Strategic selection of MDM2 inhibitors enhances the efficacy of FAK inhibition in mesothelioma based on TP53 genotype

Xuerao Ning; Thảo Thi Thanh Nguyễn; Takao Morinaga; Yuji Tada; Hideaki Shimada; Kenzo Hiroshima; Naoto Yamaguchi; Masatoshi Tagawa

doi:10.1371/journal.pone.0343551

. 2026 Feb 23;21(2):e0343551. doi: 10.1371/journal.pone.0343551

Strategic selection of MDM2 inhibitors enhances the efficacy of FAK inhibition in mesothelioma based on TP53 genotype

Xuerao Ning ^1,², Thảo Thi Thanh Nguyễn ^1,^3,⁴, Takao Morinaga ¹, Yuji Tada ⁵, Hideaki Shimada ⁶, Kenzo Hiroshima ^7,^8,⁹, Naoto Yamaguchi ², Masatoshi Tagawa ^7,^10,^*

Editor: Zu Ye¹¹

PMCID: PMC12928570 PMID: 41729969

Abstract

Mesothelioma has characteristic genetic changes including inactivation of neurofibromatosis type 2 (NF2) and deletion of the INK4A/ARF region. Cells deficient of NF2 protein (MERLIN) depend on focal adhesion kinase (FAK) for cell adhesion and FAK inhibitors suppress the cell growth. The INK4A/ARF deletion activates MDM2 functions which ubiquitinate and degrade p53, and consequently the cellular p53 levels decrease. The deletion therefore induces loss of p53 functions although a majority of mesothelioma has wild-type TP53 genotype. An MDM2 inhibitor which blocked the ubiquitination increased p53 levels, restored p53 functions and facilitated cell growth arrest. Moreover, FAK and p53 expressions were reciprocally regulated. We examined growth suppressive effects of a FAK inhibitor, defactinib, and MDM2 inhibitors, nutlin-3a and reactivation of p53 and induction of tumor cell apoptosis (RITA), with representative wild-type and mutated TP53 mesothelioma and investigated molecular changes induced by the agents. We analyzed possible combinatory effects of the inhibitors and molecular changes caused by the combination. Our study showed that defactinib inhibited cell growth and induced FAK dephosphorylation irrespective of the TP53 genotype, and that the inhibited FAK phosphorylation was not associated with MERLIN levels or with p53 up-regulation, but linked with AKT dephosphorylation. Nutlin-3a preferentially suppressed growth of wild-type TP53 cells and augment p53 expression without DNA damage, whereas RITA-mediated p53 up-regulation was linked with the damage. A combination of defactinib and the MDM2 inhibitors showed that nutlin-3a showed synergistic/additive effects in wild-type and antagonistic effects in mutated TP53 cells, whereas RITA retained synergistic activity in mutated TP53 cells. These results suggest that the therapeutic success of combined FAK and MDM2 inhibition in mesothelioma depends on the precise matching of MDM2 inhibitors with the TP53 genotypes, and highlight the need for genotype-based selection of MDM2 inhibitors.

Introduction

Malignant mesothelioma developed in the pleural cavity is invasive and often resistant to conventional treatments. A majority of the patients received platinum-based chemotherapy in combination with pemetrexed but the prognosis remained poor with a median survival period about 12 months [1]. The patients often become resistant to the agents and a second-line drug is currently unavailable. Recent clinical studies however showed that immune checkpoint inhibitors were as effective as the first-line chemotherapeutic agents [2–4] although the inhibitors produced significant adverse effects. Nevertheless, the immune checkpoint inhibitors did not produce better therapeutic effects than chemotherapy in relapsed mesothelioma patients [4]; consequently, a novel agent is required for further improvement of the treatment strategy.

Malignant mesothelioma has 2 characteristic genetic abnormalities, inactivation of neurofibromatosis type 2 (NF2) [5] and deletion of the INK4A/ARF region which includes p16^INK4A and p14^ARF genes [6]. The NF2 gene encodes an ezrin-radixin-moesin-like protein, MERLIN, which functions as a tumor suppressor and mediates contact inhibition through several signaling processes [7]. A recent study also demonstrated that mesothelioma cells deficient of MERLIN expression were susceptible to an inhibitor of focal adhesion kinase (FAK), a non-receptor-type tyrosine kinase localized to focal contacts [8]. FAK plays a role in cell proliferation and the inhibitor in particular suppressed growth of MERLIN-deficient cells since the MERLIN-negative cells were more dependent on the FAK signaling for the cell adhesion and proliferation than the positive cells. These data suggested that a FAK inhibitor was a candidate for mesothelioma treatments since mesothelioma was often defective of MERLIN expression. On the other hand, a different study showed that an expression level of E-cadherin, which also mediates cell adhesion, was linked with resistance to a FAK inhibitor in MERLIN-deficient cells [9]. Several clinical studies with FAK inhibitors were conducted for mesothelioma patients [10–12], but a possible association of susceptibility to FAK inhibitors with an expression level of MERLIN or other molecules was not well understood.

The deletion of INK4A/ARF region results in defective p53 activities even though a majority of mesothelioma has the wild-type TP53 genotype. Loss of the p14 gene, mapped in the deleted region, augments the MDM2 function which degrades wild-type p53 through enhanced p53 ubiquitination [13]. The deletion was frequently found in clinical specimens of mesothelioma and consequently, the majority showed functional loss or deficiency of p53 actions. An inhibitor of the MDM2 function can stabilize p53 protein by blocking the degradation process and increase the level, which activates p53-mediated pathways. A small-sized inhibitor interfering a binding between p53 and MDM2, for example nutlin-3a and reactivation of p53 and induction of tumor cell apoptosis (RITA), in fact up-regulated a p53 expression level and stimulated the p53 pathways; consequently, the inhibitor induced growth inhibition and apoptotic cell death in human tumors [14–16]. The inhibitors and a combination with the first-line agent also showed anti-tumor effects on mesothelioma [17]. Activation of the p53-mediated pathway is thereby a possible therapeutic strategy for mesothelioma with wild-type TP53 gene. On the other hand, effects of up-regulation of mutated p53 on tumor cell growth remained unclear.

Several previous studies showed a possible interaction between FAK and p53, and a reciprocal regulation between the molecules [18,19]. Knock-down of FAK expression increased p53 levels [18] and up-regulation of p53 suppressed FAK levels [19]. In addition, MERLIN played a certain role in p53 up-regulation through inhibition of the MDM2 function [20]. These previous data collectively suggested that a combination of a FAK inhibitor and a MDM2 inhibitor augmented p53 expression levels and induced growth inhibition even in MERLIN-positive mesothelioma. In fact, a FAK inhibitor augmented growth inhibitory effects of a p53-activating chemical agent in mesothelioma with wild-type TP53 genotype [21]. Nevertheless, a growth suppressive activity of a FAK inhibitor in terms of MERLIN expression and TP53 genotype was not well studied, and possible combinatory effects of a FAK inhibitor and a MDM2 inhibitor with respect to the TP53 genotype remained uncharacterized. In this study, we investigated growth suppressive activity of defactinib (also known as VS-6063), a FAK inhibitor used for clinical studies, and MDM2 inhibitors, nutlin-3a and RITA, and further examined a possible combinatory effect on mesothelioma cells with different MERLIN expression levels and TP53 genotypes.

Methods

Cells and agents

Human mesothelioma cells, NCI-H28, MSTO-211H, NCI-H2052, NCI-H226 and NCI-H2452 cells, and SV40-T antigen-expressing immortalized cells of mesothelium origin, Met-5A cells, were purchased from American Type Culture Collection (Manassas, VA, USA). Mesothelioma with mutated TP53 genotype, EHMES-1 (R273S) and JMN-1B (G245S) cells, and that with wild-type TP53 gene, EHMES-10 cells, were provided by Dr. Hironobu Hamada (Hiroshima University, Japan) [22]. Cells were cultured with in RPMI-1640 medium supplemented with 10% fetal calf serum, and were confirmed to be negative for mycoplasma. The genotype of TP53 was wild-type in NCI-H28, MSTO-211H, NCI-H2052, NCI-H226 and NCI-H2452 cells, but p53 protein of NCI-H2452 cells was truncated (Fig 1, S1 Table). Chemicals used in the present study, defactinib (VS-6063), nutlin-3a (S8059) and RITA (NSC 652287) were purchased from Selleck Chemicals (Houston, TX, USA).

Fig 1 — Tubulin was used as a loading control. The expression of each molecule was quantified with ImageJ software (NIH, Bethesda, MD, USA) with tubulin intensity as a normalized control (see S3 Table).

Viable cell detection assay

Cells seeded in 96-well plates (1 × 10³ cells per well) were treated with an agent alone or in a combination of 2 agents for 72 hrs. Cell viability was determined with a cell-counting WST kit (Wako, Osaka, Japan) with absorbance at 450 nm using a microplate reader (Model 620, Bio-Rad, Hercules, CA, USA). The relative viability was calculated based on the absorbance without any treatments, an absorbance of cells treated with an agent/(an absorbance of cells untreated – an absorbance of medium only) x 100 (WST assay). Half maximal inhibitory concentration (IC₅₀) values and combinatory effects were examined with the CalcuSyn software (Ver2, Biosoft, Cambridge, UK). Combination index (CI) values at respective fractions affected (Fa) points, which showed relative suppression levels of the cell viability, were calculated based on the cell viability test. CI < 1, CI = 1 and CI > 1 indicates synergistic, additive and antagonistic actions, respectively. Statistical analysis was performed with one-way analysis of variance.

