RESEARCH
Preclinical assessment of synergistic efficacy of MELK and CDK inhibitors in adrenocortical cancer
Check for updates
Dipranjan Laha1, Robert R.C. Grant1, Prachi Mishra1, Myriem Boufragech2, Min Shen3, Ya-Qin Zhang3, Matthew D. Hall3, Martha Quezado4, Michelly Sampaio De Melo4, Jaydira Del Rivero5, Martha Zeiger1 and Naris Nilubol1*
Abstract
Background: Adrenocortical cancer (ACC) is a rare and aggressive cancer with dismal 5-year survival due to a lack of effective treatments. We aimed to identify a new effective combination of drugs and investigated their synergistic efficacy in ACC preclinical models.
Methods: A quantitative high-throughput drug screening of 4,991 compounds was performed on two ACC cell lines, SW13 and NCI-H295R, based on antiproliferative effect and caspase-3/7 activity. The top candidate drugs were pairwise combined to identify the most potent combinations. The synergistic efficacy of the selected inhibitors was tested on tumorigenic phenotypes, such as cell proliferation, migration, invasion, spheroid formation, and clonogenic- ity, with appropriate mechanistic validation by cell cycle and apoptotic assays and protein expression of the involved molecules. We tested the efficacy of the drug combination in mice with luciferase-tagged human ACC xenografts. To study the mRNA expression of target molecules in ACC and their clinical correlations, we analyzed the Gene Expres- sion Omnibus and The Cancer Genome Atlas.
Results: We chose the maternal embryonic leucine zipper kinase (MELK) inhibitor (OTS167) and cyclin-dependent kinase (CDK) inhibitor (RGB-286638) because of their potent synergy from the pairwise drug combination matrices derived from the top 30 single drugs. Multiple publicly available databases demonstrated overexpression of MELK, CDK1/2, and partnering cyclins mRNA in ACC, which were independently associated with mortality and other adverse clinical features. The drug combination demonstrated a synergistic antiproliferative effect on ACC cells. Compared to the single-agent treatment groups, the combination treatment increased G2/M arrest, caspase-dependent apopto- sis, reduced cyclins A2, B1, B2, and E2 expression, and decreased cell migration and invasion with reduced vimentin. Moreover, the combination effectively decreased Foxhead Box M1, Axin2, glycogen synthase kinase 3-beta, and B-catenin. A reduction in p-stathmin from the combination treatment destabilized microtubule assembly by tubulin depolymerization. The drug combination treatment in mice with human ACC xenografts resulted in a significantly lower tumor burden than those treated with single-agents and vehicle control groups.
*Correspondence: naris.nilubol@nih.gov
1 Surgical Oncology Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Full list of author information is available at the end of the article
☒ BMC
@ The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Conclusions: Our preclinical study revealed a novel synergistic combination of OTS167 and RGB-286638 in ACC that effectively targets multiple molecules associated with ACC aggressiveness. A phase Ib/II clinical trial in patients with advanced ACC is therefore warranted.
Keywords: Adrenocortical cancer, Maternal embryonic leucine zipper kinase, Cyclin-dependent kinase, ß-catenin, Quantitative high-throughput screening, Targeted therapy
Background
ACC is a rare and aggressive endocrine malignancy with a yearly incidence of 0.5-2 cases per million population [1, 2]. Due to late clinical presentation and the aggres- sive behavior of this tumor, patients with ACC commonly present at an advanced stage [2-4]. Unfortunately, loco- regional recurrence and distant metastasis are common (up to 50-80%) despite complete surgical resection [5, 6]. The high mortality rate in patients with advanced ACC is due to aggressive biology and a lack of effective sys- temic treatments. In the past five decades, mitotane has been the only FDA-approved treatment in patients with advanced ACC and is first-line systemic therapy alone or with etoposide, doxorubicin, and cisplatin, a poorly tol- erated and minimally efficacious regimen based on the FIRM-ACT trial [7]. Overall survival (OS) remains poor with these therapies. Thus, there is a critical need to iden- tify novel, effective treatment strategies. The comprehen- sive molecular analyses in large cohorts of patients by the European Network for the Study of Adrenal Tumors (ENSAT) and TCGA provide crucial information by identifying important mutations and dysregulated path- ways by molecular signatures [8, 9]. Approximately 40% of ACC samples in TCGA and ENSAT cohorts had path- ogenic alterations in genes involved in the Wnt/B-catenin signaling pathway and many alterations involving genes that regulate the cell cycle. To date, there have been no treatments that effectively target the key downstream molecules of these pathways.
As we previously demonstrated, quantitative high- throughput drug screening (qHTS) using the pharmaceu- tical library containing agents tested or used in humans is an effective and efficient way to identify single-drug candidates and drug combinations that could be read- ily translated to a phase Ib/II clinical trial in patients with advanced ACC [10]. In this study, the combina- tion of the maternal embryonic leucine zipper kinase (MELK) inhibitor (OTS167) and the cyclin-dependent kinase (CDK) inhibitor (RGB-286638) was identified as a novel treatment in ACC. We validated the synergy of these drugs in multiple in vitro models and comprehen- sively elucidated their mechanisms via the inhibition of multiple key downstream molecules associated with the Wnt/B-catenin signaling pathway and the induction of G2/M cell cycle arrest. Combination treatment inhibited
cell invasion and reduced the epithelial-to-mesenchymal transition (EMT) markers. Furthermore, we confirmed in in vivo systems that mice treated with OTS167 and RGB- 286638 combination had a significantly lower tumor burden than those in control groups. These results dem- onstrate the effectiveness of these agents against ACC in preclinical studies and support a further evaluation in a clinical trial.
Materials and methods
Patient samples
Human adrenocortical tissue samples were collected under the clinical protocol entitled “Prospective com- prehensive molecular analysis of endocrine neoplasms” (Clinical Trial Registration number NCT01005654). The ethical approval was granted by the Institutional Review Board, National Cancer Institute, NIH, and the NIH Office of Human Subject Research. All participants pro- vided written informed consent.
qHTS and combination matrix screening
The National Center for Advancing Translational Sci- ences (NCATS) Pharmaceutical Collection (NPC) and the Mechanism Interrogation PlatE (MIPE) library, which in total consist of 4,991 small-molecule drugs and inves- tigational compounds, were screened against SW13 and NCI-H295R cell lines. Cell viability was measured using a luciferase-coupled ATP quantitation assay (CellTiter- Glo®, Promega, Madison, WI). We described the detailed methods of quantitative high-throughput screening in the supplementary section.
A combination matrix screen was performed with a subset of active hits identified by qHTS. Plating of compounds in matrix format using acoustic droplet ejection and numerical characterization of synergy, addi- tivity, and/or antagonism was conducted as described previously [11, 12]. The detailed method of combination matrix screening was described in the supplementary section.
Gene expression profiling
We analyzed publicly available genome-wide expression data from the Gene Expression Omnibus (GEO) (NCBI gene expression and hybridization array data repository) in three cohorts (GSE33371, GSE12368, and GSE90713)
to study the differential messenger RNA (mRNA) expres- sion of genes of interest in human ACC samples com- pared to adrenocortical adenoma (ACA) and normal adrenal cortex (NC) [13]. Using data from TCGA and the European Bioinformatics Institute (E-TABM-311), we analyzed clinicopathologic correlations with mRNA expression of these genes to assess clinical relevance.
Immunohistochemistry analysis
ACC, ACA patient tissue, and human ACC xenograft tis- sues were formalin-fixed, embedded in paraffin, and used for immunohistochemistry (IHC) analysis. 5-um-thick sections were used for hematoxylin and eosin (H&E) and IHC staining according to a previously published proto- col [10]. We described the IHC techniques in the supple- mentary materials section.
Cell lines and culture conditions
Two human ACC cell lines, SW13 and NCI-H295R, were purchased from the American Type Culture Collec- tion™ (Cat # CCL-105, CRL-2128; Manassas, VA, USA) and cultured in 5% CO2 atmosphere at 37 ℃ in Dulbec- co’s Modified Eagle Medium (Cat # 11195-065, Thermo Fisher Scientific, MA, USA) supplemented with 2.5% Nu-Serum (Cat # 355100, Corning, MA, USA) and 0.1% Insulin-Transferrin-Selenium (Cat # 41400045, Thermo Fisher Scientific, MA, USA). Cell lines were authenti- cated by short tandem repeat profiling. We routinely sub- cultured every 3-5 days, depending on the degree of cell confluence.
NCI-H295R cells used to generate human ACC xeno- graft were transfected with a linearized pGL4.51[luc2/ CMV/Neo] vector (9PIE132, Promega) encoding the luciferase reporter gene luc2 (Photinus pyralis) and maintained in the above medium with up to 500 µg/mL of G-418 antibiotic (11811-023, Gibco, MA, USA) for selection.
