HER2-amplified Cell Lines Research Articles

3D layered co-culture model enhances Trastuzumab Deruxtecan sensitivity and reveals the combined effect with G007-LK in HER2-positive non-small cell lung cancer

Human epidermal growth factor receptor 2 (HER2) aberrations are observed in various cancers. In non-small cell lung cancer, genetic alterations activating HER2, mostly exon 20 insertion mutations, occur in approximately 2–4% of cases. Trastuzumab deruxtecan (T-DXd), a HER2-targeted antibody-drug conjugate has been approved as the first HER2-targeted drug for HER2-mutant lung cancer. However, some cases are not responsive to T-DXd and the primary resistant mechanism remains unclear. In this study, we assessed sensitivity to T-DXd in JFCR-007, a patient-derived HER2-mutant lung cancer cell line. Although JFCR-007 was sensitive to HER2 tyrosine kinase inhibitors, it showed resistance to T-DXd in attachment or spheroid conditions. Accordingly, we established a three-dimensional (3D) layered co-culture model of JFCR-007, where it exhibited a lumen-like structure and became sensitive to T-DXd. In addition, an in-house inhibitor library screening revealed that G007-LK, a tankyrase inhibitor, was effective when combined with T-DXd. G007-LK increased the cytotoxicity of topoisomerase-I inhibitor, DXd, a payload of T-DXd and SN-38. This combined effect was also observed in H2170, an HER2-amplified lung cancer cell line. These results suggest that the proposed 3D co-culture system may help in evaluating the efficacy of T-DXd and may recapitulate the tumor microenvironment.

Evaluating antitumor activity and response determinants to trastuzumab deruxtecan in pediatric solid tumors.

10049 Background: Trastuzumab deruxtecan (T-DXd) is an anti-HER2 antibody-drug conjugate (ADC) linked to a topoisomerase inhibitor FDA-approved for several indications, including the treatment of HER2+ breast and gastric cancer. Although HER2 amplification is uncommon in pediatric cancers, recent demonstration of T-DXd efficacy in HER2-low breast cancer patients prompted us to examine the potential clinical relevance of T-DXd in pediatrics by evaluating HER2 expression and activity of T-DXd in preclinical pediatric solid tumor models. Methods: Cell viability was assessed in a panel of histologically diverse pediatric solid tumor cell lines and 2 HER2-amplified adult cancer cell lines treated with T-DXd, payload (DXd), and a control IgG-ADC. HER2 protein expression in patient-derived xenograft (PDX) tumors was evaluated by immunohistochemistry (IHC) using 5 clinically validated anti-HER2 antibodies. RNAseq was performed on clinical (n=290) and PDX (n=184) tumors to determine relative expression of ERBB2. In vivo activity of T-DXd was evaluated in osteosarcoma (OS, n=10), Wilms tumor (WT, n=1) and malignant rhabdoid tumor (MRT, n=1) PDX models and a desmoplastic small round cell tumor (DSRCT) cell line xenograft model. Differences in tumor volume and time to disease progression was assessed and compared across treatments and models. Responses were correlated with HER2 expression by IHC. Results: In vitro, HER2-amplified control cell lines demonstrated a >30-fold reduction of IC50 when comparing T-DXd to ADC control. In contrast, the ADC control IC50 was nearly identical to T-DXd across all pediatric cancer cell lines, consistent with the absence of HER2 amplification in these models. We observed focal and membranous staining of HER2 by IHC in PDXs but was quite variable across antibodies tested: WT 0-17% HER2+ cases (n=6), MRT 0-40% (n=5), DSRCT 0-33% (n=24), OS 0-7% (n=30). ERBB2 gene expression was highest in DSRCT followed by WT, OS and MRT. In vivo efficacy studies demonstrated complete and partial responses in OS, WT, and DSRCT and improved disease control rates (OS: 67%, p=0.006, Mann-Whitney; WT: 100%; DSRCT: 100%). However, T-DXd and ADC control demonstrated similar activity in all tumor types with no consistent correlation between in vivo response and HER2 expression. Consistent with these preclinical studies, 4 patients with progressive DSRCT were treated with T-DXd via compassionate/off-label access with signs of clinical and radiographic responses. Conclusions: T-DXd shows significant preclinical antitumor activity across multiple pediatric solid tumors but little correlation with HER2 expression suggesting a mechanism of action similar to the clinical activity observed in HER2-low breast cancer. Xenograft efficacy studies and preliminary clinical experience with T-DXd in DSRCT patients warrant formal clinical trial investigation in this largely incurable disease.

Molecular Mechanism by which Coumestrol Exerts its Anticancer Activity in Triple‐Negative Inflammatory Breast Cancer

