Dieter Zopf

Inhibition of LNCaP prostate tumor growth in vivo by an antisense oligonucleotide directed against the human androgen receptor

We have shown recently that a 15-mer phosphorothioate oligodeoxynucleotide (ODNas750/15) that hybridizes to the (CAG)n polyglutamine region of mRNA encoding human androgen receptor (AR) inhibits the expression of AR in LNCaP prostate cancer cells in vitro. This AR downregulation was accompanied by significant cell growth inhibition and reduced PSA secretion. In the present study we investigated the effects of this antisense AR ODN on prostate tumor growth in vivo using a mouse xenograft model. Via subcutaneously implanted diffusion pumps, either ODNas750/15 or a scrambled control sequence ODNsr750/15 was continuously administered into LNCaP tumor–bearing male nude mice for 7 weeks. Compared with untreated control animals, treatment with ODNas750/15 resulted in significant tumor growth inhibition. Retardation of tumor growth was also significant in castrated mice, whereas the scrambled control ODN did not exert any effects. No side effects such as loss of body weight were observed at any time of treatment. ODN treatment was well tolerated and, in contrast to castration, did not induce shrinkage of mouse prostates. Both AR expression in the tumor and PSA levels in mouse serum correlated with tumor size. However, we failed to demonstrate a correlation between tumor retardation and Ki-67 antigen expression and the number of apoptotic cells, respectively. Testing of antisense-treated LNCaP cells revealed that expression levels of other proteins that contain shorter polyglutamine sequence stretches such as HDAC2, TFIID, and c-jun were not affected. The present study demonstrates that downregulation of AR with antisense ODNas750/15 causes prostate tumor growth inhibition. These results further point out the important role of the AR in prostate tumors and support further testing of AR downregulation for treatment of prostate cancer.

Regorafenib Inhibits Growth, Angiogenesis, and Metastasis in a Highly Aggressive, Orthotopic Colon Cancer Model

The combination of target-specific drugs like bevacizumab with chemotherapeutics has improved treatment efficacy in advanced colorectal cancer (CRC). However, the clinical prognosis of metastatic CRCs is still poor, and novel drugs are currently assessed with respect to their efficacies in patients with CRCs. In a phase III study, the multikinase inhibitor regorafenib (BAY 73-4506) has recently been shown to prolong survival of patients with CRCs after standard therapies failed. In the present study, the activity of regorafenib was investigated in comparison with the angiogenesis inhibitor DC101 in the highly aggressive, murine CT26 metastatic colon cancer model. While a treatment for 10 days with DC101 given at a dose of 34 mg/kg every third day significantly delayed tumor growth compared with vehicle-treated animals, regorafenib completely suppressed tumor growth at a daily oral dose of 30 mg/kg. Regorafenib also induced a stronger reduction in tumor vascularization, as longitudinally assessed in vivo by dynamic contrast-enhanced MRI (DCE-MRI) and confirmed by immunohistochemistry. In addition, regorafenib inhibited the angiogenic activity more strongly and induced a three times higher apoptosis rate than DC101. Even more important, regorafenib completely prevented the formation of liver metastases, whereas in DC101-treated animals, the metastatic rate was only reduced by 33% compared with the vehicle group. In addition, regorafenib significantly reduced the amount of infiltrating macrophages. These data show that the multikinase inhibitor regorafenib exerts strong antiangiogenic, antitumorigenic, and even antimetastatic effects on highly aggressive colon carcinomas indicative for its high potential in the treatment of advanced CRCs. Mol Cancer Ther; 12(7); 1322–31. ©2013 AACR.

Regorafenib (BAY 73‐4506): Antitumor and antimetastatic activities in preclinical models of colorectal cancer

