PRIMA-1 and PRIMA-1Met (APR-246): From Mutant/Wild Type p53 Reactivation to Unexpected Mechanisms Underlying Their Potent Anti-Tumor Effect in Combinatorial Therapies

p53 protects cells from genetic assaults by triggering cell-cycle arrest and apoptosis. Inactivation of p53 pathway is found in the vast majority of human cancers often due to somatic missense mutations in TP53 or to an excessive degradation of the protein. Accordingly, reactivation of p53 appears as a quite promising pharmacological approach and, effectively, several attempts have been made in that sense. The most widely investigated compounds for this purpose are PRIMA-1 (p53 reactivation and induction of massive apoptosis )and PRIMA-1Met (APR-246), that are at an advanced stage of development, with several clinical trials in progress. Based on publications referenced in PubMed since 2002, here we review the reported effects of these compounds on cancer cells, with a specific focus on their ability of p53 reactivation, an overview of their unexpected anti-cancer effects, and a presentation of the investigated drug combinations.


Introduction
p53, so called "the guardian of the genome", appears as a key factor in the carcinogenesis. Indeed, somatically, TP53 is the most frequently mutated gene in human cancer overall [1]. The outcome of mutations in the tumor suppressor gene p53 results in the loss of the wild-type p53 (wt-p53) activity and the gain of oncogenic functions such as resistance to apoptosis and escalation in genome instability. Indeed, cell cycle control, senescence, apoptosis, and DNA repair are deregulated due to mutations in p53. In addition, such mutations push cancer cells to acquire new properties, promoting invasion, migration, angiogenesis, proliferation, genomic instability, or drug resistance [2]. Mutant-p53 are, consequently, associated with aggressive tumor phenotypes and poor patient survival.
Finally, it clearly appeared that PRIMA-1/APR-246 are tumor suppressor molecules, inducing apoptosis by the caspases activation in varied mutant-p53 cells. If the proof of its mutant-p53 reactivation property has also been made in several cancer models, the variability of the results incites to look forward other elements likely to influence the effects of PRIMA-1/APR-246, as a cell content dependency [57], or unexpected cytotoxic mechanisms.
Likewise, the implication of SLC7A11, a key component of system xċ that imports cystine for the formation of glutathione, highlights the prominent role of glutathione content in APR-246 anti-tumor First, the impact of PRIMA-1/APR-246 on cell redox balance has been assessed. Indeed, the role of the reactive oxygen species (ROS) in the PRIMA-1/APR-246 anti-tumor activity appeared since 2009 [20], with a global consensus between the authors: an increase of the amount of ROS [20,34,37,52,53,56,59,62] and a decrease of glutathione cellular content [37,40,53,62] have been widely reported under PRIMA-1/APR-246. The importance of ROS production in the PRIMA-1/APR-246-induced cell death was highlighted using a ROS scavenger, N-acetyl cysteine (NAC), which antagonist with PRIMA-1/APR-246 and inhibit its effect on cell proliferation [20,32,37,41,52,53] and apoptosis [20,32,37,52,66]. However, the conclusions from the NAC experiments should be analyzed keeping in mind the possible adduct formation between PRIMA-1 or APR-246 and NAC, decreasing the bioavailability and the efficacy of the two molecules. Glutathione metabolism seemed important in the PRIMA-1/APR-246 anti-tumor effects, as the use of buthionine-sulfoximine (BSO), an irreversible inhibitor of γ-glutamyl cysteine-synthase (γ-GCS), potentiated their anti-tumor activities [20,37,53,66]. These studies indicate that PRIMA synergize with BSO to induce cell death irrespective of p53 status.
Likewise, the implication of SLC7A11, a key component of system xċ that imports cystine for the formation of glutathione, highlights the prominent role of glutathione content in APR-246 anti-tumor effect: SLC7A11 appears upregulated after APR-246 exposure [53], or proposed as a predictive biomarker for APR-246 sensitivity [62].
