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Most systemic therapies for prostate cancer target androgen receptor signaling. Given the ongoing disease burden and mortality from advanced prostate cancer that persist despite available therapies, novel therapeutic targets are clearly needed. To this end, we conducted a high-resolution analysis of primary prostate cancer samples across a spectrum of tumor stage using 10x Chromium scRNAseq. Within each patient, we compared the prostate tumor to its adjacent normal tissue. These samples were also compared to healthy prostate samples collected from patients whose prostates were removed in the setting of bladder cancer surgery. Joint analysis revealed a rich repertoire of immune and non-immune stroma cells. Significant alterations were apparent in the prostate tumor microenvironment compared to adjacent normal and healthy samples. One striking difference was an expansion of T-cell cluster characterized by decreased cytotoxic and an increased exhaustion gene signatures. Another difference was the significantly higher regulatory T cell (Treg) activity. There was a substantial population shift within the myeloid compartment. Specifically, tumor tissue showed a selective enrichment in M2-like macrophages which was validated by multiplex immunohistochemistry. In addition, tumor tissue showed a significant increase in the myeloid-derived suppressor cell scoring in tumor inflammatory monocytes that strongly correlated with Treg activity. The changes in both myeloid and T cell gene expression suggest an immune suppressive environment within the tumor. In addition, we performed Slide-seqV2 to generate high spatial resolution maps of cellular organization of prostate tissues. The highly detailed spatial configuration of the healthy prostate tissue showed well-organized prostate epithelial glands surrounded by immune and non-immune stromal cells. This tissue architecture was severely disrupted within the cancerous prostate. Endothelial and fibroblast populations were disorganized and dispersed throughout the invading tumor. Context-dependent differential expression gene analysis on endothelial cells showed significant upregulation of sprouting angiogenesis and vascular endothelial growth factor pathways in the tumor context. In addition, we developed an approach to characterize the coordination between tumor cells and stromal compartment via spatial ligand-receptor (L/R) interaction. Data revealed 405 significant potential communication channels with many of these interactions known to have a role in prostate tumor angiogenesis and progression. These data provide a high resolution landscape of localized prostate cancer via 10x and Slide-seq. Data showed an immunosuppressive milieu in addition to highly active endothelial angiogenic program. L/R interaction analysis revealed interactions that may serve as potential therapeutic targets. Citation Format: Taghreed Hirz, Shenglin Mei, Hirak Sarkar, Youmna Kfoury, Shulin Wu, Chin-Lee Wu, Fei Chen, Evan Z. Macosko, Douglas M. Dahl, Ninib Baryawno, David T. Scadden, Philip J. Saylor, Peter V. Kharchenko, David B. Sykes. An atlas of single-cell and spatial transcriptomics reveals alterations that correlate with human prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2510.

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.

Cancer-related mortality due to solid tumor malignancies is overwhelmingly due to the development and progression of metastases. In advanced prostate cancer, metastases most often involve the bone and generally represent incurable disease. It remains unclear what aspects of the bone marrow microenvironment make it hospitable to metastatic dissemination. Similarly, the impact of the metastatic tumor on hematopoiesis and the marrow immune response is poorly understood. We took advantage of rare spinal cord decompression surgeries to profile marrow and metastatic tumors from men with advanced prostate cancer at single-cell resolution. Our analysis contrasts the cellular composition and transcriptional states in matched samples of tumor and liquid bone marrow collected at adjacent vertebral body levels, as well as bone marrow of orthopedic patients without malignancy. Metastatic prostate cancer was associated with hematopoietic suppression and multifaceted immune distortion. There was exhaustion of specific T cell subsets, appearance of inflammatory monocytes and macrophages, and alteration of cytokine profiles. Computational analysis showed association between the presence of specific myeloid subsets and the level of T lymphocyte dysfunction in the tumor fraction. We screened for potential signaling axes that may underlie this interaction. Among them was chemokine CCL20, notably overexpressed by myeloid cells, as was its cognate CCR6 receptor, expressed on T cells. We developed a syngeneic mouse model of bone-metastatic prostate cancer to explore this observation, and demonstrated that disruption of the CCL20-CCR6 axis from either side resulted in significant prolongation of survival. Our results further indicated that this dual overexpression was associated with repressed immune responses. Overall, comparative high-resolution analysis of bone marrow reveals distinct alterations associated with prostate cancer bone metastases that may be amenable to therapeutic targeting with the goal of altering cancer progression. Citation Format: Ninib Baryawno, Youmna Kfoury, Nicolas Severe, Shenglin Mei, Karin Gustafsson, Taghreed Hirz, Thomas Brouse, Elizabeth W. Scadden, Anna A. Igolkina, Bryan D. Choi, Nikolas Barkas, John H. Shin, Philip J. Saylor, David T. Scadden, David B. Sykes, Peter V. Kharchenko, as part of the Boston Bone Metastasis Consortium. Impact of metastatic prostate cancer on human bone marrow [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA23.