Regorafenib, a novel multikinase inhibitor, has recently demonstrated overall survival benefits in metastatic colorectal cancer (CRC) patients. Our study aimed to gain further insight into the molecular mechanisms of regorafenib and to assess its potential in combination therapy. Regorafenib was tested alone and in combination with irinotecan in patient‐derived (PD) CRC models and a murine CRC liver metastasis model. Mechanism of action was investigated using in vitro functional assays, immunohistochemistry and correlation with CRC‐related oncogenes. Regorafenib demonstrated significant inhibition of growth‐factor‐mediated vascular endothelial growth factor receptor (VEGFR) 2 and VEGFR3 autophosphorylation, and intracellular VEGFR3 signaling in human umbilical vascular endothelial cells (HuVECs) and lymphatic endothelial cells (LECs), and also blocked migration of LECs. Furthermore, regorafenib inhibited proliferation in 19 of 25 human CRC cell lines and markedly slowed tumor growth in five of seven PD xenograft models. Combination of regorafenib with irinotecan significantly delayed tumor growth after extended treatment in four xenograft models. Reduced CD31 staining indicates that the antiangiogenic effects of regorafenib contribute to its antitumor activity. Finally, regorafenib significantly delayed disease progression in a murine CRC liver metastasis model by inhibiting the growth of established liver metastases and preventing the formation of new metastases in other organs. In addition, our results suggest that regorafenib displays antimetastatic activity, which may contribute to its efficacy in patients with metastatic CRC. Combination of regorafenib and irinotecan demonstrated an increased antitumor effect and could provide a future treatment option for CRC patients.

Pharmacologic activity and pharmacokinetics of metabolites of regorafenib in preclinical models

Regorafenib is an orally administered inhibitor of protein kinases involved in tumor angiogenesis, oncogenesis, and maintenance of the tumor microenvironment. Phase III studies showed that regorafenib has efficacy in patients with advanced gastrointestinal stromal tumors or treatment‐refractory metastatic colorectal cancer. In clinical studies, steady‐state exposure to the M‐2 and M‐5 metabolites of regorafenib was similar to that of the parent drug; however, the contribution of these metabolites to the overall observed clinical activity of regorafenib cannot be investigated in clinical trials. Therefore, we assessed the pharmacokinetics and pharmacodynamics of regorafenib, M‐2, and M‐5 in vitro and in murine xenograft models. M‐2 and M‐5 showed similar kinase inhibition profiles and comparable potency to regorafenib in a competitive binding assay. Inhibition of key target kinases by all three compounds was confirmed in cell‐based assays. In murine xenograft models, oral regorafenib, M‐2, and M‐5 significantly inhibited tumor growth versus controls. Total peak plasma drug concentrations and exposure to M‐2 and M‐5 in mice after repeated oral dosing with regorafenib 10 mg/kg/day were comparable to those in humans. In vitro studies showed high binding of regorafenib, M‐2, and M‐5 to plasma proteins, with unbound fractions of ~0.6%, ~0.9%, and ~0.4%, respectively, in murine plasma and ~0.5%, ~0.2%, and ~0.05%, respectively, in human plasma. Estimated free plasma concentrations of regorafenib and M‐2, but not M‐5, exceeded the IC50 at human and murine VEGFR2, suggesting that regorafenib and M‐2 are the primary contributors to the pharmacologic activity of regorafenib in vivo.

Antitumor effects of regorafenib and sorafenib in preclinical models of hepatocellular carcinoma

The purpose of this study was to investigate the antitumor activity of regorafenib and sorafenib in preclinical models of HCC and to assess their mechanism of action by associated changes in protein expression in a HCC-PDX mouse model. Both drugs were administered orally once daily at 10 mg/kg (regorafenib) or 30 mg/kg (sorafenib), which recapitulate the human exposure at the maximally tolerated dose in mice. In a H129 hepatoma model, survival times differed significantly between regorafenib versus vehicle (p=0.0269; median survival times 36 vs 27 days), but not between sorafenib versus vehicle (p=0.1961; 33 vs 28 days). Effects on tumor growth were assessed in 10 patient-derived HCC xenograft (HCC-PDX) models. Significant tumor growth inhibition was observed in 8/10 models with regorafenib and 7/10 with sorafenib; in four models, superior response was observed with regorafenib versus sorafenib which was deemed not to be due to lower sorafenib exposure. Bead-based multiplex western blot analysis was performed with total protein lysates from drug- and vehicle-treated HCC-PDX xenografts. Protein expression was substantially different in regorafenib- and sorafenib-treated samples compared with vehicle. The pattern of upregulated proteins was similar with both drugs and indicates an activated RAF/MEK/ERK pathway, but more proteins were downregulated with sorafenib versus regorafenib. Overall, both regorafenib and sorafenib were effective in mouse models of HCC, although several cases showed better regorafenib activity which may explain the observed efficacy of regorafenib in sorafenib-refractory patients.

