Most patients with KIT-mutant gastrointestinal stromal tumours (GISTs) benefit from imatinib, but treatment resistance results from outgrowth of heterogeneous subclones with KIT secondary mutations. ...Once resistance emerges, targeting KIT with tyrosine kinase inhibitors (TKIs) sunitinib and regorafenib provides clinical benefit, albeit of limited duration.
We systematically explored GIST resistance mechanisms to KIT-inhibitor TKIs that are either approved or under investigation in clinical trials: the studies draw upon GIST models and clinical trial correlative science. We subsequently modelled in vitro a rapid TKI alternation approach against subclonal heterogeneity.
Each of the KIT-inhibitor TKIs targets effectively only a subset of KIT secondary mutations in GIST. Regorafenib and sunitinib have complementary activity in that regorafenib primarily inhibits imatinib-resistance mutations in the activation loop, whereas sunitinib inhibits imatinib-resistance mutations in the ATP-binding pocket. We find that rapid alternation of sunitinib and regorafenib suppresses growth of polyclonal imatinib-resistant GIST more effectively than either agent as monotherapy.
Our data highlight that heterogeneity of KIT secondary mutations is the main mechanism of tumour progression to KIT inhibitors in imatinib-resistant GIST patients. Therapeutic combinations of TKIs with complementary activity against resistant mutations may be useful to suppress growth of polyclonal imatinib-resistance in GIST.
Ripretinib (DCC-2618) was designed to inhibit the full spectrum of mutant KIT and PDGFRA kinases found in cancers and myeloproliferative neoplasms, particularly in gastrointestinal stromal tumors ...(GISTs), in which the heterogeneity of drug-resistant KIT mutations is a major challenge. Ripretinib is a “switch-control” kinase inhibitor that forces the activation loop (or activation “switch”) into an inactive conformation. Ripretinib inhibits all tested KIT and PDGFRA mutants, and notably is a type II kinase inhibitor demonstrated to broadly inhibit activation loop mutations in KIT and PDGFRA, previously thought only achievable with type I inhibitors. Ripretinib shows efficacy in preclinical cancer models, and preliminary clinical data provide proof-of-concept that ripretinib inhibits a wide range of KIT mutants in patients with drug-resistant GISTs.
•Ripretinib broadly inhibits primary and drug-resistant KIT/PDGFRA mutants•KIT/PDGFRA inhibitor of all known activation loop mutations•In drug-resistant cancers, ripretinib blocks kinase signaling and tumor growth•Circulating tumor DNA data confirm broad inhibition of mutant KIT in GIST patients
To overcome the often multi-subclonal mutations in treatment-resistant GIST, Smith et al. design ripretinib, which targets a broad spectrum of KIT and PDGFRA mutants. Ripretinib shows efficacy in KIT and PDGFRA mutant cancer models and reduces circulating tumor DNA mutant allele frequency in two GIST patients.
Most gastrointestinal stromal tumors (GISTs) harbor mutant KIT or platelet-derived growth factor receptor alpha (PDGFRA) kinases, which are imatinib targets. Sunitinib, which targets KIT, PDGFRs, and ...several other kinases, has demonstrated efficacy in patients with GIST after they experience imatinib failure. We evaluated the impact of primary and secondary kinase genotype on sunitinib activity.
Tumor responses were assessed radiologically in a phase I/II trial of sunitinib in 97 patients with metastatic, imatinib-resistant/intolerant GIST. KIT/PDGFRA mutational status was determined for 78 patients by using tumor specimens obtained before and after prior imatinib therapy. Kinase mutants were biochemically profiled for sunitinib and imatinib sensitivity.
Clinical benefit (partial response or stable disease for > or = 6 months) with sunitinib was observed for the three most common primary GIST genotypes: KIT exon 9 (58%), KIT exon 11 (34%), and wild-type KIT/PDGFRA (56%). Progression-free survival (PFS) was significantly longer for patients with primary KIT exon 9 mutations (P = .0005) or with a wild-type genotype (P = .0356) than for those with KIT exon 11 mutations. The same pattern was observed for overall survival (OS). PFS and OS were longer for patients with secondary KIT exon 13 or 14 mutations (which involve the KIT-adenosine triphosphate binding pocket) than for those with exon 17 or 18 mutations (which involve the KIT activation loop). Biochemical profiling studies confirmed the clinical results.
