HER2-HER3 Dimers Research Articles

Deciphering HER2-HER3 Dimerization at the Single CTC Level: A Microfluidic Approach.

Simple SummaryAmong these different biomarkers, circulating tumor cells have proven to be of high interest for different types of cancer and in particular for breast cancer. Here we focus our attention on a breast cancer subtype referred as HER2-positive breast cancer, this cancer being associated with an amplification of HER2 protein at the plasma membrane of cancer cells. Combined with therapies targeting the HER2 protein, HER2-HER3 dimerization blockade further improves a patient’s outcome. In this work, we propose a new approach to CTC characterization by on-chip integrating proximity ligation assay, so that we can quantify the HER2-HER3 dimerization event at the level of single CTC.Microfluidics has provided clinicians with new technologies to detect and analyze circulating tumor biomarkers in order to further improve their understanding of disease mechanism, as well as to improve patient management. Among these different biomarkers, circulating tumor cells have proven to be of high interest for different types of cancer and in particular for breast cancer. Here we focus our attention on a breast cancer subtype referred as HER2-positive breast cancer, this cancer being associated with an amplification of HER2 protein at the plasma membrane of cancer cells. Combined with therapies targeting the HER2 protein, HER2-HER3 dimerization blockade further improves a patient’s outcome. In this work, we propose a new approach to CTC characterization by on-chip integrating proximity ligation assay, so that we can quantify the HER2-HER3 dimerization event at the level of single CTC. To achieve this, we developed a microfluidic approach combining both CTC capture, identification and HER2-HER3 status quantification by Proximity Ligation Assay (PLA). We first optimized and demonstrated the potential of the on-chip quantification of HER2-HER3 dimerization using cancer cell lines with various levels of HER2 overexpression and validated its clinical potential with a patient’s sample treated or not with HER2-targeted therapy.

Landscape of Epidermal Growth Factor Receptor Heterodimers in Brain Metastases.

Simple SummaryHER2+ breast cancer patients are treated with agents that tag HER2+ tumour cells for elimination by the immune system, down-modulate HER2 activity and/or block the formation of HER2 dimers, including the neuregulin-1 receptor, HER2-HER3. HER2-targeted therapies prolong survival by lowering the risk of relapse, but do not prevent brain metastases. The reasons for this are not fully understood. We quantified HER2-HER3 dimers in 203 brain metastases, and 34 primary breast tumour samples. Dimer frequency was relatively high in brain metastases from breast, ovarian, lung and kidney cancers, and in brain metastases compared to patient-matched breast tumours; but did not reliably correlate with HER2/HER3 expression or activation. In in vitro experiments, pertuzumab failed to suppress HER2-HER3 dimers in HER2+ breast cancer cells provided with a saturating concentration of neuregulin-1. These findings may provide insights about the differences in intracranial versus extracranial efficacy of HER2-targeted therapies.HER2+ breast cancer patients have an elevated risk of developing brain metastases (BM), despite adjuvant HER2-targeted therapy. The mechanisms underpinning this reduced intracranial efficacy are unclear. We optimised the in situ proximity ligation assay (PLA) for detection of the high-affinity neuregulin-1 receptor, HER2-HER3 (a key target of pertuzumab), in archival tissue samples and developed a pipeline for high throughput extraction of PLA data from fluorescent microscope image files. Applying this to a large BM sample cohort (n = 159) showed that BM from breast, ovarian, lung and kidney cancers have higher HER2-HER3 levels than other primary tumour types (melanoma, colorectal and prostate cancers). HER2 status, and tumour cell membrane expression of pHER2(Y1221/1222) and pHER3(Y1222) were positively, but not exclusively, associated with HER2-HER3 frequency. In an independent cohort (n = 78), BM had significantly higher HER2-HER3 levels than matching primary tumours (p = 0.0002). For patients who had two craniotomy procedures, HER2-HER3 dimer levels were lower in the consecutive lesion (n = 7; p = 0.006). We also investigated the effects of trastuzumab and pertuzumab on five different heterodimers in vitro: HER2-EGFR, HER2-HER4, HER2-HER3, HER3-HER4, HER3-EGFR. Treatment significantly altered the absolute frequencies of individual complexes in SKBr3 and/or MDA-MB-361 cells, but in the presence of neuregulin-1, the overall distribution was not markedly altered, with HER2-HER3 and HER2-HER4 remaining predominant. Together, these findings suggest that markers of HER2 and HER3 expression are not always indicative of dimerization, and that pertuzumab may be less effective at reducing HER2-HER3 dimerization in the context of excess neuregulin.

Structures of the HER2-HER3-NRG1β complex reveal a dynamic dimer interface.

