pi103 and Carcinoma--Non-Small-Cell-Lung

The third-generation of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), represented by osimertinib, has achieved remarkable clinical outcomes in the treatment of non-small-cell lung cancer (NSCLC) with EGFR mutation. However, resistance eventually emerges in most patients and the underlying molecular mechanisms remain to be fully understood. In this study, we generated an osimertinib-acquired resistant lung cancer model from a NSCLC cell line H1975 harboring EGFR L858R and T790M mutations. We found that the capacity of DNA damage repair was compromised in the osimertinib resistant cells, evidenced by increased levels of γH2AX and higher intensity of the comet tail after withdrawal from cisplatin. Pharmacological inhibiting the activity or genetic knockdown the expression of DNA-PK, a key kinase in DNA damage response (DDR), sensitized the resistant cells to osimertinib. Combination of osimertinib with the DNA-PK inhibitor, PI-103, or NU7441, synergistically suppressed the proliferation of the resistant cells. Mechanistically, we revealed that DNA-PK inhibitor in combination with osimertinib resulted in prolonged DNA damage and cell cycle arrest. These findings shed new light on the mechanisms of osimertinib resistance in the aspect of DNA repair, and provide a rationale for targeting DNA-PK as a therapeutic strategy to overcome osimertinib-acquired resistance in NSCLC.

Topics: Acrylamides; Aniline Compounds; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Line, Tumor; Chromones; DNA Damage; DNA Repair; DNA-Activated Protein Kinase; Drug Resistance, Neoplasm; Drug Synergism; ErbB Receptors; Furans; Humans; Lung Neoplasms; Morpholines; Mutation; Protein Kinase Inhibitors; Pyridines; Pyrimidines

K-RAS mutated (K-RASmut) non-small cell lung cancer (NSCLC) cells are resistant to EGFR targeting strategies. We investigated the impact of K-RAS activity irrespective of mutational status in the EGFR-independent increase in clonogenic cell survival. An analysis of the K-RAS activity status revealed a constitutively high K-RAS activity in K-RASmut NSCLC cells and also in head and neck squamous cell carcinoma (HNSCC) cells overexpressing wild-type K-RAS (K-RASwt). Similar to K-RAS-mutated cells, increased K-RAS activity in HNSCC cells overexpressing K-RASwt was associated with the stimulated production of the EGFR ligand amphiregulin and resistance to EGFR tyrosine kinase (EGFR-TK) inhibitors such as erlotinib. Expression of mutated K-RAS stimulated Akt phosphorylation and increased plating efficiency. Conversely, knockdown of K-RAS in K-RASmut NSCLC cells and in HNSCC cells presenting overexpression of K-RASwt resulted in sensitization to the anti-clonogenic activity of erlotinib. K-RAS activity results in EGFR-dependent and EGFR-independent Akt activity. The short-term treatment (2 h) of cells with EGFR-TK or PI3K inhibitors (erlotinib and PI-103) resulted in the repression of Akt activation, whereas long-term treatment (24 h) with inhibitors led to the reactivation of Akt and improved clonogenicity. The Akt re-activation was MAPK-ERK2-dependent and associated with a lack of complete response to anti-clonogenic activity of PI-103. A complete response was observed when PI-103 was combined with MEK inhibitor PD98059. Together, clonogenicity inhibition in tumor cells presenting constitutive K-RAS activity independent of K-RAS mutational status can be achieved by targeting of EGFR downstream pathways, i.e., PI3K alone or the combination of PI3K and MAPK inhibitors.

Topics: Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Clone Cells; Drug Synergism; ErbB Receptors; Erlotinib Hydrochloride; Flavonoids; Furans; Head and Neck Neoplasms; Humans; Lung Neoplasms; Mitogen-Activated Protein Kinase 1; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Pyridines; Pyrimidines; Quinazolines; ras Proteins; Signal Transduction

