HER Family Signaling Research Articles

HER3: Toward the Prognostic Significance, Therapeutic Potential, Current Challenges, and Future Therapeutics in Different Types of Cancer.

Human epidermal growth factor receptor 3 (HER3) is the only family member of the EGRF/HER family of receptor tyrosine kinases that lacks an active kinase domain (KD), which makes it an obligate binding partner with other receptors for its oncogenic role. When HER3 is activated in a ligand-dependent (NRG1/HRG) or independent manner, it can bind to other receptors (the most potent binding partner is HER2) to regulate many biological functions (growth, survival, nutrient sensing, metabolic regulation, etc.) through the PI3K-AKT-mTOR pathway. HER3 has been found to promote tumorigenesis, tumor growth, and drug resistance in different cancer types, especially breast and non-small cell lung cancer. Given its ubiquitous expression across different solid tumors and role in oncogenesis and drug resistance, there has been a long effort to target HER3. As HER3 cannot be targeted through its KD with small-molecule kinase inhibitors via the conventional method, pharmaceutical companies have used various other approaches, including blocking either the ligand-binding domain or extracellular domain for dimerization with other receptors. The development of treatment options with anti-HER3 monoclonal antibodies, bispecific antibodies, and different combination therapies showed limited clinical efficiency for various reasons. Recent reports showed that the extracellular domain of HER3 is not required for its binding with other receptors, which raises doubt about the efforts and applicability of the development of the HER3-antibodies for treatment. Whereas HER3-directed antibody-drug conjugates showed potentiality for treatment, these drugs are still under clinical trial. The currently understood model for dimerization-induced signaling remains incomplete due to the absence of the crystal structure of HER3 signaling complexes, and many lines of evidence suggest that HER family signaling involves more than the interaction of two members. This review article will significantly expand our knowledge of HER3 signaling and shed light on developing a new generation of drugs that have fewer side effects than the current treatment regimen for these patients.

KRAS mutation as a predictor of insufficient trastuzumab efficacy and poor prognosis in HER2-positive advanced gastric cancer.

Although RAS and PIK3CA mutations have been associated with resistance to anti-EGFR antibody in colorectal cancer or trastuzumab in breast cancer, their implications for trastuzumab resistance in HER2-positive advanced gastric cancer (AGC) remains unclear. We aimed to assess the relationship between trastuzumab efficacy and mutation status in the HER family signaling pathway. This study retrospectively evaluated patients with HER2-positive AGC who received first-line trastuzumab-containing chemotherapy between March 2011 and November 2015. Multiplex genotyping, including KRAS, NRAS, PIK3CA, and BRAF, was then performed using the Luminex Assay, after which KRAS amplification was measured using quantitative real-time reverse transcription-polymerase chain reaction. Thereafter, the association between genetic alterations and clinical outcomes were evaluated. KRAS mutation (MT) was detected in 6 of 77 patients (7.8%), whereas KRAS amplification was found in 15 of 67 patients (22%). No mutations in NRAS, PIK3CA, or BRAF were identified. The KRAS MT group showed significantly worse response rates (16.7% vs. 66.2%, P = 0.016), progression-free survival [median, 4.8 vs. 11.6months; hazard ratio (HR), 3.95; 95% CI, 1.60-9.76; P = 0.0029], and overall survival (11.5 vs. 23.6months; HR, 3.80; 95% CI, 1.56-9.28; P = 0.033) compared to the KRAS wild-type group. KRAS amplification had no effect on clinical outcomes. KRAS mutation was an independent prognostic factor for poor survival and might predict insufficient trastuzumab efficacy, whereas KRAS amplification showed no prognostic significance during trastuzumab treatment. Further investigations are warranted to confirm the predictive value of KRAS status in HER2-positive AGC.

Abstract P2-13-36: Comparative time course analysis of the effects of neratinib, lapatinib and tucatinib in an in vitro HER2+ breast cancer model

Abstract Introduction: Human epidermal growth factor 2 (HER2/ErbB2) is overexpressed in approximately 20% of breast cancers. The HER2-targeted tyrosine kinase inhibitors (TKIs) neratinib, lapatinib and tucatinib are all FDA-approved for the treatment of HER2-positive breast cancer. All three inhibit the HER2 kinase domain but differ in binding characteristics (the irreversible neratinib vs reversible lapatinib and tucatinib) and their affinity for other HER family members. The aim of this study was to carry out kinetic analysis of the effect of the three TKIs on HER family signalling, cellular proliferation, and apoptosis induction in a cell model of breast cancer. Methods: In this study, the anti-proliferative effects of the three TKIs were assessed in the HER2-amplified breast cancer cell line SKBR3 by acid phosphatase assay (5-day endpoint assay) and with the IncuCyte→ live-cell imaging system over 120 hours. TKI IC50 values were calculated using CalcuSyn software. Apoptotic cells were detected every 3 hours for 5 days using the Incucyte→ Caspase 3/7 dye. Western blotting was used to investigate the time course effects (15 min, 6 h, 24 h, 72 h) of the TKIs on the expression and phosphorylation of EGFR, HER2 and on their downstream effectors ERK1/2 and Akt. Results: All three TKIs displayed nanomolar activity against SKBR3 cells. Neratinib showed the greatest anti-proliferative effect (IC50 = 3.4 ± 1.1 nM), followed by tucatinib (IC50 = 37.5 ± 18.4 nM) and lapatinib (IC50 = 51.0 ± 23.0 nM). 50 nM neratinib induced higher activation of caspase 3/7 than 500 nM lapatinib or 500 nM tucatinib. Total levels of HER2 and EGFR were increased in the presence of lapatinib and tucatinib, but decreased in the presence of neratinib. The three drugs (500 nM) significantly inhibited the activation of EGFR and HER2 at 15 min. From 6 h to 72 h, treatment with 5 nM and 50 nM neratinib resulted in greater inhibition of phosphorylated HER2 and EGFR, compared to the equivalent concentration of lapatinib or tucatinib. All three TKIs (500 nM) inhibited the activity of phospho-Akt at the earliest timepoint (15 min), but a strong impact on downstream ERK1/2 phosphorylation was not observed until the 6h timepoint. 50 and 500 nM neratinib was more effective at preventing reactivation of ERK1/2 and Akt phosphorylation than either 500 nM lapatinib or tucatinib at 72 h. Conclusion: These data highlight the differences in in vitro efficacy of the three HER2-targeting TKIs currently approved for the treatment of HER2+ breast cancer in the clinic. Neratinib inhibits HER2 and EGFR at lower concentrations and for longer compared to lapatinib and tucatinib, resulting in increased apoptosis induction. This may be due to the irreversible nature of neratinib binding, as opposed to the reversible nature of lapatinib and tucatinib binding. Further comparative studies between neratinib, lapatinib and tucatinib are warranted to explore the impact of varied pre-clinical characteristics on the emergence of therapeutic resistance. Citation Format: Myra E Castel, Neil T Conlon, Naomi Walsh, Irmina Diala, Lisa Eli, John Crown, Denis M Collins. Comparative time course analysis of the effects of neratinib, lapatinib and tucatinib in an in vitro HER2+ breast cancer model [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-36.

