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ConspectusWithout question, natural products have provided the lion share of leads, if not drugs themselves, for the treatment of bacterial infections. The bacterial arms race, fueled by selection and survival pressures has delivered a natural arsenal of small molecules targeting the most essential of life processes. Antibiotics that target these critical intracellular processes face the formidable defense of both penetrating a bacterial cell membrane and avoiding efflux to exert their effect. These challenges are especially effective in Gram-negative (Gram-(-)) bacteria, which have a double membrane structure and efficient efflux systems from the combination of outer-membrane porins and inner membrane proton pumps. In this landscape of offense and defense, our clinically used antibiotics have only successfully targeted three intracellular processes for therapeutic intervention in Gram-(-) bacteria: dihydrofolate biosynthesis, transcription, and translation. Not surprisingly, such critical survival machinery is a popular target for bacterial warfare, and eight of our 14 classes of commonly used antibiotics target translation with the bacterial ribosome remaining one the most vetted targets for antimicrobial therapy. On the plus side, its anionic character attracts cationic inhibitors, which are generally more capable of penetrating the bacterial cell wall, and clinical resistance rates are usually manageable as mutation of such a highly evolved machine is difficult. On the down side, this highly evolved machine renders it difficult to inhibit selectively, and the inhibition of prokaryotic translation versus both eukaryotic cellular and mitochondrial translation is critical for clinical development and minimization of undesired toxicities.A class of natural products known as the "nucleoside antibiotics" have historically been recognized as universal inhibitors of the ribosome and can inhibit translation in prokaryotes, eukaryotes, and archaea. While they have served an essential role in dissecting the biochemical underpinnings of the enzymatic functions of the ribosome, they have not proven therapeutically useful as they target the highly conserved rRNA in the P-site and are toxic to mammalian cells. In this Account, we describe our studies on the natural product amicetin, a nucleoside antibiotic that we have demonstrated to break the rule of being a universal translation inhibitor. While the cytosine of amicetin mimics C75 of the 3'-CCA tail of the P-site tRNA akin to other nucleoside antibiotics, we advance a hypothesis that amicetin's unique interaction with the ribosomal protein uL16 exploits an untapped mechanism for selectively targeting the bacterial ribosome. A complex molecule comprised of a nucleoside, carbohydrates and amino acids, amicetin is also chemically unstable. Our initial attempts to stabilize and simplify this scaffold are presented with the ultimate goal of rebuilding the compound with improved penetrance to bacterial cells. If successful, this scaffold would demonstrate a path forward for a new class of antibiotics capable of selectively targeting the ribosomal P-site.

Prosthetic joint infection (PJI) is a well-known and persisting problem. Active release coatings have promise to provide early protection to an implant by eradicating small colony biofilm contaminants or planktonic bacteria that can form biofilm. Traditional antibiotics can be limited as active release agents in that they have limited effect against biofilms and develop resistance at sub-lethal concentrations. A unique first-in-class compound (CZ-01127) was assessed as the active release agent in a silicone (Si)-based coating to prevent PJI in a sheep model of joint space infection. Titanium (Ti) plugs contained a porous coated Ti (PCTi) region and polymer-coated region. Plugs were implanted into a femoral condyle of sheep to assess the effect of the Si polymer on cancellous bone ingrowth, the effect of CZ-01127 on bone ingrowth, and the ability of CZ-01127 to prevent PJI. Microbiological results showed that CZ-01127 was able to eradicate bacteria in the local region of the implanted plugs. Data further showed that Si did not adversely affect bone ingrowth. However, bacteria that reached the joint space (synovium) were not fully eradicated. Outcomes suggested that the CZ-01127 coating provided local protection to the implant system in a challenging model, the design of which could be beneficial for testing future antimicrobial therapies for PJI. STATEMENT OF SIGNIFICANCE: Periprosthetic joint infection (PJI) is now commonplace, and constitutes an underlying problem that patients and physicians face. Active release antibiotic coatings have potential to prevent these infections. Traditional antibiotics are limited in their ability to eradicate bacteria that reside in biofilms, and are more susceptible to resistance development. This study addressed these limitations by testing the efficacy of a unique antimicrobial compound in a coating that was tested in a challenging sheep model of PJI. The unique coating was able to eradicate bacteria and prevent infection in the environment adjacent to the implant. Bacteria that escaped into the joint space still caused infection, yet benchmark data can be used to optimize the coating and translate it toward clinical use.

