ErbB2 Tyrosine Kinase Inhibitor Research Articles

Abstract 4486: Characterization of a novel mutant selective, EGFR sparing, ErbB2 inhibitor with activity across activating mutations in systemic and CNS tumors

Abstract Alterations in ErbB2, including amplification, overexpression, insertions, and point mutations, are established oncogenic drivers in many solid tumors. These mutations are found in approximately 3-4% of breast cancers and 3% of advanced lung cancers and have emerged as mechanisms of acquired resistance to targeted therapies. ErbB2 alterations are also found in metastatic breast and lung cancers that have advanced to the brain and remain a major clinical challenge with limited therapeutic options. Currently approved ErbB2 tyrosine kinase inhibitors have inferior potency against these mutations and lack sufficient brain penetration to be an impactful treatment option for patients. Herein, we describe advanced preclinical profiling of our candidate series of brain-penetrant EGFR-sparing ErbB2 inhibitors with activity against prevalent point mutations and exon 20 YVMA insertions. Citation Format: Jennifer Fulton, Tanna Bettendorf, Abiezer Blandon, Karyn Bouhana, Richard K. Brizendine, LouAnn Cable, Mark J. Chicarelli, Michelle Crow, Brad Fell, John Fischer, Anna Guarnieri, Madison Hillman, Ravi Jalluri, Vijay Kumar, Cori A. Malinky, Rob Rieger, John Robinson, Lee Stunkard, Francis Sullivan, John I. Trujillo, Shannon Winski, Yeyun Zhou. Characterization of a novel mutant selective, EGFR sparing, ErbB2 inhibitor with activity across activating mutations in systemic and CNS tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4486.

Role of human epidermal growth factor receptor 3 in treatment resistance of anaplastic lymphoma kinase translocated non-small cell lung cancer

ABSTRACT Background ALK tyrosine kinase inhibitors (TKI) have revolutionized the treatment of ALK+ non-small cell lung cancer (NSCLC), and therapy resistance occurs in virtually all patients. Multiple TKI resistance mechanisms have been characterized, including ERBB receptor coactivation. In this study, we investigated the role of HER3 in ALK TKI resistance. Methods In vitro studies were carried out using ALK+ NSCLC cell lines H3122, H2228, and DFCI032. Pharmacological co-targeting of ALK and HER3 was investigated with ALK and ERBB TKIs, and HER3 knockdown was achieved using the CRISPR-Cas9 system. Co-localization of ALK and HER3 was investigated by immunoprecipitation (IP) and proximity ligation assay (PLA) in vitro and in vivo using six ALK+ NSCLC tumor samples. Results In all tested cell lines, combined targeting with ALK and pan-ERBB TKI resulted in marked inhibition of colony formation and long-term (72 h) downregulation of pAKT levels. HER3 knockdown resulted in multiple effects on ALK+ cell lines, including the downregulation of ALK expression and visible morphological changes (H2228). Co-immunoprecipitation (IP) and proximation ligation assay (PLA) experiments provided evidence that both ALK and HER3 could interact in vitro, and this finding was verified by PLA using ALK+ NSCLC tumors. Conclusions This study provides evidence that HER3 may mediate TKI resistance in ALK+ NSCLC. Interestingly, we were able to show that both translocated ALK and HER3 could interact. Joint targeting of ALK and HER3 could be further investigate in ALK+ NSCLC.

ErbB2pY -1248 as a predictive biomarker for Parkinson's disease based on research with RPPA technology and in vivo verification.

This study aims to reveal a promising biomarker for Parkinson's disease (PD) based on research with reverse phase protein array (RPPA) technology for the first time and in vivo verification, which gains time for early intervention in PD, thus increasing the effectiveness of treatment and reducing disease morbidity. We employed RPPA technology which can assess both total and post-translationally modified proteins to identify biomarker candidates of PD in a cellular PD model. As a result, the phosphorylation (pY-1248) of the epidermal growth factor receptor (EGFR) ErbB2 is a promising biomarker candidate for PD. In addition, lapatinib, an ErbB2 tyrosine kinase inhibitor, was used to verify this PD biomarker candidate in vivo. We found that lapatinib-attenuated dopaminergic neuron loss and PD-like behavior in the zebrafish PD model. Accordingly, the expression of ErbB2pY-1248 significantly increased in the MPTP-induced mouse PD model. Our results suggest that ErbB2pY-1248 is a predictive biomarker for PD. In this study, we found that ErbB2pY-1248 is a predictive biomarker of PD by using RPPA technology and in vivo verification. It offers a new perspective on PD diagnosing and treatment, which will be essential in identifying individuals at risk of PD. In addition, this study provides new ideas for digging into biomarkers of other neurodegenerative diseases.

