ErbB Inhibitors Research Articles

Investigating mutations at the hotspot position of the ERBB2 and screening for the novel lead compound to treat breast cancer - a computational approach.

Membrane proteins are the most common types of cancer that are active in the prognosis. Membrane proteins are a distinguishing characteristic of a cancer cell. In tumor cell therapy, the overexpressed membrane proteins are becoming ever more relevant. The 3-kinase (PI3K)/AKT phosphatidylinositol pathway is downstream triggered by different extracellular signals, and this signaling pathway activation impacts a variety of proliferation of the cellular processes like cell growth and surviving. Frequent PI3K/AKT dysregulation in human cancer has rendered proteins of this pathway desirable for diagnostic markers. Members of the ERBB family-like ERBB2 and ERBB3 activate intracellular signaling pathways such as PI3K/AKT. The mutations in these proteins dysfunctions the proteins in the downstream. Considering this importance, we have developed a computational pipeline to identify the mutation position with a highest number of mutations and to screen them for pathogenicity, stability, conservation, and structural changes using PredictSNP, iStable, ConSurf, and GROMACS simulation software respectively. Further, a virtual screening approach was initiated to find the most similar non-toxic lead compound, which could be an alternative to the currently used lapatinib. To conclude, protein-ligand dynamics were undertaken to study the actions of native and mutants with the lapatinib and the lead compound. From the overall analysis, we identified position 755 with leucine in the native condition is prone to frequent mutations. The leucine at 755th position is more prone to mutate as serine and tryptophan. Further from the computational analysis, we identified that the mutation L755S is more significant than the L755W mutation. We have witnessed CID140590176 be a potential lead compound with no toxicity. The behavior of the lead compound has shown more compactness with an increased number of intermolecular hydrogen bonds in the ERBB2 with L755S. This lead compound can be further taken for experimental validations, and we believe that this lead compound could be a potent ERBB2 inhibitor.

Peritoneal Spread of Ovarian Cancer Harbors Therapeutic Vulnerabilities Regulated by FOXM1 and EGFR/ERBB2 Signaling.

Peritoneal spread is the primary mechanism of metastasis of ovarian cancer, and survival of ovarian cancer cells in the peritoneal cavity as nonadherent spheroids and their adherence to the mesothelium of distant organs lead to cancer progression, metastasis, and mortality. However, the mechanisms that govern this metastatic process in ovarian cancer cells remain poorly understood. In this study, we cultured ovarian cancer cell lines in adherent and nonadherent conditions in vitro and analyzed changes in mRNA and protein levels to identify mechanisms of tumor cell survival and proliferation in adherent and nonadherent cells. EGFR or ERBB2 upregulated ZEB1 in nonadherent cells, which caused resistance to cell death and increased tumor-initiating capacity. Conversely, Forkhead box M1 (FOXM1) was required for the induction of integrin β1, integrin-α V, and integrin-α 5 for adhesion of cancer cells. FOXM1 also upregulated ZEB1, which could act as a feedback inhibitor of FOXM1, and caused the transition of adherent cells to nonadherent cells. Strikingly, the combinatorial treatment with lapatinib [dual kinase inhibitor of EGFR (ERBB1) and ERBB2] and thiostrepton (FOXM1 inhibitor) reduced growth and peritoneal spread of ovarian cancer cells more effectively than either single-agent treatment in vivo. In conclusion, these results demonstrate that FOXM1 and EGFR/ERBB2 pathways are key points of vulnerability for therapy to disrupt peritoneal spread and adhesion of ovarian cancer cells. SIGNIFICANCE: This study describes the mechanism exhibited by ovarian cancer cells required for adherent cell transition to nonadherent form during peritoneal spread and metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5554/F1.large.jpg.

EPEN-33. PHARMACOGENOMICS REVEALS SYNERGISTIC INHIBITION OF ERBB2 AND PI3K SIGNALING AS A THERAPEUTIC STRATEGY FOR EPENDYMOMA

Subgroups of ependymoma, especially RELA fusion-positive and posterior fossa type A tumors, are associated with poor prognosis. Curative therapeutic strategies have not yet been identified. We set up a high-throughput drug screening (HTS) pipeline to evaluate clinically established compounds (n=196) in primary ependymoma cultures (n=12). As culturing ependymoma is challenging, assay miniaturization to 1536-well microplates emerged as a key feature to process HTS despite smallest cell numbers. DNA methylation profiling showed that entity and subgroup affiliation from primary diagnosis was maintained in primary cultures, as assessed through molecular neuropathology 2.0 based classification (MNP 2.0, Capper, D. et al., Nature, 2018). A comparison of HTS data of ependymoma and other pediatric brain tumor models (n=48) revealed a remarkable chemoresistance in vitro. However, we identified Neratinib, an irreversible ERBB2 inhibitor, as the most prominent candidate which was preferentially active in a subset of the investigated ependymoma cultures (n=5). Combinatory treatment with Copanlisib, a PI3K inhibitor, was able to overcome resistance to single agent treatment using Neratinib in established cell lines of ependymoma (n=3) and 2/4 primary cultures for which combinatory treatment could be tested. Finally, we validated efficacy of Neratinib combined with Copanlisib in mice bearing ependymoma xenografts which revealed significantly reduced tumor size compared to vehicle-treated animals. In summary, our study demonstrates that HTS may reveal targeted therapies for pediatric brain tumors. Specifically, we found a synergistic interaction of Neratinib and Copanlisib for treatment of ependymoma, thereby providing a novel therapeutic approach in an otherwise largely chemoresistant entity.

