Irreversible pan-HER Inhibitor Research Articles

Multicenter phase I dose escalation and expansion study of pyrotinib in combination with camrelizumab and chemotherapy as first-line treatment for HER2-positive advanced gastric and gastroesophageal junction adenocarcinoma

Pembrolizumab plus trastuzumab and chemotherapy showed remarkable efficacy as first-line therapy for advanced HER2-positive gastric cancer. Pyrotinib is an irreversible pan-HER inhibitor. This single-arm, open-label phase 1 dose-escalation (1a) and expansion (1b) study investigated camrelizumab, an anti-PD-1 antibody, plus pyrotinib and chemotherapy as first-line treatment for advanced HER2-positive gastric and gastroesophageal junction (G/GEJ) adenocarcinoma. Between June 2020 and June 2022, 41 patients with previously untreated HER2-positive locally advanced unresectable or metastatic G/GEJ adenocarcinoma were enrolled. In phase 1a, patients underwent a 3+3 escalating dose design, receiving oral pyrotinib (240mg, 320mg, or 400mg daily), intravenous camrelizumab (200mg), and CapeOX (oxaliplatin 130mg/m2 on day 1 and capecitabine 1000mg/m2 twice daily for two weeks) every 3 weeks until progression, intolerable toxicity or consent withdrawal. The recommended phase 2 dose (RP2D) of pyrotinib was determined and used in the phase 1b. The primary endpoints were the safety, maximum tolerated dose (MTD), RP2D, and confirmed objective response rate (ORR). This trial was registered with chictr.org, number ChiCTR2000029717. Among 41 patients, 10 were in phase 1a (3at 240mg, 3at 400mg, and 4at 320mg due to one patient withdrawing consent), and 31 were in phase 1b. In phase 1a, the MTD of pyrotinib was 320mg daily due to dose-limiting toxicities (diarrhea [n=3] and vomiting [n=1]) observed at 400mg. Based on all available data, the RP2D of pyrotinib was set at 320mg. Among 41 patients, 20 patients (48.8%) developed grade ≥3 treatment-emergent adverse events (TEAEs), and four patients (9.8%) had any grade serious adverse events. No deaths occurred due to TEAEs. Among 27 patients who received the RP2D of pyrotinib and had a post-baseline tumor assessment, two patients (7.4%) achieved a confirmed complete response, and 19 patients (70.4%) achieved a confirmed partial response, resulting in a confirmed ORR of 77.8% (95% CI: 57.7-91.4). Pyrotinib plus camrelizumab and chemotherapy showed promising efficacy in the first-line treatment of advanced HER2-positive G/GEJ cancer. The safety profile was consistent with known toxicities of the agents, and no new or unexpected safety signals were identified. This study was funded by the Beijing Xisike Clinical Oncology Research Foundation (Y-HR2019-0377).

Pyrotinib versus placebo in combination with trastuzumab and docetaxel as first line treatment in patients with HER2 positive metastatic breast cancer (PHILA): randomised, double blind, multicentre, phase 3 trial

ObjectiveTo assess the efficacy and safety of pyrotinib (an irreversible pan-HER (human epidermal growth factor receptor) inhibitor), trastuzumab, and docetaxel compared with placebo, trastuzumab, and docetaxel for untreated HER2 positive...

Afatinib in patients with recurrent/metastatic (RM) squamous cell carcinoma of the head and neck (SCCHN) harboring alterations in the HER pathway: Results of the B1 cohort of the EORTC-HNCG-1559 trial (UPSTREAM).

6034 Background: Afatinib (A) is an irreversible pan-HER inhibitor that improved PFS in second line RM SCCHN after platinum (LUX-H&N1 trial). In a retrospective biomarker analysis, subgroups of pts with p16 negative, PTEN high or EGFR amplification SCCHN seem to benefit the most. Other studies suggest that A has activity in tumors with HER2 gene activation. Methods: The UPSTREAM trial is a biomarker-driven umbrella trial of targeted therapies and immunotherapy for RM SCCHN (post-platinum, ECOG 0-1, measurable disease). The B1 cohort was a phase II, randomized, open-label substudy evaluating the efficacy of afatinib (orally, 40mg/day) vs physician’s choice (ctrl) (2:1 ratio) in pts with p16 negative RM SCCHN harboring one of the following biomarkers (fresh biopsy): EGFR mutation(mut)/amplification(amp) or HER2 mut/amp or PTEN high by immunohistochemistry (IHC, H-score > 150). Only KRAS/ HRAS/ NRAS wild type tumors were included. The primary endpoint was progression-free survival rate (PFSR) at 16 weeks after randomization. A 2-stage Simon optimal design was applied to arm A (H1 40%, H0 20%, 1-sided α 10%, power 90%) with 17 and 20 pts enrolled in the first and second stage respectively. Secondary endpoints included ORR, toxicity, PFS, and OS. Results: 59 RM SCCHN pts were included in the B1 cohort (A: n = 40, ctrl: n = 19). Out of the 59 patients, EGFR amp, HER2 amp and HER2 mut were found in 6 pts (10%), 1 pt (2%) and 1 pt (2%), respectively. PTEN (IHC) was high in 97%. 55 pts were evaluable (A: n = 38, ctrl: n = 17) (median age: 62, oral cavity: 29%, oropharynx: 36%, hypopharynx: 24%, larynx: 11%). 69% of pts had received ≥ 2 lines of systemic treatment for RM disease. 80% were pretreated with cetuximab. In arm A, 13/38 patients were alive and free of objective progression at 16 weeks (34.2%, 1-sided 90%CI, 23.9±inf), meeting the predefined criteria of success. The PFSR at 16W in ctrl arm was 29.4% (5/17). 4 objective responses were observed in arm A (3 PR and 1 CR) (ORR 11%, 95%CI 2.9-24.8%) and 1 PR in ctrl arm (6%). The median duration of response under A was 5.7 mo (range: 3.7-25.9 mo). All responding patients had previously been treated with cetuximab, were PTEN high but none had EGFR or HER2 alterations. The median PFS and OS were 2.2/2.4 mo and 7.2/5.0 mo in the A/ctrl arms, respectively. Grade ≥3 drug-related AEs were reported in 31% in arm A and 12% in ctrl arm. The most frequent drug-related AE with A were diarrhea (67%, G3:8%), rash (41%, G3: 3%), fatigue (31%, no G3) and mucositis (23%, G3: 3%). Conclusions: The B1 substudy met its primary endpoint of PFSR at 16 weeks. Responses were seen in cetuximab pre-treated pts. However, the clinical activity of A in this biomarker-selected population remains limited. Translational research is ongoing to better define potential biomarkers. Clinical trial information: NCT03088059 .

