REVIEW URRENT C OPINION
Antisense oligonucleotides in cancer Daniela Castanotto and Cy A. Stein
Purpose of review Over the past several dozen years, regardless of the substantial effort directed toward developing rational oligonucleotide strategies to silence gene expression, antisense oligonucleotide-based cancer therapy has not been successful. This review focuses on the most likely reasons for this lack of success, and on the barriers that still need to be overcome to make a clinical cancer treatment reality out of the promise of antisense therapy. Recent findings Considerable progress has been made in the design and delivery of nucleic acid fragments. Chemical modifications have considerably improved oligonucleotide absorption, distribution and metabolism while at the same time reducing toxicity. Nevertheless, the delivery and the cellular uptake of these molecules are still not adequate to provide the desired therapeutic outcome. Recent therapeutic interventional phase III trials of antisense oligodeoxyribonucleotides for a cancer indication will be discussed, in addition to those studies that markedly improve the scientific understanding of the properties of these molecules. Summary We still do not have a marketed antisense oligonucleotide for a cancer indication. This is because critical aspects of the cellular, tumor pharmacology and delivery properties of these agents are still not well understood. Keywords antisense, endocytosis, oligonucleotide, phase III, phosphorothioate
INTRODUCTION Antisense oligodeoxyribonucleotides (oligos) are small pieces of DNA (usually 15–18 mer in length) that are complementary via Watson–Crick base pair hybridization to a targeted mRNA. Intracellular binding of the oligo to its mRNA target is thought to result in cleavage of the mRNA strand by the enzyme RNAse H, an enzyme which cleaves the RNA strand of an RNA-DNA duplex. The mRNA strand is hence no longer available to be translated into its encoded protein. The first cell-free antisense experiments [1] were quickly followed by a study reporting an antisense strategy designed to block gene expression in cells [2]. Interest in the field ramped up dramatically with the introduction of DNA synthesizers in the 1980s. In 1998, Fomiversen [3], an antisense oligo for the treatment of cytomegalovirus retinitis [Isis, Carlsbad, California, USA], which could cause blindness in HIV/AIDS patients, was approved by the Food and Drug Administration (FDA) for marketing. However, the drug was ultimately withdrawn as the indication essentially disappeared. To date, however, despite the expenditure of great amounts of time and money and numerous clinical trials, we still do not have a single antisense www.co-oncology.com
oligo drug that has been approved for a cancer indication. In contrast, mipomersen, an antisense agent targeted to apolipoprotein B (website: http:// www.fda.gov/newsevents/newsroom/pressannoun cements/ucm337195.htm) [4,5 ,6], has recently received FDA approval for the treatment of familial hypercholesterolemia (though the European Medicines Agency declined approval, citing safety concerns). Why have phase III trials with antisense oligos in human cancers, as opposed to studies targeting the liver, for example, not been successful (see below) when the theory that underlies them is so compelling? Why are our beautiful theories being perpetually slain by inconvenient, ugly facts? This article, in addition to reviewing recent literature &
Department of Medical Oncology and Experimental Therapeutics, City of Hope, Duarte, California, USA Correspondence to Cy A. Stein, Department of Medical Oncology and Experimental Therapeutics, City of Hope, 1500 E. Duarte Rd., Duarte, CA 91010, USA. Tel: +1 626 471 3890; fax: +1 626 471 7322; e-mail: [email protected] Curr Opin Oncol 2014, 26:584–589 DOI:10.1097/CCO.0000000000000127 Volume 26 Number 6 November 2014
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Antisense oligonucleotides in cancer Castanotto and Stein
KEY POINTS Antisense oligonucleotides can be synthesized quickly, inexpensively and are fairly nontoxic. Chemically modified oligonucleotides are metabolically stable and – at least in cells and animal systems – are effective and long lasting. Antisense therapy has wide applications against a broad number of diseases, including cancer. Effective delivery of oligonucleotides to the targeted cells and/or organs is the major barrier to achieving successful therapy for human diseases.
pertinent to the field, will also focus on a discussion of this conundrum, and will provide a way forward.
THERAPEUTIC PHASE III ANTISENSE OLIGONUCLEOTIDE TRIALS IN CANCER Currently, there are approximately 90 ongoing clinical cancer trials evaluating treatment with antisense oligonucleotides (website: https://clinicaltrials. gov/ct2/results?term=antisense+and+cancer&Search =Search), given with or without cytotoxic chemotherapy. The majority of these trials are phase I/II; for the most part, they demonstrated oligo safety and tolerability. However, only a few antisense oligos have been evaluated in phase III cancer trials, in which results have been meager at best. Custirsen (OGX-011, website: http://www. oncogenex.com/physicians/custirsen-ogx-011) is an antisense oligo targeted to clusterin, a protein implicated in prostate cancer cell survival and drug resistance. Developed by Isis in partnership with Teva/ Oncogenex (Bothell, Washington, USA), this anticlusterin mRNA oligo produced promising results in a phase II trial in castrate resistant prostate cancer (CRPC) in combination with docetaxel þ prednisone [7,8]. However, no phase III trial of any of approximately 13 different agents has ever improved overall survival (OS) versus standard docetaxel, and neither did custirsen: no significant increase in OS was demonstrated in CRPC patients (website: http://ir.onco genex.com/releasedetail.cfm?ReleaseID=842949). A similar negative outcome was achieved by Eli Lilly (Indianapolis, Indiana, USA) in collaboration with Isis when an oligo designed to silence the antiapoptotic protein survivin was evaluated [9,10]. Further, despite initially successful results in CRPC patients, in a larger follow-up phase II trial, this oligo also failed to demonstrate improvements in progression-free survival or significant prostate-specific antigen (PSA) responses [11]. The clinical development of
