Ibrutinib Potentiates Anti-Hepatocarcinogenic Efficacy of Sorafenib by Targeting EGFR in Tumor Cells and BTK in Immune Cells in the Stroma

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]

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.

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.

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.

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

provided by Drs. James Taylor (Fox Chase Center, PA, USA), Gretchen Darlington (previously at Baylor College of Medicine) and Hangxiang Wang (The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China), respectively. Cells were maintained in either DMEM or Minimum Essential Media supplemented with L- glutamine (2 mM), 10% FBS, sodium pyruvate (1 mM) and penicillin/ streptomycin/amphotericin (Thermo Fisher Scientific, Pittsburg, PA) at 37C with 5%

CO2. Sorafenib resistant Huh7 (Huh7-SR) cells, previously generated in our laboratory (20), were grown in the media supplemented with sorafenib (6 µM). Sorafenib was withdrawn from the culture media Huh7-SR cells for 2 days prior to performing experiments. Firefly expressing HCCLM3 (HCCLM3-Luc) and Hepa (Hepa-Luc) cells were generated by infecting these cells with firefly luciferase lentivirus (GeneCopoeia, Rockville, MD) followed by selection of positive clones with puromycin (5 g/ml) treatment for 4 weeks. For treatment of cells in culture, sorafenib and ibrutinib were dissolved in DMSO.

Cell survival assay HCC cells seeded into 96-well plates (3000 cells/well) were allowed to grow overnight followed by treatment with sorafenib (LC Laboratories, Woburn, MA), ibrutinib (Cayman chemicals, Ann Arbor, MI and Acorn PharmaTech, Redwood City, CA) or combination of both. Cell viability was assessed after 72 hours of drug exposure using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). Each treatment was done in quadruplicate.

Statistical analysis of drug interaction The two drugs (A and B) are considered to act synergistically if the biological response (cell survival in this study) to A (sorafenib) and B (ibrutinib) co-treatment is greater than the sum of the response to A and B alone. A two-way ANOVA was used to test this hypothesis (µboth - µneither) > (µA - µneither) + (µB - µneither), where µ is the mean response to each treatment and the vehicle control. P-values <0.05 are considered as significant synergistic interactions between the two drugs (21).

Clonogenic survival assay HCC cells were seeded in 12-well plate (1~2×104 cells/well). After 24 hours, cells were treated with sorafenib, ibrutinib, combination of both or vehicle for 5-7 days. The culture medium and drugs were replaced every other day. Cells were fixed in 4% paraformaldehyde and colony formation was visualized with 0.05% crystal violet dye.

Plasmid transfection HCC cells were placed in a 6-well plate at 3×105 cells/well. After 24 hours, cells were transfected with 2 µg of Myr-Akt-HA or wild type Akt plasmid DNA using the Lipofectamine 3000 reagent (ThermoFisher Scientific, Pittsburg, PA).

RNA interference HCC cells plated overnight in a 6-well plate at 3×105 cells/well were transfected with siEGFR (cat #M-003114-03, Dharmacon, Lafayette, CO) or control siRNA (cat# D- 001206-13, Dharmacon, Lafayette, CO) (final concentration, 50 nM) using RNAiMAX (ThermoFisher Scientific). After 48 hours, cells were treated with the drugs and cell survival was measured after 72 hours.

Spheroid formation assay HCC cells (3000 cells) were plated in 96-well Corning® Costar® Ultra-Low Attachment plates in serum-free DMEM/F-12 medium supplemented with containing 2 mM glutamine, 1mM sodium pyruvate, 1% MEM nonessential amino acid solution, 1% B- 27 supplement (Gibco, Gaithersburg, MD), 100 µg/ml penicillin G, and 100 U/ml streptomycin supplemented with 20 ng/ml epidermal growth factor (EGF) and 10 ng/ml fibroblast growth factor (FGF). After 7 days, spheroids were treated with drugs or vehicle for another 48 hours, and cell viability was measured with CellTiter-Glo Luminescent Cell Viability Assay.

RNA-sequence (RNA-seq) analysis RNA sequencing (RNA-seq) was performed in PLC/PRF/5 cells treated with sorafenib (2µM), ibrutinib (4µM), combination of both or vehicle (DMSO) for 12 hours. Total RNA was isolated with TRIzol reagent (Invitrogen, CA) following the manufacturer's instructions and quantified using Invitrogen Qubit 2.0 Fluorometer (ThermoFisher Scientific, PA). Samples with RNA Integrity (RIN) scores ≥7 were used to generate libraries using the Illumina TruSeq® Stranded mRNA LT kit (Illumina, San Diego, CA). Multiplexed libraries were sequenced on Illumina HiSeq 4000 Sequencer to achieve 17 to 20 million paired-end 150bp reads per sample. De-multiplexed and quality-filtered reads were mapped to the human genome GRCh38 using Hierarchical Indexing for Spliced Alignment of Transcripts (HISAT2) (22). Raw read counts for each gene were quantified using featureCounts software, with GENCODE v.27 transcript reference (GENCODE annotation) (23). We have submitted RNA-seq raw data to the Geo database (Accession number GSE114184).

For statistical analysis of the RNA-Seq data, genes with read counts below 5 in at least 2 out of 3 biological replicates in each treatment group were filtered out. Next, the read counts were normalized using TMM method (23). R package limma with Voom transformation (24) was used to calculate p-values for group comparisons under a linear model. The p-value cut-offs were determined by calculating the mean number of false positives (25).

