Dihydroorotate Dehydrogenase Activity Research Articles

Redistribution of Defective Mitochondria-mediated Dihydroorotate Dehydrogenase Imparts 5-Fluorouracil Resistance in Colorectal Cancer

Although 5-fluorouracil (5-FU) is the primary chemotherapy treatment for colorectal cancer (CRC), its efficacy is limited by drug resistance. Ferroptosis activation is a promising treatment for 5-FU-resistant cancer cells; however, potential therapeutic targets remain elusive. This study investigated ferroptosis vulnerability and dihydroorotate dehydrogenase (DHODH) activity using stable, 5-FU-resistant CRC cell lines and xenograft models. Ferroptosis was characterized by measuring malondialdehyde levels, assessing lipid metabolism and peroxidation, and using mitochondrial imaging and assays. DHODH function is investigated through gene knockdown experiments, tumor behavior assays, mitochondrial import reactions, intramitochondrial localization, enzymatic activity analyses, and metabolomics assessments. Intracellular lipid accumulation and mitochondrial DHODH deficiency led to lipid peroxidation overload, weakening the defense system of 5-FU-resistant CRC cells against ferroptosis. DHODH, primarily located within the inner mitochondrial membrane, played a crucial role in driving intracellular pyrimidine biosynthesis and was redistributed to the cytosol in 5-FU-resistant CRC cells. Cytosolic DHODH, like its mitochondrial counterpart, exhibited dihydroorotate catalytic activity and participated in pyrimidine biosynthesis. This amplified intracellular pyrimidine pools, thereby impeding the efficacy of 5-FU treatment through molecular competition. These findings contribute to the understanding of 5-FU resistance mechanisms and suggest that ferroptosis and DHODH are promising therapeutic targets for patients with CRC exhibiting resistance to 5-FU.

Glutathione depletion and dihydroorotate dehydrogenase inhibition actuated ferroptosis-augment to surmount triple-negative breast cancer

Ferroptosis is a promising therapeutic approach for combating malignant cancers, but its effectiveness is limited in clinical due to the adaptability and self-repair abilities of cancer cells. Mitochondria, as the pivotal player in ferroptosis, exhibit tremendous therapeutic potential by targeting the intramitochondrial anti-ferroptotic pathway mediated by dihydroorotate dehydrogenase (DHODH). In this study, an albumin-based nanomedicine was developed to induce augmented ferroptosis in triple-negative breast cancer (TNBC) by depleting glutathione (GSH) and inhibiting DHODH activity. The nanomedicine (ATO/SRF@BSA) was developed by loading sorafenib (SRF) and atovaquone (ATO) into bovine serum albumin (BSA). SRF is an FDA-approved ferroptosis inducer and ATO is the only drug used in clinical that targets mitochondria. By combining the effects of SRF and ATO, ATO/SRF@BSA promoted the accumulation of lipid peroxides within mitochondria by inhibiting the glutathione peroxidase 4 (GPX4)-GSH pathway and downregulating the DHODH-coenzyme Q (CoQH2) defense mechanism, triggers a burst of lipid peroxides. Simultaneously, ATO/SRF@BSA suppressed cancer cell self-repair and enhanced cell death by inhibiting the synthesis of adenosine triphosphate (ATP) and pyrimidine nucleotides. Furthermore, the anti-cancer results showed that ATO/SRF@BSA exhibited tumor-specific killing efficacy, significantly improved the tumor hypoxic microenvironment, and lessened the toxic side effects of SRF. This work presents an efficient and easily achievable strategy for TNBC treatment, which may hold promise for clinical applications.