Abstract PD8-07: Evaluation of Tucatinib + (Paclitaxel + Pertuzumab + Trastuzumab) followed by AC in high-risk HER2 positive (HER2+) stage II/III breast cancer: Results from the I-SPY 2 TRIAL

Abstract Background: I-SPY 2 is a multicenter, phase 2 trial using response-adaptive randomization within molecular subtypes defined by receptor status and MammaPrint (MP) risk to evaluate novel agents as neoadjuvant therapy for women with high-risk breast cancer. Tucatinib is a potent HER2 (ErbB2) tyrosine kinase inhibitor, selective for HER2 vs. epidermal growth factor receptor (EGFR) and is active vs. brain metastases. Safety and efficacy of tucatinib combined with paclitaxel, pertuzumab, and trastuzumab are unknown and were tested in a planned 10 patient (pt) safety run-in of the I-SPY 2 trial. Methods: Women with tumors ≥ 2.5cm were eligible for screening. Only pts with tumors that were HER2+ by FISH were eligible for this treatment. Treatment included tucatinib (max dose 300 mg) BID for 12 weeks with weekly paclitaxel 80 mg/m2 and trastuzumab (2 mg/kg weekly following loading), and pertuzumab (420 mg every 3 weeks following loading), followed by doxorubicin/cyclophosphamide (AC) every 2 weeks x 4. The control arm was weekly paclitaxel and trastuzumab with pertuzumab for 12 weeks followed by AC every 2 weeks x 4. All pts undergo serial MR imaging and response at 3 & 12 weeks is combined with real time pCR data to estimate, and continuously update, the predicted pCR rate for each trial arm. The goal of the trial is to identify/graduate regimens with ≥85.% Bayesian predictive probability of success (i.e. demonstrating superiority to control) in a future 300-patient phase 3 neoadjuvant trial with a pCR endpoint. This run-in arm was conducted to determine safety of combining tucatinib with paclitaxel/trastuzumab/pertuzumab, monitoring special adverse events of interest including LFT elevations and gastrointestinal toxicities.Methods: The I-SPY 2 methods have been previously published. Results: 20 pts were evaluable in tucatinib treatment arm. The control arm included 329 historical controls enrolled since April 2010. The initial tucatinib dose was 300 mg BID. After enrollment of the first 8 pts, there were 3 pts with grade 3 LFT elevations, 2 pts with grade 2/3 diarrhea, 1 pt with grade 2 neutropenia, and 1 pt with grade 3 nausea. After this safety review, the tucatinib dose was lowered to 250 mg BID. Among 5 additional pts enrolled, 3 developed grade 2/3 LFT abnormalities. The protocol was then modified to tucatinib 150 mg BID days 1-28 and then 250 mg BID days 29-84; 7 pts were treated. Safety data were reviewed after 20 pts were enrolled; the arm was then suspended due to similar LFT elevations regardless of tucatinib dose reduction or schedule. 7 of 20 pts (35%) had reversible Grade 3 or higher ALT/AST elevation (Table). No pt met criteria for Hy’s Law. In terms of efficacy, 12 of 14 evaluable pts had > 80% reduction of tumor volume by 12 weeks, measured by MRI assessment of functional tumor volume (FTV). Conclusion: The goal of the run-in arm was to determine the safety of adding tucatinib to the combination of paclitaxel/trastuzumab/pertuzumab. The addition of tucatinib resulted in unacceptable but reversible LFT elevations despite tucatinib dose reduction. Tucatinib containing therapy resulted in >80% decline in tumor volume at 12 weeks in 86% of pts. Tucatinib showed a high level of activity when combined with paclitaxel/trastuzumab/pertuzumab, but the combination is not feasible. Table: Number of pts with grade 2, 3, and 4 LFT elevations by treatment schedule (highest grade per patient per event, ALT or Treatment scheduleGrade 2 LFT elevationGrade 3 LFT elevationGrade 4 LFT elevationTucatinib 300 mg BID030Tucatinib 250 mg BID210Tucatinib 150 mg BID days 1-28 followed by 250 mg BID days 29 to 84112 Citation Format: David A Potter, Erin Roesch, Christina Yau, Ruixiao Lu, Denise Wolf, Susan Samson, Debra Stafford, Kathy S Albain, Claudine Isaacs, Meghana Trivedi, Douglas Yee, Judy Boughey, Alexandra Thomas, A. Jo Chien, Nola Hylton, Wen Li, Angela DeMichele, Jane Perlmutter, W. Fraser Symmans, Dawn L Hershman, Michelle Melisko, Laura J van 't Veer, Amy Wilson, Smita M Asare, Donald A Berry, Richard Schwab, Hope S Rugo, Laura J Esserman. Evaluation of Tucatinib + (Paclitaxel + Pertuzumab + Trastuzumab) followed by AC in high-risk HER2 positive (HER2+) stage II/III breast cancer: Results from the I-SPY 2 TRIAL [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 PD8-07.