The clinical activity of sunitinib after imatinib failure is significantly influenced by both primary and secondary mutations in the predominant pathogenic kinases, which has implications for optimization of the treatment of patients with GIST.
To evaluate the activity of imatinib in treating advanced, life-threatening malignancies expressing one or more imatinib-sensitive tyrosine kinases.
This was a phase II, open-label, single arm study. ...Patients > or = 15 years old with malignancies showing histologic or molecular evidence of expression/activation of imatinib-sensitive tyrosine kinases were enrolled. Patients were treated with 400 or 800 mg/d imatinib for hematologic malignancy and solid tumors, respectively. Treatment was continued until disease progression or unacceptable toxicity. The primary objective was to identify evidence of imatinib activity with tumor response as the primary end point.
One hundred eighty-six patients with 40 different malignancies were enrolled (78.5% solid tumors, 21.5% hematologic malignancies). Confirmed response occurred in 8.9% of solid tumor patients (4 complete, 9 partial) and 27.5% of hematologic malignancy patients (8 complete, 3 partial). Notable activity of imatinib was observed in only five tumor types (aggressive fibromatosis, dermatofibrosarcoma protuberans, hypereosinophilic syndrome, myeloproliferative disorders, and systemic mastocytosis). A total of 106 tumors were screened for activating mutations: five KIT mutations and no platelet-derived growth factor receptor mutations were found. One patient with systemic mastocytosis and a partial response to therapy had a novel imatinib-sensitive KIT mutation (D816T). There was no clear relationship between expression or activation of wild-type imatinib-sensitive tyrosine kinases and clinical response.
Clinical benefit was largely confined to diseases with known genomic mechanisms of activation of imatinib target kinases. Our results indicate an important role for molecular characterization of tumors to identify patients likely to benefit from imatinib treatment.
The additional information of this manuscript originally stated that the authors declare no competing interests. This statement was incorrect, and should instead have stated the following:M.C.H. has ...the following competing interests to declare: Equity interest at Molecular MD; Consulting at Molecular MD, Blueprint Medicines, Deciphera Pharmaceuticals; Expert Testimony at Novartis; Licensed patent with royalty payments at Novartis. The remaining authors have no competing interests to declare.The authors apologise for any convenience this may have caused.
To determine the potential of crenolanib, a potent inhibitor of PDGFRA, to treat malignancies driven by mutant PDGFRA.
The biochemical activity of crenolanib was compared with imatinib using a panel ...of PDGFRA-mutant kinases expressed in several different cell line models, including primary gastrointestinal stromal tumors (GIST) cells. The antiproliferative activity of crenolanib was also studied in several cell lines with PDGFRA-dependent growth.
Crenolanib was significantly more potent than imatinib in inhibiting the kinase activity of imatinib-resistant PDGFRA kinases (D842I, D842V, D842Y, DI842-843IM, and deletion I843). For example, crenolanib was 135-fold more potent than imatinib against D842V in our isogenic model system, with an IC(50) of approximately 10 nmol/L. The relative potency of crenolanib was further confirmed in BaF3 and primary GIST cells expressing PDGFRA D842V. In contrast, imatinib was at least 10-fold more potent than crenolanib in inhibiting the V561D mutation. For all other tested PDGFRA mutations, crenolanib and imatinib had comparable potency.
Crenolanib is a potent inhibitor of imatinib-resistant PDGFRA kinases associated with GIST, including the PDGFRA D842V mutation found in approximately 5% of GISTs. The spectrum of activity of crenolanib suggests that this drug is a type I inhibitor (inhibitor of activated conformation of kinase). Based in part on these results, a phase II clinical study of this agent to treat GIST with the PDGFRA D842V mutation has been initiated.
Multiplex Mutation Screening by Mass Spectrometry Beadling, Carol; Heinrich, Michael C; Warrick, Andrea ...
