Katja Wosikowski

Increased resistance to cytotoxic agents in ZR75B human breast cancer cells transfected with epidermal growth factor receptor

Human breast cancer cells selected for multidrug resistance frequently overexpress ligands and receptors in the epidermal growth factor (EGF) receptor family. To determine whether this overexpression contributes to the drug resistant phenotype, EGF receptor transfected ZR75B human breast cancer cells were examined. Two EGF receptor overexpressing clones were evaluated: clone 11 with > 1 x 10(6) sites, and clone 13 with 310,000 receptor sites/cell. These were compared with clone 2-neo, which was transfected with the neomycin gene only and contained 43,000 receptor sites/cell. The EGF receptor overexpressing clones and the neo transfected control clone displayed comparable growth rates. Cytotoxicity analyses were performed with doxorubicin, vinblastine, cisplatin and 5-fluorouracil to determine the sensitivity of the clones to antineoplastic drugs. The EGF receptor overexpressing clones were found to be 1.5-5.6 times more resistant to the four drugs tested. This increase in the IC50 conferred a selective advantage when grown in the presence of 2, 3 and 6 ng/ml doxorubicin. Clone 13 cells overtook a mixed population which began with clone 2-neo comprising 95% of the cells. Clone 2-neo remained the dominant clone in the absence of drug. Finally, after long-term selection of the clones with 6 ng/ml doxorubicin, clone 2-neo became fourfold more resistant than the unselected clone 2-neo, a level which was comparable to that found in the EGF receptor overexpressing clones 11 and 13. No additional increase in resistance was observed for these clones, suggesting that clone 2-neo had developed additional resistance mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced growth rate accompanied by aberrant epidermal growth factor signaling in drug resistant human breast cancer cells

We examined transforming growth factor (TGF) alpha, epidermal growth factor (EGF) and EGF receptor (EGFR) expression and signaling in three drug resistant MCF-7 human breast cancer sublines and asked whether these pathways contribute to the drug resistance phenotype. In the resistant sublines, upregulation of both TGFalpha and EGFR mRNA was observed. In an apparent contrast with upregulated growth factor and receptor gene expression, the drug resistant sublines displayed a reduced growth rate. Defects in the EGFR signaling pathway cascade were found in all examined drug resistant sublines, including altered EGF-induced Shc, Raf-1, or mitogen-activated protein kinase phosphorylation. Induction of c-fos mRNA expression by EGF was impaired in the sublines compared to parental MCF-7 cells. In contrast, the induction of the stress-activated protein kinase activity was similar in both parental and drug resistant cells. Evaluating the link between the reduced growth rate and drug resistance, serum starvation experiments were performed. These studies demonstrated that a reduced proliferative activity resulted in a marked reduction in sensitivity to cytotoxic agents in the parental MCF-7 cells. We propose that the altered EGFR levels frequently observed in drug resistant breast cancer cells are associated with perturbations in the signaling pathway that mediate a reduced proliferative rate and thereby contribute to drug resistance.

TGF-ß isoforms in cancer: Immunohistochemical expression and Smad-pathway-activity-analysis in thirteen major tumor types with a critical appraisal of antibody specificity and immunohistochemistry assay validity.

The literature on TGF-Δ in cancer including data on the expression or activation of TGF-Δ pathway components in specific tumors types is steadily growing. However, no systematic and uniform analysis exists reporting expression levels of the main TGF-Δ pathway components across the most frequent tumor types. We used a standardized immunohistochemical assay investigating TGF-Δ isoform expression and pathway activation across 13 different tumor types and corresponding non-neoplastic tissues. The study was performed on tissue microarrays allowing for the parallel analysis of a total of 1638 human tumor samples. TGF-Δ1, TGF-Δ2 and p-Smad2/3 were substantially expressed in multiple cancers widening the options for TGF-Δ isoform directed therapies. Of note, TGF-Δ antigens appear to be expressed in an individual manner pointing towards a need for patient preselection for TGF-β isoform specific treatment. Yet, a thorough investigation of antibody specificity and assay validity revealed that immunohistochemistry did not correlate with other detection methods on mRNA or protein level in all instances. As such, with the currently available means (i.e. antibodies tested) a stratification of patients within clinical trials for TGF-Δ directed antisense therapies based upon TGF-β immunohistochemistry alone has to be interpreted with caution and should be carefully evaluated in combination with other parameters.

