the remaining cell subpopulations compared to tumor plasma cells. Conclusions: Our results demonstrate a potent and selective antimyeloma effect of amiloride independent of p53 status. The present data support the investigation of amiloride as a treatment option for MM patients, either alone or in combination.