Reversible epidermal growth factor receptor (EGFR) inhibitors are the first class of small molecules to improve progression-free survival of patients with EGFR-mutated lung cancers. Second-generation EGFR inhibitors introduced to overcome acquired resistance by the T790M resistance mutation of EGFR have thus far shown limited clinical activity in patients with T790M-mutant tumors. In this study, we systematically analyzed the determinants of the activity and selectivity of the second-generation EGFR inhibitors. A focused library of irreversible as well as structurally corresponding reversible EGFR-inhibitors was synthesized for chemogenomic profiling involving over 79 genetically defined NSCLC and 19 EGFR-dependent cell lines. Overall, our results show that the growth-inhibitory potency of all irreversible inhibitors against the EGFR(T790M) resistance mutation was limited by reduced target inhibition, linked to decreased binding velocity to the mutant kinase. Combined treatment of T790M-mutant tumor cells with BIBW-2992 and the phosphoinositide-3-kinase/mammalian target of rapamycin inhibitor PI-103 led to synergistic induction of apoptosis. Our findings offer a mechanistic explanation for the limited efficacy of irreversible EGFR inhibitors in EGFR(T790M) gatekeeper-mutant tumors, and they prompt combination treatment strategies involving inhibitors that target signaling downstream of the EGFR.

Topics: Afatinib; Amino Acid Substitution; Animals; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Cell Proliferation; Cluster Analysis; Drug Resistance, Neoplasm; ErbB Receptors; Furans; Humans; Lung Neoplasms; Molecular Structure; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Pyridines; Pyrimidines; Quinazolines; Receptor, ErbB-2; Signal Transduction

Non-small cell lung cancer (NSCLC) with certain activating mutations in the epidermal growth factor receptor (EGFR) is sensitive to the small molecule EGFR tyrosine kinase inhibitors gefitinib and erlotinib, although acquired resistance eventually develops. Resistance is often mediated by acquisition of the T790M mutation in the activated EGFR allele. The aim of this study was to investigate in an EGFR tyrosine kinase inhibitor sensitive NSCLC cell line model, the impact of induced EGFR T790M expression on the cell biology and sensitivity to novel therapeutic strategies.. Doxycycline inducible EGFR T790M-mediated drug resistance was generated in the clinically relevant HCC827 NSCLC cell line. Cell fate, the activities of EGFR and downstream signaling molecules, and the sensitivity to downstream inhibition of EGFR signaling networks were examined in the presence or absence of induced EGFR T790M expression.. Inducible EGFR T790M expression generated acquired resistance to EGFR inhibitors in HCC827 cells as expected. However, induced EGFR T790M expression did not affect activity of EGFR downstream signaling pathways or cell proliferation under the conditions tested. Moreover, sensitivity to inhibition of signaling molecules downstream of EGFR was unaffected by induced EGFR T790M. Importantly, HCC827 cells remained sensitive to class I phosphatidyl-inositol-3-kinase and mammalian target of rapamycin inhibition, which provoked pronounced autophagy, without significant apoptosis.. Phosphatidyl-inositol-3-kinase /mammalian target of rapamycin inhibition is a potentially effective therapeutic strategy against NSCLC with acquired resistance to EGFR inhibition. However, the implications of drug-induced autophagy in NSCLC need further exploration.

Topics: Antineoplastic Agents; Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Furans; Gefitinib; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Mutation; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Pyridines; Pyrimidines; Quinazolines; TOR Serine-Threonine Kinases

PI-103, the first synthetic multitargeted compound which simultaneously inhibits PI3Kalpha and mammalian target of rapamycin (mTOR) shows high antitumor activity in glioma xenografts. In the present study, clear antitumor activity was observed with PI-103 treatment in two gefitinib-resistant non-small cell lung cancer (NSCLC) cell lines, A549 and H460, by simultaneously inhibiting p70s6k phosporylation and Akt phosphorylation in response to mTOR inhibition. In addition, H460 cells with activating mutations of PIK3CA were more sensitive to PI-103 than A549 cells with wild-type PIK3CA. PI-103 was found to inhibit growth by causing G0-G1 arrest in A549 and H460 cells. Western blotting showed that PI-103 induced down-regulation of cyclin D1 and E1 and simultaneously up-regulated p21 and p27, associated with arrest in the G0-G1 phase of the cell cycle. Furthermore, p53, the tumor suppressor which transcriptionally regulates p21, was also upregulated with PI-103 treatment. Collectively, our results suggest that multitargeted intervention is the most effective tumor therapy, and the cooperative blockade of PI3Kalpha and mTOR with PI-103 shows promise for treating gefitinib-resistant NSCLC.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Drug Resistance, Neoplasm; Furans; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Protein Kinases; Pyridines; Pyrimidines; Quinazolines; TOR Serine-Threonine Kinases; Tumor Suppressor Proteins