Synergistic Highly Potent Targeted Drug Combinations in Different Pheochromocytoma Models Including Human Tumor Cultures

Abstract

There are no officially approved therapies for metastatic pheochromocytomas apart from ultratrace 131I-metaiodbenzylguanidine therapy, which is approved only in the United States. We have, therefore, investigated the antitumor potential of molecular-targeted approaches in murine pheochromocytoma cell lines [monocyte chemoattractant protein (MPC)/monocyte chemoattractant protein/3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], immortalized mouse chromaffin Sdhb-/- cells, three-dimensional pheochromocytoma tumor models (MPC/MTT spheroids), and human pheochromocytoma primary cultures. We identified the specific phosphatidylinositol-3-kinase α inhibitor BYL719 and the mammalian target of rapamycin inhibitor everolimus as the most effective combination in all models. Single treatment with clinically relevant doses of BYL719 and everolimus significantly decreased MPC/MTT and Sdhb-/- cell viability. A targeted combination of both inhibitors synergistically reduced MPC and Sdhb-/- cell viability and showed an additive effect on MTT cells. In MPC/MTT spheroids, treatment with clinically relevant doses of BYL719 alone or in combination with everolimus was highly effective, leading to a significant shrinkage or even a complete collapse of the spheroids. We confirmed the synergism of clinically relevant doses of BYL719 plus everolimus in human pheochromocytoma primary cultures of individual patient tumors with BYL719 attenuating everolimus-induced AKT activation. We have thus established a method to assess molecular-targeted therapies in human pheochromocytoma cultures and identified a highly effective combination therapy. Our data pave the way to customized combination therapy to target individual patient tumors.

Trial registration: ClinicalTrials.gov NCT01371201 NCT02077933.

Copyright © 2019 Endocrine Society.

Figures

Figure 1.

Cluster-1, cluster-2, and cluster-3, with molecular-targeted therapeutic options. Cluster-1: The pseudo-hypoxic signaling cluster includes mutations in genes encoding for hypoxia-inducible factor 2-α (HIF2A), Krebs-cycle enzymes such as succinate dehydrogenase subunits [SDHx (SDHA, SDHB, SDHC, SDHD)], succinate dehydrogenase complex assembly factor-2 (SDHAF2), fumarate hydratase (FH), malate dehydrogenase 2 (MDH2), and isocitrate dehydrogenase (IDH), and including von Hippel–Lindau tumor suppressor (VHL) and egl-9 prolyl hydroxylase 1 and 2 (EGLN1/2). Cluster-2: The kinase signaling cluster comprises mutations in the rearranged-during-transfection (RET) proto-oncogene, neurofibromin 1 (NF1) tumor suppressor, H-RAS and K-RAS proto-oncogenes, transmembrane protein 127 (TMEM127), and Myc-associated factor X (MAX). Receptor tyrosine kinases (among others, RET, VEGFR, c-met) activate insulin receptor substrate 1 (IRS-1), which recruits the phosphatidylinositol-3-kinase (PI3K). PI3K activates AKT, which inhibits tuberous sclerosis proteins 1/2 (TSC1/2), leading to disinhibition/activation of the mammalian target of rapamycin (mTORC1); mTORC1 phosphorylates and activates various proteins, including p70S6 kinase (p70S6K), by which p70S6 is phosphorylated. Activated p70S6 promotes cell growth, proliferation, cell survival, and leads, among other effects, to the protein synthesis of HIF-1α, which favors angiogenesis [VEGF/platelet-derived growth factor (PDGF)] transcription, among others], invasion, and metastasis under hypoxic or pseudo-hypoxic conditions. AKT also inhibits glycogen synthase kinase 3 (GSK3). Persistent activation of PI3K leads to feedback downregulation of IRS-1, which may lead to GSK3 disinhibition/activation. The RAS/RAF/ERK pathway is also activated by tyrosine kinases (RET, among others) and activates mTORC1. NF1 mutations lead to disinhibition/activation of RAS. TMEM127 mutations lead to disinhibition/activation of mTORC1. The tumor suppressor MAX antagonizes Myc-dependent cell survival, proliferation, and angiogenesis: mutations lead to increased cell proliferation and angiogenesis. Cluster-3: The Wnt signaling cluster comprises somatic mutations in cold shock domain containing E1 (CSDE1) and the mastermind-like transcriptional coactivator 3 (MAML3) fusion genes. 🛇, pheochromocytoma-promoting loss-of-function mutation of a tumor suppressor gene; ○, pheochromocytoma-promoting gain-of-function mutation of a proto-oncogene. ↑, increase/upregulation; ⊥, inhibition; ↓, activation.

Figure 2.

Significant MPC/MTT cell viability reduction by clinically relevant doses of BYL719, everolimus, and by the combination of both after 120 h. The arithmetic means and standard deviation of at least three independent experiments are shown. @The BYL719/everolimus combination treatment was statistically synergistic in MPC cells and additive in MTT cells. *Statistically significant differences in comparison with the DMSO control (P < 0.05).

