Role of Prostate Apoptosis Response-4 Tumor Suppressor in the Survival and Growth of Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia (CLL) is characterized by accumulation of clonally expanded population of CD5⁺ CD19⁺ B lymphocytes in peripheral blood and secondary lymphoid organs, with a majority of circulating cells in a non-dividing resting stage. However CLL is no longer considered a static disease that results from simple accumulation of long-lived lymphocytes, but rather, is a dynamic process with a birth rate of about 0.1-2% of the entire CLL clone per day. The Eµ-Tcl1 mouse serves as an excellent model for the development of CLL as they develop a CLL like disease by 9-13 months of age, due to overexpression of the oncogene, T cell Leukemia 1 (Tcl1), in B cells through the Ig VH promoter and Eµ enhancer (Bichi et al. PNAS. 2002). In an adoptive transfer model, intravenous or intraperitoneal injection of primary CD5⁺CD19⁺ CLL cells from the Eµ-Tcl1 CLL mouse into recipient syngeneic mice leads to development of a CLL like disease within 3-5 weeks of transfer. We have characterized the growth of CLL cells in these mice by periodic submandibular bleeding, ultrasonography of spleen and flow cytometry. We find that Eµ-Tcl1 CLL cells express more Prostate apoptosis response-4 (Par-4), a pro-apoptotic tumor suppressor protein, than normal B-1 or B-2 cells in mice. Par-4 is silenced by promoter methylation in more than 30% of all cancers. Par-4 has been shown to be secreted and to induce apoptosis selectively in various types of cancer cells but not in normal cells. Although we find that Eµ-Tcl1 CLL cells constitutively secrete Par-4, they are resistant to Par-4 mediated apoptosis. We show that CLL cells have constitutively active B-cell receptor signaling and that inhibition of BCR signaling with FDA approved drugs (i.e. Dasatinib, Ibrutinib, and Fostamatinib) causes a decrease in Par-4 protein and mRNA levels and increases apoptosis. Interestingly, systemic Par-4 appears to inhibit CLL growth in vivo, since adoptively transferred Eµ-Tcl1 CLL cells grew better in Par-4 null mice, despite excellent Par-4 expression and secretion by the transferred cells. Thus there were three times more CLL cells in the bone marrow and twice as many in the spleens of Par-4 null mice compared to their wild type counterparts. We conclude that even though Par-4 is pro-apoptotic in the CLL microenvironment, intracellular Par-4 is either rendered inactive or is potentially pro-survival in Eµ-Tcl1 CLL cells. (Supported in part by NIH grants)