Abstract 2424: Screen for actionable ERBB3 mutations

Abstract Activating mutations and copy number variations in ERBB genes have been identified in several cancer types, and a number of cancer drugs targeting ERBB receptors have been approved for clinical use. These drugs include monoclonal antibodies (mAb) that selectively target either EGFR (ERBB1) or ERBB2 (HER2), and tyrosine kinase inhibitors (TKI) that block the kinase activity of all three kinase-competent ERBBs, i. e. EGFR, ERBB2, and ERBB4. ERBB3 is a unique member of the ERBB family in that it does not possess potent intrinsic kinase activity, and thus cannot be directly targeted with TKIs. However, it is still able to dimerize and activate the other three ERBB family members, and as a result, critically mediate oncogenic signaling as a member of heterodimeric complexes, such as the ERBB2/ERBB3 heterodimer. Thus, changes in ERBB3 structure and function may still affect responsiveness of cancer cells to ERBB TKIs. Moreover, several mAbs directly targeting ERBB3 in cancer are currently in preclinical and clinical development. To address whether ERBB3 mutations can affect the potential of ERBB3 to promote growth or affect sensitivity to ERBB inhibitors, we set up a screen for functional effects of all possible random ERBB3 missense or nonsense mutations in the context of ERBB2/ERBB3 heterodimers. To this end, the iSCREAM (in vitro screen of activating mutations) model of somatic evolution, recently developed in our laboratory, was chosen. This platform exploits randomly mutated cDNA libraries of the gene of interest created with error-prone PCR and allows the assessment of growth-advantage conferred by thousands of mutations in parallel. To set up the model for ERBB3 mutation screen, interleukin-3 (IL-3)-dependent Ba/F3 cells were engineered to express a homodimerization-incompetent ERBB2 V956R mutant together with ERBB3 constructs. The model was validated to serve as a readout for ERBB3's ability in activating ERBB2 kinase by demonstrating that cells expressing the two known transforming ERBB3 mutations, E928G and G284R, together with ERBB2 V956R survived and expanded in the absence of IL-3. However, control cells expressing ERBB2 V956R together with wild-type ERBB3 rapidly died in the absence of IL-3. The cell background with ERBB2 V956R expression will subsequently be used as a target for retroviral expression of cDNA library of randomly mutated ERBB constructs. After infection, the cell clones with activating ERBB3 mutations will be allowed to evolve and the activating mutations will be identified by ERBB3-targeted next generation sequencing. The goal is to identify activating ERBB3 mutations that are directly actionable or that provide predictive value for the use of drugs targeting ERBB signaling. Citation Format: Marika K. Koivu, Deepankar Chakroborty, Kari J. Kurppa, Klaus Elenius. Screen for actionable ERBB3 mutations [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 2424.