Western blot analysis

Cell lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was transferred to a nylon filter membrane and was hybridized with antibody against phosphorylated p53 at Ser 15 (#9284), AKT (#9272), phosphorylated AKT at Ser 473 (#9271), caspase-9 (which also detected cleaved caspase-9) (#9505), PARP (poly ADP-ribose polymerase) (which also detected cleaved PARP) (#4108), FAK (#3285), phosphorylated FAK at Tyr 397 (#3283), phosphorylated MDM2 at Ser 166 (#3521), MERLIN (D1D8, #6995), actin (#4970) (Cell Signaling, Danvers, MA, USA), phosphorylated H2AX (phosphor-2A histone family member X) at Ser 139 (#613401) (BioLegend, San Diego, CA, USA), MDM2 (#413) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (Ab-6, clone DO-1, #MS-187-P0), and tubulin-α (DM1A, Thermo Fisher Scientific) followed by an appropriate second antibody (S2 Table). The membranes were developed with the ECL system (GE Healthcare, Buckinghamshire, UK). Actin and tubulin-α were used as a loading control. We chose an optimal exposure time and selected a representative blot to visualize molecular changes for each experiment.

Statistical analysis

Statistical analyses were conducted with unpaired t-test in GraphPad Prism 6 software (GraphPad, La Jolla, CA, USA) and P-values less than 0.05 were considered as statistically significant.

Results

Expression of p53, MERLIN and FAK in mesothelioma cells

We examined 8 kinds of mesothelioma cells for the expression of p53, MDM2, MERLIN and FAK with western blot analysis (Fig 1, S3 Table). Mesothelioma with the wild-type TP53 genotype (NCI-H28, MSTO-211H, NCI-H2052, NCI-H226 and EHMES-10) expressed p53 at a lower level than those with mutated TP53 genotype (EHMES-1 and JMN-1B) since wild-type p53 was more susceptible to the ubiquitin-mediated degradation than mutated p53. NCI-H2452 cells expressed truncated p53 despite having the wild-type genotype, and consequently the p53 functions were defective. The anti-MDM2 antibody detected both full-length 90 kDa and cleaved 60 kDa molecules, and both of which molecules were able to ubiquitinate p53 [23]. Mesothelioma cells with the wild-type TP53 genotype except NCI-H2452 and EHMES-10 cells expressed 90 kDa MDM2 at a relatively high level compared with those with mutated TP53. MERLIN was positive in NCI-H28, MSTO-211H, NCI-H2452, and EHMES-10 cells, and was minimally expressed in EHMES-1 cells, whereas the expression was almost undetectable in the other cells. All the mesothelioma expressed FAK, but the level was relatively low in NCI-H2452 and EHMES-10 cells compared with that of the other cells. These data indicated that an expression level of FAK was not associated with that of MERLIN or MDM2, and was independent of TP53 genotype. In addition, a MERLIN expression level was irrelevant to the TP53 genotype and an MDM2 level.

Growth inhibitory effects of defactinib on mesothelioma

We examined a growth inhibitory activity of defactinib with the colorimetric WST assay (Fig 2A). All the 8 kinds of mesothelioma and Met-5A cells which were defective in the p53 functions due to expression of the p53-inactivating SV40-T antigen, showed similar sensitivity to defactinib. The IC₅₀ values were not different between mesothelioma cells with wild-type TP53 genotype (average ± SE; 3.85 ± 0.65 μM) and those with mutated TP53 genotype (EHMES-1 and JMN-1B) or with non-functional p53 (NCI-2452 and Met-5A) (4.52 ± 1.17) (P = 0.64). Furthermore, sensitivity to the FAK inhibitor was unrelated to the MERLIN expression levels (MERLIN-high cells: NCI-H28, MSTO-211H, NCI-H2452 and EHMES-10 IC₅₀ = 5.98 ± 1.00, MERLIN low or negative cells: NCI-H2052, NCI-H226, EHMES-1 and JMN-1B IC₅₀ = 3.47 ± 0.85) (P = 0.105) (Fig 1). We also examined growth inhibitory activity of defactinib with a dye exclusion assay, which measured viable cell numbers with a trypan blue dye, and confirm the cell growth inhibition (S1A Fig). The assay showed that defactinib inhibited the proliferations of all the cells tested.

We next examined expression of FAK, p53, AKT and MDM2 with western blot analysis (Fig 2B, S4 Table). Mesothelioma cells treated with defactinib showed dose-dependent decrease of FAK phosphorylation levels at Tyr 397, an autophosphorylation site controlling the FAK activity, whereas the FAK expression levels of the treated cells remained unchanged except MSTO-211H and NCI-H226 cells. The data confirmed that defactinib at the doses used in the experiments, 1−4 μM, inhibited a kinase activity of FAK. The defactinib treatment augmented expression of p53 or phosphorylated p53 at Ser 15 in NCI-H28, MSTO-211H, NCI-H2052, NCI-H2452, EHMES-10 and EHMES-1 cells, whereas the treatment decreased the expression in NCI-H226 and JMN-1B cells. Phosphorylation of p53 at Ser 15 is a maker for p53 stabilization and can be linked with increase of p53 expression, and the linkage between the phosphorylation and increased p53 level was detected in NCI-H28, MSTO-211H, NCI-H2452 and EHMES-1 cells. These data indicated that FAK inhibition was not always associated with increase of p53 levels or the phosphorylation levels irrespective of the TP53 genotype. AKT (protein kinase B) is responsible for many cellular processes and located down-stream of receptor-type tyrosine kinases. We found that phosphorylated AKT at Ser 473 levels, which was a marker of AKT activation, and less significantly AKT levels were down-regulated in all the cells by the defactinib treatments. The decreased AKT phosphorylation was probably attributable to the defactinib-induced growth retardation and was consequently correlated with down-regulation of FAK phosphorylation. We also found that that MDM2 levels remained unchanged but the phosphorylation was differently regulated among the cells tested; increased phosphorylation in NCI-H28 and EHMES-1 cells, and decreased in EHMES-10 and JMN-1B cells. Regulation of the MDM2 phosphorylation was thus not directly linked with inhibited FAK phosphorylation, TP53 genotype, p53 or MERLIN expression levels. Cleavages of caspase-9 was slightly enhanced in NCI-H28 cells and cleaved PARP was also minimally up-regulated in NCI-H28 and EHMES-1 cells, whereas other cells did not show the enhanced cleavages. These data collectively indicated that suppressed FAK activity by the defactinib was associated with inhibition of AKT phosphorylation, but not directly linked with phosphorylation of p53 and MDM2, or TP53 genotype. Moreover, the defactinib treatment did not induce apoptotic cell death in most of the cells tested.

Growth inhibitory effects of nutlin-3a were linked with p53 functional type

We next examined sensitivity of mesothelioma and Met-5A cells to nutlin-3a, an MDM2 inhibitor, with the WST assay and calculated the IC₅₀ values (Fig 3A). Cells with the wild-type TP53 genotype excluding EHMES-10, were more susceptible to nutlin-3a (IC₅₀ = 3.62 ± 1.04 μM) than those with mutated TP53 and non-functional p53 (33.5 ± 2.80) (P < 0.01). These data showed that nutlin-3a sensitivity of mesothelioma cells was correlated with TP53 genotype and the p53 functional status except EHMES-10 cells. A mechanism of the nutlin-3a resistance in EHMES-10 cells is currently unknown, but the IC₅₀ value of the cells was relatively close to that of mutated TP53 or non-functional p53 cells. The cells were also resistant to RITA-mediated growth inhibition (see below). These data suggested that EHMES-10 cells were insusceptible to growth inhibition or cell death probably because the p53 down-stream pathway was disturbed, in which reduced p21 induction limits cell cycle arrest.

We also examined expression and phosphorylation levels of p53 and FAK in mesothelioma cells treated with nutlin-3a (Fig 3B, S5 Table). We used representative mesothelioma cells in terms of the TP53 genotype, NCI-H28, MSTO-211H and NCI-H226 as for wild-type TP53 cells, and EHMES-1 and JMN-1B as for mutated TP53 cells. All the wild-type TP53 cells showed up-regulation of p53 levels and the phosphorylation. MSTO-211H and NCI-H226 cells down-regulated FAK phosphorylation levels but NCI-H28 cells remained unchanged in the FAK phosphorylation. Levels of phosphorylated H2AX at Se 139, a marker for DNA damage, were variable with nutlin-3a doses in the wild-type TP53 cells and the expression levels were not dose-dependent. These data suggested that p53 up-regulation by nutlin-3a was not due to DNA damage but probably due to inhibition of the MDM2 activity [13]. Mesothelioma with mutated TP53 showed different responses. Nutlin-3a-treated JMN-1B and EHMES-1cells increased the p53 level and the phosphorylated level, respectively. FAK and the phosphorylation levels were down-regulated in JMN-1B cells but those were unchanged in EHMES-1 cells. Phosphorylated H2AX levels remained constant in both mutated TP53 cells. These data collectively indicated that nutrin-3a augmented p53 expression in wild-type and mutated TP53 cells with a different manner and that the association of the p53 increase with inhibited FAK phosphorylation was dependent on cells irrespective of the TP53 genotype. In addition, nutlin-3a-mediated p53 increase and FAK dephosphorylation were not linked with MERLIN expression levels.

Growth inhibitory effects of RITA were irrelevant to TP53 genotype

We examined growth inhibitory effects of RITA, another type of the MDM2 inhibitors, with the WST assay (Fig 4A). The IC₅₀ values of mesothelioma with the wild-type TP53 genotype excluding EHMES-10 (2.50 ± 1.88 μM) were not different from those of cells with mutated TP53 and non-functional p53 (1.67 ± 1.21) (P = 0.73). EHMES-10 cells were resistant to RITA and nutlin-3a as mentioned above. Susceptibility of mesothelioma to RITA was thus dependent on the cells tested and was not associated with the TP53 genotype or with the p53 functional status.

