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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.

In mammals, lactation is a rich source of nutrients and antibodies for newborn animals. However, millions of mothers each year experience an inability to breastfeed. Exposure to several environmental toxicants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been strongly implicated in impaired mammary differentiation and lactation. TCDD and related polyhalogenated aromatic hydrocarbons are widespread industrial pollutants that activate the aryl hydrocarbon receptor (AHR). Despite many epidemiological and animal studies, the molecular mechanism through which AHR signaling blocks lactation remains unclear. We employed in vitro models of mammary differentiation to recapitulate lactogenesis in the presence of toxicants. We demonstrate AHR agonists directly block milk production in isolated mammary epithelial cells. Moreover, we define a novel role for the aryl hydrocarbon receptor repressor (AHRR) in mediating this response. Our mechanistic studies suggest AHRR is sufficient to block transcription of the milk gene β-casein. Since TCDD is a prevalent environmental pollutant that affects women worldwide, our results have important public health implications for newborn nutrition.

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.

It is demonstrated that dirhodium(II) is capable of catalyzing the chemo- and regioselective hydroamination of propargyl ureas.

[107819-90-9] C12H22N2O4S (MW 290.38) InChI = 1S/C12H22N2O4S/c1-11(2,3)17-9(15)13-8(19-7)14-10(16)18-12(4,5)6/h1-7H3,(H,13,14,15,16) InChIKey = UQJXXWHAJKRDKY-UHFFFAOYSA-N (a versatile reagent for the synthesis of acyclic and cyclic guanidines) Alternate Name: N,N′-di-Boc-S-methylisothiourea. Physical data: mp 115–121°C. Solubility: soluble in ether, ethyl acetate, dichloromethane, and commonly used organic solvents. Form Supplied in: white fluffy amorphous solid with lumps. Preparative Methods: the most commonly used procedure for the preparation of N,N′-di-Boc-S-methylisothiourea is the reaction between S-methylthiourea hemisulfate and di-tert-butyl dicarbonate in a biphasic solvent system (aq. NaHCO3 & CH2Cl2) at room temperature (eq 1).1 (1) Purification: chromatography (15% Hexanes/CHCl3) or recrystallization from hexanes/ethyl acetate. Handling, Storage, and precautions: weighing and handling should be done with appropriate exhaust ventilation. Although the reagent is stable at room temperature, storing in cool place is preferred. The containers are kept in dry and well-ventilated place with tightly closed caps. Normal preventive fire protection measures are necessary.

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.

A simple and efficient one-pot method for the synthesis of monoprotected guanidines is presented. Treatment of an acylcyanamide with chlorotrimethylsilane generates a reactive N-silylcarbodiimide capable of guanylating a variety of amines. Typically the reaction is complete in 15 min for primary and secondary aliphatic amines at rt. Hindered amines and anilines are also competent nucleophiles but require extended reaction times.

A simple and efficient one-pot method for the synthesis of monoprotected guanidines is presented. Treatment of an acylcyanamide with chlorotrimethylsilane generates a reactive N-silylcarbodiimide capable of guanylating a variety of amines. Typically the reaction is complete in 15 min for primary and secondary aliphatic amines at rt. Hindered amines and anilines are also competent nucleophiles but require extended reaction times.

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.

