Toshio Yoshizawa

A phase 1 clinical trial of the selective BTK inhibitor ONO/GS-4059 in relapsed and refractory mature B-cell malignancies

We report the results of a multicenter phase 1 dose-escalation study of the selective Bruton tyrosine kinase (BTK) inhibitor ONO/GS-4059 in 90 patients with relapsed/refractory B-cell malignancies. There were 9 dose-escalation cohorts ranging from 20 mg to 600 mg once daily with twice-daily regimens of 240 mg and 300 mg. Twenty-four of 25 evaluable chronic lymphocytic leukemia (CLL) patients (96%) responded to ONO/GS-4059, with a median treatment duration of 80 weeks; 21 CLL patients remain on treatment. Lymph node responses were rapid and associated with a concurrent lymphocytosis. Eleven of 12 evaluable patients with mantle cell lymphoma (92%) responded (median treatment duration, 40 weeks). Eleven of 31 non-germinal center B-cell diffuse large B-cell lymphoma patients (35%) responded but median treatment duration was 12 weeks due to development of progressive disease. ONO/GS-4059 was very well tolerated with 75% of adverse events (AEs) being Common Toxicity Criteria for Adverse Events version 4.0 grade 1 or grade 2. Grade 3/4 AEs were mainly hematologic and recovered spontaneously during therapy. One CLL patient experienced a grade 3 treatment-related bleeding event (spontaneous muscle hematoma) but no clinically significant diarrhea, cardiac dysrhythmias, or arthralgia were observed. No maximal tolerated dose (MTD) was reached in the CLL cohort. In the non-Hodgkin lymphoma cohort, 4 patients developed a dose-limiting toxicity, yielding an MTD of 480 mg once daily. ONO/GS-4059 has significant activity in relapsed/refractory B-cell malignancies without major drug-related toxicity. The selectivity of ONO/GS-4059 should confer advantages in combination therapies. This trial was registered at www.clinicaltrials.gov as #NCT01659255.

Abstract 857: Development of a Bruton's tyrosine kinase (Btk) inhibitor - ONO-WG-307, a potential treatment for B-cell malignancies

Purpose: Signals from B cell receptors (BCR) play a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. BCR signaling is implicated in the survival of malignant B cells and recent studies indicate that targeting Btk, an essential component of the BCR pathway, may be effective in the treatment of B-cell lymphoma. ONO-WG-307 is a highly potent and selective Btk inhibitor with an IC50 in the sub-nmol/L range. We evaluated the inhibitory effect of ONO-WG-307 alone and in combination with the chimeric type I anti-CD20 antibody rituximab, using in vitro tumor growth assays and mouse xenograft models. Methods: Two types of tumor cell lines (follicular lymphoma (FL) and activated B-cell-like (ABC) sub-type of diffuse large B cell lymphoma (DLBCL)) wereplated on to micro-titration plates (at doses up to 200 umol/L for ONO-WG-307 and 100 ug/mL for rituximab) for in vitro cytotoxic assays. The same cells were also implanted subcutaneously (SC) into female SCID mice to explore ONO-WG-307 anti-tumor activity in vivo (orally at doses up to 50 mg/kg, bid for ONO-WG-307 and intraperitoneally at doses up to 10 mg/kg, q7d). The IC50 of the in vitro cytotoxic activity was determined using a MTS assay 72 and 96 hours after incubation. Anti-tumor activity was defined as the ratio of the median tumor volume of treatment groups versus control group. The determination of combination index (CI) was calculated by the median-effect method. The CI was used to express synergism ( 1). Results: In the MTS assay, ABC-DLBCL cells were much more sensitive to ONO-WG-307 given as single agent compared with FL cell lines, however, the activity of rituximab single agent had no impact on ABC-DLBCL cell lines in contrast to its inhibitory activity against FL cell lines. When ONO-WG-307 was combined with rituximab, a moderate antagonism was observed in vitro on FL cell lines. In an ABC-DLBCL xenograft model, treatment with single agent ONO-WG-307 resulted in a dose-dependent inhibition of tumor growth and also showed moderate anti-tumor activity in a FL xenograft model. Conclusion: ONO-WG-307 is a highly potent and selective oral Btk inhibitor with evidence of efficacy in both the ABC-DLBCL and the FL cell lines and in xenograft models. These preliminary results suggest that ONO-WG-307 may be an effective therapy in the treatment of B-cell malignancies. To determine whether combination of ONO-WG-307 and rituximab is more efficacious, additional in vivo combination work is underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 857. doi:1538-7445.AM2012-857

