Abstract P120: Novel in vitro targeted combination therapies for anaplastic thyroid cancer
Authors: Kunnimalaiyaan Muthusamy , Parekh Parag , Golden Jalyn , Anderson Ying , Lai Stephen , Cabanillas Maria , Zafereo Marc , Dadu Ramona , Hofmann Marie-Claude 
Published: December 2021

Abstract
Purpose: To identify effective drug combination strategies targeting multiple oncogenic pathways in anaplastic thyroid cancer. Background: Treatments with BRAF and MEK inhibitors induce impressive tumor size reduction in most BRAF-mutated anaplastic thyroid carcinoma (ATC) patients. However, these drugs are ultimately ineffective due to resistance development, probably because ATC cells express multiple oncogenic pathways that can be used to bypass the treatments. Therefore, the identification of novel therapeutic strategies is still urgently needed. We had previously identified additional pathways/targets (TWIST1, MMP9, BCL2, TGFβR, and the stress kinase MAPK14/p38α) by reverse-phase protein arrays (RPPA) and microarray analysis. In this study, we report the in vitro effects of inhibitors specific for the identified targets used alone or in combination with BRAF inhibitors. Methods: We established mouse ATC cell lines (MCH2.2-Luc and PPA6-Luc) to graft into immunocompetent syngeneic animals. As a first step, these lines were treated in vitro for 72 hours with increasing concentrations of nine different inhibitors for the above targets, and the MTS colorimetric assay determined viability/IC50. We also tested the combinatorial effects of these compounds with BRAF inhibitors (vemurafenib or dabrafenib) and compared them to single agents’ effects. Also, we investigated the colony-forming ability and apoptotic response of these cell lines. Results: IC50 concentrations for nine different compounds were calculated. Both cell lines showed similar IC50 values, except that PPA6-Luc was more resistant to the BRAF inhibitor dabrafenib than MCH2.2-Luc. The MMP-9 inhibitor and the PARP inhibitor olaparib did not significantly affect the growth of these lines. Among three different MAPK14/p38α inhibitors tested, ralimetinib was the most effective (IC50 @ 15 mM). Importantly, venetoclax (BCL2 inhibitor) and harmine (TWIST1 inhibitor) effectively inhibited cell growth with an IC50 of 8 µM and 25 mM, respectively. Treatments using 0.1mM dabrafenib or 1mM vemurafenib in combination with 4 other drugs (5 mM each below the IC50) resulted in significant synergy only with ralimetinib (38% growth decrease) and harmine (37% growth decrease) compared to treatments with single agents. Colony-forming assays confirmed the growth suppression. Conclusions: Inhibiting specific targets previously identified by RPPA and microarray analysis demonstrated, for the first time, in differential ATC cell growth suppression. Notably, only a combination of specific inhibitors (dabrafenib combined with the TWIST1 or the MAPK14/p38α inhibitors) resulted in synergy, suggesting that these targeted combinations will be more effective in our preclinical ATC mouse model than the other drugs. By treating these combinations in a mouse model may contribute, in the future, to the development of clinical and therapeutic strategies for aggressive thyroid cancer.
Citation Format: Muthusamy Kunnimalaiyaan, Parag Parekh, Jalyn Golden, Ying Anderson, Stephen Lai, Maria Cabanillas, Marc Zafereo, Ramona Dadu, Marie-Claude Hofmann. Novel in vitro targeted combination therapies for anaplastic thyroid cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P120.