Human epidermal growth factor receptor 2 (HER2) and HER3 form a potent pro-oncogenic heterocomplex1-3 upon binding of growth factor neuregulin-1β (NRG1β). The mechanism by which HER2 and HER3 interact remains unknown in the absence of any structures of the complex. Here we isolated the NRG1β-bound near full-length HER2-HER3 dimer and, using cryo-electron microscopy, reconstructed the extracellulardomain module, revealing unexpected dynamics at the HER2-HER3 dimerization interface. We show that the dimerization arm of NRG1β-bound HER3 is unresolved because the apo HER2 monomer does not undergo a ligand-induced conformational change needed to establish a HER3 dimerization arm-binding pocket. In a structure of the oncogenic extracellular domain mutant HER2(S310F), we observe a compensatory interaction with the HER3 dimerization arm that stabilizes the dimerization interface. Both HER2-HER3 and HER2(S310F)-HER3 retain the capacity to bind to the HER2-directed therapeutic antibody trastuzumab, but the mutant complex does not bind to pertuzumab. Our structure of the HER2(S310F)-HER3-NRG1β-trastuzumab Fab complex reveals that the receptor dimer undergoes a conformational change to accommodate trastuzumab. Thus, similar to oncogenic mutations, therapeutic agents exploit the intrinsic dynamics of the HER2-HER3 heterodimer. The unique features of a singly liganded HER2-HER3 heterodimer underscore the allosteric sensing of ligand occupancy by the dimerization interface and explain why extracellular domains of HER2 do not homo-associate via a canonical active dimer interface.

A feed-forward loop between SorLA and HER3 determines heregulin response and neratinib resistance

Current evidence indicates that resistance to the tyrosine kinase-type cell surface receptor (HER2)-targeted therapies is frequently associated with HER3 and active signaling via HER2-HER3 dimers, particularly in the context of breast cancer. Thus, understanding the response to HER2-HER3 signaling and the regulation of the dimer is essential to decipher therapy relapse mechanisms. Here, we investigate a bidirectional relationship between HER2-HER3 signaling and a type-1 transmembrane sorting receptor, sortilin-related receptor (SorLA; SORL1). We demonstrate that heregulin-mediated signaling supports SorLA transcription downstream of the mitogen-activated protein kinase pathway. In addition, we demonstrate that SorLA interacts directly with HER3, forming a trimeric complex with HER2 and HER3 to attenuate lysosomal degradation of the dimer in a Ras-related protein Rab4-dependent manner. In line with a role for SorLA in supporting the stability of the HER2 and HER3 receptors, loss of SorLA compromised heregulin-induced cell proliferation and sensitized metastatic anti-HER2 therapy-resistant breast cancer cells to neratinib in cancer spheroids in vitro and in vivo in a zebrafish brain xenograft model.

HER2-HER3 Heterodimer Quantification by FRET-FLIM and Patient Subclass Analysis of the COIN Colorectal Trial.

BackgroundThe phase III MRC COIN trial showed no statistically significant benefit from adding the EGFR-target cetuximab to oxaliplatin-based chemotherapy in first-line treatment of advanced colorectal cancer. This study exploits additional information on HER2-HER3 dimerization to achieve patient stratification and reveal previously hidden subgroups of patients who had differing disease progression and treatment response. MethodsHER2-HER3 dimerization was quantified by fluorescence lifetime imaging microscopy in primary tumor samples from 550 COIN trial patients receiving oxaliplatin and fluoropyrimidine chemotherapy with or without cetuximab. Bayesian latent class analysis and covariate reduction was performed to analyze the effects of HER2-HER3 dimer, RAS mutation, and cetuximab on progression-free survival and overall survival (OS). All statistical tests were two-sided. ResultsLatent class analysis on a cohort of 398 patients revealed two patient subclasses with differing prognoses (median OS = 1624 days [95% confidence interval [CI] = 1466 to 1816 days] vs 461 days [95% CI = 431 to 504 days]): Class 1 (15.6%) showed a benefit from cetuximab in OS (hazard ratio = 0.43, 95% CI = 0.25 to 0.76, P = .004). Class 2 showed an association of increased HER2-HER3 with better OS (hazard ratio = 0.64, 95% CI = 0.44 to 0.94, P = .02). A class prediction signature was formed and tested on an independent validation cohort (n = 152) validating the prognostic utility of the dimer assay. Similar subclasses were also discovered in full trial dataset (n = 1630) based on 10 baseline clinicopathological and genetic covariates.ConclusionsOur work suggests that the combined use of HER dimer imaging and conventional mutation analyses will be able to identify a small subclass of patients (>10%) who will have better prognosis following chemotherapy. A larger prospective cohort will be required to confirm its utility in predicting the outcome of anti-EGFR treatment.

Abstract PD3-05: Co-occurring gain-of-function mutations in HER2 and HER3 cooperate to enhance HER2/HER3 binding, HER-dependent signaling, and breast cancer growth