Abstract 2021: ONO-WG-307, a novel, potent and selective inhibitor of Bruton's tyrosine kinase (Btk), results in sustained inhibition of the ERK, AKT and PKD signaling pathways

Purpose: Btk is a key regulator of B cell receptors (BCR) which play a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. BCR signaling is implicated in the survival of malignant B cells and recent studies indicate that targeting Btk may be effective in the treatment of B-cell lymphoma. Response between cell survival and BCR signaling is implicated in different types of B-cell lymphoma. Therefore, we evaluated the inhibitory effect of ONO-WG-307 on Btk-dependent signal transduction by large-scale and quantitative phosphoproteome analysis to a depth of more than 10,000 phosphorylation sites. Methods: Two tumor cell lines (sensitive and non-sensitive) were treated with ONO-WG-307. After 72h, cell viability was determined by the CellTiter-Glo Luminescent Cell Viability Assay. P-Btk was used as a marker for Btk activity determined by Western blot and Flow Cytometry. Quantitative phosphoproteomics were enabled by differential SILAC labeling of lymphoma cells. After 1 hr incubation with ONO-WG-307, the total cellular proteins were digested and phosphopeptides were enriched by a combination of strong cation exchange and immobilized metal affinity chromatography. Site-specific phosphorylations were identified and by LC-MS analysis on a LTQ-Orbitrap-Velos mass spectrometer followed by bioinformatic processing. Results: Btk is potently and selectively inhibited by ONO-WG-307 in an in vitro Btk kinase assay, with an IC50 in the subnanomolar range whereas IC50 values for other tyrosine kinases (Lck, Lyn and Fyn) were above 1 µM. The concentration required for growth inhibition for the sensitive cells was 3.59 nmol/L (IC50), whereas considerably higher concentrations were required to suppress growth of non-sensitive cells. Surprisingly, inhibition of cellular Btk and ERK phosphorylation by ONO-WG-307 levels of P-Btk and P-ERK were similar in both sensitive and non-sensitive cells. However, our quantitative phosphoproteome studies revealed selective suppression of Akt-mediated signaling and cellular protein kinase D activity in sensitive compared to non-sensitive cells.Conclusion: These results suggest that the selective ONO-WG-307 inhibition of “responder” cell growth might be due to a critical role of Btk-mediated signaling through Akt and protein kinase D. These data shed new light on the cellular mode-of-action of Btk inhibition and support the potential clinical utility of ONO-WG-307 in B cell malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2021. doi:1538-7445.AM2012-2021

Anti-tumor efficacy study of the Bruton’s tyrosine kinase (BTK) inhibitor, ONO/GS-4059, in combination with the glycoengineered type II anti-CD20 monoclonal antibody obinutuzumab (GA101) demonstrates superior in vivo efficacy compared to ONO/GS-4059 in combination with rituximab

Abstract The activated B-cell diffuse large B-cell-like lymphoma (ABC-DLBCL) correlates with poor prognosis. The B-cell receptor signaling pathway is known to be dysregulated in NHL/CLL and given BTK is a downstream mediator of BCR signaling, BTK constitutes an interesting and obvious therapeutic target. Given the high potency and selectivity of the BTK inhibitor, ONO/GS-4059, it was hypothesized that, the anti-tumor activity of ONO/GS-4059 could be further enhanced by combining it with the anti-CD20 Abs, rituximab (RTX) or obinutuzumab (GA101). ONO/GS-4059 combined with GA101 or RTX was significantly better than the respective monotherapy with tumor growth inhibition (TGI) of 90% for the GA101 combination and 86% for the RTX combination. In contrast, ibrutinib (PCI-32765) combined with RTX did not result in improved efficacy compared with respective monotherapy. Taken together these data indicate that the combination of ONO/GS-4059 with rituximab and particularly obinutuzumab may be an effective treatment for ABC-DLBCL.

Abstract 2452: ONO-4059, a novel oral Bruton's tyrosine kinase (Btk) inhibitor that demonstrates potent pharmacodynamic activity through Phosphorylated Btk (P-Btk) inhibition, in addition to effective anti-tumour activity in a TMD-8 (DLBCL) xenograft model.

