Ulrike Tontsch-Grunt

BIBF 1120: Triple Angiokinase Inhibitor with Sustained Receptor Blockade and Good Antitumor Efficacy

Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a novel treatment modality in oncology. Preclinical findings suggest that long-term clinical outcomes may improve with blockade of additional proangiogenic receptor tyrosine kinases: platelet-derived growth factor receptors (PDGFR) and fibroblast growth factor receptors (FGFR). BIBF 1120 is an indolinone derivative potently blocking VEGF receptor (VEGFR), PDGFR and FGFR kinase activity in enzymatic assays (IC(50), 20-100 nmol/L). BIBF 1120 inhibits mitogen-activated protein kinase and Akt signaling pathways in three cell types contributing to angiogenesis, endothelial cells, pericytes, and smooth muscle cells, resulting in inhibition of cell proliferation (EC(50), 10-80 nmol/L) and apoptosis. In all tumor models tested thus far, including human tumor xenografts growing in nude mice and a syngeneic rat tumor model, BIBF 1120 is highly active at well-tolerated doses (25-100 mg/kg daily p.o.), as measured by magnetic resonance imaging of tumor perfusion after 3 days, reducing vessel density and vessel integrity after 5 days, and inducing profound growth inhibition. A distinct pharmacodynamic feature of BIBF 1120 in cell culture is sustained pathway inhibition (up to 32 hours after 1-hour treatment), suggesting slow receptor off-kinetics. Although BIBF 1120 is rapidly metabolized in vivo by methylester cleavage, resulting in a short mean residence time, once daily oral dosing is fully efficacious in xenograft models. These distinctive pharmacokinetic and pharmacodynamic properties may help explain clinical observations with BIBF 1120, currently entering phase III clinical development.

Transcriptional plasticity promotes primary and acquired resistance to BET inhibition

Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL–AF9;NrasG12D-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.

Design, synthesis, and evaluation of indolinones as triple angiokinase inhibitors and the discovery of a highly specific 6-methoxycarbonyl-substituted indolinone (BIBF 1120).

Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a new treatment modality in oncology. Preclinical findings suggest that blockade of additional pro-angiogenic kinases, such as fibroblast and platelet-derived growth factor receptors (FGFR and PDGFR), may improve the efficacy of pharmacological cancer treatment. Indolinones substituted in position 6 were identified as selective inhibitors of VEGF-, PDGF-, and FGF-receptor kinases. In particular, 6-methoxycarbonyl-substituted indolinones showed a highly favorable selectivity profile. Optimization identified potent inhibitors of VEGF-related endothelial cell proliferation with additional efficacy on pericyctes and smooth muscle cells. In contrast, no direct inhibition of tumor cell proliferation was observed. Compounds 2 (BIBF 1000) and 3 (BIBF 1120) are orally available and display encouraging efficacy in in vivo tumor models while being well tolerated. The triple angiokinase inhibitor 3 is currently in phase III clinical trials for the treatment of nonsmall cell lung cancer.

Efficacy and mechanism of action of volasertib, a potent and selective inhibitor of Polo-like kinases, in preclinical models of acute myeloid leukemia

Polo-like kinase 1 (Plk1), a member of the Polo-like kinase family of serine/threonine kinases, is a key regulator of multiple steps in mitosis. Here we report on the pharmacological profile of volasertib, a potent and selective Plk inhibitor, in multiple preclinical models of acute myeloid leukemia (AML) including established cell lines, bone marrow samples from AML patients in short-term culture, and subcutaneous as well as disseminated in vivo models in immune-deficient mice. Our results indicate that volasertib is highly efficacious as a single agent and in combination with established and emerging AML drugs, including the antimetabolite cytarabine, hypomethylating agents (decitabine, azacitidine), and quizartinib, a signal transduction inhibitor targeting FLT3. Collectively, these preclinical data support the use of volasertib as a new therapeutic approach for the treatment of AML patients, and provide a foundation for combination approaches that may further improve and prolong clinical responses.

