Drugs Targeting p53 Mutations with FDA Approval and in Clinical Trials

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Simple Summary​

Mutations in the tumor suppressor p53 (p53) occur in ~50% of human cancers, the majority of which are missense mutations. Mutations in p53 not only impair the tumor suppressive function, but also confer missense mutant p53 (mutp53) with oncogenic activities independent of wild-type p53 (wtp53). Since p53 mutations are cancer-specific, several approaches targeting them have been taken to develop novel cancer therapies, including restoration or stabilization of wtp53 conformation from mutp53, rescue of p53 nonsense mutations, depletion of mutp53 proteins, and induction of p53 synthetic lethality or targeting of vulnerabilities imposed by p53 deficiencies (activated retrotransposons) or mutations (enhanced YAP/TAZ). Here, we summarize clinically available investigational and FDA-approved drugs that target p53 mutations for their mechanisms of action and activities to suppress cancer progression.

 

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Abstract​

Mutations in the tumor suppressor p53 (p53) promote cancer progression. This is mainly due to loss of function (LOS) as a tumor suppressor, dominant-negative (DN) activities of missense mutant p53 (mutp53) over wild-type p53 (wtp53), and wtp53-independent oncogenic activities of missense mutp53 by interacting with other tumor suppressors or oncogenes (gain of function: GOF). Since p53 mutations occur in ~50% of human cancers and rarely occur in normal tissues, p53 mutations are cancer-specific and ideal therapeutic targets. Approaches to target p53 mutations include (1) restoration or stabilization of wtp53 conformation from missense mutp53, (2) rescue of p53 nonsense mutations, (3) depletion or degradation of mutp53 proteins, and (4) induction of p53 synthetic lethality or targeting of vulnerabilities imposed by p53 mutations (enhanced YAP/TAZ activities) or deletions (hyperactivated retrotransposons). This review article focuses on clinically available FDA-approved drugs and drugs in clinical trials that target p53 mutations and summarizes their mechanisms of action and activities to suppress cancer progression.

 

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1. Introduction​

The tumor suppressor p53 was initially thought to be an oncogene since the originally cloned cDNA contained a missense mutation [1]. Indeed, mutant p53 (mutp53) functions as an oncogene independent of wild-type p53 (wtp53) [2,3]. Later, however, in studies using p53 cDNA without any mutations, the wtp53 was proven to be a bona fide tumor suppressor [4,5].

p53 is a transcription factor and regulates the transcription of numerous downstream target genes involved in apoptosis, cell cycle arrest, senescence, DNA repair, and cellular metabolism, thereby functioning as a tumor suppressor [6]. The protein level and activity of wtp53 remain low in non-stressed conditions mainly through degradation by MDM2 [7]. Under genotoxic conditions, wtp53 is stabilized and activated through post-translational modifications (PTMs) by phosphorylation or acetylation to induce cell cycle arrest and/or cell death (Figure 1). Once the wtp53 function is impaired due to mutations or deletions, cells lose control of their growth, which promotes tumorigenesis [2,3].

 

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Accumulating evidence indicates that p53 is the most frequently mutated gene in human cancers, with mutations in over 50% of human cancers [8]. The majority of p53 mutations are missense mutations in the DNA-binding domain. Mutations in p53 result in the loss of function (LOS) as a transcription factor and a tumor suppressor. However, missense mutp53 frequently accumulates in tumors to promote malignant progression, metastasis, and drug resistance in a manner independent of wtp53. These oncogenic mutp53 activities are referred to as gain of function (GOF) (Figure 1). The mechanism of mutp53 GOF is mainly caused by mutp53’s ability to bind to tumor suppressors (e.g., p63, p73, MRN complex) and oncogenes (e.g., ETS2, SREBP2, NF-Y) to alter the functions of these binding partners [2,3,9]. Clinically, the presence of mutp53 in tumors is well correlated with advanced clinical stages, metastases, and poor outcomes in patients with multiple types of cancer [10,11]. Given that mutations in p53 are generally observed specifically in tumors and are rare in non-tumor tissues, mutp53 is an ideal therapeutic target for cancer therapy.

