Defactinib in Combination with Mitotane Can Be an Effective Treatment in Human Adrenocortical Carcinoma

Abstract

Adrenocortical carcinoma (ACC) is an aggressive cancer with a poor prognosis. Mitotane, the only FDA-approved treatment for ACC, targets adrenocortical cells and reduces cortisol levels. Although it remains the cornerstone of systemic therapy, its overall impact on long-term outcomes is still a matter of ongoing clinical debate. Drug repurposing is a cost-effective way to identify new therapies, and defactinib, currently in clinical trials as part of combination therapies for various solid tumours, may enhance ACC treatment. We aimed to assess its efficacy in combination with mitotane. We tested the combination of mitotane and defactinib in H295R, SW13, and mitotane-sensitive and -resistant HAC15 cells, using functional assays, transcriptomic profiling, 2D and 3D cultures, bioprinted tissues, and xenografts. We assessed drug interactions with NMR and toxicity in vivo, as mitotane and defactinib have never been previously administered together. Genomic data from 228 human ACC and 158 normal adrenal samples were also analysed. Transcriptomic analysis revealed dysregulation of focal adhesion along with mitotane-related pathways. Focal adhesion kinase (FAK) signalling was enhanced in ACC compared to normal adrenal glands, with PTK2 (encoding FAK) upregulated in 44% of tumour samples due to copy number alterations. High FAK signature scores correlated with worse survival outcomes. FAK inhibition by defactinib, both alone and in combination with mitotane, showed effective anti-tumour activity in vitro. No toxicity or drug-drug interactions were observed in vivo. Combination treatment significantly reduced tumour volume and the number of macrometastases compared to those in the mitotane and control groups, with defactinib-treated tumours showing increased necrosis in xenografts. Defactinib combined with conventionally used mitotane shows promise as a novel combination therapy for ACC and warrants further investigation.

Keywords: 3D model; adrenocortical cancer; defactinib; drug repurposing; focal adhesion signalling; targeted therapy.

Figure 1

Transcriptome analysis of mitotane’s effects in 2D and Matrigel-scaffolded 3D models of H295R adrenocortical cells. (A) Gene expression changes following mitotane treatment in in vitro 2D cultures and Matrigel-scaffolded 3D ACC spheroids. A total of 1755 genes were commonly downregulated (blue arrows), and 2124 were upregulated (red arrows), while 54 genes exhibited opposing expression patterns (red & blue arrows) between the two models. (B) Heatmap of commonly altered gene expression in 2D and 3D cultures. (C) Most significant biological processes commonly affected by mitotane treatment in 2D and 3D models.

Figure 2

Expression of focal adhesion kinase (FAK)-encoding PTK2 in human ACC. (A) Correlation between PTK2 expression and copy number alteration indicated a minor epigenetic effect in PTK2 gene expression regulation. Differentially expressed genes (B) and differentially methylated genes (C) between PTK2-high- and PTK2-low-expressing tumours (PTK2-high samples were selected with chromosomal gain or amplification of the PTK2 encoding region; PTK2-low samples were selected with diploid and shallow deletion of the PTK2 encoding region—see Supplementary Figure S2A). The exact gene lists and their function are presented in Supplementary Table S5C–F. Blue dots indicate significant, while grey dots indicate non-significant changes (D,E) The effect of FAK signature on ACC patient survival. FAK signature represents FAK activity characterised with 11 genes using ssGSEA. Patients with high FAK signature have worse progression-fee (D) and overall survival (E) in univariate and multivariate analyses (Supplementary Figure S3), respectively.

Figure 3

In vitro functional assessment of defactinib, mitotane, and the combined effect on H295R human ACC cell line. Cell proliferation and dead-cell ratio upon treatments in (A) 2D model and in (B) 3D Matrigel-scaffolded model. (C) Photomicrographs of Matrigel-scaffolded 3D models indicated smaller spheroid sizes (D) and increased spheroid numbers (E) upon combined treatment. Imaging was done by Canon Power Shot A590 IS v. 1.1.0.0 software using a ×50 objective and ×10 ocular. (F) Defactinib, mitotane, and combined treatment effect on cell migration. *: p = 0.01–0.05; **: p = 0.001–0.01; ***: p = 0.0001–0.001; ****: p < 0.0001

Figure 4

In vitro functional assessment of defactinib, mitotane and combined effect on SW13 ACC cell line. Treatment effect on viability and proliferation in 2D monolayer cultures (A) and in 3D bioprinted tissues (B). Proliferation and dead-cell ratio in 3D Matrigel-scaffolded models (C). (D) Defactinib, mitotane, and combined treatment effect on cell migration in time lapse (left side) and at the endpoint among different groups (right side). *: p = 0.01–0.05; **: p = 0.001–0.01; ***: p = 0.0001–0.001; ****: p < 0.0001.

Figure 5

Defactinib, mitotane, and combined treatment effect on H295R xenografts. Tumour volume during the experiment (A) and at termination (B). Metastases were developed during the 16-week experiments. (C) Black arrow indicates a marcometastasis in the lung. Metastases were observed during serial sectioning, indicated by the black arrow (D). (E) The number of macrometastases was decreased in the combined-treatment groups compared to controls, while mitotane itself had not such effect. (F) Necrotic area of the tumours was identified by histology and quantified by image analysis. (G) Defactinib increased the necrotic area compared to mitotane only and control group. *: p = 0.01–0.05; **: p = 0.001–0.01