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Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl–coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.

The phosphonate group is a key pharmacophore in many anti-viral, anti-microbial, and anti-neoplastic drugs. Due to its high polarity and short retention time, detecting and quantifying such phosphonate-containing drugs with LC/MS-based methods is challenging and requires 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 quantitation of the phosphonate-containing enolase inhibitor HEX in plasma and tissue at micromolar concentrations. Although mice were shown to rapidly clear HEX from circulation (over 95% in 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.

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 aninduced vulnerabilityto 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.

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., The metabolite methylthioadenosine (MTA) inhibits PRMT5. Therefore, MTA accumulation due to MTA phosphorylase (MTAP) deletion has been proposed as a vulnerability for PRMT5-targeted therapy in cancer. Here, the authors show that MTA does not accumulate in MTAP-deficient cancer cells but is secreted and metabolized by MTAP-intact cells in the tumour microenvironment.

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.

Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention. Electronic supplementary material The online version of this article (10.1007/s00262-020-02748-9) contains supplementary material, which is available to authorized users.

The maintenance of skeletal muscle mass depends on the overall balance between the rates of protein synthesis and degradation. Thus, age-related muscle atrophy and function, commonly known as sarcopenia, may result from decreased protein synthesis, increased proteolysis, or simultaneous changes in both processes governed by complex multifactorial mechanisms. Growing evidence implicates oxidative stress and reactive oxygen species (ROS) as an essential regulator of proteolysis. Our previous studies have shown that genetic deletion of CuZn superoxide dismutase (CuZnSOD, Sod1) in mice leads to elevated oxidative stress, muscle atrophy and weakness, and an acceleration in age-related phenotypes associated with sarcopenia. The goal of this study is to determine whether oxidative stress directly influences the acceleration of proteolysis in skeletal muscle of Sod1(−/−) mice as a function of age. Compared to control, Sod1(−/−) muscle showed a significant elevation in protein carbonyls and 3-nitrotyrosine levels, suggesting high oxidative and nitrosative protein modifications were present. In addition, age-dependent muscle atrophy in Sod1(−/−) muscle was accompanied by an upregulation of the cysteine proteases, calpain, and caspase-3, which are known to play a key role in the initial breakdown of sarcomeres during atrophic conditions. Furthermore, an increase in oxidative stress-induced muscle atrophy was also strongly coupled with simultaneous activation of two major proteolytic systems, the ubiquitin-proteasome and lysosomal autophagy pathways. Collectively, our data suggest that chronic oxidative stress in Sod1(−/−) mice accelerates age-dependent muscle atrophy by enhancing coordinated activation of the proteolytic systems, thereby resulting in overall protein degradation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11357-020-00200-5) contains supplementary material, which is available to authorized users.

Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). We previously identified and characterized a competitive, small molecule phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX), and its lipophilic bis-ester prodrug (POMHEX) in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma. Treatment with either HEX (150 mg/kg IV/IP) or POMHEX (20 mg/kg IV/IP) yielded tumor regression even after drug discontinuation. However, due to the poor pharmacokinetics of esterase-labile POMHEX, we synthesized a library of novel phosphonate prodrugs with distinct mechanisms of bioactivation and assessed their potency in D423 (ENO1-/-) cells. By conducting a prodrug structure activity relationship (SAR) study, we found that phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, while canonical McGuigan (ProTide) prodrugs were not. Other strategies, including salicylic alcohol (cycloSal) and lipid prodrugs of HEX, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over POMHEX. En route, we developed a novel class of aliphatic amine/ester prodrugs that can be broadly applied to efficiently deliver phosph(on)ate pharmacophores in cells. 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. In sum, we have developed a novel class of phosph(on)ate prodrugs that is efficiently bioactivated in cells in vitro. Our prodrug SAR study disputes the common notion that ProTides are universally advantageous promoieties on phosph(on)ate pharmacophores and we provide mechanistic rationale for this observation with HEX. Finally, we show that the cycloSal prodrug yields efficient intracellular delivery of HEX in vitro, with a mechanism of bioactivation consistent with the GBM microenvironment, making this promoiety promising for further evaluation in vivo. Citation Format: Victoria C. Yan, Cong-Dat Pham, Elliot S. Ballato, Kristine L. Yang, Sunada Khadka, Yasaman Barektain, Prakriti Shrestha, Theresa Tran, Anton H. Poral, Mykia Washington, Sudhir Raghavan, Barbara Czako, Federica Pisaneschi, Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Jeffrey J. Ackroyd, Dimitra K. Georgiou, Steven W. Millward, Florian L. Muller. Prodrugs of a 1-hydroxy-2-oxopiperidin-3-yl phosphonate enolase inhibitor for the treatment of ENO1-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3097.

