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P7170, a novel inhibitor of mTORC1/mTORC2 and Activin receptor-like Kinase 1 (ALK1) inhibits the growth of non small cell lung cancer.

Venkatesha VA, Joshi A, Venkataraman M, Sonawane V, Bhatia D, Tannu P, Bose J, Choudhari S, Srivastava A, Pandey PK, Lad VJ, Sangana R, Ahmed T, Damre A, Deore V, Sahu B, Kumar S, Sharma S, Agarwal VR - Mol. Cancer (2014)

Bottom Line: P7170 at a well-tolerated daily dose of 20 mg/kg significantly inhibited the growth of NSCLC xenografts independent of different mutations (EGFR, KRAS, or PIK3CA) or sensitivity to erlotinib.Pharmacokinetic-pharmacodynamic (PK-PD) analysis showed sub-micro molar tumor concentrations along with mTORC1/C2 inhibition.Our results provide evidence of antitumor activity of P7170 in the erlotinib -sensitive and -insensitive models of NSCLC.

View Article: PubMed Central - PubMed

Affiliation: Piramal Life Sciences Ltd, # 1 Nirlon Complex, Off: Western Express Highway, Goregaon (East), Mumbai, Maharashtra 400063, India. veena_828@yahoo.com.

ABSTRACT

Background: Lung cancer is the major cause of cancer-related deaths and many cases of Non Small Cell Lung Cancer (NSCLC), a common type of lung cancer, have frequent genetic/oncogenic activation of EGFR, KRAS, PIK3CA, BRAF, and others that drive tumor growth. Some patients though initially respond, but later develop resistance to erlotinib/gefitinib with no option except for cytotoxic therapy. Therefore, development of novel targeted therapeutics is imperative to provide improved survival benefit for NSCLC patients. The mTOR cell survival pathway is activated in naïve, or in response to targeted therapies in NSCLC.

Methods: We have discovered P7170, a small molecule inhibitor of mTORC1/mTORC2/ALK1 and investigated its antitumor efficacy using various in vitro and in vivo models of human NSCLC.

Results: P7170 inhibited the phosphorylation of AKT, S6 and 4EBP1 (substrates for mTORC2 and mTORC1) levels by 80-100% and growth of NSCLC cells. P7170 inhibited anchorage-independent colony formation of NSCLC patient tumor-derived cells subsistent of disease sub-types. The compound also induced apoptosis in NSCLC cell lines. P7170 at a well-tolerated daily dose of 20 mg/kg significantly inhibited the growth of NSCLC xenografts independent of different mutations (EGFR, KRAS, or PIK3CA) or sensitivity to erlotinib. Pharmacokinetic-pharmacodynamic (PK-PD) analysis showed sub-micro molar tumor concentrations along with mTORC1/C2 inhibition.

Conclusions: Our results provide evidence of antitumor activity of P7170 in the erlotinib -sensitive and -insensitive models of NSCLC.

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Related in: MedlinePlus

P7170 inhibited PI3K/mTOR signaling and the growth of erlotinib-resistant NSCLC cell lines. (A) Exponentially growing H460 cells were seeded in petridishes (2 × 106 cells/dish) and after cell attachment overnight, cells were serum starved for 14-16 h. Cells were then treated for 1 h with P7170 followed by 0.5 h incubation in the presence of 20% fetal bovine serum. Cells were trypsinized and lysed before electrophoresis of cellular extracts for western blot analyses of proteins of interest. (B) Quantification of pS6 and p4EBP1 protein levels in H460 cells after acquisition and analyses of immunofluorescence signals in the Cellomics Array scan platform. H460 cells were seeded in 96-well plates and treated with 0.1 or 1 μM of P7170 for 16 h; drug-containing media was removed and the cells were fixed and stained with antibodies to phosphorylated form of human S6 and 4EBP1 proteins and secondary antibodies conjugated to DyLight 549. Statistical significance values are as follows: *p < 0.05, **p < 0.01, ***p < 0.001. (C) IC50 of erlotinib or P7170 in various NSCLC cell lines after 48 h of treatment.
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Fig1: P7170 inhibited PI3K/mTOR signaling and the growth of erlotinib-resistant NSCLC cell lines. (A) Exponentially growing H460 cells were seeded in petridishes (2 × 106 cells/dish) and after cell attachment overnight, cells were serum starved for 14-16 h. Cells were then treated for 1 h with P7170 followed by 0.5 h incubation in the presence of 20% fetal bovine serum. Cells were trypsinized and lysed before electrophoresis of cellular extracts for western blot analyses of proteins of interest. (B) Quantification of pS6 and p4EBP1 protein levels in H460 cells after acquisition and analyses of immunofluorescence signals in the Cellomics Array scan platform. H460 cells were seeded in 96-well plates and treated with 0.1 or 1 μM of P7170 for 16 h; drug-containing media was removed and the cells were fixed and stained with antibodies to phosphorylated form of human S6 and 4EBP1 proteins and secondary antibodies conjugated to DyLight 549. Statistical significance values are as follows: *p < 0.05, **p < 0.01, ***p < 0.001. (C) IC50 of erlotinib or P7170 in various NSCLC cell lines after 48 h of treatment.

