HER2 Kinase Research Articles

Relationship of HER2 Alteration and Microsatellite Instability Status in Colorectal Adenocarcinoma.

BackgroundThe impact of HER2 somatic mutations in colorectal carcinoma (CRC) has not been well studied and its relationship with microsatellite instability‐high (MSI‐H) is yet to be fully elucidated.Materials and MethodsFrom February 2017 to February 2020, the data of patients with CRC who underwent next‐generation sequencing and had detailed record of clinicopathological information were investigated. HER2 alteration and its relationship with MSI‐H were analyzed.ResultsAmong 731 patients who underwent sequencing, 55 patients (7.5%) had HER2 alteration, including 29 (4.0%) with HER2 somatic mutations, 24 (3.3%) with HER2 gene amplification, and 2 patients (0.2%) with both HER2 mutations and amplification. R678Q was the most common mutated kinase domain, and no HER2 kinase domain in‐frame insertions/deletions were found in HER2 mutated cases. MSI‐H was found in 5.2% of our cohort and 36.8% of MSI‐H patients had HER2 mutation. For HER2 mutated cases, 48.3% were MSI‐H, whereas none of the HER2 amplification cases were MSI‐H. MSI‐H patients with HER2 mutation had significantly worse median progression‐free survival for programmed death‐1 (PD‐1) antibody than those without HER2 alteration (p = .036).ConclusionHigh MSI‐H rate was found in HER2 mutated cases, but no MSI‐H was found in HER2 amplification cases. MSI‐H patients with HER2 mutated had worse progression‐free survival for PD‐1 antibody than those without.Implications for PracticeThis study highlights the high microsatellite instability‐high (MSI‐H) rate in HER2 mutated cases but no MSI‐H in HER2 amplification cases. Moreover MSI‐H patients with HER2 mutated had worse progression‐free survival for programmed death‐1 antibody than those without. Further research to explore the internal relationship between HER2 alteration and MSI‐H is needed.

Concurrent mutations associated with trastuzumab-resistance revealed by single cell sequencing.

HER2-positive breast cancer patients benefit from HER2-targeted therapies, among which the most commonly used is trastuzumab. However, acquired resistance typically happens within one year. The cellular heterogeneity of it is less clear. Here we generated trastuzumab-resistant cells in two HER2-positive breast cancer cell lines, SK-BR-3 and BT-474. Cells at different time points during the resistance induction were examined by exome sequencing to study changes of genomic alterations over time. Single cell-targeted sequencing was also used to identify resistance-associated concurrent mutations. We found a rapid increase of copy number variation (CNV) regions and gradual accumulation of single nucleotide variations (SNVs). On the pathway level, non-synonymous SNVs for SK-BR-3 cells were enriched in the MAPK signaling pathway, while for BT-474 cells they were enriched in mTOR and PI3K-Akt signaling pathways. However, all of the three signaling pathways were in the downstream of the HER2 kinase. Putative trastuzumab-resistance-associated SNVs included AIFM1 P548L and ERBB2 M833R in SK-BR-3 cells, and ADAMTS19 V451L,OR5M9 D230N, COL9A1 R627T, andITGA7 H911Q in BT-474 cells. Single-cell-targeted sequencing identified several concurrent mutations. By validation, we found that concurrent mutations (AIFM1 P548L and IL1RAPL2 S546C in SK-BR-3 cells, MFSD11 L242I and ANAPC4 E16K in BT-474 cells) led to a decrease of trastuzumab sensitivity. Taken together, our study revealed a common pathway level trastuzumab-resistance mechanism for HER2-positive breast cancer cells. In addition, our identification of concurrent SNVs associated with trastuzumab-resistance may be indicative of potential targets for the treatment of trastuzumab-resistant breast cancer patients.

The Molecular Mechanisms of Cardiotoxicity Induced by HER2, VEGF, and Tyrosine Kinase Inhibitors: an Updated Review.

In recent decades, there has been a revolutionary decrease in cancer-related mortality and an increase in survival due to the introduction ofnovel targeted drugs. Nevertheless, drugs targeting human epidermal growth factor receptor 2 (HER-2), angiogenesis, and other tyrosine kinases also comewith unexpected cardiac side effects, including heart failure, hypertension, arterial thrombosis, and arrhythmias, and have mechanisms that are unlike thoseof classic chemotherapeutic agents. In addition, it is challenging to address some problems, as the existing guidelines need to be more specific, and furtherlarge-scale clinical trials and experimental studies are required to confirm the benefit of administering cardioprotective agents to patients treated withtargeted therapies. Therefore, an improved understanding of cardiotoxicity becomes increasingly important to minimize the pernicious effects and maximizethe beneficial effects of targeted agents. "Cardiotoxicity", "targeted drugs", "HER2", "trastuzumab", "angiogenesis inhibitor", "VEGF inhibitor" and "tyrosine kinase inhibitors" are usedas keywords for article searches. In this article, we report several targeted therapies that induce cardiotoxicity and update knowledge of the clinical evidence, molecularmechanisms, and management measures.