Biofilm-impaired tissue is a significant factor in chronic wounds such as diabetic foot ulcers. Most, if not all, anti-biotics in clinical use have been optimized against planktonic phenotypes. In this study, an in vitro assessment was performed to determine the potential efficacy of a unique first-in-class series of antibiofilm antibiotics and compare outcomes to current clinical standards of care. The agent, CZ-01179, was formulated into a hydrogel and tested against mature biofilms of a clinical isolate of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa ATCC 27853 using two separate methods. In the first method, biofilms were grown on cellulose discs on an agar surface. Topical agents were spread on gauze and placed over the biofilms for 24 h. Biofilms were quantified and imaged with confocal and scanning electron microscopy. In the second method, biofilms were grown on bioabsorbable collagen coupons in a modified CDC biofilm reactor. Coupons were immersed in treatment for 24 h. The first method was limited in its ability to assess efficacy. Efficacy profiles against biofilms grown on collagen were more definitive, with CZ-01179 gel eradicating well-established biofilms to a greater degree compared to clinical standards. In conclusion, CZ-01179 may be a promising topical agent that targets the biofilm phenotype. Pre-clinical work is currently being performed to determine the translatable potential of CZ-01179 gel.

We present data demonstrating the natural product mimic, zinaamidole A (ZNA), is a modulator of metal ion homeostasis causing cancer-selective cell death by specifically inducing cellular Zn(2+)-uptake in transformed cells. ZNA’s cancer selectivity was evaluated using metastatic, patient-derived breast cancer cells, established human breast cancer cell lines, and three-dimensional organoid models derived from normal and transformed mouse mammary glands. Structural analysis of ZNA demonstrated that the compound interacts with zinc through the N(2)-acyl-2-aminoimidazole core. Combination treatment with ZnSO(4) strongly potentiated ZNA’s cancer-specific cell death mechanism, an effect that was not observed with other transition metals. We show that Zn(2+)-dyshomeostasis induced by ZNA is unique and markedly more selective than other known Zn(2+)interacting compounds such as clioquinol. The in vivo bioactivity of ZNA was also assessed and revealed that tumor-bearing mice treated with ZNA had improved survival outcomes. Collectively, these data demonstrate that the N(2)-acyl-2aminoimidazole core of ZNA represents a powerful chemotype to induce cell death in cancer cells concurrently with a disruption in zinc homeostasis.

The intramolecular hydroamination of a guanidine on an eneyne unit affords a guanidine-substituted diene capable of reacting with dienophiles. These substrates undergo [4+2]-cycloaddition reactions to generate a series of complex cyclic- and spirocyclic-guanidines. Select substrates can further undergo a ring opening-elimination cascade that ultimately reveals a vinyl-2-aminoimidazole. As such this cascade reaction may find application in the synthesis of oroidin-type natural products and their analogues.

The bis-guanidinium ion family of natural products are revered for their utility in the study of ion channel physiology. While many congeners have been isolated with various oxidation and sulfation patterns, only two members of this family have been isolated bearing a carbon-carbon bond at C11, namely 11-saxitoxinethanoic acid and zetekitoxin AB. Herein we described a synthetic platform capable of efficiently targeting (+)-saxitoxin and 11-saxitoxinethanoic acid with an embedded C11 carbon-carbon bond. We demonstrate that this strategy enables direct enolate coupling in both an inter- and intramolecular fashion to create the C11-C15 carbon-carbon bond.

A variety of polyamines have been synthesized from their corresponding amino alcohols through a 3-step process. The synthesis predicates that the most likely impurities are of higher molecular weight and that distillation is effective at producing highly pure materials. This sequence is scalable, requires no protecting groups or chromatography, and provides the requisite polyamines in moderate to good yields.