Carboxy ester prodrugs are widely employed to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can mask problematic chemical features that prevent cellular uptake and may enable tissue-specific compound delivery. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, limiting their therapeutic potential. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes as microbial esterase-specific promoieties have not been described. Here we identify the bacterial esterases, GloB and FrmB, that activate carboxy ester prodrugs in

Remdesivir is a nucleoside monophosphoramidate prodrug that has been FDA approved for coronavirus disease 2019 (COVID-19). However, the clinical efficacy of remdesivir for COVID-19 remains contentious, as several trials have not found statistically significant differences in either time to clinical improvement or mortality between remdesivir-treated and control groups. Similarly, the inability of remdesivir to provide a clinically significant benefit above other investigational agents in patients with Ebola contrasts with strong, curative preclinical data generated in rhesus macaque models. For both COVID-19 and Ebola, significant discordance between the robust preclinical data and remdesivir’s lackluster clinical performance have left many puzzled. Here, we critically evaluate the assumptions of the models underlying remdesivir’s promising preclinical data and show that such assumptions overpredict efficacy and minimize toxicity of remdesivir in humans. Had the limitations of in vitro drug efficacy testing and species differences in drug metabolism been considered, the underwhelming clinical performance of remdesivir for both COVID-19 and Ebola would have been fully anticipated.

A recently published article by Li et al. titled, "Key Metabolic Enzymes in Remdesivir Activation in Lung Cells," validates the canonical McGuigan enzymes (CES1, CTSA, HINT1) involved in remdesivir's (RDV's) bioactivation using bioinformatic and biochemical approaches (1).….

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.

Remdesivir (RDV, GS-5734), the first FDA-approved antiviral for the treatment of COVID-19, is a single diastereomer monophosphoramidate prodrug of an adenosine analogue. It is intracellularly metabolized into the active triphosphate form, which in turn acts as a potent and selective inhibitor of multiple viral RNA polymerases. RDV has broad-spectrum activity against members of the coronavirus family, such as SARS-CoV-2, SARS-CoV, and MERS-CoV, as well as filoviruses and paramyxoviruses. To assess the potential for off-target toxicity, RDV was evaluated in a set of cellular and biochemical assays. Cytotoxicity was evaluated in a set of relevant human cell lines and primary cells. In addition, RDV was evaluated for mitochondrial toxicity under aerobic and anaerobic metabolic conditions, and for the effects on mitochondrial DNA content, mitochondrial protein synthesis, cellular respiration, and induction of reactive oxygen species. Last, the active 5'-triphosphate metabolite of RDV, GS-443902, was evaluated for potential interaction with human DNA and RNA polymerases. Among all of the human cells tested under 5 to 14 days of continuous exposure, the 50% cytotoxic concentration (CC

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.

Carboxy ester prodrugs have been widely employed as a means to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can successfully mask problematic chemical features that prevent cellular uptake and can be used to target delivery of compounds to specific tissues. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, curbing their potential therapeutic applications. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes due to a paucity of information about the selectivity of microbial esterases. Here we identify the bacterial esterases, GloB and FrmB, that are required for carboxy ester prodrug activation in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, which revealed several promoieties likely to be serum esterase-resistant while still being microbially labile. These studies lay the groundwork for structure-guided design of anti-staphyloccal promoieties and expand the range of molecules to target staphyloccal pathogens.

