Selective AXL Research Articles

Abstract B027: BGB324, a selective small molecule inhibitor of AXL receptor tyrosine kinase, enhances immune checkpoint inhibitor efficacy

Abstract The AXL receptor tyrosine kinase is expressed by several tumor types and is associated with poor overall survival in patients. AXL signaling is an important regulator of tumor cell plasticity related to epithelial-to-mesenchymal transition (EMT) and stem cell traits that drive metastasis, drug resistance and immune evasion. AXL is also expressed on several cells associated with the inflammatory tumor immune microenvironment including natural killer (NK) cells, dendritic cells and tumor-associated macrophages. Signaling via AXL is a key suppressor of the anti-tumor innate immune response. Hence, AXL signaling contributes uniquely to tumor cell intrinsic and microenvironmental anti-tumor immune suppression mechanisms in cancer. We evaluated whether blocking AXL signaling with BGB324, a selective clinical-stage small molecule AXL kinase inhibitor, enhanced the effect of immune checkpoint blockade in aggressive adenocarcinomas that display limited immunogenicity. Immune therapy with anti-PD-1/anti-PD-L1 or anti-CTLA-4/anti-PD-1 increased AXL and EMT-marker expression in the murine lung cancer (Lewis lung, LL2) and mammary adenocarcinoma (4T1) syngeneic models, and correlated with a lack of response to immune checkpoint therapy. Combination treatment with BGB324 (50 mg/kg bid) significantly enhanced responsiveness to anti-PD-1/anti-PD-L1 or anti-CTLA-4/anti-PD-1 treatment (10 mg/kg of each, 6 doses for LL2; 4 doses for 4T1) in mice bearing established LL2 or 4T1 tumors respectively. BGB324 in combination with anti-PD-1/anti-PD-L1 or anti-CTLA-4/anti-PD-1 enhanced tumor infiltration of cytotoxic T lymphocytes (CTLs). Increased CTLs were also detected in spleens from animals responding to treatment. BGB324 + anti-CTLA-4/anti-PD-1 combination treatment increased the number of NK cells, macrophages and polymorphonuclear neutrophils, but decreased the number of tumor-associated myeloid-derived suppressor cells (MDSC). In the 4T1 model, the combination of BGB324 + anti-CTLA-4/anti-PD-1 resulted in durable primary tumor clearance in 23% of treated mice versus 5.6% obtained with anti-CTLA-4/anti-PD-1 alone (p = 0.0157). In a separate study, BGB324 + anti-CTLA-4 therapy treated resulted in 22% long-term primary tumor clearance while no response was observed with anti-CTLA4 treatment alone. The extensive metastasis to the lung, liver and spleen characteristic of this model was concomitantly abrogated in the animals responding to the combination treatment. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity These findings along with the favorable safety profile and clinical activity of BGB324 in ongoing monotherapy clinical trials, support a rationale for clinical testing of BGB324 in combination with immune checkpoint inhibitors in cancer patients. Citation Format: Katarzyna Wnuk-Lipinska, Kjersti Davidsen, Magnus Blø, Linn Hodneland, Agnete Engelsen, Jing Kang, Maria Lie, Sebastien Bougnaud, Kristina Aguilera, Lavina Ahmed, Agata Rybicka, Elina Milde, Paulina Deyna, Anna Boniecka, Oddbjørn Straume, Salem Chouaib, Rolf Brekken, Gro Gausdal, James Lorens. BGB324, a selective small molecule inhibitor of AXL receptor tyrosine kinase, enhances immune checkpoint inhibitor efficacy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B027.

