G1202R Mutation Research Articles

Highly sensitive and accurate detection of ALK-TKI resistance mutations by oligoribonucleotide interference-PCR (ORNi-PCR)-based methods

BackgroundPatients with anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) are treated with ALK tyrosine kinase inhibitors (TKIs). Although most patients benefit from ALK-TKIs, the development of resistance mutations is common and results in NSCLC recurrence. To identify ALK-TKI-resistant NSCLC at the early recurrent phase, highly sensitive and accurate methods for the detection of mutations are essential. ObjectiveThe aim of this study was to establish highly sensitive, accurate, cost-effective, and clinically practical methods for the detection of two frequent ALK-TKI resistance mutations, ALK G1202R and L1196M, by liquid biopsy. MethodsThe efficacy of oligoribonucleotide interference-PCR (ORNi-PCR) was examined by first optimizing experimental conditions to specifically amplify the ALK-TKI resistance mutant DNA corresponding to ALK G1202R and L1196M mutations. ORNi-PCR was then combined with droplet digital PCR (ddPCR) or real-time PCR to detect these mutations in cell-free DNA (cfDNA) extracted from NSCLC patients. ResultsORNi-PCR followed by ddPCR/real-time PCR detected 1–10 copy(s) of G1202R and L1196M DNA in model cfDNA. These mutations in patients’ cfDNA were identified using ORNi-PCR-based methods, whereas conventional ddPCR failed to detect them. ConclusionORNi-PCR followed by ddPCR/real-time PCR enables highly sensitive and accurate detection of ALK mutations by liquid biopsy. Although the clinical data are limited, our results show that these methods are potentially useful for identifying ALK-TKI-resistant NSCLC at the early recurrent phase.

Discovery of orally bioavailable ALK PROTACs based ceritinib against ALK positive cancers

Anaplastic lymphoma kinase (ALK) fusion genes promote a variety of human malignancies. Although several ALK inhibitors have significantly improved disease prognosis in patients with ALK positive cancers, the persistent emergence of acquired drug-resistant mutations remain the major problem in clinic treatment. Adoption of new therapeutic strategies such as proteolysis targeting chimera (PROTAC) to overcome drug resistance in BTK/AR-related cancers have shown promising prospect. Herein, we reported the integrate ALK PROTACs through overall optimization of linker, revealed that subtle structural differences can lead to significant activity difference, indicating the key role of conformation of PROTACs in inducing the formation of E3-PROTAC-target protein ternary complexes. A series of rigid ALK PROTACs were developed through conjugation of Ceritinib and thalidomide, orally bioavailable PROTAC 4B (F = 14.22 %) was obtained by overall optimization of molecular properties. 4B effectively induced long lasting degradation of ALK fusion proteins and strong repression of downstream pathway in Karpas 299 cells (DC50 = 119.33 nM, Dmax = 97.1 %) and showed comparable anti-proliferative activity to Ceritinib (IC50 = 3.11 ± 0.08 nM vs IC50 = 1.31 ± 0.43 nM). Furthermore, 4B significantly inhibited the growth of Karpas 299 xenografts in vivo with TGI of 49.5 % and showed superior anti-proliferative activity against G1202R mutation to Ceritinib (IC50 = 52.82 nM vs IC50 = 109.5 nM). Overall, 4B is expected to be a potential treatment for ALK-driven malignancies.

TKI type switching overcomes ROS1 L2086F in ROS1 fusion-positive cancers

ROS1 tyrosine kinase inhibitors (TKIs) are highly effective in ROS1-positive non-small cell lung cancer, but resistance remains a challenge. We investigated the activity of various TKIs against wildtype and mutant ROS1, focusing on the emerging L2086F resistance mutation. Using Ba/F3 and NIH3T3 cell models, CRISPR/Cas9-edited isogenic wildtype and mutant patient-derived cell lines, and in vivo tumor growth studies, we compared type I TKIs (crizotinib, entrectinib, taletrectinib, lorlatinib, and repotrectinib) to type II TKIs (cabozantinib and merestinib) and the type I FLT3 inhibitor gilteritinib. The ROS1 L2086F mutant kinase showed resistance to type I TKIs, while type II TKIs retained activity. Gilteritinib inhibited both wildtype and L2086F mutant ROS1 but was ineffective against the G2032R mutation. Structural analyses revealed distinct binding poses for cabozantinib and gilteritinib, explaining their efficacy against L2086F. Clinical cases demonstrated cabozantinib’s effectiveness in patients with TKI-resistant, ROS1 L2086F mutant NSCLCs. This study provides the first comprehensive report of ROS1 L2086F in the context of later-generation TKIs, including macrocyclic inhibitors. While cabozantinib effectively inhibits ROS1 L2086F, its multi-kinase inhibitor nature highlights the need for more selective and better-tolerated TKIs to overcome kinase-intrinsic resistance. Gilteritinib may offer an alternative for targeting ROS1 L2086F with distinct off-target toxicities, but further studies are required to fully evaluate its potential in this setting.

Efficacy and Safety of Taletrectinib in Chinese Patients With ROS1+ Non-Small Cell Lung Cancer: The Phase II TRUST-I Study.