Abstract A109: Targeting pyrimidine biosynthesis as a metabolic vulnerability in brain metastasis

Abstract Metastasis, the spread of cancer cells from one part of the body to another, is the leading cause of death among cancer patients. Lung-to-Brain metastasis remains a largely understudied disease that possesses a dismal prognosis due to the lack of effective therapies. We previously identified purine metabolism as a metabolic vulnerability of brain metastases. Here, we sought to identify whether pyrimidine biosynthesis is also a metabolic dependency of metastatic brain tumours. We identify dihydroorotate dehydrogenase (DHODH) as a cancer-selective targetable vulnerability. DHODH is an essential enzyme that is located within the inner membrane of the mitochondria which has a primary role in producing pyrimidine metabolites through the de novo uridine biosynthesis pathway and also contributes to the proper functioning of the electron transport chain. Knock-out of DHODH with CRISPR-Cas9 or pharmacological inhibition with BAY2402234 decreased cell viability, sphere formation, and proliferation of patient-derived lung-to-brain (LBM) metastasis cells in vitro. Furthermore, suppression of DHODH expression or BAY2402234 treatment reduced growth of intracranial LBM tumours and significantly extended survival. Mechanistically, we identify interfering with DHODH activity abrogates normal mitochondrial function leading to cleavage of gasdermin E and induction of pyroptosis. Taken together, this study highlights the promise of DHODH inhibitors in targeting brain metastasis. Citation Format: Daniel Mobilio, Sheila K Singh, Shawn C Chafe. Targeting pyrimidine biosynthesis as a metabolic vulnerability in brain metastasis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A109.

Pyruvate dehydrogenase is a potential mitochondrial off-target for gentamicin based on in silico predictions and in vitro inhibition studies

During the drug development process, organ toxicity leads to an estimated failure of one-third of novel chemical entities. Drug-induced toxicity is increasingly associated with mitochondrial dysfunction, but identifying the underlying molecular mechanisms remains a challenge. Computational modeling techniques have proven to be a good tool in searching for drug off-targets. Here, we aimed to identify mitochondrial off-targets of the nephrotoxic drugs tenofovir and gentamicin using different in silico approaches (KRIPO, ProBis and PDID). Dihydroorotate dehydrogenase (DHODH) and pyruvate dehydrogenase (PDH) were predicted as potential novel off-target sites for tenofovir and gentamicin, respectively. The predicted targets were evaluated in vitro, using (colorimetric) enzymatic activity measurements. Tenofovir did not inhibit DHODH activity, while gentamicin potently reduced PDH activity. In conclusion, the use of in silico methods appeared a valuable approach in predicting PDH as a mitochondrial off-target of gentamicin. Further research is required to investigate the contribution of PDH inhibition to overall renal toxicity of gentamicin.

Protein S-glutathionylation and sex dimorphic effects on hydrogen peroxide production by dihydroorotate dehydrogenase in liver mitochondria.

Dihydroorotate dehydrogenase (DHODH) oxidizes dihydroorotate to orotate for pyrimidine biosynthesis, donating electrons to the ubiquinone (UQ) pool of mitochondria. DHODH has a measurable rate for hydrogen peroxide (H2O2) production and thus contributes to cellular changes in redox tone. Protein S-glutathionylation serves as a negative feedback loop for the inhibition of H2O2 by several α-keto acid dehydrogenases and respiratory complexes in mitochondria, as well as ROS sources in liver cytoplasm. Here, we report this redox signaling mechanism also inhibits H2O2 production by DHODH in liver mitochondria isolated from male and female C57BL6N mice. We discovered that low amounts of the glutathionylation catalyst, disulfiram (50-500nM), almost abolished H2O2 production by DHODH in mitochondria from male mice. Similar results were collected with diamide, however, higher doses (1000-5000μM) were required to elicit this effect. Disulfiram and diamide also significantly suppressed H2O2 production by DHODH in female liver mitochondria. However, liver mitochondria from female mice were more resistant to disulfiram or diamide-mediated inhibition of H2O2 genesis when compared to samples from males. Analysis of the impact of disulfiram and diamide on DHODH activity revealed that both compounds inhibited the dehydrogenase directly, however the effect was less in female mice. Additionally, disulfiram and diamide impeded the use of dihydroorotate fueled oxidative phosphorylation in mitochondria from males and females, although samples collected from female rodents displayed more resistance to this inhibition. Taken together, our findings demonstrate H2O2 production by DHODH can be inhibited by glutathionylation and sex can impact this redox modification.

Identification of a dihydroorotate dehydrogenase inhibitor that inhibits cancer cell growth by proteomic profiling.

