LAUSR

Cytarabine (Ara-C) is a nucleotide analog and a cornerstone of standard chemotherapy in acute myeloid leukemia (AML). Ara-C is phosphorylated to its active metabolite Ara-CTP where it becomes incorporated into replicating DNA strands. Ara-CTP is deaminated to an inactive uridine metabolite by cytidine deaminase (CDA) and CDA overexpression is a clinically relevant mechanism of resistance to Ara-C. Here, we report the preclinical efficacy of a novel L-nucleoside analog 5-fluorotroxacitabine (5FTRX) in AML (Figure 1A). In a panel of 31 leukemia and lymphoma cell lines, 5FTRX reduced growth and viability with a median IC50 of 200 nM (range 12nM [EOL-1] to 6 mM [TF-1]) (Figure 1B and Online Supplementary Figure S1). 5FTRX also reduced the clonogenic growth of primary AML cells (Figure 1C; see Online Supplementary Tables S1 and S2 for patients' characteristics), thus demonstrating an ability to target leukemic progenitors. In contrast, normal hematopoietic cells were more resistant to 5FTRX (Figure 1D). 5FTRX is phosphorylated via monophosphate (MP) and diphosphate (DP) to the triphosphate (TP) before being incorporated into nascent DNA strands, leading to subsequent chain termination and cell death. AML cell lines MV4-11 and THP-1 or human peripheral blood mononuclear cells (PBMC) were incubated with 5FTRX and analyzed for the intracellular concentrations of 5FTRX and phosphorylated nucleotides (Online Supplementary Table S3). Detectible concentrations of MP, DP and TP were observed in PBMC and AML cells confirming bioactivation to the active TP in cells. To test whether 5FTRX damaged DNA in AML cells, we measured changes in phosphorylated H2AX (pH2AX) after 5FTRX treatment. 5FTRX increased pH2AX at concentrations associated with loss of viability (Online Supplementary Figure S2), indicating a likely chain termination during the proliferation of these cells. CDA overexpression is a mechanism of resistance to Ara-C. We investigated the effects of increased CDA on the anti-neoplastic action of 5FTRX. Wild-type HEK293 and HEK293 cells over-expressing CDA were treated with increasing concentrations of 5FTRX and Ara-C (Figure 2A and Online Supplementary Figure S3A). As expected, overexpression of CDA rendered cells approximately 7-fold more resistant to Ara-C (Figure 2B). Treatment of these cells with the CDA inhibitor tetrahydrouridine (THU) restored sensitivity to Ara-C (Online Supplementary Figure S3B). Interestingly, overexpression of CDA increased sensitivity to 5FTRX by approximately 10-fold (Figure 2C). Further, co-treatment with 5FTRX and THU returned sensitivity of the cells to 5FTRX towards baseline (Online Supplementary Figure S3C). Increased CDA expression did not change the growth rate of these cells (Online Supplementary Figure S4). Potentially, overexpression of CDA increased degradation of endogenous cytidine nucleotide pools enhancing the incorporation of 5FTRX-monophosphate into DNA. Of note, prior studies have reported that increased CDA expression increases the sensitivity of cells to troxacitabine. 5FTRX overcomes drug resistance induced by CDA overexpression, but CDA expression did not fully explain 5FTRX sensitivity in AML cells (Online Supplementary Figure S5A and B), indicating additional mechanisms of resistance and sensitivity. Therefore, we sought to understand potential mechanisms of resistance to 5FTRX. We selected populations of THP-1 cells resistant to 5FTRX (THP1-5FR: 66-fold resistant to 5FTRX) and Ara-C (THP1-ACR; 35-fold resistant to Ara-C) by treating parental THP-1 cells with increasing concentrations of the drugs for up to 4 months (Figure 2D). THP1-5FR and THP1-ACR were cross-resistant to Ara-C and 5FTRX, respectively, suggesting that similar mechanisms were responsible for conferring resistance. Downregulation of dCK is known to confer resistance to Ara-C, so we examined dCK expression in the resistant cell lines. Indeed, dCK levels were reduced in THP1-5FR and THP1-ACR resistant cell populations (Figure 2E). To test whether the reduced dCK was functionally important for resistance to 5FTRX, we analyzed the metabolites of 5FTRX in THP1-5FR cells. THP1-5FR cells treated with 5FTRX showed decreased phosphorylated species, consistent with a reduced ability of dCK to generate the monophosphate species (Figure 2F). Moreover, the reported dCK inhibitor 15C rendered THP-1 cells resistant to 5FTRX and Ara-C with IC50 values >50 mM (Figure 2G). To assess the future potential for 5FTRX to be included in standard treatment regimens for AML treatment, the anti-proliferative effects of 5FTRX were examined in combination with Ara-C, azacytidine or doxorubicin in MV4-11 and THP-1 cells. In both cell types, 5FTRX exhibited strong synergy in combination with azacytidine or doxorubicin. The combination of 5FTRX and AraC was additive in MV4-11 cells, whereas in THP-1 cells, a moderately synergistic interaction was observed (Online Supplementary Table S5 and Online Supplementary Figure S6A-F). Finally, we examined the preclinical efficacy and toxicity of 5FTRX in mouse models of leukemia. In mice xenografted with MV4-11 cells, 5FTRX produced dramatic and sustained tumor regressions and showed a marked superiority over Ara-C at its maximum tolerated dose (Figure 3A) without evidence of toxicity or body weight loss (Online Supplementary Figure S7). Furthemore, we assessed the activity of 5FTRX at a reduced frequency of treatment (qD x5 instead of BIDx5) in vivo using MV411 cells. Sustained tumor regressions were observed at all doses (Figure 3C) without loss of body weight (Online Supplementary Figure S8). The OCI-AML2 model displayed even more sensitivity to 5FTRX, with tumor regression observed at both 100 mg/kg and 30 mg/kg doses. Mice remained tumor free for 45 days after termination of treatment (Figure 3B) and did not display any signs of toxicity (Online Supplementary Figures S9-S11). We determined the optimal treatment schedule by delivering a total dose of 150 mg/kg at different time points: a single dose of 150 mg/kg; three daily doses of 50 mg/kg; or five daily doses of 30 mg/kg. All regimens led to tumor regressions, although tumor regrowth varied between doses (Figure 3D). The maximal response was observed at five daily doses of 30 mg/kg. To test whether the tumors remained sensitive to the drug, tumors were allowed to regrow to approximately 1,000 mm from the 5x30 mg/kg dose group, and then challenged with the same schedule of 5FTRX treatment. Despite the larger size of these tumors at this re-treatment stage, they still showed pronounced regression (Figure 3D), confirming they remained sensitive to the agent.