Nukinori Akiyama

The Fibroblast Growth Factor Receptor Genetic Status as a Potential Predictor of the Sensitivity to CH5183284/Debio 1347, a Novel Selective FGFR Inhibitor

The FGF receptors (FGFR) are tyrosine kinases that are constitutively activated in a subset of tumors by genetic alterations such as gene amplifications, point mutations, or chromosomal translocations/rearrangements. Recently, small-molecule inhibitors that can inhibit the FGFR family as well as the VEGF receptor (VEGFR) or platelet-derived growth factor receptor (PDGFR) family displayed clinical benefits in cohorts of patients with FGFR genetic alterations. However, to achieve more potent and prolonged activity in such populations, a selective FGFR inhibitor is still needed. Here, we report the identification of CH5183284/Debio 1347, a selective and orally available FGFR1, FGFR2, and FGFR3 inhibitor that has a unique chemical scaffold. By interacting with unique residues in the ATP-binding site of FGFR1, FGFR2, or FGFR3, CH5183284/Debio 1347 selectively inhibits FGFR1, FGFR2, and FGFR3 but does not inhibit kinase insert domain receptor (KDR) or other kinases. Consistent with its high selectivity for FGFR enzymes, CH5183284/Debio 1347 displayed preferential antitumor activity against cancer cells with various FGFR genetic alterations in a panel of 327 cancer cell lines and in xenograft models. Because of its unique binding mode, CH5183284/Debio 1347 can inhibit FGFR2 harboring one type of the gatekeeper mutation that causes resistance to other FGFR inhibitors and block FGFR2 V564F–driven tumor growth. CH5183284/Debio 1347 is under clinical investigation for the treatment of patients harboring FGFR genetic alterations. Mol Cancer Ther; 13(11); 2547–58. ©2014 AACR.

Mechanism of Oncogenic Signal Activation by the Novel Fusion Kinase FGFR3-BAIAP2L1

Recent cancer genome profiling studies have identified many novel genetic alterations, including rearrangements of genes encoding FGFR family members. However, most fusion genes are not functionally characterized, and their potentials in targeted therapy are unclear. We investigated a recently discovered gene fusion between FGFR3 and BAI1-associated protein 2-like 1 (BAIAP2L1). We identified 4 patients with bladder cancer and 2 patients with lung cancer harboring the FGFR3–BAIAP2L1 fusion through PCR and FISH assay screens. To investigate the oncogenic potential of the fusion gene, we established an FGFR3–BAIAP2L1 transfectant with Rat-2 fibroblast cells (Rat-2_F3-B). The FGFR3–BAIAP2L1 fusion had transforming activity in Rat2 cells, and Rat-2_F3-B cells were highly tumorigenic in mice. Rat-2_F3-B cells showed in vitro and in vivo sensitivity in the selective FGFR inhibitor CH5183284/Debio 1347, indicating that FGFR3 kinase activity is critical for tumorigenesis. Gene signature analysis revealed that FGFR3–BAIAP2L1 activates growth signals, such as the MAPK pathway, and inhibits tumor-suppressive signals, such as the p53, RB1, and CDKN2A pathways. We also established Rat-2_F3-B-ΔBAR cells expressing an FGFR3–BAIAP2L1 variant lacking the Bin–Amphiphysin–Rvs (BAR) dimerization domain of BAIAP2L1, which exhibited decreased tumorigenic activity, FGFR3 phosphorylation, and F3-B-ΔBAR dimerization, compared with Rat-2_F3-B cells. Collectively, these data suggest that constitutive dimerization through the BAR domain promotes constitutive FGFR3 kinase activation and is essential for its potent oncogenic activity. Mol Cancer Ther; 14(3); 704–12. ©2015 AACR.