Figure 3.

Significant MPC/MTT cell viability reduction by sunitinib, everolimus, and by the combination of both after 120 h. The arithmetic means and SD of at least three independent experiments are shown. @Sunitinib/everolimus combination treatment was statistically synergistic in both cell lines. *Statistically significant differences in comparison with the DMSO control (P < 0.05).

Figure 4.

Significant MPC/MTT cell viability reduction by niraparib, by temozolomide, and by the combination of both after 72 h. @The niraparib/temozolomide combination was statistically synergistic in MTT cells but not in MPC cells. *Statistically significant differences in comparison with the DMSO control (P < 0.05).

Figure 5.

Cell cycle analysis via flow cytometric analysis. Significant induction of a G1-phase cell cycle arrest in MPC cells by BYL719, everolimus, or sunitinib alone and (even stronger) by the BYL719/everolimus and sunitinib/everolimus combination treatments at 24 h of treatment. Significant induction of a G1-phase cell cycle arrest in MTT cells by everolimus (10 nM) and sunitinib (1 µM/2 µM) and (even stronger) by the BYL719/everolimus and sunitinib/everolimus combination treatment at 24 h of treatment. The arithmetic means and SD of at least three independent experiments are shown. *Statistically significant differences in comparison with the DMSO control (P < 0.05).

Figure 6.

Mean caspase-3/-7 activity in percent ± SD in MPC/MTT cell lines after 24 h of incubation with different drugs. In both cell lines, significant apoptosis induction was observed after BYL719 treatment alone, after BYL719/everolimus combination treatment, and (to a lesser extent) after sunitinib/everolimus combination treatment. The arithmetic means and SD of at least three independent experiments are shown. *Statistically significant differences in comparison with the DMSO control (P < 0.05).

Figure 7.

Western blot analysis of MPC/MTT cell lines after 24 or 48 h of incubation with different drugs. Inhibition of p70S6K, S6, 4EBP1, and GSK3 signaling, attenuation of everolimus-induced AKT activation, IRS-1 upregulation, and cyclin D1/D3 downregulation after BYL719/everolimus combination treatment. A representative blot of three independently performed experiments is shown.

Figure 8.

Significant reduction of cell viability after BYL719 and everolimus treatment in immortalized mouse chromaffin WT and Sdhb−/− cells at 48 and 72 h, with a synergistic effect (@) of the combination in both cell lines at 72 h and a substantially stronger effect of BYL719 alone in the Sdhb−/− compared with the WT cells. ∆Significant difference between WT and Sdhb−/−. *Statistically significant differences in comparison with the DMSO control (P < 0.05).

Figure 9.

Significant MPC/MTT spheroid shrinkage or complete spheroid collapse after fractionated treatment with 5 µM BYL719, even stronger shrinkage, as compared with BYL719 alone, or an early complete collapse of the spheroids in several experiments after fractionated treatment with the BYL719/everolimus combination and spheroid growth inhibition after fractionated treatment with 25 nM everolimus. Spheroids were treated with different concentrations of BYL719 and everolimus after completed spheroid formation (day 4) and on days 7, 11, and 14 (fractionated treatment). Growth was monitored over 18 d. The arithmetic means and all single values of each experiment of at least three independent experiments are shown. *Statistically significant different results in comparison with the DMSO control (P < 0.05).

Figure 10.

Significant reduction of human PCC primary culture viability (n = 6) by clinically relevant doses of BYL719, everolimus, and the combination of BYL719 plus everolimus at 72 h of treatment, showing a synergistic effect (@) of 5 µM BYL719 plus 10 nM everolimus. A significant reduction of human PCC primary culture viability (n = 6) by sunitinib and the combination of sunitinib plus everolimus is seen at 72 h. Sunitinib and everolimus did not act synergistically. The arithmetic means and all single values of six human PCC cultures from six different patients are shown. *Statistically significant different results in comparison with the DMSO control (P < 0.05).

Figure 11.

Substantial reduction of human PCC primary culture viability (n = 5) by cabozantinib, entinostat, and niraparib, but not temozolomide, at 72 h of treatment. The arithmetic means and all single values of five human PCC primary cultures from five patients are shown. *Statistically significant different results in comparison with the DMSO control (P < 0.05).

Figure 12.

Western blot analysis of the human primary cultures from patients 2, 3, and 5 after 24 h of incubation with different drugs. There was strong synaptophysin expression in all primary cultures. There was strong inhibition of mTORC1/p70S6K and 4EBP1 signaling by everolimus and all combination treatments, attenuation of everolimus-induced AKT activation by the combination treatment in the primary cultures from patients 3 and 5 and downregulation of cyclin D3, CDK1 cdc2 (patient 3), chk1 (patients 3 and 5), and p53 by the combination treatment. GSK3 showed no clear differences, most likely due to suboptimal incubation times.