Abstract ERBB2, the gene encoding HER2, is mutated in 2-4% of breast cancers. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against breast cancers harboring HER2 activating mutations, suggesting these tumors depend on HER2 signaling. Co-occurring HER2 and HER3 (ERBB3) mutations have been reported in patients who respond to neratinib (Hanker et al., Cancer Discov. 2017) suggesting the possibility of cooperativity of both oncogenes. Co-expression of the mutant intracellular domains of HER2 and HER3 in HEK293 cells enhanced phosphorylation of HER3 and ERK compared to expression of either mutant alone, which was blocked by 100 nM neratinib. Interrogation of TCGA, METABRIC, Project GENIE, and Foundation Medicine datasets revealed that gain-of-function mutations in ERBB2 and ERBB3 co-occur with a statistically significant frequency. For example, in GENIE, ERBB2 mutations co-occur with mutations in ERBB3 (8.3% of ERBB2-mutant vs 2.3% of ERBB2 WT; q=1.37x10-10). We hypothesized that co-occurring mutations in HER2 and HER3 cooperate to enhance HER2 signaling and dependence and breast cancer progression. Thirty-four unique breast cancers were found to harbor co-occurring mutations in HER2 and HER3, the most common of which were ERBB2L755S/ERBB3E928G (n=10), ERBB2V777L/ERBB3E928G(n=6), and ERBB2L869R/Q/ERBB3E928G (n=4). Using co-immunoprecipitation assays with HER2 and HER3 antibodies in transfected HEK293 cells, we found that co-expression of HER3E928G with wild type (WT) HER2, or co-expression of HER2L755S or HER2L869R with HER3WT, slightly increased HER2-HER3 dimerization. However, binding was strongest between double mutants. This was accompanied by the highest levels of Y1289 p-HER3 in cells expressing both HER3E928G and each HER2L755S, HER2V777L, or HER2L869R compared to cells expressing each HER2 or HER3 mutant with a respective WT heterodimer partner. Structural modeling of the HER2L869R/HER3E928G double-mutant predicted that the HER3 mutation, located at the dimer interface, may enhance heterodimerization of the kinase domains through decreased bulk and electrostatic repulsion. We also noted that the HER2L755S mutation is predicted to be in close proximity to HER3E928G (<4 Å) and may impact binding affinity. Investigation of the structural basis for the enhanced binding of other double mutants is in progress. MCF7 “knock-in” cells incorporating HER2L755S, HER2V777L, or HER2L869R (or HER2WT) were stably transduced with HER3E928G or HER3WT. Co-expression of double mutants strongly enhanced estrogen-independent growth in 3D Matrigel over cells expressing either mutant alone. We are currently testing inhibitors of HER2/HER3 signaling, including neratinib ± trastuzumab, trastuzumab + pertuzumab, and the ERBB1-3 antibody mixture Sym013, to determine therapeutic strategies to block the cooperative growth induced by co-occurring HER2 and HER2 mutations. Conclusions: Co-expression of mutant HER2 and mutant HER3 promotes HER2/HER binding, HER3 phosphorylation, and breast tumor cell proliferation. We aim to identify therapeutic vulnerabilities for patients with co-occurring HER2 and HER3 mutations. Citation Format: Hanker AB, Koch JP, Ye D, Sliwoski G, Sheehan J, Kinch LN, Red Brewer M, He J, Miller VA, Lalani AS, Cutler, Jr. RE, Croessmann S, Zabransky DJ, Meiler J, Arteaga CL. Co-occurring gain-of-function mutations in HER2 and HER3 cooperate to enhance HER2/HER3 binding, HER-dependent signaling, and breast cancer growth [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD3-05.

Abstract LB-217: Microenvironment mediated mechanisms of resistance to HER2 inhibitors differ between HER2+ breast cancer subtypes

Abstract Clinical responses of HER2+ breast cancers to tyrosine kinase inhibitors (TKIs) are often limited by de novo and acquired resistance. Recent studies have shown that the tumor microenvironment has a substantial capacity to generate signals and create niches that enable otherwise sensitive cells to avoid therapeutic control. We describe here the use of MicroEnvironment MicroArrays (MEMAs) to identify specific signals from the tumor microenvironment that reduce sensitivity to small molecule HER2 targeted TKIs. We show here that responses to these signals differ between HER2+ cells that express transcriptional signatures associated with basal (HER2E) or luminal (L-HER2+) HER2+ subtypes. Specifically, we show that HER2E cell lines are rescued from the TKIs lapatinib and neratinib by hepatocyte growth factor (HGF), while L-HER2+ cell lines are rescued by neuregulin1-β1 (NRG1β). We further show that these divergent responses are due to differential use of the PI3K and MAPK pathways, and expression of receptors HER3 and MET. This is supported by transcriptomics of human tumors performed by The Cancer Genome Atlas. HGF and NRG1β are expressed in the stroma of primary mammary tumors, but can also be highly expressed in common sites of both HER2E and L-HER2+ metastasis. We show that NRG1β mediated resistance in L-HER2+ lines is reversed by inhibiting HER2-HER3 dimers with pertuzumab, while HGF mediated resistance in HER2E lines is reversed with the MET inhibitor crizotinib. These studies suggest that L-HER2+ and HER2E tumors should be treated as separate diseases and that treatments should be tailored to counter microenvironment mediated resistance mechanisms that may differ between metastatic locations based on local production and action of growth factors. Citation Format: Spencer Watson, Joe Gray, James Korkola, Mark Dane, Laura Hesier, Koei Chin, Gordon Mills. Microenvironment mediated mechanisms of resistance to HER2 inhibitors differ between HER2+ breast cancer subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-217. doi:10.1158/1538-7445.AM2017-LB-217