Coumestrol (Cou) is a phytoestrogen present in soy and clover sprouts which is structurally similar to estrogen (E2) and has a cytotoxic effect in breast cancer cell lines. Nevertheless, the molecular mechanism by which Cou can exert its effect in the most aggressive breast cancer subtype, triple‐negative inflammatory breast cancer (TN‐IBC), is still unknown. Several studies show that TN‐IBC cell lines are unresponsive to hormonal therapies, but E2 can activate signaling pathways involved in pro‐oncogenic phenotypes such as motility and invasion. Therefore, the objective of this study was to determine if Cou has an anti‐cancer effect in TN‐IBC cell lines and the molecular mechanism that Cou exerts for its anti‐cancer activity using 2D cell culture models. For this, a dose‐response curve of Cou in 2D models of TN‐IBC cell lines was generated to determine the half‐maximal inhibitory concentration (IC50). Also, relative cell viability was performed in ER‐positive, triple‐negative, TN‐IBC, and HER2‐amplified IBC cell lines. Functional assays were performed in TN‐IBC cell lines to determine the effect of E2 alone, Cou alone, and in a combination of treatments on cell viability, migration, and invasion. Also, the effect on phosphorylation of downstream kinases was analyzed by a proteome profile human phospho‐kinase array. An RNA‐seq was performed in TN‐IBC cell lines to identify transcriptome changes after Cou treatment. Preliminary observations demonstrated that Cou treatment using the IC50 (13µM) caused a decrease in cell viability in TN‐IBC cell lines. Similarly, Cou treatment reduces triple‐negative non‐IBC viability. Also, in comparison with E2, Cou decreases migration and invasion in TN‐IBC cell lines. Finally, Cou treatment reduces the phosphorylation of kinases that promote pro‐oncogenic phenotypes and upregulate genes involved in breast cancer phenotype suppression. In summary, the data gathered in this study will guide us in elucidating a signaling pathway affected by Cou’s anti‐cancer activity. More importantly, it will open the opportunities to design more effective targeted therapeutic strategies to improve the prognosis and survival rates of IBC patients.

Abstract P234: ORIC-114, an orally bioavailable, irreversible kinase inhibitor, has superior brain penetrant properties and enhanced potency in preclinical studies of HER2-positive breast cancer

Abstract Amplification of human epidermal growth factor receptor 2 (HER2) is an oncogenic driver found in approximately 25% of breast cancer. Despite the arsenal of HER2-directed therapies available to patients, more than 50% of patients with HER2 amplification eventually develop central nervous system (CNS) metastases over the course of their disease indicating a clear medical need for brain penetrant therapies in this patient population. ORIC-114 is a brain penetrant, orally bioavailable, irreversible small molecule inhibitor that was designed to target exon20 insertions in epidermal growth factor receptor (EGFR) and HER2. ORIC-114 is highly selective for the EGFR/HER2 family of receptors, reducing the risk for off-target kinase liabilities. In biochemical assays, ORIC-114 displayed low nanomolar potency on HER2. To explore the application of ORIC-114 in the HER2-amplified tumor setting, a cell viability screen was performed against a panel of human breast cancer lines containing both HER2-amplified and non HER2-amplified cell lines. ORIC-114 demonstrated greater than 100-fold enhanced cellular potency on HER2-amplified cancer cell lines relative to non-amplified cancer cell lines. Notably, ORIC-114 cellular EC50s in HER2-amplified breast cancer cell lines were below 30 nM and more potent than both lapatinib and tucatinib, two FDA-approved tyrosine kinase inhibitors for the treatment of HER2-positive breast cancer. ORIC-114 was designed to incorporate physicochemical properties suitable to cross the blood-brain barrier and has exhibited good brain penetration across multiple preclinical species. To further investigate the brain penetrant attributes of ORIC-114 in the context of HER2-positive breast cancer with brain metastases, key features were assessed relative to tucatinib, which has demonstrated clinical efficacy in this setting. In contrast to tucatinib, ORIC-114 displayed minimal impact on efflux transporters as it was only a weak substrate for P-glycoprotein (P-gp) and was not a substrate for breast cancer associated protein (BCRP) in vitro. In addition, ORIC-114 demonstrated superior in vivo brain penetration relative to tucatinib as measured by free brain-to-plasma ratio in mouse. Consequently, ORIC-114 free brain concentrations in rodents were greater than tucatinib, even when the active metabolites of tucatinib were considered. Taken together, these data further establish ORIC-114 as a selective, irreversible, and brain penetrant EGFR/HER2 inhibitor, making it a promising therapeutic candidate for development in patients with HER2-positive tumors including those with CNS metastases. A Clinical Trial Application for ORIC-114 is anticipated in the second half of 2021. Citation Format: Melissa R. Junttila, Jason E. Long, Robert Warne, Sunghwan Kim, Younho Lee, Hwan Kim, Juhee Kang, Jiyoon Seok, Jihye Yoo, Youngyi Lee, Dong-Hyuk Seo Seo, Jung Beom Son, Daekwon Kim, Hwan Geun Choi, Nam Doo Kim, Tatiana Zavorotinskaya, Chelsea Chan, Matthew Panuwat, Jessica Sun, Jae H. Chang, Lori S. Friedman. ORIC-114, an orally bioavailable, irreversible kinase inhibitor, has superior brain penetrant properties and enhanced potency in preclinical studies of HER2-positive breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P234.

A novel treatment strategy for lapatinib resistance in a subset of HER2-amplified gastric cancer

BackgroundGastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Human epidermal growth factor receptor 2 (HER2) amplification occurs in approximately 13–23% of all GC cases and patients with HER2 overexpression exhibit a poor prognosis. Lapatinib, a dual EGFR/HER2 tyrosine kinase inhibitor, is an effective agent to treat HER2-amplified breast cancer but it failed in gastric cancer (GC) clinical trials. However, the molecular mechanism of lapatinib resistance in HER2-amplified GC is not well studied.MethodsWe employed an unbiased, genome-scale screening with pooled CRISPR library on HER2-amplified GC cell lines to identify genes that are associated with resistance to lapatinib. To validate the candidate genes, we applied in vitro and in vivo pharmacological tests to confirm the function of the target genes.ResultsWe found that loss of function of CSK or PTEN conferred lapatinib resistance in HER2-amplified GC cell lines NCI-N87 and OE19, respectively. Moreover, PI3K and MAPK signaling was significantly increased in CSK or PTEN null cells. Furthermore, in vitro and in vivo pharmacological study has shown that lapatinib resistance by the loss of function of CSK or PTEN, could be overcome by lapatinib combined with the PI3K inhibitor copanlisib and MEK inhibitor trametinib.ConclusionsOur study suggests that loss-of-function mutations of CSK and PTEN cause lapatinib resistance by re-activating MAPK and PI3K pathways, and further proved these two pathways are druggable targets. Inhibiting the two pathways synergistically are effective to overcome lapatinib resistance in HER2-amplified GC. This study provides insights for understanding the resistant mechanism of HER2 targeted therapy and novel strategies that may ultimately overcome resistance or limited efficacy of lapatinib treatment for subset of HER2 amplified GC.