Abstract PD8-01: Comprehensive genomic profiling (CGP) of metastatic invasive lobular carcinomas reveals heterogeneity in immune biomarkers and resistance alterations across biopsy sites

Abstract Introduction Metastatic breast cancer is a clinically challenging disease with poor outcomes. Invasive lobular carcinoma (ILC) is a rarer subtype of breast cancer with distinct patterns of metastasis, including frequent GI and female reproductive (FR) metastases (mets). Due to their relative rarity, little is known about the genomic characteristic of ILC across met sites. Here we explore the genomic landscape of 1909 ILC, stratified by met site, with an examination of immune checkpoint inhibitor (ICPI) biomarkers, therapeutic alterations, and resistance mutations. Methods CGP was carried out at Foundation Medicine with hybrid capture for exons from up to 395 cancer-related genes plus select introns from up to 31 genes (PMID 24142049). Tumor mutational burden (TMB) was determined on 0.8-1.1 Mb (PMID: 28420421). Ventana PD-L1 IC staining (SP142; positive >= 1% staining) was available for a subset of samples. 1071 breast-biopsied and 1909 met-biopsied ILC and 6926 breast-biopsied and 1901 met-biopsied IDC were available for analysis. Results High TMB (>= 10 muts/mb) and PD-L1 IC staining are associated with response to ICPI. ILC mets overall had a greater rate of high TMB relative to IDC mets (21% v 9%, p = 7E-25) and breast-biopsied (breast) ILC (21% v 10%; p = 9E-15) with the highest frequency in GI (23%) and skin (21%). PD-L1 IC+ rates were lower in ILC mets (18%) relative to IDC mets (34%) and breast ILC (31%), but were variable across sites, with relatively high rates of positivity in GI (48%), skin (29%), and FR (18%) mets, and no positive staining in bone mets (0/37). Alterations in PIK3CA were higher in ILC mets (58%) relative to IDC mets (34%) and generally exhibited a similar frequency across ILC met sites, with modestly lower prevalence in skin (48%, p = 0.005). Pathogenic alterations in BRCA1/2 were observed in 4.8% of ILC mets overall, with a lower frequency in GI mets (1.3%, p = 0.03). A comparison of ILC breast biopsies to ILC mets revealed 19 genes with higher prevalence in at least one ILC met site, most with known roles in therapy resistance (eg ESR1, NF1, RB1, KRAS, ERBB2, BRAF), though significant heterogeneity was observed across sites. Met-enriched (ME) alterations were highest in ILC from the liver (71%) and lowest in FR (33%). ERBB2 mutations, which are may be targetable with HER2 kinase inhibitors, were predominantly found in liver mets (21%) with significantly lower prevalence in skin (11%), bone (10%), GI (3%), and FR (3%). ESR1 alterations were common in most ILC sites, with the highest prevalence in liver (26%) and low frequency in FR (4%). While FR ILC harbored few ME alterations, the rare alterations were primarily found in NF1 (5%) and NCOR1 (5%). Conclusions CGP revealed significant heterogeneity in ILC mets across tissues. ICPI biomarkers were variable across sites with the highest frequency in ILC GI mets, offering additional potential treatment avenues for these tumors. Alterations in PIK3CA were common in ILC mets with high prevalence across sites, suggesting utility for PIK3CA inhibitors. Therapy-resistant alterations were common in ILC mets but varied across sites. Notably, ERBB2 alterations were most prevalent in ILC liver mets, but less common at other sites. The high prevalence of therapeutic and resistance alterations suggests value in profiling metastatic lesions. breast_ILCmet_ILCbreast_IDCmet_IDCgi_ILCliver_ILCfemale_repr_ILCbone_ILCskin_ILCsample count (N)1071190969261901154639114268199ICPI biomarkerTMB-H (>=10muts/mb)10.2%21.0%4.4%9.2%23.4%18.9%14.0%19.0%21.1%PDL1-IC+ SP142 (subset)31.5% (39/124)17.8% (38/213)57.3% (480/837)34.2% (54/158)47.6% (10/21)11.6% (8/69)18.2% (2/11)0% (0/37)28.6% (6/21)Therapy AssociatedPIK3CA50.0%57.6%29.3%34.3%59.1%57.3%62.3%60.1%48.2%BRCA1/24.9%4.8%9.2%7.7%1.3%4.1%7.0%5.2%5.5%Met-EnrichedESR1_mut3.8%18.3%2.2%17.0%20.8%25.5%3.5%11.9%7.5%ERBB2_mut9.7%12.5%2.0%2.7%3.2%20.7%2.6%9.7%11.1%ARID1A_mut7.5%12.2%4.0%5.5%14.3%11.4%7.9%9.0%10.6%NF1_mut4.4%8.7%3.5%4.2%5.8%8.5%5.3%10.4%8.0%RB1_mut2.7%6.3%4.3%3.5%7.8%5.9%2.6%4.5%11.6%KRAS2.3%5.5%3.9%3.6%5.2%6.7%2.6%6.3%2.0%PTEN_del3.1%4.1%5.7%4.7%1.9%4.7%4.4%1.9%8.0%FGFR20.7%3.1%2.9%2.6%1.3%4.1%1.8%4.1%2.0%NCOR1_mut1.2%2.9%1.1%1.1%3.2%3.0%5.3%1.9%1.0%SMAD42.0%2.9%1.2%1.9%1.9%4.7%1.8%2.6%1.5%BRAF1.6%2.1%1.2%1.4%0.0%1.4%2.6%4.5%2.5%FOXP1_mut0.4%1.6%0.3%0.3%2.6%1.1%0.0%1.9%1.0%APC_mut0.9%1.4%1.1%1.1%0.6%0.6%1.8%3.4%2.0%SOX90.3%1.3%0.5%0.4%0.0%1.1%0.0%1.5%0.5%CASP8_mut0.0%0.8%0.5%0.5%0.0%1.1%0.9%1.1%1.0%PTPN11_mut0.1%0.7%0.2%0.3%0.0%0.6%1.8%1.5%1.0%TERT_mut0.2%0.6%0.4%0.7%2.6%0.0%0.0%1.1%0.5%ALK_mut0.1%0.4%0.2%0.4%2.6%0.0%0.9%0.4%0.5%KMT2D_RE0.1%0.3%0.4%0.3%1.9%0.2%0.0%0.0%0.5%any_ME32.5%60.1%30.1%42.4%53.9%71.4%33.3%53.4%52.3% Citation Format: Ethan Sokol, Dexter X Jin, Jeffrey Ross, Garrett M Frampton, Steffi Oesterreich. Comprehensive genomic profiling (CGP) of metastatic invasive lobular carcinomas reveals heterogeneity in immune biomarkers and resistance alterations across biopsy sites [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 PD8-01.