Real-time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) analysis Total RNA was isolated from cells using TRIzol reagent, treated with DNase 1 and reverse transcribed into cDNA using high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). qRT-PCR was performed using 0.01-0.1μg cDNA with SYBR Green in a thermocycler. The fold difference in target gene mRNA levels was calculated using the ΔΔCT method and normalized to 18S rRNA or β-actin mRNA. The primer sequences are provided in the Supplementary Table 1.

Flow cytometry Cells at distinct phases of cell cycle were distinguished by staining DNA with propidium iodide (PI) and quantified by flow cytometry. HCC cells treated with the drugs alone, in combination or vehicle for 24 hours, were collected, washed with PBS and fixed in ice- cold 70% ethanol and stored at -20C overnight. The cells were centrifuged, washed with phosphate-buffered saline (PBS) and resuspended in 0.5 ml PBS. To the single cell suspension, 50 µl of RNase A (1 mg/ml in PBS) was added and incubated at 37C for 30 minutes, followed by the addition of 50 µl of propidium iodide (PI) (500 µg/ml in PBS) with gentle mixing and incubation in the dark at room temperature for 15 minutes and subjected to flow cytometry using a LSRII flow cytometer (BD Biosciences, CA). Cell apoptosis was determined with PI and Annexin V double staining using Annexin V/Dead Cell Apoptosis kit (Invitrogen) following manufacturer’s instruction.

Mouse strains, animal husbandry and treatment NSG (NOD scid Il2r-/-) mice were purchased from Target Validation Shared Resource (TVSR) core facility at the Ohio State University and C57BL/6N mice were purchased from Charles River Laboratories. All animals were housed in a temperature-controlled room under a 12-hour light / 12-hour dark cycle and under helicobacter-free conditions and fed normal chow diet. All animal studies were reviewed and approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee. Both male and female mice were used for experiments. For drug treatment, ibrutinib stock (40 mg/ml in DMSO) was diluted with 10% Hydroxypropyl beta-Cyclodextrin (HPBCD) (CTD Holdings, Alachua, FL) to the final concentration of 4 mg/ml immediately before injection. Sorafenib was diluted in Cremophor EL/ethanol/water (1:1:6) to 3 mg/ml before delivery through oral gavage. For subcutaneous xenografts, 10-12 weeks old NSG mice were injected subcutaneously with HCCLM3 (5x106) cells into the right flank. When tumor volume reaching 150mm3, mice were randomized into 4 groups and injected intraperitoneally with ibrutinib (20 mg/kg/day), sorafenib (15 mg/kg/alternate day) administrated through oral gavage, combination of both and respective vehicles (DMSO in HPBCD for ibrutinib and Cremophor EL/ethanol/water for sorafenib), respectively. Tumor volumes based on digital caliper measurements were calculated by

the ellipsoidal formula (1/2(length x width2)). After 4 weeks of treatment, mice were euthanized and tumor tissues were collected, weighed and photographed.

For orthotopic model, 10-12 weeks old NSG or C57BL/6N mice were injected with 1 million of HCCLM3-Luc or 0.05 millions of Hepa-Luc cells into the left liver lobe, and the mice were monitored by IVIS Lumina (Caliper Life Sciences) every week. Once the luciferase signal was detected, mice were randomized into 4 groups and treated with the vehicle, sorafenib (15 mg/kg/alternate day), ibrutinib (20 mg/kg/day) or their combination as described above. Survival was determining using Kaplan-Meier curve.

Bioluminescent Imaging

Mice were anesthetized with 2% isoflurane and 100% O2 with a flow rate of 2 L/min, injected with luciferin (150 mg/kg, intraperitoneally) and imaged with 1 min exposure from the dorsal perspective using an IVIS Lumina (Caliper Life Sciences, Hopkinton, MA). Acquisition and analysis of images were performed by using the Living Image Software.

Analysis of systemic and tissue toxicity in mice C57BL/6N mice were treated with the vehicle, sorafenib (15 mg/kg/alternate day), ibrutinib (20 mg/kg/day) or their combination for 4 weeks. Serum collected from mice by cardiac puncture and analyzed with the Vet Axcel Chemistry Analyzer (Alfa Wasserman, West Caldwell, NJ) for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine kinase (CK), blood urea nitrogen (BUN), albumin and globulin.

Western blot analysis 7x105 HCC cells were plated overnight in 60mm dish and treated with sorafenib and ibrutinib alone or in combination for 1 hour or 24 hours. Whole cell extracts were prepared in cell lysis buffer (CLB) (cat#9803, Cell signaling technology, Beverly, MA) containing protease inhibitor cocktails (Sigma, St. Louis, MO). The cell lysates were incubated at 4oC for 10 minutes and centrifuged at 4oC for 10 minutes to collect clear