Abstract 945: Trastuzumab deruxtecan (T-DXd) sensitivity in various levels of HER2 expressing gastric cancer cells

Abstract Trastuzumab, a humanized anti-HER2 monoclonal antibody, has shown clinically significant benefits for HER2-positive GC patients. Also, all anti-HER2 agents approved by the FDA are used for HER2-positive. However, since HER2-positive is only 10-20% of GC, targeted therapies for non-HER2-positive GC is needed. To overcome these limitations, antibody-drug conjugates (ADCs), which directly deliver cytotoxic drugs to cancer cells, have been developed. T-DXd (Daiichi Sankyo, Japan), an anti-HER2 ADC, can induce the death of neighboring cells because it has a bystander killing effect due to the high membrane permeability of the payload. However, biomarkers for predicting drug response have not yet been studied other than HER2 overexpression/amplification. In this study, we investigated the correlation between HER2 expression level and anti-tumor effect of T-DXd in a 49 GC cell line panel and the HER2-associated molecules related to T-DXd sensitivity. For HER2 status, we analyzed multiple genetic and molecular characteristics using WES, SISH, IHC, flow cytometry, and immunoblotting. GC cell lines (n=49) were divided into 3 groups according to the HER2 expression level through flow cytometry using 4 breast cancer cell lines (SK-BR-3, ZR-75-1, MCF-7, and HCC1937) for quantitative control. Groups with HER2 over- and moderate/low-expression were characterized with higher expression levels compared to ZR-75-1 (HER2 2+) and HCC1937 (HER2-negative), respectively. HER2 non-expression had lower expression levels compared to HCC1937. The sensitivity of T-DXd was evaluated in 49 GC cell lines by cell viability assay. The inhibition rate of T-DXd was calculated at 10 µg/ml. Also, expression levels of RTKs were determined by immunoblotting, and HER2 extracellular domain (ECD) and NRG1 expression were evaluated by ELISA. GC cell lines were divided into 6 HER2 over-, 26 HER2 moderate/low-, and 17 HER2 non-expression cell lines. As expected, HER2 over-expression cell lines were sensitive to T-DXd (5/6, 83.3%). T-DXd also had anti-tumor effects in the HER2 moderate/low-expression cell lines (12/26, 46.2%). There were correlations between HER2 expression level and anti-tumor effect of T-DXd in both HER2 over and moderate/low-expression cell lines. HER2 over and moderate/low-expression cell lines with high HER2 ECD expression were sensitive to T-DXd. NRG1 had relatively low expression in all GC cell lines and was not correlated with T-DXd. Additionally, even HER2 non-expression cell lines were sensitive to T-DXd (10/17, 58.8%). We found that HER2 non-expression cell lines had an RTK amplification (9/17, 52.9%) and among those cell lines, MET amplified cells showed sensitivity to T-DXd. Currently, the evaluation of the underlying mechanisms is ongoing. Our results indicate that T-DXd shows efficacy in HER2-amplified GC cell lines, HER2 moderate/low-expression, and some cell lines with HER2 non-expression. Citation Format: Seo Young Yu, Juin Park, Woo Sun Kwon, Inhye Jeong, Sun Kyoung Kang, Hyun Joo Bae, Tae Soo Kim, Hyun Cheol Chung, Sun Young Rha. Trastuzumab deruxtecan (T-DXd) sensitivity in various levels of HER2 expressing gastric cancer cells [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 945.

Abstract PS10-06: Comparative analysis of anti-proliferative effects and gene profiling of lapatinib, neratinib, and tucatinib

Abstract Introduction: Human epidermal growth factor 2 (HER2/ERBB2) is frequently amplified or mutated across various cancer types. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. All three TKIs bind and inhibit the kinase domain of HER2 but differ both in the mechanism of binding and in specificity for other HER family members. Direct comparisons to differentiate the pre-clinical efficacy of the three TKIs have been limited to small-scale studies and novel biomarkers of response to further define appropriate patient populations are required. Methods: In this study, the anti-proliferative effects of the three TKIs were compared using a 115-cancer cell line panel, including 12 breast cancer cell lines and 22 cell lines harbouring point mutations or amplifications of EGFR, HER2, or HER3. Hierarchical clustering analysis was carried out to compare the IC50 “fingerprint” of the three TKIs to 168 other anti-cancer agents. Novel markers of TKI sensitivity and resistance were identified through cross-analysis of each drug response profile with mutation, copy number variation, and gene expression data. Results: All three TKIs were effective against HER2-positive breast cancer models; neratinib showed the most potent activity, followed by tucatinib and lapatinib respectively (Table 1). Neratinib displayed the greatest anti-proliferative activity in HER2-mutant and EGFR-mutant cell lines. Clustering analysis revealed that the anti-proliferative profile of tucatinib was most similar to trastuzumab, while neratinib and lapatinib were most like other HER family inhibitors. Mutation and gene expression analysis identified potential markers of response for each TKI. High expression of four genes (HER2, VTCN1, CDK12, and RAC1) correlated with response to all three TKIs. DNA damage repair genes were significantly associated with resistance to the HER2-targeted TKIs. BRCA2 mutation was correlated with neratinib and tucatinib response, and high gene expression of ATM, BRCA2, and BRCA1 were all associated with neratinib resistance. Conclusions: Neratinib was the most effective HER2-targeted TKI against HER2-amplified, -mutant, and EGFR-mutant cell lines. This analysis revealed possible mechanisms that may be exploited using combinatorial strategies involving CDK inhibitors, immunotherapies, and targeting DNA repair pathways. Table: IC50 values for neratinib, lapatinib, and tucatinib in the HER2+ breast cancer cell linesIC50 values (nM)Cell linesNeratinibLapatinibTucatinibAU-56520294125BT-4745926229HCC195413814262122MDA-MB-453306228445928SKBR3715222 Citation Format: Neil T Conlon, Jeffrey J Kooijman, Suzanne JC van Gerwen, Winfried R Mulder, Guido JR Zaman, Irmina Diala, Lisa D Eli, Alshad S Lalani, John Crown, Denis Collins. Comparative analysis of anti-proliferative effects and gene profiling of lapatinib, neratinib, and tucatinib [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 PS10-06.

