ELSEVIER
Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs
A
Pharmacological research
Preclinical evaluation of the antitumoral efficacy of Wee1 inhibitor AZD1775 in adrenocortical carcinoma
Check for updates
Emma Nozza a,b, Emanuela Espositoª, Genesio Di Muroª, Sonia Di Baria,b, Rosa Catalano ª, Gianluca Lopez c,d, Valentina Vaira “,e, Anna Maria Barbieri ª, Giusy Marraª, Federica Mangili f, Donatella Treppiedi , Michele Battistine,8, Guido Di Dalmazih,1, Serena Palmieri f, Emanuele Ferrante , Giovanna Mantovani a,f,*, Erika Peverelli a,f,”
a Department of Clinical Sciences and Community Health, Dipartimento di Eccellenza 2023-2027, University of Milan, Milan, Italy
b PhD Program in Experimental Medicine, University of Milan, Milan, Italy
” Division of Pathology, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy
d Department of Biomedical, Surgical, and Dental Sciences, University of Milan, Milan, Italy
e Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy Endocrinology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy
% Center for Preclinical Research, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy
h Division of Endocrinology and Diabetes Prevention and Care, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy
i Department of Medical and Surgical Sciences, Alma Mater University of Bologna, Bologna, Italy
ARTICLE INFO
Keywords: Adrenal Wee1 AZD1775 EDP-M in vivo tumour xenograft cortisol
ABSTRACT
Current therapy for advanced adrenocortical carcinoma (ACC) is represented by EDP-M (etoposide, doxorubicin, cisplatin + mitotane), but its efficacy is limited and new approaches are needed. Our previous in vitro findings showed that AZD1775, an inhibitor of the G2/M checkpoint gatekeeper Wee1, reduces proliferation and in- creases apoptosis in ACC cell models. The compensatory upregulation of Myt1, a Wee1-redundant kinase, has been involved in the onset of AZD1775 resistance in other tumour types. Aim of this study was to investigate in vitro and in vivo the effects of AZD1775 alone or combined with EDP-M, and to explore the onset of molecular mechanisms of resistance. In vitro experiments in human ACC cell line NCI-H295R demonstrated that coincu- bation of AZD1775 and EDP-M exerted synergistic effects in reducing cell viability and proliferation and additive effects in inhibiting cortisol secretion. In NCI-H295R xenografts in nude mice, AZD1775 demonstrated an antitumor efficacy comparable to EDP-M, without synergistic effects. Myt1 upregulation was observed after Wee1 silencing or inhibition by AZD1775 in NCI-H295R and patient-derived ACC cells, but not in mice tumours after 22-days treatment with AZD1775 and/or EDP-M. Interestingly, Myt1 increase after AZD1775 treatment in primary ACC cells was reverted by EDP-M cotreatment. Overall, our data in in vitro and in vivo preclinical ACC models support AZD1775 as a promising ACC therapeutic option, and its combination with EDP-M as a useful strategy to enhance drug efficacy, reduce cortisol secretion, prevent drug resistance and minimize side effects by reducing the therapeutic dosage.
1. Introduction
Adrenocortical carcinoma (ACC) is a rare malignancy originating from adrenal cortex, with an estimated incidence of approximately 1-2
cases per million individuals per year, and a slight female predominance [1,2]. Although overall prognosis remains poor, with a median survival of 3-4 years, outcomes vary significantly according to the European Network for the Study of Adrenal Tumours (ENSAT) stage, ranging from
Abbreviations: ACC, Adrenocortical carcinoma; BrdU, 5-bromo-2-deoxyuridine; CDK1, Cyclin-dependent kinase 1; CDX, Cell-derived Xenograft; DMF, N’N’- dimethylformamide; DMSO, Dimethyl sulfoxide; EDP-M, Etoposide, doxorubicin, cisplatin, and mitotane; ENSAT, European Network for the Study of Adrenal Tu- mours; ESMO, European Society for Medical Oncology; IHC, Immunohistochemistry; IQR, Interquartile Range; OS, Overall Survival; PFS, Progression Free Survival; STR, Short Tandem Repeat; SiRNA, Small interfering RNA.
* Correspondence to: Via F. Sforza, 35, Milan 20122, Italy. E-mail addresses: giovanna.mantovani@unimi.it (G. Mantovani), erika.peverelli@unimi.it (E. Peverelli).
https://doi.org/10.1016/j.phrs.2025.107997
Received 7 August 2025; Received in revised form 3 October 2025; Accepted 14 October 2025
Available online 15 October 2025
1043-6618/ 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
65 to 82 % 5-year survival in stage I, 58-68 % in stage II, 41-55 % in stage III, and only 10-20 % in stage IV. Despite a modest improvement in survival over the past two decades, clinical outcomes remain highly heterogeneous and difficult to predict, largely due to the wide variability in clinical presentation [3]. Complete surgical resection remains the cornerstone of treatment for ACC; however, recurrence is common, with reported rates ranging from 21 % to 91 %, depending on the cohort and criteria used. Moreover, no effective targeted therapies are currently available [4]. According to ENSAT and European Society for Medical Oncology (ESMO) guidelines, the combination of etoposide, doxoru- bicin, and cisplatin with the adrenolytic agent mitotane (EDP-M regimen) is recommended as first-line therapy for patients with advanced ACC [5,6]. Despite being the current standard of care, the EDP-M regimen is associated with significant toxicity and limited clin- ical benefit in many patients [7,8]. Over 50 % of patients with advanced ACC experience disease progression within six months of initiating first-line therapy, highlighting the urgent need for more effective and better-tolerated treatment options [2].
NCI-H295R cells, deriving from a primary ACC tumour, are the gold- standard cell line for studies testing the antimitotic effects of new po- tential drugs, alone or in combination with EDP-M, in both in vitro ex- periments and in vivo models as cell-derived xenografts (CDX) in mice [9-11].
Interestingly, we have recently demonstrated the efficacy of AZD1775, a Wee1 kinase inhibitor, in decreasing cell proliferation and viability, and increasing apoptosis in 3 different ACC cell lines, including NCI-H295R, and in ACC primary cultures [12]. Wee1 protein kinase is a key G2/M checkpoint gatekeeper that mediates inhibitory phosphory- lation of cyclin-dependent kinase 1 (CDK1) at Tyr15 residue, thus keeping inactive the CDK1- cyclin B1 complex, allowing DNA damage repair before mitotic entry. Wee1 overexpression has been described in several solid tumours, correlating to poor clinical outcomes [13,14]. Notably, we also reported an upregulation of Wee1 protein expression in ACC compared to normal adrenal gland [12]. Ultimately, increased CDK1 activity due to Wee1 inhibition by AZD1775 induces tumour cell death via mitotic catastrophe.
AZD1775 has been tested in several cancers and is currently involved in phase I and II clinical trials on ovarian, cervical, and head-and-neck carcinomas either as monotherapy or in combination with different DNA-damaging agents, as reviewed by Ghelli Luserna Di Rorà et al., 2020 [15].
Recently, the combination of classical chemotherapeutics with tar- geted therapies or immunotherapies is growing interest as a new strat- egy to overcome the limitations of EDP-M adverse effects and to enhance its efficiency. In ACC patients, Turla et al. tested the feasibility of combining EDP-M and megestrol acetate, to ameliorate patients’ con- dition and obtain a higher dosage tolerability of EDP-M [16]. Another case study from 2022 [17], described the efficacy of EDP-M plus sinti- limab, an immunosuppressor of the PD-1 pathway. Remarkably, AZD1775 could improve the efficiency of DNA damaging agents due to its inhibitory activity on Wee1, involved in DNA damage response pathways [18]. For this reason, it has been involved in multiple combinatory clinical and in vitro studies with doxorubicin, cisplatin, etoposide, gemcitabine, olaparib, and other genotoxic chemotherapeu- tics on solid and hematic tumours giving positive results [19-23] To date, AZD1775 has not been evaluated in combination with the EDP-M regimen, either in vitro, in vivo in murine models, or in patients with adrenocortical carcinoma.