Purpose: The B-cell receptor (BCR) pathway plays a central role in signal transduction pathways that regulate survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is a key regulator of the BCR signaling pathway and abberant BCR signaling has been implicated in the survival of malignant B-cells. Recent studies indicate that targeting Btk may be effective in the treatment of B-cell malignancies. ONO-4059 is a highly potent and selective oral Btk inhibitor with an IC50 in the sub-nmol/L range. Previous studies with ONO-4059 (formerly known as ONO-WG-307; AACR 2012) demonstrated that Btk-mediated signaling through Akt and PKD is a critical role for the survival of activated B-cell-like (ABC) sub-type of diffuse large B cell lymphoma (DLBCL) cell lines [TMD-8]. It was also of interest to determine the extent of gene expression changes induced in TMD-8 cells after treatment with ONO-4059. To understand the effects of ONO-4059 on gene transcription, we analyzed the gene expression patterns of ONO-4059 in the TMD-8 xenograft model. Methods: TMD-8 cells were implanted subcutaneously into SCID mice and ONO-4059 was administered orally, twice a day (BID) in two groups of animals, at doses of 3 and 10 mg/kg. When the mean tumour volumes reached 100-200mm3, treatment was initiated daily for 14 days. After the final treatment of ONO-4059, the total RNAs were extracted from frozen re-sected tumour tissue specimens, which comprised of 10 samples at each dose, along with 10 vehicle tissue samples. Microarray analysis was conducted by Agilent technology. Results: The inhibitory level of P-Btk achieved indicates profound anti-proliferative activity of ONO-4059 in the TMD-8 model. Gene expression studies demonstrated that ONO-4059 affects a core set of genes which contains 9 down-regulated and 8 up-regulated genes in a dose-dependent manner. CXCL-10 is the prominent regulated gene that is down-regulated after ONO-4059 treatment and CXCL-10 has been shown to be involved in the pathological processes of human disorders, such as, infectious diseases, inflammatory and autoimmune diseases as well as cancer. From a publicly available microarray database, CXCL-10 expression has been shown to be up-regulated in lymphoma patients, suggesting that CXCL-10 may be a potential biomarker for ONO-4059 in the treatment of B-cell malignancies. Conclusion: Our data show that ONO-4059 has potent anti-tumour activity in a ABC-DLBCL xenograft model and that ONO-4059 achieves active tumor exposures as measured by P-Btk in vivo. ONO-4059 is currently being developed in a Phase I clinical trial for the treatment of B-cell malignancies. Additional work is now underway to determine whether the combining ONO-4059 with other targeted agents affects CXCL-10 expression. Citation Format: Tomoko Yasuhiro, Toshio Yoshizawa, Shingo Hotta, Yuko Ariza, Yoshiko Ueda, Ryohei Kozaki, Joseph Birkett. ONO-4059, a novel oral Bruton9s tyrosine kinase (Btk) inhibitor that demonstrates potent pharmacodynamic activity through Phosphorylated Btk (P-Btk) inhibition, in addition to effective anti-tumour activity in a TMD-8 (DLBCL) xenograft model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2452. doi:10.1158/1538-7445.AM2013-2452

Abstract 2954A: A potent and selective TRK inhibitor ONO-5390556, shows potent antitumor activity against both TRK-rearranged cancers and the resistant mutants

