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Dual MET-EGFR combinatorial inhibition against T790M-EGFR-mediated erlotinib-resistant lung cancer.

Tang Z, Du R, Jiang S, Wu C, Barkauskas DS, Richey J, Molter J, Lam M, Flask C, Gerson S, Dowlati A, Liu L, Lee Z, Halmos B, Wang Y, Kern JA, Ma PC - Br. J. Cancer (2008)

Bottom Line: At 2 microM, SU 11274 had significant in vitro pro-apoptotic effect in H1975 cells, 3.9-fold (P = 0.0015) higher than erlotinib, but had no effect on the MET and EGFR-negative H520 cells.In vivo, SU11274 also induced significant tumour cytoreduction in H1975 murine xenografts in our bioluminescence molecular imaging assay.Together, our results suggest that MET-based targeted inhibition using small-molecule MET inhibitor can be a potential treatment strategy for T790M-EGFR-mediated erlotinib-resistant non-small-cell lung cancer.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Department of Medicine, Case Western Reserve University, University Hospitals Case Medical Center, Cleveland, OH 44106, USA.

ABSTRACT
Despite clinical approval of erlotinib, most advanced lung cancer patients are primary non-responders. Initial responders invariably develop secondary resistance, which can be accounted for by T790M-EGFR mutation in half of the relapses. We show that MET is highly expressed in lung cancer, often concomitantly with epidermal growth factor receptor (EGFR), including H1975 cell line. The erlotinib-resistant lung cancer cell line H1975, which expresses L858R/T790M-EGFR in-cis, was used to test for the effect of MET inhibition using the small molecule inhibitor SU11274. H1975 cells express wild-type MET, without genomic amplification (CNV = 1.1). At 2 microM, SU 11274 had significant in vitro pro-apoptotic effect in H1975 cells, 3.9-fold (P = 0.0015) higher than erlotinib, but had no effect on the MET and EGFR-negative H520 cells. In vivo, SU11274 also induced significant tumour cytoreduction in H1975 murine xenografts in our bioluminescence molecular imaging assay. Using small-animal microPET/MRI, SU11274 treatment was found to induce an early tumour metabolic response in H1975 tumour xenografts. MET and EGFR pathways were found to exhibit collaborative signalling with receptor cross-activation, which had different patterns between wild type (A549) and L858R/T790M-EGFR (H1975). SU11274 plus erlotinib/CL-387,785 potentiated MET inhibition of downstream cell proliferative survival signalling. Knockdown studies in H1975 cells using siRNA against MET alone, EGFR alone, or both, confirmed the enhanced downstream inhibition with dual MET-EGFR signal path inhibition. Finally, in our time-lapse video-microscopy and in vivo multimodal molecular imaging studies, dual SU11274-erlotinib concurrent treatment effectively inhibited H1975 cells with enhanced abrogation of cytoskeletal functions and complete regression of the xenograft growth. Together, our results suggest that MET-based targeted inhibition using small-molecule MET inhibitor can be a potential treatment strategy for T790M-EGFR-mediated erlotinib-resistant non-small-cell lung cancer. Furthermore, optimised inhibition may be further achieved with MET inhibition in combination with erlotinib or an irreversible EGFR-TKI.

