APR-246—The Mutant TP53 Reactivator—Increases the Effectiveness of Berberine and Modified Berberines to Inhibit the Proliferation of Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer. In ~75% of PDAC, the tumor suppressor TP53 gene is mutated. Novel approaches to treat cancer involve compounds called mutant TP53 reactivators. They interact with mutant TP53 proteins and restore some of their growth suppressive properties, but they may also interact with other proteins, e.g., TP63 and TP73. We examined the ability of the TP53 reactivator APR-246 to interact with eleven modified berberine compounds (NAX compounds) in the presence and absence of WT-TP53 in two PDAC cell lines: the MIA-PaCa-2, which has gain of function (GOF) TP53 mutations on both alleles, and PANC-28, which lacks expression of the WT TP53 protein. Our results indicate the TP53 reactivator-induced increase in therapeutic potential of many modified berberines.


Introduction
Pancreatic cancer is the second leading cause of death [1,2]. Most pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC) [1][2][3]. Their treatment consists of surgical resection of the affected part of the pancreas. Unfortunately, the tumor usually comes back, often due to metastasis to other organs [4,5], and once PDAC has metastasized, it is difficult, if not impossible, to successfully treat. For decades, chemotherapy has been used as treatment modality for various cancer patients [5,6] and PDAC patients. However, it is usually a palliative and not curative approach [3,[6][7][8].
Numerous types of the TP53 gene mutations have been documented in PDAC and other cancers [14][15][16][17][18][19][20][21]. Some mutations are deletions or frame shift mutations and may result in loss of either all or parts of the TP53 protein (truncations) [19]. These genetic events could result in TP53 null cells. Such cells could also result from epigenetic alterations, which suppress the expression of the TP53 gene.
Point mutations at the TP53 gene may result in a protein with an altered activity in comparison to the WT protein. In some cases, these mutant TP53 proteins may have altered growth regulatory properties and diverse biochemical effects. Such mutations are referred to as GOF (gain of function) mutations [14][15][16][17][18]. The mutant GOF TP53 proteins may or Figure 1. Diagram representing effects of APR-246, BBR and NAX compounds on TP53 activity and cell growth and apoptosis. Restoration of some of the activity of WT TP53 will have effects on expression of miRs, cell cycle regulators and apoptotic molecules. NAC, N-acetylcysteine; APR-246, 2-hydroxymethyl-2-methoxymethyl-3-quinuclidinone; MQ, 2-methylene-3-quinuclidinone; BBR, berberine; Mut-TP53, mutant TP53; green arrows, promote activity; red arrows, block activity; red X in arrow, activity is blocked.
As expected for a nutraceutical, BBR normally is not highly toxic or growth inhibitory. Thus, multiple approaches have been made to increase the effectiveness of BBR to suppress cell growth and other processes. A panel of modified BBRs has been developed (NAX compounds) [63][64][65]. Some of these modified BBRs interact with their intracellular targets more effectively than BBR [66][67][68][69][70]. For example, NAX014 inhibited βcatenin signaling at 100-fold lower concentrations than BBR [70].
In the following studies, we examined the abilities of the mutant TP53 reactivator APR-246 to increase the abilities of BBR and NAX compounds to inhibit the proliferation of two PDAC cell lines, one of which has GOF mutations in the p53 protein and the other of which is TP53 null, before and after introduction of WT-TP53.

Sources of APR-246, BBR and NAX Compounds
The sources of these compounds have been previously described. APR-246 was bought from Selleck Chemicals (Houston, TX, USA). BBR was purchased from Figure 1. Diagram representing effects of APR-246, BBR and NAX compounds on TP53 activity and cell growth and apoptosis. Restoration of some of the activity of WT TP53 will have effects on expression of miRs, cell cycle regulators and apoptotic molecules. NAC, N-acetylcysteine; APR-246, 2-hydroxymethyl-2-methoxymethyl-3-quinuclidinone; MQ, 2-methylene-3-quinuclidinone; BBR, berberine; Mut-TP53, mutant TP53; green arrows, promote activity; red arrows, block activity; red X in arrow, activity is blocked.

