Gastrointestinal Stromal Tumors Cell Lines 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.

MicroRNA-128 acts as a suppressor in the progression of gastrointestinal stromal tumor by targeting B-lymphoma Mo-MLV insertion region 1.

The critical role of microRNA-128 (miR-128) in gastrointestinal-related diseases has been documented. In the current study, we tried to clarify the specific role miR-128 in gastrointestinal stromal tumor (GIST) and the underlying mechanism. Differentially expressed genes in GIST were identified following bioinformatics analysis. Then, expression patterns of miR-128 and B-lymphoma Mo-MLV insertion region 1 (BMI-1) in clinical tissue samples and cell lines were characterized, followed by validation of their correlation. GIST-T1 cells were selected and transfected with different mimic, inhibitor, or siRNA plasmids, after which the biological functions were assayed. We identified low miR-128 and high BMI-1 expression in GIST tissues of 78 patients and 4 GIST cell lines. Ectopic expression of miR-128 or silencing of BMI-1 suppressed the malignant potentials of GIST-T1 cells. As a target of miR-128, BMI-1 re-expression could partly counteract the suppressive effect of miR-128 on the malignancy of GIST-T1 cells. Our study provided evidence that miR-128-mediated silencing of BMI-1 could prevent malignant progression of GIST, highlighting a promising anti-tumor target for combating GIST.

Abstract B028: Phosphodiesterase 3A modulators sensitize tumor cells to Bcl-2/Bcl-xL inhibitors

Abstract Phosphodiesterase 3A (PDE3A) modulators are promising anticancer agents for tumors that coexpress PDE3A and Schlafen 12 (SLFN12), including sarcomas. The cytotoxic effect of these modulators is based on their ability to form a complex between PDE3A and SLFN12, and correlates with the expression levels of the two proteins. In cases where the two proteins are only moderately expressed, PDE3A modulator efficacy may remain poor and additional therapeutic options are needed. In this study, we investigated the combinatorial effects of a PDE3A modulator, anagrelide, with 526 other oncological compounds in two PDE3A and SLFN12 expressing liposarcoma cell lines (SA4 and GOT3). Both cell lines were responsive to PDE3A modulators, as expected. Interestingly, we found synergy with seven different drug classes that can be grouped based on their protein and protein family targets: Bcl-2l/Bcl-xL, HDAC, integrin α2 (ITGA2), mTOR, NEDD-activating enzyme E1 regulatory subunit proteins, and PAK. Additional detailed drug combination synergy studies in PDE3A modulator –sensitive liposarcoma, gastrointestinal stromal tumor, melanoma, and astrocytoma cell lines indicated a sensitizing effect of PDE3A modulators to Bcl-2/Bcl-xL inhibitors. These cell lines, excluding GOT3, showed no response to Bcl-2/Bcl-xL inhibitors otherwise. Furthermore, we discovered that the sensitization effect depends on the coexpression of PDE3A and SLFN12, and that cotreatment resulted in stronger activation of programmed cell death. Our findings suggest additional combinatorial opportunities between PDE3A modulators and inhibitors of key cancer pathways that could be explored for their therapeutic value. Citation Format: Kirsi L. J. Toivanen, Astrid Murumägi, Mariliina A. Arjama, Nanna J. Merikoski, Sami I. Salmikangas, Olli P. Kallioniemi, Tom O. Böhling, Harri J. Sihto. Phosphodiesterase 3A modulators sensitize tumor cells to Bcl-2/Bcl-xL inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B028.