this oligo was subsequently suspended (website: http://media.corporate-ir.net/media_files/IROL/22/ 222170/IsisPharm-IR2013/ISIS-IR-2013.html#25/z). Similarly, the oligos Affinitak (website: http://www. prnewswire.com/news-releases/isis-pharmaceuticalsreports-third-quarter-2004-financial-results-and-high lights-75011347.html; Isis and Eli Lilly) and Lucanix (website: http://www.prnewswire.com/news-relea ses/novarx-announces-results-of-lucanix-phase-iiitherapeutic-vaccine-trial-for-maintenance-therapyin-nonsmall-cell-lung-cancer-to-be-presented-atesmo-225329441.html; NovaRx, San Diego, California, USA) reached phase III trials, but failed to show improvement in OS in nonsmall cell lung cancer patients, although Lucanix demonstrated some clinical benefit in a subset of trial patients. Another antisense oligo deemed to be promising was Geron’s (Menlo Park, California, USA) Imetelstat (GRN163L), designed to inhibit telomerase by binding to the RNA at the active site of the enzyme. However, in March 2014, GRN163L was placed on full clinical hold by the FDA because of the adverse events and hepatotoxicity observed during the clinical trial (website: http://www.geron.com/imetelstat). Several years ago, a large multicenter trial of oblimersen (also known as G3139 or Genasense), targeted to the B-cell lymphoma 2 (Bcl 2) mRNA in combination with dacarbazine, was successful in a phase III clinical trial versus single agent dacarbazine in advanced melanoma patients [12,13]. However, in a subsequent phase III clinical trial in a similar patient cohort, the positive results of the initial trial could not be reproduced, leading to cessation of further clinical investigation of this antisense agent, as previous clinical trials for other indications (e.g., chronic lymphocytic leukemia [14]) were also unsuccessful. Antisense Pharma (now Isarna Therapeutics, Munich, Germany) halted their phase III trial of trabedersen for glioma due to poor recruitment and the serious adverse events associated with the local mode of administration of the oligo (website: http:// clinicaltrials.gov/ct2/show/record/NCT00761280? term=Trabedersen+phase+3&rank=1) (website: http://www.b3cnewswire.com/20130226844/antise nse-pharma-announces-revised-development-pathfor-trabedersen.html). Finally, another recent clinical trial failure came from a large multicenter phase 1a/1b study of an antisense oligo targeting the androgen receptor (AR), a driver protein in metastatic CRPC. This oligo produced almost no meaningful changes in PSA expression in treated patients, no soft tissue responses and no significant AR depletion in biopsied tumor deposits [15 ]. However, in contrast to what has happened when oligos have been used to treat malignant disease, interesting
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results have been attained in an early clinical trial in Crohn’s disease with an antisense oligo targeting Smad-7 [16,17], and with Gene Signal-101 (Aganirsen), an oligo developed by Gene Signal to aid in corneal graft rejection [18 ], in addition to the results with mipomersen mentioned above. &
DELIVERY OF ANTISENSE PHOSPHOROTHIOATE OLIGONUCLEOTIDES Essentially, all antisense oligos that have entered clinical trials for a cancer indication have a phosphorothioate backbone. Here, one of the nonbridging oxygen atoms at phosphorus is replaced by a sulfur atom. However, the net charge on the oligo is not changed. Thus, a phosphorothioate oligo of 18 mer length bears 17 negative charges and is highly hydrophilic. However, the lipid bilayers of the cell membrane are highly hydrophobic. For decades, it has been unclear how a hydrophilic phosphorothioate oligo can pass through a hydrophobic cell membrane [in fact, they can, in a process known as gymnosis, if locked nucleic acids (LNAs) are placed at the 30 and 50 termini of the oligo [19]]. As a result, numerous efforts have been made to encapsulate phosphorothioate oligos with materials ranging from cationic lipids to dendrimers to alginate/chitosan nanoparticles [20], to a sub-50 nm nanocapsule containing tenfibgen, the carboxyterminal fibrinogen globe domain of tenascin-C [21,22], among others (note that the gymnosis process requires no carriers or conjugations). To date, however, none of these approaches has been clinically successful in cancer, and will not be further discussed.
NONSPECIFIC BIOLOGICAL PROPERTIES OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES The relatively straightforward substitution of sulfur for oxygen at phosphorus creates vital nuclease stability (although probably not enough of it). However, it also creates a molecule that is very different from the parental phosphodiester oligo. In general, phosphorothioate oligos in duplexes with their complementary mRNA targets have significantly lower melting temperatures than their isosequential phosphodiester analogs. Furthermore, independent of sequence, all phosphorothioate oligos, due to their negative charge, share physicochemical properties with heparin and other polyanionic glycosaminoglycans [23]. Phosphorothioate oligos, such as heparin: first, release fibroblast growth factor-2 (FGF2) bound to extracellular matrix on low-affinity 586
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binding sites; second, potentiate the binding of FGF2 to its FGFR1 domain IIIc cell surface receptor; third, protect the air oxidation and enzymatic digestion of FGF2; fourth, stimulate mitogenesis and tubular morphogenesis of human microvascular endothelial cells in three-dimensional collagen-1 gels; fifth, promote human umbilical vein endothelial cell growth; and sixth, promote the sprouting of microvessels from the surface of cut rat aortic rings in culture media. In addition to FGF2 [23], there are many heparin-binding proteins that are also known to bind phosphorothioate oligos with nanomolar affinity. These include CD4, platelet-derived growth factor BB homodimer [23], laminin and fibronectin [24], the B-lymphocyte cell surface integrin aMb2 (a.k.a. Mac-1 [25]), the mitochondrial voltage-dependent anion channel [26], the Ago2 PAZ (Piwi, Argonaute, Zwille) domain and the receptor for advanced glycation end products [27 ]. This list is only partial; all possible heparin-binding cell surface proteins, which also bind phosphorothioate oligo, have not yet been identified. However, in the absence of a carrier, binding to the cell surface is critical for phosphorothioate oligo uptake in cancer cells and eventual gene silencing. For example, in contrast to phosphorothioate oligos, siRNAs do not adsorb well to the cell surface, as their interbase phosphodiester linkages have insufficient affinity for cell surface heparin-binding proteins. Hence, these molecules must be delivered to cells with a carrier or be aided with a conjugate. In cancer cells, it has never been demonstrated that internalization of phosphorothioate oligos occurs in a manner other than by a combination of adsorptive endocytosis and pinocytosis (fluid phase endocytosis [28]). &
IMPROVED OLIGONUCLEOTIDE DELIVERY BY RETRO-1 In an attempt to increase phosphorothioate oligo silencing activity, Ming et al. [29 ] identified a small molecule known as Retro-1 which increased the exon skipping ability of a phosphorothioate 20 -Omethyl antisense gapmer and led to increased expression of EGFP in reporter-transfected HeLa cells. However, fairly high concentrations (tens of micromolar) were required for maximal effect. Exon skipping resulting in alternative splicing of Bcl-xL (to Bcl-xS) was also promoted by Retro-1 in tissue culture to some extent. In NIH-3T3 cells engineered to overexpress Pgp (multidrug resistance protein 1; ATP binding cassette B 1), Retro-1 promoted gene silencing by the AS 20 -O-methyl phosphorothioate gapmer, but as above, high concentrations of Retro1 were required. Increased silencing was associated &
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Antisense oligonucleotides in cancer Castanotto and Stein
with increased nuclear localization of the antisense oligo. Significantly, Retro-1 treatment of scid mice bearing human melanoma xenografts led to splice correction in a GFP reporter plasmid in these tumors as assessed by reverse transcription polymerase chain reaction. The cost was stated to consist of only minimal hepatic toxicity.