Abstract 772: Anti-tumor efficacy of the selective pan-FGFR Inhibitor BAY 1163877 in preclinical squamous cell carcinoma models of different origin

Squamous cell carcinoma (SCC) is a common subtype of lung cancer and is strongly associated with smoking. In contrast to adenocarcinomas of the lung, SCCs do not significantly harbor epidermal growth factor (EGFR) mutations or ALK, ROS1 or RET translocations, which are therapeutically tractable. SCC of the lung and SCC of the head and neck region and esophagus not only share risk factors, morphologic features and mechanisms of tumorigenesis but also frequent fibroblast growth factor receptor 1 (FGFR1) gene alterations. In addition, we found that FGFR2 and FGFR3 expression were also altered in patient derived models of SCC of various origins. FGFRs may thus represent viable therapeutic targets for the treatment of SCC. BAY 1163877, an orally available and potent inhibitor of FGFR1, FGFR2 and FGFR3, inhibited SCC cell proliferation as well as downstream FGFR signaling in vitro. When applied in vivo, BAY 1163877 was able to reduce growth of a FGFR1-overexpressing lung SCC xenograft model, a FGFR1-overexpressing esophageal SCC xenograft model, and a FGFR3-overexpressing head and neck SCC xenograft model, achieving reductions in tumor growth by 67 to 92% as compared to the vehicle control. BAY 1163877 was very well tolerated in all models performed. These data suggest that selective inhibition of FGFR1 to 3 with BAY 1163877 might have therapeutic potential for the treatment of SCC malignancies. A phase I clinical trial (NCT01976741) to determine the safety, tolerability and recommended Phase 2 dose in advanced cancer patients is ongoing. Citation Format: Melanie Heroult, Matthias Ocker, Charlotte Kopitz, Dieter Zopf, Andrea Hagebarth, Karl Ziegelbauer, Stuart Ince, Peter Ellinghaus. Anti-tumor efficacy of the selective pan-FGFR Inhibitor BAY 1163877 in preclinical squamous cell carcinoma models of different origin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 772. doi:10.1158/1538-7445.AM2015-772

Abstract B2: Regorafenib, an oral multi-kinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases, potently inhibits tumor growth and angiogenesis in patient-derived xenograft models of gastric cancer.

Background: Gastric cancer (GC) is a prevalent and deadly disease. Because the VEGFR and PDGFR signaling cascade plays a critical role in the development and progression of gastric cancer and VEGF expression is correlated with angiogenesis, tumor progression and poor prognosis in patients with GC, blocking these signaling pathways could thus have therapeutic efficacy. This study aims to investigate the potential of a combined therapeutic approach to target these pathways in preclinical models of GC with regorafenib an oral multi-kinase inhibitor targeting VEGFR1–3, PDGFR, FGFR, TIE-2, RET and KIT. Methods: Eight patient-derived GC xenograft models with different histotypes were treated orally with regorafenib or vehicle and tumor growth was monitored using caliper measurements. Tissue sections of vehicle- or regorafenib-treated xenografts were analyzed by immunohistochemistry for apoptosis, microvessel area, and cell proliferation using antibodies against cleaved PARP, CD31 and phospho-Histone3 Ser10 , respectively. Tumor lysates were analyzed by Western Blotting with respective antibodies for effects on marker proteins for proliferation, apoptosis and angiogenesis. Results: Regorafenib demonstrated significant tumor growth inhibition of 81%, 85% and 88% in the GC09-0109 model after oral administration of 5, 10 and 15 mg/kg/d, respectively. In addition potent tumor growth inhibition ranging from 72–96% was observed in seven other GC models at a dose of 10 mg/kg/d. Regorafenib significantly inhibited angiogenesis in all models by 4–12 fold compared to the vehicle-treated group, independent of their degree of vascularization, as indicated by reduced CD31 staining. Furthermore inhibition of cell proliferation by 2–4 fold was observed by reduced staining for p-histone 3 Ser10 . Increased staining for caspase-cleaved p85 PARP by 2 to 20 fold was detected, indicating that regorafenib induced apoptosis. Finally, regorafenib also induced tumor necrosis as detected by histological staining. Inhibition of angiogenesis, reduced cell proliferation and induction of apoptosis and necrosis was associated with decreased levels of p-ERK1/2, p-cdk2 Thr14/Tyr15 , p-VEGFR2 Tyr951 , p-p70S6K Thr421/Ser424 , p-S6R Ser235/236 and p-eIF4E Ser209 and elevation of cleaved caspase-3 and cleaved PARP. Conclusion: We show that regorafenib is an efficacious anti-tumor targeted agent in patient-derived gastric cancer preclinical models. Our findings support further investigation of regorafenib in the treatment of GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B2.

