ATR inhibition facilitates targeting of leukemia dependence on convergent nucleotide biosynthetic pathways

Abstract

Leukemia cells rely on two nucleotide biosynthetic pathways, de novo and salvage, to produce dNTPs for DNA replication. Here, using metabolomic, proteomic, and phosphoproteomic approaches, we show that inhibition of the replication stress sensing kinase ataxia telangiectasia and Rad3-related protein (ATR) reduces the output of both de novo and salvage pathways by regulating the activity of their respective rate-limiting enzymes, ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK), via distinct molecular mechanisms. Quantification of nucleotide biosynthesis in ATR-inhibited acute lymphoblastic leukemia (ALL) cells reveals substantial remaining de novo and salvage activities, and could not eliminate the disease in vivo. However, targeting these remaining activities with RNR and dCK inhibitors triggers lethal replication stress in vitro and long-term disease-free survival in mice with B-ALL, without detectable toxicity. Thus the functional interplay between alternative nucleotide biosynthetic routes and ATR provides therapeutic opportunities in leukemia and potentially other cancers.Leukemic cells depend on the nucleotide synthesis pathway to proliferate. Here the authors use metabolomics and proteomics to show that inhibition of ATR reduced the activity of these pathways thus providing a valuable therapeutic target in leukemia.

Nat Commun. 2017 Aug 14;8(1):241. doi: 10.1038/s41467-017-00221-3.

View publication

Structure-Guided Development of Deoxycytidine Kinase Inhibitors with Nanomolar Affinity and Improved Metabolic Stability

Abstract:

Pub_image

Recently, we have shown that small molecule dCK inhibitors in combination with pharmacological perturbations of de novo dNTP biosynthetic pathways could eliminate acute lymphoblastic leukemia cells in animal models. However, our previous lead compound had a short half-life in vivo. Therefore, we set out to develop dCK inhibitors with favorable pharmacokinetic properties. We delineated the sites of the inhibitor for modification, guided by crystal structures of dCK in complex with the lead compound and with derivatives. Crystal structure of the complex between dCK and the racemic mixture of our new lead compound indicated that the R-isomer is responsible for kinase inhibition. This was corroborated by kinetic analysis of the purified enantiomers, which showed that the R-isomer has >60-fold higher affinity than the S-isomer for dCK. This new lead compound has significantly improved metabolic stability, making it a prime candidate for dCK-inhibitor based therapies against hematological malignancies and, potentially, other cancers.

J Med Chem. 2014 Nov 26; 57(22): 9480–9494.
Published online 2014 Oct 23. doi: 10.1021/jm501124j

View publication

Deoxycytidine Kinase Augments ATM-Mediated DNA Repair and Contributes to Radiation Resistance

Abstract:

Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [18F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.

PLoS One. 2014; 9(8): e104125.
Published online 2014 Aug 7. doi: 10.1371/journal.pone.0104125

View publication

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.