Selected Articles from This Issue

Angiosarcomas (AS) are rare, aggressive tumors arising from vascular tissues, and harbor extensive genomic events such as driver gene mutations and chromosomal translocations. However, recurrent fusion products and specific genomic events that promote disease progression have thus far proven elusive. In this study, Kim and colleagues employ RNA sequencing data from human angiosarcomas and canine hemangiosarcomas—which closely mirror human AS—and identify 25 novel fusion products among the human and canine samples. Though the fusions were themselves non-recurrent, they co-occured alongside recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS, suggesting that genomic instability induced by these mutations may play a role in the genomic complexity of the disease. Further, the novel fusions were uniformly associated with increased angiogenic signaling, indicating that diverse genomic events in AS likely converge on the activation of a specific transcriptional program. Taken together, the data nominate a host of new molecular targets for investigation and clarify a unifying role for diverse genomic events in the pathogenesis of AS.Extracellular vesicles (EV) have emerged as a key mediator of intercellular signaling and have been linked to the pathogenesis of cancer. EV are loaded with different “cargo” depending on their source, and the characterization of these cargoes is important to understanding and contextualizing their role. Here, Ikeda and colleagues use proteomics to quantitatively probe the differences between EV from cancerous versus normal colon. Employing an ex vivo method for harvesting tissue-exudative EV (Te-EV) from patient samples, the authors find that the amino acid transporter CAT1 is enriched in Te-EV from colorectal cancer (CRC). CAT1-positive EV were found at higher abundance in the plasma of CRC patients and were shown to mediate CRC pathogenesis: by modulating the metabolism of arginine and nitric oxide in endothelial cells, CAT1-positive EV from CRC tissues promoted vascular cell proliferation and tubule formation. These observations identify a key mechanism underlying angiogenesis in CRC and nominate CAT1-positive EV as a potential plasma biomarker for CRC development and progression.Oncogene signaling is a potent driver of tumorigenesis and progression, but cells inherently resist oncogenic transformation via feedback loops leading to cellular senescence. Understanding how oncogene-induced senescence (OIS) is subverted by tumors is critical to the development of new methods to clinically manage cancers driven by BRAF- and RAS-activating mutations. Here, Garnett and colleagues employ a genetic screen to identify mediators of OIS in the context of BRAF activation. The authors found that Amylin, a 37-amino acid peptide that promotes glycolysis, is a key molecular determinant of the response to oncogene signaling. Amylin-ablated cells underwent a series of metabolomic and epigenetic changes that circumvented the onset of senescence in response to BRAF signaling. However, exogenous supplementation of Amylin to the cell growth medium restored the ability of cells to enter a senescent state. These data identify Amylin as a key control node governing the response to oncogene signaling in cancer cells, and suggest that Amylin treatment may hold clinical benefit for patients with oncogene-driven cancers.Acute myeloid leukemia (AML) is a genetically diverse cancer with over ten different molecular subtypes characterized by specific genomic events. EGFR pathway signaling has also been described in AML cells, but most AML cells paradoxically do not express any EGFR family members on the cell surface. In this study, Kam and colleagues address this discrepancy and identify intracellular expression of truncated ERBB2 isoforms in AML cells and myelodysplastic syndrome, an AML precursor lesion. These ERBB2 isoforms—which are also known to be pathogenic in breast cancer—mediated active signaling via classical EGFR-related pathways and were sensitive to inhibition with ERBB2 tyrosine kinase inhibitors. While ERBB2 inhibition was effective as a single agent, combination of the ERBB2 inhibitor lapatinib with the BCL-2 inhibitor venetoclax resulted in marked synergism at all tested doses. These observations nominate ERBB2 inhibitors for further investigation to complement the growing arsenal of therapeutic options for AML patients.

Selective ERBB2 and BCL2 Inhibition Is Synergistic for Mitochondrial-Mediated Apoptosis in MDS and AML Cells.

The ERBB2 proto-oncogene is associated with an aggressive phenotype in breast cancer. Its role in hematologic malignancies is incompletely defined, in part because ERBB2 is not readily detected on the surface of cancer cells. We demonstrate that truncated ERBB2, which lacks the extracellular domain, is overexpressed on primary CD34+ myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cells compared with healthy hematopoietic cells. This overexpression of ERBB2 is associated with aberrant, oncogenic signaling with autophosphorylation of multiple tyrosine sites. Like in breast cancers, ERBB2 can exist as truncated isoforms p95ERBB2 and p110ERBB2 in MDS and AML. Neutralization of ERBB2 signaling with ERBB2 tyrosine kinase inhibitors (i.e., lapatinib, afatinib, and neratinib) increases apoptotic cell death and reduces human engraftment of MDS cells in mice at 21 weeks posttransplantation. Inhibition of ERBB2 modulates the expression of multiple pro- and anti-apoptotic mitochondrial proteins, including B-cell lymphoma 2 (BCL2). Dual blockade with ERBB2 and BCL2 inhibitors triggers additional reductions of BCL2 phosphorylation and myeloid cell leukemia-1 (MCL1) expression compared with single drug treatment. Dual therapy was synergistic at all tested doses, with a dose reduction index of up to 29 for lapatinib + venetoclax compared with venetoclax alone. Notably, these agents operated together and shifted cancer cells to a pro-apoptotic phenotype, resulting in increased mitochondrial cytochrome c release and activated caspase-3-mediated cell death. IMPLICATIONS: These findings warrant study of ERBB2 and BCL2 combination therapy in patients with MDS and AML. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/5/886/F1.large.jpg.

An insertion mutation of ERBB2 enhances breast cancer cell growth and confers resistance to lapatinib through AKT signaling pathway.

ABSTRACTIn clinical practice, some breast cancer (BC) patients carry a rare ERBB2 in-frame insertion (p. Pro780_Tyr781insGlySerPro) and are resistant to anti-ERBB2 therapy. To explore the potential procarcinogenic role of this ERBB2 mutation, we conducted the present study using BC cells overexpressing wild-type (WT) ERBB2 or P780-Y781 ERBB2 [mutated (MT)]. MDA-MB-231 and MCF-7 cells were transfected with the following plasmids using a lentivirus system: negative control (ERBB2-NC), WT ERBB2 overexpression (ERBB2-WT), and P780-Y781 ERBB2 overexpression (ERBB2-MT). P780-Y781 ERBB2 conferred significant resistance to lapatinib, as assessed by cell viability and colony counts. Analysis of the cell cycle showed that the P780-Y781 ERBB2 group showed an elevated proportion of cells in S, G2, and M phases compared with WT ERBB2 when exposed to lapatinib. Following lapatinib treatment, phosphorylated AKT (p-AKT) was strongly upregulated in the P780-Y781 ERBB2 group. Among ERBB2+ patients, the P780-Y781 ERBB2 group showed increased levels of p-AKT. Furthermore, the AKT inhibitor perifosine effectively suppressed lapatinib resistance, as indicated by the lapatinib inhibition curve and results of the colony formation assay, and decreased AKT phosphorylation. Altogether, we discovered a procarcinogenic mutation of ERBB2 that enhances BC cell growth through AKT signaling and causes resistance to lapatinib. Patients with this in-frame insertion mutation of ERBB2 should be recommended other therapeutic strategies apart from ERBB2 tyrosine kinase inhibitors, in particular lapatinib.