Bypassing P-glycoprotein mediated efflux of afatinib by cyclodextrin complexation – Evaluation of intestinal absorption and anti-cancer activity

A cyclodextrin complex of afatinib, a FDA-approved pan–ErbB inhibitor and known P-gp substrate, was developed to enhance its bio-activity while also reducing p-glycoprotein (P-gp) efflux, thereby improving its transport across the intestinal membrane. A sulfobutyl ether derivative of β-cyclodextrin (SBE-β-CD, pharmaceutical grade) was selected based on preliminary phase solubility studies and molecular modeling to prepare an afatinib-cyclodextrin inclusion complex. Various solid-state characterization studies were performed to confirm complex formation and to evaluate its physicochemical properties. Results showed that complexation with SBE-β-CD enhanced afatinib's stability in PBS (pH 7.4), as well as in simulated gastric (pH 2.0) and intestinal fluids (pH 6.5). In addition, the cytotoxicity (reduced IC50 after complexation across multiple cancer cell lines with varied P-gp expression) and tumor volume retraction ability (in-vitro 3D-spheroids of P-gp overexpressing cell lines) of afatinib were both enhanced through cyclodextrin complexation. CD complexation effect of afatinib on P-gp efflux as well as transport was assessed using in-vitro permeability studies (Caco-2 and EpiIntestinal® tissue models). Results demonstrated that cyclodextrin complexation significantly increased the transport of afatinib across the gut membrane while also reducing its efflux ratio, indicating its vital role in P-gp efflux modulation.

Abstract 16383: Inhibition of Egfr Signalling Promotes Maladaptive Cardiac Remodelling in Hypertensive Heart Disease

Introduction: Epidermal growth factor (EGF) receptors (EGFRs: ERBB1-4) are activated by a family of ligands (e.g. EGF, Hb-EGF, EREG, TGFa), signaling through ERK1/2 and Akt to promote cell division and cancer. Antibody-based inhibition of ERBB2 in breast cancer can cause heart failure, but the role of other receptors and EGFR ligands in the heart, and potential cardiotoxicity of generic EGFR inhibitors is unclear. Hypothesis: We hypothesize that EGFR ligands play an important role in cardiac adaptation to hypertension, acting through EGFRs to promote adaptive remodelling. Methods & Results: EGF ligand/receptor mRNA expression was assessed in human failing hearts and normal controls (n=12/8). EGFRs were expressed at similar levels, but ligand expression differed with significant up- or downregulation of EGF/Hb-EGF vs EREG/TGFa, respectively, in failing hearts (p<0.05). EGF potently activated ERK1/2 and Akt (assessed by immunoblotting) in neonatal rat cardiomyocytes, leading to hypertrophy (p<0.05, n=4). The anti-cancer drug afatinib inhibits EGFRs. To assess the role of EGF signaling in cardiac adaptation to hypertension in vivo , C57Bl/6J mice (n=6) were treated with 0.8 mg/kg/d angiotensin II (AngII; 7d) ± 0.45 mg/kg/d afatinib. AngII promoted cardiac hypertrophy with increased left ventricular (LV) wall thickness (WT) and decreased LV internal diameter (ID; assessed by echocardiography). Afatinib enhanced AngII-induced hypertrophy with significantly increased WT:ID ratios (1.30-fold and 1.54-fold in diastole and systole, respectively; p<0.05) but inhibited AngII-induced increases in Nppb mRNA expression and cardiomyocyte cross-sectional area (208.80±9.78 vs 161.10±3.87μm 2 ; p<0.05). In contrast, Col1a1 mRNA expression was enhanced by afatinib, along with interstitial and perivascular fibrosis (3.21±0.38 vs 5.61±0.46, 0.98±0.06 vs 1.45±0.18 % area; p<0.05). Conclusion: EGFR signaling is modulated in human heart failure, promotes cardiomyocyte hypertrophy and is required for cardiac adaptation to hypertension. Since EGFR inhibition in hypertension prevents adaptive cardiomyocyte hypertrophy whilst promoting fibrosis, EGFR inhibitors are likely to cause cardiac dysfunction and be cardiotoxic in hypertensive patients.

Abstract PO-037: The conversion of induced pluripotent stem cells into cancer stem cells under pancreatic cancer microenvironment is inhibiting by lapatinib

Abstract Cancer stem cells (CSCs) are suggested to be responsible for drug resistance and aggressive phenotypes of tumors. Mechanisms that regulating CSCs are still under investigation. Our lab has established a novel method to produce CSCs from induced pluripotent stem cells (iPSCs) under cancer microenvironment using conditioned medium (CM) from cancer cell lines. By treatment iPSCs with CM, cells gain CSC characteristics. Here, we analyzed transcriptome from CSCs, which converted from iPSCs using CM from pancreatic ductal adenocarcinoma cells, and differentially expressed genes were identified. Pathway enrichment was analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG). Comparing iPSCs with CSCs converted from iPSCS showed elevated expression of genes related to ERBB2, ERBB3 and PI3K, while KEGG pathways revealed enrich of pathways known to be involved in cancer including ERBB2/ERBB3 signal pathway by conversion of iPSCs to CSCs. Inhibition of ERBB2 by lapatinib induced cell proliferation arrest and losing of tumorgenicity of converted cells. This study shows a potential involvement of ERBB2/ERBB3 pathway in CSCs generation and could lead to potentially new options for cancer treatment and prevention. Citation Format: Ghmkin Hassan, Masaharu Seno. The conversion of induced pluripotent stem cells into cancer stem cells under pancreatic cancer microenvironment is inhibiting by lapatinib [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-037.