Purpose: TRKA/NTRK1, TRKB/NTRK2 and TRKC/NTRK3 belong to the neurotrophic tyrosine kinase receptor family and signals from TRK receptors play a role in neuronal survival and differentiation through activation of MAPK and AKT downstream pathways. Recently, oncogenic rearrangements of TRKA, B and C gene were identified in a variety of cancers, including lung adenocarcinoma, colorectal cancer, glioblastoma and acute myeloid leukemia. These genomic rearrangements resulted in sustained cancer cell proliferation. Recent studies indicate that targeting TRK by Entrectinib, pan-TRK, ALK and ROS1 inhibitor, may be effective in the treatment of cancers with TRK rearrangements. However, patients treated with Entrectinib showed resistance early due to NTRK1 harboring acquired mutations, G595R and G667C. ONO-5390556 is a selective pan-TRK inhibitor and shows highly potent anti-tumor activity against TRK rearranged cancers. We evaluated the anti-tumor activity of ONO-5390556 against NTRK1 rearranged cancer cells with harboring mutated G595R and G667C. Methods: The anti-tumor activity of ONO-5390556 was evaluated in subcutaneous xenograft tumor models of KM12, human colorectal cancer cell lines expressing TPM3-TRKA. ONO-5390556 was administered orally with doses ranging between 0.2 and 2 mg/kg once a day for 14 days. In vitro cytotoxic activity, cell viability was evaluated by WST-8 in TPM3-TRKA positive cells harboring mutated G595R and G667C. Phosphorylated proteins were detected by Western blotting. Results: In KM12 xenograft model, treatment with ONO-5390556 at doses of 0.2, 0.6, 2 mg/kg once a day resulted in a dose-dependent inhibition of tumor growth with Tumor Growth Inhibition (TGI) of 44.4, 86.6 and 95.4%, respectively. Both G595R and G667C mutations conferred resistance to Entrectinib, but were sensitive to ONO-5390556 with an IC50 of 2.7 and 0.2 nmol/L, respectively. Interestingly, the inhibitory activity of ONO-5390556 sustained in these mutations compared with wild type (IC50 of 0.4 nmol/L). ONO-5390556 strongly inhibited phosphorylation of the TRKA mutated. Additionally, Erk phosphorylation remained strongly inhibited in KM12 cells harboring mutated G595R. Conclusion: The oncogenic TRK fusion proteins are attractive therapeutic targets but two mutations acquired resistance has been found in the clinic. ONO-5390556 is a highly potent and selective pan-TRK inhibitor with evidence of an excellent anti-tumor activity not only in cancer cells harboring the TRKA rearrangement but also in the two acquired mutations. These results suggest that ONO-5390556 may overcome Entrectinib-resistance mutations and become a potential role for sequential therapy with first generation TRK inhibitors. Citation Format: Ryohei Kozaki, Toshio Yoshizawa, Kohki Tsukamoto, Hikaru Kato, Kazuhito Kawabata. A potent and selective TRK inhibitor ONO-5390556, shows potent antitumor activity against both TRK-rearranged cancers and the resistant mutants. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2954A.

Abstract LB-218: Development of Axl/Mer inhibitor, ONO-9330547: preclinical evidence supporting the combination with immunotherapeutics

Purpose: Recent clinical studies have demonstrated that immunotherapeutics, such as checkpoint inhibitors are a promising approach to the treatment of cancer; however, strategies to improve its clinical efficacy are still required. Axl and Mer, member of the TAM receptor tyrosine kinase family are known to be over-expressed in various types of hematological and solid tumor cancers and have been reported as poor prognostic factors. ONO-9330547 is a highly potent and selective oral Axl/Mer inhibitor. Previous study with ONO-9330547 demonstrated both increase proportion of CD8+ T cell and alternation of immunosuppressive microenvironment in syngeneic tumor graft model (Tanaka AACR 2015). Thus, we next went on to examine whether combining ONO-9330547 with anti-PD1 antibody has the potential to improve efficacy in a mouse colon cancer MC38 syngeneic model. Methods: MC38 tumor cells, a mouse colon cancer cell line, were implanted into female C57/B6 mice. Randomization of mice occurred when mean tumor volume reached 150 mm3. ONO-9330547 was fed in a diet containing 0.0013 or 0.013% ONO-9330547 (equivalent to 2 or 20 mg/kg/day). Anti-mouse PD-1 Ab (4H2) was administered intraperitoneally at a dose of 10 mg/kg every 6 days for 28 days. Tumor volumes were determined using the formula volume ( = width2 x length)/2. Animals were euthanized when the tumors reached a maximum volume of 2,000 mm3. QPCR was used to determine the TAM ligands, Gas6 and Pros1. Results: Treatment with ONO-9330547 combined with 4H2 resulted in a significantly improved inhibition of tumor growth in the MC38 syngeneic model. In contrast, treatment of 4H2 monotherapy showed inferior anti-tumor activity compared with combination therapy. In particular, ONO-9330547 at 2 or 20 mg/kg/day combined with 4H2 resulted in tumor remission in 3/9 or 6/9 animals at day 28, respectively, whereas each monotherapy resulted in tumor remission in only 1/9 animals. Interestingly, the treatment with 4H2 significantly increased TAM ligands mRNA levels, Gas6 and Pros1 in tumor. Conclusion: Our results demonstrate that ONO-9330547, a highly potent dual Axl/Mer inhibitor, shows synergistic anti-tumor effect when combined with anti-PD-1 antibody which induces the expression of TAM ligands. These data support the concept that the immunomudulatory effect of ONO-9330547 is further enhanced when ONO-9330547 is combined with immunotherapy. Rational combination with immunotherapeutics is worth further investigation in the clinical setting. Citation Format: Toshio Yoshizawa, Kohei Tanaka, Tomoko Yasuhiro, Ryu Fujikawa, Suiho Ri, Kazuhito Kawabata. Development of Axl/Mer inhibitor, ONO-9330547: preclinical evidence supporting the combination with immunotherapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-218.

