Nobuaki Shindoh

Mutations in G protein β 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 Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ 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 Gβγ dimer. Different mutations in Gβ 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.

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.

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.

Abstract A227: Antitumor activities of ASP3026 against EML4-ALK-dependent tumor models.

EML4-ALK is an oncogenic fusion kinase, which was first identified in non-small cell lung cancer (NSCLC), and is regarded as an attractive therapeutic target for treating a subpopulation of NSCLC patients. Crizotinib, which is inhibitor for MET and ALK, was recently approved by FDA (26 August 2011) for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) that is anaplastic lymphoma kinase (ALK)-positive as detected by an FDA-approved test. We synthesized and screened chemical compounds utilizing an ALK kinase inhibition assay aimed at the EML4-ALK target for drug discovery, and found ASP3026, a novel and selective inhibitor for the ALK kinase. ASP3026 potently inhibited ALK kinase activity and was more selective than crizotinib in a Tyr-kinase panel. In an anchorage independent in vitro cell growth assay, ASP3026 inhibited the growth of NCI-H2228, a human NSCLC tumor cell line endogenously expressing EML4-ALK variant 3 and that of 3T3 cells expressing EML4-ALK variant 1, 2 and 3. The plasma and tumor concentrations of ASP3026 in mice xenografted with NCI-H2228 tumor were determined using high-performance liquid chromatography-tandem mass spectrometry. Significant tumor penetration was observed. The antitumor activities were evaluated using mice bearing subcutaneous NCI-H2228 tumor xenografts. ASP3026, (daily oral dosing for 14 days) induced dose dependent anti-tumor effects starting at 1 mg/kg with marked regression at 10, 30 and 100 mg/kg. Body weights were unaffected. Crizotinib, (twice daily oral dosing) was less potent, with growth inhibition at 10 mg/kg, and tumor regression at 30 mg/kg. A dose of 100 mg/kg of crizotinib was poorly tolerated. Resistance mutations in ALK kinase domain against crizotinib were reported following sequence analysis of tumor cells derived from crizotinib-relapsed patients. The position of the mutation is the so-called gatekeeper mutation and is thought to be one of the causes of crizotinib relapse. In an EML4-ALK driven tumor model with gatekeeper mutation, ASP3026 showed potent anti-tumor effects while crizotinib was ineffective even at 100 mg/gk qd. In summary, ASP3026 has a broad safety margin and inhibitory activity at the gatekeeper mutation. Therefore, ASP3026 may still effective in EML4-ALK fusion positive NSCLC patients, that have relapsed to crizotinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A227.

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.

Abstract 2821: Anti-tumor activity of ASP3026, – A novel and selective ALK inhibitor -

EML4-ALK is an oncogenic fusion kinase which was first identified in non-small cell lung cancer (NSCLC), and is regarded as an attractive therapeutic target for treating a subpopulation of NSCLC patients. We synthesized and screened chemical compounds utilizing an ALK kinase inhibition assay aimed at the EML4-ALK target for drug discovery, and found ASP3026, a novel and selective inhibitor for the ALK kinase. ASP3026 inhibited ALK kinase activity at an IC 50 value of 3.5 nmol/L, and showed more selective ALK inhibition in a Tyr-kinase panel than PF02341066. In an anchorage independent in vitro cell growth assay, ASP3026 inhibited the growth of NCI-H2228, a human NSCLC tumor cell line endogenously expressing EML4-ALK variant 3, with an IC 50 value of 64.8 nmol/L. This growth inhibition was accompanied with the decrease in phosphorylation of EML4-ALK protein, indicating that ASP3026 exerts its anti-proliferative activity through ALK kinase inhibition. The plasma and tumor concentrations of ASP3026 in mice xenografted with NCI-H2228 tumor after a 5-day repeated oral dosing of ASP3026 (10 mg/kg once daily) were determined using high-performance liquid chromatography-tandem mass spectrometry. Tmax values were 4 h in plasma and tumors. Cmax values at the corresponding doses were, respectively, 875 nmol/mL and 15500 nmol/g. A decrease of phophorylated EML4-ALK was confirmed 4 hours after a single administration of ASP3026 at 10 mg/kg by Western-blot analysis. The antitumor activities were evaluated using mice bearing subcutaneous NCI-H2228 tumor xenografts. ASP3026, administered as twice daily oral dosing for 14 days, induced dose dependent anti-tumor effects starting at 1 mg/kg with strong regression at 10, 30 and 100 mg/kg. No influence on body weights was observed in all dose range of ASP3026 treated-mice. In contrast, PF02341066 at twice daily oral dosing resulted in growth inhibition of NCI-H2228 xenografted tumors at 10 mg/kg, and tumor regression at 30 mg/kg. In addition, 100 mg/kg of PF02341066 was intolerable in this model. These results suggest that ASP3026 is a novel and selective ALK inhibitor, which is orally active, and will possibly target NSCLC patients possessing the EML4-ALK fusion. We are starting phase I clinical trials of ASP3026 in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2821. doi:10.1158/1538-7445.AM2011-2821