Charge variant analysis of proposed biosimilar to Trastuzumab

Trastuzumab is a humanized monoclonal antibody (mAb) employed for the treatment of HER2 Positive Breast Cancer. A HER2 overexpressing tumor cell binds to Trastuzumab and attracts immune cells which lead to induction of Antibody Dependent Cellular Cytotoxicity (ADCC) by binding to Fc receptors (CD16a or FcγRIIIa) on an effector cell, such as natural killer (NK) cells.The most commonly expressed receptor on NK cell is CD16a which binds to the Fc portion of Trastuzumab. The ligand-independent HER2–HER3 dimerization is the most potent stimulator of downstream pathways for regulation of cell growth and survival. An attempt has been made in this study to understand the impact of charge heterogeneity on the binding kinetics and potency of the monoclonal antibody. Trastuzumab has a pI range of 8.7–8.9 and is composed of mixture of acidic and basic variants beside the main peak. Ion exchange chromatography was used to isolate the acidic, basic, and main peak fractions from in-house proposed biosimilar to Trastuzumab and their activities were compared to the Innovator Trastuzumab Herclon®. Data from the mass analysis confirmed the potential modifications in both acidic and basic variant. Binding activity studies performed using Surface Plasmon Resonance (SPR) revealed that acidic variants had lesser binding to HER2 in comparison to the basic variants. Both acidic and basic variant showed no significant changes in their binding to soluble CD16a receptors. In vitro assay studies using a breast cancer cell line (BT-474) confirmed the binding potency of acidic variant to be lesser than basic variant, along with reduced anti-proliferative activity for the acidic variant of Trastuzumab. Overall, these data has provided meaningful insights to the impact of antibody charge variants on in vitro potency and CD16 binding affinity of trastuzumab.

Abstract 2961: deBouganin conjugated to trastuzumab overcomes multiple mechanisms of T-DM1 drug resistance

Abstract DeBouganin is a T-cell epitope-depleted variant of the type I Ribosome Inactivating Protein (RIP) plant toxin Bouganin. DeBouganin binds and deadenylates the sarcin/ricin loop of the 28S subunit of the ribosomal RNA leading to protein synthesis inhibition and apoptosis. To demonstrate the potential advantages of deBouganin over current small molecule payloads, deBouganin was randomly chemically conjugated to trastuzumab with a DAR of approximately 2 and compared to T-DM1 both in vitro and in vivo. The trastuzumab-deBouganin conjugate (T-deB) demonstrated a tighter IC50 range of killing and an overall greater potency in vitro against most cells lines with high levels of Her2 expression as compared to T-DM1. In addition, unlike T-DM1, T-deB was unaffected by inhibitors of multidrug resistance (MDR) and overexpression of Bcl-2 family members. Moreover, T-deB potency was unchanged by Her2-Her3 dimerization in the presence of heregulin. Contrary to T-DM1 which showed only minimal cytotoxicity, T-deB was highly potent in vitro against tumor cells with cancer stem cell (CSC) properties by preventing the formation of tumorspheres. Furthermore, in a BT-474 xenograft study, T-deB was more efficacious than T-DM1 resulting in increased survival of the T-deB treated mice. Overall, the results demonstrate the potency and efficacy of deBouganin and emphasize the importance of using payloads with different MOAs. The data suggest that deBouganin used alone or in combination with other payloads could be a highly effective cancer therapeutic that would provide prolonged clinical benefit. Citation Format: Rachelle L. Dillon, Shilpa Chooniedass, Arjune Premsukh, Gregory P. Adams, Joycelyn Entwistle, Glen C. MacDonald, Jeannick Cizeau. deBouganin conjugated to trastuzumab overcomes multiple mechanisms of T-DM1 drug resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2961.

Abstract 2963: VB7-756: a Her2-specific diabody armed with deBouganin, a plant toxin with a distinct MOA

Abstract VB7-756 is Targeted Protein Therapeutic (TPT) comprised of a de-immunized form of bouganin (deBouganin), a potent, plant-derived, type I ribosome-inactivating protein (RIP), genetically linked to the C6.5 anti-Her2 diabody via a furin protease sensitive linker. To engineer the optimal diabody TPT format, several constructs were generated to assess the best diabody-deBouganin orientation. All constructs were expressed as a soluble protein in E. coli supernatant and compared with respect to expression level, stability and potency. The optimal configuration consisted of deBouganin genetically linked to the N-terminus of the VH-VL diabody via a furin protease-sensitive linker and was termed VB7-756. VB7-756 potency was analyzed against a panel of breast cancer cell lines with disparate levels of Her-2 expression and compared to that of Trastuzumab chemically linked to either DM1 (T-DM1) or MMAE (T-MMAE). Overall, VB7-756 was more potent than T-DM1 and T-MMAE with high Her-2 expressing tumor cell lines. In contrast to T-DM1, VB7-756 potency was unaffected by the Her2-Her3 dimerization mediated by heregulin. As opposed to T-DM1 and T-MMAE, which showed only minimal cytoxicity, VB7-756 was highly potent in vitro against tumor cells with cancer stem cell properties. To further differentiate the RIP mechanism of action of deBouganin from tubulin inhibitor reagents, tumor cells that escaped T-DM1 and T-MMAE treatment were incubated in the presence of VB7-756. Results revealed that, VB7-756 was cytotoxic against cancer cells surviving T-DM1 or T-MMAE treatment suggesting that deBouganin can overcome mechanisms of resistance developed by small molecule agents. Moreover, VB7-756 was also cytotoxic against tumor initiating cancer cells evading T-DM1 or T-MMAE toxicity by preventing tumorosphere formation. Overall these results demonstrate that deBouganin's distinct MOA could overcome mechanisms of resistance affecting the efficacy of small molecule drugs. Citation Format: Shilpa Chooniedass, Rachelle L. Dillon, Arjune Premsukh, Joycelyn Entwistle, Gregory P. Adams, Peter J. Hudson, Glen C. MacDonald, Jeannick Cizeau. VB7-756: a Her2-specific diabody armed with deBouganin, a plant toxin with a distinct MOA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2963.

