Thomas Beckers

Distinct pharmacological properties of second generation HDAC inhibitors with the benzamide or hydroxamate head group

Advanced second generation inhibitors of histone deacetylases (HDAC) are currently used in clinical development. This study aimed at comparing the pharmacological properties of selected second generation HDAC inhibitors with the hydroxamate and benzamide head group, namely SAHA, LAQ824/LBH589, CI994, MS275 and MGCD0103. In biochemical assays using recombinant HDAC1, 3, 6 and 8 isoenzymes, SAHA and LAQ824/LBH589 behave as quite unselective HDAC inhibitors. In contrast, the benzamides CI994, MS275 and MGCD0103 are more selective, potent inhibitors of at least HDAC1 and HDAC3. All HDAC inhibitors induce histone H3 hyperacetylation, correlating with inhibition of proliferation, induction of cell differentiation and apoptosis. A broad cytotoxicity is seen across cell lines from different tumor entities with LAQ824/LBH589 being the most potent agents. The apoptosis inducing activity is evident in arrested and proliferating RKO colon cancer cells with inducible, heterologous p21waf1 expression, indicative for a cell‐cycle independent mode‐of‐action. Differentiation of MDA‐MB468 breast cancer cells is induced by benzamide and hydroxamate analogs. The reversibility of drug action was evaluated by pulse treatment of A549 lung cancer cells. Whereas paclitaxel induced irreversible cell cycle alterations already after 6 hr treatment, HDAC inhibitor action was retarded and irreversible after >16 hr treatment. Interestingly, pulse treatment was equally effective as continous treatment. Finally, the efficacy of LAQ824, SAHA and MS275 in A549 nude mice xenografts was comparable to that of paclitaxel at well tolerated doses. We conclude that despite a different HDAC isoenzyme inhibition profile, hydroxamate and benzamide analogs as studied display similar cellular profiles.

Primary resistance to cetuximab in a panel of patient-derived tumour xenograft models: activation of MET as one mechanism for drug resistance.

Cetuximab (Erbitux®) targets the epidermal growth factor receptor (EGFR) and is approved for treatment of colorectal and head and neck cancer. Despite wide expression of EGFR, only a subgroup of cancer patients responds to cetuximab therapy. In the present study we assessed the cetuximab response in vivo of 79 human patient-derived xenografts originating from five tumour histotypes. We analysed basic tumour characteristics including EGFR expression and activation, mutational status of KRAS, BRAF and NRAS, the expression of EGFR ligands and the activation of HER3 (ErbB3) and the hepatocyte growth factor receptor MET. Based on these results, a cetuximab response score including positive and negative factors affecting therapeutic response is proposed. Positive factors are high expression and activation of EGFR and its ligands epiregulin or amphiregulin, negative factors are markers for downstream pathway activation independent of EGFR. In cetuximab resistant NSCL adenocarcinoma LXFA 526 and LXFA 1647, overexpression due to gene amplification and strong activation of MET was identified. Knock-down of MET by siRNA in the corresponding cell lines showed that anchorage-independent growth and migration are dependent on MET. MET knock down sensitized LXFA 526L and LXFA 1647L to EGF. Combined treatments of a MET inhibitor and cetuximab were additive. Therefore, combination therapy of cetuximab and a MET inhibitor in selected lung cancer patients could be of high clinical significance.

Synthetic 2-aroylindole derivatives as a new class of potent tubulin-inhibitory, antimitotic agents.

A new class of simple synthetic antimitotic compounds based on 2-aroylindoles was discovered. (5-Methoxy-1H-2-indolyl)-phenylmethanone (1) as well as analogous 3-fluorophenyl- (36) and 3-methoxyphenyl (3) derivatives displayed high cytotoxicity of IC(50) = 20 to 75 nM against the human HeLa/KB cervical, SK-OV-3 ovarian, and U373 astrocytoma carcinoma cell lines. The inhibition of proliferation correlated with the arrest in the G2/M phase of the cell cycle. In in vitro assays with tubulin isolated from bovine brain, in general antiproliferative activity correlated with inhibition of tubulin polymerization. Thus, the antimitotic activity of 2-aroylindoles is explained by interference with the mitotic spindle apparatus and destabilization of microtubules. In contrast to colchicine, vincristine, nocodazole, or taxol, 1 did not significantly affect the GTPase activity of beta-tubulin. Interestingly, selected compounds inhibited angiogenesis in the chorioallantoic membrane (CAM) assay. In xenograft experiments, 1 was highly active after oral administration at 200 mg/kg against the human amelanocytic melanoma MEXF 989 in athymic nude mice. We conclude, that 2-aroylindoles constitute an interesting new class of antitubulin agents with the potential to be clinically developed for cancer treatment.

