8738 Unveiling Potential Therapeutic Candidates for ACTH-Silent Pituitary Neuroendocrine Tumors Through High Throughput Drug Screening
Authors: Marques Juliana M , Da Roz D' Alessandre Nathália , Guardia Gabriela D A , Craveiro Flora Ladeira , Grutzmacher Cristiane , Silva Gilberto O , Mendonca Berenice Bilharinho , Carvalho Luciani Renata Silveira , Fattahi Faranak , Galante Pedro A F , Batista Rafael Loch 
Published: October 2024

Disclosure: J.M. Marques: None. N.D. D' Alessandre: None. G.D. Guardia: None. F.L. Craveiro: None. C. Grutzmacher: None. G.O. Silva: None. B.B. Mendonca: None. L.R. Carvalho: None. F. Fattahi: None. P.A. Galante: None. R.L. Batista: None.

Introduction: ACTH-silent pituitary neuroendocrine tumours (PitNETs) are a subtype of PitNETs that arise from corticotroph cells of the anterior pituitary gland. They can show positive immunoreactivity for adrenocorticotropic hormone (ACTH) but with no clinical or laboratory evidence of hypercortisolism. ACTH-silent PitNETs display notably more aggressive behavior and treatment resistance. Currently, there is no established pharmacological therapy for this specific tumor subtype. Aim: To use high throughput drug screening to identify compounds that modulate ACTH secretion and tumor progression in ACTH-silent PitNETs. Methods: WTC11 cells (GM25256) were differentiated into pituitary using E6 with SB431542 10µM, SHH 200ng/mL, FGF8 100ng/mL and FGF10 50ng/mL until day 30. Pituitary cells were passaged in 384 well plates and treated with FDA-approved compound library with 3113 drugs at 1μM for 72h (#L1300, Selleckchem). Cells were stained with ACTH and imaged on ImageXpress® HCS. ACTH intensity was normalized to total cell number, measured by DAPI, using MetaXpress Software and compounds z-score was calculated, considering -2.5 for analysis. Gene and pathway enrichment analysis were performed and PitNET RNAseq data, publicly available (GSE207937), was used to determine target drugs based on similar target pathways and gene expression. To validate hits, we collected tumor samples from a patient with an aggressive silent ACTH PitNET. PitNET was histologically confirmed and hypercortisolism was excluded clinically and laboratory. Samples were cultured on DMEN/F12 with 10% FBS and 1% antibiotic and pituitary hormones were measured at days 3, 6 and 8. Cells were treated with 1uM of Letrozole, Linsitinib and Raloxifene for 48h and sample for RNA and hormone dosage were collected. Results: We identified 8 drugs that decreased ACTH in induced pituitary cells and selected 3 of them for validation. PitNET was successfully cultured and treated with selected drugs and ACTH secretion was detected at all periods (972.1 pg/mL to 10490 pg/mL), gonadotropins were below the detected level. Letrozole significantly reduced ACTH in the PitNET culture at day 8 (600.9 ± 25.8pg/mL, p&lt;0,001). ACTH was also decreased in Linsitinib treated cells (932 ± 12.7pg/mL, p&lt;0.05), while Raloxifene did not change ACTH secretion (980 ± 95.1pg/mL, p=0.061). Microscopically, PitNET cells aggregate in spheres and this seems to be affected by letrozole treatment, not influencing cell viability. Conclusion: We performed a high throughput drug screen using induced pituitary cells that led to the discovery of 3 compounds that might be used as an alternative for PitNET treatment. Pathway and gene expression analysis from target drugs and PitNET RNAseq may help in the understanding of drug effect in the PitNET context and improve the understanding of tumorigenesis and pharmacological treatment.

Presentation: 6/1/2024