Imatinib-resistant Gastrointestinal Stromal Tumors Research Articles

Abstract 3142: NN3201, a novel c-Kit targeting ADC, exhibits robust preclinical anti-tumor efficacy in SCLC and GIST models

Abstract c-Kit (CD117) is a type III receptor tyrosine kinase (RTK), which is known to be activated upon binding to its cognate ligand stem cell factor (SCF) to induce critical signaling pathways involved in cell proliferation, survival, and differentiation. Indicative of poor prognosis, overexpression and activating mutations of c-Kit have been implicated in a variety of human cancers including small cell lung cancer (SCLC), gastrointestinal stromal tumors (GIST), and acute myelogenous leukemia (AML). While imatinib, a small molecule c-Kit inhibitor, has been a beneficial treatment for GIST, resistance eventually develops to the drug, moreover, shows limited efficacy in c-Kit overexpressing tumors such as SCLC. Herein, we developed NN3201, a novel c-Kit targeting antibody-drug conjugate (ADC), comprised of fully human 2G4 antibody with a picomolar binding affinity to c-Kit and cytotoxic monomethyl auristatin E (MMAE) as the payload. NN3201 displayed potent in vitro cytotoxicity in various wild-type and mutant c-Kit positive SCLC and GIST cell lines with IC50 in the nanomolar range. In addition, NN3201 was rapidly internalized and inhibited c-Kit-mediated signaling., NN3201 treatment resulted in the increased proportions of the sub G1 and G2/M in the c-Kit positive cancer cell lines, indicating that release of MMAE induced cell cycle arrest. Moreover, bystander effect was confirmed by co-culturing c-Kit high and negative cell lines. Most importantly, in mouse xenograft models, NN3201 showed a remarkable tumor growth inhibition of several wild type, mutant c-Kit positive, and imatinib resistant SCLC and GIST cell lines. Enhanced anti-tumor activity and delayed tumor growth were observed in an SOC-treated (etoposide and carboplatin) SCLC xenograft model, by NN3201 in comparison to second line SOC (topotecan or irinotecan). In the GLP study, repeated intravenous administration of NN3201 was well tolerated at all doses (0.5, 1, 2 mg/kg Q3Wx3) without unscheduled death. Dose-dependent, yet transient, cytotoxicity of NN3201 was observed in the bone marrow. We confirmed HNSTD (Highest Non-Severely Toxic Dose) of NN3201 to be 2 mg/kg through exploratory and GLP repeat dose toxicity studies as no serious irreversible toxicity was observed. Accumulating data suggest that NN3201 is a promising therapeutic alternative for the treatment of SCLC and GIST regardless of wild-type or activating mutations in c-Kit. Citation Format: Chansik Kim, Jin Gu Cho, TaeMin Wi, Jiwoo Moon, Han-Jik Ko, Jina Lee, Jin Woo Park, Yeonjy Lee, Sanghee Kim, Jin-Ock Kim, Jaeyoung Song, Sun-Hwa Lee, Sang Gyu Park. NN3201, a novel c-Kit targeting ADC, exhibits robust preclinical anti-tumor efficacy in SCLC and GIST models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3142.

Ripretinib for the treatment of advanced, imatinib-resistant gastrointestinal stromal tumors.

The discovery of the constitutive activation of KIT/PDGFRA tyrosine kinases in gastrointestinal stromal tumors (GISTs) led to the development of the targeted drug imatinib. However, the inevitable development of imatinib resistance remains a major issue. Ripretinib is a novel targeted drug that inhibits the activity of a broad spectrum of drug-resistant KIT/PDGFRA mutants. It was approved in 2020 and is currently recommended in major international guidelines as fourth-line and beyond therapy for advanced GISTs. Emerging evidence shows that ripretinib is superior to sunitinib as second-line treatment for KIT exon 11-mutated GISTs due to its activity against highly heterogeneous frequently occurring secondary mutations. This review summarizes current data on the use of ripretinib to treat advanced imatinib-resistant GISTs. We also propose future research directions to improve the precision of targeted GIST treatment. This article is protected by copyright. All rights reserved.

Safety, pharmacokinetics (PK), and clinical activity of bezuclastinib + sunitinib in previously-treated gastrointestinal stromal tumor (GIST): Results from part 1 of the phase 3 Peak study.