Abstract 3999: Combination of T-DXd with the irreversible pan-HER TKI afatinib drives combination benefit in HER2-low gastric and lung tumors

Abstract Background: Trastuzumab deruxtecan (T-DXd) has been approved for advanced or metastatic breast, gastric and lung cancers. It has been postulated that irreversible tyrosine kinase inhibitors (TKIs) increase HER2 internalization, thus enhancing T-DXd efficacy. Here we report studies evaluating the combination of TKIs with T-DXd in preclinical models of HER2-low disease. Methods: Surface HER2 was measured with flow cytometry quantifying fluorescently labelled trastuzumab on the surface of HER2-low cell lines HCC38 and HCC1171 treated with afatinib, neratinib, lapatinib or tucatinib for 12 h. HER2 receptor dynamics on cell surface was also measured in NCI-N87 (HER2 IHC 3+) and HCC38 at 24, 48, 72 and 96 h post-treatment with afatinib, and recovery was measured up to 72 h after drug washout. In vivo efficacy was established with T-DXd alone and in combination with afatinib or tucatinib in two gastric patient-derived xenograft (PDX) models, HD-2 (HER2 IHC 1+) and FU (HER2 IHC 2+), and one lung PDX model, MEDI-NSCLC-04 (HER2 IHC 1+). Results: In both HER2-low cell lines, treatment with irreversible inhibitors neratinib or afatinib led to enhanced internalization with a greater than 50% surface HER2 reduction, while treatment with the reversible inhibitors lapatinib or tucatinib led to 13-35% higher surface HER2 levels. Washout experiments indicated that receptor recovery at the plasma membrane begins between 4-12h post-washout of afatinib, with complete recovery at about 72h. Tumor shrinkage was significant in HER2-low gastric PDXs when T-DXd was combined with afatinib, but not with tucatinib. In HD-2, T-DXd 10mg/kg with afatinib 20mg/kg, and T-DXd 3mg/kg with tucatinib 25mg/kg combinations resulted in 74% and -3% (with respect to T-DXd) tumor growth inhibition (TGI), respectively. In FU, T-DXd 3mg/kg with afatinib 20mg/kg, and T-DXd 3mg/kg with tucatinib 25mg/kg combinations resulted in 80.3% and 24.7% TGI, respectively. Similarly, in MEDI-NSCLC-04 PDX model, benefit was observed in 5mg/kg T-DXd combination with 20mg/kg QD afatinib (95.3% TGI with respect to monotherapy T-DXd), but not with tucatinib (0.37% TGI). Intermittent schedules (QW, MWF) were also tested for afatinib in combination with T-DXd, with the MWF showing 49.2% TGI and QW showing 33.0 %TGI. In tissue samples from MEDI-NSCLC-04 efficacy study, γH2AX was detected by IHC in T-DXd and T-DXd+afatinib combination. In western blots, a decrease in HER2 as well as pHER2 was observed in response to T-DXd and 20mg/kg afatinib treatment, but not with tucatinib. Conclusions: In HER2 low gastric and lung preclinical models, T-DXd combined with irreversible pan-HER inhibitor induced superior antitumor effects compared to single agents or the combination of T-DXd with reversible inhibitors. These data support the rationale to test combination of T-DXd and pan-HER irreversible inhibitors in the clinical setting. Citation Format: Deepa Bhavsar, Laura Kazlauskas, Flavia Michelini, Matt Griffin, Matt Wilson, Fabiola Cecchi, Liz Croydon, Kim Maratea, Elisa de Stanchina, Yelena Y. Janjigian, Maurizio Scaltriti, Theresa Proia, Jerome Mettetal. Combination of T-DXd with the irreversible pan-HER TKI afatinib drives combination benefit in HER2-low gastric and lung tumors. [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 3999.

180 (PB060) - Neratinib plus dasatinib has pre-clinical efficacy against HER2-positive breast cancer

Background: HER2-targeted therapies have revolutionised the treatment of HER2-positive breast cancer. However, de novo resistance or the emergence of acquired resistance is a persistent clinical problem. The aim of this study was to investigate neratinib, an irreversible pan-HER inhibitor, in combination with the multi-kinase inhibitor dasatinib, currently used to treat certain leukemias, as a new treatment option for patients with HER2-targetted therapy resistant HER2-positive breast cancer.

LBA19 Pyrotinib or placebo in combination with trastuzumab and docetaxel for HER2-positive metastatic breast cancer (PHILA): A randomized phase III trial

