A druggable addiction to de novo pyrimidine biosynthesis in diffuse midline glioma

Diffuse midline glioma (DMG) is a uniformly fatal pediatric cancer driven by oncohistones that do not readily lend themselves to drug development. To identify therapeutic targets for DMG, we conducted a genome-wide CRIPSR screen for DMG metabolic vulnerabilities, which revealed a DMG selective dependency on the de novo pathway for pyrimidine biosynthesis. The dependency is specific to pyrimidines as there is no selectivity for suppression of de novo purine biosynthesis. A clinical stage inhibitor of DHODH (a rate limiting enzyme in the de novo pathway) generates DNA damage and induces apoptosis through suppression of replication forks--an on target effect, as shown by uridine rescue. MALDI mass spectroscopy imaging demonstrates that BAY2402234 accumulates in brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis in vivo, and prolongs survival of mice bearing intracranial DMG xenografts. Our results highlight BAY2402234, a brain-penetrant DHODH inhibitor, as a promising therapy against DMGs.

CSIG-11. KINASE-DIRECTED INHIBITION OF PYRIMIDINE BIOSYNTHESIS IN PTEN-DEFICIENT GLIOBLASTOMA

Targeting pyrimidine biosynthesis has been a mainstay of chemotherapy in oncology, including frontline treatment of pancreatic, breast, and colorectal carcinomas. In glioblastoma, the targeting pyrimidine biosynthesis is a promising emerging approach for counteracting the effects of PTEN-deficiency in glioblastoma. PTEN loss triggers the activation of mTORC1, which in turn phosphorylates and activates the ribosomal protein kinases S6K1 and S6K2. We have previously shown that combination treatment of inhibitors targeting S6K1 and the TYRO3-AXL-MERTK receptor tyrosine kinases (TAM-RTKs) triggers cytotoxic responses in PTEN-deficient glioblastoma cells. Here we show brain-penetrant inactivation of S6K1 and TAM-RTKs using the S6K1 inhibitor LY-2584702 and the TAM-RTK inhibitor BMS-777607, which reduced glioblastoma tumor growth. Pharmacogenetic analysis of signal transduction indicated a key role for S6K2 in sustaining survival signaling in PTEN-deficient glioblastoma cells. Steady-state metabolomics revealed that combined inactivation of S6K1 and TAM-RTKs resulted in decreased nucleotide biosynthesis, and flux analysis indicated reduced flux of glucose to pyrimidines. Altogether the results indicate a kinase-directed therapeutic strategy for targeting S6K1 and TAM-RTKs to reduce pyrimidine biosynthesis and glioblastoma tumor growth.

Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

Background In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD. Results Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9. Conclusions Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure–function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus.

Inhibition of pyrimidine biosynthesis targets protein translation in AML

The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anti-cancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) by down-regulation of the multi-functional transcription factor YY1, has potent activity against AML in vivo and is well tolerated with minimal impact on normal blood development. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.

Identification of Small Molecule Inhibitors against Staphylococcus aureus Dihydroorotase via HTS

Drug-resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)—an integral step in the de novo pyrimidine biosynthesis pathway. This study sets forth a high-throughput screening (HTS) of 3000 fragment compounds by a colorimetry-based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca-asp determined the best compound to be a competitive inhibitor with a KD value of 11 µM, which is 10-fold tighter than Ca-asp. Preliminary structure–activity relationship (SAR) provides the foundation for further structure-based antimicrobial inhibitor design against S. aureus.

Integrated Metabolic and Gene Expression Profiling Reveals New Therapeutic Modalities for Rapidly Proliferating Breast Cancers

Metabolic dysregulation, although a prominent feature in breast cancer, remains undercharacterized in patient tumors. By performing untargeted metabolomics analyses on triple-negative breast cancer (TNBC) and Estrogen Receptor (ER) positive patient breast tumors, as well as TNBC patient-derived xenografts (PDXs), we identified two major metabolic groups independent of breast cancer histological subtypes: a “Nucleotide/Carbohydrate-Enriched” group and a “Lipid/Fatty Acid-Enriched” group. Cell lines grown in vivo more faithfully recapitulate the metabolic profiles of patient tumors. Integrated metabolic and gene expression analyses reveal genes that strongly correlate with metabolic dysregulation and predict patient prognosis. As a proof-of-principle, targeting Nucleotide/Carbohydrate-Enriched TNBC cell line or PDX xenografts with a pyrimidine biosynthesis inhibitor, and/or a glutaminase inhibitor, led to therapeutic efficacy. In addition, the pyrimidine biosynthesis inhibitor presents better therapeutic outcomes than chemotherapy agents in multiple murine TNBC models. Our study provides a new stratification of breast tumor samples based on integrated metabolic and gene expression profiling, which guides the selection of newly effective therapeutic strategies targeting rapidly proliferating breast cancer subsets. In addition, we develop a public, interactive data visualization portal (http://brcametab.org) based on the data generated from this study.