RESUMO
PURPOSE: Imatinib resistance in GI stromal tumors (GISTs) is primarily caused by secondary KIT mutations, and clonal heterogeneity of these secondary mutations represents a major treatment obstacle. KIT inhibitors used after imatinib have clinical activity, albeit with limited benefit. Ripretinib is a potent inhibitor of secondary KIT mutations in the activation loop (AL). However, clinical benefit in fourth line remains limited and the molecular mechanisms of ripretinib resistance are largely unknown. PATIENTS AND METHODS: Progressing lesions of 25 patients with GISTs refractory to ripretinib were sequenced for KIT resistance mutations. Resistant genotypes were validated and characterized using novel cell line models and in silico modeling. RESULTS: GISTs progressing on ripretinib were enriched for secondary mutations in the ATP-binding pocket (AP), which frequently occur in cis with preexisting AL mutations, resulting in highly resistant AP/AL genotypes. AP/AL mutations were rarely observed in a cohort of progressing GIST samples from the preripretinib era but represented 50% of secondary KIT mutations in patients with tumors resistant to ripretinib. In GIST cell lines harboring secondary KIT AL mutations, the sole genomic escape mechanisms during ripretinib drug selection were AP/AL mutations. Ripretinib and sunitinib synergize against mixed clones with secondary AP or AL mutants but do not suppress clones with AP/AL genotypes. CONCLUSION: Our findings underscore that KIT remains the central oncogenic driver even in late lines of GIST therapy. KIT-inhibitor combinations may suppress resistance because of secondary KIT mutations. However, the emergence of KIT AP/AL mutations after ripretinib treatment calls for new strategies in the development of next-generation KIT inhibitors.
Assuntos
Antineoplásicos , Neoplasias Gastrointestinais , Tumores do Estroma Gastrointestinal , Naftiridinas , Proteínas Proto-Oncogênicas c-kit , Ureia , Humanos , Trifosfato de Adenosina/metabolismo , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Mesilato de Imatinib/uso terapêutico , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/genética , Ureia/análogos & derivadosRESUMO
Circulating tumor DNA (ctDNA) from circulating free DNA (cfDNA) in GIST is of interest for the detection of heterogeneous resistance mutations and treatment monitoring. However, methodologies for use in a local setting are not standardized and are error-prone and difficult to interpret. We established a workflow to evaluate routine tumor tissue NGS (Illumina-based next generation sequencing) panels and pipelines for ctDNA sequencing in an academic setting. Regular blood collection (Sarstedt) EDTA tubes were sufficient for direct processing whereas specialized tubes (STRECK) were better for transportation. Mutation detection rate was higher in automatically extracted (AE) than manually extracted (ME) samples. Sensitivity and specificity for specific mutation detection was higher using digital droplet (dd)PCR compared to NGS. In a retrospective analysis of NGS and clinical data (133 samples from 38 patients), cfDNA concentration correlated with tumor load and mutation detection. A clinical routine pipeline and a novel research pipeline yielded different results, but known and resistance-mediating mutations were detected by both and correlated with the resistance spectrum of TKIs used. In conclusion, NGS routine panel analysis was not sensitive and specific enough to replace solid biopsies in GIST. However, more precise methods (hybridization capture NGS, ddPCR) may comprise important research tools to investigate resistance. Future clinical trials need to compare methodology and protocols.