Perinuclear localization of the protein-tyrosine phosphatase SHP-1 and inhibition of epidermal growth factor-stimulated STAT1/3 activation in A431 cells.

The SH2 domain protein-tyrosine phosphatase SHP-1 has been shown earlier to bind to the epidermal growth factor receptor and to have the capacity for receptor dephosphorylation. New bi- and tricistronic expression vectors (pNRTIS-21 and pNRTIS-33, respectively) based on the tetracycline system were constructed and employed to generate stable cell lines with inducible expression of SHP-1. Inducible overexpression of SHP-1 in A431 cells led to attenuation of epidermal growth factor (EGF) receptor autophosphorylation and of EGF-induced DNA binding of signal transducers and activators of transcription (STAT) 1 and 3. SHP-1 was localized in the cytoplasm with an enrichment in the perinuclear compartment. Association of SHP-1 with perinuclear structures may form the basis for a partial cofractionation with nuclei observed in different types of transfected cells and also with endogenous SHP-1 in U-937 cells. Treatment of SHP-1-overexpressing A431 cells or of HaCaT human keratinocytes expressing SHP-1 endogenously with the Ca2+-ionophore A23187 resulted in partial nuclear accumulation of SHP-1. Thus, SHP-1 may interact with substrates or regulatory proteins in perinuclear or nuclear structures.

Design of chimeric histone deacetylase- and tyrosine kinase-inhibitors: a series of imatinib hybrides as potent inhibitors of wild-type and mutant BCR-ABL, PDGF-Rbeta, and histone deacetylases.

Inhibitors of histone deacetylases are a new class of cancer therapeutics with possibly broad applicability. Combinations of HDAC inhibitors with the kinase inhibitor 1 (Imatinib) in recent studies showed additive and synergistic effects. Here we present a new concept by combining inhibition of protein kinases and HDACs, two independent pharmacological activities, in one synthetic small molecule. In general, the HDAC inhibition profile, the potencies, and the probable binding modes to HDAC1 and HDAC6 were similar as for 6 (SAHA). Inhibition of Abl kinase in biochemical assays was maintained for most compounds, but in general the kinase selectivity profile differed from that of 1 with nearly equipotent inhibition of the wild-type and the Imatinib resistant Abl T(315)I mutant. A potent cellular inhibition of PDGFR and cytotoxicity toward EOL-1 cells, a model for idiopathic hypereosinophilic syndrome (HES), are restored or enhanced for selected analogues (12b, 14b, and 18b). Cytotoxicity was evaluated by using a broad panel of tumor cell lines, with selected analogues displaying mean IC(50) values between 3.6 and 7.1 muM.

Dephosphorylation of p-ERK1/2 in relation to tumor remission after HER-2 and Raf1 blocking therapy in a conditional mouse tumor model.

Several studies have shown that HER‐2/neu (erbB‐2) blocking therapy strategies can cause tumor remission. However, the responsible molecular mechanisms are not yet known. Both ERK1/2 and Akt/PKB are critical for HER‐2‐mediated signal transduction. Therefore, we used a mouse tumor model that allows downregulation of HER‐2 in tumor tissue by administration of anhydrotetracycline (ATc). Switching‐off HER‐2 caused a rapid tumor remission by more than 95% within 7 d of ATc administration compared to the volume before switching‐off HER‐2. Interestingly, HER‐2 downregulation caused a dephosphorylation of p‐ERK1/2 by more than 80% already before tumor remission occurred. Levels of total ERK protein were not influenced. In contrast, dephosphorylation of p‐Akt occurred later, when the tumor was already in remission. These data suggest that in our HER‐2 tumor model dephosphorylation of p‐ERK1/2 may be more critical for tumor remission than dephosphorylation of p‐Akt. To test this hypothesis we used a second mouse tumor model that allows ATc controlled expression of BXB‐Raf1 because the latter constitutively signals to ERK1/2, but cannot activate Akt/PKB. As expected, downregulation of BXB‐Raf1 in tumor tissue caused a strong dephosphorylation of p‐ERK1/2, but did not decrease levels of p‐Akt. Interestingly, tumor remission after switching‐off BXB‐Raf1 was similarly efficient as the effect of HER‐2 downregulation, despite the lack of p‐Akt dephosphorylation. In conclusion, two lines of evidence strongly suggest that dephosphorylation of p‐ERK1/2 and not that of p‐Akt is critical for the rapid tumor remission after downregulation of HER‐2 or BXB‐Raf1 in our tumor model: (i) dephosphorylation of p‐ERK1/2 but not that of p‐Akt precedes tumor remission after switching‐off HER‐2 and (ii) downregulation of BXB‐Raf1 leads to a similarly efficient tumor remission as downregulation of HER‐2, although no p‐Akt dephosphorylation was observed after switching‐off BXB‐Raf1.