The ability to reprogram cellular metabolism, a hallmark of cancer first noted long ago (Warburg et al., 1927) and recently reappreciated, is essential for tumor progression (Hanahan and Weinberg, 2011). Although cancer-initiated metabolic reprogramming processes are promising therapeutic targets (Vander Heiden, 2011), the existence of alternative, compensatory biosynthetic pathways presents a significant challenge for developing such therapies. For example, in lipid metabolism, cancer cells scavenge extracellular lipids as an alternative to energy-requiring de novo fatty acid biosynthesis (Kamphorst et al., 2011). In amino acid metabolism, glycine and serine required for tumor growth can be produced de novo and can also be scavenged from the extracellular environment (Jain et al., 2012; Maddocks et al., 2013).
Nucleotide metabolism also involves redundant and convergent biosynthetic pathways. Deoxyribonucleotide triphosphate (dNTP) pools required for DNA replication and repair can be produced by the de novo pathway (DNP) or by the nucleoside salvage pathway (NSP;Fig. 1 A; Reichard, 1988). The DNP uses glucose and amino acids to generate ribonucleotide diphosphates (NDPs), which are converted to deoxyribonucleotide diphosphates (dNDPs) by ribonucleotide reductase (RNR). The same dNDPs can also be produced via the NSP (Reichard, 1988), starting with extracellular deoxyribonucleosides (dNs) which are imported in the cell via specialized transporters. The first enzymatic steps in the cytosolic NSP are catalyzed by two kinases: thymidine kinase 1 (TK1) phosphorylates thymidine (dT), while deoxycytidine (dC) kinase (dCK) phosphorylates dC, deoxyadenosine (dA), and deoxyguanosine (dG; Reichard, 1988). The relevance of these two NSP kinases for dNTP production in normal and malignant cells is yet to be defined. Because dN substrates for the NSP kinases are absent from most cell culture media, it has been assumed that the NSP is dispensable for DNA replication (Xu et al., 1995). However, recent in vivo findings have challenged this assumption. For example, we reported impaired hematopoiesis in dCK−/− mice due to dCTP pool deficiency, resulting in replication stress (RS), S-phase arrest, and DNA damage in hematopoietic progenitors (Toy et al., 2010; Austin et al., 2012). Analyses of dCK/TK1 double-knockout mice showed that NSP-derived dCTP synthesis is required to compensate for the inhibition of de novo dCTP production (Austin et al., 2012; Fig. 1 A). The mechanism of DNP inhibition involves allosteric regulation of RNR-mediated reduction of cytidine diphosphate (CDP) to dC diphosphate (dCDP) by dT triphosphate (dTTP) produced via TK1 from endogenous dT (Austin et al., 2012; Fig. 1 A).
Production of dNTPs by the NSP may be therapeutically relevant in cancer. For example, the ability of cancer cells to switch their dCTP synthesis from the DNP to the NSP may explain why dT given as a single dCTP-depleting agent showed limited efficacy in clinical trials (Chiuten et al., 1980; Kufe et al., 1980, 1981). If correct, this hypothesis suggests that a combination of dT (to inhibit DNP-mediated dCTP production) and a dCK inhibitor (to co-target dCTP production by the NSP) would be more efficacious in killing tumor cells than either treatment alone. Here, we investigate this possibility in the context of acute lymphoblastic leukemia (ALL). We demonstrate that co-targeting both de novo and salvage pathways for dCTP biosynthesis is well tolerated in mice and is efficacious in T-ALL and B-ALL models. We also describe a positron emission tomography (PET)–based assay to noninvasively monitor in vivo pharmacological targeting of dCTP biosynthesis in cancer cells.

Published February 24, 2014 // JEM vol. 211 no. 3 473-486
The Rockefeller University Press, doi: 10.1084/jem.20131738

View publication

Structural characterization of new deoxycytidine kinase inhibitors rationalizes the affinity-determining moieties of the molecules

Abstract:

Deoxycytidine kinase (dCK) is a key enzyme in the nucleoside salvage pathway that is also required for the activation of several anticancer and antiviral nucleoside analog prodrugs. Additionally, dCK has been implicated in immune disorders and has been found to be overexpressed in several cancers. To allow the probing and modulation of dCK activity, a new class of small-molecule inhibitors of the enzyme were developed. Here, the structural characterization of four of these inhibitors in complex with human dCK is presented. The structures reveal that the compounds occupy the nucleoside-binding site and bind to the open form of dCK. Surprisingly, a slight variation in the nature of the substituent at the 5-position of the thiazole ring governs whether the active site of the enzyme is occupied by one or two inhibitor molecules. Moreover, this substituent plays a critical role in determining the affinity, improving it from >700 to 1.5 nM in the best binder. These structures lay the groundwork for future modifications that would result in even tighter binding and the correct placement of moieties that confer favorable pharmacodynamics and pharmacokinetic properties.

Acta Crystallogr D Biol Crystallogr. 2014 Jan 1; 70(Pt 1): 68–78.
Published online 2013 Dec 24. doi: 10.1107/S1399004713025030

View publication

Development of new deoxycytidine kinase inhibitors and non-invasive in vivo evaluation using Positron Emission Tomography

Abstract:

Combined inhibition of ribonucleotide reductase and deoxycytidine kinase (dCK) in multiple cancer cell lines depletes deoxycytidine triphosphate pools leading to DNA replication stress, cell cycle arrest and apoptosis. Evidence implicating dCK in cancer cell proliferation and survival stimulated our interest in developing small molecule dCK inhibitors. Following a high throughput screen of a diverse chemical library, a structure-activity relationship study was carried out. Positron Emission Tomography (PET) using 18F-L-1-(2′-deoxy-2′-FluoroArabinofuranosyl) Cytosine (18F-L-FAC), a dCK-specific substrate, was used to rapidly rank lead compounds based on their ability to inhibit dCK activity in vivo. Evaluation of a subset of the most potent compounds in cell culture (IC50 = ∼1 – 12 nM) using the 18F-L-FAC PET pharmacodynamic assay identified compounds demonstrating superior in vivo efficacy.

