KIT ATP-Binding Pocket/Activation Loop Mutations in GI Stromal Tumor: Emerging Mechanisms of Kinase Inhibitor Escape.

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.

Plasma Sequencing for Patients with GIST-Limitations and Opportunities in an Academic Setting.

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.

Resistance to Avapritinib in PDGFRA-Driven GIST Is Caused by Secondary Mutations in the PDGFRA Kinase Domain.

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.

Inhibition of osimertinib-resistant epidermal growth factor receptor EGFR-T790M/C797S.

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.

KIT-Dependent and KIT-Independent Genomic Heterogeneity of Resistance in Gastrointestinal Stromal Tumors - TORC1/2 Inhibition as Salvage Strategy.

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.

Genomic aberrations in cell cycle genes predict progression of KIT-mutant gastrointestinal stromal tumors (GISTs).

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.

Inhibitors to Overcome Secondary Mutations in the Stem Cell Factor Receptor KIT.

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.

Indazole-Based Covalent Inhibitors To Target Drug-Resistant Epidermal Growth Factor Receptor.

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.

Insight into the Inhibition of Drug-Resistant Mutants of the Receptor Tyrosine Kinase EGFR.

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.