Angiogenesis inhibition has become a mainstay of oncology despite having fallen short of its early promise. As originally envisioned, angiogenesis inhibition would cut off the blood supply, deprive tumor cells of key nutrients, leading to their demise. In practice, while there is evidence that tumors under angiogenesis treatment do in fact exhibit some degree of metabolic stress, this is stress is not sufficient to induce significant cancer cell death. We posit that the full potential of angiogenesis inhibition can be realized by the combination of angiogenesis inhibition with emerging tumor metabolism targeting therapies. Because tumors under angiogenesis inhibition are already in a state of nutrient stress, the effects of metabolically targeted therapies such as amino acid depletion (e.g. asparginase, methionine restriction), inhibitors of stress adaption (AMPK and GCN2 inhibitors) or energy metabolism (e.g. IACS-010759, Metformin, POMHEX) stand to dramatically increase in potency whilst remaining selective for (angiogenic) tumor versus (non-angiogenic) normal tissue. Here, we provide proof-of-principal for this thesis. First, we performed metabolomic profiling of angiogenesis-inhibited tumors, which corroborates as state of nutrient stress in angiogenesis-inhibited tumors. Second, we demonstrate dramatic anti-neoplastic synergy (effectively curing of xenografted tumor-bearing mice, irrespective of initial tumor size), without enhanced adverse toxicities, between the OxPhos inhibitor IACS-010759 and the angiogenesis tyrosine kinase inhibitor, Tivozanib. The same results were recapitulated with the anti-VEGFA antibody, Avastin, and the OxPhos inhibitor could be substituted with the Enolase inhibitor HEX, with similar effects. The synergy was observed in a broad range of tumor types, even those without clear genetic susceptibilities. Together, these results suggest that Angiogenesis inhibitors synergize broadly with cancer therapies targeting metabolism, allowing the realization of the full potential of these previously disappointing drugs. Our results warrant systematic combination clinical trials between angiogenesis inhibitors and established, as well as emerging anti-metabolic cancer therapies.

Glycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are under investigation for treatment of obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase – a critical enzyme in glycolysis – and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage and induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context of Plasmodium falciparum malaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity in malaria parasite growth.

Phosphate and phosphonates containing a single PN bond are frequently used pro-drug motifs to improve cell permeability of these otherwise anionic moieties. Upon entry into the cell, the PN bond is cleaved by phosphoramidases to release the active agent. Here, we apply a novel mono-amidation strategy to our laboratory's phosphonate-containing glycolysis inhibitor and show that a diverse panel of phosphonoamidates may be rapidly generated for in vitro screening. We show that, in contrast to the canonical l-alanine or benzylamine moieties which have previously been reported as efficacious pro-drug moieties, small and long-chain aliphatic amines demonstrate greater drug release efficacy for our phosphonate inhibitor. These results expand the scope of possible amine pro-drugs that can be used as second pro-drug leave groups for phosphate or phosphonate-containing drugs.

With the rising prevalence of multidrug-resistance, there is an urgent need to develop novel antibiotics. Many putative antibiotics demonstrate promising in vitro potency but fail in vivo due to poor drug-like qualities (e.g. serum half-life, oral absorption, solubility, toxicity). These drug-like properties can be modified through the addition of chemical protecting groups, creating “prodrugs” that are activated prior to target inhibition. Lipophilic prodrugging techniques, including the attachment of a pivaloyloxymethyl group, have garnered attention for their ability to increase cellular permeability by masking charged residues and the relative ease of the chemical prodrugging process. Unfortunately, pivaloyloxymethyl prodrugs are rapidly activated by human sera, rendering any membrane permeability qualities absent during clinical treatment. Identification of the bacterial prodrug activation pathway(s) will allow for the development of host-stable and microbe-targeted prodrug therapies. Here, we use two zoonotic staphylococcal species, S. schleiferi and S. pseudintermedius, to establish the mechanism of carboxy ester prodrug activation. Using a forward genetic screen, we identify a conserved locus in both species encoding the enzyme hydroxyacylglutathione hydrolase (GloB), whose loss-of-function confers resistance to carboxy ester prodrugs. We enzymatically characterize GloB and demonstrate that it is a functional glyoxalase II enzyme, which has the capacity to activate carboxy ester prodrugs. As GloB homologs are both widespread and diverse in sequence, our findings suggest that GloB may be a useful mechanism for developing species-or genus-level prodrug targeting strategies.

While remdesivir has garnered much hope for its moderate anti-Covid-19 effects, its parent nucleoside, GS-441524, has been overlooked. Pharmacokinetic analysis of remdesivir evidences premature serum hydrolysis to GS-441524; GS-441524 is the predominant metabolite reaching the lungs. With its synthetic simplicity and in vivo efficacy in the veterinary setting, we contend that GS-441524 is superior to remdesivir for Covid-19 treatment.

