Off-target Resistance Mechanisms Research Articles

Real-World Genomic Profile of EGFR Second-Site Mutations and Other Osimertinib Resistance Mechanisms and Clinical Landscape of NSCLC Post-Osimertinib

IntroductionThe emergence of osimertinib as standard of care for EGFR-mutant NSCLC has renewed the need to understand and overcome drug resistance. We sought to understand the genomics and real-world treatment landscape of NSCLC with EGFR C797S and other on- and off-target resistance mechanisms. MethodsComprehensive genomic profiling (CGP) results from tissue or blood samples from 93,065 patients with NSCLC were queried for osimertinib EGFR second-site resistance mutations (ssEGFRms; C797, L718, G724, G796, L792). A real-world electronic health record–derived deidentified clinicogenomic database of patients with NSCLC undergoing CGP from approximately 280 U.S. cancer clinics was queried to assess post-osimertinib resistance and clinical treatment outcomes. ResultsA ssEGFRm was identified in 239 of 8845 (2.7%) EGFR-driven (L858R or exon 19 deletion) NSCLCs, most frequently C797 (71%), L718 (15%), and G724 (9.5%). ssEGFRms were not equally distributed across drivers; C797 and G724 changes strongly favored exon 19 deletion and L718, G796 and L792 favored L858R. Post-osimertinib CGP detected ssEGFRm in 19% of the cases (39 of 205); in paired pre-/post-osimertinib samples, on- and off-target resistance was largely mutually exclusive and observed in 24% and 27% of the cases, respectively. Of 391 patients with post-osimertinib treatment data, 62% received a chemotherapy-based regimen, whereas 25% received a targeted therapy or clinical study drug. Median real-world overall survival was 11.4 months from osimertinib progression. ConclusionsThe osimertinib resistance landscape is diverse with on-target ssEGFRm and off-target resistance detected in tissue and liquid biopsy. Post-osimertinib, patients are receiving primarily chemotherapy-based regimens with poor outcomes, and CGP at resistance may offer an opportunity to inform therapeutic development and improve treatment selection.

Selected Articles from This Issue

Despite the proven benefits of antibody-based therapies for HER2-amplified cancers, there remains an urgent need for novel therapeutic approaches to combat acquired and innate drug resistance. Here, O'Brien and colleagues demonstrate that tucatinib, a small molecule inhibitor of HER2, has selective activity in HER2-driven cancers. Tucatinib showed single agent and combined efficacy with trastuzumab and inhibitors of CDK4/6 in cell line xenograft models. The selective nature of tucatinib make it an ideal combination partner for HER2-based therapies. Data presented here provide insight into the optimal patient selection and combination strategies for the expanded clinical development of tucatinib.Head and neck squamous cell carcinoma (HNSCC) is a common cancer with limited treatment options. Recent clinical trials in patients with HRAS-mutant HNSCC have shown the efficacy of tipifarnib, a farnesyl transferase inhibitor (FTI) that blocks a required posttranslational modification of HRAS and other proteins. Here, Javaid and colleagues have validated HRAS as a key therapeutic target of tipifarnib and identified multiple signaling pathways that could cause treatment resistance. Combining FTI with an ERK inhibitor blocked compensatory signaling changes, enhancing the efficacy of FTI and showing that this combination could be a promising treatment strategy for patients with HRAS-mutant HNSCC.Pancreatic ductal adenocarcinoma (PDAC) is the most RAS-addicted of all cancers and development of anti-RAS therapies is a major priority. RRSP-DTB is an engineered biologic with specific RAS proteolytic activity. Vidimar and colleagues conducted a study to assess in vitro RAS degradation and in vivo activity of RRSP-DTB in RAS-driven PDAC models. As a single agent, RRSP-DTB effectively degraded RAS in cells and engaged RAS in mice leading to nearly complete regression of PDAC patient-derived xenografts without development of resistance. These results support further study of RRSP-DTB as an unprecedented RAS biodegrader for RAS-addicted tumors.Clinical outcomes in FLT3-mutant acute myeloid leukemia (AML) remain exceedingly poor due to FLT3 tyrosine kinase inhibitor (TKI) resistance. FF-10101 is the first covalent FLT3 inhibitor and recently completed phase I clinical evaluation. Here, Ferng and colleagues profile FF-10101 against a panel of FLT3 TKI resistance-mutations and off-target resistance mechanisms. FF-10101 suppresses all clinically described on-target mutations but not those that compromise covalent binding to FLT3. FF-10101 also exhibits activity in an in vitro model of cytokine-driven TKI resistance. Thus, FF-10101 shows the potential to benefit AML patients already treated with existing FLT3 TKIs or patients harboring certain FLT3 mutations.

