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Hepatocellular carcinoma (HCC) is the most prevalent, primary liver cancer and the second major cause of cancer-related death worldwide, and its incidence is rising. Sorafenib; the only FDA approved drug for the treatment of late stage HCC increases survival by only ~2.8 month. This is because sorafenib treated patients suffer from toxic side effects in addition to the intrinsic resistance of tumors to sorafenib; which is a major cause for only 2% of HCC patients responding to this drug. Seven large, randomized Phase III clinical trials investigating other molecular therapies have failed to detect significant survival benefits over sorafenib. Thus, there is an urgent need to develop novel alternative therapies for HCC. Re-purposing of the drugs already FDA approved for other cancers, is the quickest possible transition from bench side to bedside of cancer patients.Ibrutinib, an FDA approved targeted therapy for the treatment of B-cell malignancies, is an irreversible inhibitor of the Bruton’s tyrosine kinase (BTK) and IL2-inducible T-cell kinase (ITK). Currently, it is undergoing clinical trials in several solid tumors, which could be attributed to its reported immunomodulatory effects targeting the tumor microenvironment. Ibrutinib has been reported to modify the activation and function of different immune cell categories, including basophils, monocytes, MDSCs and T helper cells, indicating that ibrutinib has a broad immune modulatory effects. Immune microenvironment has crucial 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 the hepatic microenvironment immune cells is expected to be a fruitful strategy in the protection and treatment of HCC disease.Reported ibrutinib efficacy in a variety of solid tumors, and its much lower side effects compared to sorafenib especially on the liver, prompted us to investigate its anti-HCC efficacy. We demonstrated that ibrutinib exhibits anti-tumorigenic functions on both sorafenib sensitive and resistant HCC cell lines; using CellTiter-Glo® viability assay, clonogenic survival assay and western immunoblotting analysis. We illustrated that ibrutinib inhibits two key signaling pathways MAPK/ERK and Akt/mTOR in HCC cells through inhibiting the EGFR axis. MAPK pathway is also a target of sorafenib, whereas inhibition of Akt signaling is a distinct action of ibrutinib, overcoming the major reason of sorafenib resistance, as sorafenib activates Akt signaling. Activation of these signaling pathways, especially Akt/mTOR modulates cancer metabolism by regulating expression of key metabolic enzymes, thereby assisting cancer cells to generate biomass for rapid propagation. Cell cycle analysis showed that ibrutinib induced cell cycle arrest at G1 phase. Mechanistic investigation suggests that the ibrutinib-induced cell cycle arrest is due to cellular ATP depletion which is the result of ibrutinib inhibition of glycolysis and oxidative phosphorylation leading to the observed inhibition of mammalian target of rapamycin (mTOR) and its downstream targets, therefore inducing cell cycle arrest. Glycolysis and oxidative phosphorylation inhibition by ibrutinib was demonstrated by reduced extracellular acidification (ECAR) and oxygen consumption rate (OCR) using Seahorse system. In addition, ibrutinib reduced extracellular lactate level in sorafenib resistant Huh7 cells.Treatment of HCC tumor bearing mice; (DEN-injected miR-122 KO mouse model that mimics different stages of hepatocarcinogenesis in humans), with ibrutinib alone resulted in a moderate reduction of tumor formation, reducing tumor sizes and numbers, in contrast to recent reports that didn’t show any effect for ibrutinib alone on some solid tumors in-vivo, although HCC doesn’t express neither BTK nor ITK. Therefore, one of ibrutinib mechanisms is its direct anti-tumorigenic effect against HCC tumors through its demonstrated multi-kinase inhibitory effect on EGFR axis and its downstream Akt/mTOR and MAPK signaling pathways. This direct anti-tumorigenic effect was further confirmed by demonstrating that ibrutinib delayed tumor growth of highly aggressive MHCCLM3 xenograft in immunocompromised NSG mice. Finally, ibrutinib anti-tumorigenic effect was enhanced by the immune checkpoint blockade anti-PD-L1 (program cell death Ligand-1) therapy in DEN-injected miR-122 KO mice. Combination therapy resulted in significant reduction of tumor formation, reducing tumor sizes and numbers relative to ibrutinib alone. Furthermore, FACS analysis showed significant increase in the levels of cytotoxic T-cells (CD8+) in mice livers treated with ibrutinib and anti-PD-L1 combination therapy, while the mice treated with ibrutinib alone showed moderate increase of the cytotoxic T-cells levels in their livers, indicating an ibrutinib immunomodulatory effect. No significant difference was detected neither in CD4+ T-cells nor in macrophages in the livers of the treated mice with either ibrutinib alone or ibrutinib and anti-PD-L1 combination, indicating that ibrutinib was able to specifically enhance the cytotoxic T-cells population. Importantly, our in vivo experiments suggest that the treatment with ibrutinib is not associated with obvious gross toxicity, as the average body weights of the treated mice groups were comparable throughout all the in vivo experiments that has been done.mmcIn conclusion, this study provides the evidence for the therapeutic potential of combining ibrutinib and anti-PD-L1 immune checkpoint blockade as an effective and attractive strategy for treating HCC patients especially for patients with sorafenib resistant and/or liver toxicity. Our pre-clinical data can be the basis for future clinical trials of ibrutinib in combination with anti-PD-L1 for HCC, which is in dire need for new therapy.