Proteomic Characterization Identifies Clinically Relevant Subgroups of Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract, and it has high metastatic and recurrence rates. We aimed to characterize the proteomic features of GIST to understand biological processes and treatment vulnerabilities. Quantitative proteomics and phosphoproteomics analyses were performed on 193 patients with GIST to reveal the biological characteristics of GIST. Data-driven hypotheses were tested by performing functional experiments using both GIST cell lines and xenograft mouse models. Proteomic analysis revealed differences in the molecular features of GISTs from different locations or with different histological grades. MAPK7 was identified and functionally proved to be associated with tumor cell proliferation in GIST. Integrative analysis revealed that increased SQSTM1 expression inhibited the patient response to imatinib mesylate. Proteomics subtyping identified 4 clusters of tumors with different clinical and molecular attributes. Functional experiments confirmed the role of SRSF3 in promoting tumor cell proliferation and leading to poor prognosis. Our study provides a valuable data resource and highlights potential therapeutic approaches for GIST.

BGS-2456 Is a Novel Potent Covalent Inhibitor of FLT3 That Highly Discriminates Against KIT and Is Not Toxic Toward Normal Hematopoiesis in Vitro

Introduction: Class III receptor tyrosine kinases include FLT3, KIT, PDGFRA/B and CSF1R. Activating mutations in FLT3 represent the most common genetic anomalies in AML (30%). While there are numerous examples of KIT TKIs that do not inhibit FLT3 (imatinib, avapritinib, dasatinib), to date, all clinically active FLT3 TKIs (quizartinib, gilteritinib, midostaurin, sorafenib) fail to spare KIT inhibition. Preclinical studies suggest that coinhibition of FLT3 and KIT can result in non-specific myelosuppression, which can limit dose intensification and the ability to safely achieve maintenance dosing. A potent FLT3 TKI that is non-myelosuppressive may enable prolonged treatment in conjunction with chemotherapy and thereby substantially improve clinical outcomes, similar to BCR::ABL1 TKIs in Ph+ ALL (Slayton et al, JCO 2018). FF-10101 is a recently described covalent inhibitor to irreversibly bind FLT3. In vitro and in vivo studies have revealed that FF-10101 exhibits substantial activity against FLT3 and resistant mutants F691L and D835V/Y. However, it does not effectively discriminate against KIT inhibition (Yamara et al, Blood 2018). In clinical experience, a majority of responses were associated with persistent myelosuppression (Levis et al, JCO 2021 39:15_suppl, 7008). Further clinical development of FF-10101 has been abandoned. Here we describe a novel covalent inhibitor (BGS-2456) that potently targets FLT3 with outstanding selectivity against KIT. Results: To confirm that BGS-2456 is a covalent FLT3 inhibitor, we tested various Cys→Ser substitutions in FLT3-ITD. Of these, only substitution of C695 resulted in loss of activity, suggesting that this residue is critical for covalent bond formation with BGS-2456, as has also been demonstrated for FF-10101 (Yamara et al, Blood 2018). Additionally, the compound without the covalent warhead (BGS-2457) was found to be unimpacted by the C695S substitution and substantially less potent than BGS-2456 (Figure). To characterize in vitro properties of BGS-2456 and whether it achieves selectively against FLT3 relative to KIT, we performed cell viability assays of the patient-derived FLT-ITD-positive AML Molm14 cell line as well as the gastrointestinal stromal tumor cell line GIST-T1, which contains an activating juxtamembrane mutation in KIT. We compared EC50 ratios with other FLT3 TKIs and found that BGS-2456 is highly potent, with an EC50 value of ~0.2nM. Moreover, BGS-2456 displayed remarkable selectivity toward FLT3, >10-fold greater than all other TKIs tested. Kinome profiling confirmed FLT3 as the kinase that was most highly inhibited by BGS-2456 among the 300 kinases assessed, with BMX being a distant second (Figure). To assess whether the selectivity of BGS-2456 results in less myelosuppression in vitro, we performed CFU assays of healthy bone marrow. Compared to FF-10101 and gilteritinib, BGS-2456 exhibited the least amount of hematologic toxicity, facilitating in vitro proliferation and differentiation of normal hematopoietic progenitor cells even at 100x EC50 concentration against Molm14 cells. Finally, we assessed BGS-2456's ability to retain activity against the known clinically problematic drug-resistant FLT3-ITD mutants D835Y and F691L. Encouragingly, BGS-2456 retained activity against both D835Y and F691L mutants in the low nanomolar range. Conclusion: This is the first description of a potent and exquisitely specific FLT3 inhibitor that spares KIT inhibition and displays no myelosuppression in vitro at >100x EC50 concentration. The potent inhibitory effects of BGS-2456 on both D835Y and F691L mutants support its promise as a best-in-class TKI for the treatment of FLT3-mutant AML. Efforts to molecularly dissect the basis of the high degree of selectivity of BGS-2456 are ongoing.

