PhD Scientific Days 2019

Budapest, April 25–26, 2019

Direct targeting of mutant K-RAS protein with novel allele-specific inhibitors in in vitro lung adenocarcinoma models

Baranyi, Marcell

Marcell Baranyi1, Eszter Molnár1, Dominika Rittler1, Balázs Hegedűs1,2, József Tímár1
1 2nd Department of Pathology, Semmelweis University, Budapest, Hungary
2 Department of Thoracic Surgery, Ruhrlandklinik, University Duisburg-Essen, Germany

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Text of the abstract

Introduction: K-Ras is one of the most commonly mutated oncoproteins involved in tumor progression. However, until recent advances, it was considered as “undruggable” due to many unsuccessful attempts that tried to block its activity. Now this long-upheld dogma seems to be proven wrong at least for G12C mutant cases as new covalent inhibitors were designed that effectively lock G12C K-RAS in GDP-bound inactive state.
Aims: Aim of this study was to compare G12C specific inhibitors (ARS-853 and ARS-1620) in 2D and 3D cultures of different lung adenocarcinoma cell lines. Furthermore, specificity of the drugs was also tested in an uterocarcinosarcoma cell line harbouring G13C K-RAS mutation.
Method: Short-term cytotoxic effects of the drugs were investigated using SRB assay. 3D analyses were performed using spheroid growth test. Changes in K-RAS and PAN-RAS levels were investigated by western blot analyses.
Results: In line with findings in the literature, high heterogeneity was observed in response to treatment in G12C K-RAS mutant cell lines. Of note, ARS1620 were proved to be more potent than ARS-853 in all assay settings. Interestingly, western blot analyses revealed an upward shift of K-RAS protein bands when treated with covalent inhibitors. In addition, H358 (the most sensitive cell line) revealed unique changes in PAN-RAS bands. Of note, no changes were detected in G13C PF338 suggesting that these inhibitors interfere only with G12C K-RAS protein.
Conclusion: Our results confirmed previously reported potency of covalent inhibitors of G12C K-RAS protein. Of note, our findings suggest that ARS-853 and ARS-1620 are highly specific to G12C K-RAS and do not interfere with G13C mutant protein. Finally, western blot analyses revealed distinct changes in PAN-RAS and K-RAS protein levels and properties among different cell lines.

This work was supported by the Hungarian National Research, Development and Innovation Office, NVKP-16-1-2016-0020

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Doctoral School: Pathological Sciences
Program: Experimental Oncology
Supervisor: József Tímár