1,3,4-Thiadiazoline−coumarin hybrid compounds containing D-glucose/D-galactose moieties: Synthesis and evaluation of their antiproliferative activity

We synthesized a new series of 1,3,4-thiadiazoline−coumarin hybrid compounds that contain d-glucose and d-galactose moieties (9a-g) and evaluated their cytotoxic activity against breast adenocarcinoma (MCF-7), human liver cancer (HepG2), human cervical cancer (HeLa), human melanoma cancer (SK-Mel-2), and human lung cancer (LU-1) cells. To reveal their selectivity toward cancer cells, the compounds were also tested against human fibroblast cell line, MRC-5. Synthesized compounds exhibited potent cytotoxic activity against the tested cell lines with IC50 values of 1.18–11.81, 1.72–9.43, 1.98–13.16, 1.82–11.25, and 2.25–14.62 μM (against MCF-7, HepG2, HeLa, SK-Mel-2, and LU-1 cells, respectively) compared to Sorafenib, 5-FU, and DOX. The long and branched-terminal carbon chains often increased activity. Interestingly, compounds 9a-g displayed selectivity toward cancer cell lines over MRC-5 (IC50 3.93–25.55 μM). These compounds also displayed potent inhibitory activity against EGFR and HER2 kinases (IC50 0.22–0.47 and 0.13–0.35 μM, respectively) compared to the standard drug, Sorafenib (IC50 = 0.11 and 0.13 μM, respectively). Molecular docking study also employed to identify the structural features required for the EGFR/HER2 inhibitory activity of the new series.

Exon 20 YVMA insertion is associated with high incidence of brain metastasis and inferior outcome of chemotherapy in advanced non-small cell lung cancer patients with HER2 kinase domain mutations.

BackgroundChemotherapy remains the standard care for HER2 mutated advanced non-small cell lung cancer (NSCLC) even though several targeted drugs showed promising results in preliminary stages. This study aimed to investigate the association of mutation variants with clinical features and the efficacy of chemotherapy in patients with HER2 mutated advanced NSCLC.MethodsARMS-PCR was used to identify HER2 mutation in patients without common oncogenic alterations. Patients with detailed information were further enrolled for analysis of clinical features and efficacy of first line chemotherapy. Survival data was analyzed by Kaplan-Meier method and compared by log-rank test. Brain metastasis incidence was analyzed and compared by Gray’s test.ResultsYVMA insertion accounted for the majority (68.4%, 67/98) of HER2 mutation, and associated with significantly higher incidence of baseline extrathoracic metastasis (P=0.009), notably brain metastasis (P=0.004). Among 82 patients those received first line chemotherapy, YVMA insertion remarkably associated with inferior treatment outcomes, namely, a significantly shorter median progression free survival (PFS) and lower objective response rate (ORR) both in total patients (PFS: 5.2 vs. 7.7 m, P=0.038; ORR: 30.9% vs. 51.9%, P=0.09) and pemetrexed subgroup (PFS: 5.2 vs. 6.5 m, P=0.022; ORR: 31.8% vs. 60.0%, P=0.054). Multivariate analysis further established YVMA insertion as prognostic factor of worse PFS both for total patients (HR =1.578, 95% CI, 0.956–2.606) and patients received pemetrexed-based chemotherapy (HR =1.789, 95% CI, 1.013–3.160). In addition, YVMA insertion associated with higher incidence of lifetime brain metastasis (P=0.002) compared by Gray’s test, with estimated 12-month brain metastasis incidence as 40.2% compared with 3.6% in the non-YVMA group.ConclusionsYVMA insertion is associated with a higher incidence of brain metastasis, and inferior outcomes to chemotherapy than non-YVMA variants in patients with advanced NSCLC and HER2 kinase domain mutations, which emphasized the unmet need of more potent anti-cancer therapies with high blood-brain barrier (BBB) penetration capacity for patients with YVMA insertion.