Pyrotinib (pyro, an irreversible pan-HER inhibitor) plus capecitabine has demonstrated antitumor activity as 2L treatment in HER2-positive metastatic breast cancer (mBC) (Lancet Oncol 2021). Pertuzumab combined with trastuzumab (H) and docetaxel (T) is the standard 1L dual anti-HER2 therapy for HER2-positive mBC. Here, we report the efficacy and safety of dual anti-HER2 regimen of Pyro+H+T (PyroHT) compared with placebo+H+T (HT) in untreated HER2-positive mBC. Eligible pts were randomized (1:1) to receive oral Pyro (400 mg QD) or placebo, both combined with intravenous H (8 mg/kg in cycle 1 and 6 mg/kg in subsequent cycles) and T (75 mg/m2) on day 1 of each 21-day cycle. The primary endpoint was PFS per investigator (INV). Data cutoff for this prespecified interim analysis was May 25, 2022. Between May 2019 and Jan 2022, 590 eligible pts from 40 centers were randomized and treated (297 with PyroHT and 293 with HT). Median follow-up was 15.8 mo and 14.9 mo, respectively. PFS per INV of PyroHT was significantly longer than HT (median, 24.3 mo [95% CI 19.1-33.0] vs 10.4 mo [95% CI 9.3-12.3]; HR, 0.41 [95% CI 0.32-0.53]; P < 0.0001; subgroup results are in the Table), which was consistent with PFS per IRC (median, 33.0 vs 10.4 mo; HR, 0.35 [95% CI 0.27-0.46]). The most common grade ≥3 TRAEs were decreased neutrophil count (62.6% vs 65.2%), decreased WBC count (53.2% vs 50.9%), and diarrhea (46.5% vs 3.1%). Grade 3 diarrhea occurred mainly during cycle 1 and substantially decreased in cycle 2 and thereafter. No grade 4 or 5 diarrheas occurred. Serious TRAEs occurred in 24.9% vs 6.1% of pts and treatment-related deaths occurred in 0 vs 0.3% of pts, respectively.Table: LBA19Subgroup analyses of PFS per INVPyroHTHTHR (95% CI)n/NPFS (median, mo)n/NPFS (median, mo)ECOG performance status055/16427.596/15510.40.42 (0.30-0.59)144/13322.282/13810.30.39 (0.27-0.57)Prior neoadjuvant or adjuvant therapyYes49/14827.891/15310.60.43 (0.30-0.62)No50/14919.587/14010.30.38 (0.26-0.54)Prior neoadjuvant or adjuvant trastuzumabYes11/46NA32/429.30.23 (0.12-0.46)No88/25121.9146/25110.40.45 (0.34-0.59)Treatment-free interval with prior adjuvant therapy≥12 - <24 mo8/30NA25/388.30.22 (0.10-0.50)≥24 mo34/9922.258/10013.00.57 (0.37-0.87)n/N=events/patients. HRs are from unstratified analyses. Open table in a new tab n/N=events/patients. HRs are from unstratified analyses. PyroHT significantly prolonged PFS over HT in pts with HER2-positive mBC. The safety is manageable. To our knowledge, this is the second phase 3 trial to demonstrate PFS benefit from dual HER2 inhibition in 1L treatment of HER2-positive mBC.

Abstract 1237: Randomized phase II trial of pre-operative afatinib in non-metastatic head and neck squamous cell carcinoma patients: Identification of predictive biomarkers of response

Abstract Background: Epidermal growth factor receptor (EGFR) inhibitors, including monoclonal antibodies and tyrosine kinase inhibitors, have limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients. In the randomized phase II PREDICTOR trial, we aimed at identifying predictive and pharmacodynamics biomarkers of 2-4 weeks afatinib (an irreversible pan-HER inhibitor) versus no treatment in the pre-operative setting (NCT01415674). We previously reported a 59% metabolic response rate on PET imaging in the afatinib arm. We report here the evaluation of predictive genomic and transcriptomic biomarkers of afatinib efficacy. Patients and Methods: All patients (41 in the afatinib arm and 20 in the no treatment arm) underwent a pre-treatment biopsy. We performed targeted DNA sequencing using an in-house NGS panel of 571 genes on baseline biopsies from 56 patients, and RNA-sequencing (RNAseq) in 54 patients. In the afatinib arm, 26 patients had paired pre- and post-treatment tumor samples. DNA and RNA alterations were correlated with metabolic response to afatinib using PET imaging, as well as overall survival (OS). Results: Most frequent molecular alterations, including known activating mutations and/or focal amplifications for oncogenes or homozygous deletions and inactivating mutations for tumor suppressor genes, involved genome integrity (TP53 [70%]), cell cycle (CCND1 [38%], CDKN2A [32%], CDKN2B [14%]), senescence (TERT [23%]), Wnt signaling (NOTCH1 [16%]), and the PI3K pathway (PIK3CA [14%]). In the afatinib arm, metabolic response was observed in 1 out of 7 patients (14%) and in 19 out of 28 patients (68%) in the Wnt altered and unaltered groups (p = 0.03, fisher exact test), respectively. In the whole cohort of patients, homozygous deletions of both CDKN2A and CDKN2B correlated with shorter OS, with 6-year survival of 22% in the CDKN2A/B altered group and 70% in the CDKN2A/B wild-type group (p = 0.004; log-rank test). In the afatinib treated patients, using a generalized linear mixed model with a patient as random effect and a quasi-binomial family, the ratio of B cells expression levels in the post-treated versus pre-treated samples was significantly higher in responder as compared to non-responder patients (p = 0.001). Conclusions: Wnt signaling pathway alterations and treatment-related dynamic changes in B cells proportions were identified as predictive and pharmacodynamics biomarkers of afatinib efficacy. CDKN2A/B homozygous deletions were associated with a poor prognosis in HNSCC patients treated with upfront surgery. Citation Format: Grégoire Marret, Maud Kamal, Jocelyn Gal, Stéphane Temam, Jerzy Klijianenko, Jean-Pierre Delord, Caroline Hoffmann, Gilles Dolivet, Olivier Malard, Jerôme Fayette, Olivier Capitain, Caroline Even, Sébastien Vergez, Lionel Geoffrois, Frédéric Rolland, Philippe Zrounba, Laurent Laccourreye, Joël Guigay, Nicolas Aide, Valérie Bénavent, Constance Lamy, Elodie Girard, Marta Jimenez, Ivan Bièche, Christophe Le Tourneau. Randomized phase II trial of pre-operative afatinib in non-metastatic head and neck squamous cell carcinoma patients: Identification of predictive biomarkers of response [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 1237.

Abstract 3333: Pre-clinical investigation of neratinib plus dasatinib in breast cancer