Abstract P233: TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations

Abstract Fusions involving RET receptor tyrosine kinase are a common driver of tumors across different tissue types, such as lung, thyroid, colorectal, soft tissue and others. TAS0953/HM06 (hereby referred to as HM06) is a novel 2ndgeneration RET-specific inhibitor that is effective against RET solvent front (G810) and gatekeeper (V804) mutations. Here, we evaluated the efficacy of HM06 in lung and thyroid carcinomas, and soft-tissue sarcoma cell lines and PDXs derived from RET inhibitor-naive tumor samples or from tumors with acquired resistance to selpercatinib. HM06 was more effective than the RET multi-kinase inhibitors cabozantinib and vandetanib, and as effective as selpercatinib and pralsetinib in inhibiting growth of patient-derived and isogenic lung, thyroid and sarcoma cell lines (IC50=0.02-0.1 µM) harboring different RET fusions (KIF5B-RET, CCDC6-RET, TRIM33-RET, SPECCL1-RET) or activating mutations (RET C634W). Growth of non-tumor cells was up to 80-fold less sensitive to HM06 (IC50= 1.6 µM). Treatment of RET fusion-positive lung cancer cells with HM06 resulted in a dose-dependent inhibition of RET phosphorylation (Y905 and Y1062) and the downstream effectors AKT, ERK1/2, p70S6K and S6. Caspase 3/7 activity and markers of apoptosis (BIM, cleaved PARP) were induced by HM06 to a similar extent as pralsetinib and selpercatinib (dose range: 0.05-1 µM). HM06 induced changes in the core mediators of cell cycle regulation (upregulation of p27, downregulation of CCND1) and suppressed expression of MYC and ETV5. In vivo, HM06 blocked tumor growth and/or induced regression of up to 65% in seven patient-derived xenograft (PDX) models with RET fusions (five NSCLC PDXs, one sarcoma PDX and one NSCLC cell-line xenograft) to a similar extent as pralsetinib and selpercatinib. However, 6 weeks after cessation of treatment of the SPECCL1-RET-driven sarcoma PDX model, growth of tumors treated with HM06 was suppressed completely, whereas 3/5 pralsetinib-treated tumors and 1/5 selpercatinib-treated tumor regrew. Combination of HM06 and the MET inhibitor capmatinib effectively blocked growth of PDX tumors in a model that was derived from a patient sample that expressed RET fusion and METamplification, and was resistant to selpercatinib. These results suggest that HM06 may be an effective therapy for RET-driven tumors in a tissue-type agnostic manner and can effectively address common on-target and off-target resistance mechanisms such as RET G810X and V804X mutations. HM06 is currently in a phase 1 and 2 clinical trial for patients with advanced solid tumors with RET gene abnormalities (margaRET, NCT 04683250). Citation Format: Igor Odintsov, Renate I. Kurth, Kota Ishizawa, Lukas Delasos, Allan J.W. Lui, Inna Khodos, Connor J. Hagen, Qing Chang, Marissa S. Mattar, Morana Vojnic, Siddharth Kunte, Annalisa Bonifacio, Claudio Giuliano, Elisa De Stanchina, Emily Cheng, Emanuela Lovati, Marc Ladanyi, Romel Somwar. TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P233.

NTRK1 Fusions identified by non-invasive plasma next-generation sequencing (NGS) across 9 cancer types.