supernatants. The tumor lysates from snap-frozen tissues were prepared by suspension in CLB followed by sonication before centrifugation. Protein concentrations in the extracts were measured using a Bio-Rad protein assay kit (cat#500-0006) with BSA as the standard. Equal amounts of protein (10-50 g) from whole cell or tissue lysates were separated by SDS-polyacrylamide (10% or 4-20% gradient) gel electrophoresis (Bio- Rad, Hercules, CA), transferred to nitrocellulose membranes (GE Healthcare, Chicago, IL), incubated using blocking buffer (LI-COR, Lincoln, NE) followed by immunoblotting with phospho-Akt (Cell signaling #4060), total Akt (Cell signaling #9272), phospho-ERK (Cell signaling #4370), total ERK (Cell signaling #9102), phospho-EGFR (Cell signaling #3777), total EGFR (Santa Cruz #sc-03) PARP (Cell signaling #9532), BTK (Cell signaling #8547), β-actin (Santa Cruz #sc-47778), tubulin (Proteintech #66031) or GAPDH (Cell signaling #5174). Following incubation with appropriate secondary antibody (IRD-680 or IRD-800), the specific immune-reactive bands were visualized using Odyssey CLx Imaging System (LI-COR, Lincoln, NE) and quantified using Image Studio software.

Immunohistochemistry and tissue microarray (TMA) After fixation in 4% paraformaldehyde, tumor tissues were processed, embedded in paraffin, and sectioned at 4μm thickness. Hepa tumor and human HCC TMA (cat# LV242 and HLivH090BC01, USBiomax, Derwood, MD) sections were baked at 60°C for 30 minutes, dewaxed in xylene and rehydrated in graded ethanol. Antigen retrieval was processed in citrate buffer (0.01 M, pH=6.0) at 95°C for 30 minutes followed by incubation with the Ki-67 (Cell signaling #9027) or BTK antibodies (Cell signaling #8547), and color was developed by the DAB method. Images were taken by a phase contrast microscope (BX41TF, OLYMPUS) and camera (DP71, OLYMPUS) and the Ki- 67 positive tumor cells were counted using ImageJ software.

Results

Ibrutinib inhibits HCC cell proliferation and clonogenic survival by inhibiting EGFR signaling To test the anti-hepatocarcinogenic functions of ibrutinib, a TKI (tyrosine kinase

inhibitor), we determined its IC50 in seven human (HepG2, HCCLM3, PLC/PRF/5, Huh7, sorafenib-resistant Huh7 (Huh7-SR), SNU-182 and SNU-449) and two mouse (Hepa and BNL) HCC cell lines. These cell lines were differentially sensitive to ibrutinib as

demonstrated by IC50s ranging from 1 μM for PLC/PRF/5 cells to 40 μM for HepG2 cells

(Figure 1A and Table 1). Notably, both the parental Huh7 (IC50 6 μM) and Huh7-SR

(IC50 7 μM) cells (20) were sensitive to ibrutinib. Clonogenic survival of HCC cells was also inhibited by this TKI (Figure S1A).

To elucidate the underlying mechanism, we sought to identify the tyrosine kinase substrate of ibrutinib in HCC cells. Since immune cell - specific substrates of ibrutinib, e.g. BTK, ITK are not expressed in HCC cell lines (Figure S1B, C), we investigated its effects on ErbB family members that are expressed in epithelial cells. Among these, EGFR/ErbB1 is activated in primary HCC tissues and cell lines, and its upregulation is associated with poor survival of HCC patients (26). As shown in Figure 1B, ibrutinib (10 µM) treatment for 1 hour reduced EGFR (Y1086) phosphorylation in Huh7 by 70%. Notably, p-EGFR was elevated by ~50% in Huh7-SR cells, which was reduced by 45% upon ibrutinib exposure. Consistent with a previous report (27), Akt signaling was found to be activated in Huh7-SR cells relative to Huh7 cells as demonstrated by ~70% increase in Akt (S473) phosphorylation. Notably, ibrutinib treatment also blocked Akt (S473) phosphorylation in Huh7-SR cells. Phosphorylation of ERK (T202/Y204), was

also inhibited in both cell types. Notably, higher IC50s of HepG2, HCCLM3 and BNL cells correlated with elevated phospho- and total- Akt levels (Figure S1D), suggesting a role of Akt in ibrutinib resistance. Ibrutinib persistently inactivated EGFR phosphorylation even after 6 hours of drug removal (Figure S1E). These results suggest that the suppression of HCC cell survival by ibrutinib resulted from inactivation of EGFR function.

To identify upstream signaling responsible for activation of Akt and ERK in HCC cells, we assessed the effects of ibrutinib on EGF - induced EGFR (Y1086) phosphorylation in HCCLM3 cells. These cells exhibited ~4-fold increase in phospho-EGFR level as early as 30 minutes of EGF exposure that persisted even after 90 minutes (Figure 1C). Importantly, ibrutinib pre-treatment not only blocked EGF-enhanced EGFR (Y1086) phosphorylation by ~3-4 fold but also impeded Akt (S473) phosphorylation by ~2 folds in HCCLM3 cells (Figure 1C), suggesting that ibrutinib inhibited Akt signaling via blocking EGFR activation.

To confirm that EGFR is a critical target of ibrutinib in HCC cells, we knocked down EGFR expression in HCCLM3 cells by siRNA that reduced phospho-Akt (S473) and phospho-ERK (T202/Y204) levels (Figure 1D). These cells responded well to siRNA- mediated EGFR depletion without significantly affecting total Akt and ERK levels. As expected, ibrutinib treatment further reduced EGFR, Akt and ERK phosphorylation both

in the control siRNA- and EGFR-siRNA transfected cells. Notably, the IC50 of ibrutinib increased from 12 μM to 21 μM in EGFR-depleted HCCLM3 cells compared to control

cells (Figure 1E). Similarly, EGFR knockdown increased ibrutinib IC50 from 4 μM to 8 μM in PLC/PRF/5 cells (Figure S1F). Collectively, these results demonstrate that inactivation of EGFR and its downstream Akt and ERK signaling is a key mechanism by which ibrutinib suppresses HCC cell survival.