RESUMO
Gastrointestinal stromal tumors (GIST) harboring activating mutations of PDGFRA respond to imatinib, with the notable exception of the most common mutation, D842V. Avapritinib is a novel, potent KIT/PDGFRA inhibitor with substantial clinical activity in patients with the D842V genotype. To date, only a minority of PDGFRA-mutant patients treated with avapritinib have developed secondary resistance. Tumor and plasma biopsies in 6 of 7 patients with PDGFRA primary mutations who progressed on avapritinib or imatinib had secondary resistance mutations within PDGFRA exons 13, 14, and 15 that interfere with avapritinib binding. Secondary PDGFRA mutations causing V658A, N659K, Y676C, and G680R substitutions were found in 2 or more patients each, representing recurrent mechanisms of PDGFRA GIST drug resistance. Notably, most PDGFRA-mutant GISTs refractory to avapritinib remain dependent on the PDGFRA oncogenic signal. Inhibitors that target PDGFRA protein stability or inhibition of PDGFRA-dependent signaling pathways may overcome avapritinib resistance. SIGNIFICANCE: Here, we provide the first description of avapritinib resistance mechanisms in PDGFRA-mutant GIST.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Tumores do Estroma Gastrointestinal , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Humanos , Mutação , Pirazóis , Pirróis , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , TriazinasRESUMO
Precision medicine has revolutionized the treatment of patients in EGFR driven non-small cell lung cancer (NSCLC). Targeted drugs show high response rates in genetically defined subsets of cancer patients and markedly increase their progression-free survival as compared to conventional chemotherapy. However, recurrent acquired drug resistance limits the success of targeted drugs in long-term treatment and requires the constant development of novel efficient inhibitors of drug resistant cancer subtypes. Herein, we present covalent inhibitors of the drug resistant gatekeeper mutant EGFR-L858R/T790M based on the pyrrolopyrimidine scaffold. Biochemical and cellular characterization, as well as kinase selectivity profiling and western blot analysis, substantiate our approach. Moreover, the developed compounds possess high activity against multi drug resistant EGFR-L858R/T790M/C797S in biochemical assays due to their highly reversible binding character, that was revealed by characterization of the binding kinetics. In addition, we present the first X-ray crystal structures of covalent inhibitors in complex with C797S-mutated EGFR which provide detailed insight into their binding mode.
RESUMO
Sporadic gastrointestinal stromal tumors (GIST), characterized by activating mutations of KIT or PDGFRA, favorably respond to KIT inhibitory treatment but eventually become resistant. The development of effective salvage treatments is complicated by the heterogeneity of KIT secondary resistance mutations. Recently, additional mutations that independently activate KIT-downstream signaling have been found in pretreated patients-adding further complexity to the scope of resistance. We collected genotyping data for KIT from tumor samples of pretreated GIST, providing a representative overview on the distribution and incidence of secondary KIT mutations (n = 80). Analyzing next-generation sequencing data of 109 GIST, we found that 18% carried mutations in KIT-downstream signaling intermediates (NF1/2, PTEN, RAS, PIK3CA, TSC1/2, AKT, BRAF) potentially mediating resistance to KIT inhibitors. Notably, we found no apparent other driver mutations in refractory cases that were analyzed by whole exome/genome sequencing (13/109). Using CRISPR/Cas9 methods, we generated a panel of GIST cell lines harboring mutations in KIT, PTEN, KRAS, NF1, and TSC2 We utilized this panel to evaluate sapanisertib, a novel mTOR kinase inhibitor, as a salvage strategy. Sapanisertib had potent antiproliferative effects in all cell lines, including those with KIT-downstream mutations. Combinations with KIT or MEK inhibitors completely abrogated GIST-survival signaling and displayed synergistic effects. Our isogenic cell line panel closely approximates the genetic heterogeneity of resistance observed in heavily pretreated patients with GIST. With the clinical development of novel, broad spectrum KIT inhibitors, emergence of non-KIT-related resistance may require combination treatments with inhibitors of KIT-downstream signaling such as mTOR or MEK.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Tumores do Estroma Gastrointestinal/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Redes Reguladoras de Genes/efeitos dos fármacos , Heterogeneidade Genética , Humanos , Terapia de Salvação , Análise de Sequência de DNA , Transdução de SinaisRESUMO