The possible onset of resistance mechanisms is a major concern in cancer therapy and Myt1 overexpression is a common mechanism by which cancer cells can acquire resistance to Wee1 inhibitors. In breast cancer models, tumour sensitivity to AZD1775 was correlated to Myt1 kinase expression [24]. Myt1, as Wee1, phosphorylates CDK1 at Tyr15, and despite its redundancy to Wee1, its overexpression has been iden- tified as a key factor associated with reduced overall survival (OS) and progression-free survival (PFS) in patients with ACC [25]. AZD1775
treatment, suppressing Wee1 activity, upregulates Myt1 in other cancer models [24,26], but no data are available in ACC.
Aim of the present study was to test the efficacy of AZD1775 alone or in combination with EDP-M both in vitro, in NCI-H295R cells and pri- mary cultured ACC cells, and in vivo, in nude mice NCI-H295R xeno- grafts. Moreover, aiming at a possible therapeutic application of AZD1775, we investigated the activation of resistance mechanisms, focusing on the role of Myt1.
2. Materials and methods
2.1. Cell cultures and treatments
NCI-H295R adrenocortical carcinoma cells obtained from American Type Culture Collection [Cat# CRL-2128, RRID: CVCL_0458, ATCC] were harvested in vitro in DMEM:F12 1:1 [Cat D8437, Merck KGAA, Darmstadt, Germany] culture media, supplemented with 2.5 % Nu- Serum I [Cat #354352, Corning, NY, USA], 1 % ITS+Premix [Cat #355100, Corning, NY, USA], 100 U/mL penicillin and 100 µg/mL streptomycin [Cat# 17-602E, Lonza group Ltd, Basel, Swi]. NCI-H295R cells were used only younger than passage 20, and authenticated by genetic profiling with the polymorphic short tandem repeat (STR) technique [Promega, BMR Genomics Cell Profile service, Italy].
An ACC primary culture was established after surgical removal of the tumour mass: this study was approved by Milano Area 2 Ethical Com- mittee (554_2022bis), and patient who underwent adrenalectomy gave informed consent to the use of tumour sample and clinical information. No genetic variants in CTNNB1 and TP53 genes were found by NGS analysis. At first, mechanical dissociation of the fresh tumor tissue with a scalpel, and subsequently an enzymatic dissociation (2 h incubation in a 2 mg/mL Collagenase B solution in DMEM) were performed. Cell sus- pension was filtered with a 100 um pores cell strainer to eliminate ag- gregates and undigested material, then cells were grown into Advanced DMEM-F12 [Cat#12634 Gibco, ThermoFisher Scientific, Waltham, MA, USA] supplemented with 20 % foetal bovine serum (FBS) [Cat#10270 Gibco, ThermoFisher Scientific, Waltham, MA, USA], 2 mM L-glutamine [Cat#G7513 Merck KGaA, Darmstadt, Germany], 100 U/mL penicillin and 100 µg/mL streptomycin.
AZD1775, mitotane, and etoposide purchased from Target Mol Chemicals Inc. [Cat#T2077, T1199, T0132 Boston, MA] were dis- solved in dimethyl sulfoxide (DMSO), cisplatin [Cat#T1564 Boston, MA] was dissolved in N’N’-dimethylformamide (DMF). Doxorubicin bought from RayBiotech was dissolved in DMSO [Cat#331-10808-3, Peachtree Corners, GA, USA]. AZD1775 and EDP-M were tested alone or in combination: AZD1775 IC50 (1350 nM) on proliferation was exper- imentally assessed (Supplementary Figure 1), while IC50 of each single drug of EDP-M scheme was retrieved from Hantel: 15.9 uM mitotane, 1.2 uM etoposide, 9.6 uM cisplatin, and 11 uM doxorubicin [27]. Ac- cording to the experimental assay, cells were seeded alternatively in 96-well plates at 1.4*104 cell density or in 6-well plates at 3*105 cell density and upon adhesion starved for at least 2 h, then treated for 24 h in complete medium.
2.2. Western blot
NCI-H295R cells were seeded in 6-well plates at a density of 3.0 × 105 cells/well in complete medium. To obtain proteins, frozen tumour tissues were mechanically dissected while kept on ice, then lysed for 10 min in lysis buffer [Cat# 9803S, Cell Signaling Technology, Danvers, MA, USA] with the addition of protease inhibitors [Cat#05892791001 Roche, Basel, Swi], the homogenate was subse- quently centrifuged at 13000 rpm for 15 min to obtain a protein su- pernatant. Proteins were then collected in a fresh tube and quantified via BCA assay. 60 µg of proteins from each sample were loaded on an SDS/ polyacrylamide gel and transferred on a nitrocellulose membrane. Wee1 [Santa Cruz Biotechnology, Dallas, TX, USA; SC5285, LOT:G222] and
Myt1 antibodies [Cell Signaling Technology, Danvers, MA, USA; 4282S, LOT:2] were diluted 1:100 and 1:1000, respectively. Incubation of pri- mary antibodies was allowed overnight at 4℃, while anti-mouse and anti-rabbit secondary antibodies [Cell Signaling Technology, Danvers, MA, USA] were diluted at 1:2000 and incubated for 1 h. GAPDH anti- body [Ambion, Thermo Fisher Scientific, Waltham, MA, USA; AM4300, LOT:01062924], used as loading control, was diluted at 1:8000 and incubated for 1 h at room temperature. Chemiluminescence was detec- ted using ChemiDoc™M Imaging System [Bio-Rad, Hercules, CA, USA], and densitometrical analysis was carried out by the NIH ImageJ software.
2.3. Cell proliferation
Proliferative activity was evaluated via 5-bromo-2-deoxyuridine (BrdU) incorporation assay [Cat#11647229001 Roche, Basel, Swi]. Following drug treatments, BrdU was incorporated for 2 h and the kit was performed according to manufacturer’s instructions. Victor Nivo [PerkinElmer, Shelton, USA] plate reader set at 450 nm was used to detect the absorbance of samples: results were expressed as percentage of the ratio between the mean absorbance of each treatment and the one of basal cells.
2.4. Cell viability
MTT assay for cell viability was performed after treatments. Cells were incubated for 3 h at 37℃ in a 5 mg/mL MTT solution [Cat#475989 Merck KGaA, Darmstadt, Germany] diluted 1:10 in DMEM without Phenol Red. After formazan salt formation was verified through mi- croscopy, MTT was removed and DMSO revealed the colorimetric re- action. Victor Nivo [PerkinElmer, Shelton, USA] plate reader set at 560 nm was used to detect the absorbance of samples: results were expressed as percentage of the ratio between the mean absorbance of each treatment and the one of basal cells.
2.5. Wee1 silencing
NCI-H295R cells were seeded in 6-well plates at a density of 3.0 x 105 cells/well in complete medium. The day after, a 72 h Weel genetic silencing was performed using a human SMARTpool of Wee1 predesigned small interfering RNAs (siRNAs) [Dharmacon, GE Health- care Life Sciences, Chicago, IL, USA] at a concentration of 25 nM, using Lipofectamine™M RNAiMAX [Thermo Fisher Scientific, Waltham, MA, USA] as a transfection reagent. A negative control (C-) siRNA (a non- targeting siRNA sequence with no significant homology to any anno- tated human, mouse, or rat mRNA) was used for each experiment. Only experiments with knockdown efficiency ≥ 70 %, evaluated via Western blot analysis, were kept into consideration.
