Irreversible EGFR Inhibitor Research Articles

A phase 0/1 trigger trial of BDTX-1535 in patients with recurrent high-grade glioma (HGG) with EGFR alterations or fusions.

2069 Background: This Phase 0/1 clinical trial evaluates the pharmacokinetic (PK), pharmacodynamic (PD), and clinical response of recurrent high-grade glioma (HGG) patients with EGFR alterations and/or fusions to the ATP-competitive, irreversible EGFR inhibitor, BDTX-1535. Methods: Recurrent HGG patients with EGFR alterations (Arm A) or EGFR fusions (Arm B) received 5 days of BDTX-1535 (200 mg) prior to planned resection at 2-4 hours following the final dose. In the Phase 0 component of the study, total and unbound drug concentrations were measured in tumor tissue (Gadolinium enhancing and non-enhancing regions), cerebrospinal fluid (CSF), and plasma using validated LC-MS/MS methods. A PK ‘trigger’, defined as unbound drug concentration above 5-fold biochemical IC50 in Gadolinium (Gd)-nonenhancing tumor, determined eligibility for Phase 1. PD response was assessed by quantification of percentage of positive pEGFR, pERK and MIB-1 cells in the surgical tumor tissue compared to baseline pre-treatment tissue. Patients with tumors exceeding the PK threshold for unbound drug concentration were eligible for expansion phase therapeutic dosing of BDTX-1535. Results: A total of 7 patients with recurrent glioblastoma (Arm A) were enrolled in the Phase 0 component of the study. Two patients were excluded from PK analysis due to pseudoprogression. The mean unbound concentrations of BDTX-1535 in Gd-enhancing and nonenhancing tumor regions were 16.0 nM and 10.5 nM respectively. Four of five (80%) evaluable patients exceeded the PK threshold. The suppression of pEGFR and MIB1 was observed in 40% and 60% of patients, respectively. No serious adverse events were observed among patients in the Phase 1 component of the study and a clinical readout is planned in Q2 2024. Conclusions: BDTX-1535 is well-tolerated in recurrent HGG patients, achieves pharmacologically relevant concentrations in Gd-nonenhancing tumor tissue and is associated with suppression of EGFR signaling. Clinical trial information: NCT06072586 .

Efficacy and safety of sunvozertinib in treatment naïve NSCLC patients with EGFR exon20 insertion mutations.

9073 Background: Sunvozertinib (DZD9008) is a rationally designed, irreversible EGFR inhibitor targeting EGFR mutations with wild-type EGFR selectivity. Two pivotal second line studies (WU-KONG1 [NCT03974022] and WU-KONG6 [NCT05712902 and CTR20211009]) are ongoing in NSCLC patients with EGFR exon20 insertion mutations (exon20ins). Primary analysis of WU-KONG6 study demonstrated promising efficacy and safety of sunvozertinib in ≥ second line setting. Herein, we reported preliminary results of sunvozertinib in the first line setting. Methods: Two ongoing studies WU-KONG1 and WU-KONG15 (NCT05559645) enrolled treatment naïve advanced NSCLC patients with EGFR exon20ins. WU-KONG1 is a multinational phase I/II study, and WU-KONG15 is an investigator-initiated phase II study in China. EGFR mutation status was confirmed by local or central testing using tissue or cytological samples. Sunvozertinib was administered orally until disease progression or other discontinuation criteria were met. Patients who had at least one post-treatment RECIST assessment by investigators were evaluable for efficacy analysis, and all patients who received at least one dose of sunvozertinib were included in the safety analysis. Results: As of January 15, 2023, a total of 36 treatment naïve advanced NSCLC patients with EGFR exon20ins received sunvozertinib daily dosing at RP2Ds. Median age was 66.5 years, and 63.9% (23/36) were female. The baseline ECOG PS was 0 or 1. Majority of patients (33/36, 91.7%) had metastatic diseases at study entry, with 22.2% (8/36) having > 3 metastatic sites and 22.2% (8/36) having baseline brain metastasis (BM). The most frequent mutation subtypes included 769_ASV (13/36, 36.1%), 770_SVD (2/36, 5.6%) and others (21/36, 58.3%). In 26 efficacy-evaluable patients, 19 patients showed tumor response, with a best objective response rate of 73.1%, among them 14 patients had confirmed response, and another 3 patients were still on treatment and pending confirmation. In patients with BM, tumor response was also observed intracranially. Median duration of response had not been reached by the data cut-off date. Safety findings were consistent with what was observed in previous studies of sunvozertinib. The most common treatment emergent adverse events (TEAEs) included diarrhea, CPK increase, and skin rash. The majority of adverse events were CTCAE grade 1 or 2 and manageable with supportive measures. Conclusions: Consistent with second line results, sunvozertinib demonstrated promising efficacy and safety profile as monotherapy in the first line setting for patients with advanced EGFR exon20ins NSCLC. The updated data will be presented at the conference. A phase III, multinational, randomized study (WU-KONG28, NCT05668988) is ongoing to compare sunvozertinib to chemotherapy as first line treatment for EGFR exon20ins NSCLC. Clinical trial information: NCT03974022 , NCT05559645 .

