Comparison of nanoparticular hydroxyapatite pastes of different particle content and size in a novel scapula defect model.

Nanocrystalline hydroxyapatite (HA) has good biocompatibility and the potential to support bone formation. It represents a promising alternative to autologous bone grafting, which is considered the current gold standard for the treatment of low weight bearing bone defects. The purpose of this study was to compare three bone substitute pastes of different HA content and particle size with autologous bone and empty defects, at two time points (6 and 12 months) in an ovine scapula drillhole model using micro-CT, histology and histomorphometry evaluation. The nHA-LC (38% HA content) paste supported bone formation with a high defect bridging-rate. Compared to nHA-LC, Ostim® (35% HA content) showed less and smaller particle agglomerates but also a reduced defect bridging-rate due to its fast degradation The highly concentrated nHA-HC paste (48% HA content) formed oversized particle agglomerates which supported the defect bridging but left little space for bone formation in the defect site. Interestingly, the gold standard treatment of the defect site with autologous bone tissue did not improve bone formation or defect bridging compared to the empty control. We concluded that the material resorption and bone formation was highly impacted by the particle-specific agglomeration behaviour in this study.

[18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-ß-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-ß-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication.

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.

Development of new deoxycytidine kinase inhibitors and noninvasive in vivo evaluation using positron emission tomography.

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 (18)F-L-1-(2'-deoxy-2'-FluoroArabinofuranosyl) Cytosine ((18)F-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 (18)F-L-FAC PET pharmacodynamic assay identified compounds demonstrating superior in vivo efficacy.

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

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.

Cellular immune responses against CT7 (MAGE-C1) and humoral responses against other cancer-testis antigens in multiple myeloma patients.

The type I melanoma antigen gene (MAGE) proteins CT7 (MAGE-C1) and MAGE-A3 are commonly expressed in multiple myeloma (MM), and their expression correlates with increased plasma cell proliferation and poor clinical outcome. They belong to the cancer-testis antigen (CTAg) group of tumor-associated proteins, some of which elicit spontaneous immune responses in cancer patients. CT7 and MAGE-A3 are promising antigenic targets for therapeutic tumor vaccines in myeloma; therefore, it is critical to determine if they are immunogenic in MM patients. We analyzed cellular and humoral immune responses against CTAgs in patients with plasma cell dyscrasias: MM, monoclonal gammopathy of undetermined significance (MGUS), and Waldenström's macroglobulinemia (WM). Bone marrow lymphocytes from two of four untreated MM patients exhibited CT7-specific cellular immune responses as measured by an autologous cellular immunity assay, the first such immune response to CT7 to be reported in cancer patients. Sera from 24 patients were screened by ELISA for humoral immune responses to CTAgs. Two patients with MM demonstrated positive titers, one for MAGE-A1 and the other for SSX1. These data demonstrate that CTAgs, particularly CT7, are immunogenic in MM patients and merit further exploration as targets of immunological therapy in MM.

Requirement for deoxycytidine kinase in T and B lymphocyte development.

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.