2.6. NCI-H295R cells-derived xenografts in nude mice and in vivo treatment
In vivo experimental procedures, approved by the Italian Ministry of Health (number 628/2023), were performed at the Center for Preclinical Research, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan (Italy). Guidelines of the Italian Legislative Decree 26/2014 and the European Union listed in the Directive 2010/63/EU along with ARRIVE guidelines were strictly followed during the project. NCI-H295R cells at passages 16-17 grown confluent on T75 culture flask were de- tached and re-seeded the day before the injection. 1.5*107 cells were detached, resuspended in 150 uL of PBS and subcutaneously injected in CD1 athymic nude mice (female, 42-48 days old, Charles River Labo- ratories, IT) right flanks under isoflurane anaesthesia in sterile condi- tions. The number of mice needed for the study has been calculated using the G * Power software. The effect size and standard deviations have been estimated on the basis of the results obtained in in vitro
assays. The number of animals per group was calculated keeping the following parameters fixed: the
probability of a first-type error (alpha; false positives) of 5 %; a power of 0.8. The calculation indicated a number of 7 animals per group. Considering a failure rate in cell xenotransplantation of 10 %, the number of animals per group was set at 8, for a total of 32 mice injected for 4 groups. Animals were housed in individually ventilated cages [Tecniplast S.p.A., Varese, Italy] under standard environmental condi- tions on a 12 h dark/light cycle, and access to food and water ad libitum. 25 of 32 mice successfully developed a tumoral mass: its volume was quantified weekly via caliper measurement of length, width, and height, and calculated according to the I/6*L*W*H formula [28]. Once half of the xenografts reached 60 mm3 in volume, mice were distributed to obtain 4 groups with similar medians of tumour volume: untreated group was composed of 7 animals, while each treatment group was made of 6 mice. Treatment started the following day (T0). 60 mg/kg of AZD1775 (dissolved in 5 %DMSO +40 %PEG300 +5 %Tween80 +50 % NaCl) [29-31] was administered via oral gavage once daily for 22 days. EDP-M was administered according to Berruti treatment scheme for patients, adapted for preclinical mouse model administration [27] (Fig. 1): 3 days of 300 mg/kg mitotane dissolved in corn oil administered via intraperitoneal injection, followed by 1 day of intravenous 10 mg/kg doxorubicin, 1 day of intravenous 10 mg/kg etoposide and 1 day of intravenous 10 mg/kg etoposide+ 2 mg/kg cisplatin. This cycle of chemotherapeutics was repeated two times, concomitant to the first and last week of AZD1775 treatment. Tumour volume and mice body weight was measured every 5-6 days (at T0, T1, T2, T3, and the last day of treatment) (Fig. 1). Ulceration of tumours or body weight loss higher than 20 % of mice weight were set as humane endpoints, but were never reached during the experimental period. The day after the last treatment (T4) mice were anesthetized via isoflurane and sacrificed by exsangui- nation, contextually blood was collected to measure cortisol levels and tumours were excised and partially frozen and stored at -80, partially stored in formalin solution to be further included in paraffin and mounted on slides for immunohistochemical (IHC) analyses, and partially dissociated to obtain primary cell cultures. In addition, hearts and kidneys of 3 mice of each group and adrenal glands of 1 mouse per group were removed and stored in formalin solution for IHC analyses.
2.7. Tissue morphology and Immunohistochemistry (IHC)
Histological analyses were performed on formalin-fixed paraffin- embedded samples (tumour, hearts, kidneys, and adrenal glands). Briefly, 4-um thick tissue section were cut and stained for morphological (haematoxylin and eosin stain, H&E) or IHC examination as previously described [32]; then, all slides were digitalized using the Aperio scanner (Leica Microsystems, Milan, Italy). The mitotic index, presence of ne- crosis, and CD31 expression were therefore quantified in all tumour samples. Specifically, mitotic cells were scored in three high power fields per samples, whereas presence or absence of necrosis was evalu- ated as a dichotomous variable. The tumour vascularization was eval- uated as the percentage of CD31-positive cells within the tumour mass using the cytoplasmic CXCL12 algorithm optimized within ImageScope software (Leica Microsystems) as previously described [33]. To evaluate organ toxicity, haematoxylin and eosin staining was performed in a total of 13 hearts, 13 kidneys, and 4 adrenals.
2.8. Cortisol secretion
Cortisol levels were measured on samples of supernatant media of cells after 24 or 72 h treatment with AZD1775 and/or EDP-M, and on mice blood samples collected at T4. Supernatant samples were centri- fuged 10 min at 13000 rpm at 10℃ to eliminate debris, then measured with the specific immunoassay Elecsys Cortisol II [Cobas, Roche, Basel, Swi]. Blood samples collected at T4 were set aside for 30 min at room temperature after collection, then centrifuged 10 min at 4000 rpm at
Body weight and volume measurement
Body weight and volume measurement
TO
Day1: AZD1775
Day2: AZD1775
Day3: AZD1775
Day4: AZD1775
Day5: AZD1775
Day6: AZD1775
TO
Day1: mitotane
Day2: mitotane
Day3; mitotane
Day4: doxorubicin
Day5: etoposide
Day6: etoposide + cisplatin
T1
Day7: AZD1775
Day8: AZD1775
Day9: AZD1775
Day10: AZD1775
Day11: AZD1775
T1
Day7
Day8
Day9
Day10
Day11
T2
Day12: AZD1775
Day13: AZD1775
Day14: AZD1775
Day15: AZD1775
Day16: AZD1775
T2
Day12
Day13
Day14
Day15
Day16
T3
Day17: AZD1775
Day18: AZD1775
Day19: AZD1775
Day20: AZD1775
Day21: AZD1775
Day22: AZD1775
T3
Day17: mitotane
Day18: mitotane
Day19: mitotane
Day20: doxorubicin
Day21: etoposide
Day22: etoposide+cisplatin
T4
sacrifice
T4
sacrifice
AZD1775
EDP-M
Body weight and volume measurement
TO
Day1: mitotane AZD1775
Day2: mitotane AZD1775
Day3: mitotane AZD1775
Day4: doxorubicin AZD1775
Day5: etoposide AZD1775
Day6: etoposide+cisplatin AZD1775
T1
Day7: AZD1775
Day8: AZD1775
Day9: AZD1775
Day10: AZD1775
Day11: AZD1775
T2
Day12: AZD1775
Day13: AZD1775
Day14: AZD1775
Day15: AZD1775
Day16: AZD1775
T3
Day17: mitotane AZD1775
Day18: mitotane AZD1775
Day19: mitotane AZD1775
Day20: doxorubicin AZD1775
Day21: etoposide AZD1775
Day22: etoposide+cisplatin AZD1775
T4
sacrifice
AZD1775+EDP-M
10°C and supernatant was collected. This procedure was repeated a second time to obtain sera, samples were then frozen and later processed as indicated above for the analysis with Elecsys Cortisol II kit.
2.9. Statistics and synergy analyses
Synergy of drug combinations was calculated via Synergy- Finder+ online software [34]. Synergy scores were obtained from both HSA and Loewe mathematic models: a synergistic effect of treatments was confirmed by a synergy score higher than 10. Statistical analyses were performed using GraphPad Prism Software 10.4.1 [GraphPad Software Inc., San Diego, CA, USA]. In particular, proliferation, viability, and cortisol secretion of NCI-H295R cells results were analysed via Friedman test coupled with Dunn’s Uncorrected post hoc analysis. % increase of tumour volume at each time point among different treat- ments was analysed via Mann Whitney test. Cortisol level in blood, CD31 staining positivity, and Myt1 levels after treatment with AZD1775, EDP-M, and drug combination were analysed via Kruskal-Wallis test coupled with Dunn’s uncorrected post hoc analysis. Myt1 levels after Wee1 silencing were analysed via Mann Whitney test.
2.10. Data availability
The data generated in this study are available upon request from the corresponding author.
3. Results
3.1. In vitro effects of combinatory treatment of AZD1775 and EDP-M in NCI-H295R cells and ACC primary cultured cells
We previously demonstrated AZD1775 efficiency in reducing NCI- H295R cell proliferation and viability [12]. To assess the potential synergistic effect of AZD1775 and EDP-M regimen, we treated NCI-H295R and primary cultured ACC cells with single agents or their
combination. In vitro EDP-M treatment scheme on NCI-H295R was previously described [27]. We assessed the AZD1775 IC50 for cell pro- liferation (Supplementary Figure 1) to further test the drug combination.
In NCI-H295R, cell proliferation, measured as BrdU incorporation in newly synthesized DNA, was strongly hampered by AZD1775 +EDP-M treatment at all concentration tested, whereas a non-significant reduc- tion of cell proliferation was observed after AZD1775 incubation in these experimental conditions (Fig. 2A). Interestingly, the most efficient combination (AZD1775 IC50 plus EDP-M 1/2IC50) strongly reduced proliferation (-62.67(39.55)%, p < 0.0001) and this decrease was sig- nificant also compared to AZD1775 (-31.85(20.47)%, p < 0.01 vs combined treatment) and EDP-M treatments (-33.83(16.35)%, p < 0.05 vs combined treatment). To determine the potential synergism between AZD1775 and EDP-M, SynergyFinder+ tool was applied using two mathematical models, Loewe and HSA, both showing a synergy score > 10 that indicates a synergistic antimitotic effect (Loewe 16.15, HSA 17.63) (Fig. 2B). In agreement, AZD1775 +EDP-M combinations reduced cell viability (Fig. 2C), with a synergistic effect (Loewe 11.10, HSA 11.86) (Fig. 2D).