To understand the mechanisms of PRIMA-1/APR-246 associated to the redox status, several arguments have been proposed, concerning a decrease of the anti-oxydant response, or an increase of the pro-oxydant activity. The decrease of cellular glutathione appeared not caused by a decrease in the expression of the enzymes implicated in its production [37], but rather by the adducts formation between glutathione and MQ [40,62]. A greater sensitivity to oxidative stress was also explained through a downregulation or an inhibition of anti-oxidant enzymes-such as TrxR1 [34], Prx3, or GPx-1 [52]-and a dysregulation of the NFE2L2/HMOX1 axis [53]. On the contrary, the conversion of TrxR1 enzyme into a dedicated NADPH oxidase produced an increase oxidant activity [34]. The relationship between the redox balance and the p53 status remains discussed: several authors demonstrated the independency of the increase of ROS [41,56], or the decrease of glutathione [37], induced by APR-246 toward the p53 status. p53-independent mechanisms have been also proposed to elucidate the prooxidant effect of APR-246, based on the NFE2L2/HMOX1 axis [53] or on the TrxR1 enzyme [34]. On the contrary, Lambert et al. (2009) observed that APR-246 causes increased oxidation in a mutant p53-dependent manner [20]. The implication of mutant p53 proteins in the redox effects of APR-246 has been recently reported by Liu et al. (2017), considering that mutant p53 sensitized tumor cells to APR-246 induced oxidative stress, inhibiting the glutathione synthesis through the inhibition of system xċ [62]. Altogether, the most consensual results across cell lines and cell types (solid and hematologic cancers) are the production of ROS and the huge synergy with inhibitors of glutathione synthesis or cysteine transporter. Thus, PRIMA-1/APR-246" by inducing massive ROS, can trigger a p53-dependent or independent cell death.
A second alternative mechanism of action of PRIMA-1/APR-246 concerned the unfolded protein response (UPR) (Figure 1). Indeed, this phenomenon, activated in response to an accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), also called ER stress, is initially dedicated to restore normal function of the cells by degrading misfolded proteins, and increasing the production of molecular chaperones, but can end up to apoptosis if the repairing mechanisms are overtaken. This notion has been evocated considering the upregulation of the heat shock proteins (Hsp), notably concerning the expression of Hsp 70 [16,21,36,54] and Hsp 90 [12], which increased under PRIMA-1/APR-246 treatment. Moreover, PRIMA-1/APR-246 were linked to the induction of several genes associated with the UPR [53], such as XBP1 [22], GRP78, or CHOP [54]. The importance of UPR in PRIMA-1 anti-tumor impacts has been highlighted by Teoh et al. (2016) [54], knocking-down CHOP, a specific factor mediating the ER stress-induced apoptosis, that led to a significant cytotoxicity decrease in p53-null cells.
PRIMA-1/APR-246 have also been associated to various tissular, cellular, and molecular mechanisms. At the tumor tissue level, PRIMA-1 induced the exposure of tumor epithelial cell anionic phospholipids, reduced the blood vessel density and the blood perfusion in breast cancer xenografts [28]. In two non-malignant pathologies, the ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome, and the squamous metaplasia, APR-246 restored, partially, a normal epithelial organization and differentiation in keratinocytes derived from EEP patients [71,72], or in human conjunctival tissues used as a squamous metaplasia model [73]. In cancer disease, PRIMA-1 and APR-246 may induce cell death through autophagy in breast cancer and sarcoma cells [33,41]. Senescence was also reported in one glioblastoma cell line exposed to APR-246 [60]. In human normal erythrocytes, PRIMA-1 induced eryptosis, a suicidal cell death, independent of any transcription activation [74]. PRIMA-1 also favored a partial differentiation of thyroid cancer cells, restoring the expression of natrium-iodine symporter (NIS) and thyroglobulin [31]. At the molecular level, different potential targets for PRIMA-1 and APR-246 have been screened. Initially, p53 family members have been explored: Rökaeus et al. (2010) [24] demonstrated a mutant-p63-dependency for APR-246 effects on cell proliferation, apoptosis, transcriptional activation in p53-null cells, and less clearly, a mutant-p73-dependency. Of note, the restoration of wild-type p63 functions in mutant-p63 cells were confirmed by studies on the EEC syndrome [71,72]. The role of p73 has been also assessed, using knocking-down models, and revealed that its suppression decreased the pro-apoptotic effects of PRIMA-1/APR-246 [36,43,54], and limited the expression of the PRIMA-1-induced ER stress markers [54]. Anyway, the implication of p73 was not confirmed by Messina et al. (2012), which demonstrated no effect of PRIMA-1 on thyroid cancer cells without p53, but with wild-type p73 [31]. Finally, several other studies used knocking-down experiments to demonstrate some mechanisms that could be involved in the PRIMA-1/APR-246 induced cell death. In particular, they reported that NOXA [36,37], microRNA-34a [25], microRNA-29a [55], MEK (Mitogen-activated protein/extracellular signal-regulated kinase kinase) [56], and c-myc [55] were linked to the anti-tumor effects of PRIMA-1/APR-246.
All these unexpected mechanisms of action of PRIMA-1/APR-246 multiplied the possible uses of these compounds for malignant, and non-malignant, pathologies. This wide action spectrum is still enlarged combining them with other treatments.

PRIMA-1 and APR-246 in Combination with Other Anti-Cancer Therapies
According to the cancer types, PRIMA-1/APR-246 have been associated to chemotherapy, radiotherapy, or targeted therapy.
