OSU-2S, A Novel FTY720 derivative, Mediates Cytotoxicity In Chronic Lymphocytic Leukemia Through PKC Dependent Modulation of SHP1 Tyrosine Phosphatase

Abstract 2499

Despite development of numerous chemotherapeutic agents against Chronic Lymphocytic Leukemia (CLL), drug resistance remains impediment in the successful treatment of CLL. Currently several kinases including SYK, BTK, Pim and PI3K are being targeted with inhibitors for therapy in CLL. However, no phosphatase activation directed therapeutics has been described for CLL. We have recently reported FTY720, a potent immunosuppressive agent derived from fungal sphingosine analog ISP-1 to exhibit potent in-vitro and in-vivo preclinical activity against CLL and Mantle cell Lymphoma through protein phosphatase 2A (PP2A) activation dependent mechanisms. However, the immunosuppressive nature of this drug hinders its further development for clinical therapy in cancer. To overcome this limitation we have developed several derivatives of FTY720 by structure-activity relationship analysis. Here we demonstrate OSU-2S as an agent that modulates SHP1 and PP2A protein phosphatases in CLL with potential therapeutic options. OSU-2S is a novel FTY720 derivative that does not interact with sphingosine 1 phosphate receptor 1, thus lacking immunosuppressive property without compromising the cytotoxic potential. Here we have shown preclinical activity of OSU-2S in cell lines representing CLL (MEC-1), ALL (697), lymphoblastic lymphoma cell (Raji, Ramos), Mantle cell Lymphoma (Mino, Jeko), T-cells (Jurkat) and primary CLL and T cell leukemia cells. In primary CLL B-cells, OSU-2S induces activation of major caspases including caspase 3, 8, and 9 resulting in Poly (ADP-ribose) polymerase cleavage. It also induces reactive oxygen species in CLL primary cells with no modulation of Bcl-2 or Mcl-1 levels. Interestingly, OSU-2S induces phosphorylation of Ser 591 of the SHP1 phosphatase in time dependent manner. Consistent with a previously described role for pSer-591 to serve as the nuclear localization signal for SHP1, OSU-2S induced accumulation of pSer-591 SHP1 in the nuclear fraction. Interestingly, OSU-2S induced phosphorylation of Ser-591 SHP1 appears to be mediated through PKC dependent mechanism as activation of PKC with PMA induced phosphorylation of Ser-591 SHP1 and PKC inhibitor Bisindolylmaleimide inhibited OSU-2S induced Serine 591 phosphorylation of SHP1. Moreover, concentrations of Bisindolylmaleimide that prevented the SHP1 Ser-591 phosphorylation also partially rescued OSU-2S induced apoptosis of primary CLL B cells. Co-immunoprecipitation experiments revealed association of PP2A with pSer-591 SHP1 in CLL cells treated with OSU-2S indicating a potential crosstalk between these two phosphatases. Consistent with this hypothesis OSU-2S treatment resulted in activation of PP2A in primary CLL B cells. Further, administration of OSU-2S into Eμ-Tcl-1 transgenic mice with high circulating peripheral blood leukemic cells resulted in significant reduction in circulating CD19+CD5+ leukemic B cells indicating therapeutic efficacy. Ongoing studies on the biochemical basis of cross-talk between the Serine threonine (PP2A) and protein tyrosine (SHP1) phosphatases in response to OSU-2S treatment in CLL will be presented.

This work was supported by LLS-Specialized Center of Research in Leukemia and NCI-Leukemia SPORE Grants