We then examined p53 and FAK expression in RITA-treated cells with western blot analysis (Fig 4B, S6 Table). Expression levels of both p53 or the phosphorylation increased in the wild-type and mutated TP53 cells, but EHMES-1 cells decreased the p53 level despite enhanced p53 phosphorylation. Phosphorylated p53 at Ser 15 was thus not always linked with augmented p53 levels in the mutated TP53 cells as found in nutrin-3a-treated mutated TP53 cells. All the RITA-treated cells showed decreased FAK phosphorylation and increased H2AX phosphorylation levels, with the exception of MSTO-211H cells exhibiting a minor H2AX increase. These data suggested that DNA damage responses contributed to RITA-mediated p53 up-regulation and that increased p53 phosphorylation was linked with FAK dephosphorylation irrespective of the TP53 genotype.

Combinatory effects of defactinib and MDM2 inhibitors

We investigated a possible combinatory effect produced by defactinib and the MDM2 inhibitors with the WST assay. We treated mesothelioma cells with defactinib at a concentration of about 10−30% growth inhibition together with various doses of nutlin-3a (Fig 5) or RITA (Fig 6). The defactinib treatment further enhanced nutlin-3a-induced growth suppression even in nutlin-3a-resistant cells including those with mutated TP53 genotype and with truncated p53, and EHMES-10 cells (Fig 5A). Further investigations showed that the majority of CI values at Fa points between 0.25 and 0.75 was below or close to 1 in nutlin-3a-sensitive wild-type TP53 mesothelioma, whereas the corresponding CI values in 2 kinds of mutated TP53 mesothelioma cells were above 1 (Fig 5B). These data indicated that wild-type TP53 cells sensitive to nutlin-3a showed synergistic or additive growth inhibition with the FAK inhibitor but mutated TP53 cells rather showed antagonistic effects. In contrast, CI values of NCI-H2452 cells with truncated p53 were dependent on Fa points and those of nutlin-3a-resistant EHMES-10 were close to 1. We also examined growth inhibitory effects of defactinib in combination with RITA (Fig 6A). The defactinib treatment augmented RITA-induced growth suppression in all the cells tested. The CI values at the Fa points between 0.25 and 0.75 were below or about 1 in all the cells except NCI-H226 and NCI-H2452 cells, both of which showed most of CI values above 1 (Fig 6B). The CI value data indicated that the combination produced synergistic effects irrespective of the TP53 genotype but the synergism was dependent on cells used. The combination of defactinib and the MDM2 inhibitors thereby achieved synergistic or additive effects in most of wild-type TP53 cells, but the combination effects tested in mutated TP53 or non-functional p53 cells were dependent on the inhibitor and the cells tested.

We then examined molecular changes produced by the combination of defactinib and the MDM2 inhibitors in representative TP53 wild-type and mutated cells (Fig 7, S7 Table). We treated the cells with defactinib and the MDM2 inhibitors at a concentration of a little lower than the respective IC₅₀ values in the growth inhibition study to evaluate synergistic or additive effects. (Fig 2A, 3A and 4A, see the Fig 7 legend). We examined RITA only for mutated TP53 cells in the combination because the mutated cells were resistant to nutlin-3a and a high concentration of nutlin-3a could produce non-specific molecular changes. The defactinib treatment decreased FAK phosphorylation in all the cells irrespective of the TP53 genotype, and the combination with nutlin-3a did not further enhance the FAK dephosphorylation in TP53 wild-type cells. A combination with RITA however further down-regulated FAK phosphorylation in NCI-H28 and TP53 mutated cells, and the FAK phosphorylation level in these cells was lower in the combination than in a treatment with RITA or defactinib alone. The synergistic combinatory effects of defactinib and RITA on growth inhibition was therefore associated with decreased FAK activity in some mesothelioma cells, but the effects of defactinib and nutlin-3a were not directly relevant to FAK dephosphorylation in wild-type TP53 cells. Nutrin-3a-treated wild-type TP53 cells showed up-regulated p53 and the phosphorylation but the combination with defactinib did not further enhance the expressions except the p53 level in MSTO-211H cells. NCI-H226 cells showed that the combination with defactinib rather decreased the p53 expression. RITA-treated wild-type TP53 cells increased p53 and the phosphorylation, but additional defactinib did not further augment the expression levels. RITA-treated mutated TP53 cells up-regulated p53 phosphorylation, but the combination with defactinib showed different responses; decreased and unchanged levels in EHMES-1 and JMN-1B cells, respectively. The MDM2 inhibitors augmented p53 and the p53 phosphorylation, but the combination of defactinib did not always contribute to the p53 up-regulation; consequently, the p53 regulations by MDM2 inhibitors were not influenced by inhibited FAK phosphorylation.

Fig 7 — Cells were treated as follows for 24 hrs and the cell lysate were subjected to western blot analysis, which included expression levels of a phosphorylated form of FAK, p53 and AKT, and a cleaved form of PARP. We selected concentrations lower than the IC₅₀ values of each single agent to detect the molecular changes induced by the combination. Concentrations of defactinib, nutrin-3a and RITA used were as follows. NCI-H28: 2 μM defactinib, 4 μM nutlin-3a, 0.04 μM RITA; MSTO-211H: 2 μM defactinib, 1 μM nutlin-3a, 1 μM RITA; NCI-H226: 2 μM defactinib, 2 μM nutlin-3a, 0.02 μM RITA; EHMES-1: 2 μM defactinib, 0.25 μM RITA; JMN-1B: 2 μM defactinib, 1.5 μM RITA. Actin was used as a loading control. The expression of each molecule was quantified with ImageJ software (NIH, Bethesda, MD, USA) with actin intensity as a normalized control (see S7 Table).

Defactinib treatments decreased AKT phosphorylation in all the cells except MSTO-211H cells, but additional nutlin-3a did not further down-regulate the phosphorylation in wild-type TP53 cells except MSTO-211H cells. RITA did not influence AKT phosphorylation in wild-type or mutated TP53 cells except JMN-1B cells. The combination with defactinib and RITA further decreased the phosphorylation in NCI-H28 and slightly in mutated TP53 cells. PARP molecules were cleaved by defactinib or nutlin-3a, and the combination did not enhance the cleavages. Combinatory treatments of RITA and defactinib increased PARP cleavage more than a treatment with RITA or defactinib alone regardless of the TP53 genotype except in NCI-H226 cells. These data suggested that synergistic growth inhibitory effects in the combination of defactinib and nutlin-3a were not directly associated with FAK dephosphorylation, p53 phosphorylation or AKT dephosphorylation levels in wild-type TP53 cells. The combination of defactinib and RITA achieved synergistic effects in the majority of wild-type and mutated TP53 cells, and the effects were in general associated with down-regulated FAK and AKT phosphorylation, and apoptotic cell death induced.

Discussion

We showed in this study that susceptibility of mesothelioma to defactinib was not associated with MERLIN expression or the TP53 genotype, and demonstrated that a combination of defactinib and an MDM2 inhibitor produced synergistic or additive effects on cell growth inhibition in a manner determined by the TP53 status and the inhibitor type.

FAK plays a key role in adhesion of cancer cells to the extracellular matrix, and is consequently related with a metastatic ability and stem cell-like property of the cancer. Mesothelioma often had mutated NF2 gene and loss of MERLIN functions, and the cells of low-MERLIN expression were sensitive to defactinib since MERLIN-deficient cells were more dependent on FAK for the cell growth than MERLIN-high cells [8]. Nevertheless, the present study demonstrated that the sensitivity to defactinib was not linked with MERLIN expression levels. Kato et al. reported that E-cadherin but not MERLIN expression influenced sensitivity to a FAK inhibitor (VS-4718) in mesothelioma [9] and Blum et al. demonstrated that the susceptibility of cancer stem cell-enriched mesothelioma cells to defactinib was independent of MERLIN expression [24]. The present study showed that E-cadherin-low NCI-H28 and MSTO-211H cells as demonstrated in the E-cadherin study [9] were not particularly sensitive to defactinib compared with other cells (Fig 2A). The defactinib sensitivity was affected by cell adhesion and the downstream signaling of adhesion molecules, but a number of the molecules contribute to the adhesions and the signal processes. The adhesion is also influenced by the nature of extracellular matrix components in vivo. Sensitivity of mesothelioma to the FAK inhibitors is thus subjected to many factors such as the inhibitor types, and is also dependent on experimental systems in vitro and on local environmental milieux in vivo.

We further examined an effect of defactinib on p53 and MDM2 expression. The current study showed that dephosphorylation of FAK was in general associated with p53 up-regulation or the phosphorylation, but not with MDM2 and the phosphorylation levels. In addition, mutated p53 was known to be resistant to MDM2-mediated degradation [25]. We then presumed that the p53 up-regulation by defactinib was not attributable to the MDM2-mediated effects. The present data however did not deny a possible reciprocal regulation between FAK and p53 expression levels [18,19]. On the other hand, MDM2 by itself had another function to activate p53 at the transcriptional levels besides ubiquitination of p53, indicating a dual action of MDM2 on p53 expression levels [26]. Furthermore, Kim et al. demonstrated MERLIN can facilitate MDM2 degradation and subsequent augmentation of p53 expression [20]. We also found that defactinib-mediated dephosphorylation of FAK was linked with AKT dephosphorylation. Ammoun et al. showed that a MERLIN-deleted condition induced phosphorylated AKT and activated MDM2 with subsequent decrease of p53 [27]. This study however demonstrated that the AKT dephosphorylation was not influenced by MERLIN expression but suggested that he dephosphorylation was rather attributable to defactinib-induced growth inhibition since AKT phosphorylation was an active marker for cell growth [28]. These data collectively indicated that defactinib-mediated growth suppression was irrelevant to MERLIN expression, but closely associated with AKT dephosphorylation and generally with p53 augmentation. A previous study indicated that FAK inhibition induced DNA damage and augmented sensitivity to irradiation [29]. We did not precisely examine the DNA damage induction by defactinib since our experimental design was different from the previous study. We however showed that a FAK inhibitor increased susceptibility to a p53-activating DNA damage agent [21] and presume that induction of DNA damage by a FAK inhibitor is the next issue to be investigated.