Anti-tumor activity of sunvozertinib in NSCLC with EGFR sensitizing mutations after failure of EGFR TKI treatment.

9103 Background: Sunvozertinib is a rationally designed, irreversible EGFR inhibitor targeting various EGFR mutations with wild-type EGFR selectivity. Clinical studies have showed its superior efficacy than current available therapies and favorable safety profile for treating NSCLC patients with EGFR exon20 mutations. Here we reported its anti-tumor activity in patients with EGFR sensitizing mutations (EGFRm) who failed from standard EGFR TKI treatment. Methods: This is a pooled analysis of three clinical studies in patients with EGFRm or HER2m NSCLC: WU-KONG1 (multinational phase I/II study, NCT03974022), WU-KONG2 (phase I study in China, CTR20192097), and WU-KONG15 (phase II study in China, NCT05559645). Patients were enrolled to receive sunvozertinib once daily at defined doses until discontinuation criteria were met. Patients who had EGFRm NSCLC, at least one measurable lesion at baseline, received at least one dose of sunvozertinib, and underwent at least one post-treatment RECIST assessment were evaluable for efficacy analysis. Patients with EGFRm or HER2m NSCLC who failed from standard systemic therapies and received at least one dose of sunvoertinib were included in the safety analysis. Results: As of October 17, 2022, a total of 32 patients who met efficacy evaluable criteria were included in the analysis. The doses of sunvozertinib ranged from 50 mg - 400 mg. Baseline characteristics: median age was 64.5 years old; 68.8% (22/32) were female; 75.0% (24/32) ECOG PS was 1; 34.4% (11/32) had more than three metastatic sites; 43.8% (14/32) had baseline brain metastases. Patients were heavily pretreated, with a median of 5 lines (range 1 - 16) of prior therapies. All patients had been treated with at least one type of EGFR TKI, including 68.8% (22/32) had been treated with a 3rd generation EGFR TKI. The majority of patients (29/32, 90.6%) had also been treated with chemotherapy. Anti-tumor activity was observed starting from 50 mg. Per investigators’ assessment, the best objective response rate (BoR) was 21.9% (7/32). Anti-tumor activity was observed regardless of T790M mutation status. At the data cutoff date, the median duration of response and progression-free survival were 4.0 months and 5.9 months, respectively. Sunvozertinib was well-tolerated across all dose levels. The safety profile was similar to what has been previously reported. The longest treatment duration was more than 35 months (still ongoing). Conclusions: Sunvozertinib monotherapy demonstrated promising anti-tumor activity in heavily pretreated EGFRm NSCLC patients. Further clinical evaluation of sunvozertinib in this patient population is warranted. The updated data will be presented at the conference. Clinical trial information: NCT05559645 NCT03974022 and CTR20192097 .

EGFR Inhibitor CL-387785 Suppresses the Progression of Lung Adenocarcinoma.

This study aimed to explore the influence of the irreversible EGFR inhibitor CL-387785 on invasion, metastasis, and radiation sensitization of non-small cell lung cancer cells. The proliferation inhibitory rate at different time points was detected by MTT assay. The apoptosis of H1975 cells treated with CL-387785 was detected using flow cytometry. The invasion and migration of H1975 cells treated with CL-387785 were determined by Transwell assay and wound healing assay. The survival fraction (SF) of H1975 cells cultured with CL- 387785 under X-ray (0, 2, 4, 6, 8, and 10 Gy) was detected by cloning formation experiment, and the sensitization ratio (SER) was calculated by clicking the multi-target model to fit the cell survival curve. CL-387785 restrained H1975 cell proliferation in a concentration- and time-dependent manner. CL-387785 promoted H1975 cell apoptosis and reduced cell migration distance and the number of transmembrane cells. The SF treated by different concentrations of CL-387785 (10, 25, 50, and 100 nM) was all below 0 nM. The radiation SER of CL-387785 (10, 25, 50 and 100 nM) were 1.17, 1.39, 2.88, and 3.64, respectively. The invasion and metastasis of H1975 cells were restrained by irreversible EGFR inhibitor CL-387785. CL-387785 also exhibited the effect of radiotherapy sensitization.

Abstract P2-24-01: The development of covalent microtubule stabilizers for drug-resistant cancers