SMALL CHANGES IN THE SEQUENCE OF A LOCKED NUCLEIC ACID OLIGONUCLEOTIDE MAY PRODUCE DRAMATIC BIOLOGICAL EFFECTS Over the years, it has been observed that the phosphorothioate backbone, rather than acting as a basis for a platform technology, may produce idiosyncratic antisense oligo behavior in tissue culture and with respect to animal toxicity. This behavior is exquisitely dependent on oligo sequence, independent of any Watson–Crick interactions with its complementary mRNA, and is particularly pronounced when LNAs are incorporated into the 30 and 50 molecular termini. For example, Hagedorn et al. [30 ] noted that an antisense 13mer phosphorothioate LNA gapmer targeting PTEN, called seth [31], produced extensive hepatotoxicity, characterized by elevations in ALT in a 16-day mouse model. Extension of the oligo length by one base, to a 14 mer, dramatically reduced liver toxicity. The authors demonstrated that a random Forest algorithm could successfully predict the toxicity patterns of a series of antisense phosphorothioate LNA gapmers. In other experiments demonstrating that very small changes can affect the behavior of these oligos, Koch et al. [32 ] synthesized a series of antisense phosphorothioate LNA gapmers targeted to Bcl-2 that incorporated a single-base modification in the middle of the gap. Every oligo silenced Bcl-2 protein expression to the same extent. After gymnotic delivery of the oligos to 518A2 melanoma cells, the phosphorylation/dephosphorylation patterns of proteins of the PI3K/Akt/mTOR pathway were examined by western blotting. Each and every oligo produced a different pattern. &&
&&
QUANTUM MECHANICAL MODELING OF SHORT OLIGONUCLEOTIDES The mechanism of this so called property diversity of phosphorothioate LNA oligos is not well understood [32 ], but an interesting attempt has been made through quantum mechanical modeling to propose a basis for it. Briefly, the energies and atomic distributions of the frontier molecular orbitals of the oligo were calculated from an elaboration of the Schrodinger wave function. These orbitals, &&
also known as the highest occupied molecular orbitals, are responsible for the nonspecific electrostatic interactions of oligos with non-Watson–Crick targets, that is, proteins, as above. In essence, quantum mechanical modeling of an all-LNA trimer (50 -AAG30 ) demonstrates that the substitution of phosphorothioate for phosphodiester induces major changes in the molecular localization of the frontier orbitals, with a shift of the electrostatic potential to the 50 molecular terminus. The effect of a single-base mutation also produced major shifts in highest occupied molecular orbital localization. Stereochemistry at phosphorus (Rp versus Sp enantiomers) also significantly altered frontier orbital topology. Extension of the trimer oligo to a pentamer (50 -AAGAC-30 ) changed the electrostatic potential from being concentrated around the central and 50 adenosines to being more localized toward the 30 terminus. The electrostatic density on the 50 adenines was diminished. For a biologically active 7mer (all phosphodiester DNA versus all phosphodiester LNA; 50 -ATGTAGC-30 ) that targets the seed region of miR-221 and miR-222, the LNA structure was determined by quantum mechanical modeling to be bent, whereas the DNA structure was linear. A similar study on an identical sequence (an all-phosphodiester DNA-LNA gapmer with three DNA nucleotides in the gap) showed that this structure was also bent and almost ‘globular.’ This shape was ascribed to the increased flexibility of DNA versus LNA, which brought the two LNA termini close to each other. This fascinating study from first principles is reminiscent of observations made over the years when cells or organisms were treated with a series of minimally different phosphorothioate LNA oligos, and may help explain the ‘property diversity’ observed for phosphorothioate LNA gapmers. However, a cautionary note must be introduced, as the oligos evaluated in this study were much shorter than those which are likely to be clinically useful.
BARRIERS TO IN-VIVO OLIGONUCLEOTIDE EFFICACY What are the barriers to in-vivo oligo efficacy? A list has been compiled by Juliano et al. [33]; most of the problems he cites have been overcome by chemical modifications (e.g., 20 -methoxyethyl, a.k.a. MOE, LNA) that have dramatically improved oligo pharmacokinetics and pharmacodynamics. However, in our opinion, for antitumor efficacy, two hitherto unaddressed problems must be considered. They are as follows: first, binding of phosphorothioate oligos to extracellular matrix. These molecules bind with exceptionally high affinity to collagen I (Kd approximately 400 pM), which is found in the matrix of
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many if not most tumors, and to other matrix proteins (e.g., laminin, fibronectin) as well and second, inefficient endocytosis by tumor cells and release from endosomes, a critical issue as it appears that oligo uptake in tumor cells is entirely dependent on endocytosis in one form or another [28]. This is not the case in normal renal brush border cells or in cells in the central nervous system, in which a nucleic acid channel exists through which phosphorothioate oligos can transit [34,35].
CANCER CELL HYPOXIA MAY IMPACT OLIGONUCLEOTIDE DELIVERY The vascular nonhomogeneity of tumors presents a unique and challenging problem with respect to oligo uptake and gene silencing. As has been described [36], macroscopic tumors easily outgrow their blood supply, leading to tumor hypoxia. Hypoxia, in turn, causes the production of hypoxia-inducible factor 1-alpha and similar factors, which upregulate the production of proangiogenic factors, such as vascular endothelial growth factor. However, neovessels produced under the influence of vascular endothelial growth factor are fenestrated and hence leaky. Intravascular fluid, plasma proteins, and it is reasonable to assume small molecules such phosphorothioate oligos, will leak out of the vessels into the intracellular matrix. This leakage problem is most likely augmented by the high affinity of phosphorothioate oligos for matrix proteins. Subsequently, after leaking out of neovessels, the oligos would be cleared by lymphatics or postcapillary venules, bypassing the tumor cells entirely. Moreover, as leakage out of the vasculature occurs, red cells sludge, increasing tumor hypoxia and producing diminished intratumoral pH. Cells cease cycling, and endocytosis slows or stops entirely. But as tumor cells appear to be entirely dependent on endocytosis for oligo uptake, their efficacy is strongly vitiated.
