An oligoclonal antibody durably overcomes resistance of lung cancer to third‐generation EGFR inhibitors
2017; Springer Nature; Volume: 10; Issue: 2 Linguagem: Inglês
10.15252/emmm.201708076
ISSN1757-4684
AutoresMaicol Mancini, Hilah Gal, Nadège Gaborit, Luigi Mazzeo, Donatella Romaniello, Tomer Meir Salame, Moshit Lindzen, Georg Mahlknecht, Yehoshua Enuka, Dominick G. A. Burton, L. E. Roth, Ashish Noronha, Ilaria Marrocco, Dan Adreka, Raya Eilam Altstadter, Emilie Bousquet, Julian Downward, Antonio Maraver, Valery Krizhanovsky, Yosef Yarden,
Tópico(s)Lung Cancer Research Studies
ResumoResearch Article6 December 2017Open Access Source DataTransparent process An oligoclonal antibody durably overcomes resistance of lung cancer to third-generation EGFR inhibitors Maicol Mancini Maicol Mancini Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Hilah Gal Hilah Gal Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Nadège Gaborit Nadège Gaborit Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Luigi Mazzeo Luigi Mazzeo Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Donatella Romaniello Donatella Romaniello Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Tomer Meir Salame Tomer Meir Salame Department of Biological Services, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Moshit Lindzen Moshit Lindzen Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Georg Mahlknecht Georg Mahlknecht Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Yehoshua Enuka Yehoshua Enuka orcid.org/0000-0001-6327-2845 Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Dominick GA Burton Dominick GA Burton Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Lee Roth Lee Roth Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ashish Noronha Ashish Noronha Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ilaria Marrocco Ilaria Marrocco Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Dan Adreka Dan Adreka Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Raya Eilam Altstadter Raya Eilam Altstadter Department of Veterinary Resources, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Emilie Bousquet Emilie Bousquet Oncogenic Pathways in Lung Cancer, Institut de Recherche en Cancérologie de Montpellier (IRCM), Inserm U1194, Montpellier Cedex 5, France Search for more papers by this author Julian Downward Julian Downward Signal Transduction Laboratory, Francis Crick Institute, London, UK Lung Cancer Group, The Institute of Cancer Research, London, UK Search for more papers by this author Antonio Maraver Antonio Maraver Oncogenic Pathways in Lung Cancer, Institut de Recherche en Cancérologie de Montpellier (IRCM), Inserm U1194, Montpellier Cedex 5, France Search for more papers by this author Valery Krizhanovsky Valery Krizhanovsky orcid.org/0000-0002-3977-5482 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Yosef Yarden Corresponding Author Yosef Yarden [email protected] orcid.org/0000-0001-8578-0250 Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Maicol Mancini Maicol Mancini Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Hilah Gal Hilah Gal Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Nadège Gaborit Nadège Gaborit Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Luigi Mazzeo Luigi Mazzeo Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Donatella Romaniello Donatella Romaniello Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Tomer Meir Salame Tomer Meir Salame Department of Biological Services, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Moshit Lindzen Moshit Lindzen Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Georg Mahlknecht Georg Mahlknecht Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Yehoshua Enuka Yehoshua Enuka orcid.org/0000-0001-6327-2845 Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Dominick GA Burton Dominick GA Burton Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Lee Roth Lee Roth Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ashish Noronha Ashish Noronha Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ilaria Marrocco Ilaria Marrocco Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Dan Adreka Dan Adreka Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Raya Eilam Altstadter Raya Eilam Altstadter Department of Veterinary Resources, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Emilie Bousquet Emilie Bousquet Oncogenic Pathways in Lung Cancer, Institut de Recherche en Cancérologie de