Abstract Microtubule targeting agents (MTAs) are some of the most commonly used and effective chemotherapeutic agents used in the treatment of breast cancer and other solid tumors. Clinically approved MTAs include microtubule destabilizers, such as the vinca alkaloids or eribulin, as well as the taxane class of microtubule stabilizers, including paclitaxel. While MTAs are effective chemotherapeutics, the development of resistance due to drug efflux or changes in β-tubulin isotype expression are major clinical limitations to each the currently approved drugs of this class. One strategy for overcoming drug resistance is the development of drugs that covalently bind to their drug target, which has been validated in the clinic with the success of irreversible EGFR inhibitors in drug resistant non-small cell lung cancer. We have identified the C22,23-epoxytaccalonolides (taccas) as a class of plant-derived microtubule stabilizers that covalently bind to β-tubulin with a high degree of specificity and retain efficacy in drug-resistant models in vitro and in vivo. Although the taccas have potent and persistent antitumor efficacy in vivo, they suffer from a narrow therapeutic index and short serum half-life that provides optimal efficacy when targeted directly to the tumor site. We are undertaking a multipronged approach to generate synthetic tacca analogs (taccalogs) that retain the efficacy of the natural product with improved pharmacokinetic properties, including improved serum binding, to increase half-life and provide better in vivo tolerability. We are also identifying strategies to improve tumor targeting of the taccas, which is being guided by determining their in vivo distribution with radiolabeled analogs. In a complementary approach, we tested the hypothesis that non-covalent microtubule stabilizers that have increased binding affinity to tubulin share properties with the taccas, such as the ability to circumvent drug resistance and provide potent and persistent in vivo efficacy with a more tolerable therapeutic window. Our data suggest that while non-covalent microtubule stabilizers with improved binding compared to paclitaxel provide improved cellular persistence after drug washout and potent antitumor efficacy, they are not able to overcome drug resistance to some degree as covalent inhibitors, demonstrating the advantage of continuing to develop irreversible drugs such as the taccalonolides for the treatment of drug resistant disease. Citation Format: Jacob N. Essif, April Risinger. The development of covalent microtubule stabilizers for drug-resistant cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-01.

66 (PB056) - Anti-tumor activity of BDTX-1535, an irreversible CNS penetrant inhibitor of multiple EGFR extracellular domain alterations, in preclinical glioblastoma models

Background: EGFR genetic alterations, including mutation and amplification, are very common in glioblastoma multiforme (GBM), however, no targeted therapy has been approved for this disease. EGFR oncogenic mutations in GBM promote EGFR oncogenic covalent homodimer formation which leads to paradoxical activation and renders reversible inhibitors ineffective. An effective EGFR inhibitor in GBM must meet four conditions:1Highly potent and selective against a broad range of extracellular domain mutations and amplified EGFR,2Wild-type EGFR sparing,3Irreversible binding to circumvent paradoxical activation,4High CNS penetration. Our MAP discovery platform led to the identification of MasterKey inhibitor BDTX-1535, an irreversible 4th-generation CNS penetrant EGFR tyrosine kinase inhibitor (TKI) that potently and selectively targets a broad range of EGFR alterations in GBM. Materials and Methods: BDTX-1535 preclinical exposure was evaluated in multiple species in plasma and brain. Antitumor activity was assessed across a broad range of mouse PDX and allograft models, including an intracranial PDX. 2540 tumors were sequenced with the Tempus xT NGS assay. Mutational frequencies and co-expression status of oncogenic EGFR variants were assessed. The relationship between treatment history and oncogenic EGFR mutational frequency was analyzed. Results: BDTX-1535 is a small molecule that irreversibly inhibits a spectrum of EGFR alterations expressed in GBM together with EGFR amplification, while sparing inhibition of normally expressed wild type EGFR. BDTX-1535 avoids efficacy limiting paradoxical activation that can occur with reversible EGFR inhibitors. BDTX-1535 exhibits good oral bioavailability and a CNS Kpuu of 0.55 and 0.48 in rat and dog, respectively, comparing favorably with other CNS penetrant TKIs. Tumor regression and survival advantage were observed across all tumor models with EGFR amplification or mutation, including in an intracranial PDX model. EGFR mutations and amplification are commonly expressed in GBM. Consistent with published data, EGFR mutations, variants, and copy number gain were frequently co-expressed. For example, among tumors expressing EGFRvIII, ∼95% co-expressed at least one other variant, and among tumors expressing EGFRvVI, ∼45% co-expressed at least one other variant. Within a subset of GBM patients with longitudinal sequencing data, oncogenic EGFR mutations were expressed and persisted following front-line treatment. Conclusions: BDTX-1535 is a potent, CNS penetrant, irreversible small molecule EGFR inhibitor that shows efficacy against a broad range of extracellular domain mutations and EGFR amplification found in GBM patients. RWE findings demonstrate a heterogeneous EGFR mutational profile in GBM patients, with persistent prevalence over time. BDTX-1535 is currently in a phase I clinical study (NCT05256290). Conflict of interest: Ownership: Black Diamond Therapeutics Tempus Labs

Abstract LB140: CNS penetrant, irreversible inhibitors potently inhibit the family of allosteric oncogenic EGFR mutants expressed in GBM and demonstrate efficacy in patient-derived xenograft models