11537 Background: Patients with GIST often relapse after first line therapy with imatinib due to secondary resistance mutations in KIT. As the KIT mutation targeting profiles of bezuclastinib (type I TKI) and sunitinib are distinct and complementary, when combined they inhibit a broad spectrum of secondary KIT mutations. In a prior Phase 1b/2a study (NCT02401815), the combination of the original formulation (Form. A) of bezuclastinib + sunitinib had an acceptable safety profile and was associated with clinical activity at the RP2D (Wagner, JAMA Oncol 2021;7(9):1343-50). An optimized formulation (Form. B) of bezuclastinib with improved bioavailability was developed to reduce pill burden for GIST pts. PK and safety results from the Part 1a lead-in of Peak (NCT05208047) have been previously presented (Wagner, CTOS 2022, P320). Herein we intend to report comprehensive clinical results from Part 1a including duration of therapy, initial response assessment and updated safety. Methods: Peak, a randomized Phase 3, open-label study, aims to evaluate efficacy and safety of bezuclastinib + sunitinib vs sunitinib in pts with imatinib-resistant or intolerant GIST. In Part 1a of the 3-part study, bezuclastinib (Form. B) dosing was escalated in serial cohorts based on PK results until the target exposure, comparable to those achieved at the RP2D established in the Phase 1b/2a study, was achieved. Key inclusion: adult with locally advanced, metastatic and/or unresectable GIST, ≥1 measurable lesion according to modified RECIST v1.1, and ECOG PS 0 to 2. Results: Part 1a enrolled 19 pts. Five pts (Cohort 1) received the starting dose of once daily (QD) bezuclastinib 300 mg+ sunitinib 37.5 mg; dose was escalated to 600 mg with 14 pts receiving bezuclastinib 600 mg + sunitinib 37.5 mg. Median age - 60 yrs (range: 42-77); 68% male; 95% ECOG PS 0-1; 95% metastatic and 5% locally advanced. As of Sept 2022 data cutoff, the median (range) treatment duration was 6 weeks (3.1, 23.9). Due to AEs, one pt required dose reduction of bezuclastinib (Gr 3 diarrhea) and 1 pt discontinued (Gr 2 rash). The majority of TEAEs were low grade, with no ≥ Gr4. Most common TRAEs were diarrhea (37%), neutropenia (37%), ALT (32%) and AST (26%) increases. One pt experienced SAEs of Gr 2 neutropenia and pyrexia and Gr 3 thrombocytopenia. Steady state exposure in pts receiving QD doses of bezuclastinib 600 mg + sunitinib 37.5 mg in this study were similar to that at the RP2D established in the prior Phase 1b/2a study. Conclusions: Initial data from Peak shows an encouraging safety and tolerability profile with no unique safety signals when compared to the known safety profile for sunitinib monotherapy. A dose of bezuclastinib 600 mg QD + sunitinib 37.5 mg QD was confirmed for use in Peak study Part 2, for which enrollment is ongoing. Updated data from Part 1a, including response data, will be presented. Clinical trial information: NCT05208047 .

Abstract 1631: Combination of olverembatinib (HQP1351) with BCL-2 inhibitor lisaftoclax (APG-2575) overcomes resistance in gastrointestinal stromal tumors (GISTs)

Abstract GISTs are common malignant mesenchymal tumors that occur in the GI tract, with an estimated incidence rate of 1% to 2%. About 75% to 80% of patients with GIST have mutations in the KIT gene, while 5% to 10% have mutations in the platelet-derived growth factor receptor α (PDGFRA) gene. Tyrosine kinase inhibitors (TKIs) offer patients an improved quality of life but increased pharmacological resistance to TKIs is often observed. BCL-2 is expressed in >80% of GISTs, and amplification of BCL-2 and BCL-xL are common features associated with disease progression. One approach to enhance GIST eradication is to concurrently inhibit oncogenic KIT signaling while actively engaging apoptotic pathways. Olverembatinib (HQP1351) is a new, third-generation TKI that targets BCR-ABL1, KIT and PDGFRA and is currently in development for relapsed or refractory chronic myeloid leukemia and GIST. Lisaftoclax (APG-2575) is a selective BCL-2 inhibitor under development for hematologic malignancies. The purpose of this study was to evaluate whether combining a BCL-2 inhibitor, lisaftoclax, with olverembatinib enhances treatment effect on imatinib-resistant GISTs. Compared to imatinib-sensitive GIST cell lines (GIST882 and GIST-T1), increased BCL-2 protein expression was observed in imatinib-resistant GIST cells, particularly in GIST430, GIST48, and GIST48B lines. Cell-based antiproliferation studies showed additive activity of olverembatinib and lisaftoclax in imatinib-resistant GIST cells. In vivo studies using imatinib-resistant GIST430 cell-derived xenograft models further revealed that coadministration of olverembatinib at 15 mg/kg (every other day for 3 weeks) with lisaftoclax at 50 mg/kg (daily for 3 weeks) resulted in enhanced antitumor effects compared to either agent alone. Tumor growth inhibition (TGI) rates for combined treatment with olverembatinib and lisaftoclax reached 76.8%, which was superior to the TGI rate of either agent alone: 57.6% and 31.2% for olverembatinib and lisaftoclax, respectively. Regarding putative mechanisms, olverembatinib treatment was shown to downregulate BCL-xL and MCL-1 protein expression, possibly via inhibition of KIT signal pathway and downstream signal transducer and activator of transcription 3 (STAT3). Lisaftoclax triggered apoptosis by disrupting the BCL-2:BIM complex. Therefore, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly augmented cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1), thus triggering apoptosis and subsequently enhancing antitumor effects. Our results demonstrate that olverembatinib and BCL-2 inhibitor lisaftoclax have additive antitumor effects in imatinib-resistant GIST. This novel dual approach may have the potential for treating patients with GISTs whose disease has progressed after treatment with TKIs. Citation Format: Ping Min, Guangfeng Wang, Eric Liang, Bingxing Wu, Huidan Yu, Dajun Yang, Yifan Zhai. Combination of olverembatinib (HQP1351) with BCL-2 inhibitor lisaftoclax (APG-2575) overcomes resistance in gastrointestinal stromal tumors (GISTs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1631.