Taletrectinib, a highly potent, CNS-active, ROS1 tyrosine kinase inhibitor (TKI), has demonstrated high and durable response rates, high intracranial objective response rate (ORR), prolonged progression-free survival (PFS), and activity against G2032R with a favorable safety profile. We report outcomes from the pivotal TRUST-I study (ClinicalTrials.gov identifier: NCT04395677) of taletrectinib for ROS1+ non-small cell lung cancer in China. TRUST-I evaluated TKI-naїve and crizotinib-pretreated patients. The primary end point was confirmed ORR (cORR) by independent review committee; key secondary end points included duration of response (DOR), PFS, and safety. As of November 2023, 173 patients were enrolled (median age, 55 years; 58% female; 73% never smoked; TKI naїve: n = 106; crizotinib pretreated: n = 67). In TKI-naїve patients, cORR and intracranial cORR were 91% and 88%, respectively, and 52% and 73% in crizotinib-pretreated patients. In TKI-naїve patients, median DOR and median PFS were not reached (NR) with 22.1-month and 23.5-month follow-up, respectively. In crizotinib-pretreated patients, the median DOR was 10.6 months (95% CI, 6.3 months to NR; 8.4-month follow-up), and the median PFS was 7.6 months (95% CI, 5.5 to 12.0 months; 9.7-month follow-up). Eight of 12 patients (67%) with G2032R mutations responded. The most frequent treatment-emergent adverse events (TEAEs) were increased AST (76%), diarrhea (70%), and increased ALT (68%), most of which were grade 1-2. Incidences of neurologic TEAEs were low (dizziness: 23%; dysgeusia: 10%) and mostly grade 1. Discontinuations (5%) and dose reductions (19%) due to TEAEs were low. Taletrectinib continues to show high and durable overall responses, prolonged PFS, robust activity against intracranial lesions and acquired resistance mutations including G2032R, and a favorable safety profile with a low incidence of neurologic TEAEs.

Efficacy and safety of taletrectinib in patients with advanced or metastatic ROS1+ non–small cell lung cancer: The phase 2 TRUST-I study.

8520 Background: Taletrectinib, a highly potent, next-generation, central nervous system–active, selective ROS1 tyrosine kinase inhibitor (TKI), was previously reported to provide high overall and intracranial (IC) response rates, prolonged progression-free survival (PFS), and activity against G2032R with favorable tolerability. We report updated data from the TRUST-I study (NCT04395677), the largest clinical trial to date conducted in patients living with ROS1+ NSCLC. Methods: TRUST-I, a multicenter, single-arm, open-label, pivotal phase 2 study of taletrectinib conducted in China, had 2 cohorts: TKI-naïve and TKI-pretreated. The primary endpoint was objective response rate (ORR) by Independent Review Committee (IRC) per RECIST 1.1. Secondary endpoints included duration of response (DoR), PFS, overall survival (OS), and safety. Results: As of November 2023, 173 patients were enrolled (median age: 55 y; 58% female; 73% never smoked; TKI-naïve: n = 106; TKI-pretreated: n = 67). In TKI-naïve patients, 21% had prior chemotherapy, and 17% had brain metastases; in TKI-pretreated patients, 34% had prior chemotherapy, and 42% had brain metastases. In TKI-naïve patients, the ORR was 91% (95% CI: 83, 95) and IC-ORR was 88% (7/8; 95% CI: 47, 100). In TKI-pretreated patients, the ORR was 52% (95% CI: 40, 65) and IC-ORR was 75% (12/16; 95% CI: 48, 93). In patients with G2032R mutation, 8 of 12 patients (67% [95% CI: 35, 90]) responded. DoR and PFS remain durable, with median follow-up of 23.5 months in TKI-naïve patients and 9.7 months in TKI-pretreated patients (Table). Among all patients (n = 173), the most frequent treatment-emergent adverse events (TEAEs) were AST increase (76%), diarrhea (70%), and ALT increase (68%). Rates of neurologic TEAEs were low (dizziness: 23%; dysgeusia: 10%) and mostly grade 1. Discontinuation (5%) and dose reductions (19%) due to TEAEs were low. Conclusions: With longer follow-up, taletrectinib continues to show high and durable overall responses, robust intracranial and G2032R activity, and a favorable safety profile with a low incidence of neurologic AEs. An additional ongoing pivotal phase 2 study, TRUST-II (NCT04919811), is further evaluating the efficacy and safety of taletrectinib in patients in the US, Europe, and Asia. Clinical trial information: NCT04395677 . [Table: see text]

CRISPR/Cas9-edited ROS1 + non-small cell lung cancer cell lines highlight differential drug sensitivity in 2D vs 3D cultures while reflecting established resistance profiles