Cyclic and polycyclic guanidinium ion natural products have been shown to modulate a variety of important biological processes and their activities are often reliant on the unique hydrogen bond donor-acceptor topologies that these substructures display.1 While most synthetic methods to prepare guanidines rely on the addition of an amine (guanylation) of an activated thiourea or urea,2 alternative methodologies that generates peripheral C-N bonds from an intact guanidine nucleus have proven powerful for the preparation of polycyclic guanidine natural products.3a-e Our interest in the biological activity of guanidine natural products has prompted us to develop a synthetic platform that is capable of delivering cyclic guanidines having multiple ring sizes, substitution patterns, and oxidation states in short order. To this end we have been interested in the addition of guanidine N-H bonds across C-C π systems and recently reported a La(III) catalyzed tandem addition-hydroamination reaction of propargylcyanamides which required forcing conditions, and resulted in exclusive 5-exo-dig cyclization. 4 This led us to study the hydroamination of preformed di-Boc protected propargylguanidines of the type 1, in hopes of finding a 6-endo-dig selective process. Traditionally, metal catalyzed cyclizations on alkynes favor a 5-exo-dig pathway. This can be seen in Au(I) and Au(III) catalyzed cyclization of propargylcarbamates, propargylureas, and propargylamides, 5,6 as well as the Ti(IV)-amide hydroamination reactions of homopropargylamines. 7,8 Examples of cyclization of heteroatom nucleophiles onto alkynes leading to 6-endo-dig cyclization, although as synthetically significant as the 5-exo-dig products, are nevertheless much less common and usually observed with substrates in which the tether is largely sp2 hybridized. 9,10 During the preparation of this manuscript, Gin and co-workers described the AuCl3 catalyzed hydroamination of alkynes with 2-aminopyrimidines en route to the synthesis of crambedine.11 Their gold-catalyzed 6-exo-dig cyclization highlights the power of this approach in total synthesis. Achieving selectivity in these hydroaminations, particularly 5-exo-dig vs. 6-endo-dig, would present a valuable tool for the synthesis of complex guanidine containing natural products. This manuscript highlights the discovery of an unusual reactivity of dirhodium(II)-carboxylates as highly 6-endo-dig selective hydroamination catalysts in the cyclization of propargylguanidines, while Ag(I) is typically 5-exo-dig selective. We first examined the ability of traditional π-Lewis acids to catalyze the hydroamination of 1a (Table 1). Initial experiments highlighted that there were multiple reaction pathways available (entry 1). Cyclization of 1a in the presence of Cu(I) salts reliably gave a mixture of 6-endo-dig and 5-exo-dig products, 2a and 3a respectively, that are easily identified by the magnitude of either 3J or 4J coupling. From nOe experiments it was also confirmed that the 5-exo product 3a carried the Z-alkene configuration. The unanticipated product was the yne-guanidine 4, which may arise from a [1,3]-prototropic shift followed by isomerization as detailed by Gevorgyan for propargyl acetates.12 Table 1 Catalyst screen for propargylguanidine hydroamination. Ag(I) catalysis proved to be optimal for the generation of 3a with AgOAc giving a 1 : >20 ratio of 2a : 3a in 55% yield. It was further found that addition of AcOH to the AgOAc catalyzed reaction gave significant rate enhancements suggesting that protonolysis of the vinyl metal species was rate limiting, consistent with the known electrophilic hydroamination mechanism 13 Our first success in reversing the selectivity was found with gold (III) catalysts that favored the 6-endo product (entries 6-7). The sluggishness of the Au catalyzed reactions directed our attention to Rh(II). Dirhodium(II)-carboxylates have rarely been used to activate alkynes, however Murai has shown that Rh2(tfa)4 is a competent ene-ene-yne cycloisomerization catalyst and gives different product distributions than Pt(II) or Ru(II).14 Gratifyingly, Rh2(tfa)4 turned the selectivity over favoring the 6-endo product (entry 8). Quite to our surprise, given its decreased Lewis-acidity, Rh2(OAc)4 was a very selective catalyst favoring the 6-endo product 13 : 1 (entry 9). The reaction times were long and we suspected that this was due to catalyst solubility. The reaction was enhanced with Rh2(Oct)4 giving the 6-endo product in 81% isolated yield after 16 hours in > 20 : 1 (2a : 3a)selectivity as judged by 1H NMR (entry 10). Having identified a selective catalyst set, a substrate-selectivity profile was developed for the reaction (Table 2) and it was immediately apparent that the reactivity of the dirhodium(II)-carboxylates was quite unique. For example, cyclization of the p-methoxyphenyl alkyne (entry 1) with Ag(I) favored the 