Co-treatment with panitumumab and trastuzumab augments response to the MEK inhibitor trametinib in a patient-derived xenograft model of pancreatic cancer

Abstract

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations and epidermal growth factor receptor (EGFR) family signaling are drivers of tumorigenesis in pancreatic ductal adenocarcinoma (PDAC). Previous studies have demonstrated that combinatorial treatment of PDAC xenografts with the mitogen-activated protein kinase-extracellular-signal-regulated kinase (ERK) kinase1/2 (MEK1/2) inhibitor trametinib and the dual EGFR/human epidermal growth factor receptor 2 (HER2) inhibitor lapatinib provided more effective inhibition than either treatment alone. In this study, we have used the therapeutic antibodies, panitumumab (specific for EGFR) and trastuzumab (specific for HER2), to probe the role of EGFR and HER2 signaling in the proliferation of patient-derived xenograft (PDX) tumors. We show that dual anti-EGFR and anti-HER2 therapy significantly augmented the growth inhibitory effects of the MEK1/2 inhibitor trametinib in three different PDX tumors. While significant growth inhibition was observed in both KRAS mutant xenograft groups receiving trametinib and dual antibody therapy (tumors 366 and 608), tumor regression was observed in the KRAS wild-type xenografts (tumor 738) treated in the same manner. Dual antibody therapy in conjunction with trametinib was equally or more effective at inhibiting tumor growth and with lower apparent toxicity than trametinib plus lapatinib. Together, these studies provide further support for a role for EGFR and HER2 in pancreatic cancer proliferation and underscore the importance of therapeutic intervention in both the KRAS-rapidly accelerated fibrosarcoma kinase (RAF)-MEK-ERK and EGFR-HER2 pathways to achieve maximal therapeutic efficacy in patients.

Figure 1

Antibody-mediated inhibition of EGFR and HER2 in response to EGF stimulation. Phospho-RTK arrays were used to assess the relative phosphorylation of EGFR (black bars) and HER2 (lined bars) from EGF-stimulated tumor 366 cells following 90 minutes of drug treatment with DMSO, panitumumab (5 μg/ml), trastuzumab (20 μg/ml), trametinib (10 nM), or the indicated combination therapy (+ indicates drug present and − indicates drug absent). Images from the pRTK arrays used to assay relative phosphorylation level are depicted below each treatment group. Images were cropped and aligned from individual arrays exposed for the same period of time for ease of comparison with vertical bars separating individual arrays. The mean pixel density of two replicates for each phospho-antibody was quantified, normalized to control, and depicted in bar graph form. Image acquisition and analysis were performed using a Bio-Rad GS-800 calibrated densitometer and ImageQuant TL 2005 software. Significant difference in mean pixel density for each treatment group compared to control is denoted as *P < .05. Figure W1 shows the complete array images.

Figure 2

ERK and AKT signaling in a PDX-derived tumor cell line. Western blot depicting the in vitro response of tumor 366 cells following 90 minutes of drug treatment with DMSO, panitumumab (5 μg/ml), trastuzumab (20 μg/ml), trametinib (10 nM), or the indicated combination therapy (+ indicates drug present and − indicates drug absent) under EGF-stimulated or starved conditions. Starved cells were maintained in serum-free media for 4 hours before drug therapy and then given PBS after 60 minutes of drug exposure; EGF-stimulated cells (EGF +) were starved for 4 hours before drug therapy and then exposed to 100 ng/ml of human EGF after 60 minutes of drug exposure. Lysates prepared from cells exposed to each indicated treatment group were blotted for pEGFR (Y1068), pERK1/2 (T202/204), ERK, pAKT (S473), AKT, and RAN. The pERK/ERK and pAKT/AKT ratios are presented to permit quantitative comparisons between groups. Vertical bars denote the alignment of different regions of the same blot. All samples in two separate gels were obtained from one experiment and the same control was included on both gels. Image acquisition and analysis were performed using the Odyssey Infrared Imaging System and Image Studio V2.1 software.

