Inhibitors of Branched-Chain-Aminotransferase-1 (BCAT1) for the Treatment of Brain Tumors
Described is a compound capable of reducing or inhibiting (a) the biological activity of branched-chain-aminotrans-ferase-1 (BCATI) or (b) the expression of the gene encoding BCATI for use in a method of treating a neoplasia. A preferred compound is 1-(aminomethyl)cyclohexaneacetic acid (gabapentin).
The present invention provides a compound capable of reducing or inhibiting (a) the biological activity of branched-chain aminotransferase-1 (BCAT1) or (b) the expression of the gen encoding BCAT1 for use in a method of treating a brain tumor.
Malignant human glioblastomas account for the largest number of human malignant brain tumors. So far, the treatment o gliomas includes neurosurgical techniques (resection o stereotactic procedures), radiation therapy and chemotherapy The current standard of care for, e.g., astrocytic tumor involves surgical tumor resection that can be followed b chemotherapy with the oral alkylating agent temozolamide (TMZ and radiotherapy. However, glioblastomas are considered a being incurable as they fail to respond to ionising radiation chemotherapy and surgical resection. In other words, with these therapies only a very limited prolongation of the lifespan of patients can be achieved. This means that despite these therapies, the average life span after the cancer diagnosis is merely 12 to 16 months.
Thus, the technical problem underlying the present invention is to provide means for the therapy of brain tumors preferably glioblastomas or astrocytic brain tumors, which overcome the disadvantages of the current therapies and improve the survival of patients.
The solution of said technical problem is achieved b providing the embodiments characterized in the claims.
During the experiments resulting in the present invention it was found that known BACT1 inhibitors, like gabapentin, which have been used so far for example as anticonvulsant drugs represent a novel treatment option for cancer therapy, i.e. an effective therapy for neoplasias in general and in particular for the treatment of brain tumors like astrocytic brain tumors. They potentially act by targeting a molecular pathway that is not targeted by any established chemotherapy.
(a) Schematic representation of BCAA catabolism. BCAAs branched-chain amino acids; BCKAs, branched-chain ketoacids (b,c) RNA expression of BCAT1 (b) and BCAT2 (c) in 7 astrocytic gliomas (41 IDHwt and 29 IDHmut) normalized t expression in normal brain (dashed line). Data are expressed as mean±s.d. (two-tailed Student's t-test). *, P<0.05; ** P, <0.001. (d) Western blot showing expression of BCAT protein in astrocytic gliomas with IDH1 and IDH2 wildtype genes (lanes 1-5), different mutations in the IDH2 (lanes 6-7 or IDH1 (lanes 8-12) genes, and normal brain (lane 13). AII diffuse astrocytoma WHO grade II; AAIII, anaplasti astrocytoma WHO grade III; sGBIV, secondary glioblastoma WH grade IV; pGBIV, primary glioblastoma WHO grade IV; AOIII anaplastic oligodendroglioma WHO grade III. (e-h Immunohistochemical stainings of BCAT1 in an IDHwt primary glioblastoma (e), a primary glioblastoma with IDH1-R132 mutation (f), a diffuse astrocytoma with IDH1-R132C mutation (g), and an anaplastic oligodendroglioma with IDH2-R172 mutation (h). (i,j) Immunohistochemical staining of IDH1-R132 in the same tumors as in panels b and c, respectively demonstrating the complementarity of BCAT1 and IDH1-R132 staining. Scale bars: 50 μm. (k) Two-by-two table showing the significant correlation of BCAT1 protein expression an mutation status of the IDH1 and IDH2 genes in 81 gliomas (p 0.0001; Fisher's Exact Test).
(a) Western blot showing BCAT1 protein expression in IDH glioma cell lines. (b-d) Effects of hypoxia and alpha-KG o BCAT1 expression. RNA and protein expression are shown at the bottom and the top of each panel. The numbers above the Western blots indicate the fold ratios of expression relative to control cells. The mRNA expression values represent mean s.d. of triplicate samples. (b) BCAT1 is upregulated under hypoxic (1% O2) conditions. (c) BCAT1 expression is induced by supplementation of the culture media with cell-permeable dimethyl-alpha KG. (d) Knockdown by two different shRNAs o the alpha-KG-producing cytoplasmatic IDH1 leads the downregulation of BCAT1 expression.