Mentions: We evaluated the activity of P7170 in cell based assays. The phosphorylation of AKT (S473) (substrate of mTORC2) [23], S6 (indirect substrate of mTORC1), and 4EBP1 (substrate of mTORC1) were nearly completely inhibited (100%) in H460 NSCLC cells upon treatment with 50 nM P7170. In the same experiment, phosphorylation of ERK, the effector of RAF-MEK-ERK pathway was marginally decreased (10%) (Figure 1A). The kinase activities of upstream PI3K alpha and mTOR were inhibited by P7170 (IC50 = 2.2 and 4.4 nM, respectively) but potent biochemical activity of PI3K did not translate in intact cells most likely because of feedback mechanism of mTOR inhibition. P7170 is a weak PI3K inhibitor (a separate manuscript submitted). In an immunofluorescence assay, P7170 treatment caused a consistent and marked decrease in the phosphorylation of S6 with a concentration-dependent suppression of p4EBP1 in H460 cells (Figure 1B, Additional file 1: Figure S1). Longer incubation time with P7170 resulted in an enhanced inhibition of pS6 and p4EBP1 (Additional file 1: Figure S1). The effect of P7170 on cell growth was evaluated in three different NSCLC cell lines, where a dose-dependent inhibition was observed. The IC50 of P7170 in EGFR over expressing A431 (EGFR wild type) cells was 10 nM compared to 5 and 7 nM in KRAS mutant A549 and H460 cell lines, respectively. In general, P7170 showed nano molar IC50 concentrations in the growth of various NSCLC cell lines as opposed to micro molar IC50 of erlotinib (Figure 1C).Figure 1


P7170, a novel inhibitor of mTORC1/mTORC2 and Activin receptor-like Kinase 1 (ALK1) inhibits the growth of non small cell lung cancer.

Venkatesha VA, Joshi A, Venkataraman M, Sonawane V, Bhatia D, Tannu P, Bose J, Choudhari S, Srivastava A, Pandey PK, Lad VJ, Sangana R, Ahmed T, Damre A, Deore V, Sahu B, Kumar S, Sharma S, Agarwal VR - Mol. Cancer (2014)

