Quantification of Phosphonate Drugs by 1H-31P HSQC Shows That Rats Are Better Models of Primate Drug Exposure than Mice.

The phosphonate group is a key pharmacophore in many antiviral, antimicrobial, and antineoplastic drugs. Due to its high polarity and short retention time, detecting and quantifying such phosphonate-containing drugs with LC/MS-based methods are challenging and require derivatization with hazardous reagents. Given the emerging importance of phosphonate-containing drugs, developing a practical, accessible, and safe method for their quantitation in pharmacokinetics (PK) studies is desirable. NMR-based methods are often employed in drug discovery but are seldom used for compound quantitation in PK studies. Here, we show that proton-phosphorous (1H-31P) heteronuclear single quantum correlation (HSQC) NMR allows for the quantitation of the phosphonate-containing enolase inhibitor HEX in plasma and tissues at micromolar concentrations. Although mice were shown to rapidly clear HEX from circulation (over 95% in <1 h), the plasma half-life of HEX was more than 1 h in rats and nonhuman primates. This slower clearance rate affords a significantly higher exposure of HEX in rat models compared to that in mouse models while maintaining a favorable safety profile. Similar results were observed for the phosphonate-containing antibiotic, fosfomycin. Our study demonstrates the applicability of the 1H-31P HSQC method to quantify phosphonate-containing drugs in complex biological samples and illustrates an important limitation of mice as preclinical model species for phosphonate-containing drugs.

Anaplerotic nutrient stress drives synergy of angiogenesis inhibitors with therapeutics targeting tumor metabolism.

Tumor angiogenesis is a cancer hallmark, and its therapeutic inhibition has provided meaningful, albeit limited, clinical benefit. While anti-angiogenesis inhibitors deprive the tumor of oxygen and essential nutrients, cancer cells activate metabolic adaptations to diminish therapeutic response. Despite these adaptations, angiogenesis inhibition incurs extensive metabolic stress, prompting us to consider such metabolic stress as an induced vulnerability to therapies targeting cancer metabolism. Metabolomic profiling of angiogenesis-inhibited intracranial xenografts showed universal decrease in tricarboxylic acid cycle intermediates, corroborating a state of anaplerotic nutrient deficit or stress. Accordingly, we show strong synergy between angiogenesis inhibitors (Avastin, Tivozanib) and inhibitors of glycolysis or oxidative phosphorylation through exacerbation of anaplerotic nutrient stress in intracranial orthotopic xenografted gliomas. Our findings were recapitulated in GBM xenografts that do not have genetically predisposed metabolic vulnerabilities at baseline. Thus, our findings cement the central importance of the tricarboxylic acid cycle as the nexus of metabolic vulnerabilities and suggest clinical path hypothesis combining angiogenesis inhibitors with pharmacological cancer interventions targeting tumor metabolism for GBM tumors.

Generation and Validation of an Anti-Human PANK3 Mouse Monoclonal Antibody.

Coenzyme A (CoA) is an essential co-factor at the intersection of diverse metabolic pathways. Cellular CoA biosynthesis is regulated at the first committed step-phosphorylation of pantothenic acid-catalyzed by pantothenate kinases (PANK1,2,3 in humans, PANK3 being the most highly expressed). Despite the critical importance of CoA in metabolism, the differential roles of PANK isoforms remain poorly understood. Our investigations of PANK proteins as potential precision oncology collateral lethality targets (PANK1 is co-deleted as part of the PTEN locus in some highly aggressive cancers) were severely hindered by a dearth of commercial antibodies that can reliably detect endogenous PANK3 protein. While we successfully validated commercial antibodies for PANK1 and PANK2 using CRISPR knockout cell lines, we found no commercial antibody that could detect endogenous PANK3. We therefore set out to generate a mouse monoclonal antibody against human PANK3 protein. We demonstrate that a clone (Clone MDA-299-62A) can reliably detect endogenous PANK3 protein in cancer cell lines, with band-specificity confirmed by CRISPR PANK3 knockout and knockdown cell lines. Sub-cellular fractionation shows that PANK3 is overwhelmingly cytosolic and expressed broadly across cancer cell lines. PANK3 monoclonal antibody MDA-299-62A should prove a valuable tool for researchers investigating this understudied family of metabolic enzymes in health and disease.

Prodrugs of a 1-Hydroxy-2-oxopiperidin-3-yl Phosphonate Enolase Inhibitor for the Treatment of ENO1-Deleted Cancers.

Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). A previous work described the sustained tumor regression activities of a substrate-competitive phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX) (5), and its bis-pivaloyoxymethyl prodrug, POMHEX (6), in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma [Nature Metabolism 2020, 2, 1423-1426]. Due to poor pharmacokinetics of bis-ester prodrugs, this study was undertaken to identify potential non-esterase prodrugs for further development. Whereas phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, McGuigan prodrugs were not. Other strategies, including cycloSal and lipid prodrugs of 5, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over 6. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation.

Pharmacokinetics of Orally Administered GS-441524 in Dogs.

Despite being FDA-approved for COVID-19, the clinical efficacy of remdesivir (Veklury®) remains contentious. We previously pointed out pharmacokinetic, pharmacodynamic and toxicology reasons for why its parent nucleoside GS-441524, is better suited for COVID-19 treatment. Here, we assess the oral bioavailability of GS-441524 in beagle dogs and show that plasma concentrations ~24-fold higher than the EC50 against SARS-CoV-2 are easily and safely sustained. These data support translation of GS-441524 as an oral agent for COVID-19.

Impaired anaplerosis is a major contributor to glycolysis inhibitor toxicity in glioma.

BACKGROUND: Reprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. In tumors, the tricarboxylic acid (TCA) cycle generates biosynthetic intermediates and must be replenished (anaplerosis), mainly from pyruvate and glutamine. We recently described a novel enolase inhibitor, HEX, and its pro-drug POMHEX. Since glycolysis inhibition would deprive the cell of a key source of pyruvate, we hypothesized that enolase inhibitors might inhibit anaplerosis and synergize with other inhibitors of anaplerosis, such as the glutaminase inhibitor, CB-839. METHODS: We analyzed polar metabolites in sensitive (ENO1-deleted) and resistant (ENO1-WT) glioma cells treated with enolase and glutaminase inhibitors. We investigated whether sensitivity to enolase inhibitors could be attenuated by exogenous anaplerotic metabolites. We also determined the synergy between enolase inhibitors and the glutaminase inhibitor CB-839 in glioma cells in vitro and in vivo in both intracranial and subcutaneous tumor models. RESULTS: Metabolomic profiling of ENO1-deleted glioma cells treated with the enolase inhibitor revealed a profound decrease in the TCA cycle metabolites with the toxicity reversible upon exogenous supplementation of supraphysiological levels of anaplerotic substrates, including pyruvate. ENO1-deleted cells also exhibited selective sensitivity to the glutaminase inhibitor CB-839, in a manner rescuable by supplementation of anaplerotic substrates or plasma-like media PlasmaxTM. In vitro, the interaction of these two drugs yielded a strong synergistic interaction but the antineoplastic effects of CB-839 as a single agent in ENO1-deleted xenograft tumors in vivo were modest in both intracranial orthotopic tumors, where the limited efficacy could be attributed to the blood-brain barrier (BBB), and subcutaneous xenografts, where BBB penetration is not an issue. This contrasts with the enolase inhibitor HEX, which, despite its negative charge, achieved antineoplastic effects in both intracranial and subcutaneous tumors. CONCLUSION: Together, these data suggest that at least for ENO1-deleted gliomas, tumors in vivo-unlike cells in culture-show limited dependence on glutaminolysis and instead primarily depend on glycolysis for anaplerosis. Our findings reinforce the previously reported metabolic idiosyncrasies of in vitro culture and suggest that cell culture media nutrient composition more faithful to the in vivo environment will more accurately predict in vivo efficacy of metabolism targeting drugs.

Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine.

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP's substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion.

Robust detection of oncometabolic aberrations by 1H-13C heteronuclear single quantum correlation in intact biological specimens.

Magnetic resonance (MR) spectroscopy has potential to non-invasively detect metabolites of diagnostic significance for precision oncology. Yet, many metabolites have similar chemical shifts, yielding highly convoluted 1H spectra of intact biological material and limiting diagnostic utility. Here, we show that hydrogen-carbon heteronuclear single quantum correlation (1H-13C HSQC) offers dramatic improvements in sensitivity compared to one-dimensional (1D) 13C NMR and significant signal deconvolution compared to 1D 1H spectra in intact biological settings. Using a standard NMR spectroscope with a cryoprobe but without specialized signal enhancing features such as magic angle spinning, metabolite extractions or 13C-isotopic enrichment, we obtain well-resolved 2D 1H-13C HSQC spectra in live cancer cells, in ex vivo freshly dissected xenografted tumors and resected primary tumors. This method can identify tumors with specific oncometabolite alterations such as IDH mutations by 2-hydroxyglutarate and PGD-deleted tumors by gluconate. Results suggest potential of 1H-13C HSQC as a non-invasive diagnostic in precision oncology.

Bioreducible Phosphonoamidate Pro-drug Inhibitor of Enolase: Proof of Concept Study.

