BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models

Abstract

Genetic alterations in the kinase domain of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to treatment with small molecule tyrosine kinase inhibitors. Although first-generation reversible, ATP-competitive inhibitors showed encouraging clinical responses in lung adenocarcinoma tumors harboring such EGFR mutations, almost all patients developed resistance to these inhibitors over time. Such resistance to first-generation EGFR inhibitors was frequently linked to an acquired T790M point mutation in the kinase domain of EGFR, or upregulation of signaling pathways downstream of HER3. Overcoming these mechanisms of resistance, as well as primary resistance to reversible EGFR inhibitors driven by a subset of EGFR mutations, will be necessary for development of an effective targeted therapy regimen. Here, we show that BIBW2992, an anilino-quinazoline designed to irreversibly bind EGFR and HER2, potently suppresses the kinase activity of wild-type and activated EGFR and HER2 mutants, including erlotinib-resistant isoforms. Consistent with this activity, BIBW2992 suppresses transformation in isogenic cell-based assays, inhibits survival of cancer cell lines and induces tumor regression in xenograft and transgenic lung cancer models, with superior activity over erlotinib. These findings encourage further testing of BIBW2992 in lung cancer patients harboring EGFR or HER2 oncogenes.

Figures

Figure 1

BIBW2992 effectively inhibits anchorage-independent proliferation of NIH-3T3 cells ectopically expressing EGFR mutants. (a) NIH-3T3 cells expressing wild-type or mutant EGFR constructs were treated with the indicated concentrations of BIBW2992 or erlotinib and suspended in soft agar. Values shown are averages of triplicate samples normalized to absence of drug treatment; error bars represent s.d. Expression of mutant EGFR was confirmed by immunoblotting (data not shown). wt + EGF, wild-type EGFR-expressing NIH-3T3 cells supplemented with 100 ng/ml EGF. (b) Anti-phospho-EGFR Y1173 immunoblots of NIH-3T3 cell lysates treated with the indicated concentrations of BIBW2992 or erlotinib for 2 h. A representative anti-actin immunoblot is shown as a loading control.

Figure 2

Growth inhibition of lung cancer cell lines by BIBW2992 and other EGFR and/or HER2 inhibitors (a) Sensitivity of NSCLC cell lines that express EGFR WT, Exon 19 Del, L858R/T790M and HER2 INS776V to BIBW2992 and erlotinib. Dose-dependent growth inhibition was determined by MTS assay. Only HCC827 cells are sensitive to erlotinib whereas NCI-H1781 and NCI-H1975 cells are also sensitive to BIBW2992. Points, average of two independent experiments, each done in triplicate; bars, s.d. *A549 cells harbor a Kras G12S mutation. See also in Supplementary Table 4. (b) Inhibition of anchorage independent cell proliferation of various lung cancer cell lines treated with BIBW2992, lapatinib, canertinib, gefitinib or erlotinib. The IC50 values were determined as described in ‘Materials and methods’, confirmed in independent experiments and reported as nM values. H1666 express wild-type EGFR, H3255 express the EGFR L858R mutant and NCI 1975 express the EGFR L858R/T790M double mutant.

Figure 3

In vivo activity of BIBW 2992 in xenograft models. (a) Animals carrying tumors established from A431 cells were treated daily with BIBW2992 by oral gavage at indicated doses. Statistical analysis of the tumor volumes in each group was performed using one-way analysis of variance (Dunnett's multiple test) with P-values consistently below 0.01. (b) Immunohistochemical analysis of A431 tumor sections obtained 6 h after the last dose confirms in vivo modulation of phosphorylated EGFR and AKT by BIBW2992. (c) Animals carrying tumors established from NCI-N87 cells were treated daily with BIBW2992 or weekly with an intravenous bolus of 20 mg/kg trastuzumab and analysed as in a. (d) 14 days after NCI-N87 xenograft formation, animals carrying tumors were treated daily with BIBW2992 and analysed as in Figure 3a. (e) Animals carrying tumors established from H1975 cells were treated daily with BIBW2992, gefitinib, or lapatinib and analysed as in a.

Figure 4

BIBW2992 induces dramatic tumor regression in the L858R/T790M EGFR—driven lung cancer model. (a) Radiographic response induced by BIBW2992 in L858R/T790M EGFR mice. After approximately 6 weeks of doxycycline administration, tumor-bearing mice were identified by MRI and treatment with 20 mg/kg/day BIBW2992 was initiated with continuous doxycycline diet. MRI scanning was repeated 2 and 4 weeks later to monitor tumor development, and mice were sacrificed at 2 or 4 weeks after re-imaging. Representative MRI photos from one of four mice before treatment and after 2 weeks of BIBW2992 treatment are shown. H: heart area. (b) Radiographic response in L858R/T790M EGFR mice after 1 week of BIBW2992 (20 mg/kg, gavage) and rapamycin (2 mg/kg, intraperitoneally) combination treatment. Mice were on continuous doxycycline diet during treatment. Representative MRI photos from one of six treated mice are shown. H: heart area. (c) Histogram expressed as mean±s.d. illustrating the tumor regression in all treated mice in (a) and (b) at time point indicated below as evaluated by MRI. Statistical analyses were performed using Student's exact t-test. BIBW: BIBW2992 at 20 mg/kg/day for 2 weeks; Rapa: rapamycin at 2 mg/kg/day for 2 weeks; combination: BIBW2992 and rapamycin combination at above dose for 1 week; *P<0.05 for tumor regression rates of BIBW2992 alone and BIBW2992 combined with rapamycin group. (d) Immunohistochemistry staining showed inhibited EGFR and also downstream signaling after BIBW2992 alone treatment and combination therapy of BIBW2992 and rapamycin. L858R/T790M EGFR mice were treated with BIBW2992 (20 mg/kg, gavage) and rapamycin (2 mg/kg, intraperitoneally) for 48 h and lung sections were then stained with antibodies recognizing phospho-EGFR, and phospho-S6. Pictures shown are representative fields from two mice. Photographs were taken at low (×100, insets) and high magnification (×800), respectively.