Comprehensive Molecular Characterization of Adenocarcinoma of the Gastroesophageal Junction Between Esophageal and Gastric Adenocarcinomas.

To investigate the molecular characteristics of AGEJ compared with EAC and gastric adenocarcinoma. Classification of AGEJ based on differential molecular characteristics between EAC and gastric adenocarcinoma has been long-standing controversy but rarely conducted due to anatomical ambiguity and epidemiologic difference. The molecular classification model with Bayesian compound covariate predictor was developed based on differential mRNA expression of EAC (N = 78) and GCFB (N = 102) from the Cancer Genome Atlas (TCGA) cohort. AGEJ/cardia (N = 48) in TCGA cohort and AGEJ/upper third GC (N = 46 pairs) in Seoul National University cohort were classified into the EAC-like or GCFB-like groups whose genomic, transcriptomic, and proteomic characteristics were compared. AGEJ in both cohorts was similarly classified as EAC-like (31.2%) or GCFB-like (68.8%) based on the 400-gene classifier. The GCFB-like group showed significantly activated phosphoinositide 3-kinase-AKT signaling with decreased expression of ERBB2. The EAC-like group presented significantly different alternative splicing including the skipped exon of RPS24, a significantly higher copy number amplification including ERBB2 amplification, and increased protein expression of ERBB2 and EGFR compared with GCFB-like group. High-throughput 3D drug test using independent cell lines revealed that the EAC-like group showed a significantly better response to lapatinib than the GCFB-like group (P = 0.015). AGEJ was the combined entity of the EAC-like and GCFB-like groups with consistently different molecular characteristics in both Seoul National University and TCGA cohorts. The EAC-like group with a high Bayesian compound covariate predictor score could be effectively targeted by dual inhibition of ERBB2 and EGFR.

Effect of Combining EGFR Tyrosine Kinase Inhibitors and Cytotoxic Agents on Cholangiocarcinoma Cells.

PurposeThe potential of members of the epidermal growth factor receptor (ErbB) family as drug targets in cholangiocarcinoma (CCA) has not been extensively addressed. Although phase III clinical trials showed no survival benefits of erlotinib in patients with advanced CCA, the outcome of the standard-of-care chemotherapy treatment for CCA, gemcitabine/cisplatin, is discouraging so we determined the effect of other ErbB receptor inhibitors alone or in conjunction with chemotherapy in CCA cells.Materials and MethodsErbB receptor expression was determined in CCA patient tissues by immunohistochemistry and digital-droplet polymerase chain reaction, and in primary cells and cell lines by immunoblot. Effects on cell viability and cell cycle distribution of combination therapy using ErbB inhibitors with chemotherapeutic drugs was carried out in CCA cell lines. 3D culture of primary CCA cells was then adopted to evaluate the drug effect in a setting that more closely resembles in vivo cell environments.ResultsCCA tumors showed higher expression of all ErbB receptors compared with resection margins. Primary and CCA cell lines had variable expression of erbB receptors. CCA cell lines showed decreased cell viability when treated with chemotherapeutic drugs (gemcitabine and 5-fluorouracil) but also with ErbB inhibitors, particularly afatinib, and with a combination. Sequential treatment of gemcitabine with afatinib was particularly effective. Co-culture of CCA primary cells with cancer-associated fibroblasts decreased sensitivity to chemotherapies, but sensitized to afatinib.ConclusionAfatinib is a potential epidermal growth factor receptor targeted drug for CCA treatment and sequential treatment schedule of gemcitabine and afatinib could be explored in CCA patients.

Increased Seizure Susceptibility for Rats Subject to Early Life Hypoxia Might Be Associated with Brain Dysfunction of NRG1-ErbB4 Signaling in Parvalbumin Interneurons.

Neuregulin 1 (NRG1)-induced activation of ErbB4 in parvalbumin (PV) inhibitory interneurons is reported to serve as a critical endogenous negative-feedback mechanism to repress brain epileptogenesis. Here, we investigated the seizure susceptibility and the role of NRG1-ErbB4 signaling in PV interneurons in the suppression of epileptic seizures for rats subject to early life hypoxia. Neonatal postnatal day 5 (P5) rats were exposed to intermittent hypoxia (IH) or control (CON) room air for 10 days. In the prefrontal cortex (PFC) of P54 rats, we determined the impact of neonatal IH exposures on the expression of PV, NRG1, ErbB4, and phosphorylated ErbB4 (p-ErbB4) during the seizure induction. Seizure susceptibility tests with the common convulsant agent pentylenetetrazole (PEN) at P54 revealed that rats subject to neonatal hypoxia exposure developed faster and more serious epileptic seizures. Neonatal IH exposures (1) decreased the number of PV cells in the PFC of P54 rats; (2) interrupted the expression of NRG1 gene; and (3) altered the activity of NRG1 on PV interneurons in the PFC after the seizure induction. Intracerebroventricular delivery of exogenous NRG1 before seizure induction by PEN significantly reduced the seizure susceptibility for neonatal IH-exposed rats. The ErbB4 inhibitor AG1478 inhibited the exogenous NRG1's effects on seizure susceptibility. Environmental enrichment (EE) rescued the abovementioned pathophysiological alterations and significantly attenuated the epileptic seizures after the seizure induction for neonatal IH-exposed rats. Our study indicated early life hypoxia exposure might increase the seizure susceptibility for rats and contribute to pathophysiological dysfunction of NRG1-ErbB4 signaling in PV interneurons in the suppression of epileptic seizures. EE might attenuate the increased seizure susceptibility for neonatal IH-exposed rats through rescuing pathophysiological alterations of NRG1-ErbB4 signaling in PV interneurons.