IntroductionThe study of resistance-causing mutations in oncogene-driven tumors is fundamental to guide clinical decisions. Several point mutations affecting the ROS1 kinase domain have been identified in the clinical setting, but their impact requires further exploration, particularly in improved pre-clinical models. Given the scarcity of solid pre-clinical models to approach rare cancer subtypes like ROS1 + NSCLC, CRISPR/Cas9 technology allows the introduction of mutations in patient-derived cell lines for which resistant variants are difficult to obtain due to the low prevalence of cases within the clinical setting.MethodsIn the SLC34A2-ROS1 rearranged NSCLC cell line HCC78, we knocked-in through CRISPR/Cas9 technology three ROS1 drug resistance-causing mutations: G2032R, L2026M and S1986Y. Such variants are located in different functional regions of the ROS1 kinase domain, thus conferring TKI resistance through distinct mechanisms. We then performed pharmacological assays in 2D and 3D to assess the cellular response of the mutant lines to crizotinib, entrectinib, lorlatinib, repotrectinib and ceritinib. In addition, immunoblotting assays were performed in 2D-treated cell lines to determine ROS1 phosphorylation and MAP kinase pathway activity. The area over the curve (AOC) defined by the normalized growth rate (NGR_fit) dose–response curves was the variable used to quantify the cellular response towards TKIs.ResultsSpheroids derived from ROS1G2032R cells were significantly more resistant to repotrectinib (AOC fold change = − 7.33), lorlatinib (AOC fold change = − 6.17), ceritinib (AOC fold change = − 2.8) and entrectinib (AOC fold change = − 2.02) than wild type cells. The same cells cultured as a monolayer reflected the inefficacy of crizotinib (AOC fold change = − 2.35), entrectinib (AOC fold change = − 2.44) and ceritinib (AOC fold change = − 2.12) in targeting the ROS1 G2032R mutation. ROS1L2026M cells showed also remarkable resistance both in monolayer and spheroid culture compared to wild type cells, particularly against repotrectinib (spheroid AOC fold change = − 2.19) and entrectinib (spheroid AOC fold change = − 1.98). ROS1S1986Y cells were resistant only towards crizotinib in 2D (AOC fold change = − 1.86). Overall, spheroids showed an increased TKI sensitivity compared to 2D cultures, where the impact of each mutation that confers TKI resistance could be clearly distinguished. Western blotting assays qualitatively reflected the patterns of response towards TKI observed in 2D culture through the levels of phosphorylated-ROS1. However, we observed a dose–response increase of phosphorylated-Erk1/2, suggesting the involvement of the MAPK pathway in the mediation of apoptosis in HCC78 cells.ConclusionIn this study we knock-in for the first time in a ROS1 + patient-derived cell line, three different known resistance-causing mutations using CRISPR/Cas9 in the endogenous translocated ROS1 alleles. Pharmacological assays performed in 2D and 3D cell culture revealed that spheroids are more sensitive to TKIs than cells cultured as a monolayer. This direct comparison between two culture systems could be done thanks to the implementation of normalized growth rates (NGR) to uniformly quantify drug response between 2D and 3D cell culture. Overall, this study presents the added value of using spheroids and positions lorlatinib and repotrectinib as the most effective TKIs against the studied ROS1 resistance point mutations.

Repotrectinib in ROS1 Fusion–Positive Non–Small-Cell Lung Cancer

BackgroundThe early-generation ROS1 tyrosine kinase inhibitors (TKIs) that are approved for the treatment of ROS1 fusion–positive non–small-cell lung cancer (NSCLC) have antitumor activity, but resistance develops in tumors, and intracranial activity is suboptimal. Repotrectinib is a next-generation ROS1 TKI with preclinical activity against ROS1 fusion–positive cancers, including those with resistance mutations such as ROS1 G2032R.MethodsIn this registrational phase 1–2 trial, we assessed the efficacy and safety of repotrectinib in patients with advanced solid tumors, including ROS1 fusion–positive NSCLC. The primary efficacy end point in the phase 2 trial was confirmed objective response; efficacy analyses included patients from phase 1 and phase 2. Duration of response, progression-free survival, and safety were secondary end points in phase 2.ResultsOn the basis of results from the phase 1 trial, the recommended phase 2 dose of repotrectinib was 160 mg daily for 14 days, followed by 160 mg twice daily. Response occurred in 56 of the 71 patients (79%; 95% confidence interval [CI], 68 to 88) with ROS1 fusion–positive NSCLC who had not previously received a ROS1 TKI; the median duration of response was 34.1 months (95% CI, 25.6 to could not be estimated), and median progression-free survival was 35.7 months (95% CI, 27.4 to could not be estimated). Response occurred in 21 of the 56 patients (38%; 95% CI, 25 to 52) with ROS1 fusion–positive NSCLC who had previously received one ROS1 TKI and had never received chemotherapy; the median duration of response was 14.8 months (95% CI, 7.6 to could not be estimated), and median progression-free survival was 9.0 months (95% CI, 6.8 to 19.6). Ten of the 17 patients (59%; 95% CI, 33 to 82) with the ROS1 G2032R mutation had a response. A total of 426 patients received the phase 2 dose; the most common treatment-related adverse events were dizziness (in 58% of the patients), dysgeusia (in 50%), and paresthesia (in 30%), and 3% discontinued repotrectinib owing to treatment-related adverse events.ConclusionsRepotrectinib had durable clinical activity in patients with ROS1 fusion–positive NSCLC, regardless of whether they had previously received a ROS1 TKI. Adverse events were mainly of low grade and compatible with long-term administration. (Funded by Turning Point Therapeutics, a wholly owned subsidiary of Bristol Myers Squibb; TRIDENT-1 ClinicalTrials.gov number, NCT03093116.)

Repotrectinib's Clinical Benefit and Its Brain Penetration in a Patient with Meningeal Carcinomatosis from G2032R-Mutated ROS-1 Positive Non-Small Cell Lung Cancer.

In this work, we report on a clinically significant response of meningeal carcinomatosis to repotrectinib in a woman with a heavily pretreated ROS1-rearranged non-small cell lung cancer (NSCLC) that harbored the concomitant solvent front G2032R mutation. Meningeal carcinomatosis has a higher incidence in oncogene addicted NSCLC due to increased life expectancy, yet no report has ever documented the activity of repotrectinib in this context. In line with its activity, we documented the presence of the drug at potentially active concentrations in the cerebrospinal fluid. Nevertheless, the short-lived response reported by our patient highlights the importance for novel ROS1-tyrosine kinase inhibitors (TKIs) to be specifically developed to be able to penetrate the blood-brain barrier.