BACKGROUND: Activating mutations of the receptor tyrosine kinase KIT are early events in the development of most gastrointestinal stromal tumors (GISTs). Although GISTs generally remain dependent on oncogenic KIT during tumor progression, KIT mutations alone are insufficient to induce malignant behavior. This is evidenced by KIT-mutant micro-GISTs, which are present in up to one-third of normal individuals, but virtually never progress to malignancy. METHODS: We performed whole exome sequencing on 29 tumors obtained from 21 patients with high grade or metastatic KIT-mutant GIST (discovery set). We further validated the frequency and potential prognostic significance of aberrations in CDKN2A/B, RB1, and TP53 in an independent series of 71 patients with primary GIST (validation set). RESULTS: Using whole exome sequencing we found significant enrichment of genomic aberrations in cell cycle-associated genes (Fisher's Exact p = 0.001), most commonly affecting CDKN2A/B, RB1, and TP53 in our discovery set. We found a low mutational tumor burden in these 29 advanced GIST samples, a finding with significant implications for the development of immunotherapy for GIST. In addition, we found mutation of spliceosome genes in a minority of cases, implicating dysregulation of splicing as a potential cancer promoting mechanism in GIST. We next assessed the prognostic significance of CDKN2A, RB1 or TP53 mutation/copy loss in an independent cohort of 71 patients with primary GIST. Genetic events (mutation, deletion, and/or LOH) involving at least one of the three genes examined were found in 17% of the very low-risk, 36% of the low-risk, 42% of the intermediate risk, 67% of the high-risk/low mitotic-count, and in 86% of the high-risk/high mitotic-count group. The presence of cell cycle-related events was associated with a significantly shorter relapse-free survival (median 67 months versus not reached; p < 0.0001) and overall survival (Log Rank, p = 0.042). CONCLUSION: Our results demonstrate that genomic events targeting cell cycle-related genes are associated with GIST progression to malignant disease. Based on this data, we propose a model for molecular pathogenesis of malignant GIST.
RESUMO
In modern cancer therapy, the use of small organic molecules against receptor tyrosine kinases (RTKs) has been shown to be a valuable strategy. The association of cancer cells with dysregulated signaling pathways linked to RTKs represents a key element in targeted cancer therapies. The tyrosine kinase mast/stem cell growth factor receptor KIT is an example of a clinically relevant RTK. KIT is targeted for cancer therapy in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). However, acquired resistance mutations within the catalytic domain decrease the efficacy of this strategy and are the most common cause of failed therapy. Here, we present the structure-based design and synthesis of novel type II kinase inhibitors to overcome these mutations in KIT. Biochemical and cellular studies revealed promising molecules for the inhibition of mutated KIT.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mutação , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Gastrointestinais/tratamento farmacológico , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas Proto-Oncogênicas c-kit/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
The specific targeting of oncogenic mutant epidermal growth factor receptor (EGFR) is a breakthrough in targeted cancer therapy and marks a drastic change in the treatment of non-small cell lung cancer (NSCLC). The recurrent emergence of resistance to these targeted drugs requires the development of novel chemical entities that efficiently inhibit drug-resistant EGFR. Herein, we report the optimization process for a hit compound that has emerged from a phenotypic screen resulting in indazole-based compounds. These inhibitors are conformationally less flexible, target gatekeeper mutated drug-resistant EGFR-L858R/T790M, and covalently alkylate Cys797. Western blot analysis, as well as characterization of the binding kinetics and kinase selectivity profiling, substantiates our approach of targeting drug-resistant EGFR-L858R/T790M with inhibitors incorporating the indazole as hinge binder.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/farmacocinética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/genética , Humanos , Indazóis , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Simulação de Acoplamento Molecular , Mutação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Targeting acquired drug resistance represents the major challenge in the treatment of EGFR-driven non-small-cell lung cancer (NSCLC). Herein, we describe the structure-based design, synthesis, and biological evaluation of a novel class of covalent EGFR inhibitors that exhibit excellent inhibition of EGFR-mutant drug-resistant cells. Protein X-ray crystallography combined with detailed kinetic studies led to a deeper understanding of the mode of inhibition of EGFR-T790M and provided insight into the key principles for effective inhibition of the recently discovered tertiary mutation at EGFR-C797S.
