Activity of the Ubiquitin-activating Enzyme Inhibitor TAK-243 in Adrenocortical Carcinoma Cell Lines, Patient-derived Organoids, and Murine Xenografts

Abstract

Current treatment options for metastatic adrenocortical carcinoma (ACC) have limited efficacy, despite the common use of mitotane and cytotoxic agents. This study aimed to identify novel therapeutic options for ACC. An extensive drug screen was conducted to identify compounds with potential activity against ACC cell lines. We further investigated the mechanism of action of the identified compound, TAK-243, its synergistic effects with current ACC therapeutics, and its efficacy in ACC models including patient-derived organoids and mouse xenografts. TAK-243, a clinical ubiquitin-activating enzyme (UAE) inhibitor, showed potent activity in ACC cell lines. TAK-243 inhibited protein ubiquitination in ACC cells, leading to the accumulation of free ubiquitin, activation of the unfolded protein response, and induction of apoptosis. TAK-243 was found to be effluxed out of cells by MDR1, a drug efflux pump, and did not require Schlafen 11 (SLFN11) expression for its activity. Combination of TAK-243 with current ACC therapies (e.g., mitotane, etoposide, cisplatin) produced synergistic or additive effects. In addition, TAK-243 was highly synergistic with BCL2 inhibitors (Navitoclax and Venetoclax) in preclinical ACC models including patient-derived organoids. The tumor suppressive effects of TAK-243 and its synergistic effects with Venetoclax were further confirmed in a mouse xenograft model. These findings provide preclinical evidence to support the initiation of a clinical trial of TAK-243 in patients with advanced-stage ACC. TAK-243 is a promising potential treatment option for ACC, either as monotherapy or in combination with existing therapies or BCL2 inhibitors.

Significance: ACC is a rare endocrine cancer with poor prognosis and limited therapeutic options. We report that TAK-243 is active alone and in combination with currently used therapies and with BCL2 and mTOR inhibitors in ACC preclinical models. Our results suggest implementation of TAK-243 in clinical trials for patients with advanced and metastatic ACC.

FIGURE 1

NCATS screening identifies TAK-243 as one of the most potent drugs against ACC cell lines. A, CU-ACC1, CU-ACC2, and NCI-H295R cell lines were screened with the NCATS MIPE v5.0 (MIPE 5.0) library of approved and investigational drugs (n = 2,480). Drugs with Z-AUC values less than −1.5 and good-quality dose–response curves were selected. The number of drugs effective in the three cell lines is shown in the Venn diagram (left). The 21 drugs commonly effective in the three cell lines and their mechanism of action were tabulated. B, Drugs were arranged in Z-AUC order for the three cell lines tested, with red dots indicating TAK-243. The positions of mitotane, doxorubicin, and etoposide were also shown. C, Heat map of IC₅₀ values of selected drugs with highest and lowest activity against the three ACC cell lines and SW-13 (small cell carcinoma cell line of adrenocortical origin). Drug targets were listed in parentheses.

FIGURE 2

Cytotoxicity of TAK-243 on ACC cell lines. A, Concentration–response curve and IC₅₀ values of TAK-243 in ACC cell lines and SW-13. Cell viability after 72 hours under the indicated drug concentrations was measured by the CellTiter-Glo viability assay. B, Expression of ABC transporters (MDR-1, BCRP) and SLFN11 in ACC cell lines and SW-13. Proteins were extracted from each cell line and expression of SLFN11, MDR-1, and BCRP were evaluated by Western blotting. C, Effects of TAK-243 on DNA synthesis and cell cycle. CU-ACC1, CU-ACC2, and NCI-H295R cells were treated with 100 nmol/L TAK-243 for the indicated times and pulse-labeled with EdU (10 µmol/L) for 30 minutes before harvest. Cellular EdU uptake and cell cycle were analyzed by flow cytometry. D, Change in percentage of S-phase after TAK-243 treatment. E, ACC cell lines and SW-13 were treated with TAK-243 at the indicated concentrations for 24 hours, and PARP cleavage was detected by Western blotting.