Why this research is important Clinical care for localized prostate cancer in 2019 is primarily guided by a system of risk stratification that has remained unchanged for decades (PSA, tumor T-stage, Gleason grade). True advances are needed in: (1) risk stratification among primary tumors and (2) novel mechanistically rational approaches to tumors that pose the greatest clinical threat of metastatic progression. The primary prostate tumor is remarkably complex, consisting not only of tumor cells but also of extracellular matrix, vasculature, stromal cells and infiltrating immune cells. Most systemic therapies simply target the androgen receptor, a mainstay of prostate cancer therapy. Novel therapeutic targets would benefit patients with high-risk localized tumors and potentially patients with currently incurable metastatic disease. What we aim to accomplish We aim to understand, through the single-cell lens, the differences in the prostate tumor microenvironment across the spectrum of Gleason grades to identify novel cellular and molecular targets for therapeutic intervention. On the path to discovery The design of novel therapies against prostate cancer relies on a deep understanding of the identity and function of each cell type within the tumor microenvironment. Our interdisciplinary team of clinicians, surgeons, bioinformaticians and biologists will characterize changes in the cellular composition, state, and function within the prostate tumor microenvironment across the spectrum of surgical stages and grades. These efforts are largely driven by advances in single-cell sequencing and imaging technologies. Using single-cell RNA sequencing (scRNA-seq), we created the cellular anatomy of the prostate tumor and their matched-normal glandular tissues (19 patients; Gleason 6-10; T-stage 2a to 3b). We constructed an atlas comprised of ~70,000 non-erythroid viable cells collected from treatment-naïve primary prostate cancer specimens and ~75,000 cells from the matched normal prostate tissue. Analysis of transcriptional states revealed a complex tumor microenvironment, including immune cells and supporting non-immune stroma. Our analysis has allowed us to 1) create the cell-atlas of prostate tissue, 2) characterize heterogeneity of intratumoral cells of both lymphoid and myeloid cell lineages and 3) identify tumor heterogeneity among patients. We identify 8 clusters of myeloid cells and 14 clusters of lymphoid cells. A broad survey of both lineages revealed different functional states for the different clusters. Gene expression analysis will be combined with Slide- seq - a new technique that preserves the tumor architecture. Future impact of our research Our research will advance our understanding of prostate cancer biology with dual downstream goals of honing our prognostic algorithms and identifying new avenues of therapeutic intervention. Ultimately, our goal is to improve the quality and length of life of patients diagnosed with prostate cancer. Citation Format: Taghreed Hirz, Shenglin Mei, Youmna Kfoury, Chin-Lee Wu, Douglas M. Dahl, Philip Saylor, Peter Kharchenko, Ninib Baryawno, David Sykes. High-resolution mapping of human prostate tumors- guiding better risk stratification and therapeutic approaches [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3446.

