Nadège Gaborit

Inhibition of triple-negative breast cancer models by combinations of antibodies to EGFR

Breast tumors lacking expression of human epidermal growth factor receptor 2 (HER2) and the estrogen and the progesterone receptors (triple negative; TNBC) are more aggressive than other disease subtypes, and no molecular targeted agents are currently available for their treatment. Because TNBC commonly displays EGF receptor (EGFR) expression, and combinations of monoclonal antibodies to EGFR effectively inhibit other tumor models, we addressed the relevance of this strategy to treatment of TNBC. Unlike a combination of the clinically approved monoclonal antibodies, cetuximab and panitumumab, which displaced each other and displayed no cooperative effects, several other combinations resulted in enhanced inhibition of TNBC’s cell growth both in vitro and in animals. The ability of certain antibody mixtures to remove EGFR from the cell surface and to promote its intracellular degradation correlated with the inhibitory potential. However, unlike EGF-induced sorting of EGFR to lysosomal degradation, the antibody-induced pathway displayed independence from the intrinsic kinase activity and dimer formation ability of EGFR, and it largely avoided the recycling route. In conclusion, although TNBC clinical trials testing EGFR inhibitors reported lack of benefit, our results offer an alternative strategy that combines noncompetitive antibodies to achieve robust degradation of EGFR and tumor inhibition.

Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) to Analyze the Disruption of EGFR/HER2 Dimers A NEW METHOD TO EVALUATE THE EFFICIENCY OF TARGETED THERAPY USING MONOCLONAL ANTIBODIES

In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation.

Examination of HER3 targeting in cancer using monoclonal antibodies

Significance The human EGF receptor (EGFR/HER) family plays critical roles in tumor progression. Therefore, several therapies intercepting these receptors were developed and clinically approved. Importantly, patients treated with such therapeutics often develop resistance, and in some cases this resistance has been associated with activation of HER3. Potentially, HER3 blockade might overcome patient resistance. Hence, antibodies to HER3 have been developed by us and other researchers. However, it has remained unclear which antibody attributes are required for effective tumor inhibition. To address this issue, we generated several monoclonal antibodies, which were tested in vitro and in tumor-bearing animals. Our results suggest that anti-HER3 antibodies able to intercept stroma–tumor interactions, as well as accelerate HER3 degradation, might inhibit tumor growth better than other antibodies. The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells’ ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma–tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients.

Combining three antibodies nullifies feedback-mediated resistance to erlotinib in lung cancer

Triple antibody targeting of multiple receptors subverts the resistance induced by single-agent therapies in lung cancer. Triple teaming cancer Patients with lung cancer that is driven by mutant epidermal growth factor receptor (EGFR) are resistant to EGFR-targeted kinase inhibitors, such as erlotinib. Mancini et al. found that resistance may be overcome by inhibiting erlotinib-induced feedback activation of other EGFR family members and the receptor tyrosine kinase MET. In culture and in mice, combining the three antibodies targeting three different EGFR family members inhibited the growth of tumor cells that survived treatment with erlotinib or a single antibody targeting only EGFR. Thus, attacking triple targets subverts the resistance-mediating network rewiring induced by single-agent therapies. Despite initial responses to targeted kinase inhibitors, lung cancer patients presenting with primary epidermal growth factor receptor (EGFR) mutations acquire resistance, often due to a second-site mutation (T790M). However, clinical trials found no survival benefits in patients treated with a monoclonal antibody (mAb) to EGFR that should block activation of the mutated receptor and thus bypass resistance to molecules that target the catalytic or ATP-binding site. Using cell lines with the T790M mutation, we discovered that prolonged exposure to mAbs against only the EGFR triggered network rewiring by (i) stimulating the extracellular signal–regulated kinase (ERK) pathway; (ii) inducing the transcription of HER2 (human epidermal growth factor receptor 2) and HER3, which encode other members of the EGFR family, and the gene encoding HGF, which is the ligand for the receptor tyrosine kinase MET; and (iii) stimulating the interaction between MET and HER3, which promoted MET activity. Supplementing the EGFR-specific mAb with those targeting HER2 and HER3 suppressed these compensatory feedback loops in cultured lung cancer cells. The triple mAb combination targeting all three receptors prevented the activation of ERK, accelerated the degradation of the receptors, inhibited the proliferation of tumor cells but not of normal cells, and markedly reduced the growth of tumors in mice xenografted with cells that were resistant to combined treatment with erlotinib and the single function-blocking EGFR mAb. These findings uncovered feedback loops that enable resistance to treatment paradigms that use a single antibody and indicate a new strategy for the treatment of lung cancer patients.

Emerging anti-cancer antibodies and combination therapies targeting HER3/ERBB3.