To gain mechanistic insight at the gene expression level, we queried the effects of ibrutinib on the transcriptome of PLC/PRF/5 cells. We chose these cells as they are

highly sensitive to ibrutinib (IC50 1 μM) (Figure 1A). RNA-seq analysis of these cells treated with ibrutinib (1 μM) for 12 hours showed that significant (P<0.001) upregulation (>1.5 fold) of 170 genes and downregulation (>1.5 fold) of 225 genes compared to DMSO controls. Expression of several genes that regulate cell proliferation, migration, stemness and apoptosis were significantly altered by ibrutinib treatment (Figure 1F). Effects of Ibrutinib on expressions of key genes were confirmed by qRT-PCR (Figure 1G). These include downregulated genes involved in cell proliferation (JUN, FOS,

EGR1 and S100A9), migration/invasion (CXCL5, CEMIP/KIAA1199 and SERPINE1), fibrosis (CTGF), stemness (SOX2 and SOX9). Among the upregulated genes are the tumor suppressor LHPP that encodes a histidine phosphatase (28), and differentiation promoting genes (CEBPA and HMGCS2). Intriguingly, CEMIP is an EGFR interacting protein that stabilizes activated EGFR to promote cell survival, epithelial - mesenchymal transition (EMT) and drug resistance (29,30).

Ibrutinib co-operates with sorafenib to reduce HCC cell survival There is a lot of impetus to develop novel combinations to improve therapeutic efficacy of sorafenib, a first-line treatment for advanced HCCs. We, therefore, examined if ibrutinib could sensitize HCC cells to sorafenib. To this end, we first treated multiple HCC cell lines with both drugs at concentrations ranging from 0.25- to 2- fold of

respective IC50s. The combination treatment decreased cell viability more significantly compared to individual drugs (Figures 2A, B and S2A-F). We determined the degree of synergy between sorafenib and ibrutinib using the widely accepted Chou-Talalay combination index (CI) method (31). A (CI) value < 1 indicates that the drugs are acting synergistically; a lower CI value indicates a greater degree of synergy. CI values for sorafenib - ibrutinib combinations were <1 in all human and mouse HCC cell lines tested (Figure 2C and Table 1), suggesting that these two drugs co-operatively suppressed HCC cell survival. Among these cell lines, PLC/PRF/5, SNU-449 and HepG2 cells were more sensitive to the co-treatment (CI<0.5) (Table 1). Importantly, the two TKIs also inhibited proliferation of sorafenib resistant Huh7-SR and HCCLM3 cells and ibrutinib resistant HepG2 cells more effectively than individual drugs. Statistical analysis confirmed significant synergy between the two TKIs in inhibiting HCC cell survival (data for 3 cell lines shown in the Supplementary Table 2).

Next, we tested whether ibrutinib can synergize with regorafenib, recently FDA- approved as a second-in-line therapy for HCC patients who progress on sorafenib. Notably, ibrutinib exhibited synergy in combination with regorafenib in all three cell lines

tested (Huh7, PLC/PRF/5 and HCCLM3), as demonstrated by much lower IC50 of the combination compared to individual TKIs (Figure S2G-I). This is not surprising

considering the fact that regorafenib is structurally identical to sorafenib except for one fluorine atom in the central phenyl ring (32).

FACS analysis of propidium iodide (PI) - stained cells revealed a substantial increase in sub-G1 cell population in PLC/PRF/5, HCCLM3 and HepG2 cells with concomitant decrease in G1 population upon ibrutinib-sorafenib co-treatment at their respective IC50s (Figures 2D, S3A, S3C). In PLC/PRF/5 and HCCLM3 cells, each drug increased sub- G1 population, however, the effect was more pronounced with the drug combination. In contrast, sub-G1 population increased only after TKI co-treatment in HepG2 cells (Figure S3C). Intriguingly, the population of HepG2 cells at G1 increased without significant changes in sub-G1 when treated at half of the IC50 dose (Figure S3D), suggesting that the cells were arrested at G1 at lower drug concentrations. Elevated cleaved PARP levels in PLC/PRF/5 cells and Annexin/PI positive HCCLM3 cells (Figures 2E, S3B) confirmed enhanced apoptosis induced by this drug combination.

Investigation of the molecular mechanism underlying synergy revealed that the ibrutinib - sorafenib combination reduced the phosphorylation of Akt in HCC cells when compared to ibrutinib only (Figures 2F, S4A). Akt signaling, critical for chemo-resistance in HCC (33), is highly activated in sorafenib resistant cells (27,34). To confirm the role of Akt pathway in mediating the co-operation of these two TKIs, Akt signaling was activated in Huh7 cells by transfecting the wild type (WT-Akt) or constitutively active, myristoylated Akt (Myr-Akt) expression vector, followed by drug treatment. As expected, Myr-Akt was stabilized and the phospho-/total-Akt ratio increased 3-fold compared to WT-Akt in Huh7 cells (Figure S4B). Assessment of cell viability after 72 hours of drug exposure showed that Myr-Akt expressing cells became resistant individual drugs (IC50 of each TKI could not be reached at the drug concentrations tested) (Figures 2G, H). Notably, CI values at different concentrations of the two drugs in combination increased in Myr-Akt compared to WT-Akt expressing cells (Figure 2I), suggesting that the constitutively active Akt abrogated drug synergy.