Cellular proliferation assay
The effects of drug treatments on cell proliferation were quantitated by CyQuant assay (Cat # C7026, Invitrogen, MA, USA). SW13 (3×103) and NCI-H295R (6x103) cells were plated in 96-well black plates (Cat # 353219, Costar®, Corning, NY, USA). After 24 h, the culture medium containing vehicle control (dimethyl sulfoxide up to 0.125%), OTS167, RGB-286638, or the combination of OTS167 and RGB-286638 were added at various con- centrations. The medium with the drugs or vehicle was replaced every 48 h. Fluorescence intensity was deter- mined using a microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 485 nm/538 nm. We repeated the experiment with consistent results at least three times.
We used the automated computerized algorithm (Chou-Talalay method) to assess the synergistic effi- cacy. Efficacy indicated by the combination index (CI) was compared to cells treated with a single drug. CI<1 indicated synergy; CI = 1 indicated an additive effect; and CI>1, indicated an antagonistic effect [14].
Three-dimensional multicellular aggregates (MCA)
Compared to monolayer cell culture that lacks the tumor microenvironment, MCAs recapitulate the in vivo envi- ronment by growing solid, 3-dimensional tumors in vitro more accurately as MCAs contain different areas affected by various degrees of oxygenation, nutrients, and drug exposure [15]. SW13 (6x104 cells/0.5 ml) and NCI- H295R (1 × 105 cells/0.5 ml) cells, which form multicellu- lar aggregates (MCA) or tumor spheroids, were plated in ultra-low cluster 24-well plates (Cat No # 3473, Costar®, Corning, NY, USA). The anticancer activity of OTS167, RGB-286638, and the combination of OTS167 and RGB- 286638 were tested in MCAs that mimic solid tumors in vitro, in ACC cell lines according to standard protocol. The detailed methods are described in the supplementary materials section.
Clonogenic assay
Cells were seeded in triplicate in 6-well plates (1000 cells/ well) and allowed to grow for 7-10 days. The cells were then treated with drug(s) alone or in combination or with the vehicle in complete media for 12 to 14 days. Growth media with vehicle or drug(s) were replaced every 48-72 h. The cells were fixed with 0.4% buffered para- formaldehyde and then stained with 0.5% crystal violet in methanol for 10 min. The colonies were counted and photographed using a ChemiDoc system (Bio-Rad).
Caspase-3/-7 activation assay
To check caspase-3/7-mediated apoptosis, cells were plated in 96-well plates and treated for 24 to 48 h with various concentrations of the drug combination. The cas- pase-3/-7 activity was measured using the Caspase-Glo® 3/7 assay (Cat # G8091, Promega, USA), according to the manufacturer’s instructions. The method of treatment for caspase-3 and -7 activation and analysis are described in the supplementary materials section.
Cell cycle assay
SW13 (3×104) and NCI-H295R (2×105) cells were plated in a 100-mm dish with 10 mL of culture medium and treated 24 h later. After 24 to72 hours, cells were trypsinized, washed with phosphate-buffered saline (PBS), and fixed with ice-cold 70% ethanol. Cells were then resuspended in PBS with ribonuclease A (100 mg/ mL) and propidium iodide (PI) (0.05 mg/ml) for
fluorescence-activated cell sorting analysis using the Canto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Data were generated for at least 20,000 events per sample. The cell cycle of the gated PI distri- bution was analyzed using ModFit software (Verity Soft- ware House, Inc., Topsham, ME, USA).
Western blot analysis
Cell lysates were prepared from the cells after treatment with drug(s) or vehicle control. The protein concentration was determined using the Pierce™ BCA assay kit solu- tion (Cat # 23227, Thermo Fisher Scientific, Waltham, MA, USA). An equal amount of proteins from different treatment conditions was used for western blot experi- ments. The western blot techniques are described in the supplementary materials section. Because the treatments directly affected ACC cell cycles and the doubling time of SW13 and NCI-H295R cells are approximately 24 and 48 h, respectively, the optimal time to capture the changes in molecular signaling was shorter in SW13 (24- 48 h) than in NCI-H295R (48-72 h) before ACC cells underwent treatment-related apoptosis.
Cellular migration and invasion assay
To determine the effects of a single drug or combina- tion drugs on the migratory and invasive capacity of ACC cells, we performed cellular migration and invasion assays according to the manufacturer’s instruction (Cat # 354578, Cat # 3544880, BD Bioscience, San Joes, CA, USA). ACC cells were plated in six-well plates in a trip- licate manner and treated with varying concentrations of OTS167, RGB-286638, the combination of OTS167 and RGB-286638, and vehicle control for 24 h and 48 h, respectively. The methods of cellular migration and inva- sion assay are described in the supplementary materials section.
Immunofluorescence analysis
2 × 105 SW13 and NCI-H295R cells were plated on glass coverslips, allowed to attach overnight, and treated for 48 to 72 h. Cells were fixed with 4% paraformaldehyde, permeabilized in 0.25% Triton-X, and incubated over- night with primary antibodies. DNA was stained with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were obtained by fluorescence microscopy with 40 x magnification and collected using Carl Zeiss ZEN Software (Zeiss, Germany).
Separation of polymerized and depolymerized tubulin
Tubulin polymerization and depolymerization assay was performed to check the effects of drug treatments on
the tubulin polymerization process in ACC cells. There- fore, SW13 (1 x 106) and NCI-H295R (1 x 106) cells were plated in a 100 mm dish with 10 mL of culture medium. Cells were treated with drug(s) and vehicle control for 48 h for SW13 and 72 h for NCI-H295R cells. The method of separation of polymerized and depolymerized tubulin assays is described in the supplementary materials section.
Oligo small interfering RNA (siRNA)-mediated transfection
SW13 and NCI-H295R cells were transfected with small interfering RNA (siRNA) specific for MELK (4390824, assay ID s386, Thermo Fisher Scientific, MA, USA) or control siRNA (4390843, Thermo Fisher Scientific, MA, USA) using Lipofectamine RNAiMAX (13778-015, Inv- itrogen; Thermo Fisher Scientific, Inc., MA, USA). After 48 h of transfection, Western Blot was performed to check the transfection efficiency of MELK knockdown and target proteins.
In vivo study
The protocol designed to study the in vivo efficacy of OTS167 and RGB-286638 in mice with human ACC xenografts was approved by the National Cancer Insti- tute, National Institutes of Health (NIH), Animal Care and Use Committee. Mice were maintained accord- ing to NIH Animal Research Advisory Committee guidelines. A total of 5 x 106 NCI-H295R cells with luciferase reporter in Corning® Matrigel® Matrix (Cat # 354234, Corning, NY, USA) were injected into each flank of a Nup/Nup mouse (two xenografts per mouse). After 21 days, mice were randomized into four groups by the treatment. We selected the dosages of OTS167 and RGB-286638 based on prior publications that showed in vivo efficacy with no signs of treatment- related toxicities in mice [16, 17]. Treatments included: Group 1: 0.1% DMSO as vehicle control; Group 2: daily (Monday-Friday) OTS167 (10 mg/kg) via intraperito- neal injection; Group 3: RGB-286638 (20 mg/kg) using an intravenous injection (IV) via tail vein three times (Monday, Wednesday, Friday) weekly for two weeks, followed by RGB-286638 drugs (6 mg/100 ul) loaded ALZET pumps with 0.25 ul/hour delivery rate (Model 1002, Alzet, Cupertino, CA, USA) Group 4: the combi- nation of OTS167 (10 mg/kg) and RGB-286638 (20 mg/ kg), following the above protocol for drug administra- tions. The duration of treatment was five weeks. Mice received daily health monitoring, and mouse weight was recorded weekly. In brief, the details of in vivo imaging studies are described in the supplementary materials.