Abstract 5036: Correlation of preclinical antitumor activity of regorafenib in CRC-PDX xenografts with gene expression and clinical parameters of the primary tumor

Regorafenib is a small molecule inhibitor of multiple transmembrane and intracellular kinases involved in normal cellular functions and in pathologic processes such as oncogenesis, tumor angiogenesis, metastasis, and tumor immunity. Regorafenib is approved for the treatment of patients with metastatic colorectal cancer (CRC) who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an anti-VEGF therapy, and, if RAS wild-type, an anti-EGFR therapy or with locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) who have been previously treated with imatinib mesylate and sunitinib malate. Recently an overall survival benefit has been shown in patients with hepatocellular carcinoma (HCC) who had previously been treated with sorafenib (RESORCE). OncoTrack is an Innovative Medicines Initiative (IMI) sponsored project with the goal to improve the basis for identification of biomarkers based on the mechanisms of action of therapies approved for this indication. For this purpose, a panel of CRC-PDX xenografts was generated by the OncoTrack project. At the time of the analysis reported here fifty xenografts had been treated with regorafenib at a dose of 10 mg/kg/d or with vehicle for 24 days. The analysis of tumor growth rates (TGR) showed pronounced differences between different tumors and between vehicle and regorafenib treated models. The relative antitumor activity (relative TGR) of regorafenib varied between -0,13 (good response) and 0.0 (no response). Investigations of relative TGR in relationship to (non-) clinical parameters of the primary tumor such as age, gender, sidedness and tumor histology identified a marginally significant (p= 0.04) better response in tumors from younger patients. No other correlations were detected to this end, which may be due to the small sample number. To correlate antitumor activity of regorafenib with gene expression, RNA was isolated from sections of selected vehicle and regorafenib treated xenografts and hybrized on Affymetrix HuGene-2.1_st human transcriptome arrays. Expression profiles were subsequently analyzed using the Random Forests algorithm to identify gene expression signatures predicting response to regorafenib. The best signatures did not perform better than signatures derived after randomizing responses, i.e. no predictive signature could be identified. Further studies with larger samples sizes are necessary to improve the outcome of such an approach; however one should acknowledge that to date no predictive gene signatures could be identified for multikinase inhibitors, which may be intrinsic to their complex mechanism of action. The research reported here received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement 115234 (OncoTrack). Citation Format: Henrik Seidel, Jens Hoffmann, Ralf Lesche, Sylvia Grunewald, David Henderson, Dieter Zopf. Correlation of preclinical antitumor activity of regorafenib in CRC-PDX xenografts with gene expression and clinical parameters of the primary tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5036. doi:10.1158/1538-7445.AM2017-5036

Abstract 5244: Effects of chronic low dose aspirin treatment in the mouse AOM/DSS model of colon carcinogenesis

