Lavina Ahmed

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

Strong Expression of Hypoxia-Inducible Factor-1α (HIF-1α) Is Associated with Axl Expression and Features of Aggressive Tumors in African Breast Cancer.

Purpose Inhibition of hypoxia-inducible factor (HIF) and Axl receptor tyrosine kinase is being evaluated for targeted therapy in solid tumors. Both HIF-1α and Axl influence tumor growth and metastatic potential, and they have been linked to treatment failure in many cancers. However, there is a lack of reports on HIF-1α expression in African breast cancer, which has a poor prognosis, and novel treatment targets must therefore be established. Here, we aimed to evaluate HIF-1α in relation to Axl expression, angiogenesis markers, and other tumor characteristics in a series of African breast cancer. Methods Using immunohistochemistry, we examined 261 invasive breast cancers on tissue microarrays for HIF-1α and Axl as well as several other markers, and a subset of 185 cases had information on VEGF (vascular endothelial growth factor) expression, microvessel density (MVD), proliferating microvessel density (pMVD) and vascular proliferation index (VPI) for important comparisons. Results Strong HIF-1α expression was associated with increased Axl (p = 0.007), VEGF (p<0.0005), and p53 (p = 0.032) expression, as well as high tumor cell proliferation by Ki-67 (p = 0.006), and high tumor grade (p = 0.003). Tumors with strong HIF-1α expression had significantly higher MVD (p = 0.019) and higher pMVD (p = 0.027) than tumors with weak expression. Conclusions High HIF-1α expression is significantly associated with Axl and VEGF expression, and with markers of poor prognosis in this series of breast cancer, suggesting HIF-1α and Axl as potential therapeutic targets in African breast cancer.

Increased tumor cell expression of Axl is a marker of aggressive features in breast cancer among African women

Axl, a receptor tyrosine kinase belonging to the Tyro/Axl/Mer (TAM) family, has been shown to be overexpressed in breast cancer with poor outcome. Moreover, Axl was associated with a basal‐like phenotype (BLP) in these tumors. Our aim was to investigate Axl expression in breast cancers from an African population since these tumors are known to be aggressive and have a high frequency of the basal‐like phenotype. We studied 170 paraffin‐embedded breast carcinoma cases by tissue microarrays and immunohistochemical methods. In total, 128 tumor cases (75%) had strong Axl expression and 42 cases (25%) had weak or negative staining. Strong expression of Axl was associated with high tumor grade (p < 0.0005), estrogen receptor (ER) negativity (p = 0.024), p53 expression (p = 0.004), P‐cadherin positivity (p = 0.017), and basal‐like phenotypic profiles BLP2 (p = 0.033) and BLP3 (p = 0.022). In addition, Axl overexpression also showed an association with markers of tumor cell proliferation and tumor angiogenesis. In conclusion, our findings indicate strong expression of Axl in a high proportion of breast cancer cases among African women and associations with markers of aggressive features, indicating poor prognosis. These findings suggest Axl as a potential therapeutic target in this population.

Clear Cell Renal Cell Carcinoma is linked to Epithelial‐to‐Mesenchymal Transition and to Fibrosis

Clear cell renal cell carcinoma (ccRCC) represents the most common type of kidney cancer with high mortality in its advanced stages. Our study aim was to explore the correlation between tumor epithelial‐to‐mesenchymal transition (EMT) and patient survival. Renal biopsies of tumorous and adjacent nontumorous tissue were taken with a 16 g needle from our patients (n = 26) undergoing partial or radical nephrectomy due to ccRCC. RNA sequencing libraries were generated using Illumina TruSeq® Access library preparation protocol and TruSeq Small RNA library preparation kit. Next generation sequencing (NGS) was performed on Illumina HiSeq2500. Comparative analysis of matched sample pairs was done using the Bioconductor Limma/voom R‐package. Liquid chromatography‐tandem mass spectrometry and immunohistochemistry were applied to measure and visualize protein abundance. We detected an increased generic EMT transcript score in ccRCC. Gene expression analysis showed augmented abundance of AXL and MMP14, as well as down‐regulated expression of KL (klotho). Moreover, microRNA analyses demonstrated a positive expression correlation of miR‐34a and its targets MMP14 and AXL. Survival analysis based on a subset of genes from our list EMT‐related genes in a publicly available dataset showed that the EMT genes correlated with ccRCC patient survival. Several of these genes also play a known role in fibrosis. Accordingly, recently published classifiers of solid organ fibrosis correctly identified EMT‐affected tumor samples and were correlated with patient survival. EMT in ccRCC linked to fibrosis is associated with worse survival and may represent a target for novel therapeutic interventions.

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.

Abstract P2-04-08: BGB324, a selective small molecule inhibitor of the receptor tyrosine kinase AXL, enhances immune checkpoint inhibitor efficacy in mammary adenocarcinoma

The AXL receptor tyrosine kinase is associated with poor overall survival in breast cancer. 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. AXL is expressed on several cells associated with the tumor immune microenvironment including natural killer cells, dendritic cells and tumor-associated macrophages. AXL is required for tumor immune evasion in mammary adenocarcinoma models and EMT-mediated resistance to cytotoxic T cell and natural killer (NK)-cell mediated cell killing. Hence AXL signaling contributes uniquely to both tumor cell intrinsic and microenvironmental anti-tumor immune suppression mechanisms in breast cancer. We evaluated whether blocking AXL signaling with BGB324, a selective clinical-stage small molecule Axl kinase inhibitor, enhances the effect of immune checkpoint blockade in the aggressive mammary adenocarcinoma (4T1) syngeneic (Balb/C) mouse modelthat display limited immunogenicity. Immune therapy with anti-CTLA-4/anti-PD-1 increased AXL and EMT-marker expression in 4T1 tumors, and correlated with lack of response to immune therapy. Combination treatment 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 (CTLs). Enhanced presence of CTLs was also detected in spleens from animals responding to treatment. BGB324 + anti-CTLA-4/anti-PD-1 treatment increased the number of NK cells, macrophages and polymorphonuclear neutrophils, but decreased the number of mMDSC. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity. Together with recent results in other tumor types that support a prominent role for AXL in resistance to immune therapy and encouraging results from ongoing clinical trials with BGB324, support combining BGB324 with immune checkpoint inhibitors to improve treatment of breast cancer. Citation Format: Lorens JB, Lipinska KW, Davidsen K, Blo M, Hodneland L, Engelsen A, Kang J, Lie MK, Bougnaud S, Aguilera K, Ahmed L, Rybicka A, Naevdal EM, Deyna P, Boniecka A, Straume O, Chouaib S, Brekken RA, Gausdal G. BGB324, a selective small molecule inhibitor of the receptor tyrosine kinase AXL, enhances immune checkpoint inhibitor efficacy in mammary adenocarcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-08.