Abstract Background: HER2-targeted therapies have revolutionized the prognosis of HER2-positive (HER2+) breast cancer (BC). However, the emergence of acquired resistance or de novo resistance are persistent clinical problems. The objective of this study was to examine neratinib (NER), an irreversible pan-HER inhibitor approved for early-stage and metastatic HER2+ BC, and dasatinib (DAS), a multi-kinase inhibitor used to treat certain leukemias, as a new treatment for HER2-targeted therapy resistant HER2+ BC and triple negative BC (TNBC). Methods: The anti-proliferative effect of NER plus DAS was screened in a panel of 20 breast cancer cell lines using a 5-day acid phosphatase assay. This panel included 12 HER2+ (3 trastuzumab (TRAS) sensitive (SKBR3, BT474, EFM192A), 2 acquired TRAS resistant (BT474-T, SKBR3-T), 3 innately TRAS resistant (HCC1954, HCC1569, JIMT1), and 4 acquired neratinib resistant cell lines (HCC1954-N, HCC1569-N, SKBR3-N, BT474-N), 6 triple negative (BT20, HCC1143, HCC1937, MDA-MB- 157/231/468), and 2 estrogen receptor positive (ER+) cell lines (MCF7, ZR-75-1). Drug synergy was examined using Combenefit and Calcusyn software. Combination Index (CI) values &amp;lt; 1 indicated synergy, &amp;gt; 1 antagonism. Neratinib IC50 values &amp;lt; 150nM were considered physiologically relevant. Prolonged exposure in vitro efficacy was tested in these cell lines by treating twice weekly with NER +/- DAS until resistance emerged. Cell confluence was then quantified using a crystal violet-based method. The safety and efficacy of NER (10 mg/kg) plus DAS (15 mg/kg) was investigated in vivo in HER2+ HCC1954 xenografts in BALB/c nude mice. Results: NER was highly effective against HER2+ BC cell lines regardless of TRAS sensitivity (IC50 values range 1-172 nM), showed moderate activity in TNBC (IC50 value range 31 nM - &amp;gt;1 μM), and did not achieve &amp;lt;1 μM activity in ER+ BC cell lines. NER resistant cell lines had IC50 values greater than 150 nM DAS alone displayed IC50 values from 8-135 nM in 4/6 TNBC cell lines, with all 6 TNBC cell lines sensitive to either the single agents or the combination at concentrations less than 100 nM. DAS enhanced the anti-proliferative activity of NER in the HCC1954, HCC1569, JIMT1 cell lines (CI values = 0.27-0.62). The NER/DAS combination overcame NER resistance in all acquired NER-resistant cell line models and no antagonism was observed in any cell line tested. Matrix assays confirmed synergy at low nanomolar concentrations of both drugs. NER plus DAS had a greater long term in vitro anti-proliferative effect over NER in HCC1954, HCC1569, and JIMT1 cells, preventing or delaying the development of NER resistance. NER plus DAS was well tolerated in mice for over 60 days. The combination was better than single agents and showed prolonged anti-tumor effect against HCC1954 xenografts in vivo (p=0.02). Conclusions: This study suggests that the addition of DAS to NER may have potential as a new treatment strategy for BC, particularly for HER2+ BC. Citation Format: Neil T. Conlon, Sandra Roche, Fiona O'Neill, Justine Meiller, John Crown, Denis M. Collins. Pre-clinical investigation of neratinib plus dasatinib in breast cancer [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 3333.

Selected Articles from This Issue

Neratinib is an irreversible pan-HER inhibitor approved for patients with HER2 amplified breast cancer. MutHER trial evaluated the antitumor activity of neratinib and neratinib in combination with fulvestrant for HER2 non-amplified metastatic breast cancer harboring activating mutations in HER2, a population otherwise not eligible for HER2-targeted therapy. The study demonstrated the antitumor effect of neratinib, especially in invasive lobular breast cancer. Interestingly, responses were observed with the addition of trastuzumab to neratinib upon disease progression, suggesting dual HER2 blockade may be important in targeting HER2 mutation. Further investigation of trastuzumab in combination with neratinib is therefore warranted.Fusions involving the NTRK1/2/3 genes have been identified in many tumor types, several of which often metastasize to the CNS. Entrectinib is a potent TRK inhibitor that was specifically designed to penetrate and remain in the CNS. Demetri and colleagues present the latest results of an integrated analysis of three phase I/II trials: entrectinib continued to induce deep and durable systemic and intracranial responses across tumor types and was well tolerated. Entrectinib could address the need for a CNS-active treatment in NTRK fusion-positive solid tumors; therefore, encouraging broader NTRK fusion testing would improve patient access to effective, personalized treatments.Concurrent radiation therapy (RT) with cetuximab, an anti-EGFR monoclonal antibody (mAb), is a standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, control in high-risk disease is suboptimal. CTLA-4+ T regulatory cells (Treg) dampen cellular immunity and correlate negatively with clinical outcomes. Ferris and colleagues conducted a phase I trial to evaluate the safety and preliminary efficacy of the CTLA-4 targeting mAb, ipilimumab, with standard, fixed cetuximab-RT. They identified the recommended phase II dose and observed acceptable PFS and OS without cytotoxic chemotherapy. High expression of coinhibitory receptors PD1/LAG3/CD39 on baseline tumor infiltrating Treg was marginally associated with worse PFS. This is the first trial to combine CTLA-4 targeting with definitive cetuximab-RT in HNSCC, integrating immune checkpoint inhibitors into curative-intent RT platforms.Otani and colleagues used immunofluorescence staining and RNA-seq to uncover a significant induction of MDSC cell populations into brains of tumor-bearing mice treated with oHSV. This was mediated by NOTCH induction. In human patients, NOTCH signaling also correlated with a higher myeloid cell infiltration. The tumor infiltrating myeloid cells further spread NOTCH signaling in tumors and also created a CCL-2 feed-forward loop to induce more myeloid cells into the tumors. Increased CCL2 was also observed in serum of GBM patients treated with oHSV. Blockade of NOTCH signaling rescued these immunosuppressive effects and enhanced antitumor immunity.

Discovery of N-(3-bromo-1H-indol-5-yl)-quinazolin-4-amine as an effective molecular skeleton to develop reversible/irreversible pan-HER inhibitors

Pan-HER inhibitors exhibit extensive biological activity and offer unique advantages and usually bind to targets in an irreversible manner. Owing to the off-target toxicity of irreversible inhibitors, reversible pan-HER inhibitors are desirable. Herein, we describe the process of N-(ring structure fused phenyl)quinazoline-4-amine-based design, synthesis, and biological evaluation of pan-HER inhibitors in vitro and in vivo. Compound C5, with the molecular skeleton of N-(3-bromo-1H-indol-5-yl)-quinazolin-4-amine, displayed irreversible binding just like other effective pan-HER inhibitors. To our surprise, compound C6, which possessed the same skeleton, was found to be a high-strength reversible pan-HER inhibitor. This compound was capable of inhibiting HER1s (such as EGFR T790M/L858R and WT), HER2, and HER4 and can be considered as a breakthrough in the development of pan-HER inhibitors. Altogether, N-(3-bromo-1H-indol-5-yl)-quinazolin-4-amine can serve as an effective molecular skeleton for developing both reversible and irreversible pan-HER inhibitors in the following discovery of antitumor drugs.

