Expression of JAZ in Multiple Myeloma and Its Potential Role as A Novel Molecular Target.

Abstract 2862

Poster Board II-838

Multiple myeloma (MM) is an incurable hematologic tumor caused by malignant transformation of plasma cells. JAZ (just another zinc finger protein) was previously identified in our laboratory as a unique ZFP that preferentially binds to double-stranded (ds) RNA rather than dsDNA. The JAZ gene is localized to the human chromosome 5q35-ter, which is a specific chromosomal region at which deletions and translocations occur in different hematologic malignancies including multiple myeloma. The NCI Cancer Genome Anatomy Project data base search reveals that a validated SNP (single nucleotide polymorphism) exists for JAZ at an evolutionarily conserved, 3'-untranslated, regulatory region of JAZ mRNA. This specific SNP exists only in the bone marrow cancer but not in the normal tissue, suggesting a potential role for JAZ in hematologic malignancies. Importantly, we recently discovered JAZ as a novel direct, positive regulator of p53 transcriptional activity. The mechanism involves direct binding to p53's C-terminal (negative) regulatory domain to activate “latent” p53 in response to non-genotoxic stress signals. Moreover, we found that interleukin-3 growth factor withdrawal upregulates JAZ expression in factor-dependent hematopoietic cells in association with activation of the p53 tumor suppressor and induction of apoptotic cell death, indicating that the expression of JAZ is important in the stress response. Thus, to examine the role of JAZ expression in hematologic malignancies, we carried out an immunohistochemistry (IHC) study of JAZ expression in murine and human bone marrow cells and in normal and malignant hematologic tissues and cell lines. The affinity-purified rabbit polyclonal antibody JAZ111 was used and its specificity was verified by the peptide inhibition and also using a commercially available monoclonal antibody against JAZ. Results reveal that JAZ is differentially expressed in different types or stages of hematopoietic cells. For instance, morphologically, JAZ appears to be (relatively) abundantly expressed in plasma cells in normal bone marrow samples and such observation was verified by co-staining with a CD38 antibody. Interestingly, results of JAZ111 staining of an MM tissue microarray (24 cases/48 cores, 13 MM and 11 normal tissues) reveal that in ∼50 % of the MM samples the expression of JAZ is substantially down-regulated compared to the normal tissue controls. This supports the notion that JAZ may play a tumor suppressor role. However, there are exceptions that JAZ was found to be highly or over-expressed in some MM samples on the microarray and other regular individual sample slides, suggesting that JAZ may be latent or inactivated in these cases. Co-staining of the MM samples with a p53 antibody shows that expression of p53 is low, which agrees with the notion that p53 expressed in the MM samples is usually the wild type but in a latent state since the p53 gene has been reported to be rarely mutated and the p53 pathway remains intact in multiple myeloma. Thus, we hypothesize that activation or reactivation of JAZ in the MM cells which express abundant but latent JAZ may induce p53 activation to arrest or kill MM cells. We have explored JAZ as a potentially novel molecular target in multiple myeloma by identifying small molecules that bind and activate JAZ. Using a high-throughput, “molecular docking” strategy, we have screened approximately 240,000 small molecules for their ability to interact with JAZ. Based on the Lipinski Rules for Drug Likeness (molecular characteristics favorable for absorption and permeability), we identified ∼70 putative “drug-like” binding molecules with high scores and obtained ∼40 of them from the NCI Developmental Therapeutics Program. We performed the cell viability study, flow cytometry and Western blot analysis to test their effect on the MM cell lines. Results demonstrate that several of the “candidate” JAZ-targeting compounds can potently induce growth arrest and/or cell death in association with p53 activation. Therefore, while further in vitro and in vivo characterization remains to be carried out, the JAZ-“targeting” compounds point the way to develop a potentially novel therapeutic strategy targeting JAZ to treat multiple myeloma.