CONCLUSION Antisense oligonucleotides have been evaluated in the clinic for cancer indications for many years, but thus far, no drug has been FDA approved. That is because, as detailed above, this very promising therapeutic field is trapped in a vicious circle instigated by very well protected tumor cells. Our lack of understanding oligo delivery to tumors in vivo has resulted in the equivocal phase III clinical trial results observed with not only antisense oligos, but with all types of oligonucleotide strategies. Success will come, we contend, not by repeating previously unsuccessful experiments, but only when 588
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these complex problems are understood and overcome. This, in turn, can only be brought about through meticulous scientific research. Acknowledgements None. Conflicts of interest The authors report no financial conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Paterson B, Roberts B, Kuff E. Structural gene identification and mapping by DNA-mRNA hybrid arrested cell free translation. Proc Natl Acad Sci U S A 1977; 74:4370–4374. 2. Zamecnik P, Stephenson ML. Inhibiton of Rous sarcoma virus replication and transformation by aspecifric oligodeoxynucleotide. Proc Natl Acad Sci U S A 1978; 75:280–284. 3. Piascik P. Fomiversen sodium approved to treat CMV retinitis. J Am Pharm Assoc (Wash) 1999; 39:84–85. 4. Hovingh K, Besseling J, Kastelein J. Efficacy and safety of mipomersen sodium (Kynamro). Expert Opin Drug Saf 2013; 12:568–579. 5. Crooke S, Geary R. Clinical pharmacological properties of mipomersen & (Kynamro), a second generation antisense inhibitor of apolipoprotein B. Br J Clin Pharmacol 2013; 76:269–276. This is an interesting article on the first approved systemic antisense therapeutic agent. 6. Gebhard C, Huard G, Kritikou E, Tardif J. Apolipoprotein B antisense inhibition: update on mipomersen. Curr Pharm Des 2013; 19:3132–3142. 7. Saad F, Hotte S, North S, et al. Randomized phase II trial of Custirsen (OGX011) in combination with docetaxel or mitoxantrone as second-line therapy in patients with metastatic castrate-resistant prostate cancer progressing after first-line docetaxel: CUOG trial P-06c. Clin Cancer Res 2011; 17:5765– 5773. 8. Blumenstein B, Saad F, Hotte S, et al. Reduction in serum clusterin is a potential therapeutic biomarker in patients with castration-resistant prostate cancer treated with custirsen. Cancer Med 2013; 2:468–477. 9. Talbot DC, Ranson M, Davies J, et al. Tumor surviving is downregulated by the antisense oligonucleotide LY2181308: a proof-of-concept, first-in-human dose study. Clin Cancer Res 2010; 16:6150–6158. 10. Erba HP, Sayar H, Juckett M, et al. Safety and pharmacokinetics of the antisense oligonucleotide (ASO) LY2181308 as a single-agent or in combination with idarubicin and cytarabine in patients with refractory or relapsed acute myeloid leukemia (AML). Invest New Drugs 2013; 4:1023–1034. 11. Wiechno P, Somer BG, Mellado B, et al. A randomised phase 2 study combining LY2181308 sodium (survivin antisense oligonucleotide) with first-line docetaxel/prednisone in patients with castration-resistant prostate cancer. Eur Urol 2014; 65:516–520. 12. Agarwala S, Keilholz U, Gilles E, et al. LDH correlation with survival in advanced melanoma from two large, randomized trials (Oblimersen GM301 and EORTC 18951). Eur J Cancer 2009; 45:1807–1814. 13. Bedikian A, Millward M, Pehamberger H, et al. Bcl-2 antisense (oblimersen sodium) plus dacarbazine in patients with advanced melanoma: the Oblimersen Melanoma Study Group. J Clin Oncol 2006; 24:4738–4745. 14. Cheson B. Oblimersen for the treatment of patients with chronic lymphocytic leukemia. Ther Clin Risk Manag 2007; 3:855–870. 15. Bianchini D, Omlin A, Pezaro C, et al. First-in-human phase I study of EZN& 4176, a locked nucleic acid antisense oligonucleotide to exon 4 of the androgen receptor (AR) mRNA in patients with castration-resistant prostate cancer. Br J Cancer 2013; 109:2579–2586. This article describes the not untypical results observed after systemic delivery of an antisense oligonucleotide. 16. Zorzi F, Angelucci E, Sedda S, et al. Smad7 antisense oligonucleotide-based therapy for inflammatory bowel diseases. Dig Liver Dis 2013; 45:552–555. 17. Monteleone C, Fantini M, Onali S, et al. Phase I clinical trial of Smad7 knockdown using antisense oligonucleotide in patients with active Crohn’s disease. Mol Ther 2012; 20:870–876. 18. Cursiefen C, Viaud E, Bock F, et al. Aganirsen antisense aligonucleotide eye & drops inhibit keratitis-induced corneal neovascularization and reduce need for transplantation: the I-CAN study. Ophthalmology 2014; 121:1683–1692. This article describes an interesting novel use for antisense oligonucleotides, although not for a cancer indication.
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Antisense oligonucleotides in cancer Castanotto and Stein 19. Stein C, Hansen B, Lai J, et al. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res 2010; 38:e3. 20. Borges O, Cordeiro-da-Silva A, Tavares J, et al. Immune response by nasal delivery of hepatitis B surface antigen and codelivery of a CpG ODN in alginate coated chitosan nanoparticles. Eur J Pharm Biopharm 2008; 69:405–416. 21. Trembley J, Unger G, Tobolt D, et al. Systemic administration of antisense oligonucleotides simultaneously targeting CK2alpha and alpha’ subunits reduces orthotopic xenograft prostate tumors in mice. Mol Cell Biochem 2011; 356:21–35. 22. Trembley J, Wang G, Unger G, et al. Proteins kinase CK2 in health and disease: CK2: a key player in cancer biology. Cell Mol Life Sci 2009; 66:1858–1867. 23. Stein C, Wu S, Voskresenskiy A, et al. G3139, an anti-Bcl-2 antisense oligomer that binds heparin-binding growth factors and collagen I, alters in-vitro endothelial cell growth and tubular morphogenesis. Clin Cancer Res 2009; 15:2797–2807. 24. Khaled Z, Benimetskaya L, Khan T, et al. Multiple mechanisms may contribute to the cellular antiadhesive effects of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res 1996; 24:737–745. 25. Benimetskaya L, Loike J, Khaled Z, et al. Mac-1 (CD11b/CD18) is a cell surface oligodeoyxnucleotide binding protein. Nat Med 1997; 3:414–420. 26. Lai J, Tan W, Benimetskaya L, et al. A pharmacologic target of G3139 in melanoma cells may be the mitochondrial VDAC. Proc Natl Acad Sci U S A 2006; 103:7494–7499. 27. Sirois C, Jin T, Miller A, et al. RAGE is a nucleic acid receptor that promotes & inflammatory responses to DNA. J Exp Med 2013; 210:2447–2463. This article describes in some detail the characteristics of phosphorothioate oligo binding to a heparin-binding protein. 28. Juliano R, Ming X, Nakagawa O. Cellular uptake and intracellular trafficking of antisense and siRNA oligonucleotides. Bioconjug Chem 2012; 23:147–157.
