Acute myeloid leukemia (AML) is an incurable disease with 5 year survival rates of 10% in patients over 60 years. Poor tolerance to chemotherapy, chemo resistance and high rate of relapse warrants less toxic and more effective regimens in AML. OSU-2S is a novel non-immunosuppressive derivative of FTY720, a sphingosine analogue. The promising in-vitro and in-vivo activity of OSU-2S against a number of leukemias and lymphomas, and other malignancies such as hepatocellular carcinoma, impelled us to evaluate the activity of OSU-2S in AML. The potent cytotoxic activity of the OSU-2S (5µM, 24hrs) in AML cell lines HL-60, MV411 and MOLM13 (n=3; HL-60: p=0.008; MV411: p=0.04; MOLM13: p=0.0094) encouraged us to evaluate the effect of OSU-2S in primary leukemic cells from AML patients. OSU-2S (5µM, 24 and 48 hrs) demonstrated significant cytotoxic activity against AML cells, including high risk FLT3-ITD mutated AMLs, (n=13, p<0.0001, mean difference in viability= -65.46) in a dose dependent manner (dose trend p<0.0001 at 24 and 48hrs). While OSU-2S induced caspase activation in primary AML cells as evidenced by Poly (ADP-ribose) polymerase cleavage, its cytotoxic effect is independent of caspase activation (n=8, p=0.0016), as demonstrated by comparable cytotoxicity even in the presence of a broad spectrum caspase inhibitor Q-VD-OPH. Moreover, OSU-2S also significantly increased the levels of reactive oxygen species production in primary AML cells (n=8, p=0.003, mean difference in ROS production= 33.5).
Interaction of AML cells with the bone marrow stromal environment plays a critical role in leukemic cell survival and proliferation, thus contributing to resistance to chemotherapy. To effectively mimic this interactive environment which would be encountered in the patients, bone marrow stromal cells (MSCs) were cultured and expanded from AML patients to develop autologous co-culture systems with AML cells. The autologous stromal cells mediated significant protective effect on AML cells (n=3, p=0.022, mean difference in viability with MSC=21.05), which was compromised in the presence of OSU-2S (5µM) (n=3, p=0.01, mean difference in viability= -64.36), indicating lack of stromal protection mediated resistance to OSU-2S. To circumvent any unintended off target effects of OSU-2S on normal cells, we synthesized an immunoliposomal (ILP) nanoparticle formulation targeting CD33 (SIGLEC-3), a transmembrane receptor which is highly expressed on myeloid progenitor cells and AML. OSU-2S encapsulated immunoliposomes (OSU-2S-CD33-ILP) showed significant cytotoxicity as compared to empty-CD33-ILPs in primary cells (n=5 AML, p=0.0005, mean difference in viability= -48.63) as well as AML cell lines (n=3, MOLM13: p=0.002; MV411: p=0.004). Importantly, OSU-2S-CD33-ILP selectively depleted CD33 positive myeloid population (n=3, p=0.0011, mean difference in % viable population= -43.39) without compromising the CD33 negative non targeted lymphoid population (n=3, p=0.013, mean difference in % viable population= 29.56) in primary AML cell cultures. Similarly, selective cytotoxicity ablated CD33+ MOLM13 and MV411 AML cell lines but not CD33 non targeted Jurkat cell line, which is sensitive to the free (naked) drug. In summary, OSU-2S mediated potent cytotoxic activity against primary AML cells that is not compromised in the presence of autologous bone marrow stromal cells from AML patients. Further, CD33 targeted delivery of OSU-2S has promising selective activity against CD33+ but not CD33- cells. Ongoing studies are evaluating the efficacy of free OSU-2S and OSU-2S-CD33-ILP formulations in-vivo. [This work was supported by NIH-R01-CA197844-01, P50-CA140158, Lauber Funds for Immunotherapy in AML and Robert J. Anthony Leukemia Fund]
Chen:The Ohio State University: Patents & Royalties: OSU-2S patent.
Asterisk with author names denotes non-ASH members.