Pharmacological Study Of KIT Kinase Inhibitors Against Gastrointestinal Stromal Tumors

Gastrointestinal Stromal Tumors(GIST)is one of the most common tumors originating from the gastrointestinal mesothelial tissue.Most gastrointestinal stromal tumors are caused by overexpression or gain-of-function mutations in the KIT gene.At present,KIT kinase has become an important target for molecular targeted therapy of tumors.The long-term use of the first-line therapeutic drug imatinib causes a variety of drug resistance mutations,so the development of drugs that can overcome imatinib resistance has very important clinical significance.Leukemia is a blood cell cancer that proliferates abnormally in abnormal cells in the bone marrow and blood.STAT5 is highly activated in FLT3 ITD positively mediated acute myeloid leukemia and BCR-ABL fused chronic myeloid leukemia,and is one of the most important transcription factors in these two hematological diseases.However,the current STAT5 inhibitor pimozide has low activity,so the development of a potent STAT5 inhibitor has a very important significance for the treatment of myeloid leukemia.We devide the paper into four parts,mainly talking about the pharmacological study of KIT inhibitors in gastrointestinal stromal tumors and the mechanism of STAT5 inhibitors.Chapter 1:Discovery and study of highly selective KIT V559D inhibitors against gastrointestinal stromal tumorsKIT kinase V559D mutation is the most prevalent primary gain-of-function mutation in Gastrointestinal Stromal Tumors(GIST).Here we reported a highly selective KIT V559D inhibitor CHMFL-KIT-031,which displayed about 10-20 fold selectivity over KIT WT in the biochemical assay,and 15 fold in cell exanimation.It also displayed 15-400-fold selectivity over other primary mutants such as L576P and secondary mutants including T6701,V654A(ATP binding pocket)as well as N822K and D816V(activation loop).CHMFL-KIT-031 showed potent inhibitory efficacy for KIT V559D mediated signaling pathways in cell and anti-tumor activity in vivo.Its superior selectivity would make it a good pharmacological tool for further dissection of KIT V559D mediated pathology in the GIST.Chapter 2:Axitinib Overcomes Multiple Imatinib Resistance KIT Mutations in the Gastrointestinal Stromal Tumor ModelKIT kinase overexpression and gain-of-function mutations are the critical pathogenesis of gastrointestinal stromal tumors(GIST).Introduction of Imatinib has remarkably improved patients' survival but primary and acquired point mutations rendered drug resistance still remain challenging.Although the multiple kinase inhibitors such as Sunitinib and Regorafenib that were approved latterly for GIST could overcome some of the primary and secondary mutations,the limited overall response of these two drugs and the different drug targets profile associated toxicity are still limitation for GIST treatment and hence the new target therapy with different mutants sensitivity spectrum is still urgently needed in the precision medicine era.Through the drug repositioning approach,we have found that Axitinib,which is approved for the renal cell carcinoma(RCC),exhibited potent activities against a variety of KIT associated primary and secondary mutations.Especially it displayed better activity against KIT V559D/A/G and L576P primary gain-of-function mutations than Imatinib,Regorafenib and Sunitinib.In addition,it could also overcome Imatinib resistant KIT T6701 and V654A mutants.It inhibits these two mutations with GI50 values of 108 and 5nM,IC50 values of 51.8 and 4.56 nM.Axitinib displayed great in vitro and in vivo efficacies in the KIT and,KIT T670I and/V654A mutants GIST preclinical models.Given the fact that the toxicity and pharmacokinetic profile of Axitinib in human has been well characterized,our results would provide the basis for extension of Axitinib to the GIST patients who may not respond to or tolerate the current therapies.Chapter 3:Nintedanib Overcomes Multiple Imatinib Resistance KIT Mutations and Drug Resistance caused by FGFR Activation in Gastrointestinal Stromal Tumor ModelsAxitinib can overcome multiple resistance to imatinib mutations in the GIST model.However,for certain mutations such as T670I,axitinib is not very effective.Moreover,a part of imatinib resistance in gastrointestinal stromal tumors is caused by the activation of FGFR signaling pathway.Such resistance cannot be overcome by axitinib.After screening a set of clinically approved inhibitors,we discovered another drug,Nintedanib,which is more effective than axitinib against imatinib resistant KIT primary and secondary mutations.Especially for T6701,Nintedanib showed a stronger pharmacodynamic effect than Axitinib(G150<3 nM,IC50=1.65 nM).It also showed strong in vivo inhibitory effects on GIST-5R,a preclinical model of gastrointestinal stromal tumors(GI50=9 nM).It exhibited a dose-dependent inhibition on the mouse allograft tumor model.Its tumor inhibition rate reached 44.6%at a dose of 25 mg/kg/d.In addition,Nintedanib is also a FGFR2 inhibitor and has a strong effect on FGFR2.We found that it can overcome the imatinib resistance caused by the activation of FGFR signaling pathway on GIST-T1(GI50=490nM).Our results provide an important solution for the study of drug resistance in gastrointestinal stromal tumors and a new treatment plan,which is clinically significant.Chapter 4:Development and research of STAT5 inhibitors for acute and chronic myeloid leukemiaSTAT5 is activated in the FLT3 ITD-positive mediated AML and BCR-ABL fusion CML.It is one of the most important signaling factors in these two hematological diseases.However,the activity of present STAT5 inhibitor pimozide is relatively weak.It is of great clinical significance to develop an inhibitor against STAT5.We conducted a series of studies through computer-aided drug design,drug chemistry and biochemistry.Then we discovered a efficient STAT5 inhibitor,LXX-5-111,that inhibits the activity of FLT3-ITD positive cell lines MOLM13,MOLM14,and MV4-11.It also induced apoptosis and arrested cell cycle progression in the G0/G1 phase.We demonstrated that LXX-5-111 can bind to STAT5 and inhibits its activity in vitro.Our results provide new options for the treatment of FLL3-ITD-positive AML and BCR-ABL-fused CML.