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.
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 7,000 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.
According to the project plan, the initial phase involved the execution of “assay optimization and transfer to a 384 well format”. The project has successfully validated the screenable assays for selecting the hits during the drug screening in monolayer culture cell lines, followed by a 3D culture.
The project is currently transferring assays to screening, a critical step that sets the foundation for subsequent studies. Following this, the project will move on to hit validation, using the parental hPheo1 cell line to exclude compounds that affect readouts, thereby ruling out nonspecific activity. Additionally, a follow-up study will be conducted (microarray gene expression and seahorse analysis, and in vivo studies using “Zebrafish model”) to track changes induced by the identified drugs.
If the SDHB part is successful, we plan to introduce other mutations associated with the hypoxic gene expression pathway and malignancy (SDHA/D, VHL, MAX, RET, CSDE1) into hPheo1 using CRISPR-Cas9 technology. These newly generated variants of hPheo1 would then undergo drug screening and subsequent experiments as described above.
In a concurrent investigation, we pursue our secondary objective, which involves the detection of somatic mutations in PPGLs in liquid biopsies 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 somatic mutations in cfDNA. Our approach uses of two sets of mutation-specific primers and Sanger sequencing.