Next-Generation cDNA Screening for Oncogene and Resistance Phenotypes

There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo

Adsorption of benzene derivatives on allophane

The adsorption properties of benzene derivatives from water on allophane, extracted from soil, have been investigated by UV and FTIR spectroscopic measurements. Allophane adsorbs benzoic acid, phthalic acid, benzaldehyde, ethyl benzoate, and diethyl phthalate. Benzoic acid, phthalic acid, and benzaldehyde formed carboxylate anions on the positive sites of the hydrated alumina surface of allophane. In the case of adsorption from an acidic solution (pH 2), a small amount of a neutral species of benzoic acid was detected on the allophane. Ethyl benzoate and diethyl phthalate were adsorbed by an interaction between their carbonyl groups and the hydroxyl groups of the allophane. It was confirmed that allophane has an adsorption ability for the benzene derivatives that are not only ionic but also polar molecules. Allophane was found to be available as an absorbent for use in water purification by a simple procedure.ArticleAPPLIED CLAY SCIENCE. 43(2):160-163 (2009)journal articl

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Adsorption of benzene derivatives on allophane

The adsorption properties of benzene derivatives from water on allophane, extracted from soil, have been investigated by UV and FTIR spectroscopic measurements. Allophane adsorbs benzoic acid, phthalic acid, benzaldehyde, ethyl benzoate, and diethyl phthalate. Benzoic acid, phthalic acid, and benzaldehyde formed carboxylate anions on the positive sites of the hydrated alumina surface of allophane. In the case of adsorption from an acidic solution (pH 2), a small amount of a neutral species of benzoic acid was detected on the allophane. Ethyl benzoate and diethyl phthalate were adsorbed by an interaction between their carbonyl groups and the hydroxyl groups of the allophane. It was confirmed that allophane has an adsorption ability for the benzene derivatives that are not only ionic but also polar molecules. Allophane was found to be available as an absorbent for use in water purification by a simple procedure.ArticleAPPLIED CLAY SCIENCE. 43(2):160-163 (2009)journal articl

Identification of a novel oncogenic mutation of FGFR4 in gastric cancer

AbstractGastric cancer remains one of the leading causes of cancer death worldwide. Despite intensive investigations of treatments over the past three decades, the poor prognosis of patients with unresectable advanced or recurrent gastric cancer has not significantly changed, and improved therapies are required. Here, we report the identification of an oncogenic mutation in FGFR4 in a human gastric tumour that leads to constitutive activation of its product, FGFR4. The G636C-FGFR4 tyrosine kinase domain mutation was found in 1 of 83 primary human gastric tumours. The G636C mutation increased FGFR4 autophosphorylation, and activated FGFR4 downstream signalling molecules and enhanced anchorage-independent cell growth when expressed in NIH/3T3 cells. 3D-structural analysis and modelling of FGFR4 suggest that G636C destabilizes an auto-inhibitory conformation and stabilizes an active conformation, leading to increased kinase activation. Ba/F3 cell lines expressing the G636C-FGFR4 mutant were significantly more sensitive to ASP5878, a selective FGFR inhibitor, than the control. Oral administration of ASP5878 significantly inhibited the growth of tumours in mice engrafted with G636C-FGFR4/3T3 cells. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncoprotein. These findings support the therapeutic targeting of FGFR4 in gastric cancer.</jats:p

Abstract A170: Preclinical antitumor activity of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, in bladder cancer harboring FGFR3-fusion or -mutation

Abstract Background: Fibroblast growth factor (FGF) / FGF receptor (FGFR) gene aberrations such as amplification, mutation and fusion are associated with many types of human cancers. Recently, FGFR3 fusions (FGFR3-TACC3 and FGFR3-BAIAP2L1), which have potent oncogenic activity, have been discovered in bladder and lung cancer. FGFR kinase inhibitors are expected to be a targeted therapy for bladder cancer harboring FGFR3 gene alternations. A phase I clinical trial of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, is ongoing (NCT02038673). Method: We tested selectivity of ASP5878 among 128 kinases and sensitivity of ASP5878 on cell proliferation of bladder cancer cell lines. FGFR3 and ERK phosphorylation in FGFR3-dependent bladder cancer cell lines were evaluated with sandwich ELISA or Western blotting. In vivo antitumor effects of ASP5878 were examined in subcutaneously implanted bladder cancer cell lines in nude mice. Results: Among 128 kinases, only 9 kinases including wild-type FGFR1-4 and FGFR3/4 mutants were inhibited more than 50% by ASP5878 (200 nmol/L). The IC50 values of ASP5878 against FGFR1, 2, 3 and 4 kinases are 0.47, 0.60, 0.74 and 3.5 nmol/L, respectively. In addition, ASP5878 suppressed cell growth in several cancer cell lines harboring FGF / FGFR gene alternations. Among 25 bladder cancer cell lines, ASP5878 selectively inhibited cell proliferation of UM-UC-14 [FGFR3 (S249C) positive], RT-112 (FGFR3-TACC3 positive), RT-4 (FGFR3-TACC3 positive) and SW780 (FGFR3-BAIAP2L1 positive). FGFR3 and ERK phosphorylation in UM-UC-14 and RT-112 cell lines were inhibited by ASP5878 in a concentration-dependent manner. Furthermore, ASP5878 inhibited cell proliferation of gemcitabine-resistant RT-112 cells and adriamycin-resistant UM-UC-14 cells. Once-daily oral administration of ASP5878 induced tumor regression at 1 and 3 mg/kg in UM-UC-14 and RT-112 xenograft models, respectively, without body weight loss. Conclusions: These findings suggest that ASP5878 has the potential to be an oral targeted therapy against bladder cancer harboring FGFR3-TACC3 fusion or FGFR3 point mutation even after the acquisition of chemoresistance. Citation Format: Tomoyuki Suzuki, Aya Kikuchi, Taisuke Nakazawa, Masateru Iizuka, Ayako Nakayama, Minoru Kameda, Nobuaki Shindoh, Tadashi Terasaka, Masaaki Hirano, Sadao Kuromitsu. Preclinical antitumor activity of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, in bladder cancer harboring FGFR3-fusion or -mutation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A170.</jats:p