Detection of resistance mutations in patients with anaplastic lymphoma kinase-rearranged lung cancer through liquid biopsy.

Tyrosine kinase inhibitors (TKIs) significantly improve clinical outcomes in patients with non-small cell lung cancer due to anaplastic lymphoma kinase (ALK) gene rearrangement. However, the rate of relapse with TKIs is high owing to the development of resistance mutations during treatment. Repeated biopsies during disease progression are crucial for elucidating the molecular mechanisms underlying the development of resistance to ALK inhibitors. Analysis of cell-free DNA (cfDNA) obtained from plasma is a novel approach for tumor genotyping. In this mixed prospective and retrospective observational cohort study, we investigated the clinical feasibility of continuous quantitative monitoring of ALK-acquired mutations in plasma obtained from patients with ALK+ non-small cell lung cancer by using a highly sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay. We enrolled nine patients, including three treatment-naïve patients recently diagnosed with ALK+ non-small cell lung cancer via tissue biopsy and expected to receive ALK TKIs and six patients already receiving ALK TKIs. Plasma samples were collected from these patients every 3 months. cfDNA was extracted from 66 samples during the study period, and 10 ALK mutations were simultaneously evaluated. The numbers of samples showing the G1202R, C1156Y, G1269A, F1174L, T1151ins, and I1171T mutations were 32, 16, 5, 4, 1, and 1, respectively. The L1196M, L1152R, V1180L, and S1206Y mutations were not detected. Correlation analyses between progression-free survival and the time from treatment initiation (or treatment modification) to the detection of resistance mutations revealed that although resistance mutations may occur before a drug change becomes necessary, there is a duration during which the disease does not progress. Our findings suggest that real-time quantitative monitoring of ALK resistance mutations during the response period could provide a time course of changes while acquiring resistance mutations. This information would be beneficial for designing an appropriate treatment strategy.

HGG-12. INFANT HEMISPHERIC GLIOMA: CLINICAL AND MOLECULAR CHARACTERISTICS AND RESULTS OF TARGETED THERAPY

Abstract We analyzed an infant cohort consisting of 14 patientswith hemispheric gliomas (hHGG, n=10 and DIGG, n=4) diagnosed at D. Rogachev Center between 2017and 2022. The hHGG cohort included nine females and one male ranging in age from one week to 22 months. RTK-fusions were detected in all (ROS1 - 4, ALK - 2,NTRK1 – 2, NTRK3 - 1, and EGFR - 1). In one of thesecases, the diagnosis was made retrospectively in a patient initially diagnosed with anaplastic ependymoma who died during treatment. Out of the nine remaining hHGG patients, four (ROS1 - 3, NTRK3-1) experienced rapid progressive disease (PD) duringconventional chemotherapy and one pt (ROS1) after RT. These five pts received targeted therapy with entrectinib with initial good responses in all cases (CR-2, PR-3); one of these patients (ROS1) experienced PD after 7 months of targeted therapy and died of disease. Molecular genetic analysis after the second operationin this case revealed an acquired ROS1 G2032R mutation. Treatment-related adverse events on entrectinib therapy were detected in two patients and included bone fractures and neutrophil count decrease in one case and decrease left ventricular ejection fraction in another patient. Median duration of targeted therapy was 7 months (range, 3-33). From those pts who received only chemotherapy – 2 pts are alive with CR and 1 pt died from complication of CT. All patients with DIGG are alive without signs of progression after surgery alone. Median follow-up time is 19 months (range, 5-64). Molecular genetic analysis revealed MAPK-pathway alterations in each case (rearrangements of NTRK1-1, ROS-1, RAF1-1, BRAF-1), respectively. Patients with infant hHGG demonstrate an aggressive course on conventional therapy, especially in cases with ROS1-positive tumors. Targeted therapy may be an effective treatment option but required close monitoring for toxicity and resistance.

Discovery of imidazo[1,2-b]pyridazine macrocyclic derivatives as novel ALK inhibitors capable of combating multiple resistant mutants

Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (TKI) often loses effectiveness against non-small cell lung malignancies (NSCLCs) with ALK gene rearrangements (ALK+). 19 novel imidazo[1,2-b]pyridazine macrocyclic derivatives were designed, synthesized, and tested for their biological activities in an effort to develop ALK inhibitors that would overcome second-generation ALK-TKIs, particularly the G1202R mutation and the lorlatinib-resistant L1196M/G1202R double mutations. Of all the target substances, O-10 had the most effective enzymatic inhibitory activity, with IC50 values for ALKWT, ALKG1202R, and ALKL1196M/G1202R of 2.6, 6.4, and 23 nM, respectively. O-10, on the other hand, reduced the growth of ALK-positive Karpas299, BaF3-EML4-ALKG1202R, and BaF3-EML4-ALKL1196M/G1202R cells with IC50 values of 38, 52, and 64 nM, respectively. This was equally effective to the reference drug Repotrectinib (IC50 = 40, 164, and 208 nM). The kinase selectivity profile, liver microsome stability test and in vivo pharmacokinetic properties in SD rats of compound O-10 were further evaluated. O-10 was regarded as an effective ALK inhibitor for the treatment of mutations overall.