A combinatory treatment of defactinib and nutlin-3a produced synergistic or additive inhibitory effects on growth of wild-type but not mutated TP53 cells. Since nutlin-3a did not produce DNA damage response, the nutrin-3a-induced p53 up-regulation and subsequent cell growth inhibition was probably due to inhibition of the p53-MDM2 binding. The nutlin-3a-mediated FAK dephosphorylation was not always linked with p53 phosphorylation or with TP53 genotype. These data again showed that reciprocal inhibition between p53 and FAK dephosphorylation was dependent on cell types irrespective of the TP53 genotype. RITA achieved growth inhibitory effects on mesothelioma regardless of TP53 genotype and a mechanism of the inhibition was different from that of nutlin-3a. Both agents were developed as an inhibitor for p53 and MDM2 interactions, but RITA additionally induced DNA damage responses. Several studies showed that RITA produced cytotoxic effects even in mutated TP53 cells and the susceptibility to RITA was linked with DNA damage [30,31]. Furthermore, the present study suggested that down-regulated FAK phosphorylation irrespective of TP53 genotype was attributable to the damage. A previous study also showed that inhibition of FAK activity induced DNA damage responses [29] but it remains unknown whether the RITA-induced DNA damage directly influenced FAK activity. Our study implied that RITA-induced DNA damage was associated with a reciprocal linkage between p53 phosphorylation and FAK dephosphorylation rather than the inhibition of the p53-MDM2 interaction. Chk2 can be involved in RITA-mediated DNA damage since Chk2-defective cells were resistant to RITA- but not nutlin-3a-mediated growth inhibition [32]. We speculate that the ATM-Chk2 pathway contributes to RITA-mediated activation of p53 and subsequent growth suppression. Our previous data showed that a DNA-damaging agent achieved combinatory cytotoxicity with defactinib in wild-type TP53 mesothelioma cells through down-regulated FAK phosphorylation and p53 up-regulation [21]. Ou et al. also reported that combination of nutlin-3a and shRNA for FAK produced additive combinatory effects on growth inhibition of wild-type TP53 mesothelioma cells through activation of p53 pathways [33]. The present results were consistent with those previous studies and supported the reciprocal regulation between FAK and p53.

Mesothelioma has histologically 3 types, epithelioid, sarcomatoid, and biphasic, and these subtypes have different TP53 genetic alterations. Epithelioid subtype tends to retain wild-type genotype, whereas sarcomatoid and biphasic types are more likely to harbor the mutations. The present study therefore indicated that the combination of nutlin-3a and defactinib was effective for the epithelioid subtype, while the combination of RITA and defactinib was more suitable for sarcomatoid and biphasic types. Moreover, the sensitivity to defactinib was not linked with the histological types. On the other hand, recent studies have demonstrated that immune checkpoint inhibitors and antiangiogenic agents achieved good clinical effects [34,35]. Expression levels of PD-L1 and VEGF (vascular endothelial growth factor) in mesothelioma in vivo can be one of the markers to predict the therapeutic efficacy. Regulation of PD-L1 or VEGF expression through the p53 and FAK signaling pathways also suggests that a possible combination of FAK/MDM2 inhibitors with an immune checkpoint inhibitor or an antiangiogenic agent may represent next directions to pursue [36,37]. In addition, these small molecule agents need to show high affinity binding to the targets, extend the half-life and enhance the tissue distribution in order to minimize off-target risks and to improve clinical feasibilities. Recent pharmaceutical and clinical developments may enable such nanomolar-level potency in FAK and MDM2 inhibitors [38–40] and facilitate personalized medicine based on patient stratification.

The present study is a preclinical proof-of-concept to explore the synergy between p53 stabilization and FAK inhibition. We showed that a combination of defactinib and a MDM2 inhibitor achieved synergistic or additive inhibitory effects on mesothelioma cell growth, and a potential contribution of FAK and p53 signal pathways to the suppressive effects was associated with selection of MDM2 inhibitors and the TP53 genotypes (S1B Fig).

Supporting information

S1 Fig

(A) Defactinib-mediated inhibition of mesothelioma cell proliferation with a dye exclusion assay. Cells were seeded in 6 well-plates and treated with defactinib for 24–72 hrs. They were then stained with 0.4% trypan blue solution (Sigma-Aldrich, St. Louis, MO) for 3 minutes at room temperature. The number of stained and unstained cells was counted and the assay were tested in triplicate. (B) A schema of the present study. Mesothelioma cells often have deletion of INK4A/ARF region and MERLIN inactivation. These characteristics induce up-regulated MDM2 expression with subsequent p53 down-regulation. An MDM2 and a FAK inhibitor can augment p53 expression and induce growth inhibition. The current study indicated that nutlin-3a and RITA, a representative MDM2 inhibitor, up-regulated p53 expression via a different mechanism and that the growth inhibition by defactinib, a FAK inhibitor, was unrelated MERLIN expression. A combination of the MDM2 inhibitor and the FAK inhibitor achieved synergistic or additive inhibitory effects, and the effects were linked with the AKT signaling. Nevertheless, the growth inhibitory activity was also subjected to the kind of the MDM2 inhibitor and cells used.

(PDF)

pone.0343551.s001.pdf^{(250.7KB, pdf)}

S2 Fig. (Figure 1 all).

Original blots which were used for Fig 1. Arrows indicate the target molecules. The name of cells was shown in abbreviations. H28: NCI-H28, 211H: MSTO-211H, H2052: NCI-H2052, H226: NCI-H226, H2452: NCI-H2452. EH-10: EHMES-10, EH-1: EHMES-1.

(PDF)

pone.0343551.s002.pdf^{(116.4KB, pdf)}

S3 Fig. (Figure 2B FAK).

Original blots which were used for Fig 2B FAK expression. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. We did not use a photo of 8 μM defactinib treatments. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s003.pptx^{(989.5KB, pptx)}

S4 Fig. (Figure 2B p-FAK).

Original blots which were used for Fig 2B phosphorylated FAK expression. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. We did not use a photo of 8 μM defactinib treatments. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s004.pptx^{(1.3MB, pptx)}

S5 Fig. (Figure 2B p53).

Original blots which were used for Fig 2B p53 expression. Arrows indicate the target molecules (NCI-H2452 had a truncated p53). The name of cells was shown in the abbreviations.

(PPTX)

pone.0343551.s005.pptx^{(809.1KB, pptx)}

S6 Fig. (Figure 2B p-p53).

Original blots which were used for Fig 2B phosphorylated p53 expression. Arrows indicate the target molecules (NCI-H2452 had a truncated p53). The name of cells was shown in the abbreviations. We did not use a photo of 8 μM defactinib treatments. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s006.pptx^{(1.7MB, pptx)}

S7 Fig. (Figure 2B AKT).

Original blots which were used for Fig 2B AKT expression. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. We did not use a photo of 8 μM defactinib treatments.

(PPTX)

pone.0343551.s007.pptx^{(783.1KB, pptx)}

S8 Fig. (Figure 2B p-AKT).

Original blots which were used for Fig 2B phosphorylated AKT expression. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. We did not use a photo of 8 μM defactinib treatments. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s008.pptx^{(1.4MB, pptx)}

S9 Fig. (Figure 2B MDM2).

Original blots which were used for Fig 2B MDM2 expression. Arrows indicate the target molecules (both 90 kDa and 60 kDa molecules). The name of cells was shown in the abbreviations.

(PPTX)

pone.0343551.s009.pptx^{(1.8MB, pptx)}

S10 Fig. (Figure 2B p-MDM2).

Original blots which were used for Fig 2B phosphorylated MDM2 expression. Arrows indicate the target molecules (90 kDa). The name of cells was shown in the abbreviations. We did not use a photo of 8 μM defactinib treatments.

(PPTX)

pone.0343551.s010.pptx^{(1.4MB, pptx)}

S11 Fig. (Figure 2B Caspase-9).

Original blots which were used for Fig 2B caspase-9 and the cleaved caspase-9 expressions. The antibody detected the cleaved form. Arrows indicate the target molecules (both original and cleaved molecules). The name of cells was shown in the abbreviations. We did not use a photo of 8 μM defactinib treatments. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s011.pptx^{(1.6MB, pptx)}

S12 Fig. (Figure 2B PARP).

Original blots which were used for Fig 2B PARP and the cleaved PARP expressions. The antibody detected the cleaved form. Arrows indicate the target molecules (both original and cleaved molecules). The name of cells was shown in the abbreviations. We did not use a photo of 8 μM defactinib treatments. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s012.pptx^{(1.3MB, pptx)}

S13 Fig. (Figure 2B Tubulin).

Original blots which were used for Fig 2B tubulin expression. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. We did not use a photo of 8 μM defactinib treatments.

(PPTX)

pone.0343551.s013.pptx^{(732.3KB, pptx)}

S14 Fig. (Figure 3B p53 and p-p53).

Original blots which were used for Fig 3B p53 and phosphorylated p53 expressions. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s014.pptx^{(931.3KB, pptx)}

S15 Fig. (Figure 3B FAK and p-FAK).

Original blots which were used for Fig 3B FAK and phosphorylated FAK expressions. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s015.pptx^{(1.1MB, pptx)}

S16 Fig. (Figure 3B p-H2AX).

Original blots which were used for Fig 3B p-H2AX expression. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. We did not use a photo of 15 μM nutlin-3a treatments. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s016.pptx^{(1,009.6KB, pptx)}

S17 Fig. (Figure 3B Actin).

Original blots which were used for Fig 3B actin expression. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. We did not use a photo of 15 μM nutlin-3a treatments.

(PPTX)

pone.0343551.s017.pptx^{(791.4KB, pptx)}

S18 Fig. (Figure 4B p53 and p-p53).

Original blots which were used for Fig 4B p53 and phosphorylated p53 expressions. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s018.pptx^{(807.9KB, pptx)}

S19 Fig. (Figure 4B FAK and p-FAK).