ERK Signal Suppression and Sensitivity to CH5183284/Debio 1347, a Selective FGFR Inhibitor

Drugs that target specific gene alterations have proven beneficial in the treatment of cancer. Because cancer cells have multiple resistance mechanisms, it is important to understand the downstream pathways of the target genes and monitor the pharmacodynamic markers associated with therapeutic efficacy. We performed a transcriptome analysis to characterize the response of various cancer cell lines to a selective fibroblast growth factor receptor (FGFR) inhibitor (CH5183284/Debio 1347), a mitogen-activated protein kinase kinase (MEK) inhibitor, or a phosphoinositide 3-kinase (PI3K) inhibitor. FGFR and MEK inhibition produced similar expression patterns, and the extracellular signal–regulated kinase (ERK) gene signature was altered in several FGFR inhibitor–sensitive cell lines. Consistent with these findings, CH5183284/Debio 1347 suppressed phospho-ERK in every tested FGFR inhibitor–sensitive cell line. Because the mitogen-activated protein kinase (MAPK) pathway functions downstream of FGFR, we searched for a pharmacodynamic marker of FGFR inhibitor efficacy in a collection of cell lines with the ERK signature and identified dual-specificity phosphatase 6 (DUSP6) as a candidate marker. Although a MEK inhibitor suppressed the MAPK pathway, most FGFR inhibitor–sensitive cell lines are insensitive to MEK inhibitors and we found potent feedback activation of several pathways via FGFR. We therefore suggest that FGFR inhibitors exert their effect by suppressing ERK signaling without feedback activation. In addition, DUSP6 may be a pharmacodynamic marker of FGFR inhibitor efficacy in FGFR-addicted cancers. Mol Cancer Ther; 14(12); 2831–9. ©2015 AACR.

Acquired JHDM1D-BRAF fusion confers resistance to FGFR inhibition in FGFR2- amplified gastric cancer

FGFR2 gene is frequently amplified in gastric cancer. Recently, targeting FGFR2 has drawn attention as a form of gastric cancer therapy, and FGFR-selective inhibitors have shown promising efficacy in clinical studies. Because overcoming acquired resistance is a common problem with molecular targeting drugs, we investigated a resistant mechanism of FGFR inhibitors using the gastric cancer cell line SNU-16, which harbors FGFR2 amplification. We established single-cell clones of FGFR inhibitor–resistant SNU-16 (AZD-R) by continuous exposure to AZD4547, a selective FGFR inhibitor. To screen the genetic alterations acquired in AZD-R, we ran a comparative genomic hybridization assay and found an amplification of Chr7q34 region. The chromosomal breakpoints were located between the 12th and the 13th exon of jumonji C domain containing histone demethylase 1 homolog D (JHDM1D) and between the 3rd and the 4th exon of BRAF. We sequenced cDNA of the AZD-R clones and found fusion kinase JHDM1D-BRAF, which has previously been identified in primary ovarian cancer. Because JHDM1D–BRAF fusion lacks a RAS-binding domain, the dimerization of JHDM1D–BRAF was enhanced. A cell growth inhibition assay using MEK inhibitors and RAF-dimer inhibitors indicated the dependence of AZD-R clones for growth on the MAPK pathway. Our data provide a clinical rationale for using a MEK or RAF dimer inhibitor to treat FGFR2-amplified gastric cancer patients who have acquired resistance through the JHDN1D–BRAF fusion. Mol Cancer Ther; 17(10); 2217–25. ©2018 AACR.

Abstract 123: Mechanism of oncogenic signal activation by the novel fusion kinase FGFR3-BAIAP2L1