The significance of the neuregulin-1/ErbB signaling pathway and its effect on Sox10 expression in the development of terminally differentiated Schwann cells in vitro

Purpose The purpose of this study was to explore the significance of the neuregulin-1/ErbB signaling pathway and its effect on Sox10 expression in the course of the differentiation of mouse bone marrow mesenchymal stem cells into Schwann-like cells in vitro. Materials and methods The experiment was conducted with three groups—control, TAK 165, and HRG-off. In the control group, we used the classical induction method of adding β-ME, RA, FSK, b-FGF, PDGF, and neuregulin (HRG); the cells were collected on the 7th day. Using the same basic protocol as the control group, the specific ErbB2 inhibitor mubritinib (TAK 165) was added to block the neuregulin-1/ErbB pathway in the TAK 165 group, while HRG was not added in the HRG-off group. We detected the degree of differentiation of stem cells into Schwann-like cells by using RT-PCR to examine the expression of Sox10, NRG-1, ErbB2, ErbB3, and ErbB4 and by using immunofluorescence staining to examine the Schwann cell marker S100B, Glial Fibrillary Acidic Protein (GFAP) and P75. Results Our results showed that the proliferation of Schwann cells was reduced and apoptosis was increased in the TAK 165 group and the HRG-off group. Sox10 was stably expressed and NRG-1, ErbB2, and ErbB3 increased in the control group. However, the expression of Sox10 in the TAK 165 group was obviously decreased at the end of induced differentiation; meanwhile, the degree of stem cell differentiation also decreased. Conclusions the neuregulin-1/ErbB signaling pathway plays an important role in the differentiation of bone marrow mesenchymal stem cells into Schwann-like cells and can promote the maintenance of Sox10 表达。

Abstract 1912: Study on the role of targeting ERBB and its potential resistance mechanism in biliary tract cancer

Abstract Purpose: Biliary tract cancer (BTC) is a kind of malignant digestive system tumors without a targeted drug approved in the clinic. A high proportion of gene mutations and abnormal expressions of ERBB pathway have been reported in BTC. However, there are few studies on targeting ERBB in BTC. In this study, we aim to explore the role of targeting ERBB and its potential resistance mechanisms in BTC. Materials and Methods: We used the tumor tissue samples of BTC patients to establish patient-derived cell lines (PDCs), and perform the biological characteristics of PDCs. At the first, Western Blot was used to select the PDCs with different ERBB2 expressions as in vitro models. Secondly, colony formation assay was to verify the sensitivity of inhibitors and Western blot was to confirm the change of signaling pathways in PDCs. Then, whole exome sequencing (WES) and transcriptome sequencing (RNA-seq) were integrated to explore the potential resistance mechanisms of ERBB inhibitors. Moreover, stable PDCs with overexpression and knockdown established by lentiviral vectors were used. Finally, PDC xenografts were used to further confirm the hypothesis from the in vitro study. Results: Twenty-two BTC PDCs were successfully established in this study. According to ERBB2 expression, nine PDCs were selected for the sensitivity evaluation of ERBB inhibitors. Four PDCs of them are sensitive to three ERBB inhibitors, while the other five PDCs are not. Furthermore, colony formation assay has demonstrated the difference of colonies between sensitive cell lines and resistant cell lines for all three ERBB inhibitors. Meanwhile, Western Blot showed that inhibition activity is negatively correlated with phosphorylation of ERBB2 in PDCs. These results demonstrated that decreasing ERBB activity could inhibit the proliferation of BTC PDCs. Further analysis showed that all four sensitive PDCs highly expressed ERBB2, while four resistant PDCs carried with low ERBB2 expression levels. HCC783 was the only one PDC with high ERBB2 expression as well as resistance to ERBB inhibitors and was considered as a primary resistant PDC for follow-up study. RNA-seq analysis results showed that SMARCA1 was significantly lower (p < 0.01) and the gene transcriptional spectrum were greatly changed in resistant PDCs compared with sensitive PDCs, suggesting that SMARCA1 may have been involved in resistance mechanisms of ERBB inhibitors in BTC. Importantly, stable PDCs were established with overexpression and knockdown of SMARCA1 and the colony formation assay with them confirmed this hypothesis in vitro. The results demonstrated that a resistant cell line by overexpression of SMARCA1 could make sensitive to ERBB inhibitors, while a sensitive cell line by knockdown of SMARCA1 could become relatively resistant to ERBB inhibitors. The exploration of resistance mechanisms triggered by SMARCA1 and the in vivo validation with PDC xenografts are undergoing. Conclusions: In this study, the PDC model, drug screening and colony formation assay were integrated to verify that ERBB inhibitors could prevent the proliferation of BTC cells for the first time. Furthermore, WES and RNA-seq combined with stable PDC models were used to discover and validate that SMARCA1 is one of the potential resistance mechanisms of ERBB inhibitors in BTC. Citation Format: Feiling Feng, Qingbao Cheng, Dadong Zhang, Chen Liu, Xiaoya Xu, Bin Li, Huizhen Wang, Yong Yu, Bin Li, Xiaobing Wu, Jun Zhou, Kaijian Chu, Zhenghua Xie, Qingxiang Gao, Lei Xiong, Fugen Li, Bin Yi, Xiaoqing Jiang. Study on the role of targeting ERBB and its potential resistance mechanism in biliary tract cancer [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 1912.