Ibrutinib and sorafenib combination inhibits survival of liver cancer stem cells (CSCs) in vitro Suppression of SOX2 and SOX9 expression (Figures 1F, G) led us to hypothesize that the ibrutinib - sorafenib combination could inhibit CSC properties of HCC cells. The tumor spheroid model has been widely used to elucidate the mechanism of chemoresistance (35,36), and to uncover CTC characteristics that are not manifested in monolayer cultures. To determine the effect of the sorafenib - ibrutinib combination on CSC properties of HCC cells, we first established HCC spheroid cultures in ultra-low attachment plates (Figure 3A) and measured expressions of CSC marker genes, e.g. CD13, CD44, CD133, SOX2, SOX9, EPCAM and KLF4 in self-renewing spheroids. As expected, compared to monolayers, expressions of these genes were highly upregulated in PLC/PRF/5 spheroids (Figure 3B). Survival analysis revealed increased resistance of spheroids to both drugs compared to monolayers (Figure 3C, D). Nevertheless, both drugs co-operatively (CI<1) inhibited the growth of HCC spheroids (Figure 3E), which correlated with marked decrease in expressions of CD13, CD44, KLF4, SOX2 and SOX9 by this TKI combination compared to the vehicle controls (Figure 3F). This drug combination also inhibited survival of Hepa and HCCLM3 spheroids (Figure S4C, D). These results suggest that this combination therapy could potentially reduce HCC tumor recurrence by suppressing CTC survival.

Ibrutinib and sorafenib combination inhibits the growth of human HCC xenografts and increases median survival of NSG mice To validate our in vitro findings, we tested the efficacy of the ibrutinib - sorafenib combination therapy in NSG (immunodeficient) mice bearing subcutaneous HCCLM3 xenograft tumors. We chose HCCLM3 cells because these cells form tumors within a short time with ~100% penetrance. When the tumor volume reached ~150 mm3, we started treating mice with the drugs or vehicle and measured body weights and tumor volumes weekly. To determine drug synergy, we treated mice with sorafenib at 15 mg/kg/alternate day and ibrutinib at 20 mg/kg/day (Figure 4A), which are much less than usual doses of sorafenib (37,38) and ibrutinib (39,40) used to treat tumor-bearing mice. The loss of body weight in mice due to tumor burden was <5% and it was not

significantly affected by the drug treatment (Figure S4E). After 4 weeks of treatment, tumor volume was significantly reduced by the TKI combination compared to the vehicle controls or single drugs (Figure 4B, C). Although decrease in tumor weight was significant in mice treated with sorafenib (35%) or ibrutinib (36%), co-treatment showed more pronounced decrease (60%) in tumor weight (Figure 4D). Scoring Ki-67 positive cells showed significant decrease in proliferating tumor cells in combination therapy compared to the monotherapy (Figure 4E, F). Immunoblot analysis showed that the treatment with each drug reduced phospho-EGFR/Akt/ERK levels in tumors, which was more prominent in tumors developed in mice treated with both drugs (Figure S4F, G).

Monitoring NSG mice bearing orthotopic HCCLM3-Luc xenografts showed that tumor growth was significantly (P=0.011) inhibited by the ibrutinib-sorafenib combination compared to the vehicle (Figure 5A, B). Notably, the median survival of mice increased from 27 to 40 days (P=0.0007) upon treatment with both drugs compared to vehicle controls (Figure 5C).

Ibrutinib and sorafenib act in concert to increase median survival of immune competent mice bearing orthotopic Hepa tumors Since ibrutinib exhibits immunomodulatory functions, we next tested therapeutic efficacy of these drugs in immunocompetent C57BL/6N mice bearing orthotopic tumors developed by transplanting Hepa-Luc cells. These cells form tumors within 1-2 weeks when inoculated in the livers (Figure 6A) (41). When the tumor volume reached ~150 mm3, we started treating mice with the drugs as described in Figure 5A and monitored their survival. Notably, tumor-bearing mice treated with the TKI combination significantly (P=0.0004) prolonged survival compared to the vehicle control (Figure 6B); the median survival increased from 8 to 38 days in mice treated with both drugs.

Next, we determined systemic toxicity of this treatment regimen (Figure 4A) by monitoring body weights and assessing organ functions in C57BL/6N mice. Body weights of these mice were not significantly affected by these drugs alone or in combination (Figure S5A). The serum levels of liver enzymes e.g. alkaline phosphatase

(ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and cardiac enzyme e.g. creatine kinase (CK) were not significantly affected by the TKIs alone or in combination (Figure S5B-E). Only blood urea nitrogen (BUN) level was slightly decreased with single drugs; however, it was not further affected by co-treatment (Figure S5F). Unaltered serum albumin and globulin levels indicate that the protein synthesis capacity of the liver was not compromised by these TKIs in mice (Figure S5G, H). Microscopic examination of hematoxylin and eosin (H&E) - stained tissue sections did not detect any significant difference in the structure of liver, lung, spleen and kidney among the drug-treated and control mice (Figure S5I-L).