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Recent cancer genome profiling studies have identified many novel genetic alterations, including rearrangements of genes encoding fibroblast growth factor receptor (FGFR) family members. However, most fusion genes are not functionally well characterized, and the oncogenicity of some fusions as well as their potential sensitivity to targeted therapy are still unclear. In a previous study, we investigated a recently discovered gene fusion between FGFR3 and BAI1-associated protein 2-like 1 (BAIAP2L1). The FGFR3-BAIAP2L1 fusion gene was identified in 4 bladder cancer patients and 2 lung cancer patients via screens involving PCR and a break-apart fluorescence in situ hybridization assay. The functional analysis of FGFR3-BAIAP2L1 transfectant in Rat-2 fibroblast cells (Rat-2\_F3-B) indicated that FGFR3-BAIAP2L1 forms a dimer via the Bin-Amphiphysin-Rvs (BAR) BAIAP2L1 dimerization domain that constitutively activates its FGFR3 kinase activity. CH5183284/Debio 1347*, a selective FGFR inhibitor, effectively inhibits growth of Rat-2\_F3-B both in vitro and in vivo, indicating that the FGFR3 kinase activity is critical for tumorigenic activity of this fusion. These results indicate a potential application of FGFR inhibitors to treat FGFR3-BAIAP2L1fusion gene positive patients. In this study, we further elucidate the mechanism of tumorigenic potential of FGFR3-BAIAP2L1A by a comprehensive gene expression analysis of 4 cell lines (Rat-2\_mock, Rat-2\_FGFR3, Rat-2\_F3-B, and Rat-2\_BAIAP2L1) using RNA sequencing. We identified 143 up-regulated and 67 down-regulated genes specifically engaged by FGFR3-BAIAP2L1. Our gene signature analysis with this gene set revealed that FGFR3-BAIAP2L1 activates growth signals, such as the mitogen-activated protein kinase pathway, and inhibits tumor-suppressive signals, such as the p53, RB1, and CDKN2A pathways. Analysis by Western blot in xenograft tissues confirmed the activation and inactivation status of those pathways. These data suggest that a concurrent regulation of an oncogenic pathway and a tumor-suppressive pathway results in the tumorigenic potential of FGFR3-BAIAP2L1. *CH5183284/Debio 1347 was discovered by Chugai Pharmaceutical Co., Ltd. and is being developed by Debiopharm International S.A. under an exclusive worldwide license. Citation Format: Yoshito Nakanishi, Nukinori Akiyama, Toshiyuki Tsukaguchi, Toshihiko Fujii, Yasuko Satoh, Hideaki Mizuno, Nobuya Ishii, Masahiro Aoki. Mechanism of oncogenic signal activation by the novel fusion kinase FGFR3-BAIAP2L1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 123. doi:10.1158/1538-7445.AM2015-123

Abstract 2533: Design and preclinical profile of CH5183284/Debio 1347, a novel orally available and selective FGFR inhibitor acting on a gatekeeper mutant of FGFR2

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases (RTKs) comprises four members (FGFR1-4). FGFRs regulate multiple biological processes, such as cell proliferation, migration, apoptosis, and differentiation. Various genetic alterations, as well as overexpression, drive activation of both kinase activity of the receptors and the pathway signaling, which is associated with tumor growth and survival. Therefore, the FGFR family represents an attractive therapeutic target for treating cancer. Here, we report the discovery and the pharmacological profiles of CH5183284/Debio 1347, an orally available and selective inhibitor of FGFR1, 2, and 3. The lead compound CH5183284/Debio 1347 was identified from our original compound library by a high throughput screening program. Chemical modifications, which were guided by 3D-modeling analyses of the lead series and FGFRs, led to identifying an inhibitor that is selective to FGFR1, FGFR2, and FGFR3 (IC50: 9.3 nM, 7.6 nM, and 22 nM), but does not effectively inhibit FGFR4 (IC50: 290 nM), KDR (IC50: 2,100 nM) nor other 34 kinases. To evaluate kinase selectivity further, we used a KINOMEscan panel consisting of 442 kinases including some mutated forms of kinases. At 100 nM, CH5183284/Debio 1347 only bound to 5 kinases in the panel including FGFR1, FGFR2, and FGFR3 (over 80% inhibition to an ATP analog binding). An X-ray crystal structure analysis showed CH5183284/Debio 1347 binding to the ATP-binding site of FGFR1 in DFG-in mode and its interaction with both the hinge region and the backpocket of the protein. CH5183284/Debio 1347 also showed antitumor activity against cancer cell lines harboring genetic alterations in FGFRs in vitro and in xenograft models in mice. In addition, the unique ability of CH5183284/Debio 1347 to inhibit a relevant FGFR2 gatekeeper mutant (V564F) was documented in cellular phosphorylation assays, in vitro cell proliferation assays, and in vivo efficacy studies in a xenograft mouse model. The X-ray crystal structure analysis and modeling studies of the inhibitors, including NVP-BGJ398 and AZD4547, with FGFRs that were used to guide the SAR strategy in the optimization program will be presented and discussed, together with the unique susceptibility of CH5183284/Debio 1347 against a gatekeeper mutant of FGFR2. These findings underline the therapeutic potential of CH5183284/Debio 1347 in patients with FGFR genetic alterations and are the basis for an ongoing early clinical trial in several cancer types. Citation Format: Hirosato Ebiike, Naoki Taka, Yoshito Nakanishi, Nukinori Akiyama, Fumie Sawamura, Kenji Morikami, Masayuki Matsushita, Masayuki Ohmori, Kyouko Takami, Ikumi Hyohdoh, Masami Kohchi, Tadakatsu Hayase, Hiroki Nishii, Nobuya Ishii, Hiroharu Matsuoka. Design and preclinical profile of CH5183284/Debio 1347, a novel orally available and selective FGFR inhibitor acting on a gatekeeper mutant of FGFR2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2533. doi:10.1158/1538-7445.AM2014-2533