[Image: see text] 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.

Summary: The plasticity of a preexisting regulatory circuit compromises the effectiveness of targeted therapies, and leveraging genetic vulnerabilities in cancer cells may overcome such adaptations. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is characterized by oxidative phosphorylation (OXPHOS) deficiency caused by fumarate hydratase (FH) nullizyogosity. To identify metabolic genes that are synthetically lethal with OXPHOS deficiency, we conducted a genetic loss-of-function screen and found that phosphogluconate dehydrogenase (PGD) inhibition robustly blocks the proliferation of FH mutant cancer cells both in vitro and in vivo. Mechanistically, PGD inhibition blocks glycolysis, suppresses reductive carboxylation of glutamine, and increases the NADP+/NADPH ratio to disrupt redox homeostasis. Furthermore, in the OXPHOS-proficient context, blocking OXPHOS using the small-molecule inhibitor IACS-010759 enhances sensitivity to PGD inhibition in vitro and in vivo. Together, our study reveals a dependency on PGD in OXPHOS-deficient tumors that might inform therapeutic intervention in specific patient populations. : Loss-of-function genetics screen reveals a synthetically lethal interaction between OXPHOS inhibition and phosphogluconate dehydrogenase (PGD) inactivation. Sun et al. provide an example of targeting tumor metabolism in a genetically predefined context to maximize therapeutic impact and propose PGD as a therapeutic target for fumarate hydratase-deficient HLRCC. Keywords: synthetic lethality, PGD, OXPHOS, tumor metabolism, metabolic vulnerability, fumarate hydratase, redox homeostasis, functional genomics, hereditary leiomyomatosis renal cell carcinoma, pentose phosphate pathway

Mono-amidated P(V) pro-drugs predominately contribute to the vastly improved delivery of phosphate and phosphonate-containing anti-viral/cancer nucleotide analogues. However, synthetic approaches towards their formation are often harsh and unreliable, which may hamper the identification of novel, more effective amine pro-drugs. Here, we show that direct mono-amidation of structurally complex phosphonic and phosphoric acids may be accomplished in as quickly as seconds by re-purposing the PPh3/DIAD redox pair. Where the triphenylphosphine oxide byproduct is often cited as a vulnerability, we use its formation as an asset. Juxtaposing the anionic nature of the generated mono-amidated product, the desired product may be isolated with a single water extraction. Compared to state-of-the-art strategies towards phosphoramidates, our approach is mild, reliable, and enables access to a variety of aliphatic and benzylic amines for pro-drug attachment.

Phosphate and phosphonates containing a single P-N bond are frequently used pro-drug motifs to improve cell permeability of these otherwise anionic moieties. Upon entry into the cell, the P-N bond is cleaved by phosphoramidases to release the active agent. Here, we apply a novel mono-amidation strategy to our laboratory’s phosphonate-containing glycolysis inhibitor and show that a diverse panel of phosphonoamidates may be rapidly generated for in vitro screening. We show that, in contrast to the canonical L-alanine or benzylamine moieties which have previously been reported as efficacious pro-drug moieties, small aliphatic amines demonstrate greater drug release efficacy for our phosphonate inhibitor. These results expand the scope of possible amine pro-drugs that can be used as second pro-drug leave groups for phosphate or phosphonate-containing drugs.Abstract Figure