Abstract P5-10-05: HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers

Abstract HER2 is amplified in about 20% of breast cancers. HER3 is as essential as HER2 for maintaining cell viability in HER2+ breast cancer cells. Inhibition of HER2 tyrosine kinase activity results in upregulation of HER3 transcription and phosphorylation. We sought to identify HER3 binding partners upon pharmacological inhibition of HER2 using the irreversible pan HER inhibitor neratinib. We immunoaffinity-purified HER3 from HER2+ BT474 cells treated ± neratinib. Following immunoprecipitation using a HER3 antibody, binding partners were released under reducing conditions. Mass spectrometry experiments identified non-muscle myosin IIA (NMIIA) increased upon inhibition of HER2 with neratinib and decreased under DMSO control treatment from HER3 immunoprecipitates. To validate the presence of NMIIA, we performed immunoprecipitation experiments in HER2+ BT474 and MDA-MB-453 cells using a HER3 antibody. Immunoblots showed increased NMIIA levels upon treatment with 200nM neratinib for 24 hours in both cell lines. Myosin heavy chain 9 (MYH9) gene encodes the protein NMIIA. NMIIA localizes to actin stress fibers and has been implicated in regulation of cell contractility and stress fiber organization.To test if there is an interaction between HER3 and NMIIA, we immunoprecipitated NMIIA from BT474 and MDA-MB-453 cells using an NMIIA antibody. The products were analyzed using immunoblots. The results indicated that HER3 levels were increased upon inhibition of HER2 with 200 nM neratinib for 24 hours.We next examined long term overall survival of primary breast cancer patients who have high and low levels of gene expression for MYH9 from the METABRIC cohort. We observed that patients with high levels of MYH9 have a statistically significant worse overall survival versus patients who express low levels of MYH9.We next sought to examine overall levels of HER3 and MYH9 mRNA and protein upon treatment with neratinib in HER2+ breast cancer whole cell lysates. We observed that mRNA and protein levels of both HER3 and MYH9 increased upon HER2 inhibition with 24 hours of neratinib treatment. To further investigate the role of NMIIA, we knocked out NMIIA in BT474 and MDA-MB-453 using inducible lentiviral shRNA targeting MYH9. This system utilizes the Tet-On 3G induction system and is a highly controlled system that consists of an inducible RNA polymerase II promoter. In the presence of doxycycline, the TRE3G promoter is bound and activated by the constitutively expressed Tet-On 3G trans-activator protein. Non-targeting shRNA was used as control and cells were selected in 1-2 ug/ml puromycin. Western blot analysis demonstrated that cells infected with lentiviral shRNA targeting MYH9 treated with doxycycline had reduced protein expression of NMIIA. BT474 and MDA-MB-453 transduced with shMYH9 were used to perform growth, migration, and invasion assays. The results indicated that MYH9 knockdown + neratinib treatment suppresses BT474 and MDA-MB-453 growth, migration, and invasion. Currently, in vivo xenograft experiments examining BT474 and MDA-MB-453 tumor growth ±NMIIA shRNA in the presence or absence of neratinib treatment are underway. In conclusion, we have identified that NMIIA mRNA and protein levels are increased in the presence of neratinib treatment. Our data reveals that HER2 inhibition increases NMIIA to promote HER2+ breast cancer growth. Citation Format: Samar M Alanazi, Rosalin Mishra, Hima Patel, Long Yuan, Mary Kate Kilroy, Joan T Garrett. HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-05.

Abstract CT026: A phase II trial of neratinib (NER) or NER plus fulvestrant (FUL) (N+F) in HER2 mutant, non-amplified (HER2mut) metastatic breast cancer (MBC): Part II of MutHER

Abstract Introduction: The irreversible pan-HER inhibitor NER showed modest single agent activity for HER2mut MBC in Part I of MutHER trial. In Part II, we hypothesized that (1) N+F would improve activity in estrogen receptor positive (ER+) HER2mut MBC due to ER-HER2 crosstalk and (2) dual HER2 blockade by adding trastuzumab at disease progression (PD) could overcome resistance. Methods: Pts with ER+HER2mut MBC were enrolled to 2 cohorts (FUL treated or naive) to receive N+F with diarrhea prophylaxis. ER- pts received NER in an exploratory ER- cohort. Trastuzumab was added at PD if approved by insurance. Simon's Minimax 2-stage phase II design with the primary endpoint of clinical benefit rate (CBR: rates of complete/partial response [CR/PR] plus stable disease [SD] &amp;gt;24 weeks [wks]), with anticipated vs null hypothesis being CBR of 55% vs 35% (FUL treated) or 65% vs 40% (FUL naïve) with 80% power, 1 sided 0.05 alpha, was used. Secondary endpoints included progression free survival (PFS) and adverse events (AEs). Serial blood samples were analyzed for circulating tumor DNA (ctDNA) by Guardant360 for concomitant mutations, HER2mut variant allele frequency (VAF) dynamics, and resistance mechanisms. Results: Between Sep. 2015 and Oct. 2020, 40 pts with HER2mut MBC were enrolled, completing the 1st stage of each ER+ cohort. 35 pts (21 FUL treated, 10 FUL naïve, 4 ER-) were evaluable for response, with median age 63 (35-82) years, 3 (0-12) prior MBC regimen, lobular BC in 13 (37%) and visceral mets in 32 (91%) pts. 21 (68%) ER+ pts had prior CDK4/6 inhibitor. All but 1 pt has come off study due to PD. Table 1 shows the efficacy by cohort. Further enrollment is closed per protocol. Adding trastuzumab at PD induced CB in 4 (3 PR, 1 SD≥24 wks) of 5 pts (1 ER-, 4 ER+), with PFS 28 (95% CI 18~NA) wks. Common AEs across cohorts were diarrhea (G3 21%) and fatigue (G3 5%). No G4 AEs. ctDNA HER2mut was detected in 72% (23/32) baseline (BL) samples tested. In pts with paired samples, HER2mut VAF decreased at C1D15/C2D1 from BL in 75% (15/20) and rose in 89% (16/18) at PD. Acquired HER2mut, including the T798I gatekeeper mutation, were detected in 2 pts at PD. Mutations in TP53 (53%), PIK3CA (43%), and CDH1 (35%) were common, but none significantly associated with PFS in all or ER+ pts. Conclusions: NER, or N+F, is active for HER2mut MBC with good tolerability. Adding trastuzumab at PD induced further response, supporting dual HER2 blockade for HER2mut MBC. Table 1.EfficacyCohortFUL treatedFUL naïveER-Best Response, n evaluablen = 21n = 10n = 4CR, n100PR, n431SD (≥ 24 wks), n300SD (&amp;lt; 24 wks), n1030PD, n343CBR, n with CB/total n evaluable, % (95% CI)8 of 20*, 40% (19~64%)3 of 10, 30% (7~65%)1 of 4, 25% (0.6~81%)mPFS (95% CI), wks, ITT (n)24 (16~31) wks, (n = 24)20 (8~NA) wks, (n = 11)8.5 (8~NA) wks, (n = 5)*20 of 21 pts are evaluable for CBR in the FUL treated Cohort as 1 pt had SD as best response and treatment is still ongoing. ITT (intent to treat) population is used for mPFS estimate. Citation Format: Cynthia X. Ma, Jingqin Luo, Rachel A. Freedman, Timothy Pluard, Julie Nangia, Janice Lu, Frances Valdez-Albini, Melody Cobleigh, Jason Jones, Nancy U. Lin, Eric Winer, P. Kelly Marcom, Shana Thomas, Jill Anderson, Brittney Haas, Kimberly M. Hamann, Richard Bryce, Alshad S. Lalani, Lisa Carey, Matthew Goetz, Feng Gao, Gretchen Kimmick, Mark Pegram, Matthew J. Ellis, Ron Bose. A phase II trial of neratinib (NER) or NER plus fulvestrant (FUL) (N+F) in HER2 mutant, non-amplified (HER2mut) metastatic breast cancer (MBC): Part II of MutHER [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 CT026.