Abstract Oncogenic EGFR mutations occur in approximately 50% of glioblastomas (GBM) and largely reside in the extracellular domain. Prior attempts to reposition current generation EGFR inhibitors to treat GBM likely failed due to poor brain penetration and an inability to potently target the full spectrum of oncogenic mutations. As EGFR oncogenic mutations are found to be co-expressed in many GBMs, it is important that an inhibitor be broadly active against the entire family of relevant EGFR mutants. Additionally, a successful inhibitor would require a pharmacokinetic (PK) profile that allows for sufficient penetration of the blood brain barrier to elicit robust target engagement of the brain tumor. Using these design principles, we designed a series of highly potent molecules exemplified by BDTX-507. This molecule is an irreversible inhibitor of EGFR with antiproliferative IC50's less than 10 nM against the spectrum of GBM-relevant EGFR mutations. PK/PD studies demonstrated sustained pERK suppression exceeding 24 hours following a single QD dose. Furthermore, when dosed in GBM xenografts, including an intracranial Viii PDX (GBM6), robust tumor regressions and improved survival were observed. Emerging from this series were two advanced compounds, BDTX-700 and BDTX-1535, which also demonstrated potent inhibition of the GBM EGFR spectrum, selectivity v. wild-type EGFR, and an excellent CNS PK profile. BDTX-1535 is currently being evaluated in IND-enabling studies for future clinical evaluation in GBM patients. Citation Format: Matthew O'Connor, Matt Lucas, Darlene Romashko, Sara Rasmussen, Tai-An Lin, Nigel Waters, Raffaele Fiorenza, Iwona Wrona, Deborah Chen, Theodore Nicolaides, David R. Raleigh, Tomoko Ozawa, George Trainor, Luca Arista, Alexander Flohr, Giorgio Ottaviani, Chris Roberts, Elizabeth Buck. CNS penetrant, irreversible inhibitors potently inhibit the family of allosteric oncogenic EGFR mutants expressed in GBM and demonstrate efficacy in patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB140.

Amphiregulin-EGFR Autocrine Signaling Mediates Neutrophil Elastase-Triggered Prostate Cancer Progression

Abstract Neutrophil elastase (NE) is a serine protease stored in neutrophil azurophilic granules. Growing evidence indicates that NE is intimately involved in the activities of proinflammatory cytokines / chemokines, growth factors, and cell surface receptors. These molecular regulations can modulate innate immune responses as well as directly promote cancer cell outgrowth. To date, however, little is known regarding the molecular mechanisms underlying the stimulatory properties of NE in cancer cells. Here we examine NE effects on prostate cells, demonstrating that NE triggers proliferative signals and cell migration in six prostate cell lines representing the spectrum of prostate cell malignancy, including normal prostatic epithelium, benign prostatic hypertrophy, and metastatic prostate cancer. Using ERK activation as a read-out, we show that NE promotes ERK phosphorylation in a dose dependent manner, and time course study further reveal a sustained ERK activation upon NE treatment. Western blot evaluation demonstrates strong EGFR expression in cell lines derived from normal and benign prostatic gland, and preferential expression in hormone resistant versus hormone responsive cells. In agreement with EGFR-dependent mitogenic signaling, activation of ERK is abrogated by siRNA-mediated EGFR knockdown, as well as by pretreatment of cells with irreversible EGFR inhibitor AG1478. Importantly, NE evokes cancer cell migration at a lower range of NE concentrations relative to nonneoplastic cells. In prostate cells, from a total of seven EGFR ligands, amphiregulin (AREG) is predominantly expressed, and the addition of NE results in the release of AREG. Moreover, AREG gene silencing by siRNA or inhibition of AREG biological activity by neutralizing antibody, prevents NE-induced ERK phosphorylation and cell migration. Together, our study reveals a distinct and essential role of AREG-EGFR signaling axis in NE-triggered prostatic cellular response.

BEBT-109, a pan-mutant-selective EGFR inhibitor with potent antitumor activity in EGFR-mutant non-small cell lung cancer

EGFR mutation-positive NSCLC tumors are highly heterogeneous, therefore, exploring an agent simultaneously targeting multiple EGFR mutations may be valuable for clinical practice. Compared with osimertinib, BEBT-109 shows more sensitive and extensive antitumor activity in EGFR mutant NSCLC, while sparing wild-type EGFR cell lines. Meanwhile, unlike the metabolite of osimertinib AZ5104, the main metabolites of BEBT-109 are found lacking in activity against wild-type EGFR cell lines. Preclinical and clinical studies demonstrate a unique pharmacokinetic profiles of BEBT-109 with rapid absorption and quick in vivo clearance without accumulation, which are conducive to minimizing the off-target toxicity of the covalent irreversible EGFR inhibitor. Oral administration of BEBT-109 induces tumor regression in EGFR exon 20 insertion xenografts, and even tumor disappearance in PC-9, HCC827 and H1975 xenograft models. Furthermore, in clinical trials, the objective responses were observed in NSCLC patients with EGFR T790M mutation in the first and second dosing cohorts. These findings demonstrate that BEBT-109, a potent pan-mutant-selective EGFR inhibitor with improved pharmacokinetic properties, might offer a promising new option for the treatment of multiple mutant-EGFR-driven NSCLC.

SAT-138 Neutrophil Elastase Promotes Proliferative Signals in Prostate Cells Through EGFR and DDR1