Trastuzumab-deBouganin Conjugate Overcomes Multiple Mechanisms of T-DM1 Drug Resistance.

The development of antibody drug conjugates has provided enhanced potency to tumor-targeting antibodies by the addition of highly potent payloads. In the case of trastuzumab-DM1 (T-DM1), approved for the treatment of metastatic breast cancer, the addition of mertansine (DM1) to trastuzumab substantially increased progression-free survival. Despite these improvements, most patients eventually relapse due to complex mechanisms of resistance often associated with small molecule chemotherapeutics. Therefore, identifying payloads with different mechanisms of action (MOA) is critical for increasing the efficacy of targeted therapeutics and ultimately improving patient outcomes. To evaluate payloads with different MOA, deBouganin, a deimmunized plant toxin that inhibits protein synthesis, was conjugated to trastuzumab and compared with T-DM1 both in vitro and in vivo. The trastuzumab-deBouganin conjugate (T-deB) demonstrated greater potency in vitro against most cells lines with high levels of Her2 expression. In addition, T-deB, unlike T-DM1, was unaffected by inhibitors of multidrug resistance, Bcl-2-mediated resistance, or Her2-Her3 dimerization. Contrary to T-DM1 that showed only minimal cytotoxicity, T-deB was highly potent in vitro against tumor cells with cancer stem cell properties. Overall, the results demonstrate the potency and efficacy of deBouganin and emphasize the importance of using payloads with different MOAs. The data suggest that deBouganin could be a highly effective against tumor cell phenotypes not being addressed by current antibody drug conjugate formats and thereby provide prolonged clinical benefit.

Engineering multivalent antibodies to target heregulin-induced HER3 signaling in breast cancer cells

The use of antibodies in therapy and diagnosis has undergone an unprecedented expansion during the past two decades. This is due in part to innovations in antibody engineering that now offer opportunities for the production of “second generation” antibodies with multiple specificities or altered valencies. The targeting of individual components of the human epidermal growth factor receptor (HER)3-PI3K signaling axis, including the preferred heterodimerization partner HER2, is known to have limited anti-tumor effects. The efficacy of antibodies or small molecule tyrosine kinase inhibitors (TKIs) in targeting this axis is further reduced by the presence of the HER3 ligand, heregulin. To address these shortcomings, we performed a comparative analysis of two distinct approaches toward reducing the proliferation and signaling in HER2 overexpressing tumor cells in the presence of heregulin. These strategies both involve the use of engineered antibodies in combination with the epidermal growth factor receptor (EGFR)/HER2 specific TKI, lapatinib. In the first approach, we generated a bispecific anti-HER2/HER3 antibody that, in the presence of lapatinib, is designed to sequester HER3 into inactive HER2-HER3 dimers that restrain HER3 interactions with other possible dimerization partners. The second approach involves the use of a tetravalent anti-HER3 antibody with the goal of inducing efficient HER3 internalization and degradation. In combination with lapatinib, we demonstrate that although the multivalent HER3 antibody is more effective than its bivalent counterpart in reducing heregulin-mediated signaling and growth, the bispecific HER2/HER3 antibody has increased inhibitory activity. Collectively, these observations provide support for the therapeutic use of bispecifics in combination with TKIs to recruit HER3 into complexes that are functionally inert.

Combination of Antibody That Inhibits Ligand-Independent HER3 Dimerization and a p110α Inhibitor Potently Blocks PI3K Signaling and Growth of HER2+ Breast Cancers