Dihydroorotate dehydrogenase (DHODH) is a central enzyme of the de novo pyrimidine biosynthesis pathway and is a promising drug target for the treatment of cancer and autoimmune diseases. This study presents the identification of a potent DHODH inhibitor by proteomic profiling. Cell-based screening revealed that NPD723, which is reduced to H-006 in cells, strongly induces myeloid differentiation and inhibits cell growth in HL-60 cells. H-006 also suppressed the growth of various cancer cells. Proteomic profiling of NPD723-treated cells in ChemProteoBase showed that NPD723 was clustered with DHODH inhibitors. H-006 potently inhibited human DHODH activity in vitro, whereas NPD723 was approximately 400 times less active than H-006. H-006-induced cell death was rescued by the addition of the DHODH product orotic acid. Moreover, metabolome analysis revealed that H-006 treatment promotes marked accumulation of the DHODH substrate dihydroorotic acid. These results suggest that NPD723 is reduced in cells to its active metabolite H-006, which then targets DHODH and suppresses cancer cell growth. Thus, H-006-related drugs represent a potentially powerful treatment for cancer and other diseases.

Identifying Mechanisms of Resistance to DHODH Inhibition in Cancer

All transcriptionally active and rapidly dividing cells require high amounts of pyrimidines for DNA replication and repair and RNA synthesis. The only two known mechanisms by which cells can acquire pyrimidines are the de novo pyrimidine biosynthesis pathway and the pyrimidine salvage pathway. The rate-limiting enzyme in de novo synthesis is dihydroorotate dehydrogenase (DHODH), a critical metabolic enzyme that converts dihydroorotate (DHO) to orotate, which is then converted to the pyrimidine precursor uridine monophosphate (UMP). The rate-limiting enzyme in the salvage pathway is uridine-cytidine kinase 2 (UCK2), a ribonucleoside kinase that converts uridine to UMP and cytidine to cytidine monophosphate (CMP). Under physiologic conditions, salvage pathway substrates are limiting, and many cancer types, most notably acute myeloid leukemia (AML), are highly dependent on DHODH activity for proliferation and survival. Based on these observations, several DHODH inhibitors have been developed as anti-cancer therapeutics and have been tested clinically. Unfortunately, to date, these drugs have failed to show significant anti-cancer activity in early-phase clinical trials. In order to understand how leukemia cells that are highly sensitive to DHODH inhibitors can bypass their dependence on DHODH activity, we conducted positive-selection whole-genome CRISPR-activation drug resistance screens in TF-1 human AML cells using BAY 2402234, a highly potent and highly specific DHODH inhibitor. In preliminary studies, we found that 1 nM BAY 2402234 fully inhibits TF-1 cells whereas TF-1 cells that overexpress high levels of DHODH require 50 nM BAY 2402234 to achieve the same effect. Importantly, the effects of 1 nM and 50 nM BAY 2402234 are fully rescued by addition of uridine to the culture media, demonstrating that the anti-leukemic activity of BAY 2402234 is on-target at both drug doses. To perform the drug resistance screens, we stably expressed nuclease-deactivated Cas9 fused to a transcriptional activator (dCas9-VP64) in TF-1 cells and then infected the cells with the human whole-genome Calabrese sgRNA lentiviral library. We then passaged the cells in media containing vehicle control (DMSO), 1 nM or 50 nM BAY 2402234 for 18 days, collecting cells on day 0 and day 18 of drug treatment. Genomic DNA was then extracted and was subjected to next generation sequencing to assess the relative abundance of each sgRNA in each sample. We reasoned that activation of genes that enhance de novo pathway activity at the level of, or downstream of, DHODH would promote resistance to 1 nM BAY 2402234 but not 50 nM BAY 2402234, whereas activation of genes that act through de novo pathway-independent mechanisms would promote resistance to both 1 nM and 50 nM BAY 2402234. As predicted, sgRNAs targeting DHODH conferred resistance to 1 nM but not 50 nM BAY 2402234 whereas sgRNAs targeting UCK2 conferred resistance to both 1 and 50 nM BAY 2402234 (Figure 1). A number of other known salvage pathway genes scored in both screens, including uracil phosphoribosyltransferase (UPRT) and the nucleoside transporters SLC4A1 and SLC29A1. Unexpectedly, the screens also identified carbamoyl-phosphate synthetase 2/aspartate transcarbamoylase/dihydroorotase (CAD) as a potential mechanism of resistance to DHODH inhibition. CAD is a trifunctional multi-domain enzyme that acts upstream of DHODH, catalyzing the first three steps in de novopyrimidine biosynthesis. How upregulation of CAD activity can bypass the requirement for DHODH activity is not clear, although CAD is a very large enzyme complex about which very little is known besides its de novo pyrimidine synthesis activity. The screens also identified several genes that are not known to play a role in pyrimidine metabolism as potential resistance mechanisms. These include the serine-threonine kinase MARK3, the serine-threonine phosphatase CTDSPL2, and the RNA-binding protein NANOS2. Finally, the screens identified several genes whose upregulation sensitized cells to BAY 2402234, including the urokinase receptor PLAUR, the adaptor protein BCAR3, the scaffold protein SHOC2, and the deoxynucleoside phosphohydrolase SAMHD1. Studies are currently underway to validate the results of the CRISPRa screens and elucidate the mechanisms by which the candidate resistance genes help cells to bypass their requirement for DHODH activity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

