Identification of new drugs for the treatment of malignant pheochromocytomas and paragangliomas (PPGLs)
This project is focused on the identification of potential therapeutic drugs for the treatment of malignant Pheochromocytoma and Paraganglioma (PPGLs). The project is structured around two primary aims:
Aim #1: to use the human pheochromocytoma cell line (hPheo1) that will be genetically modified (by introducing mutations associated with malignancy, i.e., SDHB) to screen libraries of drugs already approved for other indications regarding their effect on hPheo1 (drug repurposing),
Aim #2: to analyze whether the somatic mutations in human pheochromocytomas can be identified in liquid biopsies (e.g., cell-free DNA). This would help regarding the choice of the drug identified under Aim #1 to be the most effective one for individual patients diagnosed with malignant PPGL.
Progress regarding aim #1: The project has initiated a collaboration with the Chemical Biology Consortium Sweden (CBCS) at SciLifeLab, focusing initially on the SDHB gene. After the successful knockdown of the SDHB gene in the hPheo1 cell line using CRISPR-cas9, the mutated cell line has been characterized using various techniques such as DNA sequencing, DDPCR, western blot, and microarray gene expression analysis.
The current goal is to identify approved drugs that specifically target mutated pheochromocytoma cells without affecting wild-type cells. This involves screening a collection of about 8,000 clinically active compounds, including approximately 1,200 FDA-approved drugs.
The project is conducting extensive dose-response experiments using both 3D and 2D cell cultures to validate potential hits. Cell viability and microscopic imaging are being used as assays to assess the effectiveness of these compounds.
The project commenced with the transfer of assays to prescreening, a crucial step for establishing the hit threshold. Subsequently, the primary screening experiment was conducted, testing the entire set of compounds at a concentration of 10µM. Based on the previously set hit threshold, 704 compounds were selected.
The project then advanced to hit validation, which was performed at three different concentrations of the selected compounds. Compounds were deselected if they exhibited less than 40% cell inhibition and showed the same or higher effect in wild-type (WT) cells. From this process, 22 compounds were chosen as candidates for dose-response curves. The dose-response results revealed that six compound batches and five individual structures demonstrated a significant difference in cytotoxicity between the two cell lines, with higher levels of cell killing observed in the knockdown (KD) cells compared to the WT cells. The remaining compounds either showed similar levels of cell killing in both lines or greater activity in the WT cells.
Based on these findings, potential candidates for further follow-up experiments were selected, considering both functional annotations and structural analogues.
Following this, the project will proceed with testing the analogues at various concentrations. Subsequently, hit validation of the resulting compounds from the screen and analogue testing will be performed. This includes gene expression analysis, Western blotting, and in vivo studies.
Progress regarding aim #2: In a concurrent line of investigation, we pursued our secondary objective, which involved the detection of somatic mutations in PPGLs in order to facilitate mutation‐specific drug treatment. Circulating free DNA (cfDNA) extracted from patients with PPGLs is anticipated to mirror the mutation status of the susceptibility genes. While examining frozen blood samples from pheochromocytoma tumors, we were able to successfully pinpoint different somatic mutations in cfDNA. Our approach involved the use of two sets of mutation-specific primers and Sanger sequencing.