Abstract 566: BGB324, a selective small molecule inhibitor of the receptor tyrosine kinase AXL, enhances immune checkpoint inhibitor efficacy

Abstract Signaling via the AXL receptor tyrosine kinase is a key suppressor of the anti-tumor innate immune response. AXL is expressed on several cells associated with the tumor immune microenvironment including natural killer cells, dendritic cells and tumor-associated macrophages. AXL is also an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) that drives tumor immune evasion and resistance to cytotoxic T cell-mediated cell killing. Hence AXL signaling contributes uniquely to both tumor cell intrinsic and microenvironmental anti-tumor immune suppression mechanisms. We therefore evaluated whether blocking AXL signaling with BGB324, a selective clinical-stage small molecule Axl kinase inhibitor, enhances the effect of immune checkpoint blockade in syngeneic cancer mouse models that display limited immunogenicity. We conducted studies in the aggressive mammary adenocarcinoma (4T1) syngeneic (Balb/C) mouse model. We found that AXL expression increased in 4T1 tumors treated with anti-CTLA-4/anti-PD-1 and correlated with lack of response to immune therapy. Combination with BGB324 (50 mg/kg bid) significantly enhanced responsiveness to anti-CTLA-4/anti-PD-1 treatment (10 mg/kg of each, 4 doses) in Balb/C mice bearing established 4T1 tumors. The combination of BGB324 + anti-CTLA-4/anti-PD-1 resulted in durable primary tumor clearance in 23% of treated mice versus 5.6% obtained with anti-CTLA-4/anti-PD-1 alone (p = 0.0157). In a separate study, BGB324 + anti-CTLA-4 treated resulted in 22% long-term primary tumor clearance while no response was observed with anti-CTLA4 treatment alone. The extensive metastasis to the lung, liver and spleen characteristic of this model were concomitantly abrogated in the animals responding to the combination treatment. In addition, BGB324 + anti-CTLA-4/anti-PD-1 treated tumors displayed enhanced infiltration of cytotoxic T lymphocytes. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity. In conclusion, targeting AXL signaling represents a unique opportunity to address multiple tumor immune suppression mechanisms. Our results support combining the clinical-stage AXL inhibitor, BGB324, with immune checkpoint inhibitors to improve treatment of human cancers. Citation Format: Gro Gausdal, Kjersti Davidsen, Katarzyna Wnuk-Lipinska, Kathleen Wiertel, Jing Kang, Agnete Engelsen, Sébastien Bougnaud, Monica Hellesøy, Magnus Blø, Lavina Ahmed, Linn Hodneland, Sergej Kiprijanov, Oddbjørn Straume, Rolf A. Brekken, James B. Lorens. BGB324, a selective small molecule inhibitor of the receptor tyrosine kinase AXL, enhances immune checkpoint inhibitor efficacy. [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 566.

A first-in-patient phase I study of BGB324, a selective Axl kinase inhibitor in patients with refractory/relapsed AML and high-risk MDS.

2561Background: The RTK Axl represents an independent prognostic factor and therapeutic target in AML. BGB324 is an orally available selective potent Axl inhibitor, which has antileukemic activity in preclinical models of AML. Methods: A standard 3 + 3 dose escalation study was performed to identify the maximum tolerated dose of BGB324 in patients with previously treated AML or high risk MDS. BGB324 was administered as an oral loading dose on days one and two followed by a reduced daily maintenance. Three dose levels were explored 400/100mg, 600/200mg and 900/300mg Results: To date, 13 patients with AML and 3 patients with MDS were treated in the first three dose levels. 6 patients remain on treatment and 11 are evaluable. Therapy has been well-tolerated and the MTD has not yet been reached. The majority of adverse events reported have been Grade 1 and 2. The most common related adverse events are diarrhea and fatigue. One patient with pre-existing cardiac conduction abnormalities discontinued study treat...