The triple angiokinase inhibitor nintedanib directly inhibits tumor cell growth and induces tumor shrinkage via blocking oncogenic receptor tyrosine kinases

The triple-angiokinase inhibitor nintedanib is an orally available, potent, and selective inhibitor of tumor angiogenesis by blocking the tyrosine kinase activities of vascular endothelial growth factor receptor (VEGFR) 1–3, platelet-derived growth factor receptor (PDGFR)-α and -β, and fibroblast growth factor receptor (FGFR) 1–3. Nintedanib has received regulatory approval as second-line treatment of adenocarcinoma non–small cell lung cancer (NSCLC), in combination with docetaxel. In addition, nintedanib has been approved for the treatment of idiopathic lung fibrosis. Here we report the results from a broad kinase screen that identified additional kinases as targets for nintedanib in the low nanomolar range. Several of these kinases are known to be mutated or overexpressed and are involved in tumor development (discoidin domain receptor family, member 1 and 2, tropomyosin receptor kinase A (TRKA) and C, rearranged during transfection proto-oncogene [RET proto oncogene]), as well as in fibrotic diseases (e.g., DDRs). In tumor cell lines displaying molecular alterations in potential nintedanib targets, the inhibitor demonstrates direct antiproliferative effects: in the NSCLC cell line NCI-H1703 carrying a PDGFRα amplification (ampl.); the gastric cancer cell line KatoIII and the breast cancer cell line MFM223, both driven by a FGFR2 amplification; AN3CA (endometrial carcinoma) bearing a mutated FGFR2; the acute myeloid leukemia cell lines MOLM-13 and MV-4-11-B with FLT3 mutations; and the NSCLC adenocarcinoma LC-2/ad harboring a CCDC6-RET fusion. Potent kinase inhibition does not, however, strictly translate into antiproliferative activity, as demonstrated in the TRKA-dependent cell lines CUTO-3 and KM-12. Importantly, nintedanib treatment of NCI-H1703 tumor xenografts triggered effective tumor shrinkage, indicating a direct effect on the tumor cells in addition to the antiangiogenic effect on the tumor stroma. These findings will be instructive in guiding future genome-based clinical trials of nintedanib.

The novel BET bromodomain inhibitor BI 894999 represses super-enhancer-associated transcription and synergizes with CDK9 inhibition in AML

Bromodomain and extra-terminal (BET) protein inhibitors have been reported as treatment options for acute myeloid leukemia (AML) in preclinical models and are currently being evaluated in clinical trials. This work presents a novel potent and selective BET inhibitor (BI 894999), which has recently entered clinical trials (NCT02516553). In preclinical studies, this compound is highly active in AML cell lines, primary patient samples, and xenografts. HEXIM1 is described as an excellent pharmacodynamic biomarker for target engagement in tumors as well as in blood. Mechanistic studies show that BI 894999 targets super-enhancer-regulated oncogenes and other lineage-specific factors, which are involved in the maintenance of the disease state. BI 894999 is active as monotherapy in AML xenografts, and in addition leads to strongly enhanced antitumor effects in combination with CDK9 inhibitors. This treatment combination results in a marked decrease of global p-Ser2 RNA polymerase II levels and leads to rapid induction of apoptosis in vitro and in vivo. Together, these data provide a strong rationale for the clinical evaluation of BI 894999 in AML.

Abstract B79: BI 894999, a novel BET inhibitor: Treatment of hematological malignancies by repression of super-enhancer driven oncogenes

