Targeting pheochromocytoma/paraganglioma with polyamine inhibitors

Abstract

Background: Pheochromocytomas (PCCs) and paragangliomas (PGLs) are neuroendocrine tumors that are mostly benign. Metastatic disease does occur in about 10% of cases of PCC and up to 25% of PGL, and for these patients no effective therapies are available. Patients with mutations in the succinate dehydrogenase subunit B (SDHB) gene tend to have metastatic disease. We hypothesized that a down-regulation in the active succinate dehydrogenase B subunit should result in notable changes in cellular metabolic profile and could present a vulnerability point for successful pharmacological targeting.

Methods: Metabolomic analysis was performed on human hPheo1 cells and shRNA SDHB knockdown hPheo1 (hPheo1 SDHB KD) cells. Additional analysis of 115 human fresh frozen samples was conducted. In vitro studies using N¹,N¹¹-diethylnorspermine (DENSPM) and N¹,N¹²- diethylspermine (DESPM) treatments were carried out. DENSPM efficacy was assessed in human cell line derived mouse xenografts.

Results: Components of the polyamine pathway were elevated in hPheo1 SDHB KD cells compared to wild-type cells. A similar observation was noted in SDHx PCC/PGLs tissues compared to their non-mutated counterparts. Specifically, spermidine, and spermine were significantly elevated in SDHx-mutated PCC/PGLs, with a similar trend in hPheo1 SDHB KD cells. Polyamine pathway inhibitors DENSPM and DESPM effectively inhibited growth of hPheo1 cells in vitro as well in mouse xenografts.

Conclusions: This study demonstrates overactive polyamine pathway in PCC/PGL with SDHB mutations. Treatment with polyamine pathway inhibitors significantly inhibited hPheo1 cell growth and led to growth suppression in xenograft mice treated with DENSPM. These studies strongly implicate the polyamine pathway in PCC/PGL pathophysiology and provide new foundation for exploring the role for polyamine analogue inhibitors in treating metastatic PCC/PGL. PRéCIS: Cell line metabolomics on hPheo1 cells and PCC/PGL tumor tissue indicate that the polyamine pathway is activated. Polyamine inhibitors in vitro and in vivo demonstrate that polyamine inhibitors are promising for malignant PCC/PGL treatment. However, further research is warranted.

Keywords: DENSPM; DESPM; Paraganglioma (PGL); Pheochromocytoma (PCC); Polyamine; SDHB.

Fig. 1.

hPheo1 WT and hPheo1 SDHB KD cell lines. (a) Quantitative Real Time-PCR analysis in hPheo1 WT(■) and hPheo1 SDHB KD (□) are shown; mRNA level were normalized with GAPDH. Results show mean ± SD, n = 3. (b) Total-cell extracts were prepared and analyzed by western blot for SDHB protein expression. Note decreased expression of SDHB in both experiments.

Fig. 2.

Polyamine pathway and altered succinate. Comparison of succinate level in hPheo1 WT and hPheo1 SDHB KD are represented on the left and PCC/PGL tumor tissues of non-SDHx mutated samples and SDHx mutated samples are compared on the right side. Y-axis represents relative intracellular metabolite levels respective to normal samples. Succinate levels are elevated in cells carrying SDHx mutation and tumor tissue samples. Significant increases for spermidine and spermine were observed in human SDHx-mutated PCC/PGLs; a similar pattern was observed in hPheo1 SDHB KD cells, although spermidine did not reach statistical significance in cell line analysis.

Fig. 3.

A–D: (A–B). Polyamine analog DENSPM was used for treatment of BJ fibroblasts (....), hPheo1 WT (——) and hPheo1 SDHB KD (—) cells at 10 nM–75 nM. (A) The quantitative analysis of the colony-forming assay is represented in left panel. (B) Representative images of colony forming assay for BJ fibroblasts, hPheo1 WT, and hPheo1 SDHB KD cells. All experiments were performed in triplicate and represented as mean ± SD. (C–D). Polyamine analog DESPM was used for treatment of BJ fibroblasts (....), hPheo1 WT (——) and hPheo1 SDHB KD (—) cells at 10 nM–50 nM. (C) The quantitative analysis of the colony-forming assay was represented in left panel. (D) Representative images of colony forming assay for BJ fibroblast, hPheo1 WT, and hPheo1 SDHB KD. All experiments were performed in triplicate and represented as mean ± SD. Note that DENSPM dose response curves showed IC₅₀ value for hPheo1WT at 75 nM, whereas hPheo1 SDHB KD showed a response at 40 nM (3B). Also note that BJ fibroblasts showed no sensitivity towards DENSPM at this concentration range (3B). For DESPM, the dose response data demonstrated a very low IC₅₀ for hPheo1 SDHB KD at 10 nM, whereas hPheo1 WT showed an IC₅₀ of 50 nM (3C and 3D).

Fig. 4.

A–C. DENSPM treatment in hPheo1 WT and hPheo1 SDHB KD xenograft models. (A) Four cohorts of male mice (Taconic C.B-Igh-1b/IcrTac-Prkdcscid; n = 6–10 per cohort) were administered vehicle control (PBS) or DENSPM (60 mg/kg) via i.p. injection, daily over 6 weeks to generate tumor growth curves of cell line xenografts. Tumor volumes are displayed as means ± SD (***p < .001). (B) Weights of tumors isolated at endpoint after 45 days of treatment. Tumor weights are displayed as means ± SD (***p < .001). (C) Representative three-dimensional images of the tumors obtained through MRI volumetric analysis in four animal cohorts as indicated, before and after treatment with either PBS or DENSPM. Note that DENSPM administration resulted in a reduction of tumor volume, with tumor growth inhibition (TGI) reaching 75% and 86% in hPheo1 WT and hPheo1 SDHB KD cohorts, respectively. DENSPM significantly reduced tumor volume, with a TGI of ~66% in both cohorts by day 16. Tumor weight in treated animals was also significantly reduced compared to vehicle-injected mice (4B).

Fig. 5.

Graphic summary of key results. Polyamine (PA) pathway is overactive in cells carrying SDHB mutations and in PCC/PGL SDHx-mutated tumor tissue samples. Treatment with polyamine pathway inhibitors preferentially suppresses growth of hPheo1 SDHB KD and hPheo1 WT compared to control cells in culture and in a mouse xenograft model.