Jürgen Moll

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.

RHAMM deficiency disrupts folliculogenesis resulting in female hypofertility

The postnatal mammalian ovary contains the primary follicles, each comprising an immature oocyte surrounded by a layer of somatic granulosa cells. Oocytes reach meiotic and developmental competence via folliculogenesis. During this process, the granulosa cells proliferate massively around the oocyte, form an extensive extracellular matrix (ECM) and differentiate into cumulus cells. As the ECM component hyaluronic acid (HA) is thought to form the backbone of the oocyte-granulosa cell complex, we deleted the relevant domain of the Receptor for HA Mediated Motility (RHAMM) gene in the mouse. This resulted in folliculogenesis defects and female hypofertility, although HA-induced signalling was not affected. We report that wild-type RHAMM localises at the mitotic spindle of granulosa cells, surrounding the oocyte. Deletion of the RHAMM C-terminus in vivo abolishes its spindle association, resulting in impaired spindle orientation in the dividing granulosa cells, folliculogenesis defects and subsequent female hypofertility. These data reveal the first identified physiological function for RHAMM, during oogenesis, and the importance of this spindle-associated function for female fertility.

Spindle Misorientation of Cerebral and Cerebellar Progenitors Is a Mechanistic Cause of Megalencephaly

Summary Misoriented division of neuroprogenitors, by loss-of-function studies of centrosome or spindle components, has been linked to the developmental brain defects microcephaly and lissencephaly. As these approaches also affect centrosome biogenesis, spindle assembly, or cell-cycle progression, the resulting pathologies cannot be attributed solely to spindle misorientation. To address this issue, we employed a truncation of the spindle-orienting protein RHAMM. This truncation of the RHAMM centrosome-targeting domain does not have an impact on centrosome biogenesis or on spindle assembly in vivo. The RHAMM mutants exhibit misorientation of the division plane of neuroprogenitors, without affecting the division rate of these cells, resulting against expectation in megalencephaly associated with cerebral cortex thickening, cerebellum enlargement, and premature cerebellum differentiation. We conclude that RHAMM associates with the spindle of neuroprogenitor cells via its centrosome-targeting domain, where it regulates differentiation in the developing brain by orienting the spindle.

The IGF1R/INSR Inhibitor BI 885578 Selectively Inhibits Growth of IGF2-Overexpressing Colorectal Cancer Tumors and Potentiates the Efficacy of Anti-VEGF Therapy

Clinical studies of pharmacologic agents targeting the insulin-like growth factor (IGF) pathway in unselected cancer patients have so far demonstrated modest efficacy outcomes, with objective responses being rare. As such, the identification of selection biomarkers for enrichment of potential responders represents a high priority for future trials of these agents. Several reports have described high IGF2 expression in a subset of colorectal cancers, with focal IGF2 amplification being responsible for some of these cases. We defined a novel cut-off value for IGF2 overexpression based on differential expression between colorectal tumors and normal tissue samples. Analysis of two independent colorectal cancer datasets revealed IGF2 to be overexpressed at a frequency of 13% to 22%. An in vitro screen of 34 colorectal cancer cell lines revealed IGF2 expression to significantly correlate with sensitivity to the IGF1R/INSR inhibitor BI 885578. Furthermore, autocrine IGF2 constitutively activated IGF1R and Akt phosphorylation, which was inhibited by BI 885578 treatment. BI 885578 significantly delayed the growth of IGF2-high colorectal cancer xenograft tumors in mice, while combination with a VEGF-A antibody increased efficacy and induced tumor regression. Besides colorectal cancer, IGF2 overexpression was detected in more than 10% of bladder carcinoma, hepatocellular carcinoma and non-small cell lung cancer patient samples. Meanwhile, IGF2-high non-colorectal cancer cells lines displayed constitutive IGF1R phosphorylation and were sensitive to BI 885578. Our findings suggest that IGF2 may represent an attractive patient selection biomarker for IGF pathway inhibitors and that combination with VEGF-targeting agents may further improve clinical outcomes. Mol Cancer Ther; 16(10); 2223–33. ©2017 AACR.

Abstract 4558: In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091

The programmed cell death-1 (PD-1) receptor provides inhibitory checkpoint signals to activated T cells upon binding to its ligands, PD-L1 and PD-L2, which are expressed on antigen-presenting cells and cancer cells leading to suppression of T-cell effector function and tumor immune evasion. Blockade of the PD-1 axis using either anti-PD-1 or anti-PD-L1 approved monoclonal antibodies (mAbs) results in improved T-cell effector function and anti-tumor immune responses. Durable tumor responses occur in 15-30% of cancer patients. BI 754091, a humanized IgG4 mAb with high affinity against hPD-1 blocks the interaction between PD-1 and PD-L1 or PD-L2. BI 754091 was characterized in a panel of binding, blocking and functional cell-based assays. In addition, efficacy and safety was assessed in mice and in cynomolgus monkeys, respectively. The ability of BI 754091 to stimulate cytokine production in exhausted human T cells in vitro was tested in an autologous assay system with antigen-specific memory CD4+ T cells being re-stimulated by antigen-pulsed dendritic cells in the presence of BI 754091 or isotype control. Under these assay conditions the majority of T cells co-expressed the exhaustion markers PD-1 and LAG-3 on their surface. Furthermore, PD-L1 and PD-L2 were expressed on the dendritic cells. At the end of the experiment supernatants were harvested and analyzed for IFNγ secretion as a measure for T-cell activation. BI 754091 showed a potent dose dependent T-cell activation. The average fold increase of IFNγ was 7.9 as compared to isotype control, with an average EC50 of 0.9 nM. The in vivo activity of BI 754091 was determined in MC-38 tumor-bearing mice, using a mouse strain where parts of the extracellular domain of murine PD-1 was replaced by the corresponding human PD-1 domain (C57BL/6NTac-PDCD1tm(PDCD1)Arte mice). A dose of 10 mg/kg BI 754091, given either as single treatment or in a twice weekly schedule, induced significant tumor growth inhibition (median TGI of 83% and 90%, respectively) and complete responses (CRs) in some tumors (3 CRs out of 10 and 2 CRs out of 10, respectively). BI 754091 binds to PD-1 from cynomolgus monkeys with comparable affinities as to human PD-1, thus allowing pharmacokinetic and toxicological assessment in this species. Repeated high doses of BI 754091 were well tolerated without adverse immune-related effects. BI 754091 is currently undergoing clinical investigations (NCT02952248). Citation Format: Markus Zettl, Melanie Wurm, Otmar Schaaf, Inigo Tirapu, Sven Mostbock, Markus Reschke, Stephan-Michael Schmidbauer, Lee Frego, Ivo C. Lorenz, Michael Thibodeau, Diann Blanset, Elisa Oquendo Cifuentes, Jurgen Moll, Norbert Kraut, Eric Borges, Anne Vogt, Jonathon Sedgwick, Irene C. Waizenegger. In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091 [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 4558.