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MET inhibition with SU11274 successfully induced in vivo tumour response in EGFR-TKI-resistant H1975 cells in murine xenograft model assessed by multimodal molecular imaging. (A) In vivo tumour xenografts for H1975-luc cells were established as described in the Materials and Methods section in 6-week old nude mice. Daily SU11274 (100 μg per xenograft) treatment was administered to the H1975-luc lung cancer tumour xenografts in nude mice as described. DMSO diluent control was included for comparison. Imaging was performed using a Xenogen IVIS 200 System cooled CCD camera at indicated times. (a) Representative BLI digital pictures of nude mouse from each of the treatment conditions are illustrated. SU11274 significantly inhibited L858R/T790M-EGFR expressing H1975-luc in vivo tumorigenesis within the treatment durations (6 days). (b) Mean values of relative BLI flux of each group are plotted here (H1975-luc). N=4 per treatment group. Error bar, s.e.m. (*), P=0.025 for H1975-luc. Representative tumour xenograft micrographs from H1975-luc (c) cell line under haematoxylin and eosin (H&E) staining are also shown here for the control and SU11274 treatment animals. Magnification × 100 (inset, × 200). (d) SU11274 inhibited HGF-driven signalling activation in H1975 cells. H1975 cells were stimulated with HGF (50 ng ml−1, 15 min) and inhibited by MET inhibitor SU11274 (1 μM, 4 h) in vitro, and analysed with 7.5% SDS–PAGE and immunoblotting with the indicated antibodies as described in Materials and Methods. (B, C) Magnetic resonance imaging (MRI) and microPET molecular imaging studies of MET inhibition of H1975 in vivo xenograft. H1975 in vivo xenografts were established as above for treatment with either diluent control (N=2) or SU11274 (N=2). The nude mice with H1975 xenografts were subjected to MRI and microPET imaging as described in the Materials and Methods section at 0, and 24 h with the MET inhibitor SU11274 treatment or diluent control. (B) Examples of the transverse sections of high-resolution MRI images of the tumour xenografts at baseline between the two treatment groups were shown here for illustration (left). The MRI tumour volumes were analysed digitally with the calculated tumour volume changes at the indicated time intervals (0 and 24 h) plotted. Comparing with baseline, the control xenograft tumour volume increased by 105.1±8.3% at 24 h, whereas the SU11274-treated xenografts increased by 120.2±16.2% at 24 h. The MRI tumour volumes changes at 24 h post-treatment between the two groups were not statistically significant (*P=0.360). Error bar, s.e.m. Quantitative microPET radiotracer uptake of the H1975 tumour xenografts at 60 min of radiotracer tail-vein infusion in the animals' pretreatment baseline (0 h) and post-MET-TKI treatment at 24 h is shown graphically (right). N=2 in each treatment group: Control and SU11274. Error bar, s.e.m. Representative co-registered pictures of the microPET/MRI (low-resolution) images of each xenograft from the two treatment groups are shown in (C). SU11274 induced early tumour metabolic response, as early as 24-h post-TKI treatment, with statistically significant inhibition of glucose metabolism as evident in the decrease in microPET uptake signal intensity by 45% (P=0.0226) in SU11274-treated xenografts, when compared with diluent control. The degree of increase in the glucose uptake in the H1975 tumour xenograft in diluent control is also consistent with the average rate of the xenograft growth (increase of 50.8% BLI flux per day) as reflected in the bioluminescence imaging.
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fig3: MET inhibition with SU11274 successfully induced in vivo tumour response in EGFR-TKI-resistant H1975 cells in murine xenograft model assessed by multimodal molecular imaging. (A) In vivo tumour xenografts for H1975-luc cells were established as described in the Materials and Methods section in 6-week old nude mice. Daily SU11274 (100 μg per xenograft) treatment was administered to the H1975-luc lung cancer tumour xenografts in nude mice as described. DMSO diluent control was included for comparison. Imaging was performed using a Xenogen IVIS 200 System cooled CCD camera at indicated times. (a) Representative BLI digital pictures of nude mouse from each of the treatment conditions are illustrated. SU11274 significantly inhibited L858R/T790M-EGFR expressing H1975-luc in vivo tumorigenesis within the treatment durations (6 days). (b) Mean values of relative BLI flux of each group are plotted here (H1975-luc). N=4 per treatment group. Error bar, s.e.m. (*), P=0.025 for H1975-luc. Representative tumour xenograft micrographs from H1975-luc (c) cell line under haematoxylin and eosin (H&E) staining are also shown here for the control and SU11274 treatment animals. Magnification × 100 (inset, × 200). (d) SU11274 inhibited HGF-driven signalling activation in H1975 cells. H1975 cells were stimulated with HGF (50 ng ml−1, 15 min) and inhibited by MET inhibitor SU11274 (1 μM, 4 h) in vitro, and analysed with 7.5% SDS–PAGE and immunoblotting with the indicated antibodies as described in Materials and Methods. (B, C) Magnetic resonance imaging (MRI) and microPET molecular imaging studies of MET inhibition of H1975 in vivo xenograft. H1975 in vivo xenografts were established as above for treatment with either diluent control (N=2) or SU11274 (N=2). The nude mice with H1975 xenografts were subjected to MRI and microPET imaging as described in the Materials and Methods section at 0, and 24 h with the MET inhibitor SU11274 treatment or diluent control. (B) Examples of the transverse sections of high-resolution MRI images of the tumour xenografts at baseline between the two treatment groups were shown here for illustration (left). The MRI tumour volumes were analysed digitally with the calculated tumour volume changes at the indicated time intervals (0 and 24 h) plotted. Comparing with baseline, the control xenograft tumour volume increased by 105.1±8.3% at 24 h, whereas the SU11274-treated xenografts increased by 120.2±16.2% at 24 h. The MRI tumour volumes changes at 24 h post-treatment between the two groups were not statistically significant (*P=0.360). Error bar, s.e.m. Quantitative microPET radiotracer uptake of the H1975 tumour xenografts at 60 min of radiotracer tail-vein infusion in the animals' pretreatment baseline (0 h) and post-MET-TKI treatment at 24 h is shown graphically (right). N=2 in each treatment group: Control and SU11274. Error bar, s.e.m. Representative co-registered pictures of the microPET/MRI (low-resolution) images of each xenograft from the two treatment groups are shown in (C). SU11274 induced early tumour metabolic response, as early as 24-h post-TKI treatment, with statistically significant inhibition of glucose metabolism as evident in the decrease in microPET uptake signal intensity by 45% (P=0.0226) in SU11274-treated xenografts, when compared with diluent control. The degree of increase in the glucose uptake in the H1975 tumour xenograft in diluent control is also consistent with the average rate of the xenograft growth (increase of 50.8% BLI flux per day) as reflected in the bioluminescence imaging.