Montpellier (IRCM), Inserm U1194, Montpellier Cedex 5, France Search for more papers by this author Julian Downward Julian Downward Signal Transduction Laboratory, Francis Crick Institute, London, UK Lung Cancer Group, The Institute of Cancer Research, London, UK Search for more papers by this author Antonio Maraver Antonio Maraver Oncogenic Pathways in Lung Cancer, Institut de Recherche en Cancérologie de Montpellier (IRCM), Inserm U1194, Montpellier Cedex 5, France Search for more papers by this author Valery Krizhanovsky Valery Krizhanovsky orcid.org/0000-0002-3977-5482 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Yosef Yarden Corresponding Author Yosef Yarden [email protected] orcid.org/0000-0001-8578-0250 Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Author Information Maicol Mancini1, Hilah Gal2, Nadège Gaborit1, Luigi Mazzeo1, Donatella Romaniello1, Tomer Meir Salame3, Moshit Lindzen1, Georg Mahlknecht1, Yehoshua Enuka1, Dominick GA Burton2, Lee Roth1, Ashish Noronha1, Ilaria Marrocco1, Dan Adreka1, Raya Eilam Altstadter4, Emilie Bousquet5, Julian Downward6,7, Antonio Maraver5, Valery Krizhanovsky2 and Yosef Yarden *,1 1Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel 2Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel 3Department of Biological Services, Weizmann Institute of Science, Rehovot, Israel 4Department of Veterinary Resources, Weizmann Institute of Science, Rehovot, Israel 5Oncogenic Pathways in Lung Cancer, Institut de Recherche en Cancérologie de Montpellier (IRCM), Inserm U1194, Montpellier Cedex 5, France 6Signal Transduction Laboratory, Francis Crick Institute, London, UK 7Lung Cancer Group, The Institute of Cancer Research, London, UK *Corresponding author. Tel: +972 8 934 3974; Fax: +972 8 934 2488; E-mail: [email protected] EMBO Mol Med (2018)10:294-308https://doi.org/10.15252/emmm.201708076 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Synopsis EGFR kinase blockers effectively inhibit a fraction of lung tumors, but second and third site EGFR mutations drive relapses. Acquired resistance to the blockers can be delayed or prevented in animal models by a mixture of three antibodies targeting EGFR/HER1, HER2 and HER3. A mixture containing the clinically approved cetuximab and trastuzumab, in combination with an anti-HER3 antibody, overcomes resistance to first-generation EGFR inhibitors. Unlike third-generation inhibitors (e.g., osimertinib), which cause cell death, the mixture of antibodies accelerates receptor endocytosis and arrests tumor cell growth. The mixture of three antibodies overcomes C797S, an osimertinib-resistant mutation, and synergizes with osimertinib. Introduction Somatic mutations in the gene encoding the epidermal growth factor receptor (EGFR) are detected in approximately 12% of non-small cell lung cancers (NSCLCs) from Caucasian patients and in 30–40% of NSCLCs from Asian patients (Lynch et al, 2004; Paez et al, 2004; Pao et al, 2004). All oncogenic mutations locate to the catalytic, tyrosine-specific kinase domain of EGFR. Two aberrations, the L858R mutation and the exon 19 deletion (del746-750), represent the vast majority of activating EGFR mutations. Treatment of patients with gefitinib or erlotinib, two reversible tyrosine kinase inhibitors (TKIs), is associated with response rates that are superior to treatment with chemotherapy (Mok et al, 2009). However, despite initial drug activity, all patients acquire resistance within approximately 1 year (Mok et al, 2009; Rosell et al, 2009). The most common mechanism (> 50%) of acquired resistance involves a secondary mutation, T790M (Kobayashi et al, 2005; Pao et al, 2005; Oxnard et al, 2011). Two strategies have been developed to overcome EGFR TKI resistance. The first involves synthesis of novel compounds (Zhou et al, 2009), including irreversible TKIs, which covalently conjugate to cysteine 797 of EGFR. For example, afatinib (Gilotrif™) covalently binds with EGFR and HER2 (Regales et al, 2009). However, inhibition of wild-type EGFR and relatively high drug doses required for inhibition of T790M-EGFR (Yap et al, 2010) limit afatinib application. Secondly, a combination of afatinib and cetuximab (an anti-EGFR monoclonal antibody) overcomes T790M-mediated resistance (Regales et al, 2009; Keating, 2014). A study that combined afatinib and cetuximab, and recruited patients who acquired TKI resistance, showed an overall response rate of 29%, but this result