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

Comparative analysis of drug response and gene profiling of HER2-targeted tyrosine kinase inhibitors

BackgroundHuman epidermal growth factor 2 (HER2/ERBB2) is frequently amplified/mutated in cancer. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. Direct comparisons of the preclinical efficacy of the TKIs have been limited to small-scale studies. Novel biomarkers are required to define beneficial patient populations.MethodsIn this study, the anti-proliferative effects of the three TKIs were directly compared using a 115 cancer cell line panel. Novel TKI response/resistance markers were identified through cross-analysis of drug response profiles with mutation, gene copy number and expression data.ResultsAll three TKIs were effective against HER2-amplified breast cancer models; neratinib showing the most potent activity, followed by tucatinib then lapatinib. Neratinib displayed the greatest activity in HER2-mutant and EGFR-mutant cells. High expression of HER2, VTCN1, CDK12, and RAC1 correlated with response to all three TKIs. DNA damage repair genes were associated with TKI resistance. BRCA2 mutations were correlated with neratinib and tucatinib response, and high expression of ATM, BRCA2, and BRCA1 were associated with neratinib resistance.ConclusionsNeratinib was the most effective HER2-targeted TKI against HER2-amplified, -mutant, and EGFR-mutant cell lines. This analysis revealed novel resistance mechanisms that may be exploited using combinatorial strategies.

Trastuzumab upregulates programmed death ligand-1 expression through interaction with NK cells in gastric cancer

BackgroundThe predictive significance of programmed death ligand 1 (PD-L1) for programmed death 1 (PD-1) inhibitors remains unclear in gastric cancer (GC) due to the dynamic alteration by treatments. We aimed to elucidate the effects of trastuzumab (Tmab) on PD-L1 expression in GC.MethodsPD-L1 expression was evaluated by multicolour flow cytometry analysis after co-culturing GG cell lines and immune cells with Tmab. IFN-γ in the co-culture experiments was quantified. Immunohistochemistry (IHC) for PD-L1 expression using clinical samples was also performed to confirm PD-L1 alteration by Tmab.ResultsPD-L1 expression was significantly upregulated by Tmab in HER2-amplified GC cell lines co-cultured with peripheral blood mononuclear cells (PBMCs). PD-L1 upregulation by Tmab was also observed in the GC cells co-cultured with NK cells in time-dependent manner, but not with monocytes. IFN-γ concentration in conditioned media from co-cultured PBMCs and NK cells with Tmab was significantly higher and anti-IFN-γ significantly suppress the Tmab-induced PD-L1 upregulation. IHC also suggested PD-L1 upregulation after Tmab treatment.ConclusionsTmab can upregulate PD-L1 expression on GC cells through interaction with NK cells. These results suggest clinical implications in the assessment of the predictive significance of PD-L1 expression for PD-1 inhibitors.

Abstract 1962: Preclinical characterization of tucatinib in HER2-amplified xenograft and CNS implanted tumors

Abstract Tucatinib is an investigational, oral, small molecule tyrosine kinase inhibitor that is highly selective for the kinase domain of HER2, without significant inhibition of EGFR. Recently, HER2CLIMB (NCT02614794), a pivotal, randomized, international, double-blind trial that evaluated tucatinib or placebo in combination with trastuzumab and capecitabine in patients with HER2+ metastatic breast cancer (MBC) with or without brain metastases, after progression with trastuzumab, pertuzumab, and ado-trastuzumab emtansine (T-DM1), showed superior progression-free and overall survival in patients treated on the tucatinib arm. Adverse events in the tucatinib arm were primarily low grade and occurred at rates similar to what was observed in the placebo arm. In this report, we characterize the in vitro and in vivo activity of tucatinib alone and in combination with T-DM1 in HER2-amplified preclinical models. In vitro assays demonstrate that tucatinib potently suppresses HER2-meditated signaling pathways, including phosphorylation of HER2, HER3, AKT and ERK. Ex vivo analysis of tucatinib-treated xenografts shows similar suppression of signal transduction pathways. Tucatinib inhibits the proliferation of HER2+ breast cancer (BC) cell lines in vitro with single digit nanomolar potencies, but was inactive in blocking cell proliferation of BC cell lines lacking amplified HER2. In HER2-amplified BC cell line derived- and patient-derived xenograft models, tucatinib suppresses growth of tumors as a single agent and shows improved activity when combined with T-DM1. To evaluate activity and drug penetration in HER2+ metastatic CNS tumors, we developed and characterized a red-shifted luciferase-expressing BT-474 BC cell line which was stereotaxically implanted into the brain. In vivo bioluminescence imaging was used to evaluate the efficacy of tucatinib and T-DM1 in CNS tumors as single agents and in combination. Histological assessment of treated tumors suggests that tucatinib can penetrate the blood-brain tumor barrier to suppress HER2 signaling. These data are further supported by a drug penetration study of CNS-implanted BT-474 tumors. Quantitative autoradiography analysis demonstrates that 14C-labeled tucatinib penetrates the CNS tumor mass with drug concentration levels exceeding measurements in normal brain regions. These data demonstrate that tucatinib is a uniquely selective and highly potent inhibitor of HER2 signaling, with activity against HER2+ breast cancer xenograft and brain metastasis models. Tucatinib is effective against implanted CNS tumors, suggesting that therapeutically relevant drug concentrations are achievable. These preclinical findings are consistent with clinical data showing tucatinib activity in patients with HER2+ MBC with brain metastases and support the continued development of tucatinib in patients with HER2+ BC. Citation Format: Devra J. Olson, Anita Kulukian, Janelle D. Taylor, Margo C. Zaval, Albina Nesterova, Kelly M. Hensley, Michelle L. Ulrich, Nicole S. Stevens, Scott R. Peterson. Preclinical characterization of tucatinib in HER2-amplified xenograft and CNS implanted tumors [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 1962.