Assuntos
Receptores ErbB/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Sítios de Ligação , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Humanos , Cinética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Simulação de Acoplamento Molecular , Fosforilação , Mutação Puntual , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologiaRESUMO
Gastrointestinal stromal tumors (GIST) exhibit a strong oncogenic dependency on KIT and KIT inhibitors confer long lasting disease stabilization in the majority of patients. Nonetheless, KIT inhibition alone does not cure GIST as a subset of GIST cells evade apoptosis and eventually develop resistance. Inhibitors of Apoptosis Proteins (IAPs) may confer resistance to drug-induced apoptosis. We observed that the mRNA and protein of IAPs XIAP (BIRC4) and survivin (BIRC5) were highly expressed in primary GIST tumors and cell line models. Amplification of the respective gene loci (BIRC2, BIRC3, BIRC4, BIRC5) was detected in 47% of GIST studied by SNP arrays. Whole exome analyses revealed a mutation of SMAC(DIABLO) in a heavily pretreated patient. Both, survivin (rank 62-92/11.194 tested proteins) and XIAP (rank 106-557/11.194) were found to be essential proteins for survival in a synthetic lethality screen. Expression of XIAP and survivin decreased upon KIT inhibition and may play a role in KIT-regulated pro-survival signaling. SMAC-mimetic treatment with LCL161 and TL32711 reduced cIAP1 and XIAP expression. Survivin inhibitor YM155 lead to transcriptional repression of BIRC5/survivin (YM155) and induced apoptosis. Combinational treatment with KIT inhibitors (imatinib, regorafenib) enhanced the proapoptotic effect. These findings support the combination of KIT inhibition with IAP antagonists in GIST.
Assuntos
Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Mesilato de Imatinib/uso terapêutico , Proteínas Inibidoras de Apoptose/genética , Inibidores de Proteínas Quinases/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation-dependent signaling of protein kinases. Herein the structure-based design, synthesis, and evaluation of pleckstrin homology (PH) domain-dependent covalent-allosteric inhibitors (CAIs) of the kinase Akt is reported. These inhibitors bind covalently to a distinct cysteine of the kinase and thereby stabilize the inactive kinase conformation. These modulators exhibit high potency and selectivity, and represent an innovative approach for chemical biology and medicinal chemistry research.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Ligação Competitiva , Humanos , Modelos MolecularesRESUMO
Positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) is frequently used for visualizing gastrointestinal stromal tumors (GIST), which are highly glucose-avid tumors. Dramatic metabolic responses following imatinib treatment indicate a high, KIT-dependent glucose turnover which has been particularly helpful for predicting tumor response to imatinib. The glucose analogue 2-deoxyglucose (2DG) inhibits glucose metabolism in cancer cells that depend on aerobic glycolysis for ATP production. We show that 2DG inhibits proliferation in both imatinib-sensitive and imatinib-resistant GIST cell lines at levels that can be achieved clinically. KIT-negative GIST48B have 3-14-fold higher IC50 levels than KIT-positive GIST cells indicating that oncogenic KIT may sensitize cells to 2DG. GIST sensitivity to 2DG is increased in low-glucose media (110 mg/dl). 2DG leads to dose- and glucose dependent inhibition of KIT glycosylation with resultant reduction of membrane-bound KIT, inhibition of KIT-phosphorylation and inactivation of KIT-dependent signaling intermediates. In contrast to imatinib, 2DG caused ER-stress and elicited the unfolded protein response (UPR). Mannose but not pyruvate rescued GIST cells from 2DG-induced growth arrest, suggesting that loss of KIT integrity is the predominant effect of 2DG in GIST. Additive anti-tumoral effects were seen with imatinib and BH3-mimetics. Our data provide the first evidence that modulation of the glucose-metabolism by 2DG may have a disease-specific effect and may be therapeutically useful in GIST.
Assuntos
Compostos de Anilina/farmacologia , Apoptose/efeitos dos fármacos , Desoxiglucose/farmacologia , Tumores do Estroma Gastrointestinal/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Compostos de Anilina/agonistas , Animais , Apoptose/genética , Linhagem Celular Tumoral , Desoxiglucose/agonistas , Sinergismo Farmacológico , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Glicosilação/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-kit/genética , Transdução de Sinais/genética , Sulfonamidas/agonistasRESUMO
Acquired resistance to imatinib is a significant problem for the clinical management of gastrointestinal stromal tumor (GIST) patients, and second-line small molecules have shown limited efficacy in this setting. We have recently demonstrated that a monoclonal antibody targeting KIT could potentially bypass imatinib resistance in preclinical models of GIST.