Abstract 3198: Hyperactive c-Met and ErbB signaling detected in a sub-group of ovarian patient tumors: Patient sub-group may benefit from c-Met and pan-HER combination therapy

Abstract Background: Sub-groups of breast, lung, and colon cancer patients with genetic variants associated with dysregulated ErbB signaling benefit from treatment with ErbB targeted therapies. No sub-groups of ovarian cancer patients with actionable ErbB genetic variants have yet been found. Measurement of biological factors other than genetic variants, such as ErbB signaling activity, may identify ovarian cancer patients likely to benefit from ErbB targeted therapies. We have previously reported studies detecting dysregulated ErbB and c-Met signaling activity in 20%-25% of HER2-negative breast cancer patient tumors using the CELx Multi-Pathway (MP) Signaling Function Test. To determine whether ErbB or c-Met signaling dysregulation is involved in ovarian cancer, the CELx MP test was adapted to analyze ovarian tumor cells. The current study set out to: 1) characterize c-Met and ErbB family signaling activity in ovarian patient tumor cells and ovarian tumor cell lines; 2) evaluate in vivo response to pan-HER and c-Met inhibitors in an ovarian tumor xenograft model. Methods: For the ex vivo studies, a set of fresh tumor specimens from 15 ovarian cancer patients (90% high-grade serous, 10% endometrioid) and a set of 12 ovarian cancer cell lines was obtained. Cell samples were cultured from each patient specimen. Real-time live cell response to specific ErbB and c-Met agonists (NRG1b, EGF, or HGF) with or without an antagonist (2C4, a HER2 dimerization inhibitor or tepotinib, a c-Met kinase inhibitor) was measured and quantified using an xCELLigence impedance biosensor (Agilent Technologies). Signaling activity above a previously established cut-off value was used to identify abnormal levels of EGFR, HER2 and c-Met signaling activity. For the xenograft study, OVCAR-4, an ovarian cancer cell line, determined to have abnormal HER2, EGFR, and c-Met signaling with the CELx MP Test, was studied. 40 female NSG mice were injected with two million OVCAR-4 cells. Mice were randomly assigned to either a control group that received Captisol excipient or a treatment group that received neratinib, tepotinib, or neratinib and tepotinib for 16 days. Results: Of the patient cell and cell lines samples tested ex vivo with the CELx MP test, 4 of 27 (15%; 95% CI=6%-32%) had hyperactive HER2 and c-Met signaling pathways. In the xenograft study, there was no significant difference in tumor volume between the control and tepotinib-treated or neratinib groups after 16 days of treatment. Tumor volumes relative to tumor size before treatment were reduced by 40% in the tepotinib + neratinib treated group. Conclusions: These findings suggest that a significant sub-group of ovarian cancer patients have abnormal ErbB and c-Met signaling activity that may respond to treatment with a combination of ErbB and c-Met inhibitors. A clinical trial to evaluate treatment response of this patient sub-set to combined c-Met and pan-HER inhibitors is warranted. Citation Format: Stefano Rossetti, Catherine Kuzmicki, Aaron Broege, Joanna Sabat, Salmaan Khan, Ian MacNeil, Brian Sullivan, Lance Laing. Hyperactive c-Met and ErbB signaling detected in a sub-group of ovarian patient tumors: Patient sub-group may benefit from c-Met and pan-HER combination therapy [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 3198.

Abstract 5636: Restoration of tumor immunosurveillance in tasmanian devil facial tumor disease

Abstract Transmissible cancer is a rare, but mechanistically not understood form of cancer that can directly spread between individuals. At least six species in the animal kingdom harbor clonal cancers that spread horizontally. These diseases include the fatal devil facial tumor disease (DFTD) in Tasmanian devils, a sexually transmitted sarcoma in dogs, and leukemia-like cancers in mollusks. DFTD is an allogeneic graft of Schwann cell origin, which is transmitted by direct transfer of cancer cells from one individual to another as a result of biting behavior. Two strains of DFTD, DFT1 and DFT2, emerged independently in the population of Tasmanian devils. It is essential to gain mechanistic understanding of horizontal cancers to understand how cancer cells escape host immune cells. We designed an integrative systems biology approach using primary biopsies, established Tasmanian devil tumor and control cell lines and mouse xenograft model. Specifically, we combined pharmacological drug screening, transcriptomics, epigenetics, proteomics and comparative pathology to unveil aberrant signaling pathways in DFTD. We found that DFT1 tumor cells express high levels of phosphorylated ERBB2 and ERBB3 tyrosine kinase receptor and they are exquisitely vulnerable to ERBB kinase inhibitors. Blockade of the hyperactive ERBB-STAT3 axis with ERBB inhibitor sapitinib or with the STAT3 inhibitor DR-1-55 led to recovery of MHC-I expression, suggesting that immune escape could be antagonized by blockade of hyperactive ERBB-STAT3 action. In contrary to DFT1, progression of DFT2 is known to be associated with PDGF receptor amplification. This proposed an attractive pharmacological approach of targeted inhibition of PDGFR by broad kinase inhibitor imatinib. We validated in xenograft mouse model that targeted inhibition of PDGFR results in the efficient reduction of tumor growth as well as restoration of MHC-I gene expression. In summary, we identified a critical role for hyperactive tyrosine kinase-STAT signaling whose pharmacological inhibition resulted in arrested growth and possible restoration of immune cell recognition. We validated that DFT2 displays a distinct oncogene mechanism than DFT1, but pharmacologic inhibition of hyperactive driver kinase pathway leads to similar restoration of MHC-I expression. This link between the hyperactive pathway and MHC-I-mediated tumor immunosurveillance in both DFTD strains provides mechanistic insights into horizontal transmissibility and suggests a strategy to save Tasmanian devils from extinction. Citation Format: Anna Orlova, Lindsay Kosack, Alexandra M. Popa, Csilla Viczenczova, Alexander Lercher, Elvin D. de Araujo, Patrick Gunning, Richard Moriggl, Andreas Bergthaler. Restoration of tumor immunosurveillance in tasmanian devil facial tumor disease [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 5636.

