SHP2 Allosteric Inhibitor Batoprotafib Inhibits Proliferation Of Imatinib-resistant Gastrointestinal Stromal Tumor (GIST) And The Comprehensive Analyses Of Treatment Strategies For GIST

Gastrointestinal stromal tumor(GIST)is the most common mesenchymal tumor in gastrointestinal tract,mainly driven by KIT or PDGFRA mutations,accounting for about 1-3%of all gastrointestinal malignancies.Imatinib is the first-line treatment for metastatic or unresectable GIST,bringing a median survival benefit of more than 5 years.However,more than 80%of patients experience disease progression after 18 months of therapy due to secondary mutations in KIT.Thus,the mechanism of drug-resistance and treatment of GIST warrants further investigation.For resectable GIST,surgery is the primary treatment option,but there are still some controversies about the perioperative management of this tumor.The current study aimed to conduct research through the following two parts.Part Ⅰ SHP2 allosteric inhibitor Batoprotafib inhibits proliferation of Imatinibresistant GISTBackground The drug-resistant mutations of KIT seriously threaten the life of patients with GIST.Although TKI therapy has brought significant survival benefit,it can easily lead to KIT drug-resistant mutations.At present,the research and development of GISTtargeted drugs is mainly carried out around KIT itself,and the research on the downstream signaling pathway of KIT needs to be strengthened.In view of the complexity of KIT drugresistant mutations,exploring the key downstream targets of KIT and developing new inhibitors is expected to be the key to breakthroughs in the treatment of advanced drugresistant GIST.The non-receptor tyrosine phosphatase SHP2,which is the first recognized proto-oncogene in the PTP family,can be recruited by a variety of RTKs to transduce membrane signals.However,the role of SHP2 in GIST,which is a typical RTK-driven tumor,is unknown.This study will for the first time explore the role of SHP2 in the development and drug resistance of GIST,elucidate its regulatory mechanism,and further verify the inhibitory effect of Batoprotafib,a new allosteric inhibitor of SHP2,on Imatinibresistant GIST induced by KIT mutation.Methods In this study,the function of SHP2 in GIST was firstly studied at the cellular level using CCK-8 assay,colony forming assay,flow cytometry and other techniques.Subsequently,two drug-resistant GIST cell lines with the highest frequency of secondary mutation(GIST-T1/V654A and GIST-T1/D816H)were constructed using CRISPR-Cas9 technology.The function and molecular mechanisms of SHP2 in GIST was explored by WB and Co-IP methods.Finally,the tumor suppression effect of Batoprotafib on Imatinibresistant GIST was evaluated at the cell and animal levels.Results Knocking out the SHP2 coding gene PTPN11 or treating the cells with the SHP2 allosteric inhibitor Batoprotafib significantly promoted cell apoptosis and inhibit cell proliferation and colony formation of GIST-T1 cells.In the subcutaneous tumorigenesis model of GIST-T1 cells in nude mice,knocking out PTPN 11 or oral administration of Batoprotafib significantly inhibited the growth rate of the cells.After the cells were treated with different concentrations of Imatinib,Sunitinib or Batoprotafib for 6 hours,WB detection was performed.In Imatinib-sensitive cells(T1 and 882),as the concentration of Imatinib or Sunitinib increased,the phosphorylation levels of p-SHP2Y542 and pMAPKT202/Y204 were significantly inhibited along with the inhibition of p-KITY719,but it had not been observed in Imatinib/Sunitinib-resistant cells(GIST-T1/D816H).Co-IP results showed that SHP2 was activated and recruited by KIT as a docking molecule to form a protein complex with Grb2 and SOS 1.SHP2 inhibitor Batoprotafib could prevent the formation of KIT-SHP2-Grb2-SOS1 complex from further activating RAS/MAPK pathway in both sensitive-or resistant-cells.Further results verified that Batoprotafib had good tumor suppression effects on GIST-T1/V654A and GIST-T1/D816H in vitro and in vivo.Conclusion The non-receptor tyrosine phosphatase SHP2 can be activated by the GISTspecific driver gene KIT as an adapter protein to recruit Grb2 and SOS1,which is essential for the activation of the downstream RAS/MAPK pathway,one of the key signals in the regulation of growth and drug resistance of GIST.Batoprotafib,a novel allosteric inhibitor of SHP2,showed significant capability of tumor suppression on both Imatinibsensitive/resistant GIST cells,providing new evidence for non-TKI targeted therapy research on advanced drug-resistant GIST.Part Ⅱ The perioperative management of GIST1.Comparison of prognosis between neoadjuvant imatinib plus surgery and upfront surgery for GISTBackground Significant survival benefit of adjuvant imatinib therapy has been observed in gastrointestinal stromal tumor(GIST).However,the impact of neoadjuvant imatinib on prognosis of GIST remains unclear.This meta-analysis