Abstract 2729: FGFR genetic alterations as a potential predictor of the sensitivity to CH5183284/Debio 1347, a selective FGFR inhibitor with a novel chemical scaffold

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The fibroblast growth factor receptors (FGFR) are tyrosine kinases that are constitutively activated in a subset of tumors by genetic alterations such as gene amplification, point mutation, or chromosomal translocation/rearrangement. Recently, small-molecule inhibitors that can inhibit the FGFR family as well as the VEGFR or PDGFR family showed some clinical benefits in FGFR genetically altered patient populations. However, to achieve more potent and prolonged efficacy in such populations, a selective FGFR inhibitor is still needed. Here, we report identifying CH5183284/Debio 1347, a selective and orally available FGFR1, FGFR2, and FGFR3 inhibitor that has a unique chemical scaffold as a FGFR inhibitor. By interacting with unique residues in the ATP binding site of FGFR1, FGFR2, or FGFR3, CH5183284/Debio 1347 selectively inhibits FGFR1, FGFR2, and FGFR3 (IC50: 9.3 nM, 7.6 nM, and 22 nM), but does not effectively inhibit FGFR4 (IC50: 290 nM ), KDR (IC50: 2,100 nM) or other 34 kinases. At 100 nM, CH5183284/Debio 1347 only binds to 5 kinases in the KinomeScan panel, including FGFR1, FGFR2, and FGFR3. Consistent with its high selectivity for FGFR enzymes, CH5183284/Debio 1347 does not lead to significant changes in blood pressure in telemetry-instrumented rats. In addition, CH5183284/Debio 1347 has a preferential antitumor activity against cancer cells with FGFR genetic alterations in a panel of 327 cancer cell lines. Among them, 4 cancer cell lines have copy number variations (CNV) of FGFR1 (>2.2 fold), 2 cancer cell lines have chromosomal translocation of FGFR1 (FGFR1OP-FGFR1), 6 cancer cell lines have CNV of FGFR2 (>2.2 fold), 3 cancer cell lines have point mutation of FGFR2 (S252W, K310R, N549K), 3 cancer cell lines have chromosomal translocation of FGFR3 (FGFR3-TACC3, FGFR3-BAIAP2L1), and 2 cancer cell lines have point mutation of FGFR3 (S249C, Y373C). This preferential efficacy against cancers harboring genetic alterations in FGFR was also confirmed in mouse xenograft studies. These findings warrant further investigation of CH5183284/Debio 1347 in patients harboring FGFR genetic alterations. Clinical studies have been initiated. Citation Format: Yoshito Nakanishi, Nukinori Akiyama, Toshiyuki Tsukaguchi, Yukako Tachibana-Kondo, Toshihiko Fujii, Kiyoaki Sakata, Hitoshi Sase, Takehito Isobe, Yasuko Sato, Kenji Morikami, Hidetoshi Shindoh, Toshiyuki Mio, Hirosato Ebiike, Naoki Taka, Yuko Aoki, Nobuya Ishii. FGFR genetic alterations as a potential predictor of the sensitivity to CH5183284/Debio 1347, a selective FGFR inhibitor with a novel chemical scaffold. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2729. doi:10.1158/1538-7445.AM2014-2729