Abstract B014: BGB324, a selective small molecule inhibitor of the receptor tyrosine kinase AXL, enhances immune checkpoint inhibitor efficacy

Abstract Signaling via the AXL receptor tyrosine kinase is a key suppressor of anti-tumor innate immune response. AXL is expressed on several cells associated with the suppressive tumor immune microenvironment including natural killer cells, dendritic cells and tumor-associated macrophages. AXL is also an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) that contributes to anti-tumor immune evasion. Hence AXL signaling contributes uniquely to tumor intrinsic and microenvironmental immune suppression in tumors. We therefore evaluated whether blocking AXL signaling with BGB324, a selective clinical-stage small molecule Axl kinase inhibitor, enhances the effect of immune checkpoint blockade in syngeneic cancer mouse models that display limited immunogenicity. We measured the effect of BGB324 in combination with anti-CTLA-4 and anti-PD-1 in the mammary adenocarcinoma 4T1/Balb/C syngeneic mouse model. BGB324 (50 mg/kg bid) significantly enhanced responsiveness to anti-CTLA-4/anti-PD-1 treatment (10 mg/kg of each, 4 doses) in Balb/C mice bearing 4T1 tumors. The combination of BGB324 + anti-CTLA-4/anti-PD-1 resulted in complete tumor clearance in 46.1 % of mice versus complete tumor clearance in 11.7 % of the mice treated with anti-CTLA-4/anti-PD-1 (p = 0.0087). BGB324 + anti-CTLA-4/anti-PD-1 treated tumors displayed enhanced CD8+ T cell tumor infiltration. Combination of BGB324 with immune checkpoint inhibitors is being evaluated in additional models, and detailed interrogation of AXL-dependent immune effector cell activity in tumors is in progress. In conclusion, AXL inhibition represents a unique opportunity to target anti-tumor immune suppressive mechanisms and supports clinical translation of BGB324 in combination with cancer immunotherapy in human cancer. Citation Format: Gro Gausdal, Kjersti Davidsen, Katarzyna Wnuk-Lipinska, Kathleen Wiertel, Monica Hellesøy, Magnus Blø, Lavina Ahmed, Linn Hodneland, Sergej Kiprijanov, Rolf A Brekken, James B Lorens. BGB324, a selective small molecule inhibitor of the receptor tyrosine kinase AXL, enhances immune checkpoint inhibitor efficacy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B014.

Abstract B78: Warfarin blocks Gas6-mediated Axl activation required for pancreatic tumor plasticity and metastasis

Abstract Warfarin, a vitamin K antagonist anti-coagulant in clinical use for over 50 years, is reported to exert anti-cancer effects. We hypothesized that the molecular mechanism underlying the observed anti-tumor effects of warfarin is unrelated to generalized anti-coagulation, but rather due to inhibition of the Axl receptor tyrosine kinase. Activation of Axl by its ligand Gas6, a vitamin K-dependent protein, is inhibited at doses of warfarin that do not affect coagulation. Here we document that inhibiting Gas6-mediated Axl activation with low dose warfarin blocks pancreatic cancer progression and spread. Warfarin and other Axl-targeting agents inhibit tumor progression and block spontaneous metastasis in multiple murine models of pancreatic cancer. Warfarin inhibited Axl-dependent tumor cell migration, invasiveness and proliferation while increasing apoptosis and sensitivity to chemotherapy. We demonstrate that Axl signaling is necessary for pancreatic tumor cell epithelial plasticity which is potently reversed by warfarin or selective Axl inhibition in vitro and in vivo. We anticipate Axl is a critical driver of pancreatic cancer progression and its inhibition with low dose warfarin or Axl-selective targeting agents may significantly improve outcome in patients. Citation Format: Amanda Kirane, Kathleen W. Ludwig, Gry Haaland, Tone Sandal, Renata Ranaweera, Jason E. Toombs, Laura A. Sullivan, Miao Wang, Noah Sorrelle, Sean P. Dineen, Michael T. Dellinger, James B. Lorens, Rolf A. Brekken. Warfarin blocks Gas6-mediated Axl activation required for pancreatic tumor plasticity and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B78.