Sources of APR-246, BBR and NAX Compounds
The sources of these compounds have been previously described. APR-246 was bought from Selleck Chemicals (Houston, TX, USA). BBR was purchased from MilliporeSigma (Saint Louis, MO, USA). Dr. Paolo Lombardi provided the NAX compounds (Naxospharma (Novate Milanese, Italy).   These experiments were repeated 4 times, and similar results were observed. * = p < 0.05, NS, not statistically significant.

Statistical Analysis
MTT experiments were set up as described [77][78][79] in triplicate, with a series of 11 serial 2-fold dilutions as well as an untreated control. The means and standard deviation of the samples were calculated by GraphPad Prism software (San Diego, CA, USA). Statistical significance of the comparison of two means of IC50s of two different culture conditions (+/− 12.5 nM APR-246) or clonogenicity of two different cell types was calculated using the GraphPad QuickCalcs software using an unpaired t test with a 95%

Statistical Analysis
MTT experiments were set up as described [77][78][79] in triplicate, with a series of 11 serial 2-fold dilutions as well as an untreated control. The means and standard deviation of the samples were calculated by GraphPad Prism software (San Diego, CA, USA). Statistical significance of the comparison of two means of IC 50s of two different culture conditions (+/− 12.5 nM APR-246) or clonogenicity of two different cell types was calculated using the GraphPad QuickCalcs software using an unpaired t test with a 95% confidence interval.

Effects of the Mutant TP53 Reactivator on Two PDAC Cell Lines
The effects of the mutant TP53 reactivator were examined on two PDAC cell lines: MIA-PaCa-2, which contains GOF TP53 genes on both alleles [71], and PANC-28, which does not express detectable levels of the TP53 protein [73]. Into these cells, we introduced either a vector containing the WT-TP53 cDNA [79] or the control parental vector (pLXSN) lacking any insert [80]. Both vectors encode resistance to G418.
Then, the combination of APR-246 with 11 structurally different NAX compounds was examined in MIA-PaCa-2 + WT-TP53 and MIA-PaCa-2 + pLXSN. The low dose of APR-246 did not lower the IC 50 of NAX012 in either MIA-PaCa-2 + WT-TP53 or MIA-PaCa-2 + pLXSN cells ( Figure 3C,D), and had only a mild effect on the sensitivity of MIA-PaCa-2 + pLXSN to NAX014 as the IC 50 decreased 1.1-fold ( Figure 4A). In contrast, the presence of APR-246 had a more significant effect on the sensitivity of In the presence of APR-246, the sensitivity of MIA-PaCa-2 + pLXSN cells to NAX035 decreased only slightly (1.1-fold), but in MIA-PaCa-2 + WT-TP53 cells, the TP53 reactivator reduced the NAX035 IC50 almost 19-fold ( Figure 4C,D).
Addition of the low dose of APR-246 had a mild effect on the sensitivity of both MIA-PaCa-2 + pLXSN MIA-PaCa-2 + WT-TP53 cells to NAX038 as the IC 50 decreased, respectively, 1.2-and 1.3-fold ( Figure 5A,B).
A dose of APR-246 had a similar mild effect on the sensitivity of MIA-PaCa-2 + pLXSN to NAX042 ( Figure 5, Panel C). In contrast, APR-246 had a more significant effect on the sensitivity of MIA-PaCa-2 + WT-TP53 cells to NAX042 as the IC 50 decreased 23-fold, from 300 to 13 nM ( Figure 5D).
APR-246 decreased the sensitivity of MIA-PaCa-2 + pLXSN cells to NAX053 only 1.5-fold ( Figure 6, Panel A), but it had a more significant on the sensitivity of MIA-PaCa-2 + WT-TP53 cells to NAX053 as the IC 50 decreased 100-fold from 200 to 2 nM ( Figure 6B).
The low dose of APR-246 had an effect on the sensitivity of MIA-PaCa-2 + pLXSN cells to NAX075 as the IC 50 decreased >4-fold from >2000 to 500 nM ( Figure 7C), but it had a more significant effect on the sensitivity of MIA-PaCa-2 + WT-TP53 cells to NAX075 as the IC 50 decreased 357-fold from 1000 to 2.8 nM ( Figure 7D). A dose of APR-246 had a similar mild effect on the sensitivity of MIA-PaCa-2 + pLXSN to NAX042 ( Figure 5, Panel C). In contrast, APR-246 had a more significant effect on the sensitivity of MIA-PaCa-2 + WT-TP53 cells to NAX042 as the IC50 decreased 23fold, from 300 to 13 nM ( Figure 5D).
APR-246 decreased the sensitivity of MIA-PaCa-2 + pLXSN cells to NAX053 only 1.5fold ( Figure 6, Panel A), but it had a more significant on the sensitivity of MIA-PaCa-2 + WT-TP53 cells to NAX053 as the IC50 decreased 100-fold from 200 to 2 nM ( Figure 6B). The low dose of APR-246 had an effect on the sensitivity of MIA-PaCa-2 + pLXSN cells to NAX060 as the IC 50 decreased two-fold from 800 to 400 nM ( Figure 7, Panel A), but it had a more significant effect on the sensitivity of MIA-PaCa-2 + WT-TP53 cells to NAX060 as the IC 50 decreased 110-fold from 220 to 2 nM ( Figure 7B). The low dose of APR-246 had an effect on the sensitivity of MIA-PaCa-2 + pLXSN cells to NAX075 as the IC50 decreased >4-fold from >2000 to 500 nM ( Figure 7C), but it had a more significant effect on the sensitivity of MIA-PaCa-2 + WT-TP53 cells to NAX075 as the IC50 decreased 357-fold from 1000 to 2.8 nM ( Figure 7D).
Similarly, APR-246 had a stronger effect on the sensitivity of MIA-PaCa-2 + pLXSN than of MIA-PaCa-2 + WT-TP53 cells to NAX111, as the IC 50 for this compound decreased three-fold in the former cells and only 1.3-fold in the latter cells ( Figure 8C,D).
In summary, the addition of a low dose of APR-246 could increase the cytotoxic effects of certain NAX compounds on MIA-PaCa-2 cells. The most significant effect-over 100-fold increase in sensitivity to NAX-was observed with MIA-PaCa-2 + WT-TP53 with NAX060.
Surprisingly, APR-246 has stronger effects on MIA-PaCa-2 + pLXSN than of MIA-PaCa-2 + WT-TP53 when combined with NAX077 and NAX111. Similarly, APR-246 had a stronger effect on the sensitivity of MIA-PaCa-2 + pLXSN than of MIA-PaCa-2 + WT-TP53 cells to NAX111, as the IC50 for this compound decreased three-fold in the former cells and only 1.3-fold in the latter cells ( Figure 8C,D).
In summary, the addition of a low dose of APR-246 could increase the cytotoxic effects of certain NAX compounds on MIA-PaCa-2 cells. The most significant effect-over 100-fold increase in sensitivity to NAX-was observed with MIA-PaCa-2 + WT-TP53 with NAX060.