Biofilm-related infection is among the worst complication to prosthetic joint replacement procedures; once established on the implant surface, biofilms show strong recalcitrance to clinical antibiotic therapy, frequently requiring costly revision procedures and prolonged systemic antibiotics for their removal. A well-designed active release coating might assist host immunity in clearing bacterial contaminants within the narrow perioperative window and ultimately prevent microbial colonization of the joint prosthesis. A first-in-class compound (CZ-01127) was tested as the active release agent in a silicone (Si) coating using an in vitro dynamic flow model of surgical site contamination and compared with analogous coatings containing clinical gold-standard antibiotics vancomycin and gentamicin; the CZ-01127 coating outperformed both vancomycin and gentamicin coatings and was the only to decrease the methicillin-resistant Staphylococcus aureus (MRSA) inocula below detectable limits for the first 3 days. The antimicrobial activity of CZ-01127, and for comparison vancomycin and gentamicin, were characterized against both planktonic and biofilm MRSA using the minimum inhibitory concentration (MIC) assay, serial passages, and serial dilution tests against established biofilms grown with a CBR 90 CDC biofilm reactor. Despite a similar MIC (1 µg/ml) and behavior in a 25-day serial passage analysis, CZ-01127 displayed much greater bactericidal activity against established biofilms and was the only to decrease biofilm colony forming units (CFUs) below detectable limits at the highest concentration tested (500 µg/ml). Coating release profiles were characterized using ATR-FTIR and displayed burst release kinetics within the decisive period of the perioperative window suggesting the silicon carrier is broadly useful for screening antibiotic compound for local delivery applications. Statement of Significance With an aging population, an increasing number of people are undergoing total joint replacement procedures in which diseased joint tissues are replaced with permanent metallic implants. Some of these procedures are burdened by costly and debilitating infections. A promising approach to prevent infections is the use of an antimicrobial coating on the surface of the implant which releases antibiotics into the surgical site to prevent infection. In this study, we tested a new antibiotic compound formulated in a silicone coating. Data showed that this compound was more effective at killing pathogenic methicillin resistant Staphylococcus aureus (MRSA) bacteria than two clinical gold-standard antibiotics—vancomycin and gentamicin—and could be a promising agent for antimicrobial coating technologies.

Histone lysine demethylases (KDMs) are 2-oxoglutarate-dependent dioxygenases (2-OGDDs) that regulate gene expression by altering chromatin structure. Their dysregulation has been associated with many cancers. We set out to study the catalytic and inhibitory properties of human KDM4A, KDM4B, KDM5B, KDM6A and KDM6B, aiming in particular to reveal which of these enzymes are targeted by cancer-associated 2-oxoglutarate (2-OG) analogues. We used affinity-purified insect cell-produced enzymes and synthetic peptides with trimethylated lysines as substrates for the in vitro enzyme activity assays. In addition, we treated breast cancer cell lines with cell-permeable forms of 2-OG analogues and studied their effects on the global histone methylation state. Our data show that KDMs have substrate specificity. Among the enzymes studied, KDM5B had the highest affinity for the peptide substrate but the lowest affinity for the 2-OG and the Fe2+ cosubstrate/cofactors. R-2-hydroxyglutarate (R-2HG) was the most efficient inhibitor of KDM6A, KDM4A and KDM4B, followed by S-2HG. This finding was supported by accumulations of the histone H3K9me3 and H3K27me3 marks in cells treated with the cell-permeable forms of these compounds. KDM5B was especially resistant to inhibition by R-2HG, while citrate was the most efficient inhibitor of KDM6B. We conclude that KDM catalytic activity is susceptible to inhibition by tumorigenic 2-OG analogues and suggest that the inhibition of KDMs is involved in the disease mechanism of cancers in which these compounds accumulate, such as the isocitrate dehydrogenase mutations.

Scope: Lipotoxicity-induced endothelial dysfunction is an important vascular complication associated with diabetes. Clinical studies support the vascular benefits of blueberry anthocyanins, but the underlying mechanism is unclear. The hypothesis that metabolites of blueberry anthocyanins attenuate lipotoxicity-induced endothelial dysfunction was tested. Methods and results: Human aortic endothelial cells (HAECs) were treated for 6 h with either: (i) the parent anthocyanins (malvidin-3-glucoside and cyanidin-3-glucoside); or (ii) the blueberry metabolites (hydroxyhippuric acid, hippuric acid, benzoic acid-4-sulfate, isovanillic acid-3-sulfate, and vanillic acid-4-sulfate), at concentrations known to circulate in humans following blueberry consumption. For the last 5 h HAECs were treated with palmitate or vehicle. HAECs treated with palmitate displayed elevated reactive oxygen species generation, increased mRNA expression of NOX4, chemokines, adhesion molecules, and I?Ba, exaggerated monocyte binding, and suppressed nitric oxide production. Of note, the damaging effects of palmitate were ameliorated in HAECs treated with blueberry metabolites but not parent anthocyanins. Further, important translational relevance of these results was provided by our observation that palmitate-induced endothelial dysfunction was lessened in arterial segments that incubated concurrently with blueberry metabolites. Conclusion: The presented findings indicate that the vascular benefits of blueberry anthocyanins are mediated by their metabolites. Blueberries might complement existing therapies to lessen vascular complications.

IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas. Gliomas with IDH mutations show increased sensitivity to radiation in concert with glutaminase inhibition, offering a new approach to treating these tumors.

Human d-3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme in the serine biosynthetic pathway, is genomically amplified in tumors including breast cancer and melanoma. In PHGDH-amplified cancer cells, knockdown of PHGDH is not fully rescued by exogenous serine, suggesting possible additional growth-promoting roles for the enzyme. Here we show that, in addition to catalyzing oxidation of 3-phosphoglycerate, PHGDH catalyzes NADH-dependent reduction of α-ketoglutarate (AKG) to the oncometabolite d-2-hydroxyglutarate (d-2HG). Knockdown of PHGDH decreased cellular 2HG by approximately 50% in the PHGDH-amplified breast cancer cell lines MDA-MB-468 (normal concentration 93 μM) and BT-20 (normal concentration 35 μM) and overexpression of PHGDH increased cellular 2HG by over 2-fold in non-PHGDH-amplified MDA-MB-231 breast cancer cells, which normally display very low PHGDH expression. The reduced 2HG level in PHGDH knockdown cell lines can be rescued by PHGDH re-expression, but not by a catalytically inactive PHGDH mutant. The initial connection between cancer and d-2HG involved production of high levels of d-2HG by mutant isocitrate dehydrogenase. More recently, however, elevated d-2HG has been observed in breast cancer tumors without isocitrate dehydrogenase mutation. Our results suggest that PHGDH is one source of this d-2HG.

Bis -aryloxadiazoles are common scaffolds in medicinal chemistry due to their wide range of biological activities. Previously, we identified a 1,2,4-bis-aryloxadiazole that blocks mammary branching morphogenesis through activation of the aryl hydrocarbon receptor (AHR). In addition to defects in mammary differentiation, AHR stimulation induces toxicity in many other tissues. We performed a structure activity relationship (SAR) study of 1,2,4-bis-aryloxadiazole to determine which moieties of the molecule are critical for AHR activation. We validated our results with a functional biological assay, using desmosome formation during mammary morphogenesis to indicate AHR activity. These findings will aid the design of oxadiazole derivative therapeutics with reduced off-target toxicity profiles.

The CDC biofilm reactor is a robust culture system with high reproducibility in which biofilms can be grown for a wide variety of analyses. Multiple material types are available as growth substrates, yet data from biofilms grown on biologically relevant materials is scarce, particularly for antibiotic efficacy against differentially supported biofilms. In this study, CDC reactor holders were modified to allow growth of biofilms on collagen, a biologically relevant substrate. Susceptibility to multiple antibiotics was compared between biofilms of varying species grown on collagen versus standard polycarbonate coupons. Data indicated that in 13/18 instances, biofilms on polycarbonate were more susceptible to antibiotics than those on collagen, suggesting that when grown on a complex substrate, biofilms may be more tolerant to antibiotics. These outcomes may influence the translatability of antibiotic susceptibility profiles that have been collected for biofilms on hard plastic materials. Data may also help to advance information on antibiotic susceptibility testing of biofilms grown on biologically relevant materials for future in vitro and in vivo applications.

A synthesis of the 1,2-isoxazolidine fragment of the potent voltage gated sodium channel blocker, zetekitoxin AB is described. The synthesis utilizes an intramolecular nitrone –olefin 1,3-dipolar cycloaddition to establish the stereochemistry of the cis-1,2-isoxazolidine. The oxidative cleavage of an all anti-triol with the excision of the central carbon is central to using α-D-glucopyranoside as a traceless stereochemical template. This route furnishes a suitably protected synthon for the synthesis of zetekitoxin AB.

Cationic Rh(II) complexes are able to catalyze the regioselective hydroamination of propargyl ureas in a 6-endo fashion. This transformation permits access to interesting substitution patterns of dihydropyrimidines, which have found use as nucleotide exchange factor inhibitors.

A concise synthesis of cytimidine was developed utilizing tandem Cu-mediated N-aryl amidations followed by global deprotection. This sequence exploits a regioselective coupling of an iodobenzamide with a halopyrimidine that allows the union of three fragments in a single synthetic manipulation and will permit the efficient and rapid diversification of the cytimidine core.