Given that the main function of BTK is in the immune cells, we assessed the influence of ibrutinib on the immune cells in the tumor microenvironment. Indeed, immunohistochemical analysis of Hepa orthotopic tumor sections showed enrichment of BTK+ cells in the tumor stroma (Figure 6C, D). Importantly, the BTK+ cell population was profoundly decreased in mice subjected to combination therapy compared to monotherapy or vehicle. Immunoblot results of the representative tumor lysates showed that ibrutinib treatment reduced BTK protein level, which was more significant in mice treated with both drugs (Figure 6E, F). These results indicate that ibrutinib and sorafenib combination therapy is effective in suppressing HCC growth in the liver microenvironment of immune - competent mice.

To determine the relevance of these findings to human HCC patients, we probed two human primary HCC tissue microarrays (TMAs) with a BTK-specific antibody. Results showed that BTK-expressing (BTK+) cells were enriched in the stroma of a subset of HCCs, whereas few such cells were detected only in the sinusoids of normal livers (Figures 6G, S6A, B). Although, some BTK+ cells were recruited in the benign inflamed liver tissues adjacent to the HCCs (Figure S6C), enrichment of these cells was significantly higher (P=0.29) in the tumor microenvironment (Figure 6H). Scoring two TMAs revealed that 27% of HCCs (23 of 86) and only 4% (1 of 24) benign livers exhibited score 3. These results clearly demonstrate that BTK+ immune cells are recruited in the tumor stroma of a subset of HCC patients. Collectively, these results

implicate BTK expressing immune cells in HCC pathogenesis and provide rationale for ibrutinib - sorafenib combination therapy.

Discussion

Although systemic treatment with sorafenib is a standard of care option in advanced- stage HCC patients, its overall survival benefit is limited, with < 3 months improvement in the SHARP study (42) and median overall survival of 12.3 months in the REFLECT study (5). Targeted therapy directed towards crucial survival pathways in both cancer cells and tumor microenvironment is an emerging treatment strategy in cancer. In the present study, we have demonstrated that ibrutinib exhibits anti-tumorigenic effects on both sorafenib sensitive and resistant HCC cell lines by irreversibly inactivating EGFR signaling. Ibrutinib also inactivates BTK in immune cells, and thereby modulating the tumor microenvironment. EGFR, a receptor tyrosine kinase (RTK), is stimulated by multiple ligands resulting in the activation of various signaling pathways that primarily control cell proliferation, differentiation and survival. EGFR signaling plays a key role in liver generation after acute and chronic liver damage. Also, it plays a key role in cirrhosis and HCC. Furthermore, HCC metastasis and production of inflammatory cytokines have been shown to be regulated by EGFR regulated pathways (43). Unlike other clinically approved EGFR inhibitors directed towards its kinase domain, ibrutinib can irreversibly inactivate both the wild type and kinase-mutant EGFR (44,45). Inhibition of EGFR, Akt and ERK signaling in HCC cells by ibrutinib treatment holds great therapeutic potential since activation of these signaling pathways is associated with the tumorigenic potential of various cancers including HCC (26).

Activation of Akt signaling is involved in sorafenib resistance (27). Ability of ibrutinib to suppress EGFR, Akt and ERK pathways suggests a great potential for ibrutinib to overcome sorafenib resistance. More importantly, ibrutinib and sorafenib combination therapy inhibited HCC cell survival of highly aggressive and sorafenib resistant HCCLM3 cells in NSG mice. This was accomplished at a dose much lower than the

dose of each drug alone without causing notable systemic toxicity to the mice. These data obtained in preclinical models suggest that ibrutinib - sorafenib combination therapy will be effective as a first line of treatment for patients with sorafenib resistance often associated with activated EGFR/Akt/ERK signaling. The improved survival (27 to 40 days) of the immune cell - deficient NSG mice bearing orthotopic HCC-LM3 xenografts with this combination therapy indicates that the inhibition of EGFR signaling in the tumor cells is a key the mechanism of action of ibrutinib.

Currently, ibrutinib is undergoing clinical trials in several solid tumors, because of its immunomodulatory effects targeting the tumor microenvironment. Several reports underscore the relevance of understanding the role of tumor-infiltrating immune cells including B cells in hepatocarcinogenesis (46-48). Ibrutinib inhibits the function of all BTK expressing hematopoietic cells e.g. B cells, basophils, monocytes, MDSCs and NK cells. In fact, it inhibits T helper cells as well due to its action on ITK (13,16). Thus, ibrutinib has broad immune modulatory effects (19,49). Immune microenvironment plays a critical role in the pathogenesis of HCC due to its prototypical inflammation-associated nature where prolonged hepatitis accounts for approximately 90% of the HCC burden. Therefore, targeting immune cells in the tumor microenvironment is expected to be a rewarding strategy in the treatment of HCC. Indeed, although not significant (P=0.059), ibrutinib alone increased median survival of immune-competent C57BL/6N mice bearing orthotopic Hepa tumors from 8 days to 21 days, which was increased to 38 days in combination with sorafenib (P=0.004). Immunohistochemical analysis showed that infiltration of BTK+ immune cells in human primary HCCs and mouse Hepa orthotopic tumors. Precipitous reduction in BTK+ cells in the Hepa tumor stroma upon combination therapy implicates their role in modulating HCC growth. However, the role of BTK+ immune cells in hepatocarcinogenesis has not been elucidated. Nonetheless, the efficacy of ibrutinib in potentiating anti-HCC action of sorafenib both in NSG and C57BL/6N mouse models suggests that it functions by inhibiting distinct tyrosine kinase signaling both in the tumor cells and immune cells in the tumor microenvironment. Studies are ongoing to determine the cell type-specific role of BTK in HCC development using cell type specific knockout mice. It has been reported that infiltration of PD-1high B

cells in the stroma is associated with poor prognosis of HCC patients (50). It would be of interest to monitor PD-1 expression in BTK+ cells before and after drug treatment.

