Targeting Kinases that Phosphorylate the KSHV LANA Chromatin Binding Domain

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DESCRIPTION (provided by applicant): KSHV is associated with the endothelial malignancy Kaposi sarcoma (KS) and the B cell cancers primary effusion lymphoma (PEL) and multicentric Castleman disease. The incidence of these cancers is increased in individuals who are immunocompromised such as those undergoing organ transplantation and those who are infected with human immunodeficiency virus 1 (HIV-1) and have AIDS. Treatments for KSHV associated malignancies remain sub-optimal and new treatment approaches are needed. The Kaposi sarcoma associated herpesvirus (KSHV) encoded LANA protein is expressed in all KSHV infected cells and KSHV associated malignancies. LANA is essential for replication and retention of KSHV genomes in infected cells and also provides cell growth and cell survival functions. The critical roles played by LANA in driving KSHV associated cell proliferation make LANA an attractive target for strategies to treat KSHV associated disease. LANA tethers KSHV genomes to cell chromatin through a chromatin binding domain. This function is essential for the maintenance of the viral genomes in infected cells. Post-translational modification of proteins by phosphorylation is a mechanism by which protein function is frequently regulated. We noted that the LANA chromatin binding domain contains serine and threonine residues that could be subject to phosphorylation. Investigation revealed that, while mutation of these residues to alanine prevented chromatin binding, conversion to phosphomimetic glutamic acid residues restored chromatin binding. Based on this evidence for a role of phosphorylation in LANA function, we undertook a screen to identify kinases that phosphorylate LANA. This screen started with 289 human kinases that were purified in active form from recombinant yeast. In a series of assays we subsequently identified four kinases from this collection that phosphorylated the critical serine and threonine residues in the LANA chromatin binding domain. Preliminary experiments using an inhibitor to one of these kinases revealed that kinase inhibition affected both LANA interaction with chromatin and the levels of LANA present in the cell. The promising nature of these observations has led us to propose a more in depth analysis of the outcome of inhibition of the four identified kinases in KSHV infected PEL and endothelial cell cultures. The goal of this application is to determine the optimal strategy for inhibitor mediated intervention in KSHV disease by examining the effects of inhibitors of the four identified kinases and, where available, of non-specific inhibitors of these kinases that are already in use in the clinic and could potentially be more rapidly transitioned into pre-clinical and clinical protocols for KSHV associated disease. We will examine the effect of kinase inhibition on KSHV genome maintenance, KSHV infected PEL and endothelial cell growth and viability and induction of markers of cell cycle arrest and apoptosis. We will also determine the mechanism of inhibitor induced LANA instability.