Activating fusions of the NTRK1, NTRK2 and NTRK3 genes are drivers of carcinogenesis and proliferation across a broad range of tumour types in both adult and paediatric patients. Recently, the FDA granted tumour-agnostic approvals of TRK inhibitors, larotrectinib and entrectinib, based on significant and durable responses in multiple primary tumour types. Unfortunately, testing rates in clinical practice remain quite low. Adding plasma next-generation sequencing of circulating tumour DNA (ctDNA) to tissue-based testing increases the detection rate of oncogenic drivers and demonstrates high concordance with tissue genotyping. However, the clinical potential of ctDNA analysis to identify NTRK fusion-positive tumours has been largely unexplored. We retrospectively reviewed a ctDNA database in advanced stage solid tumours for NTRK1 fusions. NTRK1 fusion events, with nine unique fusion partners, were identified in 37 patients. Of the cases for which clinical data were available, 44% had tissue testing for NTRK1 fusions; the NTRK1 fusion detected by ctDNA was confirmed in tissue in 88% of cases. Here, we report for the first time that minimally-invasive plasma NGS can detect NTRK fusions with a high positive predictive value. Plasma ctDNA represents a rapid, non-invasive screening method for this rare genomic target that may improve identification of patients who can benefit from TRK-targeted therapy and potentially identify subsequent on- and off-target resistance mechanisms.

CtDNA resistance landscape of lazertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI).

9601 Background: While EGFR mutant ( EGFRm) non-small cell lung cancer (NSCLC) patients usually experience improved clinical benefit with EGFR TKIs, most eventually progress. Understanding mechanisms of resistance (MoR) may allow for more personalized treatment. Lazertinib is an irreversible third generation EGFR TKI for which MoR are unknown. Obtaining sufficient tumor tissue for genotyping at progression is often difficult. Therefore, we utilized plasma ctDNA from patients treated with lazertinib to explore MoR. Methods: Plasma samples from 47 NSCLC patients in the phase 2 trial of lazertinib (NCT03046992) were collected at screening and progressive disease (PD) and underwent ctDNA NGS of 74 genes using Guarant360. All patients were positive for an EGFR Ex19del or L858R ( EGFRm) and T790M by tissue testing at screening. Acquired, nonsynonymous, characterized mutations detected in a PD sample but not in the screening sample from the respective patient were considered putative MoR, excluding aneuploidy. Patients with detectable plasma EGFRm and/or T790M at screening were evaluable. Results: ctDNA was detected in 47 (100%) screening samples and 43/45 (96%) PD samples (two failed sequencing). An EGFRm was detected in 85% of patients at screening (n = 40), 38 of which had PD ctDNA results and were included in analysis. T790M was detected in 30 patients at screening and subsequently not detected at PD in 21 of these patients, 55% of all 38 included patients. Among the ten patients with T790M detected at PD, on-target MoR were detected in 7 (18% of all included patients) including EGFR C797S (n = 3, 8%), EGFR amplification (n = 3, 8%), and EGFR T854A (n = 1, 3%). All C797S were in cis with T790M. No on-target MoR were detected in patients without T790M detected at PD. Off-target MoR were seen in 34% of patients (13/38) including mutations in PIK3CA (13%; 2 E545K, 2 E542K, 1 E81K), ERBB2 (5%; 1 D769H, 1 V777L), KRAS (3%; 1 G12C), and BRAF (3%; 1 G469A). Gene amplifications were detected in CCND1 (n = 1, 3%) , CCNE1 (n = 2, 5%) , ERBB2 (n = 1, 3%) , FGFR1 (n = 1, 3%) , MET (n = 4, 11%) , and PIK3CA (n = 1, 3%), with some patients having multiple MoR. Conclusions: The spectrum of MoR identified in this cohort of patients treated with lazertinib is similar to that reported in other third generation EGFR TKIs, but with some differences in frequencies. The most common resistance mechanisms are T790M loss and PIK3CA alterations which may address the mechanism of action. Our findings suggest putative MoR of lazertinib and show that ctDNA NGS is an effective way to identify MoR in patients progressing on targeted therapy. Clinical trial information: NCT03046992 .