Cortisol secretion, commonly reduced in ACC patients after mitotane therapy [35,36], was assessed in the culture media of NCI-H295R, a cortisol-secreting cell line (Fig. 2E). Combined treatment with AZD1775 and EDP-M induced a stronger effect on cortisol secretion inhibition compared to single treatments. Interestingly, the incubation with the lower dose of EDP-M (1/4 IC50) was not effective alone on cortisol in- hibition, but significantly reduced hormone release after coincubation with AZD1775. A synergistic effect of EDP-M with AZD1775 was revealed by HSA model (Fig. 2F).
These experiments were replicated in primary cultured cell derived from a surgically removed ACC. Due to the low number of available cells, we tested only the effect of AZD1775 1/2IC50 or EDP-M 1/4IC50, or drug combination, on cell proliferation, viability and cortisol secretion. We observed a strong reduction of both cellular proliferation and viability after single treatments (Fig. 3A-B), with no further decrease after cotreatment. Cortisol secretion of the ACC primary culture was
| AZD1775 1/2IC50 + | + | + | |||||
| AZD1775 IC50 | + | + | + | ||||
| EDP-M 1/4|C50 | + | + | + | ||||
| EDP-M 1/2/C50 | + | + | + | ||||
A
B
:
40
30
30
20
*
20
30
150
Synergy Score
10
10
20
*
0
0
10
10
cell proliferation (% vs. untreated)
Synergy Score
20
-10
0
100
$
30
20
-10
# 40 -30 -20 7.95 7.95 -30 50 EDP-M 9.975 1.343 EDP-M 3.975 1.343 0.6715 AZD1775 0.6715 AZD1775 0 U T T T T T 0 0 0 0 Loewe Model Synergy Score 16.15 HSA Model Synergy Score 17.63 C D **** 20 20 **** 15 15 10 10 30 **** Synergy Score & Synergy Score 5 20 150 0 0 10 \* -5 -5 0 cell viability (% vs. untreated) -10 -10 $$
-15
15
-10
100
=
-20
20
1
:20
7.95
7.95
-30
50
EDP-M
3.975
1.343
EDP-M
3.975
1.343
0.6715
AZD1775
0.6715
AZD1775
0
0
0
0
0
T
T
T
AZD1775 1/2IC50
+
+
+
Loewe Model Synergy Score 11.10
HSA Model Synergy Score 11.86
AZD1775 IC50
+
+
+
EDP-M 1/4IC50
+
+
+
EDP-M 1/2IC50
+
+
+
E
F
20
20
15
15
10
10
30
Synergy Score
5
Synergy Score
5
20
0
0
7
10
5
5
6-
-10
-10
·
-15
-15
-10
cortisol secretion (ng/ml)
5.
-20
-20
-20
7.95
7.95
4-
#
-30
$$ 3\. EDP-M 3.975 1.343 3.975 1.343 2 0.6715 AZD1775 EDP-M 0.6715 AZD1775 L 0 1\. 0 0 0 0 T T T Loewe Model Synergy Score HSA Model Synergy Score 10.56 AZD1775 1/2IC50 \+ \+ \+ AZD1775 IC50 \+ \+ \+ EDP-M 1/4IC50 \+ \+ \+ EDP-M 1/2IC50 7.51 \+ \- \+ \+ </figure> <!-- PageNumber="5" --> <!-- PageBreak --> <!-- PageHeader="E. Nozza et al." --> <!-- PageHeader="Pharmacological Research 221 (2025) 107997" --> <figure> <figcaption>Fig. 3. Primary ACC cell culture in vitro experiments. A) Cell proliferation, via BrdU incorporation assessed after 72 h treatment with AZD1775, EDP-M, or com- bination in complete medium. Results of a single experiment (5 technical replicates) were plotted as % of cell proliferation vs untreated sample. B) Cell viability, assessed via MTT test, after 72 h treatment with AZD1775, EDP-M, or combination in complete medium. Result of a single experiment (5 technical replicates) were plotted as % of cell viability vs untreated sample. C) Cortisol secretion measured after 72 h treatment, plotted as ng/ml from cell supernatant, result of n = 1 experiment (2 technical replicates).</figcaption> A 1.0 B 0.4 C 50 absorbance values (450nm) 0.8 absorbance values (560nm) 0.3 cortisol (ng/ml) 40 0.6 30 0.2 0.4 20 0.2 0.1 10 0.0 0.0 0 AZD1775 1/2IC50 \- \+ \- \+ \- \+ \- \+ \- \+ \- \+ EDP-M 1/4|C50 AZD1775 1/2IC50 EDP-M 1/4IC50 AZD1775 1/2IC50 \- \- \+ \+ \- \- \+ \+ EDP-M 1/4IC50 \- \- \+ \+ </figure> <figure> <figcaption>Fig. 4. In vivo efficacy of AZD1775, EDP-M, and their combination on NCI-H295R xenografts. A-F) Percentage of tumour volume increase over time, plotted as median and IQR of n = 7 mice in the untreated group and n = 6 mice in each treatment group. A-C) Comparison of untreated vs treated tumours. D-F) Comparison in pairs of different treatments between each other. Statistical analysis performed via GraphPad Prism Software, using Mann-Whitney test. * p < 0.05, ** p < 0.01 vs untreated. G) Mice body weight measured over time. H) Cortisol secretion in serum, expressed as single ng/ml value for each mouse. Statistical analysis performed via GraphPad Prism Software, using Kruskal-Wallis test coupled with Dunn's Uncorrected post hoc analysis. * p < 0.05, ** p < 0.01 vs untreated.</figcaption> A B C 400 400 400 ** \* ** % volume increase % volume increase 300 % volume increase 300 \* 300 0.073 200 \* 200 200 \* 100 100 100 0 0 0 TO T1 T2 T3 T4 TO T1 T2 T3 T4 TO T1 T2 T3 T4 D E F 300 300 300 % volume increase % volume increase 200 200 T % volume increase I 200 100 I 100 100 I 0 0 0 TO T1 T2 T3 T4 TO T1 T2 T3 T4 TO T1 T2 T3 T4 G H ** \* untreated 35 40 AZD1775 mice weight (g) 30 cortisol (ng/mL) 30 EDP-M ₮ 1 25 E 20 \- AZD1775+EDP-M 20 10 15 0 T T T T TO T1 T2 T3 T4 AZD1775 \- \+ \- \+ EDP-M \- \- \+ \+ </figure> <!-- PageNumber="6" --> <!-- PageBreak --> <!-- PageHeader="E. Nozza et al." --> <!-- PageHeader="Pharmacological Research 221 (2025) 107997" --> quantified in the supernatant of cells treated for proliferation assess- ment, after 72 h of treatment. Interestingly, only the combined treat- ment induced a strong inhibition of cortisol production (-71 % vs untreated) (Fig. 3C). 3.2. In vivo efficacy of AZD1775, EDP-M, and their combination in NCI- H295R xenografts in nude mice We further assessed the efficacy of AZD1775 as single treatment or in combination with EDP-M in vivo in NCI-H295R xenografts in nude mice. <figure> <figcaption>Fig. 5. IHC analyses on NCI-H295R xenograft mice. A-B) Evaluation of necrosis in tumour tissues, graph indicating % of necrotic tumours and representative images. C) IHC evaluation of mitosis % in NCI-H295R tumour xenografts: cut off level at 70 %. D) Representative images of hearts, kidneys and adrenals of NCI-H295R xenograft mice after in vivo treatment. E-F) Evaluation of intratumoral vascularization, graph indicating % of CD31 positive cells and representative images. Sta- tistical analysis performed via GraphPad Prism Software, using Kruskal-Wallis test coupled with Dunn's Uncorrected post hoc analysis. * p < 0.05, ** p < 0.01 vs. untreated.</figcaption> A 150 D absence of necrosis % necrotic tumors presence of necrosis HEART KIDNEY ADRENAL 29% 83% 100 67% 50% untreated 50 0 T 1 T 1 4 AZD1775 EDP-M \- \+ \- \+ 200um 200wm \- \- \+ \+ \- B untreated AZD1775 AZD1775 \- EDP-M EDP-M AZD1775+EDP-M AZD1775+EDP-M C E \* 150 <70% mitotic index ** 86% 33% 20% 40% \>70% mitotic index % tumors % CD31 positive cells (H-score) 15 ** 100 10 50 5 0 1 AZD1775 EDP-M \- \+ \+ 1 \- \- \+ \+ 0 U AZD1775 EDP-M \- \+ \- \+ \- \- \+ \+ F untreated AZD1775 EDP-M AZD1775+EDP-M e V 100um 100um 100um 100um </figure> <!-- PageNumber="7" --> <!-- PageBreak --> <!-- PageHeader="E. Nozza et al." --> <!