Abstract P6-03-20: The development and characterization of novel HER2-positive breast cancer models of acquired neratinib resistance

Abstract Introduction: Neratinib is an irreversible, small molecule pan-HER tyrosine kinase inhibitor that has shown clinical activity against HER2-amplified (HER2+) and HER2-mutated breast cancer. However, there are limited data on potential mechanisms of resistance to neratinib. This study aimed to develop HER2+ breast cancer cell line models of acquired neratinib resistance, and to elucidate the effects of acquired resistance on HER family signalling and drug response. As neratinib has activity in both estrogen receptor positive (ER+) and ER- HER2+ breast cancer, neratinib-resistant cell lines were generated and characterised from breast cancer cell lines of both subtypes. Methods: HER2+, ER- HCC1569 cells were continuously exposed to 150 nM neratinib for six months to generate the neratinib-resistant HCC1569 (HCC1569-N) cell line. HER2+ ER+ BT474 cells were continuously exposed to escalating doses of 5-80 nM neratinib for six months to generate the BT474-N cell line. Sensitivity to HER2-targeted therapies (neratinib, lapatinib and trastuzumab) and chemotherapeutics (5’dFUR, carboplatin, and vinorelbine) were assessed by 5-day acid phosphatase based proliferation assay. IC50 and combination index (CI) values were calculated using CalcuSyn software. Total and phosphorylated levels of HER2, EGFR, ERK, and Src were assessed by Western blotting following neratinib treatment (150 nM), to determine changes in HER2 signalling. Results: The HCC1569-N and BT474-N cells had a 50.3-fold and 105-fold increase in neratinib resistance. In addition, both cell lines developed cross-resistance to lapatinib and were resistant at clinically relevant concentrations (> 1 µM). HCC1569-Par cells are innately resistant to trastuzumab, which was not altered in HCC1569-N cells. However, neratinib resistance also caused resistance to trastuzumab in the BT474-N cell line, compared to its trastuzumab-sensitive parental cell line (Table 1). The HCC1569-N cell line did not show any significant difference in sensitivity to 5’dFUR, carboplatin, or vinorelbine compared to parental HCC1569, but the addition of vinorelbine to neratinib was synergistic (CI value = 0.28 ± 0.19). HCC1569-N cells displayed no changes in total protein or phosphorylated levels of HER2 or EGFR compared to parental cells. Additionally, neratinib treatment still resulted in inhibition of HER2, EGFR, and downstream ERK signalling, indicating a lack of reliance on HER2 signalling in HCC1569-N cells. Phosphorylated Src levels were elevated (p = 6.3 × 10−4) in HCC1569-N compared to parental cells. Conclusions: Neratinib resistance in HCC1569-N and BT474-N cells confers cross-resistance to other HER2-targeted therapies. Increased Src activity may be implicated in the neratinib-resistant HER2+ ER- phenotype. Cell line10 µg/mL Trastuzumab (%Growth)NeratinibIC50 valueLapatinibIC50 valueHCC1569-Par97.6 ± 2.57.8 ± 5.8653.0 ± 144.0HCC1569-N92.7 ± 2.9393.0 ± 77.1> 1000BT474-Par44.7 ± 1.81.2 ± 0.323.9 ± 19.6BT474-N97.8 ± 3.3126.8 ± 53.5> 1000 Citation Format: Neil T Conlon, Alacoque Browne, Laura Breen, Alex J Eustace, John Crown, Denis M Collins. The development and characterization of novel HER2-positive breast cancer models of acquired neratinib resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-20.

Abstract P4-10-31: Patterns of protein expression and signaling assessed by reverse phase protein array (RPPA) in estrogen-receptor negative (ER-), HER2-amplified breast cancer (BC) that was primary-refractory to preoperative (preop) HER2-directed therapy