ASP5878, a Novel Inhibitor of FGFR1, 2, 3, and 4, Inhibits the Growth of FGF19-Expressing Hepatocellular Carcinoma

Abstract Hepatocellular carcinoma is an aggressive cancer with poor prognosis. Fibroblast growth factor 19, a member of the fibroblast growth factor family, is a ligand for fibroblast growth factor receptor 4. Moreover, it plays a crucial role in the progression of hepatocellular carcinoma. ASP5878 is a novel inhibitor of fibroblast growth factor receptors 1, 2, 3, and 4 that is under development. It inhibits fibroblast growth factor receptor 4 kinase activity with an IC50 of 3.5 nmol/L. ASP5878 potently suppressed the growth of the fibroblast growth factor 19–expressing hepatocellular carcinoma cell lines Hep3B2.1-7, HuH-7, and JHH-7. In the Hep3B2.1-7 cell line, ASP5878 inhibited the phosphorylation of fibroblast growth factor receptor 4 and its downstream signaling molecules as well as induced apoptosis. Oral administration of ASP5878 at 3 mg/kg induced sustained tumor regression in a subcutaneous xenograft mouse model using Hep3B2.1-7. In HuH-7, an orthotopic xenograft mouse model, ASP5878 induced complete tumor regression and dramatically extended the survival of the mice. These results suggest that ASP5878 is a potentially effective therapeutic agent for hepatocellular carcinoma patients with tumors expressing fibroblast growth factor 19. Mol Cancer Ther; 16(1); 68–75. ©2016 AACR.</jats:p

Abstract A172: Preclinical antitumor activity of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, in FGF19-expressing hepatocellular carcinoma

Abstract Background: Fibroblast growth factor (FGF) / FGF receptor (FGFR) gene aberrations such as amplification, mutation and fusion are associated with many types of human cancers. Recently, FGF19 overexpression was observed in approximately 50% of hepatocellular carcinoma (HCC) patients. The FGF19-FGFR4 signaling has been implicated in the development of HCCs in mice. FGFR4 kinase inhibitors are expected to be a targeted therapy for FGF19-expressing HCC. A phase I clinical trial of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, is ongoing (NCT02038673). Method: We tested selectivity of ASP5878 among 128 kinases and sensitivity of ASP5878 on cell proliferation of HCC cell lines. Activation of FRS2 and ERK, downstream molecules of FGFR signaling, and PARP cleavage in FGF19 expressing HCC cell lines were evaluated with Western blotting. In vivo antitumor effects of ASP5878 were examined in HCC subcutaneous xenograft and orthotopic inoculation mouse models. Finally, plasma levels of FGF19 were measured after dosing ASP5878. Results: Among 128 kinases, only 9 kinases including FGFR1-4 and FGFR3/4 mutations were inhibited more than 50% by ASP5878 (200 nmol/L). The IC50 values of ASP5878 against FGFR1, 2, 3 and 4 kinases were 0.47, 0.60, 0.74 and 3.5 nmol/L, respectively. ASP5878 inhibited cell proliferation of HCC cell lines with FGF19 overexpression. IC50 values were 8.5, 27, and 21 nmol/L in Hep3B2.1-7, HuH-7 and JHH-7, respectively. ASP5878 inhibited activation of downstream signaling molecules, FRS2 and ERK, and induced apoptosis in Hep3B2.1-7 cells. Oral dosing of ASP5878 at 3 mg/kg induced sustained tumor regression in the Hep3B2.1-7 subcutaneous xenograft model, which was poorly responsive to sorafenib. In an HuH-7 orthotopic inoculation mouse model, ASP5878 induced complete tumor regression and dramatically extended the survival. In addition, oral dosing of ASP5878 reduced plasma levels of FGF19 in the HuH-7 subcutaneous xenograft model.Conclusion: These results suggest that ASP5878 is a potentially effective therapeutic agent for FGF19-expressing HCC. Citation Format: Takashi Futami, Hidetsugu Okada, Rumi Kihara, Tatsuya Kawase, Ayako Nakayama, Tomoyuki Suzuki, Minoru Kameda, Nobuaki Shindoh, Tadashi Terasaka, Masaaki Hirano, Sadao Kuromitsu. Preclinical antitumor activity of ASP5878, a novel inhibitor of FGFR1, 2, 3 and 4, in FGF19-expressing hepatocellular carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A172.</jats:p