Thymidylate Synthase Inhibition with the Novel Fluoropyrimidine FdUMP[10] Is Highly Effective Against Acute Lymphoblastic Leukemia

Abstract 1505

Despite significant improvements in cure rates in pediatric patients, outcomes in adults with acute lymphoblastic leukemia (ALL) remain dismal with an estimated 5-year survival of less than 10% for patients over the age of 60. Compared to children, ALL in adults is characterized by an increased incidence of poor prognostic cytogenetics, lower complete remission rates, and higher relapse rates. Once relapse occurs in adults, the outcome is truly dismal, with a 6% salvage rate with current therapies. Initial treatment of ALL combines essentially all currently used agents, leaving very few therapies once relapse occurs. There is a clear need for better therapeutic options for these patients.

5-Fluorouracil (5-FU) is a fluoropyrimidine (FP) prodrug that must first be converted to 5-fluoro-2'-deoxyuridine-5'-O-monophosphate (FdUMP) in order to inhibit thymidylate synthase (TS) and shut down tumor thymidine synthesis. It can also be metabolized to a ribose metabolite that interferes with RNA processing and leads to toxicity in any transcriptionally active cell. The profound GI toxicities of 5-FU and lack of potency against ALL has lead to the abandonment of TS inhibition in the treatment of ALL. In contrast, FdUMP[10] is a oligodeoxynucleotide containing 10 FdUMP moieties linked by phosphodiester bonds. FdUMP[10] has profound activity against preclinical models of acute myeloid leukemia (AML), with minimal toxicities. The human T cell ALL cell line, Jurkat, was extremely sensitive to FdUMP[10], with an IC₅₀of 5.4nM (95% CI 4.609–6.417). We sought to determine the activity of FdUMP[10] against additional preclinical ALL models.

In vitro, FdUMP[10] exhibited remarkable activity against the human T and B Cell ALL cell lines, DG75, Molt-4, CCRF-CEM, and SUP-B15 with an average IC₅₀ value of 1.83nM (Range 0.21–4.1nM). In two murine ALL cell lines driven by expression of BCR-ABL, FdUMP[10] exhibited even greater activity, with an average IC₅₀ value of 0.662nM (range 0.125–1.2nM). For comparison we also tested 5-FU and cytarabine in the same assay against DG75, MOLT-4 and a BCR-ABL driven murine line. In all cases FdUMP[10] was the most potent agent and was more than 1000 times more potent than 5-FU despite having only 10 times the FP content. In vivo, FdUMP[10] treatment provided a statistically significant increase in survival in a BCR-ABL driven, syngeneic ALL mouse model (p=0.0002 by log rank test). In a separately derived syngeneic model expressing the T315I variant of BCR-ABL, treatment with FdUMP[10] significantly prolonged survival (p=0.0013).

In AML models exposure to FdUMP[10] resulted in apoptosis. To assess if ALL cells undergo a similar apoptotic response we exposed human and mouse ALL cells to FdUMP[10] and assessed for apoptosis induction via propidium iodide (PI) and Annexin V staining. As we found previously in AML models, FdUMP[10] exposure resulted in a robust induction of Annexin V and PI staining consistent with an apoptotic response. The level of apoptosis with FdUMP[10] could not be reproduced by 5-FU even when used at 100 times higher concentration.

To determine the effect of FdUMP[10] on thymidylate synthase (TS) activity in ALL, Jurkat cells were exposed to 10nM FdUMP[10] or 100nM 5- FU and TS activity was assayed. We found a profound and prolonged inhibition with FdUMP[10] compared to 5-FU, despite the identical amount of fluoropyrimidine.

FdUMP[10] is synergistic with doxorubicin and cytarabine in AML models. To assess whether FdUMP[10] could synergize with targeted therapy in ALL, Baf-3 cells expressing BCR-ABL were exposed to the tyrosine kinase inhibitor nilotinib plus FdUMP[10]. The combination was synergistic, with combinatory index values of 0.7113 to 0.4803. In preliminary data in vivo, the combination of FdUMP[10] and nilotinib resulted in a greater reduction in leukemic burden then nilotinib alone.

In summary, FdUMP[10] exhibited remarkable activity against human and murine ALL cells in vitro and in vivo by inducing apoptosis and profound TS inhibition, and showing a synergistic benefit when combined with nilotinib. These data demonstrate that TS is a valid target in ALL cells. This fact combined with previous studies demonstrating a favorable toxicity profile, make FdUMP[10] a promising candidate for treatment of ALL.