Oncogenic Fusions May Be Frequently Present at Resistance of EGFR Tyrosine Kinase Inhibitors in Patients With NSCLC: A Brief Report

IntroductionDespite initial benefit, virtually all patients suffering from EGFR-mutant NSCLC experience acquired resistance to tyrosine kinase inhibitors (TKIs), driven by multiple mechanisms. Recent reports have identified oncogenic kinase fusions as off-target resistance mechanisms; however, these alterations have been rarely investigated at EGFR TKIs progression. MethodsPatients with EGFR-mutated metastatic NSCLC (N = 62) with tissue and plasma biopsies at EGFR TKI progression between January 2015 and June 2019, at a French hospital and optionally before progression, were identified from the prospective MATCH-R study (NCT02517892). Postprogression biopsy samples were analyzed for gene fusions using targeted gene panel sequencing, whole-exome sequencing, RNA sequencing, and comparative genomic hybridization array. ResultsSix gene fusions were detected in tumor progression biopsies under an EGFR TKI from 62 consecutive patients (9.7%) with EGFR-mutated advanced NSCLC. Among 31 patients progressing to first- or second-generation EGFR TKIs, one (3%) had an Eukaryotic translation initiation factor 4 gamma 2–GRB2 associated binding protein 1 (EIF4G2-GAB1) fusion. Among 31 patients progressing to the third-generation osimertinib, five (16%) presented oncogene fusions of fibroblast growth factor receptor 3–transforming acidic coiled-coil containing protein 3 (FGFR3-TACC3) (n = 2), kinesin family member 5B–Ret proto-oncogene (KIF5B-RET) (n = 1), striatin–anaplastic lymphoma kinase (STRN-ALK) (n = 1), and zinc finger DHHC-Type palmitoyltransferase 20–Thr790Met (ZDHHC20-BRAF) (n = 1) transcripts. Out of two patients that received osimertinib at first-line, one acquired an FGFR3-TACC3 fusion at progression. In all patients, fusions co-occurred with the original activating EGFR mutation; however, among four patients with an acquired T790M mutation, three (75%) lost the T790M mutation. ConclusionsOncogenic fusions at the time of EGFR TKI resistance were identified at a relatively high frequency, mainly after the third-generation TKI osimertinib. Patients progressing to EGFR TKIs may have a new opportunity for targeted therapy when oncogenic fusions are identified.

Lorlatinib Treatment Elicits Multiple On- and Off-Target Mechanisms of Resistance in ALK-Driven Cancer.

: Targeted therapy changed the standard of care in ALK-dependent tumors. However, resistance remains a major challenge. Lorlatinib is a third-generation ALK inhibitor that inhibits most ALK mutants resistant to current ALK inhibitors. In this study, we utilize lorlatinib-resistant anaplastic large cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cell lines in vitro and in vivo to investigate the acquisition of resistance and its underlying mechanisms. ALCL cells acquired compound ALK mutations G1202R/G1269A and C1156F/L1198F in vitro at high drug concentrations. ALCL xenografts selected in vivo showed recurrent N1178H (5/10 mice) and G1269A (4/10 mice) mutations. Interestingly, intracellular localization of NPM/ALKN1178H skewed toward the cytoplasm in human cells, possibly mimicking overexpression. RNA sequencing of resistant cells showed significant alteration of PI3K/AKT and RAS/MAPK pathways. Functional validation by small-molecule inhibitors confirmed the involvement of these pathways in resistance to lorlatinib. NSCLC cells exposed in vitro to lorlatinib acquired hyperactivation of EGFR, which was blocked by erlotinib to restore sensitivity to lorlatinib. In neuroblastoma, whole-exome sequencing and proteomic profiling of lorlatinib-resistant cells revealed a truncating NF1 mutation and hyperactivation of EGFR and ErbB4. These data provide an extensive characterization of resistance mechanisms that may arise in different ALK-positive cancers following lorlatinib treatment. SIGNIFICANCE: High-throughput genomic, transcriptomic, and proteomic profiling reveals various mechanisms by which multiple tumor types acquire resistance to the third-generation ALK inhibitor lorlatinib.