Abstract 5252: Inhibition of FGFR3-BAIAP2L1 fusion kinase oncogenic potential by CH5183284/Debio 1347, a compound that inhibits FGFR3 kinase activity constitutively activated by BAIAP2L1 BAR domain dimerization

Advances in next-generation sequencing technologies have made possible to identify more efficiently novel fusion proteins in cancer. Among them, fusion kinases are well known as potent oncogenes and promising therapeutic targets for cancer patients. Recently, several FGFR fusion genes, such as FGFR1-TACC1, FGFR2-CCDC6, FGFR3-TACC3, and FGFR3-BAIAP2L1, have been identified in GBM, bladder cancer, and breast cancer as well as other tumor types. In this study, we focused on the FGFR3-BAIAP2L1 (F3-B) gene fusion and investigated its prevalence in clinical samples, tumorigenic activity, mechanism of constitutive activation, and sensitivity to CH5183284/ Debio 1347. We screened cancer tissue panels by RT-PCR for F3-B mRNA and identified F3-B positive specimens in bladder cancer (4.3%: 2/47) and lung cancer (1.2%: 1/83). All these F3-B fusions were confirmed by cDNA sequencing. To expand this study further, we established an FGFR3 break apart FISH assay. In a larger bladder cancer panel, we found 2 additional positive cases (2.2%: 2/89). To investigate the role of F3-B in tumors, we established Rat-2 transfectants with full length F3-B and assessed tumorigenic activity of these cells in in vitro and in vivo settings. Rat-2/F3-B cells acquired sphere forming activity even without FGF1 in vitro, and efficiently formed tumors when subcutaneously inoculated the cells into nude mice. Consistent with these observations, FGFR3 or BAIAP2L1siRNA blocked the proliferation of F3-B positive SW780 cells. Interestingly, the selective FGFR inhibitor, CH5183284/Debio 1347, effectively inhibited the in vivo tumor growth of Rat-2/F3-B and SW780 cells, indicating that F3-B oncogenic activity is depending on FGFR kinase activity. To confirm this, we established Rat-2/F3-B-ΔBAR, which lacks dimerization domain of BAIAP2L1 (BAR domain: Bin-Amphiphysin-Rvs). Phosphorylation of FGFR3 in Rat-2/F3-B-ΔBAR cells was lowered as compared to that in Rat-2/F3-B cells. Furthermore, Rat-2/F3-B-ΔBAR cells exhibited lower spheroid formation activity and slower tumor growth compared with Rat-2/F3-B cells. Taking this information together, the constitutive dimerization though BAR domain is essential for F3-B fusion to exert its potent oncogene activity in tumors. These findings underline the oncogenic potential of FGFR3-BAIAP2L1 gene fusion and warrant further clinical evaluation of the therapeutic potential of CH5183284/Debio 1347 in patients harboring this genetic alteration. Citation Format: Nukinori Akiyama, Yoshito Nakanishi, Toshiyuki Tsukaguchi, Yasuko Satoh, Yasue Nagata, Tsutomu Takahashi, Yukari Nishito, Motoki Ashihara, Nobuya Ishii, Masahiro Aoki. Inhibition of FGFR3-BAIAP2L1 fusion kinase oncogenic potential by CH5183284/Debio 1347, a compound that inhibits FGFR3 kinase activity constitutively activated by BAIAP2L1 BAR domain dimerization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5252. doi:10.1158/1538-7445.AM2014-5252