Abstract 3757 Background:The majority of patients with chronic myeloid leukemia (CML) achieve durable remissions with ABL tyrosine kinase inhibitor (TKI) therapy. However, a subset of CML patients develop resistance to ABL TKIs, either through BCR-ABL kinase-dependent (e.g. kinase domain point mutations) or BCR-ABL kinase-independent (e.g. overexpression of LYN) mechanisms. Recent studies have demonstrated that the presence of BCR-ABL in CML cells result in overexpression of the protein phosphatase 2A (PP2A) inhibitor SET, which in turn prevents PP2A from performing its regulatory role in deactivating signaling proteins through dephosphorylation. Further, loss of PP2A function accounts for increased and sustained BCR-ABL activity in CML blast crisis cells and activation of PP2A via treatment with Forskolin or FTY720 reduces clonogenicity of CML cells, regardless of their sensitivity to ABL TKIs. Therefore, given the central role of PP2A in regulating both BCR-ABL kinase-dependent and kinase-independent downstream signaling, pharmacological activation of PP2A may provide a novel approach to the development of CML therapeutics, particularly for patients with imatinib-resistant CML. Results:Here, we report a novel, specific, cell penetrating peptide known as OP449 that binds to SET and antagonizes SET’s inhibition of PP2A. Initial in vitro studies of OP449 in human (K562) and murine (32Dp210) CML cell lines revealed a robust (45%) increase in PP2A activity after treatment with 1μM OP449 when compared to vehicle treated cells. OP449 was more potent than the PP2A activator FTY720 (5μM; 20% increase in PP2A activity compared to vehicle treated control). In comparative cytotoxicity studies, treatment with OP449 consistently resulted in reduced proliferation and increased apoptosis compared to FTY720 in K562 (IC50: 2.5μM vs. 5μM, respectively) and LAMA (IC50: 1.25μM vs. 2.5μM, respectively) cells. Consistent with the role of OP449 as a PP2A activator, its growth inhibitory effects in CML cells were rescued by treatment with okadaic acid, a PP2A inhibitor, indicating that OP449 specifically activates PP2A. Furthermore, the cytotoxic effects of OP449 are more selective for BCR-ABL-positive cells (K562, LAMA, 32Dp210 IC50 range: 1.0 –2.5μM) relative to parental 32D cells (IC50: 5μM). Importantly, TKI-resistant mutant CML cells were also sensitive to OP449 at similar doses (32Dp210T315I cells IC50: 2.5μM). Similarly, in primary CML CD34+ cells, we noted CFU-GM colony formation was reduced by 75% and 98% with 2.5 μM and 5 μM OP449, respectively, while no toxicity was observed in normal human CD34+ cells at concentration of 5 μM. Intriguingly, the combination of OP449 with ABL TKIs (imatinib 1.0μM, nilotinib 50nM, or dasatinib 5nM) further reduced proliferation and increased apoptosis in K562 cells and in primary CD34+ CML cells combination of 2.5 μM OP449 with nilotinib (50nM) reduced colony formation by 98%, which was more than treatment with each drug alone. These data support the potential use of OP449 in combination with ABL TKIs.Mechanistically, the amount of SET protein remained unchanged with OP449 treatment, although levels of CIP2a, another PP2A inhibitor, were reduced significantly. OP449 also inhibited BCR-ABL-mediated downstream signaling, and induced dephosphorylation and degradation of BCR-ABL, STAT5, AKT, ERK, and p38/MAPK in CD34+ CML cells and K562 cells within 6 hr of treatment. Lastly, in vivo toxicity studies did not identify any toxicity of OP449 in non-leukemic mice up to 10mg/kg/day. In a 32Dp210 xenograft model, mice treated with OP449 survived slightly but significantly longer than vehicle-treated mice (p=0.05). In a murine CML bone marrow transplantation model, increasing doses of OP449 correlated with improved survival, reduced WBC count, and reduced GFP+ leukemic cells. Combination treatment of OP449 with nilotinib showed further reduced leukemia burden as compared to monotherapy. Conclusions:Taken together, these data demonstrate that OP449 is selectively cytotoxic to CML cells both in vitro and in vivo by antagonizing SET and increasing PP2A activity, thus restoring PP2A's tumor suppressor function. Furthermore, these data validate SET antagonism as a target for development of new CML therapeutics for treatment of CML patients with resistance to ABL TKIs either alone or in combination with ABL TKIs. Disclosures:Oddo:Oncotide: Employment, Research Funding. Vitek:Oncotide: Employment, Equity Ownership. Christensen:Oncotide: Employment, Equity Ownership. Druker:Bristol-Myers-Squibb: OHSU has clinical trial contracts with Novartis and Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead.; Novartis: OHSU has clinical trial contracts with Novartis and Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead.; MolecularMD: Equity Ownership, verbatim: OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Commi; Cylene: Consultancy; Ariad pharmaceuticals: Consultancy.