<p>Second-generation EGFR and ErbB tyrosine kinase inhibitors as first-line treatments for non-small cell lung cancer</p>

The discovery that mutations in the EGFR gene are present in up to 50% of patients with lung adenocarcinoma, and the development of highly efficacious EGFR tyrosine kinase inhibitors (TKIs), has revolutionized the way this common malignancy is treated. Three generations of EGFR TKIs are now approved for use in EGFR mutation-positive non-small cell lung cancer (NSCLC); the first-generation agents erlotinib, gefitinib, and icotinib; the second-generation ErbB family blockers afatinib and dacomitinib; and most recently, osimertinib, a third-generation EGFR TKI. The second-generation agents have demonstrated impressive efficacy relative to both standard platinum-based chemotherapy and first-generation EGFR TKIs, significantly improving response and progression-free and overall survival. Data from real-world studies suggest that afatinib is as effective and well tolerated in routine clinical practice as it is in clinical studies and is effective in patients with certain uncommon EGFR mutations, patients with brain metastases, and older patients. Few real-world data are available for dacomitinib in the first-line setting. Afatinib and dacomitinib have similar safety profiles, with acne/skin dryzness, diarrhea, stomatitis, and paronychia the most common adverse events (AEs) reported in clinical and real-world studies. Numerous studies have shown that tolerability-guided dose reductions can help manage afatinib-related AEs without reducing efficacy. As the number of therapeutic options for advanced NSCLC increases, the optimal choice for first-line treatment will be determined by considering patient factors such as the presence of brain metastases, the type of EGFR mutation, tolerability, and subsequent therapy options for long-term treatment.

Intestinal epithelial potassium channels and CFTR chloride channels activated in ErbB tyrosine kinase inhibitor diarrhea.

Diarrhea is a major side effect of ErbB receptor tyrosine kinase inhibitors (TKIs) in cancer chemotherapy. Here, we show that the primary mechanism of ErbB TKI diarrhea is activation of basolateral membrane potassium (K+) channels and apical membrane chloride (Cl-) channels in intestinal epithelia and demonstrate the efficacy of channel blockers in a rat model of TKI diarrhea. Short-circuit current in colonic epithelial cells showed that the TKIs gefitinib, lapatinib, and afatinib do not affect basal secretion but amplify carbachol-stimulated secretion by 2- to 3-fold. Mechanistic studies with the second-generation TKI afatinib showed that the amplifying effect on Cl- secretion was Ca2+ and cAMP independent, was blocked by CF transmembrane conductance regulator (CFTR) and K+ channel inhibitors, and involved EGFR binding and ERK signaling. Afatinib-amplified activation of basolateral K+ and apical Cl- channels was demonstrated by selective membrane permeabilization, ion substitution, and channel inhibitors. Rats that were administered afatinib orally at 60 mg/kg/day developed diarrhea with increased stool water from approximately 60% to greater than 80%, which was reduced by up to 75% by the K+ channel inhibitors clotrimazole or senicapoc or the CFTR inhibitor (R)-BPO-27. These results indicate a mechanism for TKI diarrhea involving K+ and Cl- channel activation and support the therapeutic efficacy of channel inhibitors.

Dysfunctional ErbB2, an EGF receptor family member, hinders repair of airway epithelial cells from asthmatic patients