| 0 | 0.1 | 0 | 0.1 | 0.1 | 0 | 0 | 0 | -0.1 | 0 | 10000.0 | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 0.3 | 0.2 | 0 | 0.2 | 0.1 | 0.3 | 0.1 | 0 | 0.1 | 5000.0 | |
| 0 | 0.2 | 0.2 | 0.3 | 0.3 | 0.2 | 0.4 | 0.5 | 0.2 | -0.1 | 2500.0 | |
| 0.1 | 0 | 0.2 | 1.2 | 0.5 | 0.7 | 0.3 | 0.5 | 0.5 | -0.1 | 1250.0 | |
| 0.1 | 0.1 | 0.4 | 1.8 | 1.5 | 0.5 | 1.1 | 1.4 | 1.5 | 0.5 | 625.0 | |
| 0.3 | 0 | 1.2 | 4.1 | 4.8 | 4.7 | 6.3 | 11.7 | 10.9 | 11.4 | -312.5 | |
| 0.4 | 0.2 | 2.3 | 1.7 | 1.7 | 7.6 | 28.4 | 42.5 | 51.2 | 62.3 | 156.25 | |
| 0.5 | 0.1 | 1.9 | 0.9 | 2 | 24.1 | 45.4 | 57.4 | 16.7 | 85.7 | -78.13 | |
| 0.5 | 0.6 | 1.6 | 0.6 | 6 | 28.6 | 58.3 | 77.4 | 92 | 99.4 | -39.06 | |
| 0.6 | 0.1 | 1.4 | 0.9 | 7.7 | 28.5 | 55.1 | 80.4 | 85.5 | 97.4 | 0.0 | |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0000.0 |
|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 5000.0 |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2500.0 |
| 0 | 0 | 0 | 0.01 | 0.01 | 0.01 | 0 | 0.01 | 0.01 | 0 | 1250.0 |
| 0 | 0 | 0 | 0.01 | 0.01 | 0 | 0.01 | 0.01 | 0.01 | 0 | 625.0 |
| 0 | 0 | 0 | 0.03 | -0.03 | -0.07 | -0.05 | 0 | 0 | 0 | -312.5 |
| 0 | 0 | 0.01 | 0.01 | -0.06 | -0.21 | -0.27 | -0.2 | -0.11 | 0 | 156.25 |
| 0 | 0 | 0 | 0 | -0.06 | -0.04 | -0.1 | -0.23 | -0.69 | 0 | -78.13 |
| 0 | 0.01 | 0 | 0 | -0.02 | 0 | 0.03 | -0.03 | 0.06 | 0.02 | -39.06 |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.0 |
A
B
Dose Response
HSA Synergy
A RGB-286638(nM) A
RGB-286638(nM)
5000.0 2500.0 1250.0 625.0 312.5 156.25 78.13 39.06 19.53
0.0
5000.0 2500.0 1250.0 625.0 312.5 156.25 78.13 39.06 19.53 0.0
OTS167(nM)
OTS167(nM)
C
NCI-H295R
NCI-H295R
%Inhibition
40
40-
0
OTS167
%Inhibition
Cisplatin
0
· RGB-286638
Cisplatin
-40
Doxorubicin
-40
E Doxorubicin
-80
Etoposide
A Etoposide
Mitotane
-80
-120
Streptozocin
℮ Mitotane
Streptozocin
-10 -9 -8 -7 -6 -5 -4 Log[Compound], M
-120
-10 -9
-8
-7 -6 -5 -4
Log[Compound], M
SW13
SW13
%Inhibition
40
40-
0
· OTS167
%Inhibition
0
· RGB-286638
-40
Cisplatin
E Doxorubicin
Cisplatin
Etoposide
-40
a Doxorubicin
-80
0
A Mitotane
-80
0
e Etoposide
-120
Streptozocin
A Mitotane
-10 -9 -8 -7 -6 -5 -4
-120
Streptozocin
Log[Compound], M
-10 -9 -8 -7 -6 -5 -4
Log[Compound], M
Fig. 1 Drug candidates identified through high throughput drug screening in ACC: A 10 x 10 combination matrix screening data demonstrating the drug synergy between OTS167 and RGB-286638. B HSA synergy of OTS167 and RGB-286638 drugs. C Dose-response curve comparing OTS167 (left panel) and RGB-286638 (right panel) with the current standard of care chemotherapy: cisplatin, doxorubicin, etoposide, mitotane, and streptozocin in NCI-H295R and SW13 cells
Statistical analysis
Statistical analyses were performed using SPSS version 25.0 for Windows (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA). Statistical analysis methods are described in the supplementary materials section.
Results
qHTS identified effective combination therapy in ACC
The results of qHTS produced 85 compounds with efficacy>80% at concentrations <1 µM in both cell lines. An orthogonal assay to measure caspase-3/-7
activation identified 30 of these 85 compounds that were then tested in pairwise combination matrices (Supplementary Table 1). The combination of OTS167 and RGB-286638 showed a potent synergistic effect based on the highest single agent (HSA) and Bliss syn- ergy model scores at concentrations in the nanomolar range (Fig. 1A, B). We then compared the single-agent activity of these drugs to current systemic therapy for
patients with advanced ACC. Each had higher efficacy at equal or lower concentrations than individual treat- ment EDP-M or streptozocin in both cell lines (Fig. 1C).
Expressions of MELK, CDK 1 and 2, and partnering cyclins are associated with poor clinical outcomes in ACC Because OTS167 is a MELK inhibitor, we aimed to determine if ACC overexpresses MELK mRNA in
A
GSE33371
B
ACA
ACC
MELK mRNA Expression
GSE12368
C
positive cells(%)
positive cells (%)
5.
MELK mRNA Expression
*
25-
20
25
5000
MELK
MELK
Ki67
4
4000
ACA (n=10)
ACC(n=10)
3000
ACA(n=10)
ACC(n=10)
3
2.
2000
100 um
100 um
1
1000
50-
40
0
0
30
NC (n=10)
ACA (n=22)
ACC (n=33)
NC (n=6)
ACA (n=16)
ACC (n=12)
Ki67
Ki67
20
10
0
0
100
200
300
100 pm
100 jim
MELK
D
E
F
Overall Survival by mRNA Expression
1.0
Overall Survival by mRNA Expression
Disease Free Survival by mRNA Expression
Low MELK
Percent Survival
1.0
1.0
0.8
High MELK
Low MELK-
Percent Survival
Low MELK
0.8
High MELK
Percent Survival
0.8
Low MELK
nHigh MELK
0.6
censored
Low MELK.
High MELK-
0.6
censored
0.6
Low MELK-
p<0.01
censored
p<0.01
High MELK
censored
0.4
0.4
censored
0.4
p<0.01
High MELK-
censored
0.2
0.2
0.2
0.0
0.0
0.0
.00
2.50
5.00
7.50
10.00 12.50
.00
2.00
4.00
6.00
8.00
10.00
.00
2.50
5.00
7.50
10.00 12.50
Follow up Duration (Years)
Follow up Duration (Years)
Follow up Duration (Years)
G
MELK Expression by Stage
H
MELK Expression by T Stage
I
MELK Expression by Lymph Node Status
J
MELK Expression by Vital Status
1500
6-
6
*
mRNA Expression
mRNA Expression
mRNA Expression
mRNA Expression
6-
1000-
4
4
4.
2-
2-
2-
500
0
0
0-
L
0
-2
-2
-2
Stage I-II
Stage III-IV
T1-T2
T3-T4
Negative
Positive
Alive Dead
human samples. MELK mRNA expression was sig- nificantly higher in ACC compared to ACA and NC in two GEO cohorts (Fig. 2A). Next, we analyzed MELK protein expression and its relation to Ki67, a marker of proliferation commonly used as a prog- nostic indicator in ACC, by IHC in human adrenal samples from the NIH cohort and found a linear and, positive correlation (Spearman’s correlation coeffi- cient 0.5049, p= 0.04) (Fig. 2B, C) [18, 19]. Analysis of the TCGA and E-TABM-311 databases showed that the estimated overall survival (OS) of patients with ACC that overexpressed MELK was shorter than patients with low MELK mRNA expression (TCGA: 4.3 years vs. 10.8 years, p<0.001; E-TABM-311: 3.1 years vs. 7.7 years, p= 0.004) (Fig. 2D, E). Moreo- ver, in the TCGA cohort, overexpression of MELK was associated with shorter disease-free survival (DFS) (3.6 years vs. 8.6 years, p<0.001), more advanced tumor stage and overall stage, lymph node metastasis, and death during follow-up (Fig. 2F-J).
Because RGB-286638 targets the kinase activity of mul- tiple CDKs, we analyzed mRNA expressions of CDK1/2 and their associated cyclins in the GEO cohorts. Previ- ous work has shown overexpression of CDK1 and CDK2 in ACC [10]. Here, the additional independent data set (GSE90713) confirmed that CDK1 mRNA expression, but not CDK2, was significantly higher in ACC compared to NC (Supplementary Fig. 1A). We also found a significant positive linear correlation between the mRNA expression of MKI67 and both CDK1 and CDK2 (Supplementary Table 2). Because CDK inhibitors do not change the pro- tein expression of CDKs, and measuring CDK activity as a marker of treatment response is impractical, we studied the expression of the partnering cyclins and their clinical relevance in ACC retrospectively. We found overexpres- sion of cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin B2 (CCNB2), and cyclin E2 (CCNE2) mRNA in ACC compared to NC and ACAwith the exception of CCNE2 in GSE90713 (Supplementary Fig. 1B-E). Next, we con- firmed the positive linear correlations between the mRNA expression of MKI67 vs. CCNA2, CCNB1, CCNB2, and CCNE2, suggesting their role in tumor progression (Sup- plementary Table 2). From the TCGA dataset, we con- firmed that patients with CCNA2, CCNB1, CCNB2, and CCNE2 overexpression had significantly shorter OS, DFS, higher overall stage, and higher T stage compared to patients with low mRNA expressions (Supplementary Fig. 2A-D, 3A-C). We also found that MELK expression strongly correlated with CDK1, CDK2, CCNA2, CCNB1, CCNB2, and CCNE2 mRNA expressions (Supplementary Table 3).