Novel thiazoline–coumarin hybrid compounds containing sugar moieties: synthesis, biological evaluation and molecular docking study as antiproliferative agents

1,3-Thiadiazoline–coumarin hybrid compounds containing d-glucose and d-galactose moieties have the cytotoxicity against MCF-7, HepG2, HeLa, SK-Mel-2, and LU-1 cells and also EGFR and HER2 kinases. Molecular docking showed key roles in enhancing potency against both enzymes.

SATB1-Mediated Upregulation of the Oncogenic Receptor Tyrosine Kinase HER3 Antagonizes MET Inhibition in Gastric Cancer Cells.

MET-amplified gastric cancer cells are extremely sensitive to MET inhibition in vitro, whereas clinical efficacy of MET inhibitors is disappointing. The compensatory activation of other oncogenic growth factor receptors may serve as an underlying mechanism of resistance. In this study, we analyzed the role of HER receptors, in particular HER3 and its ligand heregulin, in this respect. This also included the chromatin-organizer protein SATB1, as an established regulator of HER expression in other tumor entities. In a panel of MET-amplified gastric carcinoma cell lines, cell growth under anchorage-dependent and independent conditions was studied upon inhibitor treatment or siRNA-mediated knockdown. Expression analyses were performed using RT-qPCR, FACS, and immunoblots. Signal transduction was monitored via antibody arrays and immunoblots. As expected, MET inhibition led to a growth arrest and inhibition of MAPK signaling. Strikingly, however, this was accompanied by a rapid and profound upregulation of the oncogenic receptor HER3. This finding was determined as functionally relevant, since HER3 activation by HRG led to partial MET inhibitor resistance, and MAPK/Akt signaling was even found enhanced upon HRG+MET inhibitor treatment compared to HRG alone. SATB1 was identified as mediator of HER3 upregulation. Concomitantly, SATB1 knockdown prevented upregulation of HER3, thus abrogating the HRG-promoted rescue from MET inhibition. Taken together, our results introduce the combined HER3/MET inhibition as strategy to overcome resistance towards MET inhibitors.

Accuracy of endoscopic ultrasound-guided needle aspiration specimens for molecular diagnosis of non-small-cell lung carcinoma.

BACKGROUNDEndoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) are highly sensitive for diagnosing and staging lung cancer. In recent years, targeted therapy has shown great significance in the treatment of non-small cell lung carcinoma (NSCLC). Using these minimally invasive techniques to obtain specimens for molecular testing will provide patients with a more convenient diagnostic approach.AIMTo evaluate the feasibility and accuracy of tissue samples obtained using EUS-FNA and EBUS-TBNA for molecular diagnosis of NSCLC.METHODSA total of 83 patients with NSCLC underwent molecular testing using tissues obtained from EUS-FNA or EBUS-TBNA at the Tianjin Medical University Cancer Hospital from January 2017 to June 2019. All enrolled patients underwent chest computed tomography or positron emission tomography/computed tomography prior to puncture. We detected abnormal expression of EGFR, KRAS, MET, HER2, ROS1 and anaplastic lymphoma kinase protein. Two patients failed to complete molecular testing due to insufficient tumor tissue. The clinical features, puncture records, molecular testing results and targeted treatment in the remaining 81 patients were summarized.RESULTSIn a total of 99 tissue samples obtained from 83 patients, molecular testing was successfully completed in 93 samples with a sample adequacy ratio of 93.9% (93/99). Biopsy samples from two patients failed to provide test results due to insufficient tumor tissue. In the remaining 81 patients, 62 cases (76.5%) were found to have adenocarcinoma, 11 cases (13.6%) had squamous cell carcinoma, 3 cases (3.7%) had adenosquamous carcinoma and 5 cases (6.2%) had NSCLC-not otherwise specified. The results of molecular testing showed EGFR mutations in 21 cases (25.9%), KRAS mutations in 9 cases (11.1%), ROS-1 rearrangement in 1 case (1.2%) and anaplastic lymphoma kinase-positive in 5 cases (6.2%). Twenty-four patients with positive results received targeted therapy. The total effectiveness rate of targeted therapy was 66.7% (16/24), and the disease control rate was 83.3% (20/24).CONCLUSIONTissue samples obtained by EUS-FNA or EBUS-TBNA are feasible for the molecular diagnosis of NSCLC and can provide reliable evidence for clinical diagnosis and treatment.

Novel benzimidazole derivatives as anti-cervical cancer agents of potential multi-targeting kinase inhibitory activity

Multi-target EGFR, HER2, VEGFR-2 and PDGFR is an improved strategy for the treatment of solid tumors. This work deals with synthesis of an array of new 6-benzoyl benzimidazole derivatives utlizing1-(6-benzoyl-2-(3,4-dimethoxyphenyl)-1H benzo[d] imidazol-1-yl)propan-2-one (1) as a starting compound. The new compounds were screened as cytotoxic agents against cervical cancer cells (Hela) and Doxorubicin served as a reference drug. Most of the tested compounds showed promising anticancer activity in addition to their safety towards the normal cell line. The most potent candidates were evaluated as EGFR, HER2, PDGFR-β and VEGFR2 inhibitors in comparison to Erlotinib. Compounds 9 and 13 exhibited promising suppression effects. Also, the latter compounds exhibited their ability to induce cellular apoptosis alongside cell cycle arrest at the G2/M phase and accumulation of cells in pre-G1 phase. Molecular docking analysis suggested that compounds 2c, 3f, 9, 12 and 13 tightly interacts with the amino acid residues in the active binding site of HER2 kinase.