Abilities of Low Doses of BBR or NAX060 to Increase the Cytotoxicity of APR-246 and Decrease Clonogenicity of MIA-PaCa-2 Cells Containing and Lacking WT-TP53
In subsequent experiments, we tested a promising combination: APR-246, and NAX060, and APR-246 with BBR as the benchmark, in terms of their abilities to influence the clonogenicity of the MIA-PaCa-2 cells containing and lacking WT-TP53. This time, we used different concentrations of APR-246 and low doses of BBR or NAX060 (Figure 9). In subsequent experiments, we tested a promising combination: APR-246, and NAX060, and APR-246 with BBR as the benchmark, in terms of their abilities to influence the clonogenicity of the MIA-PaCa-2 cells containing and lacking WT-TP53. This time, we used different concentrations of APR-246 and low doses of BBR or NAX060 (Figure 9). APR-246 inhibited the clonogenicity of MIA-PaCa-2 + pLXSN and MIA-PaCa-2 + WT-TP53 cells in a dose-dependent fashion. Furthermore, the addition of a low dose of berberine resulted in increased suppression of growth ( Figure 9A). The effects of APR-246 were greater when the MIA-PaCa-2 + WT-TP53 cells were treated with higher concentrations of APR-246 and BBR than in MIA-PaCa-2 + pLXSN cells. Higher APR-246 concentrations had stronger effects on MIA-PaCa-2 + WT-TP53 than control cells which lacked WT TP53.
A summary of the effects of combination of APR-246, BBR an NAX compounds in both MIA-PaCa-2 + pLXSN and MIA-PaCa-2 + WT-TP53 cells is presented in Figure 10. Included in this figure as well are the structures of BBR and the NAX compounds. Likewise, a low dose of NAX060 suppressed clonogenicity in both MIA-PaCa-2 + pLXSN and MIA-PaCa-2 + WT-TP53 cells ( Figure 9B). As observed previously ( Figures  3A,B and 7A,B), NAX060 suppressed growth to a greater extent than BBR, and a higher level of growth inhibition was observed in MIA-PaCa-2 + WT-TP53 than in MIA-PaCa-2 + WT-TP53 cells.
A summary of the effects of combination of APR-246, BBR an NAX compounds in both MIA-PaCa-2 + pLXSN and MIA-PaCa-2 + WT-TP53 cells is presented in Figure 10. Included in this figure as well are the structures of BBR and the NAX compounds.

