Bo Hansen

Knockdown of survivin (BIRC5) causes apoptosis in neuroblastoma via mitotic catastrophe

BIRC5 (survivin) is one of the genes located on chromosome arm 17q in the region that is often gained in neuroblastoma. BIRC5 is a protein in the intrinsic apoptotic pathway that interacts with XIAP and DIABLO leading to caspase-3 and caspase-9 inactivation. BIRC5 is also involved in stabilizing the microtubule-kinetochore dynamics. Based on the Affymetrix mRNA expression data, we here show that BIRC5 expression is strongly upregulated in neuroblastoma compared with normal tissues, adult malignancies, and non-malignant fetal adrenal neuroblasts. The over-expression of BIRC5 correlates with an unfavorable prognosis independent of the presence of 17q gain. Silencing of BIRC5 in neuroblastoma cell lines by various antisense molecules resulted in massive apoptosis as measured by PARP cleavage and FACS analysis. As both the intrinsic apoptotic pathway and the chromosomal passenger complex can be therapeutically targeted, we investigated in which of them BIRC5 exerted its essential anti-apoptotic role. Immunofluorescence analysis of neuroblastoma cells after BIRC5 silencing showed formation of multinucleated cells indicating mitotic catastrophe, which leads to apoptosis via P53 and CASP2. We show that BIRC5 silencing indeed resulted in activation of P53 and we could rescue apoptosis by CASP2 inhibition. We conclude that BIRC5 stabilizes the microtubules in the chromosomal passenger complex in neuroblastoma and that the apoptotic response results from mitotic catastrophe, which makes BIRC5 an interesting target for therapy.

Biological activity and biotechnological aspects of locked nucleic acids

Locked nucleic acid (LNA) is one of the most promising new nucleic acid analogues that has been produced under the past two decades. In this chapter, we have tried to cover many of the different areas, where this molecule has been used to improve the function of synthetic oligonucleotides (ONs). The use of LNA in antisense ONs, including gapmers, splice-switching ONs, and siLNA, as well as antigene ONs, is reviewed. Pharmacokinetics as well as pharmacodynamics of LNA ONs and a description of selected compounds in, or close to, clinical testing are described. In addition, new LNA modifications and the adaptation of enzymes for LNA incorporation are reviewed. Such enzymes may become important for the development of stabilized LNA-containing aptamers.

Silencing of gene expression by gymnotic delivery of antisense oligonucleotides.

Antisense oligodeoxyribonucleotides have been used for decades to achieve sequence-specific silencing of gene expression. However, all early generation oligonucleotides (e.g., those with no other modifications than the phosphorothioate backbone) are inactive in vitro unless administered using a delivery vehicle. These delivery vehicles are usually lipidic but can also be polyamines or some other particulate reagent. We have found that by employing locked nucleic acid (LNA) phosphorothioate gap-mer nucleic acids of 16 mer or less in length, and by carefully controlling the plating conditions of the target cells and duration of the experiment, sequence-specific gene silencing can be achieved at low micromolar concentrations in vitro in the absence of any delivery vehicle. This process of naked oligonucleotide delivery to achieve gene silencing in vivo, which we have termed gymnosis, has been observed in many both adherent and nonadherent cell lines against several different targets genes.

Aurora kinases in childhood acute leukemia: the promise of aurora B as therapeutic target.

We investigated the effects of targeting the mitotic regulators aurora kinase A and B in pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Aurora protein expression levels in pediatric ALL and AML patient samples were determined by western blot and reverse phase protein array. Both kinases were overexpressed in ALL and AML patients (P<0.0002), especially in E2A-PBX1-translocated ALL cases (P<0.002), compared with normal bone-marrow mononuclear cells. Aurora kinase expression was silenced in leukemic cell lines using short hairpin RNAs and locked nucleic acid-based mRNA antagonists. Aurora B knockdown resulted in proliferation arrest and apoptosis, whereas aurora A knockdown caused no or only minor growth delay. Most tested cell lines were highly sensitive to the AURKB-selective inhibitor barasertib–hydroxyquinazoline–pyrazol–anilide (AZD1152-HQPA) in the nanomolar range, as tested with an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. But most importantly, primary ALL cells with a high aurora B protein expression, especially E2A-PBX1-positive cases, were sensitive as well. In adult AML early clinical trials, clear responses are observed with barasertib. Here we show that inhibition of aurora B, more than aurora A, has an antiproliferative and pro-apoptotic effect on acute leukemia cells, indicating that particularly targeting aurora B may offer a new strategy to treat pediatric ALL and AML.

The synergism of MCL1 and glycolysis on pediatric acute lymphoblastic leukemia cell survival and prednisolone resistance

In vitro and in vivo resistance to prednisolone are predictive for an adverse prognosis in pediatric precursor B-acute lymphoblastic leukemia. Causes of resistance are still poorly understood. In this study, we observed that prednisolone exposure of prednisolone-sensitive patients’ leukemic cells decreased anti-apoptotic MCL1 protein levels by 2.9-fold, while MCL1 protein expression in prednisolone-resistant leukemic patients’ cells was unaffected (P<0.01). Locked nucleic acid oligonucleotides directed against MCL1 reduced MCL1 protein levels by 82±16% (P<0.05) in leukemic cells, decreased proliferation by 9-fold and sensitized to prednisolone up to 80.8-fold, compared to a non-silencing-control locked nucleic acid (P<0.05). Remarkably, we discovered that MCL1-silencing up-regulated the glucose consumption of leukemic cells by 2.5-fold (P<0.05), suggesting a potential rescue mechanism mediated by glycolysis. Targeting glycolysis by 2-deoxyglucose synergistically inhibited leukemic survival by 23.2-fold in MCL1-silenced cells (P<0.05). Moreover, 2-deoxyglucose and MCL1 locked nucleic acid concomitantly sensitized leukemic cells to prednisolone compared to MCL1 locked nucleic acid or 2-deoxyglucose alone (P<0.05). In conclusion, these results indicate the need to target both MCL1 and glycolysis simultaneously to inhibit leukemic survival and sensitize acute leukemia patients towards prednisolone.

