Ml-12 mechanisms of cell death inhibition through change in subcellular localization of pim-1 by pim1 gene mutation recurrently found in primary central nervous system lymphoma

Abstract BACKGROUNDS In a study of Next Generation Sequencing in primary central nervous system lymphoma (PCNSL), we have previously reported several mutations of high frequency, in comparison with systemic diffuse large B cell lymphoma (DLBCL)s. Consequences of these specific mutations in PCNSL are unknown. In this study, we have analyzed the functional consequence of mutations in the PIM1 gene, observed in 100% of PCNSL patients, which encodes a serine/threonine kinase and is known to drive tumorigenesis in several malignancies. METHODS Four most frequent mutations of PIM1 in PCNSL, S77N, K115N, P216S, L275F, were chosen from our previous study, and each mutant was generated by site directed mutagenesis in PIM1 cDNA cloned in an expression vector. Resulting vectors were transiently transfected into human cancer cell lines. Functional studies were carried out using various biochemical methods. RESULTS Among the four mutants, increased phosphorylation of BCL-2 associated death promoter (BAD) at Ser112, which is a phosphorylation target of Pim-1, was observed by expression of K115N mutant compared with wild type PIM1 in Hela and Nagai cells expressing endogeneous BAD. Decreased cell death under campthothecin treatment was also observed in K115N mutant expressing Nagai cells compared with wild type PIM1-expressed cells. We also observed a significant shift in subcellular localization of Pim-1 carrying K115N mutant; from the nucleus, main sublocalization for wild type Pim-1, into the cytosol determined by immunocytochemistry and immunoblotting of nuclear and cytosolic fraction of the cells. Augmented cytosolic localization of Pim-1 carrying K115N mutant was suppressed by inhibition of glycosylation. DISCUSSION It is suggested that PIM1 K115N mutant may drive chemoresistance through increased BAD phosphorylation that suppresses cell death compared with wild-type PIM1 through modification of its subcellular localization, which might be regulated by its glycosylation status.