Background: Bromodomain and extra-terminal (BET) protein inhibitors comprising the family members BRD2, 3, 4 and T are being extensively studied as treatment options in human haematological malignancies as well as in solid cancers. BRD4 is a key epigenetic regulator playing an important role in activating p-TEFb and governing expression of various oncogenes including MYC by contributing to multi-protein complexes forming so-called super-enhancers. BI 894999 is a novel, potent and selective orally bioavailable inhibitor of the BET family which has recently entered clinical trials. Results: Analysis of BI 8949999s activity in cell proliferation assays has revealed that it is a highly potent drug, particularly in hematological cell lines (n>40) such as MM, AML and lymphoma. Compound profiling using the BROMOscanTM assay revealed high selectivity for BRD2/3/4 and BRDT. On the cellular level, BI 894999 treatment leads to G1 arrest and subsequent apoptosis. Importantly, the compound also displayed excellent activity (low NM range) on 18/20 primary patient derived AML samples tested ex vivo. Potent tumor growth inhibition of this compound has been demonstrated in disseminated xenograft models of AML (MV-4-11, THP-1) and MM (MOLP-8), and in a transgenic model (Vk*MYC MM) as single agent or in combination with established and investigational therapeutic agents. When comparing the epigenetic regulator BI 894999 to other drugs, active in AML and also targeting chromatin pathways such as histone acetylation or DNA methylation, BI 894999 is found to regulate a very distinct set of genes. As already known, MYC expression and MYC target genes are affected by BET inhibitor treatment, as well as multiple other cancer relevant transcriptional signatures. HEXIM1, a negative regulator of p-TEFb was shown at RNA and protein level to be highly induced by BI 894999 in all cell lines tested so far and was verified as an excellent PD biomarker in extended PK/PD analyses. Detailed molecular analyses employing BRD4 ChIP-seq in MV-4-11 AML cells confirmed the published data that the strong repressive effect of BET inhibition on MYC expression is at least partly mediated by the displacement of BRD4 from a distant 39 MYC super-enhancer region. In addition, we have identified multiple other oncogene driving super-enhancers in AML cells. Conclusion: BI 894999 is a potent BET inhibitor which is currently being evaluated in clinical trials. This compound is highly active in MM and AML cell lines and patient samples, as well as in in vivotumor models. HEXIM1 was identified as a robust PD biomarker. Finally, we have demonstrated that BI 894999 is active by antagonizing super-enhancer driven oncogene expression such as MYC. Citation Format: Ulrike Tontsch-Grunt, Fabio Savarese, Daniel Gerlach, Davide Gianni, Anke Baum, Dirk Scharn, Harald Engelhardt, Onur Kaya, Norbert Schweifer, Thomas Gerstberger, Norbert Kraut. BI 894999, a novel BET inhibitor: Treatment of hematological malignancies by repression of super-enhancer driven oncogenes. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B79.

Abstract 1082: BI 811283, a potent inhibitor of the mitotic kinase Aurora B, shows dose- and schedule-dependent efficacy in human cancer xenograft models

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background. The serine/threonine kinase Aurora B is involved in the regulation of several mitotic processes, including chromosome condensation, congression and segregation as well as cytokinesis. The essential functions of Aurora B and its overexpression in many cancer types render this protein kinase an attractive target for therapeutic intervention. BI 811283, a potent inhibitor of this key mitotic regulator, inhibits proliferation of a wide range of cultured human cancer cells at low nanomolar concentrations by inducing polyploidy, senescence and apoptosis. Methods. BomTac:NMRI-Foxn1nu mice were grafted subcutaneously with NCI-H460 non-small cell lung carcinoma (mutant KRAS, wild-type p53), HCT 116 colon carcinoma (mutant KRAS, wild-type p53) or BxPC-3 pancreas adenocarcinoma cells (wild-type KRAS, mutant p53). Treatment was initiated when the tumors had reached a volume of ∼50 mm3. BI 811283 was injected intravenously once or twice weekly as a single bolus or b.i.d. Alternatively, the compound was administered once-weekly by a continuous 24 h infusion via subcutaneously implanted osmotic mini-pumps. Multiple dose levels and dosing schedules were evaluated. Results. In models of human non-small cell lung cancer, colon carcinoma and pancreas carcinoma, multiple cycles of treatment with BI 811283 at total weekly doses of 20 to 75 mg/kg resulted in dose-dependent inhibition of tumor growth or tumor regression. Continuous s.c. infusion at 20 mg/kg over 24 h once-weekly was clearly superior to all bolus injection schedules delivering weekly doses up to 75 mg/kg. Furthermore, regression of large tumors (350 mm3) was induced in the HCT 116 colon carcinoma model. Biomarker analyses of HCT 116 tumors revealed that therapeutic doses of BI 811283 inhibited phosphorylation of histone H3, a direct substrate of Aurora B. Histological examination showed an accumulation of enlarged, multinucleated cells in accordance with the expected mechanism of action. Conclusions. BI 811283 has demonstrated potent antitumor activity in multiple cancer models at well-tolerated doses; treated tumors show hallmarks of Aurora B inhibition. Continuous infusion over 24 h provides a superior therapeutic index compared with bolus administration. The compound is currently under investigation in Phase I clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1082.