Interactions between Berberine (BBR), Chemically Modified BBRs (NAX Compounds) and APR-246 in PANC-28 + pLXSN and PANC-28 + WT-TP53 Cells
Next, we determined whether the effects of BBR and NAX compounds on the growth of PDAC cells could be enhanced by the low concentration (12.5 nM) of mutant TP53 reactivator APR-246 also in PANC-28 cells containing or lacking WT TP53.
Addition of APR-246 had a mild effect on the sensitivity of PANC + pLXSN as the IC 50 on the combination of BBR and APR-246 decreased only 1.1-fold, from 1600 to 1500 nM ( Figure 11A). In contrast, when PANC-28 + WT-TP53 cells were treated with APR-246, the IC 50 for BBR decreased 55.6-fold from 1000 to 18 nM ( Figure 11B). These results are summarized in Table 2.   Then, we tested the same panel of compounds as in the case of MIA-PaCa-2 cells on PANC-28 cells. The addition of APR-246 had no or only moderate effects on the sensitivity of PANC-28 + pLXSN to NAX compounds (Figures 11-16). The best results for PANC-28 + pLXSN cells were obtained in the combination with NAX012: APR-246 lowered the IC 50 for this compound four-fold ( Figure 11C).

Interactions between Berberine (BBR), Chemically Modified BBRs (NAX Compounds) and APR-246 in PANC-28 + pLXSN and PANC-28 + WT-TP53 Cells
Next, we determined whether the effects of BBR and NAX compounds on the growth of PDAC cells could be enhanced by the low concentration (12.5 nM) of mutant TP53 reactivator APR-246 also in PANC-28 cells containing or lacking WT TP53.
Then, we tested the same panel of compounds as in the case of MIA-PaCa-2 cells on PANC-28 cells. The addition of APR-246 had no or only moderate effects on the sensitivity of PANC-28 + pLXSN to NAX compounds (Figures 11-16). The best results for PANC-28 + pLXSN cells were obtained in the combination with NAX012: APR-246 lowered the IC50 for this compound four-fold ( Figure 11C).      In turn, in the case of PANC-28 + WT-TP53 cells, the low dose of APR-246 was more effective in reducing the IC 50 for almost all the tested NAX compounds (Figures 11-16). The only exception was NAX054, whose IC 50 remained unchanged by APR-246 ( Figure 14D). This is, however, in agreement with our previous studies showing that NAX054 did not have any significant effects on PANC-28 cells [81].
The most impressive influence of the low dose of APR-246 was observed in the case of NAX077 as the IC 50 for this compound in PANC-28 + WT-TP53 cells decreased 1000-fold from 1500 to 1.5 nM ( Figure 16B). All data are summarized in Table 2.  In turn, in the case of PANC-28 + WT-TP53 cells, the low dose of APR-246 was more effective in reducing the IC50 for almost all the tested NAX compounds (Figures 11-16). The only exception was NAX054, whose IC50 remained unchanged by APR-246 ( Figure  14D). This is, however, in agreement with our previous studies showing that NAX054 did not have any significant effects on PANC-28 cells [81].
The most impressive influence of the low dose of APR-246 was observed in the case of NAX077 as the IC50 for this compound in PANC-28 + WT-TP53 cells decreased 1000fold from 1500 to 1.5 nM ( Figure 16B). All data are summarized in Table 2.