Abstract 4630: Development of selective and potent CDK8 inhibitors that increase NK cell activity, which translates in tumor surveillance

Background: Cyclin-dependent kinase 8 (CDK8) is part of the mediator complex that can either positively or negatively influence transcription. CDK8 is known to phosphorylate signal transducer and activator of transcription 1 (STAT1) at the position Ser727. STAT1 activity is regulated by JAK-mediated phosphorylation of tyrosine701 which leads to dimerization, nuclear translocation and IFN-γ induced phosphorylation mediated by CDK8. Introduction of an alanine mutation at the phosphorylation site STAT1-S727 results in enhanced NK cell cytotoxicity accompanied by increased levels of perforin and granzyme B (Putz et al. 2013). Method: Here we present the discovery and development of potent and selective CDK8 inhibitors guided by crystallography. The inhibitory effect of optimized compounds BI 9811 and BI 1347 on STAT1 phosphorylation and perforin release was investigated in the human NK cell line NK-92MI. Direct effects on cancer cells were furthermore analyzed in a broad panel of cell lines. The compound BI 1347 was profiled in vivo in the orthotopic B16-F10 melanoma mouse model. Results: Highly potent and selective CDK8 inhibitors were identified with an IC50 of below 10 nM in a biochemical kinase assay, which translated in a potent down regulation of the STAT1- Ser727 signal and in increased perforin and granzyme B secretion. BI 9811 and BI 1347 were highly selective for CDK8, as tested in a broad kinase panel and showed no cytotoxic activity on NK cells and most cancer cell lines, which distinguishes this compound class from published CDK8 inhibitors. A representative molecule out of this compound class demonstrated in vivo biomarker modulation and survival increase in the murine B16-F10 melanoma mouse model. Conclusion: We developed potent CDK8 inhibitors that show activation of NK cells that translates into biomarker modulation (pSTAT1Ser727) and in vivo efficacy. Citation Format: Marco H. Hofmann, Harald Engelhardt, Sebastian Carotta, Heribert Arnhof, Dirk Scharn, Marc Kerenyi, Moritz Mayer, Gerhard Gmaschitz, Georg Egger, Christian Engelhardt, Michael Sanderson, Maria A. Impagnatiello, Renate Schnitzer, Mark Pearson, Darryl McConnell, Norbert Kraut, Jurgen Moll. Development of selective and potent CDK8 inhibitors that increase NK cell activity, which translates in tumor surveillance [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 4630. doi:10.1158/1538-7445.AM2017-4630

Abstract B241: Targeting the mitotic checkpoint with NMS-P153, a novel MPS1 kinase inhibitor.

Aneuploidy is one of the major hallmarks of cancer and is a unique property of tumoral cells. Thus, targeting the molecular mechanisms required for the growth of aneuploid cells might be expected to yield more cancer cell specific therapeutic approaches. The Spindle Assembly Checkpoint (SAC) is a mitotic mechanism required for proper chromosomal segregation, ensuring that cells do not divide until all sister chromatids correctly align to the metaphase plate. It is a complex machinery tightly regulated by a number of members which are rarely mutated in tumors. Rather, checkpoint components have been found to be upregulated in aneuploid tumor cells, suggesting a potential tumor “addiction” to SAC functions. MPS1 kinase, (also known as TTK) is an essential SAC enzyme aberrantly overexpressed in a wide range of tumors and necessary for tumor cell proliferation. We previously reported the identification of NMS-P715 as a selective and orally bioavailable MPS1 inhibitor capable of promoting mitotic acceleration, thereby causing aneuploidy and cancer cell death both in vitro and in vivo. Supported by the proof that MPS1 small-molecule inhibitors display the expected mechanism of action and promote tumor growth inhibition in preclinical cancer models, further medicinal chemistry efforts were undertaken and led to the discovery of NMS-P153 as an early candidate product. NMS-P153 is highly selective, and compared to NMS-P715, shows improved in vitro enzymatic potency on MPS1 (IC50 of 3 nM), 10-fold increased mitotic override activity, longer dissociation half-life and better mouse and rat PK parameters, leading to impressive tumor growth inhibition associated with tumor regression in efficacy studies. Proliferation data performed on a large panel of cell lines (>100) have been obtained and will be essential to decipher the most sensitive genetic backgrounds to chemical abrogation of MPS1 function. In addition, single and repeated toxicology studies in rat are being carried out to establish therapeutic safety margins. Inhibiting the spindle assembly checkpoint by targeting MPS1 kinase could represent a promising new approach to cancer therapy. 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 B241.