Abstract 2937: Preclinical evaluation of new therapeutic strategies on SDHB invalidated clones from human pheochromocytoma cells

Abstract

Abstract Introduction: Malignant paraganglioma/pheochromocytoma (MPP) are very rare neuroendocrine tumors with heterogeneous prognostic and no gold-standard treatment. MPP can be associated with germline mutations at SDHB gene which encode for a TCA enzyme, the succinate dehydrogenase that catalyzes the oxidation of succinate to fumarate. When mutated, SDHB losses its function, leading to succinate accumulation by inhibiting 2-oxoglutarate dependent dioxygenases involved in methylation and angiogenesis. Thus alkylating agents, antiagniogenic and demethylating drugs are potential strategies for MPP patients. Aim: This project aimed to establish preclinical models of SDHB invalidated human cell line and to characterize new therapeutic strategies. Material & Methods: Invalidation of SDHB gene has been performed using the CRISPR-Cas9 technology on a human pheochromocytoma cell line (hPHEO1). Isogenic clones have been characterized at the genetic, cellular, proteic, and enzymatic levels through sequencing, immunohistochemistry, western-blotting and enzymatic assays, respectively. The antiproliferative effects of three drugs (temozolomide, sunitinib, azacytidine) have been explored and half maximal inhibitory concentrations (IC50) have been determined on the parental cell line and the 4 clones using the WST1® assay. Results: Four heterozygote isogenic clones have been obtained with 3 different genetic alterations (deletion-insertion). Expression of Ki67 was increased in the SDHB+/- clones. SDHB expression was partially reduced in clones 1 and 2 and almost totally suppressed in clones 3 and 4. For temozolomide and sunitinib, IC50 were significantly lower in SDHB+/- clones (~140 µM for temozolomide and ~1.4 µM for sunitinib) compared to the parental cell line (~1300 µM for temozolomide and ~6 µM for sunitinib) whereas IC50 were similar for azacytidine (~3.5 µM). No significant difference was observed between clones. Conclusion: In this study we obtained 4 SDHB invalidated isogenic clones from the human hPHEO1 cell line, pretty useful for preclinical evaluations in this rare disease. In agreement with literature clones were more sensitive to temozolomide but also to sunitinib. Metabolomic characterization of hPHEO1 and SDHB+/- clones is currently under investigation at baseline and after IC50 treatment of the 3 therapeutic strategies. Drugs’ combinations will be studied in a further step. Citation Format: Constance Lamy, Julien Hadoux, Sylvere Durand, Abir Alghuzlan, Julie Riviere, Deborah Lefevre, Amaury Bongers, Jean-Yves Scoazec, Eric Baudin, Angelo Paci, Sophie Broutin. Preclinical evaluation of new therapeutic strategies on SDHB invalidated clones from human pheochromocytoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2937.