Frank C. Bennett

Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.

Effect of dose and plasma concentration on liver uptake and pharmacologic activity of a 2′-methoxyethyl modified chimeric antisense oligonucleotide targeting PTEN

The role of dose and plasma concentration on liver tissue uptake and resulting antisense pharmacology using a chemically modified antisense oligonucleotide (ASO) targeting PTEN was assessed in mice. A single bolus s.c. dose of 60 mg/kg in mice showed a time-dependent reduction in liver PTEN mRNA that was maximal at 48-72 h and returned to near control levels by 20 days after administration. These pharmacodynamics are in good agreement with liver concentrations of ASO and are consistent with slow elimination (t(1/2)=8 days) of the PTEN ASO from Balb/C mouse liver. As expected, highest ASO concentrations in liver resulted from the s.c. slow infusion at all doses tested. Unexpectedly, the liver EC(50) for the 24-h s.c. slow infusion was approximately twofold higher than the two bolus routes of administration. Based on plasma concentration analysis it appears that 1-2 microg/mL ASO plasma concentration is a threshold that, if exceeded, results in robust antisense effects and below which there is reduced or complete loss of antisense pharmacology in liver even though bulk uptake in the organ is improved. Co-administration of a nonsense ASO competed for liver uptake, but unexpectedly increased pharmacodynamic response for the active oligonucleotide (ISIS 116847) supporting inhibition of a nonproductive bulk uptake pathway while simultaneously improving productive uptake (pharmacodynamics). This competition effect was similar whether the nonsense oligonucleotide was co-administered with ASO or administered up to 24 h prior to active ASO injection.

Intracerebral Infusion of Antisense Oligonucleotides Into Prion-infected Mice

Mice deficient for the cellular prion protein (PrPC) do not develop prion disease; accordingly, gene-based strategies to diminish PrPC expression are of interest. We synthesized a series of chemically modified antisense oligonucleotides (ASOs) targeted against mouse Prnp messenger RNA (mRNA) and identified those that were most effective in decreasing PrPC expression. Those ASOs were also evaluated in scrapie-infected cultured cells (ScN2a) for their efficacy in diminishing the levels of the disease-causing prion protein (PrPSc). When the optimal ASO was infused intracerebrally into FVB mice over a 14-day period beginning 1 day after infection with the Rocky Mountain Laboratory (RML) strain of mouse prions, a prolongation of the incubation period of almost 2 months was observed. Whether ASOs can be used to develop an effective therapy for patients dying of Creutzfeldt–Jakob disease remains to be established.

Pharmacokinetic and Pharmacodynamic Investigations of ION-353382, a Model Antisense Oligonucleotide: Using Alpha-2-Macroglobulin and Murinoglobulin Double-Knockout Mice

To investigate the pharmacokinetics (PKs) and pharmacodynamics (PDs) for ION-353382, an antisense oligonucleotide (ASO) targeting scavenger receptor class B type I (SRB1) mRNA, using alpha-2-macroglobulin (A2M), murinoglobulin double-knockout (DKO), and wild-type mice. Wild-type and DKO homozygous mice were administered a single subcutaneous injection of ION-353382 at 0, 5, 15, 30, and 60 mg/kg. Mice were sacrificed at 72 h with plasma and organs harvested. Both liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA) were used to determine ASO exposure with real-time PCR for SRB1 expression. Immunohistochemistry was evaluated to explore hepatic uptake of ASOs. The total plasma protein binding and profiling was assessed. Finally, two-dimensional gel electrophoresis identified protein expression differences. PK exposures were comparable between wild-type and DKO mice in plasma, liver, and kidney, yet a near twofold reduction in EC50 was revealed for DKO mice based on an inhibitory effect liver exposure response model. Total plasma protein binding and profiling revealed no major dissimilarities between both groups. Plasma proteome fingerprinting confirmed protein expression variations related to A2M. Histological examination revealed enhanced ASO distribution into hepatocytes and less nonparenchymal uptake for DKO mice compared to wild-type mice. Knocking out A2M showed improved PD activities without an effect on total plasma and tissue exposure kinetics. Binding to A2M could mediate ASOs to nonproductive compartments, and thus, decreased binding of ASOs to A2M could potentially improve ASO pharmacology.

Abstract 3302: Antisense oligonucleotide depletion of the mitotic kinesin Eg5 by direct delivery to the brain could be a useful strategy for treating glioma tumors.

To date, all approved chemotherapeutic agents which target the mitotic cell division interfere with spindle microtubule dynamics, leading to mitotic arrest and apoptosis. While effective, these drugs are subject to resistance mechanisms and they are also associated with a variety of side effects, including neurotoxicity. Their use in treating nervous system tumors is therefore not warranted. One strategy to target mitosis, without damaging microtubules in non-dividing neurons, would be to inhibit key mitotic components, such as the mitotic kinesin Eg5, which is required for establishing a normal bipolar mitotic spindle. We have shown that glioblastoma cells depleted for Eg5 arrest in the next mitosis. After a prolonged arrest, they may slip out and become multinucleated, which will likely prevent further successful divisions or they may go into apoptosis. Further, mitotic arrest and induction of apoptosis in Eg5 depleted glioblastoma cells occur independent of p53, Rb-signalling and the PI3K-pathway suggesting that Eg5 is a potential therapeutic target for glioblastoma patients with different underlying genetic abnormalities We have also tested the clinical feasibility of using a cell cycle targeting antisense oligonucleotide based therapy delivered directly to the central nervous system (CNS) as a novel treatment for glioblastoma tumors. This work has demonstrated that intraventricular administration of ASOs can efficiently target cells in the CNS and be delivered to glioma-initiating neural stem cells transplanted into the cortex of naive mice as well as to glioblastoma tumors in a genetically predisposed mouse model. This strategy is therefore a potential route of administration for treating glioblastoma tumors which originate in the CNS. Direct targeting of mitotic components in the brain should have a limited toxicity to non-cycling neurons and as a benefit, as long as the blood-brain barrier is intact direct CNS delivery should have minimal dose-limiting toxicity outside of the CNS. Ongoing studies will determine the effect of Eg5 inhibition on glioblastoma growth in vivo. Citation Format: Cecilia C. Krona, Jihane Boubaker, Dinorah Friedmann-Morvinski, Alex Wong, Melissa McAlonis-Downes, Erich Koller, Aneeza S. Kim, Gene Hung, Frank Rigo, Seung Chun, Benjamin Vitre, Frank Bennett, Inder Verma, Don W. Cleveland. Antisense oligonucleotide depletion of the mitotic kinesin Eg5 by direct delivery to the brain could be a useful strategy for treating glioma tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3302. doi:10.1158/1538-7445.AM2013-3302

Therapeutic suppression of tau kinases in the CNS with antisense oligonucleotides

to target pathogenic tau in 4-, 8-, and 18-month-old P301L tau transgenic mice, a model of AD and FTD, that have an onset of NFT pathology at 6 months of age. After up to 10 months of treatment, the degree of NFT pathology was determined by histological analysis. Results:All age groups, NFT pathology was significantly reduced in treated compared to non-immunized mice. Similarly, phosphorylation of tau at pathological sites was reduced. In addition, increased astrocytosis was found in the oldest treated group. Conclusions: Our data suggests that tau-targeted immunization slows the progression of NFT pathology in a mouse model of tau pathology.