29. Ming X, Carver K, Fisher M, et al. The small molecule Retro-1 enhances the pharmacological actions of antisense and splice switching oligonucleotides. Nucleic Acids Res 2013; 41:3673–3687. This article describes one of the first, if not the first, examples of a small molecule that increases antisense oligo silencing. 30. Hagedorn P, Yakimov V, Ottosen S, et al. Hepatotoxic potential of therapeutic && oligonucleotides can be predicted from their sequence and modification pattern. Nucleic Acid Ther 2013; 23:302–310. This article demonstrates that the hepatotoxicity of some LNA oligonucleotides is predictable from their sequence, and refutes the notion that this chemical modification has unusually high toxicity. 31. Seth P, Siwkowski A, Allerson C, et al. Short antisense oligonucleotides with novel 20 -40 conformationally restricted nucleoside analogues show improved potency without increased toxicity in animals. J Med Chem 2009; 52:10–13. 32. Koch T, Shim I, Lindow M, et al. Quantum mechanical studies of DNA and && LNA. Nucleic Acid Ther 2014; 24:139–148. This article provides the first data on the quantum mechanical modeling of LNAcontaining oligos, and shows, at least for shorter ones, that the wave function of the highest occupied molecular orbital (the frontier orbital) is delocalized over the entire molecule. The article also demonstrates the striking differences between oligos that are different chemically in only subtle ways. 33. Juliano R, Alam M, Dixit V, Kang H. Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res 2008; 36:4158–4171. 34. Leal-Pinto E, Teixeira A, Tran B, et al. Presence of the nucleic acid channel in renal brush-border membranes: allosteric modulation by extracellular calcium. Am J Physiol Renal Physiol 2005; 289:F97–106. 35. Shi F, Gounko N, Wang X, et al. In situ entry of oligonucleotides into brain cells can occur through a nucleic acid channel. Oligonucleotides 2007; 17:122– 133. 36. Jain R. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005; 307:58–62. &
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Antisense oligonucleotides in cancer Daniela Castanotto and Cy A. Stein
Purpose of review Over the past several dozen years, regardless of the substantial effort directed toward developing rational oligonucleotide strategies to silence gene expression, antisense oligonucleotide-based cancer therapy has not been successful. This review focuses on the most likely reasons for this lack of success, and on the barriers that still need to be overcome to make a clinical cancer treatment reality out of the promise of antisense therapy. Recent findings Considerable progress has been made in the design and delivery of nucleic acid fragments. Chemical modifications have considerably improved oligonucleotide absorption, distribution and metabolism while at the same time reducing toxicity. Nevertheless, the delivery and the cellular uptake of these molecules are still not adequate to provide the desired therapeutic outcome. Recent therapeutic interventional phase III trials of antisense oligodeoxyribonucleotides for a cancer indication will be discussed, in addition to those studies that markedly improve the scientific understanding of the properties of these molecules. Summary We still do not have a marketed antisense oligonucleotide for a cancer indication. This is because critical aspects of the cellular, tumor pharmacology and delivery properties of these agents are still not well understood. Keywords antisense, endocytosis, oligonucleotide, phase III, phosphorothioate
INTRODUCTION Antisense oligodeoxyribonucleotides (oligos) are small pieces of DNA (usually 15–18 mer in length) that are complementary via Watson–Crick base pair hybridization to a targeted mRNA. Intracellular binding of the oligo to its mRNA target is thought to result in cleavage of the mRNA strand by the enzyme RNAse H, an enzyme which cleaves the RNA strand of an RNA-DNA duplex. The mRNA strand is hence no longer available to be translated into its encoded protein. The first cell-free antisense experiments [1] were quickly followed by a study reporting an antisense strategy designed to block gene expression in cells [2]. Interest in the field ramped up dramatically with the introduction of DNA synthesizers in the 1980s. In 1998, Fomiversen [3], an antisense oligo for the treatment of cytomegalovirus retinitis [Isis, Carlsbad, California, USA], which could cause blindness in HIV/AIDS patients, was approved by the Food and Drug Administration (FDA) for marketing. However, the drug was ultimately withdrawn as the indication essentially disappeared. To date, however, despite the expenditure of great amounts of time and money and numerous clinical trials, we still do not have a single antisense www.co-oncology.com
oligo drug that has been approved for a cancer indication. In contrast, mipomersen, an antisense agent targeted to apolipoprotein B (website: http:// www.fda.gov/newsevents/newsroom/pressannoun cements/ucm337195.htm) [4,5 ,6], has recently received FDA approval for the treatment of familial hypercholesterolemia (though the European Medicines Agency declined approval, citing safety concerns). Why have phase III trials with antisense oligos in human cancers, as opposed to studies targeting the liver, for example, not been successful (see below) when the theory that underlies them is so compelling? Why are our beautiful theories being perpetually slain by inconvenient, ugly facts? This article, in addition to reviewing recent literature &
Department of Medical Oncology and Experimental Therapeutics, City of Hope, Duarte, California, USA Correspondence to Cy A. Stein, Department of Medical Oncology and Experimental Therapeutics, City of Hope, 1500 E. Duarte Rd., Duarte, CA 91010, USA. Tel: +1 626 471 3890; fax: +1 626 471 7322; e-mail: [email protected] Curr Opin Oncol 2014, 26:584–589 DOI:10.1097/CCO.0000000000000127 Volume 26 Number 6 November 2014
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Antisense oligonucleotides in cancer Castanotto and Stein
KEY POINTS Antisense oligonucleotides can be synthesized quickly, inexpensively and are fairly nontoxic. Chemically modified oligonucleotides are metabolically stable and – at least in cells and animal systems – are effective and long lasting. Antisense therapy has wide applications against a broad number of diseases, including cancer. Effective delivery of oligonucleotides to the targeted cells and/or organs is the major barrier to achieving successful therapy for human diseases.
pertinent to the field, will also focus on a discussion of this conundrum, and will provide a way forward.
THERAPEUTIC PHASE III ANTISENSE OLIGONUCLEOTIDE TRIALS IN CANCER Currently, there are approximately 90 ongoing clinical cancer trials evaluating treatment with antisense oligonucleotides (website: https://clinicaltrials. gov/ct2/results?term=antisense+and+cancer&Search =Search), given with or without cytotoxic chemotherapy. The majority of these trials are phase I/II; for the most part, they demonstrated oligo safety and tolerability. However, only a few antisense oligos have been evaluated in phase III cancer trials, in which results have been meager at best. Custirsen (OGX-011, website: http://www. oncogenex.com/physicians/custirsen-ogx-011) is an antisense oligo targeted to clusterin, a protein implicated in prostate cancer cell survival and drug resistance. Developed by Isis in partnership with Teva/ Oncogenex (Bothell, Washington, USA), this anticlusterin mRNA oligo produced promising results in a phase II trial in castrate resistant prostate cancer (CRPC) in combination with docetaxel þ prednisone [7,8]. However, no phase III trial of any of approximately 13 different agents has ever improved overall survival (OS) versus standard docetaxel, and neither did custirsen: no significant increase in OS was demonstrated in CRPC patients (website: http://ir.onco genex.com/releasedetail.cfm?ReleaseID=842949). A similar negative outcome was achieved by Eli Lilly (Indianapolis, Indiana, USA) in collaboration with Isis when an oligo designed to silence the antiapoptotic protein survivin was evaluated [9,10]. Further, despite initially successful results in CRPC patients, in a larger follow-up phase II trial, this oligo also failed to demonstrate improvements in progression-free survival or significant prostate-specific antigen (PSA) responses [11]. The clinical development of