Co-targeting of ACK1 and KIT triggers additive anti-proliferative and -migration effects in imatinib-resistant gastrointestinal stromal tumors

Most gastrointestinal stromal tumors (GIST) harbor mutated receptor tyrosine kinase (RTK) KIT/PDGFRA, which provides an attractive therapeutic target. However, a majority of GISTs ultimately develop resistance to KIT/PDGFRA inhibitor imatinib, multiple therapeutic targets will be identified as a reasonable strategy in imatinib-resistant GISTs. Biological mechanisms of non-RTK activated CDC42 associated kinase 1 (ACK1) are still unclear, which has been found to be activated in GISTs. In the current report, ACK1 overexpression is demonstrated in GIST cell lines and biopsies. RNA-seq analysis and immunoblotting show that ACK1 expression is dependent on imatinib treatment time in GIST-T1 cell line. The colocalization/complex of KIT and ACK1 in GIST cells are observed, and ACK1 activation is in a partially KIT and CDC42 dependent manner. Treatment with a specific ACK1 inhibitor AIM-100 or ACK1 siRNA, mildly suppresses cell viability, but markedly inhibits cell migration in imatinib sensitive and in imatinib resistant GIST cell lines, which is associated with inactivation of PI3K/AKT/mTOR and RAF/MAPK signaling pathways, and inhibition of epithelial-mesenchymal transition, evidencing upregulation of E-cadherin and downregulation of ZEB1, N-cadherin, vimentin, snail, and/or β-catenin after treatment with AIM-100 or ACK1/CDC42 shRNAs. Combination inhibition of ACK1 and KIT results in additive effects of anti-proliferation and pro-apoptosis as well as cell cycle arrest, and inhibition of invasiveness and migration in vitro and in vivo, compared to either intervention alone through dephosphorylation of KIT downstream intermediates (AKT, S6, and MAPK). Our data suggest that co-targeting of ACK1 and KIT might be a novel therapeutic strategy in imatinib-resistant GIST.

Development of Highly Sensitive Digital Droplet PCR for Detection of cKIT Mutations in Circulating Free DNA That Mediate Resistance to TKI Treatment for Gastrointestinal Stromal Tumor (GIST).

Mutations in cKIT or PDGFRA are found in up to 90% of patients with gastrointestinal stromal tumors (GISTs). Previously, we described the design, validation, and clinical performance of a digital droplet (dd)PCR assay panel for the detection of imatinib-sensitive cKIT and PDFGRA mutations in circulating tumor (ct)DNA. In this study, we developed and validated a set of ddPCR assays for the detection of cKIT mutations mediating resistance to cKIT kinase inhibitors in ctDNA. In addition, we cross-validated these assays using next generation sequencing (NGS). We designed and validated five new ddPCR assays to cover the most frequent cKIT mutations mediating imatinib resistance in GISTs. For the most abundant imatinib-resistance-mediating mutations in exon 17, a drop-off, probe-based assay was designed. Dilution series (of decreasing mutant (MUT) allele frequency spiked into wildtype DNA) were conducted to determine the limit of detection (LoD). Empty controls, single wildtype controls, and samples from healthy individuals were tested to assess specificity and limit of blank (LoB). For clinical validation, we measured cKIT mutations in three patients and validated results using NGS. Technical validation demonstrated good analytical sensitivity, with a LoD ranging between 0.006% and 0.16% and a LoB ranging from 2.5 to 6.7 MUT fragments/mL. When the ddPCR assays were applied to three patients, the abundance of ctDNA in serial plasma samples reflected the individual disease course, detected disease activity, and indicated resistance mutations before imaging indicated progression. Digital droplet PCR showed good correlation to NGS for individual mutations, with a higher sensitivity of detection. This set of ddPCR assays, together with our previous set of cKIT and PDGFRA mutations assays, allows for dynamic monitoring of cKIT and PDGFRA mutations during treatment. Together with NGS, the GIST ddPCR panel will complement imaging of GISTs for early response evaluation and early detection of relapse, and thus it might facilitate personalized decision-making.