J Med Chem. 2013 Sep 12; 56(17): 6696–6708.
Published online 2013 Aug 15. doi: 10.1021/jm400457y

View publication

Nucleoside salvage pathway kinases regulate hematopoiesis by linking nucleotide metabolism with replication stress

Abstract:

Nucleotide deficiency causes replication stress (RS) and DNA damage in dividing cells. How nucleotide metabolism is regulated in vivo to prevent these deleterious effects remains unknown. In this study, we investigate a functional link between nucleotide deficiency, RS, and the nucleoside salvage pathway (NSP) enzymes deoxycytidine kinase (dCK) and thymidine kinase (TK1). We show that inactivation of dCK in mice depletes deoxycytidine triphosphate (dCTP) pools and induces RS, early S-phase arrest, and DNA damage in erythroid, B lymphoid, and T lymphoid lineages. TK1(-/-) erythroid and B lymphoid lineages also experience nucleotide deficiency but, unlike their dCK(-/-) counterparts, they still sustain DNA replication. Intriguingly, dCTP pool depletion, RS, and hematopoietic defects induced by dCK inactivation are almost completely reversed in a newly generated dCK/TK1 double-knockout (DKO) mouse model. Using NSP-deficient DKO hematopoietic cells, we identify a previously unrecognized biological activity of endogenous thymidine as a strong inducer of RS in vivo through TK1-mediated dCTP pool depletion. We propose a model that explains how TK1 and dCK “tune” dCTP pools to both trigger and resolve RS in vivo. This new model may be exploited therapeutically to induce synthetic sickness/lethality in hematological malignancies, and possibly in other cancers.

J Exp Med. 2012 Nov 19;209(12):2215-28. doi: 10.1084/jem.20121061. Epub 2012 Nov 12.

View publication

Stratification of Nucleoside Analog Chemotherapy Using 1-(2′-Deoxy-2′-18F-Fluoro-β-D-Arabinofuranosyl)Cytosine and 1-(2′-Deoxy-2′-18F-Fluoro-β-L-Arabinofuranosyl)-5-Methylcytosine PET

Abstract:

The ability to measure tumor determinants of response to nucleoside analog (NA) chemotherapy agents such as gemcitabine and related compounds could significantly affect the management of several types of cancer. Previously we showed that the accumulation in tumors of the new PET tracer 1-(2′-deoxy-2′-18F-fluoro-β-D-arabinofuranosyl)cytosine (18F-FAC) is predictive of responses to gemcitabine. 18F-FAC retention in cells requires deoxycytidine kinase (dCK), a rate-limiting enzyme in the deoxyribonucleoside salvage metabolism and in gemcitabine conversion from an inactive prodrug to a cytotoxic compound. The objectives of the current study were to determine whether 18F-FAC tumor uptake is also influenced by cytidine deaminase (CDA), a determinant of resistance to gemcitabine; to develop a new PET assay using 18F-FAC and the related probe 1-(2′-deoxy-2′-18F-fluoro-β-L-arabinofuranosyl)-5-methylcytosine (L-18F-FMAC) to profile tumor lesions for both dCK and CDA enzymatic activities; and to determine whether this PET assay can identify the most effective NA chemotherapy against tumors with differential expression of dCK and CDA.
Methods

Isogenic murine leukemic cell lines with defined dCK and CDA activities were generated by retroviral transduction. A cell viability assay was used to determine the sensitivity of the isogenic cell lines to the dCK-dependent NA prodrugs gemcitabine and clofarabine. In vitro enzymatic and cell-based tracer uptake assays and in vivo PET with 18F-FAC and L-18F-FMAC were used to predict tumor responses to gemcitabine and clofarabine.

Results

dCK and CDA activities measured by kinase and tracer uptake assays correlated with the sensitivity of isogenic cell lines to gemcitabine and clofarabine. Coexpression of CDA decreased the sensitivity of dCK-positive cells to gemcitabine treatment in vitro by 15-fold but did not affect responses to clofarabine. Coexpression of CDA decreased 18F-FAC but not L-18F-FMAC, phosphorylation, and uptake by dCK-positive cells. 18F-FAC and L-18F-FMAC PET estimates of the enzymatic activities of dCK and CDA in tumor implants in mice were predictive of responses to gemcitabine and clofarabine treatment in vivo.

