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Acute myeloid leukemia (AML), the most common acute leukemia in adults, is an aggressive hematologic malignancy resulting in bone marrow failure with a poor outcome; overall survival is approximately 25% at five years. Treatment options, in particular for the elderly population, are limited. Induction chemotherapy of cytarabine and an anthracycline (7+3) remains unchanged standard of care since its introduction in the early 1970s and there is a high medical need for new therapies (Yates et al. Cancer Chemother Rep 1973). DHODH is a key enzyme in the de novo pyrimidine synthesis converting dihydroorotate to orotate. Using a HOXA9 driven phenotypic screen to overcome differentiation arrest in myeloid cells, we have recently identified DHODH as a surprising novel target to overcome differentiation blockade in AML (Sykes et al. Cell 2016). Differentiation therapy already showed its enormous clinical benefit potential in the small subset of patients diagnosed with acute promyelocytic leukemia (APL) following treatment with all-trans retinoic acid with five-year survival exceeding 85% and should be considered the ultimate therapeutic goal for all AML subsets (Lo-Coco et al. NEJM 2013). Here, we disclose for the first time the structure and functional characterization of the novel DHODH inhibitor BAY 2402234. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro, it potently inhibits proliferation of AML cell lines in the sub-nanomolar to low-nanomolar range. BAY 2402234 induces differentiation of AML cell lines also in a sub-nanomolar to low-nanomolar range, demonstrating the anticipated mode of action in cellular mechanistic assays. In vivo, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in several subcutaneous and disseminated AML xenografts as well as AML patient-derived xenograft (PDX) models. Target engagement of the novel DHODH inhibitor BAY 2402234 can be observed by increase of tumoral and plasma dihydroorotate levels after treatment with the inhibitor. Consistent with the in vitro data BAY 2402234 induces AML differentiation in vivo as detected by upregulation of differentiation cell surface markers in xenograft and PDX models after treatment with the inhibitor. Furthermore, differentiation-associated transcriptomic changes were evident following a single administration of BAY 2402234 in vivo. The start of clinical investigations of BAY 2402234 is planned for early 2018. Citation Format: Andreas Janzer, Stefan Gradl, Sven Christian, Katja Zimmermann, Claudia Merz, Hanna Meyer, Timo Stellfeld, Judith Guenther, Detlef Stoeckigt, Henrik Seidel, Pascale Lejeune, Michael Bruening, Ashley Eheim, Thomas Mueller, Ralf Lesche, Martin Michels, Andrea Haegebarth, Marcus Bauser, Sherif El Sheikh, Steven Ferrara, David Sykes, David Scadden. BAY 2402234: A novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of myeloid malignancies [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 DDT02-04.

Immune checkpoint blockade using antibodies targeting the cell surface expressed proteins CTLA-4, PD-1 and PD-L1 has revolutionized cancer care and its clinical impact in several indications has prompted a search for complementary immunostimulatory approaches that can further increase the efficacy of these drugs. Mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1; HPK1), a serine/threonine kinase expressed exclusively in hematopoietic cell lineages, mediates a negative feedback signal downstream of T-cell receptor stimulation. Its activity is enhanced by PGE2 and TGFβ, factors commonly present in the tumor microenvironment. Mice deficient for MAP4K1 or expressing a kinase-dead variant of MAP4K1 exhibit enhanced T-cell function, including increased anti-tumor immunity. We developed the small molecule inhibitor BAY-405 that displays potent nanomolar MAP4K1 inhibition in biochemical and cellular assays, good kinase selectivity, and in vivo exposure after oral dosing. Pharmacological inhibition of MAP4K1 enhances T-cell immunity and overcomes the suppressive impact of PGE2, TGFβ and CD4+ T-regulatory cells. Single agent treatment of tumor-bearing mice results in suppression of tumor outgrowth in several syngeneic models. This is accompanied by an increase in the anti-tumor T-cell response, dependent on an intact T-cell compartment, while not involving direct anti-tumor cytotoxicity. Inhibition of MAP4K1 in conjunction with PD-L1 blockade results in further suppression of tumor outgrowth. Moreover, we found that MAP4K1 is expressed in both PD-L1-high and PD-L1-low human cancers. In summary, our data show that selective inhibition of MAP4K1 by means of small molecule drugs may be used to expand the patient population responding to immune checkpoint inhibition. Citation Format: Gabriele Leder, Rafael Carretero, Jeffrey Mowat, Sandra Berndt, Roland Neuhaus, Nuria Aiguabella Font, Ulf Boemer, Oliver von Ahsen, Uwe Eberspaecher, Judith Guenther, Mareike Grees, Corinna Link, Barbara Nicke, Daniel Baumann, Martina Schaefer, Mine Oezcan-Wahlbrink, Nicolas D. Werbeck, Ingo Hartung, Bertolt Kreft, Rienk Offringa. Enhancement of anti-tumor T-cell immunity by means of an oral small molecule targeting the intracellular immune checkpoint MAP4K1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1722.