Studies examining many different cancers have demonstrated that inflammation plays a critical role in tumor progression, in part through the release of proteases from stromal cells that function to either remodel the tumor microenvironment or to directly stimulate cancer cells to grow. One specific protease, neutrophil elastase (NE), has been shown to be a critical regulator of cancer growth in several mouse models. Accordingly, our laboratory demonstrated that NE, most likely from granulocytic myeloid-derived suppressor cells, potentially promotes prostate cancer progression in several different in-vivo and in-vitro models. To date, however, little is known regarding the mechanisms utilized by NE to promote tumor growth. It has been suggested that NE might cleave epidermal growth factor (EGF) or transforming growth factor-α from the cell surface to induce activation of EGFR/ERK signal transduction in an autocrine fashion. Alternatively, NE has been shown to enter into early endosomes to degrade insulin receptor substrate-I, ultimately resulting in phosphoinositol 3-kinase hyperactivity and subsequent tumor cell proliferation. Here we demonstrate that NE triggered proliferative signals in six prostate cell lines representing the spectrum of prostate cell differentiation, including normal prostatic epithelium, benign prostatic hypertrophy, and metastatic prostate cancer. Focusing on ERK signaling, we found that the stimulatory effect of NE on ERK phosphorylation was dose dependent and was abrogated by small interfering RNA induced EGFR knockdown, as well as by pretreatment of cells with irreversible EGFR inhibitor AG1478. Unlike EGF, however, NE-initiated EGFR phosphorylation was minimal. Thus, while EGFR appears to be critical for NE-induced ERK activation, perhaps it is not extensively activated directly by NE. Notably, discoidin domain receptor-1 (DDR1) was strongly expressed in normal prostate epithelium cells, but gradually decreased and had little expression in benign and metastatic prostate cancer cells sequentially. Nevertheless, similar to EGFR knockdown, silencing of DDR1 in all cell types inhibited NE mediated pERK upregulation, suggesting that DDR1 may also be important for NE-induced action. Together, our data suggest that NE, in concert with low level signals from the EGFR and DDR1, play an important role in promoting prostate cell proliferation both in normal and cancerous prostate epithelial cells.

Discovery of a Novel, Selective and Irreversible Inhibitor (Abivertinib) of Mutated EGFR and T790M-induced Resistance for the Treatment of NSCLC

Three chemical series of heterocyclic small molecules were designed and synthesized as the 3rd generation EGFR inhibitors. Detailed structure-activity relationship (SAR) studies and lead optimization (from 1a to 4d) were descripted. With the support of molecular modeling and evaluation through biochemical and cellular assays, liver microsomal/ plasma/ blood stability studies, preliminary rat PK screening and in vivo animal models, pyrrolopyrimidine-based compound 4d (abivertinib, AC0010), a novel potent and highly selective irreversible EGFR inhibitor, was discovered. [Display omitted] This compound has been being clinically investigated in several countries including China, the United States, and Europe. Structurally distinct from those irreversible pyrimidine-based EGFR inhibitors (rociletinib, and recently approved osimertinib), abivertinib (AC0010) has demonstrated significant clinical benefit with good safety profiles (through a different metabolic pathway and possibly different resistance mechanism compared to both osimertinib and rociletinib) in NSCLC patients who had progressed on previous therapy, which provides an alternative therapeutic agent for NSCLC patients who develop acquired resistance to first generation EGFR TKIs.

In vivo efficacy studies of novel quinazoline derivatives as irreversible dual EGFR/HER2 inhibitors, in lung cancer xenografts (NCI-H1975) mice models.

Lung cancer is the most common cancer and leading cause of cancer-related deaths worldwide. The first-generation reversible, ATP-competitive inhibitors gefetinib and elotinib showed good clinical responses in lung adenocarcinoma tumors (NSCLC). But almost all patients developed resistance to these inhibitors over time. Such resistance of EGFR inhibitors was frequently linked to the acquired L858R and T790M point mutations in the kinase domain of EGFR. To overcome these resistance problems, the second and the third generation inhibitors have been discovered. FDA approved afatinib, the second generation irreversible inhibitor and osimitinib, the third generation irreversible EGFR inhibitors for the treatments of NSCLC. We identified new covalent quinazoline inhibitors (E)-N-(4-(3-chloro-4-fluorophenylamino)-7-(2-ethoxyethoxy)quinazolin-6-yl)-4-(dimethylamino)but-2-enamide (6d) and (E)-N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-7-(2-ethoxyethoxy)quinazolin-6-yl)-4-(dimethyl-amino)but-2-enamide (6h) that exhibited potent EGFR kinase inhibitory activities on L858R and T790M mutations. The compound 6h showed selectivity similar to AZD9291 (osimertinib) in mutated and wild type tumor cell lines. In vitro cell assay 6d and 6h were better than afatinib and osimertinib. In vivo antitumor efficacy studies of these compounds were done in NCI-H1975 mice xenografts.

Abstract P1-19-07: Results from plasmaMATCH trial treatment cohort B: A phase II trial of neratinib plus fulvestrant in ER positive breast cancer or neratinib alone in ER negative breast cancer in patients with a HER2 mutation identified via ctDNA screening (CRUK/15/010)

