FMS-like Tyrosine Kinase 3 (FLT3) mutation is associated with poor survival in AML. The specific Anexelekto/MER Tyrosine Kinase (AXL) inhibitor ONO-7475 kills FLT3-mutant acute myeloid leukemia cells with targets including Extracellular-signal Regulated Kinase (ERK) and Myeloid Cell Leukemia 1 (MCL1). ERK and MCL1 are known resistance factors for Venetoclax (ABT-199), a popular drug for AML therapy, prompting the investigation of the efficacy of ONO-7475 in combination with ABT-199 in vitro and in vivo. ONO-7475 synergizes with ABT-199 to potently kill FLT3-mutant acute myeloid leukemia cell lines and primary cells. ONO-7475 is effective against ABT-199-resistant cells including cells that overexpress MCL1. Proteomic analyses revealed that ABT-199-resistant cells expressed elevated levels of pro-growth and anti-apoptotic proteins compared to parental cells, and that ONO-7475 reduced the expression of these proteins in both the parental and ABT-199-resistant cells. ONO-7475 treatment significantly extended survival as a single agent in vivo using acute myeloid leukemia cell lines and PDX models. Compared to ONO-7474 monotherapy, the combination of ONO- 7475/ABT-199 was even more potent in reducing leukemic burden and prolonging survival of mice in both model systems. These results suggest the ONO-7475/ABT-199 combination may be effective for acute myeloid leukemia therapy.

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

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), additivity (=1) or antagonism (>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

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.

3731 Purpose: ONO-WG-307 is a small molecule inhibitor that covalently binds to Btk. 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. The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis and new therapies, preferably chemo-sparing therapies, or as add-on to existing treatment regimens are required to help treat patients with ABC-DLBCL. Therefore, Btk constitutes an interesting therapeutic target, thus the activity of ONO-WG-307 was evaluated in an ABC-DLBCL xenograft model. Methods: Tumor cells (TMD-8) were implanted subcutaneously into female SCID mice. Tumors were allowed to grow to a volume of 100–200 mm3 before the mice were randomized into groups based on tumor size. ONO-WG-307 was administered orally at doses up to 10 mg/kg bid. Tumors were measured two or three times weekly after initiation of treatment, and tumor volumes were determined using the formula volume (=width2xlength)/2. Animals were euthanized when the tumors reached a maximum volume of 2,000 mm3 or after a maximum period of 2 months. In parallel, an exploratory pharmacodynamic marker of Btk inhibition (Phosphorylated-Btk [P-Btk]) was also investigated in vivo. Results: Treatment with ONO-WG-307 resulted in a dose-dependent inhibition of tumor growth in a TMD-8 xenograft model. Furthermore, parallel analysis of a pharmacodynamic marker, P-Btk, supported that Btk was inhibited and the level of P-Btk inhibition was correlated with the decreased tumor volumes observed in the TMD-8 model. Conclusion: ONO-WG-307 is a highly potent and selective oral Btk inhibitor with evidence of efficacy in the ABC-DLBCL xenograft model, with Btk inhibition further supported using a PD marker. Given the need to treat and overcome disease resistance especially in ABC-DLBCL, the use of a Btk inhibitor is a novel, mechanistic approach to treating B cell malignancies. Additional work is underway, combining ONO-WG-307 with chemotherapy and other targeted agents. Disclosures: No relevant conflicts of interest to declare.