45P Long-term efficacy of neratinib in HER2-positive early-stage breast cancer: Overall survival and central nervous system outcomes from the phase III ExteNET trial

Neratinib (Nerlynx®) is an irreversible pan-HER inhibitor that significantly improves invasive disease-free survival (iDFS) vs placebo as extended adjuvant therapy in HER2-positive (HER2+) early-stage breast cancer (eBC) after trastuzumab-based therapy. The phase III ExteNET trial (NCT00878709) showed an absolute iDFS benefit of 2.5% and distant disease-free survival benefit of 1.7% with neratinib after 5 years’ follow-up. The brain is a common first site of metastasis after HER2-directed adjuvant therapy for eBC.

Abstract PS4-07: Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference

Abstract Background: Neratinib is a potent, irreversible pan-HER inhibitor that inhibits the ErbB family members EGFR, HER2, and HER4 and downstream signal transduction of these receptors. Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks druggable levels of receptors for estrogen, progesterone and HER2 and therefore challenging to treat. There is evidence that some cases of TNBC have activated signaling pathways mediated by ErbB family members that may contribute to aggressive behavior. The purpose of this preclinical study was to identify and validate kinases whose targeting may enhance the antitumor activity of neratinib in TNBC cell lines. Methods: In vitro proliferation assays were used to evaluate the efficacy of neratinib in TNBC cell lines. Baseline and post-neratinib-treatment expression of EGFR and phosphorylated EGFR (phospho-EGFR) were assessed via Western blot analysis in 18 TNBC cell lines. Reverse-phase protein array (RPPA) was used to profile and validate the signaling networks induced by neratinib. To identify potential targets or pathways that may synergize with neratinib treatment, we performed high-throughput RNA interference (HT RNAi) screening using a 709-kinome library. CellTiter-Blue, sulforhodamine B, and soft-agar assays were performed to evaluate the antiproliferative effect of neratinib alone and with target inhibitor. Mammary fat pad xenograft models were used to evaluate the efficacy of neratinib alone or with inhibitor in vivo. Results: In vitro proliferation assays showed that the half-maximal inhibitory concentration (IC50) of neratinib in tested TNBC cell lines ranged from 0.16 µM to 1.25 µM. RPPA and Western blot analyses revealed that the efficacy of neratinib correlated with phospho-EGFR expression levels across the TNBC cell lines tested (R2 = 0.3245). Among the tested TNBC cell lines, SUM149 cells (PIK3CA wild-type) were selected for high throughput RNAi screening because this cell line has high EGFR expression and is moderately sensitive to neratinib (IC50 = 0.35 µM). We identified the 40 most relevant kinase targets by the sensitivity index analysis, and further pathway analysis identified PI3K/AKT/mTOR (drug: everolimus) and MAPK (drug: trametinib) as major canonical pathways whose targeting enhanced the cytotoxic effect of neratinib. Everolimus (mTOR inhibitor) produced a strong antiproliferative effect when combined with neratinib in most tested TNBC cell lines (12 of 15 cell lines; combination index [CI] values, 0.1-0.5) and was more effective in PIK3CA-mutated compared to wildtype cell lines. Trametinib (MEK inhibitor) showed a moderate antiproliferative effect (effective in 10 of 15 cell lines; CI values, 0.2-0.9). Synergistic antitumor effects of neratinib combined with everolimus or with trametinib were also observed in anchorage-independent growth conditions (P &amp;lt; 0.05). In vivo experiments demonstrated that neratinib plus everolimus and neratinib plus trametinib combinations inhibited tumor growth in the SUM149 xenograft model for than single drug (neratinib, 42.3% growth inhibition; everolimus, 29.7%; trametinib, 47.1%; neratinib plus everolimus, 69.7%; neratinib plus trametinib, 77.7%; P &amp;lt; 0.0001). Conclusion: Combining neratinib with everolimus or with trametinib enhanced the antitumor effects of these drugs in TNBC regardless of PIK3CA mutation status, and clinical investigations evaluating these combination regimens for the treatment of TNBC are warranted. Citation Format: Jangsoon Lee, Troy Pearson, Huey Liu, Jon A. Fuson, Toshiaki Iwase, Irmina Diala, Alshad S. Lalani, Lisa D. Eli, Debu Tripathy, Bora Lim, Naoto T. Ueno. Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-07.