MicroRNA-409-5p Inhibits GIST Tumorigenesis and Improves Imatinib Resistance by Targeting KDM4D Expression.

Gastrointestinal stromal tumors (GISTs) can rapidly proliferate through angiogenesis. Previous studies indicated the potential influence of microRNA on the progression of tumor immature angiogenesis. This study aimed to explore the specific mechanism by which microRNA-409-5p (miR-409-5p) contributes to GIST. To identify genes potentially involved in the development and progression of GIST, the differences of miR-409-5p between tumors and adjacent tissues were first analyzed. Following this analysis, target genes were predicted. To further investigate the function of miRNA in GIST cells, two GIST cell lines (GIST-T1 and GIST882) were transfected with lentiviruses that stably expressed miR-409-5p and scrambled miRNA (negative control). Later, the cells were subjected to Western blotting and ELSA to determine any differences in angiogenesis-related genes. In GISTs, there was a decrease in the expression levels of miR-409-5p compared to the adjacent tissues. It was observed that the upregulation of miR-409-5p in GIST cell lines effectively inhibited the proteins hypoxia-inducible transcription factor 1β (HIF1β) and vascular endothelial growth factor A (VEGF-A). Further investigations revealed that miR-409-5p acted as an inhibitor of angiogenesis by binding to the 3'-UTR of Lysine-specific demethylase 4D (KDM4D) mRNA. Moreover, the combination of miR-409-5p with imatinib enhanced its inhibitory effect on angiogenesis. This study demonstrated that the miRNA-409-5p/KDM4D/HIF1β/VEGF-A signaling pathway could serve as a novel target for the development of therapeutic strategies for the treatment of imatinib-resistance in GIST patients.

Characterization of a Human Gastrointestinal Stromal Tumor Cell Line Established by SV40LT-Mediated Immortalization.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the digestive tract and originate from the interstitial cells of Cajal (ICC), which is the pacemaker for peristaltic movement in the gastrointestinal tract. Existing GIST cell lines are widely used as cell models for in vitro experimental studies because the mutation sites are known. However, the immortalization methods of these cell lines are unknown, and no Chinese patient-derived GIST cell lines have been documented. Here, we transfected simian virus 40 large T antigen (SV40LT) into primary GIST cells to establish an immortalized human GIST cell line (ImGIST) for the first time. The ImGIST cells had neuronal cell-like irregular radioactive growth and retained the fusion growth characteristics of GIST cells. They stably expressed signature proteins, maintained the biological and genomic characteristics of normal primary GIST cells, and responded well to imatinib, suggesting that ImGIST could be a potential in vitro model for research in GIST to explore the molecular pathogenesis, drug resistance mechanisms, and the development of new adjuvant therapeutic options.