Abstract Background: ER- HER2+ BC is comprised of a heterogeneous mix of intrinsic BC subtypes which have differential responsiveness to preop HER2-directed therapies combined with chemotherapy. Patients (pts) with ER- HER2+ BC whose disease progressed while on or who had no response to preop HER2-directed therapy are uncommon and the mechanisms of resistance within this primary-refractory disease are unknown. We utilized RPPA-based protein pathway activation mapping to map the drug target signaling architecture in pts’ primary-refractory ER- HER2+ BCs to begin to assess mechanisms of resistance. Methods: Eight primary-refractory ER- HER2+ FFPE BC tissues obtained from 5 pts (preop biopsies n=4 and/or post-treatment surgical resection n=4) underwent laser capture microdissection to isolate pure tumor epithelium followed by RPPA-based analysis of the levels and/or activation/phosphorylation of approximately 130 key signaling proteins known to be involved in tumorigenesis and metastatic progression. Results: Pts were all treated with preop trastuzumab, pertuzumab, docetaxel, and carboplatin (TCHP) with no response and/or with progression of disease. Three pts also received preop doxorubicin/cyclophosphamide without response. RPPA-based analysis revealed that post-TCHP surgical samples had higher activation of a number of drug targets including receptor tyrosine kinases (RTK) such as MET and ALK, as well as HER family signaling proteins phospho-HER3 and phospho-EGFR compared to the matched pre-TCHP samples. Conclusions: In these ER- HER2+ BCs that were primary-refractory to preop TCHP, in those pts with patient-matched pre/post treatment samples, activation of several potential resistance pathways such as alternative RKT signaling as well as EGFR and HER3 signaling was observed. These data are hypothesis-generating and require exploration in additional ER- HER2+ primary-refractory tissues. Citation Format: Mariaelena Pierobon, Maren K Levin, Elisa Baldelli, Leslie SL Kim, Lance Liotta, Emanuel F Petricoin, Joyce O'Shaughnessy. Patterns of protein expression and signaling assessed by reverse phase protein array (RPPA) in estrogen-receptor negative (ER-), HER2-amplified breast cancer (BC) that was primary-refractory to preoperative (preop) HER2-directed therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-31.

Evaluation of the HER/PI3K/AKT Family Signaling Network as a Predictive Biomarker of Pathologic Complete Response for Patients With Breast Cancer Treated With Neratinib in the I-SPY 2 TRIAL.

In the I-SPY 2 TRIAL (Investigation of Serial Studies to Predict Your Therapeutic Response With Imaging and Molecular Analysis 2), the pan-erythroblastic oncogene B inhibitor neratinib was available to all hormone receptor (HR)/human epidermal growth factor receptor 2 (HER2) subtypes and graduated in the HR-negative/HER2-positive signature. We hypothesized that neratinib response may be predicted by baseline HER2 epidermal growth factor receptor (EGFR) signaling activation/phosphorylation levels independent of total levels of HER2 or EGFR proteins. Complete experimental and response data were available for between 130 and 193 patients. In qualifying analyses, which used logistic regression and treatment interaction analysis, 18 protein/phosphoprotein, 10 mRNA, and 12 DNA biomarkers that related to HER family signaling were evaluated. Exploratory analyses used Wilcoxon rank sum and t tests without multiple comparison correction. HER pathway DNA biomarkers were either low prevalence or nonpredictive. In expression biomarker analysis, only one gene (STMN1) was specifically associated with response to neratinib in the HER2-negative subset. In qualifying protein/phosphoprotein analyses that used reverse phase protein microarrays, six HER family markers were associated with neratinib response. After analysis was adjusted for HR/HER2 status, EGFR Y1173 (pEGFR) showed a significant biomarker-by-treatment interaction (P = .049). Exploratory analysis of HER family signaling in patients with triple-negative (TN) disease found that activation of EGFR Y1173 (P = .005) and HER2 Y1248 (pHER2) (P = .019) were positively associated with pathologic complete response. Exploratory analysis in this pEGFR/pHER2-activated TN subgroup identified elevated levels of estrogen receptor α (P < .006) in these patients. Activation of HER family phosphoproteins associates with response to neratinib, but only EGFR Y1173 and STMN1 appear to add value to the graduating signature. Activation of HER2 and EGFR in TN tumors may identify patients whose diseases respond to neratinib and implies that there is a subset of patients with TN disease who paradoxically exhibit HER family signaling activation and may achieve clinical benefit with neratinib; this concept must be validated in future studies.

Profiling of protein-protein interactions via single-molecule techniques predicts the dependence of cancers on growth-factor receptors.

The accumulation of genetic and epigenetic alterations in cancer cells rewires cellular signalling pathways through changes in the patterns of protein-protein interactions (PPIs). Understanding these patterns may facilitate the design of tailored cancer therapies. Here, we show that single-molecule pull-down and co-immunoprecipitation techniques can be used to characterize signalling complexes of the human epidermal growth-factor receptor (HER) family in specific cancers. By analysing cancer-specific signalling phenotypes, including post-translational modifications and PPIs with downstream interactions, we found that activating mutations of the epidermal growth-factor receptor (EGFR) gene led to the formation of large protein complexes surrounding mutant EGFR proteins and to a reduction in the dependency of mutant EGFR signalling on phosphotyrosine residues, and that the strength of HER-family PPIs is correlated with the strength of the dependence of breast and lung adenocarcinoma cells on HER-family signalling pathways. Furthermore, using co-immunoprecipitation profiling to screen for EGFR-dependent cancers, we identified non-small-cell lung cancers that respond to an EGFR-targeted inhibitor. Our approach might help predict responses to targeted cancer therapies, particularly for cancers that lack actionable genomic mutations.

