Characterization of CENPM Isoform Expression, Intracellular Localization, and Potential Functional Interactions In Mature B-Cell Malignancies

Abstract 2496

The lymphoid-specific PANE1 human minor histocompatibility antigen (mHAg) is overexpressed in primary chronic lymphocytic leukemia (CLL) cells. The PANE1 mHAg peptide is encoded by an alternative transcript of the centromere protein M (CENPM) gene, and may be a desirable target for immunotherapy due to its lymphoid specificity, and hence its potential to instigate a graft-versus-leukemia effect with absent or mitigated graft-versus-host disease. While its immunotherapeutic potential is compelling, little is presently known about the expression, cellular localization, and functional role of CENPM isoforms. To gain further insight into CENPM isoform expression and function in B-lymphoid and other malignancies, we assessed these characteristics of the CENPM transcript encoding a 58 amino acid isoform encompassing the mHAg (referred to as CENPM-S), and the longer canonical 180 amino acid CENPM isoform (referred to as CENPM-L). These two isoforms share a common 46 amino acid C-terminus, and differ in that the N-terminal 134 amino acids of CENPM-L are substituted in CENPM-S by a 12 amino acid sequence containing the mHAg peptide, which is derived from a novel exonization event within the fifth intron of the canonical genomic sequence. The CENPM-L isoform has been shown to be a critical component of the centromeric protein complex on which the transient assembly of the kinetochore occurs during mitosis. We determined that the novel CENPM-S mHAg-encoding exon and its adjacent predicted alternative promoter (distinct from that of CENPM-L) is derived from a LINE2 non-LTR retrotransposon insertion. To better assess the types of lymphoid malignancies that could potentially serve as targets of a CENPM-S-specific immune response, we examined a diverse panel of primary CLL cells, lymphoid malignancies and lymphoma cell lines for the expression of CENPM-S and CENPM-L via quantitative real-time PCR (qRT-PCR). In accord with our previous results, highest levels of CENPM-S were observed in primary CLL cells. Within lymphoma cell lines, moderate levels of CENPM-S were seen in mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) cells, and expression was detectable in only one out of four multiple myeloma cell lines surveyed. Within a panel of human lymphoma tumor samples, highest levels of CENPM-S were observed in marginal zone lymphoma, DLBCL, MCL, follicular lymphoma, and one peripheral T-cell lymphoma sample. Strikingly, the highest levels of CENPM-S and the lowest levels of CENPM-L were always observed specifically in primary CLL cells, and such high ratios of CENPM-S to CENPM-L expression were not observed in any other B-cell malignancy surveyed. These results suggest a profound transcriptional switch from the upstream canonical promoter to the putative LINE2 element-proximal CENPM-S alternative promoter, which occurs exclusively in CLL cells. In order to further elucidate the cellular localization and potential function of the CENPM-S and CENPM-L isoforms, we transiently transfected vectors encoding FLAG-tagged CENPM isoforms in HeLa cells. Transfected cells were subjected to subcellular fractionation followed by Western blot with anti-FLAG antibodies, which indicated CENPM-L localization to cytoplasmic, membrane, soluble nuclear, and cytoskeletal fractions, in accord with our observations of endogenous CENPM-L in Granta-519 MCL cells using CENPM-L-specific monoclonal antibody. In contrast, CENPM-S exhibited localization in cytoplasm and membrane and was present only weakly in the soluble nuclear fraction. Preliminary immunofluorescence microscopy data are in agreement with these patterns of intracellular localization. We are presently assessing CENPM isoforms for impact on cell cycle, proliferation, and apoptosis, in order to better understand their potential involvement in biological processes underlying CLL and other non-Hodgkins lymphomas. To further elucidate the potential functional role of CENPM-S in mature B-lymphocytes and mature B-lymphoid malignancies, we performed yeast two-hybrid library screening with a human bone marrow cDNA library to reveal putative interacting proteins. The non-muscle myosin protein, myosin IIA, was revealed as a strong candidate CENPM-S interacting protein. We have recently validated via sandwich ELISA an interaction between endogenous myosin IIA (within Granta-519 MCL cell extract) and recombinant CENPM-S.