There is an unmet need to develop better therapeutic options in the first-line setting when patients are most fit to receive therapy, to improve the survival and response rate in HCC. Although data with PD-1 inhibitors show improved results for HCC treatment in the second-line setting, in recently completed clinical trials, PD1 inhibitors failed to improve HCC patient survival in the first-in line setting. Additionally, not all patients are candidates for immune therapies, either due to history of orthotopic liver transplant or autoimmune disorders. Thus, developing novel combination therapy to improve survival benefit of sorafenib is important and relevant. Our data in preclinical models clearly show that ibrutinib may potentiate anti-HCC therapeutic efficacy of sorafenib.

Authors’ contribution

K.G. conceived the project; C.H L., K.E. and K.G. wrote the paper; C.H.L., N.W., K.E. and K.G. designed experiments; C.H.L., N.W., K.E., D.L. and P.H. performed experiments, C.H.L., N.W., K.E., P.H., D.L., Y.L., X.Z., J.M.B., X.B. and W.N.K. analyzed data, C.H.L. generated the figures and A.N., X.B. and W.N.K. edited the paper.

Acknowledgements

We thank Dr. Amy Johnson and Dr. Hui-Lung Sun for kindly providing ibrutinib and Akt plasmids, respectively. We thank Jianmin Zhu and Pietrzak Maciej for their assistance with flow cytometry and RNA-seq analysis, respectively. We thank analytical cytometry, target validation, and small animal imaging shared resources at The Ohio State University Comprehensive Cancer Center (supported by P30CA016058 from NCI) for providing FACS analysis, NSG mice and bioluminescence imaging of tumors.

References

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Table 1. IC50 and CI (combination index) when FA (fraction affected) is 50%

Cell line Ibrutinib (IC50) Sorafenib (IC50) CI at 0.5 FA

PLC/PRF/5 1 µM 1.4 µM 0.36

Hepa 1.7 µM 1.4 µM 0.53

SNU-449 3 µM 4 µM 0.39

Huh7 6 µM 3 µM 0.64

Huh7-SR 7 µM 12 µM 0.73

SNU-182 16 µM 5 µM 0.83

HCCLM3 24 µM 12 µM 0.69

BNL 32 µM 9 µM 0.64

HepG2 40 µM 4 µM 0.43

Figure Legends

Figure.1. Effect of ibrutinib (IBT) on HCC cell survival and EGFR, Akt and ERK phosphorylation. (A) HCC cell lines were incubated with ibrutinib at concentrations ranging from 0.5–60 μM for 72 hours, and cell viability was determined using CellTiter- Glo kit, which measures cellular ATP level (n=4/each cell line, data are mean ± SD). (B) Huh7 and Huh7-SR cells were treated with ibrutinib for 1 hour, and the lysates were subjected to western blotting with specific antibodies. (C) HCCLM3 cells were treated with EGF (20 ng/ml) along with ibrutinib in DMSO or DMSO alone for the indicated times, and phospho-/total- EGFR and Akt levels were determined by immunoblotting. The signals were quantified using Image Studio software. (D) HCCLM3 cells were transfected with EGFR-siRNA (siEGFR) or control siRNA (siCon) for 48 hours followed by ibrutinib (24 μM) or vehicle treatment for 1 hour and the lysates were subjected to immunoblotting, and (E) cell viability of siRNA transfected cells treated with ibrutinib at indicated concentrations for 72 hours was measured (*p<0.05, relative to siCon, n=4, t- test, data are mean ± SD). (F) The heatmap depicting expression of several critical genes whose expression was significantly altered in IBT treated PLC/PRF/5 cells, as identified by RNA-seq analysis. (G) qRT-PCR validation of selected genes in PLC/PRF/5 cells treated with ibrutinib (2 μM) for 24 hours (*p<0.05, relative to DMSO, n=3, t-test, data are mean ± SD).