Glucose and glutamine are abundant nutrients required for cell growth, yet how cells sense and adapt to changes in their levels is not well understood. The MondoA transcription factor forms a heterocomplex with its obligate partner Mlx to regulate approximately 75% of glucose-dependent transcription. By mediating glucose-induced activation of thioredoxin-interacting protein (TXNIP), MondoA:Mlx complexes directly repress glucose uptake. We show here that glutamine inhibits transcriptional activation of TXNIP by triggering the recruitment of a histone deacetylase-dependent corepressor to the amino terminus of MondoA. Therefore, in the presence of both glucose and glutamine, TXNIP expression is low, which favors glucose uptake and aerobic glycolysis; the Warburg effect. Consistent with MondoA functioning upstream of TXNIP, MondoA knockdown reduces TXNIP expression, elevates glucose uptake and stimulates cell proliferation. Although glutamine has many intracellular fates, a cell permeable analog of a tricarboxylic acid cycle (TCA) intermediate, alpha-ketoglutarate, also blocks the transcriptional activity of MondoA at the TXNIP promoter and stimulates glucose uptake. Together our data suggest that glutamine-dependent mitochondrial anapleurosis dictates glucose uptake and aerobic glycolysis by blocking MondoA:Mlx-dependent transcriptional activation of TXNIP. We propose that this previously unappreciated coordination between glutamine and glucose utilization defines a metabolic checkpoint that restricts cell growth when subthreshold levels of these essential nutrients are available.

An intramolecular 1,3-dipolar cycloaddition has efficiently constructed the A-ring portions of the cylindrospermopsin alkaloids. A nitro-aldol addition of an elaborated nitroalkane to a pyrimidine aldehyde followed by an intramolecular reductive guanidinylation has enabled the syntheses of all three alkaloids in this family in 18–19 steps. We report the first asymmetric synthesis of cylindrospermopsin, unambiguously assigning its absolute configuration.

[reaction: see text] The Cu(I)-catalyzed cycloaddition of alkynes and azides (click reaction) provides a robust method for the construction of macrocyclic small molecules via an intramolecular macrocycloaddition. A three-subunit system has been used to explore the tolerance of this macrocycloaddition to variations of stereochemistries and substituents.

A concise stereoselective total synthesis of (+)-saxitoxin is described. A silver(I)-initiated hydroamination cascade constructs the bicyclic guanidinium ion core from a alkynyl bisguanidine. This sequence creates two C-N bonds, one C-O bond, and three rings and forms a single stereoisomer in a single synthetic transformation. This process enabled us to complete the synthesis of (+)-saxitoxin in 14 steps from N-Boc-l-serine methyl ester.

A cascade silver(I)-catalyzed hydroamination/Michael addition sequence has been developed to deliver highly substituted bicyclic guanidines. This transformation gives rise to geometrically and constitutionally stable ene–guanidines and generates a remote stereocenter with moderate to high diastereoselectivity.

The total synthesis of (±)-heliannuol D and its epimer has been completed in 9 steps and 12% overall yield from 2-methylanisole. The benzoxepane moiety of the title compound, a common structural feature in the heliannuol family of natural products, is prepared by a biomimetic opening of an epoxide by a phenol.

Mutations in IDH1 and IDH2, the genes coding for isocitrate dehydrogenases 1 and 2, are common in several human cancers, including leukemias, and result in overproduction of the (R)-enantiomer of 2-hydroxyglutarate [(R)-2HG]. Elucidation of the role of IDH mutations and (R)-2HG in leukemogenesis has been hampered by a lack of appropriate cell-based models. Here, we show that a canonical IDH1 mutant, IDH1 R132H, promotes cytokine independence and blocks differentiation in hematopoietic cells. These effects can be recapitulated by (R)-2HG, but not (S)-2HG, despite the fact that (S)-2HG more potently inhibits enzymes, such as the 5′-methylcytosine hydroxylase TET2, that have previously been linked to the pathogenesis of IDH mutant tumors. We provide evidence that this paradox relates to the ability of (S)-2HG, but not (R)-2HG, to inhibit the EglN prolyl hydroxylases. Additionally, we show that transformation by (R)-2HG is reversible.