Epigenetic targeting of neuropilin-1 prevents bypass signaling in drug-resistant breast cancer

Human epidermal growth factor receptor 2 (HER2)-amplified breast cancers are treated using targeted antibodies and kinase inhibitors, but resistance to these therapies leads to systemic tumor recurrence of metastatic disease. Herein, we conducted gene expression analyses of HER2 kinase inhibitor-resistant cell lines as compared to their drug-sensitive counterparts. These data demonstrate the induction of epithelial–mesenchymal transition (EMT), which included enhanced expression of fibroblast growth factor receptor 1 (FGFR1) and axonal guidance molecules known as neuropilins (NRPs). Immunoprecipitation of FGFR1 coupled with mass spectroscopy indicated that FGFR1 forms a physical complex with NRPs, which is enhanced upon induction of EMT. Confocal imaging revealed that FGFR1 and NRP1 predominantly interact throughout the cytoplasm. Along these lines, short hairpin RNA-mediated depletion of NRP1, but not the use of NRP1-blocking antibodies, inhibited FGFR signaling and reduced tumor cell growth in vitro and in vivo. Our results further indicate that NRP1 upregulation during EMT is mediated via binding of the chromatin reader protein, bromodomain containing 4 (BRD4) in the NRP1 proximal promoter region. Pharmacological inhibition of BRD4 decreased NRP1 expression and ablated FGF-mediated tumor cell growth. Overall, our studies indicate that NRPs facilitate aberrant growth factor signaling during EMT-associated drug resistance and metastasis. Pharmacological combination of epigenetic modulators with FGFR-targeted kinase inhibitors may provide improved outcomes for breast cancer patients with drug-resistant metastatic disease.

Antitumor activity, multitarget mechanisms, and molecular docking studies of quinazoline derivatives based on a benzenesulfonamide scaffold: Cell cycle analysis

The in vitro cytotoxicity of some substituted quinazolinones, 1–15, was evaluated using NCI (10 µM) in a full NCI 59–cell line panel assay. Relative to the reference drug, imatinib (PCE = 20/59), compounds 3, 4, 7, 9, and 10 exhibited remarkable antitumor activity against the tested cell lines, with positive cytotoxic effects (PCE) of 29/59, 18/59, 17/59, 44/59, and 24/59 respectively. Enzymatic inhibitory assay conducted on 3, 4, 9, and 10 as the most potent antitumor agents against EGFR, HER2 and CDK9 kinases, and COX-2 enzyme. Compound 3 possessed good COX-2 inhibitory activity (IC50 = 0.775 μM) compared to the reference drug, celecoxib (IC50 = 0.153 μM). Compounds 4 and 9 were closely potent to the reference compounds against EGFR and (HER2) tyrosine kinases, with IC50 values of 90.17 (and 131.39 for HER2) for 4 and 145.35 (and 129.07 for HER2) nM for 9; the reference drugs in this case, namely, gefitinib and erlotinib, exhibited IC50 values of 55.58 (90) and 110 (79.28) nM against the EGFR and (HER2) tyrosine kinases, respectively. Compound 4 was approximately similar potent against CDK9 kinase (IC50 = 67.04 nM) like the reference compound, dinaciclib (IC50 = 53.12 nM). Compound 9 induced cytotoxicity in the MCF–7 cell line (GI % at 10.0 μM = 47%) through pre-G1 apoptosis, thereby inhibiting cell growth at the G2/M phase. Molecular docking models of 3 and 4 with COX-2, EGFR, and CDK9 were conducted to determine their binding mode within the putative binding pockets.

The G Protein-Coupled Receptor PAC1 Regulates Transactivation of the Receptor Tyrosine Kinase HER3.

Peptide G protein-coupled receptors (GPCRs) for pituitary adenylate cyclase activating polypeptide (PACAP) regulate the growth of non-small cell lung cancer (NSCLC) cells. PACAP binds with high affinity to PAC1, which causes transactivation of receptor tyrosine kinases (RTK) for the EGFR and HER2 but its effect on HER3 is unknown. Using 3 NSCLC cell lines (NCI-H358, NCI-H441, and Calu-3), proteins for EGFR, HER2, HER3, and PAC1 were detected. The increase in EGFR tyrosine phosphorylation caused by PACAP was blocked by the EGFR tyrosine kinase inhibitor (TKI) gefitinib, or PACAP(6-38), a PAC1 antagonist. The increase in HER2 tyrosine phosphorylation caused by PACAP was inhibited by trastuzumab, a monoclonal antibody (mAb) for HER2, or PACAP(6-38). The increase in HER3 tyrosine phosphorylation caused by PACAP was inhibited by HER3 mAb3481 or PACAP(6-38). Immunoprecipitation experiments indicated the PACAP addition to Calu-3 cells resulted in the formation of EGFR/HER3 and HER2/HER3 heterodimers. Addition of the HER3 agonist neuregulin (NRG)-1 increased HER3 tyrosine phosphorylation in non-small-cell lung cancer (NSCLC) cells. PACAP or NRG-1 increased the proliferation of NSCLC cells, whereas PACAP(6-38), gefitinib, trastuzumab, or mAb3481 inhibited proliferation. The results indicate that PAC1 regulates the proliferation of NSCLC cells as a result of transactivation of the EGFR, HER2, and HER3.

