EGFR Exon 20 Insertion Mutations Display Sensitivity to Hsp90 Inhibition in Preclinical Models and Lung Adenocarcinomas.

PURPOSE: EGFR exon 20 insertions account for up to 10% of all EGFR mutations in lung adenocarcinomas, representing the third most common cluster of mutations. The management of advanced cancers with these mutations remains elusive, without an approved inhibitor. EXPERIMENTAL DESIGN: Preclinical models of a representative set of EGFR exon 20 insertion mutations to evaluate the efficacy of different inhibitors and description of the clinical outcome of an advanced lung cancer. RESULTS: We show that select first-, second-, and third-generation EGFR inhibitors are unable to deter common EGFR exon 20 insertion mutants in concentrations that spare the wild-type kinase. Nonetheless, EGFR exon 20 insertion mutants associate with the Hsp90 chaperone system. We exploit this vulnerability to show that the nongeldanamycin Hsp90 inhibitor luminespib (formerly AUY922) degrades EGFR exon 20 mutations, downstream targets, and induces apoptosis. In addition, a patient whose EGFR inhibitor-insensitive lung adenocarcinoma harbored an EGFR exon 20 insertion mutation had a confirmed radiographic response to luminespib. CONCLUSIONS: The report confirms that EGFR exon 20 mutations are dependent on Hsp90 and are readily inhibited by the Hsp90 inhibitor luminespib; a treatment strategy that has been pursued in a confirmatory clinical trial (NCT01854034) for this group of lung adenocarcinomas that currently represent an unmet clinical need in precision oncology.

Tumor 5-FU-related mRNA Expression and Efficacy of Oral Fluoropyrimidines in Adjuvant Chemotherapy of Colorectal Cancer.

BACKGROUND: It has not been elucidated whether the clinical efficacy of oral fluoropyrimidines for adjuvant chemotherapy of colorectal cancer varies with tumor biological characteristics. PATIENTS AND METHODS: A multicenter randomized trial was performed comparing oral tegafur/gimeracil/oteracil (S-1) and uracil-tegafur/ leucovorin (UFT/LV) as adjuvant therapy for stage III colorectal cancer. Postoperative survival was compared based on the 5-FU-related mRNA levels in cancer tissues. RESULTS: Among patients with tumor expressing dihydropyrimidine dehydrogenase (DPD) mRNA within the 66.7th percentile (lower 2/3) of all cases, overall survival (OS) was significantly better in the S-1 than in the UFT/LV group. In the S-1 group, patients with low DPD-expressing tumors had significantly better OS than those with highly expressing tumors. Patients with low thymidine synthase (TS)-expressing tumors had significantly better OS than those with highly expressing tumors. CONCLUSION: The efficacy of oral fluoropyrimidines as adjuvant chemotherapy for colorectal cancer may be influenced by the level of 5-FU-related mRNA in cancer tissues.

Preclinical rationale for use of the clinically available multitargeted tyrosine kinase inhibitor crizotinib in ROS1-translocated lung cancer.

INTRODUCTION: Most clinically available small-molecule kinase inhibitors are multi-targeted and can inhibit multiple kinases. Our driving hypothesis was that one of these multi-targeted tyrosine kinase inhibitors (TKIs) would have antiproliferative activity against ROS1 translocated non-small-cell lung cancer (NSCLC). METHODS: We selected NSCLC cell lines--A549 (KRAS G12S), NCI-H3255 (EGFR L858R), NCI-H3122 (EML4-ALK E13;A20), and HCC78 (SLC34A2-ROS1)-to evaluate the antiproliferative effects of submicromolar concentrations of the multitargeted TKIs imatinib, sorafenib, erlotinib, and crizotinib. RESULTS: Imatinib and sorafenib were unable to significantly inhibit proliferation of the aforementioned cell lines. Erlotinib only inhibited EGFR mutated NCI-H3255, as expected. Crizotinib displayed dose-dependent inhibition of anaplastic lymphoma kinase translocated NCI-H3122 and also ROS1--translocated HCC78. The SLC34A2-ROS1 translocated HCC78 cell line had phosphorylated levels of ROS1, AKT, and ERK inhibited by submicromolar doses of crizotinib, and subsequently underwent apoptosis. CONCLUSIONS: The ROS1-translocated HCC78 cell line was sensitive to inhibition by the multitargeted ALK/MET/RON/ROS1 inhibitor crizotinib. Preclinical data supports the clinical development of crizotinib for ROS1-translocated NSCLC.

Feasibility study of adjuvant chemotherapy with S-1 (TS-1; tegafur, gimeracil and oteracil potassium) for colorectal cancer.

BACKGROUND/AIMS: The aim of this study is to evaluate feasibility and safety of 3-week method (3-week administration and 1-week withdrawal) for colorectal cancer as adjuvant chemotherapy with an oral anticancer drug, S-1. METHODOLOGY: Forty-two patients with stage II or III colorectal cancer who underwent curative resection in our hospital during a one year period in 2005 were enrolled in the preliminary pilot study. Between 2006 and 2007, 104 patients with stage II or III colorectal cancer who underwent curative resection in our hospital were chosen and were randomly divided into two groups, 3-week method or 4-week method (4-week administration and 2-week withdrawal) for a prospective randomized trial. RESULTS: The one-year completion rate in the 3-week method group was 98% (50/51) which was significantly better than that in the 4-week method group, 68% (36/53) (p=0.035). There were no grade 3 or 4 adverse reactions in both laboratory and clinical findings in the pilot study and in the prospective randomized trial. CONCLUSIONS: Three-week method of S-1 administration had good feasibility, easily manageable toxicity, high accumulated dose in one year and good compliance. The 3-week method with S-1 may be a standard adjuvant chemotherapy schedule for colorectal cancer.