N-Heterocyclic 3-Pyridyl Carboxamide Inhibitors of DHODH for the Treatment of Acute Myelogenous Leukemia.

Acute myelogenous leukemia (AML), a disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway; however, small molecule DHODH inhibitors were recently shown to induce differentiation in multiple AML subtypes. Using virtual screening and structure-based drug design approaches, a new series of N-heterocyclic 3-pyridyl carboxamide DHODH inhibitors were discovered. Two lead compounds, 19 and 29, have potent biochemical and cellular DHODH activity, favorable physicochemical properties, and efficacy in a preclinical model of AML.

Abstract 5492: Activity of RP7214, a novel, selective, and potent small molecule inhibitor of DHODH, in AML

Abstract Background: Dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme in pyrimidine synthesis, is overexpressed in certain cancers and impedes tumor cell proliferation via cell cycle arrest. RP7214 is a novel and potent DHODH inhibitor that inhibited DHODH activity in enzyme and PHA induced HWB/PBMC proliferation with IC50 and EC50 values of 7.8 & 2.5/0.60 nM respectively. Herein, we describe the efficacy of RP7214 as a single agent and in combination with standard of care drugs in preclinical models of AML Methods: Cell viability of AML cell lines (U937, HL-60, THP-1, and KG-1) following incubation with RP7214 as a single agent or in combination with Azacytidine or Venetolcax for 72 h was determined in an MTT-based assay. Cell surface expression of CD11b in THP-1 and human primary bone marrow AML mononuclear cells in presence of RP7214 was evaluated by flow cytometry. Apoptosis was evaluated following incubation of cell lines with compound for 48 or 72 h, subsequent staining with Annexin-V-PE and 7-AAD, and analysis by flow cytometry. Anti-tumor potential of RP7214 as single agent was tested in subcutaneous MV-4-11 human leukemia xenograft model. Results: RP7214 inhibited AML cell proliferation in all the four cell lines tested with GI50 ranging from 2.4 to 7.6 µM. Expression of the CD11b marker on THP-1 cell surface was enhanced by 40% following incubation with 5 µM compound; effect of which was reduced to 15% in the presence of 300 µM Uridine. Incubation of primary human bone marrow AML mononuclear cells with 10 µM of RP7214 increased the CD11b+ cells by > 15 %. Combination of 3 µM-RP7214 and 1 µM-Azacytidine inhibited cell proliferation by > 25% (P < 0.05) compared to single agents in MV-4-11 cells. Similarly, combination of 3 µM-RP7214 with 3 nM Venetoclax demonstrated 15% (P < 0.05) inhibition of cell growth compared to single agents in MV-4-11 cell line. RP7214 at 3, 10, and 30 mg/kg BID demonstrated significant anti-tumor activities in both tumor size and tumor weight in subcutaneous MV-4-11 human leukemia xenograft model. Conclusions: Results demonstrate the utility of RP7214 as a single agent and in combination with standard of care drugs in inducing apoptosis and promoting differentiation to fully mature cells in AML. A Phase 2a trial in AML patients is planned. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Swaroop Vakkalanka. Activity of RP7214, a novel, selective, and potent small molecule inhibitor of DHODH, in AML [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 5492.