93P - KRAS status and HER2 targeted treatment in advanced gastric cancer

Trastuzumab targeted on HER2 has been shown to confer overall survival benefit adding to fluoropyrimidine (Fp) plus CDDP in HER2-positive advanced gastric cancer (AGC). HER2 is known to make the formation of heterodimer with EGFR(HER1), HER3 and HER4. HER2 containing heterodimer activates the common downstream of HER family such as MAPK pathway via RAS. RAS and BRAF status has been established as predictive biomarkers for anti EGFR treatment in metastatic colorectal cancer. However it remains unclear that the implications for these status and HER2 targeted treatment in AGC. In this study we attempted to assess the relationship with the efficacy of trastuzumab including treatment and mutational status of HER family signaling pathway. Out of 100 patients received Fp plus CDDP with trastuzumab as 1st-line between March 2011 and November 2015, total 77 patients with sufficient specimen for DNA extraction were enrolled in this analysis. Multiplex genotyping of HER family common downstream was performed on archival samples using Luminex Assay (MEBGEN and GENOSEARCH Mu-PACK, MBL) for KRAS and NRAS including exon 2, 3 and 4, PIK3CA and BRAF. Tumor response was re-assessed by the investigator retrospectively by RECIST1.1. KRAS mutation of exon2 was detected in only 6 patients of 77 patients (7.8 %). No mutations were found in NRAS, PIK3CA and BRAF in this HER2 positive AGC series. An overall RR and the disease control rate (DCR) in KRAS wild type (WT) vs. mutant type (MT) were following, RR; 66.2% vs. 16.7%, DCR; 87.3% vs. 66.7%, respectively (CR2/0, PR 45/1, SD 15/3, PD 9/2). The median PFS and OS in KRAS WT vs. MT were as followed, 8.9 months (m) vs. 3.6 m and 20.8 m vs. 10.3 m, respectively. KRAS MT showed extremely shorter PFS and OS compared with KRAS WT (P = 0.00008 and 0.0008). Our data suggested HER2-positive AGC harbored KRAS mutation at the low frequency. KRAS mutation might predict poor prognosis as receiving HER2 targeted treatment. Further investigation was warranted to confirm the predictive value of KRAS status in HER2-positive AGC treated with trastuzumab to fluoropyrimidine plus CDDP. We will present additional analysis of KRAS amplification in this cohort at the convention.

Abstract P3-06-15: Evaluation of HER family protein signaling network as a predictive biomarker for pCR for breast cancer patients treated with neratinib in the I-SPY 2 trial

Abstract Background: We hypothesize that response to the pan-ERBB inhibitor, neratinib (N), may be predicted by pre-treatment HER2-EGFR signaling. In the I-SPY 2 TRIAL, N graduated in the HR-/HER2+ signature. All patients received at least standard chemotherapy. For HER2+ patients, N was administered in place of trastuzumab. We evaluated 18 HER family signaling proteins as biomarkers of N response using reverse phase protein microarray (RPMA) data from pre-treatment LCM purified tumor epithelium. Methods: 168 patients (N: 106, concurrent controls: 62) had RPMA and pCR data. 18 biomarkers relating to HER family signaling were evaluated: AKT S473, AKT T308, EGFR, EGFR Y1068, EGFR Y1148, EGFR Y1173, EGFR Y992, ERBB2, ERBB2 Y1248, ERBB3 total, ERBB3 Y1289, ERK1/2 T202/Y204, Heregulin, mTOR, mTOR S2448, PI3K p85 Y458/p55 Y199, PTEN S380, and SHC Y317. We assessed association between biomarker and response in the N and control arms alone (likelihood ratio test), and relative performance between arms (biomarker x treatment interaction) using a logistic model. Analysis was also performed adjusting for HR/HER2 status. In an exploratory analysis, we selected the marker with the greatest interaction (phosphorylated EGFR (Y1173)) to dichotomize patients optimally based on the data and assessed it in the context of the graduating signature by adding the EGFR Y1173-High patients to the HR-/HER2+ subtype and evaluating the treatment effect in this ‘biomarker-positive’ group. Our study is exploratory with no claims for generalizability of the data and does not account for multiplicities. Statistical calculations are descriptive (e.g. p-values are measures of distance with no inferential content). Results: 7 HER pathway markers (EGFR Y1068, EGFR Y1173, EGFR Y992, ERBB2 total, ERBB2 Y1248, ERBB3 Y1289, SHC Y317) are associated with response in the N but not the control arm. However, the difference in performance between arms did not reach significance by permutation testing. Adjusting for HR/HER2 status, EGFR Y1173 shows a significant biomarker x treatment interaction (p = 0.049). In an exploratory analysis, we dichotomized patients by their EGFR Y1173 levels and evaluated the distribution of pCR rates (Table 1). Neratinib (n=106)Control (n=62)EGFR Y1173 Low (n=31)EGFR Y1173 High (n=75)EGFR Y1173 Low (n=29)EGFR Y1173 High (n=33)HR-/HER2+ (n=28)0 / 412 / 181 / 11 / 5Not HR-/HER2+ (n=140)3 / 2724 / 575 / 285 / 28Table 1 OR between EGFR Y1173 groups in the N relative to control arm is 10.1. When EGFR Y1173 High patients are added to the graduating HR-/HER2+ subset, the OR associated with treatment is 3.2 and is comparable to that in the HR-/HER2+ signature (OR: 2.1), while increasing the prevalence of biomarker-positive patients by ∼50%. Evaluation of EGFR Y1173 under the I-SPY 2 Bayesian model is pending. Conclusion: Our sample size is too small to draw definitive conclusions. Our exploratory analysis reveals that HER family phosphoproteins associate with response to N, but only phosphorylated EGFR Y1173 appears to add value to the graduating signature. Given that this biomarker would expand the patient population that may benefit, it merits evaluation in other ongoing trials of neratinib. Citation Format: Julia D Wulfkuhle, Christina Yau, Denise M Wolf, Rosa I Gallagher, Ashish Sanil, Lamorna Brown-Swigart, Susan Flynn, Gillian Hirst, I-SPY 2 TRIAL Investigators, Meredith Buxton, Angela DeMichele, Nola Hylton, William F Symmans, Laura van't Veer, Douglas Yee, Melissa Paoloni, Laura Esserman, Donald Berry, Minetta C Liu, John W Park, Emanuel F Petricoin III. Evaluation of HER family protein signaling network as a predictive biomarker for pCR for breast cancer patients treated with neratinib in the I-SPY 2 trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-15.