ABSTRACt Cancer progression depends on stepwise accumulation of oncogenic mutations and a select group of growth factors essential for tumor growth, metastasis and angiogenesis. Agents blocking the epidermal growth factor receptor (EGFR, also called HER1 and ERBB1) and the co-receptor called HER2/ERBB2 have been approved over the last decade as anti-cancer drugs. Because the catalytically defective member of the family, HER3/ERBB3, plays critical roles in emergence of resistance of carcinomas to various drugs, current efforts focus on antibodies and other anti-HER3/ERBB3 agents, which we review herein with an emphasis on drug combinations and some unique biochemical features of HER3/ERBB3.

CRISPR‐assisted receptor deletion reveals distinct roles for ERBB2 and ERBB3 in skin keratinocytes

While the epidermal growth factor receptor (EGFR) is an established regulator of skin development and homeostasis, the functions of the related tyrosine kinase receptors ERBB2 and ERBB3 in this tissue have only recently been examined. Previously reported, skin‐specific deletion of each of these receptors in mice resulted in similar defects in keratinocyte proliferation and migration, resulting in impaired wound healing and tumorigenesis. Because both ERBB2 and ERBB3 are targets for treating an array of cancer types, it is important to examine the consequences of receptor inhibition in human keratinocytes. Here, we employed the CRISPR/Cas9 technology to generate HaCaT cells (an established human keratinocyte cell line) lacking ERBB2 or ERBB3. HaCaT clones lacking ERBB2 or ERBB3 showed comparable reductions in cell proliferation as assessed by BrdU staining. Apoptosis, in contrast, was reduced in ERBB3‐deficient HaCaT cells only. Assessment of cell migration using a wound healing (scratch) assay showed that the closure of the wound gaps was completed by 48 h in mock and in ERBB3 knockout clones. In contrast, this process was considerably delayed in ERBB2 knockout clones, and a complete closure of the gap in the latter cells did not occur before 72 h. In conclusion, both ERBB2 and ERBB3 are essential for normal proliferation of skin keratinocytes, but in contrast to ERBB3, ERBB2 is essential for migration of human keratinocytes. These observations might bear significance to patient adverse effects of therapeutic agents targeting ERBB2 and ERBB3.

An oligoclonal antibody durably overcomes resistance of lung cancer to third‐generation EGFR inhibitors

Epidermal growth factor receptor (EGFR) mutations identify patients with lung cancer who derive benefit from kinase inhibitors. However, most patients eventually develop resistance, primarily due to the T790M second‐site mutation. Irreversible inhibitors (e.g., osimertinib/AZD9291) inhibit T790M‐EGFR, but several mechanisms, including a third‐site mutation, C797S, confer renewed resistance. We previously reported that a triple mixture of monoclonal antibodies, 3×mAbs, simultaneously targeting EGFR, HER2, and HER3, inhibits T790M‐expressing tumors. We now report that 3×mAbs, including a triplet containing cetuximab and trastuzumab, inhibits C797S‐expressing tumors. Unlike osimertinib, which induces apoptosis, 3×mAbs promotes degradation of the three receptors and induces cellular senescence. Consistent with distinct mechanisms, treatments combining 3×mAbs plus sub‐inhibitory doses of osimertinib synergistically and persistently eliminated tumors. Thus, oligoclonal antibodies, either alone or in combination with kinase inhibitors, might preempt repeated cycles of treatment and rapid emergence of resistance.

Abstract A52: Extracellular matrix regulation of ErbB3 in a subset of wild-type BRAF/NRAS melanoma

While the incidence of melanoma increases each year, treatment options for melanoma patients wild-type for both BRAF and NRAS (WT-WT) remain limited. The tumor microenvironment has been highly implicated in the promotion of tumor growth and proliferation; however, its precise role in melanoma is not well understood. Extracellular matrix (ECM) proteins within the tumor microenvironment have been shown to regulate many cellular pathways, including the activation of several growth factor receptors. The receptor ErbB3 is involved in the regulation of melanocyte homeostasis, and elevated ErbB3 expression in malignant melanomas correlates with reduced patient survival. In our study we demonstrate that, in a subgroup of WT-WT cells, an interaction between melanoma cells and ECM proteins within the tumor microenvironment promotes the activation of the NRG1/ErbB3 pathway, leading to phosphorylation of the downstream target AKT and increased cell growth. Furthermore, knockdown of NRG1 reduced cell viability, which was associated with decreased phosphorylation of ErbB3, its co-receptor ErbB2 and AKT. Analogous effects were obtained targeting ErbB3 with either small interfering RNAs or the neutralizing ErbB3 monoclonal antibodies, huHER-8 and NG33. Additionally, pertuzumab-mediated inhibition of ErbB2 heterodimerization decreased AKT phosphorylation, cell growth in vitro and xenograft growth in vivo. These findings are supportive of the crucial role of the ECM in regulating tumor growth through the activation of ErbB3 signaling, and establish a basis for targeting the NRG1-ErbB3-ErbB2 axis as a novel treatment strategy in a subset of malignant cutaneous melanoma. Citation Format: Sheera Rosenbaum, Claudia Capparelli, Lisa D. Berman-Booty, Nadege Gaborit, Tingting Zhan, Michael A. Davies, Yulius Y. Setiady, Yosef Yarden, Andrew E. Aplin. Extracellular matrix regulation of ErbB3 in a subset of wild-type BRAF/NRAS melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A52.