Genetic and genomic data increasingly point to the airway epithelium as critical to asthma pathogenesis. Epithelial growth factor (EGF) family members play a fundamental role in epithelial differentiation, proliferation, and repair. Although expression of erythroblastosis oncogene B2 (ErbB2) mRNA, an EGF family receptor, was reported to be lower in asthmatic patients, little is understood about its functional role. We sought to determine whether decreased ErbB2 activation in freshly isolated human airway epithelial cells (HAECs) from asthmatic patients associated with impaired wound closure invitro. An invitro scratch-wound model of air-liquid interface cultured and freshly isolated HAECs were compared between HAECs from healthy control subjects (HCs) and asthmatic patients in relation to ErbB2. Freshly brushed HAECs from asthmatic patients had impaired ErbB2 activation compared with those from HCs. In an invitro scratch-wound model, HAECs from asthmatic patients showed delayed wound closure compared with HAECs from HCs. Cell proliferation, as assessed based on [3H] thymidine incorporation after wounding, and expression or activation of ErbB2 and cyclin D1 at the leading edge of the wound were lower in HAECs from asthmatic patients and HCs. Aselective ErbB2 tyrosine kinase inhibitor, mubritinib, impaired wound closure and decreased cyclin D1 expression in healthy HAECs, with less effect on cells from asthmatic patients, supporting diminished activity in asthmatic patients. These results implicate a primary defect in the ErbB2 pathway as constraining epithelial repair processes in asthmatic patients. Restoration of homeostatic ErbB2 function should be considered a novel asthma therapeutic target.

ErbB4 tyrosine kinase inhibition impairs neuromuscular development in zebrafish embryos.

Tyrosine kinase inhibitors are widely used in the clinic, but limited information is available about their toxicity in developing organisms. Here, we tested the effect of tyrosine kinase inhibitors targeting the ErbB receptors for their effects on developing zebrafish (Danio rerio) embryos. Embryos treated with wide-spectrum pan-ErbB inhibitors or erbb4a-targeting antisense oligonucleotides demonstrated reduced locomotion, reduced diameter of skeletal muscle fibers, and reduced expression of muscle-specific genes, as well as reduced motoneuron length. The phenotypes in the skeletal muscle, as well as the defect in motility, were rescued both by microinjection of human ERBB4 mRNA and by transposon-mediated muscle-specific ERBB4 overexpression. The role of ErbB4 in regulating motility was further controlled by targeted mutation of the endogenous erbb4a locus in the zebrafish genome by CRISPR/Cas9. These observations demonstrate a potential for the ErbB tyrosine kinase inhibitors to induce neuromuscular toxicity in a developing organism via a mechanism involving inhibition of ErbB4 function.

Integrative Kinome Profiling Identifies mTORC1/2 Inhibition as Treatment Strategy in Ovarian Clear Cell Carcinoma.

Purpose: Advanced-stage ovarian clear cell carcinoma (OCCC) is unresponsive to conventional platinum-based chemotherapy. Frequent alterations in OCCC include deleterious mutations in the tumor suppressor ARID1A and activating mutations in the PI3K subunit PIK3CA In this study, we aimed to identify currently unknown mutated kinases in patients with OCCC and test druggability of downstream affected pathways in OCCC models.Experimental Design: In a large set of patients with OCCC (n = 124), the human kinome (518 kinases) and additional cancer-related genes were sequenced, and copy-number alterations were determined. Genetically characterized OCCC cell lines (n = 17) and OCCC patient-derived xenografts (n = 3) were used for drug testing of ERBB tyrosine kinase inhibitors erlotinib and lapatinib, the PARP inhibitor olaparib, and the mTORC1/2 inhibitor AZD8055.Results: We identified several putative driver mutations in kinases at low frequency that were not previously annotated in OCCC. Combining mutations and copy-number alterations, 91% of all tumors are affected in the PI3K/AKT/mTOR pathway, the MAPK pathway, or the ERBB family of receptor tyrosine kinases, and 82% in the DNA repair pathway. Strong p-S6 staining in patients with OCCC suggests high mTORC1/2 activity. We consistently found that the majority of OCCC cell lines are especially sensitive to mTORC1/2 inhibition by AZD8055 and not toward drugs targeting ERBB family of receptor tyrosine kinases or DNA repair signaling. We subsequently demonstrated the efficacy of mTORC1/2 inhibition in all our unique OCCC patient-derived xenograft models.Conclusions: These results propose mTORC1/2 inhibition as an effective treatment strategy in OCCC. Clin Cancer Res; 24(16); 3928-40. ©2018 AACR.

Pulse Afatinib for ERBB2 Exon 20 Insertion–Mutated Lung Adenocarcinomas

IntroductionGenomic aberrations involving the erb-b2 receptor tyrosine kinase 2 gene (ERBB2) are driver oncogenes in approximately 2% of lung adenocarcinomas. However, the use of daily dosing of ERBB2 tyrosine kinase inhibitors (TKIs)—including afatinib—has been fraught with plasma concentrations that barely achieve preclinical model inhibition, significant patient-reported toxicities, and limited clinical activity. We hypothesized that alternative dosing strategies could improve tolerability and efficacy. MethodsWe profiled lung cancer cell lines against TKIs and retrospectively evaluated the toxicity of and response to pulse afatinib (280 mg once weekly) in lung cancers with ERBB2 mutations. ResultsAn ERBB2 exon 20 insertion–mutated lung cancer cell line had a 50% inhibitory concentration in response to afatinib that was higher than the reported plasma concentration of afatinib, 40 mg daily. Three patients with advanced ERBB2-mutated lung adenocarcinomas were treated with off-label pulse afatinib. The 280-mg weekly dose was well tolerated with no reported rash and minimal diarrhea. One TKI-naive patient achieved a partial response for 5 months and another achieved stable disease for 11 months. ConclusionsPulse afatinib at a weekly dosing scheme induced antitumor activity in ERBB2 exon 20 insertion–mutated lung adenocarcinomas. Future clinical trials of alternative dosing schemes of ERBB TKIs as monotherapy or in combination with other therapies are warranted for ERBB2-mutated tumors.