HER3 as a potential prognostic biomarker in pancreatic cancer.

295 Background: HER 3 is revealing itself to be increasingly important in HER family signaling. Understanding the role of HER 3 in pancreatic cancer may shed light on targeted therapy development. Methods: A large human pancreatic cancer tumor tissue microarray (TMA) was established at our institution from 165 patients with pancreatic cancer who underwent surgical resection. The TMA contains two cores from each patient. Clinical information of these patients has been prospectively maintained in a clinical database. Immunohistochemistry (IHC) was performed on the TMA using a monoclonal antibody against HER 3. An independent pathologist blinded to the patient data applied the H score to grade the expression level of HER 3. The association between expression levels of HER 3 and survival were evaluated by the Kaplan-Meyer product limit method and log-rank test, while the optimal cut-off for each marker was determined using the Martingale residuals from Cox proportional hazard models. Results: Out of 165 patients, 136 patients’ tumors were identified to have adequate tumor content for IHC analysis. The group with higher HER 3 levels at the cell membrane (n=50)(H score ≥150) had a superior overall survival compared to the low HER 3 membrane expression group (n=86) (22.5 months vs. 17 months; p=0.036). Interestingly, the group with higher expression levels at both the cell membrane and in the cytosol (n=37) (H score ≥190) had a more striking survival difference compared to the lower group (n=99) (26 months vs. 15 months; p= 0.035). Conclusions: Patients with high HER 3 expression either at the cell membrane only or at both the membrane and in the cytosol have longer survival, perhaps indicating a lower heregulin and inactive HER 3 signaling. Additional biomarker analyses are ongoing. The functional role of HER 3 in pancreatic cancer is being further explored to assist clinical evaluation of HER 3 targeting in pancreatic cancer.

HER2-family signalling mechanisms, clinical implications and targeting in breast cancer.

Approximately 20 % of human breast cancers (BC) overexpress HER2 protein, and HER2-positivity is associated with a worse prognosis. Although HER2-targeted therapies have significantly improved outcomes for HER2-positive BC patients, resistance to trastuzumab-based therapy remains a clinical problem. In order to better understand resistance to HER2-targeted therapies in HER2-positive BC, it is necessary to examine HER family signalling as a whole. An extensive literature search was carried out to critically assess the current knowledge of HER family signalling in HER2-positive BC and response to HER2-targeted therapy. Known mechanisms of trastuzumab resistance include reduced receptor-antibody binding (MUC4, p95HER2), increased signalling through alternative HER family receptor tyrosine kinases (RTK), altered intracellular signalling involving loss of PTEN, reduced p27kip1, or increased PI3K/AKT activity and altered signalling via non-HER family RTKs such as IGF1R. Emerging strategies to circumvent resistance to HER2-targeted therapies in HER2-positive BC include co-targeting HER2/PI3K, pan-HER family inhibition, and novel therapies such as T-DM1. There is evidence that immunity plays a key role in the efficacy of HER-targeted therapy, and efforts are being made to exploit the immune system in order to improve the efficacy of current anti-HER therapies. With our rapidly expanding understanding of HER2 signalling mechanisms along with the repertoire of HER family and other targeted therapies, it is likely that the near future holds further dramatic improvements to the prognosis of women with HER2-positive BC.

Targeting of HER Family Signaling Pathways in Gastric Cancer

Targeting of HER Family Signaling Pathways in Gastric Cancer

Abstract P2-09-08: Therapeutic potential of the dual HER1/2 inhibitor AZD8931 in circumventing endocrine resistance

Abstract Background: Crosstalk between ER and HER-family signaling pathways has been suggested to play a role in the development of endocrine resistance. Preclinical studies have shown that AZD8931, a dual tyrosine kinase inhibitor (TKI) of HER1 (EGFR) and HER2, elicits an equipotent inhibition of HER1, HER2, and HER3 signaling, and is consequently more effective in blocking ligand-dependent HER signaling than the dual HER 1/2 TKI lapatinib. Using in vitro and in vivo models we investigated AZD8931's therapeutic potential in enhancing endocrine therapy and in overcoming the growth of tumor cells resistant to tamoxifen (Tam). Materials and Methods: The effect of different TKIs (AZD8931 and lapatinib) on endocrine therapy [Tam and Fulvestrant (Ful)] was tested in ER+ MCF7 and T47D parental cells and their Tam-resistant derivatives (TamRes). In vitro growth, proliferation, and apoptosis were assessed using an in situ cell cytometer, EDU incorporation, and Annexin V-FITC/c-PARP, respectively. HER ligands in the parental and TamRes cells were profiled using RNASeq analysis of these lines. Western blot analysis was used to analyze the effect of AZD8931, lapatinib, and the HER1 TKI gefitinib on HER 1/2 pathway activation upon ligand stimulation. Nude mice with transplantable MCF7/TamRes xenografts at 200 mm3 were randomized to continued Tam, Tam+AZD8931, Fulvestrant (Ful), or Ful+AZD8931 treatments. Results: We found that neither lapatinib nor AZD8931 significantly enhanced endocrine sensitivity in parental MCF7 breast cancer cells, although AZD8931 did enhance endocrine sensitivity in parental T47D cells. Furthermore, AZD8931 combined with either Tam or Ful inhibited cell growth more profoundly than lapatinib in the T47D TamRes cell model, and was significantly, though modestly, more potent than lapatinib in the MCF7 TamRes model when combined with Tam. In both TamRes models, AZD8931 significantly inhibited cell proliferation and induced apoptosis, with the highest effects seen in combination with Ful. Interestingly, multiple HER ligands are upregulated in both MCF7 and T47D TamRes models, which could explain the superiority of AZD8931 over lapatinib in these models. Indeed, in EGF and heregulin (HRG) stimulated conditions, AZD8931 more potently inhibited HER signaling (i.e., phosphorylated (p) levels of HER1/2, MAPK, and AKT) than lapatinib or gefitinib. Finally, AZD8931 significantly delayed the growth of MCF7 TamRes xenografts in the presence of continued Tam or Ful. These tumors were also inhibited by Ful alone, but the strongest inhibition was achieved by Ful and AZD8931 in combination. However, despite the marked tumor growth delay, no tumor regression was found in any of these treatments. Conclusion: This study provides evidence that AZD8931 has greater inhibitory efficacy than lapatinib in endocrine resistant models that are dependent on ligand activation of the HER pathway. Although the AZD8931 combination with Ful robustly slowed growth of TamRes tumors in vivo, the absence of tumor regression suggests that additional escape pathways are also involved and should also be targeted to fully circumvent tamoxifen resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-08.