First-in-human phase I study of ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-β2), in subjects with open-angle glaucoma undergoing glaucoma filtration surgery

Purpose To evaluate the safety and tolerability of intravitreal ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-β2), in patients with primary open angle glaucoma (POAG) undergoing trabeculectomy (TE; glaucoma filtration surgery). Methods In this prospective phase I trial glaucoma patients scheduled for TE with mitomycin C (MMC) received a single intravitreal injection of ISTH0036 at the end of surgery in escalating total doses of 6.75 μg, 22.5 μg, 67.5 μg or 225 μg, resulting in calculated intraocular ISTH0036 concentrations in the vitreous humor of approximately 0.3 μM, 1 μM, 3 μM or 10 μM after injection, respectively. Outcomes assessed included: type and frequency of adverse events (AEs), intraocular pressure (IOP), numbers of interventions post trabeculectomy, bleb survival, visual acuity, visual field, electroretinogram (ERG), slit lamp biomicroscopy and optic disc assessment. Results In total, 12 patients were treated in the 4 dose groups. Main ocular AEs observed were corneal erosion, corneal epithelium defect, or too high or too low IOP, among others. No AE was reported to be related to ISTH0036. All other safety-related analyses did not reveal any toxicities of concern, either. The mean medicated preoperative IOP at decision time-point for surgery was 27.3 mmHg +/- 12.6 mmHg (SD). Mean IOP (±SD) for dose levels 1, 2, 3, and 4 were at Day 43 9.8 mmHg ± 1.0 mmHg, 11.3 mmHg ± 6.7 mmHg, 5.5 mmHg ± 3.0 mmHg and 7.5 mmHg ± 2.3 mmHg SD; and at Day 85 9.7 mmHg ± 3.3 mmHg, 14.2 mmHg ± 6.5 mmHg, 5.8 mmHg ± 1.8 mmHg and 7.8 mmHg ± 0.6 mmHg, respectively. In contrast to IOP values for dose levels 1 and 2, IOP values for dose levels 3 and 4 persistently remained below 10 mmHg throughout the observation period. Conclusion This first-in-human trial demonstrates that intravitreal injection of ISTH0036 at the end of TE is safe. Regarding IOP control, single-dose ISTH0036 administration of 67.5 μg or 225 μg at the time of TE resulted in IOP values persistently < 10 mmHg over the three month postoperative observation period.

Abstract 3741: Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma

Transforming growth factor beta (TGF-β) isoforms are the primary mediators for TGF-β signaling via TGF-β receptors and downstream phosphorylation/dephosphorylation cascade. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Mouse 4T1 mammary carcinoma cell line is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone. Considering rather challenging preclinical evaluation of antitumor activity in tumor models, mouse 4T1 mammary carcinoma model has been widely used in the literature for evaluation of TGF-β antagonists. In this report, we describe the efficacy of ISTH0047 - a potent and selective TGF-β2 antisense oligonucleotide - in murine 4T1 primary tumors and lung metastasis following tumor cell injection into the mammary fat pad (orthotopic tumor model) of syngeneic Balb/c mice. Consistent with literature data generated with other classes of TGF-β antagonists (e.g., small-molecule kinase inhibitors, antibodies or TGF-β trap agents), although limited antitumor activity was demonstrated on primary tumor growth, marked and statistically significant decrease of lung metastasis number was observed upon subcutaneous administrations of ISTH0047. In addition, side by side comparison with murine surrogates of CTLA-4 or PD-1 antibodies indicated similar efficacy of all test items on lung metastasis in this model. Taken together, these encouraging results pave the way for in-depth preclinical evaluation of both ‘seed and soil’ theory and efficacy of combination regimen (immunomodulation) for better clinical outcome. Citation Format: Katja Wosikowski, Kathy Hasenbach, Jutta Petschenka, Diana Barea Roldan, Sebastian Kreiter, Ugur Sahin, Guillaume Serin, Julie-Orlane Redon, Marc Hillairet de Boisferon, Francis Bichat, Hanna Kohonen, Michel Janicot. Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3741.

