Preclinical assessment of synergistic efficacy of MELK and CDK inhibitors in adrenocortical cancer.

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 clonogenicity, 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 Expression 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 apoptosis, 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 ß-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. 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.

Tumor mutational burden presents limiting effects on predicting the efficacy of immune checkpoint inhibitors and prognostic assessment in adrenocortical carcinoma.

BACKGROUND: Adrenocortical carcinoma (ACC) is a highly malignant urologic cancer and tends to metastasize. Although immune checkpoint inhibitors (ICIs) bring a glimmer of light to conquer ACC, only a fraction of patients have benefit from ICIs treatment. It is well known that tumor mutational burden (TMB) is closely associated with the efficacy and response rate of immunotherapy. However, its roles in ACC were not investigated. METHODS: Using somatic mutations data of 92 ACC samples in TCGA database, we calculated their TMB values by the 'maftools' package in R software (Ver 3.6.3). To explore the roles of TMB in ICIs therapy, we have addressed this issue from three perspectives. First, the effects of TMB levels on tumor immune microenvironment (TIM) were analyzed through CIBERSORT algorithm, ssGSEA method and TIMER web server. Second, we investigated the expressive correlations between TMB level and five pivotal immune checkpoints based on Pearson coefficient. Third, the difference in TIDE score between high- and low-TMB groups was compared. The prognostic value of TMB was also evaluated. Besides, GSEA was performed to determine the changes in the activities of signaling pathways caused by TMB. RESULTS: TMB values in ACC samples were not high. The average of total mutation counts in each sample was only 21.5. High TMB could lead metabolic reprogramming and poor survival outcomes. However, it was unable to affect the infiltration levels of lymphocytes, and failed to facilitate the activities of immune-related pathways. Regarding immune checkpoints (ICs), only PD-L1 upregulation could result in a good prognosis, and TMB level did not correlate with the expressions of other ICs except for LAG3. There was no significant difference in TIDE score between high- and low-TMB groups. Combining the present results and previous study, we speculated that inadequate stimulation for neoantigens formation, intrinsic immune-resistance and special genomic alterations were three possible reasons for TMB limiting functions in TIM and ICIs. Besides, TMB was toughly applied in clinical practice due to its high cost of determination and non-universal definition of high TMB. CONCLUSIONS: TMB presents limiting effects on prediction for ICIs efficacy and prognostic assessment for ACC patients.

Contribution of Therapeutic Monitoring in the Assessment of Toxic Adverse Effects of Mitotane: a Case Report.

Mitotane provided serious side effects and low doses seemed to be tolerated. Determination of mitotane concentration in plasma is recommended. We report the case of toxic plasma levels with low doses of mitotane in a 47-year-old man with adrenocortical cancer.

Mitotane therapy in adrenocortical cancer induces CYP3A4 and inhibits 5α-reductase, explaining the need for personalized glucocorticoid and androgen replacement.

CONTEXT: Mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane] is the first-line treatment for metastatic adrenocortical carcinoma (ACC) and is also regularly used in the adjuvant setting after presumed complete removal of the primary tumor. Mitotane is considered an adrenolytic substance, but there is limited information on distinct effects on steroidogenesis. However, adrenal insufficiency and male hypogonadism are widely recognized side effects of mitotane treatment. OBJECTIVE: Our objective was to define the impact of mitotane treatment on in vivo steroidogenesis in patients with ACC. SETTING AND DESIGN: At seven European specialist referral centers for adrenal tumors, we analyzed 24-h urine samples (n = 127) collected from patients with ACC before and during mitotane therapy in the adjuvant setting (n = 23) or for metastatic ACC (n = 104). Urinary steroid metabolite excretion was profiled by gas chromatography/mass spectrometry in comparison with healthy controls (n = 88). RESULTS: We found a sharp increase in the excretion of 6ß-hydroxycortisol over cortisol (P < 0.001), indicative of a strong induction of the major drug-metabolizing enzyme cytochrome P450 3A4. The contribution of 6ß-hydroxycortisol to total glucocorticoid metabolites increased from 2% (median, interquartile range 1-4%) to 56% (39-71%) during mitotane treatment. Furthermore, we documented strong inhibition of systemic 5α-reductase activity, indicated by a significant decrease in 5α-reduced steroids, including 5α-tetrahydrocortisol, 5α-tetrahydrocorticosterone, and androsterone (all P < 0.001). The degree of inhibition was similar to that in patients with inactivating 5α-reductase type 2 mutations (n = 23) and patients receiving finasteride (n = 5), but cluster analysis of steroid data revealed a pattern of inhibition distinct from these two groups. Longitudinal data showed rapid onset and long-lasting duration of the observed effects. CONCLUSIONS: Cytochrome P450 3A4 induction by mitotane results in rapid inactivation of more than 50% of administered hydrocortisone, explaining the need for doubling hydrocortisone replacement in mitotane-treated patients. Strong inhibition of 5α-reductase activity is in line with the clinical observation of relative inefficiency of testosterone replacement in mitotane-treated men, calling for replacement by 5α-reduced androgens.