Predictive gene signatures for bevacizumab and cetuximab as well as cytotoxic agents.

©2012 Dustri-Verlag Dr. K. Feistle ISSN 0946-1965 DOI 10.5414/CPP50070 e-pub: December 28, 2011 Correspondence to Prof. Dr. Heinz-Herbert Fiebig Oncotest GmbH, Institute for Experimental Oncology, Am Flughafen 12 – 14, 79108 Freiburg, Germany Fiebig@Oncotest.de

Pyrrolo[3,4-c]-β-carboline-diones as a novel class of inhibitors of the platelet-derived growth factor receptor kinase

Members of the structurally diverse family of beta-carbolines have previously been shown to exhibit a wide range of biological activities. A novel synthetic strategy for generation of beta-carbolines was developed, allowing imido-beta-carbolines to be created in three steps from known compounds. The compounds were screened for inhibition of platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation in Swiss 3T3 fibroblasts. A number of the newly synthesized beta-carbolines with moderate to potent inhibitory activity were revealed. The most active derivative, 2,3-dihydro-8,9-dimethoxy-5-(2-methylphenyl)-1H,6H-pyrrolo[3, 4-c]pyrido¿3,4-bĭndole-1,3-dione 2ee, inhibited purified PDGF receptor kinase and PDGF-receptor autophosphorylation in intact cells with IC(50) values of 0.4 and 2.6 microM, respectively. Dione 2ee also inhibited PDGF-stimulated DNA synthesis in Swiss 3T3 fibroblasts with an IC(50) of 3.2 microM. The compound had no effect on Src or epidermal growth factor (EGF) receptor kinase activity and a six-seven-fold higher IC(50) for inhibition of basic fibroblast growth factor (bFGF)-stimulated tyrosine phosphorylation or Kit/stem cell factor (SCF) receptor autophosphorylation, indicating a reasonable extent of kinase specificity. Thus, beta-carbolines present a new lead of tyrosine kinase inhibitors with the capacity to selectively interfere with PDGF receptor signal transduction and PDGF-dependent cell growth.

Development of Tubulin Inhibitors as Antimitotic Agents for Cancer Therapy

ABSTRACT: Cancer represents one of the most threatening diseases of mankind. Within the last decade, our understanding of malignant cell growth and regulation of the cell cycle machinery has offered several new molecular targets with the promise of higher selectivity in human cancer therapy. Within mitosis, αβ-tubulin heterodimers, building up the mitotic spindle, are still an attractive target in the development of anticancer drugs. In the following article, we review the recent advances in the development of tubulin interfering agents. These agents are divided according to their mode of action into colchicine site binder, vinca -alkaloid related drugs and those interacting with the Taxol binding site and functioning as stabilising agents. Since clinically used compounds such as Paclitaxel or Vincristine are facing severe disadvantages, namely a small therapeutic window, restrictions in bioavailability and solubility, a complex synthesis and most importantly development of drug resistance in patients, special emphasis is laid on the development of synthetic small molecule tubulin inhibitors (SMTIs). These new agents offer promise for the rational design of new chemotherapeutic drugs by their simple structures and potential broad applicability in 2 nd and 3 rd line standard chemotherapy regimens towards resistant tumours. In this regard, most SMTIs are not P-glycoprotein substrates. Most tumours can only grow beyond a critical size by inducing the formation of new blood vessels, a process called neovascularisation. SMTIs as well as natural tubulin inhibitors have been described to interfere with this angiogenic process and some, like combretastatin A4 phosphate, are even described as selectively damaging tumour vasculatures. Finally, the present review emphasises the preclinical and clinical status of tubulin inhibitors in cancer therapy.

Chimeric tyrosine kinase-HDAC inhibitors as antiproliferative agents.

Combined treatment with tyrosine kinase inhibitors (TKi) and additional drugs is emerging as a promising strategy for cancer therapy. TKi and histone-deacetylase inhibitors (HDI) are two classes of anti-tumor agents with distant mechanisms of action. We have designed and synthesized chimeric compounds, which comprise structural elements of the TKi imatinib, and of prototypical HDI compounds. These compounds retain TKi activity similar to imatinib, exemplified by the inhibition of the platelet-derived growth factor receptor, and c-Kit kinase in intact cells. In addition, the chimeric compounds have in vitro and cellular HDI activity, and potently inhibit growth of cancer cell lines, including that of imatinib-resistant cell lines. Chimeric molecules with combined TKi and HDI activity may simplify combination treatment and be applicable to overcome clinical resistance to TKi single-agent therapy.