Clinical and molecular characteristics of Chinese non-small cell lung cancer patients with ERBB2 transmembrane domain mutations.

Transmembrane domain (TMD) mutations of ERBB2 have previously been reported in lung cancer patients in addition to well‐studied kinase domain (KD) mutations, which may stabilize ERBB2 heterodimerization with other EGFR family members and favor a kinase active conformation. However, the frequency and clinical significance of ERBB2 TMD mutations in Chinese population is unknown. We prospectively analyzed the next‐generation sequencing data of 34 368 Chinese lung cancer patients with different sample types, including tumor tissue, plasma, cerebrospinal fluid, and pleural effusion. Patients' clinical characteristics and treatment history were retrieved from the database for further evaluation. Our findings show that ERBB2 V659/G660 mutations were detected at a frequency of 0.13% (45/34 368), of which the most frequent was V659D/E (88.9%), with a trend in nonsmokers and male. Moreover, 18% of patients (8/45) showed EGFR and/or ERBB2 amplification, whereas nine patients presented EGFR L858R or exon19 deletion. Interestingly, novel ERBB3 TMD mutation I646R was found coexisting in three patients with ERBB2 V659D and one patient with ERBB2 G660D, which might influence its heterodimerization with ERBB2 and further activate ERBB2. Four ERBB2 TMD mutation‐positive patients received afatinib monotherapy or combination therapy, but showed variable responses. One patient with V659E responded well to ERBB2 inhibitor lapatinib plus capecitabine as well as subsequent afatinib treatment upon progression. Our study provides valuable insights into the distribution of ERBB2 TMD mutations by employing the largest Asian lung cancer cohort thus far. Patients with ERBB2 TMD mutations who received afatinib, a pan‐ERBB inhibitor, demonstrated mixed responses, posing the urgent need to develop more effective therapeutic strategy for patients who carry ERBB2 TMD mutations.

ErbB1-dependent signalling and vesicular trafficking in primary afferent nociceptors associated with hypersensitivity in neuropathic pain

Effective analgesic treatment for neuropathic pain remains an unmet need, so previous evidence that epidermal growth factor receptor inhibitors (EGFRIs) provide unexpected rapid pain relief in a clinical setting points to a novel therapeutic opportunity. The present study utilises rodent models to address the cellular and molecular basis for the findings, focusing on primary sensory neurons because clinical pain relief is provided not only by small molecule EGFRIs, but also by the anti-EGFR antibodies cetuximab and panitumumab, which are unlikely to access the central nervous system in therapeutic concentrations. We report robust, rapid and dose-dependent analgesic effects of EGFRIs in two neuropathic pain models, matched by evidence with highly selective antibodies that expression of the EGFR (ErbB1 protein) is limited to small nociceptive afferent neurons. As other ErbB family members can heterodimerise with ErbB1, we investigated their distribution, showing consistent co-expression of ErbB2 but not ErbB3 or ErbB4, with ErbB1 in cell bodies of nociceptors, as well as providing evidence for direct molecular interaction of ErbB1 with ErbB2 in situ. Co-administration of selective ErbB1 and ErbB2 inhibitors produced clear evidence of greater-than-additive, synergistic analgesia; highlighting the prospect of a unique new combination therapy in which enhanced efficacy could be accompanied by minimisation of side-effects. Peripheral (intraplantar) administration of EGF elicited hypersensitivity only following nerve injury and this was reversed by local co-administration of selective inhibitors of either ErbB1 or ErbB2. Investigating how ErbB1 is activated in neuropathic pain, we found evidence for a role of Src tyrosine kinase, which can be activated by signals from inflammatory mediators, chemokines and cytokines during neuroinflammation. Considering downstream consequences of ErbB1 activation in neuropathic pain, we found direct recruitment to ErbB1 of an adapter for PI 3-kinase and Akt signalling together with clear Akt activation and robust analgesia from selective Akt inhibitors. The known Akt target and regulator of vesicular trafficking, AS160 was strongly phosphorylated at a perinuclear location during neuropathic pain in an ErbB1-, ErbB2- and Akt-dependent manner, corresponding to clustering and translocation of an AS160-partner, the vesicular chaperone, LRP1. Exploring whether neuronal ion channels that could contribute to hyperexcitability might be transported by this vesicular trafficking pathway we were able to identify Nav1.9, (Nav1.8) and Cav1.2 moving towards the plasma membrane or into proximal axonal locations – a process prevented by ErbB1 or Akt inhibitors. Overall these findings newly reveal both upstream and downstream signals to explain how ErbB1 can act as a signalling hub in neuropathic pain models and identify the trafficking of key ion channels to neuronal subcellular locations likely to contribute to hyperexcitability. The new concept of combined treatment with ErbB1 plus ErbB2 blockers is mechanistically validated as a promising strategy for the relief of neuropathic pain.