Abstract 1893: Activating NRAS mutations are associated with FLT3-independent resistance to the clinically active FLT3 inhibitor AC220 in vitro

Abstract Background: Clinical outcomes associated with acute myeloid leukemia (AML) have not improved substantially in decades. Activating FMS-like tyrosine kinase-3 (FLT3) mutations are detected in ∼30% of AML cases with internal tandem duplication (ITD) mutations conferring worse prognosis. Recently, the potent FLT3 inhibitor AC220 has achieved composite complete remission in 45% of patients with relapsed/refractory FLT3-mutant AML (Cortes et al, EHA 2011). Translational studies from our laboratory implicate reactivation of FLT3-ITD through kinase domain mutations in the majority AC220 relapse cases (Smith et al, ASH 2011). Given that less than half of treated patients initially achieve deep remissions on AC220, we hypothesize that off-target (non-FLT3 mediated) resistance is responsible for primary resistance to AC220, and may mediate loss of response in a subset of patients. Results: To model and identify putative mechanisms of off-target resistance to AC220 in vitro, we cultured the FLT3-ITD+ patient derived cell lines MV4;11 and Molm14 in escalating concentrations of AC220. To date, we have isolated 8 Molm14 subclones that are resistant to at least 20nM AC220, which is >60-fold the IC50 of the parental line (0.3nM), while efforts to generate resistant MV4;11 cells have repeatedly failed. Analysis of FLT3 signaling revealed persistent FLT3 phosphorylation in 5 clones in the presence of 10nM AC220. Sequencing of the FLT3 kinase domain identified acquired secondary activation loop mutations suggesting on-target acquired resistance. The remaining 3 clones demonstrated off-target resistance as evidenced by the absence of a FLT3 mutation and loss of FLT3 phosphorylation with 10nM AC220 treatment. Despite FLT3 inhibition, the clones failed to undergo apoptosis and maintained ERK phosphorylation when treated with AC220. Sequencing of K- and NRAS revealed an NRAS G12C mutation in 2 clones and an NRAS Q61K mutation in the third clone, both of which are known activating RAS mutations. The AC220 IC50 values for the G12C mutant clones were determined to be 134nM and 329nM AC220 and the Q61K mutant was one of the most resistant of all clones with an IC50 of >1000nM AC220. Further, all 3 NRAS mutant clones showed increased sensitivity to the MEK inhibitor PD0325901 compared to the parental cells and on-target resistant clones. Conclusions: Generation of 8 independent AC220-resistant AML cell lines revealed evidence for on- or off-target resistance mechanisms. We have identified oncogenic RAS as a putative mechanism of acquired AC220 off-target resistance in vitro, suggesting that pathologic activation of the RAS/ERK pathway may result in failure of clinically active FLT3 inhibitor therapy in AML patients. Assessment for activating RAS mutations in patient samples with primary and acquired resistance to AC220 and other clinically effective FLT3 inhibitors is currently ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1893. doi:1538-7445.AM2012-1893

High-frequency transposition for determining antibacterial mode of action

Connecting bacterial growth inhibitors to molecular targets at the whole-cell level is a major impediment to antibacterial development. Herein we report the design of a highly efficient and versatile bacteriophage-based mariner transposon delivery system in Staphylococcus aureus for determining inhibitor mode of action. Using bacteriophage-mediated delivery of concatameric minitransposon cassettes, we generated nonclonal transposant libraries with genome-wide insertion-site coverage in either laboratory or methicillin-resistant strain backgrounds and screened for drug resistance in situ on a single agar plate in one step. A gradient of gene-target expression levels, along with a correspondingly diverse assortment of drug-resistant phenotypes, was achieved by fitting the transposon cassette with a suite of outward-facing promoters. Using a panel of antibiotics, we demonstrate the ability to unveil not only an inhibitor's molecular target but also its route of cellular entry, efflux susceptibility and other off-target resistance mechanisms.