OTS167 and RGB-286638 combination treatment synergistically inhibits cell proliferation, MCAs, and colony formation
We studied the antiproliferative effect of OTS167 and RGB-286638 alone and in combination in monolayer cell culture and MCAs. Dose-dependent antiproliferative and cytotoxic effects occurred at clinically achievable con- centrations after treatment. The combination treatment resulted in a significantly higher antiproliferative effect in both cell lines compared to controls. After testing multi- ple-dose combinations, we found that the CI was<1 by the Chou-Talalay method, consistent with synergistic activity (Fig. 3A and Supplementary Table 4).
The combination treatment reduced colony formation more effectively than the single-drug or vehicle groups (P<0.001)(Fig. 3B).
MCAs mimic three-dimensional tumors more accu- rately than a monolayer culture. The combination of OTS167 and RGB-286638 was more effective than the single-drug treatment in SW13, with the disintegration of MCAs after two weeks (Fig. 3C). MCAs of NCI-H295R with combination treatment present as small and scat- tered foci compared to controls (Fig. 3C).
OTS167 and RGB-286638 combination treatment induces caspase-dependent apoptosis
We evaluated caspase-3/-7 activity, which revealed co- treatment with OTS167 and RGB-286638 in different dose combinations increased caspase-dependent apop- tosis as compared to controls in both cell lines (Fig. 4A). Validation studies by Western Blot and immunofluores- cence showed higher cleaved caspase-3 expression in both cell lines with combination treatment (Fig. 4B and Supplementary Fig. 4).
OTS167 and RGB-286638 combination treatment induces G2/M cell cycle arrest
We observed an increased ratio of cells in G2/M arrest with combination treatment compared to single-drug or vehicle control (Fig. 4C).
Next, we validated the effects of combination treatment on the prognostically significant cyclins (Fig. 4D). The combination treatment downregulated protein expres- sion of these cyclins and upregulated the expressions of p21Cip1 and p27Kip1 compared to controls in both cell lines (Fig. 4D and E).
OTS167 and RGB-286638 combination treatment inhibits cell migration, invasion, and EMT
ACC-related mortality is often caused by progressive metastasis as cancer cells migrate and invade the meta- static sites [20]. We found that OTS167 and RGB-286638
A
SW13
· VC
NCI-H295R
VC
6.25nM OTS167
1×10
8
6×107
6.25nM OTS167
12.5nM OTS167
RFU
12.5nM RGB-286638
8×10
12.5nM OTS167
4×107
25nM RGB-286638
RFU
6×107
6.25nM RGB-286638
2×107
6.25nM OTS167 +
12.5nM RGB-286638
12.5nM RGB-286638
4×107
O
6.25nM OTS167
12.5nM OTS167 +
2×107
+6.25nM RGB-286638
0
8
25nM RGB-286638
8
12.5nM OTS167+
H
0
1
2
3
4
5
6
0
12.5nM RGB-286638
0
1
2
3
4
5
6
B
Days
C
Days
OTS167
OTS167
OTS167+ RGB-
VC
VC
RGB-
VC
OTS167
286628
286628
286638
RGB-
286638
RGB.
6.25nM
12.5nM
6.25nM+12.5nM
286638
RGB-
OTS167 +
286638
RGB-
OTS167 +
SW13
SW13
NCI-H295R
12.5nM
25nM
6.25nM+12.5nM
SW13
NCI-H295R
No. of colonies
100
VC
100-
IVCHOTS167
OTS167
RGB-286638
No. of colonies
80
OTS167+RGB-286638
/well
80
RGB-286638
OTS167+RGB-286638
6.25nM
6.25nM
6.25nM+6.25nM
/well
60
60
ns
40
40
20
20
NCI-H295R
0
0
P
5
5
1.25439
12.5nM
12.5nM+12.5nM
o
.25+1
12.5nM
0.625+1.
5
Concentration (nM)
Concentration (nM)
combination treatment decreased ACC migration and invasion compared to controls and single-drug in both cell lines (Fig. 5A and B). Next, we studied the EMT markers due to their association with cellular invasion and migration [21]. The combination treatment down- regulated N-cadherin and vimentin in both ACC cell lines compared to control groups (Fig. 5C). We validated the lower vimentin expression by immunofluorescence analysis (Fig. 5D).
OTS167 and RGB-286638 combination treatment downregulates MELK and key molecules in the ß-catenin pathway
ß-catenin (CTNNB1) overexpression is a key oncogenic signaling pathway in ACC [9]. We first evaluated the correlations between the mRNA expression of genes involved in Wnt/B-catenin signaling and the mRNA expression of MELK in the TCGA cohort. The mRNA expression of FOXM1 positively and strongly correlated with MELK (Supplementary Table 5). Next, we con- firmed that ACC overexpressed FOXM1 in two GEO
cohorts (Supplementary Fig. 5A). FOXM1 mRNA expres- sion analyses in TCGA revealed that overexpression was associated with statistically shorter OS, DFS, higher over- all stage and T stage, and positive lymph nodes (Supple- mentary Fig. 5B-F).
We then confirmed significant positive correlations in mRNA expression between CTNNB1 and other genes that regulate the ß-catenin signaling pathway (Supple- mentary Table 5). Compared to control groups, the com- bination of OTS167 and RGB-286638 downregulated FOXM1, AXIN2, GSK-3a/B, and ß-catenin protein in SW13 and NCI-H295R cells (Fig. 6). We performed at least three repeated experiments with consistent results. We found that OTS167 downregulated MELK expres- sion in a dose- and time-dependent manner (Supplemen- tary Fig. 6A). We then treated cells with siMELK, which resulted in downregulation of FOXM1, AXIN2, GSK-3B, and ß-catenin, suggesting that MELK is the upstream regulator of these molecules in the Wnt/ß-catenin signal- ing pathway in ACC (Supplementary Fig. 6B).
SW13
NCI-H295R
A
VC
OTS167
B
RGB-286638
0.0025
OTS167+RGB-286638
Caspase 3/7
VC
OTS167
Caspase 3/7
glo activity
glo activity
0.00025
RGB-286638
SW13
NCI-H295R
0.0020
0.00020
OTS167+RGB-286638
48h
48h
72h
0.0015
**
ns
0.00015
24h
**
OTS167
6.25 -
6.25 . +
12.5 - +
12.5 - +
0.0010
0.00010
RGB-286638
12.5
12.5 +
- 12.5+
- 12.5 +
ns
**
0.0005
**
ns
0.00005
Cleaved caspase 3(17Kd)>
0.0000
0.00000
GAPDH(37Kd)
60
5
5
2
6.25+150
152.50
12 645
.2+25
25+50
5
6.25
6.25+6.25
25+25
C
6
12.5+15
D
SW13
NCI-H295R
24h
48h
OTS167
6.25
6.25
RGB-286638
12.5
12.5
C
Cyclin A2 (55kd)
SW13
NCI-H295R
Cyclin B1 (48kd)
% of cell population
24h
48h
% of cell population
48h
72h
Cyclin B2 (45kd)
100
G2/M
100
G2/M
GAPDH (38kd)
S
S
E
G1
50
G1
50
24h
48h
48h
72h
OTS167
6.25
6.25
12.5
12.5
RGB-286638
12.5
12.5
12.5
12.5
p21cip1 (18Kd)
0
0
VC
OTS167
RGB-286638
OTS167+RGB-286638
VC
OTS167
RGB-286638
OTS167+RGB-286638
VC
OTS167
RGB-286638
OTS167+RGB-286638
VC
OTS167
RGB-286638
OTS167+RGB-286638
p27Kip1((22Kd)
GAPDH(37Kd)
OTS167 and RGB-286638 combination treatment decreases stathmin expression and microtubule stability
Stathmin (STMN1) regulates microtubule dynamics for cell cycle regulation and has strong and statistically significant correlation coefficients with CDK1, CDK2, and their partnering cyclins molecules (Supplementary Table 6). We found that human ACC samples overex- pressed STMN1 compared to NC and ACA in a GEO cohort (Fig. 7A). A previous study showed that STMN1 overexpression in ACC was associated with adverse prognostic clinical features and shorter OS [22]. We validated this finding in an independent cohort using the E-TABM-311 database. STMN1 mRNA expression is higher in ACC samples in the E-TABM-311 cohort as compared to NC, adrenocorticotropic hormone-inde- pendent macronodular adrenal hyperplasia (AIMAH), and ACA (p<0.001) (Fig. 7B). In addition, STMN1 over- expression was significantly associated with stage IV dis- ease and shorter OS (Fig. 7C, D).
