The Poison Oligonucleotide F10 Is Efficiently Taken Up By, and Highly Effective Against, Acute Lymphoblastic Leukemia While Sparing Normal Hematopoietic Cells

Abstract The development of imatinib to target the BCR-ABL kinase in chronic phase (CP) CML has changed the natural history of the disease. This success generated much enthusiasm for the approach in the more genetically complex acute lymphoblastic leukemia (ALL). Unfortunately, the durable responses seen in CP CML are not reproduced in BCR-ABL positive ALL. Indeed, all previous attempts to target a single oncogenic pathway in acute leukemias have resulted in transient responses with frequent relapse. An alternative approach is to use agents that target “final common pathways”, i.e. processes that must be accomplished to produce additional leukemia cells regardless of driving mutations. In this paradigm, agents are not judged by differential expression of a target, but by the degree of differential uptake. One pathway to exploit is the known increased uptake of oligonucleotides by ALL cells. F10 is a poison deoxy-oligonucleotide that is a 10mer of the thymidylate synthase (TS) inhibitory 5-fluorouracil (5-FU) metabolite, 5-fluoro-2'-deoxyuridine-5'-O-monophosphate. We sought to determine the uptake and activity of F10 against preclinical ALL models. Using fluorescently labeled F10, we determined that uptake by the human ALL cell lines DG75 and SUP-B15 was rapid and had a profound temperature dependence consistent with an active process. Both cell lines demonstrated increased uptake compared to normal, lineage- depleted marrow cells from C57Bl/6 mice. Using a syngeneic, BCR-ABL-expressing murine ALL model, we confirmed rapid uptake of F10 in vivo. Furthermore, preliminary experiments suggested normal human HSCs have decreased uptake compared to primary patient samples. Consistent with this decreased uptake, F10 treatment did not alter the ability of human HSCs to engraft in immunodeficient mice. F10 treatment resulted in robust induction of apoptosis that could not be equaled by 100 fold more 5-FU. IC50 values for F10 against B6 ALL, Jurkat, DG75, Molt-4, CCRF-CEM, and SUP-B15 were in the picomolar to nanomolar range, with an average value of 2.15 nM (range 0.12 to 5.4 nM). For comparison we also tested 5-FU, doxorubicin, and cytarabine. In all cases, F10 was the most potent agent; over 1000 times more potent than 5-FU. In vivo, F10 treatment was associated with a significant increase in survival in a BCR-ABL driven, syngeneic ALL mouse model (p= 0.0001 by log rank test). Survival was dose-dependent, with median survival extended by 3, 9 or 15 days for 4, 6, or 9 doses, respectively. F10 also protected mice from leukemia-induced weight loss during and for several days after treatment. To confirm the in vivo activity of F10, we treated a separately derived syngeneic model expressing the T315I variant of BCR-ABL. As in the previous model, F10 significantly prolonged survival (p=0.0013). To further extend these results and confirm F10's activity against human ALL cells, we treated DG75, Jurkat, and SUP-B15 xenograft models. As in the murine models, F10 caused regression of disease with several animals cured and resulted in a significant survival benefit (p=0.0112). As the uptake and mechanism of action of F10 is unique we sought to determine the in vivo efficacy of F10 on ALL previously treated with cytarabine. When mice injected with previously treated ALL cells were treated with cytarabine a median survival benefit of only one day (11 vs 12 days) was observed. In contrast F10 treatment resulted in a median survival of 31 days, similar to the survival seen in previously untreated ALL. F10 exposure results in trapped topoisomerase I (Topo1) cleavage complexes in vitro. To determine if this occurs in vivo, we treated leukemic C57Bl/6 or nude mice harboring ALL xenografts with F10. After 24 hours, leukemia cells were harvested and assayed for trapped Topo1 complexes. F10 treatment resulted in detectable trapped Topo1 complexes in both the xenograft and syngeneic models. In summary, F10 exhibited remarkable activity against human and murine ALL cells in vitro and in vivo by inducing apoptosis and trapping Topo1 complexes. These data demonstrate agents that target “final common pathways” but with differential uptake can be safe and effective, even against genetically complex and aggressive leukemias. Disclosures: Pardee: Salzburg Therapeutics: Membership on an entity's Board of Directors or advisory committees. Gmeiner: Salzburg Therapeutics: Membership on an entity's Board of Directors or advisory committees.