Abstract Background: HER2 mutations occur in approximately 3% of HER2 non-amplified cancers and include missense substitutions and indels within the tyrosine kinase domain resulting in oncogenic HER2 activation. Neratinib, an irreversible EGFR, HER2 and ERBB4 tyrosine kinase inhibitor, has demonstrated activity in pre-clinical HER2 mutant models, and in a prior phase I trial with HER2 mutations identified through tumour testing. The plasmaMATCH trial Cohort B assessed the efficacy of neratinib in BC patients with a HER2 mutation identified in ctDNA testing. Methods: The plasmaMATCH trial was an open-label, multi-centre, multi-cohort platform trial, consisting of ctDNA testing in ~1000 patients with advanced BC. Patients with a HER2 mutation identified via ctDNA testing were registered to Cohort B. Patients were treated with 240mg neratinib once daily. Patients with ER positive BC were also treated with fulvestrant 500mg intramuscularly on Cycle 1 Days 1 and 15, and Cycle 2 onwards every 28 days. The primary endpoint for Cohort B was confirmed objective response rate as defined by RECIST v1.1. Using a single stage A’Hern design with a target response rate of 25%, unacceptable response rate of 5%, alpha=5% and power=80%, at least 3 responses out of 16 evaluable patients were required to infer efficacy. Results: Following ctDNA testing, 21 patients enrolled in Cohort B (58% of patients with HER2 mutations identified in ctDNA testing). Eighteen (86%) were ER positive, 2 (10%) were HER2 amplified, and 18 (86%) had visceral metastases. The most common HER2 mutations detected in baseline plasma were L755S (47.6%), V777L (19%), and S310F (14.3%). In the 20 evaluable patients, confirmed response rate was 25.0% (95%CI 8.7-49.1%, 5/20) (first 16 evaluable patients: 4/16, 25.0% (95%CI 7.3-52.4)). One patient had a complete response, ongoing at 29 months duration, and three additional patients had unconfirmed partial responses. Median progression free survival was 5.4 months (IQR 3.4-9.1) and median duration of response was 5.7 months (IQR 3.7-9.7 months) with 3 patients continuing on treatment. The most common clinically significant grade 3 or 4 adverse events were diarrhoea (20%) and hypertension (15%). Conclusions: Neratinib, with or without fulvestrant, was active in advanced BC patients with HER2 mutations identified in ctDNA testing, meeting the pre-specified threshold for efficacy. Citation Format: Andrew M Wardley, Lucy Kilburn, Sarah Kernaghan, Iain Macpherson, Richard D Baird, Rebecca Roylance, Peter Stephens, Olga Oikonomidou, Jeremy P Braybrooke, Mark Tuthill, Jacinta Abraham, Matthew C Winter, Belinda Kingston, Katie Wilkinson, Judith M Bliss, Alistair Ring, Nicholas Turner, on behalf of the plasmaMATCH Trial Management Group. Results from plasmaMATCH trial treatment cohort B: A phase II trial of neratinib plus fulvestrant in ER positive breast cancer or neratinib alone in ER negative breast cancer in patients with a HER2 mutation identified via ctDNA screening (CRUK/15/010) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-07.

Mutant-Selective Irreversible EGFR Inhibitor, Naquotinib, Inhibits Tumor Growth in NSCLC Models with EGFR-Activating Mutations, T790M Mutation, and AXL Overexpression.

First- and second-generation EGFR tyrosine kinase inhibitors (TKI) are effective clinical therapies for patients with non-small cell lung cancer (NSCLC) harboring EGFR-activating mutations. However, almost all patients develop resistance to these drugs. The EGFR T790M mutation of EGFR is the most predominant mechanism for resistance. In addition, activation of AXL signaling is one of the suggested alternative bypassing pathways for resistance to EGFR-TKIs. Here, we report that naquotinib, a pyrazine carboxamide-based EGFR-TKI, inhibited EGFR with activating mutations, as well as T790M resistance mutation while sparing wild-type (WT) EGFR. In in vivo murine xenograft models using cell lines and a patient-derived xenograft model, naquotinib induced tumor regression of NSCLC with EGFR-activating mutations with or without T790M resistance mutation, whereas it did not significantly inhibit WT EGFR signaling in skin. Furthermore, naquotinib suppressed tumor recurrence during the treatment period of 90 days. In addition, unlike erlotinib and osimertinib, naquotinib inhibited the phosphorylation of AXL and showed antitumor activity against PC-9 cells overexpressing AXL in vitro and in vivo Our findings suggest that naquotinib has therapeutic potential in patients with NSCLC with EGFR-activating mutations, T790M resistance mutation, and AXL overexpression.

Brain accumulation of osimertinib and its active metabolite AZ5104 is restricted by ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein)

Osimertinib is an irreversible EGFR inhibitor registered for advanced NSCLC patients whose tumors harbor recurrent somatic activating mutations in EGFR (EGFRm+) or the frequently occurring EGFR-T790M resistance mutation. Using in vitro transport assays and appropriate knockout and transgenic mouse models, we investigated whether the multidrug efflux transporters ABCB1 and ABCG2 transport osimertinib and whether they influence the oral availability and brain accumulation of osimertinib and its most active metabolite, AZ5104. In vitro, human ABCB1 and mouse Abcg2 modestly transported osimertinib. In mice, Abcb1a/1b, with a minor contribution of Abcg2, markedly limited the brain accumulation of osimertinib and AZ5104. However, no effect of the ABC transporters was seen on osimertinib oral availability. In spite of up to 6-fold higher brain accumulation, we observed no acute toxicity signs of oral osimertinib in Abcb1a/1b;Abcg2 knockout mice. Interestingly, even in wild-type mice the intrinsic brain penetration of osimertinib was already relatively high, which may help to explain the documented partial efficacy of this drug against brain metastases. No substantial effects of mouse Cyp3a knockout or transgenic human CYP3A4 overexpression on oral osimertinib pharmacokinetics were observed, presumably due to a dominant role of mouse Cyp2d enzymes in osimertinib metabolism. Our results suggest that pharmacological inhibition of ABCB1 and ABCG2 during osimertinib therapy might potentially be considered to further benefit patients with brain (micro-)metastases positioned behind an intact blood-brain barrier, or with substantial expression of these transporters in the tumor cells, without invoking a high toxicity risk.