Epigenetic modifiers frequently harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Histone methyltransferase KMT2D (a COMPASS-like enzyme, also called MLL4) is among the most highly inactivated epigenetic modifiers in lung cancer. Here we show that lung-specific loss of Kmt2d promotes lung tumorigenesis in mice and upregulates pro-tumorigenic programs, including glycolysis. Pharmacological inhibition of glycolysis preferentially impedes growth of human lung cancer cells bearing KMT2D-inactivating mutations. Mechanistically, Kmt2d loss widely impairs epigenomic signals for super-enhancers/enhancers, including the super-enhancer for the circadian rhythm repressor Per2. Loss of Kmt2d decreases expression of PER2, which regulates multiple glycolytic genes. These findings indicate that KMT2D is a lung tumor suppressor and KMT2D deficiency confers a therapeutic vulnerability to glycolytic inhibitors. SIGNIFICANCE: Lung cancer is the leading cause of cancer deaths. The overall survival rate for lung cancer patients remains low despite recent therapeutic advances, and a majority of lung cancer patients lack a druggable target. Therefore, there is a great need for a better understanding of the molecular mechanisms driving lung cancer. Epigenetic modifiers are frequently lost in lung cancer, but how this provokes lung tumorigenesis remains unclear. We show that KMT2D, which is recurrently mutated in lung cancer, is a lung tumor suppressor. Our results uncover a tumor-promoting epigenetic mechanism by which KMT2D-inactivating mutations induce aberrant metabolic reprogramming via super-enhancer impairment in lung cancer. Our findings support a glycolysis-inhibitory approach as a therapeutic intervention strategy against KMT2D-mutant lung cancer.

Glycolysis inhibition remains aspirational in cancer therapy. We recently described a promising phosphonate inhibitor of Enolase for cancers harboring deletions in ENO1. Here, we describe the application of nitroheterocycle pro-drugs capitalizing on tumor hypoxia. These bioreducible pro-drugs exhibit up to 14-fold greater potency under hypoxic conditions compared to normoxia and exhibit robust stability in biological fluids. Our work provides strong proof-of-concept for using bioreduction as a pro-drug delivery strategy in the context of Enolase inhibition.

Chemotherapy is central to oncology, perceived to operate only on prolific cancerous tissue. Yet, many non-neoplastic tissues are more prolific compared with typical tumors. Chemotherapies achieve sufficient therapeutic windows to exert antineoplastic activity because they are prodrugs that are bioactivated in cancer-specific environments. The advent of precision medicine has obscured this concept, favoring the development of high-potency kinase inhibitors. Inhibitors of essential mitotic kinases exemplify this paradigm shift, but intolerable on-target toxicities in more prolific normal tissues have led to repeated failures in the clinic. Proliferation rates alone cannot be used to achieve cancer specificity. Here, we discuss integrating the cancer specificity of prodrugs from classical chemotherapeutics and the potency of mitotic kinase inhibitors to generate a class of high-precision cancer therapeutics.

Background Immunotherapy has increasingly become a staple in cancer treatment. However, substantial limitations in the durability of response highlight the need for more rational therapeutic combinations. The aim of this study is to investigate how to make tumor cells more sensitive to T-cell-based cancer immunotherapy. Methods Two pairs of melanoma patient-derived tumor cell lines and their autologous tumor-infiltrating lymphocytes were utilized in a high-throughput screen of 850 compounds to identify bioactive agents that could be used in combinatorial strategies to improve T-cell-mediated killing of tumor cells. RNAi, overexpression, and gene expression analyses were utilized to identify the mechanism underlying the effect of Topoisomerase I (Top1) inhibitors on T-cell-mediated killing. Using a syngeneic mouse model (n = 5 per group), the antitumor efficacy of the combination of a clinically relevant Top1 inhibitor, liposomal irinotecan (MM-398), with immune checkpoint inhibitors was also assessed. All statistical tests were two-sided. Results We found that Top1 inhibitors increased the sensitivity of patient-derived melanoma cell lines (n = 7) to T-cell-mediated cytotoxicity (P < .001, Dunnett’s test). This enhancement is mediated by TP53INP1, whose overexpression increased the susceptibility of melanoma cell lines to T-cell cytotoxicity (2549 cell line: P = .009, unpaired t test), whereas its knockdown impeded T-cell killing of Top1 inhibitor–treated melanoma cells (2549 cell line: P < .001, unpaired t test). In vivo, greater tumor control was achieved with MM-398 in combination with α-PD-L1 or α-PD1 (P < .001, Tukey’s test). Prolonged survival was also observed in tumor-bearing mice treated with MM-398 in combination with α-PD-L1 (P = .002, log-rank test) or α-PD1 (P = .008, log-rank test). Conclusions We demonstrated that Top1 inhibitors can improve the antitumor efficacy of cancer immunotherapy, thus providing the basis for developing novel strategies using Top1 inhibitors to augment the efficacy of immunotherapy.