Discovery of AZD8931, an Equipotent, Reversible Inhibitor of Signaling by EGFR, HER2, and HER3 Receptors.

Deregulation of HER family signaling promotes proliferation and tumor cell survival and has been described in many human cancers. Simultaneous, equipotent inhibition of EGFR-, HER2-, and HER3-mediated signaling may be of clinical utility in cancer settings where the selective EGFR or HER2 therapeutic agents are ineffective or only modestly active. We describe the discovery of AZD8931 (2), an equipotent, reversible inhibitor of EGFR-, HER2-, and HER3-mediated signaling and the structure-activity relationships within this series. Docking studies based on a model of the HER2 kinase domain helped rationalize the increased HER2 activity seen with the methyl acetamide side chain present in AZD8931. AZD8931 exhibited good pharmacokinetics in preclinical species and showed superior activity in the LoVo tumor growth efficacy model compared to close analogues. AZD8931 is currently being evaluated in human clinical trials for the treatment of cancer.

Preclinical studies with neratinib in triple-negative breast cancer.

1054 Background: Currently, one of the most urgent problems in breast cancer therapeutics is the validation of targeted therapy for triple-negative breast cancer (TNBC). Neratinib is an irreversible small molecule inhibitor that targets the kinase activity of EGFR, HER2 and HER4. As one of these targets, EGFR, is expressed at high levels in a subset of TNBC, we hypothesised that neratinib is a potential treatment for at least some patients with TNBC. Methods: The antiproliferative effects of neratinib were investigated in a panel of 35 breast cancer cells lines including 9 luminal, 12 HER2-positive and 14 triple-negative (TN). Baseline levels of total HER proteins were determined using flow cytometry. Results: IC50 values for neratinib across the panel of breast cancer cell lines ranged from &lt; 0.001 - 1.9 µM. HER2-positive cell lines were significantly more sensitive to neratinib than HER2-negative cells (p = 0.008, Student’s t-test). For the 14 TN cell lines, IC50 values ranged from 0.03 – 1.9 µM. TN cell lines defined as basal-type A were significantly more sensitive to neratinib than those designated basal-type B (p = 0.02, Student’s t-test). Treatment with neratinib resulted in a reduction in HER-family signalling as detected by decreased Akt and ERK phosphorylation. Consistent with the finding that basal type A TN cells were more sensitive to neratinib than type B, significantly higher levels of EGFR expression were found in the A subtype compared to the B subtype (p = 0.02, Student’s t-test). To determine a possible predictive marker of response for neratinib, levels of all 4 HER proteins were related to response in the basal A subgroup. Baseline levels of HER3 expression were found to correlate significantly with response to neratinib (p = 0.02, r = -0.875, n = 7, Spearman), i.e., cell lines with the highest levels of HER3 expression had the lowest IC50values within this cohort. Conclusions: Our results suggest that neratinib is a potential novel therapeutic option for patients with basal A type triple negative breast cancer. HER3 may act as a predictive marker of response for this subgroup of patients. Acknowledgements: The authors thank SFI for funding MTCI (SRC award, 08/SRC/B1410 to MTCI) and an SFI Short Term Travel Fellowship to MM, for funding this work.

Abstract 4446: Activation of HER Family signaling as a mechanism of acquired resistance to ALK Inhibitors in EML4-ALK-positive Non-Small Cell Lung Cancer.

Abstract Purpose: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) such as crizotinib show marked efficacy in patients with non-small cell lung cancer positive for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion protein. However, acquired resistance to these agents has already been described in treated patients, and the mechanisms of such resistance remain largely unknown. Experimental Design: We established lines of EML4-ALK-positive H3122 lung cancer cells that are resistant to the ALK inhibitor TAE684 (H3122/TR cells) and investigated their resistance mechanism with the use of immunoblot analysis, ELISA, reverse transcription and real-time PCR analysis, and an annexin V binding assay. We isolated EML4-ALK-positive lung cancer cells (K-3) from a patient who developed resistance to crizotinib and investigated their characteristic. Results: The expression of EML4-ALK was reduced at the transcriptional level, whereas phosphorylation of the epidermal growth factor receptor (EGFR), HER2, and HER3 was up-regulated, in H3122/TR cells compared with those in H3122 cells. This activation of HER family proteins was accompanied by increased secretion of EGF. Treatment with an EGFR-TKI induced apoptosis in H3122/TR cells, but not in H3122 cells. The TAE684-induced inhibition of extracellular signal-regulated kinase (ERK) and STAT3 phosphorylation observed in parental cells was prevented by exposure of these cells to exogenous EGF, resulting in a reduced sensitivity of cell growth to TAE684. K-3 cells also manifested HER family activation accompanied by increased EGF secretion. Conclusions: EGF-mediated activation of HER family signaling is associated with ALK-TKI resistance in lung cancer positive for EML4-ALK. Citation Format: Junko Tanizaki, Isamu Okamoto, Takafumi Okabe, Kazuko Sakai, Kaoru Tanaka, Hidetoshi Hayashi, Hiroyasu Kaneda, Ken Takezawa, Kazuto Nishio, Kazuhiko Nakagawa. Activation of HER Family signaling as a mechanism of acquired resistance to ALK Inhibitors in EML4-ALK-positive Non-Small Cell Lung Cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4446. doi:10.1158/1538-7445.AM2013-4446

Abstract 2090: Targeting HER family signaling in gastric cancer: The selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, is efficacious in primary models of HER family driven disease.