Abstract 1991: Axl overexpression drives migration and invasion in colon cancer

Abstract Receptor tyrosine kinases (RTK) of the TAM family have been described as oncogenes. Axl and Mertk, two members of the TAM family, are also potent negative regulators of the inflammatory response. Inflammation is an enabling characteristic of cancer hallmarks and can promote tumorigenesis and tumor progression. Genetic ablation of Axl and Mertk in mice, results in heightened inflammation in the gut and increased susceptibility to inflammation-driven colon cancer (azoxymethane/dextran sodium sulfate treatment paradigm). We sought to understand the role of Axl and Mertk, individually, in colon cancer cells to identify the key TAM oncogene in this disease. Here we show that Axl, but not Mertk, was highly expressed and correlated with grade of metastatic colon cancer. Silencing Axl expression in RKO-AS45-1 and HCT116 colon cancer cells had no significant effect on proliferation and survival, but markedly decreased migration and invasion. R428, a selective Axl small molecule inhibitor, also drastically reduced migration in these colon cancer cell lines. Analyses of Axl knockout mice revealed that single, genetic ablation of this TAM RTK does not increase susceptibility to DSS-induced colitis, in contrast to that observed in Axl and Mertk double knockout mice. These results are consistent with a redundant function of Axl and Mer in the regulation of the inflammatory response. Our findings suggest that the selective targeting of Axl in colon cancer might disable the oncogenic function of TAM RTKs, while sparing the protective, anti-inflammatory function of this RTK family in this disease. Citation Format: Diana J. Uribe, Lidia Bosurgi, Nicola Gagliani, Leonel Joannas, Adam Watson, Jesse D. Martinez, Carla V. Rothlin, Sourav Ghosh. Axl overexpression drives migration and invasion in colon cancer. [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 1991. doi:10.1158/1538-7445.AM2014-1991

Abstract 1747: BGB324, a selective small molecule Axl kinase inhibitor to overcome EMT-associated drug resistance in carcinomas: Therapeutic rationale and early clinical studies

Abstract Axl is a member of the TAM (Tyro3, Axl and Mer) family of receptor tyrosine kinases that regulate multiple cellular responses including cell survival, proliferation, and migration. Axl expression is predictive of poor patient overall survival in a variety of human cancers including triple negative breast (TNBC), pancreatic ductal adenocarcinoma (PDA) and non-small cell lung cancer (NSCLC). Axl expression is induced by the epithelial-to-mesenchymal transition (EMT) gene program in cancer cells and Axl signaling is required to maintain EMT-associated features including invasiveness, metastasis, stem cell-like traits and resistance to targeted inhibitors and other chemotherapeutic agents. BGB324/R428 is an oral, selective small molecule inhibitor of Axl that recently was evaluated in early clinical safety studies in healthy volunteers. Treatment with BGB324 up to and including 1.5 gms daily (per os) was established as being safe and well tolerated. The endpoints of the study included pharmacokinetics and safety. Bioavailability was increased in the presence of food and systemic exposure increased dose proportionately. At the highest dose the apparent elimination half-life approached four days presenting a range of dosing options. We evaluated the effects of BGB324 in preclinical models of TNBC, PDA and NSCLC, including 2D/3D cell culture and mouse xenograft models, in combination with targeted and chemotherapeutic agents. BGB324 treatment of mesenchymal carcinoma cells blocked invasiveness and enhanced chemotherapeutic efficacy. BGB324 abrogated the tumor initiation capacity of TNBC cells, an activity associated with cancer stem cells. BGB324 treatment blocked the emergence of EMT-associated acquired resistance to erlotinib in human NSCLC xenografts. Furthermore, combination treatment of BGB324 with chemotherapy inhibited the growth of human NSCLC xenografts and significantly prolonged survival in orthotopic and genetically engineered mouse models of PDA. Collectively, these data suggest that the first-in-class selective Axl inhibitor BGB324 can overcome EMT-related acquired therapeutic resistance and enhance the efficacy of multiple anti-cancer strategies. Together with the results of results of the early clinical safety studies, this provides a rationale for further clinical studies. Citation Format: Katarzyna Wnuk-Lipinska, Crina Tiron, Gro Gausdal, Tone Sandal, Robin Frink, Stefan Hinz, Monica Hellesøy, Lavina Ahmed, Hallvard Haugen, Xiao Liang, Magnus Blø, David Micklem, Murray Yule, John Minna, Longen Zhou, Rolf Brekken, James Lorens. BGB324, a selective small molecule Axl kinase inhibitor to overcome EMT-associated drug resistance in carcinomas: Therapeutic rationale and early clinical studies. [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 1747. doi:10.1158/1538-7445.AM2014-1747