Abstract A09: ErbB3/ErbB2 complexes as a therapeutic target in a subset of wild-type BRAF/NRAS cutaneous melanomas

The treatment options remain poor for melanoma patients who are wild-type for both BRAF and NRAS (WT/WT). ErbB3 regulates melanocyte homeostasis and elevated ErbB3 expression in malignant melanomas correlates with reduced patient survival; however, the effect of selectively targeting ErbB3 in melanoma remains poorly characterized. We demonstrate that a subgroup of WT/WT melanomas display high basal phosphorylation of ErbB3 that is associated with autocrine production of the ErbB3 ligand, neuregulin-1 (NRG1). In phospho-ErbB3-high WT/WT cell lines, knockdown of NRG1 reduced cell viability that was associated with decreased phosphorylation of ErbB3, its co-receptor ErbB2 and its downstream target, AKT. Analogous effects were obtained targeting ErbB3 with either small interfering RNAs or the neutralizing ErbB3 monoclonal antibodies, huHER3-8 and NG33. Additionally, pertuzumab-mediated inhibition of ErbB2 heterodimerization decreased AKT phosphorylation, cell growth in vitro and xenograft growth in vivo. Our study demonstrates that targeting ErbB3-ErbB2 signaling in a cohort WT/WT melanomas leads to tumor growth reduction. These findings are supportive of targeting the NRG1-ErbB3-ErbB2 axis as a novel treatment strategy in a subset of malignant cutaneous melanoma. Citation Format: Claudia Capparelli, Sheera Rosenbaum, Lisa D. Berman-Booty, Nadege Gaborit, Tingting Zhan, Michael A. Davies, Yulius Y. Setiady, Yosef Yarden, Andrew E. Aplin. ErbB3/ErbB2 complexes as a therapeutic target in a subset of wild-type BRAF/NRAS cutaneous melanomas. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A09.

Abstract C44: Targeting neuregulin1-ErbB3 signaling in wild-type BRAF/NRAS melanoma.

Melanoma is the deadliest form of skin cancer due to its highly metastatic nature and refractoriness to standard chemotherapies. At present, few therapy options exist for wild-type BRAF/NRAS (WT/WT) melanoma patients. We examined the role of the neuregulin (NRG1)-ErbB3 signaling pathway in WT/WT melanoma cells since it is emerging as an important player in melanocyte homeostasis and melanoma pathogenesis. NRG1 has been shown to promote melanoblast proliferation and survival. ErbB3 is elevated in malignant melanoma specimens and cell lines and its expression correlates with reduced patient survival. The purpose of this study was to determine the role of the NRG1-ErbB3 signaling pathway in WT/WT melanoma cells. We demonstrated that in a subgroup of WT/WT cell lines, expressing high level of ErbB3 and NRG1, neuregulin is able to activate ErbB3 in basal conditions. Knockdown of NRG1 in high NRG1-expressing cells led to reduction of cell viability. This effect was associated with a decrease in the phosphorylation of ErbB3 and ErbB2 receptors as well as downstream AKT and ERK1/2 signaling. All these effects were rescued by exogenous addition of NRG1. We obtained analogous effects targeting ErbB3 with either small interfering RNAs or with clinical grade ErbB3 monoclonal antibodies. In these WT/WT cell lines with high expression of NRG1, inhibition of ErbB2-ErbB3 dimerization with pertuzumab caused a decrease in AKT phosphorylation and cell growth, indicating that ErbB3 signaling is dependent on ErbB2. Synergistic effects on cell growth as well as on AKT and ERK1/2 signaling were observed targeting ErbB2 and ErbB3 combining both antibodies. Our study demonstrates that targeting ErbB3 and ErbB2 signaling in a cohort WT/WT melanoma cell lines leads to tumor growth reduction. We propose that NRG1 activation of ErbB3 signaling promotes progression in these tumors and that these findings may form the basis for the use of agents targeting the NRG1-ErbB3 axis as novel treatment strategies in a subset of malignant melanoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C44. Citation Format: Claudia Capparelli, Nadege Gaborit, Yosef Yarden, Andrew Aplin. Targeting neuregulin1-ErbB3 signaling in wild-type BRAF/NRAS melanoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C44.