Combination of pimitespib (TAS-116) with sunitinib is an effective therapy for imatinib-resistant gastrointestinal stromal tumors.

Despite the effectiveness of imatinib, most gastrointestinal stromal tumors (GISTs) develop resistance to the treatment, mainly due to the reactivation of KIT tyrosine kinase activity. Sunitinib, which inhibits the phosphorylation of KIT and vascular endothelial growth factor (VEGF) receptor, has been established as second-line therapy for GISTs. The recently-developed heat shock protein 90 (HSP90) inhibitor pimitespib (PIM; TAS-116) demonstrated clinical benefits in some clinical trials; however, the effects were limited. The aim of our study was therefore to clarify the effectiveness and mechanism of the combination of PIM with sunitinib for imatinib-resistant GISTs. We evaluated the efficacy and mechanism of the combination of PIM with sunitinib against imatinib-resistant GIST using imatinib-resistant GIST cell lines and murine xenograft models. In vitro analysis demonstrated that PIM and sunitinib combination therapy strongly inhibited growth and induced apoptosis in imatinib-resistant GIST cell lines by inhibiting KIT signaling and decreasing auto-phosphorylated KIT in the Golgi apparatus. In addition, PIM and sunitinib combination therapy enhanced antitumor responses in the murine xenograft models compared to individual therapies. Further analysis of the xenograft models showed that the combination therapy not only downregulated the KIT signaling pathway but also decreased the tumor microvessel density. Furthermore, we found that PIM suppressed VEGF expression in GIST cells by suppressing protein kinase D2 and hypoxia-inducible factor-1 alpha, which are both HSP90 client proteins. In conclusion, the combination of PIM and sunitinib is effective against imatinib-resistant GIST via the downregulation of KIT signaling and angiogenic signaling pathways.

Deubiquitylation of Rab35 by USP32 promotes the transmission of imatinib resistance by enhancing exosome secretion in gastrointestinal stromal tumours.

Imatinib is a tyrosine kinase inhibitor that is widely used to combat gastrointestinal stromal tumours (GISTs). However, secondary resistance to imatinib is an important challenge in GIST treatment. Recent studies have demonstrated that cancer-derived nanosized exosomes play a key role in intercellular communication, but little is known about the roles of exosomes in imatinib-resistant GISTs. Here, we reveal that exosomes released from imatinib-resistant GISTs transmit drug resistance to imatinib-sensitive tumours. By using iTRAQ technology, we demonstrate that Ras-related protein Rab-35 (Rab35) is upregulated differentially in imatinib-resistant GISTs. Loss of Rab35 decreases exosome secretion, thereby hampering the transmission of imatinib resistance to sensitive tumours. Mechanistically, we showed that the ubiquitin‒proteasome system is involved in elevated Rab35 expression and that ubiquitin-specific protease 32 (USP32), a deubiquitylating enzyme, is bound to Rab35. Further experiments demonstrate that this protease protects Rab35 from proteasomal degradation by reducing Lys48 (K48)-ubiquitination. Additionally, we found that the transcription factor ETV1, which is a lineage survival factor in GISTs, promotes USP32 expression. Collectively, our results reveal that exosomes transmit imatinib resistance in GISTs and that deubiquitylation plays a key role in regulating the transmission process. The USP32-Rab35 axis provides a potential target for interventions to reduce the occurrence of imatinib resistance in GISTs.

Sunitinib therapy for imatinib-resistant and/or intolerant gastrointestinal stromal tumors: comparison of safety and efficacy between standard and reduced dosage regimens.