Abstract 1746: Novel and selective Axl inhibitors

Abstract Axl is a member of the TAM tyrosine kinase family and is activated by products of Gas6 and Protein S genes. Axl is overexpressed or overactive in breast, renal, endometrial, ovarian, thyroid, non-small cell lung carcinoma, uveal melanoma as well as in myeloid leukemias. It has also been demonstrated that Axl expression is upregulated in drug-resistant cancer cells. Knock-down of Axl by RNAi repressed tumor formation in MDA-231 breast carcinoma model. Downregulation of Axl expression by RNAi led to a decrease of K562/ADR and MCF-7/ADR cells invasion, proliferation, and increased cell chemosensitivity in vitro. In a xenograft model of MDR cells, downregulation of Axl enhanced the anticancer activity of chemotherapeutic drugs. Therefore, Axl is a potential target for the development of chemotherapeutic agents for cancer therapy. During our endeavor of discovering and developing Axl kinase inhibitors for cancer therapy, we have discovered a series of potent and selective Axl inhibitors. These new compounds have demonstrated nM potency against Axl and good selectivity against other kinases in a selected panel of kinases. Many of these new compounds can reduce the pAkt levels in several cancer cell lines in a dose dependent manner and demonstrated inhibition of cell proliferation at sub-micromolar concentration in thymidine incorporation assay. Several selected compounds have demonstrated efficacy in colony formation assays. The most promising compounds are now being selected for in vivo proof-of-concept studies. The pharmaceutical properties of these compounds together with ADME profile were also evaluated and will be reported. These new Axl inhibitors represent a new approach for cancer therapy. Citation Format: Zaihui Zhang, Rick Li, Erica Lee, Yuxiang Hu, Jun Yan, Jasbinder Sanghera. Novel and selective Axl inhibitors. [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 1746. doi:10.1158/1538-7445.AM2014-1746

Abstract B30: Selective small molecule AXL inhibitor BGB324 overcomes acquired drug resistance in non-small cell lung carcinoma models.

Abstract Axl is a member of the TAM (Tyro3, Axl and Mer) family of receptor tyrosine kinases that regulate multiple cellular responses including cell survival, proliferation, and migration. Axl expression is associated with a variety of human cancers including non-small cell lung carcinoma (NSCLC), and is predictive of poor patient overall survival. Axl is induced by the epithelial-to-mesenchymal transition (EMT) gene program in cancer cells. Axl signaling is required to maintain EMT-associated features including invasiveness, metastasis, and can confer resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), as well as other chemotherapeutic agents. BGB324 is a potent, reversible and selective small molecule inhibitor of Axl that has recently entered into phase I clinical trials. We evaluated the effects of BGB324 on NSCLC cells in in vitro 3D assays and in mouse xenograft models, in combination with targeted and chemotherapeutic agents. BGB324 in combination with EGFR TKI erlotinib demonstrated a synergistic anti-proliferation effect on NCI-H1299 (mesenchymal, EGFR wild-type, erlotinib-resistant) human NSCLC cells in 3D culture. In mouse xenograft models using NCI-H1299 cells BGB324 treatment significantly enhanced the antitumor acitivity of Docetaxel. Similarly, using the mesenchymal NSCLC cell line A549 (EGFR wild-type), the combination of BGB324 with either erlotinib or anti-VEGF agent bevacizumab showed synergistic or additive anti-tumour activity. Furthermore, in HCC827 (EGFR mutant, erlotinib-sensitive) human NSCLC xenograft model, addition of BGB324 treatment strikely delayed the emergence of acquired resistance to erlotinib. Taken together, these data suggest the first-in-class selective Axl inhibitor BGB324 can overcome acquired resistance in in vivo models of NSCLC. Studies in human NSCLC patient-derived xenograft models and mechanisms of action are in progress. Citation Format: Katarzyna Wnuk-Lipinska, Gro Gausdal, Tone Sandal, Robin Frink, Stefan Hinz, Monica Hellesøy, Lavina Ahmed, Hallvard Haugen, Hallvard Haugen, Liang Xiao, Magnus Blø, David Micklem, John Minna, Rolf Brekken, Longen Zhou, James Lorens. Selective small molecule AXL inhibitor BGB324 overcomes acquired drug resistance in non-small cell lung carcinoma models. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B30.