Multiple intratumoral sources of kit ligand promote gastrointestinal stromal tumor.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the Kit or PDGFRA receptor. While highly effective, tyrosine kinase inhibitors (TKIs) are not curative. The natural ligand for the Kit receptor is Kit ligand (KitL), which exists in both soluble and membrane-bound forms. While KitL is known to stimulate human GIST cell lines in vitro, we used a genetically engineered mouse model of GIST containing a common human KIT mutation to investigate the intratumoral sources of KitL, importance of KitL during GIST oncogenesis, and contribution of soluble KitL to tumor growth in vivo. We discovered that in addition to tumor cells, endothelia and smooth muscle cells produced KitL in KitV558Δ/+ tumors, even after imatinib therapy. Genetic reduction of total KitL in tumor cells of KitV558Δ/+ mice impaired tumor growth in vivo. Similarly, genetic reduction of tumor cell soluble KitL in KitV558Δ/+ mice decreased tumor size. By RNA sequencing, quantitative PCR, and immunohistochemistry, KitL expression was heterogeneous in human GIST specimens. In particular, PDGFRA-mutant tumors had much higher KitL expression than Kit-mutant tumors, suggesting the benefit of Kit activation in the absence of mutant KIT. Serum KitL was higher in GIST patients with tumors resistant to imatinib and in those with tumors expressing more KitL RNA. Overall, KitL supports the growth of GIST at baseline and after imatinib therapy and remains a potential biomarker and therapeutic target.

TATA-box-binding protein-associated factor 15 is a novel biomarker that promotes cell proliferation and migration in gastrointestinal stromal tumor.

Gastrointestinal stromal tumor (GIST) is a common neoplasm with high rates of recurrence and metastasis, and its therapeutic efficacy is still not ideal. There is an unmet need to find new molecular therapeutic targets for GIST. TATA-box-binding protein-associated factor 15 (TAF15) contributes to the progress of various tumors, while the role and molecular mechanism of TAF15 in GIST progression are still unknown. To explore new molecular therapeutic targets for GIST and understand the biological role and underlying mechanisms of TAF15 in GIST progression. Proteomic analysis was performed to explore the differentially expressed proteins in GIST. Western blotting and immunohistochemical analysis were used to verify the expression level of TAF15 in GIST tissues and cell lines. Cell counting kit-8, colony formation, wound-healing and transwell assay were executed to detect the ability of TAF15 on cell proliferation, migration and invasion. A xenograft mouse model was applied to explore the role of TAF15 in the progression of GIST. Western blotting was used to detect the phosphorylation level and total level of RAF1, MEK and ERK1/2. A total of 1669 proteins were identified as differentially expressed proteins with 762 upregulated and 907 downregulated in GIST. TAF15 was selected for the further study because of its important role in cell proliferation and migration. TAF15 was significantly over expressed in GIST tissues and cell lines. Overexpression of TAF15 was associated with larger tumor size and higher risk stage of GIST. TAF15 knockdown significantly inhibited the cell proliferation and migration of GIST in vitro and suppressed tumor growth in vivo. Moreover, the inhibition of TAF15 expression significantly decreased the phosphorylation level of RAF1, MEK and ERK1/2 in GIST cells and xenograft tissues, while the total RAF1, MEK and ERK1/2 had no significant change. TAF15 is over expressed in GIST tissues and cell lines. Overexpression of TAF15 was associated with a poor prognosis of GIST patients. TAF15 promotes cell proliferation and migration in GIST via the activation of the RAF1/MEK/ERK signaling pathway. Thus, TAF15 is expected to be a novel latent molecular biomarker or therapeutic target of GIST.

Abstract 3887: ATP-binding pocket substitutions as secondary or tertiary in-cis mutations are major on-target ripretinib resistance mechanisms in gastrointestinal stromal tumor

Abstract Introduction: Ripretinib (Rip) is a kinase inhibitor with broad preclinical activity against mutant KIT. Based on the INVICTUS trial, Rip was approved for patients with Gastrointestinal Stromal Tumors (GIST) after treatment with 3 or more kinase inhibitors. Most patients in this ≥4th-line setting progress on Rip within one year. Here, we characterized Rip-progressing GIST samples to identify resistance mechanisms. Methods: Progressing lesions in 25 patients were analyzed by NGS after Rip failure. KIT mutations (muts) were recapitulated by gene editing. Activities of Rip, sunitinib (SU), and novel TKIs were characterized by cell viability assays and immunoblot. Mutagenesis experiments were performed using ENU. SU- and Rip-resistant cell lines were pooled and treated with various regimens, with clonal composition deconvoluted by cDNA-based amplicon sequencing. Results: 19/25 Rip-progressing GISTs displayed muts in the ATP-binding pocket (ATP-BP; e13/14). Four of these had pre-existing muts in the activation loop (AL; e17/18), which were confirmed to be in-cis with the primary and ATP-BP muts (triple in cis; TIC). Mutagenesis screens using a double KIT-mutant GIST line (e11 + AL) as a starting point confirmed that TIC-muts are a predominant escape mechanism when treated with Rip. A GIST subline (T1-triple) with TIC-muts in e11, e18 (A829P), and e13 (V654A) was highly resistant to Rip (GR50 > 2µM). Structural analyses of ATP-BP muts suggest steric interference and loss of van der Waals interactions that impede Rip binding and thereby confer Rip resistance. Another 3/25 Rip-progressing GISTs harbored pathogenic KIT muts in e9 only. A novel GIST cell line with primary KIT e9 mut (T1-e9) was 14-fold less sensitive to Rip than isogenic e11-driven cells (GR50 = 115 vs 8nM). Notably, adding a typical AL mut to T1-e9 (T1-e9-N822K) sensitized the cells to Rip (GR50 = 20nM). Immunoblots showed >95% inhibition of phospho-KIT in AL-mutant cell lines at Rip 100nM, whereas inhibition was weaker in T1-e9 (77%) and T1-V654A (46%), and absent in T1-triple. A compound screen of FDA-approved kinase inhibitors identified Nintedanib (NIN) as the most active compound against T1-triple. Clonal outgrowth assays of mixed cultures revealed SU (93% inhibition), and a weekly switch between Rip and either SU (96%) or NIN (79%) as the most effective inhibitors of pooled cell growth. Conclusions: KIT e9 primary muts and e13/14 (ATP-BP) secondary muts are enriched in post-progression biopsies following Rip treatment. KIT TIC-muts are novel frequent events driving GIST clinical progression and confer a high degree of resistance. Strategies to overcome resistance may include combinations or sequences of approved drugs, and novel drugs that more efficiently inhibit TIC-mutant KIT. Citation Format: Thomas Mühlenberg, Johanna Falkenhorst, Tom Schulz, Benjamin S. Fletcher, Alina Teuber, Dawid Krzeciesa, Isabella Klooster, Jonas Lategahn, Wen-Bin Ou, Meijun Lundberg, Margaret von Mehren, Susanne Grunewald, Alicia I. Tüns, Mehdi Brahmi, Michael C. Heinrich, Cesar Serrano, Hans-Ulrich Schildhaus, Sonja Sievers, Jürgen Treckmann, Lydia Wilson, Chandrajit P. Raut, Adrian Marino-Enriquez, Suzanne George, Daniel Rauh, Jonathan A. Fletcher, Sebastian Bauer. ATP-binding pocket substitutions as secondary or tertiary in-cis mutations are major on-target ripretinib resistance mechanisms in gastrointestinal stromal tumor. [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 3887.

Abstract 4872: DCC-3116, a first-in-class selective ULK1/2 inhibitor of autophagy, in combination with the KIT inhibitor ripretinib induces complete regressions in GIST preclinical models

Abstract Background: Cancer cells activate autophagy as an adaptive stress response (ASR) mechanism to therapies targeting the RTK/RAS/MAPK/PI3K pathways, limiting antitumor response. Autophagy is initiated through ULK1/2 kinases and is triggered by inhibitors of the MAPK and PI3K pathways. Most gastrointestinal stromal tumors (GIST) are driven by mutations in KIT kinase. KIT signals through MAPK/PI3K pathways, suppressing ULK1/2 kinases and autophagy1,2,3. Inhibition of mutant KIT reverses this suppression, activating autophagy and cancer cell survival. Approved therapies for GIST include imatinib, sunitinib, regorafenib, ripretinib, and avapritinib. Treatment with these inhibitors is initially successful, but drug resistance can develop either through KIT secondary mutations or ASR pathways including autophagy. DCC-3116 is a selective, potent, first-in-class investigational inhibitor of the ULK1/2 in clinical development in combination with targeted therapies that activate the autophagic ASR pathway. Herein we demonstrate that ULK1/2 and autophagy are activated upon treatment with ripretinib in KIT mutant GIST models A combination of ripretinib with DCC-3116 inhibits autophagy in vitro and leads to complete tumor regressions in preclinical models of GIST. Methods: Inhibition of ULK1/2 in cell assays was measured using an ELISA for the ULK substrate phospho-ATG13 (pATG13). Autophagic flux was measured by monitoring mCherry/GFP tagged LC3 protein in GIST cells. Xenograft studies were performed at CROs. Results: Ripretinib treatment led to the activation of ULK1/2 by 2-3-fold in mutant KIT GIST cell lines. DCC-3116 inhibited both ripretinib-induced and basal pATG13 with IC50 values of 12-32 nM. Treatment of GIST-T1 cells with ripretinib increased autophagic flux 3-fold. DCC-3116 potently inhibited flux with an IC50 value of 38 nM. Ripretinib also induced pATG13 and autophagic flux (1.5-2.5-fold) in multiple imatinib-resistant cell lines, which was inhibited by DCC-3116 with IC50 values between 8-189 nM. In a GIST T1 PK/PD model, DCC-3116 inhibited ULK1/2-mediated pATG13. The combination of DCC-3116 with ripretinib resulted in complete tumor regressions in comparison to single agent treatment in GIST preclinical models. Conclusions: These data demonstrate preclinically that, like other receptor tyrosine kinase inhibitors1, ripretinib activates ULK1/2-mediated autophagy as an ASR resistance mechanism which is inhibited by DCC-3116, providing the rationale to study the combination of DCC-3116 with ripretinib in GIST patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors (NCT04892017).

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.

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.

MITF Downregulation Induces Death in Human Mast Cell Leukemia Cells and Impairs IgE-Dependent Degranulation.

Activating mutations in KIT (CD117) have been associated with several diseases, including gastrointestinal stromal tumors and mastocytosis. Rapidly progressing pathologies or drug resistance highlight the need for alternative treatment strategies. Previously, we reported that the adaptor molecule SH3 binding protein 2 (SH3BP2 or 3BP2) regulates KIT expression at the transcriptional level and microphthalmia-associated transcription factor (MITF) expression at the post-transcriptional level in human mast cells and gastrointestinal stromal tumor (GIST) cell lines. Lately, we have found that the SH3BP2 pathway regulates MITF through miR-1246 and miR-5100 in GIST. In this study, miR-1246 and miR-5100 were validated by qPCR in the SH3BP2-silenced human mast cell leukemia cell line (HMC-1). MiRNA overexpression reduces MITF and MITF-dependent target expression in HMC-1. The same pattern was observed after MITF silencing. In addition, MITF inhibitor ML329 treatment reduces MITF expression and affects the viability and cell cycle progression in HMC-1. We also examine whether MITF downregulation affected IgE-dependent mast cell degranulation. MiRNA overexpression, MITF silencing, and ML329 treatment reduced IgE-dependent degranulation in LAD2- and CD34+-derived mast cells. These findings suggest MITF may be a potential therapeutic target for allergic reactions and deregulated KIT mast-cell-mediated disorders.

SH3BP2 Silencing Increases miRNAs Targeting ETV1 and Microphthalmia-Associated Transcription Factor, Decreasing the Proliferation of Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Gain of function in receptor tyrosine kinases type III, KIT, or PDGFRA drives the majority of GIST. Previously, our group reported that silencing of the adaptor molecule SH3 Binding Protein 2 (SH3BP2) downregulated KIT and PDGFRA and microphthalmia-associated transcription factor (MITF) levels and reduced tumor growth. This study shows that SH3BP2 silencing also decreases levels of ETV1, a required factor for GIST growth. To dissect the SH3BP2 pathway in GIST cells, we performed a miRNA array in SH3BP2-silenced GIST cell lines. Among the most up-regulated miRNAs, we found miR-1246 and miR-5100 to be predicted to target MITF and ETV1. Overexpression of these miRNAs led to a decrease in MITF and ETV1 levels. In this context, cell viability and cell cycle progression were affected, and a reduction in BCL2 and CDK2 was observed. Interestingly, overexpression of MITF enhanced cell proliferation and significantly rescued the viability of miRNA-transduced cells. Altogether, the KIT-SH3BP2-MITF/ETV1 pathway deserves to be considered in GIST cell survival and proliferation.

Microfluidic-based dynamic BH3 profiling predicts anticancer treatment efficacy

Precision medicine is starting to incorporate functional assays to evaluate anticancer agents on patient-isolated tissues or cells to select for the most effective. Among these new technologies, dynamic BH3 profiling (DBP) has emerged and extensively been used to predict treatment efficacy in different types of cancer. DBP uses synthetic BH3 peptides to measure early apoptotic events (‘priming’) and anticipate therapy-induced cell death leading to tumor elimination. This predictive functional assay presents multiple advantages but a critical limitation: the cell number requirement, that limits drug screening on patient samples, especially in solid tumors. To solve this problem, we developed an innovative microfluidic-based DBP (µDBP) device that overcomes tissue limitations on primary samples. We used microfluidic chips to generate a gradient of BIM BH3 peptide, compared it with the standard flow cytometry based DBP, and tested different anticancer treatments. We first examined this new technology’s predictive capacity using gastrointestinal stromal tumor (GIST) cell lines, by comparing imatinib sensitive and resistant cells, and we could detect differences in apoptotic priming and anticipate cytotoxicity. We then validated µDBP on a refractory GIST patient sample and identified that the combination of dactolisib and venetoclax increased apoptotic priming. In summary, this new technology could represent an important advance for precision medicine by providing a fast, easy-to-use and scalable microfluidic device to perform DBP in situ as a routine assay to identify the best treatment for cancer patients.

Ferroptosis Induction and YAP Inhibition as New Therapeutic Targets in Gastrointestinal Stromal Tumors (GISTs).

Simple SummaryDespite targeted therapy with tyrosine kinase inhibitors (TKIs), unresectable or metastatic human GISTs generally relapse under treatment. Thus, alternative therapeutic approaches are needed to overcome GIST resistance. Ferroptosis, a cell death driven by iron-dependent phospholipid peroxidation, is an emerging therapeutic approach in cancers. However, the potential of ferroptosis induction in GISTs remains largely unknown. This study shows that GIST cell lines are highly sensitive to the type II ferroptosis inducer, RSL3. We also provide evidence that inhibition of YAP by verteporfin (VP) promotes ferroptosis in GIST cells while, surprisingly, CA3 mediates ferroptosis independently of YAP inhibition. Finally, we highlight a positive correlation between the highly expressed transferrin receptor protein 1 (TFRC), GISTs with elevated mitotic counts or higher risk, and with YAP expression/activation in human GIST tissue microarrays (TMA). Taken together, our results suggest that induction of ferroptosis and/or modulation of YAP activity offer promising perspectives for GIST treatment.GISTs are sarcomas of the gastrointestinal tract often associated with gain-of-function mutations in KIT or PDGFRA receptor genes. While most GISTs initially respond to tyrosine kinase inhibitors, relapses due to acquired resistance frequently occur. The induction of ferroptosis, an iron-dependent form of non-apoptotic cell death, emerged as a novel therapeutic approach in cancers and remains poorly characterized in GISTs. We studied hallmarks of ferroptosis, i.e., lipid peroxidation, iron and glutathione content, and GPX4 protein expression in imatinib-sensitive (GIST882) and -resistant (GIST48) GIST cell lines. GIST cells were highly sensitive to the induction of ferroptosis by RSL3, which was reversed by liproxstatin and deferoxamine. Lipid peroxidation and ferroptosis were mediated by VP and CA3 in GIST cells through a significant decrease in antioxidant defenses. Moreover, VP, but surprisingly not CA3, inhibited a series of target genes downstream of YAP in GIST cells. The ferroptosis marker TFRC was also investigated by immunohistochemistry in GIST tissue arrays. TFRC expression was observed in all samples. High TFRC expression was positively correlated with high-risk GISTs, elevated mitotic count, and YAP nuclear localization, reflecting YAP activation. This study highlights ferroptosis as a novel cell death mechanism in GISTs, and a potential therapeutic target to overcome resistance to tyrosine kinase inhibitors.

The microphthalmia-associated transcription factor is involved in gastrointestinal stromal tumor growth.

Gastrointestinal stromal tumors (GISTs) are the most common neoplasms of mesenchymal origin, and most of them emerge due to the oncogenic activation of KIT or PDGFRA receptors. Despite their relevance in GIST oncogenesis, critical intermediates mediating the KIT/PDGFRA transforming program remain mostly unknown. Previously, we found that the adaptor molecule SH3BP2 was involved in GIST cell survival, likely due to the co-regulation of the expression of KIT and Microphthalmia-associated transcription factor (MITF). Remarkably, MITF reconstitution restored KIT expression levels in SH3BP2 silenced cells and restored cell viability. This study aimed to analyze MITF as a novel driver of KIT transforming program in GIST. Firstly, MITF isoforms were characterized in GIST cell lines and GIST patients' samples. MITF silencing decreases cell viability and increases apoptosis in GIST cell lines irrespective of the type of KIT primary or secondary mutation. Additionally, MITF silencing leads to cell cycle arrest and impaired tumor growth in vivo. Interestingly, MITF silencing also affects ETV1 expression, a linage survival factor in GIST that promotes tumorigenesis and is directly regulated by KIT signaling. Altogether, these results point to MITF as a key target of KIT/PDGFRA oncogenic signaling for GIST survival and tumor growth.

Methyltransferase-like 3 (METTL3) mediated N6-methyladenosine (m6A) modifications facilitate mir-25-3p maturation to promote gastrointestinal stromal tumors (GISTs) progression.

Methyltransferase-like 3 (METTL3) is an RNA N6-methyladenosine (m6A) methyltransferase, which plays a critical role in micorRNA (miRNAs) processing and maturation, but it is still unclear whether METTL3 regulated miRNAs participates in the regulation of cancer aggressiveness in gastrointestinal stromal tumors (GISTs). This study was designed to investigate this issue, and uncover the potential underlying mechanisms. the expression of METTL3 in GISTs tissues and cell lines were determined by RT-qPCR and Western blot. Cell proliferation and migration were assessed by colony formation, CCK-8 and Transwell. The mRNA expression of all proteins was detected by RT-qPCR, and tumor xenograft study was applied to confirm the effect of METTL3 on GISTs development in vivo. In our study, we showed that METTL3 was significantly upregulated in GISTs tissues and cell lines. Functional experiments demonstrated that overexpression of METTL3 promoted GISTs cell malignant biological behavior and tumor growth in vitro and in vivo, and conversely, silencing of METTL3 had opposite effects and suppressed GISTs progression. Further mechanistical experiments verified that METTL3 promoted the maturation of miR-25-3p in an m6A-dependent manner. Similar to METTL3, miR-25-3p was also validated as an oncogene to promote cancer development in GISTs. Finally, our rescuing experiments hinted that silencing of miR-25-3p abrogated the tumor-initiating effects of METTL3 overexpression on GISTs. Collectively, those results indicated that METTL3 played an oncogenic role in GISTs through positively modulating the miR-25-3p in an m6A-dependent manner, and we firstly discussed how the METTL3/m6A/miR-25-3p axis affected GISTs development.