Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0135 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Ibrutinib potentiates anti-hepatocarcinogenic efficacy of sorafenib by targeting EGFR in tumor cells and BTK in immune cells in the stroma Cho-Hao Lin1,2, Khadija H. Elkholy1,2, Nissar A. Wani2,3, Ding Li1,2, Peng Hu1,2, Juan M. Barajas1,2, Lianbo Yu2,4, Xiaoli Zhang2,4, Samson T. Jacob2,3, Wasif N. Khan6, Xue-Feng Bai1,2, Anne M. Noonan2,5 and Kalpana Ghoshal1,2* 1 Department of Pathology, 2 Comprehensive Cancer Center, 3 Department of Cancer Biology and Genetics, 4 Department of Biomedical Informatics, 5 Department of Internal Medicine, The Ohio State University, Columbus, OH and 6 Department of Microbiology & Immunology and Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL Authors’ e-mail address Cho-hao Lin: [email protected] Khadija Elkholy: [email protected] Nissar A. Wani: [email protected] Ding Li: [email protected] Peng Hu: [email protected] Juan M. Barajas: [email protected] Lianbo Yu: [email protected] Xiaoli Zhang: [email protected] Samson Jacob: [email protected] Wasif N. Khan: [email protected] Xue-Feng Bai: [email protected] Anne Noonan: [email protected] Kalpana Ghoshal: [email protected] 1 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0135 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Keywords: Hepatocellular cancer, Akt, ERK, regorafenib, drug synergy, combination index, CEMIP, LHPP *Corresponding author: Kalpana Ghoshal: 420 W 12th Avenue, Columbus Ohio 43210, Ph: 614-292-8865, e-mail: [email protected] Running title: sorafenib/Ibrutinib combination therapy for HCC Conflict of interest: no conflict Financial Support: This work was supported, in part, by grants R01CA193244 (Kalpana Ghoshal) and R01CA086978 (Samson T. Jacob and Kalpana Ghoshal) from the National Cancer Institute, a seed grant from Translational Therapeutics program of OSUCCC and Intramural Funding Program from Sylvester Comprehensive Cancer Center, the University of Miami (Wasif N. Khan). Juan M. Barajas was a Howard Hughes Medical Institute Gilliam fellow. 2 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0135 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Hepatocellular carcinoma (HCC), the most prevalent primary liver cancer, is a leading cause of cancer-related death worldwide because of rising incidence and limited therapy. Although treatment with sorafenib or lenvatinib is the standard of care in advanced-stage HCC patients, the survival benefit from sorafenib is limited due to low response rate and drug resistance. Ibrutinib, an irreversible tyrosine kinase inhibitor (TKI) of TEC (e.g. BTK) and ErbB (e.g. EGFR) families, is an approved treatment for B cell malignancies. Here, we demonstrate that ibrutinib inhibits proliferation, spheroid formation and clonogenic survival of HCC cells including sorafenib resistant cells. Mechanistically, ibrutinib inactivated EGFR and its downstream Akt and ERK signaling in HCC cells, and downregulated a set of critical genes involved in cell proliferation, migration, survival and stemness, and upregulated genes promoting differentiation. Moreover, ibrutinib showed synergy with sorafenib or regorafenib, a sorafenib congener by inducing apoptosis of HCC cells. In vivo, this TKI combination significantly inhibited HCC growth and prolonged survival of immune-deficient mice bearing human HCCLM3 xenograft tumors and immune competent mice bearing orthotopic mouse Hepa tumors at a dose that did not exhibit systemic toxicity. In immune competent mice, the ibrutinib- sorafenib combination reduced the numbers of BTK+ immune cells in the tumor microenvironment. Importantly, we found that the BTK+ immune cells were also enriched in the tumor microenvironment in a subset of primary human HCCs. Collectively, our findings implicate BTK signaling in hepatocarcinogenesis and support clinical trials of the sorafenib-ibrutinib combination for this deadly disease. 