Basically, the chemotherapy drugs which lead to DNA damages and interfere with DNA synthesis were supposed to trigger p53 activation, and thus, to synergize with PRIMA-1/APR-246 in mutant-p53 cells. Several therapeutic agents have been associated to PRIMA-1/APR-246 in cell cultures, reporting additive or synergic effects. Among the pyrimidine analogs, a synergic anti-tumor effect of the treatment combination has been proven for 5-FU in head and neck [26], lung [8], oesophageal cancer [48], whereas, for gemcitabine, the association showed a synergic effect in ovarian cells [40], but an additive one in pancreatic cells [32], as well as for aracytin, in acute myeloid leukemia (AML) cells, where the time schedule and a pre-exposure to APR-246 seemed important [30]. For the purine analogs, the use of fludarabin with PRIMA-1/APR-246 had additive or synergic effects according to the tested B-CLL (Chronic lymphocytic leukemia) [11] or AML (Acute myeloid leukemia cell lines) [30], without any cross-resistance between PRIMA-1 and fludarabin [13]. Several intercalating agents demonstrated an increased cell toxicity when associated to PRIMA-1/APR-246, as doxorubicin in breast cancer (enhanced effect [12,45]), lung cancer (enhanced effect [8], or synergic effect [17]), thyroid cancer (enhanced effect [31,47]), pancreatic cancer (enhanced effect [67], or synergic effect [32]), myeloma (additive effect [36]), ovarian cancer (synergic effect and restoration of sensitivity in resistant cell line [40]). In this context, epirubicin had also synergic anti-tumor effects in esophageal adenocarcinoma cell lines [48], and daunorubicin in AML primary cultures [30]. Similarly, with topo-isomerase inhibitors, the results of their association with PRIMA-1/APR-246 varied as a synergic effect was obtained in lung, colon and osteosarcoma cell lines with camptothecin [8], but not in pancreatic cancer with irinotecan [32]. Platinum salts were also combined with PRIMA-1/APR-246, in particular cisplatin. A sensitization to cisplatin was observed with in thyroid cancer cells [31,47], a synergic cell toxicity was proven in colon [8], lung [8], head and neck cancer [26], oesophageal [62], pancreatic [32], and ovarian cancers. Besides, it has been reported that PRIMA-1/APR-246 restored sensitivity to cisplatin-resistant cell lines [35,40,51]. Anti-tumoral efficacy of these DNA-targeted associations has also been tested in vivo, for APR-246+5FU in esophageal cancer xenografts [48], for PRIMA-1/APR-246+Cisplatin 5FU in lung cancer [8], esophageal cancer [48], and ovarian cancer [40] xenografts. Independently of DNA damages, taxanes combined with PRIMA-1/APR-246 provided various results. PRIMA-1/APR-246 enhanced the anti-tumor effects of paclitaxel in lung cancer and osteosarcoma cell lines [8], and showed an additive effect with docetaxel in breast cancer cells [61], or a synergic one with taxol in head and neck cancer cells [26], whereas no sensitization was observed in thyroid cancer cells with taxol [31] or in ovarian cancer cells with docetaxel [40]. Among the spindle poisons, a synergic anti-tumor effect was demonstrated combining APR-246 to vinblastine in colon cancer cells [8], and to epirubicin in esophageal cancer cells [48]. Lastly, the associations of APR-246 with dexamethasone enhanced the cytotoxic effect in Waldenström cells [43] and in myeloma cell lines or xenografts [36]. In pre-clinical studies, 3-BrPA, a halogenated pyruvate derivative and an alkylating agent, depleting the cellular ATP pool and inhibiting glycolysis, has been associated to PRIMA-1. The association led to an enhanced anti-proliferative effect in mutant KRAS (Kirsten rat sarcoma viral oncogene homolog) lung cancer and melanoma cells [59], and in mutant-p53 bladder cancer cells [44].
As the ionizing radiation causes DNA damage, and, indirectly, a p53-activation, the association of PRIMA-1 or APR-246 with radiotherapy seems promising, although it remains little explored. The only study which focused on this combination reported a decrease of the surviving fraction, and of the clonogenic survival when prostate cancer cells were exposed to APR-246 for 24 h, with an irradiation occurring five hours after the beginning of APR-246 treatment [18]. APR-246 sensitized to irradiation the mutant-p53, and p53-null cells, but had no impact on wt-p53 cells. The mechanism of the radiosensitization of p53-null cells remained unexplained and could implicate the oxidative stress.
