In Vivo Evaluation of Fibroblast Growth Factor Receptor Inhibition in Mouse Xenograft Models of Gastrointestinal Stromal Tumor.

Advanced gastrointestinal stromal tumors (GIST) are typically treated with tyrosine kinase inhibitors, and imatinib is the most commonly used standard of care in first line treatments. The use of this and other tyrosine kinase inhibitors is associated with objective tumor responses and prolongation of progression-free and overall survival, but the treatment of metastatic disease is non-curative due to the selection or acquisition of secondary mutations and the activation of alternative kinase signaling pathways, leading to resistance and disease progression after an initial response. The present preclinical study evaluated the potential use of the fibroblast growth factor receptor inhibitors infigratinib and dovitinib alone or in combination with the mitogen-activated protein kinase inhibitor binimetinib in mouse models of GIST with different sensitivity or resistance to imatinib. Patient- and cell-line-derived GIST xenografts were established by bilateral, subcutaneous transplantation of human GIST tissue in female adult nu/nu NMRI mice. The mice were treated with dovitinib, infigratinib, or binimetinib, either alone or in combination with imatinib. The safety of treated animals was assessed by well-being inspection and body weight measurement. Antitumor effects were assessed by caliper-based tumor measurement. H&E staining and immunohistochemistry were used for assessing anti-mitotic and pro-apoptotic activity of the experimental treatments. Western blotting was used for assessing effects of the agents on kinase signaling pathways. Anti-angiogenic activity was assessed by measuring tumor vessel density. Dovitinib was found to have antitumor efficacy in GIST xenografts characterized by different imatinib resistance patterns. Dovitinib had better efficacy than imatinib (both at standard and increased dose) and was found to be well tolerated. Dovitinib had better efficacy in a KIT exon 9 mutant model, highlighting a role of patient selection in clinical GIST trials with the agent. In a model with KIT exon 11 and 17 mutations, dovitinib induced tumor necrosis, most likely due to anti-angiogenic effects. Additive effects combining dovitinib with binimetinib were limited.

Integrating Duodenal Sampling in a Human Mass Balance Study to Quantify the Elimination Pathways of JNJ-53718678, a Respiratory Syncytial Virus Fusion Protein Inhibitor.

INTRODUCTION: The study objective was to characterize the excretion and metabolic profile of the respiratory syncytial virus fusion protein inhibitor, JNJ-53718678. Prior animal and in vitro studies suggested three main elimination pathways: N-glucuronidation to M8; CYP(3A4) metabolism leading to circulating metabolites M5, M12, M19 and M37; and JNJ-53718678 biliary excretion. To gain insight into the relative contribution of JNJ-53718678 and M8 biliary excretion, duodenal fluid sampling was incorporated into this mass balance study. METHODS: A single oral dose of 500 mg 14C-JNJ-53718678 was administered to six healthy male subjects. Four hours after study drug intake, gallbladder contraction was stimulated and duodenal fluid samples were collected. JNJ-53718678, its key circulating metabolites and total radioactivity (TR) were quantified in plasma, feces, urine and duodenal fluid. Safety was monitored throughout. RESULTS: JNJ-53718678 and M12 represented 47.4% and 17.8%, respectively, of TR area under the curve (AUC)∞ in plasma. M37 (9.6%), M19 (5.2%), M5 (4.3%) and M8 (1.4%) were minor metabolites; 70.6% of TR was recovered in feces and 19.9% in urine. Duodenal fluid concentrations (% of TR) were highest for JNJ-53718678 (11.6%) followed by M8 (10.4%), M5 (5.9%) and M12 (1.1%). In feces, 10-16% of TR was JNJ-53718678, 5-8% M5, < 1% M12 and < 1% M8. N-glucuronidation to M8 and direct biliary excretion of JNJ-53718678 represented 7% and 8% of drug clearance, respectively. JNJ-53718678 was safe and well tolerated. CONCLUSIONS: JNJ-53718678 is primarily eliminated through CYP3A4-mediated metabolism. By integrating duodenal sampling, N-glucuronidation was confirmed as another metabolic pathway despite the low amount of M8 excreted in urine and feces. TRIAL REGISTRATION: Eudract no. 2016-002664-14.

Cabozantinib Is Active against Human Gastrointestinal Stromal Tumor Xenografts Carrying Different KIT Mutations.