5-exo product, however with poor selectivity, consistent with the ability of this substituent to competitively stabilize a vinyl cation for 6-endo cyclization. In contrast, Rh(II) gave exclusively the 6-endo product 2b. Cyclization of electron poor aryl alkynes showed the opposite trend, with Ag(I) giving high 5-exo selectivity while Rh(II) was more modestly selective for the 6-endo product 2c (entry 2). Alkyl substituted alkynes gave the 6-endo products exclusively with Rh(II) (entries 3-7). This is in direct contrast to the Ag(I) catalyzed reactions, where modest selectivities for the 5-exo products are seen when the alkyne termini are electronically indistinguishable. Importantly for subsequent annulations and access to polycyclic skeletons, Rh2(Oct)4 tolerates useful functional groups (e.g. alcohols and alkyl chlorides, entries 4-7). These primary alkyl chlorides were also cleanly cyclized to the 5-exo-dig isomers 2g,h with Ag(I). Substitution is also permitted at the propargylic position wherein good selectivities are seen with Ag(I) for the 5-exo product and with Rh(II) for the 6-endo product (entry 8). Substituents on the guanidine nitrogen had modest effects on selectivity, with the 6-endo product always favored under Rh(II) catalysis (entries 9,10). The t-butyl alkyne 1l (entry 11) reacted well in the Ag(I) catalyzed process but was unreactive toward Rh(II), presumably due to the size of the Rh(II) catalyst. Table 2 Substrate scope for the Rh(II)-catalyzed hydroamination of propargylguanidines. The resultant cyclic eneguanidines contain a rich functional group handle for further manipulations (Scheme 1). For example bromination proceeds to give the 5-bromodihydropyrimidine 5 the structure of which was further confirmed by X-ray crystallography (Eq. 1). Oxidative cyclization of pendant nucleophiles gives the spirocyclic hemiaminal 6, reminiscent of the crambescidins (Eq. 2). Deprotection of the 5-membered ene-guanidine 2a under acidic conditions was accompanied by isomerization to give the 2-aminoimidazole 7 (Eq. 3). Reduction was also cleanly performed on 2b to give tetrahydropyrimidine 8 (Eq. 4). Scheme 1 Transformations of the resultant ene-guanidines. Examples of alkyne activation by dirhodium(II)-carboxylates are quite rare.8,15 Given the fact that reactions of internal alkynes have only been observed with Rh2(tfa)4 16 and that Rh2(Oct)4 is not able to activate alkynes independently,8 we were surprised that the dirhodium(II)-alkylcarboxylates were competent catalysts for this transformation especially at room temperature.17 This was especially true for the cyclization of electron deficient alkynes such as the pCF3Ph substituted alkyne 1c. We were even more surprised by the observation that 1c reacted ten times faster than the p-methoxy substituted alkyne 1b. Regardless of the poor π-Lewis aciditiy of these catalysts, this suggested that coordination of the alkyne was not rate limiting. Addition of 3 equivalents of acetic acid changed the selectivity from 5:1 to 1:1.7 (2c:3c, table 3 compare entries 1 and 2) suggesting that protonation of a vinyl-rhodium intermediate might be involved in the product determining step. The use of Rh2(tfa)4 as a catalyst also favored the 5-exo-dig product with 1c (entry 3). Taken together this may suggest a Curtin-Hammett scenario wherein the initial cyclization to form a 5- or 6-membered vinyl-rhodium anion is highly reversible and that perturbation of this pre-equilibrium by stabilizing the kinetically favored 5-exo-dig intermediate leads to poor selectivity.18 Table 3 Condition effects on selectivity. Mechanistically it is difficult to rationalize oxidation state changes in the dirhodium(II)-carboxylates upon arriving at a vinylrhodium intermediate (in this case the formation of Rh(I) in the dimer). This prompted us to examine better defined catalysts known to proceed through vinyl-rhodium intermediate.19 Although sluggish, Wilkinson’s catalyst promoted the cyclization and favored the 6-endo product in 10:1 selectivity (entry 4). Cationic Rh(I) however reversed the selectivity favoring the 5-exo product in 10:1 selectivity (entry 5). Again this may support the necessity for the initial cyclization event to be reversible in order to access the 6-endo product. More importantly this suggests access to more defined vinyl-rhodium intermediates that might be exploited for cascade reactivity. In conclusion we have demonstrated the unique ability of dirhodium(II)-carboxylates to catalyze the 6-endo-dig selective hydroamination of propargylguanidines. The resultant cyclic eneguanidines contain a rich latent functional group for the preparation of skeletally diverse cyclic guanidine natural product substructures. Studies to further understand the reactivity of dirhodium(II)-carboxylates and the application of these products to more complex targets are ongoing and will be reported in due course.

