12422 Linsitinib Inhibits Proliferation And Induces Apoptosis Of IGF-1R And TSH-R Expressing Cells

Abstract Disclosure: M. Luffy: None. A. Ganz: None. J. Ropertz: None. R. Douglas: Employee; Self; Sling Therapeutics. R. Zeidan: Employee; Self; Sling Therapeutics. J. Kent: Employee; Self; Sling Therapeutics. J. Wolf: None. G.J. Kahaly: Grant Recipient; Self; Sling Therapeutics. Background: The insulin-like growth factor 1 receptor (IGF-1R) and the thyrotropin receptor (TSH-R) are expressed on orbital target cells and thyrocytes. Targeting these pathways effectively improves thyroid eye disease (TED). Methods: The inhibitory effect of Linsitinib, a small molecule targeting the IGF-1R (Sling Therapeutics, Ann Arbor, MI, USA), on cell proliferation was investigated in an IGF-1R expressing cell line, NCI-H295R (ATCC, Manassas, VA, USA) and a Chinese Hamster Ovary (CHO) cell line overexpressing the TSH-R (QuidelOrtho, San Diego, CA, USA). An IGF-1R antagonist, Teprotumumab (BioVision, Milpitas, CA, USA) served as control. Both cell lines were plated in a 96-well format and treated with four concentrations of both compounds for 24 hours. After addition of tetrazolium, absorbance was measured (GloMax luminometer, Promega, Madison, WI, USA). Furthermore, the half-maximal inhibitory concentration (IC50) of TSH-R-Ab induced stimulation (stimulatory monoclonal antibody, mAb, M22, RSR, Cardiff, UK) of the TSH-R cell line was evaluated with a cell-based bioassay for blocking TSH-R-Ab (Thyretain TBI, QuidelOrtho). Cells were treated with ten rising concentrations of either Linsitinib, Linsitinib + Metformin, Teprotumumab, or a blocking TSH-R mAb (K1-70, RSR). The Kruskal-Wallis and/or the two-tailed Mann-Whitney tests were used for statistical comparisons. Results: Linsitinib strongly inhibited the proliferation of both cell lines at the following concentrations: 31,612.5 ng/mL (-78%, P=0.0031, IGF-1R cell line and -75%, P=0.0059, TSH-R cell line), as well as at 63,225 ng/mL (-73%, P=0.0073, IGF-1R cell line and -73%, P=0.0108, TSH-R cell line). Linsitinib induced morphologically confirmed apoptosis of both cell lines across all tested concentrations (Luna-FL cell counter, Logos Biosystems, Annandale, VA, USA). Compared to Teprotumumab, Linsitinib inhibited markedly more the proliferation of the IGF-1R cell line at all concentrations (Linsitinib versus Teprotumumab, P=0.0286). Teprotumumab inhibition ranged from -21 to -31% of the IGF-1R cell line across all concentrations, while it was significant only at 15,806.25 ng/mL with the TSH-R cell line (-15%, P=0.0396). In