Abstract 1916: Targeting Warburg effect to overcome lapatinib resistance in esophageal adenocarcinoma

Abstract Background: A major target for esophageal adenocarcinoma (EAC) therapies is the human epidermal growth factor receptor 2 (HER2). EAC overexpresses HER2 and lapatinib, a dual tyrosine kinase inhibitor blocking HER1 and HER2 pathways fails to improve patient survival. Molecular mechanisms of this lapatinib resistance are still unclear. We therefore explored the role of glycolytic enzyme pyruvate kinase M2 (PKM2), a key regulator of Warburg effect in lapatinib resistance mechanism of EAC cells. Methods: First we established lapatinib-resistant OE19 (LPR-OE19) cells from OE19 EAC cells and characterized it. We then investigated the comparative cell growth inhibition, apoptotic and lactate production effects of HER2 inhibitor lapatinib and PKM2 inhibitor shikonin either alone or in combinations, both in OE19 and LPR-OE19 cells with and without knockdown of PKM2 and HSP40 by siRNAs. We then explored the antitumor efficacy following lapatinib and shikonin mono and combination therapies in murine subcutaneous xenograft model of human EAC. Results: Lapatinib-resistant OE19 (LPR-OE19) cells were established from OE19 cells by intermittent exposure to increasing concentrations of lapatinib for a period of total six months. Lapatinib resistant LPR-OE19 cells showed downregulation of HSP40 both at protein and mRNA levels whereas showed upregulation of PKM2 only at protein level. LPR-OE19 cells showed significantly reduced sensitivity to lapatinib induced cell growth inhibition, apoptosis and decreased cell migration compared to parent OE19 cells. Interestingly, augmented cell growth inhibition, apoptosis and decreased cell migration were observed in LPR-OE19 cells compared to parent OE19 cells when lapatinib was combined with shikonin. More interestingly, knockdown of PKM2 in LPR-OE19 cells abolished the reduced sensitivity of lapatinib induced cell growth inhibition and also abolished augmented cell growth inhibition response when lapatinib and shikonin were combined. Contrary to that, knockdown of HSP40 in OE19 cells enhanced PKM2 expression leading to significantly reduced sensitivity to lapatinib induced growth inhibition with augmented growth inhibition when lapatinib and shikonin were combined. Moreover, LPR-OE19 cells showed enhanced lactate production compared to parent OE19. Interestingly, PKM2 and HSP40 co-localize with each other in the cell nucleus suggesting that PKM2 bound to molecular chaperone HSP40. In subcutaneous xenografts using LPR-OE19 cells, average net tumor growth after two weeks in different therapy groups was 494.72 mm3 in control, 403.11 mm3 after lapatinib (p=0.241), 339.48 mm3 after shikonin (p=0.0478), and 141.59 mm3 after shikonin plus lapatinib (p=0.0003). Conclusions: In conclusion, these data suggest that combination therapy with lapatinib and glycolytic inhibitor shikonin should be tested as a potent option for lapatinib resistant HER2-positive EAC patients and could be a novel treatment strategy for specific EAC. In addition, it is suggested that targeting HSP40-PKM2 interaction as an innovative therapeutic approach to overcome lapatinib resistance in EAC. Citation Format: Md Sazzad Hassan, Samantha Holmes, Victoria Makuru, Chloe Johnson, Urs von Holzen. Targeting Warburg effect to overcome lapatinib resistance in esophageal adenocarcinoma [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 1916.

Hsp90 inhibition and co‐incubation with pertuzumab induce internalization and degradation of trastuzumab: Implications for use of T‐DM1

The receptor tyrosine kinase HER2 is associated with a number of human malignancies and is an important therapeutic target. The antibody‐drug conjugate trastuzumab emtansine (T‐DM1; Kadcyla®) is recommended as a first‐line treatment for patients with HER2‐positive metastatic breast cancer. T‐DM1 combines the antibody‐induced effects of the anti‐HER2 antibody trastuzumab (Herceptin®) with the cytotoxic effect of the tubulin inhibitor mertansine (DM1). For DM1 to have effect, the T‐DM1‐HER2 complex has to be internalized and the trastuzumab part of T‐DM1 has to be degraded. HER2 is, however, considered endocytosis‐resistant. As a result of this, trastuzumab is only internalized to a highly limited extent, and if internalized, it is rapidly recycled. The exact reasons for the endocytosis resistance of HER2 are not clear, but it is stabilized by heat‐shock protein 90 (Hsp90) and Hsp90 inhibitors induce internalization and degradation of HER2. HER2 can also be internalized upon activation of protein kinase C, and contrary to trastuzumab alone, the combination of two or more anti‐HER2 antibodies can induce efficient internalization and degradation of HER2. With intention to find ways to improve the action of T‐DM1, we investigated how different ways of inducing HER2 internalization leads to degradation of trastuzumab. The results show that although both Hsp90 inhibition and activation of protein kinase C induce internalization of trastuzumab, only Hsp90 inhibition induces degradation. Furthermore, we find that antibody internalization and degradation are increased when trastuzumab is combined with the clinically approved anti‐HER2 antibody pertuzumab (Perjeta®).