Original blots which were used for Fig 4B FAK and phosphorylated FAK expressions. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s019.pptx^{(985.2KB, pptx)}

S20 Fig. (Figure 4B p-H2AX and Actin).

Original blots which were used for Fig 4B p-H2AX and actin expressions. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. We did not use a photo of 6 μM RITA treatments. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s020.pptx^{(1.3MB, pptx)}

S21 Fig. (Figure 7 FAK).

Original blots which were used for Fig 7 FAK expression. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. Abbreviations. NT: no treatment shown as (-) in Fig 7, Def: defactinib, Nut: nutlin-3a, N + D: nutlin-3a+defactinib, R + D: RITA+defactinib.

(PPTX)

pone.0343551.s021.pptx^{(641.6KB, pptx)}

S22 Fig. (Figure 7 p-FAK).

Original blots which were used for Fig 7 phosphorylated FAK expression. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. Abbreviations used for treatment were shown in S21 Fig.

(PPTX)

pone.0343551.s022.pptx^{(743.9KB, pptx)}

S23 Fig. (Figure 7 p53).

Original blots which were used for Fig 7 p53 expression. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. Abbreviations used for treatment were shown in S21 Fig.

(PPTX)

pone.0343551.s023.pptx^{(701.4KB, pptx)}

S24 Fig. (Figure 7 p-p53).

Original blots which were used for Fig 7 phosphorylated p53 expression. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. Abbreviations used for treatment were shown in S21 Fig. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s024.pptx^{(754.6KB, pptx)}

S25 Fig. (Figure 7 AKT and p-AKT).

Original blots which were used for Fig 7 AKT and phosphorylated AKT expressions. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. Abbreviations used for treatment were shown in S21 Fig. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s025.pptx^{(1MB, pptx)}

S26 Fig. (Figure 7 PARP).

Original blots which were used for Fig 7 PARP and cleaved PARP expressions. The antibody detected the cleaved form. Arrows indicate the target molecules (both original and cleaved molecules). The name of cells was shown in the abbreviations. Abbreviations used for treatment were shown in S21 Fig. In some of the blots, we used the same blot to detect others molecules without stripping the blot and consequently showed the target molecules by the arrows.

(PPTX)

pone.0343551.s026.pptx^{(927.2KB, pptx)}

S27 Fig. (Figure 7 Actin).

Original blots which were used for Fig 7 actin expression. Arrows indicate the target molecules. The name of cells was shown in the abbreviations. Abbreviations used for treatment were shown in S21 Fig.

(PPTX)

pone.0343551.s027.pptx^{(518.4KB, pptx)}

S1 Table. Information of cells used in the study.

(DOCX)

pone.0343551.s028.docx^{(20.2KB, docx)}

S2 Table. Information of antibody used in the study.

Dilution of primary antibody and information of secondary antibody used in the study.

(DOCX)

pone.0343551.s029.docx^{(17.3KB, docx)}

S3 Table. Molecular expression levels in Fig 1.

Expression of the molecules in Fig 1 was quantified with ImageJ software (NIH, Bethesda, MD, USA). The intensity of target protein bands was normalized to the intensity of tubulin as a loading control. Respective protein expression levels of NCI-H28 were used as a standard (expressed as 1.00).

(DOCX)

pone.0343551.s030.docx^{(17.2KB, docx)}

S4 Table. Molecular expression levels in Fig 2B.

Expression of the molecules in Fig 2B was quantified with ImageJ software (NIH, Bethesda, MD, USA). The intensity of target protein bands was normalized to the intensity of tubulin as a loading control. Respective protein expression levels of untreated cells were used as a standard (expressed as 1.00).

(DOCX)

pone.0343551.s031.docx^{(26.6KB, docx)}

S5 Table. Molecular expression levels in Fig 3B.

Expression of the molecules in Fig 3B was quantified with ImageJ software (NIH, Bethesda, MD, USA). The intensity of target protein bands was normalized to the intensity of actin as a loading control. Respective protein expression levels of untreated cells were used as a standard (expressed as 1.00).

(DOCX)

pone.0343551.s032.docx^{(19.7KB, docx)}

S6 Table. Molecular expression levels in Fig 4B.

Expression of the molecules in Fig 4B was quantified with ImageJ software (NIH, Bethesda, MD, USA). The intensity of target protein bands was normalized to the intensity of actin as a loading control. Respective protein expression levels of untreated cells were used as a standard (expressed as 1.00).

(DOCX)

pone.0343551.s033.docx^{(19.7KB, docx)}

S7 Table. Molecular expression levels in Fig 7.

Expression of the molecules in Fig 7 was quantified with ImageJ software (NIH, Bethesda, MD, USA). The intensity of target protein bands was normalized to the intensity of actin as a loading control. Respective protein expression levels of untreated cells were used as a standard (expressed as 1.00).

(DOCX)

pone.0343551.s034.docx^{(23KB, docx)}

Abbreviations

NF2: neurofibromatosis type 2
FAK: focal adhesion kinase
RITA: reactivation of p53 and induction of tumor cell apoptosis (RITA)
IC₅₀: half maximal inhibitory concentration
CI: combination index
Fa: fractions affected
SE: standard error
VEGF: vascular endothelial growth factor.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was supported by Grants-in-Aid from Japan Society for the Promotion of Science (JSPS KAKENHI Grant number: 21K08199) to MT and a Grant-in-aid from the Nichias Corporation to KH and MT. The fund bodies were not involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. We obtained a grant from Nichias Corporation. It is not a pharmaceutical company but a company making industrial products for building, automobiles and pipes (see http://www.nichias.co.jp/). The grant is as a kind of their mécénat activities, corporate social contributions, which is aimed to assist for medical research for intractable cancer treatments. We are thereby irrelevant to any employments, consultancy, patents or products in development or marketed products of the company.

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PLoS One. doi: 10.1371/journal.pone.0343551.r001

Decision Letter 0

Zu Ye

26 Sep 2025

Dear Dr. Tagawa,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: No

**********

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The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors investigated the combination effect of FAK and MDM2 inhibitors using mesothelioma cell lines. They showed that FAK inhibitor defactinib inhibited cell growth and induced FAK dephosphorylation irrespective of the TP53 genotype, and that the inhibited FAK phosphorylation was not associated with MERLIN levels or with p53 up-regulation but linked with AKT dephosphorylation. This article may include some interesting findings. However, the following issues should be addressed.

#1. NCI-H226 is used as a mesothelioma cell line. The website of ATCC shows that this line exhibits epithelial morphology that was isolated from the lungs of a male with squamous cell carcinoma. The website of cBioPortal also shows that the type of this line is lung squamous cell carcinoma. The authors should confirm that what type of cell line NCI-H226 is.

#2. The authors concluded that a combination of defactinib and the MDM2 inhibitors in general produced synergistic or additive effects to inhibit growth of mesothelioma. However, as they describe, these effects were different depending on MDM2 inhibitors (nultin-3a or RITA) and cell lines tested. This study lacks impact.

#3. The part of “Discussion” is redundant.

Reviewer #2: Mesothelioma has characteristic genetic changes including inactivation of neurofibromatosis type 2 (NF2) and deletion of the INK4A/ARF region. This study analyzed the combinatory effects of the FAK and MDM2 inhibitors and that consequent signaling pathway changes. The results showed that combination of FAK inhibitor defactinib and the MDM2 inhibitors in general produced synergistic or additive effects to inhibit growth of mesothelioma, but these effects were influenced by MDM2 inhibitors and cell lines tested. However, there are some concerns as follows:

1. The FAK inhibitor defactinib and the MDM2 inhibitor nutlin-3a were at the micromolar scale, however, the small molecular inhibitor drugs are usually at 10-20 nanomole scale. How about the future drug development?

2. The combination effects were mutation and cell line specific. How to combine the findings of this study with the subgroup mesothelioma patients?

3. The immune check point inhibitors have made great success in the treatment of mesothelioma patients. How about the influence of both FAK and MDM2 inhibitors to the expression level of PD-L1 in mesothelioma cells?

4. The antiangiogenic therapy has been proved valuable in relieving pleural effusion. How about the influence of both FAK and MDM2 inhibitors to the expression level of VEGF in mesothelioma cells?

5. There is no proper statistics in the figures and figure legends. All the WB results should have statistics of gray values.

**********

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Reviewer #2: No

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PLoS One. 2026 Feb 23;21(2):e0343551. doi: 10.1371/journal.pone.0343551.r002

Author response to Decision Letter 1

12 Nov 2025

Replies to Reviewers

(A) Comments from Reviewer #1

(1) NCI-H226 is used as a mesothelioma cell line. The website of ATCC shows that this line exhibits epithelial morphology that was isolated from the lungs of a male with squamous cell carcinoma. The website of cBioPortal also shows that the type of this line is lung squamous cell carcinoma. The authors should confirm that what type of cell line NCI-H226 is.

[Reply]

Thank you very much for your comment. The ATCC website also showed that this cell line was mesothelioma in addition to lung carcinoma. As mentioned by the reviewer, cBioPortal showed that the cell line was classified as lung carcinoma. The Biohippo and the Cellosaurus websites however mentioned that NCI-H226 was mesothelioma cell line (pleural epithelioid mesothelioma). The confusion or the ambiguity came from several reasons. For example, the cells were derived from pleural effusion of a lung cancer patient and histology of the two cancer types was overlapped. Nevertheless, genetic markers supported that NCI-H226 cells were rather mesothelioma. NCI-H226 cells showed wild-type TP53 genotype, deletion of the INK4A/ARF region and mutated NF2 gene (according to Cancer Cell Line Encyclopedia, Catalogue of Somatic Mutations in Cancer, and Cancer Dependency Map). Moreover, NCI-H226 cells strongly positive for mesothelin which was a characteristic protein marker of mesothelium and mesothelioma, but non-small cell lung cancer was negative for mesothelin. While some studies classified the NCI-H226 cell line as lung carcinoma based on sources like ATCC, many others used it as a model for mesothelioma. Given its genetic profile and mesothelin expression, we consider it more appropriate to regard NCI-H226 as a mesothelioma cell line, in line with the majority of previous publications.