First-time in-human study of VMD-928, an allosteric and irreversible TrkA selective inhibitor, in patients with solid tumors or lymphoma.

TPS3146 Background: Tropomysin receptor kinase A (TrkA) is a protein encoded by the NTRK1 gene. NTRK fusions involving the kinase domain are oncogenic for multiple tumor types and larotrectinib was recently approved for advanced solid tumors harboring NTRK gene fusions. Larotrectinib, an ATP-competitive, reversible pan-TrkA/B/C inhibitor, has shown impressive response rates in patients harboring these fusions; however, resistance can develop due to acquired ATP-site mutations. This has been previously identified in other oncogenic driver kinases such as ALK and EGFR treated with ATP-competitive kinase inhibitors. A newly approved allosteric ALK/EGFR inhibitor brigatinib was able to clinically overcome acquired resistance of many ATP-competitive ALK/EGFR inhibitors (1). Also, irreversible EGFR inhibitors such as afatinib (ATP-competitive) were active against tumors resistant to first-generation inhibitors (2), although their efficacy can be compromised by acquired ATP-site mutations (3). VMD-928 is the first oral small-molecule TrkA (NTRK1) selective inhibitor with dual allosteric and irreversible mechanisms of action. It inhibits TrkA non-competitively at an allosteric (non-ATP) site and has no resistance in vitro to acquired ATP-site mutations such as G667C. VMD-928 in vitro has little or no activity against 348 other kinases including TrkB (NTRK2) and TrkC (NTRK3). We are conducting the first time in human phase 1 trial of oral VMD-928, a novel allosteric and irreversible TrkA selective inhibitor. Methods: This is an open label, Phase 1 study investigating the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of oral VMD-928 in adults with advanced solid tumors or lymphoma (NCT03556228). In part 1 of the study, an accelerated titration scheme will be utilized to determine the recommended phase 2 dose and evaluate PK / PD of VMD-928. In part 2, expansion cohorts including patients with thymic, pancreatic, triple-negative breast carcinoma, or solid tumors with TrkA alterations will be accrued to further evaluate safety and efficacy. Part 3 of the study will characterize the biologically active dose. The study is open and accruing patients at City of Hope. Clinical trial information: NCT03556228.

Determination of BPI15086 and its metabolite in human plasma by ultra-high performance liquid chromatography-MS/MS and its application to a pharmacokinetic study.

Aim: BPI15086 is a potent, irreversible mutant-selective inhibitor of both EGFR (EGFR tyrosine kinase inhibitor) and the T790M resistance mutations tyrosine kinase. A simultaneous quantification method of BPI15086 and its main metabolite in human plasma using LC-MS/MS is documented and fully validated in this study. Methodology & results: Plasma samples were extracted and chromatographed on an Acquity ultra-high performance liquid chromatography BEH C18 column with a gradient elution. Detection was performed on a Sciex 5500 QTRAP® mass spectrometer using positive electrospray ionization. The results indicated that the method had excellent sensitivity and specificity. Conclusion: For the first time a sensitive and robust ultra-high performance liquid chromatography-MS/MS method was established and validated of BPI15086 in human plasma, this method was successfully applied in a first-in-human Phase I clinical trial studying the pharmacokinetics of the BPI15086 tablet in Chinese non-small-cell lung cancer patients.

Abstract 825: Identification of molecular therapeutic targets that enhance the antitumor activity of neratinib in breast cancer cells

Abstract Background: Aberrant expression of members of the ErbB family of receptor tyrosine kinases and its downstream pathways is known to play pivotal roles in breast cancer malignancy. HER2-positive breast cancer has an aggressive nature that is driven by HER2 gene amplification. Inflammatory breast cancer is a rare but the most aggressive clinical subtype of breast cancer. Triple-negative breast cancer is an aggressive receptor subtype of breast cancer. More than 30% of cases of each of these groups express EGFR. Emerging therapeutic resistance continues to be a challenge in recurrent and/or metastatic disease. Neratinib is a potent irreversible EGFR, HER2, and HER4 tyrosine kinase inhibitor that blocks signal transduction generated via these receptors. In this study, we sought to identify the optimal targets in different subtypes of breast cancer and synergistic partners to maximize the antitumor effect of neratinib against breast cancer cells in vitro and in vivo. Methods: Expression of EGFR and HER2 was assessed via Western blot in 31 breast cancer cell lines, including HER2-targeted drug-resistant cell lines (14 triple-negative breast cancer and 10 HER2-positive). The antiproliferative effects of neratinib were measured using cell proliferation and anchorage-independent colony-formation (soft agar) assays. A reverse-phase protein array was used to profile and validate the signaling networks induced by neratinib-based treatment. To identify the synergistic targets, RNA interference screening of a 779-kinome library was performed under multicellular tumor spheroid culture conditions. Results: In vitro proliferation data demonstrated that neratinib had a nanomolar range of half-maximal inhibitory concentrations in most of the tested cell lines, including T-DM1-resistant HER-2 breast cancer cell lines. Neratinib inhibited colony formation in a dose-dependent manner (P = 0.0001). Reverse-phase protein array data revealed that the efficacy of neratinib correlated with phosphorylated EGFR (R2 = 0.6055) or phosphorylated HER2 (R2 = 0.6734) expression level. Statistical analysis identified the 25 most relevant targets in the RNA interference screening, which we will validate for combinational antitumor effects with neratinib in in vitro and in vivo studies. Conclusion: We determined the efficacy of neratinib and identified target molecules to enhance neratinib efficacy in different breast cancer subtype cell lines with ErbB alterations. Our data inform new combined treatment strategies with neratinib for breast cancer that will lead to innovative designs using optimized neratinib combinations in clinical trials. Citation Format: Bora Lim, Jangsoon Lee, Troy Pearson, Liu Huey, Minha Hwang, Kuicheon Choi, Francesca Avogadri-Connors, Richard E. Cutler, Debu Tripathy, Naoto T. Ueno. Identification of molecular therapeutic targets that enhance the antitumor activity of neratinib in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 825.