The Hippo Pathway Transducers YAP1/TEAD Induce Acquired Resistance to Trastuzumab in HER2-Positive Breast Cancer.

Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer, leading to improved overall survival. However, acquired resistance inevitably occurs. We aimed to identify, quantify, and assess the mechanisms of acquired resistance to trastuzumab. We established an acquired trastuzumab-resistant model in vitro from BT-474, a trastuzumab-sensitive, HER2-amplified breast-cancer cell line. A multi-omic strategy was implemented to obtain gene, proteome, and phosphoproteome signatures associated with acquired resistance to trastuzumab in HER2-positive breast cancer, followed by validation in human clinical samples. YAP1 dephosphorylation and TEAD2 overexpression were detected as significant alterations in the Hippo pathway in trastuzumab-resistant breast cancer. Because of the emerging role of these proteins as mediators of normal growth and tumorigenesis, we assessed the exogenous modulation of their activity, either by in vitro gene silencing or by pharmacological inhibition of the YAP1/TEAD complexes, both in vitro and in vivo. Moreover, we identified increased signaling through the Hippo pathway in human samples after progression following trastuzumab treatment. Finally, YAP1/TAZ nuclear accumulation in malignant cells in HER2 breast tumor was significantly associated with worse progression-free and overall survival in metastatic HER2-positive breast-cancer patients. Our results suggest the involvement of Hippo signaling in acquired trastuzumab resistance in breast cancer. Additionally, we provide novel evidence for a potential breast-cancer treatment strategy based on dual targeting of HER2 and Hippo pathway effectors, which may improve the antitumor activity of trastuzumab and help overcome resistance.

Abstract B003: Combinatorial therapies of neratinib for HER2-amplified cancer

Abstract Activation of HER2 signaling by amplification or overexpression of ERBB2 (HER2) is associated with the development and progression of breast cancer. Neratinib is an irreversible, pan-HER tyrosine kinase inhibitor which selectively inhibits EGFR, HER2 and HER4. In this preclinical study, we explored the therapeutic efficacy of neratinib in combination with other clinically relevant targeted agents for HER2-amplified breast cancer in in vitro and in vivo models. HER2-amplified cell lines BT-474, SK-BR-3, and HCC-1954 were sensitive to neratinib with low nanomolar IC50s. Cell viability and colony formation assays demonstrated synergistic anti-proliferative activity of neratinib in combination with multiple targeted agents, including the MEK inhibitor trametinib, the PI3Ka inhibitor alpelisib, the mTOR inhibitors everolimus and sapanisertib, and CDK4/6 inhibitor palbociclib. We tested combinatorial efficacy of neratinib with everolimus, trametinib, and palbociclib in vivo on five HER2-amplified PDXs (two breast, two colorectal and one gastric-esophageal junction cancer), four derived from patients with previous HER2-targeted therapy. The combination of neratinib with everolimus or trametinib led to at least a 100% increase in median time for tumor doubling in 25% (1 of 4) and 60% (3 of 5) models respectively and was associated with decreased tumor volumes in 50% (2 of 4) and 20% (1 of 5) of the models respectively. The combination of neratinib with palbociclib significantly enhanced antitumor efficacy compared to single agent treatment in all five PDX models. The combination achieved treatment/control volume ratios of 0.03-0.16 and increased time for tumor doubling more than 100% in all five models. Taken together, our results provide solid preclinical evidence for targeting combinatorial therapies of neratinib for HER2-amplified cancer. Citation Format: Ming Zhao, Stephen Scott, Kurt Evans, Erkan Yuca, Turcin Saridogan, Mehmet E Demirhan, Bryce P Kirby, Scott Kopetz, Irmina Diala, Alshad S Lalani, Sarina A Piha-Paul, Funda Meric-Bernstam. Combinatorial therapies of neratinib for HER2-amplified cancer [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 B003. doi:10.1158/1535-7163.TARG-19-B003

Synthesis and Enhanced Cellular Uptake In Vitro of Anti-HER2 Multifunctional Gold Nanoparticles.