RESUMO
Gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT or platelet-derived growth factor receptor α(PDGFRA), which can be therapeutically targeted by tyrosine kinase inhibitors (TKI) such as imatinib. Despite long-lasting responses, most patients eventually progress after TKI therapy. The calcium-dependent chloride channel DOG1 (ANO1/TMEM16A), which is strongly and specifically expressed in GIST, is used as a diagnostic marker to differentiate GIST from other sarcomas. Here, we report that loss of DOG1 expression occurs together with loss of KIT expression in a subset of GIST resistant to KIT inhibitors, and we illustrate the functional role of DOG1 in tumor growth, KIT expression, and imatinib response. Although DOG1 is a crucial regulator of chloride balance in GIST cells, we found that RNAi-mediated silencing or pharmacologic inhibition of DOG1 did not alter cell growth or KIT signaling in vitro. In contrast, DOG1 silencing delayed the growth of GIST xenografts in vivo. Expression profiling of explanted tumors after DOG1 blockade revealed a strong upregulation in the expression of insulin-like growth factor-binding protein 5 (IGFBP5), a potent antiangiogenic factor implicated in tumor suppression. Similar results were obtained after selection of imatinib-resistant DOG1- and KIT-negative cells derived from parental DOG1 and KIT-positive GIST cells, where a 5,000-fold increase in IGFBP5 mRNA transcripts were documented. In summary, our findings establish the oncogenic activity of DOG1 in GIST involving modulation of IGF/IGF receptor signaling in the tumor microenvironment through the antiangiogenic factor IGFBP5.
Assuntos
Canais de Cloreto/genética , Tumores do Estroma Gastrointestinal/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteínas de Neoplasias/genética , Animais , Anoctamina-1 , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/metabolismo , Tumores do Estroma Gastrointestinal/metabolismo , Tumores do Estroma Gastrointestinal/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mesilato de Imatinib , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Mutação , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Ácido Niflúmico/farmacologia , Nitrobenzoatos/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the gastrointestinal tract and arises from the interstitial cells of Cajal. It is characterized by expression of the receptor tyrosine kinase CD117 (KIT). In 70-80% of GIST cases, oncogenic mutations in KIT are present, leading to constitutive activation of the receptor, which drives the proliferation of these tumors. Treatment of GIST with imatinib, a small-molecule tyrosine kinase inhibitor, inhibits KIT-mediated signaling and initially results in disease control in 70-85% of patients with KIT-positive GIST. However, the vast majority of patients eventually develop resistance to imatinib treatment, leading to disease progression and posing a significant challenge in the clinical management of these tumors. Here, we show that an anti-KIT monoclonal antibody (mAb), SR1, is able to slow the growth of three human GIST cell lines in vitro. Importantly, these reductions in cell growth were equivalent between imatinib-resistant and imatinib-sensitive GIST cell lines. Treatment of GIST cell lines with SR1 reduces cell-surface KIT expression, suggesting that mAb-induced KIT down-regulation may be a mechanism by which SR1 inhibits GIST growth. Furthermore, we also show that SR1 treatment enhances phagocytosis of GIST cells by macrophages, indicating that treatment with SR1 may enhance immune cell-mediated tumor clearance. Finally, using two xenotransplantation models of imatinib-sensitive and imatinib-resistant GIST, we demonstrate that SR1 is able to strongly inhibit tumor growth in vivo. These results suggest that treatment with mAbs targeting KIT may represent an alternative, or complementary, approach for treating GIST.