this oligo was subsequently suspended (website: http://media.corporate-ir.net/media_files/IROL/22/ 222170/IsisPharm-IR2013/ISIS-IR-2013.html#25/z). Similarly, the oligos Affinitak (website: http://www. prnewswire.com/news-releases/isis-pharmaceuticalsreports-third-quarter-2004-financial-results-and-high lights-75011347.html; Isis and Eli Lilly) and Lucanix (website: http://www.prnewswire.com/news-relea ses/novarx-announces-results-of-lucanix-phase-iiitherapeutic-vaccine-trial-for-maintenance-therapyin-nonsmall-cell-lung-cancer-to-be-presented-atesmo-225329441.html; NovaRx, San Diego, California, USA) reached phase III trials, but failed to show improvement in OS in nonsmall cell lung cancer patients, although Lucanix demonstrated some clinical benefit in a subset of trial patients. Another antisense oligo deemed to be promising was Geron’s (Menlo Park, California, USA) Imetelstat (GRN163L), designed to inhibit telomerase by binding to the RNA at the active site of the enzyme. However, in March 2014, GRN163L was placed on full clinical hold by the FDA because of the adverse events and hepatotoxicity observed during the clinical trial (website: http://www.geron.com/imetelstat). Several years ago, a large multicenter trial of oblimersen (also known as G3139 or Genasense), targeted to the B-cell lymphoma 2 (Bcl 2) mRNA in combination with dacarbazine, was successful in a phase III clinical trial versus single agent dacarbazine in advanced melanoma patients [12,13]. However, in a subsequent phase III clinical trial in a similar patient cohort, the positive results of the initial trial could not be reproduced, leading to cessation of further clinical investigation of this antisense agent, as previous clinical trials for other indications (e.g., chronic lymphocytic leukemia [14]) were also unsuccessful. Antisense Pharma (now Isarna Therapeutics, Munich, Germany) halted their phase III trial of trabedersen for glioma due to poor recruitment and the serious adverse events associated with the local mode of administration of the oligo (website: http:// clinicaltrials.gov/ct2/show/record/NCT00761280? term=Trabedersen+phase+3&rank=1) (website: http://www.b3cnewswire.com/20130226844/antise nse-pharma-announces-revised-development-pathfor-trabedersen.html). Finally, another recent clinical trial failure came from a large multicenter phase 1a/1b study of an antisense oligo targeting the androgen receptor (AR), a driver protein in metastatic CRPC. This oligo produced almost no meaningful changes in PSA expression in treated patients, no soft tissue responses and no significant AR depletion in biopsied tumor deposits [15 ]. However, in contrast to what has happened when oligos have been used to treat malignant disease, interesting
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results have been attained in an early clinical trial in Crohn’s disease with an antisense oligo targeting Smad-7 [16,17], and with Gene Signal-101 (Aganirsen), an oligo developed by Gene Signal to aid in corneal graft rejection [18 ], in addition to the results with mipomersen mentioned above. &
DELIVERY OF ANTISENSE PHOSPHOROTHIOATE OLIGONUCLEOTIDES Essentially, all antisense oligos that have entered clinical trials for a cancer indication have a phosphorothioate backbone. Here, one of the nonbridging oxygen atoms at phosphorus is replaced by a sulfur atom. However, the net charge on the oligo is not changed. Thus, a phosphorothioate oligo of 18 mer length bears 17 negative charges and is highly hydrophilic. However, the lipid bilayers of the cell membrane are highly hydrophobic. For decades, it has been unclear how a hydrophilic phosphorothioate oligo can pass through a hydrophobic cell membrane [in fact, they can, in a process known as gymnosis, if locked nucleic acids (LNAs) are placed at the 30 and 50 termini of the oligo [19]]. As a result, numerous efforts have been made to encapsulate phosphorothioate oligos with materials ranging from cationic lipids to dendrimers to alginate/chitosan nanoparticles [20], to a sub-50 nm nanocapsule containing tenfibgen, the carboxyterminal fibrinogen globe domain of tenascin-C [21,22], among others (note that the gymnosis process requires no carriers or conjugations). To date, however, none of these approaches has been clinically successful in cancer, and will not be further discussed.
NONSPECIFIC BIOLOGICAL PROPERTIES OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES The relatively straightforward substitution of sulfur for oxygen at phosphorus creates vital nuclease stability (although probably not enough of it). However, it also creates a molecule that is very different from the parental phosphodiester oligo. In general, phosphorothioate oligos in duplexes with their complementary mRNA targets have significantly lower melting temperatures than their isosequential phosphodiester analogs. Furthermore, independent of sequence, all phosphorothioate oligos, due to their negative charge, share physicochemical properties with heparin and other polyanionic glycosaminoglycans [23]. Phosphorothioate oligos, such as heparin: first, release fibroblast growth factor-2 (FGF2) bound to extracellular matrix on low-affinity 586
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binding sites; second, potentiate the binding of FGF2 to its FGFR1 domain IIIc cell surface receptor; third, protect the air oxidation and enzymatic digestion of FGF2; fourth, stimulate mitogenesis and tubular morphogenesis of human microvascular endothelial cells in three-dimensional collagen-1 gels; fifth, promote human umbilical vein endothelial cell growth; and sixth, promote the sprouting of microvessels from the surface of cut rat aortic rings in culture media. In addition to FGF2 [23], there are many heparin-binding proteins that are also known to bind phosphorothioate oligos with nanomolar affinity. These include CD4, platelet-derived growth factor BB homodimer [23], laminin and fibronectin [24], the B-lymphocyte cell surface integrin aMb2 (a.k.a. Mac-1 [25]), the mitochondrial voltage-dependent anion channel [26], the Ago2 PAZ (Piwi, Argonaute, Zwille) domain and the receptor for advanced glycation end products [27 ]. This list is only partial; all possible heparin-binding cell surface proteins, which also bind phosphorothioate oligo, have not yet been identified. However, in the absence of a carrier, binding to the cell surface is critical for phosphorothioate oligo uptake in cancer cells and eventual gene silencing. For example, in contrast to phosphorothioate oligos, siRNAs do not adsorb well to the cell surface, as their interbase phosphodiester linkages have insufficient affinity for cell surface heparin-binding proteins. Hence, these molecules must be delivered to cells with a carrier or be aided with a conjugate. In cancer cells, it has never been demonstrated that internalization of phosphorothioate oligos occurs in a manner other than by a combination of adsorptive endocytosis and pinocytosis (fluid phase endocytosis [28]). &
IMPROVED OLIGONUCLEOTIDE DELIVERY BY RETRO-1 In an attempt to increase phosphorothioate oligo silencing activity, Ming et al. [29 ] identified a small molecule known as Retro-1 which increased the exon skipping ability of a phosphorothioate 20 -Omethyl antisense gapmer and led to increased expression of EGFP in reporter-transfected HeLa cells. However, fairly high concentrations (tens of micromolar) were required for maximal effect. Exon skipping resulting in alternative splicing of Bcl-xL (to Bcl-xS) was also promoted by Retro-1 in tissue culture to some extent. In NIH-3T3 cells engineered to overexpress Pgp (multidrug resistance protein 1; ATP binding cassette B 1), Retro-1 promoted gene silencing by the AS 20 -O-methyl phosphorothioate gapmer, but as above, high concentrations of Retro1 were required. Increased silencing was associated &
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Antisense oligonucleotides in cancer Castanotto and Stein
with increased nuclear localization of the antisense oligo. Significantly, Retro-1 treatment of scid mice bearing human melanoma xenografts led to splice correction in a GFP reporter plasmid in these tumors as assessed by reverse transcription polymerase chain reaction. The cost was stated to consist of only minimal hepatic toxicity.
