Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication

Abstract

Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies.

Figure 1.

dC salvage via dCK prevents dT-induced lethal RS in T-ALL cells. (A) Allosteric control of DNP dCTP production by dT via dTTP. (B) Effects of dT treatment (24 h) on dCTP and dTTP pools. Values represent mean ± SEM. (C) CEM cell cycle analysis after treatment with vehicle or 50 µM dT ± 2.5 µM dC for 24 h. (D) CEM cell cycle analysis after treatment with 50 µM hydroxyurea, 15 µM 5-fluorouracil, or 1.6 µM cisplatin for 24 h ± 2.5 µM dC. (E and F) Representative immunoblots of dCK and actin expression (E) and dCK kinase assay (F) in CEM dCK^wt (scrambled shRNA) cells and dCK^low (shRNA against dCK) cells. Values are mean ± SEM. ***, P < 0.001. (G) dCTP levels in CEM dCK^wt and dCK^low cells treated for 24 h with vehicle or 50 µM dT ± 2.5 µM dC. Values are mean ± SEM. ***, P < 0.001. (H) Cell cycle analysis of CEM dCK^low cells treated with vehicle or 50 µM dT ± 2.5 µM dC for 24 h. (I) Representative immunoblots detecting Chk1, pChk1 (Ser345), Chk2, pChk2 (Thr68), dCK, and actin in CEM dCK^wt and dCK^low cells treated with vehicle or 50 µM dT in the presence of 2.5 µM dC for 24, 48, and 72 h. (J) pH2A.X (Ser139) and DNA content (DAPI) in CEM dCK^wt and dCK^low cells treated with vehicle or 50 µM dT in the presence of 2.5 µM dC for 24 h. (K) Representative images and quantification of the COMET assay conducted on CEM dCK^wt and dCK^low cells 48 h after treatment with vehicle or 50 µM dT in the presence of 2.5 µM dC. Values represent the mean Olive Tail Moment ± SEM from 100 cells per image × 4 images/group; n = 2 independent experiments. ***, P < 0.001. Bars, 50 µm. (L) Annexin V staining of CEM dCK^wt and dCK^low cells after treatment with vehicle, 2.5 µM dC, 50 µM dT, or dC + dT for 72 h. All values are mean ± SEM from at least three replicates/data point. ***, P < 0.001. All data are representative of n = 3 independent experiments, unless indicated.

Figure 2.

Treatment with dT triggers a metabolic switch to NSP-mediated dCTP biosynthesis in T-ALL cells and up-regulates the NSP. (A) Schematic of the [U-¹³C]-glucose and [U-¹³C/¹⁵N]-dC stable isotope labeling approach used to determine the source (DNP or NSP) of the free dCTP pool and of the dCTP incorporated into the DNA of CEM cells treated with various dT concentrations. (B) dCTP derived from [U-¹³C]-glucose (DNP) and [U-¹³C/¹⁵N]-dC (NSP) in the free dCTP pool and incorporated into the DNA of CEM cells after 12 h of incubation with stable isotope–labeled DNP and NSP precursors, in the presence or absence of dT. Values are the mean of absolute peak area/10³ cells ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with 0 µM dT control. Data are representative of n = 2 independent experiments. (C) Quantification of dCK kinase activity in CEM cells at baseline and after 8 h of treatment with 50 µM dT. Data are representative of n = 2 independent experiments. Values are mean ± SEM. ***, P < 0.001. (D) Quantification of the uptake of ³H-labeled dC by CEM cells at baseline and after 4 h of treatment with 50 µM dT. Data are representative of n = 2 independent experiments. Values represent mean ± SEM. ***, P < 0.001.

Figure 3.

In vivo, salvage of endogenous dC rescues T-ALL cells from RS induced by dT treatment. (A) Left axis: plasma dT levels in NSG mice treated with 2 g/kg dT (single dose). Values are mean ± SEM from n = 3 mice/time point; n = 2 independent experiments. Right axis: dTTP concentrations from CEM dCK^wt and dCK^low tumors at various time points after single-dose dT (2 g/kg) treatment. Values are mean ± SEM, n = 4 mice/time point; n = 2 independent experiments. (B) Representative immunoblot (n = 3 independent experiments) showing pChk1 (Ser345) levels at various time points in bilateral s.c. CEM dCK^wt and dCK^low tumors implanted in NSG mice treated with 2 g/kg dT (single-dose). (C) dCTP concentrations from CEM dCK^wt and dCK^low tumors at various time points after single-dose dT (2 g/kg) treatment. Values are mean ± SEM, n = 5 mice/time point; n = 2 independent experiments. ***, P < 0.001. (D) Schematic of experimental design for quantifying the incorporation of [U-¹³C/¹⁵N]-dC into the DNA of dCK^wt and dCK^low CEM tumors 4 h after single-dose treatment with 2 g/kg dT or vehicle. (E) Quantification of the LC/MS/MS-MRM data for labeled dCTP incorporation into the DNA. Data are mean ± SEM of n = 6 mice/group; n = 2 independent experiments. **, P < 0.01. (F) Schematic of the in vivo PET assay of dCK activity. (G) ¹⁸F-L-FAC uptake in s.c. CEM dCK^wt and dCK^low tumor xenografts 4 h after vehicle or dT injection. Values represent the mean percent decrease in ¹⁸F-FAC signal relative to dCK^wt vehicle ± SEM, n = 4 mice/group; n = 2 independent experiments. **, P < 0.01; ***, P < 0.001.

