Bishr Omary

Mo1772 Kinase Inhibition Normalizes Mutation-Mediated Keratin Filament Disruption and Predisposition to Apoptosis and Liver Injury

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.

M1994 Molecular Factors Involved in Determining Genetic Susceptibility to Mallory-Denk Body Formation

Background:Mallory-Denk bodies (MDBs) are hepatocyte inclusions associated with alcoholic and nonalcoholic steatohepatitis, and some forms of drug-induced liver injury. Understanding the biological significance of MDBs and devising means to alter their formation has clinical relevance to protein aggregation diseases beyond the liver. Feeding mice 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) produces, in a strain-selective manner, inclusions resembling human MDBs. Aims: We hypothesized that proteomic comparison of livers from MDBprone C57BL/6 mice and MDB-resistant C3H mice, before and after DDC treatment, will illuminate the molecular mechanisms regulating susceptibility of C57BL/6 mice to MDB formation. Methods: 2-dimensional (2D) differential-in-gel electrophoresis and mass spectrometry of liver protein homogenates resulted in the identification of >200 charged isoforms with >2 fold differences between the two strains. The results for some of the proteins were validated in control and DDC-treated livers, and isolated hepatocytes using 1D/2D gel analysis followed by immunoblotting. Enzymatic activities were also measured. Results: Proteins involved in cellular homeostasis and survival, such as carbonic anhydrase 3 (CA3) and GAPDH are expressed at lower levels in C57BL/6 livers. Whereas DDC treatment strongly induces CA3 and GAPDH expression in isolated C3H hepatocytes, it has no effect in C57BL/ 6 hepatocytes. There is significantly higher expression of reactive oxygen species-generating cytochrome P450 and detoxifying glutathione-S-transferase enzymes, and lower expression of the antioxidant enzyme peroxiredoxin 6 in the C57BL/6 livers, indicating a state of elevated oxidative stress. Furthermore, the livers, but not other organs, of untreated and DDC-fed C3H mice have a 2-fold higher expression of nucleoside diphosphate kinase B (NDPK-B), a multifunctional protein involved in regulating intracellular energy pools and protection from oxidative stress. Additionally, isolated C3H hepatocytes have 6-fold higher NDPK expression and activity relative to C57BL/6 hepatocytes, indicating that differences in total liver NDPK levels are accounted for by differences in hepatocytes. Conclusions: Genetic predisposition to MDB formation is likely related, at least in part, to deficiencies in systems involved in regulating intracellular homeostasis, energy generation and protection from oxidative stress. NDPK may be an upstream effector functioning in a strain-dependent and tissue-selective manner to limit MDB formation. Similar molecular determinants might contribute to the genetic differences in human liver diseases that are associated with MDB formation.