Abilities of Low Doses of BBR or NAX060 to Increase the Cytotoxicity of APR-246 and Decrease Clonogenicity of PANC-28 Cells Containing and Lacking WT-TP53
We compared the effectiveness of low doses of NAX060 and BBR to influence the clonogenicity of the PANC-28 cells containing and lacking WT-TP53 in the presence of increasing concentrations of APR-246 ( Figure 17). APR-246 inhibited the colony formation of PANC-28 + WT-TP53 cells in a dosedependent fashion. In contrast, APR-246 had less effects on PANC-28 + pLXSN cells.
Furthermore, the addition of a low dose of BBR resulted in increased suppression of growth in PANC-28 + pLXSN cells ( Figure 17A). However, the addition of increasing concentrations of APR-246 did not result in a significant decrease in clonogenicity in APR-246 inhibited the colony formation of PANC-28 + WT-TP53 cells in a dosedependent fashion. In contrast, APR-246 had less effects on PANC-28 + pLXSN cells.
Furthermore, the addition of a low dose of BBR resulted in increased suppression of growth in PANC-28 + pLXSN cells ( Figure 17A). However, the addition of increasing concentrations of APR-246 did not result in a significant decrease in clonogenicity in PANC-28 + pLXSN cells. The effects of APR-246 were greater when the PANC-28 + WT-TP53 cells were treated with higher concentrations of APR-246 and BBR than in PANC-28 + pLXSN cells.
In contrast, NAX060 had some effects on PANC-28 + pLXSN cells that were greater at higher doses of APR-246. Likewise, a low dose of NAX060 suppressed clonogenicity in both PANC-28 + pLXSN and PANC-28 + WT-TP53 cells ( Figure 17B). As observed previously ( Figure 15C), NAX060 suppressed growth to a greater extent than BBR (Figure 17A), and a higher level of growth inhibition was observed in PANC-28 + WT-TP53 cells than in PANC-28 + pLXSN cells.