Nanoparticle carriers offer the possibility of enhanced delivery of therapeutic payloads in tumor tissues due to tumor-selective accumulation through the enhanced permeability and retention effect (EPR). Gold nanoparticles (AuNP), in particular, possess highly appealing features for development as nanomedicines, such as biocompatibility, tunable optical properties and a remarkable ease of surface functionalization. Taking advantage of the latter, several strategies have been designed to increase treatment specificity of gold nanocarriers by attaching monoclonal antibodies on the surface, as a way to promote selective interactions with the targeted cells—an approach referred to as active-targeting. Here, we describe the synthesis of spherical gold nanoparticles surface-functionalized with an anti-HER2 antibody-drug conjugate (ADC) as an active targeting agent that carries a cytotoxic payload. In addition, we enhanced the intracellular delivery properties of the carrier by attaching a cell penetrating peptide to the active-targeted nanoparticles. We demonstrate that the antibody retains high receptor-affinity after the structural modifications performed for drug-conjugation and nanoparticle attachment. Furthermore, we show that antibody attachment increases cellular uptake in HER2 amplified cell lines selectively, and incorporation of the cell penetrating peptide leads to a further increase in cellular internalization. Nanoparticle-bound antibody-drug conjugates retain high antimitotic potency, which could contribute to a higher therapeutic index in high EPR tumors.

NRF2 through RPS6 Activation Is Related to Anti-HER2 Drug Resistance in HER2-Amplified Gastric Cancer.

Despite the clinical advantage of the combination of trastuzumab and platinum-based chemotherapy in HER2-amplified tumors, resistance will eventually develop. The identification of molecular mechanisms related to primary and acquired resistance is needed. We generated lapatinib- and trastuzumab-resistant clones deriving from two different HER2-amplified gastric cancer cell lines. Molecular changes such as protein expression and gene-expression profile were evaluated to detect alterations that could be related to resistance. Functional studies in vitro were corroborated in vivo. The translational relevance of our findings was verified in a patient cohort. We found RPS6 activation and NRF2 to be related to anti-HER2 drug resistance. RPS6 or NRF2 inhibition with siRNA reduced viability and resistance to anti-HER2 drugs. In knockdown cells for RPS6, a decrease of NRF2 expression was demonstrated, suggesting a potential link between these two proteins. The use of a PI3K/TORC1/TORC2 inhibitor, tested in vitro and in vivo, inhibited pRPS6 and NRF2 expression and caused cell and tumor growth reduction, in anti-HER2-resistant models. In a cohort of HER2-amplified patients treated with trastuzumab and chemotherapy, a high level of NRF2 at baseline corresponds with worse progression-free survival. NRF2 through the PI3K/AKT/mTOR/RPS6 pathway could be a potential effector of resistance to anti-HER2 drugs in our models. RPS6 inhibition decreases NRF2 expression and restores sensitivity in HER2-amplified gastric cancer in vitro and in vivo. High NRF2 expression in gastric cancer patients predicts resistance to treatment. RPS6 and NRF2 inhibition could prevent resistance to anti-HER2 drugs.

Abstract P6-17-11: The small molecule inhibitor of HER2, tucatinib, has potent and highly selective activity in preclinical modes of HER2-driven cancer

Abstract Background: Pharmacologically targeting the HER2 oncoprotein provides clinical benefit for patients with HER2-amplified cancers. However, a significant number of patients do not respond to the currently approved HER2-targeted therapies, despite carrying the HER2-alteration. Small molecule inhibitors of HER2, that target other receptor tyrosine kinases such as EGFR (i.e. lapatinib), are approved and provide some clinical benefit but are often associated with increased toxicity. Tucatinib (ARRY-380) is an orally available, potent, highly selective small molecule inhibitor of the HER2 kinase. In this study, we assessed the in vitroand in vivoactivity of tucatinib, relative to approved HER2-targeting molecules, in a panel of molecularly characterized breast cancer cell lines. Materials and Methods:The growth inhibitory activity of tucatinib, trastuzumab and lapatinib were evaluated in a panel of 48 breast cancer cell lines molecularly characterized at baselineby genomic (array-CGH) and proteomic (Reverse Phase Protein Array; RPPA) profiling. IC50values for tucatinib and lapatinibwere determined from direct cell counts using a Cellavista Cell Imaging System. Trastuzumab activity was measured as % inhibition of cell growth at fixed concentrations. In vivoefficacy of tucatinib was assessed in cell line xenograft models of HER2+/ER- and HER2+/ER+ breast cancers as a single agent or in combination with targeted therapies for breast cancer. Results: A broad range of IC50values (3.2nM to >10μM), was seen for tucatinib with a high degree of selectivity for the HER2-amplfied sub-type. High levels of total and phosphorylated HER2 (pHER2) accompanied by high levels of pEGFR and pHER3 enriched for sensitivity to tucatinib, confirming that HER2-driven cancers may be uniquely sensitive to tucatinib. The response profile for lapatinib was less clean, with responses also observed in HER2-low/EGFR-high cell lines. Sensitivity to tucatinib was also observed in HER2-amplified cell lines that were either de novoor acquired resistant to trastuzumab. Single agent tucatinib induced tumor regressions in a xenograft model of HER2+/ER+ breast cancer. Tumor regressions were further enhanced by combination with trastuzumab. The combination of tucatinib plus trastuzumab was as efficacious and better tolerated than trastuzumab plus docetaxel or trastuzumab plus pertuzumab plus docetaxel. The triple combination of tucatinib plus hormonal blockade (fulvestrant) and CDK4/6 inhibition (abemaciclib) also induced robust tumor regressions, without significant body weight loss. Discussion: These preclinical data highlight the potential of the HER2-selective small molecule inhibitor, tucatinib, to provide benefit to patients with HER2-amplifed cancers. Furthermore, our biomarker analysis of response to tucatinib has identified a HER2-driven signature within the HER2-amplfied sub-type that selects for sensitivity to tucatinib. Selecting patients based on this profile may further enrich for individuals most likely to benefit from tucatinib-based therapies. Citation Format: O'Brien NA, Conklin D, McDermott M, Luo T, Ayala R, Issakhanian S, Salgar S, Hurvitz S, Slamon DJ. The small molecule inhibitor of HER2, tucatinib, has potent and highly selective activity in preclinical modes of HER2-driven cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-17-11.