Abstract PD3-03: Continued efficacy of neratinib in patients with HER2-positive early-stage breast cancer: Final overall survival analysis from the randomized phase 3 ExteNET trial

Abstract Background: Neratinib (NERLYNX®) is an irreversible pan-HER inhibitor that significantly improves invasive disease-free survival (iDFS) compared with placebo when given as extended adjuvant therapy in patients with HER2-positive (HER2+) early breast cancer after trastuzumab-based adjuvant therapy. In the phase 3 ExteNET trial, an absolute iDFS benefit of 2.5% and distant disease-free survival (DDFS) benefit of 1.7% were observed with neratinib after 5 years’ follow-up. As reflected in the approved indication by the European Medicines Agency (EMA), patients with hormone receptor-positive (HR+) disease who initiated neratinib treatment within 1 year of completing trastuzumab (HR+/≤1 year) experienced an absolute iDFS benefit of 5.1% and DDFS benefit of 4.7% at 5 years. In HR+/≤1 year patients with residual disease after neoadjuvant therapy, absolute 5-year iDFS and DDFS benefits of 7.4% and 7.0%, respectively, were observed. Here we report the final protocol-defined, event-driven analysis of overall survival (OS) from ExteNET, and provide descriptive analyses of subgroups of primary interest according to the EU label and current clinical practice in early-stage HER2+ disease. Methods: ExteNET was a multicenter, randomized, double-blind, placebo-controlled phase 3 trial of women with early-stage HER2+ breast cancer who had completed neoadjuvant or adjuvant trastuzumab plus chemotherapy (NCT00878709). Patients were randomly assigned to oral neratinib 240 mg/day or placebo for 1 year. Hazard ratios (HR) for OS were estimated from Cox proportional hazards models, and survival rates by the Kaplan-Meier method. The OS analysis was event-driven and powered for the intention-to-treat (ITT) population with a target of 248 events. Descriptive analyses were performed in the HR+/≤1 year subgroup per the approved indication in the EU, and in higher-risk patients, i.e. HR+/≤1 year who have residual disease after neoadjuvant therapy [i.e. those who did not achieve a pathologic complete response (pCR)]. Cut-off date: July 2019. Results: 2840 patients were randomized to study treatment (1420 per group). After a median follow-up of 8.1 years, 127 (8.9%) and 137 (9.6%) patients in the neratinib and placebo ITT groups had died, respectively. The 8-year OS rates were 90.1% (95% CI, 88.3–91.6) in the neratinib group and 90.2% (95% CI, 88.4–91.7) in the placebo group (absolute difference at 8 years -0.1%; stratified HR=0.95; 95% CI, 0.75–1.21; p=0.6914). A positive trend was seen in the prespecified HR+ subgroup (n=1631; absolute difference at 8 years 1.5%; HR=0.80; 95% CI, 0.58–1.12), and within this population, descriptive analyses suggested greater benefits with neratinib in the HR+/≤1 year subgroup (n=1334; absolute difference at 8 years 2.1%; HR=0.79; 95% CI, 0.55–1.13) and in the HR+/≤1 year subset with no pCR after neoadjuvant therapy (n=295; absolute difference at 8 years 9.1%; HR=0.47; 95% CI, 0.23–0.92). No new safety signals were reported with this long-term follow-up to 8 years. Conclusions: In this final OS analysis of ExteNET, there were fewer deaths with neratinib than placebo in the ITT population, but the results did not reach statistical significance. Analyses showed greater OS improvements with neratinib in subgroups including HR+/≤1 year, and HR+/≤1 year with residual disease after neoadjuvant therapy. These findings are consistent with the results based on the primary endpoint of iDFS, and support the use of neratinib in clinical practice in these patients. Citation Format: Frankie Ann Holmes, Beverly Moy, Suzette Delaloge, Stephen Chia, Bent Ejlertsen, Janine Mansi, Hiroji Iwata, Michael Gnant, Mark Buyse, Carlos Barrios, Tajana Silovski, Robert Separovic, Anna Bashford, Angel Guerrero-Zotano, Neelima Denduluri, Debra Patt, Erhan Gokmen, Ira Gore, John Smith, Richard Bryce, Feng Xu, Alvin Wong, Miguel Martin, Arlene Chan. Continued efficacy of neratinib in patients with HER2-positive early-stage breast cancer: Final overall survival analysis from the randomized phase 3 ExteNET trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD3-03.

Abstract 4205: The role of non-muscle myosin IIA in HER2+ breast cancers

Abstract HER2 is amplified in about 20% of breast cancers. HER3 plays a critical role in HER2 mediated tumorigenesis. It is now clear that inhibition of HER2 tyrosine kinase activity results in upregulation of HER3 transcription and phosphorylation. We sought to identify HER3 binding partners upon pharmacological inhibition of HER2 using the irreversible pan HER inhibitor neratinib. We immunoaffinity-purified HER3 from HER2+ BT474 cells treated ± neratinib. Following immunoprecipitation using a HER3 antibody, binding partners were released under reducing conditions. Mass spectrometry experiments identified non-muscle myosin IIA (NMMIIA) increased upon inhibition of HER2 with neratinib and decreased under DMSO control treatment from HER3 immunoprecipitates. To confirm the presence of NMMIIA, we performed immunoprecipitation experiments in BT474 and MDA-MB-453 cells using a HER3 antibody. Immunoblots showed increased NMMIIA levels upon treatment with 200nM neratinib for 24 hours in both cell lines. Myosin heavy chain 9 (MYH9) gene encodes the protein nonmuscle myosin of class II, isoform A (NMMIIA). NMMIIA localizes to actin stress fibers and has been implicated in regulation of cell contractility and stress fiber organization To further investigate the role of NMMIIA, we used blebbistatin, a myosin IIa inhibitor, in growth assays using BT474 and MDA-MB-453 breast cancer cells in vitro. The combination of blebbistatin and neratinib significantly decreased the proliferation in both cell lines compared to neratinib or blebbistatin treatment alone. Additionally, we knocked out myosin IIa in BT474 cells using myosin IIa specific siRNA to examine the effect of myosin IIa silencing on HER3 and Myosin IIa protein levels in the presence or absence of neratinib treatment. Results indicated that in BT474 cells knockdown of NMMIIA resulted in both decreased HER3 and Myosin IIa protein expression and that neratinib treatment rescued this effect. We next examined long term overall survival of primary breast cancer patients who have high and low levels of MYH9 gene expression from the METABRIC cohort. We observed that patients with high levels of MYH9 have a statistically significant worse overall survival versus patients who express low levels of MYH9. Currently, further experiments are ongoing to determine the mechanism of action of the Myosin IIA/HER3 complex, which could be an adaptive response to HER2 inhibitors in HER2+ breast cancers and to evaluate the role of myosin IIa on proliferation, migration, and invasion of HER2+ breast cancer cells. In conclusion, we have identified that neratinib treatment results in increased NMMIIA bound to HER3. Further studies to determine if NMMIIA could play a role in invasion of HER2+ breast cancer cells and/or resistance to HER2 inhibitors are ongoing. Citation Format: Samar M. Alanazi, Rosalin Mishra, Long Yuan, Hima Patel, Joan Garrett. The role of non-muscle myosin IIA in HER2+ breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4205.