Sunitinib therapy for patients with imatinib-resistant and/or intolerant gastrointestinal stromal tumors (GISTs) often causes severe adverse events (AEs) that lead to treatment discontinuation. We retrospectively reviewed the clinical records of imatinib-resistant and/or intolerant GIST patients who underwent sunitinib therapy in our institutions between 2007 and 2020. Forty-one patients were enrolled and divided into two groups on the basis of the starting dosage: the standard dosage group (50mg/day, 21 patients) and the reduced dosage group (37.5mg/day, 20 patients). Tolerability, safety and clinical efficacy of the two groups were compared. Three patients (14%) in the standard dosage group and another three (15%) in the reduced dosage group (P=1.000) discontinued sunitinib therapy because of AEs. The incidences of grade 3 or more severe treatment-related AEs were 90 and 75%, respectively (P=0.238). Two possible treatment-related deaths were noted in the standard dosage group. Clinical efficacy was comparable between the two groups: median time to treatment failure and overall survival were 4.5months [interquartile range (IQR), 3.6-9.0] and 13.7months (IQR, 7.5-22.9) in the standard dosage group and 4.6months (IQR, 2.7-17.0) and 13.4months (IQR, 9.3-36.8) in the reduced dosage group, respectively. The reduced dosage of 37.5mg sunitinib tended to decrease toxicity and the incidences of severe AEs and treatment-related deaths. This reduced dosage regimen showed equivalent clinical efficacy including patient survival. The reduced dosage of 37.5mg sunitinib can be adopted as an alternative therapy for patients with imatinib-resistant and/or intolerant GISTs.

Establishment of the prediction model and biological mechanism exploration for secondary imatinib-resistant in gastrointestinal stromal tumor

A gastrointestinal stromal tumor (GIST) is mostly driven by the auto-activated, mutant KIT receptor tyrosine kinase gene or by the platelet-derived growth factor receptor alpha. Inhibition of KIT-signaling is the primary molecular target therapy for GIST, which is performed by the drug imatinib clinically. However, more than half of advanced or metastatic GIST develop secondary resistance to imatinib within 2 years after initiation of treatment, and the mechanism of acquired imatinib-resistant in GIST remains unclear. Therefore, we designed the present study, and firstly analyzed the gene expression profile of imatinib-resistant and sensitive GIST from GEO DataSet and identified 44 differential expressed genes. Then, a model including nine genes with their expressed coefficients was identified as a risk score to predict imatinib-resistant GIST. Internal and external validation of the prediction model was performed through the ROC curve, and the area under the curve was 0.967 (95%CI 0.901–1.000) and 0.917 (95%CI 0.753–1.000), separately. Lastly, the effect of immune, m6A, pyroptosis, and ferroptosis-related genes on imatinib-resistant GIST was also assessed because DNA replication was the most enriched biological function of DEGs after functional annotation, pathway enrichment, and protein-protein interaction network analyses. In conclusion, the present study established a novel model to predict secondary imatinib-resistant GIST. Meanwhile, the bioinformatic mining results provided potential and promising targets for imatinib-resistant therapy.

97P Development of a cas9-expressing gastrointestinal stromal tumor cell line T1-1M-R

Gastrointestinal stromal tumors (GIST) are the most frequent mesenchymal malignancies of the gastrointestinal tract. Pathogenesis of GIST is mostly associated with KIT tyrosine kinase gene mutations. Therefore, tyrosine kinases inhibitor imatinib mesylate (Gleevec) is the first-line GIST treatment. Nevertheless, GIST patients are facing secondary resistance to imatinib within 2 years of ongoing treatment. One of the possible mechanisms related to imatinib resistance is the FGF/FGFR autocrine loop, thus, FGF and FGFR might be prospective targets for imatinib-resistant GIST treatment.

Hematologic toxicities of sunitinib in patients with gastrointestinal stromal tumors: a systematic review and meta-analysis.

Sunitinib offers a significant survival benefit to patients with imatinib-resistant gastrointestinal stromal tumors (GIST). However, the incidence and risk of sunitinib-induced hematologic toxicities in such a population are often overlooked and have not been well characterized. This meta-analysis was performed to assess the summary incidence and risk of hematologic toxicities secondary to sunitinib in patients with GIST. Searches were performed in PubMed, Embase, Cochrane Library, and Web of Science as well as ClinicalTrials.gov to identify relevant studies up to April 2022. Studies with adequate safety profile, including anemia, neutropenia, and thrombocytopenia, were included to calculate the pooled incidence, relative risk (RR), and corresponding 95% confidence intervals (CIs). This study was registered with PROSPERO under number CRD42022328202. A total of 2593 patients from 13 studies were included in the present meta-analysis. For patients with GIST assigned to sunitinib, the overall incidences of all-grade anemia, neutropenia, and thrombocytopenia were 26.2% (95% CI, 14.9-39.4%), 41.8% (95% CI, 29.0-55.1%), and 36.4% (95% CI, 22.8-51.1%), respectively. Regarding high-grade (grades 3 and 4) events, there were 4.7% (95% CI, 3.8-5.6%) for anemia, 9.3% (95% CI, 5.6-13.7%) for neutropenia and 5.0% (95% CI, 2.9-7.3%) for thrombocytopenia. Compared to placebo arms, sunitinib was related to an increased risk of high-grade neutropenia with an RR of 10.39 (95% CI, 1.53-70.72; p = 0.017). Sunitinib carries a relatively high incidence of hematologic toxicities and a substantial increased risk of high-grade neutropenia in patients with GIST. Appropriate prevention and management seem to be inevitable.