ERBB2 Targeting Increases Cardiomyocyte β1-Adrenergic Receptor Density

Objective: To determine the underlying pathways that promote cardiotoxicity following anti-ERBB2 cancer therapeutics. Background: Epidermal growth factor receptor 2 (EGFR2/ERBB2) is a member of EGFR (ERBB) family represented by ERBB 1 through 4 that either homo- or hetero-dimerize to signal. ERBB2 has constitutively active confirmation but explicitly needs to hetero-dimerize with either ERBB1 or ERBB4 to mediate signaling and cardiac hypertrophy. Less is known about the mechanisms through which ERBB2 mediates the adaptive cardiac hypertrophy given that anti-ERBB2 therapeutics results in cardiac dilation of the otherwise healthy heart. Methods: We generated transgenic (Tg) mice with cardiomyocyte-specific overexpression of microRNA-7 (miRNA-7) that targets ERBB2. Echocardiography, immunohistochemistry and β-adrenergic receptor (βAR) density were assessed upon aging and transverse aortic constriction (TAC). To determine whether inhibition of ERBB2 has similar outcomes, C57Bl/6 mice were administered ERBB2-specific inhibitor AG825. Also, βAR density was assessed in end-stage human heart failure patient samples with Adriamycin cardiotoxicity. Results: Loss in ERBB2 expression due to miRNA-7 targeting or ERRB2 inhibition by AG 825 results in cardiac dilation. Loss of ERBB2 in miRNA-7 Tg mice leads to increased fibrosis post-TAC. Selective significant increase in β1AR density is observed in miRNA-7 Tg mice or in mice following ERBB2 inhibition by AG825 and in the human heart failure patient samples due to Adriamycin cardiotoxicity. Conclusions: Cardiomyocyte ERBB2 expression and function is required for maintaining the normal cardiac function. Targeting ERBB2 leads to selective and significant increase in β1ARs that may promote cardiac dilation and heart failure.

Characterization of ERBB2 alterations in non-small cell lung cancer.

e21553 Background: ERBB2 alteration (mutation and/or amplification) is associated with poor survival in non-small cell lung cancer (NSCLC) and is commonly reported as a resistance mechanism to EGFR tyrosine kinase inhibitors. Several clinical trials are ongoing for the management of ERBB2-altered NSCLC. Here we report the prevalence of different ERBB2 alterations in NSCLC. Methods: We retrospectively analyzed ERBB2 alterations in NSCLC tumors that underwent next-generation sequencing (NGS) with Caris Life Sciences. De-identified pathological and molecular information was collected. ERBB2 copy number of greater than 6 was defined as amplification; mutations were classified among 7 groups based on their mechanism of action. Results: A total of 12946 NSCLC tumors with ERBB2 NGS results were available. Among them, 12492 had ERBB2 copy number alteration (CNA) data available. 321 tumors (2.5%) had ERBB2 alteration (mutation or amplification). Among them, ERBB2 was mutated in 197 tumors (1.5%) and amplified in 134 (1.1%) tumors. Type of ERBB2 mutation, respective median age and distribution among sex is in the table. 10 tumors with ERBB2 mutation (7.46%) also showed ERBB2 amplification. Six percent of tumors (8/135) with ERBB2 exon 20 insertion mutations had co-occurring ERBB2 amplification. One tumor for each extracellular domain (ECD) and transmembrane group had co-occurring ERBB2 amplification. Eight tumors with ERBB2 mutation had an overlapping EGFR L858R or exon19del mutation. Seven of these 8 tumors were from the ECD group (7/21) and all had mutations in the S310 locus. One tumor was from the transmembrane group. Conclusions: Exon 20 insertion mutation is the most commonly found ERBB2 mutation among NSCLC, a few of them had co-occurring ERBB2 amplification. One third of tumors with extracellular domain ERBB2 mutation had EGFR mutation. Tarloxitinib (NCT03805841), trastuzumab deruxtucan (NCT03505719), pyrotinib (NCT02500199), poziotinib (NCT03318939) are just a few of the novel ERBB2 inhibitors available in clinical trials. It will be critical to utilize next generation sequencing to effectively capture uncommon mutations and amplifications in ERBB2 so that patients may be offered therapy directly targeted to their genomic alterations. [Table: see text]

Translational analysis of esophageal adenocarcinoma (EAC) patients treated with oxaliplatin and capecitabine (Xelox) +/- the dual ErbB inhibitor AZD8931 in the DEBIOC study.