Abstract P2-03-08: Preclinical evaluation of the PI3Kα/δ inhibitor, copanlisib in HER2+ breast cancer: A proof of concept study

Abstract Purpose: The PI3K-AKT-mTORC1/C2 pathway is frequently activated in HER2+ breast cancer and upregulation of this pathway is a key mechanism of trastuzumab resistance. However, attempts to indirectly target this pathway by using the allosteric mTOR inhibitor everolimus have had limited clinical success. Here, we present the results of a preclinical study of the PI3K α/δ (dominant) inhibitor copanlisib alone and in combination with T-DM1, in HER2 amplified cell lines including a model with acquired resistance to trastuzumab. Method: Anti-proliferative, apoptotic, cell cycle, and intracellular signaling effects of copanlisib alone and in combination with T-DM1 were evaluated in a panel of HER2 amplified (ER+ or ER-), and HER2 amplified/PIK3CA mutated cell lines, as well as trastuzumab-resistant breast cancer cell lines. Results: 1) Copanlisib inhibited PI3K, mTOR and their downstream signaling molecules in HER2 amplified/PIK3CA WT or PIK3CA mutated as well as in trastuzumab-resistant cell lines, 2) interestingly, copanlisib also inhibited RAS-MAPK signaling in the earlier time points, 3) copanlisib caused a strong differential growth inhibition in HER2 amplified BC cell lines by 3D-ON-TOP clonogenic assay and real-time monitoring in an IncuCyte Zoom. Inhibition was greater when copanlisib was combined with T-DM1, 4) administration of copanlisib induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependent manner, 5) unlike everolimus copanlisib blocked HIF1α accumulation in hypoxic condition and this blocking effect was reversed by prior treatment with the proteasome inhibitor, carfilzomib and 6) copanlisib also attenuated HER2 amplified cell migration, an important phenotypic feature for metastasis. Conclusions: Copanlisib is highly effective (blocks proliferation, induces apoptosis, and inhibits PI3K and its downstream signaling targets) in HER2 amplified breast cancer cell lines including trastuzumab-resistant and PIK3CA mutated cell lines. The addition of copanlisib to T-DM1 might represent an improved treatment strategy for patients with refractory metastatic HER2+ breast cancer. Citation Format: De P, Carlson JH, Dey N, Leyland-Jones B. Preclinical evaluation of the PI3Kα/δ inhibitor, copanlisib in HER2+ breast cancer: A proof of concept study [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-03-08.

Abstract B66: The conditional requirement of HER3 in HER2-amplified breast cancers

Abstract The amplification and overexpression of HER2 underlies the pathogenesis of a large subset of breast, gastric, and esophageal cancers as well as smaller subsets of many other types of cancer. A substantial body of work has confirmed that overactive HER2 signaling is etiologically linked with the pathogenesis, progression and metastasis of these cancers. HER3, the preferred heterodimerization partner of HER2, is known to be critical for HER2-amplified tumorigenesis, and this has been thought to be due to its unique ability to activate PI3K/Akt pathway. This has fueled efforts to develop HER3 targeting agents to combine with HER2 inhibitors for the treatments of these cancers, and many such agents are in the preclinical and clinical pipelines. We have been modeling HER2-HER3 signaling co-targeting in experimental models to foresee the activities and limitations of this clinical strategy and have identified potentially significant escape mechanisms that limit the therapeutic potential of this approach. This appears to be largely due to the mistaken attribution of PI3K/Akt signaling to HER3 and the dogma that HER2 cannot activate PI3K by itself. The functions of HER3 are not entirely understood and may be partly redundant with HER2, enabling HER2 to escape the requirement for HER3. In this regard, we found that HER3-knock out HER2-overexpressing cells form tumors, and some of these tumors exhibit increased HER2 phosphorylation and Akt activation. We validated, in three different HER2-amplified cancer cell lines, that phosphorylated HER2 drives PI3K-mediated Akt activation in a HER3-independent manner. Interestingly, we found that HER2 interacts with the regulatory subunit of PI3K in these cells. We characterized this interaction and identified a tyrosine in the C-terminal tail of HER2 involved in PI3K binding. Most importantly, in vivo work demonstrates that mutation of this tyrosine reduces the transforming and tumorigenic potential of HER2-amplified cancer cells. This work highlights that the activation of PI3K/Akt signaling by HER3 may be an important but not essential function and may ultimately be redundant with the functional capabilities of massively overexpressed HER2. Therefore, building a better understanding of the molecular mechanisms that underlie HER2-HER3-induced tumorigenesis is essential for the development of more effective therapeutic strategies. Citation Format: Ana Ruiz-Saenz, Courtney Dreyer, Veronica Steri, Marcia Campbell, Nathaniel Gulizia, Mark Moasser. The conditional requirement of HER3 in HER2-amplified breast cancers [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B66.