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., Barekatain et al. demonstrate that hydrogen–carbon heteronuclear single quantum correlation (HSQC) spectra, obtained using a standard NMR spectroscope, can detect tumours with specific oncometabolite alterations including IDH1 mutant glioblastoma, suggesting the feasibility of this method as a diagnostic tool.

Extensive efforts have been made to use non-invasive 1H magnetic resonance (MR) spectroscopy to quantify metabolites that are diagnostic of specific disease states. Within the realm of precision oncology, these efforts have largely centered on quantifying 2-hydroxyglutarate (2-HG) in tumors harboring isocitrate dehydrogenase 1 (IDH1) mutations. As many metabolites have similar chemical shifts, the resulting 1H spectra of intact biological material are highly convoluted, limiting the application of 1H MR to high abundance metabolites. Hydrogen-Carbon Heteronuclear single quantum correlation 1H-13C HSQC is routinely employed in organic synthesis to resolve complex spectra but has received limited attention for biological studies. Here, we show that 1H-13C HSQC offers a dramatic improvement in sensitivity compared to one-dimensional (1D) 13C NMR and dramatic signal deconvolution compared to 1D 1H spectra in an intact biological setting. Using a standard NMR spectroscope without specialized signal enhancements 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. We demonstrate that this method can readily identify tumors with specific genetic-driven oncometabolite alterations such as IDH mutations with elevation of 2-HG as well as PGD-homozygously deleted tumors with elevation of gluconate. These data support the potential of 1H-13C HSQC as a non-invasive diagnostic tool for metabolic precision oncology.

Tumors lack a well-regulated vascular supply of O2 and often fail to balance O2 supply and demand. Net O2 tension within many tumors may not only depend on O2 delivery but also depend strongly on O2 demand. Thus, tumor O2 consumption rates may influence tumor hypoxia up to true anoxia. Recent reports have shown that many human tumors in vivo depend primarily on oxidative phosphorylation (OxPhos), not glycolysis, for energy generation, providing a driver for consumptive hypoxia and an exploitable vulnerability. In this regard, IACS-010759 is a novel high affinity inhibitor of OxPhos targeting mitochondrial complex-I that has recently completed a Phase-I clinical trial in leukemia. However, in solid tumors, the effective translation of OxPhos inhibitors requires methods to monitor pharmacodynamics in vivo. Herein, 18F-fluoroazomycin arabinoside ([18F]FAZA), a 2-nitroimidazole-based hypoxia PET imaging agent, was combined with a rigorous test-retest imaging method for non-invasive quantification of the reversal of consumptive hypoxia in vivo as a mechanism-specific pharmacodynamic (PD) biomarker of target engagement for IACS-010759. Neither cell death nor loss of perfusion could account for the IACS-010759-induced decrease in [18F]FAZA retention. Notably, in an OxPhos-reliant melanoma tumor, a titration curve using [18F]FAZA PET retention in vivo yielded an IC50 for IACS-010759 (1.4 mg/kg) equivalent to analysis ex vivo. Pilot [18F]FAZA PET scans of a patient with grade IV glioblastoma yielded highly reproducible, high‐contrast images of hypoxia in vivo as validated by CA-IX and GLUT-1 IHC ex vivo. Thus, [18F]FAZA PET imaging provided direct evidence for the presence of consumptive hypoxia in vivo, the capacity for targeted reversal of consumptive hypoxia through the inhibition of OxPhos, and a highly-coupled mechanism-specific PD biomarker ready for translation.