Mentions: To further test the role of MET inhibition in EGFR-TKI-resistant lung cancer in vivo, we developed stable luciferase-expressing H1975 lung cancer cells using lentivirus transduction. These cells were used in an in vivo xenograft model coupled with multimodal molecular imaging for non-invasive monitoring of xenograft growth and tumour response to TKI. Daily treatment with the MET inhibitor SU11274 caused statistically significant interval retardation of the xenograft tumour growth of H1975 cells with a ninefold reduction (P=0.0251) in the xenograft growth, when compared with the diluent control, during the treatment period (Figure 3A-a,b). At the end of treatment period, SU11274-treated H1975 xenograft tumour BLI flux remained essentially unchanged at 104% (P=0.0251), when compared to 905% as seen in the diluent control (Figure 3A-a,b). Histological analysis of the tumour xenografts harvested at the end of the experiment confirmed the presence of intense tumour necrosis in the SU11274-treated animals, but not in the diluent control (Figure 3A-c). Both EGFR and MET signal pathways are functional and ligand-sensitive in the erlotinib-resistant H1975 cells. HGF stimulated downstream signal path activation in AKT (survival) and the mitogen-activated protein kinase ERK1/2 (proliferation–differentiation), as surrogate markers for MET inhibition were both abrogated in the H1975 cells by SU11274 treatment in vitro (Figure 3A-d). Furthermore, we also showed that SU11274 inhibition effectively induced erlotinib-resistant A549 xenograft cytoreduction in vivo (Supplementary Figure 1).