Chapter 5:Locked Nucleic Acid: Properties and Therapeutic Aspects

In 1978 Zamecnik and Stephenson1 showed for the first time that messenger RNA (mRNA) repression could be achieved by single-stranded oligonucleotides (ONs). This mechanistic approach for gene inhibition was later called the antisense (AS) principle. The simplicity of this new principle captivated ma...

Abstract SY31-02: Locked nucleic acid (LNA)-modified antisense oligonucleotides as anticancer agents: Using high-affinity antisense molecules in the laboratory and in the clinic

The inhibition of cellular processes associated with the malignant pheonotype is the goal many oncolytics. In the antisense oligonucleotide (ASO) approach, Watson and Crick base-pairing rules serve as the basis of rational drug design to create single-stranded oligonucleotides that are complementary and bind to RNAs critical for the malignant phenotype. The targets for antisense approaches can be mRNAs that encode for disease related proteins like those that control tumor growth, cell division, or survival. More recently, RNA targets in oncology have been expanded to include microRNAs. A successful oncolytic oligonucleotide must effectively bind and inhibit a target mRNA or miRNA that is critical for the malignant transformation or survival. Target identification presents the same challenge in antisense therapeutics as any other class of oncolytics, but drug design is based the known sequence of the target RNA. The first clinical trials to use antisense oligonucleotides in oncology produced only modest efficacy in clinical trials, but the therapeutic potential of these initial antisense drugs may have been hampered by their low stability and low binding affinity. Advancements in oligonucleotide chemistry have produced oligonucleotides with increased stability and increased affinities. This talk will focus on the use of oligonucleotides with the locked nucleic acid (LNA) modification. In these antisense constructs, some of the nucleotides in the sequence have a modification of the ribose sugar that includes a methylene bridge between the 2’ and 4’ positions. This bridge functions to lock the ribose into a (C3’-endo) configuration: a configuration that is optimized for binding to its cognate nucleotide. LNA-modified oligonucleotides bind to their target RNAs with higher affinities than most other oligonucleotide chemistries. One measure of affinity is the melting temperature (T m ) for binding to its complementary sequence. For each LNA-modified nucleotide added to an ASO sequence, T m can increase T m by over 5°. For example, an LNA-modified oligonucleotide currently in clinical development, miravirsen (SPC3649), with 9 LNA-modified nucleotides has a T m of approximately 80° demonstrating that LNA-modified oligonucleotides have sufficient affinities to bind to and inhibit RNA function. Affinities like these have translated into increase potency for the inhibition of target RNAs in nonclinical models and should yield therapeutic benefit more robust than those seen with earlier generations of oligonucleotide therapeutics. Other drug-like properties of antisense therapeutics have also been improved by including LNA-modified nucleotides. For example, the pharmacokinetic properties of antisense oligonucleotides support their use in clinical medicine. Antisense oligonucleotides are single stranded and bind to proteins in circulation and on cell surfaces. After parenteral administration, antisense oligonucleotide are bound first to plasma proteins, limiting glomerular filtration, and then later the antisense oligonucleotides appear bound to cell surface (proteins), allowing them to be internalized in cells. Delivery into cells and tissues is achieved without the need for formulations more complex than just aqueous solutions. LNA modifications increase the metabolic stability in both circulation and inside of cells resulting in stable tissue concentrations and prolonged activities. This long tissue residence and stability, allows for constant exposure in tumor cells with dosing as infrequent as weekly or fortnightly. Again, this represents an improvement over antisense drugs used in previous oncology trials. Using in vitro and in vivo laboratory models it has been possible to demonstrate that treatment with LNA-modified oligonucleotides produces reductions in target gene expression and ultimately tumor growth that are sequence-, concentration-, and duration-of-therapy-dependent. Taken together the properties of LNA-modified oligonucleotides make them excellent candidates for inhibiting key RNA targets and at this time there are multiple LNA-modified oligonucleotides in clinical trials in oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY31-02. doi:10.1158/1538-7445.AM2011-SY31-02

Targeting sphingosine kinase 1 with LNA oligonucleotides in gastric cancer.

Background Gastric cancer is the fourth most common cancer worldwide. Despite advances in diagnosis of gastric cancer, the prognosis at advanced stage of disease with the current chemotherapeutic treatment strategies remains poor. Hence, new agents and molecular targets for gastric cancer therapy are desperately needed. Sphingosine kinase 1 represents a promising novel target for anti-cancer therapy. However, the most common used small molecule inhibitors of SphK are unspecific inhibitors of SphK1. Here we investigated the effect of targeted downregulation of SphK1 by locked nucleic acid antisense oligonucleotides (LNA-ASO) in gastric cancer cell lines.