Abstract Despite an overall decline, gastric cancer incidence remains high in Asian countries including China, Japan and South Korea. Five-year survival rates continue to improve in early stage disease through an aggressive combination of surgery and chemo/radiotherapy, primarily using fluoropyrimidine and platinum agent combinations. In advanced disease, the TOGA trial provided clinical validation of a role for HER2 in driving tumor progression in a subset of gastric cancer (GC) patients. However, recent clinical studies with EGFR targeting antibodies in GC have failed to show promise (EXPAND and REAL3 trials). Nonetheless, the dismal prognosis associated with late stage disease and the frequent implication of dysregulated HER family signaling in GC necessitate further investigation. AZD8931 is an orally bioavailable, highly selective and potent small molecule inhibitor of EGFR, HER2 and HER3 signaling(1). To evaluate the potential utility of this agent in clinical GC, we examined HER family gene amplification and expression in GC tissue samples and sought to correlate these profiles with response to AZD8931 in GC tumor xenograft models. High Her2 expression (IHC3+ or IHC2+/FISH+ve) was confirmed in 17.4% of tumours (20/115). Importantly, high level EGFR (44.4%, 48/108) and HER3 (89.3%, 92/103) protein expression was also detected in our cohort of Chinese GC samples. Gastric cancer cell panel screening (n=25 lines) identified a number of AZD8931-sensitive cell lines with a range of HER family expression profiles (GI50 range 0.02 to 0.35μM). In the NUGC3 xenograft model (high EGFR expression), twice-daily oral dosing of AZD8931 elicited potent, dose-responsive anti-tumour activity at well-tolerated doses (98% TGI at 6mg/kg/bid, 108% TGI at 12.5mg/kg/bid, all P&amp;lt;0.0001) and anti-tumour efficacy correlated well with pharmacodynamic modulation of phospho-HER signaling. To investigate the translational relevance of these data, primary GC models with varying HER family protein expression profiles were identified and established. Within these models, AZD8931 anti-tumor efficacy correlated broadly with strong HER pathway activation (0 to 83% TGI at 12.5mg/kg/bid). We further evaluated the activity of AZD8931 in combination with cisplatin/5-FU, 1st line standard of care chemotherapies commonly used in the treatment of gastric cancer. Using a primary GC xenograft model, AZD8931 demonstrated additive benefit when used at well tolerated doses (minimal body weight loss). These data support the potential clinical utility of AZD8931 for the therapeutic treatment of gastric cancers exhibiting strong HER pathway signaling. 1 Hickinson et al. Clin.Cancer Res 2010; Feb 15. Citation Format: Paul R. Gavine, Liang Xie, Lin Zhang, Jing Lv, Lu Han, XiaoLu Yin, Yuan Jie Liu, Xinying Su, Shuqiong Fan, Teresa Klinowska. Targeting HER family signaling in gastric cancer: The selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, is efficacious in primary models of HER family driven disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2090. doi:10.1158/1538-7445.AM2013-2090

Abstract 912: Targeting HER family signaling in low HER2-expressing breast cancer: activity of the selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, in models of low HER2-expressing disease.

Abstract Effective therapies for women with low-HER2 expressing breast cancer remain a significant unmet clinical need. We have detected significant phosphorylated-HER and HER2:HER3 dimer expression in clinical breast cancer samples without HER2 amplification suggesting that HER signaling may play a role in these tumors. It is our hypothesis that ligand-driven signaling is the major route of HER family activation in the absence of HER2 amplification. AZD8931 is an orally bioavailable and highly selective small molecule inhibitor of EGFR, HER2 and HER3 signaling, which shows greatest potency when HER signaling is ligand-driven(1). In a range of low HER2 breast cancer cell lines, AZD8931 inhibited heregulin-driven proliferation (GI50 range 0.05 to 0.38μM) HER2:HER3 signaling, HER3:PI3K interaction and downstream signaling. In vivo, twice-daily oral dosing of AZD8931 showed significant monotherapy anti-tumor efficacy in a xenograft model of low HER2 breast cancer (MDA-MB-175VII) at well-tolerated doses (90% TGI at 12.5 mg/kg/bid). We further evaluated the activity of AZD8931 in combination with paclitaxel, a commonly used standard of care chemotherapy for patients with advanced breast cancer expressing low levels of HER2. Pre-clinically, the combination of AZD8931 with paclitaxel showed at least additive activity in vitro in a range of breast cell lines and additive efficacy in vivo in a BT474 (high HER2) xenograft model at well tolerated doses (AZD8931 12.5 mg/kg/bid TGI 63%; paclitaxel 7.5mg/kg/qw TGI 41%; combination TGI 95%). Combination work in the low HER2 xenograft model is ongoing. These data demonstrate that AZD8931 inhibits ligand-driven HER family receptor activation, receptor dimerization, PI3K interaction and downstream signaling leading to anti-tumor activity in vivo. These data also support the potential clinical utility of AZD8931 for the therapeutic treatment of low HER2 expressing breast cancers in combination with paclitaxel. 1 Hickinson et al. Clin.Cancer Res (2010) 16:1159-69. Citation Format: Gayle Marshall, Susan Ashton, Georgina Speake, Celina D'Cruz, Michael Grondine, Cath Trigwell, Graham Bigley, Garry Beran, Katy Lynaugh, Teresa C. Klinowska. Targeting HER family signaling in low HER2-expressing breast cancer: activity of the selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, in models of low HER2-expressing disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 912. doi:10.1158/1538-7445.AM2013-912

Abstract 4455: Studies of early adaptive resistance in mutant-EGFR and EML4-ALK addicted non-small cell lung cancer in escape against small molecule inhibitors.