Figure 2. Effect of ibrutinib and sorafenib co-treatment on HCC cell survival. (A) Huh7 and (B) Huh7-SR cells were incubated with various concentrations of sorafenib and ibrutinib for 72 hours and cell viability was assessed using CellTiter-Glo assay (*P<0.05, relative to Combo, n=4, one-way ANOVA, data are mean ± SD). (C) Combination index (CI) analysis of multiple HCC cell lines treated with sorafenib and ibrutinib at different concentrations. Trend lines indicate CI values at any given effect (Fa, fraction affected), and a CI value < 1 indicates synergism. The dotted line indicates a reference point of a CI value of 1. (D) PLC/PRF/5 cells treated with sorafenib (2 μM) and ibrutinib (4 μM) for 24 hours were subjected to flow cytometry after staining with propidium iodide (PI) (*p<0.05, relative to DMSO, n=3, one-way ANOVA, data are mean

± SD). (E) PLC/PRF/5 cells were treated with sorafenib (2 μM) and ibrutinib (4 μM) for 24 hours, and PARP levels were analyzed by immunoblotting. (F) Immunoblotting of phospho- and total- EGFR, Akt, and ERK in HCCLM3 cells treated with either sorafenib (12 µM) or ibrutinib (24 µM) alone or in combination (Combo) for 1 hour. (G, H) Huh7 cells transfected with wild type Akt (WT-Akt) or myristoylated Akt (Myr-Akt) for 48 hours followed by treatment with IBT and SOR at the indicated concentration for 72 hours, and cell viability was determined (*p<0.05, relative to Combo, n=4, one-way ANOVA, data are mean ± SD). (I) The combination index plots were generated in WT-Akt- and Myr- Akt-transfected Huh7 cells. The dotted line indicates a reference point of a CI value of 1.

Figure 3. Sorafenib and ibrutinib combination treatment regulates CSC properties in HCC spheroids. (A) PLC/PRF/5 cells were cultured in ultra-low attachment plate for 9 days to form spheroids. The images were captured with microscope at 200-fold magnification. (B) PLC/PRF/5 cells were cultured in ultra-low attachment plate for 9 days to form spheroids, followed by qRT-PCR analysis of cancer stem cell markers (*p<0.05, relative to monolayer, n=3, t-test, data are mean ± SD). (C) Spheroid or monolayer cultures were treated with the drugs at the indicated concentrations and cell viability was measured after 48 hours (*p<0.05, relative to Combo, n=4, one-way ANOVA, data are mean ± SD). (D) Images of PLC/PRF/5 spheroids after drug treatment for 48 hours. (Magnification: 100X) (E) Combination index plot in drug-treated PLC/PRF/5 spheroid and monolayer cultures. (F) qRT-PCR analysis of cancer stem cell markers in drug treated PLC/PRF/5 cells (*p<0.05, relative to DMSO spheroids, n=3, one-way ANOVA, data are mean ± SD)

Figure 4. Effect of ibrutinib, sorafenib and their combination on subcutaneous HCCLM3 xenograft growth in NSG mice. (A) Schematic of the treatment protocol. NSG mice were injected subcutaneously with 5x106 HCCLM3 cells. (n=9~10 mice/group, data are mean ± SD) and tumor growth was monitored weekly. (B) The volume of tumors following ibrutinib (IBT), sorafenib (SOR) and their combination (Combo) therapy for 4 weeks. The tumors image (C) and tumor weights (D) after drug treatment for 4 weeks (n=9~10, one-way ANOVA, data are mean ± SD).

(E) Photographs of Ki-67 stained tumor sections. (Magnification: 200X and 400X (inset)) (F) Quantification of Ki-67 positive cells using ImageJ software (n=9~10, one-way ANOVA, data are mean ± SD).

Figure 5. Effect of ibrutinib, sorafenib alone and their combination on orthotopic HCCLM3 xenograft growth and survival of mice. (A) NSG mice were injected orthotopically with 106 HCCLM3-Luc cells (n=9~10 mice/group) and tumor growth was monitored weekly by bioluminescent imaging. Representative images of each treatment group at indicated times are shown. (B) Luciferase activity (photons/second) of each group was quantified using Living Image software (n=9~10 mice/group, one-way ANOVA, data are mean ± SD). (C) The Kaplan-Meier survival curves of HCCLM3-Luc tumor bearing mice following treatments (n=9~10 mice/group, log-rank test).

Figure 6. Effect of ibrutinib - sorafenib combination on survival of immune- competent mice bearing orthotopic Hepa-Luc tumors. (A) C57BL/6N mice were injected orthotopically with 5x104 Hepa-Luc cells (n=7 mice/group) and tumor growth was monitored weekly by imaging. Representative picture and bioluminescence image of the Hepa-Luc tumor are shown. (B) The Kaplan-Meier survival curves of Hepa-Luc tumor bearing mice following drug treatment (n=7 mice/group, log-rank test). (C) C57BL/6N mice were injected orthotopically with 5x104 Hepa-Luc cells and treated with drugs for 14 days. BTK immunohistochemistry of Hepa-Luc tumor sections is shown. (Magnification: 200X and 400X). (D) Quantification of BTK positive cells using ImageJ software (n=6, one-way ANOVA, data are mean ± SD). (E) Immunoblotting and (F) quantification of BTK protein expression in Hepa-Luc tumor (n=5~6, one-way ANOVA, data are mean ± SD). (G) BTK immunohistochemistry of human HCC TMA (Magnification: 100X and 400X). (H) Distribution and quantification of human liver (n=24) and HCC (n=86) TMA samples with IHC scores (t-test, data are mean ± SD).

Ibrutinib potentiates anti-hepatocarcinogenic efficacy of sorafenib by targeting EGFR in tumor cells and BTK in immune cells in the stroma

Cho-Hao Lin, Khadija H. Elkholy, Nissar A Wani, et al.

Mol Cancer Ther Published OnlineFirst October 3, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-19-0135

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2019/10/03/1535-7163.MCT-19-0135.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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