4539 Background: The Dual Erb B Inhibition in Oesophago-gastric Cancer (DEBIOC) trial reported an acceptable safety profile for neoadjuvant Xelox +/- AZD8931 but limited efficacy. We utilized EAC patient samples from DEBIOC to evaluate the impact of neoadjuvant Xelox +/-AZD8931 on biological pathways using a unique software driven solution. Methods: 24 pre-treatment FFPE EAC biopsies and 17 matched surgical resection specimens were transcriptionally profiled using the Almac Diagnostics Xcel Array. Gene expression data was analyzed using the Almac claraT total mRNA report V3.0.0, reporting on 92 gene expression signatures and 7337 single genes associated with 10 key biologies. Paired Wilcoxon tests (5% significance level) were used to evaluate changes in claraT scores pre- and post-treatment. EGFR and Her2 expression were assessed by IHC and FISH. Results: 15 patients received Xelox+AZD8931 and 9 Xelox alone. Hierarchical clustering of biopsies identified 4 major clusters: Inflammation active, Genomic Instability active, EGFR & MAPK active, and EMT & Angiogenesis active. Comparison of signature scores pre- and post- neoadjuvant treatment demonstrated a significant reduction in scores relating to DNA damage repair (DDR) deficiency (Almac DNA Damage assay, p< 0.0001; BRCAness Profile, p= 0.0025; HRD Gene Signature, p< 0.0001; BRCA1ness Signature, p= 0.0004) and a significant increase in angiogenesis signatures (Almac Angiogenesis Assay, p= 0.0002; Angio Predictive G model, p= 0.0228; Angiogenesis Signature A, p= 0.0034) and EMT signatures (EMT Signature, p= 0.0031, EMT Enrichment Score, p= 0.0013, Pan-Can EMT Signature B, p= 0.0001). Comparing pre- and post-treatment signature scores in patients treated with Xelox +/-AZD8931 revealed a significant reduction in EGFR Sensitivity Signature ( p= 0.0088), ERBB2-specific Gene Expression Signature ( p= 0.0127) and Hallmark PI3K-AKT-MTOR Signaling ( p= 0.0195) in those treated with Xelox + AZD8931 in keeping with the mechanism of action of AZD8931. Downregulation of AKT signaling was confirmed in AZD8931 treated and resistant cell lines. Conclusions: We report the use of a novel software tool to apply 92 gene expression signatures to EAC biopsy and resection specimens from the DEBIOC trial to provide insight into mechanisms of action. Neoadjuvant treatment was associated with a reduction in DDR deficiency and an increase in angiogenesis and EMT signatures whilst a reduction in EGFR, Her2 and AKT pathways was noted with AZD8931 treatment.

New Treatment Strategies for Metastatic Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at an advanced stage, with systemic therapy being the mainstay of treatment. Survival continues to be limited, typically less than 1year. The PDAC microenvironment is characterized by a paucity of malignant epithelial cells, abundant stroma with predominantly immunosuppressive T cells and myelosuppressive-type macrophages (M2), and hypovascularity. The current treatment options for metastatic PDAC are modified (m)FOLFIRINOX /FOLFIRINOX or nab-paclitaxel and gemcitabine in patients with good performance status (PS) (ECOG 0-1/KPS 70-100%) and gemcitabine with or without a second agent for those with ECOG PS 2-3. New therapies are emerging, and the current guidelines endorse both germline and somatic testing in PDAC to evaluate actionable findings. Important themes related to new therapeutic approaches include DNA damage repair strategies, immunotherapy, targeting the stroma, and cancer-cell metabolism. Targeted therapy alone (outside small genomically defined subsets) or in combination with standard cytotoxic therapy, thus far, has proven disappointing in PDAC; however, novel therapies are evolving with increased integration of genomic profiling along with a better understanding of the tumor microenvironment and immunology. A small but important sub-group of patients have some of these agents available in the clinics for use. Olaparib was recently approved by the US Food and Drug Administration for maintenance therapy in germline BRCA1/2 mutated PDAC following demonstration of survival benefit in a phase 3 trial. Pembrolizumab is approved for patients with defects in mismatch repair/microsatellite instability. PDAC with wild-type KRAS represents a unique subgroup who have enrichment of potentially targetable oncogenic drivers. Small-molecule inhibitors including ERBB inhibitors (e.g., afatinib, MCLA-128), TRK inhibitors (e.g., larotrectinib, entrectinib), ALK/ROS inhibitor (e.g., crizotinib), and BRAF/MEK inhibitors are in development. In a small subset of patients withthe KRASG12C mutation,a KRASG12C inhibitor, AMG510, and other agents are being investigated. Major efforts are underway to effectively target the tumor microenvironment and to integrate immunotherapy into the treatment of PDAC, and although thus far the impact has been modest to ineffective, nonetheless, there is optimism that some of the challenges will be overcome.

BRAF mutant colorectal cancer: ErbB2 expression levels as predictive factor for the response to combined BRAF/ErbB inhibitors

BackgroundColorectal cancer (CRC) is a heterogeneous disease with a complex biology and a wide number of altered genes such as BRAF, KRAS and PIK3CA. Advances with new-targeted therapies have been achieved and available treating options have prolonged patient’s survival. However, BRAF-mutated CRC patients remain unresponsive to available therapies with RAF inhibitors (RAFi) alone or combined with ErbB inhibitors (ErbBi). These unmet needs require further exploitation of oncogenic signaling in order to set up individualized treatments.MethodsTo this end, we tested the efficacy of single agent or combined treatments using the BRAFi, vemurafenib and two different ErbBi: panitumumab and afatinib in CRC cells characterized by different molecular phenotypes.ResultsCombination strategies with BRAFi and ErbBi achieved a better response in BRAFV600E mutated cells expressing high levels of ErbB2.ConclusionsOur findings support the importance of ErbB2 evaluation in BRAF-mutated CRC patients and its role as a positive predictor factor of response to BRAFi/ErbBi combination.