MTH1 is a hydrolase responsible for sanitization of oxidized purine nucleoside triphosphates to prevent their incorporation into replicating DNA. Early tool compounds published in the literature inhibited the enzymatic activity of MTH1 and subsequently induced cancer cell death; however recent studies have questioned the reported link between these two events. Therefore, it is important to validate MTH1 as a cancer dependency with high quality chemical probes. Here, we present BAY-707, a substrate-competitive, highly potent and selective inhibitor of MTH1, chemically distinct compared to those previously published. Despite superior cellular target engagement and pharmacokinetic properties, inhibition of MTH1 with BAY-707 resulted in a clear lack of in vitro or in vivo anticancer efficacy either in mono- or in combination therapies. Therefore, we conclude that MTH1 is dispensable for cancer cell survival.

DHODH is a key enzyme in the biosynthesis of pyrimidines and recent studies have renewed interest in this old anti-cancer target. Here, we disclose the discovery of 4-triazolosalicylamides as inhibitors of DHODH and their structure activity relationship (SAR). The hit cluster was discovered during a phenotypic high throughput screen (HTS) of 2.5 million compounds where proliferation of H460 lung cancer cells was used as read-out. DHODH was successfully identified as the molecular target by comparing the activity profile of the hits in a panel of cell lines to a set of inhibitors with known pharmacological activity. The hit compounds showed good cellular potency but had undesirable DMPK properties. Interestingly, the compounds are non-ionizable in contrast to many other DHODH inhibitors and show no potency shift from biochemical to cellular assays. Structural modifications lead to compounds with sub-nanomolar potency in cellular assays and increased metabolic stability enabling the proof of concept in vivo xenograft experiments. Further optimization guided by lipophilicity efficiency and identification of metabolic hot spots resulted in molecules with low clearance and improved solubility. BAY 2402234 was selected as the clinical candidate after side by side comparison of a number of promising compounds. It shows great oral bioavailability, target engagement in all preclinical species tested, induces differentiation in AML models, and has excellent activity in a variety of leukemia models. A clinical phase I study has been initiated in patients with myeloid malignancies. (NCT03404726) Citation Format: Stefan N. Gradl, Thomas Mueller, Steven Ferrara, Sherif El Sheikh, Andreas Janzer, Han-Jie Zhou, Anders Friberg, Judith Guenther, Martina Schaefer, Timo Stellfeld, Knut Eis, Michael Kroeber, Duy Nguyen, Claudia Merz, Michael Niehues, Detlef Stoeckigt, Sven Christian, Katja Zimmermann, Pascal Lejeune, Michael Bruening, Hanna Meyer, Vera Puetter, David T. Scadden, David B. Sykes, Henrik Seidel, Ashley Eheim, Martin Michels, Andrea Haegebarth, Marcus Bauser. Discovery of BAY 2402234 by phenotypic screening: A human Dihydroorotate Dehydrogenase (DHODH) inhibitor in clinical trials for the treatment of myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2.

Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is frequently characterized by aberrant activation of both B-Cell Receptor (BCR) & TLR/MYD88 signaling pathways. Constitutive BCR signaling via Bruton's tyrosine kinase (BTK) and PI3K pathways leads to downstream activation of NF-κB and AKT signaling. In addition, IRAK4 mediated activation of the TLR/MYD88 pathway further activates NF-κB signaling and pro-survival pathways. Simultaneous blockade of TLR/MYD88 signaling via IRAK4 inhibition in combination with pharmacological blockade of PI3K/BCR signaling pathways may therefore provide a novel treatment strategy in ABC-DLBCL. BAY 1830839 is a novel small molecule inhibitor of IRAK4 identified by a medicinal chemistry optimization program. Key features of the compound are high potency (IC50 of 3 nM) in a biochemical assay, excellent kinase selectivity and a good overall PK profile making the compound a valuable tool for in vivo studies. In vitro, treatment of IRAK4 inhibitor BAY 1830839 in combination with BTK inhibitors or copanlisib, a pan class I PI3K inhibitor with predominant activity towards PI3Kα and PI3Kδ, synergistically inhibited NF-κB activation and cell viability in human ABC-DLBCL cell lines. In vivo, IRAK4 inhibition alone did not exhibit anti-tumor effects but in combination treatment with ibrutinib, a covalent inhibitor of BTK, synergistic anti-tumor activity with significantly improved efficacy over ibrutinib monotherapy was observed in the human ABC-DLBCL xenograft models TMD-8 and OCI-LY10 (MYD88mut/CD79A/Bmut). Moreover, IRAK4 inhibitor BAY 1830839 showed synergistic anti-tumor activity in combination with copanlisib with significant improvement of copanlisib monotherapy efficacy in the ABC-DLBCL PDX models LY2988 and LY2266 (MYD88mut/CD79A/Bmut and MYD88wt/CD79A/Bwt, respectively). In addition, the combination of IRAK4 inhibition with pharmacological blockade of PI3K-/ BCR signaling led to reduced activity of the downstream pro-survival STAT3 pathway and IL-6/IL-10 production as detected in tumor xenografts, validating our biological rationale and the expected mechanism of action. In summary, IRAK4 inhibition in combination with pharmacological blockade of PI3K or BCR signaling blocks pro-survival NF-κB & JAK-STAT pathway activation and subsequent IL-6/IL-10 production. Enhancing activity of clinically used PI3K or BTK inhibitors by combination with IRAK4 inhibition indicates a potential new treatment approach for ABC-DLBCL patients progressing on Standard of Care therapy. Citation Format: Martin Lange, Antje Margret Wengner, Ulrich Bothe, Ulf Boemer, Reinhard Nubbemeyer, Holger Siebeneicher, Holger Steuber, Judith Guenther, Lisette Potze, Nicole Schmidt, Oliver Politz, Wolf-Dietrich Doecke, Eleni Lagkadinou, Thomas M. Zollner, Franz von Nussbaum, Dominik Mumberg, Andreas Steinmeyer, Michael Brands, Karl Ziegelbauer. Preclinical evaluation of a novel interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitor in combination with PI3K inhibitor copanlisib or BTK inhibitors in ABC-DLBCL [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 1887.

Mitochondria are key regulators of both energy supply and apoptotic cell death. The mitochondrial electron transport chain (ETC) consists of four enzyme complexes that transfer electrons from NADH to oxygen. During electron transfer, the ETC pumps protons into the inter-membrane space, generating a gradient across the inner mitochondrial membrane that is used by Complex V to drive ATP synthesis. Recent publications have shown that tumor cells harboring specific mutations (LKB1, mIDH and others) are more sensitive to Complex I inhibition, potentially providing an opportunity for selectively targeting tumor cells. Based on a high throughput screen (HTS), we identified new, albeit moderately active, lead structures with cross reactivity between mouse and human Complex 1. SAR elaboration of the lead structure allowed for optimization of the potency, although compounds still suffered from low metabolic stability. Further improvement of the in vitro and in vivo PK properties finally permitted in vivo animal studies. Herein, we report for the first time the preclinical profile and structure of a highly active, optimized, human/mouse cross-reactive Complex I inhibitor that allowed for the further investigation into the therapeutic potential of Complex I inhibition in cancer. Citation Format: Jeffrey Mowat, Sven Christian, Carolyn Sperl, Alexander Ehrmann, Stephan Menz, Judith Guenther, Roman Hillig, Marcus Bauser, Andrea Haegebarth, Wolfgang Schwede. Identification and optimization of a highly active, cross reactive Complex-1 inhibitor [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 3248. doi:10.1158/1538-7445.AM2017-3248

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions and therefore represent a promising model system for discovery of phenotypes specifically expressed in stressed, hypoxic and nutrient-depleted conditions. We here show the setup of a 3D cell culture compatible high-content screening system for the identification of substances that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. This setup was used in a pilot screen to identify tool compounds and subsequently in a larger screening approach covering approximately 500000 compounds from the Bayer substance library. The resulting hit list could be subdivided into two different categories based on the identification of two distinct phenotypes. Mode of action studies identified the first category as inhibitors of the mitochondrial respiratory chain, being critically dependent on extracellular glucose concentrations. On the other hand the second hit category could be identified as modifiers of fatty acid metabolism. The identified hits offer the opportunity for further characterization in vivo, particularly in combination with drugs targeting the well perfused, proliferating tumor areas, such as chemotherapy. Taken together the data presented here show for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. Citation Format: Carsten Wenzel, Sven Christian, Carolyn Algire, Wolfgang Schwede, Roland Neuhaus, Judith Guenther, Ningshu Liu, Sebastian Raese, Karsten Parczyk, Stefan Prechtl, Patrick Steigemann. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 317. doi:10.1158/1538-7445.AM2015-317