Mo1774 BCL6B Inhibits Hepatocellular Carcinoma Metastases In Vitro and in Mice

Abstract

Objective: Mutations in members of the cytoskeletal intermediate filament proteins (IFs), keratin polypeptides 8 and 18 (K8/K18), predispose their carriers to acute and chronic liver disease progression. Keratin associated diseases are among .70 human diseases that are caused or predisposed to by IF mutations, with the defining feature in many of these diseases being disruption of the normal IF organization within the cytoplasm. In contrast to microfilaments and microtubules, no drugs are available for IFs that can modulate their organization and potentially provide a sorely lacking therapy. Methods: We used an imagingbased drug screening platform consisting of lung carcinoma A549 cells transduced with lentiviruses containing GFP-tagged K18 R90C and wild-type K8. The cells were screened against several compound libraries, and a select compound was further tested in cell culture and in livers of mice that express mutant K18 in order to define its mechanism of action. Results: Screening of a 320-compound kinase inhibitor library showed that the inhibitors of protein kinase C (PKC) and tyrosine kinases, particularly MIDOSTAURIN, led to significant correction of the defective organization of K18 R90C puncta towards normal filaments after 24 h with a maximal effect at 48 h. The findings were confirmed using multiple MIDOSTAURIN concentrations and in another cell line, Hacat, that expresses mutant K14. While MIDOSTAURIN diminished the expression of the mutant keratin 14 in Hacat cells, the effect in the A549 cells was independent of K18 expression but causes changes in K18 phosphorylation that are known to regulate filament formation. MIDOSTAURIN significantly prevented interferon and FAS ligand-induced apoptosis in A549 cells. The protective effect occurs after 48 h but not after 1 h treatment with MIDOSTAURIN, suggesting that the drug effect is more likely to be related to the correction of filament organization. In mice, administration of MIDOSTAURIN is well tolerated and its administration prior to exposure to FAS results in dramatic protection from liver injury. Conclusion: The kinase inhibitor MIDOSTAURIN alleviates cytoskeletal structural defects caused by K18 R90C mutation and also protects from apoptosis in cultured and in the livers of mice exposed to FAS ligand. MIDOSTAURIN may be a potential therapeutic for IF diseases and, notably, it is presently undergoing clinical trials in patients with acute myeloid leukemia and gastrointestinal stromal tumors.