PRIMA-1 and APR-246 have been also associated to several targeted therapies, with a current clinical use or at a pre-clinical stage. Combined with the PARP-inhibitor, olaparib, APR-246 sensitized lung cancer cell lines to the targeted therapy, independently of p53 status [50]; besides, the combination restored the sensitivity to olaparib in mutant-p53 cells that were previously olaparib-resistant [50]. In breast cancer cell lines, the combination had a cytotoxic synergic effect in mutant p53 cells [61]. With the mTOR inhibitor, rapamycin, APR-246 had a cytotoxic synergic effect in a mutant-p53 AML cell line and in primary cultures [53]. With BRAF (v-Raf (Rapidly Accelerated Fibrosarcoma) viral oncogene homolog B) enzyme inhibitor, vemurafenib, APR-246 overcame acquired resistance to vemurafenib in melanoma cell lines and in xenografts, with a cytotoxic and proapototic synergic effect [58]. Strikingly and similarly, p53 reactivation by APR-246 also broke intrinsic and acquired resistance and synergized with the MEK inhibitor pimasertib to induce massive apoptosis in NRAS-mutant melanoma cells with wild-type or mutant-p53, identifying MITF/Bcl-2 as a key mechanism underlying resistance of mutant-NRAS melanoma cells to apoptosis by MEK inhibitors and propose clinically relevant drug combinations able to prevent or reverse it [64]. Combined with a tyrosine kinase inhibitor, erlotinib, PRIMA-1 synergized in mutant-p53 head and neck cancer [26] and pancreas cancer cells [32]. The anti-tumoral effect of PRIMA-1/APR-246 was enhanced when combined to the proteasome inhibitor, bortezomib, in mutant-p53 pancreas cancer cells [32], in wt-p53 Waldenström cells [43] and in myeloma cells, independently of p53 status, with a restoration to sensitivity in bortezomib-resistant cells [54]. According to the essential role of ROS production and glutathione content in PRIMA-1/APR-246 efficacy, associations between inhibitors of glutathione synthesis or cysteine transporter and PRIMA-1/APR-246 appear particularly relevant: thus, an inhibitor of the system xċ, (cystine/glutamate antiporter), sulfasalazine, had a synergic anti-tumor effect with APR-246 in mutant p53 oesophageal adenocarcinoma cells and xenografts [62]. Considering targeted therapies in pre-clinical development, an enhanced cytotoxic effect has been observed in p53-mutant cancer cell cultures when PRIMA-1/APR-246 was combined to tunicamycin (ER stress inducer) in myeloma cells [54], or to wortmannin (PI3K inhibitor) in AML cells [53]. A synergic effect was found for nutlin-3 (MDM2 inhibitor) associated with PRIMA-1 in pancreatic cancer cells [32]. In vivo, an increase of the anti-tumor impacts has been observed when PRIMA-1 was associated to Deazaneplanocin A (a negative regulator of polycomb group actions that inhibits histone methyltransferase activity) in mutant-p53 thyroid cancer xenografts [38], and with 2aG4 (a monoclonal anti-body that binds specifically to the surface of tumor blood vessels and disrupts tumor vasculature) in breast cancer xenografts [28].
Altogether, these multiple efficient associations between PRIMA-1 or APR-246 and anti-cancer treatments make conceivable to treat many malignant diseases, and in particular, tumor sub-types, currently associated with poor prognosis because of genetic profile (mutant p53, KRAS, or BRAF) or acquired resistance to treatment (doxorubicin, cisplatin, olaparib, bortezomib, or vemurafenib).

Conclusions, Perspectives, and Clinical Impacts
In conclusion, PRIMA-1 and APR-246 appeared as important molecules with an anti-tumor effect in many cancer types. Its main cellular mechanism of action is the induction of apoptosis, mediated by the caspase activation. PRIMA-1 as well as APR-246 triggers an upregulation of genes involved in cell cycle control and apoptosis in mutant-p53 and wild-type p53 cancer cells. Anyway, 15 years after their discovery, it clearly appears that PRIMA-1/APR-246 have also p53-independent effects, as oxidative and ER stress, which emphasize their efficacies and extend their possible clinical uses, on tumor cells, independently of the p53 status. Combined with chemotherapies, ionizing radiations or targeted therapies, PRIMA-1 and APR-246 could offer new perspectives to treat the more aggressive tumor sub-types such as mutant-cKIT metastatic melanoma, HPV (Human papillomavirus)-positive head and neck squamous cell carcinoma, and anaplastic thyroid cancer. The first-in-human study (NCT00900614) that demonstrates, clinically, a good tolerance to the drug and a favorable pharmacokinetic profile, and biologically, an increased apoptosis with upregulation of p53 target genes, concludes to the safety of APR-246 use in hematologic malignancies and prostate cancer [75]. Three clinical trials are currently recruiting, with the objectives to test the safety and efficacy of APR-246 treatment in advanced oesophageal carcinoma (NCT02999893), high grade serous ovarian cancer (NCT02098343), and mutant p53 hematologic myeloid malignant disease (NCT03072043). Finally, a phase I/II study to investigate the safety and clinical activity of APR-246 in combination with a BRAF inhibitor in patients with mutant-BRAF unresectable metastatic melanoma resistant to anti-BRAF/anti-MEK inhibitors is starting.