Several strategies to target p53 mutations have been taken (Figure 1). The first strategy is to directly target missense mutp53 to restore the activity of wtp53 or stabilize the wtp53 conformation. Drugs or compounds that have this function are referred to as reactivators. The most representative drug in this group is APR-246 (eprenetapopt/PRIMA-1MET), which is in several phase 2 or 3 clinical trials. Although the tumor suppressive effects of APR-246 in mutp53-carrying tumors in mouse models have been shown to be successful, it is not yet approved by the Food and Drug Administration (FDA) [12]. The second strategy is to induce degradation or depletion of missense mutp53, which capitalizes upon the addiction of cancer cells to mutp53 and potentially restores the activities of some tumor suppressors, including p63 and p73, whose functions have been suppressed by mutp53. Drugs or compounds employed for this strategy include HSP90 inhibitors or statins, cholesterol-lowering drugs that are shown to induce degradation of mutp53, leading to tumor suppression [13,14]. The third strategy is to induce cell death specifically in cancer cells with p53 deletions or mutations, so called p53 synthetic lethality [15]. This strategy often targets vulnerabilities imposed by p53 deficiency or mutp53 GOF, instead of directly targeting mutp53. Drugs or compounds used for this strategy include Wee1 inhibitors or inhibitors of DNA damage response signaling (e.g., ATR or Chk1/2 inhibitors) [16,17,18]. Other strategies include wtp53 rescue of non-sense mutations in p53 [19,20], inhibition of retrotransposon activated upon p53 deficiency [19], and inhibition of YAP/TAZ function activated by mutp53 [21,22]. Here, we have summarized updated information about drugs developed for the aforementioned strategies that are approved by the FDA or are in clinical trials, including their mechanisms of action and activities to suppress cancer progression (Table 1).

 

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Drugs or compounds employed for this strategy include HSP90 inhibitors or statins, cholesterol-lowering drugs that are shown to induce degradation of mutp53, leading to tumor suppression

Wonderful finding!

 

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2. Drugs Targeting p53 Mutations​

2.1. Restoration or Stabilization of wtp53 Conformation from Missense mutp53​

Most mutp53 lose the activity of wtp53 as a transcription factor and a tumor suppressor. However, accumulated evidence indicates that the tumor suppressive activity can be restored under specific conditions including temperature shift, exposure to synthetic peptides from 53BP2-derived “CDB3” and p53 C-terminal domain “Peptide 46”, and insertion of second-site mutations or an N-terminal deletion [36,37,38]. Many investigators have attempted to discover small molecule compounds that restore the wtp53 conformation, transcriptional activity, and tumor suppressive activity [2]. For example, CP-31398 is one of the earliest mutp53-reactivating compounds that can stabilize active confirmation of p53 and promote p53-mediated tumor suppression [39,40]. Additionally, JC744, an aminobenzothiazole analog, was recently identified as a new compound that could specifically bind to and stabilize the p53Y220C mutant in vitro in the nanomolar range, through a narrow surface pocket induced by p53Y220C [41]. However, only a few compounds are in clinical trials. These include APR-246 (eprenetapopt/PRIMA-1MET), phenethyl isothiocyanate (PEITC), and arsenic trioxide (ATO/Trisenox).

2.1.1. APR-246 (Eprenetapopt, PRIMA-1MET)​

PRIMA-1 was identified as a small molecule compound that suppressed the growth of Saos-2 osteosarcoma cell line expressing p53R273H [12]. PRIMA-1 was shown to restore the p53’s sequence-specific DNA-binding and growth-suppressing activities in multiple cancer cell lines with different p53 mutants, including R110L, V157F, R175H, L194F, R213Q/Y234H, G245V, R248Q, R273C, R273H/P309S, R280K, and R282W. Thus, PRIMA-1 and its methylated analog PRIMA-1MET(also known as eprenetapopt or APR-246) rescue the p53’s transcriptional activity from both DNA contact and structural p53 mutants [12,42]. In mouse models, APR-246 successfully inhibits tumor progression of multiple cancer cell lines, including osteosarcoma Saos2 exogenously expressing p53R273H, small cell lung cancer GLC16 (p53R273L) and DMS53 (p53S241F), and colon cancer DLD-1 (p53S241F) [12,42,43]. Additionally, Fransson et al. [44] demonstrated synergetic effects of APR-246 with DNA-damaging drugs (cisplatin, carboplatin, doxorubicin) using 10 primary ovarian cancer cells from patients. As a mechanism of action, APR-246 is converted to the biologically active methylene quinuclidinone (MQ) compound that covalently binds to cysteine residues in the core domain of mutp53 to promote refolding and restoration of wtp53’s DNA-binding activity [45]. Specifically, among 10 cysteine residues in p53 [46], two residues at C124 and C277 appear to be crucial for the APR-246-mediated functional restoration of p53R175H [47].

 

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A phase 1b clinical trial (NCT04383938) was performed to examine the safety and efficacy of APR-246 combined with pembrolizumab, an immune checkpoint inhibitor, for patients with advanced/metastatic solid tumors [23]. Due to insufficient sample sizes (37 patients evaluated), a formal assessment of the treatment efficacy was not executed, but the combination was well tolerated and did not cause unmanageable adverse effects. An investigation comparing the therapy response between p53-mutated vs. wild-type tumors was not yet made in this study.