We next evaluated the effect of OTS167 and RGB- 286638 on total STMN1 and p-STMN1 expression.
Compared to controls, combination drug treatment decreased total and p-STMN1 expression in both ACC cell lines (Fig. 7E). STMN1 destabilizes microtubules and is negatively regulated by phosphorylation during mitosis by CDKs. STMN1 has two distinct activities: 1) reduce microtubule polymer by sequestration of tubulin and 2) direct binds to microtubule and promotes depo- lymerization of microtubules and preventing the micro- tubule assembly required for cells to build the mitotic spindle [23]. In contrast, STMN1 phosphorylation weak- ens STMN1-tubulin binding and increases the concen- tration of cytoplasmic tubulin for microtubule assembly [24]. The combination treatment increased depolymer- ized tubulin in ACC cells, consistent with the expected effect caused by decreased p-STMN1. In NCI-H295R cells, we also observed an upregulation of polymerized a-tubulin, possibly from decreased microtubule polymer sequestration due to downregulation of total STMN1 [25] (Fig. 7F). Furthermore, we assessed the treatment effect on cytoskeletal structure by evaluating a-tubulin expression and pattern of microtubule distribution using
A
B
C
VC
OTS167
RGB- 286638
OTS167 +
RGB-286638
SW13
NCI-H295R
Migration (%)
150
SW13 NCI-H295R
Migration Assay NCI-H295R SW13
100
24h
48h
48h
72h
50
OTS167
6.25
+
+
+
+
6.25nM
12.5nM
6.25
6.25+12.5nM
12.5
- 12.5
0
¥
n
OTS167+RGB-286638
,
OTS167+RGB-286638
RGB-286628
- 12.5
+
12.5 +
12.5 +
12.5
+
N-Cadherin (127Kd)
Vimentin (55Kd)
12.5nM
12.5nM
12.5+12.5nM
Invasion Assay NCI-H295R SW13
Invasion (%)
150
GAPDH (37Kd)
SW13 NCI-H295R
100
50
SW13
6.25nM
12.5nM
6.25+12.5nM
0
A
4
OTS167+RGB-286638
N-cadherin/GAPDH
1.5
OTS167+RGB-286638
Vimentin/GAPDH
1.5-
1.0
1.0
12.5nM
12.5nM
12.5+12.5nM
0.5
0.5
D
0.0
VC
OTS167
RGB-286638
VC
OTS167
RGB-286638
OTS167 + RGB-286638
0.0
VC
OTS167
RGB-286638
OTS167+RGB-286638
JC
OTS167
RGB-286638
OTS167 + RGB-286638
SW13
NCI-H295R
Vimentin
DAPI
Vimentin/ DAPI
Vimentin
Vimentin/ DAPI
DAPI
VC
NCI-H295R
OTS167
286638
N-cadherin/GAPDH
1.5-
Vimentin/GAPDH
1.5
1.0
1.0
RGB-
0.5
0.5
OTS167+ RGB-
0.0
OTS167 RGB-286638
0.0
JC
VC
OTS167
RGB-286638
VC
OTS167
VC
286638
OTS167+RGB-286638
OTS167 + RGB-286638
RGB-286638
OTS167+RGB-286638
OTS167
RGB-286638
OTS167 + RGB-286638
| SW13 | NCI-H295R | |||
|---|---|---|---|---|
| 24h | 48h | 48h | 72h | |
| OTS167 - | 6.25 - + | - 6.25 - + | - 12.5 - + | - 12.5 - + |
| RGB-286628 - | - 12.5 + | - - 12.5 + | - 12.5 + | - - 12.5 + |
| FOXM1 (110Kd) | ||||
| ß-catenin (92Kd) | ||||
| GSK3a/B(51/46Kd) | ||||
| H3(17Kd) | ||||
| Axin2(98kd) | ||||
| H3(17Kd) | ||||
Fig. 6 Drug combination inhibits multiple molecules in Wnt/B-catenin signaling pathway in ACC: SW13 and NCI-H295R cells were treated with OTS167, RGB-286638, and a combination of OTS167 and RGB-286638. Western blot analysis for ß-catenin, AXIN2, GSK3a/ß, and FOXM1 expression in SW13 cells treated for 24-48 h and NCI-H295R cells treated for 48-72 h with OTS167, RGB-286638, and OTS167 and RGB-286638 combined is shown
A
B
E-TABM-311
C
GSE33371
STMN1 Expression by Stage **
D
STMN1 mRNA Expression
STMN1 mRNA Expression
4.0-
**
10-
mRNA Expression
8
Overall Survival by mRNA Expression
I
1.0
8
6
Percent Survival
Low STMN1
3.5-
0.8
High STMN1
6
Low STMN1-
censored
₮
4.
I
0.6
High STMN1-
censored
3.0-
4
0.4
p=0.032
2.
2
0.2
2.5
0
0
0.0
NC(n=10)
ACA (n=22)
ACC (n=33)
NC (n=4)
AIMAH (n=10)
ACA (n=58)
ACC (n=34)
Stage I
Stage II
Stage III
Stage IV
.00
2.00
4.00
6.00
8.00
10.00
Follow up Duration (Years)
SW13
NCI-H295R
a-tubulin
DAPI
a-tubulin/DAPI
a-tubulin
DAPI
a-tubulin/DAPI
G
VC
OTS167
RGB-286638
.
OTS167
+RGB-286638
Fig. 7 Drug combination inhibits STMN1 expression in ACC: A mRNA expression of STMN1 in NC (n= 10), ACA (n=22), and ACC (n=35) in the GSE33371 data set. B mRNA expression of STMN1 in NC (n=4), AIMAH (n= 10), ACA (n=58), and ACC (n=34) in the E-TABM-311 cohort. C Differential mRNA expression of STMN1 by tumor stages in E-TABM-311 ACC patient samples (p <0.01). D Kaplan-Meier survival curve representing the OS in ACC patient samples from E-TABM-311 with high and low STMN1 expression. High STMN1 expression (red); low STMN1 expression (blue) (*p <0.05). E STMN1 expression in SW13 cells treated for 24-48 h and NCI-H295R cells treated for 48-72 h with OTS167, RGB-286638, and OTS167 and RGB-286638 combined, analyzed by Western blot analysis. F Expression of depolymerized and polymerized tubulin in SW13 cells treated for 24 h and NCI-H295R cells treated for 48 h with OTS167, RGB-286638, and OTS167 and RGB-286638 combined. G Immunofluorescence images of tubulin structure. SW13 and NCI-H295R cells treated with OTS167, RGB-286638, and OTS167 and RGB-286638 combined for 24 and 48 h respectively were labeled with a-tubulin (green), and DAPI (blue) for nuclei staining. Images were taken under confocal microscopy at 20X magnification
| E | SW13 | NCI-H295R | ||
|---|---|---|---|---|
| 24h | 48h | 48h | 72h | |
| OTS167 | 6.25 | 6.25 - + | - 12.5- - | + 12.5 - + |
| RGB-286638 | 12.5+ | 12.5 + | - - 12.5 - | + - 12.5+ |
| Stathmin1(19Kd) GAPDH (37kd) | ||||
| phosho-Stathmin1(17Kd) | ||||
| GAPDH (37kd) | ||||
| F | SW13 | NCI- H295R | |
|---|---|---|---|
| OTS167 | - 6.25 . + | 12.5 . + | |
| RGB-286638 | - - 12.5 + | . - 12.5 + | |
| a-tubulin (Depolymerized)(50Kd) | |||
| H3(17Kd) | |||
| a-tubulin (Polymerized)(50Kd) | |||
| H3(17Kd) | |||
immunofluorescence (Fig. 7G). While untreated SW13 and NCI-H295R cells showed normal microtubule distri- bution expanding throughout the cytoplasm, ACC cells treated with OTS167, RGB-286638, and the combination
drugs exhibited irregular and disorganized microtubule structure and distribution (Fig. 7G). The antibody details are included in supplementary table 7.
OTS167 and RGB-286638 combination treatment reduces tumor burden in an NCI-H295R human ACC xenograft model
We assessed the anti-tumor efficacy of the combination therapy in vivo using a subcutaneously implanted human ACC xenograft model. We included in vivo study schema in Fig. 8A. Mice treated with the combination of OTS167 and RGB-286638 had markedly lower tumor burden measured by luciferase activity and tumor volume after five weeks compared to single-drug and vehicle control groups (Fig. 8B-E). We observed no significant change in animal weight or well-being during the treatment and observation period, suggesting that there were no serious treatment-related toxicities (Fig. 8F).
