Nikunj Satani

The intricate mechanisms of neurodegeneration in prion diseases

Prion diseases are a group of infectious neurodegenerative diseases with an entirely novel mechanism of transmission, involving a protein-only infectious agent that propagates the disease by transmitting protein conformational changes. The disease results from extensive and progressive brain degeneration. The molecular mechanisms involved in neurodegeneration are not entirely known but involve multiple processes operating simultaneously and synergistically in the brain, including spongiform degeneration, synaptic alterations, brain inflammation, neuronal death and the accumulation of protein aggregates. Here, we review the pathways implicated in prion-induced brain damage and put the pieces together into a possible model of neurodegeneration in prion disorders. A more comprehensive understanding of the molecular basis of brain degeneration is essential to develop a much needed therapy for these devastating diseases.

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.

SF2312 is a natural phosphonate inhibitor of enolase

Despite being critical 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 for the treatment of cancers with deletion of Enolase 1, we modeled the synthetic tool compound inhibitor, Phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analogue of PhAH, with the hydroxamic nitrogen linked to the alpha-carbon 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 back-bone-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 nM inhibitor of Enolase.

Is Immunomodulation a Principal Mechanism Underlying How Cell-Based Therapies Enhance Stroke Recovery?

Inflammation within the brain and in peripheral tissues contributes to brain injury following ischemic stroke. Therapeutic modulation of the inflammatory response has been actively pursued as a novel stroke treatment approach for decades, without success. In recent years, extensive studies support the high potential for cell-based therapies to become a new treatment modality for stroke and other neurological disorders. In this review, we explore different types of cellular therapies and discuss how they modulate central and peripheral inflammatory processes after stroke. Apart from identifying potential targets for cell therapy, we also discuss paracrine and immunomodulatory mechanisms of cell therapy.

Erratum: Tumor Evolution of Glioma-Intrinsic Gene Expression Subtypes Associates with Immunological Changes in the Microenvironment (Cancer Cell (2017) 32(1) (42–56.e6)(S1535610817302532)(10.1016/j.ccell.2017.06.003))

Department of Genomic Medicine, Department of Radiation Oncology, Department of 8 Bioinformatics and Computational Biology, Department of Cancer Systems Imaging, 9 Department of Pathology, Department of Cancer Biology, Department of Neurosurgery, The 10 University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; University of 11 Texas-Houston Graduate School in Biomedical Sciences, Houston, TX 77030, USA; Cancer 12 Cell Biology Programme, Seve Ballesteros Foundation Brain Tumor Group, Centro Nacional de 13 Investigaciones Oncológicas, CNIO, 28029 Madrid, Spain; Departments of Neurology and 14 Neurosurgery, Henry Ford Hospital, Detroit, MI 48202, USA; Oncology Graduate School 15 Amsterdam, VU University Medical Center, 1081 HV Amsterdam, The Netherlands; Institute 16 for Refractory Cancer Research, Samsung Medical Center, Seoul 06351, Korea; Department 17 of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and 18 Technology, Sungkyunkwan University, Seoul 06351, Korea; Department of Neurosurgery 19 Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 135-710, Korea. 20 21 * These authors contributed equally 22 †Co-senior authors 23 24 Correspondence: rverhaak@mdanderson.org 25 26

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.

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.

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.

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

Abstract 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.

Eradication of ENO1-deleted Glioblastoma through Collateral Lethality

Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We recently demonstrated that SF2312, a natural product phosphonate antibiotic, is a potent inhibitor of the glycolytic enzyme Enolase with potential utility for the collateral lethality-based treatment of Enolase-deficient glioblastoma (GBM). However, phosphonates are anionic at physiological pH, limiting cell and tissue permeability. Here, we show that addition of pivaloyloxymethyl (POM) groups to SF2312 (POMSF) dramatically increases potency, leading to inhibition of glycolysis and killing of ENO1-deleted glioma cells in the low nM range. But the utility of POMSF in vivo is dose-limited by severe hemolytic anemia. A derivative, POMHEX, shows equipotency to POMSF without inducing hemolytic anemia. POMHEX can eradicate intracranial orthotopic ENO1-deleted tumors, despite sub-optimal pharmacokinetic properties. Taken together, our data provide in vivo proof-of-principal for collateral lethality in precision oncology and showcase POMHEX as a useful molecule for the study of glycolysis in cancer metabolism.