Abstract Lung cancer is the second most common malignancy in the United States with the highest cancer-mortality rate. Molecular targeted therapy in oncogene addicted non-small cell lung cancer (NSCLC) has gain landmark proof-of-principle success in clinical utility in recent years. Prime examples are EGFR targeted therapy against mutated EGFR tumors using tyrosine kinase inhibitors (TKIs) such as erlotinib/gefitinib, and also ALK targeted therapy against EML4-ALK tumors using ALK inhibitor crizotinib. Superior response rates and progression free survival outcomes were observed with the use of targeted therapeutics even in first line setting. Nonetheless, targeted therapeutics against mutant-EGFR and EML4-ALK tumors invariably develop an acquired tumor resistance to the TKIs. Mutational resistant mechanism has been well-established. Alternative signal pathway activation such as MET/HGF in both EGFR and ALK TKI resistance, or HER family signaling activation in EML4-ALK resistance have also been reported. We have recently begun studies focusing on the non-mutational mechanisms of tumor resistance under early time window of TKI treatment. Using HCC827 and PC9 cells as mutant EGFR addiction model, as well as H3122 and H2228 NSCLC cells as EML4-ALK addicted model, we have uncovered a novel early adaptive TKI resistance that can be evident as early as 7-14 days under initial TKI treatment. The early adaptive tumor cells displayed remarkable tumor resistance against the TKIs with 100-fold higher IC50. In mutant-EGFR against erlotinib, there was an upregulated mitochondrial antiapoptotic/prosurvival BCL-2/BCL-xL signaling dependence that is MET-independent. The early resistant cells were adaptive, highly reversible and adopted a quiescent state during TKI-induced resistance. On the other hand, our studies of early TKI resistance in EML4-ALK fusion cells revealed that while the adaptive resistance to TAE684 was also reversible, it appears to be not dependent on BCL-2/BCL-xL upregulation. We found that in both targeted TKI early resistance, the tumor survivor cells exhibited epithelial-mesenchymal transition (EMT) markers changes with decreased E-cadherin expression and increased vimentin expression. Our studies also provided in vitro and in vivo evidence that these early adaptive TKI resistant tumor cells can be targetable for prevention or eradication of tumor resistance emergence, such as utilizing BCL-2/BCL-xL pathway inhibitors, such as ABT-737, in combination with EGFR TKI in mutant-EGFR addicted NSCLC. Further preclinical model studies will focus on investigating the molecular and genomic changes and gene network regulation in the early adaptive resistant tumor cells, to enable a new paradigm of novel therapeutic targets discovery to overcome early tumor resistance emergence and improve long term outcome of targeted therapy in lung cancer. Citation Format: Lihong Yin, Ivy Shi, Wei Zhang, Rakesh Bagai, Yan Feng, Patrick C. Ma. Studies of early adaptive resistance in mutant-EGFR and EML4-ALK addicted non-small cell lung cancer in escape against small molecule inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4455. doi:10.1158/1538-7445.AM2013-4455

Abstract P3-04-02: Protein pathway activation mapping of I-SPY 1 biopsy specimens identifies new network focused drug targets for patients with HR+/HER2− tumors

Abstract Background: Profiling protein signaling activation in cancer is critical as these proteins represent targets for new molecular therapeutics. The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane- based chemotherapy that longitudinally collected biopsy specimens and molecular and clinical/pathological characterization. Methods: Tumor epithelium was procured by Laser Capture Microdissection from 149 pretreatment frozen biopsy specimens (T1) and 102 frozen biopsy specimens (T2, 1–4 days post-chemotherapy). Reverse Phase Protein Microarray technology was used to quantitatively measure the activation of 39 and 100 key signaling proteins in the T1 and T2 biopsies, respectively, including numerous drug targets for Phase I-III and FDA-cleared therapeutics. Associations between protein activation and response to chemotherapy (RCB 0/1 vs 2/3) and relapse-free survival (RFS) were evaluated for the HR+/HER2− patient population at each time point and for changes between T1 and T2. Significance thresholds for response and RFS were as follows: Wilcoxon rank sum test p &amp;lt; 0.05; Cox proportional hazard model, likelihood ratio p &amp;lt; 0.05. Results: We focused our analysis on protein changes in patients who had poor clinical outcomes (Table 1) We observed systemic activation of HER family signaling and downstream AKT activation in tumors from patients who do not respond (RCB2/3) and/or whom recurred. Increased ERBB2 and ERBB4 levels were observed at T1 and T2, respectively, from patients who did not respond to neoadjuvant therapy. Dynamic changes in ERBB3 and AKT phosphorylation/activation were revealed between the T1 and T2 time points, which were associated with recurrence. Increased phosphorylation of MARCKS, a known PKC substrate, was seen in the T1 biopsy in the non-response cohort. Conclusions: Our analysis revealed activation/increased expression of HER family proteins along with downstream AKT activation in HR+/HER2− patients who have poor clinical response. These events, if validated, point to potential treatment options that could be rationally considered for non-responding HR+/HER2− patients with a number of investigational agents that target ERBB3 and AKT signaling in the clinic today. Increasing HER2 protein expression in a HER2− cohort may appear contradictory, however these findings may point to important subtle increases in HER2 that have important clinical considerations. Given the known clinical benefit from HER2 directed therapy in HER2− patients seen in other studies, our findings may have clinical relevance if validated. Further validation is required to explore the significance of these ongoing findings with the hope that the analysis could lead to molecularly rationalized therapies for patients with HR+/HER2− tumors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-02.