Abstract 4022: Preclinical intracranial activity of NVL-655 in an alectinib-resistant patient-derived model harboring EML4-ALK fusion with G1202R mutation

Abstract Introduction: NVL-655 is a brain-penetrant, ALK-selective tyrosine kinase inhibitor (TKI) designed to maintain activity against ALK and ALK mutations that confer resistance to currently approved therapies, and to avoid neurological adverse events and dose-limiting toxicities associated with TRK inhibition. Brain metastases are common in ALK-positive non-small cell lung cancer (NSCLC), with an incidence of ~40% at diagnosis and ~60% after progressive disease on the first-generation ALK TKI (crizotinib) due to its limited brain penetration. Second- (ceritinib, alectinib, brigatinib, and ensartinib) and third-generation (lorlatinib) ALK TKIs have higher intracranial responses than crizotinib but are still limited by emergence of resistance mutations, most commonly G1202R-containing single and compound mutations. We previously reported that NVL-655 has intracranial activity in a preclinical model of Ba/F3 EML4-ALK v1 G1202R/L1196M. Here we further demonstrate the intracranial activity of NVL-655 in a preclinical patient-derived model harboring EML4-ALK v3 G1202R. Methods: The YU-1077 cell line was established from a patient with ALK-positive NSCLC after relapse on alectinib and was confirmed to harbor EML4-ALK v3 G1202R alteration. ALK TKIs (crizotinib, ceritinib, alectinib, brigatinib, ensartinib, lorlatinib, and NVL-655) were profiled against YU-1077 cells in 3-day viability assays and signaling assays. YU-1077 cells were intracranially implanted in Balb/c nude mice and treated with NVL-655 orally twice daily. Brain tumor burden was monitored weekly using magnetic resonance imaging (MRI). Results: NVL-655 suppressed the viability of YU-1077 cells bearing EML4-ALK v3 G1202R (IC50 < 1 nM) with potency >10-fold that of lorlatinib. NVL-655 at ≤10 nM fully inhibited ALK signaling as measured by ALK, ERK, and S6 phosphorylation, compared to ≥100 nM of lorlatinib required to achieve similar inhibition. Other ALK TKIs tested had limited activity against YU-1077 cells in viability and signaling assays, consistent with the G1202R mutation conferring resistance to first- and second-generation ALK TKIs. In mice intracranially implanted with YU-1077 cells, MRI scans confirmed rapid tumor regression upon treatment with 0.5 and 1.5 mg/kg NVL-655. Conclusion: Patient-derived models are widely viewed as among the most disease-relevant models for evaluating new therapies and drug resistance. Using a xenograft model derived from an alectinib-relapsed patient, we showed that NVL-655 had high intracranial activity against brain tumors bearing the ALK G1202R mutation that confers resistance to multiple ALK TKIs. NVL-655 is being evaluated in a Phase 1/2 clinical trial for patients with advanced NSCLC and other solid tumors harboring ALK rearrangement or activating ALK mutation (ALKOVE-1): NCT05384626. [J. Lee and M. Yu contributed equally.] Citation Format: Jii Bum Lee, Mi Ra Yu, Mi Ran Yun, You Won Lee, Seung Yeon Oh, Eun Ji Lee, Anupong Tangpeerachaikul, Henry E. Pelish, Byoung Chul Cho. Preclinical intracranial activity of NVL-655 in an alectinib-resistant patient-derived model harboring EML4-ALK fusion with G1202R mutation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4022.

Abstract 3400: TY-2136b, a next generation ROS1/TRK/ALK inhibitor, potently inhibits kinase and cell proliferation activities of tumor cells bearing drug-dependent acquired mutations

Abstract Although tyrosine kinase inhibitors (TKIs) with activity against ALK, ROS1, or TRKA-C have significantly improved clinical benefits in patients with diverse tumors harboring ALK, ROS1, or NTRKs rearrangements, drug resistance will be developed and subsequent therapy overcoming acquired resistance remains limited. The resistance is mainly caused by the adaptive mutations evolved in the structural region of ROS1/TRK/ALK kinases, especially solvent front substitutions such as ROS1 G2032R/TRKA G595R/ALK G1202R mutations. Next-generation TKIs targeting these mutations could potentially address this unmet medical need. Currently, several inhibitors, including TPX-0005, are under development in phase I/II clinical trials. Here, we report the finding of a novel small molecule, TY-2136b, which has been identified through a systemic approach against acquired ROS1/TRK/ALK mutations. Kinase assay results suggest that TY-2136b grants similar potency to TPX-0005 inhibiting ROS1 G2032R mutant activity (IC50 1.6 nM vs 2.4 nM ), confers significantly stronger potency than LOXO-101 inhibiting TRKA activity with G595R substitution (IC50 0.8 mM vs 460.1 nM ). The cell proliferation assay results with Ba/F3 cells suggest that TY-2136b is similar potent as TPX-0005 inhibiting cell proliferation of the Ba/F3 strain expressing mutant ROS1 bearing G2032R mutation, a major resistance mutation. TY-2136b also shows potent inhibition towards ROS1, ERK and AKT phosphorylation and downstream signaling in Ba/F3-CD74-ROS1-G2032R cells. Meanwhile, TY-2136b, TPX-0005, and TRK-selective second-generation LOXO-195 inhibitors had similar activity against TRKA G595R and TRKC G623R resistance mutations, but TY-2136b was better than TPX-0005 and LOXO-195 against TRKA G595R/F589L dual mutations in vitro. In vivo studies, TY-2136b showed dose-dependent anti-tumor effect at the dose of 5, 10, and 20 mg/kg, bid, in xenograft tumor models carrying ROS1 G2032R and TRKA G595R mutation, and was more effective than Crizotinib and LOXO-101 at testing dose, and showed better efficacy than TPX-0005 in higher dose. Next, in vivo activity of TY-2136b was examined with xenograft model of KM-12 expressing TPM3-NTRK1 fusion proteins. The results show that TY-2136b and TPX-0005 demonstrate more effectiveness than LOXO-195, and the animals tolerated TY-2136b better than TPX-0005. In the ALK-G1202R xenograft model, TY-2136b showed a significant anti-tumor effect in a certain dose-dependent manner. Taken together, our preclinical data demonstrate that TY-2136b can treat cancers caused by ROS1/TRK/ALK mutations and overcome drug resistance due to acquired solvent-front mutations. Currently, TY-2136b is under first-in-human clinical investigations in the US and China. * To Whom Correspondence should be addressed to: Jun Li, Chengshan Niu and Yusheng Wu Citation Format: Chengshan Niu, Apeng Liang, Yuge Dou, Kaige Ji, Meihua Li, Yanchao Zhao, Yan Zhang, Zhongwei Guo, Aishen Gong, Mingyu Jiang, Shaoqing Chen, Xiugui Chen, Jun Li, Yusheng Wu. TY-2136b, a next generation ROS1/TRK/ALK inhibitor, potently inhibits kinase and cell proliferation activities of tumor cells bearing drug-dependent acquired mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3400.