Abstract 4776: Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer

Abstract Purpose: Molecularly targeted therapy has enabled outstanding advances in cancer treatment. Whereas various anti-HER2 drugs have been developed, trastuzumab is still the only anti-HER2 drug presently available for gastric cancer. Among HER2 targeting drug, afatinib and neratinib inhibit the activation of all HER family protein, and are called pan-HER inhibitors. In this study, we examined the effect of these pan-HER inhibitors to gastric cancer cells. Materials and Methods: We determined the molecular profiles of 12 gastric cancer cell lines. Protein level of HER2 and down-signal pathway molecules were analyzed by Western blotting, and copy number assay or gene expression assay were performed using qPCR. To detect HER2 mutation, we also performed direct-sequence of these cell lines. And next, we examined the antitumor effect of the pan-HER inhibitors afatinib and neratinib in those cell lines, in vitro and in vivo. In addition, we analyzed HER2 alteration in 123 primary gastric cancers resected from Japanese patients to clarify possible candidates with the potential to respond to these drugs. Results: HER2 was amplified in 5 out of 12 gastric cancer cell lines. Gene expression or protein level of HER2 were generally correlated with the copy number of HER2. HER2 mutation was found in one cell line, ECC10, at kinase domain (L755S). In the drug sensitivity analysis, both afatinib and neratinib showed an anti-tumor effect in all the HER2 amplified cell lines both in vitro and in vivo except MKN7 cell line. When the molecular profiles of the cells were compared based on the drug sensitivities, we found that cancer cells with lower mRNA expression levels of IGFBP7, a tumor suppressor gene by inhibiting the activation of IGF-1R, were less sensitive to pan-HER inhibitors. A combination therapy consisting of pan-HER inhibitors and an IGF-1R inhibitor, picropodophyllin, demonstrated a notable synergistic effect. Regarding HER2 alteration in 123 clinical samples, we found 19 cases of HER2 amplification and 3 cases of oncogenic mutations. Conclusion: Afatinib and neratinib are promising therapeutic options for the treatment of HER2-amplified gastric cancer. In addition to HER2 amplification, IGFBP7 might be a biomarker of sensitivity to these drugs, and IGF-1R-targeting therapy can overcome drug insensitiveness in HER2-amplified gastric cancer. Citation Format: Takahiro Yoshioka, Kazuhiko Shien, Yuta Takahashi, Eisuke Kurihara, Kei Namba, Yusuke Ogoshi, Hidejiro Torigoe, Hiroki Sato, Hiromasa Yamamoto, Junichi Soh, Toshiyoshi Fujiwara, Shinichi Toyooka. Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4776.

Abstract A163: Effective targeting of HER2-amplified cancers with trastuzumab in combination with T cells expressing a novel Antibody-Coupled T-cell Receptor (ACTR)

Abstract HER2 gene amplification occurs in 20-30% of aggressive breast and gastric cancers, and is associated with poor prognosis. The standard of care for HER2-positive early breast cancer includes the HER2-targeting antibody, trastuzumab, administered with chemotherapy. In patients with advanced HER2-positive disease, the standard of care as first-line therapy includes dual blockade of HER2 with trastuzumab and pertuzumab, an antibody that binds to an alternative epitope of HER2. Despite the clinical success of targeting HER2, many patients are intrinsically resistant or develop resistance during treatment regardless of continued expression of HER2, highlighting the need for novel therapeutic paradigms. Engineered cell therapies, including chimeric antigen receptor (CAR) T cells, have demonstrated promising clinical activity in advanced cancers. Despite success, CAR-T cells have shown substantial toxicity, and in initial clinical trials of HER2-amplified cancer CAR-T cells demonstrated acute toxicities, presumably due to "on target/off tumor" activity. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T-cell therapy developed to mediate antitumor activity when administered with tumor-targeting antibodies. The ACTR construct is composed of the extracellular domain of the high-affinity CD16 (FCGR3A) V158 variant fused with costimulatory and CD3ζ intracellular signaling domains. ACTR is engineered to engage the Fc domain of human IgG1 antibodies when opsonized to target cells, which delivers an activation signal to the engineered T cells. Two distinct ACTR variants, ACTR087 and ACTR707, are currently being evaluated clinically in combination with rituximab in patients with B cell lymphoma (NCT02776813, NCT03189836). The ACTR707 variant was selected from a panel of constructs based on its strong performance in preclinical tests to assess activity not only in hematologic cancers, but also in solid tumors. When combined with trastuzumab, ACTR707 mediated potent cytotoxicity against HER2-amplified tumor cell lines in vitro. Cytokine response and proliferation of ACTR707 was also observed in the presence of trastuzumab and target cells. ACTR707 activity was specific to trastuzumab-treated HER2-positive cells as no cytotoxicity was observed in the absence of trastuzumab, or with a nontargeting antibody. In HER2-amplified xenograft models, such as the N87 gastric cancer model, ACTR707 and trastuzumab combination treatment mediated tumor regressions, demonstrating significantly enhanced activity compared to trastuzumab alone. Finally, the activity of ACTR707 on a panel of normal cells representing normal tissue expression of HER2 was tested in the presence of trastuzumab. ACTR707 in combination with trastuzumab demonstrated marginal cytotoxicity on these cells. In contrast, trastuzumab-based CAR-T cells mediated potent cytotoxicity against these cells. Taken together, our data demonstrate that ACTR707 in combination with trastuzumab has robust antitumor activity in vitro and in vivo. Furthermore, this combination has differential cytotoxicity on HER2-amplified tumor cells compared to HER2-expressing nonmalignant cells, suggesting that a favorable therapeutic index may be established in the clinical treatment of HER2-amplified cancers. Citation Format: Katie M. O'Callaghan, John Shin, Eugene Choi, Greg Motz, Casey B. Judge, Heather A. Huet, Birgit C. Schultes, Seth A. Ettenberg. Effective targeting of HER2-amplified cancers with trastuzumab in combination with T cells expressing a novel Antibody-Coupled T-cell Receptor (ACTR) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A163.