SMALL CHANGES IN THE SEQUENCE OF A LOCKED NUCLEIC ACID OLIGONUCLEOTIDE MAY PRODUCE DRAMATIC BIOLOGICAL EFFECTS Over the years, it has been observed that the phosphorothioate backbone, rather than acting as a basis for a platform technology, may produce idiosyncratic antisense oligo behavior in tissue culture and with respect to animal toxicity. This behavior is exquisitely dependent on oligo sequence, independent of any Watson–Crick interactions with its complementary mRNA, and is particularly pronounced when LNAs are incorporated into the 30 and 50 molecular termini. For example, Hagedorn et al. [30 ] noted that an antisense 13mer phosphorothioate LNA gapmer targeting PTEN, called seth [31], produced extensive hepatotoxicity, characterized by elevations in ALT in a 16-day mouse model. Extension of the oligo length by one base, to a 14 mer, dramatically reduced liver toxicity. The authors demonstrated that a random Forest algorithm could successfully predict the toxicity patterns of a series of antisense phosphorothioate LNA gapmers. In other experiments demonstrating that very small changes can affect the behavior of these oligos, Koch et al. [32 ] synthesized a series of antisense phosphorothioate LNA gapmers targeted to Bcl-2 that incorporated a single-base modification in the middle of the gap. Every oligo silenced Bcl-2 protein expression to the same extent. After gymnotic delivery of the oligos to 518A2 melanoma cells, the phosphorylation/dephosphorylation patterns of proteins of the PI3K/Akt/mTOR pathway were examined by western blotting. Each and every oligo produced a different pattern. &&
&&
QUANTUM MECHANICAL MODELING OF SHORT OLIGONUCLEOTIDES The mechanism of this so called property diversity of phosphorothioate LNA oligos is not well understood [32 ], but an interesting attempt has been made through quantum mechanical modeling to propose a basis for it. Briefly, the energies and atomic distributions of the frontier molecular orbitals of the oligo were calculated from an elaboration of the Schrodinger wave function. These orbitals, &&
also known as the highest occupied molecular orbitals, are responsible for the nonspecific electrostatic interactions of oligos with non-Watson–Crick targets, that is, proteins, as above. In essence, quantum mechanical modeling of an all-LNA trimer (50 -AAG30 ) demonstrates that the substitution of phosphorothioate for phosphodiester induces major changes in the molecular localization of the frontier orbitals, with a shift of the electrostatic potential to the 50 molecular terminus. The effect of a single-base mutation also produced major shifts in highest occupied molecular orbital localization. Stereochemistry at phosphorus (Rp versus Sp enantiomers) also significantly altered frontier orbital topology. Extension of the trimer oligo to a pentamer (50 -AAGAC-30 ) changed the electrostatic potential from being concentrated around the central and 50 adenosines to being more localized toward the 30 terminus. The electrostatic density on the 50 adenines was diminished. For a biologically active 7mer (all phosphodiester DNA versus all phosphodiester LNA; 50 -ATGTAGC-30 ) that targets the seed region of miR-221 and miR-222, the LNA structure was determined by quantum mechanical modeling to be bent, whereas the DNA structure was linear. A similar study on an identical sequence (an all-phosphodiester DNA-LNA gapmer with three DNA nucleotides in the gap) showed that this structure was also bent and almost ‘globular.’ This shape was ascribed to the increased flexibility of DNA versus LNA, which brought the two LNA termini close to each other. This fascinating study from first principles is reminiscent of observations made over the years when cells or organisms were treated with a series of minimally different phosphorothioate LNA oligos, and may help explain the ‘property diversity’ observed for phosphorothioate LNA gapmers. However, a cautionary note must be introduced, as the oligos evaluated in this study were much shorter than those which are likely to be clinically useful.
BARRIERS TO IN-VIVO OLIGONUCLEOTIDE EFFICACY What are the barriers to in-vivo oligo efficacy? A list has been compiled by Juliano et al. [33]; most of the problems he cites have been overcome by chemical modifications (e.g., 20 -methoxyethyl, a.k.a. MOE, LNA) that have dramatically improved oligo pharmacokinetics and pharmacodynamics. However, in our opinion, for antitumor efficacy, two hitherto unaddressed problems must be considered. They are as follows: first, binding of phosphorothioate oligos to extracellular matrix. These molecules bind with exceptionally high affinity to collagen I (Kd approximately 400 pM), which is found in the matrix of
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many if not most tumors, and to other matrix proteins (e.g., laminin, fibronectin) as well and second, inefficient endocytosis by tumor cells and release from endosomes, a critical issue as it appears that oligo uptake in tumor cells is entirely dependent on endocytosis in one form or another [28]. This is not the case in normal renal brush border cells or in cells in the central nervous system, in which a nucleic acid channel exists through which phosphorothioate oligos can transit [34,35].
CANCER CELL HYPOXIA MAY IMPACT OLIGONUCLEOTIDE DELIVERY The vascular nonhomogeneity of tumors presents a unique and challenging problem with respect to oligo uptake and gene silencing. As has been described [36], macroscopic tumors easily outgrow their blood supply, leading to tumor hypoxia. Hypoxia, in turn, causes the production of hypoxia-inducible factor 1-alpha and similar factors, which upregulate the production of proangiogenic factors, such as vascular endothelial growth factor. However, neovessels produced under the influence of vascular endothelial growth factor are fenestrated and hence leaky. Intravascular fluid, plasma proteins, and it is reasonable to assume small molecules such phosphorothioate oligos, will leak out of the vessels into the intracellular matrix. This leakage problem is most likely augmented by the high affinity of phosphorothioate oligos for matrix proteins. Subsequently, after leaking out of neovessels, the oligos would be cleared by lymphatics or postcapillary venules, bypassing the tumor cells entirely. Moreover, as leakage out of the vasculature occurs, red cells sludge, increasing tumor hypoxia and producing diminished intratumoral pH. Cells cease cycling, and endocytosis slows or stops entirely. But as tumor cells appear to be entirely dependent on endocytosis for oligo uptake, their efficacy is strongly vitiated.