We examined the effects of LJM716, an HER3 (ERBB3) neutralizing antibody that inhibits ligand-induced and ligand-independent HER3 dimerization, as a single agent and in combination with BYL719, an ATP competitive p110α-specific inhibitor, against HER2-overexpressing breast and gastric cancers. Treatment with LJM716 reduced HER2-HER3 and HER3-p85 dimers, P-HER3 and P-AKT, both in vitro and in vivo. Treatment with LJM716 alone markedly reduced growth of BT474 xenografts. The combination of LJM716/lapatinib/trastuzumab significantly improved survival of mice with BT474 xenografts compared with lapatinib/trastuzumab (P = 0.0012). LJM716 and BYL719 synergistically inhibited growth in a panel of HER2+ and PIK3CA mutant cell lines. The combination also inhibited P-AKT in HER2-overexpressing breast cancer cells and growth of HER2+ NCI-N87 gastric cancer xenografts more potently than LJM716 or BYL719 alone. Trastuzumab-resistant HER2+/PIK3CA mutant MDA453 xenografts regressed completely after 3 weeks of therapy with LJM716 and BYL719, whereas either single agent inhibited growth only partially. Finally, mice with BT474 xenografts treated with trastuzumab/LJM716, trastuzumab/BYL719, LJM716/BYL719, or trastuzumab/LJM716/BYL719 exhibited similar rates of tumor regression after 3 weeks of treatment. Thirty weeks after treatment discontinuation, 14% of mice were treated with trastuzumab/LJM716/BYL719, whereas >80% in all other treatment groups were sacrificed due to a recurrent large tumor burden (P = 0.0066). These data suggest that dual blockade of the HER2 signaling network with an HER3 antibody that inhibits HER2-HER3 dimers in combination with a p110α-specific inhibitor in the absence of a direct HER2 antagonist is an effective treatment approach against HER2-overexpressing cancers.

Model-Based Analysis of HER Activation in Cells Co-Expressing EGFR, HER2 and HER3

The HER/ErbB family of receptor tyrosine kinases drives critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation, and trafficking to quantitatively link receptor expression levels to dimerization and activation. We parameterized the model with a comprehensive set of HER phosphorylation and abundance data collected in a panel of human mammary epithelial cells expressing varying levels of EGFR/HER1, HER2 and HER3. Although parameter estimation yielded multiple solutions, predictions for dimer phosphorylation were in agreement with each other. We validated the model using experiments where pertuzumab was used to block HER2 dimerization. We used the model to predict HER dimerization and activation patterns in a panel of human mammary epithelial cells lines with known HER expression levels in response to stimulations with ligands EGF and HRG. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1-HER1 and HER1-HER2 dimers, and not HER1-HER3 dimers, ii) HER1-HER2 and HER2-HER3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2-HER3 dimer is largely responsible for HER3 activation. The model can be used to predict phosphorylated dimer levels for any given HER expression profile. This information in turn can be used to quantify the potencies of the various HER dimers, and can potentially inform personalized therapeutic approaches.

Abstract 5461: A HER3 antibody that blocks ligand-independent HER2-HER3 dimerization sensitizes to HER2 and PI3K inhibitors .

Abstract We hypothesized that complete inhibition of HER3 is required for the optimal action of HER2 or PI3K/Akt inhibitors against HER2+ tumors. The HER3 monoclonal antibody LJM716 (LJM) inhibits ligand-induced and ligand-independent dimerization of HER3. Treatment with LJM inhibited P-HER3 and P-Akt in a panel of HER2+ breast cancer cell lines. In BT474 and SKBR3 cells, LJM inhibited HER2-HER3 dimers as assessed by coprecipitation of HER2 with HER3 antibodies. Using a DUAL antibody proximity base assay, formalin-fixed sections of HER2+ xenografts treated with LJM showed a reduction of HER2-HER3 dimers and P-HER3 compared to untreated controls. Treatment with LJM alone also induced regression of established BT474 xenografts. Mice with BT474 xenografts were treated with lapatinib and trastuzumab (Lap/T), LJM and trastuzumab (LJM/T) or lapatinib, trastuzumab and LJM716 (Lap/T/LJM). After 3 weeks of treatment, mice from the 3 groups exhibited nearly complete tumor regression (volume <25 mm3). At 28 weeks of follow-up with no treatment, 8/12 mice in the Lap/T group and 5/7 mice in the LJM/T exhibited tumor regrowth (≥200 mm3), whereas only 2 of 15 mice in the Lap/T/LJM group did so (p=0.0046, Log-rank test). We expanded our studies to examine the combination of LJM716 with the p110α (PI3Kα) specific inhibitor BYL719 (BYL). LJM in combination with BYL inhibited growth in monolayer and 3-D Matrigel and S473 P-Akt in HER2+ breast cancer cells with mutations in the PI3K pathway better than either single agent. MDA-MB-453 xenografts regressed completely after 3 weeks of therapy with BYL/LJM whereas either single agent inhibited xenograft growth only partially. All mice bearing BT474 xenografts treated with T/LJM, T/BYL, LJM/BYL or T/LJM/BYL exhibited tumor regression after 24 days of treatment. Twenty weeks after treatment discontinuation, only 1/8 mice in the T/LJM/BYL exhibited tumor re-growth whereas >80% of mice in all other groups did so (p=0.0009, Log-rank test). Finally, we hypothesized that LJM would be at least equivalent to the HER2 antibody pertuzumab (P) in combination with T against HER2+ tumors. Inhibition of growth in vitro and P-Akt were superior with LJM/T compared to P/T in vitro. Mice bearing BT474 xenografts treated with either LJM/T or P/T had a similar nearly complete tumor regression after 4 weeks of treatment. We are currently monitoring potential tumor regrowth in these mice. In summary, treatment with LJM inhibited HER2-HER3 dimers, P-HER3 and P-Akt in HER2+ breast cancer cells with PI3K pathway mutations. As a single agent, LJM markedly inhibited HER2+ xenograft growth. Treatment with LJM in combination with Lap/T improved survival of mice with HER2+ xenografts compared to Lap/T. Further, LJM sensitized breast cancer cells and xenografts to a p110α-specific inhibitor. Finally, the HER3 antibody in combination with T inhibited PI3K/Akt and xenograft growth as well as the combination of P/T. Citation Format: Joan T. Garrett, Cammie R. Sutton, Carl Uli Bialucha, Seth A. Ettenberg, Jerry Wallweber, Lisa DeFazio-Eli, Carlos L. Arteaga. A HER3 antibody that blocks ligand-independent HER2-HER3 dimerization sensitizes to HER2 and PI3K 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 5461. doi:10.1158/1538-7445.AM2013-5461