-- PageHeader="Pharmacological Research 221 (2025) 107997" --> Fig. 4 illustrates tumour growth curves expressed as percentage of tumour volume increase from treatment start. We found that either single or combined treatments significantly reduced tumour growth. The increase of tumour volume after 22 days of treatment with AZD1775 (+124.5(54.5)%), EDP-M (+153.5(110.8)%), AZD1775 +EDP-M (+145.5(71.5)%) was halved compared to the growth of the untreated group (+268[99]%) (p <0.01, p <0.05 and p <0.01, respectively). The antitumoral efficacy of AZD1775 was comparable to those of EDP-M alone and combined treatment. It is noteworthy that, during the time of treatment, no mice were sacrificed due to tumour ulceration or significant weight loss (>20 % loss in body weight). Combination therapy induced about 13 % of weight loss at the end of treatment, slightly higher than AZD1775 (-5 %) and EDP-M (+1 %) alone (Fig. 4G). After sacrifice, tumour cortisol levels in mice blood were quantified (Fig. 4H). In agreement with in vitro results in NCI-H295R cells (Fig. 2E), both EDP-M alone and AZD1775 +EDP-M combination significantly lowered cortisol secretion (EDP-M 12.35(3.72) ng/ml, p < 0.05; AZD1775 +EDP-M 10.85(3.16) ng/ml, p < 0.01 vs untreated 20.18 (5.9) ng/mL), whereas no effect on serum cortisol levels was observed after AZD1775 treatment. ### 3.3. AZD1775 treatment increased tumour necrosis and reduced tumour mitotic index and vascularization Cytotoxic and anti-mitotic effects of AZD1775, EDP-M, and their combination were investigated by IHC assessment of necrosis and mitotic index. Necrosis was detected only in 2 out of 5 untreated mice; on the opposite, 5 out of 6 tumours deriving from mice belonging to AZD1775 group, and 4 of 6 of EDP-M group, presented necrotic areas (Fig. 5A-B). Only two tumours showed a percentage of necrotic area higher than 50 %, one was found in AZD1775 group and the other one in the EDP-M group. In addition, tumours derived from mice treated with EDP-M exhibited reduced percentage of mitosis compared to untreated mice (median mitotic index EDP-M 40 vs. untreated 78). As shown in Fig. 5C, all but 1 tumors of the untreated group displayed a mitotic index higher than 70 %, whereas in treated groups the majority of tumors showed a mitotic index lower than 70 % (4/6 in AZD1775, 4/5 in EDP-M, 3/5 in AZD1775 +EDP-M). No histological alteration was observed in mice hearts, kidneys, nor adrenal glands after HE staining in tumours deriving from all groups, suggesting the absence of toxicity (Fig. 5D). IHC analysis of tumour vascularization was based on CD31 positive staining. Tumours of the untreated group had a significantly higher CD31 positivity (7.87 %) compared to treated mice (4.36 % AZD1775 p < 0.01, 3.28 % EDP-M p < 0.01, 3.52 % AZD1775 +EDP-M, p < 0.05). Reduction of tumour vascularization in treated animals suppor the eficacy of treatment in controlling xenograft growth. ### 3.4. In vitro and in vivo investigation of resistance mechanisms onset Since Myt1 has been involved in the onset of resistance mechanisms to AZD1775 therapy [26], we investigated Myt1 expression levels after AZD1775 treatment in in vitro and in vivo models. 24 h treatment with AZD1775 significantly reduced Wee1 (0.51-fold (0.51) vs untreated, p < 0.01) and increased Myt1 (3.42-fold (3.19) vs untreated, p < 0.01) expression in NCI-H295R cells (Fig. 6A&B). A comparable Myt1 increase was also observed after combined treatment with AZD1775 +EDP-M, but not after EDP-M alone. In agreement, an upregulation of Myt1 was observed after 72 h of Wee1 genetic silencing (4.41-fold increase vs negative control NCI-H295R <figure> <figcaption>Fig. 6. Onset of resistance in different ACC models treated with AZD1775, EDP-M or combination. A-B) Protein expression of Wee1 (A) and Myt1 (B) in NCI-H295R. 24 h in vitro treatment, n = 5. C) Protein expression of Myt1 after 72 h in vitro genetic silencing of Wee1 (25 nM) in NCI-H295R, n = 6. Immunoblot of one representative experiment is shown. Statistical analysis performed via GraphPad Prism Software, using Mann-Whitney test. D-E). Protein expression of Myt1 and GAPDH of primary ACC culture as representative immunoblot. 24 h in vitro treatment of AZD1775, EDP-M, and combination (D) and 72 h in vitro genetic silencing of Wee1 (25 nM, E) with fold change over control sample.) F-G) Protein expression of Wee1 (F) and Myt1 (G) of mice xenograft after 22 days of in vivo treatment, n = 6 untreated and AZD1775 treated mice, n = 5 EDP-M and AZD1775 +EDP-M treated mice. Statistical analysis performed via GraphPad Prism Software, using Kruskal- Wallis test coupled with Dunn's Uncorrected post hoc analysis. * p < 0.05 vs. C- siRNA, ** p < 0.01 vs. untreated.</figcaption> C- siRNA Wee1 siRNA ** A 0.056 B C ns AZD1775 IC50 8 \* \- \+ \+ ns ** 2.0- EDP-M 1/2IC50 \- \+ \+ ** 6- 6 WEE1 Wee1 expression 1.5- Myt1 expression Myt1 expression WEE1 4- 4 1.0- MYT1 MYT1 2- 2\. 0.5. GAPDH 0.0 0 GAPDH T 0 T AZD1775 IC50 EDP-M 1/2IC50 \- \+ AZD1775 IC50 EDP-M 1/2IC50 C- SİRNA Wee1 siRNA \+ \+ \+ . \+ \+ \- \+ \+ ACC PRIMARY CULTURE MICE XENOGRAFTS D E F G ns ns 1 41.33 0.70 0 1 0.40 ns 1.5- ns 4- WEE1 ns ns MYT1 Wee1 expression Myt1 expression 3- 1 1.31 1.0- MYT1 2 GAPDH 0.5 1- . GAPDH · AZD1775 IC50 \- \+ \- \+ 0 0.0 EDP-M 1/2IC50 \+ \+ C- siRNA Wee1 siRNA AZD1775 \+ \- \+ \- EDP-M AZD1775 \+ \+ \+ \+ EDP-M \+ \+ </figure> <!-- PageNumber="8" --> <!-- PageBreak --> <!-- PageHeader="E. Nozza et al." --> <!-- PageHeader="Pharmacological Research 221 (2025) 107997" --> transfected cells, p < 0.05) (Fig. 6C), strongly suggesting that the reduction of the expression or function of Wee1 induces a compensatory upregulation of Myt1. These results were replicated in primary cultured ACC cells, where both pharmacological inhibition and genetic silencing of Wee1 induced Myt1 upregulation (Fig. 6D-E). In contrast with NCI-H295R cells, the AZD1775 effect on Myt1 increase was completely reverted by coincu- bation with EDP-M (Fig. 6D). To assess whether Myt1 upregulation might represent a possible mechanism of resistance to AZD1775 treatment in vivo, we measured Myt1 protein levels in tumour tissues derived from control and treated mice. No change in both Wee1 and Myt1 protein levels was observed after 22 days treatments (Fig. 6F-G), suggesting the absence of resistance mechanism onset based on Myt1 or Wee1 expression changes in in vivo model after prolonged treatment. ## 4. Discussion In the present study, we provided evidence of the antitumoral effects of the Wee1 inhibitor AZD1775 both in vitro and in a preclinical ACC mouse model. Indeed, we found that AZD1775 inhibits ACC xenograft growth similarly to EDP-M, the first-line treatment for advanced ACC. Interestingly, AZD1775 reduced tumour vascularization, induced intra- tumoral necrosis, and was generally well tolerated. Furthermore, AZD1775 tested in combination with EDP-M showed comparable out- comes to treatment with either AZD1775 or EDP-M alone. However, in vitro co-treatment with AZD1775 and EDP-M demonstrated synergistic effects, significantly reducing proliferation and viability of NCI-H295R cells. The discrepancy observed between the in vitro and the in vivo response to treatment could be explained at several levels: first, the huge difference of the two models, including also the presence of a complex tumour microenvironment in mice xenografts. Moreover, mice treat- ment protocol, with drugs systemically administered and the interfer- ence of mitotane on AZD1775 metabolism through sustained activation of CYP3A4 [19,37], could result in a differential efficiency of the com- bination between AZD1775 and EDP-M in vivo. At last, drug concen- trations could represent another variable, indeed the in vitro synergistic effect was detectable only at low