Comparative Pharmacology of a Bis-Pivaloyloxymethyl Phosphonate Prodrug Inhibitor of Enolase after Oral and Parenteral Administration.

Metabolically labile prodrugs can experience stark differences in catabolism incurred by the chosen route of administration. This is especially true for phosph(on)ate prodrugs, in which successive promoiety removal transforms a lipophilic molecule into increasingly polar compounds. We previously described a phosphonate inhibitor of enolase (HEX) and its bis-pivaloyloxymethyl ester prodrug (POMHEX) capable of eliciting strong tumor regression in a murine model of enolase 1 (ENO1)-deleted glioblastoma following parenteral administration. Here, we characterize the pharmacokinetics and pharmacodynamics of these enolase inhibitors in vitro and in vivo after oral and parenteral administration. In support of the historical function of lipophilic prodrugs, the bis-POM prodrug significantly improves cell permeability of and rapid hydrolysis to the parent phosphonate, resulting in rapid intracellular loading of peripheral blood mononuclear cells in vitro and in vivo. We observe the influence of intracellular trapping in vivo on divergent pharmacokinetic profiles of POMHEX and its metabolites after oral and parenteral administration. This is a clear demonstration of the tissue reservoir effect hypothesized to explain phosph(on)ate prodrug pharmacokinetics but has heretofore not been explicitly demonstrated.

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.

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.

Mesenchymal Stem Cell Derived Extracellular Vesicles for Repairing the Neurovascular Unit after Ischemic Stroke.

Ischemic stroke is a debilitating disease and one of the leading causes of long-term disability. During the early phase after ischemic stroke, the blood-brain barrier (BBB) exhibits increased permeability and disruption, leading to an influx of immune cells and inflammatory molecules that exacerbate the damage to the brain tissue. Mesenchymal stem cells have been investigated as a promising therapy to improve the recovery after ischemic stroke. The therapeutic effects imparted by MSCs are mostly paracrine. Recently, the role of extracellular vesicles released by these MSCs have been studied as possible carriers of information to the brain. This review focuses on the potential of MSC derived EVs to repair the components of the neurovascular unit (NVU) controlling the BBB, in order to promote overall recovery from stroke. Here, we review the techniques for increasing the effectiveness of MSC-based therapeutics, such as improved homing capabilities, bioengineering protein expression, modified culture conditions, and customizing the contents of EVs. Combining multiple techniques targeting NVU repair may provide the basis for improved future stroke treatment paradigms.

Aurora kinase inhibition sensitizes melanoma cells to T-cell-mediated cytotoxicity.

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.

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.

The 3S Enantiomer Drives Enolase Inhibitory Activity in SF2312 and Its Analogues.

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.

Aspirin in stroke patients modifies the immunomodulatory interactions of marrow stromal cells and monocytes.

BACKGROUND AND OBJECTIVE: Most stroke patients are prescribed aspirin (ASA) to adjust blood coagulability. Marrow stromal cells (MSCs) are being tested in clinical trials for stroke patients who likely are prescribed aspirin. One of the principal mechanisms of action of MSCs and ASA is modulation of the inflammatory response, including those mediated by monocytes (Mo). Thus, here we tested if aspirin can modify anti-inflammatory properties of MSCs or Mo alone, and in combination. METHODS: Mo were isolated at 24 h of stroke onset from ischemic stroke patients with NIHSS ranging from 11 to 20 or from healthy controls. Human bone marrow-derived MSCs from healthy subjects were used at passage 3. Mo, MSCs, and MSCs-Mo co-cultures were exposed to ASA at clinically relevant doses. The secretome profile of inflammatory mediators was measured using Magpix multiplex cytokine array. Viability was measured using MTT assay. Linear mixed effect model was used for statistical analysis. RESULTS: Overall Mo from control subjects exposed to ASA showed increased secretion of IL-1RA, IL-8, MCP-1, and TNF-α and Mo from stroke patients showed greater release of IL-1RA and MCP-1. In MSCs-Mo co-cultures, ASA added to co-cultures of control Mo reduced fractalkine secretion while it increased the fractalkine secretion when added to Mo from stroke patients. In addition, in co-cultures independent of Mo origin, ASA reduced IL-6, IL-8, MCP-1, and TNF-α. CONCLUSIONS: Aspirin in acute stroke patients may modulate the secretome profile of Mo and MSCs, thus potentially modulating immune and inflammatory responses associated with stroke. Our results suggest that stroke trials involving the use of intravenous MSCs should consider the effect of aspirin as a confounding factor.