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/patologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/imunologia , Pirimidinas/uso terapêutico , Animais , Anticorpos Monoclonais/farmacologia , Benzamidas , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fagocitose/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The KIT-inhibitor imatinib mesylate (IM) has greatly improved the treatment of metastatic gastrointestinal stromal tumors (GIST). IM exhibits strong antiproliferative effects but fails to induce sufficient levels of apoptosis resulting in low pathologic complete remission rates and a high rate of secondary progression in the metastatic setting. Upregulation of p53 by MDM2 inhibitors has been shown to induce apoptosis in p53 wildtype tumors. Analyzing a series of 62 mostly untreated, localized and metastatic GIST we detected a low rate (3%) of inactivating p53 mutations, thus providing a rationale for further exploration of p53-directed therapeutic strategies. To this end, we studied nutlin-3, an inhibitor of the p53 antagonist MDM2, and RITA, a putative p53 activator, in GIST cell lines. Nutlin-3 effectively induced p53 at therapeutically relevant levels, which resulted in moderate antiproliferative effects and cell cycle arrest in p53 wildtype GIST cell lines GIST430, GIST48 and GIST48B. P53 reactivation substantially improved the apoptotic response after effective KIT inhibition with sunitinib and 17-AAG in IM-resistant cell lines. The commonly used imatinib-sensitive cell lines GIST882 and GIST-T1 were shown to harbor defective p53 and therefore failed to respond to nutlin-3 treatment. RITA induced p53 in GIST48B, followed by antiproliferative effects and a strong induction of apoptosis. Surprisingly, GIST-T1 was also highly sensitive to RITA despite lacking functional p53. This suggested a more complex, p53-independent mechanism of action for the latter compound. No antagonistic effects from p53-activating drugs were seen with any drug combination. Our data provide first evidence that modulation of the MDM2/p53 pathway may be therapeutically useful to improve the apoptotic response of KIT-inhibitory drugs in the treatment of naïve GIST, with p53 mutation status being a predictive factor of response.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/metabolismo , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Supressora de Tumor p53/genética , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Benzamidas , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias Gastrointestinais/tratamento farmacológico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Mesilato de Imatinib , Imidazóis/farmacologia , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Piperazinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Pirimidinas/farmacologia , Análise de Sequência de DNA , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Soft-tissue sarcomas are a group of malignant tumours whose clinical management is complicated by morphological heterogeneity, inadequate molecular markers and limited therapeutic options. Receptor tyrosine kinases (RTKs) have been shown to play important roles in cancer, both as therapeutic targets and as prognostic biomarkers. An initial screen of gene expression data for 48 RTKs in 148 sarcomas showed that ROR2 was expressed in a subset of leiomyosarcoma (LMS), gastrointestinal stromal tumour (GIST) and desmoid-type fibromatosis (DTF). This was further confirmed by immunohistochemistry (IHC) on 573 tissue samples from 59 sarcoma tumour types. Here we provide evidence that ROR2 expression plays a role in the invasive abilities of LMS and GIST cells in vitro. We also show that knockdown of ROR2 significantly reduces tumour mass in vivo using a xenotransplantation model of LMS. Lastly, we show that ROR2 expression, as measured by IHC, predicts poor clinical outcome in patients with LMS and GIST, although it was not independent of other clinico-pathological features in a multivariate analysis, and that ROR2 expression is maintained between primary tumours and their metastases. Together, these results show that ROR2 is a useful prognostic indicator in the clinical management of these soft-tissue sarcomas and may represent a novel therapeutic target.