Abstract 1080: Molecular and cellular pharmacology of BI 811283, a potent inhibitor of Aurora B kinase

Background. The serine/threonine kinase Aurora B is involved in the regulation of several mitotic processes, including chromosome condensation, congression and segregation as well as cytokinesis. These essential functions of Aurora B and its overexpression in many cancer types render this protein kinase an attractive target for anticancer drug development. Methods. BI 811283 was profiled in enzymatic kinase assays as well as in proliferation assays on various human cancer cell lines. Cell cycle status was assessed by DNA content analysis (Cellomics ArrayScan, FACScalibur). Histone H3 phosphorylation was determined by immunofluorescence (Cellomics ArrayScan). Apoptosis was detected by Western blotting for cleaved PARP and microscopic enumeration of DAPI-stained cells showing nuclear fragmentation. Senescent cells were identified by staining for SA-s-Gal activity. Results. BI 811283 inhibited human Aurora B kinase activity with an IC 50 value of 9 nM, Aurora A and C kinases with 70 nM and 17 nM, respectively. In a panel of 46 additional kinases representative of the human kinome, BI 811283 at 1000 nM inhibited 7/46 kinases by more than 50%. EC 50 values for inhibition of proliferation of >20 human cancer cell lines were in the range of 2 to 14 nM. In the non-small cell lung cancer cell line NCI-H460, treatment with BI 811283 resulted in a rapid ( 80%, paralleled by a marked increase in cell volume. An increase of cleaved poly (ADP-ribose) polymerase and a concomitant increase in the fraction of cells with nuclear fragmentation from Conclusions. BI 811283 is a potent and selective Aurora kinase inhibitor that inhibits proliferation of cancer cells independent of tissue origin or oncogenome status. Treated cells exhibit a polyploid phenotype characteristic for Aurora B inhibition and show hallmarks of senescence as well as a slow onset of apoptosis in a small fraction of cells. In vivo activity of BI 811283 has been demonstrated in multiple cancer xenograft models in nude mice (see accompanying poster). Phase I clinical trials are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1080.

Pharmacological Profile of BI 847325, an Orally Bioavailable, ATP-Competitive Inhibitor of MEK and Aurora Kinases.

Although the MAPK pathway is frequently deregulated in cancer, inhibitors targeting RAF or MEK have so far shown clinical activity only in BRAF- and NRAS-mutant melanoma. Improvements in efficacy may be possible by combining inhibition of mitogenic signal transduction with inhibition of cell-cycle progression. We have studied the preclinical pharmacology of BI 847325, an ATP-competitive dual inhibitor of MEK and Aurora kinases. Potent inhibition of MEK1/2 and Aurora A/B kinases by BI 847325 was demonstrated in enzymatic and cellular assays. Equipotent effects were observed in BRAF-mutant cells, whereas in KRAS-mutant cells, MEK inhibition required higher concentrations than Aurora kinase inhibition. Daily oral administration of BI 847325 at 10 mg/kg showed efficacy in both BRAF- and KRAS-mutant xenograft models. Biomarker analysis suggested that this effect was primarily due to inhibition of MEK in BRAF-mutant models but of Aurora kinase in KRAS-mutant models. Inhibition of both MEK and Aurora kinase in KRAS-mutant tumors was observed when BI 847325 was administered once weekly at 70 mg/kg. Our studies indicate that BI 847325 is effective in in vitro and in vivo models of cancers with BRAF and KRAS mutation. These preclinical data are discussed in the light of the results of a recently completed clinical phase I trial assessing safety, tolerability, pharmacokinetics, and efficacy of BI 847325 in patients with cancer. Mol Cancer Ther; 15(10); 2388–98. ©2016 AACR.