Protective Effects of Autologous Bone Marrow Mononuclear Cells After Administering t-PA in an Embolic Stroke Model.

Tissue plasminogen activator (t-PA) is the only FDA-approved drug for acute ischemic stroke but poses risk for hemorrhagic transformation (HT). Cell therapy has been investigated as a potential therapy to improve recovery after stroke by the modulation of inflammatory responses and the improvement of blood-brain barrier (BBB) integrity, both of which are associated with HT after t-PA. In our present study, we studied the effect of autologous bone marrow mononuclear cells (MNCs) in an embolic stroke model. We administered MNCs in a rat embolic stroke 2 h after administering t-PA. We observed that even though autologous MNCs did not alter the incidence of HT, they decreased the severity of HT and reduced BBB permeability. One possible mechanism could be through the inhibition of MMP3 released by astrocytes via JAK/STAT pathway as shown by our in vitro cell interaction studies.

The Effect of Topoisomerase I Inhibitors on the Efficacy of T-Cell-Based Cancer Immunotherapy.

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.

HSP90 inhibition enhances cancer immunotherapy by upregulating interferon response genes.

T-cell-based immunotherapies are promising treatments for cancer patients. Although durable responses can be achieved in some patients, many patients fail to respond to these therapies, underscoring the need for improvement with combination therapies. From a screen of 850 bioactive compounds, we identify HSP90 inhibitors as candidates for combination with immunotherapy. We show that inhibition of HSP90 with ganetespib enhances T-cell-mediated killing of patient-derived human melanoma cells by their autologous T cells in vitro and potentiates responses to anti-CTLA4 and anti-PD1 therapy in vivo. Mechanistic studies reveal that HSP90 inhibition results in upregulation of interferon response genes, which are essential for the enhanced killing of ganetespib treated melanoma cells by T cells. Taken together, these findings provide evidence that HSP90 inhibition can potentiate T-cell-mediated anti-tumor immune responses, and rationale to explore the combination of immunotherapy and HSP90 inhibitors.Many patients fail to respond to T cell based immunotherapies. Here, the authors, through a high-throughput screening, identify HSP90 inhibitors as a class of preferred drugs for treatment combination with immunotherapy.

Tumor Evolution of Glioma-Intrinsic Gene Expression Subtypes Associates with Immunological Changes in the Microenvironment.

We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single-cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4+ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Longitudinal transcriptome analysis showed that expression subtype is retained in 55% of cases. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence associated with CD8+ T cell enrichment. Frequency of M2 macrophages detection associated with short-term relapse after radiation therapy.

Genomic deletion of malic enzyme 2 confers collateral lethality in pancreatic cancer.

The genome of pancreatic ductal adenocarcinoma (PDAC) frequently contains deletions of tumour suppressor gene loci, most notably SMAD4, which is homozygously deleted in nearly one-third of cases. As loss of neighbouring housekeeping genes can confer collateral lethality, we sought to determine whether loss of the metabolic gene malic enzyme 2 (ME2) in the SMAD4 locus would create cancer-specific metabolic vulnerability upon targeting of its paralogous isoform ME3. The mitochondrial malic enzymes (ME2 and ME3) are oxidative decarboxylases that catalyse the conversion of malate to pyruvate and are essential for NADPH regeneration and reactive oxygen species homeostasis. Here we show that ME3 depletion selectively kills ME2-null PDAC cells in a manner consistent with an essential function for ME3 in ME2-null cancer cells. Mechanistically, integrated metabolomic and molecular investigation of cells deficient in mitochondrial malic enzymes revealed diminished NADPH production and consequent high levels of reactive oxygen species. These changes activate AMP activated protein kinase (AMPK), which in turn directly suppresses sterol regulatory element-binding protein 1 (SREBP1)-directed transcription of its direct targets including the BCAT2 branched-chain amino acid transaminase 2) gene. BCAT2 catalyses the transfer of the amino group from branched-chain amino acids to α-ketoglutarate (α-KG) thereby regenerating glutamate, which functions in part to support de novo nucleotide synthesis. Thus, mitochondrial malic enzyme deficiency, which results in impaired NADPH production, provides a prime 'collateral lethality' therapeutic strategy for the treatment of a substantial fraction of patients diagnosed with this intractable disease.

Mesenchymal stromal cell secretomes are modulated by suspension time, delivery vehicle, passage through catheter, and exposure to adjuvants.