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.

A methodology to generate 2-thio- and 2-oxoimidazoles through an addition−cyclization−isomerization reaction of propargylcyanamides with thiol and alcohol nucleophiles is described. In general, the reaction sequence allows for the rapid formation of highly substituted 2-thio- and 2-oxoimidazoles in good to excellent yields.

This review will focus on the ability of the 2-aminoimidazole to occupy a unique subset of chemical space which makes it an ideal pharmacophore for the development of small molecule collections for discovery based research. These observations rely both on the use of 2-aminoimidazoles as building blocks in medicinal chemistry as well as the recent discovery of alkaloids from sponges of the genus Leucetta which exhibit a diverse range of biological activities around a relatively limited structural core. The preparation of these compounds will also be highlighted. In particular, marine natural products derived from sponges have provided valuable leads for therapeutic small molecules (3, 4). Surprisingly the large majority of these compounds have been isolated from organisms of the class Dermospongiae. In the mid 1980's chemists noted that the other major sponge class, Calcarea, had rarely been subject to chemical investigations. A flurry of efforts through the mid-1990's helped to establish biogenetic relationships among these sponges. Isolated to explore these inter- connections and not necessarily for specific biological responses the activities of these natural products have remained largely uncovered. Since these initial inves- tigations, an emerging structural class has recurrently been identified through bioassay guided isolation which contains the 2-aminoimidazole core. From the viewpoint of small molecule discovery this review will highlight alkaloids isolated from Leucetta sp. This small skeletal family has been shown to interrogate an incredibly diverse range of biological processes and thus represents an important discovery scaffold for both medicinal and discovery based research.

A valuable pharmacophore, the 2-aminoimidazole moiety, can be accessed with a variety of substitution patterns through an addition–hydroamination–isomerization sequence (see scheme; R1,R4,R5=alkyl; R3=alkyl, aryl; R2=H, alkyl, aryl). The synthesis of the propargyl cyanamide precursors through a three-component coupling enables the preparation of this important heterocyclic core structure in just three steps.

A valuable pharmacophore, the 2-aminoimidazole moiety, can be accessed with a variety of substitution patterns through an addition-hydroamination-isomerization sequence (see scheme; R(1),R(4),R(5)=alkyl; R(3)=alkyl, aryl; R(2)=H, alkyl, aryl). The synthesis of the propargyl cyanamide precursors through a three-component coupling enables the preparation of this important heterocyclic core structure in just three steps.

Aromatic bisabolene derivatives were prepared by two methods involving cross-coupling of organozinc reagents. The first synthesis of (+/-)-glandulone A (10), as well as syntheses of (+/-)-curcuhydroquinone (8) and (+/-)-curcuquinone (9), were accomplished via coupling of a secondary alkyl zinc reagent (1,5-dimethyl-4-hexenylzinc halide, 18) to protected bromohydroquinones using Pd(dppf)Cl(2) as catalyst. Coupling of arylzinc halides with alkenyl triflate 16 using Pd(PPh(3))(4) catalyst provided a number of bisabolene derivatives and led to syntheses of dehydro-alpha-curcumene (2), (+/-)-curcuphenol (3), and (+/-)-elvirol (13). A high-yield synthesis of the (+/-)-heliannuol D precursor 29 is also reported using this method.