Next questions for the medical treatment of gastrointestinal stromal tumor.

Since its approval in 2002, imatinib remains the standard first-line treatment for patients with advanced gastrointestinal stromal tumors (GISTs). Overall, all the drugs approved for patients who have developed secondary resistance to imatinib are less effective than imatinib in first-line. Even if, overall survival of patients with advanced GIST has improved over time the last 20 years, imatinib-resistant GIST remains therefore a difficult-to-treat cancer. The aim of this review is to elaborate on the potential strategies to improve outcome for patients with imatinib-refractory disease. New-generation potent KIT and PDGFRA inhibitors such as ripretinib and avapritinib developed for the treatment of GIST have shown very promising clinical activity in patients with highly refractory disease. However, both failed to improve outcome in comparison with standard of care in earlier lines settings. Clinical trials investigating the efficacy of multikinase inhibitor with highly specific KIT inhibitors are currently ongoing. Targeting the microenvironment of GIST may also represent a promising approach and is investigated in several clinical studies. Imatinib-refractory GIST still represent a therapeutic challenge. It is likely that only combination therapies with new generation of tyrosine kinase inhibitors (TKIs) and/or immune-oncology agents might potentially result in an enhanced therapeutic efficacy compared with current standard of care.

Abstract 147: Bezuclastinib is a differentiated KIT inhibitor that exhibits unique selectivity to KIT A-loop mutations, minimal brain penetration, and favorable pharmacokinetic properties in preclinical models

Abstract KIT is a well characterized oncogenic driver, mutated in up to 80% of gastro-intestinal stromal tumors (GIST). Front-line imatinib is effective in many patients but durable responses in the setting of imatinib failure are rarely achieved due to complex secondary mutations that arise in the KIT ATP-binding pocket (exons 13/14) or the activation (A)loop (exons 17/18). Second-line sunitinib is active against exon 13/14 mutations but not A-loop mutations and identifying inhibitors that target the spectrum of possible mutations without incurring off-target toxicities related to broad-spectrum kinase inhibition has been challenging. Bezuclastinib (CGT9486) is a novel type I tyrosine kinase inhibitor that was designed as a potent and selective inhibitor of KIT A-loop mutations to address this unmet medical need. Herein, we present nonclinical data on the differentiated selectivity profile of bezuclastinib compared with other KIT inhibitors. We show that bezuclastinib is a low nanomolar inhibitor of KIT A-loop mutants which uniquely avoids targeting closely related kinases with known liabilities (e.g., PDGFRA, PDGFRB, CSF1R, and KDR) versus other approved inhibitors. In vivo pharmacokinetic/pharmacodynamic (PKPD) relationships have been evaluated utilizing either HMC1.2 human mast cell leukemia tumors for KIT phosphorylation or a Ba/F3-KIT-D816V splenomegaly model. In these models, plasma exposure associated with 50% reduction in splenomegaly or KIT phosphorylation was observed at 3-10 mg/kg/day corresponding to exposures 1.5-2.2 µg∙hr/mL. This correlated well with plasma exposure required to cause significant tumor growth inhibition in a GIST patient-derived xenograft model harboring an A-loop mutation (~1.3 µg∙hr/mL). Maximal efficacy in the PKPD models and tumor regression in GIST disease models was observed in exposure ranges of 19-36 or 11-44 µg∙hr/mL, respectively, which corresponds to clinically reached exposures in a recent GIST combination study of bezuclastinib with sunitinib. Importantly, in nonclinical studies, we also show that bezuclastinib has minimal brain penetration, a preferred feature of an anti-KIT molecule due to CNS-related adverse events observed in this targeted class. We present a tissue distribution study which demonstrates a brain:plasma ratio <0.1 following a 3-day administration at a dose that correlates with clinical exposure. This attractive selectivity and nonclinical safety profile, combined with early clinical data in advanced solid tumors, supports the potential for a best-in-class KIT inhibitor. Bezuclastinib is currently under clinical investigation in advanced SM (Phase 2, Apex NCT04996875), non-advanced SM (Phase 2, Summit), and imatinib-resistant GIST (Phase 3, Peak). Citation Format: Anna Guarnieri, Karyn Bouhana, LouAnn Cable, Rob Rieger, Leyla Haygood, John Robinson, Shannon Winski. Bezuclastinib is a differentiated KIT inhibitor that exhibits unique selectivity to KIT A-loop mutations, minimal brain penetration, and favorable pharmacokinetic properties in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 147.