was comparable in T790M-positive and in T790M-negative tumors (Janjigian et al, 2014; Pirazzoli et al, 2014). The pharmacological limitations of second-generation inhibitors might be overcome by the newest, third-generation inhibitors, such as osimertinib, CO-1686 (rociletinib), and HM61713 (reviewed in Costa & Kobayashi, 2015; Mancini & Yarden, 2015). Osimertinib inhibits T790M-EGFR, while sparing wild-type EGFR (Liao et al, 2015). In a phase I trial, osimertinib demonstrated manageable tolerability and 51% response rate among T790M mutant tumors (Janne et al, 2015). Subsequent studies not only demonstrated that osimertinib has significantly greater efficacy than chemotherapy (Mok et al, 2017), but also confirmed its ability to provide a high overall response rate, as well as confer durable response (Yang et al, 2017). Nevertheless, patients treated with osimertinib acquire resistance, due to several mechanisms, including emergence of C797S mutations (Eberlein et al, 2015; Thress et al, 2015). Other mechanisms include aberrant expression of NRAS and KRAS (Eberlein et al, 2015), or activation of MET and HER2, a kin of EGFR (Planchard et al, 2015). Unlike the mutation-prone intracellular domain of EGFR, the extracellular domain is rarely mutated, which predicts low incidence of resistance to anti-EGFR monoclonal antibodies (mAbs). We previously examined this strategy and uncovered compensatory loops that increase transcription of HER2 and HER3 in response to an anti-EGFR mAb (Mancini et al, 2015). Preventing this by means of a triple combination of mAbs to EGFR, HER2 and HER3 (hereinafter, 3×mAbs) robustly inhibited tumor growth. The present study compares 3×mAbs and osimertinib. While in animal models, both treatments effectively inhibited growth of T790M-positive tumors, when tested in vitro and in animals osimertinib-induced apoptosis, whereas 3×mAbs caused cellular senescence and weak apoptosis. Importantly, prolonged exposure to osimertinib promoted emergence of resistant tumor cells, including C797S-EGFR expressing cells, which remained sensitive to 3×mAbs. Congruent with distinct mechanisms of action of 3×mAbs and osimertinib, treatments of tumor-bearing mice with 3×mAbs plus a sub-inhibitory dose of osimertinib durably prevented tumor relapses after ending all treatments. Taken together, these observations offer a new NSCLC treatment strategy, potentially able to overcome many, if not all resistance-conferring EGFR kinase mutations. Results Combining trastuzumab and cetuximab with an anti-HER3 antibody strongly inhibits erlotinib-resistant tumors EGFR's intracellular part presents mutations responsible for recurring TKI resistance (Camidge et al, 2014), but the ectodomain is rarely mutated, such that anti-EGFR antibodies might overcome mutation-driven resistance to TKIs. We previously examined this scenario using two NSCLC cell lines (Mancini et al, 2015): patient-derived H1975 cells expressing a double mutant of EGFR, L858R, and T790M, and the PC9ER cell line, a derivative of PC9 (del746-750 EGFR), which acquired the T790M mutation (de Bruin et al, 2014). Inhibition of both animal models required simultaneous treatment with three homemade mAbs, to EGFR, HER2, and HER3 (Mancini et al, 2015). Similarly, we now report that combining two clinically approved mAbs, cetuximab (anti-EGFR) and trastuzumab (anti-HER2), with a murine anti-HER3 (mAb33), partly inhibited in vitro growth of PC9ER and H1975 cells (Fig EV1A) and almost completely prevented tumorigenic growth of PC9ER cells in animals (Fig 1A). Moreover, this effect persisted at least 30 days post-treatment. In similarity to the murine anti-EGFR antibodies we previously tested (Mancini et al, 2015), cetuximab was more effective in vivo than singly applied anti-HER2 or anti-HER3 antibodies. In conclusion, the therapeutic activities of cetuximab and trastuzumab can be augmented by adding an anti-HER3 antibody, such that the oligoclonal mixture of two humanized antibodies and a murine mAb persistently inhibits TKI-resistant NSCLC models. Click here to expand this figure. Figure EV1. A combination of three antibodies inhibits erlotinib-resistant lung cancer cells in vitro and in animals and downregulates both EGFR and phospho-EGFR PC9ER (upper panel) and H1975 cells (lower panel) were grown in RPMI-1640 (2% serum) and exposed for 4 days to the indicated antibodies (20 μg/ml) against EGFR (cetuximab; CTX), HER2 (trastuzumab; TRZ), or HER3 (mAb33). Whenever antibody mixtures were applied, the total antibody concentration remained constant. Cell survival was assessed using the MTT colorimetric assay. Data are means ± SD. **P < 0.01, and ***P < 0.001; n = 3; one-way ANOVA with Tukey's test. PC9 cells were cultured for 4 days with increasing concentrations of either TKIs (erlotinib, osimertinib, or CO-1686) or the triple antibody combination (CTX, TRZ, and mAb33). Metabolic activity was determined using the MTT assay. Data are means ± SD values from three experiments. PC9ER cells were treated for 24 h with saline, vehicle (DMSO), the indicated TKIs (each at 10 nM), mAb565 (20 μg/ml) against EGFR, cetuximab (CTX; 20 μg/ml), and two different antibody mixtures (3×mAbs): murine (M; mAbs 565, N12 and mAb33; each at 20 μg/ml) and partly human (H; CTX, TRZ and mAb33; each at 20 μg/ml). Thereafter, cells were analyzed using flow cytometry for surface expression levels of EGFR, HER2, and HER3. Normalized data are means ± SEM of two independent experiments. H1975 cells (3 × 106 cells per animal) were subcutaneously grafted in the flanks of CD1-nu/nu mice, which were subsequently randomized and subjected to the following treatments: erlotinib (50 mg/kg/day), osimertinib (5 mg/kg/day), or 3×mAbs (CTX, TRZ, and mAb33; 0.2 mg/mouse/injection; administered twice a week). Shown is immunohistochemical staining for KI67 in paraffin-embedded sections using specific antibodies. Scale bars, 100 μm. CD1-nu/nu mice harboring H1975 NSCLC xenografts were treated as indicated for 10 days. Thereafter, tumors were harvested, embedded in paraffin, and stained with antibodies specific to EGFR, EGFR-L858R, and phospho-EGFR (Y1068). Scale bars, 100 μm. Comparison of body weights (averages ± SD) of groups of eight CD1-nu/nu mice harboring H1975 xenografts and treated with either erlotinib (50 mg/kg/day), osimertinib (5 mg/kg/day), or a mixture of three mAbs (3×mAbs; CTX, TRZ, and mAb33; 0.2 mg/mouse/injection). Note that TKIs were daily administered using oral gavage, while the triple antibody combination was injected intraperitoneally once every 3 days. PC9ER cells (3 × 106 cells per animal) were subcutaneously grafted in the flanks of CD1-nu/nu mice. Animals were randomized into groups of six mice after tumors became palpable. Erlotinib (50 mg/kg/day) and osimertinib (5 mg/kg/day) were daily administered using oral gavage, while the triple antibody combination (3×mAbs; CTX, TRZ, and mAb33; 0.2 mg/mouse/injection) was administered intraperitoneally once every 3 days. Shown are results of body mass composition analyses (mean ± SD) of the fraction of fat mass (left) and lean mass (right) on day 20 of treatment. Mice harboring no tumors represent an internal control. One-way ANOVA with Tukey's test. Download figure Download PowerPoint Figure 1. Both third-generation TKIs and a triple mAb mixture inhibit erlotinib-resistant tumors, but their mechanisms of action might differ A. PC9ER cells (4 × 106 cells per animal) were subcutaneously implanted in CD1-nu/nu mice. Thereafter, tumor-bearing mice were randomized into groups of 9–10 animals that were later treated with the indicated antibodies (0.2 mg/mouse/injection) once every 3 days, for 30 days. Thereafter, tumor growth was followed without any further treatment. Data are means ± SEM from nine mice in each group. CTX, cetuximab; TRZ, trastuzumab; 33, a monoclonal anti-human HER3 antibody; PBS, saline control. B. Metabolic activity of PC9ER and H1975 cells cultured for 4 days in the presence of increasing concentrations of erlotinib, osimertinib, CO-1686 (rociletinib), or the triple antibody combination (CTX, TRZ, and mAb33). Data are means ± SD values from three experiments. C. PC9ER cells were treated overnight with the indicated TKIs, or with 3×mAbs, and whole-cell extracts were prepared. Cleared extracts were electrophoresed, and resolved proteins were transferred onto filters. Filters were immunoblotted for the indicated proteins or for their phosphorylated forms. Blots are representative of two independent experiments. D. H1975 NSCLC cells (3 × 106 cells per animal) were subcutaneously grafted in the flanks of CD1-nu/nu mice. Animals were randomized into groups of eight mice after tumors became palpable. Erlotinib (50 mg/kg/dose) and osimertinib (5 mg/kg/dose) were daily administered using oral gavage, whereas the triple antibody combination (3×mAbs; CTX, TRZ, and mAb33; 