Abstract P4-14-26: Phase I expansion of S-222611, a reversible inhibitor of EGFR and HER2, in advanced solid tumors, including HER2-positive breast cancer patients with brain metastases

Abstract BACKGROUND S-222611 is an oral, reversible ErbB tyrosine kinase inhibitor of EGFR and HER2 with potent pre-clinical activity. MTD was not reached during the dose-escalation phase, (maximum dose 1600 mg QD). PK and efficacy data supported a daily dose of 800 mg. An expansion cohort of patients has been treated to further explore safety and efficacy. METHODS Subjects with advanced solid tumors expressing EGFR and/or overexpressing HER2 were enrolled. S-222611 800 mg daily was administered until disease progression or unacceptable toxicity. RESULTS 76 patients were included in this phase 1 expansion cohort with a variety of tumor types. Dose reduction was required because of adverse events in 15 patients; the most frequent of which being diarrhea and elevated bilirubin. Two patients discontinued treatment due to drug- related adverse events. Of the 25 patients with HER2-positive metastatic breast cancer (MBC), 4 partial responses were observed, and prolonged stable disease (≥ 6 months) was observed in 3 additional patients. These 25 patients had received prior HER2-directed therapy as shown in Table 1. Table 1. Prior therapies received by patients with HER2-positive MBCPrior therapyn (%)Trastuzumab22 (88)T-DM13 (12)Lapatinib16 (64)Chemotherapy23 (92) Six of these patients had brain metastases, in whom 1 intracranial response and 2 prolonged stable disease (≥ 6 months) were observed (Table 2). Table 2. HER2-positive MBC patients with brain metastases - best overall response to S-222611Pts #HER2 IHCBrain metastasesBest overall response (RECIST 1.1)Patient 13+Target lesionPRPatient 23+Target lesionSD (≥12 M)Patient 33+Target lesionSD (6.0 M)Patient 43+Non-target lesionSD (4.7 M)Patient 53+Non-target lesionSD (3.3 M)Patient 63+Non-target lesionNE The patient showing intracranial response was previously treated with lapatinib and capecitabine after diagnosis of BM. CONCLUSIONS S-222611 was well tolerated at a dose of 800 mg once daily. Anti-tumour activity, including shrinkage of brain metastases, was evident in a heavily pre-treated population of patients with HER2-positive breast cancer. Citation Format: Baird RD, Arkenau H-T, Deva S, Cresti N, Garcia-Corbacho J, Hogarth L, Frenkel E, Kawaguchi K, Arimura A, Donaldson K, Posner J, Sarker D, Jodrell D, Plummer R, Spicer J, Italiano A. Phase I expansion of S-222611, a reversible inhibitor of EGFR and HER2, in advanced solid tumors, including HER2-positive breast cancer patients with brain metastases. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-14-26.

Afatinib: a second-generation EGF receptor and ErbB tyrosine kinase inhibitor for thetreatment of advanced non-small-cell lung cancer.

First-generation reversible EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) changed our understanding of advanced non-small-cell lung cancer biology and behavior. The presence of sensitizing EGFRmutations in advanced non-small-cell lung cancer defines a subset of patients with a better prognosis and sensitivity to EGFR-TKIs with a better response rate, progression-free survival, quality of life and symptom control than with chemotherapy in the first-line therapy setting. However, current EGFR-TKIs show minimal responses in EGFRwild-type patients or with acquired TKI resistance mediated through the EGFR T790M allele. Afatinib is an irreversible pan-ErbB-TKI, active against wild-type EGFR, sensitizing and T970M-mutant EGFR, ErbB2 and ErbB4 receptors, and represents a step change between reversible first-generation and future irreversible highly specific third-generation EGFR-TKIs. Here, we review the clinical development of afatinib through the LUX-Lung trials portfolio highlighting benefits and toxicities.

