Identification of a novel oncogenic mutation of FGFR4 in gastric cancer.

Gastric 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.

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

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.

ASP5878, a selective FGFR inhibitor, to treat FGFR3-dependent urothelial cancer with or without chemoresistance.

FGF/FGFR gene aberrations such as amplification, mutation and fusion are associated with many types of human cancers including urothelial cancer. FGFR kinase inhibitors are expected to be a targeted therapy for urothelial cancer harboring FGFR3 gene alternations. ASP5878, a selective inhibitor of FGFR1, 2, 3 and 4 under clinical investigation, selectively inhibited cell proliferation of urothelial cancer cell lines harboring FGFR3 point mutation or fusion (UM-UC-14, RT-112, RT4 and SW 780) among 23 urothelial cancer cell lines. Furthermore, ASP5878 inhibited cell proliferation of adriamycin-resistant UM-UC-14 cell line harboring MDR1 overexpression and gemcitabine-resistant RT-112 cell line. The protein expression of c-MYC, an oncoprotein, in gemcitabine-resistant RT-112 cell line was higher than that in RT-112 parental cell line and ASP5878 decreased the c-MYC expression in both RT-112 parental and gemcitabine-resistant RT-112 cell lines. Once-daily oral administration of ASP5878 exerted potent antitumor activities in UM-UC-14, RT-112 and gemcitabine-resistant RT-112 xenograft models without affecting body weight. These findings suggest that ASP5878 has the potential to be an oral targeted therapy against urothelial cancer harboring FGFR3 fusion or FGFR3 point mutation after the acquisition of gemcitabine- or adriamycin-resistance.

Mutations in G protein ß subunits promote transformation and kinase inhibitor resistance.

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gß subunits have not been defined. Here we demonstrate that recurrent mutations in the Gß proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gßγ dimer. Different mutations in Gß proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

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.

Histone deacetylase inhibitors from microorganisms: the Astellas experience.

Histone deacetylase (HDAC) inhibitors, such as trichostatin A and trapoxin, which were first found in microorganisms, potently and selectively inhibit HDAC enzymes. They have made a strong contribution to research on HDACs, chromatin control, abnormal epigenetic control in various diseases and the significance of acetylation in posttranslational modification. Recently, HDAC inhibitors have been focused on as potential drugs for the treatment of several diseases, including cancer, although trichostatin A and trapoxin show no effects in animal models because of their metabolic instability in vivo. Chemical modification has been conducted in order to overcome this drawback. We discovered the microbial metabolites FK228 (also known as FR901228, romidepsin, depsipeptide, NSC-630176 and NSC-630176D) and YM753 (spiruchostatin A). Both compounds have bicyclic structures and represent a novel structural class of HDAC inhibitor. The enzyme and tumor cell growth inhibitory activities of FK228 were found to be very potent. It also showed potent HDAC inhibitory activity in vivo. FK228 is the first potent HDAC inhibitor to undergo clinical development as a potential treatment for solid and hematological cancers. Due to its dramatic effect in patients with refractory cutaneous T-cell lymphoma (CTCL), in October 2004 the US Food & Drug Administration (FDA) granted fast-track status to FK228 as monotherapy for the treatment of CTCL in patients who have relapsed following, or become refractory to, another systemic therapy. Thus HDAC inhibitors such as FK228 and YM753 have potential as tools for life science studies and also as therapeutic agents for various intractable diseases.

YM753, a novel histone deacetylase inhibitor, exhibits antitumor activity with selective, sustained accumulation of acetylated histones in tumors in the WiDr xenograft model.

Histone deacetylase (HDAC) inhibitors have been shown to have antitumor activity in vitro and in vivo. Various studies related to their antitumor activity and mechanism of action have been reported for HDAC inhibitors, but the relationship of their antitumor effects to their pharmacodynamic and pharmacokinetic properties in vivo has not ever fully characterized. We report here the discovery of a novel cyclic-peptide-based HDAC inhibitor, YM753. YM753 is a bacteria-derived natural product containing a disulfide bond. It potently inhibited HDAC enzyme with an IC50 of 2.0 nM in the presence of dithiothreitol. YM753 was rapidly converted to a reduced form in tumor cells, and then induced accumulation of acetylated histones, followed by p21WAF1/Cip1 expression, tumor cell growth inhibition and tumor-selective cell death. In an in vitro washout study, YM753 showed prolonged accumulation of acetylated histones in WiDr human colon carcinoma cells. In vivo YM753 dosing of mice harboring WiDr colon tumor xenografts significantly inhibited the tumor growth via sustained accumulation of acetylated histones in the tumor tissue. In a pharmacokinetic study, YM753 rapidly disappeared from the plasma, but its reduced form remained in the tumor tissue. Moreover, the accumulation of acetylated histones induced by YM753 was tumor tissue selective compared to several normal tissues. This study provides evidence that YM753 has antitumor activity that is the result of selective, sustained accumulation of acetylated histones in tumor tissues despite rapid disappearance of the drug from the plasma. These results suggest that the novel HDAC inhibitor, YM753 has attractive pharmacodynamic and pharmacokinetic properties giving it potential as an antitumor agent.

Three new cyclostellettamines, which inhibit histone deacetylase, from a marine sponge of the genus Xestospongia.

Three new cyclostellettamines, cyclostellettamine G (1), dehydrocyclostellettamines D (2), and E (3), were isolated together with the known cyclostellettamine A (4) from a marine sponge of the genus Xestospongia as histone deacetylase inhibitors. Their structures were determined by spectral and chemical methods. They inhibit histone deacetylase derived from K562 human leukemia cells with IC(50) values ranging from 17 to 80 microM.