Abstract 4788: Preclinical evaluation of E-191, a highly selective EGFR T790M inhibitor

Abstract Objective: Patients of non-small cell lung cancer (NSCLC) with activating EGFR mutations initially respond to the first generation reversible EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib. However, most of the patients acquire resistance within 10~16 months. The most common mechanism of resistance is the EGFR T790M mutation. In addition, selectivity of the EGFR inhibitors is important, as inhibition of EGFR wild type can lead to dose limiting toxicities, including rash and diarrhea. This may be the reason why the second generation irreversible EGFR inhibitors, such as afatinib, which also inhibit EGFRWT, is not widely used. Here, we disclose our clinical candidate compound E-191 as a novel irreversible inhibitor that potently inhibits the resistance mutant forms of EGFR with a good selectivity sparing EGFRWT. Method: The anti-proliferative activity of E-191 was evaluated in NCI-H1975 (EGFRT790M/L58R), HCC827 (EGFRd746-750), and A431 (EGFRWT) cell lines. Daily oral administration of E-191 at 3 doses was used to evaluate the in vivo anti-tumor activity in three cell-derived tumor xenograft (CDX) models, NCI-H1975, HCC827, and Ba/F3 (EGFRT790M /d746-750). The in vivo selectivity was evaluated in the A431 (EGFRWT) CDX model. Effects on EGFR signaling pathway were assessed by western blot analysis of the downstream effector proteins p-EGFR (Y1068), p-AKT (Ser473) and p-ERK (Thr202/Tyr204). Result: E-191 displayed potent anti-proliferative activity in the EGFR mutant cell lines NCI-H1975 (IC50 = 22 nM) and HCC827 (IC50=1.9 nM), and a much less potent activity in the EGFRWT A431 cell line (IC50 = 531 nM). E-191 has about 24 fold selectivity for EGFRT790M over EGFRWT. E-191 potently inhibited tumor growth at all doses tested (P<0.001) in the NCI-H1975, HCC827 and Ba/F3 (EGFRT790M /d746-750) models, while significantly less so in the A431 model. Western blot analysis of the tumor samples in the NCI-H1975 model study showed that E-191 inhibited p-EGFR (Y1068), p-AKT (Ser473), and p-ERK (Thr202/Tyr204), indicating that the tumor growth inhibition was through inhibition of the resistant mutant EGFR. Conclusion: We have demonstrated with in vitro and in vivo preclinical models that E-191 is a selective inhibitor of the resistance mutant forms of EGFR, potently inhibiting EGFRT790M but not EGFRWT. These results make us believe that E-191 has a good potential in the clinic for the treatment of resistant EGFR driven NSCLC. Citation Format: Liu Xile, Lihong Hu, Charles Z Ding, Weifeng Mao, Liqing Wang, Xiaohe Shi, Chaofeng Long, Xiaoxin Chen, Haijun Li, Xing Liu, Zhuowei Liu. Preclinical evaluation of E-191, a highly selective EGFR T790M inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4788.

YL143, a novel mutant selective irreversible EGFR inhibitor, overcomes EGFRL858R, T790M -mutant resistance in vitro and in vivo.

YL143 was identified as a novel wild‐type sparing EGFRT790M inhibitor with good pharmacokinetic properties. It potently suppresses EGFRL858R/T790M with an 50% inhibitory concentration (IC50) value of 2.0 ± 0.3 nmol/L, but is approximately 92‐folds less potent against EGFRWT kinase. YL143 suppresses cellular proliferation and induces G0/G1 phase arrest and apoptosis in H1975 cells with EGFRL858R/T790M mutation at 30 nmol/L. It also exhibits acceptable pharmacokinetics (PK) parameters with an oral bioavailability value of 25.0% after oral administration in rats and exhibits promising antitumor efficacy in a gefitinib‐resistant human H1975 xenografted model after oral administration of 30 mg/kg/day. These data supported that YL143 could be a promising lead compound for overcoming clinical EGFRT790M resistance of patients with non‐small‐cell lung cancer (NSCLC).