Figure 4.

dCK mediates resistance to dT in T-ALL cells in vivo. (A) Serial secreted Gaussia luciferase measurements of peripheral blood from NSG mice bearing CEM dCK^wt or dCK^low s.c. tumors (n = 6 mice/condition) treated every 12 h with vehicle or 2 g/kg dT starting at day 7 after tumor implantation until day 13. Values represent mean ± SEM; n = 2 independent experiments. **, P < 0.01; ***, P < 0.001, compared with dCK^low vehicle at the indicated time point. (B) CEM dCK^wt and dCK^low tumors from vehicle or dT-treated mice from A. (C) Tumor weights (in milligrams) from A. Values represent the mean ± SEM; n = 2 independent experiments. ***, P < 0.001.

Figure 5.

Development of DI-39, a small molecule dCK inhibitor which synergizes with inhibition of de novo dCTP biosynthesis in leukemic cells. (A) Schematic illustrating the development of DI-39, beginning with high-throughput screen (HTS) of a 90,000-compound library, which provided the initial hit DI-0120. Further structural activity relationship (SAR) yielded 80 novel compounds including DI-39. (B) Chemical structure of DI-39. (C) LC/MS/MS-MRM measurements of DI-39 in CEM cells exposed to 1 µM drug for indicated periods of time. Cells were washed three times after 60 min (indicated by vertical line) and cellular drug retention was measured again 60 min later. Values represent mean ± SEM. (D) IC₅₀ value of DI-39 determined by percent inhibition of ³H-dC uptake by CEM cells. Values represent mean ± SEM. (E) 2.1 Å crystal structure of dCK with bound DI-39 and uridine diphosphate (UDP). (F) Intracellular dCTP concentrations in cultured CEM dCK^wt cells treated with vehicle, 50 µM dT, 1 µM DI-39, or DI-39 + dT for 24 h. Values represent the mean ± SEM; n = 2 independent experiments. ***, P < 0.001. (G) Representative immunoblots detecting Chk1, pChk1 (Ser345), and actin in CEM cells treated with vehicle, 1 mM dT, 100 nM DI-39, or DI-39 + dT in the presence of 2.5 µM dC for 24 h. (H) Annexin V staining of CEM cells treated for 72 h with indicated concentrations of DI-39 and dT in the presence of 2.5 µM dC. Values are mean ± SEM; n = 2 independent experiments. ***, P < 0.001 compared with 50 µM dT. (I) Annexin V staining of L1210-10 dCK-null cells treated for 72 h with indicated concentrations of DI-39. Values represent the mean percentage of cells staining positive for Annexin V ± SEM; n = 2 independent experiments. (J) Representative immunoblots of Jurkat, MOLT-4, RSR4;11, NALM-6, and TF-1 leukemia cells treated with vehicle, 1 mM dT, 100 nM DI-39, or DI-39 + dT in the presence of 2.5 µM dC for 24 h (NALM-6) or 72 h (Jurkat, MOLT-4, RSR4;11, and TF-1). (K) Annexin V staining of the same panel of leukemia cell lines as in J treated for 72 h with vehicle, 1 mM dT, 100 nM DI-39, or DI-39 + dT. Cultures were supplemented with 2.5 µM dC. Values represent mean percentage of cells staining positive for Annexin V ± SEM; n = 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

DI-39 inhibits dCK activity in vivo, as determined by ¹⁸F-FAC PET, and promotes RS when combined with dT. (A) Pharmacokinetic profile of DI-39. C57BL/6 mice were dosed with DI-39 via intraperitoneal injection. Dose formulation: 10% DMSO and 40% Captisol (SBE-β-CD, a polyanionic variably substituted sulfobutyl ether of β-cyclodextrin; Stella and He, 2008) in water. Approximated values of the area under the curve (AUC), clearance rate (CL), half-life (T_1/2), maximum concentration in the plasma (C_max), and time to reach the maximum concentration (T_max) were calculated using Boomer/Multi-Forte PK Functions for Excel. Values represent the mean ± SD, n = 4/time point; n = 2 independent experiments. (B) LC/MS/MS-MRM quantification of DI-39 concentrations in plasma and CEM tumors at various time points after treatment. See Materials and methods for details. Values represent the mean ± SD, n = 4/group. (C) Schematic illustration of the ¹⁸F-FAC PET/CT study to determine in vivo dCK inhibition by DI-39 in CEM s.c. xenografts. (D) Time course of in vivo ¹⁸F-FAC PET/CT scans to determine dCK inhibition by DI-39 (single intraperitoneal injection, 50 mg/kg). Values represent the mean percent decrease in ¹⁸F-FAC signal ± SD, n = 4 mice/group; n = 2 independent experiments. (E) Percent incorporation of [U-¹³C/¹⁵N]-dC into the DNA of CEM xenografts 5.5 h after single-dose treatment with vehicle, 50 mg/kg DI-39, 2 g/kg dT, or DI-39 + dT. Mice were pulsed with the stable isotope-labeled dC for 30 min before sacrifice. Values represent mean ± SEM, n = 4/group; n = 2 independent experiments. **, P < 0.01; ***, P < 0.001. (F) Representative immunoblots of pChk1 (Ser345), Chk1, and actin in tumor tissues collected from mice 6 h after treatment with 50 mg/kg DI-39, 2 g/kg dT, or both agents; n = 3 independent experiments.