Responses to the Selective Bruton’s Tyrosine Kinase (BTK) Inhibitor Tirabrutinib (ONO/GS-4059) in Diffuse Large B-cell Lymphoma Cell Lines

Bruton’s tyrosine kinase (BTK) is a key regulator of the B-cell receptor signaling pathway, and aberrant B-cell receptor (BCR) signaling has been implicated in the survival of malignant B-cells. However, responses of the diffuse large B-cell lymphoma (DLBCL) to inhibitors of BTK (BTKi) are infrequent, highlighting the need to identify mechanisms of resistance to BTKi as well as predictive biomarkers. We investigated the response to the selective BTKi, tirabrutinib, in a panel of 64 hematopoietic cell lines. Notably, only six cell lines were found to be sensitive. Although activated B-cell type DLBCL cells were most sensitive amongst all cell types studied, sensitivity to BTKi did not correlate with the presence of activating mutations in the BCR pathway. To improve efficacy of tirabrutinib, we investigated combination strategies with 43 drugs inhibiting 34 targets in six DLBCL cell lines. Based on the results, an activated B-cell-like (ABC)-DLBCL cell line, TMD8, was the most sensitive cell line to those combinations, as well as tirabrutinib monotherapy. Furthermore, tirabrutinib in combination with idelalisib, palbociclib, or trametinib was more effective in TMD8 with acquired resistance to tirabrutinib than in the parental cells. These targeted agents might be usefully combined with tirabrutinib in the treatment of ABC-DLBCL.

Abstract 3727: Plasma inhibitory activity (PIA) is a possible pharmacodynamic marker for clinical development of a next generation pan-TRK inhibitor, ONO-5390556

Background: Colorectal cancer and Mammary analogue secretory carcinoma harboring Neurotrophic tyrosine receptor kinase (NTRK/TRK) gene rearrangements developed resistance to Entrectinib, ALK/ROS/TRK inhibitor. We previously demonstrated that the next generation pan-TRK inhibitor,ONO-5390556 may potently overcome Entrectinib-resistance mutations. Furthermore, inhibition of Trk phosphorylation in tumors has excellent correlation with the in vivo anti-tumor effect by ONO-5390556 (AACR2016, Kozaki et al). Purpose: Accurate measurement of target inhibition in a phase 1 clinical trial is critical to informing selection of appropriate doses for ONO-5390556 in more advanced clinical trials. Plasma inhibitory activity (PIA) assay has recently been performed as a surrogate assay in clinical trials of FLT3, MET and ALK kinase inhibitors. The assay employs the incubation of Tyrosine Kinase expressing cell lines in aliquots of plasma collected at various time points from patients treated with TKI. Herein, we report the preclinical evaluation of PIA assay in accordance with an inhibition of TRK phosphorylation in tumors. Methods: KM12 cells, human colorectal cancer cell lines expressing TPM3-TRKA, were implanted subcutaneously into nude mice. Mice were randomized when the mean tumor volume was 150-600 mm3. Tumors and blood were collected from mice, 2, 4, 7 and 24 hours after the single treatment of ONO-5390556 with the doses of 0.06 and 0.6 mg/kg. The collected tumors were disrupted and phosphorylated TrkA in tumors was detected by Electrochemiluminescence (ECL). In a PIA assay, KM12 was incubated in plasma from the collected blood for 4 hours and phosphorylated TrkA in the cells was detected by ECL.. Results: Treatment with ONO-5390556 at doses of 0.06, 0.6 mg/kg resulted in a significant inhibition of Trk phosphorylation in tumors up to 24 hours. Compared to inhibition of P-TRK in tumors, the inhibition of P-TRK in PIA sustained until 7 hours but rapidly decreased at 24 hours after administration. The levels of both tumors and PIA showed in a dose-dependent inhibition and an excellent correlation until 7 hours. Inhibition of TRK phosphorylation (PIA/Tumor, %)0.06 mg/kg (2,4,7,24h) : 56.3/41.4 (PIA/Tumor, %), 51.9/53.5, 43.0/66.5, -5.5/51.30.6 mg/kg (2,4,7,24h) : 90.2/84.3, 88.4/93.0, 87.4/95.7, 8.3/83.3Conclusion: We have validated the PIA assay that correlates with the inhibition of P-TRK in tumors. Our results demonstrated that the potential utility of PIA as a PD marker may contribute to determining the effective dose of ONO-5390556 in the clinical development. Citation Format: Ryohei Kozaki, Ryu Fujikawa, Hikaru Kato, Natsuka Goto, Toshio Yoshizawa. Plasma inhibitory activity (PIA) is a possible pharmacodynamic marker for clinical development of a next generation pan-TRK inhibitor, ONO-5390556 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3727. doi:10.1158/1538-7445.AM2017-3727