Neoechinulin a impedes the progression of rotenone-induced cytotoxicity in PC12 cells.

Neoechinulin A, an indole alkaloid from marine fungi, can protect PC12 cells from the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP(+)), a Parkinson disease-inducing neurotoxin, by ameliorating downstream events resulting from mitochondrial complex I inactivation. However, the cytoprotective mechanisms remained unclear. In this study, by using rotenone, another parkinsonian-inducing neurotoxin targeting mitochondrial complex I, we investigated the cytoprotective mechanism of neoechinulin A. Rotenone-induced cell death was associated with accelerated glucose consumption, and excess glucose supplementation in the culture medium almost completely suppressed cell death, suggesting that glucose deficiency in the medium is critical for triggering cell death in this model. Co-treatment with neoechinulin A, but not neoechinulin A pre-treatment before rotenone exposure, significantly impeded cell death by rotenone. Although the presence of neoechinulin A did not affect the accelerated glycolytic turnover in rotenone-treated cells, it paradoxically decreased ATP levels in the cells, suggesting increased ATP consumption. Although the link between the decreased ATP levels and cytoprotection is not clear at present, it suggests that neoechinulin A may ameliorate rotenone toxicity by activating a cytoprotective machinery that requires ATP.

Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool.

Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.

Podophyllotoxin directly binds a hinge domain in E2 of HPV and inhibits an E2/E7 interaction in vitro.

Podophyllotoxin (PT), a strong cytotoxic agent from berberidaceae, has been known to inhibit tubulin polymerization. Although PT has been used for developing anticancer drugs as one of seed compounds, clinical treatment by itself has been unsuccessful because of the side effects, except one example in the treatments of warts. In this study, we screened peptides binding to PT with T7 phage display clonings in order to obtain more information about molecular mechanism of the action. A selected phage clone has a specific amino acid sequence to be SVPSRRRPDGRTHRSSRG. A homology search by protein database BLAST showed that this sequence had a similarity to a hinge domain (HD) of E2 protein in human papillomavirus (HPV) type 1a which is known to cause plantar warts. Surface plasmon resonance (SPR) analysis showed that PT bound to a recombinant HPV 1a E2 protein giving a K(D)=24.1microM which has compared with those of other domains of E2 protein. Also we demonstrated whether PT inhibited HD interaction or not. E7 protein of HPV has been known to be an oncoprotein and was reported to interact with HD of E2 protein. We demonstrated that an E2/E7 interaction was inhibited by the addition of PT in this report. And we showed the bindings of PT to other types of HPV. Our results suggest that PT is potential as a tool for clarifying the molecular mechanism of HPV.

A PhSeCl-mediated allylic oxidation of prenyl moiety: a convenient method for the construction of 3-isopenten-2-ol unit.

A phenylselenenyl chloride (PhSeCl)-mediated allylic oxidation to give allylically rearranged alcohol has been developed. A possible mechanism for the present reaction is generation of allylic selenide from prenyl moiety via [1,3]-sigmatropic rearrangement, followed by oxidation and [2,3]-sigmatropic rearrangement to afford 3-isopenten-2-ol.

Inhibitory effect of sulfoquinovosyl diacylglycerol on prokaryotic DNA polymerase I activity and cell growth of Escherichia coli.

We isolated the glycolipids fraction from spinach (Spinacia oleracea L.) and found that the fraction inhibited the activities of prokaryotic DNA polymerase I from Escherichia coli (E. coli) and cell growth of E. coli. The fraction contained mainly three glycolipids, monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG), and purified SQDG inhibited these activities, however, purified MGDG and DGDG had no influence. In the tested strains of E. coli, SQDG inhibited the cell proliferation of the JM109 strain. It could be considered that a SQDG-containing thylakoid membrane in plant chloroplasts might have anti-bacterial activity.

New beauveriolides produced by amino acid-supplemented fermentation of Beauveria sp. FO-6979.

Five new beauveriolides were isolated from the acetone extracts of Beauveria sp. FO-6979 mycelia fermented in amino acid-supplemented media. The structures were elucidated by spectroscopic studies including NMR experiments and chemical degradation. All the beauveriolides are cyclodepsipeptides consisting of one 3-hydroxy-4-methyl fatty acid, two L-amino acids and one D-amino acid in common. Beauveriolide VII with the structure of cyclo-[3-hydroxy-4-methyloctanoyl-L-phenylalanyl-L-alanyl-D-valyl] inhibited lipid droplet formation and cholesteryl ester synthesis in macrophages, but the other beauveriolides showed only slight or almost no effect on lipid droplet formation.

Selective production of fungal beauveriolide I or III by fermentation in amino acid-supplemented media.

Beauveriolides I and III, cyclic depsipeptides composed of L-Phe, L-Ala, D-Leu and (3S,4S)-3-hydroxy-4-methyloctanoic acid, and L-Phe, L-Ala, L-allo-Ile and (3S,4S)-3-hydroxy-4-methyloctanoic acid, respectively, were previously isolated from the culture broth of fungal Beauveria sp. FO-6979 as inhibitors of macrophage foam cell formation. To improve the production of these compounds by fermentation, the culture conditions were studied. The production of both beauveriolides was increased five to ten folds by fermentation in the culture media containing tryptone. Further study revealed that addition of L-Leu/L-Ile, but not D-Leu/D-allo-Ile, to the culture medium yielded a high and selective production of beauveriolide I or III. As a result, regardless of their separation difficulty due to the similar physico-chemical properties, a large amount of beauveriolide I or III was prepared from the culture broth obtained from L-Leu- or L-Ile-supplemented fermentation, respectively, by one step purification using silica gel column chromatography.