Assessment of salivary free cortisol levels by liquid chromatography with tandem mass spectrometry (LC-MS/MS) in patients treated with mitotane.

OBJECTIVE: Mitotane is an adrenocytolytic agent used in adrenocortical carcinoma, inducing adrenal insufficiency, requiring replacement treatment. Such therapy is not easy to monitor because of mitotane interference. Salivary cortisol reflects a free fraction of plasma cortisol and may be useful in such patients. DESIGN: The aim of our study was to evaluate salivary cortisol by HPLC coupled to tandem-mass spectrometry (LC-MS/MS) and by an electrochemiluminescence immunoassay (ECLIA) in patients treated with mitotane. We enrolled 6 patients receiving mitotane and 2 Addison disease patients as negative controls and determined salivary cortisol rhythm. We also determined the salivary cortisol rhythm in 8 healthy subjects. Salivary samples (n=112) were assayed by ECLIA, using Roche Modular E170, and by LC-MS/MS. RESULTS: The mean values obtained by ECLIA were significantly higher than those obtained by LC-MS/MS in the mitotane group (p<0.001). In fact, in the group measured by LC-MS/MS, we observed several peaks eluting at a retention time different from the cortisol group, presumably due to cortisol-like analogues. In Addison disease, since steroidogenesis is absent, salivary cortisol values measured by the two methods did not show any significant difference (p=0.61). CONCLUSIONS: Salivary cortisol measured by LC-MS/MS is a selective method, excluding cortisol analogues accumulating in treated patients. Therefore, LC-MS/MS offers an effective system to monitor replacement therapy in mitotane treated patients.

Assessment of serum-free cortisol levels in patients with adrenocortical carcinoma treated with mitotane: a pilot study.

OBJECTIVE: Mitotane treatment in adrenocortical carcinoma (ACC) results in unreliable measurement of serum total cortisol (TC) levels because of an elevation in corticosteroid-binding globulin (CBG). DESIGN: The use of a newly-developed serum-free cortisol (FC) assay was assessed to investigate the characteristics of a more valid measure of cortisol status. PATIENTS: Sixty-two serum samples from patients with ACC treated with mitotane were studied. Different subgroups were studied according to mitotane levels (<14, 14-20 and >20 mg/dl), hydrocortisone replacement treatment, presence of Cushing's syndrome (CS) and adrenocorticotrophin (ACTH) levels. MEASUREMENTS: Serum FC was measured using a newly-developed assay, TC, CBG and plasma ACTH using conventional laboratory kits; TC-to-CBG (Free cortisol index, FCI, nmol/mg) and TC-to-FC (TFR) ratios were calculated. RESULTS: CBG levels were elevated and positively correlated to mitotane levels. FC was positively related to TC and FCI in nearly all subgroups studied. Plasma ACTH was negatively related to parameters of cortisol levels in the total samples studied. In the 'target' subgroup with normal ACTH levels and mitotane levels 14-20 mg/dl, no correlation of plasma ACTH with any parameter studied was seen, and FC suggested over-replacement with hydrocortisone treatment in the subgroup with CS. CONCLUSIONS: FC measurement may offer additional information in the follow-up of patients on mitotane, especially when there is a history of CS which invalidates the use of acute changes in plasma ACTH as a parameter of hydrocortisone replacement. These preliminary data suggest that it may prove useful as a biochemical marker when TC or FCI are invalidated by mitotane treatment or plasma ACTH is suppressed by hypercortisolaemia. Larger studies are needed to substantiate the clinical utility of FC measurement in specific groups of patients.