Abstract 788: A novel, potent and selective pan-Trk inhibitor ONO-5390556, demonstrates therapeutic efficacy in cancer cells harboring the TrkA rearrangement

Purpose: Fusion genes including EML4-ALK, which have been recognized as driver mutations in cancers, encode chimeric proteins with constitutive kinase activity, which promote tumor cell survival. Patients with ALK-Positive Non-Small Cell Lung Cancer have demonstrated remarkable clinical responses to the ALK tyrosine kinase inhibitor, Crizotinib. TrkA/NTRK1,TrkB/NTRK2 and TrkC/NTRK3 belong to the neurotrophic tyrosine kinase receptor family. Signals from Trk receptors play a role in neuronal survival and differentiation through several signal cascades. Recently, oncogenic rearrangements of TrkA, B and C genes were identified in a variety of cancers, including lung adenocarcinoma, colorectal cancer, glioblastoma and acute myeloid leukemia. Therefore, the oncogenic Trk fusion proteins are attractive therapeutic targets that warrant further investigation. ONO-5390556 is a novel, potent and selective pan-Trk inhibitor with an IC50 in the low nM range. We evaluated the anti-tumor activity of ONO-5390556 against TrkA rearranged cancer cells in vitro and in vivo. Methods: Phospho-proteins were detected by Western blotting. In a proliferation assay, cells were treated with ONO-5390556 at concentrations ranging from 0.03 to 100 nM for 72h. Cell viability was determined by WST-8 assay. In a KM12 xenograft model, cells were implanted subcutaneously into female nude mice. Mice were randomized when the mean tumor volume was 100-200 mm3. ONO-5390556 was administered orally with doses ranging between 0.1 and 1 mg/kg twice daily (BD) for 14 days. Tumor volumes were measured twice a week after initiation of treatment, and tumor volumes were determined using the formula volume ( = width2 x length)/2. Results: ONO-5390556 inhibited TrkA autophosphorylation and cell proliferation in BaF3 murine pro B cell lines transformed with a TrkA fusion gene with an IC50 of 0.4 and 0.2 nM, respectively. Similarly, in KM12, human colorectal cancer cell lines harboring TPM3-TrkA fusions, both the autophosphorylation of TrkA and the cell proliferation were strongly inhibited by ONO-5390556 with an IC50 of 0.2 and 0.3 nM, respectively. Interestingly, TrkA autophosphorylation remained strongly inhibited even 24 hours after a washout of ONO-5390556 in KM12, which suggests ONO-5390556 has tight-binding properties. In the KM12 Xenograft model, treatment with ONO-5390556 at doses of 0.1, 0.3 and 1mg (BD) for 14 days resulted in a dose-dependent inhibition of tumor growth with Tumor Growth Inhibition (TGI) of 48.0, 89.2 and 95.6%, respectively. Conclusion: ONO-5390556 is a highly potent and selective pan-Trk inhibitor with evidence of an excellent anti-tumor activity in cancer cells harboring the TrkA rearrangement. These preliminary results suggest that ONO-5390556 may be an effective therapeutic option in a wide variety of cancers including lung and colorectal cancers with Trk rearrangements. Citation Format: Kohki Tsukamoto, Toshio Yoshizawa, Ryohei Kozaki, Kazuhito Kawabata. A novel, potent and selective pan-Trk inhibitor ONO-5390556, demonstrates therapeutic efficacy in cancer cells harboring the TrkA rearrangement. [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 788. doi:10.1158/1538-7445.AM2015-788