Two clinical trials for p53-mutated myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) with 20–30% marrow blasts (oligoblastic AML) (phase 1b/2, NCT03072043) [24] and for p53-mutated MDS/AML, including cases with more than 30% blasts (phase 2, NCT03588078) [25,48], were performed to test the safety and efficacy of APR-246 in combination with azacitidine. In the NCT03072043 trial, the response rates for MDS and oligoblastic AML were 73% and 64%, respectively [24]. In the NCT03588078 trial, the response rates for MDS and AML were 62% and 33%, respectively [25]. The combination treatment was well tolerated. In both studies, responding patients had significant reductions in the frequency of p53 variant alleles, determined by negativity of next generation sequencing (NGS). Additionally, a phase 2 trial (NCT03931291) was conducted to investigate the efficacy and safety of APR-246 in combination with azacitidine for p53-mutated MDS or AML patients as post-hematopoietic stem-cell transplantation (HCT) maintenance therapy [26]. This treatment was also well tolerated with an acceptable safety profile. Importantly, the 1-year relapse-free survival (RFS) was improved to 60%, as compared with a previous report showing a 1-year RFS of approximately 30% for p53-mutated MDS patients [49]. APR-246 is currently in a phase 3 clinical trial (NCT03745716) to investigate the possible additive effects of azacitidine with APR-246 on inhibiting the progression of p53-mutated MDS. Additional clinical trials testing the efficacy of APR-246 in combination with other drugs on high grade serous ovarian cancer (HGSOC) and myeloid malignancies have also been underway (NCT02098343, NCT03268382, NCT04214860). However, there is currently no clinical trial testing the effects of APR-246 as a single agent.

 

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2.1.2. Phenethyl Isothiocyanate (PEITC)​

PEITC is extracted from cruciferous vegetables (e.g., watercress) and has been suggested to have anti-cancer effects [50]. Epidemiological studies also support the preventive effects of dietary isothiocyanates in different types of human cancers [51,52,53]. Aggarwal et al. [54] demonstrated that PEITC inhibits viable proliferation of cancer cells expressing p53R175H (Sk-Br-3, AU565) and p53R175L (HOP92) more effectively than those with wtp53, p53 null, and DNA contact mutp53 (p53R248W, p53R273H, p53R280K). As a mechanism, PEITC enhances zinc-mediated refolding of p53R175H or restoration of intact p53 structure. Moreover, PEITC induces reactive oxygen species (ROS) by impairing the GSH antioxidant system, which greatly contributes to mutp53 reactivation and inhibition of the growth of cells and tumors [54]. However, it remains unclear how PEITC restores the intact p53 structure of p53R175H or R175L in a manner dependent on zinc and whether PEITC can reactivate other zinc-binding and conformational mutp53.

A phase 2 clinical trial (NCT01790204) has been performed to examine whether PEITC-containing juice from watercress could reduce the number of oral cells having p53 mutations (not specified for p53R175H or R175L); however, the outcome of this study has not yet been reported.

 

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2.1.3. Arsenic Trioxide (ATO/Trisenox)​

Arsenic trioxide (ATO, also known as Trisenox) is an FDA-approved drug to treat acute promyelocytic leukemia (APL) that is characterized by the expression of PML-RARα fusion protein [55,56]. ATO induces degradation of PML-RAR alpha, differentiation of APL cells, mitochondrial oxidative stress and apoptosis, repression of c-fos, and upregulation of p53 [55,56]. Intriguingly, ATO is shown to induce PirH2-mediated ubiquitination and degradation of both DNA contact and structural mutp53 (see Section 2.2.3) [57].

Recently, Chen et al. [58] demonstrated that ATO rescues p53 activity from structural mutp53 through promotion of p53 folding by covalently binding to multiple cysteines in p53. ATO is selected through a series of screens. These include (1) inhibition of the growth of NCI-60 cell lines carrying structural p53 mutations (p53R175H, p53R175L, p53G245S, p53R249S), (2) in silico studies to identify the ability to bind multiple cysteines, and (3) biochemical approaches to select compounds with refolding potential using a p53 conformation-specific antibody (PAb1620). ATO restores the p53’s ability to transactivate p53 downstream targets including CDKN1A, PUMA, and MDM2 in cells expressing p53R175H, p53R249S, p53G245S, and p53R282W, but not in cells expressing p53R248Q and p53R273H. In vivo mouse model studies show that ATO inhibits tumor growth of xenografts (p53R175H) and PDXs (p53R282W). Moreover, of the 25 most frequent p53 mutations covering 40.87% of p53 missense mutations, ATO rescues intact p53 structure from mutp53, except DNA contact mutants (S241F, R248L/Q/W, R273C/H/L) and some structural mutants (V157F, R158H, Y205C, Y220C) that are distant from the ATO-binding site [58]. Currently, several clinical trials to examine the effects of ATO on inhibiting p53-mutated cancers (MDS, AML, refractory solid tumors, recurrent and metastatic ovarian and endometrial cancer) are underway in China (NCT03855371, NCT04869475, NCT04489706, NCT04695223). However, the outcomes of these studies have not yet been reported.