Dual MET-EGFR combinatorial inhibition against T790M-EGFR-mediated erlotinib-resistant lung cancer.

Tang Z, Du R, Jiang S, Wu C, Barkauskas DS, Richey J, Molter J, Lam M, Flask C, Gerson S, Dowlati A, Liu L, Lee Z, Halmos B, Wang Y, Kern JA, Ma PC - Br. J. Cancer (2008)

MET inhibition with SU11274 successfully induced in vivo tumour response in EGFR-TKI-resistant H1975 cells in murine xenograft model assessed by multimodal molecular imaging. (A) In vivo tumour xenografts for H1975-luc cells were established as described in the Materials and Methods section in 6-week old nude mice. Daily SU11274 (100 μg per xenograft) treatment was administered to the H1975-luc lung cancer tumour xenografts in nude mice as described. DMSO diluent control was included for comparison. Imaging was performed using a Xenogen IVIS 200 System cooled CCD camera at indicated times. (a) Representative BLI digital pictures of nude mouse from each of the treatment conditions are illustrated. SU11274 significantly inhibited L858R/T790M-EGFR expressing H1975-luc in vivo tumorigenesis within the treatment durations (6 days). (b) Mean values of relative BLI flux of each group are plotted here (H1975-luc). N=4 per treatment group. Error bar, s.e.m. (*), P=0.025 for H1975-luc. Representative tumour xenograft micrographs from H1975-luc (c) cell line under haematoxylin and eosin (H&E) staining are also shown here for the control and SU11274 treatment animals. Magnification × 100 (inset, × 200). (d) SU11274 inhibited HGF-driven signalling activation in H1975 cells. H1975 cells were stimulated with HGF (50 ng ml−1, 15 min) and inhibited by MET inhibitor SU11274 (1 μM, 4 h) in vitro, and analysed with 7.5% SDS–PAGE and immunoblotting with the indicated antibodies as described in Materials and Methods. (B, C) Magnetic resonance imaging (MRI) and microPET molecular imaging studies of MET inhibition of H1975 in vivo xenograft. H1975 in vivo xenografts were established as above for treatment with either diluent control (N=2) or SU11274 (N=2). The nude mice with H1975 xenografts were subjected to MRI and microPET imaging as described in the Materials and Methods section at 0, and 24 h with the MET inhibitor SU11274 treatment or diluent control. (B) Examples of the transverse sections of high-resolution MRI images of the tumour xenografts at baseline between the two treatment groups were shown here for illustration (left). The MRI tumour volumes were analysed digitally with the calculated tumour volume changes at the indicated time intervals (0 and 24 h) plotted. Comparing with baseline, the control xenograft tumour volume increased by 105.1±8.3% at 24 h, whereas the SU11274-treated xenografts increased by 120.2±16.2% at 24 h. The MRI tumour volumes changes at 24 h post-treatment between the two groups were not statistically significant (*P=0.360). Error bar, s.e.m. Quantitative microPET radiotracer uptake of the H1975 tumour xenografts at 60 min of radiotracer tail-vein infusion in the animals' pretreatment baseline (0 h) and post-MET-TKI treatment at 24 h is shown graphically (right). N=2 in each treatment group: Control and SU11274. Error bar, s.e.m. Representative co-registered pictures of the microPET/MRI (low-resolution) images of each xenograft from the two treatment groups are shown in (C). SU11274 induced early tumour metabolic response, as early as 24-h post-TKI treatment, with statistically significant inhibition of glucose metabolism as evident in the decrease in microPET uptake signal intensity by 45% (P=0.0226) in SU11274-treated xenografts, when compared with diluent control. The degree of increase in the glucose uptake in the H1975 tumour xenograft in diluent control is also consistent with the average rate of the xenograft growth (increase of 50.8% BLI flux per day) as reflected in the bioluminescence imaging.
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fig3: MET inhibition with SU11274 successfully induced in vivo tumour response in EGFR-TKI-resistant H1975 cells in murine xenograft model assessed by multimodal molecular imaging. (A) In vivo tumour xenografts for H1975-luc cells were established as described in the Materials and Methods section in 6-week old nude mice. Daily SU11274 (100 μg per xenograft) treatment was administered to the H1975-luc lung cancer tumour xenografts in nude mice as described. DMSO diluent control was included for comparison. Imaging was performed using a Xenogen IVIS 200 System cooled CCD camera at indicated times. (a) Representative BLI digital pictures of