Identification of Novel Imatinib-Resistant Genes in Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are common ICC precursor sarcomas, which are considered to be a potential malignant mesenchymal tumor driven by specific KIT or PDGFRA signals in the gastrointestinal tract. The standard treatment for GIST without metastasis is surgical resection. GIST with metastasis is usually treated with tyrosine kinase inhibitors (TKIs) only but cannot be cured. The TKI imatinib is the main drug of GIST drug therapy. In adjuvant therapy, the duration of imatinib adjuvant therapy is 3 years. It has been proved that imatinib can improve the overall survival time (OS). However, many GIST patients develop drug resistance due to the long-term use of imatinib. We were forced to look for new strategies to treat GIST. The purpose of the current academic work is to study the drug-resistant genes of imatinib and their potential mechanisms. A total of 897 differentially expressed genes (DEGs) were found between imatinib-sensitive cell line GIST882 and imatinib-resistant cell line GIST430 by RNA sequencing (RNA-seq). After analyzing the DEGs, 10 top genes were selected (NDN, FABP4, COL4A1, COLEC11, MEG3, EPHA3, EDN3, LMO3, RGS4, and CRISP2). These genes were analyzed by RT-PCR, and it was confirmed that the expression trend of FABP4, COL4A1, and RGS4 in different imatinib-resistant cell lines was in accord with the GEO database. It is suggested that these genes may play a potential role in the clinical diagnosis and treatment of imatinib resistance in GIST.

N6-methyladenosine modification regulates imatinib resistance of gastrointestinal stromal tumor by enhancing the expression of multidrug transporter MRP1.

N6-methyladenosine (m6A) is a frequently occurring mRNA modification, which regulates mRNA stability, splicing, and translation. However, its role in drug resistance of gastrointestinal stromal tumor (GIST) is not known. Here, we report that m6A modification levels are elevated in imatinib-resistant GIST cells and tissues, and that methyltransferase METTL3 is one of the main protein responsible for this aberrant modification. Increased METTL3 levels contributed to imatinib resistance and worse progression-free survival of GIST patients. Mechanistic studies revealed that METTL3-mediated m6A modification of the 5'UTR of the multidrug transporter MRP1 mRNA promoted drug resistance of GIST by stimulating MRP1 mRNA translation, via binding with YTHDF1 and eEF-1. Further, METTL3 transcription in imatinib resistant GIST cells was activated by ETV1, leading to the increased m6A methylation of MRP1 mRNA. This is the first report showing a novel regulatory mechanism whereby ETV1, METTL3, and the YTHDF1/eEF-1 complex mediate the translation of MRP1 mRNA in an m6A-dependent manner to regulate the intracellular concentration of imatinib and drug resistance of GIST. These findings highlight MRP1 as a new potential therapeutic target for imatinib resistance of GIST.

New Tyrosine Kinase Inhibitors for the Treatment of Gastrointestinal Stromal Tumors.

This article critically revisits novel data on tyrosine kinase inhibitors that have shown clinical activity in the treatment of gastrointestinal stromal tumor (GIST). GIST therapeutic development exploits the oncogene addiction to KIT or PDGFRA receptor tyrosine kinases. Based on this principle, two new drugs were approved in 2020: ripretinib in GIST patients after progression to all standard treatments and avapritinib, the first agent ever active in the multiresistant PDGFRA D842V-mutant GIST. Additionally, cabozantinib has also shown encouraging activity in imatinib-resistant GIST patients. Finally, novel agents targeting NTRK-driven GIST have emerged as a breakthrough for the treatment of a subset of KIT/PDGFRA wild-type GIST patients. GIST is a paradigmatic tumor model for the rational and successful development of molecularly targeted agents directed against driver mutations in cancer.

Identifying Risk Factors Associated with Survival and Drug-Related Toxicities in Imatinib-Resistant Gastrointestinal Stromal Tumor (GIST) Patients Treated with Sunitinib

Identifying Risk Factors Associated with Survival and Drug-Related Toxicities in Imatinib-Resistant Gastrointestinal Stromal Tumor (GIST) Patients Treated with Sunitinib

Circ-CCS enhances autophagy during imatinib resistance of gastrointestinal stromal tumor by regulating miR-197-3p/ATG10 signaling.

