Research On Action And Mechanism Of PDGFR Inhibitor JND6250 To Overcome PDGFRA-D842V Mutant Resistance In Vitro And In Vivo

Platelet-derived growth factor receptors（PDGFR）belong to the type III tyrosine kinase（RTK）family.The abnormal activation of PDGFR mediated by fusion,point mutation and overexpression have been proved to be a reliable therapeutic target for a variety of tumors,which is the main driven factor of gastrointestinal stromal tumors（GIST）,leukemia and other tumors.PDGFR inhibitors such as imatinib,sunitinib and regorafenib have significantly improved survival time and quality of patients with GIST and other tumors.However,the clinical medicines for patients harboring PDGFR resistant mutations,especially PDGFRA-D842x mutations,are still limited.Although avapritinib as a fourth-line drug can overcome the D842x mutant resistance（approved in China,2021/3/31）,it possesses expensive price（1533 RMB/pill）and patients’clinical treatment options are still limited.Therefore,there is an urgent need to develop novel PDGFR inhibitors with independent intellectual property rights to overcome D842x mutant resistance,provide other options and reduce the burden for Chinese patients’treatment.In this study,we successfully constructed six Ba/F3 stable cell models harboring different TEL/FIP1L1-PDGFRA-WT and D842x mutants through electro transfection method,which were identified by cell viability,protein expression and gene sequencing.We screened the anti-proliferative activtities of the compouds desigened by our group using CCK-8 assays,and found an excellent lead compound of JND6250 with an IC₅₀value of 19.00±5.00 and 62.00±22.00 n M against Ba/F3-TEL-PDGFRA-WT and Ba/F3-TEL-PDGFRA-D842V cells,respectively.Based on FRET assay,JND6250 displayed the IC₅₀values of 7.12±7.14 n M and45.20±14.99 n M against PDGFRA-WT and PDGFRA-D842V proteins,respectively,however the control drug imatinib and sunitinib showed resistance to PDGFRA-D842V protein(IC₅₀>1000 n M).Through flow cytometry assay,we found that JND6250 could induce G0/G1 phase arrest（18.68%,14.83%,24 h）and apoptosis（20.61%,24.71%,48 h）at 200 n M after JND6250 treatment in Ba/F3-TEL/FIP1L1-PDGFRA-D842V cells.Through Western Blot assay,we found that JND6250 can dose-dependably inhibit phosphorylation of PDGFRαand downstream signaling proteins such as STAT5,AKT,and ERK from12.5 to 200 n M after JND6250 treatment for 4 hrs.in H1703,Ba/F3-TEL-PDGFRA-D842V and Ba/F3-FIP1L1-PDGFRA-D842V stable cells.JND6250 can decrease the expression level of cell cycle related proteins such as Cyclin E1,CDK4（24 h）and activated the apoptosis-related proteins such as PARP,Caspase-3,Caspase-9（48 h）at 50 n M after treatment in Ba/F3-FIP1L1-PDGFRA-D842V cell.We also unexpectedly found that JND6250 could selectively increase the stability of TEL-PDGFRα-D842x protein level in time and concentration dependent manner.We further found that JND6250 inhibited the growth of xenografted tumors with a tumor growth inhibition rates（TGI）of 27.10%and 57.51%at 50 mg/kg and 100mg/kg,respectively,in Ba/F3-TEL-PDGFRA-D842V mouse xenografted model.We have not found obvious side effects such as weight loss during the experiments period.Through the Western Blot assay in tumor tissues,we found that JND6250 could suppress the activation of PDGFR downstream signaling proteins such as AKT,ERK even at the low dose of 50 mg/kg.In summary,in this study,we identified JND6250 as a novel structural PDGFR inhibitor,which can overcome the PDGFRA-D842x mutant resistance in vitro and in vivo.In this paper,we systematically studied the antitumor effect and mechanism of PDGFR inhibitor JND6250 in vitro and in vivo,provided an important research basis for the development of next generation PDGFR inhibitors.