HER2-Mediated Internalization of Cytotoxic Agents in ERBB2 Amplified or Mutant Lung Cancers.

Amplification of and oncogenic mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody-drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy. SIGNIFICANCE: T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2. This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy.See related commentary by Rolfo and Russo, p. 643.This article is highlighted in the In This Issue feature, p. 627.

In This Issue

See article, [p. 198][1] . </br>![][2]</img> In breast cancer, some mutations in ERBB2 (encoding HER2) are associated with oncogenic addiction to HER2 signaling. Building on a phase II trial of single-agent treatment with the irreversible pan-HER inhibitor neratinib in patients with ERBB2 -

Abstract A046: Pre-clinical assessment of neratinib sensitivity and biomarkers of response

Abstract Introduction: Neratinib is an irreversible pan-HER inhibitor that has shown clinical activity against HER2-amplified (HER2+) and HER2-mutated cancers. Alteration in HER2 is currently the only biomarker of sensitivity to neratinib. To examine other potential biomarkers of sensitivity, we evaluated neratinib as a single agent in a panel of 108 cancer cell lines from 25 different primary tumour sites. In order to determine possible biomarkers of response, correlation analysis was performed to assess the relationship between genomic alterations or gene expression levels of oncogenes and neratinib response. Methods: Sensitivity to neratinib was assessed in 108 cancer cell lines (Oncolines™) by ATPlite assay after 72-hour treatment. Pearson correlation analysis was carried out between the gene expression profile of 361 relevant cancer genes for 94 of the 108 cell lines (CCLE) and neratinib sensitivity (10logIC50 values). The neratinib gene expression profile was then compared to &amp;gt; 160 reference compound profiles to exclude false positives. The mutation and copy number status of established oncogenes and tumour suppressor genes was sourced from COSMIC cell lines project and CCLE and compared to neratinib IC50 values by ANOVA and t-test. Results: Neratinib had a potent anti-proliferative effect across numerous cancer types, achieving an IC50 value of &amp;lt; 500 nM in 18 of the 108 cell lines; including breast, lung, pancreatic, colorectal, bladder, head and neck, and cervical cancers. Overall, no significant difference was observed between solid and haematological cancers. As expected, ERBB2-amplification, over-expression, and mutation correlated with neratinib response. However, sensitivity was also significantly correlated with EGFR mutations/ amplification/over-expression, amplification of MRPS28 (p=5.2 x 10-5), mutations in BRCA2 (p=0.02) and FBXW7 (p=0.008), and with both CNVs and mutations in SMAD4 (p=0.007) and PTEN (p=0.02). Gene expression of ERBB2 and RAC1, which can be activated downstream of HER2, positively correlated to neratinib response compared to reference compounds. Conclusions: Neratinib displayed broad-spectrum pre-clinical efficacy across numerous cancer types outside of HER2+ breast cancer. This study identified several novel markers of response to neratinib, which warrant further validation. Citation Format: Neil T Conlon, Jeffrey J Kooijman, Guido JR Zaman, Irmina Diala, Alshad Lalani, John Crown, Denis M Collins. Pre-clinical assessment of neratinib sensitivity and biomarkers of response [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A046. doi:10.1158/1535-7163.TARG-19-A046

P2.03-20 Potent in Vitro Activity of Tarloxotinib for HER2 Exon 20 Mutations in Lung Cancer and Mechanism of Acquired Resistance

Oncogenic HER2 (ERBB2) mutations are present in 2-3% of lung adenocarcinoma. No targeted therapy is currently approved for HER2-mutated lung cancers, and clinical trials using novel irreversible pan-HER inhibitors are underway. However, all of these irreversible pan-HER inhibitors are also active against wild-type (WT) EGFR, resulting in dose limiting toxicities. Tarloxotinib is a novel clinical-stage prodrug that releases a potent, irreversible pan-HER inhibitor (tarloxotinib-E) selectively in pathologically hypoxic regions of tumors. In this study, we evaluated tarloxotinib-E activity against various HER2 exon20 insertion mutations and explored the resistance mechanisms to tarloxotinib-E. We introduced WT HER2 or HER2 activating mutations, including A775_G776insYVMA, G776delinsVC, and P780_Y781insGSP into Ba/F3 cells by retroviral transfection. Growth inhibitory assays were performed in these Ba/F3 cells and in H1781 cells (G776delinsVC). Tarloxotinib-E resistant clones were established by exposing these Ba/F3 cells to 200nM of tarloxotinib-E after treatment with N-ethyl-N-nitrosourea. Acquired resistant cells to tarloxotinib-E were also developed from H1781 cells via chronic exposure to increasing concentrations of tarloxotinib-E. HER2 secondary mutations were detected by direct sequencing. Tarloxotinib-E displayed potent activity against WT and mutant HER2 Ba/F3 cells and H1781 cells. Furthermore, the IC50 of tarloxotinib (prodrug) for wild-type HER2 was >100 times higher than that of tarloxotinib-E (active drug). So far, we established 12 tarloxotinib-E resistant clones from Ba/F3 models (6 with A775_G776insYVMA and 6 with G776delinsVC), all of which harbored C805S secondary mutation (corresponding to C797S of the EGFR). Tarloxotinib-E exhibited potent activity for all HER2 exon 20 mutations. We identified secondary HER2 C805S mutation as a common mechanism of acquired resistance, consistent with the covalent binding mode of tarloxotinib via this residue. Additional resistant clones are currently being evaluated.