Discussion
APR-246 is a drug that has been clinically evaluated in patients with certain types of cancer. Berberine (BBR) is a nutraceutical that has been examined for many types of health problems including cancer. Modified BBRs have been developed that have enhanced effects in certain pre-clinical cancer models. Some studies have indicated that some effects of BBR are TP53 dependent [69]. The possible effects of combining the mutant TP53 reactivator APR-246 and NAX compounds on PDAC cells containing and lacking WT TP53 activity have not been previously described.
The MIA-PaCa-2 cell line contains GOF mutant TP53. The addition of WT-TP53 increased the sensitivity of MIA-PaCa-2 cells to a low dose of APR-246. BBR showed some cytotoxic effects on MIA-PaCa-2 + pLXSN and MIA-PaCa-2 + WT-TP53. The addition of a low dose of APR-246 did not significantly change the effect of BBR. Similar results were observed with NAX012. A summary of these results is presented in Figure 18.
The differences in chemical modifications of the BBR backbone structures of the various NAX are presented in Figures 10 and 18. The chemical modifications of the NAX compounds have (un)substituted (hetero) aromatic moieties bonded to position 13 of the parent alkaloid BBR skeleton through a hydrocarbon (C) linker of variable lengths. The structures of the NAX compounds are presented in Figures 10 and 18 and [78,83].
NAX012 and NAX042 have additional phenyl moieties with hydrocarbon (C) linkers of different lengths. NAX012 has a 3 C linker and has been prepared as iodide salt. NAX042 has a 4 C linker and has been prepared as chloride salt. NAX042 was more effective in suppressing proliferation of MIA-PaCa-2 cells than NAX012. NAX012 and NAX014 suppressed proliferation of MIA-PaCa-2 cells moderately; however, the addition of WT-TP53 resulted in increased sensitivity to the combination of NAX014 and APR-246 substantively, while in the case of NAX012 it has no effect. Clearly, the number of C in the linkers present in the NAX012 or NAX014 compounds and the presence of a chlorine atom on the added monophenyl present in NAX014 can affect their ability to inhibit PDAC growth. of BBR are TP53 dependent [69]. The possible effects of combining the mutant TP53 reactivator APR-246 and NAX compounds on PDAC cells containing and lacking WT TP53 activity have not been previously described.
The MIA-PaCa-2 cell line contains GOF mutant TP53. The addition of WT-TP53 increased the sensitivity of MIA-PaCa-2 cells to a low dose of APR-246. BBR showed some cytotoxic effects on MIA-PaCa-2 + pLXSN and MIA-PaCa-2 + WT-TP53. The addition of a low dose of APR-246 did not significantly change the effect of BBR. Similar results were observed with NAX012. A summary of these results is presented in Figure 18.  NAX035 and NAX053 have two phenyl moieties (benzhydryl) with C linkers of different lengths. NAX035 has a 2 C linker while NAX035 has a 3 C linker. Both compounds were effective in suppressing the proliferation of MIA-PaCa-2 cells. The addition of a low dose of APR-246 exhibited similar sensitizing effects on treatment of MIA-PaCa-2 + WT-TP53 cells to both NAX035 and NAX053.
NAX075 and NAX077 have additional one heterocycle (pyridine) with C linkers of different lengths. NAX075 has a 3 C linker, while NAX077 has a 4 C linker. These compounds are similar in structure. They had minimal effects on suppressing the proliferation on MIA-PaCa-2 + pLXSN cells. The addition of a low dose of APR-246 had more substantial effects on NAX075 than NAX077 treatment of MIA-PaCa-2 + WT-TP53 cells.
NAX038 and NAX054 have multiple electron-releasing substituents on the introduced monophenyl moiety. NAX038 was effective in suppressing the proliferation of MIA-PaCa-2 + pLXSN and MIA-PaCa-2 + WT-TP53 cells. In contrast, NAX054 with three methoxy (OCH 3 ) groups on the added phenyl moiety did not suppress proliferation of either MIA-PaCa-2 + pLXSN or MIA-PaCa-2 + WT-TP53 cells. The addition of a low dose of APR-246 had modest on the effects of NAX038 on either MIA-PaCa-2 + pLXSN or MIA-PaCa-2 + WT-TP53 cells. A low dose of APR-246 was modest on the effects on NAX054 in MIA-PaCa-2 + WT-TP53 cells.
NAX014, NAX060 and NAX111 have multiple electron-withdrawing substituents on the introduced monophenyl moiety. NAX060 and NAX111 had similar effects on MIA-PaCa-2 cells. However, introduction of WT-TP53 increased the effects of addition of a low concentration of APR-246 when added with NAX060, but not NAX111.
In terms of clonogenicity, the effects of APR-246 were increased when WT-TP53 was present in MIA-PaCa-2 + WT-TP53 cells. The effects of BBR by itself were not different in the absence of APR-246. However, the effects of NAX060 were different in the absence of APR-246 in the presence and absence of WT-TP53. As observed by MTT analysis, NAX060 was more potent than BBR in suppressing growth.
These modifications of the BBR core structure had different effects on suppression of MIA-PaCa-2 growth and sensitivity in combination with APR-246. It is possible that the different modifications of the BBR core structure in the NAX compounds affect the ability of the molecules to interact with the promoter regions of key genes involved in regulation of cell growth, induction of apoptosis and other important biochemical processes.
APR-246 did not have significant effects on colony formation of PANC-28 + pLXSN. However, the addition of WT-TP53 sensitized the PANC-28 + WT-TP53 to APR-246. The colony formation of PANC-28 + pLXSN cells was sensitive to BBR and NAX060. The addition of WT-TP53 sensitized the PANC-28 + WT-TP53 cells more to the combination of APR-246 and NAX060 than to BBR.
In terms of clonogenicity, NAX060 was more effective in suppressing colony formation than BBR. This agrees with what we observed by MTT growth assays. The addition of WT-TP53 increased the sensitivity of PANC-28 cells to APR-246.
Our studies point to the value of knowing what type of TP53 mutation(s) present in cancer patients that may be treated with APR-246 and next-generation-related compounds. These compounds may not have a significant effect on cells that have deleted or silenced TP53 expression. While this observation may be obvious, there are other TP53-related molecules such as TP63 and TP73, which could be stabilized by the APR-246 compound in PANC-28 + pLXSN cells. However, we observed that treatment of PANC-28 + pLXSN was not sensitive to the combination of BBR and APR-246. In contrast, PANC-28 + WT-TP53 were highly sensitive to the combination of APR-246 and BBR. Our studies indicate that APR-246 can interact with certain modified BBRs to increase their cytotoxicity on PDAC cells. These interactions are often increased in the presence of WT TP53.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.

Conflicts of Interest:
The authors declare no conflict of interest.