Figure 3

In vivo response of PDX tumors to trametinib and antibody treatment. (A, C, and E) In vivo response of three different established PDAC tumors (100-500 mm³ before starting therapy) to treatment with vehicle control (black bar), trametinib (Tra), panitumumab (P), or trametinib plus panitumumab (Tra + P) (treated mice, hatched bars). The number of mice in each treatment group is indicated in parentheses. An MRI was obtained just before the start of treatment to establish an index tumor volume for each mouse. Subsequent interval MRI studies were used to assess the change in tumor volume while on treatment (see Figure W2). To calculate the relative change in tumor volume (fold change) for each tumor, the interval tumor volumes were divided by the index tumor volume and linear regression was used to model a line of best fit for the tumor fold change data plotted relative to time. The slope of that line of best fit demonstrates the tumor growth rate for each treatment group expressed as the fold change per week on treatment. Mean fold change per week plus the standard error of the mean are displayed for each treatment group as bar graphs. Significance is denoted as *P < .05. Following the initial treatment period, a group of mice treated with trametinib was maintained on trametinib. Mice in the panitumumab (P) group were switched to panitumumab plus trastuzumab (P + T) and mice in the trametinib plus panitumumab (Tra + P) group were switched to combined treatment with panitumumab, trastuzumab, and trametinib (Tra + P + T) for an additional 2 to 4 weeks (open bars, Figure W2). (B, D, and F) Representative MRI images of control and triple therapy–treated (Tra + P + T) tumors obtained just before the onset of treatment (Start Rx) and at the conclusion of treatment just before sacrifice (End Rx). The tumors are outlined and these images depict the axial slice with the largest cross-sectional area in each tumor.

Figure 4

Comparison of in vivo response of PDX tumors to trametinib plus lapatinib versus trametinib plus antibody treatment. (A and B) In vivo response of two different PDAC tumors to treatment with vehicle control, trametinib plus lapatinib (Tra + L), trametinib plus pertuzumab (Tra + PZ), or trametinib plus panitumumab plus trastuzumab (Tra + P + T). Tumors were allowed to grow to a starting volume of 100 to 500 mm³ before the onset of treatment. Mice were treated with vehicle control or drug therapy for 4 weeks (the number of mice in each treatment group is indicated in parentheses). Initial tumor volume was assessed by MRI before the start of dosing and subsequent weekly MRI assessments were carried out to calculate the change in relative tumor volume (see also Figure W3). The mean relative tumor volume ± the standard error of the mean over time is plotted in line graph form for each treatment group. Linear regression was used to model a line of best fit for the tumor fold change data plotted relative to time. The slope of that line of best fit that served as an estimate of the tumor growth rate for each treatment group expressed as fold change per week on treatment was determined as described in the Materials and Methods section. The mean fold change per week ± the standard error of the mean are displayed for each treatment group as bar graphs. Significance is denoted as *P < .05.

Figure 5

Relative phosphorylation of RTKs and MAPKs in different PDX tumors. Extracts were prepared from PDX tumors following 4 to 6 weeks of in vivo treatment with vehicle control, panitumumab plus trastuzumab (P + T), trametinib (Tra), or triple therapy (Tra + P + T) and analyzed on duplicate arrays. Bar graphs depict relative pEGFR (pan Y), pHER2 (pan Y), pERK1 (T202/Y204), pAKT2 (S474), pJNK-pan (T183/Y185, T221/Y223), and p70S6K (T421/S424) levels in treated tumor 366 (A), tumor 608 (B), and tumor 738 (C) xenografts. Array images from duplicate arrays were used to calculate the mean pixel density for each phospho-protein under each treatment condition. Images were cropped and aligned from individual arrays exposed for the same period of time for ease of comparison with vertical bars separating individual arrays. All phosphorylation values for each treatment group are presented normalized to control for each phospho-protein assessed. Differences in relative phosphorylation were compared between groups with significance denoted as *P < .05. Image acquisition and analysis were as described above in Figure 1. Figure W4 shows the complete array images.