Dihydroorotate dehydrogenase inhibition acts synergistically with tyrosine kinase inhibitors to induce apoptosis of mantle cell lymphoma cells.

Mantle cell lymphoma (MCL) is a non‐Hodgkin lymphoma that remains incurable with the treatment options available today. In the present study, we have identified the dihydroorotate dehydrogenase (DHODH), an essential enzyme for the de novo biosynthesis of pyrimidine‐based nucleotides, to be overexpressed in MCL in comparison to healthy peripheral blood mononuclear cells (PBMC). In vitro inhibition of the DHODH activity using a newly developed DHODH inhibitor, namely (R)‐HZ05, can induce MCL cell death in the nanomolar range independently than the P53 status of the investigated cell lines. Moreover, the combination of (R)‐HZ05 with tyrosine kinase inhibitor shows the synergistic activity on cell death. Pre‐clinical investigation on the efficacy of (R)‐HZ05 shows that it can be prolonged animal lifespan similar to ibrutinib. (R)‐HZ05 use in combination with tyrosine kinase inhibitor demonstrated a superior efficacy on tumor burden reduction and survival than either drug alone. We have demonstrated that the depletion of the pyrimidine nucleotide pool, using DHODH inhibitor, represents a new therapeutic strategy that may benefit MCL patients.

Identification of Dihydroorotate Dehydrogenase Inhibitors─Indoluidins─That Inhibit Cancer Cell Growth.

Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis and is a promising cancer treatment target. This study reports the identification of indoluidin D and its derivatives as inhibitors of DHODH. Cell-based phenotypic screening revealed that indoluidin D promoted myeloid differentiation and inhibited the proliferation of acute promyelocytic leukemia HL-60 cells. Indoluidin D also suppressed cell growth in various other types of cancer cells. Cancer cell sensitivity profiling with JFCR39 and proteomic profiling with ChemProteoBase revealed that indoluidin D is a DHODH inhibitor. Indoluidin D inhibited human DHODH activity in vitro; the DHODH reaction product orotic acid rescued indoluidin D-induced cell differentiation. We synthesized several indoluidin D diastereomer derivatives and demonstrated that stereochemistry was vital to their molecular activity. The indoluidin D derivative indoluidin E showed similar activity to its parent compound and suppressed tumor growth in a murine lung cancer xenograft model. Hence, indoluidin D and its derivatives selectively inhibit DHODH and suppress cancer cell growth.

Abstract 1256: JNJ-74856665, a novel DHODH inhibitor, mediates potent anti-leukemic activity and differentiation in vitro and in vivo