Spontaneous and pronase‐induced HER2 truncation increases the trastuzumab binding capacity of breast cancer tissues and cell lines

A subgroup of HER2-overexpressing breast tumours co-expresses p95(HER2), a truncated HER2 receptor that retains a functional HER2 kinase domain but lacks the extracellular domain, thus impairing trastuzumab binding. We evaluated p95(HER2) expression in 99 frozen breast carcinoma samples by western blot analysis. The HER2-positive cell line BT474 treated with pervanadate or pronase was used as a positive control for p95(HER2) expression. Immunohistochemistry was performed on parallel formalin-fixed, paraffin-embedded sections of the same case series using antibodies directed against either the intra- or extra-cellular binding domain of HER2. In particular, biotinylated trastuzumab (BiotHER) was used to evaluate the binding capacity of the humanized antibody. To avoid a subjective evaluation of the score values and the percentage of immunostained cells, the slides were scanned and automatically analysed. The number of cases with HER2 overexpression (score 3+) and HER2 gene amplification was higher in the p185(HER2)-positive/p95(HER2)-positive samples than in the p185(HER2)-positive/p95(HER2)-negative group. Automated analysis confirmed a significantly higher percentage of 3+ scored cells in p95(HER2)-positive cases. Conversely, the percentage of 2+ scored cells was higher inp95(HER2)-negative cases. The status of the HER2 extracellular domain was then studied using flow cytometry on BT474 cells after pronase enzymatic digestion using trastuzumab and pertuzumab, while the presence of HER2-HER3 dimers was studied using a proximity-ligation assay. In vitro experiments showed that short-term pronase digestion of BT474 cells produced two HER2 fragments (of 95 and 150 kDa, detectable in tissue specimens as well), increased the binding affinity of trastuzumab, reduced the rate of HER2-HER3 dimers, and did not interfere with pertuzumab-binding capacity. In conclusion, the presence of p95(HER2 as detected by western blot analysis does not compromise the immunohistochemical detection of HER2. Our data suggest that a reduction of the receptor steric hindrance as induced by enzymatic shedding may facilitate the binding capacity of trastuzumab.

Differential action of small molecule HER kinase inhibitors on receptor heterodimerization: Therapeutic implications

Deregulation of ErbB/HER receptor tyrosine kinases has been linked to several types of cancer. The mechanism of activation of these receptors includes establishment of receptor dimers. Here, we have analyzed the action of different small molecule HER tyrosine kinase inhibitors (TKIs) on HER receptor dimerization. Breast cancer cell lines were treated with distinct TKIs and the formation of HER2-HER3 dimers was analyzed by coimmunoprecipitation and western blot or by Förster resonance energy transfer assays. Antibody-dependent cellular cytotoxicity was analyzed by measuring the release of lactate dehydrogenase and cell viability. Lapatinib and neratinib interfered with ligand-induced dimerization of HER receptors; while pelitinib, gefitinib, canertinib or erlotinib did not. Moreover, lapatinib and neratinib were able to disrupt previously formed receptor dimers. Structural analyses allowed the elucidation of the mechanism by which some TKIs prevent the formation of HER receptor dimers, while others do not. Experiments aimed at defining the functional importance of dimerization indicated that TKIs that impeded dimerization prevented down-regulation of HER2 receptors, and favored the action of trastuzumab. We postulate that TKIs that prevent dimerization and down-regulation of HER2 may augment the antitumoral action of trastuzumab, and this mechanism of action should be considered in the treatment of HER2 positive tumors which combine TKIs with antireceptor antibodies.

Abstract 710: Gene expression profile of the Wnt signaling pathway in trastuzumab resistant breast cancer cells