Figure 7.

Pharmacological co-targeting of DNP and NSP dCTP production is effective against T-ALL cells in vivo. (A) Representative images of CEM xenografts isolated from mice treated with vehicle, 2 g/kg dT, 50 mg/kg DI-39, or DI-39 + dT every 12 h beginning at day 7 after inoculation and continuing to day 14. n = 6 mice/group; n = 2 independent experiments. (B) Tumor weights from A. Values represent mean ± SEM; n = 2 independent experiments, n = 6 mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Representative images and quantification of TUNEL staining of tumor samples from A. Bars, 50 µm. Values represent mean ± SEM, n = 6 mice/group. ***, P < 0.001. (D) Representative FACS plots and quantification of eGFP + CEM leukemia cells in the BM of NSG mice treated with vehicle, 2 g/kg dT, 50 mg/kg DI-39, or DI-39 + dT. Mice (n = 6/group) were treated every 12 h beginning at day 3 after inoculation with 1.0 × 10⁶ CEM cells. Values represent mean ± SEM; n = 2 independent experiments. **, P < 0.01; ***, P < 0.001.

Figure 8.

Pharmacological co-targeting of the DNP and NSP is efficacious against primary mouse p185^BCR-ABLArf^−/− Pre-B ALL cells, while sparing the hematopoietic progenitor pool. (A) Annexin V staining of p185^BCR-ABL Arf^−/− pre–B cells after 48-h treatment with vehicle, 200 µM dT, 100 nM DI-39, or DI-39 + dT in the presence of 2.5 µM dC. Values are mean ± SEM; n = 2 independent experiments. **, P < 0.01. (B) Representative BLIs of mice (n = 6/group) treated with vehicle, 2 g/kg dT, 50 mg/kg DI-39, or DI-39 + dT at day 14 after intravenous injection of 2.0 × 10⁴ pre–B leukemia cells/mouse. (C) Quantification of BLI from in BM and spleen. *, P < 0.05; **, P < 0.01. (D) Representative FACS analyses and quantification of CD19⁺ leukemic cells in the BM of treated mice. **, P < 0.01; ***, P < 0.001. (E) Quantification of Lineage⁻ Sca-1⁺ c-Kit⁺ (LSK) populations from treated mice. **, P < 0.01. (F) LSK cells from BM of treated mice were analyzed for expression of CD34 and Flt3 to identify and quantify long-term (LT, CD34⁻, Flt3⁻), short-term (ST, CD34⁺, Flt3⁻), and multipotent progenitor (MPP, CD34⁺, Flt3⁺) stem cells. (G and H) Body weights (G), as well as RBC, hemoglobin, platelet, and neutrophil measurements (H) of NSG mice (n = 6/group) treated with vehicle, 2 g/kg dT, 50 mg/kg DI-39, or DI-39 + dT every 12 h for 7 d. Data represent mean ± SEM. All data are representative of at least two independent experiments.

Figure 9.

Assessment of potential toxicity of the DI-39/dT combination therapy and model. (A) Representative FACS staining of pH2A.X and data quantification in EryA (CD71⁺/high forward scatter) erythroblasts to estimate endogenous (for dCK^−/− mice, n = 4 mice/group) or potential pharmacologically induced (DI-39 + dT) genotoxic stress. NSG mice (n = 5 mice/group) were treated with vehicle or combination of 50 mg/kg DI-39 and 2 g/kg dT every 12 h for 8 d. Values are mean ± SEM. **, P < 0.01. (B) Representative FACS plots and quantification of micronucleated erythrocytes indicative of endogenous (for dCK^−/− mice) or potential pharmacologically induced (DI-39 + dT) genotoxic stress. Values represent mean ± SEM from n = 2 independent experiments. *, P < 0.05; ***, P < 0.001. (C and D) Proposed rationale for explaining the selectivity of the combination therapy for leukemia cells relative to normal hematopoietic progenitors (see text for details).