Despite prolonged progression free survival with the introduction of novel therapies for Chronic lymphocytic leukemia (CLL) currently available therapies are not curative in all patients and patients who relapse generally respond poorly. Identification of new agents that work in resistant patients represents a major focus of investigation. Constitutive phosphorylation of proteins associated with prolonged survival and defective apoptosis is a recurrent theme in CLL and other cancers. While inhibition of kinases is actively pursued, pharmacological activation of protein phosphatases is less explored and represents a paradigm shift for potential therapeutic modulation of aberrantly phosphorylated proteins in CLL. We have identified FTY720, an FDA approved immunosuppressant, to mediate cytotoxicity through a protein phosphatase 2A (PP2A) activation dependent mechanism in CLL. Despite its potent pre-clinical activity in multiple heme-malignancies including CLL, mantle cell lymphoma, chronic myeloid leukemia and acute lymphoid leukemia, the immunosuppressive property of FTY720 precluded its further development for clinical application in heme-malignancies. We therefore developed a non-immunosuppressive FTY720 derivative, OSU-2S, with potent cytotoxic activity. This proposal will pursue mechanistic, pharmacological and translational development of OSU-2S in B cell malignancies. Specifically, in Aim 1 we propose to identify the direct target of OSU-2S and determine the mechanistic basis of OSU-2S induced apoptosis with emphasis on phosphatases in CLL. To overcome potential off target effects of OSU-2S in-vivo, we generated CLL tumor antigen (ROR1) targeted immunoliposomal delivery formulations (2A2-OSU-2S-ILP) and CLL mouse models expressing human ROR1 antigen on leukemic B cells. In Aim 2, we propose to maximize the therapeutic index of 2A2-OSU-2S-ILP by a) determining maximum tolerable doses and exposures of 2A2-OSU-2S-ILP in human ROR1+Tcl1 CLL mice; b) characterizing the in-vivo relationships between 2A2-OSU-2S-ILP pharmacokinetics and pharmacodynamic (PK/PD) modulation of pSHP1 in bone marrow, spleen, circulating WBCs and other tissues in E-ROR1-Tcl1 mice, and c) using PK/PD modeling and simulation to design optimal dose regimens of 2A2-OSU-2S-ILP that maximize pSHP1 induction while minimizing dose levels and OSU-2S exposure in non-diseased tissues. We will validate the in-vivo therapeutic efficacy of the human ROR1 targeted OSU-2S formulations in our human ROR1+ CLL mouse model in Aim 3. At completion of this project we will have generated sufficient mechanistic and pharmacological data with OSU-2S to justify its transition to early clinical trials for CLL. We are poised to accomplish this through our patented OSU-2S, novel CLL targeted delivery formulation and unique animal models exclusively developed to meet our needs to perform a comprehensive pre-clinical evaluation. To accomplish this goal we have assembled a highly interactive team with expertise in proteomics, translational medicine, drug development, mouse modeling, drug delivery, and PK/PD modeling and simulation.