BACKGROUND AIMS: Extensive animal data indicate that mesenchymal stromal cells (MSCs) improve outcome in stroke models. Intra-arterial (IA) injection is a promising route of delivery for MSCs. Therapeutic effect of MSCs in stroke is likely based on the broad repertoire of secreted trophic and immunomodulatory cytokines produced by MSCs. We determined the differential effects of exposing MSCs to different types of clinically relevant vehicles, and/or different additives and passage through a catheter relevant to IA injections. METHODS: MSCs derived from human bone marrow were tested in the following vehicles: 5% albumin (ALB), 6% Hextend (HEX) and 40% dextran (DEX). Each solution was tested (i) alone, (ii) with low-dose heparin, (iii) with 10% Omnipaque, or (iv) a combination of heparin and Omnipaque. Cells in vehicles were collected directly or passed through an IA catheter, and MSC viability and cytokine release profiles were assessed. RESULTS: Cell viability remained above 90% under all tested conditions with albumin being the highest at 97%. Viability was slightly reduced after catheter passage or exposure to heparin or Omnipaque. Catheter passage had little effect on MSC cytokine secretion. ALB led to increased release of angiogenic factors such as vascular endothelial growth factor compared with other vehicles, while HEX and DEX led to suppression of pro-inflammatory cytokines such as interleukin-6. However, when these three vehicles were subjected to catheter passage and/or exposure to additives, the cytokine release profile varied depending on the combination of conditions to which MSCs were exposed. DISCUSSION: Exposure of MSCs to certain types of vehicles or additives changes the profile of cytokine secretion. The activation phenotype of MSCs may therefore be affected by the vehicles used for these cells or the exposure to the adjuvants used in their administration.

ENOblock Does Not Inhibit the Activity of the Glycolytic Enzyme Enolase.

Inhibition of glycolysis is of great potential for the treatment of cancer. However, inhibitors of glycolytic enzymes with favorable pharmacological profiles have not been forthcoming. Due to the nature of their active sites, most high-affinity transition-state analogue inhibitors of glycolysis enzymes are highly polar with poor cell permeability. A recent publication reported a novel, non-active site inhibitor of the glycolytic enzyme Enolase, termed ENOblock (N-[2-[2-2-aminoethoxy)ethoxy]ethyl]4-4-cyclohexylmethyl)amino]6-4-fluorophenyl)methyl]amino]1,3,5-triazin-2-yl]amino]benzeneacetamide). This would present a major advance, as this is heterocyclic and fully cell permeable molecule. Here, we present evidence that ENOblock does not inhibit Enolase enzymatic activity in vitro as measured by three different assays, including a novel 31P NMR based method which avoids complications associated with optical interferences in the UV range. Indeed, we note that due to strong UV absorbance, ENOblock interferes with the direct spectrophotometric detection of the product of Enolase, phosphoenolpyruvate. Unlike established Enolase inhibitors, ENOblock does not show selective toxicity to ENO1-deleted glioma cells in culture. While our data do not dispute the biological effects previously attributed to ENOblock, they indicate that such effects must be caused by mechanisms other than direct inhibition of Enolase enzymatic activity.

SF2312 is a natural phosphonate inhibitor of enolase.

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.

Cryopreservation of Bone Marrow Mononuclear Cells Alters Their Viability and Subpopulation Composition but Not Their Treatment Effects in a Rodent Stroke Model.

The systemic administration of autologous bone marrow (BM) derived mononuclear cells (MNCs) is under investigation as a novel therapeutic modality for the treatment of ischemic stroke. Autologous applications raise the possibility that MNCs could potentially be stored as a banked source. There have been no studies that investigate the effects of cryopreservation of BM-MNCs on their functional abilities in stroke models. In the present study, C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAo) for 60 minutes and then divided into two treatment groups: fresh MNCs versus cryopreserved MNCs. BM-MNCs were collected at 22 hours after MCAo and were stored in liquid nitrogen for 12 months in cryopreserved MNCs group. BM-MNCs cellular viability, composition, and phenotype of the various subpopulations of mice BM-MNCs were evaluated by flow cytometry, and the behavioral recovery of stroke animals was tested with freshly harvested MNCs versus cryopreserved MNCs by corner test and ladder rung test. We found that long-term cryopreservation negatively impacts the cellular viability of bone marrow MNCs. Cryopreservation also alters the cellular composition of various subpopulations within the MNCs. However, despite the changes observed in cryopreserved cells, both fresh and frozen MNCs have similar beneficial effect on behavioral and histological outcomes.