Abstract PR07: Novel oncogenic mutations in the beta subunit of heteromeric G-proteins identified by functional cDNA library screening.

Although next-generation sequencing can delineate the genetic alterations within a primary tumor specimen, it can be difficult to distinguish the small number of driver mutations from the large number of passenger mutations. To overcome this issue, we developed a system for identifying oncogenic alterations directly from tumor cells. In this system, retroviral cDNA libraries built from cancer cell lines and directly from primary cancer samples are transduced into BaF3 cells, an IL3-dependent B cell line. Transformation by oncogenes promotes IL3-independent survival, allowing for the isolation of individual transformed clones and sequencing of the integrated cDNA. In the past, we identified CRLF2 as a novel oncogene in acute lymphoblastic leukemia (Yoda et al. PNAS 2010). We have improved the method and demonstrated 100% sensitivity for isolating well-characterized oncogenes, including EGFR, HER2, RAS and ALK (Shindoh et al. PLoS One 2012). Recently, we isolated a mutated GNB1 K89E allele from a cDNA library generated from a primary blastic plasmacytoid dendritic cell neoplasm (BPDCN). BPDCN is a rare and aggressive leukemia with a dismal prognosis. GNB1 encodes the beta subunit of the heterotrimeric G-protein, a binding complex that transduces signals from G-protein coupled receptors to multiple downstream pathways. Gain-of-function mutations have been reported in alpha subunits of the G-protein, however, the contributions of beta subunits to cancer remains undefined. To investigate downstream signaling from GNB1 K89E, we performed gene expression profiling and mass spectrometry (MS)-based phosphoproteomics and found significant activation of RAS/MAPK and PI3K/AKT pathways in GNB1 K89E-expressing cells compared to isogenic cells expressing wild-type GNB1. To target GNB1 K89E signaling, we screened kinase inhibitors using a multiplex panel of small molecules and found selective sensitivity of GNB1 K89E cells to MEK and pan-PI3-kinase inhibitors. Next, we transduced GNB1 alleles into bone marrow cells from Cdkn2a-deficient mice and transplanted into wild-type recipient mice. Within 4 months after transplantation, all mice (n=10) that received bone marrow transduced with GNB1 K89E developed a lethal dendritic cell malignancy, confirming the transforming effects of GNB1 K89E in vivo. A search of published cancer mutations identified four cases with GNB1 I80T/N in chronic lymphocytic leukemia or B-cell lymphomas, five cases with GNB1 K57E/T in myeloid malignancies, one case of GNB1 K89E in acute lymphoblastic leukemia, and two cases with GNB2 M101T/V in ovarian cancer. All of these alleles promoted GM-CSF-independent growth in human TF1 cells. Interestingly, the mutated codons are all located on the GNB1 molecular surface that is critical for interactions between GNB1 and both alpha subunits and downstream effectors. Immunoprecipitation followed by MS demonstrated that GNB1 K89E and I80T mutants failed to bind inhibitory G alpha subunits GNAI2 and GNAI3 as well as GNA11 that are bound by wild-type GNB1. Thus, gain-of-function mutations in G-protein beta subunits occur across a broad range of malignancies, can drive in vivo transformation, and activate targetable downstream kinases by modifying essential interactions with partner proteins. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR07. Citation Format: Akinori Yoda, Guillaume Adelmant, Nobuaki Shindoh, Bjoern Chapuy, Yuka Yoda, Oliver Weigert, Nadja Kopp, Shuo-Chieh Wu, Sunhee S. Kim, Huiyun Liu, Trevor Tivey, Jeffrey W. Tyner, Jason Gotlib, Michael W. Deininger, Shannon Turley, Jarrod A. Marto, Andrew A. Lane, David M. Weinstock. Novel oncogenic mutations in the beta subunit of heteromeric G-proteins identified by functional cDNA library screening. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR07.

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.

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

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.

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

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.