Assuntos
Biomarcadores Tumorais/metabolismo , Tumores do Estroma Gastrointestinal/enzimologia , Leiomiossarcoma/enzimologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Neoplasias Uterinas/enzimologia , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Movimento Celular , Intervalo Livre de Doença , Feminino , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/mortalidade , Tumores do Estroma Gastrointestinal/patologia , Tumores do Estroma Gastrointestinal/terapia , Perfilação da Expressão Gênica/métodos , Terapia Genética , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Leiomiossarcoma/genética , Leiomiossarcoma/mortalidade , Leiomiossarcoma/patologia , Leiomiossarcoma/terapia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise Multivariada , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Modelos de Riscos Proporcionais , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Fatores de Tempo , Transfecção , Carga Tumoral , Neoplasias Uterinas/genética , Neoplasias Uterinas/mortalidade , Neoplasias Uterinas/patologia , Neoplasias Uterinas/terapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gastrointestinal stromal tumors (GIST) are caused by activating mutations in the KIT or PDGFRA receptor tyrosine kinase genes. Although >85% of GIST patients treated with the small-molecule inhibitor imatinib mesylate (Gleevec) achieve disease stabilization, complete remissions are rare and a substantial proportion of patients develop resistance to imatinib over time. Upregulation of soluble, non-chromatin-bound histone H2AX has an important role in imatinib-induced apoptosis of GIST cells. Additionally, H2AX levels in untreated GIST are maintained at low levels by a pathway that involves KIT, phosphoinositide 3-kinase, and the ubiquitin-proteasome system. In this study, we asked whether bortezomib-mediated inhibition of the ubiquitin-proteasome machinery could lead to upregulation of histone H2AX and GIST cell death. We show that bortezomib rapidly triggers apoptosis in GIST cells through a combination of mechanisms involving H2AX upregulation and loss of KIT protein expression. Downregulation of KIT transcription was an underlying mechanism for bortezomib-mediated inhibition of KIT expression. In contrast, the nuclear factor-kappaB signaling pathway did not seem to play a major role in bortezomib-induced GIST cell death. Significantly, we found that bortezomib would induce apoptosis in two imatinib-resistant GIST cell lines as well as a short-term culture established from a primary imatinib-resistant GIST. Collectively, our results provide a rationale to test the efficacy of bortezomib in GIST patients with imatinib-sensitive or -resistant tumors.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Tumores do Estroma Gastrointestinal/metabolismo , Pirazinas/farmacologia , Bortezomib , Linhagem Celular Tumoral , Citometria de Fluxo , Imunofluorescência , Tumores do Estroma Gastrointestinal/genética , Expressão Gênica/efeitos dos fármacos , Histonas/biossíntese , Histonas/efeitos dos fármacos , Humanos , Imunoprecipitação , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Proto-Oncogênicas c-kit/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Inadequate response to cisplatin-based chemotherapy is associated with poor prognosis in patients with advanced malignant testicular germ cell tumours (TGCTs), especially of the nonseminomatous type. Novel chemotherapeutic agents have failed so far to significantly improve the outcome of such patients. The majority of these tumours express low levels of p53, and TP53 mutations are rarely observed. Murine double minute 2 (Mdm2) inhibitors enhance apoptosis in tumours harbouring wild-type p53. OBJECTIVE: We sought to investigate the potential therapeutic value of Mdm2 in TGCT-derived cell lines with the histology of nonseminoma. DESIGN, SETTING, AND PARTICIPANTS: The Mdm2 inhibitor nutlin-3 was evaluated alone and in combination with cisplatin in a panel of germ cell tumour (GCT)-derived cell lines (embryonal carcinomas, being the nonseminomatous stem-cell component) with wild-type (NT2 and 2102EP cells) and mutant (NCCIT cells) p53 status. MEASUREMENTS: Biological consequences of Mdm2 inhibition were determined by analysis of the p53 pathway, cell proliferation, and apoptosis. RESULTS AND LIMITATIONS: Nutlin-3 exhibited significant activity (IC50 2.8 µM) in NT2 and 2102EP (wild-type p53) but not in p53-mutant NCCIT cells (<10% inhibition at 10 µM). At concentrations beyond 500 nM, additive effects were seen for the combination of nutlin-3 and cisplatin in NT2 and 2102EP cells but not in NCCIT cells. This correlated with the induction of p53 and its target p21, suggesting an on-target effect of nutlin-3. Moreover, nutlin-3 (5 µM) and cisplatin (0.5 µM) additively induced caspase cleavage and apoptosis in NT2 cells and 2102-EP cells but not in p53-mutant NCCIT cells. CONCLUSIONS: These results provide strong evidence for further development of pharmacologic Mdm2 inhibition for the treatment of patients suffering from high-risk nonseminomatous TGCT with wild-type p53 status.