Although early detection and treatment of colorectal cancer (CRC) have improved in recent years, it remains a significant healthcare problem with high morbidity and mortality. Published data indicate that long-term intake of low dose aspirin reduces the risk of CRC, however the molecular mechanisms underlying this effect are still unclear. The aim of this study was to investigate the efficacy of low dose equivalent aspirin treatment in a mouse colon carcinogenesis model, including evaluation of potential mechanistic contributors. An initial PK/PD study in healthy mice indicated that aspirin 25 mg/kg/day given via drinking water had a similar PD effect - reduction in plasma thromboxane B2 (TXB2), as an indicator of cyclooxygenase-1 (COX-1) inhibition - as did 5-day low-dose aspirin treatment (100 mg/day) in healthy human volunteers. Male and female C57BL/6J mice were treated with the carcinogen azoxymethane (AOM) 10 mg/kg once i.p. combined with three 5-day cycles (at 14 day intervals) of dextran sodium sulphate (DSS) 2% in drinking water. Aspirin treatment was started concomitant with AOM administration and continued for 12 weeks, at which time the data outlined below were obtained. Aspirin 25 mg/kg/day significantly reduced tumor burden in 3 independent experiments (total tumor number reduced by 43 - 51%; tumor area per mouse reduced by 59 - 65%; mean tumor area reduced by 22 - 27%). Increasing the dose to 50 mg/kg/day did not show better inhibition of tumorigenesis. Aspirin-induced reduction in tumorigenesis in this model was accompanied by reduction in systemic plasma TXB2 by about 70%, indicating reduced platelet activation. TXB2 production by colon explants was also reduced, by 30-50%. Aspirin treatment reduced inflammatory activity (fecal blood; IL-1β and IL-6 production by colon explant cultures; iNOS-positive tumor macrophage infiltration). Immunohistological analysis of tumor tissue showed no reduction in proliferation (BrdU staining) by aspirin but there was a trend towards increased apoptosis (caspase-3 positive cells) and a significant reduction in CD31-positive microvessel density. Aspirin treatment did not result in significant modulation of the Wnt/β-catenin, NF-κB or HIF-1 pathways as assessed by mRNA expression of several of their target genes in tumor tissue. The results show that aspirin can inhibit tumor development in this established model of colon carcinogenesis, primarily by reducing tumor number with a less marked effect on the size of individual tumors. This effect is associated with COX-1 inhibition of a similar magnitude to that seen with low dose aspirin in humans. The influence of aspirin on intestinal inflammation and platelet activation as well as on tumor cell apoptosis and tumor angiogenesis might all contribute to the beneficial effects seen in this model. Additional studies are planned to further elucidate aspirin9s mechanism of action in inhibiting tumor development. Citation Format: Nadine Rohwer, Anja Kuehl, Dieter Zopf, Fiona M. McDonald, Karsten-Heinrich Weylandt. Effects of chronic low dose aspirin treatment in the mouse AOM/DSS model of colon carcinogenesis. [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 5244.

Abstract 1052: Refametinib (BAY 86-9766): An allosteric inhibitor of MEK1/2, potently inhibits tumor growth in patient-derived xenograft models of gastric cancer.

Background: Gastric cancer (GC) is a prevalent and deadly disease. Because the Ras/Raf/MEK/ERK signaling cascade plays a critical role in signal transduction pathways that regulate growth factor response, angiogenesis, tumor cell proliferation, and apoptosis, blocking this signaling pathway could thus have therapeutic efficacy. This study aims to target this pathway in preclinical models of GC with refametinib (BAY 86-9766, RDEA119), an orally available, allosteric inhibitor of MEK1/2. Methods: Eight patient-derived GC xenograft models with different histotypes were treated orally with refametinib or vehicle and tumor growth was monitored using caliper measurements. Tissue sections of vehicle- or refametinib-treated xenografts were analyzed by immunohistochemistry for cell proliferation, apoptosis and microvessel area, using antibodies against p-Histone H3Ser10, cleaved PARP and CD31, respectively. Tumor lysates were analyzed by Western Blotting with antibodies for effects on marker proteins for proliferation, apoptosis and angiogenesis.Results: Refametinib demonstrated significant tumor growth inhibition ranging from 66-87% in eight GC models at a dose of 15 mg/kg/d. Refametinib inhibited cell proliferation by 2-4 fold as observed by reduced staining for p-Histone H3Ser10. Increased staining for caspase-cleaved p85 PARP by 1.5 to 9 fold was detected, indicating that refametinib induced apoptosis. Furthermore the compound significantly inhibited angiogenesis in 2 of 8 models by 3-fold compared to the vehicle-treated group as indicated by reduced CD31 staining. Reduced cell proliferation and induction of apoptosis were associated with decreased levels of p-ERK1/2, p-cdk2Thr14/Tyr15, p-p90RSKSer359/363, p-p70S6KThr421/Ser424, p-4EBP1Thr70, p-S6RSer235/236 and p-eIF4ESer209 and elevation of p-AktSer473, Bim and cleaved PARP. Conclusion: We show that single-agent refametinib has significant anti-tumor activity in patient-derived gastric cancer preclinical models. Our findings support further investigation of refametinib either in monotherapy or in combination with inhibitors of the PI3K-AKT-mTOR pathway in the treatment of GC. Citation Format: Huynh T. Hung, Richard Ong, Khee Chee Soo, Florian Puehler, Arne Scholz, Dominik Mumberg, Dieter Zopf, Karl Ziegelbauer. Refametinib (BAY 86-9766): An allosteric inhibitor of MEK1/2, potently inhibits tumor growth in patient-derived xenograft models of gastric cancer. [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 1052. doi:10.1158/1538-7445.AM2013-1052