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease characterized by uncontrolled clonal expansion of hematopoietic progenitor cells (myeloid blasts). Disease remission can be achieved with standard induction chemotherapy, but refractory and relapsed disease remains a challenge with a 5-year overall survival rate of approximately 25%. Induction of differentiation is an alternative approach to AML therapy. Dihydroorotate dehydrogenase (DHODH) catalyzes the ubiquinone-mediated oxidation of dihydroorotate (DHO) to orotate in the de novo pyrimidine synthesis pathway. Pre-clinical findings demonstrated that DHODH is a metabolic vulnerability in AML and a new target for differentiation therapy. Given the genetic complexity of myeloid malignancies, future use of DHODH inhibitors in combination with selected antineoplastic agents (e.g., azacitidine) with nonoverlapping mechanisms of action may address specific aspects of the complex pathogenesis of AML and MDS (myelodysplastic syndrome), ultimately improving patients' outcomes.JNJ74856665 is an orally available, potent and selective DHODH inhibitor. JNJ-74856665 inhibited the biochemical activity of human DHODH with a 50% inhibitory concentration (IC50) in the subnanomolar range and in vitro inhibited proliferation of MOLM-13, OCI-AML3, HL60 and THP-1 AML cell lines with low nanomolar IC50 values. Mechanism of action studies using these four cell lines demonstrated induction of CD11b and CD14 differentiation marker mRNA levels. Differentiation was accompanied by cell cycle arrest and apoptosis induction. Importantly, addition of excess uridine to the tissue culture conditions abrogated the anti-proliferative, differentiation and apoptosis inducing activity of JNJ-74856665, indicating on target activity. In vivo, JNJ-74856665 mediated significant activity in decreasing leukemic burden and increasing life span across subcutaneous (MOLM-13 and OCI-AML3) and disseminated (MOLM-13) xenografts, respectively. Pharmacodynamic investigations demonstrated target engagement as evidenced by upregulation of the DHODH substrate dihydroorotate in plasma and induction of differentiation markers in tumor. Combination of JNJ-74856665 with azacitidine in vivo demonstrated lack of antagonism in the anti-leukemic activity.The results described herein demonstrate that JNJ-74856665 exerts potent anti-leukemic activity in vitro and in vivo and warrant further investigation. To that end, a phase 1 clinical trial assessing the safety and efficacy of JNJ-74856665 in patients with AML and MDS is planned to initiate shortly. Citation Format: Christine Pietsch, Scott Kuduk, Xiaochun Zhang, Tammy Bush, Faraz Kazmi, Edgar Jacoby, Zhuming Zhang, Lindsey DeRatt, Weixue Wang, Aaron Patrick, Andrew Steele, Friedericke Pastore, Tony Greway, Nikki Daskalakis, Kathryn Bradford, Kathryn Packman, Gilles Bignan, James Edwards, Ricardo Attar, Yusri Elsayed, Jacqueline Bussolari, Ulrike Philippar. JNJ-74856665, a novel DHODH inhibitor, mediates potent anti-leukemic activity and differentiation in vitro and in vivo [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 1256.

FSMP-08. TARGETING PYRIMIDINE SYNTHESIS ACCENTUATES MOLECULAR THERAPY RESPONSE IN GLIOBLASTOMA STEM CELLS

Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase (CAD) or the critical downstream enzyme dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity in vitro. Higher expression of pyrimidine synthesis genes portends poor prognosis of patients with glioblastoma. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.

Effective deploying of a novel DHODH inhibitor against herpes simplex type 1 and type 2 replication

Emergence of drug resistance and adverse effects often affect the efficacy of nucleoside analogues in the therapy of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Host-targeting antivirals could therefore be considered as an alternative or complementary strategy in the management of HSV infections. To contribute to this advancement, here we report on the ability of a new generation inhibitor of a key cellular enzyme of de novo pyrimidine biosynthesis, the dihydroorotate dehydrogenase (DHODH), to inhibit HSV-1 and HSV-2 in vitro replication, with a potency comparable to that of the reference drug acyclovir. Analysis of the HSV replication cycle in MEDS433-treated cells revealed that it prevented the accumulation of viral genomes and reduced late gene expression, thus suggesting an impairment at a stage prior to viral DNA replication consistent with the ability of MEDS433 to inhibit DHODH activity. In fact, the anti-HSV activity of MEDS433 was abrogated by the addition of exogenous uridine or of the product of DHODH, the orotate, thus confirming DHODH as the MEDS433 specific target in HSV-infected cells. A combination of MEDS433 with dipyridamole (DPY), an inhibitor of the pyrimidine salvage pathway, was then observed to be effective in inhibiting HSV replication even in the presence of exogenous uridine, thus mimicking in vivo conditions. Finally, when combined with acyclovir and DPY in checkerboard experiments, MEDS433 exhibited highly synergistic antiviral activity. Taken together, these findings suggest that MEDS433 is a promising candidate as either single agent or in combination regimens with existing direct-acting anti-HSV drugs to develop new strategies for treatment of HSV infections.

Inhibition of canine distemper virus replication by blocking pyrimidine nucleotide synthesis with A77 1726, the active metabolite of the anti-inflammatory drug leflunomide.

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

Selective eradication of pluripotent stem cells by inhibiting DHODH activity.