CONCLUSION Antisense oligonucleotides have been evaluated in the clinic for cancer indications for many years, but thus far, no drug has been FDA approved. That is because, as detailed above, this very promising therapeutic field is trapped in a vicious circle instigated by very well protected tumor cells. Our lack of understanding oligo delivery to tumors in vivo has resulted in the equivocal phase III clinical trial results observed with not only antisense oligos, but with all types of oligonucleotide strategies. Success will come, we contend, not by repeating previously unsuccessful experiments, but only when 588
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these complex problems are understood and overcome. This, in turn, can only be brought about through meticulous scientific research. Acknowledgements None. Conflicts of interest The authors report no financial conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Paterson B, Roberts B, Kuff E. Structural gene identification and mapping by DNA-mRNA hybrid arrested cell free translation. Proc Natl Acad Sci U S A 1977; 74:4370–4374. 2. Zamecnik P, Stephenson ML. Inhibiton of Rous sarcoma virus replication and transformation by aspecifric oligodeoxynucleotide. Proc Natl Acad Sci U S A 1978; 75:280–284. 3. Piascik P. Fomiversen sodium approved to treat CMV retinitis. J Am Pharm Assoc (Wash) 1999; 39:84–85. 4. Hovingh K, Besseling J, Kastelein J. Efficacy and safety of mipomersen sodium (Kynamro). Expert Opin Drug Saf 2013; 12:568–579. 5. Crooke S, Geary R. Clinical pharmacological properties of mipomersen & (Kynamro), a second generation antisense inhibitor of apolipoprotein B. Br J Clin Pharmacol 2013; 76:269–276. This is an interesting article on the first approved systemic antisense therapeutic agent. 6. Gebhard C, Huard G, Kritikou E, Tardif J. Apolipoprotein B antisense inhibition: update on mipomersen. Curr Pharm Des 2013; 19:3132–3142. 7. Saad F, Hotte S, North S, et al. Randomized phase II trial of Custirsen (OGX011) in combination with docetaxel or mitoxantrone as second-line therapy in patients with metastatic castrate-resistant prostate cancer progressing after first-line docetaxel: CUOG trial P-06c. Clin Cancer Res 2011; 17:5765– 5773. 8. Blumenstein B, Saad F, Hotte S, et al. Reduction in serum clusterin is a potential therapeutic biomarker in patients with castration-resistant prostate cancer treated with custirsen. Cancer Med 2013; 2:468–477. 9. Talbot DC, Ranson M, Davies J, et al. Tumor surviving is downregulated by the antisense oligonucleotide LY2181308: a proof-of-concept, first-in-human dose study. Clin Cancer Res 2010; 16:6150–6158. 10. Erba HP, Sayar H, Juckett M, et al. Safety and pharmacokinetics of the antisense oligonucleotide (ASO) LY2181308 as a single-agent or in combination with idarubicin and cytarabine in patients with refractory or relapsed acute myeloid leukemia (AML). Invest New Drugs 2013; 4:1023–1034. 11. Wiechno P, Somer BG, Mellado B, et al. A randomised phase 2 study combining LY2181308 sodium (survivin antisense oligonucleotide) with first-line docetaxel/prednisone in patients with castration-resistant prostate cancer. Eur Urol 2014; 65:516–520. 12. Agarwala S, Keilholz U, Gilles E, et al. LDH correlation with survival in advanced melanoma from two large, randomized trials (Oblimersen GM301 and EORTC 18951). Eur J Cancer 2009; 45:1807–1814. 13. Bedikian A, Millward M, Pehamberger H, et al. Bcl-2 antisense (oblimersen sodium) plus dacarbazine in patients with advanced melanoma: the Oblimersen Melanoma Study Group. J Clin Oncol 2006; 24:4738–4745. 14. Cheson B. Oblimersen for the treatment of patients with chronic lymphocytic leukemia. Ther Clin Risk Manag 2007; 3:855–870. 15. Bianchini D, Omlin A, Pezaro C, et al. First-in-human phase I study of EZN& 4176, a locked nucleic acid antisense oligonucleotide to exon 4 of the androgen receptor (AR) mRNA in patients with castration-resistant prostate cancer. Br J Cancer 2013; 109:2579–2586. This article describes the not untypical results observed after systemic delivery of an antisense oligonucleotide. 16. Zorzi F, Angelucci E, Sedda S, et al. Smad7 antisense oligonucleotide-based therapy for inflammatory bowel diseases. Dig Liver Dis 2013; 45:552–555. 17. Monteleone C, Fantini M, Onali S, et al. Phase I clinical trial of Smad7 knockdown using antisense oligonucleotide in patients with active Crohn’s disease. Mol Ther 2012; 20:870–876. 18. Cursiefen C, Viaud E, Bock F, et al. Aganirsen antisense aligonucleotide eye & drops inhibit keratitis-induced corneal neovascularization and reduce need for transplantation: the I-CAN study. Ophthalmology 2014; 121:1683–1692. This article describes an interesting novel use for antisense oligonucleotides, although not for a cancer indication.
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Antisense oligonucleotides in cancer Castanotto and Stein 19. Stein C, Hansen B, Lai J, et al. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res 2010; 38:e3. 20. Borges O, Cordeiro-da-Silva A, Tavares J, et al. Immune response by nasal delivery of hepatitis B surface antigen and codelivery of a CpG ODN in alginate coated chitosan nanoparticles. Eur J Pharm Biopharm 2008; 69:405–416. 21. Trembley J, Unger G, Tobolt D, et al. Systemic administration of antisense oligonucleotides simultaneously targeting CK2alpha and alpha’ subunits reduces orthotopic xenograft prostate tumors in mice. Mol Cell Biochem 2011; 356:21–35. 22. Trembley J, Wang G, Unger G, et al. Proteins kinase CK2 in health and disease: CK2: a key player in cancer biology. Cell Mol Life Sci 2009; 66:1858–1867. 23. Stein C, Wu S, Voskresenskiy A, et al. G3139, an anti-Bcl-2 antisense oligomer that binds heparin-binding growth factors and collagen I, alters in-vitro endothelial cell growth and tubular morphogenesis. Clin Cancer Res 2009; 15:2797–2807. 24. Khaled Z, Benimetskaya L, Khan T, et al. Multiple mechanisms may contribute to the cellular antiadhesive effects of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res 1996; 24:737–745. 25. Benimetskaya L, Loike J, Khaled Z, et al. Mac-1 (CD11b/CD18) is a cell surface oligodeoyxnucleotide binding protein. Nat Med 1997; 3:414–420. 26. Lai J, Tan W, Benimetskaya L, et al. A pharmacologic target of G3139 in melanoma cells may be the mitochondrial VDAC. Proc Natl Acad Sci U S A 2006; 103:7494–7499. 27. Sirois C, Jin T, Miller A, et al. RAGE is a nucleic acid receptor that promotes & inflammatory responses to DNA. J Exp Med 2013; 210:2447–2463. This article describes in some detail the characteristics of phosphorothioate oligo binding to a heparin-binding protein. 28. Juliano R, Ming X, Nakagawa O. Cellular uptake and intracellular trafficking of antisense and siRNA oligonucleotides. Bioconjug Chem 2012; 23:147–157.
29. Ming X, Carver K, Fisher M, et al. The small molecule Retro-1 enhances the pharmacological actions of antisense and splice switching oligonucleotides. Nucleic Acids Res 2013; 41:3673–3687. This article describes one of the first, if not the first, examples of a small molecule that increases antisense oligo silencing. 30. Hagedorn P, Yakimov V, Ottosen S, et al. Hepatotoxic potential of therapeutic && oligonucleotides can be predicted from their sequence and modification pattern. Nucleic Acid Ther 2013; 23:302–310. This article demonstrates that the hepatotoxicity of some LNA oligonucleotides is predictable from their sequence, and refutes the notion that this chemical modification has unusually high toxicity. 31. Seth P, Siwkowski A, Allerson C, et al. Short antisense oligonucleotides with novel 20 -40 conformationally restricted nucleoside analogues show improved potency without increased toxicity in animals. J Med Chem 2009; 52:10–13. 32. Koch T, Shim I, Lindow M, et al. Quantum mechanical studies of DNA and && LNA. Nucleic Acid Ther 2014; 24:139–148. This article provides the first data on the quantum mechanical modeling of LNAcontaining oligos, and shows, at least for shorter ones, that the wave function of the highest occupied molecular orbital (the frontier orbital) is delocalized over the entire molecule. The article also demonstrates the striking differences between oligos that are different chemically in only subtle ways. 33. Juliano R, Alam M, Dixit V, Kang H. Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res 2008; 36:4158–4171. 34. Leal-Pinto E, Teixeira A, Tran B, et al. Presence of the nucleic acid channel in renal brush-border membranes: allosteric modulation by extracellular calcium. Am J Physiol Renal Physiol 2005; 289:F97–106. 35. Shi F, Gounko N, Wang X, et al. In situ entry of oligonucleotides into brain cells can occur through a nucleic acid channel. Oligonucleotides 2007; 17:122– 133. 36. Jain R. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005; 307:58–62. &
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