nude mouse from each of the treatment conditions are illustrated. SU11274 significantly inhibited L858R/T790M-EGFR expressing H1975-luc in vivo tumorigenesis within the treatment durations (6 days). (b) Mean values of relative BLI flux of each group are plotted here (H1975-luc). N=4 per treatment group. Error bar, s.e.m. (*), P=0.025 for H1975-luc. Representative tumour xenograft micrographs from H1975-luc (c) cell line under haematoxylin and eosin (H&E) staining are also shown here for the control and SU11274 treatment animals. Magnification × 100 (inset, × 200). (d) SU11274 inhibited HGF-driven signalling activation in H1975 cells. H1975 cells were stimulated with HGF (50 ng ml−1, 15 min) and inhibited by MET inhibitor SU11274 (1 μM, 4 h) in vitro, and analysed with 7.5% SDS–PAGE and immunoblotting with the indicated antibodies as described in Materials and Methods. (B, C) Magnetic resonance imaging (MRI) and microPET molecular imaging studies of MET inhibition of H1975 in vivo xenograft. H1975 in vivo xenografts were established as above for treatment with either diluent control (N=2) or SU11274 (N=2). The nude mice with H1975 xenografts were subjected to MRI and microPET imaging as described in the Materials and Methods section at 0, and 24 h with the MET inhibitor SU11274 treatment or diluent control. (B) Examples of the transverse sections of high-resolution MRI images of the tumour xenografts at baseline between the two treatment groups were shown here for illustration (left). The MRI tumour volumes were analysed digitally with the calculated tumour volume changes at the indicated time intervals (0 and 24 h) plotted. Comparing with baseline, the control xenograft tumour volume increased by 105.1±8.3% at 24 h, whereas the SU11274-treated xenografts increased by 120.2±16.2% at 24 h. The MRI tumour volumes changes at 24 h post-treatment between the two groups were not statistically significant (*P=0.360). Error bar, s.e.m. Quantitative microPET radiotracer uptake of the H1975 tumour xenografts at 60 min of radiotracer tail-vein infusion in the animals' pretreatment baseline (0 h) and post-MET-TKI treatment at 24 h is shown graphically (right). N=2 in each treatment group: Control and SU11274. Error bar, s.e.m. Representative co-registered pictures of the microPET/MRI (low-resolution) images of each xenograft from the two treatment groups are shown in (C). SU11274 induced early tumour metabolic response, as early as 24-h post-TKI treatment, with statistically significant inhibition of glucose metabolism as evident in the decrease in microPET uptake signal intensity by 45% (P=0.0226) in SU11274-treated xenografts, when compared with diluent control. The degree of increase in the glucose uptake in the H1975 tumour xenograft in diluent control is also consistent with the average rate of the xenograft growth (increase of 50.8% BLI flux per day) as reflected in the bioluminescence imaging.
Mentions: To further test the role of MET inhibition in EGFR-TKI-resistant lung cancer in vivo, we developed stable luciferase-expressing H1975 lung cancer cells using lentivirus transduction. These cells were used in an in vivo xenograft model coupled with multimodal molecular imaging for non-invasive monitoring of xenograft growth and tumour response to TKI. Daily treatment with the MET inhibitor SU11274 caused statistically significant interval retardation of the xenograft tumour growth of H1975 cells with a ninefold reduction (P=0.0251) in the xenograft growth, when compared with the diluent control, during the treatment period (Figure 3A-a,b). At the end of treatment period, SU11274-treated H1975 xenograft tumour BLI flux remained essentially unchanged at 104% (P=0.0251), when compared to 905% as seen in the diluent control (Figure 3A-a,b). Histological analysis of the tumour xenografts harvested at the end of the experiment confirmed the presence of intense tumour necrosis in the SU11274-treated animals, but not in the diluent control (Figure 3A-c). Both EGFR and MET signal pathways are functional and ligand-sensitive in the erlotinib-resistant H1975 cells. HGF stimulated downstream signal path activation in AKT (survival) and the mitogen-activated protein kinase ERK1/2 (proliferation–differentiation), as surrogate markers for MET inhibition were both abrogated in the H1975 cells by SU11274 treatment in vitro (Figure 3A-d). Furthermore, we also showed that SU11274 inhibition effectively induced erlotinib-resistant A549 xenograft cytoreduction in vivo (Supplementary Figure 1).