0.2 mg/mouse/injection) and saline (vehicle) were administered intraperitoneally once every 3 days. Data are means ± SEM values. The broken horizontal line marks the initial tumor volume. E. H1975 NSCLC cells (3 × 106 cells per animal) were subcutaneously grafted in the flanks of four groups of CD1-nu/nu mice. Animals were subjected to the following treatments: erlotinib (50 mg/kg/a daily treatment), osimertinib (5 mg/kg/a daily treatment), or 3×mAbs (CTX, TRZ and mAb33; 0.2 mg/mouse/injection) administered twice a week. Immunohistochemical staining for KI67 in paraffin-embedded sections was performed (see Fig EV1D), and the results are presented in box and whisker plots, where the ends of the box are the upper and lower quartiles, the median is marked by a line inside the box and the whiskers mark the highest and lowest values. Depicting quantifications of KI67 staining using 6–8 sections/tumor. Statistical calculations refer to the control group as reference. ***P < 0.001; ****P < 0.0001; n = 5; one-way ANOVA with Tukey's test. F, G. H1975 NSCLC cells (3 × 106 cells per animal) were subcutaneously grafted in the flanks of three groups of CD1-nu/nu mice. Animals were subjected to erlotinib treatment (50 mg/kg/dose), which continued until tumors reached 800 mm3. Thereafter, each group received one of the following treatments: erlotinib (50 mg/kg/dose), osimertinib (5 mg/kg/dose) or 3×mAbs (CTX, TRZ and mAb33; 0.2 mg/mouse/injection) administered as in (D). Data are means ± SEM from seven mice in each group. Also shown are tumors harvested from each group of animals. Scale bar, 1 cm. Source data are available online for this figure. Source Data for Figure 1 [emmm201708076-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Third-generation TKIs and 3×mAbs comparably inhibit TKI-resistant tumors, but their biological effects are distinct Toward in vivo comparisons of 3×mAbs and a third-generation TKI, we examined effects on metabolic activity and EGFR phosphorylation. As predicted, the third-generation TKIs completely inhibited metabolic activity of PC9, PC9ER, and H1975 cells (Figs 1B and EV1B). In contrast, 3×mAbs achieved only partial (< 50%) inhibition of metabolic activity, even at relatively high concentrations. Unlike erlotinib, which exerted no consistent effect on EGFR phosphorylation, both third-generation inhibitors we tested, osimertinib and CO-1686 (Sequist et al, 2015), strongly reduced EGFR phosphorylation in PC9ER cells, and this was associated with parallel decreases in downstream phosphorylation of AKT and ERK (Fig 1C). Interestingly, 3×mAbs only partly reduced phospho-EGFR signals, an effect attributable to EGFR degradation. Accordingly, the antibodies strongly reduced pERK. However, 3×mAbs spared the basally active AKT. In contrast to the antibodies, the TKIs caused no detectable EGFR degradation, but both inhibitors elevated HER3 (and HER2) surface expression, while 3×mAbs induced down-regulation of surface EGFR, HER2, and HER3 (Fig EV1C), consistent with the reported effect on receptor endocytosis (Mancini et al, 2015). In conclusion, the set of in vitro assays uncovered remarkable differences between 3×mAbs and osimertinib: While the former reduced surface expression of the target receptors and inhibited pERK, it only partly inhibited metabolism and did not significantly affect pAKT. In contrast, the irreversible TKI strongly inhibited pEGFR, pAKT, pERK, and cellular metabolism, but it up-regulated surface HER3 and HER2. Next, we compared the ability of 3×mAbs and osimertinib to inhibit tumor growth in mice. Interestingly, both treatments effectively inhibited tumorigenic growth of H1975 cells, but osimertinib achieved an earlier effect (Fig 1D). As expected, both osimertinib and 3×mAbs strongly reduced expression of KI67, a proliferation antigen (Figs 1E and EV1D). The inhibitory effects were reflected also by another test, which administered the two drugs to animals already bearing relatively large H1975 tumors (Fig 1F and G). Immunohistochemical analyses of excised tumors confirmed, on the one hand, the ability of osimertinib to strongly inhibit EGFR phosphorylation and, on the other hand, the ability of 3×mAbs to downregulate EGFR abundance in tumors (Fig EV1E). To address potential toxicities, we analyzed body weights. While animals treated with 3×mAbs gained weight in the course of the experiment (45 days), mice treated with osimertinib displayed