Glycolysis inhibition remains aspirational in cancer therapy. We recently described a promising phosphonate inhibitor of enolase for cancers harboring homozygous deletions of ENO1. Here, we describe the application of a nitroheterocycle phosphonoamidate pro-drug pair to capitalize on tumor hypoxia. This bioreducible prodrug exhibits greater-than 2-fold potency under hypoxic conditions compared to normoxia and exhibits robust stability in biological fluids. Our work provides strong in vitro proof-of-concept for using bioreduction as a pro-drug delivery strategy in the context of enolase inhibition.

An enolase inhibitor for the targeted treatment of ENO1-deleted cancers.

Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We have previously identified a subset of cancers harbouring homozygous deletion of the glycolytic enzyme enolase (ENO1) that have exceptional sensitivity to inhibition of its redundant paralogue, ENO2, through a therapeutic strategy known as collateral lethality. Here, we show that a small-molecule enolase inhibitor, POMHEX, can selectively kill ENO1-deleted glioma cells at low-nanomolar concentrations and eradicate intracranial orthotopic ENO1-deleted tumours in mice at doses well-tolerated in non-human primates. Our data provide an in vivo proof of principle of the power of collateral lethality in precision oncology and demonstrate the utility of POMHEX for glycolysis inhibition with potential use across a range of therapeutic settings.

An In Vivo Screen Identifies PYGO2 as a Driver for Metastatic Prostate Cancer.

Advanced prostate cancer displays conspicuous chromosomal instability and rampant copy number aberrations, yet the identity of functional drivers resident in many amplicons remain elusive. Here, we implemented a functional genomics approach to identify new oncogenes involved in prostate cancer progression. Through integrated analyses of focal amplicons in large prostate cancer genomic and transcriptomic datasets as well as genes upregulated in metastasis, 276 putative oncogenes were enlisted into an in vivo gain-of-function tumorigenesis screen. Among the top positive hits, we conducted an in-depth functional analysis on Pygopus family PHD finger 2 (PYGO2), located in the amplicon at 1q21.3. PYGO2 overexpression enhances primary tumor growth and local invasion to draining lymph nodes. Conversely, PYGO2 depletion inhibits prostate cancer cell invasion in vitro and progression of primary tumor and metastasis in vivo In clinical samples, PYGO2 upregulation associated with higher Gleason score and metastasis to lymph nodes and bone. Silencing PYGO2 expression in patient-derived xenograft models impairs tumor progression. Finally, PYGO2 is necessary to enhance the transcriptional activation in response to ligand-induced Wnt/ß-catenin signaling. Together, our results indicate that PYGO2 functions as a driver oncogene in the 1q21.3 amplicon and may serve as a potential prognostic biomarker and therapeutic target for metastatic prostate cancer.Significance: Amplification/overexpression of PYGO2 may serve as a biomarker for prostate cancer progression and metastasis. Cancer Res; 78(14); 3823-33. ©2018 AACR.

Targeting YAP-Dependent MDSC Infiltration Impairs Tumor Progression.

UNLABELLED: The signaling mechanisms between prostate cancer cells and infiltrating immune cells may illuminate novel therapeutic approaches. Here, utilizing a prostate adenocarcinoma model driven by loss of Pten and Smad4, we identify polymorphonuclear myeloid-derived suppressor cells (MDSC) as the major infiltrating immune cell type, and depletion of MDSCs blocks progression. Employing a novel dual reporter prostate cancer model, epithelial and stromal transcriptomic profiling identified CXCL5 as a cancer-secreted chemokine to attract CXCR2-expressing MDSCs, and, correspondingly, pharmacologic inhibition of CXCR2 impeded tumor progression. Integrated analyses identified hyperactivated Hippo-YAP signaling in driving CXCL5 upregulation in cancer cells through the YAP-TEAD complex and promoting MDSC recruitment. Clinicopathologic studies reveal upregulation and activation of YAP1 in a subset of human prostate tumors, and the YAP1 signature is enriched in primary prostate tumor samples with stronger expression of MDSC-relevant genes. Together, YAP-driven MDSC recruitment via heterotypic CXCL5-CXCR2 signaling reveals an effective therapeutic strategy for advanced prostate cancer. SIGNIFICANCE: We demonstrate a critical role of MDSCs in prostate tumor progression and discover a cancer cell nonautonomous function of the Hippo-YAP pathway in regulation of CXCL5, a ligand for CXCR2-expressing MDSCs. Pharmacologic elimination of MDSCs or blocking the heterotypic CXCL5-CXCR2 signaling circuit elicits robust antitumor responses and prolongs survival.