DLBCL is the most common type of non-Hodgkin lymphoma. It is an aggressive and fast growing tumor with two major molecular subtypes: germinal center (GCB) and activated B-cell like (ABC). While the majority of patients are 60 years or older, DLBCL can occur at any age. Despite a cure rate of around 50% the need for novel therapies remains high, especially for relapsed/refractory DLBCL patients not eligible for stem cell transplant. DHODH is a key enzyme in the de novo pyrimidine synthesis converting dihydroorotate to orotate. We recently discovered its role in AML differentiation (Sykes et al 2016, Cell) and we are investigating the novel DHODH inhibitor BAY 2402234 in an ongoing phase I study in myeloid malignancies (NCT03404726). Further screening of non-leukemia cancer types identified DLBCL as highly responsive to DHODH inhibition in preclinical studies. Here, we disclose for the first time the functional preclinical characterization of BAY 2402234 in DLBCL. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro it potently inhibits proliferation of DLBCL cell lines in the sub-nanomolar to low-nanomolar range. The anti-proliferative effects can be rescued by uridine supplementation which bypasses DHODH via the salvage pathway and demonstrates the on-target specificity of the inhibitor. In vivo, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in subcutaneous models derived from patient-derived xenograft (PDX) and cell lines representing various DLBCL subtypes, including GCB and ABC. Dose dependent target engagement and drug exposure of BAY 2402234 could be observed by increases in plasma dihydroorotate levels and unbound plasma drug levels after treatment with the inhibitor. Based on preclinical data presented herein we plan to start clinical investigations of BAY 2402234 in patients with DLBCL in early 2019. Citation Format: Ashley Eheim, Sven Christian, Hanna Meyer, Detlef Stoeckigt, Claudia Merz, Katja Zimmermann, Marcus Bauser, Andrea Haegebarth, Steven Ferrara, David B. Sykes, David T. Scadden, Stefan Gradl, Andreas Janzer. BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of diffuse large B-cell lymphoma (DLBCL) [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 3597.

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.

The prognosis for adults diagnosed with acute myeloid leukemia (AML) remains poor, with a five-year survival of only 25%. This prognosis is even more dismal in older patients who are not well enough to receive standard induction chemotherapy. Speaking to the need for new therapies is the fact that our therapeutic backbone - a combination of cytarabine and an anthracycline - remains unchanged since 1973. The promise of differentiation therapy was realized in the small subset of patients diagnosed with acute promyelocytic leukemia (APL). Here, treatment in the form of all-trans retinoic acid (ATRA) and arsenic trioxide inverted the survival curve; where APL was once the worst form of myeloid leukemia, it now carries the best prognosis, with a five-year survival exceeding 85%. The goal of this study was thus to develop differentiation therapy for patients with non-promyelocytic AML with the question: “Can we identify small molecules that overcome myeloid differentiation arrest?” A phenotypic differentiation screen in a HOXA9 driven leukemia model followed by target deconvolution, identified DHODH as an unexpected target for overcoming differentiation arrest in AML. We used 2 potent small molecule inhibitors of DHODH to validate this initial finding: Brequinar, a known DHODH inhibitor and an in house compound BAY DHODHi. In several in vitro experiments we demonstrated induction of AML differentiation in a dose dependent fashion. Interestingly, these effects could be completely rescued by addition of uridine, confirming target specificity. Treating mice in multiple genetically diverse AML in vivo models with a DHODH inhibitor led to tumor growth reduction and AML differentiation. Expression analysis of leukemia cells explanted from mice xenografts treated with a DHODH inhibitor demonstrate an early onset of differentiation markers indicating a direct role of DHODH with the onset of differentiation in vivo. The mechanism for selective vulnerability of leukemia cells to DHODH inhibition remains under investigation. Despite the observation that DHODH is expressed in all cells, normal and malignant, mice can tolerate DHODH inhibitor therapy for more than 100 days without weight-loss or other concerning side-effects. Thus, our pre-clinical studies point towards DHODH as a new metabolic target in the differentiation treatment of AML. Hopefully, small molecule DHODH inhibitors will provide a much-needed differentiation therapy for patients with acute myeloid leukemia. Citation Format: Andreas Janzer, David Sykes, Stefan Gradl, Steven Ferrara, Sven Christian, Claudia Merz, Henrik Seidel, Andreas Bernthaler, Ralf Lesche, Mathias Wawer, David T. Scadden. Inhibitors of the enzyme dihydroorotate dehydrogenase, overcome the differentiation blockade in acute myeloid leukemia [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 3086. doi:10.1158/1538-7445.AM2017-3086