(2) The authors concluded that a combination of defactinib and the MDM2 inhibitors in general produced synergistic or additive effects to inhibit growth of mesothelioma. However, as they describe, these effects were different depending on MDM2 inhibitors (nultin-3a or RITA) and cell lines tested. This study lacks impact.

[Reply]

Thank you for your comment. We understand the concern that the combinatory effects varied and were to some extent dependent on the type of MDM2 inhibitors and the mesothelioma cell lines used, which may reduce the overall impact of the study. Initially, we considered focusing on nutlin-3a in wild-type p53 cells to simplify our conclusions. However, we expanded the study to include RITA and mutated p53 cells to explore the broader applicability of the combination. While both nutlin-3a and RITA inhibit the MDM2-p53 interaction, RITA also induces DNA damage, which can elevate p53 levels independently. The combinatory effects were therefore influenced by the inhibitors. Our results showed that both MDM2 inhibitor and defactinib produced synergistic or additive inhibitory effects in most of wild-type p53 cells except for NCI-H226 cells treated at a low RITA concentration. In mutated p53 cells treated with nutlin-3a, the combinatory effects were antagonistic. In the case of RITA-treated cells, the combination produced synergistic effects in irrespective of the TP53 genotype except NCI-226 cells as mention above and H2452 cells with truncated p53.

Furthermore, the effects of MDM2 inhibitors on mutated p53 is influenced by the p53 structure and the MDM2-mediated effects could be altered in mutated p53 cells. Even if the MDM2 inhibitors increases expression levels of mutated p53, the up-regulation would rather support for cell survival but not growth inhibition. The combinatory effects in mutated p53 cells could be therefore different from those in wild-type p53 cells. Nevertheless, the combination of RITA and defactinib achieved synergistic effects in mutated p53 cells. On the other hand, NCI-H2452 cells with truncated p53 and EHMES-10 cells with resistance to nutrin-3a and RITA showed different responses from wild-type p53 cells. The extended experiments thus revealed that the combinatory effects were influenced by MDM2 inhibitors and cells used although the combination produced synergistic or additive effects in most of the cases. We have revised the manuscript to clarify our conclusions and better reflect the scope and limitations of the study. Please see the revised part on page 4-5 line 67-71, on page 8 line 110-111 in unmarked manuscript, and Discussion in marked manuscript.

(3) The part of “Discussion” is redundant.

[Reply]

Thank you very much for your comment. We have revised the Discussion section to enhance clarity and eliminate redundancy. Please refer to the revised parts on page 22-27 in marked manuscript.

(B) Comments from Reviewer #2

(1) The FAK inhibitor defactinib and the MDM2 inhibitor nutlin-3a were at the micromolar scale, however, the small molecular inhibitor drugs are usually at 10-20 nanomole scale. How about the future drug development?

[Reply]

Thank you very much for your insightful point. As the reviewer pointed out, small-molecule inhibitor drugs used in clinical settings typically exhibit efficacy at nanomolar concentrations, whereas the compounds used in our study were applied at micromolar levels. The present study is preclinical and intended to demonstrate proof-of-concept to explore a strategy for up-regulating endogenous wild-type p53 levels. While some molecular-targeted agents are effective at micromolar concentrations, such doses may increase off-target risks in clinical applications. Future drug development should therefore aim for high target affinity binding, extended half-life and enhanced tissue distribution to improve clinical feasibilities. We added a statement regarding this possible future direction in Discussion of the revised manuscript (See on page 27 line 433-436 with references, No 38 and 39 in unmarked manuscript).

(2) The combination effects were mutation and cell line specific. How to combine the findings of this study with the subgroup mesothelioma patients?

[Reply]

Thank you very much for your valuable comment. The reviewer referred to the histological subtypes of mesothelioma-epithelioid, sarcomatoid, and biphasic-as well as their associated genetic alterations. In general, the epithelioid subtype tends to retain wild-type TP53 genotype, whereas sarcomatoid and biphasic types are more likely to harbor TP53 mutations; consequently, the epithelioid subtype is more susceptible to combination therapy aimed at up-regulating wild-type p53. On the other hand, RITA increases susceptibility to the combination even in cells with mutated p53. Based on our findings, we suggest that the combination of nutlin-3a and defactinib is effective for the epithelioid subtype, while the combination of RITA and defactinib is more suitable for sarcomatoid and biphasic types. We discussed these possible relationships between histological subtypes and combination effects in the revised manuscript (page 26-27 line 422-427 in unmarked manuscript). In addition, sensitivity to defactinib was not linked to TP53 mutation status (page 27 line 427 in unmarked manuscript).

(3) The immune check point inhibitors have made great success in the treatment of mesothelioma patients. How about the influence of both FAK and MDM2 inhibitors to the expression level of PD-L1 in mesothelioma cells?

[Reply]

Thank you very much for highlighting the importance of immune checkpoint inhibitors in mesothelioma treatment. We fully agree with the reviewer’s point. Although PD-L1 expression has been widely studied, its predictive value in mesothelioma patients receiving immune checkpoint inhibitors remains inconclusive at this moment as far as we are aware. Nevertheless, FAK inhibition may reduce PD-L1 expression and p53 is known to down-regulates PD-L1 expression. We therefore believe that a potential combination of FAK inhibitors, MDM2 inhibitors and immune check point inhibitors represents an interesting research direction. We however presume that investigating PD-L1 expression following treatment with FAK and MDM2 inhibitors will be difficult to include in this study since it may fall outside the scope of the present study. Such analyses may be the next investigation because it requires separate, dedicated studies. We hope that the future studies, possibly including in vivo models or patient-derived samples, will clarify the impact of these inhibitors on PD-L1 expression and their potential synergy with immune checkpoint therapies. We appreciate the reviewer’s suggestion and have included this point in the revised Discussion section on page 27 line 428-433 with references in unmarked manuscript.

(4) The antiangiogenic therapy has been proved valuable in relieving pleural effusion. How about the influence of both FAK and MDM2 inhibitors to the expression level of VEGF in mesothelioma cells?

[Reply]

Thank you very much for your comment. As the reviewer mentioned, recent studies indicated that inhibition of angiogenesis was effective, particularly when combined with chemotherapy and immunotherapy. Nevertheless, the clinical value of VEGF expression as a predictive marker remains unclear at this time. On the other hand, FAK is involved in VEGF regulation and angiogenesis, and p53 up-regulation can negatively regulate VEGF transcription. These findings suggest that FAK and MDM2 inhibitors can potentially enhance efficacy of the antiangiogenesis therapy. We however believe that analyzing the VEGF expression following the treatments is important but may fall beyond the scope of the current work at this moment. We consider this a promising direction for future research and have mentioned the potential combination with antiangiogenesis therapy in revised Discussion section (page 27 line 428-433 and references in unmarked manuscript).

(5) There is no proper statistics in the figures and figure legends. All the WB results should have statistics of gray values.

[Reply]

We appreciate the reviewer’s comment regarding statistical analysis of the Western blot data. In our study, we performed densitometric quantification using ImageJ and normalized each band to the corresponding loading control. We think at this moment that it is unfortunately not feasible to repeat all the analyses from the original materials due to the large number of samples. We conducted multiple Western blot experiments to obtain clear and consistent data, and selected representative results for presentation in the figures. We therefore are confident in the accuracy and reproducibility of the data. While we acknowledge that statistical testing across biological replicates is ideal, the scope and limitations of our dataset make this approach challenging. We have clarified this point in the revised manuscript and added a note in the figure legend (Figure 1, 2B, 3B, 4B, 7B) to explain the methodology. We hope this explanation addresses the reviewer’s concern and respectfully request understanding given the constraints of our study.

Attachment

Submitted filename: Comments for reviewers 2025-11-5.docx

pone.0343551.s036.docx^{(44.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0343551.r003

Decision Letter 1

Zu Ye

22 Dec 2025

Dear Dr. Tagawa,

Please submit your revised manuscript by Feb 05 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Zu Ye, Ph.D.

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #3: No

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #3: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #3: Yes

**********

Reviewer #1: The authors revised the manuscript based on the comments by the reviewers and the manuscript became much better.

Reviewer #3: This is a systematic but incremental in vitro study with some remaining significant methodological and analytical concerns. Is not that easy to draw sound valid conclusions from these experimental schemes. On the good side it is using a comprehensive cell line panel with varied TP53, INK4/ARF genotypes. They testing both nutlin-3a and RITA (which also induces DNA damage). Though it is clear that none of these molecules are really in clinical progress and there are more potent MDM2 inhibitors like idasanutlin.

Results are also not really surprising for the basics, like RITA works in mutant p53 cells, induces DNA damage. FAK-p53 is confusing. Synergy mechanism unclear authors admit not linked to expected molecular changes. This study is refinement rather than original novel findings since there is previous work on p53-activating agents, FAK inhibitors and so on.

The immunblots and statistics in paper is an issue. This is not a matter of scope and limitations it's fundamental scientific rigor. The study needs substantial strengthening before publication.

Main concerns

Immunonblotting is not easy methodology, and I think authors did a good job, showing all blots, full scan in supplement. But there are major issues with MDM2, gH2AX, dirty antibodies and comparison issues. Control for antibodies. Also, authors state they conducted multiple immunoblot experiments but show only representative results. Response to reviewer 2 is to some extent inadequate "it is unfortunately not feasible to repeat all the analyses". They claim confidence in accuracy and reproducibility without providing statistics. This becomes more of an issue given concerns below. In general a better approach is statistical analysis of western blots from multiple independent experiments, time and dose response. Add blot statistics from ≥3 independent experiments. Quantify blots with error bars and statistical tests where effect is limited or vague/unclear. Clarify blotting methodology approach and add time course and dose experiments (not just 24 hrs).