ACC xenografts treated with the drug combination had lower expressions of ß-catenin and vimentin with higher expression of cleaved caspase-3 by IHC as com- pared to control groups, which validated in vitro findings (Fig. 8G).
Discussion
We aimed to address a lack of effective systemic treat- ments for patients with advanced ACC. We used a drug repurposing approach by performing a qHTS campaign using a comprehensive library comprising 4,991 experi- mental and approved drugs against two ACC cell lines. To facilitate clinical trial development, we prioritized drugs with known toxicity profiles, pharmacokinet- ics, and pharmacodynamics in humans with efficacy against ACC cells at clinically achievable concentra- tions. Because ACC is often refractory to systemic ther- apy, we aimed to identify synergistic drug combinations and study synergistic mechanisms to improve patient selection, treatment efficacy, and outcome. We identi- fied OTS167 and RGB-286638 from the combination matrix drug screening as they demonstrated potent syn- ergy. OTS167 and RGB-286638 each had higher efficacy against ACC cells than the current chemotherapy for ACC. OTS167 (OncoTherapy Science, Japan) is an orally available potent small-molecule inhibitor of MELK. It is currently in Phase I clinical trials in patients with breast cancers and leukemia. RGB-286638 (Aggenix AG, Ger- many) is a multi-CDK small molecule inhibitor with additional activity against tyrosine kinases and serine/ threonine kinases and was well tolerated with prolonged disease stabilization in Phase I clinical trials in patients with solid tumors [17]. We chose the combination of MELK (OTS167) and CDK (RGB-286638) inhibitors because 1) ACC overexpressed MELK, CDK1, CDK2, and partnering cyclins, and 2) these therapeutic targets of OTS167 and RGB-286638 were associated with advanced ACC and independently associated with shorter OS and DFS in several databases, suggesting their role in ACC
progression [10]. Compared to our previous study that manually identified the effective combination based on mechanisms of action [26], we utilized high-throughput technology with pairwise combination matrix screen- ing to identify the synergy regardless of their mecha- nisms. In this study, we showed that the combination treatment was more effective against in vitro and in vivo ACC cell proliferation than single-drug treatments. We confirmed that the drug combination induced G2/M cell cycle arrest and caspase-dependent apoptosis. Because RGB-286638 inhibits the enzymatic activity of CDKs, we assessed the expression of other cell cycle regulators as these can be clinically used to aid patient selection and assess treatment response. CDK1 is the key determinant of mitotic progression, while CDK2 plays a pivotal role in DNA replication. Encoded by the cdc2 gene, CDK1 is activated by and complexes with cyclins A2, B1, and B2 to phosphorylate key proteins leading to mitosis [27, 28]. CDK2 and A- and E-type cyclins form active kinase com- plexes to phosphorylate proteins involved in DNA repli- cation, which are inactivated by the cell cycle checkpoints p21Cip1 and p27Kip1 [29]. Dysregulation of CDK1, CDK2, and their partner cyclins has been observed in multiple solid cancers with aggressive behavior [30-33]. Similar to the overexpression of CDK1 and CDK2 in ACC, we con- firmed associations between CCNA2, CCNB1, CCNB2, and CCNE2 mRNA overexpression and adverse clinical features. We demonstrated that the drug combination effectively decreased cyclin expression, induced p21Cipl and p27Kip1, and caused G2/M cell cycle arrest, suggest- ing that the combination of OTS167 and RGB-286638 preferentially targets the molecules associated with ACC aggressiveness.
One of the key mechanisms leading to cancer metas- tasis is the EMT-related cellular invasion [34]. The EMT has also been implicated in the generation of cancer stem cells and in chemo- and radio-resistance [35]. The molecular characteristics of EMT include the downregu- lation of epithelial markers such as E-cadherin and the upregulation of mesenchymal markers such as vimentin and N-cadherin. The combination of OTS167 and RGB- 286638 effectively decreased ACC cell migration and invasion and reduced vimentin and N-cadherin expres- sions, possibly via the downregulation of the Wnt/B- catenin signaling pathway [36].
MELK has been shown to activate mitotic regulatory genes by activating FOXM1 in other cancers [37]. We analyzed TCGA data and confirmed a positive linear cor- relation between MELK and FOXM1 mRNA expressions, suggesting the MELK-dependent activation of FOXM1 also occurs in ACC. We validated this hypothesis by showing that FOXM1 overexpression is associated with adverse clinical features in ACC. Additionally, 41% of
A
OTS167 (IP):5days/week (10mg/kg/BW)
Week 0
Week 3
Week 4
Week 5
Week 6
Week 7
Week 8
Bilateral SubQ injections NCI-H295R-Luc
IVIS Imaging and grouping
IVIS Imaging
IVIS Imaging
IVIS Imaging
IVIS Imaging
(i) Final Imaging (ii) Caliper measurement (iii) Tumors collection
RGB-286638 (IV): 3days/week (20mg/kg/BW)
RGB-286638 (Alzet pumps) (20mg/kg/BW)
IP: Intraperitoneal injection.
IV: Intravenous injection.
B
C
Control
Fold Change from Baseline
D
Fold change from base line
3000
.30
Group 1
5000
Group 1
*
Group 2
OTS167
4000-
Group 2
2000
*
Group 3
Group 4
-
3000
Group 3
286638
2000
Group 4
1000
RGB-
1000
*
*
RGB-286638
0
0
OTS167+
-1000
0
1
2
3
4
5
Time (Weeks)
Group 1
Group 2
Group 3
Group 4
1
G
Group 1
Group 2
Group3
Group 4
B-catenin
E
F
Tumor Volume (mm3)
Vimentin
600
*
Avg Mice weight (g)
40
*
Caspase 3
Group 1
Group 2
Group 3
Group 4
Cleaved
400
30
20
…
200
Beta-catenin Expression(%)
Cleaved caspase3 expression(%)
300
Vimentin Expression(%)
300-
400
10
200
200
300
200
0
100
100
100
Group 1
Group 2
Group 3
Group 4
0
0123450123A50123A50123AS
0
Group 1
Group 2
Group J
Group 4
Group
Group 2
Group 3
Group 4
Group
Group 2
Group 3
Group 4
Time (Weeks)
ACC samples in TCGA harbored alterations of the Wnt/ ß-catenin pathway. Preclinical studies of glioma cells showed that FOXM1 directly interacts with ß-catenin and promotes nuclear translocation [38]. Downregula- tion of the ß-catenin pathway using a doxycycline-induc- ible shRNA plasmid inhibited ACC cell proliferation, induced apoptosis, and inhibited in vivo ACC tumor growth [39]. Although these findings suggested that the Wnt/B-catenin pathway is a promising therapeutic tar- get in ACC, current therapies do not address this key oncogenic driver. The novel combination of OTS167 and RGB-286638 downregulated multiple clinically relevant key molecules in the Wnt/B-catenin pathway, such as FOXM1, Axin2, GSK3-B, and nuclear and cytoplasmic ß-catenin.
In addition to the role of FOXM1 in cell cycle pro- gression, a recent study showed that FOXM1 is a mas- ter regulator of hepatocellular carcinoma metastasis by inducing EMT and increasing cell migration by transcriptionally activating STMN1 [40]. The altered cytoskeleton caused by the microtubule destabilizing activity of STMN1 increases cell motility and plays a critical role in cell migration, invasion [41], and pro- liferation. STMN1 also regulates cell cycle progression by controlling tubulin polymerization and depolym- erization, and its inhibition results in the G2/M cell cycle arrest [42]. Previous work showed that STMN1 overexpression in ACC promoted a more aggressive in vitro phenotype and was associated with shorter OS in the TCGA cohort [22]. Once we confirmed the positive correlations between STMN1 mRNA expres- sion to FOXM1, MELK, CDK1, and CDK2, we vali- dated the STMN1 overexpression in advanced ACC and the association with shorter disease-specific survival from the E-TABM-311 cohort. Our work demonstrated that the combination of OTS167 and RGB-286638 decreased total and p-STMN1, resulting in increased depolymerized a-tubulin in ACC cells. Because STMN1 sequestered tubulin polymer, the increased polymerized microtubule in ACC treated with OTS167 and RGB-286638 was consistent with a previous report [25]. Our data suggest that this drug combination inhibited MELK and FOXM1, resulting in the downregulation of total and p-STMN1, affect- ing microtubule dynamics. We confirmed that the treatments with OTS167 and RGB-286638 caused disorganized microtubule and irregular cytoskeletal distribution in ACC cells. Such effects on the ACC cytoskeleton can lead to decreased motility, invasion, and mitotic spindle formation and resulting in G2/M cell cycle arrest.