3 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0135 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Liver cancer is an aggressive disease; there has been a dramatic increase in incidence and mortality in the last several decades worldwide (1,2). Between 1990 and 2015, there was a ~75% increase in liver cancer incidence globally (3). In 2015, incidence of liver cancer was 854,000 cases and 810,000 patients died of this disease. Hepatocellular carcinoma (HCC) which accounts for ~90% of all primary liver cancers, and is the fourth major cause of cancer-related death worldwide and the fastest growing cause of cancer related mortality in the United States (2). Hepatitis B or hepatitis C virus infection, alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD), a complication of obesity, are the major risk factors for HCC (2). High mortality rate is, in part, due to late diagnosis of this aggressive tumor in patients with chronic liver disease. Sorafenib is a widely used first-line treatment of HCC since 2007 (2). As a pan-kinase inhibitor, sorafenib strongly inhibits proangiogenic VEGFRs, PDGFRβ, as well as pro- proliferative kinases such as RAF, c-KIT and FLT-3 (4). However, patients treated with sorafenib had a response rate of 2-9% and an overall survival time of only 10.9 to 12.3 months. Patients also frequently developed drug resistance. Recently approved targeted therapies, e.g. lenvatinib in the first-line setting, improved clinical outcomes such as response rate and progression-free survival, but did not increase the median overall survival (5). Regorafenib and cabozantanib approved in the second-line setting have both shown improvement in median overall survival (6,7). The US Food and Drug Administration (FDA) recently approved PD-1 checkpoint antibodies, Nivolumab (8), and Pembrolizumab (9) in HCC patients who have been previously treated with sorafenib. However, only a subset of patients responded to this type of immunotherapy. Furthermore, transplant recipients on immune suppression are not candidates for immunotherapy. More importantly, the first-line trials of the checkpoint inhibitors in HCC patients failed to improve overall survival of HCC patients compared to best supportive care (10). Thus, there is an unmet need to develop better therapeutic options in the first- line setting to improve the survival and response rate in HCC. 4 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0135 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Ibrutinib (PCI-32765), is the first-in-class small molecule inhibitor targeting Bruton’s tyrosine kinase (BTK) (11), a key non-receptor tyrosine kinase in the B-cell receptor signaling pathway, and critical for the survival of malignant B cells. The FDA designated ibrutinib as a breakthrough therapy, and it was approved for the treatment of relapsed and refractory mantle cell lymphoma (MCL), relapsed and refractory chronic lymphocytic leukemia (CLL) and Waldenstrom’s Macroglobulinemia (12). Ibrutinib irreversibly inhibits Tyr-223 kinase activity by covalently binding to Cys-481 adjacent to the ATP binding pocket of BTK (13). Ibrutinib exhibits both anti-tumorigenic and immunomodulatory and anti-tumorigenic functions because of its ability to inhibit tyrosine kinases e.g. BTK, ITK, BLK, TEC, BMX and JAK3 expressed in innate and adaptive immune cells (13) and ErbB family members, e.g. EGFR/ErbB1, Her2/ErbB2 and ErbB4 (14), predominantly expressed in epithelial cells. Several reports demonstrated ibrutinib’s efficacy in preclinical models of a variety of solid tumors (15). Ibrutinib exhibits lasting tumor suppression by activating CD8+ T cells (16-18) and suppressing myeloid-derived suppressor cells (MDSCs) (19). Currently, ibrutinib in combination with chemo- or immuno-therapy is being assessed in clinical trials in several solid tumors. In this study, we assessed the anti-tumorigenic efficacy of ibrutinib alone and in combination with sorafenib in vitro and in vivo and determined the underlying molecular mechanisms. We found that ibrutinib co-operates with sorafenib by inactivating its substrate EGFR in tumor cells and BTK in immune cells in the tumor microenvironment. Our study also demonstrated that the BTK positive immune cells are enriched in the tumor stroma in a subset of primary human HCCs. Materials and Methods Cell culture and drug treatment HCC cell lines: HepG2, Hep3B, PLC/PRF/5, SNU-182, SNU-449 and BNL 1ME A.7R.1 (BNL) were obtained from the ATCC. Huh-7, Hepa1-6 (Hepa) and HCCLM3 cells were 5 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019