doses of EDP-M, that were not tested in mice. Moreover, in ACC primary cultured cells the combined treatment was more efficient in reducing cortisol secretion compared to single agents and did not induce the upregulation of Myt1, that is recognized to mediate acquired resistance to AZD1775 treatment. The development of new therapies against ACC is urgently needed. Patients with advanced ACC, only partially benefit from EDP-M therapy, despite bearing important side effects. Combination therapies are considered promising in ACC management. Coupling EDP-M with tar- geted agents, such as linsitinib, or RTK inhibitors, such as cabozantinib, or even immune checkpoint inhibitors, such as sintilimab, could in- crease single agent drug efficacy [17,38,39]. Previous findings from our group demonstrated that AZD1775 administration inhibited prolifera- tion and viability in different ACC in vitro models [12]. AZD1775 acts as pro-apoptotic agent on ACC cells, by inducing their premature mitosis due to the abrogation of DNA damage checkpoint. Since Wee1 regulates the G2 checkpoint in response to DNA damage, its inhibitor AZD1775 can potentiate the DNA-damage response caused by cytotoxic chemo- therapies. Indeed, AZD1775 was already tested in p53-deficient colon, cervical, lung, and pancreatic cells lines, where it enhanced the anti- tumor efficacy of various DNA-damaging agents, including doxorubicin and cisplatin [39-41]. Moreover, AZD1775 has been tested in phase II clinical trials on various other solid cancers, demonstrating few side effects and a promising therapeutic potential [19,42,43]. In vitro results in NCI-H295R cells indicated a synergistic effect be- tween AZD1775 and sub-effective concentrations of EDP-M (i.e. quarter or half of its IC50) in reducing cellular proliferation and viability, along with an additive effect in reducing cortisol secretion. In primary cultured ACC cells, both AZD1775 and EDP-M alone exerted a strong reduction of cell proliferation, thus no synergic effect was noticeable at these concentrations. However, strong inhibition of cortisol secretion was observed only after combined treatment, suggesting a synergic anti- secretory effect of AZD1775 +EDP-M. It is of interest to note that these cell models have a different genetic background, since NCI-H295R are characterized by a large deletion in the TP53 locus and an activating CTNNB1 mutation [44], whereas primary cultured ACC cells showed no genetic variants in TP53 and CTNNB1 genes. We can hypothesize that the synergistic effect observed in NCI-H295R cells can be due to the p53 deficiency in NCI-H295R cells, as demonstrated in other cancer types. Further studies are required to deeply investigate the impact of genetic background of ACC on the responsiveness to either AZD1775 and the combination with EDP-M, in order to identify predictive biomarkers useful for patients' selection. In vivo xenograft growth and vascularization were hampered in a comparable manner by AZD1775 or EDP-M alone, as well as by cotreatment. Moreover, although the small animal number of each group did not allow to reach statistical significance, IHC analyses demonstrated that AZD1775 was the most effective drug in inducing intra-tumoral necrosis, showing a threefold increase compared to un- treated tumours. Conversely, EDP-M exhibited the strongest antimitotic activity, suggesting that the two drugs act through distinct mechanisms to control tumour growth. Plasma cortisol levels were significantly reduced following treatment with EDP-M or the combination therapy, highlighting the pivotal role of EDP-M in controlling cortisol excess. Although we observed a discrepancy of results obtained from primary cells and in vivo models, our data show that both preclinical models are affected by AZD1775 +EDP-M combination. Notably, histopathological analyses of mice hearts, kidneys, and adrenals revealed no signs of toxicity following in vivo treatments, consistent with previous preclinical studies and clinical trials that have reported the combination of AZD1775 and chemotherapy to be safe and well tolerated [19,45]. The observation that daily administration to mice of AZD1775 at a dose of 60 mg/kg was efficient in reducing tumour growth could constitute a starting point for future clinical trials on ACC, since it could be translated into an administration dose of 340 mg in humans, already tested as tolerable in previous trials on solid tumours [46,47]. Since resistance mechanisms are often the reason of poor therapeutic efficacy or tumour recurrence in ACC [48], we investigated both in vitro and in vivo the onset of possible molecular mechanisms mediating AZD1775 resistance. The onset of resistance to Wee1 inhibitors has been attributed to the upregulation of Myt1, a redundant kinase activated in case of Wee1 absence or impairment, and considered a possible pitfall of AZD1775 treatment in other cancer types [24]. Moreover, ACC patients over- expressing Myt1 in tumoral tissues showed reduced OS [26], suggesting that this protein is relevant in ACC tumour growth. In our in vitro setting, in both NCI-H295R cells and patient-derived primary ACC cells, Myt1 protein levels were upregulated after both AZD1775 treatment and Wee1 genetic silencing, demonstrating that this compensatory mecha- nism is active also in ACC. However, Myt1 was not altered after cotreatment with AZD1775 and EDP-M in patient-derived ACC cells, suggesting that the combined therapy can overcome Myt1-dependent resistance mechanisms in this cell model. Surprisingly, no alterations of Myt1 expression were found in tu- moral tissues derived from AZD1775 and/or EDP-M treated compared to control mice, in contrast with in vitro experiments results. We can hy- pothesize a possible transient compensatory activation of Myt1 after Wee1 inhibition, measured in cell lines models, that is not maintained after the long-term treatment in mice. In addition, it is worth noting that in an in vivo model the effects of the drugs can be modulated by several mechanisms, including the tumour microenvironment-mediated effects, leading to results that are unpredictable in an in vitro model. A similar discrepancy in Myt1 overexpression upon AZD1775 treatment between in vitro and in vivo models has been described for nonsmall cell lung <!-- PageNumber="9" --> <!-- PageBreak --> <!-- PageHeader="E. Nozza et al." --> <!-- PageHeader="Pharmacological Research 221 (2025) 107997" --> cancer [49]. Further experiments testing the levels of Myt1 in vivo at earlier time point, as well as re-exposure of treated mice to a second cycle of treatment could be useful to gain insights in the in vivo mech- anisms of acquired resistance to AZD1775. Despite the variety of ACC preclinical models employed to test AZD1775 efficacy, a potential limitation of the present study is the use of NCI-H295R cell line and primary cultured cells derived from one patient as cell models representing the heterogeneity of ACC pathology. Although NCI-H295R cell line is considered the gold standard human ACC cell model, further similar studies on other ACC cell lines with different genetic background and derivation from both primary tumour and metastasis and, even better, the establishment of patient derived xenograft mouse models, could give deeper insight into AZD1775 effi- cacy in controlling ACC tumoral growth. Another limitation to the generalizability of the study is that it did not consider gender/sex issues. In conclusion, AZD1775 was efficient in reducing in vitro and in vivo growth of ACC tumours. Its application in monotherapy resulted safe and comparable to EDP-M in mice. The combination therapy AZD1775 +EDP-M might allow dose reduction of conventional thera- pies, at least in the subset of patients experiencing severe side effects, while maintaining antitumoral efficacy and cortisol secretion inhibition. Our data, if confirmed by further preclinical and clinical evidence, could support the use of this drug combination to prevent the onset of Myt1- mediated acquired resistance to AZD1775. ### CRediT authorship contribution statement Esposito Emanuela: Writing - original draft, Investigation, Formal analysis, Data curation, Conceptualization. Nozza Emma: Writing - review & editing, Writing - original draft, Investigation, Formal anal- ysis, Data curation, Conceptualization. Ferrante Emanuele: Writing - review & editing, Resources. Di Bari Sonia: Investigation, Data cura- tion. Di Muro Genesio: Writing - original draft, Investigation, Data curation. Peverelli Erika: Writing - review & editing, Writing - original draft, Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization. Lopez Gian- luca: Writing - review & editing, Writing - original draft, Methodology, Investigation. Mantovani Giovanna: Writing - review & editing, Validation, Supervision, Resources, Project administration, Methodol- ogy, Funding acquisition, Conceptualization. Catalano Rosa: Investi- gation, Formal analysis, Data curation. Anna Maria Barbieri: Investigation. Vaira Valentina: Writing - original draft, Investigation, Formal analysis. Mangili Federica: Investigation. Marra Giusy: Investigation. Battistin Michele: Writing - original draft, Resources, Investigation. Treppiedi Donatella: Investigation. Palmieri Serena: Writing - review & editing, Writing - original draft, Resources. Di Dalmazi Guido: Writing - review & editing, Writing - original draft, Resources. ## Declaration of Competing Interest The authors declare they have no conflict of interests. ## Acknowledgements This research was funded by an AIRC (Associazione Italiana Ricerca Cancro) grant to E.P. (IG 2021-25920), by two Progetti di Ricerca di Interesse Nazionale (PRIN) grants to E.P. (P20227KXJK and 2022CZR88M), by Ricerca Corrente Funds from the Italian Ministry of Health to Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, and was supported by European Network for the Study of Adrenal Tu- mours (ENS@T) and COST Action CA20122-Harmonizing clinical care and research on adrenal tumours in European countries (HARMO- NISATION). The authors would like to thank the patient. ## Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.phrs.2025.107997. ## Data availability Data will be made available on request. ## References [1] J. Crona, F. Beuschlein, Adrenocortical carcinoma - towards genomics guided clinical care, Nat. Rev. Endocrinol. 15 (2019). [2] M. Fassnacht, S. Puglisi, O. Kimpel, M. Terzolo, Adrenocortical carcinoma: a practical guide for clinicians, Lancet Diabetes Endocrinol. 13 (5) (2025 May) 438-452. [3] M. Daher, J. Varghese, S.K. Gruschkus, C. Jimenez, S.G. Waguespack, S. Bedrose, et al., Temporal trends in outcomes in patients with adrenocortical carcinoma: a multidisciplinary Referral-center experience, J. Clin. Endocrinol. Metab. 107 (5) (2022 Apr 19) 1239-1246. [4] S. Sidhu, Ip Glover, Soon Zhao, Robinson, Current management options for recurrent adrenocortical carcinoma, Onco Targets Ther. (2013 Jun) 635. [5] M. Fassnacht, G. Assie, E. Baudin, G. Eisenhofer, C. de la Fouchardiere, H.R. Haak, et al., Adrenocortical carcinomas and malignant phaeochromocytomas: ESMO-EURACAN clinical practice guidelines for diagnosis, treatment and follow- up, Ann. Oncol. 31 (11) (2020 Nov) 1476-1490. [6] M. Fassnacht, O.M. Dekkers, T. Else, E. Baudin, A. Berruti, R.R. de Krijger, et al., European society of endocrinology clinical practice guidelines on the management of adrenocortical carcinoma in adults, in collaboration with the european network for the study of adrenal tumors, Eur. J. Endocrinol. 179 (4) (2018 Oct) G1-G46. [7] P. Debets, K.M.A. Dreijerink, A. Engelsman, M. Dahele, H.R. Haak, R.V. Steenaard, et al., Impact of EDP-M on survival of patients with metastatic adrenocortical carcinoma: a population-based study, Eur. J. Cancer 196 (2024 Jan 1). [8] M. Laganà, S. Grisanti, D. Cosentini, V.D. Ferrari, B. Lazzari, R. Ambrosini, et al., Efficacy of the EDP-M scheme plus adjunctive surgery in the management of patients with advanced adrenocortical carcinoma: the brescia experience, Cancers (Basel) 12 (4) (2020). [9] M. Tamburello, A. Abate, E. Rossini, R.M. Basnet, D. Zizioli, D. Cosentini, et al., Preclinical evidence of progesterone as a new pharmacological strategy in human adrenocortical carcinoma cell lines, Int J. Mol. Sci. 24 (7) (2023 Apr 1). [10] L. Wang, Y. Lyu, Y. Li, K. Li, H. Wen, C. Feng, et al., Correction: ASXL1 promotes adrenocortical carcinoma and is associated with chemoresistance to EDP regimen, (Original article: Aging (Albany NY) (2021), 13(18), (22286-22297), (PMID: 34536950), (PMCID: PMC8507286), (10.18632/aging.203534)), Aging Impact J. LLC 15 (2023) 11690-1. [11] D.R. Szabó, K. Baghy, P.M. Szabó, A. Zsippai, I. Marczell, Z. Nagy, et al., Antitumoral effects of 9-cis retinoic acid in adrenocortical cancer, Cell. Mol. Life Sci. 71 (5) (2014 Mar) 917-932. [12] E. Esposito, G. Marra, R. Catalano, S. Maioli, E. Nozza, A.M. Barbieri, et al., Therapeutic potential of targeting the FLNA-regulated Wee1 kinase in adrenocortical carcinomas, Int J. Cancer [Internet] 156 (6) (2025 Mar 15) 1256-1271. Available from: (https://onlinelibrary.wiley.com/doi/10.1002/ij c.35239). [13] S.E. Mir, P.C. De Witt Hamer, P.M. Krawczyk, L. Balaj, A. Claes, J.M. Niers, et al., In silico analysis of kinase expression identifies WEE1 as a gatekeeper against mitotic catastrophe in glioblastoma, Cancer Cell 18 (3) (2010) 244-257. [14] L.M. Murrow, S.V. Garimella, T.L. Jones, N.J. Caplen, S. Lipkowitz, Identification of WEE1 as a potential molecular target in cancer cells by RNAi screening of the human tyrosine kinome, Breast Cancer Res Treat. 122 (2) (2010 Jul) 347-357. [15] A. Ghelli Luserna Di Rorà, C. Cerchione, G. Martinelli, G. Simonetti, A WEE1 family business: regulation of mitosis, cancer progression, and therapeutic target, in: Journal of Hematology and Oncology, 13, BioMed Central Ltd, 2020. [16] A. Turla, M. Laganà, A. Abate, V. Cremaschi, M. Zamparini, M. Chittò, et al., Feasibility and activity of megestrol acetate in addition to etoposide, doxorubicin, cisplatin, and mitotane as First-Line therapy in patients with Metastatic/ Unresectable adrenocortical carcinoma with low performance status, Cancers (Basel) 15 (18) (2023 Sep 1). [17] Z. Zhang, N. Liu, Q. Li, EDP-M plus sintilimab in the treatment of adrenocortical carcinoma: a case report, Transl. Cancer Res 11 (6) (2022 Jun 1) 1829-1835. [18] T.B. Garcia, J.C. Snedeker, D. Baturin, L. Gardner, S.P. Fosmire, C. Zhou, et al., A Small-Molecule inhibitor of WEE1, AZD1775, synergizes with olaparib by impairing homologous recombination and enhancing DNA damage and apoptosis in acute leukemia, Mol. Cancer Ther. 16 (10) (2017 Oct) 2058-2068. [19] S. Leijen, R.M.J.M. Van Geel, A.C. Pavlick, R. Tibes, L. Rosen, A.R.A. Razak, et al., Phase I study evaluating WEE1 inhibitor AZD1775 as monotherapy and in combination with gemcitabine, cisplatin, or carboplatin in patients with advanced solid tumors, J. Clin. Oncol. 