The Journal of molecular diagnostics : JMD,
09/2011, Letnik:
13, Številka:
5
Journal Article
Recenzirano
Odprti dostop
There is an immediate and critical need for a rapid, broad-based genotyping method that can evaluate multiple mutations simultaneously in clinical cancer specimens and identify patients most likely ...to benefit from targeted agents now in use or in late-stage clinical development. We have implemented a prospective genotyping approach to characterize the frequency and spectrum of mutations amenable to drug targeting present in urothelial, colorectal, endometrioid, and thyroid carcinomas and in melanoma. Cancer patients were enrolled in a Personalized Cancer Medicine Registry that houses both clinical information and genotyping data, and mutation screening was performed using a multiplexed assay panel with mass spectrometry–based analysis to detect 390 mutations across 30 cancer genes. Formalin fixed, paraffin-embedded specimens were evaluated from 820 Registry patients. The genes most frequently mutated across multiple cancer types were BRAF , PIK3CA , KRAS , and NRAS . Less common mutations were also observed in AKT1 , CTNNB1 , FGFR2 , FGFR3 , GNAQ , HRAS , and MAP2K1 . Notably, 48 of 77 PIK3CA -mutant cases (62%) harbored at least one additional mutation in another gene, most often KRAS . Among melanomas, only 54 of 73 BRAF mutations (74%) were the V600E substitution. These findings demonstrate the diversity and complexity of mutations in druggable targets among the different cancer types and underscore the need for a broad-spectrum, prospective genotyping approach to personalized cancer medicine.
Sorafenib has substantial clinical activity as third- or fourth-line treatment of imatinib- and sunitinib-resistant gastrointestinal stromal tumors (GIST). Because sorafenib targets both ...angiogenesis-related kinases (VEGFR) and the pathogenetic kinases found in GIST (KIT or PDGFRA), the molecular basis for sorafenib efficacy in this setting remains unknown. We sought to determine the spectrum of activity of sorafenib against different mutant kinases associated with drug-sensitive and drug-resistant GIST. We compared the activity of imatinib and sorafenib against transiently expressed mutant forms of KIT and PDGFRA, including various secondary mutations that have been identified in imatinib-resistant or sunitinib-resistant GISTs. We also examined these drugs against four GIST cell lines, three of which are imatinib resistant. In our in vitro studies, we determined that sorafenib inhibited imatinib-resistant mutations in exons encoding the ATP/drug-binding pocket and in exons encoding the activation loop, with the exception of substitutions at KIT codon D816 and PDGFRA codon 842. Notably our data indicate that sorafenib is more effective than imatinib or sunitinib for inhibiting the kinase activity of drug-resistant KIT mutants (as assessed by biochemical IC(50)). We hypothesize that a major determinant of the efficacy of sorafenib for treatment of advanced GIST is the activity of this agent against KIT or PDGFRA-mutant kinases. These results have implications for the further development of treatments for drug-resistant GIST.
Activating KIT mutations are a hallmark of human systemic mast cell disease and have also been identified in gastrointestinal stromal tumors, AML, melanoma, and seminoma. Traditionally, ...constitutively active KIT mutations have been targeted by tyrosine kinase inhibitors; however, currently available inhibitors have limited activity against resistant mutations and sustainable responses are often not achieved. Our goal has been to identify a synergistic drug combination that increases the effectiveness of KIT inhibitors. A recent study in chronic myelogenous leukemia reported enhanced anti-proliferative/pro-apoptotic effects when combining a BCR-ABL inhibitor with inhibitors targeting a novel Wnt/Ca++/Calcineurin/NFAT pathway. Based on shared downstream signaling mechanisms between BCR-ABL and KIT kinases we hypothesized that a similar combination would work to target KIT mutant kinases.
