Potent siRNA Inhibitors of Ribonucleotide Reductase Subunit RRM2 Reduce Cell Proliferation In vitro and In vivo

Abstract

Purpose: Ribonucleotide reductase (RR) is a therapeutic target for DNA replication^dependent diseases such as cancer. Here, a potent small interfering RNA (siRNA) duplex against the M2 subunit of RR (RRM2) is developed and shown to reduce the growth potential of cancer cells both in vitro and in vivo. Experimental Design: Three anti-RRM2 siRNAs were identified via computational methods, and the potency of these and additional ''tiling'' duplexes was analyzed in cultured cells via cotransfections using a RRM2-luciferase fusion construct. Knockdown of RRM2 by the best duplex candidates was confirmed directly by Western blotting. The effect of potent duplexes on cell growth was investigated by a real-time cell electronic sensing assay. Finally, duplex performance was tested in vivo in luciferase-expressing cells via whole animal bioluminescence imaging. Results: Moderate anti-RRM2 effects are observed from the three duplexes identified by computational methods. However, the tiling experiments yielded an extremely potent duplex (siR2B+5). This duplex achieves significant knockdown of RRM2 protein in cultured cells and has pronounced antiproliferative activity. S.c. tumors of cells that had been transfected with siR2B+5 preinjection grew slower than those of control cells. Conclusions: An anti-RRM2 siRNA duplex is identified that exhibits significant antiproliferative activity in cancer cells of varying human type and species (mouse, rat, monkey); these findings suggest that this duplex is a promising candidate for therapeutic development.Ribonucleotide reductase (RR) catalyzes the conversion of ribonucleoside 5 ¶-diphosphates into their corresponding 2 ¶-deoxyribonucleotides and is a rate-limiting step in the pathway for the production of 2 ¶-deoxyribonucleoside 5 ¶-triphosphates that are necessary for DNA replication. Human RR consists of two subunits, RRM1 and RRM2, and the expression of both proteins is required for enzymatic activity. RRM1 and RRM2 are encoded by different genes on separate chromosomes, and their mRNAs are differentially expressed throughout the cell cycle. The cellular RRM1 protein level is kept relatively stable through the entire cell cycle, whereas RRM2 is only expressed during the late G 1 /early S phase when DNA replication occurs (1).RR has long been an important target for controlling pathologies that depend on DNA replication. Inhibition of RR activity has been tested as potential therapy in anticancer, antiviral, antibacterial, and other antiparasitic settings. A recent review on RR inhibitors as anticancer agents has appeared (2). Numerous small-molecule inhibitors, such as hydroxyurea (approved for human use) and triapine (3), interact with subunit RRM2. These small molecules are not completely specific to the RRM2 protein, however, and work continues on the design of more specific and effective inhibitors (4).A recent approach to RR down-regulation involves the use of nucleic acid -based, gene-specific inhibition. Yen et al. (5) and Lee et al. (6) have shown that antisense...

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“…More recently, clofarabine and troxacitabine have been evaluated in clinical trials for various cancers (13 that these tumors synthesize their DNA primarily via the de novo pathway rather than by using the DCKdependent nucleoside salvage pathway. In turn, this may indicate that these patients could respond to therapies that interfere with the de novo pathway, including conventional drugs like hydroxyurea-and siRNA-based approaches that block the function of key enzymes in the de novo pathway such as ribonucleotide reductase (30)(31)(32).…”

Correlating animal and human phase Ia/Ib clinical data with CALAA-01, a targeted, polymer-based nanoparticle containing siRNA

“…Vial 2 contains (iv) siRNA designed to reduce the expression of the M2 subunit of ribonucleotide reductase (RRM2). RRM2 is an established anticancer target (7,8), and i.v. administration of CALAA-01 inhibits the growth of human tumor xenografts in mice by inhibiting the expression of RRM2 (9) (SI Appendix, Fig.…”

Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA

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“…The siRNA sequence is a potent inhibitor of the M2 subunit of ribonucleotide reductase (RRM2) (34) and is active in mouse, monkey, and human cells (ref. 34 and SI Fig. 7).…”

“…This sequence has been shown to be a potent inhibitor of the RRM2 protein and exhibits significant antiproliferative activity in cancer cells of varying human types and species (mouse, rat, and monkey) (34). Antiproliferation data for human and monkey cancer cell lines are given in SI Fig.…”