The pro-tumorigenic activity of p38γ overexpression in nasopharyngeal carcinoma.

It is urgent to identify and validate biomarkers for early diagnosis and efficient treatment of nasopharyngeal carcinoma (NPC). Recent studies have proposed p38 gamma (p38γ) as a cyclin-dependent kinase (CDK)-like kinase that phosphorylates retinoblastoma (Rb) to promote cyclins expression and tumorigenesis. Here the Gene Expression Profiling Interactive Analysis (GEPIA) database and results from the local NPC tissues demonstrate that p38γ is significantly upregulated in NPC tissues, correlating with poor overall survival. Furthermore, p38γ mRNA and protein expression is elevated in established NPC cell lines (CNE-1 HONE-1 and CNE-2) and primary human NPC cells, but low expression detected in human nasal epithelial cells. In established and primary NPC cells, p38γ depletion, using the shRNA strategy or the CRISPR/Cas9 gene-editing method, largely inhibited cell growth, proliferation and migration, and induced significant apoptosis activation. Contrarily, ectopic p38γ overexpression exerted opposite activity and promoted NPC cell proliferation and migration. Retinoblastoma (Rb) phosphorylation and cyclin E1/A expression were decreased in NPC cells with p38γ silencing or knockout, but increased after p38γ overexpression. Moreover, mitochondrial subcellular p38γ localization was detected in NPC cells. Significantly, p38γ depletion disrupted mitochondrial functions, causing mitochondrial depolarization, reactive oxygen species production, oxidative injury and ATP depletion in NPC cells. In vivo, intratumoral injection of adeno-associated virus-packed p38γ shRNA potently inhibited primary human NPC xenograft growth in nude mice. In p38γ shRNA virus-injected NPC xenograft tissues, p38γ expression, Rb phosphorylation, cyclin E1/A expression and ATP levels were dramatically decreased. Taken together, we conclude that p38γ overexpression is required for NPC cell growth, acting as a promising therapeutic target of NPC.

GNE-493 inhibits prostate cancer cell growth via Akt-mTOR-dependent and -independent mechanisms.

GNE-493 is a novel PI3K/mTOR dual inhibitor with improved metabolic stability, oral bioavailability, and excellent pharmacokinetic parameters. Here GNE-493 potently inhibited viability, proliferation, and migration in different primary and established (LNCaP and PC-3 lines) prostate cancer cells, and provoking apoptosis. GNE-493 blocked Akt-mTOR activation in primary human prostate cancer cells. A constitutively-active mutant Akt1 restored Akt-mTOR activation but only partially ameliorated GNE-493-induced prostate cancer cell death. Moreover, GNE-493 was still cytotoxic in Akt1/2-silenced primary prostate cancer cells. Significant oxidative stress and programmed necrosis cascade activation were detected in GNE-493-treated prostate cancer cells. Moreover, GNE-493 downregulated Sphingosine Kinase 1 (SphK1), causing ceramide accumulation in primary prostate cancer cells. Daily single dose GNE-493 oral administration robustly inhibited the growth of the prostate cancer xenograft in the nude mice. Akt-mTOR inactivation, SphK1 downregulation, ceramide level increase, and oxidative injury were detected in GNE-493-treated prostate cancer xenograft tissues. Together, GNE-493 inhibited prostate cancer cell growth possibly through the Akt-mTOR-dependent and -independent mechanisms.

Crystal structure of EGFR T790M/C797S/V948R in complex with EAI045.

Lung cancer is the leading cause of cancer deaths. Epidermal growth factor receptor (EGFR) kinase domain mutations are a common cause of non-small cell lung cancers (NSCLCs), a major subtype of lung cancers. Patients harboring most of these mutations respond well to the anti-EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib initially, but soon develop resistance to them in about half of the cases due to the emergence of the gatekeeper mutation T790M. The third-generation TKIs such as AZD9291, HM61713, CO-1686 and WZ4002 can overcome T790M through covalent binding to the EGFR kinase through Cys 797, but ultimately lose their efficacy upon emergence of the C797S mutation that abolishes the covalent bonding. Therefore to develop new TKIs to overcome EGFR drug-resistant mutants harboring T790M/C797S is urgently demanded. EAI001 and EAI045 are a new type of EGFR TKIs that bind to EGFR reversibly and not relying on Cys 797. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR L858R/T790M and L858R/T790M/C797S. Here we report the crystal structure of EGFR T790M/C797S/V948R in complex with EAI045, and compare it to EGFR T790M/V948R in complex with EAI001. The complex structure reveals why EAI045 binds tighter to EGFR than does EAI001, and why EAI001 and EAI045 prefer binding to EGFR T790M. The knowledge may facilitate future drug development studies targeting this very important cancer target.

Structural pharmacological studies on EGFR T790M/C797S.

Drug-resistance is a major challenge in targeted therapy of EGFR mutated non-small cell lung cancers (NSCLCs). The third-generation irreversible inhibitors such as AZD9291, CO-1686 and WZ4002 can overcome EGFR T790M drug-resistance mutant through covalent binding through Cys 797, but ultimately lose their efficacy upon emergence of the new mutation C797S. To develop new reversible inhibitors not relying on covalent binding through Cys 797 is therefore urgently demanded. Gö6976 is a staurosporine-like reversible inhibitor targeting T790M while sparing the wild-type EGFR. In the present work, we reported the complex crystal structures of EGFR T790M/C797S + Gö6976 and T790M + Gö6976, along with enzyme kinetic data of EGFR wild-type, T790M and T790M/C797S. These data showed that the C797S mutation does not significantly alter the structure and function of the EGFR kinase, but increases the local hydrophilicity around residue 797. The complex crystal structures also elucidated the detailed binding mode of Gö6976 to EGFR and explained why this compound prefers binding to T790M mutant. These structural pharmacological data would facilitate future drug development studies.