8 Oral - Safety and preliminary clinical activity of NVL-520, a highly selective ROS1 inhibitor, in patients with advanced ROS1 fusion-positive solid tumors

8 Oral - Safety and preliminary clinical activity of NVL-520, a highly selective ROS1 inhibitor, in patients with advanced ROS1 fusion-positive solid tumors

Abstract 3336: Preclinical activity of NVL-520 in ROS1-driven cancer models with diverse fusion partners and kinase-domain mutations

Abstract The ROS1 receptor tyrosine kinase can be aberrantly activated by gene rearrangements that generate oncogenic ROS1 kinase fusions. ROS1 fusions have been detected in diverse adult and pediatric cancers including up to 2% of non-small cell lung cancers (NSCLC), 1-9% of cholangiocarcinomas, and 0.5-1% of gliomas and glioblastomas. There are over 50 known fusion partners of ROS1. Notable examples include CD74, EZR, SDC4, and SLC34A2 in NSCLC; GOPC exons 1-3 (short form or GOPC(S)) in cholangiocarcinoma; and GOPC exons 1-7 (long form or GOPC(L)) in glioblastoma. Crizotinib and entrectinib are FDA-approved ROS1 tyrosine kinase inhibitors (TKIs), but clinical emergence of ROS1 resistance mutations S1986F/Y, F2004C/I/V, L2026M, G2032R, and D2033N restricts their therapeutic utility. Due to the diversity of ROS1 fusion partners and kinase-domain mutations, development of novel ROS1 inhibitors that retain potency against fusion variants and resistance mutations would be beneficial to address these clinical needs. NVL-520 is a novel, brain-penetrant ROS1-selective inhibitor that exhibits preclinical activity against ROS1 alterations, including resistance mutations, while also sparing inhibition of TRKB. TRKB inhibition in the central nervous system has been implicated in adverse events observed with FDA-approved dual TRK/ROS1 inhibitor entrectinib and FDA-approved ALK inhibitor lorlatinib. We previously reported the activity of NVL-520 in several cell line and xenograft models bearing CD74-ROS1, EZR-ROS1, and SDC4-ROS1 fusions, with a wild-type kinase domain or a G2032R mutation. Here we report a broader comparative characterization of NVL-520 and other ROS1 inhibitors against a diverse set of ROS1 fusion partners and kinase-domain mutations. NVL-520 showed potent suppression of proliferation and/or ROS1 signaling in Ba/F3, NIH3T3, or human cancer cell lines expressing CD74-, EZR-, SLC34A2-, or GOPC(L)-ROS1 fusions. NVL-520 also retained potent inhibition of Ba/F3 cells expressing ROS1 fusions with F2004C, F2004V, G2101A, or G2032R mutations. To further demonstrate the broad activity of NVL-520, we utilized NIH3T3 cells expressing CD74- or EZR-ROS1 fusions with G2032R or F2004C mutations. NVL-520 robustly suppressed colony formation and downstream effector signaling in the ROS1 G2032R and F2004C resistance mutant context. Lastly, NVL-520 demonstrated inhibition of ROS1 signaling in a human glioblastoma cell line U118MG, which harbors the GOPC(L)-ROS1 fusion. In conclusion, the preclinical profile of NVL-520 supports its potential to address a medical need for patients with a diverse array of ROS1 fusion partners and kinase-domain mutations, both in NSCLC and in other cancers such as glioblastoma. NVL-520 is being evaluated in a Phase 1/2 clinical trial for patients with advanced ROS1-positive NSCLC or other solid tumors (NCT05118789). Citation Format: Anupong Tangpeerachaikul, Clare Keddy, Katelyn Nicholson, Monika Davare, Henry E. Pelish. Preclinical activity of NVL-520 in ROS1-driven cancer models with diverse fusion partners and kinase-domain mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3336.