We found that combining a KIT inhibitor with a calcineurin phosphatase inhibitor led to a synergistic decrease (average combination index (CI) values less than 1.0) in cell viability as well as an increase in apoptosis in KIT-mutant cell lines including six mastocytosis cell lines. For example, we found that 1uM of cyclosporine A (CSA) plus 20nM of dasatinib in the p815 cell line consistently reduced cell viability by 50% compared to dasatinib alone. This decrease in cell viability corresponded to a 40 fold increase in capsase 3/7 activity (Figure 1). Similar results were obtained using the other five mastocytosis cell lines. Display omitted
The activity of NFAT transcription factors is known to be regulated by the phosphatase activity of calcineurin. To confirm our results were calcineurin- and NFAT-dependent, we repeated the previous combination experiments with the NFAT specific inhibitors, rocaglamide and tributylhexadecylphosphonium bromide (THPB). We observed a synergistic decrease (average CI value 0.81 for rocaglamide and 0.43 for THPB) in cell viability and increase in apoptosis when combining these NFAT-specific and KIT kinase inhibitors, indicating NFAT as an important mediator of the observed synergy. Next we characterized NFAT in KIT mutant mast cells and found constitutive NFAT activation in each cell line, as indicated by nuclear localization and dephosphorylation under unstimulated conditions. NFAT-dependent transcriptional activity, as measured by a luciferase reporter system, decreased following treatment with either calcineurin or NFAT inhibitors. Unexpectedly, NFAT-dependent transcriptional activity also decreased following treatment with a number of KIT inhibitors. However, only calcineurin inhibitors and not KIT inhibitors, completely blocked NFAT activation following calcium influx, suggesting a unique mechanism by which KIT inhibition affects NFAT-dependent transcriptional activity. To determine downstream effectors of the observed synergy, we measured changes in RNA expression following mono- and combination therapy. RNA-Seq revealed synergistic decreases in a number of genes including members of the JAK-STAT and MAPK pathways, which are druggable targets downstream of this transcriptional program. Further validation studies of these targets are currently underway and will be reported later.
The constitutive activation of NFAT has not been previously reported in KIT mutant cell lines. Further, our data suggest a point of convergence between KIT and NFAT signaling pathways, possibly through the interaction of NFAT with a DNA binding partner. Importantly, our results indicate that combination therapy using an NFAT inhibitor and a KIT kinase inhibitor results in synergistic killing of KIT mutant mast cells. However, existing calcineurin phosphatase inhibitors such as CSA have known chronic long-term toxicities, including immunosuppression. To identify alternative drug targets we used RNA-Seq and found that simultaneous calcineurin and KIT inhibition modulated members of the JAK-STAT and MAPK pathways. Preliminary data indicates that targeting these pathways in combination with dasatinib achieves the same synergistic effect on KIT mutant cancer cells. We are currently testing the potential of these alternative combination therapies for the treatment of advanced mast cell disease.
Heinrich: Onyx: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; MolecularMD: Consultancy, Equity Ownership; AROG: Research Funding.
There is an immediate and critical need for a rapid, broad-based genotyping method that can evaluate multiple mutations simultaneously in clinical cancer specimens and identify patients most likely ...to benefit from targeted agents now in use or in late-stage clinical development. We have implemented a prospective genotyping approach to characterize the frequency and spectrum of mutations amenable to drug targeting present in urothelial, colorectal, endometrioid, and thyroid carcinomas and in melanoma. Cancer patients were enrolled in a Personalized Cancer Medicine Registry that houses both clinical information and genotyping data, and mutation screening was performed using a multiplexed assay panel with mass spectrometry-based analysis to detect 390 mutations across 30 cancer genes. Formalin fixed, paraffin-embedded specimens were evaluated from 820 Registry patients. The genes most frequently mutated across multiple cancer types were BRAF, PIK3CA, KRAS, and NRAS. Less common mutations were also observed in AKT1, CTNNB1, FGFR2, FGFR3, GNAQ, HRAS, and MAP2K1. Notably, 48 of 77 PIK3CA-mutant cases (62%) harbored at least one additional mutation in another gene, most often KRAS. Among melanomas, only 54 of 73 BRAF mutations (74%) were the V600E substitution. These findings demonstrate the diversity and complexity of mutations in druggable targets among the different cancer types and underscore the need for a broad-spectrum, prospective genotyping approach to personalized cancer medicine.
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