Neuregulin 1 as an endogenous regulator of nicotinic acetylcholine receptors in adult major pelvic ganglion neurons

We investigated whether endogenous neuregulin 1 (NRG1) is released in a soluble form (called sNRG1) and upregulates expression of nicotinic acetylcholine receptor (nAChR) in autonomic major pelvic ganglion (MPG) neurons of adult rats. To elicit the release of sNRG1, either the hypogastric nerve or the pelvic nerve was electrically stimulated. Then, the MPG-conditioned medium (CM) was subjected to western blotting using an antibody directed against the N-terminal ectodomain of NRG1. Both sympathetic and parasympathetic nerve activation elicited the release of sNRG1 from MPG neurons in a frequency-dependent manner. The sNRG1 release was also induced by treatment of MPG neurons with either high KCl or neurotrophic factors. The biological activity of the released sNRG1 was detected by tyrosine phosphorylation (p185) of the ErbB2 receptors in MPG neurons. When MPG neurons were incubated for 6 h in the CM, the protein level of the nAChR α3 subunit and ACh-induced current (IACh) density were significantly increased. The CM-induced changes in IACh was abolished by a selective ErbB2 tyrosine kinase inhibitor. Taken together, these data suggest that NRG1 functions as an endogenous regulator of nAChR expression in adult MPG neurons.

Phase I expansion of S-222611, a reversible inhibitor of EGFR and HER2, in advanced solid tumors, including patients with brain metastases.

2511 Background: S-222611 is an oral, reversible ErbB tyrosine kinase inhibitor of EGFR and HER2 with potent pre-clinical activity. MTD was not reached during dose-escalation, even at the maximum d...

Examining the protective role of ErbB2 modulation in human-induced pluripotent stem cell-derived cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are being used as an in vitro model system in cardiac biology and in drug discovery (e.g., cardiotoxicity testing). Qualification of these cells for use in mechanistic investigations will require detailed evaluations of cardiomyocyte signaling pathways and cellular responses. ErbB signaling and the ligand neuregulin play critical roles in survival and functional integrity of cardiac myocytes. As such, we sought to characterize the expression and activity of the ErbB family of receptors. Antibody microarray analysis performed on cell lysates derived from maturing hiPSC-CMs detected expression of ∼570 signaling proteins. EGFR/ErbB1, HER2/ErbB2, and ErbB4, but not ErbB3 receptors, of the epidermal growth factor receptor family were confirmed by Western blot. Activation of ErbB signaling by neuregulin-1β (NRG, a natural ligand for ErbB4) and its modulation by trastuzumab (a monoclonal anti-ErbB2 antibody) and lapatinib (a small molecule ErbB2 tyrosine kinase inhibitor) were evaluated through assessing phosphorylation of AKT and Erk1/2, two major downstream kinases of ErbB signaling, using nanofluidic proteomic immunoassay. Downregulation of ErbB2 expression by siRNA silencing attenuated NRG-induced AKT and Erk1/2 phosphorylation. Activation of ErbB signaling with NRG, or inhibition with trastuzumab, alleviated or aggravated doxorubicin-induced cardiomyocyte damage, respectively, as assessed by a real-time cellular impedance analysis and ATP measurement. Collectively, these results support the expanded use of hiPSC-CMs to examine mechanisms of cardiotoxicity and support the value of using these cells in early assessments of cardiotoxicity or efficacy.

Afatinib demonstrates remarkable activity against HER2-amplified uterine serous endometrial cancer in vitro and in vivo.

Background:Uterine serous carcinomas (USCs) are an aggressive form of uterine cancer that may rely on HER2/neu amplification as a driver of proliferation. The objective of this paper is to assess the sensitivity of USC cell lines with and without HER2/neu gene amplification to afatinib, an irreversible ErbB tyrosine kinase inhibitor, and to test the efficacy of afatinib in the treatment of HER2-amplified USC xenografts.Methods:Eight of fifteen primary USC cell lines (four with HER2 amplification and four without) demonstrating similar in vitro growth rates were treated with scalar concentrations of afatinib. Effects on cell growth, signalling and cell cycle distribution were determined by flow cytometry assays. Mice harbouring xenografts of HER2/neu-amplified USC were treated with afatinib by gavage to determine the effect on tumour growth and overall survival.Results:Primary chemotherapy-resistant USC cell lines harbouring HER2/neu gene amplification were exquisitely sensitive to afatinib exposure (mean±s.e.m. IC50=0.0056±0.0006 μM) and significantly more sensitive than HER2/neu-non-amplified USC cell lines (mean±s.e.m. IC50=0.563±0.092 μM, P<0.0001). Afatinib exposure resulted in abrogation of cell survival, inhibition of HER2/neu autophosphorylation and S6 transcription factor phosphorylation in HER2/neu overexpressing USC and inhibited the growth of HER2-amplified tumour xenografts improving overall survival (P=0.0017).Conclusions:Afatinib may be highly effective against HER2/neu-amplified chemotherapy-resistant USC. The investigation of afatinib in patients harbouring HER2/neu-amplified USC is warranted.