Fibroblast–tumor cell signaling limits HER2 kinase therapy response via activation of MTOR and antiapoptotic pathways

Despite the implementation of multiple HER2-targeted therapies, patients with advanced HER2+ breast cancer ultimately develop drug resistance. Stromal fibroblasts represent an abundant cell type in the tumor microenvironment and have been linked to poor outcomes and drug resistance. Here, we show that fibroblasts counteract the cytotoxic effects of HER2 kinase-targeted therapy in a subset of HER2+ breast cancer cell lines and allow cancer cells to proliferate in the presence of the HER2 kinase inhibitor lapatinib. Fibroblasts from primary breast tumors, normal breast tissue, and lung tissue have similar protective effects on tumor cells via paracrine factors. This fibroblast-mediated reduction in drug sensitivity involves increased expression of antiapoptotic proteins and sustained activation of the PI3K/AKT/MTOR pathway, despite inhibition of the HER2 and the RAS-ERK pathways in tumor cells. HER2 therapy sensitivity is restored in the fibroblast cocultures by combination treatment with inhibitors of MTOR or the antiapoptotic proteins BCL-XL and MCL-1. Expression of activated AKT in tumor cells recapitulates the effects of fibroblasts resulting in sustained MTOR signaling and poor lapatinib response. Lapatinib sensitivity was not altered by fibroblasts in tumor cells that exhibited sustained MTOR signaling due to a strong gain-of-function PI3KCA mutation. These findings indicate that in addition to tumor cell-intrinsic mechanisms that cause constitutive PI3K/AKT/MTOR pathway activation, secreted factors from fibroblasts can maintain this pathway in the context of HER2 inhibition. Our integrated proteomic-phenotypic approach presents a strategy for the discovery of protective mechanisms in fibroblast-rich tumors and the design of rational combination therapies to restore drug sensitivity.

Molecular selectivity design of mitogen-inducible gene-derived phosphopeptides between oncogenic HER kinases

Mitogen-inducible gene (MIG) is a natural negative regulator of the oncogenic HER kinase signaling by binding at the activation interface of kinase domain to disrupt the kinase dimerization. In this study, we systematically examine the binding structures, dynamics and energetics of MIG region 2 to four HER kinases based on their crystal or modeled complex structures, and identify an 8-mer phosphopeptide segment pYpY from the core strand sequence of MIG region 2 as the binding hotspot of MIG protein to HER kinases. We demonstrate that the small pYpY phosphopeptide can partially restore the binding affinity of full-length MIG protein, but exhibit a moderate selectivity over different HER kinases (S = 2.3-fold). In addition, the two phosphotyrosine residues pTyr394 and pTyr395 play an essential role in MIG–HER binding; dephosphorylation of them would fully eliminate the binding capability. A machine evolution algorithm is used to optimize the wild-type pYpY phosphopeptide, aiming to simultaneously improve affinity for these kinases and to maximize the affinity gap between different kinases. Consequently, a population is computationally evolved as selective phosphopeptide candidates; the dissociation constants of four representatives with HER kinases are systematically determined using binding affinity analysis, from which their selectivity is derived. The designed pYpYp3 phosphopeptide possesses a high selectivity over different HER kinases (S = 4.8-fold) and satisfactory affinity profile to these kinase (KD = 140–1000 μM). Structural analysis observes that the global binding modes of pYpYp3 to different kinases are roughly consistent, but its local conformation may vary considerably, thus conferring specificity to the phosphopeptide.

Specific Roles of HSP27 S15 Phosphorylation Augmenting the Nuclear Function of HER2 to Promote Trastuzumab Resistance.

Trastuzumab (TZMB) is widely used as first line therapy for breast cancer (BC) patients overexpressing human epidermal growth factor receptor 2 (HER2). Despite its clinical benefits, many patients suffer from primary or secondary resistance to this drug within one year. As diverse molecular mechanisms occur contemporaneously during the resistance development, we focused on elucidating the role of heat shock protein 27 (HSP27) in TZMB-resistance, as this protein simultaneously regulates the function of diverse client molecules that are involved in the resistance mechanism. By extensively utilizing TZMB-refractory breast cancer cell lines transduced with diverse phosphovariants of HSP27, our study newly revealed that specific phosphorylation of HSP27 at S15 promoted its S78 phosphorylation and served as key mediator to promote direct interactions that increase the stability of HER2 and protein kinase B (AKT). This phosphorylation promoted nuclear translocation of HER2, enhancing the distinct nuclear function of HER2 that promoted AKT activation and cyclin D1 expression. Co-administration of TZMB and a functional inhibitor of HSP27, J2, significantly reduced the S15/78 phosphorylation of HSP27, which downregulated HER2 and its downstream signals, sensitizing TZMB-refractory cell, and JIMT1-xenograft mouse models to TZMB. Collectively, p-HSP27S15 could serve as a valuable predictive marker and also a therapeutic target for TZMB-resistance.

Tucatinib versus placebo added to trastuzumab and capecitabine for patients with previously treated HER2+ metastatic breast cancer with brain metastases (HER2CLIMB).

