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Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine.

ABSTRACT: Cytotoxic drugs, such as nucleoside analogs and toxins, commonly suffer from off-target effects. One approach to mitigate this problem is to deliver the cytotoxic drug selectively to the intended site. While for toxins this can be achieved by conjugating the cell-killing moiety to a targeting moiety, it is not an option for nucleoside analogs, which rely on intracellular enzymes to convert them to their active triphosphorylated form. To overcome this limitation, and achieve site-targeted activation of nucleoside analogs, we fused the coding region of a prodrug-activating enzyme, deoxycytidine kinase (dCK), to affinity reagents that bind to the Her2 cell surface protein. We evaluated dCK fusions to an anti-Her2 affibody and Designed Ankyrin Repeat Protein (DARPin) for their ability to kill cancer cells by promoting the activation of the nucleoside analog fludarabine. Cell staining and flow cytometry experiments with three Her2 positive cancer cell lines (BT-474-JB, JIMT-1 and SK-OV-3) indicate dCK fusions binding and cellular internalization. In contrast, these reagents bind only weakly to the Her2 negative cell line, MCF-7. Cell proliferation assays indicate that SK-OV-3 and BT-474-JB cell lines exhibit significantly reduced proliferation rates when treated with targeting-module fused dCK and fludarabine, compared to fludarabine alone. These findings demonstrate that we have succeeded in delivering active dCK into the Her2-positive cells, thereby increasing the activation of fludarabine, which ultimately reduces the dose of nucleoside analog needed for cell killing. This strategy may help establish the therapeutic index required to differentiate between healthy tissues and cancer cells.

SUBMITTER: Koduvayur SP

PROVIDER: S-EPMC4900609 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine.

Koduvayur Sujatha P SP Su Ying Y Kay Brian K BK Lavie Arnon A

PloS one 20160609 6

Cytotoxic drugs, such as nucleoside analogs and toxins, commonly suffer from off-target effects. One approach to mitigate this problem is to deliver the cytotoxic drug selectively to the intended site. While for toxins this can be achieved by conjugating the cell-killing moiety to a targeting moiety, it is not an option for nucleoside analogs, which rely on intracellular enzymes to convert them to their active triphosphorylated form. To overcome this limitation, and achieve site-targeted activat ...[more]

PMID: 27280468

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Project description:BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. Implementation of high-dose cytarabine (araC) into induction therapy became standard-of-care for all newly diagnosed younger MCL patients. However, many patients relapse even after araC-based regimen. Molecular mechanisms responsible for araC resistance in MCL are unknown and optimal treatment strategy for relapsed/refractory MCL patients remains elusive. METHODS: Five araC-resistant (R) clones were derived by long-term culture of five MCL cell lines (CTRL) with increasing doses of araC up to 50 microM. Illumina BeadChip and 2-DE proteomic analysis were used to identify gene and protein expression changes associated with araC resistance in MCL. In vitro cytotoxicity assays and experimental therapy of MCL xenografts in immunodeficient mice were used to analyze their relative responsiveness to a set of clinically used anti-MCL drugs. Primary MCL samples were obtained from patients at diagnosis and after failure of araC-based therapies. RESULTS: Marked downregulation of deoxycytidine-kinase (DCK) mRNA and protein expression was identified as the single most important molecular event associated with araC-resistance in all tested MCL cell lines and in 50% primary MCL samples. All R clones were highly (20-1000x) cross-resistant to all tested nucleoside analogs including gemcitabine, fludarabine and cladribine. In vitro sensitivity of R clones to other classes of clinically used anti-MCL agents including genotoxic drugs (cisplatin, doxorubicin, bendamustine) and targeted agents (bortezomib, temsirolimus, rituximab) remained unaffected, or was even increased (ibrutinib). Experimental therapy of immunodeficient mice confirmed the anticipated loss of anti-tumor activity (as determined by overall survival) of the nucleoside analogs gemcitabine and fludarabine in mice transplanted with R clone compared to mice transplanted with CTRL cells, while the anti-tumor activity of cisplatin, temsirolimus, bortezomib, bendamustine, cyclophosphamide and rituximab remained comparable between the two cohorts. CONCLUSIONS: Acquired resistance of MCL cells to araC is associated with downregulation of DCK, enzyme of the nucleotide salvage pathway responsible for the first phosphorylation (=activation) of most nucleoside analogs used in anti-cancer therapy. The data suggest that nucleoside analogs should not be used in the therapy of MCL patients, who relapse after failure of araC-based therapies.

Dataset Information

Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine.

Publications

Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine.

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