Background: Mammary gland branching morphogenesis is a highly regulated developmental process often disrupted in breast cancer. Results: A chemical genetic screen in primary three-dimensional culture revealed that activation of the aryl hydrocarbon receptor promotes desmosomes to block branching. Conclusion: Down-regulation of desmosomes is required for proper mammary branching morphogenesis. Significance: Desmosomes are a novel mechanism through which exposure to environmental pollutants may affect mammary development., During the process of branching morphogenesis, the mammary gland undergoes distinct phases of remodeling to form an elaborate ductal network that ultimately produces and delivers milk to newborn animals. These developmental events rely on tight regulation of critical cellular pathways, many of which are probably disrupted during initiation and progression of breast cancer. Transgenic mouse and in vitro organoid models previously identified growth factor signaling as a key regulator of mammary branching, but the functional downstream targets of these pathways remain unclear. Here, we used purified primary mammary epithelial cells stimulated with fibroblast growth factor-2 (FGF2) to model mammary branching morphogenesis in vitro. We employed a forward chemical genetic approach to identify modulators of this process and describe a potent compound, 1023, that blocks FGF2-induced branching. In primary mammary epithelial cells, we used lentivirus-mediated knockdown of the aryl hydrocarbon receptor (AHR) to demonstrate that 1023 acts through AHR to block branching. Using 1023 as a tool, we identified desmosomal adhesion as a novel target of AHR signaling and show that desmosomes are critical for AHR agonists to block branching. Our findings support a functional role for desmosomes during mammary morphogenesis and also in blocking FGF-induced invasion.

Syntheses of the reported structures of kealiinines B and C have been executed. An intermolecular electrophile-induced cyclization of a pendant arene on an ene–guanidine affords the tetracyclic, oxidized naphthimidazole cores.

A synthetic strategy to establish five contiguous stereocenters, in a stereocontrolled manner, on the core structure of pactamycin is described. This sequence exploits the use of a Lewis acid mediated epoxide opening cascade to set the relative configuration of the C4–C5 diol while reversing the configuration at C7. This sequence provides the oxygenated core of pactamycin in just 11 steps.

The efficient synthesis of an A-ring synthon for the marine hepatatoxin cylindrospermopsin has been achieved. The key step features an intramolecular oxazinone N-oxide/alkene dipolar cycloaddition resulting in the establishment of the three contiguous stereogenic centers in the A-ring from one pre-existing stereogenic center in a single step.

Materials and methods To analyze the catalytic activity of PHGDH on possible alternative substrates, we conducted in vitro biochemical assays on recombinant human PHGDH. The reaction rate in the reductive direction with various substrates was monitored based on NADH consumption, measured by absorbance at 340 nm. The identity and chirality of products were analyzed by liquid chromatography-mass spectrometry (LC-MS) and by gas chromatography-mass spectrometry (GC-MS) after (R)-2-butanol derivatization. To analyze the intracellular function of PHGDH, we generated stable PHGDH knockdown cell lines, using two shRNA sequences. Metabolite levels in the knockdown cells were analyzed by LC-MS [4], and DNA and histone methylation was analyzed by immunoblotting.