1005 Background: Tucatinib (TUC) is an investigational, highly selective HER2 kinase inhibitor. HER2CLIMB (NCT02614794) showed clinically meaningful and statistically significant improvements in overall survival (OS) and progression free survival (PFS) in all pts, prolongation of PFS in pts with brain metastases (BM), and objective response rate (ORR) when TUC was added to trastuzumab (T) and capecitabine (C). Primary methods and outcomes have been reported previously (Murthy NEJM 2019). We report the results of exploratory efficacy analyses in pts with BM. Methods: All pts with HER2+ metastatic breast cancer (MBC) enrolled in HER2CLIMB had a baseline brain MRI. Pts with BM were eligible and classified as untreated, treated stable, or treated and progressing. Pts were randomized 2:1 to receive TUC or placebo, in combination with T and C. Efficacy analyses in pts with BM at baseline were performed by applying RECIST 1.1 to the brain based on investigator evaluation. CNS-PFS (progression in the brain or death) and OS were evaluated in BM pts overall. Intracranial (IC) confirmed ORR (ORR-IC) and IC duration of response (DOR-IC) were evaluated in BM pts with measurable IC disease. After isolated brain progression, pts could continue study therapy after local treatment until second progression, and time from randomization to second progression or death was evaluated. Results: Overall, 291 pts (48%) had BM at baseline: 198 (48%) in the TUC arm and 93 (46%) in the control arm. There was a 68% reduction in risk of CNS-PFS in the TUC arm (HR: 0.32; 95% CI: 0.22, 0.48; P < 0.0001). Median CNS-PFS was 9.9 mo in the TUC arm vs 4.2 mo in the control arm. Risk of death overall was reduced by 42% in the TUC arm (OS HR: 0.58; 95% CI: 0.40, 0.85; P = 0.005). Median OS was 18.1 mo vs 12.0 mo. ORR-IC was higher in the TUC arm (47.3%; 95% CI: 33.7, 61.2) vs the control arm (20.0%; 95% CI: 5.7, 43.7). Median DOR-IC was 6.8 mo (95% CI: 5.5, 16.4) vs 3.0 mo (95% CI: 3.0, 10.3). In pts with isolated brain progression who continued study therapy after local treatment (n = 30), risk of second progression or death was reduced by 67% (HR: 0.33; 95% CI: 0.11, 1.02), and median PFS from randomization was 15.9 mo vs 9.7 mo, favoring the TUC arm. Conclusions: In pts with heavily previously treated HER2+ MBC with BM, TUC in combination with T and C doubled the ORR-IC, reduced risk of IC progression or death by two thirds and reduced risk of death by nearly half. If approved, TUC in combination with T and C has the potential to become a new standard of care in pts with HER2+ MBC with and without BM. Clinical trial information: NCT02614794 .

Masterkey-01: Phase I/II, open-label multicenter study to assess safety, tolerability, pharmacokinetics, and antitumor activity of BDTX-189, an inhibitor of allosteric ErbB mutations, in patients with advanced solid malignancies.

TPS3665 Background: A significant unmet need exists for drugs targeting allosteric ErbB mutations (non-canonical mutations outside the ATP binding site). Current EGFR and HER2 tyrosine kinase inhibitors or mAbs have limited antitumor activity against allosteric mutations, resulting in toxicity before adequate drug exposure (Connell and Doherty, 2017). BDTX-189 is a potent and selective orally available irreversible inhibitor targeting unique oncogenic driver mutations of ErbB kinases in EGFR and HER2, while sparing WT EGFR. Preclinical studies demonstrated antitumor activity across a range of allosteric ErbB mutants, including extracellular domain allosteric mutations of HER2 as well as EGFR and HER2 kinase domain exon 20 insertions (Buck, 2019). This first-in-human trial (NCT04209465) is aimed to determine the recommended phase 2 dose (RP2) and schedule (Phase 1, P1), and evaluate the efficacy (Phase 2, P2) of BDTX-189. P1 primary objective is to determine the RP2 dose and schedule of monotherapy BDTX-189. Secondary objectives include assessment of safety, tolerability, pharmacokinetics (PK), pharmacodynamic (PD) effects in tumor, and preliminary efficacy. The P2 primary objective is to assess antitumor activity of monotherapy BDTX-189. Methods: The study will enroll patients (pts) ≥18 yrs with histologically or cytologically confirmed locally advanced or metastatic solid tumors with no standard therapy available or for whom standard therapy is unsuitable or intolerable. P1 dose-escalation will use a BOIN design (Yuan, 2016) and will enroll ≤ 88 pts with allosteric HER2 or HER3 mutation; EGFR or HER2 exon 20 insertion mutation; HER2 amplified or overexpressing tumor; or EGFR exon 19 deletion or L858R mutation. BDTX-189 will be dosed orally (PO) initially QD in 3 wk cycles. Regimen optimization will use PK, PD and safety data and may explore a BID schedule. An expansion cohort of ≤12 pts will further evaluate safety and preliminary efficacy of BDTX-189 prior to P2. P2, utilizing a Simon 2-stage design, will enroll ≤100 pts with NSCLC with EGFR or HER2 exon 20 insertion mutations (cohort 1); breast cancer with an allosteric ErbB mutation (cohort 2); tumors (except breast) with S310F/Y mutation (cohort 3); and other allosteric ErbB mutations not defined in cohorts 1-3 (cohort 4). Assessments include safety, tolerability, DLTs, evaluation of MTD, PK, PD, and preliminary antitumor activity. Enrollment began 1/2020. Clinical trial information: NCT04209465 .