First-in-human phase I results of APG-2449, a novel FAK and third-generation ALK/ ROS1 tyrosine kinase inhibitor (TKI), in patients (pts) with second-generation TKI-resistant ALK/ROS1+ non–small cell lung cancer (NSCLC) or mesothelioma.

9071 Background: Investigational agent APG-2449 is a novel, orally active FAK inhibitor and a third-generation ALK/ROS1 TKI that has shown potent activity against a range of ALK-resistant mutations, including G1202R, L1196M, V1180L, E1210K, S1206F, G1269A, F1174L, I1171S, and C1156Y in preclinical NSCLC, mesothelioma, and other solid tumor models. Methods: This dose escalation and dose expansion study evaluated APG-2449 in patients with second-generation TKI-resistant ALK/ROS1+ NSCLC or mesothelioma. APG-2449 was administered orally once daily at the assigned doses on a 28-day cycle using a “3+3” dose escalation design under fasted/fed conditions. Study aims were to assess safety/tolerability, recommended phase 2 dose (RP2D), pharmacokinetics (PK), pharmacodynamics (PD), and efficacy. Results: As of the data cutoff date of December 30, 2021, 84 pts (median age 52 [range 21-78] years; 42% female) with NSCLC or mesothelioma enrolled were treated with APG-2449 at doses ranging from 150 to 1,500 mg. PK analyses indicated an approximately dose-proportional increase in plasma exposure under fed conditions across dose levels tested. Cerebrospinal fluid PK analyses showed that APG-2449 was brain-penetrant. Low-fat meals increased APG-2449 Cmax and AUC by approximately 40% to 80% compared to fasting conditions. Based on PK, biomarker, efficacy, and safety results, the RP2D was determined to be 1,200 mg. Four partial responses (PRs) were observed in 14 ALK+ pts resistant to second-generation TKIs treated at the RP2D. Another pt with the G1202R mutation following alectinib treatment had tumor shrinkage of 27.9%. Among 8 pts with brain metastases, 1 complete response and 3 PRs were observed intracranially. In 10 TKI-naïve pts, the overall response rate was 80% (ALK+, 6/8; ROS1+, 2/2) and the disease control rate (DCR) was 100%. Preliminary biomarker data showed decreased FAK phosphorylation in peripheral blood mononuclear cells and increased IFN-γ levels in serum after multiple doses of APG-2449. No dose-limiting toxicity was observed. A total of 66 (78.6%) pts experienced treatment-related adverse events (TRAEs). The most frequent TRAEs included elevated blood creatinine (33.3%), ALT (25.0%), and AST (19.0%) levels and gastrointestinal disorders: nausea (22.6%), vomiting (17.9%), and diarrhea (13.1%). Only 6 (7.1%) TRAEs were grade ≥ 3. Conclusions: APG-2449 has a favorable safety and PK profile and was well tolerated in 84 subjects. Preliminary efficacy was observed in pts whose disease was resistant to second-generation TKIs, especially among those with brain metastases, and in TKI-naïve pts. Biomarker data indicated potential target engagement on FAK and immunomodulatory effects of APG-2449. Clinical trial information: NCT03917043.

The efficacy and safety of taletrectinib in patients with TKI-naïve or crizotinib-pretreated ROS1-positive non–small cell lung cancer (NSCLC).

8572 Background: Taletrectinib (AB-106 / DS-6051b) is a next-generation, potent, CNS- penetrant, selective ROS1 tyrosine kinase inhibitor. The ongoing TRUST study (NCT04395677) is a multicenter, open-label, single-arm, Phase 2 study of taletrectinib in Chinese ROS1-positive NSCLC patients who are TKI -naïve or crizotinib-pretreated. Here we present the updated efficacy and safety results of the study. Methods: The eligible ROS1-positive NSCLC patients were enrolled into either TKI-naïve or crizotinib-pretreated cohorts, and treated with taletrectinib 600mg once daily. The study endpoints included overall response rate (ORR), duration of response (DOR), disease control rate (DCR), overall intracranial response rate (IC-ORR), progression-free survival (PFS), and safety profile. Results: As of the data cutoff date of September 7, 2021, 61 of the 86 stage IV patients enrolled in the study have at least three postbaseline tumor assessment of which 40 patients in the TKI-naïve cohort, and 21 patients in the crizotinib-pretreated cohort (50% patients having at least one prior chemotherapy). In the TKI -naïve cohort, the confirmed ORR by investigators per RECIST 1.1 was 90.0%: [95%CI: 76.3%; 97.2%] (36/40); and the DCR was 95% [95%CI: 83.1%; 99.4%] (38/40). In the crizotinib-pretreated cohort, the confirmed ORR by investigators was 47.6% [95%CI: 25.7%; 70.2%] (10/21); and DCR was 76.2%: [52.8%; 91.8%] (16/21). The mDOR and mPFS are not reached yet for both cohorts. Of 6 patients having brain metastasis and measurable target brain lesions at baseline, the intracranial ORR and IC-DCR were 83.3% and 100%, respectively. Of 4 patients with ROS1 G2032R mutation, 3 patients achieved partial response (PR), and 1 patient achieved stable disease (SD). The most common treatment-related adverse events (TRAEs) include diarrhea, nausea, vomiting, transaminase elevation, anemia, neutrophil count decrease, etc. were Grade 1 or 2, and the most common AEs (below 10%) were ALT/AST increased but reversible. Conclusions: Taletrectinib demonstrated meaningful clinical efficacy in both TKI-naïve and crizotinib-pretreated ROS1 positive NSCLC patients. In particular, taletrectinib showed clinical effectiveness in patients with ROS1 secondary G2032 mutations and patients with brain metastasis. Taletrectinib was well tolerated in this patient population. Clinical trial information: NCT04395677.