34 (36) (2016 Dec 20) 4371-4380. [20] D. Chen, X. Lin, J. Gao, L. Shen, Z. Li, B. Dong, et al., Wee1 inhibitor AZD1775 combined with cisplatin potentiates anticancer activity against gastric cancer by increasing DNA damage and cell apoptosis, Biomed. Res Int 2018 (2018). [21] S.J. Hartman, S.M. Bagby, B.W. Yacob, D.M. Simmons, M. MacBeth, C.H. Lieu, et al., WEE1 inhibition in combination with targeted agents and standard <!-- PageNumber="10" --> <!-- PageBreak --> <!-- PageHeader="E. Nozza et al." --> <!-- PageHeader="Pharmacological Research 221 (2025) 107997" --> chemotherapy in preclinical models of pancreatic ductal adenocarcinoma, Front Oncol. 11 (2021 Mar 25). [22] A. Lallo, K.K. Frese, C.J. Morrow, R. Sloane, S. Gulati, M.W. Schenk, et al., The combination of the PARP inhibitor olaparib and the WEE1 inhibitor AZD1775 as a new therapeutic option for small cell lung cancer, Clin. Cancer Res. 24 (20) (2018 Oct 15) 5153-5164. [23] M.R.W. de Jong, M. Langendonk, B. Reitsma, P. Herbers, M. Lodewijk, M. Nijland, et al., WEE1 inhibition synergizes with CHOP chemotherapy and radiation therapy through induction of premature mitotic entry and DNA damage in diffuse large B- cell lymphoma, Ther. Adv. Hematol. 11 (2020 Jan), 204062071989837. [24] C.W. Lewis, A.B. Bukhari, E.J. Xiao, W.S. Choi, J.D. Smith, E. Homola, et al., Upregulation of MyT1 promotes acquired resistance of cancer cells to WEE1 inhibition, Cancer Res 79 (23) (2019 Dec 1) 5971-5985. [25] C. Shao, Y. Wang, M. Pan, K. Guo, T.F. Molnar, F. Kocher, et al., The DNA damage repair-related gene PKMYT1 is a potential biomarker in various malignancies, Transl. Lung Cancer Res 10 (12) (2021 Dec 1) 4600-4616. [26] S. Sokhi, C.W. Lewis, A.B. Bukhari, J. Hadfield, E.J. Xiao, J. Fung, et al., Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors, Front Cell Dev. Biol. 11 (2023). [27] C. Hantel, S. Jung, T. Mussack, M. Reincke, F. Beuschlein, Liposomal polychemotherapy improves adrenocortical carcinoma treatment in a preclinical rodent model, Endocr. Relat. Cancer 21 (3) (2014) 383-394. [28] M.M. Tomayko, C.P. Reynolds, Determination of subcutaneous tumor size in athymic (nude) mice, Cancer Chemother. Pharm. 24 (1989) 148-154. [29] Y.Y. Lee, Y.J. Cho, S. won Shin, C. Choi, J.Y. Ryu, H.K. Jeon, et al., Anti-Tumor effects of Wee1 kinase inhibitor with radiotherapy in human cervical cancer, Sci. Rep. 9 (1) (2019 Dec 1). [30] K. Murakami, Y. Kita, T. Sakatani, A. Hamada, K. Mizuno, K. Nakamura, et al., Antitumor effect of WEE1 blockade as monotherapy or in combination with cisplatin in urothelial cancer, Cancer Sci. 112 (9) (2021 Sep 1) 3669-3681. [31] S. Bi, Q. Wei, Z. Zhao, L. Chen, C. Wang, S. Xie, Wee1 inhibitor AZD1775 effectively inhibits the malignant phenotypes of esophageal squamous cell carcinoma in vitro and in vivo, Front Pharm. 10 (JULY) (2019). [32] M.V. Russo, A. Faversani, S. Gatti, D. Ricca, A. Del Gobbo, S. Ferrero, et al., A new mouse avatar model of Non-Small cell lung cancer, Front Oncol. 5 (2015 Mar 3). [33] I. Righi, V. Vaira, L.C. Morlacchi, G.A. Croci, V. Rossetti, F. Blasi, et al., Immune checkpoints expression in chronic lung allograft rejection, Front Immunol. 12 (2021 Aug 13). [34] S. Zheng, W. Wang, J. Aldahdooh, A. Malyutina, T. Shadbahr, Z. Tanoli, et al., SynergyFinder plus: toward better interpretation and annotation of drug combination screening datasets, Genom. Proteom. Bioinforma. 20 (3) (2022 Jun 1) 587-596. [35] C.R. Corso, A, Acco, C. Bach, S.J.R. Bonatto, B.C. de Figueiredo, L.M. de Souza, Pharmacological profile and effects of mitotane in adrenocortical carcinoma, in: British Journal of Clinical Pharmacology, 87, John Wiley and Sons Inc, 2021, pp. 2698-2710. [36] C.S. Winther, F.K. Nielsen, M. Hansen, B. Styrishave, Corticosteroid production in H295R cells during exposure to 3 endocrine disrupters analyzed with LC-MS/MS, Int J. Toxicol. 32 (3) (2013 May) 219-227. [37] M. Kroiss, M. Quinkler, W.K. Lutz, B. Allolio, M. Fassnacht, Drug interactions with mitotane by induction of CYP3A4 metabolism in the clinical management of adrenocortical carcinoma, Clin. Endocrinol. (Oxf. ) 75 (5) (2011 Nov) 585-591. [38] C. Pak, S. Yoon, J.L. Lee, T. Yun, I. Park, Current status and future direction in the treatment of advanced adrenocortical carcinoma, in: Current Oncology Reports, 26, Springer, 2024, pp. 307-317. [39] H. Hirai, T. Arai, M. Okada, T. Nishibata, M. Kobayashi, N. Sakai, et al., MK-1775, a small molecule Wee1 inhibitor, enhances antitumor efficacy of various DNA- damaging agents, including 5-fluorouracil, Cancer Biol. Ther. 9 (7) (2010 Apr 1) 514-522. [40] H. Hirai, Y. Iwasawa, M. Okada, T. Arai, T. Nishibata, M. Kobayashi, et al., Small- molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents, Mol. Cancer Ther. 8 (11) (2009 Nov) 2992-3000. [41] N.V. Rajeshkumar, E. De Oliveira, N. Ottenhof, J. Watters, D. Brooks, T. Demuth, et al., MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts, Clin. Cancer Res. 17 (9) (2011 May 1) 2799-2806. [42] S. Fu, S. Yao, Y. Yuan, R.A. Previs, A.D. Elias, R.D. Carvajal, et al., Multicenter phase II trial of the WEE1 inhibitor adavosertib in refractory solid tumors harboring CCNE1 amplification, J. Clin. Oncol. [Internet] 41 (2022) 1725-1734. Available from: (https://doi). [43] K.N. Moore, S.K. Chambers, E.P. Hamilton, L.M. Chen, A.M. Oza, S.A. Ghamande, et al., Adavosertib with chemotherapy in patients with primary Platinum-Resistant ovarian, fallopian tube, or peritoneal cancer: an Open-Label, Four-Arm, phase II study, Clin. Cancer Res. 28 (1) (2022 Jan 1) 36-44. [44] S. Sigala, E. Rossini, A. Abate, M. Tamburello, S.R. Bornstein, C. Hantel, An update on adrenocortical cell lines of human origin, in: Endocrine, 77, Springer, 2022, pp. 432-437. [45] K. Do, D. Wilsker, J. Ji, J. Zlott, T. Freshwater, R.J. Kinders, et al., Phase I study of single-agent AZD1775 (MK-1775), a wee1 kinase inhibitor, in patients with refractory solid tumors, J. Clin. Oncol. 33 (30) (2015 Oct 20) 3409-3415. [46] A. Nair, M.A. Morsy, S. Jacob, Dose translation between laboratory animals and human in preclinical and clinical phases of drug development, in: Drug Development Research, 79, Wiley-Liss Inc., 2018, pp. 373-382. [47] M. Johnson, D. Kaschek, D. Ghiorghiu, S. Lanke, K. Miah, H. Schmidt, et al., Population pharmacokinetic modeling of adavosertib (AZD1775) in patients with solid tumors, J. Clin. Pharm. 64 (11) (2024 Nov 1) 1419-1431. [48] L.S. Kirschner, Review: emerging treatment strategies for adrenocortical carcinoma: a new hope, J. Clin. Endocrinol. Metab. 91 (2006) 14-21. [49] Y. Takashima, E. Kikuchi, J. Kikuchi, M. Suzuki, H. Kikuchi, M. Maeda, et al., Bromodomain and extraterminal domain inhibition synergizes with WEE1- inhibitor AZD1775 effect by impairing nonhomologous end joining and enhancing DNA damage in nonsmall cell lung cancer, Int J. Cancer 146 (4) (2020 Feb 15) 1114-1124. <!-- PageNumber="11" -->