Pluripotent stem cells (PSCs), such as embryonic stem cells and induced pluripotent stem cells, give rise to all kinds of functional cells, making them promising for successful application in regenerative medicine. However, there is concern that a PSC-derived differentiated cell population may form teratomas when used for cell therapy if the population contains undifferentiated PSCs. Therefore, for the success of regenerative medicine, it is crucial to establish methods that induce complete PSC differentiation and eliminate the contamination of PSCs. Here, I show that the dihydroorotate dehydrogenase (DHODH) inhibitor brequinar (BRQ) induced cell cycle arrest, cell death, and stemness loss in mouse PSCs (mPSCs), whereas it was less toxic against normal tissue-specific stem cells and differentiating cells. I demonstrate that BRQ-pretreated mPSCs did not form teratomas after being transplanted into NOD/SCID mice. Moreover, BRQ administration to teratoma-bearing mice prevented tumor growth and decreased PSC marker levels in the tumor without any visible effects in the differentiated germ layer cells and the mice. Collectively, these data suggested that DHODH inhibitors such as BRQ can be indispensable in the fundamental methods of PSC-based therapy.

Mitochondrial ubiquinol oxidation is necessary for tumour growth.

The mitochondrial electron transport chain (ETC) is necessary for tumor growth1–6 and its inhibition has demonstrated efficacy in combination with immuno-, antiangiogenic, or oncogene targeted-therapies7–9. Furthermore, brain and lung human tumors display robust glucose oxidation by mitochondria10,11. However, it is not fully understood why a functional ETC is necessary for tumor growth in vivo. ETC function is coupled to the generation of ATP, i.e. oxidative phosphorylation (OXPHOS) and the production of TCA cycle metabolites. Mitochondrial complex I and II donate electrons to ubiquinone resulting in the generation of ubiquinol. This allows regeneration of NAD+ and FAD, necessary cofactors for oxidative TCA cycle flux. Mitochondrial complex III oxidizes ubiquinol back to ubiquinone to allow complexes I and II to function. The ubiquinone generated by complex III is also used as an electron acceptor by the only mitochondrial enzyme essential for de novo pyrimidine synthesis, dihydroorotate dehydrogenase (DHODH). We observed impaired tumor growth in cancer cells lacking mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX) 12 that also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II, or DHODH diminished the tumor growth of AOX expressing cancer cells deficient in mitochondrial complex III, highlighting the necessity of ubiquinone as an electron acceptor for tumor growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX) 13 targeted to the mitochondria or cytosol still failed to grow tumors. This suggests that NAD+ regeneration is not sufficient to drive tumor growth in vivo. Collectively, our findings indicate that tumor growth requires the ETC to oxidize ubiquinol which is essential to drive oxidative TCA cycle and DHODH activity.

Abstract C044: RP7214, a small molecule inhibitor of dihydroorotate dehydrogenase (DHODH), potentiates activity of Gilteritinib and Cytarabine in preclinical models of AML

Abstract Background: Interventions that promote differentiation of immature blasts have been shown to be a promising therapeutic strategy for the treatment of AML. Inhibition of dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme in pyrimidine synthesis, promotes AML cell differentiation besides impeding tumor cell proliferation via cell cycle arrest. RP7214 is a novel and potent DHODH inhibitor that inhibited DHODH activity in enzyme and PHA induced HWB/PBMC proliferation with IC50 and EC50 values of 7.8 & 2.5/0.60 nM respectively. The objective of this study was to evaluate the effect of combining RP7214 with Gilteritinib, a small molecule inhibitor of FLT-3, or cytarabine in preclinical models of AML. Methods: Cell growth following incubation with RP7214 (1 μM), Gilteritinib (0.25-1.5 µM), or a combination of both agents was determined in the AML lines, THP-1 and U-937. Apoptosis was evaluated following incubation of cell lines with compounds for 72 h, subsequent staining with Annexin-V-PE and 7-AAD, and analysis by flow cytometry. For cell cycle, cells were incubated with compounds for 72 h, fixed with 70% ethanol, incubated at 4°C for 3 h, and stained with Propidium Iodide prior to analysis by flow cytometry. AKT phosphorylation was determined by Western blotting. In vivo efficacy was evaluated in a MV-4-11 mouse xenograft model. Results: In the AML cell lines (THP-1 and U-937), addition of RP7214 potentiated Gilteritinib activity manifested by a significant (P<0.05) growth inhibition when compared to the activity of the individual agents. Similarly, the combination (3 µM RP7214 + 0.75 µM Gilteritinib) led to a 50% increase in the number of apoptotic cells besides increasing cells in the G2/M phase by 31%. Combining RP7214 (3 µM) and Gilteritinib (0.1 µM) reduced AKT phosphorylation by 54% when compared to Gilteritinib in the THP-1 cell line. In the MV-4-11 mouse xenograft model, RP7214 when administered orally at 30 mg/kg/BID for 21 days demonstrated significant (P<0.001) anti-tumor activity both as a single agent and in combination with Cytarabine with tumor growth inhibitions of 37 and 73 %, respectively. No adverse events or body weight changes were observed throughout the study period. Conclusions: Inhibition of DHODH by RP7214 represents a unique therapeutic strategy in AML that accentuates the effect of approved and standard of care drugs such as Gilteritinib and Cytarabine. The compound is currently being tested in Phase-1 enabling studies with human trials expected to commence in 2020. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Kumar V. Penmetsa, Swaroop Vakkalanka. RP7214, a small molecule inhibitor of dihydroorotate dehydrogenase (DHODH), potentiates activity of Gilteritinib and Cytarabine in preclinical models of AML [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C044. doi:10.1158/1535-7163.TARG-19-C044