Evaluation of Lorlatinib Cerebrospinal Fluid Concentrations in Relation to Target Concentrations for Anaplastic Lymphoma Kinase (ALK) Inhibition.

Lorlatinib is a third‐generation, brain‐penetrant anaplastic lymphoma kinase (ALK) and c‐ros oncogene 1 (ROS1) tyrosine kinase inhibitor (TKI) with robust intracranial activity in patients with ALK‐ or ROS1‐positive non‐small cell lung cancer (NSCLC). Data from the ongoing open‐label, single‐arm, multicenter, phase‐1/2 study of lorlatinib in patients with metastatic ALK‐ or ROS1‐positive NSCLC were used to further investigate the potential brain penetration of lorlatinib. Patients received escalating lorlatinib doses (10–200 mg once daily or 35–100 mg twice daily) or the approved dosing (100 mg daily). Plasma was collected from all patients, and cerebrospinal fluid (CSF) was collected at baseline and during the study from 5 patients with suspected or confirmed leptomeningeal carcinomatosis or carcinomatous meningitis. For those 5 patients, lorlatinib concentrations ranged from 2.64 to 125 ng/mL in the CSF and from 12.7 to 457 ng/mL in the plasma; free plasma concentrations ranged from 4.318 to 155.385 ng/mL. The CSF/free plasma ratio was 0.77 (R2 = 0.96 and P < .001). Using a post‐hoc population pharmacokinetic model, the average steady‐state unbound plasma concentration of lorlatinib was derived and the CSF concentration was estimated for all patients. Known minimum efficacy concentrations (Ceff) for wild‐type and mutated (L1196M and G1202R) ALK were used to derive central nervous system (CNS) Ceff. Estimated CNS concentrations exceeded the derived CNS Ceff values in all patients for wild‐type ALK and the ALK L1196M mutation, and in 35.8% of patients for the ALK G1202R mutation. Projected lorlatinib CNS concentrations were consistent with the high intracranial response rates reported in clinical trials and provide further evidence of the potent CNS penetration of lorlatinib.

EML4-ALK G1202R mutation induces EMT and confers resistance to ceritinib in NSCLC cells via activation of STAT3/Slug signaling

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene occurs in approximately 5% of non-small-cell lung cancers (NSCLCs). The development of ALK tyrosine kinase inhibitors (ALK-TKIs) is a major advance in treating NSCLC with the ALK fusion gene. Nevertheless, acquired resistance to ALK-TKIs ultimately limits their use. A prevalent mechanism of drug resistance in kinases occurs through the mutation of G1202R in ALK. However, the mechanisms underlying G1202R resistance to ceritinib are not fully understood. Here, we demonstrated that the expression of EML4-ALK G1202R mutation in A549 cells induced an epithelial-mesenchymal transition (EMT) phenotype and significantly increased the migration and invasion abilities. These phenomena may be due to the upregulation of signal transducer and activator of transcription 3 (STAT3), accompanied by the elevated expression of Slug in EML4-ALK G1202R mutant cells. Furthermore, the combination of ALK and STAT3 inhibitors restored the sensitivity of EML4-ALK G1202R mutant cells to ceritinib. In conclusion, these data indicate that the EML4-ALK G1202R mutation mediates the EMT phenotype by activating the STAT3/Slug signaling pathway, resulting in resistance to ceritinib, and that the combination of STAT3 and ALK inhibitors may overcome ALK mutation-driven drug resistance in the clinic.

Clinical utility of next-generation sequencing-based ctDNA testing for common and novel ALK fusions

ObjectivesLiquid biopsy for plasma circulating tumor DNA (ctDNA) next-generation sequencing (NGS) can detect ALK fusions, though data on clinical utility of this technology in the real world is limited. Materials and Methods: Patients with lung cancer without known oncogenic drivers or who had acquired resistance to therapy (n = 736) underwent prospective plasma ctDNA NGS. A subset of this cohort (n = 497) also had tissue NGS. We evaluated ALK fusion detection, turnaround time (TAT), plasma and tissue concordance, matching to therapy, and treatment response. Results: ctDNA identified an ALK fusion in 21 patients (3%) with a variety of breakpoints and fusion partners, including EML4, CLTC, and PON1, a novel ALK fusion partner. TAT for ctDNA NGS was shorter than tissue NGS (10 vs. 20 days; p < 0.001). Among ALK fusions identified by ctDNA, 93% (13/14, 95% CI 66%–99%) were concordant with tissue evaluation. Among ALK fusions detected by tissue NGS, 54% (13/24, 95% CI 33%–74%) were concordant with plasma ctDNA. ctDNA matched patients to ALK-directed therapy with subsequent clinical response, including four patients matched on the basis of ctDNA results alone due to inadequate or delayed tissue testing. Serial ctDNA analysis detected MET amplification (n = 2) and ALK G1202R mutation (n = 2) as mechanisms of acquired resistance to ALK-directed therapy. Conclusion: Our findings support a complementary role for ctDNA in detection of ALK fusions and other alterations at diagnosis and therapeutic resistance settings.