slower rates of weight gain (Fig EV1F). In addition, only small differences in favor of fat accumulation in antibody-treated animals were observed when using fat/lean analyses (Fig EV1G). In summary, treatments using osimertinib and 3×mAbs are comparably effective and safe when tested in mice, but the TKI achieves faster kinetics, probably due to complete inhibition of the AKT survival pathway. Third-generation TKIs strongly induce apoptosis of erlotinib-resistant cells In line with a TKI-specific effect on cell growth and survival, we observed a decrease in S-phase cells and a parallel increase in the fraction of cells found in the G0/G1 phase of the cell cycle (Fig 2A). Moreover, prolonged incubation of PC9ER cells with osimertinib-induced caspase-3 cleavage, a hallmark of cells undergoing programmed death, but treatment with 3×mAbs was associated with very weak caspase cleavage (Fig 2B). Additional experiments, which are presented in Fig EV2A, employed another marker of apoptosis, namely BIM, which is essential for the action of EGFR kinase inhibitors (Gong et al, 2007). The results obtained further supported our conclusion that osimertinib induces stronger cell death signals than the very weak apoptosis effect observed after treatment with 3×mAbs. Probing osimertinib-treated cells for another apoptosis marker, namely annexin V, further supported the notion that the TKI more strongly induced cell death than the antibodies (Figs EV2B and 2C). To help distinguish between early and late apoptosis, we used 7-amino-actinomycin D (7-AAD). Early apoptotic cells exclude 7-AAD, while late-stage apoptotic cells stain positively, due to passage of the dye into the nucleus. Interestingly, whereas a cytotoxic agent, cisplatin, increased primarily late apoptosis, osimertinib increased both early and late apoptosis, and combining cisplatin and 3×mAbs increased late apoptosis (Fig 2C), in line with a recent study (Ellebaek et al, 2016). Consistent with the in vitro observations, widespread caspase-3 cleavage was observed in H1975 and in PC9ER xenografts already 4 days after osimertinib treatment (Fig 2D and E). In summary, the third-generation TKI, more than 3×mAbs, induces apoptosis of erlotinib-resistant cells both in vitro and in animals. Figure 2. Unlike 3×mAbs, osimertinib induces apoptosis of erlotinib-resistant NSCLC cells PC9ER cells were treated for 24 h with increasing concentrations of 3×mAbs or osimertinib, or with the respective vehicles (saline or DMSO). Following incubation with BrdU (60 min), cells were fixed and subjected to BrdU and PI staining. Shown are cell cycle distributions of one representative experiment that used cytometry and 100,000 cells/sample. The experiment was repeated three times. PC9ER cells were treated for the indicated time intervals with osimertinib (0.5 μM) or 3×mAbs (TRZ, CTX, and mAb 33; each at 20 μg/ml). Alternatively, cells were treated for 48 h with an irrelevant immunoglobulin G (Irr-IgG), cetuximab (CTX, 20 μg/ml), osimertinib, or 3×mAbs. Cell extracts were prepared, electrophoresed, and immunoblotted for caspase-3 and its cleaved form. The locations of caspase-3 and two cleaved forms are indicated. GAPDH was used as an equal loading control. Blots are representative of two experiments. PC9ER cells were treated for 48 h with the following agents: saline (PBS), cetuximab (CTX, 20 μg/ml), 3×mAbs (CTX, TRZ, and mAb33, each at 20 μg/ml), osimertinib, CO-1686, cisplatin (1 μM), and a mixture of 3×mAbs and cisplatin. Shown are results of an apoptosis assay performed using an annexin V/7-AAD kit (BioLegend, Inc.). Quantification of the fractions of early and late apoptotic cells is shown (see Fig EV2B). L, low drug concentration (0.01 μM); H, high drug concentration (0.5 μM). The experiment was repeated three times. Mice bearing H1975 tumor xenografts were treated for the indicated time interval with either vehicle, 3×mAbs (0.2 mg/mouse/dose), or with osimertinib (5 mg/kg/dose). Whole tumor extracts were immunoblotted for caspase-3. Note that each lane represents a single tumor. Immunohistochemical staining for cleaved caspase-3 performed on paraffin-embedded sections derived from xenografts of either PC9ER or H1975 cells. Two weeks after tumor inoculation, mice were randomized (3–4 mice/group) and treated for 12 days either with vehicle, 3×mAbs (CTX, TRZ, and mAb33; 0.2 mg/mouse/injection, on
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