Abstract Introduction: We are interested in understanding mechanisms that lead to Trastuzumab (Herceptin) resistance in HER2 overexpressing breast tumors. In this study, we examined the role of the Wnt/β-catenin signaling pathway in Trastuzumab resistance. In addition to the Wnt pathway genes, we examined gene expression profiles using 44K gene chip array and EMT associated genes in Trastuzumab resistant cells. Methods: Wnt pathway gene profiles were examined by Agilent chip (4×44K) and Wnt/β-catenin specific array. HER2 overexpressing breast cancer cells, SKBR3 and trastuzumab resistant clone, SKBR3 100-8 cells were used as cell models. The EMT (Epithelial to Mesenchymal Transition) markers Snail, Slug, Twist and E-Cadherin were examined by both real-time PCR and western blot analysis. Cell invasiveness was determined by invasive assay. HER2-HER3 dimerization was examined using immunoprecipitation and immunoblotting procedures. Results: We established a Trastuzumab resistant cell line from the HER2 overexpressing parental SKBR3 cell line. The resistant clone called SKBR3/100-8 is insensitive to 100µg/ml of Trastuzumab. It has a mesenchymal or more basal-like morphology. It overexpresses HER2 protein and mRNA more than the parental line SKBR3. This resistant clone shows increased dimerization of HER2 with HER3. The SKBR3 100-8 cells were found to overexpress EGFR1. It is more invasive compared to wt SKBR3 and shows a significant increase in EMT marker, SLUG, but a decrease in Snail and E-cadherin compared to the wtSKBR3 cells. The resistant clone shows increase in phospho-Akt, pGSK3β and a decrease in β-Catenin. Agilent chip (4×44K) array (containing 84 Wnt pathway genes) identified the following Wnt pathway genes to be upregulated by 2 or more fold in the resistant clone: AXIN1, CSNK1A1, CSNK1D, CTBP2, DAAM1, c-JUN, TLE1, WNT3 and WNT6. Conversely, the following genes were downregulated by 2 or more fold in the resistant clone: AES, CCND3, CTNNB1, FZD1, FZD4, FZD5, PITX2, PPP2CA, PPP2R1A, WIF1, WNT11, and WNT4. The resistant clone also increased Nanog and VEGF significantly. TGFβ treatment in wtSKBR3 increased SLUG, Snail, Twist, and decreased E-Cadherin. The cells showed more mesenchymal morphology than wtSKBR without TGFβ treatment. TGFβ treatment in wtSKBR3 upregulated: c-Jun, C-Myc, CCND1, FosL1, LEF1, and TCF-1. TGFβ also induced Met, MMP1, MMP7, MMP9, MMP26, Nanog, NOS2, POU5F1, PPARd, Survivin, uPAR and VEGF. In contrast, TGFβ upregulated only c-Jun, Met, Nanog, and VEGF in SKBR3 100-8 cells. Conclusion: Wnt/β-catenin signaling is highly involved in the trastuzumab resistant breast cancer cells. The resistant signature involves c-Jun, Met, Nanog and VEGF. These results suggest a very specific signature that needs further studies for target therapy for the Trastuzumab resistant tumors and implies the use of anti VEGF therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 710. doi:10.1158/1538-7445.AM2011-710

Abstract 4564: huHER3-8, a novel humanized anti-HER3 antibody that inhibits exogeneous ligand-independent proliferation of tumor cells

Abstract HER3 (ErbB3) is a member of the human epidermal growth factor receptor (HER) tyrosine kinase family that includes EGFR and HER2, two well-known targets for oncology therapy. Kinase signaling of the HER family is initiated by ligand binding that induces receptor homodimerization or heterodimerization and transphosphorylation of the intracellular domain. In this regards, HER3 is unique among the HER kinase family members. Despite being able to bind ligand, HER3 does not possess a catalytically active kinase domain. To turn on the signaling pathway, HER3 needs to form a heterodimer with another HER family member. However, this does not make HER3 inferior with respect to HER pathway signaling, since dimerization of HER3 with HER2 elicits one of the most powerful oncogenic signals. Similar to EGFR and HER2, HER3 expression and activity have been linked to the pathogenesis of various cancers including breast, ovarian and melanoma, and has been associated with resistance to several cancer therapies. The significance of HER3 in cancer pathogenesis prompted us to develop HER3 antagonistic antibodies. A large number of HER3 antibodies were generated by immunizing mice with HER3 expressing cell lines, and screened for the ability to bind human and monkey HER3. Additionally, since HER3 and activated HER3 (phospho-HER3) were detected in the majority of primary as well as metastatic breast cancer tissues by immunohistochemistry staining, antibodies were screened for their ability to inhibit the HER3 ligand-induced proliferation as well as the basal proliferation of breast cancer cell lines. Three HER3 antibodies, HER3-3, HER3-8 and HER3-10, were exceptionally potent in inhibiting the basal proliferation of four breast cancer cell lines that constitutively express phospho-HER3, where other HER3 antibodies (i.e. U1-59 and MM-121) had little or no activity. In SKBR3 cells, these HER3 antibodies inhibited cell proliferation, comparable to the activity of trastuzumab although the HER3 expression level was thirty-fold lower than the HER2 level. All three HER3 antibodies recognized overlapping epitopes. However, only HER3-8 and HER3-10 antibodies strongly competed for the binding of HER3 ligand and inhibited HER2-HER3 dimerization. These HER3 antibodies were also very potent in inhibiting ligand-induced HER3 signaling and tumor cell proliferation. Because of its best overall activities, HER3-8 was humanized by variable domain resurfacing technology. Humanized HER3-8 retained all of the biological properties and activities of muHER3-8. In conclusion, we have generated a novel humanized HER3 antibody, huHER3-8 that is not only active in inhibiting exogenous ligand-induced HER3 signaling and tumor cell growth, but also effective in inhibiting the basal proliferation of HER3-positive tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4564. doi:10.1158/1538-7445.AM2011-4564