I understand the issue of repeating many experiments and that some are repeated within the paper from figure to figure. However, regardless of this there are major issues remaining.

1.There is a major issue with MDM2 (figure 2), as it is shown in main figure, it looks like MDM2 and p-MDM2 bands are similar in size and overlap, but they are not looking at the full scan the bands don’t fully match. It is not possible to deduce what we are looking at or that the MDM2 bands are what they are. Also no size markers for the main figure. In supplement is clear non-modified MDM2 bands do not correspond to sizes of p-MDM2. And, it is not clear that this is actually MDM2 to begin with. What is the evidence that p90, p60 bands as shown is a actually MDM2?

2.Second major issue concerns comparability of basal gH2AX (figure 3 versus figure 4, respective cell line untreated condition). The level of gH2AX in untreated cells (respective cells) varies a lot between experiments for the same cell line. Presumably this could be to different exposure times,between lines/experiments though this does not seem to be an issue for p53. This makes it very difficult to judge and compare gH2AX. Besides, the antibody is dirty, many bands.

3.Third major issue, how come there is such increase in p-p53 upon Nutlin treatment same almost as with RITA? It looks quite a lot, though corresponding to total levels. Figure 3 vs 4. This nutlin effect on p53-p is not really that apparent in figure 7 e g in NCI H28, this really contracts within the paper. Similar to gH2AX.

4.Drugs and cell lines. IC50 values in 1-6 micromolar range is not so good actually, effects vary dramatically between cell lines (though expected). EHMES-10 resistant to both MDM2 inhibitors it looks like and NCI-H226 shows unusual responses in my opinion. Variability limits generalizability and clinical translation.

5.Direct comparisons difficult since only one exposure duration tested.

6.Figure 7: Complex dosing scheme makes interpretation difficult

7.Discussion is still somewhat redundant despite revisions

8.Abstract conclusion is very vague just says "effects depend on genotype and inhibitor type"

9. The title is not really what the data shows, it is a bit confusing.

**********

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Reviewer #1: No

Reviewer #3: No

**********

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PLoS One. 2026 Feb 23;21(2):e0343551. doi: 10.1371/journal.pone.0343551.r004

Author response to Decision Letter 2

3 Feb 2026

Replies to Reviewer #3

Major concerns

[Reply]

We appreciate the reviewer’s constructive comments regarding our immunoblotting methodology. We have addressed the specific concerns regarding MDM2 and γH2AX (p-H2AX) in the subsequent itemized responses as below. Here, we clarify the rationale for our choice of treatment duration and dosage.

For defactinib, a concentration of 8 µM resulted in excessive cell death, which interfered with the detection of clear FAK bands. Furthermore, while the treatment beyond 24 hrs effectively induced FAK dephosphorylation, prolonged exposure led to significant cytotoxicity. Consequently, we selected a concentration range of 1–4 µM and a 24-hrs incubation period to demonstrate FAK inhibition without compromising cell viability.

Regarding nutlin-3a and RITA, the concentrations used in Figures 3B (nutlin-3a; 0, 5 and 10 microM) and 4B (RITA; 0, 1.5, 3 microM) were optimized to ensure p53 induction without significant toxicity. Since p53 expression levels remained consistent for 24 hrs after the defactinib treatment and across 24, 48, and 72 hrs of the nutlin-3a/RITA treatment, we standardized the incubation period to 24 hrs. This duration was also applied to the combination treatments to maximize p53 stability while minimizing defactinib-induced cell death, thereby ensuring the reliability of the immunoblotting results. We therefore conducted preliminary experiments and selected the optimal condition regarding treatment time and dose.

Comment (1) 1 There is a major issue with MDM2 (figure 2), as it is shown in main figure, it looks like MDM2 and p-MDM2 bands are similar in size and overlap, but they are not looking at the full scan the bands don’t fully match. It is not possible to deduce what we are looking at or that the MDM2 bands are what they are. Also no size markers for the main figure. In supplement is clear non-modified MDM2 bands do not correspond to sizes of p-MDM2. And, it is not clear that this is actually MDM2 to begin with. What is the evidence that p90, p60 bands as shown is a actually MDM2?

[Reply]

Thank you for pointing out the inconsistencies regarding the MDM2 and p-MDM2 blots. We realized our mistakes of p-MDM blots in Figure 2B and Supplementary Figure 10 (the corresponding original full blot) and noticed labeling errors in both of the figures.

Specifically, the bands previously labeled as 90 kDa and 60 kDa in Supplementary Figure 10 were incorrect. Upon re-examination of molecular weight markers and additional sample blots (see below), the band previously indicated as 90 kDa was a non-specific signal appearing above 100 kDa, while the band previously labeled as 60 kDa is the authentic p-MDM2 at 90 kDa. Authentic 60 kDa p-MDM2 bands were quite low in the intensity with almost background levels because 60 kDa MDM-2 molecules lost the phosphorylation site at Ser 166 and thereby the anti-p-MDM2 antibody used in the study could not detect 60 kDa p-MDM2. We then removed the 60 kDa p-MDM2 arrows in the revised Figure 2B and Supplementary Figure 10.

We have up-loaded new Figure 1, 2, Supplementary Figure 10 and Supplementary Table 4 to address your concerns.

(a) new Figure 2B & Supplementary Figure 10: We have replaced the p-MDM2 blots with the correct 90 kDa bands and added molecular weight markers as requested. We have also revised Supplementary Figure 10 legend (line 737 in page 45). The identification of the 90 kDa band as MDM2/p-MDM2 is based on the manufacturer's datasheet (Santa Cruz Biotechnology and Cell Signaling) and its alignment with the protein standards.

(b) new Supplementary Table 4: We have updated the data. We removed the non-specific (previously "90 kDa") data and provided the intensity data for the authentic 90 kDa p-MDM2 (previously "60 kDa"). Despite these labeling corrections, the trend of p-MDM2 levels in response to defactinib treatment remained consistent with our original findings; the intensity data of non-specific (previously “90 kDa”) bands were similar to those of authentic 90 kDa p-MDM2 (previously "60 kDa"). The overall conclusion of the study thereby remains unchanged.

(c) Size markers: We added the size markers in MDM2 and p-MDM2 blots of revised Figure 2B and also in Figure 1 to ensure clarity.

We apologize for the confusion caused by the initial mislabeling.

Comment (2) 2 Second major issue concerns comparability of basal gH2AX (figure 3 versus figure 4, respective cell line untreated condition). The level of gH2AX in untreated cells (respective cells) varies a lot between experiments for the same cell line. Presumably this could be to different exposure times,between lines/experiments though this does not seem to be an issue for p53. This makes it very difficult to judge and compare gH2AX. Besides, the antibody is dirty, many bands.

[Reply A]

Thank you for your comment. The reviewer is correct that the level of p-H2AX in untreated cells appeared different between Figure 3B and 4B even for the same cell line. This discrepancy is due to different exposure times, which were optimized to capture the specific effects of each inhibitor. We needed to show whether nutlin-3a or RITA induced DNA damage, and consequently we had to use a different exposure time depending on the property of the inhibitor.

(a) Figure 3B (nutlin-3a): All the cells showed relatively high expression levels even in untreated cells. Since nutlin-3a did not induce DNA damage, the p-H2AX signals were inherently weak across all the samples. We intentionally exposed the filters for a long period until the basic signals in untreated cells became clearly detectable in order to confirm whether the western blotting was technically successful and to demonstrate the lack of p-H2AX induction, A short exposure showed no p-H2AX signal in all the lanes, which made it difficult to judge whether the western blots were properly conducted.

(b) Figure 4B (RITA): In contrast, RITA is a potent inducer of DNA damage. We used a shorter exposure time to prevent the strong signals in RITA-treated cells from reaching saturation. Consequently, the basal levels in untreated cells (with the exception of MSTO-211H) appeared undetectable under these conditions. MSTO-211H cells showed less sensitivity to RITA-induced DNA damage compared with other cells. To visualize their response, we applied a longer exposure specifically to this cell line. We therefore added sentences in Figure 3B and 4B legends as follows to explain more in detail. We also revised Results section to explain results of RITA-treated MSTO-211H cells.

(a) Figure 3 legend (lines 640-642 in page 40)

For phosphorylated H2AX, the blots were exposed for a longer period to ensure the detection of basal signals in untreated cells. The basal intensity therefore differed from that of the RITA experiments in Figure 4B where shorter exposures were used.

(b) Figure 4 Legend (lines 651-652 in page 40)

For phosphorylated H2AX of MSTO-211H cells, the blot was exposed for a longer period to ensure the detection of basal signals in untreated cells.

(Before the revision)

All the RITA-treated cells showed decreased FAK phosphorylation levels and increased phosphorylation of H2AX.

(After the revision)

All the RITA-treated cells showed decreased FAK phosphorylation and increased H2AX phosphorylation levels, with the exception of MSTO-211H cells exhibiting a minor H2AX increase.

[Reply B]

Regarding the "dirty" appearance and multiple bands mentioned by the reviewer, this was also a result of the extended exposure times required for the nutlin-3a experiments (as seen in Supplementary Figure 16, which is for Figure 3B p-H2AX). The high background and non-specific bands emerged only under prolonged exposure to visualize the weak basal signals. For nutlin-3a treatment, we needed to expose the filters for a long period to obtain the signal in untreated cells since nutlin-3a did not induce DNA damage. In contrast, the blots in Supplementary Figure 20 (RITA treatment, used for Figure 4B p-H2AX) showed a cleaner background due to the shorter exposure time which enabled us to detect the DNA damage signal. We used the same antibody lot from BioLegend for all experiments, and we would like to maintain that the extra bands were an artifact of overexposure rather than a lack of antibody specificity.