P7170 inhibited PI3K/mTOR signaling and the growth of erlotinib-resistant NSCLC cell lines. (A) Exponentially growing H460 cells were seeded in petridishes (2 × 106 cells/dish) and after cell attachment overnight, cells were serum starved for 14-16 h. Cells were then treated for 1 h with P7170 followed by 0.5 h incubation in the presence of 20% fetal bovine serum. Cells were trypsinized and lysed before electrophoresis of cellular extracts for western blot analyses of proteins of interest. (B) Quantification of pS6 and p4EBP1 protein levels in H460 cells after acquisition and analyses of immunofluorescence signals in the Cellomics Array scan platform. H460 cells were seeded in 96-well plates and treated with 0.1 or 1 μM of P7170 for 16 h; drug-containing media was removed and the cells were fixed and stained with antibodies to phosphorylated form of human S6 and 4EBP1 proteins and secondary antibodies conjugated to DyLight 549. Statistical significance values are as follows: *p < 0.05, **p < 0.01, ***p < 0.001. (C) IC50 of erlotinib or P7170 in various NSCLC cell lines after 48 h of treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: P7170 inhibited PI3K/mTOR signaling and the growth of erlotinib-resistant NSCLC cell lines. (A) Exponentially growing H460 cells were seeded in petridishes (2 × 106 cells/dish) and after cell attachment overnight, cells were serum starved for 14-16 h. Cells were then treated for 1 h with P7170 followed by 0.5 h incubation in the presence of 20% fetal bovine serum. Cells were trypsinized and lysed before electrophoresis of cellular extracts for western blot analyses of proteins of interest. (B) Quantification of pS6 and p4EBP1 protein levels in H460 cells after acquisition and analyses of immunofluorescence signals in the Cellomics Array scan platform. H460 cells were seeded in 96-well plates and treated with 0.1 or 1 μM of P7170 for 16 h; drug-containing media was removed and the cells were fixed and stained with antibodies to phosphorylated form of human S6 and 4EBP1 proteins and secondary antibodies conjugated to DyLight 549. Statistical significance values are as follows: *p < 0.05, **p < 0.01, ***p < 0.001. (C) IC50 of erlotinib or P7170 in various NSCLC cell lines after 48 h of treatment.
Mentions: We evaluated the activity of P7170 in cell based assays. The phosphorylation of AKT (S473) (substrate of mTORC2) [23], S6 (indirect substrate of mTORC1), and 4EBP1 (substrate of mTORC1) were nearly completely inhibited (100%) in H460 NSCLC cells upon treatment with 50 nM P7170. In the same experiment, phosphorylation of ERK, the effector of RAF-MEK-ERK pathway was marginally decreased (10%) (Figure 1A). The kinase activities of upstream PI3K alpha and mTOR were inhibited by P7170 (IC50 = 2.2 and 4.4 nM, respectively) but potent biochemical activity of PI3K did not translate in intact cells most likely because of feedback mechanism of mTOR inhibition. P7170 is a weak PI3K inhibitor (a separate manuscript submitted). In an immunofluorescence assay, P7170 treatment caused a consistent and marked decrease in the phosphorylation of S6 with a concentration-dependent suppression of p4EBP1 in H460 cells (Figure 1B, Additional file 1: Figure S1). Longer incubation time with P7170 resulted in an enhanced inhibition of pS6 and p4EBP1 (Additional file 1: Figure S1). The effect of P7170 on cell growth was evaluated in three different NSCLC cell lines, where a dose-dependent inhibition was observed. The IC50 of P7170 in EGFR over expressing A431 (EGFR wild type) cells was 10 nM compared to 5 and 7 nM in KRAS mutant A549 and H460 cell lines, respectively. In general, P7170 showed nano molar IC50 concentrations in the growth of various NSCLC cell lines as opposed to micro molar IC50 of erlotinib (Figure 1C).Figure 1

Bottom Line: P7170 at a well-tolerated daily dose of 20 mg/kg significantly inhibited the growth of NSCLC xenografts independent of different mutations (EGFR, KRAS, or PIK3CA) or sensitivity to erlotinib.Pharmacokinetic-pharmacodynamic (PK-PD) analysis showed sub-micro molar tumor concentrations along with mTORC1/C2 inhibition.Our results provide evidence of antitumor activity of P7170 in the erlotinib -sensitive and -insensitive models of NSCLC.

View Article: PubMed Central - PubMed

Affiliation: Piramal Life Sciences Ltd, # 1 Nirlon Complex, Off: Western Express Highway, Goregaon (East), Mumbai, Maharashtra 400063, India. veena_828@yahoo.com.

ABSTRACT

Background: Lung cancer is the major cause of cancer-related deaths and many cases of Non Small Cell Lung Cancer (NSCLC), a common type of lung cancer, have frequent genetic/oncogenic activation of EGFR, KRAS, PIK3CA, BRAF, and others that drive tumor growth. Some patients though initially respond, but later develop resistance to erlotinib/gefitinib with no option except for cytotoxic therapy. Therefore, development of novel targeted therapeutics is imperative to provide improved survival benefit for NSCLC patients. The mTOR cell survival pathway is activated in naïve, or in response to targeted therapies in NSCLC.

Methods: We have discovered P7170, a small molecule inhibitor of mTORC1/mTORC2/ALK1 and investigated its antitumor efficacy using various in vitro and in vivo models of human NSCLC.

Results: P7170 inhibited the phosphorylation of AKT, S6 and 4EBP1 (substrates for mTORC2 and mTORC1) levels by 80-100% and growth of NSCLC cells. P7170 inhibited anchorage-independent colony formation of NSCLC patient tumor-derived cells subsistent of disease sub-types. The compound also induced apoptosis in NSCLC cell lines. P7170 at a well-tolerated daily dose of 20 mg/kg significantly inhibited the growth of NSCLC xenografts independent of different mutations (EGFR, KRAS, or PIK3CA) or sensitivity to erlotinib. Pharmacokinetic-pharmacodynamic (PK-PD) analysis showed sub-micro molar tumor concentrations along with mTORC1/C2 inhibition.

Conclusions: Our results provide evidence of antitumor activity of P7170 in the erlotinib -sensitive and -insensitive models of NSCLC.

Show MeSH
Related in: MedlinePlus