FIGURE 3

Activity of TAK-243 on ACC PDOs. Concentration–response curves and IC₅₀ values of TAK-243 for ACC PDOs. The indicated concentrations of TAK-243 were administered to organoids derived from patients with ACC for 72 hours and cell viability was determined. Representative brightfield images of organoids were also shown (20x magnification, scale bar 50 µm).

FIGURE 4

TAK-243 inhibits ubiquitylation and induces UPR in ACC cell lines. A, Time course for ubiquitylated proteins and free ubiquitin in cells treated with TAK-243 (500 nmol/L, 2 or 4 hours). The indicated proteins were evaluated by Western blotting. B, Quantitation of the data shown in A. C, Concentration response of ubiquitylated proteins and free ubiquitin in cells treated with the indicated concentrations of TAK-243 for 2 hours. The indicated proteins were evaluated by Western blotting. D, Quantitation of the data shown in B. E, UPR induced by TAK-243 (500 nmol/L, 2 or 4 hours). The samples were from the same cellular lysates as in A, and GAPDH blots were intentionally reused for presentation. The indicated proteins were evaluated by Western blotting.

FIGURE 5

Synergy between TAK-243 and mitotane. A, Top: The indicated ACC cell lines were treated with TAK-243 with or without mitotane for 72 hours. Bottom: CI values were calculated and plotted. B, Enhanced induction of apoptosis by the TAK-243 + mitotane combination was measured as caspase-3 cleavage. C, Enhanced UPR induction by the TAK-243 + mitotane combination. CU-ACC2 cells were treated with or without mitotane (40 µmol/L) for 24 hours before adding the indicated concentrations of TAK-243 for 4 hours. UPR proteins were evaluated by Western blotting.

FIGURE 6

NCATS screening for synergistic drug combinations with TAK-243 in the CU-ACC1 and NCI-H295R cell lines. One hundred and seven drugs were tested in combination with TAK-243. A, Overall result of the screen with drugs listed in ExcessHSA order. The BCL2 inhibitors (S55746, Navitoclax and Venetoclax) and the mTOR inhibitors (Everolimus and Vistusertib) were indicated as red dots. B and C, Independent validation of the NCATS data for the BCL2 and mTOR inhibitors. ACC cell lines were treated with TAK-243 at the specified concentrations for 72 hours with or without Venetoclax (B) and Everolimus (C). Cell viability was assessed by CellTiter-Glo. Error bars represent SD of triplicates; CI values were also plotted.

FIGURE 7

Synergy between TAK-243 and Venetoclax in organoids generated from patient-derived xenograft (PDXO). A, Representative brightfield images of PDXOs (20x magnification, scale bar 50 µm). B, Phenotypic characteristics of PDXOs. Expression of biomarkers routinely used to diagnose ACC, including inhibin-α, SF-1, INSM-1, and β-catenin in ACC-PDX-derived organoids using immunofluorescence assay. C, Top: The indicated ACC PDXOs were treated with TAK-243 with or without Venetoclax for 72 hours. Bottom: Plots of the corresponding CI values.

FIGURE 8

TAK-243 inhibits tumor growth in ACC xenograft models and synergizes with the BCL2 inhibitor Venetoclax. A, H295R xenografts were treated with TAK-243 (10 and 20 mg/kg) intraperitoneally twice weekly and mean tumor volume was evaluated. Error bars indicate SEM (n = 5); tumor volume for each mouse on day 29 was shown individually on the right. B, Xenograft tissues collected on day 29 (H&E staining, immunostaining for anti-multi-ubiquitin and anti-cleaved caspase-3 antibodies). Top panel: control, bottom panel: TAK-243 (20 mg/kg) group. Scale bar: 200 µm. C, Left: CU-ACC1 xenografts were treated with TAK-243 (10 mg/kg) intraperitoneally twice weekly, venetoclax (100 mg/kg) orally five times weekly, and a combination of TAK-243 and venetoclax, and mean tumor volume was evaluated. Error bars indicate SEM (n = 5); tumor volume of each mouse on day 29 was shown separately. Right: Tumor weight of each treatment group on day 29. Horizontal lines indicate mean values and error bars indicate SEM.