OSU-2S, a Novel PKC Activator, Mediates PKC Dependent Cell Death, Differentiation and Cell Cycle Arrest in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a near incurable disease with 5-year survival rates of 10% in older patients. Poor tolerance to chemotherapy, chemo resistance and high rate of relapse warrants less toxic and more effective regimens in AML. AML leads to a block in normal myeloid differentiation, leading to an accumulation of undifferentiated blasts. Thus, therapies directed towards promoting these cells to differentiate re-establishes the ability of AML cells to undergo cell death, reducing the leukemic population. Such differentiation therapy with all-trans retinoic acid (ATRA) has been successful in acute promyelocytic leukemia (APL), but not in other AML subtypes resistant to ATRA, thus presenting a promising and unexploited avenue for targeted therapies. There is accumulating evidence that Protein Kinase C (PKC) activation induces myeloid differentiation. However, since most PKC activators are either tumor promoters or have inconclusive activity, their potential for use in AML has yet to be realized. OSU-2S is a non-immunosuppressive derivative of FTY720 (a sphingosine analogue with anti-tumorigenic properties) and has previously been reported to directly activate PKC to mediate cytotoxicity, showing promising in vitro and in vivo activity against a number of leukemias and lymphomas, and other malignancies such as hepatocellular carcinoma. This impelled us to evaluate the activity of OSU-2S in causing cell death and differentiation in AML. We found that OSU-2S resulted in increased activation of PKC in HL-60 cells. In addition to effecting significant dose-dependent cytotoxicity against AML cell lines HL-60, MV4-11 and MOLM-13 [mean decrease in viability with OSU-2S in HL-60: 85±7% (p=0.08), MV4-11: 74±20% (p=0.02), MOLM-13: 60±25% (p=0.04)], and in primary samples from leukemic patients, including high risk FLT3-ITD mutated AMLs (65±8% decrease in viability with OSU-2S, p To further gain insight into the mechanism of cell death and differentiation effected by OSU-2S, we performed RNA-Sequencing analysis of total RNA isolated from MV4-11 cells treated with OSU-2S. Ingenuity Pathway Analysis on these transcripts showed significant changes in gene expression affecting cell cycle regulation and proliferation (p=10-13). Cell cycle analysis of MV4-11, HL-60 and U-937 cells by PI staining followed by flow cytometry confirmed a decrease in cells in G2/M phase of the cell cycle associated with increased cells in S but not Go/G1 phase (69.63% decrease in G2/M phase cells with OSU-2S vs vehicle control, p=0.04). Current ongoing studies in cell line-derived xenografted mouse models of AML indicate favorable therapeutic effect of OSU-2S in providing survival advantage in AML. Tumor burden monitoring by bioluminescent imaging shows reduction in tumor burden following OSU-2S treatment. Our results show promising pre-clinical efficacy of OSU-2S against AML, and warrants further studies to establish clinical efficacy as a potential therapeutic agent. Ongoing studies are also directed towards studying the involvement and role of PKC and other target molecules in the cell death and differentiation effected by OSU-2S. Disclosures Walker: Gilead: Research Funding. Mims: Novartis: Honoraria. Chen: The Ohio State University: Patents & Royalties: OSU-2S. Byrd: Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; Janssen: Research Funding; Genentech: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S. Vasu: Boehringer-Ingelheim: Research Funding; Stemline Therapeutics: Research Funding.