Gerhard Siemeister

Improved Cellular Pharmacokinetics and Pharmacodynamics Underlie the Wide Anticancer Activity of Sagopilone

Sagopilone (ZK-EPO) is the first fully synthetic epothilone undergoing clinical trials for the treatment of human tumors. Here, we investigate the cellular pathways by which sagopilone blocks tumor cell proliferation and compare the intracellular pharmacokinetics and the in vivo pharmacodynamics of sagopilone with other microtubule-stabilizing (or tubulin-polymerizing) agents. Cellular uptake and fractionation/localization studies revealed that sagopilone enters cells more efficiently, associates more tightly with the cytoskeleton, and polymerizes tubulin more potently than paclitaxel. Moreover, in contrast to paclitaxel and other epothilones [such as the natural product epothilone B (patupilone) or its partially synthetic analogue ixabepilone], sagopilone is not a substrate of the P-glycoprotein efflux pumps. Microtubule stabilization by sagopilone caused mitotic arrest, followed by transient multinucleation and activation of the mitochondrial apoptotic pathway. Profiling of the proapoptotic signal transduction pathway induced by sagopilone with a panel of small interfering RNAs revealed that sagopilone acts similarly to paclitaxel. In HCT 116 colon carcinoma cells, sagopilone-induced apoptosis was partly antagonized by the knockdown of proapoptotic members of the Bcl-2 family, including Bax, Bak, and Puma, whereas knockdown of Bcl-2, Bcl-X(L), or Chk1 sensitized cells to sagopilone-induced cell death. Related to its improved subcellular pharmacokinetics, however, sagopilone is more cytotoxic than other epothilones in a large panel of human cancer cell lines in vitro and in vivo. In particular, sagopilone is highly effective in reducing the growth of paclitaxel-resistant cancer cells. These results underline the processes behind the therapeutic efficacy of sagopilone, which is now evaluated in a broad phase II program.

Characterization of novel MPS1 inhibitors with preclinical anticancer activity

Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A synergistic interaction between paclitaxel and MPS1 inhibitors could also be demonstrated in vivo, as the combination of these agents efficiently reduced the growth of tumor xenografts and exerted superior antineoplastic effects compared with either compound employed alone. Altogether, these results suggest that MPS1 inhibitors may exert robust anticancer activity, either as standalone therapeutic interventions or combined with microtubule-targeting chemicals.

The oral multitarget tumour growth inhibitor, ZK 304709, inhibits growth of pancreatic neuroendocrine tumours in an orthotopic mouse model

Background and aims: Current systemic therapies for neuroendocrine tumours (NETs) do not provide sufficient control of tumour growth. However, efficient evaluation of novel drugs is hindered by the lack of a suitable preclinical animal model. Here an orthotopic mouse model of pancreatic NET is established and used to study the action of ZK 304709, a first in class, oral multitarget tumour growth inhibitor. ZK 304709 is an inhibitor of cyclin-dependent kinases (Cdks) 1, 2, 4, 7 and 9, vascular endothelial growth factor receptor-type kinases (VEGF-RTKs) 1–3 and platelet-derived growth factor receptor-type kinase β (PDGF-RTKß). Methods: BON and QGP-1 human NET cells were used to study proliferation, survival and cell cycle distribution in vitro. For induction of orthotopic NETs, BON cells were injected into the pancreas of NMRInu/nu mice. Primary tumour growth and metastatic spread were recorded after 9 weeks, and apoptosis, microvessel density and lymphatic vessel density were determined. Results: ZK 304709 dose-dependently suppressed proliferation and colony formation of NET cells. Direct effects on NET cells were consistent with Cdk inhibition and involved G2 cell cycle arrest and apoptosis induction, which was associated with reduced expression of MCL1 (myeloid cell leukaemia sequence 1), survivin and hypoxia-inducible factor 1α (HIF1α). Apoptosis similarly occurred in vivo in ZK 304709-treated orthotopic BON tumours, resulting in a 80% reduction of primary tumour growth. In contrast, treatment with lanreotide or 5-fluorouracil and streptozotocin failed to inhibit tumour gowth. ZK 304709 also reduced tumour microvessel density, implicating antiangiogenic mechanisms. Conclusion: BON orthotopic tumours provide an informative model for preclinical drug evaluation in NETs. In this model, ZK 304709 achieved efficacious tumour growth control via induction of apoptosis and inhibition of tumour-induced angiogenesis.

Identification of Atuveciclib (BAY 1143572), the First Highly Selective, Clinical PTEFb/CDK9 Inhibitor for the Treatment of Cancer

Selective inhibition of exclusively transcription‐regulating PTEFb/CDK9 is a promising new approach in cancer therapy. Starting from lead compound BAY‐958, lead optimization efforts strictly focusing on kinase selectivity, physicochemical and DMPK properties finally led to the identification of the orally available clinical candidate atuveciclib (BAYu20051143572). Structurally characterized by an unusual benzyl sulfoximine group, BAYu20051143572 exhibited the best overall profile inu2005vitro and inu2005vivo, including high efficacy and good tolerability in xenograft models in mice and rats. BAYu20051143572 is the first potent and highly selective PTEFb/CDK9 inhibitor to enter clinical trials for the treatment of cancer.

Abstract 321: Synergistic activity of the ATR inhibitor BAY 1895344 in combination with DNA damage inducing and DNA repair compromising therapies in preclinical tumor models

The DNA damage response (DDR) system consists of complex signalling pathways that secure the integrity of the genome in eukaryotic cells. DDR pathway activation follows recognition of DNA damage and results in cell cycle arrest, suppression of general translation, induction of DNA repair, cell survival or even cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR, such as ATR (ataxia telangiectasia and Rad3-related). ATR responds to a broad spectrum of DNA damages, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress. Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in tumors with increased DNA damage, deficiency in DDR or replication stress. The potential of combining ATR kinase inhibitor with DNA damage inducing or DNA repair compromising anti-cancer therapeutics was studied in preclinical tumor models. We assessed the novel ATR kinase inhibitor (ATRi) BAY 1895344 in combination with external beam radiation therapy (EBRT), poly ADP ribose polymerase (PARP) inhibition or anti-androgen (AA) therapy. In cellular anti-proliferation assays as well as in tumor xenograft studies we could demonstrate synergistic activity of BAY 1895344 in combination treatment with the PARP inhibitor AZD-2281 in the homologous recombination (HR) defective breast cancer model MDA-MB-436, and with the non-steroidal AA darolutamide in the hormone-dependent prostate cancer model LAPC-4. Strong synergistic anti-tumor activity of BAY 1895344 could be further demonstrated in combination with EBRT inducing long-lasting tumor growth inhibition in the colorectal cancer xenograft model LOVO. The mechanism-based potential of combining DNA damage induction by EBRT with ATRi BAY 1895344 suggests a potential new treatment option for radiation therapy-resistant patients. Furthermore, the inhibition of parallel DDR pathways, as a combination of ATRi BAY 1895344 with a PARP inhibitor, indicates novel treatment opportunities in breast cancer patients with homologous recombination deficiencies, as does the synergism of BAY 1895344 and AA darolutamide therapy in hormone-dependent prostate cancer patients. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965). Citation Format: Antje Margret Wengner, Gerhard Siemeister, Ulrich Luecking, Julien Lefranc, Kirstin Meyer, Eleni Lagkadinou, Bernard Haendler, Pascale Lejeune, Dominik Mumberg. Synergistic activity of the ATR inhibitor BAY 1895344 in combination with DNA damage inducing and DNA repair compromising therapies in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 321.