Dijkhuizen, Christa

Drayer, Lyndsay

Korthuis, Patrick

Smelt, Maaike

van den Berg, Anke

van der Wekken, Anthonie

Vilachã, Juliana F.

English

Hanze UAS repository

 SLC34A2-ROS1 L2026M+G2032R confers resistance to ROS1 tyrosine kinase inhibitors in Ba/F3 cells through a reduced ATP binding pocket volume  C. Dijkhuizen1, P. Korthuis1, J. Vilacha2, M. Smelt1, A.J. Van Der Wekken3, A. van den Berg4, L. Drayer5  1 Ins tute For Life Science And Technology, Hanze University of Applied Sciences, 9747 AS - Groningen/NL 2 Faculty Of Science And Engineering, Molecular Dynamics — Groningen Biomolecular Sciences And Biotechnology, University of Groningen, 9747 AG - Groningen/NL 3 Pulmonary Oncology, University of Groningen - UMCG - University Medical Center Groningen, 9700 RB - Groningen/NL 4 Faculty Of Medical Sciences, University of Groningen - UMCG - University Medical Center Groningen, 9713 AV - Groningen/NL 5 Ins tute For Life Science And Technology, Hanze University of Applied Sciences, 9747AS - Groningen/NL  Background The ROS1 G2032R muta on is the most common on-target resistance muta on in crizo nib treated ROS1-posi ve lung cancer pa ents. The aim of our study was to inves gate resistance mechanisms in SCL34A2-ROS1G2032R posi ve Ba/F3 cells against second line treatment with lorla nib.  Methods Ba/F3 SLC34A2-ROS1G2032R cells were subjected to N-ethyl-N-nitrosourea (ENU) mutagenesis and clones were selected upon treatment with 1000 nM lorla nib for 4 weeks. Resistant clones were analyzed for presence of on-target resistant muta ons using Sanger sequencing. In addi on, we generated subclones expressing SLC34A2-ROS1L2026M+G2032R and SLC34A2-ROS1L2026M in Ba/F3 cells. Sensi vity to ROS1 TKIs was determined by measuring cell viability and ROS1 phosphoryla on. Molecular Dynamic simula ons of the ATP binding pocket were performed for all ROS1 variants.  Results The ENU-screen of 41 lorla nib resistant clones revealed one with a muta on in the kinase domain: L2026M. Cell viability assays of the ENU-induced resistant cell line and the Ba/F3 cells transfected with the mutant SCL34A2-ROS1 fusion gene constructs revealed a decreased sensi vity of SLC34A2-ROS1L2026M+G2032R cells for lorla nib, crizo nib, entrec nib and repotrec nib compared to the single mutants. Consistent with these findings, we observed phosphoryla on of ROS1 fusion protein in the double mutant cells which was not inhibited upon treatment with ROS1 TKIs. The single mutant cells showed as expected a clear reduc on in phosphorylated ROS1 fusion protein . Molecular modeling to unravel the effect of the muta ons demonstrated that the volume of the ATP-binding pocket was reduced in single and double mutants compared to wild type. The double L2026M+G2032R mutant displayed the smallest pocket.  Conclusions We iden fied a novel on-target muta on a er inducing lorla nib resistance in SLC34A2-ROS1G2032R Ba/F3 cells. This SLC34A2-ROS1L2026M+G2032R cell line was also resistant to crizo nib, entrec nib and repotrec nib. The resistance can be explained by a smaller ATP binding pocket in the mutated ROS1 fusion protein preven ng effec ve binding of the inves gated TKIs.  Clinical trial iden fica on Editorial acknowledgement Legal en ty responsible for the study The authors.  Funding The Netherlands Organiza on for Scien fic Research (NWO) Doctoral Grant for Teachers to C. Dijkhuizen Applied Research SIA (regie orgaan SIA) HBO-Postdoc Grant to M. Smelt.  Disclosure A.J. Van Der Wekken: Financial Interests, Ins tu onal, Funding: AstraZeneca, Boehringer Ingelheim, Pfizer, Roche, Takeda; Non-Financial Interests, Ins tu onal, Advisory Board: AstraZeneca, Lilly, Roche, Takeda; Non-Financial Interests, Ins tu onal, Speaker, Consultant, Advisor: AstraZeneca, BMS, Lilly, Pfizer, Roche; Financial Interests, Ins tu onal, Other, involved in Clinical Studies: AstraZeneca, Amgen, Blueprint Medicin, Nuvalent, Novar s, Merck, Pfizer, Roche, Takeda. All other authors have declared no conflicts of interest.  

SLC34A2-ROS1 L2026M+G2032R confers resistance to ROS1 tyrosine kinase inhibitors in Ba/F3 cells through a reduced ATP binding pocket volume

https://research.hanze.nl/ws/files/73260720/Abstract_ESMO_Madrid_2023_C.O.Dijkhuizen.pdf

SLC34A2-ROS1 L2026M+G2032R confers resistance to ROS1 tyrosine kinase inhibitors in Ba/F3 cells through a reduced ATP binding pocket volume

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