Abstract 717: Novel potent antisense oligonucleotides targeting transforming growth factor beta1 (TGF-β1)

Transforming growth factor beta (TGF-β) is a key member of a large family of cytokines, which play critical, pleiotropic roles in the pathophysiology of various human diseases, such as cancer, inflammation, autoimmune disease, and cirrhosis/fibrosis. TGF-β1, -2 and -3 isoforms are cytokines encoded by different genes but sharing strong sequence and structure homology. They function as the primary mediators of TGF-β signaling via both non-canonical and canonical signaling pathways. In Oncology, TGF-β isoforms are associated with a wide range of biological processes such as tumor cell invasion and migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Hence, blocking the TGF-β signaling pathway may have a multifold therapeutic benefit in Oncology, although therapeutic relevance of the respective TGF-β isoforms remains poorly documented. In order to evaluate the specific biological relevance of TGF-β1 isoform in cancer, we have initiated an extensive discovery program for identification of antisense oligodeoxynucleotide constructs selectively inhibiting expression of the TGF-β1 ligand. Based on the sequence of the human TGF-β1 mRNA, more than 150 Locked Nucleic Acid (LNA)-modified gapmers were designed, synthesized and tested in cell-based assays. Highly potent and selective TGF-β1 constructs were identified and selected based on efficient suppression of TGF-β1 mRNA/protein expression in different human and rodent tumor cell lines, and in human Peripheral Blood Mononuclear Cells (PBMCs). Effective target downregulation was demonstrated after lipofectamine aided-transfection (sub-nM concentration range), but also in the absence of any transfection agent (gymnotic delivery) at sub-µM concentrations. Selected TGF-β1 specific oligonucleotides were further tested in relevant animal xenograft models and strong target inhibition was also observed following systemic administration. Further details of this discovery program will be discussed and pharmacology properties and features of potent and selective TGF-β1 antagonists will be presented. Citation Format: Frank Jaschinski, Hanna Korhonen, Stephan Braun, Katja Wosikowski, Michel Janicot. Novel potent antisense oligonucleotides targeting transforming growth factor beta1 (TGF-β1). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 717. doi:10.1158/1538-7445.AM2014-717

Abstract 3126: Renca RCC syngeneic model to evaluate efficacy of novel antisense oligonucleotides targeting TGF-β isoforms

Transforming growth factor beta (TGF-β represents a family of cytokines, which function as the primary mediators for TGF-β signaling via TGF-β receptor type II (TβIIR) and both non-canonical and canonical downstream signaling pathways. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Hence, optimal preclinical evaluation of efficacy of TGF-β antagonists is challenging. Isarna Therapeutics has designed and developed selective and potent LNA-modified antisense oligonucleotides targeting the various TGF-β isoforms. In order to adequately evaluate selected preclinical development candidates, Oncodesign has developed customized experimental Renca tumor models in syngeneic and/or immunodeficient mice. The Renca cell line was established from a murine transplantable renal adenocarcinoma of spontaneous origin, and has been used under various experimental conditions: (1) subcutaneous tumor model by inoculating cells into the flanks of the animals; (2) the pulmonary metastatic tumor model by an intravenous injection of cells into the tail vein; and (3) the orthotopic tumor model by injecting cells into the renal subcapsule (and subsequent pulmonary metastasis). Outcome of this development program and preliminary results for selected TGF-β antisense oligonucleotides will be presented and discussed. Citation Format: Hanna Korhonen, Julie-Orlane Redon, Damien France, Guillaume Serin, Francis Bichat, Frank Jaschinski, Katja Wosikowski, Michel Janicot. Renca RCC syngeneic model to evaluate efficacy of novel antisense oligonucleotides targeting TGF-β isoforms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2014-3126