Axl-Targeted Cancer Imaging with Humanized Antibody h173

The tyrosine kinase receptor Axl is overexpressed in various types of cancer and correlated with cancer malignancy. Selective Axl blockade reduces tumor growth and metastasis. The purpose of this study was to examine whether the humanized anti-Axl antibody humanized 173 (h173) labeled with near-infrared fluorescence (NIRF) dye Cy5.5 could be applied as a molecular imaging probe for NIRF imaging of Axl expression in tumor models. NIRF dye Cy5.5 was conjugated to h173 or human normal immunoglobulin G (hIgG) control through amino groups. The resulting probes were evaluated in both A549 (Axl positive) and NCI-H249 (Axl negative) lung cancer xenografts through in vivo NIRF imaging. Ex vivo imaging and probe distribution assay were also carried out to confirm the in vivo imaging results. After conjugation, binding activity of h173-Cy5.5 was determined to be 97.75 % ± 2.09 % of the unmodified h173. In vitro fluorescence-activated cell sorting (FACS) and fluorescence microscopy analysis validated the specific binding of h173 toward Axl-positive A549 cells. h173-Cy5.5 was then applied to image Axl expression in vivo. In A549 (Axl positive) cancer xenografts, the tumor uptake of h173-Cy5.5 was significantly higher than that of the hIgG-Cy5.5 control (P < 0.05) at late time points (1, 2, 3, 4, and 7 days). On the contrary, in NCI-H249 (Axl negative) cancer xenografts, the tumor uptake of both hIgG-Cy5.5 and h173-Cy5.5 was low and showed no significant difference (P > 0.05) at all time points examined. Ex vivo imaging and immunofluorescence staining analysis further validated the in vivo imaging results. Collectively, all in vitro, in vivo, and ex vivo data suggested that h173-Cy5.5 could serve as a valid probe for Axl-targeted cancer imaging, which could therefore aid in tumor diagnosis, prognosis, and treatment monitoring.

High-Throughput High-Content Imaging Assays for Identification and Characterization of Selective AXL Pathway Inhibitors

Receptor tyrosine kinases (RTKs) regulate a wide range of important biological activities, including cell proliferation, differentiation, migration, and apoptosis. Abnormalities in RTKs are involved in numerous diseases, including cancer and other proliferative disorders. AXL belongs to the TAM (Tyso3, AXL, and Mer) family of RTKs. The AXL signaling pathway represents an attractive target for the treatment of diseases, such as cancer. Using phospho-AKT as readout, a high-throughput 384-well cell-based assay was established in the NCI-H1299 human non-small cell lung carcinoma cell line to evaluate compound potency in inhibiting AXL pathway activation. In addition, a counter screen assay was established in the same cellular background to differentiate AXL kinase inhibitors from AXL receptor antagonists, which block the interaction of AXL and its natural ligand GAS6. These cell-based functional assays are useful tools in the identification and optimization of small molecules and biological reagents for potential therapeutics for the treatment of GAS6/AXL-related diseases.