aimed to compare the prognostic impact between upfront surgery and neoadjuvant imatinib plus surgery on GIST.Methods A comprehensive literature search was performed to identify eligible studies up to Sep 30,2021,through PubMed,Embase,Web of Science,and Cochrane Library.Studies compared the impact of upfront surgery and neoadjuvant imatinib plus surgery on diseasefree(DFS)or overall survival(OS)in patients with GIST were selected.Results Seven eligible studies with 17171 patients were included.Among them,five studies involved a total of 642 cases of rectal GIST.The reduction rates of tumor size in rectal and mixed site GIST were 33%and 29.8%,respectively.Neoadjuvant imatinib was not significantly associated with DFS compared with no-neoadjuvant therapy in rectal GIST(HR:0.71,95%CI:0.35-1.41).The OS of rectal GIST was significantly improved by neoadjuvant imatinib compared with no-neoadjuvant therapy(HR:0.36,95%CI:0.170.75).Conclusions Neoadjuvant imatinib therapy contributed to tumor shrinkage and R0 resection of rectal GIST.Neoadjuvant imatinib plus surgery significantly improved overall survival of rectal GIST in comparison with upfront surgery.2.Comparison among endoscopic,laparoscopic and open resection for relatively small gastric GIST(<5 cm)Background Endoscopic resection(ESR)is a novel minimally invasive procedure for superficial tumors.Its safety,efficiency and outcome for gastric GISTs(gGISTs)less than 5cm remains unclear compared to laparoscopic resection(LAR)and open resection(ONR).The current network meta-analysis aimed to review and analyze the available evidence of this question.Methods PubMed,Embase,Cochrane Library and Web of Science databases were searched to identify eligible studies published up to July 6,2020.The perioperative and long-term oncological outcomes among ESR,LAR and ONR for gGIST(<5cm)were estimated through the Bayesian network meta-analysis with a random effect model.Results Fifteen studies with 1631 patients were included.ESR was associated with shorter operative time(MD:-36,95%CI:-55--16),a higher rate of positive margin(OR:5.1×1010,95%CI:33-2.5×1032)and less costs(MD:-1×104,95%CI:-1.6×104--4.4×103)but similar time to resume flatus(MD:0.52,95%CI:-0.16-1.10)and diet(MD:-3.50,95%CI:-5.60--1.60)compared to LAR.A higher rate of total complications(OR:11,95%CI:1.20-140)was observed in patients received ESR compared to patients received LAR.After excluding perforation from total complication category,the difference of complication between ESR and LAR disappeared(OR:0.87,95%CI:0.22-2.30).The recurrence rate(OR:1.30,95%CI:0.40-4.50)and disease-free survival(DFS)(HR:1.26,95%CI:0.60-2.63)showed no significant difference between ESR and LAR.ESR was associated with better or equivalent perioperative and long-term outcomes compared to ONR except for positive margin.Subgroup analysis(<2cm and 2-5cm)showed no significant different results among these three procedures either.Conclusion ESR was showed to be a safe and efficient alternative procedure to both LAR and ONR for gGISTs less than 2cm and within 2-5cm,respectively,without worsening the oncologic outcomes.This optimistic result needs to be further evaluated due to the retrospective nature of the enrolled studies which might lead to potential bias.However,preoperative assessment of tumor site is of importance for the determination of procedures regarding the increased incidence of positive margin related to ESR.3.Comparison of prognosis between microscopically positive and negative surgical margins for primary GISTBackground This meta-analysis aimed to determine the prognostic impact of microscopically positive margins(R1)on primary GISTs.Methods A literature search was performed using PubMed,Embase,Web of Science,and Cochrane Library for studies up to November 23,2020.The pooled disease-free survival(DFS)and overall survival(OS)between R1 and negative margins(R0)were estimated using a random-effects model.Results Twenty studies with 6465 patients were included.Compared with R0 resection,R1 was associated with poor DFS in patients who did not receive adjuvant Imatinib(HR:1.62,95%CI:1.26-2.09).This negative impact of R1 disappeared with the use of adjuvant Imatinib(HR:1.23,95%CI:0.95-1.60).R1 was related to poor DFS in gastric GISTs(HR:2.15,95%CI:1.15-5.02),which was attenuated in the subgroup of adjuvant Imatinib(HR:2.24,95%CI:0.32-15.60).Rectal GIST with R1 margin who even received adjuvant Imatinib still had poor DFS(HR:3.79,95%CI:1.27-11.31).Patients who underwent R1 resection had similar OS compared with those underwent R0 resection regardless of the use of adjuvant Imatinib.Conclusion R1 was associated with poor DFS for primary GISTs,which was attenuated by adjuvant therapy with Imatinib.Similar result was observed in the gastric GISTs subgroup.Rectal GIST patients with R1 resection had poor DFS even when they received adjuvant Imatinib.The R1 margin did not influence the OS of GISTs.