Comment (3) 3.Third major issue, how come there is such increase in p-p53 upon Nutlin treatment same almost as with RITA? It looks quite a lot, though corresponding to total levels. Figure 3 vs 4. This nutlin effect on p53-p is not really that apparent in figure 7 e g in NCI H28, this really contracts within the paper. Similar to gH2AX.

[Reply]

Thank you for this critical observation regarding the p-p53 levels. We understand the reviewer's concern about the apparent discrepancy between Figure 3B and Figure 7. The reviewer mentioned that p-p53 in the wild-type TP53 cells treated with nutlin-3a significantly increased as much as that in the cells treated with RITA, and questioned that the nutlin-3a-mediated up-regulation of p-p53 was not significant in Figure 7. The difference in p-p53 induction levels between these figures is primarily due to the different concentrations of nutlin-3a used in each experiment, as detailed below.

(a) Concentrations: In Figure 3B, we aimed to demonstrate the maximum biological effect of nutlin-3a as a single agent. In contrast, for the combination studies in Figure 7, we intentionally used nutlin-3a at concentrations lower than the IC50 values in growth inhibitory study (4 miroM of nutlin-3a for NCI-H28, 1 microM for MSTO-211H and 2 microM for NCI-H226). All the nutlin-3a concentrations used in Figure 7 were thus lower than those in Figure 3B. Please see the Figure 7 legend in line 671-674 page 41-42, which mentioned the concentrations in detail. We also explained the reason for different concentrations used in Figure 7 in the legend in page 669-671 in page 41. In that sense, the situation is the same as p-H2AX in Figure 3B and 4B, which used different exposure times.

(b) Rationale for Figure 7: We selected these lower doses to avoid a "ceiling effect," where a high dose of nutlin-3a alone might saturate the p53 response, thereby masking any synergistic or additive effects when combined with defactinib. Consequently, the up-regulation of p-p53 in Figure 7 appears less pronounced than in Figure 3B.

(c) Mechanism of up-regulation: While nutlin-3a increases p53 levels by inhibiting MDM2-mediated degradation, RITA can further enhance p53 phosphorylation through the induction of DNA damage and subsequent kinase activation. Furthermore, many factors influence the augmentation levels of p53 in cells treated with MDM2 inhibitors such as stability and affinity of the inhibitors, and susceptibility to ubiquitination in respective cells. The expression levels of p-p53 are therefore the outcomes of these factors involved. As shown in Supplementary Tables 5 (nutlin-3a) and 6 (RITA), the phosphorylated p53 levels varies depending on the cells and the inhibitor used. Phosphorylated p53 levels mediated by nutlin-3a were not always similar to those by RITA.

Comment (4) 4 Drugs and cell lines. IC50 values in 1-6 micromolar range is not so good actually, effects vary dramatically between cell lines (though expected). EHMES-10 resistant to both MDM2 inhibitors it looks like and NCI-H226 shows unusual responses in my opinion. Variability limits generalizability and clinical translation.

[Reply]

Thank you very much for your thoughtful comment regarding the drug sensitivities and clinical translation. We agree that the observed variability and the micromolar-range IC50 values are critical points.

(a) Variability and TP53 genotype: A key finding of this study is that sensitivity of nutlin-3a was dependent on the TP53 genotype. We showed that wild-type TP53 cells were sensitive, whereas mutated or non-functional p53 cells (NCI-H2452, Met-5A cells) were resistant (line 234-236 in page 15). This suggests that the INK4A/ARF deletion with wild-type TP53, often found in the majority of mesothelioma, could serve as a biomarker for MDM2 inhibitor sensitivity. We presume that identifying such "resistant" and "sensitive" profiles is not a limitation but rather a step toward patient stratification and personalized medicine. In contrast, sensitivity to defectinib was not linked with the TP53 genotype and sensitivity range of defectinib (Figure 2A) was not as great as that of nutlin-3a (in Figure 3A). Furthermore, sensitivity to RITA was not linked with the TP53 genotype but can be influenced by induction of DNA damage responses. Drug sensitivity is influenced by many factors such as genetic alterations and modifications of the target proteins; consequently, the sensitivity will be variable depending on properties of inhibitors and target cells. This study indicated that the sensitivity was subjected the agents and cells tested although the TP53 genotype can play a role in the sensitivity.

(b) EHMES-10 NCI-H226 cells: As the reviewer noted, EHMES-10 cells exhibited resistance to both inhibitors despite being wild-type TP53. We mentioned this in the Results section and suggested that a defect in downstream p53 pathways (e.g., impaired p21 induction) may result in reduced cell cycle arrest and contribute to this phenotype. We modified the sentence to explain more (lines 240–242 in page 15). Regarding NCI-H226, its response to nutlin-3a was relatively consistent with other wild-type TP53 cells; however, we acknowledge that differences in the RITA sensitivity probably due to some kinds of protein modification levels or other compensatory pathways, both of which might influence DNA damage responses and inhibition of MDM2 activity.

(c) Clinical translation: We acknowledge that the potency of the inhibitors used in this study will be lower than that of current small-molecule inhibitors that operate in the nanomolar range. We agree that high-dose applications increase the risk of off-target effects. This study however serves as a preclinical proof-of-concept to explore the synergy between p53 stabilization and FAK inhibition. These findings provide a rationale for developing next-generation agents with higher affinity and better pharmacological profiles. We revised the Discussion section and explained more (line 417-421 and line 422-423 in page 26). We also added reference 40 for idasanutlin which was mentioned by the reviewer.

(d) Future drug development: We would like to contend that "negative" results or variability are informative. Our data suggest that while this combination is promising for epithelioid type mesothelioma with wild-type TP53 genotype, different strategies will be required for sarcomatoid or biphasic types which often have mutated TP53 genotype (line 407-411 in page 25-26). We believe that variability of drug sensitivity will not be negative in terms of clinical applications but rather stimulate further investigations. We mentioned these in Discussion as above.

Comment (5) 5 Direct comparisons difficult since only one exposure duration tested.

[Reply]

Thank you very much for your valuab

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PLoS One. doi: 10.1371/journal.pone.0343551.r005

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Strategic selection of MDM2 inhibitors enhances the efficacy of FAK inhibition in mesothelioma based on TP53 genotype

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig

(PDF)

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S2 Fig. (Figure 1 all).

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S3 Fig. (Figure 2B FAK).

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S4 Fig. (Figure 2B p-FAK).

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S5 Fig. (Figure 2B p53).

Original blots which were used for Fig 2B p53 expression. Arrows indicate the target molecules (NCI-H2452 had a truncated p53). The name of cells was shown in the abbreviations.

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S6 Fig. (Figure 2B p-p53).

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S7 Fig. (Figure 2B AKT).

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S8 Fig. (Figure 2B p-AKT).

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S9 Fig. (Figure 2B MDM2).

Original blots which were used for Fig 2B MDM2 expression. Arrows indicate the target molecules (both 90 kDa and 60 kDa molecules). The name of cells was shown in the abbreviations.

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S10 Fig. (Figure 2B p-MDM2).

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S11 Fig. (Figure 2B Caspase-9).

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S12 Fig. (Figure 2B PARP).

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S13 Fig. (Figure 2B Tubulin).

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S14 Fig. (Figure 3B p53 and p-p53).

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S15 Fig. (Figure 3B FAK and p-FAK).

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S16 Fig. (Figure 3B p-H2AX).

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S17 Fig. (Figure 3B Actin).

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S18 Fig. (Figure 4B p53 and p-p53).

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S19 Fig. (Figure 4B FAK and p-FAK).

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S20 Fig. (Figure 4B p-H2AX and Actin).

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S21 Fig. (Figure 7 FAK).

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S22 Fig. (Figure 7 p-FAK).

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S23 Fig. (Figure 7 p53).

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S24 Fig. (Figure 7 p-p53).

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S25 Fig. (Figure 7 AKT and p-AKT).

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S26 Fig. (Figure 7 PARP).

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S27 Fig. (Figure 7 Actin).

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S1 Table. Information of cells used in the study.

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S2 Table. Information of antibody used in the study.

Dilution of primary antibody and information of secondary antibody used in the study.

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S3 Table. Molecular expression levels in Fig 1.

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S4 Table. Molecular expression levels in Fig 2B.

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S5 Table. Molecular expression levels in Fig 3B.

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S6 Table. Molecular expression levels in Fig 4B.

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S7 Table. Molecular expression levels in Fig 7.

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Strategic selection of MDM2 inhibitors enhances the efficacy of FAK inhibition in mesothelioma based on TP53 genotype

Xuerao Ning

Thảo Thi Thanh Nguyễn

Takao Morinaga

Yuji Tada

Hideaki Shimada

Kenzo Hiroshima

Naoto Yamaguchi

Masatoshi Tagawa

Roles

Abstract

Introduction

Methods

Cells and agents

Fig 1. Expression levels of p53, MDM2, MERLIN and FAK in mesothelioma cells with western blot analysis.

Viable cell detection assay

Western blot analysis

Statistical analysis

Results

Expression of p53, MERLIN and FAK in mesothelioma cells

Growth inhibitory effects of defactinib on mesothelioma

Fig 2. Growth inhibition and molecular changes induced by defactinib.

Growth inhibitory effects of nutlin-3a were linked with p53 functional type

Fig 3. Growth inhibition and molecular changes induced by nutlin-3a.

Growth inhibitory effects of RITA were irrelevant to TP53 genotype

Fig 4. Growth inhibition and molecular changes induced by RITA.

Combinatory effects of defactinib and MDM2 inhibitors

Fig 7. Expression level of the molecules associated with FAK and p53 in mesothelioma cells treated with defactinib and/or the MDM2 inhibitor, nutlin-3a or RITA.

Discussion

Supporting information

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Zu Ye

Roles

Author response to Decision Letter 1

Decision Letter 1

Zu Ye

Roles

Author response to Decision Letter 2

Decision Letter 2

Zu Ye

Roles

Acceptance letter

Zu Ye

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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