Abstract C275: CEP-40783: A potent and selective AXL/c-Met inhibitor for use in breast, non-small cell lung (NSCLC), and pancreatic cancers.

Abstract The oncogenic receptor tyrosine kinases AXL and c-Met are over-expressed and constitutively activated in a variety of human cancers. Activation of these receptors contributes to multiple steps in tumor progression by promoting cancer cell migration and invasion, enhancing tumor angiogenesis, facilitating cancer cell survival and tumor growth, and contributing to tumor resistance to several chemotherapy and molecular-targeted therapeutic agents. CEP-40783 is an orally active, potent and selective AXL and c-Met kinase inhibitor, with enzyme IC50 values of 7 nM and 12 nM, respectively. In AXL-transfected 293GT cells, CEP-40783 was 27-fold more active compared to recombinant enzyme with an IC50 value of 0.26 nM. Comparably high cellular potency was observed in NCI-H1299 human NSCL cells. CEP-40783 also demonstrated superior activity against c-Met in GTL-16 cells (IC50 = 6 nM). The increased inhibitory activity of CEP-40783 in cells could be attributed to its extended residence time on both AXL and c-Met, consistent with a Type II mechanism. The prolonged residence time of CEP-40783 at the target may provide for improved in vivo efficacy, selectivity and therapeutic index. Additionally, CEP-40783 showed high kinome selectivity against 298 kinases with an S90 of 0.04 (fraction of kinases showing &amp;gt;90% inhibition at 1 µM). In PK/PD studies, CEP-40783 showed dose- and time-dependent inhibition of AXL phosphorylation using NCI-H1299 NSCL xenografts with ∼80% target inhibition at 0.3 mg/kg 6 h post dose and complete target inhibition to &amp;gt;90% inhibition at 1 mg/kg between 6-24 h, while a 10 mg/kg po dose resulted in complete AXL inhibition up to 48 h post dosing. In AXL/NIH3T3 xenografts, 0.3 mg/kg po resulted in complete tumor regressions. CEP-40783 was also efficacious in reducing spontaneous lymph node and pulmonary metastatic tumor burden in the MDA-MB-231-luc and 4T1-luc orthotopic breast cancer models, respectively, at 10 and 30 mg/kg po. PK/PD evaluation of the c-Met activity of CEP-40783 (10, 30, 55 mg/kg po qdX5d) showed significant to complete inhibition of c-Met phosphorylation in GTL-16 gastric carcinoma xenografts. Efficacy studies in GTL-16 xenografts demonstrated significant anti-tumor efficacy (tumor stasis and regressions) at 10 and 30 mg/kg po. In EBC-1 NSCL xenografts, administration of CEP-40783 (3, 10 and 30 mg/kg, po qd) resulted in dose-related efficacy, with tumor stasis at 3 mg/kg, tumor regressions and &amp;gt;96% TGI at 10 mg/kg. In all studies CEP-40783 was well tolerated with no compound-related body weight loss. CEP-40783 demonstrated potent AXL and c-Met pharmacodynamic and anti-tumor efficacy in established tumor xenograft models, having potential therapeutic utility in multiple human tumor types in which c-Met and AXL activity play a critical role in tumor formation, local invasion and metastasis. Studies in primary human tumorgrafts are in progress. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C275. Citation Format: Sheila Miknyoczki, Mangeng Cheng, Robert Hudkins, Thelma Angeles, Lisa Aimone, Jean Husten, Jie Qian, Seetha Murthy, Thomas Conners, Robert Bendesky, Amy Landis, Jennifer Grobelny, Hong Chang, Bruce Dorsey, Mark Ator, Bruce Ruggeri. CEP-40783: A potent and selective AXL/c-Met inhibitor for use in breast, non-small cell lung (NSCLC), and pancreatic cancers. [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 C275.

R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors.