Maria Impagnatiello

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.

Abstract 2330: BI5: a novel SMAC mimetic that triggers tumor cell death and potentiates PD-1 mediated cancer therapy

Background: Inhibitors of apoptosis proteins (IAPs) regulate cellular apoptosis by interfering with the proteolytic activities of caspases. IAP inhibitors (SMAC mimetics) have been developed to restore the defective apoptosis that characterizes many tumour cells. Emerging evidence demonstrates that IAPs are critical components of immune-modulatory pathways that control innate and adaptive immunity. Accordingly, SMAC mimetics hold the promise of both inducing tumour cell killing and stimulating the immune system to recognize and eliminate dying tumour cells. Here we show that BI5 primes immune components and synergises with PD-1 checkpoint inhibitors to promote eradication of syngeneic tumors. Methods: Here we report the efficacy and modulation of the immune response by a potent and selective SMAC mimetic, BI5. We characterised the effect of BI5 on tumor growth inhibition as a single agent and in combination with an anti-PD-1 antibody in syngeneic mouse tumor models. A detailed 17-colour multi-color flow cytometry analysis was used to investigate the mechanisms by which the SMAC mimetic interacts with anti-PD-1 therapy in vivo. Results: Treatment of the syngeneic mouse tumor models MBT-2 and EMT-6 with the SMAC mimetic in combination with an anti-PD-1 antibody results in remarkable tumor regressions in vivo. Importantly, the combined effect of the SMAC mimetic and anti-PD-1 on tumor growth was dependent on the adaptive immune system in vivo. Mechanistic studies show that degradation of IAP triggers tumor cell death, which leads to a potent activation of dendritic cells in the draining lymph nodes and a subsequent influx of T and NK cells into the tumor microenvironment. Interestingly, in the presence of the SMAC mimetic alone, an induction of PD-1 expression on tumor-infiltrating CD8+ T cells was observed, which in turn resulted in the exhaustion of these cells and tumor outgrowth. In the presence of the anti-PD-1 antibody, T cells are reactivated leading to potent and long term tumor eradication. Conclusion: We show that our SMAC mimetic leads to a potent induction of immunogenic cell death and sets up a “virtuous cycle” by potentiating dendritic cell and T cell mediated immune responses that further promote induction of cell death. These effects are potentiated by checkpoint inhibitors, leading to long term tumor control. Tumours with minimal T-cell infiltration are poorly responsive to PD-1 monotherapy. These studies indicate that SMAC mimetics, such as BI5, represent promising and tolerated combination partners for checkpoint inhibitors in patients that lack a strong immune inflammatory signature. Citation Format: Markus Reschke, Maria Antonietta Impagnatiello, Ulrich Reiser, Dirk Scharn, Walter Spevak, Alexander Savchenko, Andreas Wernitznig, Martina Sykora, Rebecca Langlois, Elisabeth Zier, Daniel Zach, Sabine Kallenda, Pilar Garin-Chesa, Jens Quant, Mark Pearson, Darryl McConnell, Norbert Kraut, Juergen Moll. BI5: a novel SMAC mimetic that triggers tumor cell death and potentiates PD-1 mediated cancer therapy [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 2330. doi:10.1158/1538-7445.AM2017-2330

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