Abstract 2413 Somatic mutations in IDH1 and IDH2 are common in normal karyotype AML as well as in other clonal myeloid disorders and several solid tumors. Mutant IDH overproduces the R-enantiomer of 2-hydroxyglutarate, (R)-2HG (Dang, et al Nature 462: 739, 2009), which is hypothesized to alter the epigenetic landscape of cancer cells by inhibiting the activity of α-ketoglutarate-dependent enzymes, including the TET family of 5-methylcytosine hydroxylases and the jumonji-domain-containing family of histone demethylases. There is considerable interest in developing inhibitors of mutant IDH in the hope that, by decreasing (R)-2HG production in cancer cells, their epigenetic regulation can be restored. However, there is, to date, no evidence that transformation by mutant IDH is reversible or that inhibiting production of (R)-2HG has any effect on cancers harboring IDH mutations. Herein we report that in two different myeloid transformation assays, transformation by (R)-2HG and mutant IDH1 is reversible by removal of (R)-2HG. We previously reported that stable expression of a tumor-derived mutant IDH1 (IDH1R132H) induces growth factor independence and blocks EPO-induced differentiation in the human TF-1 erythroleukemia cell line, and that treatment of TF-1 cells with a cell-permeable form of (R)-2HG, TFMB-(R)-2HG, is sufficient to recapitulate this phenotype (Late Breaking Abstract #LBA-4, ASH 2011). We have extended these studies and found that transformation by TFMB-(R)-2HG is reversible and that this reversibility is influenced by the duration and intensity (dose) of (R)-2HG exposure. We developed a second myeloid transformation assay using a murine myeloid leukemia cell line that is transformed by expression of a HoxB8-ER fusion protein when cultured in the presence of estrogen. Upon estrogen withdrawal, the cells undergo monocytic differentiation and apoptosis. We expressed wild-type IDH1 or IDH1R132H in the cells and found that cells expressing wild-type IDH1 differentiate normally, but cells expressing IDH1R132H do not upregulate monocytic markers CD11b/Mac1 and Gr1 upon estrogen withdrawal. Furthermore, treatment of the IDH1R132H-expressing cells with an inhibitor of mutant IDH1 restores their ability to undergo monocytic differentiation upon estrogen withdrawal. Our findings suggest that continued exposure to (R)-2HG is required to maintain the cellular changes induced by mutant IDH, and further suggest that targeting (R)-2HG production may have therapeutic efficacy in the treatment of cancers harboring IDH mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract LBA-4 Somatic mutations in IDH1 and IDH2 occur frequently in clonal myeloid disorders and result in the neomorphic ability of IDH to convert α-ketoglutarate (2-OG) to the R-enantiomer of 2-hydroxyglutarate (R-2HG) (Dang, et al Nature 462: 739, 2009). 2OG is an essential cofactor for many metabolic enzymes, including the TET family of 5-methylcytosine hydroxylases and the EglN family of prolyl-4-hydroxylases, and 2HG has been shown to inhibit several 2OG-dependent dioxygenases in vitro, including TET2 (Xu, et al Cancer Cell 19: 17, 2011; Figueroa, et al Cancer Cell 18: 1, 2010). We recently showed that the (S) enantiomer of 2HG (S-2HG), but not the (R) enantiomer of 2HG (R-2HG), inhibits the EglN prolyl-4-hydroxylases (Koivunen, et al. Submitted for publication). Moreover, we found that R-2HG can act as a cofactor to promote the hydroxylase activity of EglN1, EglN2 and EglN3. We hypothesized that the qualitatively different effects of R- and S-2HG on the EglN prolyl-4-hydroxylases might influence their transforming activities. In order to elucidate the role of mutant IDH, and R- and S-2HG, in myeloid leukemia, we developed a myeloid transformation assay using TF-1 cells. TF-1 is a human erythroleukemia cell line that requires GM-CSF for growth and undergoes erythrocytic differentiation when stimulated with erythropoietin (EPO). We expressed wild-type IDH1 (WTIDH1), a tumor-derived mutant IDH1 (IDH1R132H), or a catalytically inactive IDH1R132H variant (IDH1R132H/3DN) in TF-1 cells. As expected, cells expressing IDH1R132H, but not cells expressing WTIDH1 or IDH1R132H/3DN, had dramatically elevated levels of 2HG. Furthermore, we found that expression of IDH1R132H, but not WTIDH1 or IDH1R132H/3DN, conferred growth factor-independence to TF-1 cells (Figure 1a), and blocked their EPO-induced differentiation (Figure 1b). In order to determine whether transformation of TF-1 cells by IDH1R132H is mediated by 2HG, we treated TF-1 cells with cell-permeable esterified R-2HG or S-2HG. R-2HG recapitulated the growth and differentiation phenotypes of IDH1R132H expression in a dose-dependent manner. In contrast, S-2HG did not induce these phenotypes at any concentration tested. Next, we examined the effect of loss of TET2 on TF-1 cells. We infected TF-1 cells with shRNAs targeting TET1 or TET2 and found that knockdown of TET2, but not TET1, induced growth factor-independence and blocked EPO-induced differentiation similarly to expression of IDH1R132H or treatment with R-2HG. Interestingly, we found that transformation by IDH1R132H and TET2 knockdown were reversed by inhibition of EglN1 (Figure 2), suggesting that R-2HG, but not S-2HG, transforms leukemic cells by inhibiting targets such as TET2 while preserving, and possibly enhancing, EglN activity. These findings further suggest that therapeutic targeting of EglN prolyl-4-hydroxylase activity might be effective in the treatment of IDH1-mutant and TET2-mutant myeloid leukemias. Disclosures: Kaelin: Fibrogen: Consultancy, Equity Ownership.