Conclusion

These findings support the utility of PET-based phenotyping of tumor nucleoside metabolism for guiding the selection of NA prodrugs.

J Nucl Med. Author manuscript; available in PMC 2012 Aug 31.
Published in final edited form as:
J Nucl Med. 2012 Feb; 53(2): 275–280.
doi: 10.2967/jnumed.111.090407

View publication

Novel Positron Emission Tomography Probes Specific for Deoxycytidine Kinase

Abstract:

Deoxycytidine kinase (dCK) is a rate-limiting enzyme in the deoxyribonucleoside salvage pathway and a critical determinant of therapeutic activity for several nucleoside analog pro-drugs. We have previously reported the development of 18F-FAC, (1-(2′-deoxy-2′-18F-fluoro-β-D-arabinofuranosyl) cytosine), a new probe for PET imaging of dCK activity in immune disorders and certain cancers. The objective of the current study was to develop PET probes with improved metabolic stability and specificity for dCK. Towards this goal, several candidate PET probes were synthesized and evaluated in vitro and in vivo.
Methods

High pressure liquid chromatography was used to analyze the metabolic stability of 18F-FAC and of several newly-synthesized analogs with the natural D-enantiomeric sugar configuration or the corresponding unnatural L-configuration. In vitro kinase and uptake assays were used to determine the affinity of the 18F-FAC L-nucleoside analogs for dCK. The biodistribution of selected L- analogs in mice was determined by microPET/CT imaging.

Results

Candidate PET probes were selected using the following criteria: low susceptibility to deamination, high affinity for purified recombinant dCK, high uptake in dCK expressing cell lines and biodistribution in mice reflective of the tissue expression pattern of dCK. Amongst the ten newly-developed candidate probes, 1-(2′-deoxy-2′-18F-fluoro-β-L-arabinofuranosyl) cytosine (L-18F-FAC) and 1-(2′-deoxy-2′-18F-fluoro-β-L-arabinofuranosyl)-5-methylcytosine (L-18F-FMAC) most closely matched the selection criteria. The selection of L-18F-FAC and L-18F-FMAC was validated by showing that these two PET probes can be used to image animal models of leukemia and autoimmunity.

Conclusion

Promising in vitro and in vivo data warrant biodistribution and dosimetry studies of L-18F-FAC and L-18F-FMAC in humans.

J Nucl Med. Author manuscript; available in PMC 2011 Jun 22.
Published in final edited form as:
J Nucl Med. 2010 Jul; 51(7): 1092–1098.
Published online 2010 Jun 16. doi: 10.2967/jnumed.109.073361

View publication

Requirement for deoxycytidine kinase in T and B lymphocyte development

Abstract:

Deoxycytidine kinase (dCK) is a rate-limiting enzyme in deoxyribonucleoside salvage, a metabolic pathway that recycles products of DNA degradation. dCK phosphorylates and therefore activates nucleoside analog prodrugs frequently used in cancer, autoimmunity, and viral infections. In contrast to its well established therapeutic relevance, the biological function of dCK remains enigmatic. Highest levels of dCK expression are found in thymus and bone marrow, indicating a possible role in lymphopoiesis. To test this hypothesis we generated and analyzed dCK knockout (KO) mice. dCK inactivation selectively and profoundly affected T and B cell development. A 90-fold decrease in thymic cellularity was observed in the dCK KO mice relative to wild-type littermates. Lymphocyte numbers in the dCK KO mice were 5- to 13-fold below normal values. The severe impact of dCK inactivation on lymphopoiesis was unexpected given that nucleoside salvage has been thought to play a limited, “fine-tuning” role in regulating deoxyribonucleotide triphosphate pools produced by the de novo pathway. The dCK KO phenotype challenges this view and indicates that, in contrast to the great majority of other somatic cells, normal lymphocyte development critically requires the deoxyribonucleoside salvage pathway.
Keywords: combined immunodeficiency, deoxyribonucleoside salvage, dNTP metabolism, immune development, T and B lymphocytes

Proc Natl Acad Sci U S A. 2010 Mar 23; 107(12): 5551–5556.
Published online 2009 Dec 31. doi: 10.1073/pnas.0913900107

View publication