Katarzyna Wnuk-Lipinska

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

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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 Hellesoy, Lavina Ahmed, Hallvard Haugen, Xiao Liang, Magnus Blo, 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

Small-Molecule Inhibition of Axl Targets Tumor Immune Suppression and Enhances Chemotherapy in Pancreatic Cancer

Activation of the receptor tyrosine kinase Axl is associated with poor outcomes in pancreatic cancer (PDAC), where it coordinately mediates immune evasion and drug resistance. Here, we demonstrate that the selective Axl kinase inhibitor BGB324 targets the tumor-immune interface to blunt the aggressive traits of PDAC cells in vitro and enhance gemcitibine efficacy in vivo Axl signaling stimulates the TBK1-NFκB pathway and innate immune suppression in the tumor microenvironment. In tumor cells, BGB324 treatment drove epithelial differentiation, expression of nucleoside transporters affecting gemcitabine response, and an immune stimulatory microenvironment. Our results establish a preclinical mechanistic rationale for the clinical development of Axl inhibitors to improve the treatment of PDAC patients.Significance: These results establish a preclinical mechanistic rationale for the clinical development of AXL inhibitors to improve the treatment of PDAC patients. Cancer Res; 78(1); 246-55. ©2017 AACR.

Abstract 626: BGB324, a selective small molecule inhibitor of receptor tyrosine kinase AXL, abrogates tumor intrinsic and microenvironmental immune suppression and enhances immune checkpoint inhibitor efficacy in lung and mammary adenocarcinoma models

The AXL receptor tyrosine kinase is associated with poor overall survival in a wide spectrum of cancers including lung and breast adenocarcinomas. AXL signaling is an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) and stem cell traits that drive metastasis and drug resistance. Signaling via AXL is also a key suppressor of the anti-tumor innate immune response, and AXL is expressed on several cells associated with the tumor immune microenvironment including natural killer (NK) cells and tumor-associated macrophages. Hence AXL resides uniquely at the nexus between tumor and microenvironmental anti-tumor immune suppression mechanisms. We report that BGB324, a selective clinical-stage small molecule Axl kinase inhibitor, enhances the effect of immune checkpoint blockade in aggressive adenocarcinoma models with limited immunogenicity by targeting both tumor intrinsic and microenvironmental immune suppression. Immune therapy with anti-CTLA4/PD1 in the 4T1 model increased AXL and EMT-marker expression correlating with a lack of response. Combination with BGB324 resulted in durable primary tumor clearance versus anti-CTLA4/PD1 alone. In a separate study, BGB324 + anti-CTLA4 treatment resulted in significant 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 the 4T1 model was not detected in animals responding to the combination treatment. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity. In the LL2 Lewis Lung model, BGB324 in combination with anti-PD1/PDL1 significantly prevented tumor growth compared to treatment with anti-PD1/PDL1. Tumors from mice treated with BGB324 in combination with immune checkpoint inhibitors displayed reduced EMT traits, altered cytokine expression, enhanced tumor infiltration of effector cells and decreased number of mMDSC. Also, BGB324 significantly reduced IL10 secretion by isolated human macrophages and enhanced human NK-cell mediated NSCLC tumor cell lysis. Collectively these results support a prominent role for AXL in resistance to immune therapy and support clinical translation of combining BGB324 with immune checkpoint inhibitors to improve cancer treatment. Citation Format: Katarzyna Wnuk-Lipinska, Kjersti Davidsen, Magnus Blo, Agnete Engelsen, Jing Kang, Linn Hodneland, Maria Lie, Sebastien Bougnaud, Kristina Aguilera, Lavina Ahmed, Agata Rybicka, Eline Milde Naevdal, Paulina Deyna, Anna Boniecka, Straume Oddbjorn, Salem Chouaib, Rolf Brekken, Gro Gausdal, James B. Lorens. BGB324, a selective small molecule inhibitor of receptor tyrosine kinase AXL, abrogates tumor intrinsic and microenvironmental immune suppression and enhances immune checkpoint inhibitor efficacy in lung and mammary adenocarcinoma models [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 626. doi:10.1158/1538-7445.AM2017-626

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

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 Blo, Linn Hodneland, Agnete Engelsen, Jing Kang, Maria Lie, Sebastien Bougnaud, Kristina Aguilera, Lavina Ahmed, Agata Rybicka, Elina Milde, Paulina Deyna, Anna Boniecka, Oddbjorn 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

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, Sebastien Bougnaud, Monica Hellesoy, Magnus Blo, Lavina Ahmed, Linn Hodneland, Sergej Kiprijanov, Oddbjorn 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.