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.

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.

Introduction: High failure rates of new investigational drugs have impaired the development of breast cancer therapies. One challenge is that excellent activity in preclinical models, such as established cancer cell lines, does not always translate into improved clinical outcomes for patients. New preclinical models, which better replicate clinically-relevant attributes of cancer, such as chemoresistance, metastasis and cellular heterogeneity, may identify novel anti-cancer mechanisms and increase the success of drug development. Methods: Metastatic breast cancer cells were obtained from pleural effusions of consented patients whose disease had progressed. Normal primary human breast cells were collected from a reduction mammoplasty and immortalized with human telomerase. The patient-derived cells were characterized to determine their cellular heterogeneity and proliferation rate by flow cytometry, while dose response curves were performed for chemotherapies to assess resistance. A screen was developed to measure the differential activity of small molecules on the growth and survival of patient-derived normal breast and metastatic, chemoresistant tumor cells to identify selective anti-cancer compounds. Several hits were identified and validated in dose response assays. One compound, C-6, was further characterized for its effect on cell cycle and cell death in cancer cells. Results: Patient-derived cells were found to be more heterogeneous, with reduced proliferation rates and enhanced resistance to chemotherapy compared to established cell lines. A screen was subsequently developed that utilized both tumor and normal patient-derived cells. Several compounds were identified, which selectively targeted tumor cells, but not normal cells. Compound C-6 was found to inhibit proliferation and induce cell death in tumor cells via a caspase-independent mechanism. Conclusions: Short-term culture of patient-derived cells retained more clinically relevant features of breast cancer compared to established cell lines. The low proliferation rate and chemoresistance make patient-derived cells an excellent tool in preclinical drug development.

The development of anti-cancer therapeutics that target non-apoptotic pathways has received increasing attention from the scientific community in recent years. The prevalence of chemoresistance and the characterization of defective apoptotic pathways in cancer has promoted interest in the expansion of current therapeutic regimens to include drugs that induce non-apoptotic cell death. From a phenotypic screen we have identified a small molecule named C6 that induces caspase-independent, non-apoptotic cell death in chemoresistant patient-derived breast cancer cells. Additionally, C6 is also selectively cytotoxic against cancer cells compared to normal mammary epithelial cells. In an effort to characterize this small molecule's mechanism of action and identify relevant biological pathways that might be used as therapeutic drug targets, we have utilized a photoaffinity pull-down strategy to identify biological binding partners of C6. Our photoaffinity pull-down studies have revealed Mitsugumin 23 (MG23), an endoplasmic reticulum-bound transmembrane protein capable of ion channel formation, as a binding partner for C6. Additionally, we have identified a metabolic component of C6-induced cell death through the use of mitochondrial respiration measurements, metabolomic analyses, and mitochondrial transmission electron microscopy (TEM) imaging. Metabolic studies have identified mitochondrial respiration defects, excess lactic acid production, and gross changes in mitochondrial morphology as a result of C6 treatment. Collectively, our findings suggest a role for ionic imbalance and subsequent metabolic disruption in this form of caspase-independent cell death. Our ongoing work is focused on further mechanistic characterization of this non-apoptotic pathway as a potential target for breast cancer drug development. Citation Format: Rachel M. Vaden, Keith M. Gligorich, Dawne N. Shelton, Guoying Wang, Cindy B. Matsen, Ryan E. Looper, Matthew S. Sigman, Bryan E. Welm. Targeting nonapoptotic cell death in chemoresistant patient-derived breast cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B05.

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.