In BriefThe co-deletion ofMTAPin theCDKN2Alocus is a frequent event in diverse cancers including glioblastoma. Recent publications report that significant accumulations of the MTAP substrate, methylthioadenosine (MTA), can sensitizeMTAP-deleted cancer cells to novel inhibitors of PRMT5 and MAT2A for targeted therapy against tumors with this particular genetic alteration. In this work, using comprehensive metabolomic profiling, we show that MTA is primarily secreted, resulting in exceedingly high levels of extracellular MTAin vitro. We further show that primary human glioblastoma tumors minimally accumulate MTAin vivo, which is likely explained by the metabolism of MTA byMTAP-competent stromal cells. Together, these data challenge whether the metabolic conditions required for therapies to exploit vulnerabilities associatedMTAPdeletions are present in primary human tumors, questioning their translational efficacy in the clinic.HighlightsMethylthioadenosine (MTA) is elevated inMTAP-deleted cancer cellsin vitro, which provides a selective vulnerability to PRMT5 and MAT2A inhibitorsAccumulation of MTA inMTAP-deleted cancer cells is predominately extracellular, suggesting active secretion of MTA.MTAP-deleted primary human glioblastoma tumors show minimal intratumoral elevations of MTA, which is likely explained by secretion and metabolism byMTAP-competent stromal cells.SUMMARYHomozygous deletion of theCDK2NAlocus frequently results in co-deletion of methylthioadenosine phosphorylase (MTAP) in many fatal cancers such as glioblastoma multiforme (GBM), resulting in elevations of the substrate metabolite, methylthioadenosine (MTA). To capitalize on such accumulations, therapeutic targeting of protein arginine methyltransferase 5 (PRMT5) and methionine adenosyl transferase (MAT2A) are ongoing. While extensively corroboratedin vitro, the clinical efficacy of these strategies ultimately relies on equally significant accumulations of MTA in human tumors. Here, we show thatin vitroaccumulation of MTA is a predominately extracellular phenomenon, indicating secretion of MTA fromMTAP-deleted cells. In primary human GBMs, we find that MTA levels are not significantly higher inMTAP-deleted compared toMTAP-intact tumors or normal brain tissue. Together, these findings highlight the metabolic discrepancies betweenin vitromodels and primary human tumors and should thus be carefully considered in the development of the precision therapies targetingMTAP-homozygous deleted GBM.

We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3S,5S)-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization; thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3S)-MethylSF2312 was up to 2000-fold more potent than (3R)-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3S enantiomer of SF2312. Novel X-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3S,5S)-enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3S,5S)-enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3S,5S)-SF2312 is the single active enantiomer of inhibitor SF2312.

Despite being crucial for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme enolase 2 (ENO2) for the treatment of cancers with deletion of ENO1 (encoding enolase 1), we modeled the synthetic tool compound inhibitor phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analog of PhAH, in which the hydroxamic nitrogen is linked to Cα by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure-based search revealed that our hypothesized backbone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low-nanomolar inhibitor of enolase.

Epigenetic modifiers often harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Here we show that lung-specific loss of the gene encoding the histone methyltransferase MLL4 (alias KMT2D; a COMPASS-like enzyme), which is ranked the most highly inactivated epigenetic modifier in lung cancer, strongly promotes lung adenocarcinoma in mice. Mll4 loss upregulated tumor-promoting programs, including glycolysis. The pharmacological inhibition of glycolysis preferentially impeded tumorigenic growth of human lung cancer cells bearing MLL4-inactivating mutations. Mll4 loss widely impaired epigenomic signals for super-enhancers and enhancers, including the super-enhancer for the circadian rhythm repressor gene Per2, and decreased Per2 expression. Per2 downregulated several glycolytic pathway genes. These findings uncover a distinct tumor-suppressive epigenetic mechanism in which MLL4 enhances Per2-mediated repression of pro-tumorigenic glycolytic genes via super-enhancer activation to suppress lung adenocarcinoma tumorigenesis and also implicate a glycolysis-targeting strategy as a therapeutic intervention for the treatment of MLL4- mutant lung cancer.

SUMMARY Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on conditional and melanocyte-specific deletion of KMT2D. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways, including glycolysis. KMT2D deficiency aberrantly upregulates glycolysis enzymes, intermediate metabolites, and glucose consumption rates. Mechanistically, KMT2D loss causes genome-wide reduction of H3K4me1-marked active enhancer chromatin states. Enhancer loss and subsequent repression of IGFBP5 activates IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. Pharmacological inhibition of glycolysis and insulin growth factor (IGF) signaling reduce proliferation and tumorigenesis preferentially in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating an increased use of the glycolysis pathway for enhanced biomass needs via enhancer reprogramming, thus presenting an opportunity for therapeutic intervention through glycolysis or IGF pathway inhibitors., In Brief Through an in vivo epigenome-focused pooled RNAi screen, Maitituoheti et al. identify KMT2D as a tumor suppressor in melanoma. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways by reduction of H3K4me1-marked active enhancers, conferring sensitivity to glycolysis and IGFR inhibitors in melanoma with KMT2D-inactivating mutations., Graphical Abstract