Drivers of EMT and Immune Evasion

The heterogeneity of tumor cells and the complexity of the surrounding microenvironment make the process of predicting patient outcome and selection of the most suitable treatment regimen very difficult. Over the past decades, many biomarkers have been evaluated for prognostic value. The advent of immune therapy as a frontline treatment for some cancers has moved immune phenotyping into the forefront of biomarker and predictive marker research. Here, we review some of the regulatory mechanisms of the host immune response and epithelial plasticity and highlight their potential as biomarkers of the hallmark of immune evasion.

Abstract 4888: Axl receptor signaling in required for stem cell traits and metastasis in breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Epithelial-to-mesenchymal transition (EMT) endows carcinoma cells with migratory, survival and stem cell-like attributes that facilitate therapeutic resistance and metastasis. Expression of the Axl receptor tyrosine kinase in aggressive breast cancer correlates with EMT, poor survival and is prevalent in patient metastases. Induction of EMT in immortalized mammary epithelial cells by EMT-transcription factors, TGFbeta or hypoxia upregulates Axl and establishes an autocrine-signaling loop with its ligand, Gas6. Axl receptor signaling is required to maintain EMT-related invasive, drug resistance and cancer stem cell (CSC) traits of malignant breast cancer cells. Targeting Axl signaling with RNAi or pharmacological agents blocks the EMT/CSC gene program and inhibits malignant functions including invasiveness, drug resistance, mammosphere formation, in vivo tumor initiation, and spontaneous metastasis in orthotopic breast cancer models. These results suggest that Axl expression is requisite for the maintenance of EMT and stem cell-like traits during malignant progression. The EMT program is characteristic of normal adult mammary epithelial stem/progenitor cells. Congruently, we show that the Axl receptor expressed on mammary epithelial stem/progenitor cells, and Axl signaling is necessary for mammary epithelial multipotent progenitor activity. Thus Axl receptor signaling represents a novel regulatory pathway linking normal mammary stem/progenitor cells and breast cancer stem cells. Hence, clinical Axl inhibitors represent a novel therapeutic avenue to target EMT/CSC traits of aggressive breast cancer. Citation Format: Crina Tiron, Fanny Pelissier, Katarzyna Wnuk-Lipinska, Ingunn Stefansson, Reeta Virtakoivu, Masaru Miyano, Tone Sandal, David Micklem, James Garbe, Martha Stampfer, Johanna Ivaska, Lars Akslen, Mark LaBarge, James Lorens. Axl receptor signaling in required for stem cell traits and metastasis in breast cancer. [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 4888. doi:10.1158/1538-7445.AM2013-4888

Abstract C76: Axl signaling is required for stem cell traits and metastasis in breast cancer

Epithelial-to-mesenchymal transition (EMT) endows carcinoma cells with migratory, survival and stem cell-like attributes that facilitate therapeutic resistance and metastasis. Expression of the Axl receptor tyrosine kinase in breast cancer correlates with EMT, poor survival and is prevalent in patient metastases. Axl upregulation in mammary epithelial cells by EMT-transcription factors, TGFbeta or hypoxia, establishes an autocrine-signaling loop with its ligand, Gas6. Inhibition of Axl signaling blocks EMT/ cancer stem cell traits including invasiveness, drug resistance, mammosphere formation, in vivo tumor initiation, and prevents spontaneous metastasis in orthotopic breast cancer models. Congruently, we show that Axl is expressed on mammary epithelial stem/progenitor cells and regulates multipotent progenitor activity. Thus Axl signal transduction represents a novel regulatory pathway linking normal mammary stem/progenitor cells and breast cancer stem cell activity. Clinical Axl-inhibitors represent a novel therapeutic opportunity to treat aggressive breast cancer. Citation Format: Crina Tiron, Fanny Pelissier, Katarzyna Wnuk-Lipinska, Ingunn Stefansson, Reeta Virtakoivu, Masaru Miyano, Tone Sandal, David Micklem, James Garbe, Martha Stampfer, Johanna Ivaska, Lars Akslen, Mark LaBarge, James Lorens. Axl signaling is required for stem cell traits and metastasis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C76.