In the majority of gastrointestinal stromal tumors (GIST), oncogenic signaling is driven by KIT mutations. Advanced GIST is treated with tyrosine kinase inhibitors (TKI) such as imatinib. Acquired resistance to TKI is mainly caused by secondary KIT mutations, but can also be attributed to a switch of KIT dependency to another receptor tyrosine kinase (RTK). We tested the efficacy of cabozantinib, a novel TKI targeting KIT, MET, AXL, and vascular endothelial growth factor receptors (VEGFR), in patient-derived xenograft (PDX) models of GIST, carrying different KIT mutations. NMRI nu/nu mice (n = 52) were bilaterally transplanted with human GIST: UZLX-GIST4 (KIT exon 11 mutation, imatinib sensitive), UZLX-GIST2 (KIT exon 9, imatinib dose-dependent resistance), or UZLX-GIST9 (KIT exon 11 and 17 mutations, imatinib resistant). Mice were grouped as control (untreated), imatinib (50 mg/kg/bid), and cabozantinib (30 mg/kg/qd) and treated orally for 15 days. Cabozantinib resulted in significant tumor regression in UZLX-GIST4 and -GIST2 and delayed tumor growth in -GIST9. In all three models, cabozantinib inhibited the proliferative activity, which was completely absent in UZLX-GIST4 and significantly reduced in -GIST2 and -GIST9. Increased apoptotic activity was observed only in UZLX-GIST4. Cabozantinib inhibited the KIT signaling pathway in UZLX-GIST4 and -GIST2. In addition, compared with both control and imatinib, cabozantinib significantly reduced microvessel density in all models. In conclusion, cabozantinib showed antitumor activity in GIST PDX models through inhibition of tumor growth, proliferation, and angiogenesis, in both imatinib-sensitive and imatinib-resistant models. Mol Cancer Ther; 15(12); 2845-52. ©2016 AACR.

Therapeutic Efficacy Assessment of CK6, a Monoclonal KIT Antibody, in a Panel of Gastrointestinal Stromal Tumor Xenograft Models.

We evaluated the efficacy of CK6, a KIT monoclonal antibody, in a panel of human gastrointestinal stromal tumor (GIST) xenograft models. Nude mice were bilaterally transplanted with human GIST xenografts (four patient derived and two cell line derived), treated for 3 weeks, and grouped as follows: control (untreated); CK6 (40 mg/kg, 3× weekly); imatinib (50 mg/kg, twice daily); sunitinib (40 mg/kg, once daily); imatinib + CK6; sunitinib + CK6 (same doses and schedules as in the single-agent treatments). Tumor volume assessment, Western blot analysis, and histopathology were used for evaluation of efficacy. Statistical analysis was performed using Mann-Whitney U (MWU) and Wilcoxon matched-pairs tests. CK6 as a single agent only reduced tumor growth rate in the UZLX-GIST3 model (P = .053, MWU compared to control), while in none of the other GIST models an effect on tumor growth rate was observed. CK6 did not result in significant anti-proliferative or pro-apoptotic effects in any of the GIST models, and moreover, CK6 did not induce a remarkable inhibition of KIT activation. Furthermore, no synergistic effect of combining CK6 with tyrosine kinase inhibitors (TKIs) was observed. Conversely, in certain GIST xenografts, anti-tumor effects seemed to be inferior under combination treatment compared to single-agent TKI treatment. In the GIST xenografts tested, the anti-tumor efficacy of CK6 was limited. No synergy was observed on combination of CK6 with TKIs in these GIST models. Our findings highlight the importance of using relevant in vivo human tumor xenograft models in the preclinical assessment of drug combination strategies.

Pazopanib, a Receptor Tyrosine Kinase Inhibitor, Suppresses Tumor Growth through Angiogenesis in Dedifferentiated Liposarcoma Xenograft Models.

INTRODUCTION: The rarity of dedifferentiated liposarcoma (DDLPS) and the lack of experimental DDLPS models limit the development of novel therapeutic strategies. Pazopanib (PAZ) is a tyrosine kinase inhibitor that is approved for the treatment of non-adipocytic advanced soft tissue sarcoma. The activity of this agent has not yet been properly explored in preclinical liposarcoma models nor in a randomized phase Ш clinical trial in this entity. The aim of the present study was to investigate whether PAZ had antitumor activity in DDLPS models in vivo. MATERIAL AND METHODS: We established two patient-derived DDLPS xenograft models (UZLX-STS3 and UZLX-STS5) through implantation of tumor material from sarcoma patients in athymic nude NMRI mice. An animal model of the SW872 liposarcoma cell line was also used. To investigate the efficacy of PAZ in vivo, mice bearing tumors were treated for 2 weeks with sterile water, doxorubicin (1.2 mg/kg, intraperitoneally, twice per week), PAZ [40 mg/kg, orally (p.o.), twice per day], or PAZ plus doxorubicin (same schedules as for single treatments). RESULTS: Patient-derived xenografts retained the histologic and molecular features of DDLPS. PAZ significantly delayed tumor growth by decreasing proliferation and inhibited angiogenesis in all models tested. Combining the angiogenesis inhibitor with an anthracycline did not show superior efficacy. CONCLUSION: These results suggest that PAZ has potential antitumor activity in DDLPS primarily through antiangiogenic effects and therefore should be explored in clinical trials.

Phosphoinositide 3-kinase inhibitors combined with imatinib in patient-derived xenograft models of gastrointestinal stromal tumors: rationale and efficacy.

INTRODUCTION: The PI3K signaling pathway drives tumor cell proliferation and survival in gastrointestinal stromal tumor (GIST). We tested the in vivo efficacy of three PI3K inhibitors (PI3Ki) in patient-derived GIST xenograft models. EXPERIMENTAL DESIGN: One hundred and sixty-eight nude mice were grafted with human GIST carrying diverse KIT genotypes and PTEN genomic status. Animals were dosed orally for two weeks as follows: control group (untreated); imatinib (IMA); PI3Ki (BKM120-buparlisib, BEZ235, or BYL719) or combinations of imatinib with a PI3Ki. Western blotting, histopathology, and tumor volume evolution were used for the assessment of treatment efficacy. Furthermore, tumor regrowth was evaluated for three weeks after treatment cessation. RESULTS: PI3Ki monotherapy showed a significant antitumor effect, reflected in tumor volume reduction or stabilization, inhibitory effects on mitotic activity, and PI3K signaling inhibition. The IMA+PI3Ki combination remarkably improved the efficacy of either single-agent treatment with more pronounced tumor volume reduction and enhanced proapoptotic effects over either single agent. Response to IMA+PI3Ki was found to depend on the KIT genotype and specific model-related molecular characteristics. CONCLUSION: IMA+PI3Ki has significant antitumor efficacy in GIST xenografts as compared with single-agent treatment, resulting in more prominent tumor volume reduction and enhanced induction of apoptosis. Categorization of GIST based on KIT genotype and PI3K/PTEN genomic status combined with dose optimization is suggested for patient selection for clinical trials exploring such combinations.

Characterization and assessment of the sensitivity and resistance of a newly established human gastrointestinal stromal tumour xenograft model to treatment with tyrosine kinase inhibitors.

BACKGROUND: Acquired resistance to tyrosine kinase inhibitors (TKIs) in gastrointestinal stromal tumours (GISTs) is most commonly caused by secondary KIT or PDGFRA mutations. In this study we characterize a newly established GIST xenograft model, UZLX-GIST9, and evaluate the in vivo response of the model to standard TKIs (imatinib, sunitinib, and regorafenib). METHODS: Tumour fragments from a metastatic lesion of a GIST patient clinically progressing after treatment with imatinib, sunitinib and regorafenib were engrafted in a nude, immunodeficient mouse. Upon sequential passaging from mouse to mouse, tumour fragments were collected for histopathological and molecular characterization. The sensitivity of the model to treatment with TKIs was evaluated in 28 mice [passage 2 (n = 8), passage 4 (n = 20), 41 tumours]. Mice were grouped as follows: control (untreated), imatinib (50 mg/kg/BID), imatinib (100 mg/kg/BID), sunitinib (40 mg/kg/QD), and regorafenib (30 mg/kg/QD). After three weeks of oral treatment, tumours were collected for subsequent analysis. The efficacy of treatment was assessed by tumour volume, histopathology and Western immunoblotting. RESULTS: UZLX-GIST9 maintains the same typical morphological features and immunohistochemical characteristics as the original patient biopsy and expresses CD117 and DOG1. The KIT mutational profile (p.P577del + W557LfsX5+ D820G) remains the same as the original tissue sample originating from an intraspinal metastatic site. Three week treatment with different TKIs showed that the model is resistant to imatinib. Sunitinib induces tumour growth delay and regorafenib reduces the tumour burden by 30% as compared to control animals. While none of the TKIs had a significant effect on cell proliferation or cell survival, a remarkable increase of necrosis and significant reduction of microvessel density was observed under sunitinib and regorafenib. Western immunoblotting showed a mild reduction in KIT and AKT activation only in regorafenib treated tumours. CONCLUSIONS: We established a novel human GIST xenograft, UZLX-GIST9, harbouring KIT exon 11 and 17 mutations and maintaining the pheno-and genotype of the original tumour. UZLX-GIST9 shows different levels of response to standard TKIs. This model will help to study TKI resistance and to explore novel treatment approaches for patients with TKI-resistant GIST.

Frequent mono-allelic loss associated with deficient PTEN expression in imatinib-resistant gastrointestinal stromal tumors.

Insufficiency of phosphatase and tensin homolog (PTEN) occurs in numerous tumor types and has been implicated as a resistance mechanism to receptor tyrosine kinase-targeted therapies in human cancer. In this study, we have performed a comprehensive molecular and immunohistochemical characterization of PTEN in 58 imatinib-naïve and 54 imatinib-treated gastrointestinal stromal tumors (GISTs). The findings were correlated with clinicopathological data. At the genomic level, PTEN was affected mainly by mono-allelic loss, which was significantly less frequent in imatinib-naïve vs imatinib-resistant tumors (9% vs 39%, P<0.001). Neither PTEN mutations nor PTEN promoter hyper-methylation were found. By immunohistochemistry, PTEN depletion was clearly related to GIST progression. Low PTEN protein expression was common (50%) and often paralleled with total immunonegativity in imatinib-resistant tumors. The abnormal PTEN protein expression correlated with PTEN loss at the genomic level (P=0.001). In addition, the effect of small interfering RNA (siRNA) PTEN knockdown on KIT signaling was examined in GIST-T1 and GIST430 cell lines, in the absence or presence of a dual PI3K/mTOR inhibitor NVP-BEZ235, alone or in combination with imatinib. In both cell lines, siRNA silencing of PTEN resulted in the substantial upregulation of PI3K-AKT and MAPK pathways. The MAPK hyperactivation was further potentiated by NVP-BEZ235 in the imatinib-sensitive GIST-T1 cells; yet, this effect was counteracted efficiently by combined treatment. In the imatinib-resistant GIST430 cells, neither NVP-BEZ235 alone or in combination with imatinib yielded sufficient inhibition of hyper-phosphorylated MAPK and downstream intermediate S6 protein. In conclusion, depleted PTEN expression associated with mono-allelic PTEN loss occurs frequently in imatinib-resistant GIST and might serve as a biomarker for stratifying patients for optimal treatment. In vitro, the PTEN insufficiency leads to hyperactivation of AKT and MAPK pathways in tumor cells. Novel therapies targeting multiple components of the integrated KIT receptor signaling pathways in imatinib-resistant GIST warrant further studies.

A potent combination of the novel PI3K Inhibitor, GDC-0941, with imatinib in gastrointestinal stromal tumor xenografts: long-lasting responses after treatment withdrawal.

PURPOSE: Oncogenic signaling in gastrointestinal stromal tumors (GIST) is sustained via PI3K/AKT pathway. We used a panel of six GIST xenograft models to assess efficacy of GDC-0941 as single agent or in combination with imatinib (IMA). EXPERIMENTAL DESIGN: Nude mice (n = 136) were grafted bilaterally with human GIST carrying diverse KIT mutations. Mice were orally dosed over four weeks, grouped as follows: (A) control; (B) GDC-0941; (C) imatinib, and (D) GDC+IMA treatments. Xenografts regrowth after treatment discontinuation was assessed in groups C and D for an additional four weeks. Tumor response was assessed by volume measurements, micro-PET imaging, histopathology, and immunoblotting. Moreover, genomic alterations in PTEN/PI3K/AKT pathway were evaluated. RESULTS: In all models, GDC-0941 caused tumor growth stabilization, inhibiting tumor cell proliferation, but did not induce apoptosis. Under GDC+IMA, profound tumor regression, superior to either treatment alone, was observed. This effect was associated with the best histologic response, a nearly complete proliferation arrest and increased apoptosis. Tumor regrowth assays confirmed superior activity of GDC+IMA over imatinib; in three of six models, tumor volume remained reduced and stable even after treatment discontinuation. A positive correlation between response to GDC+IMA and PTEN loss, both on gene and protein levels, was found. CONCLUSION: GDC+IMA has significant antitumor efficacy in GIST xenografts, inducing more substantial tumor regression, apoptosis, and durable effects than imatinib. Notably, after treatment withdrawal, tumor regression was sustained in tumors exposed to GDC+IMA, which was not observed under imatinib. Assessment of PTEN status may represent a useful predictive biomarker for patient selection.

The HSP90 inhibitor, AT13387, is effective against imatinib-sensitive and -resistant gastrointestinal stromal tumor models.

The majority of gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT, an HSP90 client protein. Further secondary resistance mutations within KIT limit clinical responses to tyrosine kinase inhibitors, such as imatinib. The dependence of KIT and its mutated forms on HSP90 suggests that HSP90 inhibition might be a valuable treatment option for GIST, which would be equally effective on imatinib-sensitive and -resistant clones. We investigated the activity of AT13387, a potent HSP90 inhibitor currently being evaluated in clinical trials, in both in vitro and in vivo GIST models. AT13387 inhibited the proliferation of imatinib-sensitive (GIST882, GIST-T1) and -resistant (GIST430, GIST48) cell lines, including those resistant to the geldanamycin analogue HSP90 inhibitor, 17-AAG. Treatment with AT13387 resulted in depletion of HSP90 client proteins, KIT and AKT, along with their phospho-forms in imatinib-sensitive and -resistant cell lines, irrespective of KIT mutation. KIT signaling was ablated, whereas HSP70, a marker of HSP90 inhibition, was induced. In vivo, antitumor activity of AT13387 was showed in both the imatinib-sensitive, GIST-PSW, xenograft model and a newly characterized imatinib-resistant, GIST430, xenograft model. Induction of HSP70, depletion of phospho-KIT and inhibition of KIT signaling were seen in tumors from both models after treatment with AT13387. A combination of imatinib and AT13387 treatment in the imatinib-resistant GIST430 model significantly enhanced tumor growth inhibition over either of the monotherapies. Importantly, the combination of AT13387 and imatinib was well tolerated. These results suggest AT13387 is an excellent candidate for clinical testing in GIST in combination with imatinib.

The Novel HSP90 inhibitor, IPI-493, is highly effective in human gastrostrointestinal stromal tumor xenografts carrying heterogeneous KIT mutations.

PURPOSE: KIT activity is crucial for gastrointestinal stromal tumors (GIST). Imatinib (IMA) and sunitinib (SUN) are very effective KIT-inhibitors in patients with advanced GIST but have no curative potential. We evaluated the efficacy of the novel HSP90 inhibitor IPI-493 alone, or in combination with IMA or SUN in GIST xenografts with KIT mutations. EXPERIMENTAL DESIGN: Nude mice (n = 98) were grafted bilaterally with human GIST carrying KIT exon 11 (GIST-PSW), KIT exon 9 (GIST-BOE), or double, KIT imatinib-sensitive exon 11 and imatinib-resistant exon 17 mutations (GIST-48). Mice were divided into six treatment groups and dosed orally for 15 days as follows: (i) control group, sterile water; (ii) IMA alone; (iii) SUN alone; (iv) IPI-493 alone; (v) IPI-493+IMA; and (vi) IPI-493+SUN. RESULTS: Treatment with IPI-493 resulted in tumor growth stabilization, variable proliferation arrest, induction of apoptosis and necrosis, and downregulation of KIT and its signaling cascade, especially in the GIST-BOE model. Significant reduction of vessel density was observed with IPI-493 treatment, and was equal to SUN treatment in GIST-PSW and GIST-BOE xenografts. IPI-493 treatment effects were enhanced in combination with TKIs, especially with IPI-493+SUN. In our hands, IPI-493 showed dose-dependent liver damages. CONCLUSIONS: When administered as a single agent in a xenograft model, the HSP90 inhibitor IPI-493 has consistent antitumor activity and induces KIT downregulation in GISTs with heterogeneous KIT mutations. IPI-493 synergizes with TKIs that are commonly used for the treatment of advanced or IMA-resistant GIST. The antitumor response of IPI-493 is particularly enhanced in combination with SUN.