Abstract C042: Inhibition of Dihydroorotate Dehydrogenase (DHODH) by RP7214 attenuates growth and promotes differentiation of AML cell lines

Abstract Background: Dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme in pyrimidine synthesis, is overexpressed in certain cancers; blockade of which impedes tumor cell proliferation via cell cycle arrest. RP7214 is a novel and potent DHODH inhibitor that inhibited DHODH activity in enzyme and PHA induced HWB/PBMC proliferation with IC50 and EC50 values of 7.8 & 2.5/0.60 nM respectively. The objective of this study was to evaluate the single-agent activity of RP7214 on AML cell differentiation and growth. Methods: Viability of representative AML cell lines (U937, HL-60, THP-1, and KG-1) following incubation with RP7214 for 72 h as well as rescue in the presence of 300 µM Uridine was determined in an MTT-based assay. For differentiation experiments, THP-1 cells were incubated with RP7214 in complete media for 72 h. RNA was isolated, reverse transcribed to cDNA, and real-time PCR was performed to analyze CD11b gene expression. In addition, cell surface expression of CD11b in THP-1 cells incubated with RP7214 and the effect-reversal in presence of Uridine was evaluated by flow cytometry. Apoptosis and cell cycle were determined following incubation with RP7214 for 72 and 48 h respectively. Results: RP7214 inhibited AML cell proliferation in all the four cell lines tested with GI50 ranging from 2.4 to 7.6 µM. Addition of 300 µM Uridine caused a rightward shift with GI50 >10 µM. RT-PCR analysis indicated an 80-fold increase in mRNA expression of the differentiation marker CD11b in THP-1 cells treated with 3 µM RP7214. Similarly, expression of the CD11b marker on THP-1 cell surface was enhanced by 40% following incubation with 5 µM compound; effect of which was reduced to 15% in the presence of 300 µM Uridine. RP7214 dose-dependently induced apoptosis (30-50% at 3 µM) and arrested cycle progression in the “S-phase” (40-45% at 3 µM) in U937 and THP-1 cells. Conclusions: Results demonstrate the utility of RP7214 as a potent DHODH inhibitor in AML acting via suppression of cellular pyrimidine pools and promoting differentiation to fully mature cells. The compound is currently being evaluated in IND-enabling tolerability studies with Phase-1 trial in AML expected to commence in 2020. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Swaroop Vakkalanka. Inhibition of Dihydroorotate Dehydrogenase (DHODH) by RP7214 attenuates growth and promotes differentiation of AML cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C042. doi:10.1158/1535-7163.TARG-19-C042

STEM-22. TARGETING PYRIMIDINE SYNTHESIS ACCENTUATES MOLECULAR THERAPY RESPONSE IN GLIOBLASTOMA STEM CELLS

Abstract Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamyolase, dihydroorotase (CAD) or the critical downstream enzyme, dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity. Higher expression of pyrimidine synthesis genes portend poor prognosis of glioblastoma patients. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.