Drug resistance in gastrointestinal stromal tumors (GISTs) is connected with autophagy activation. Accumulating data demonstrates the critical role of circular RNAs (circRNAs) dysregulation in this development. To explore the possible function of hsa_circ_0092306 (circ-CCS) in GIST imatinib resistance. Quantitative real-time reverse transcription PCR (RT-qPCR) was used to determine the expression levels of circ-CCS and miR-197-3p. The vitality and apoptosis of cells were determined using the Cell Counting Kit-8 and TUNEL assays, respectively. Western blot analysis was used to evaluate the relative protein expression. A dual-luciferase reporter assay was used to validate the link between circ-CCS, miR-197-3p, and ATG10. Comparisons of two groups were analyzed using Student's t tests, and analysis of variance (ANOVA) with Tukey's post hoc test was used to compare three or more groups. Circulating-CCS expression was considerably increased in the serum of imatinib-resistant GIST patients (P < 0.001). Circulating-CCS deficiency decreased cell proliferation and autophagy in GIST-882 and GIST-T1 cells, but promoted apoptosis (P < 0.05). Additionally, circ-CCS was predominantly found in the cytoplasm. Mechanically, circ-CCS targeted miR-197-3p, which may influence autophagy by downregulating ATG10, in order to modulate GIST cells' malignant tendencies. Moreover, silencing miR-197-3p reversed the effect of circ-CCS knockdown on apoptosis and autophagy in GIST cells. By modulating the miR-197-3p/ATG10 axis, circ-CCS increased imatinib resistance in GIST cells, establishing a potential target for reversing medication resistance in such patients.

Abstract P257: Preclinical data identifies bezuclastinib as a differentiated KIT inhibitor with unique selectivity to KIT D816V and minimal evidence of brain penetration

Abstract KIT is a well characterized oncogenic driver of gastro-intestinal stromal tumors (GIST) and systemic mastocytosis (SM), and targeted therapeutics have offered valuable treatment options for these patients. Durable responses in imatinib-resistant GIST are rarely achieved due to secondary mutations that occur in exons 13/14 and 17/18. Exon 13/14 mutations can be addressed with sunitinib treatment, but identifying inhibitors that additionally target exon 17/18 mutations without incurring off-target toxicities related to broad-spectrum kinase inhibition has been challenging. Similarly, agents targeting KIT D816V mutations have shown activity in the treatment of advanced SM, but toxicities such as cognitive effects and intracranial hemorrhage may limit dosing and availability of these therapies. Bezuclastinib (CGT9486) was designed to inhibit exon 17/18 KIT mutations (including KIT D816V) with selectivity versus other closely-related kinases with known liabilities, such as PDGFRα, PDGFRβ, KIT wt, VEGFR2 (KDR), and CSF1R (FMS). Herein, we are presenting results from cell-based kinase profiling assays, which demonstrate that bezuclastinib has a significant and unique selectivity to KIT D816V vs. these aforementioned kinases when tested head-to-head against other clinically relevant compounds in GIST or SM. Additionally, a similar selectivity profile was observed for a broader panel of kinases, ion channels, transporters, and enzymes, which will be presented here. Importantly, we also show that bezuclastinib has minimal brain penetration. In a tissue distribution study performed in rats, bezuclastinib shows a brain:plasma ratio &amp;lt;0.1 following 3 day administration at 25 mg/kg, a dose that closely correlates with clinical exposure. This was supported functionally by assessing neurobehavioral effects of bezuclastinib at dose levels up to 100 mg/kg which showed no CNS related effects. Minimal brain penetration is a preferred feature of an anti-Kit molecule due to CNS-related adverse events observed in these indications. This attractive selectivity and nonclinical safety profile, combined with early clinical data (1) in advanced solid tumors, supports the potential for a best-in-class KIT inhibitor. Bezuclastinib is currently under clinical investigation in advanced SM with additional clinical studies planned in non-advanced SM and imatinib-resistant GIST. 1. Jonathan Trent, William D. Tap, Rashmi Chugh, Gabriel Tinoco, Athanasios Tsiatis, Paul Severson, Kerry Inokuchi, Chao Zhang, Glenn Michelson, Andrew J. Wagner. The Potent and Selective KIT Inhibitor PLX9486 Dosed in Combination With Sunitinib Demonstrates Promising Progression Free Survival (PFS) in Patients With Advanced Gastrointestinal Stromal Tumor (GIST): Final Results of a Phase 1/2 Study. Abstract Presented at the Connective Tissue Oncology Society Virtual Meeting; November 20, 2020. Citation Format: Anna Guarnieri, Mark Chicarelli, LouAnn Cable, Francis Sullivan, Howard Ball, Shannon Winski, John Robinson. Preclinical data identifies bezuclastinib as a differentiated KIT inhibitor with unique selectivity to KIT D816V and minimal evidence of brain penetration [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P257.