The increased caspase-dependent apoptosis in ACC xenografts treated with OTS167 and RGB-286638
validated the in vitro synergy of this combination. We also confirmed the loss of cytoplasmic and nuclear expression of ß-catenin in human ACC xenografts. Thus, we believe that the combination of OTS167 and RGB-286638 addresses the key molecular characteris- tics involved in ACC tumorigenesis and metastasis. This regimen should be studied in patients with advanced ACC, especially tumors with higher expression of Ki67, MELK, CDK1, CDK2, their partnering cyclins, FOXM1, STMN1, and CTNNB1. In addition to in vivo validation of anti-tumor efficacy of the combination treatment in human ACC xenografts, we found that mice tolerated the combination treatment well with no signs of sys- temic toxicities until the endpoint was met at week 5. Nevertheless, a phase Ib dose-escalation of OTS167 and RGB-286638 in patients with advanced ACC with care- ful monitoring of major organ functions is still required to assess safety and tolerability and to identify the rec- ommended phase II dose.
Conclusions
In summary, we addressed an urgent need to identify effective systemic treatment in patients with advanced ACC by identifying a potent antineoplastic drug combi- nation through qHTS in ACC in vitro. We validated the in vitro and in vivo efficacy of OTS167 and RGB-286638 and discovered that this drug combination effectively targeted MELK, FOXM1, STMN1, and multiple clini- cally significant molecules in the Wnt/ß-catenin signaling pathway, resulting in an antiproliferative and cytotoxic effect, inhibition of cell migration and invasion, and clo- nogenicity. A clinical trial to assess the safety and efficacy of these compounds is warranted.
Abbreviations
ACC: Adrenocortical Carcinoma; MELK: Maternal embryonic leucine zipper kinase; CDK: Cyclin-dependent kinase; FOXM1: Forkhead box protein M1; GEO: Gene Expression Omnibus; TCGA: The Cancer Genome Atlas; GSK3-B: Glycogen synthase kinase 3 beta; qHTS: Quantitative high-throughput drug screening; EMT: Epithelial to mesenchymal transition; OS: Overall survival; ENSAT: European Network for the Study of Adrenal Tumors; ACA: Adreno- cortical adenoma; NC: Normal cortex; E-TABM-311: European Bioinformatics Institute; IHC: Immunohistochemistry; PBS: Phosphate buffer saline.
Supplementary Information
The online version contains supplementary material available at https://doi. org/10.1186/s13046-022-02464-5.
Additional file 1. Supplementary methods.
Additional file 2: Supplementary Figure 1. CDKs and cyclin molecules are overexpressed in ACC: A mRNA expression of CDK1 and CDK2 (NC vs. ACC) from the publicly available data set GSE90713 (NC = 5, ACC = 57) (p < 0.05). B mRNA expression of CCNA2, C mRNA expression of CCNB1, D mRNA expression of CCNB2, and E mRNA expression of CCNE2, from
the GSE33371, GSE12638, and GSE90713 data sets. Supplementary Figure 2. Cyclin molecule overexpression correlates with poor prognosis in ACC: A Kaplan-Meier survival curve representing the OS in the TCGA ACC cohort with low and high expression of CCNA2, CCNB1, and CCNB2. B Kaplan-Meier survival curve representing the overall DFS in the TCGA ACC cohort with low and high expression of CCNA2, CCNB1, and CCNB2. C, D Differential mRNA expression of CCNA2, CCNB1, and CCNB2 by over all stages and T stage in the TCGA ACC cohort. All data were analyzed by two-tailed unpaired Student’s t-test or one-way ANOVA. Supplemen- tary Figure 3. Cyclin E2 is associated with poor prognosis of patients with ACC: A, B Kaplan-Meier survival curve representing the overall and DFS in the TCGA ACC cohort with high and low CCNE2expression. High CCNE2 expression (red); low CCNE2 expression (blue) (*p < 0.01). C CCNE2 expression by over all stage and T stage in the TCGA ACC cohort (*p < 0.05). Supplementary Figure 4. Cleaved Caspase 3 expression in SW13 and NCI-H295R cells treated with OTS167, RGB-286638, and OTS167 and RGB-286638 combined for 24 and 48 hours respectively. Cells were stain with cleaved caspase 3 conjugated with Alexa Fluor® 555 Conjugate (Red). Actin filaments and nuclei were labeled with Alexa Fluor 488 phalloidin (green) and DAPI respectively. The images were taken under confocal microscopsy (Zeiss) at 63X. Supplementary Figure 5. FOXM1 is overex- pressed in advanced tumor stage and correlated with poor prognosis of ACC patient: A mRNA expression of FOXM1 in the publicly available data sets GSE33371 (NC vs. ACC, ACA vs. ACC) (p < 0.001) and GSE12368 (NC vs. ACC, ACA vs. ACC) (p < 0.05). B, C Kaplan-Meier survival curve and DFS curve of patients with ACC, stratified by FOXM1 expression level. D, E, F Differential expression of FOXM1 mRNA by stage, tumor grade status, and lymph node status. Supplementary Figure 6. MELK inhibition affects B-catenin pathway in ACC cells: A SW13 and NCI-H295R cells were treated with OTS167. Expression of MELK in SW13 and NCI-H295R cells treated with OTS167 for 24-72 hours. B Expression of ß-catenin, FOXM1, GSK-3B, and Axin 2 in siMELK and siNegative transfected cells.
Additionalfile 3: Supplementary Table 1. List of 30 selected drugs for combination drug screening in ACC cell lines. Supplementary Table 2. Correlations between mRNA expression of CDKs and cyclin molecules with MKI67 in human ACC from the TCGA cohort Supplementary Table 3. Correlations between mRNA expression of CDKs and cyclin molecules with MELK in human ACC samples from the TCGA cohort. Sup- plementary Table 4. OTS167 and RGB-286638 combination shows syner- gistic activity in ACC cells. The combination index (CI) was calculated using the Chou-Talalay method. The CI is determined by the following range: CI < 1, synergistic; CI = 1, additive; CI > 1, antagonistic. Supplementary Table 5. Correlations between mRNA expression of MELK and ß-catenin regulatory molecules in human ACC samples from the TCGA cohort Sup- plementary Table 6. Correlations between mRNA expression of CDKs and cyclin molecules with STMN1 in human ACC samples from the TCGA cohort Supplementary Table 7. List of Antibodies.
Acknowledgements
The authors are thankful to the Central Animal Research Facility and FACS core facility of the National Cancer Institute, NIH; and the NIH Fellows Editorial Board for their editorial assistance.
Authors’ contributions
Study concept and design: DL, MB, NN. Experiments and data acquisition: DL, RG, PM, MB, MS, YZ, MQ, MSDM, JDR, NN. Data analysis and interpreta- tion: DL, RG, PM, MB, MS, YZ, MZ, NN. Manuscript preparation: DL, RG, PM, MB, MS, YZ, MQ, MSDM, JDR, MZ, NN. The author(s) read and approved the final manuscript.
Funding
Open Access funding provided by the National Institutes of Health (NIH). Funding was provided by the NIH intramural grant (#ZIA BC 011286) and the Intramural Research Programs of NCATS.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
Datasets are described in the material and method section.
Declarations
Ethics approval and consent to participate
Human adrenocortical tissue samples were collected under a protocol designed to evaluate the molecular biology of endocrine neoplasms (clinicaltrials.gov, NCT01005654), which was approved by the Institutional Review Board, National Cancer Institute (NCI), National Institutes of Health (NIH), and the NIH Office of Human Subject Research. All participants provided written informed consent. All animal experiments were approved by the National Institutes of Health, Bethesda, USA.
Consent for publication
The authors provide consent for publication.
Competing interests
The authors declare no competing interests.
Author details
1 Surgical Oncology Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 2Department of Molecular Biosciences, College of Natural Sciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA. 3National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA. 4Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. 5 Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Received: 9 May 2022 Accepted: 10 August 2022 Published online: 23 September 2022
References
1. Wooten MD, King DK. Adrenal cortical carcinoma. Epidemiology and treatment with mitotane and a review of the literature. Cancer. 1993;72(11):3145-55.
2. Kebebew E, Reiff E, Duh QY, Clark OH, McMillan A. Extent of disease at presentation and outcome for adrenocortical carcinoma: have we made progress? World J Surg. 2006;30(5):872-8.
3. Kerkhofs TM, Verhoeven RH, Van der Zwan JM, Dieleman J, Kerstens MN, Links TP, et al. Adrenocortical carcinoma: a population-based study on incidence and survival in the Netherlands since 1993. Eur J Cancer. 2013;49(11):2579-86.
4. Fassnacht M, Dekkers OM, Else T, Baudin E, Berruti A, de Krijger R, 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. 2018;179(4):G1-46.
5. Fassnacht M, Kroiss M, Allolio B. Update in adrenocortical carcinoma. J Clin Endocrinol Metab. 2013;98(12):4551-64.
6. Wang S, Chen SS, Gao WC, Bai L, Luo L, Zheng XG, et al. Prognostic Factors of Adrenocortical Carcinoma: An Analysis of the Surveillance Epidemiology and End Results (SEER) Database. Asian Pac J Cancer Prev. 2017;18(10):2817-23.
7. Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, et al. Com- bination chemotherapy in advanced adrenocortical carcinoma. N Engl J Med. 2012;366(23):2189-97.
8. Assie G, Letouze E, Fassnacht M, Jouinot A, Luscap W, Barreau O, et al. Integrated genomic characterization of adrenocortical carcinoma. Nat Genet. 2014;46(6):607-12.
9. Zheng S, Cherniack AD, Dewal N, Moffitt RA, Danilova L, Murray BA, et al. Comprehensive Pan-Genomic Characterization of Adrenocortical Carci- noma. Cancer Cell. 2016;29(5):723-36.
10. Nilubol N, Boufragech M, Zhang L, Gaskins K, Shen M, Zhang YQ, et al. Synergistic combination of flavopiridol and carfilzomib targets com- monly dysregulated pathways in adrenocortical carcinoma and has biomarkers of response. Oncotarget. 2018;9(68):33030-42.
11. Martinez NJ, Rai G, Yasgar A, Lea WA, Sun H, Wang Y, et al. A High- Throughput Screen Identifies 2,9-Diazaspiro[5.5]Undecanes as Inducers
of the Endoplasmic Reticulum Stress Response with Cytotoxic Activity in 3D Glioma Cell Models. PLoS One. 2016;11(8):e0161486.
12. Mathews Griner LA, Guha R, Shinn P, Young RM, Keller JM, Liu D, et al. High-throughput combinatorial screening identifies drugs that cooper- ate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells. Proc Natl Acad Sci U S A. 2014;111(6):2349-54.
13. Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2002;30(1):207-10.
14. Chou TC. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 2010;70(2):440-6.
15. Ravi M, Paramesh V, Kaviya SR, Anuradha E, Solomon FD. 3D cell culture systems: advantages and applications. J Cell Physiol. 2015;230(1):16-26.
16. Maes A, Maes K, Vlummens P, De Raeve H, Devin J, Szablewski V, et al. Maternal embryonic leucine zipper kinase is a novel target for diffuse large B cell lymphoma and mantle cell lymphoma. Blood Cancer J. 2019;9(12):87.
17. Cirstea D, Hideshima T, Santo L, Eda H, Mishima Y, Nemani N, et al. Small-molecule multi-targeted kinase inhibitor RGB-286638 trig- gers P53-dependent and -independent anti-multiple myeloma activity through inhibition of transcriptional CDKs. Leukemia. 2013;27(12):2366-75.
18. Beuschlein F, Weigel J, Saeger W, Kroiss M, Wild V, Daffara F, et al. Major prognostic role of Ki67 in localized adrenocortical carcinoma after com- plete resection. J Clin Endocrinol Metab. 2015;100(3):841-9.
19. Wang Y, Lee YM, Baitsch L, Huang A, Xiang Y, Tong H, et al. MELK is an oncogenic kinase essential for mitotic progression in basal-like breast cancer cells. Elife. 2014;3:e01763.
20. Jiang WG, Sanders AJ, Katoh M, Ungefroren H, Gieseler F, Prince M, et al. Tissue invasion and metastasis: Molecular, biological and clinical perspec- tives. Semin Cancer Biol. 2015;35(Suppl):S244-75.
21. Bulzico D, Faria PAS, Maia CB, de Paula MP, Torres DC, Ferreira GM, et al. Is there a role for epithelial-mesenchymal transition in adrenocortical tumors? Endocrine. 2017;58(2):276-88.
22. Aronova A, Min IM, Crowley MJP, Panjwani SJ, Finnerty BM, Scognamiglio T, et al. STMN1 is Overexpressed in Adrenocortical Carcinoma and Promotes a More Aggressive Phenotype In Vitro. Ann Surg Oncol. 2018;25(3):792-800.
23. Howell B, Larsson N, Gullberg M, Cassimeris L. Dissociation of the tubulin- sequestering and microtubule catastrophe-promoting activities of oncoprotein 18/stathmin. Mol Biol Cell. 1999;10(1):105-18.
24. Amayed P, Pantaloni D, Carlier MF. The effect of stathmin phosphorylation on microtubule assembly depends on tubulin critical concentration. J Biol Chem. 2002;277(25):22718-24.
25. Howell B, Deacon H, Cassimeris L. Decreasing oncoprotein 18/stathmin levels reduces microtubule catastrophes and increases microtubule polymer in vivo. J Cell Sci. 1999;112(Pt 21):3713-22.
26. Mueller-Klieser W. Multicellular spheroids. A review on cellular aggregates in cancer research. J Cancer Res Clin Oncol. 1987;113(2):101-22.
27. Bellanger S, de Gramont A, Sobczak-Thepot J. Cyclin B2 suppresses mitotic failure and DNA re-replication in human somatic cells knocked down for both cyclins B1 and B2. Oncogene. 2007;26(51):7175-84.
28. Okamoto K, Sagata N. Mechanism for inactivation of the mitotic inhibitory kinase Wee1 at M phase. Proc Natl Acad Sci U S A. 2007;104(10):3753-8.
29. Asghar U, Witkiewicz AK, Turner NC, Knudsen ES. The history and future of targeting cyclin-dependent kinases in cancer therapy. Nat Rev Drug Discov. 2015;14(2):130-46.
30. Scaltriti M, Eichhorn PJ, Cortes J, Prudkin L, Aura C, Jimenez J, et al. Cyclin E amplification/overexpression is a mechanism of trastuzumab resistance in HER2+ breast cancer patients. Proc Natl Acad Sci U S A. 2011;108(9):3761-6.
31. Karst AM, Jones PM, Vena N, Ligon AH, Liu JF, Hirsch MS, et al. Cyclin E1 deregulation occurs early in secretory cell transformation to promote formation of fallopian tube-derived high-grade serous ovarian cancers. Cancer Res. 2014;74(4):1141-52.
32. Kuhn E, Bahadirli-Talbott A, Shih IM. Frequent CCNE1 amplification in endometrial intraepithelial carcinoma and uterine serous carcinoma. Mod Pathol. 2014;27(7):1014-9.
33. Li M, He F, Zhang Z, Xiang Z, Hu D. CDK1 serves as a potential prognostic biomarker and target for lung cancer. J Int Med Res. 2020;48(2):300060519897508.
34. Son H, Moon A. Epithelial-mesenchymal Transition and Cell Invasion. Toxicol Res. 2010;26(4):245-52.
35. Zheng HC. The molecular mechanisms of chemoresistance in cancers. Oncotarget. 2017;8(35):59950-64.
36. Salomon A, Keramidas M, Maisin C, Thomas M. Loss of beta-catenin in adrenocortical cancer cells causes growth inhibition and reversal of epithelial-to-mesenchymal transition. Oncotarget. 2015;6(13):11421-33.
37. Joshi K, Banasavadi-Siddegowda Y, Mo X, Kim SH, Mao P, Kig C, et al. MELK-dependent FOXM1 phosphorylation is essential for proliferation of glioma stem cells. Stem Cells. 2013;31(6):1051-63.
38. Zhang N, Wei P, Gong A, Chiu WT, Lee HT, Colman H, et al. FoxM1 pro- motes beta-catenin nuclear localization and controls Wnt target-gene expression and glioma tumorigenesis. Cancer Cell. 2011;20(4):427-42.
39. Gaujoux S, Hantel C, Launay P, Bonnet S, Perlemoine K, Lefevre L, et al. Silencing mutated beta-catenin inhibits cell proliferation and stimu- lates apoptosis in the adrenocortical cancer cell line H295R. PLoS ONE. 2013;8(2):e55743.
40. Liu J, Li J, Wang K, Liu H, Sun J, Zhao X, et al. Aberrantly high activation of a FoxM1-STMN1 axis contributes to progression and tumorigenesis in FoxM1-driven cancers. Signal Transduct Target Ther. 2021;6(1):42.
41. Ni PZ, He JZ, Wu ZY, Ji X, Chen LQ, Xu XE, et al. Overexpression of Stathmin 1 correlates with poor prognosis and promotes cell migration and proliferation in oesophageal squamous cell carcinoma. Oncol Rep. 2017;38(6):3608-18.
42. Rubin CI, Atweh GF. The role of stathmin in the regulation of the cell cycle. J Cell Biochem. 2004;93(2):242-50.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
Ready to submit your research? Choose BMC and benefit from:
· fast, convenient online submission
· thorough peer review by experienced researchers in your field
· rapid publication on acceptance
· support for research data, including large and complex data types
· gold Open Access which fosters wider collaboration and increased citations
· maximum visibility for your research: over 100M website views per year
At BMC, research is always in progress.
BMC
Learn more biomedcentral.com/submissions