Bottom Line: At 2 microM, SU 11274 had significant in vitro pro-apoptotic effect in H1975 cells, 3.9-fold (P = 0.0015) higher than erlotinib, but had no effect on the MET and EGFR-negative H520 cells.In vivo, SU11274 also induced significant tumour cytoreduction in H1975 murine xenografts in our bioluminescence molecular imaging assay.Together, our results suggest that MET-based targeted inhibition using small-molecule MET inhibitor can be a potential treatment strategy for T790M-EGFR-mediated erlotinib-resistant non-small-cell lung cancer.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Department of Medicine, Case Western Reserve University, University Hospitals Case Medical Center, Cleveland, OH 44106, USA.

ABSTRACT
Despite clinical approval of erlotinib, most advanced lung cancer patients are primary non-responders. Initial responders invariably develop secondary resistance, which can be accounted for by T790M-EGFR mutation in half of the relapses. We show that MET is highly expressed in lung cancer, often concomitantly with epidermal growth factor receptor (EGFR), including H1975 cell line. The erlotinib-resistant lung cancer cell line H1975, which expresses L858R/T790M-EGFR in-cis, was used to test for the effect of MET inhibition using the small molecule inhibitor SU11274. H1975 cells express wild-type MET, without genomic amplification (CNV = 1.1). At 2 microM, SU 11274 had significant in vitro pro-apoptotic effect in H1975 cells, 3.9-fold (P = 0.0015) higher than erlotinib, but had no effect on the MET and EGFR-negative H520 cells. In vivo, SU11274 also induced significant tumour cytoreduction in H1975 murine xenografts in our bioluminescence molecular imaging assay. Using small-animal microPET/MRI, SU11274 treatment was found to induce an early tumour metabolic response in H1975 tumour xenografts. MET and EGFR pathways were found to exhibit collaborative signalling with receptor cross-activation, which had different patterns between wild type (A549) and L858R/T790M-EGFR (H1975). SU11274 plus erlotinib/CL-387,785 potentiated MET inhibition of downstream cell proliferative survival signalling. Knockdown studies in H1975 cells using siRNA against MET alone, EGFR alone, or both, confirmed the enhanced downstream inhibition with dual MET-EGFR signal path inhibition. Finally, in our time-lapse video-microscopy and in vivo multimodal molecular imaging studies, dual SU11274-erlotinib concurrent treatment effectively inhibited H1975 cells with enhanced abrogation of cytoskeletal functions and complete regression of the xenograft growth. Together, our results suggest that MET-based targeted inhibition using small-molecule MET inhibitor can be a potential treatment strategy for T790M-EGFR-mediated erlotinib-resistant non-small-cell lung cancer. Furthermore, optimised inhibition may be further achieved with MET inhibition in combination with erlotinib or an irreversible EGFR-TKI.

Show MeSH
Related in: MedlinePlus