(a) Schematic drawing of exons 1 to 4 of BCATI showing the exon structure of the three transcripts T1, T4 and T6. The two alternative promoters 1 and 2 are shown in the enlarge sections on the lower left and lower right, respectively. (b qRT-PCR quantification of RNA expression of transcripts T1, T and T6 in astrocytic gliomas and a pool of RNAs from normal brain tissues (n=23). Values represent mean±s.d. o triplicate samples. (c) DNA-methylation patterns detected i promoters 1 (left) and 2 (right) by massarray analysis o bisulfite-PCR amplicons A1 to A8 in normal brain an astrocytic gliomas WHO grade II-IV. *, IDHwt anaplasti astrocytoma. (d-f), Extent of DNA methylation in normal brain (Nbr), IDHwt and IDHmut tumors in (d) average of all CpGs i promoter 2 (two-tailed Student's t-test, P<0.0001) (e) CpG4, (P=0.0003) and (f) CpG6 in promoter 1 (P<0.0001). (g Correlation of CpG6 methylation grade and BCAT1 T1 expression (h,i) Knockdown of HEY1 in HEK293T cells (h) results in upregulation of BCAT1 expression (i). Values represent mean s.d. (n=3). (j,k) ChIP assays showing preferred binding o HEY1 to the DMR (amplicon c1) as compared to control amplicon in promoter 1 (c2), about 5kb upstream of BCAT1, and unrelate positive (BHLH15) and negative (PTPRD) controls. (j) Flag-HEY construct. (k) HEY1-Flag construct. qRT-PCR was performed i triplicate and ratios of bound to input are shown a mean±s.d.
(a,b) NMR spectra of U-87MG (a) and U-373MG (b) cells after treatment with control (−) or 20 mM gabapentin (+) for 2 hours. Difference spectra are shown near the top of the panels. Upon inhibitor treatment levels of valine (Val) leucine (Leu) and isoleucine (Ile) increased by factors 1.09 1.38, 1.19, respectively, in U-87MG cells and by factors 1.83 2.18% and 2.32, respectively, in U-373MG cells. (c) Glutamat release by glioma cells at 6 and 12 hours after start o treatment with control (−) or with BCAT1 inhibition (+) by 2 mM gabapentin (n=3). (d) Tandem-MS analysis of amino acid concentrations in culture media of BCAT1 knockdown and control U-87MG cells 8 days after lentiviral transduction. Values ar shown as difference to the media starting concentrations Positive and negative values indicate amino acid release an uptake, respectively (n=6). (e) Intracellular amino acid concentrations of the same cells as in (d) (n=6). (f) Western blot showing downregulation of HADH upon BCAT1 knockdown. (g Relative depletion of cholesterol and very long chain fatt acids in BCAT1 knockdown vs. control U-87MG cells (n=3). Dat are expressed as mean±s.d. *, P<0.05; **, P<0.01; ** P<0.001.
(a,b) Immuno-fluorescence labeling of alpha-tubulin in control (a) and BCAT1 knockdown (b) U-87MG cells. blue: DAPI; scal bar: 50 μm. (c) Sequential images showing the permeation of U-87MG cell through a 5×11×300 μm microchannel over a period of 9 hours; scale bar: 50 μm. (d) BCAT1 knockdown significantly inhibits the invasion potential of U-87MG cell compared to nontarget shRNA transduced cells. Results indicate the mean±s.d. of three independent experiments. P=0.0146.
(a-d) Cell proliferation and cell cycle analyses of glioma cells upon BCAT1 suppression. Proliferation of cells was examined using the Click-iT® EdU cell proliferation assay Cell cycle analysis was performed after DNA staining with propidium iodide. The DNA distribution is shown for living cells. Values in graphs represent mean±s.d. for n=3. nt nontarget shRNA. *, P<0.05; **, P<0.01; *** P<0.001 compared to respective controls. (a) Treatment with the BCAT inhibitor gabapentin for 20 hours suppressed proliferation of glioma cell lines in a concentration-dependent manner relative to control cells treated with solvent only. (b) Cell cycle analysis of gabapentin-treated glioma cells showed a accumulation of cells in 01-phase. (c) Knockdown of BCAT caused a significant reduction of cell proliferation relative to samples treated with nontarget shRNA (nt) in all three glioma cell lines and (d) resulted in the accumulation of the G1-arrest marker CDKN1B/p27KIP1. Cell cycle analysis showed significant increases of the proportions of cells in G1 phase (e) BCAT1 knockdown results in decreased phosphorylation o AKT. (f-i) Cross-sections of tumors induced by intracrania injection of U-87MG glioblastoma cells into CD-1 nude mice Hematoxylin-eosin staining is shown for mice injected with (f control nontarget-shRNA or (g) BCAT1-shRNA. (h) Quantification of tumor volumes (n=5 mice for each group, P=0.0091).
(a) Gene structure of BCAT1 showing 11 exons and the three transcripts T1 (ENST00000261192), T4 (ENST00000539282) and T (ENST00000538118) which originate in two different promoters The regions around the transcription start sitzes (TSS) an exon 5 are enlarged to show primer locations. (b) Agarose ge image of PCR products amplified from an IDHwt glioblastom (left) and a pool of normal brain RNAs from 23 individual (right) using the reverse primer and the transcript-specific exon 1 primers. Band sizes match the expected sizes of the respective spliced mRNAs.
Western Blot analysis of total and phosphorylated protein of the mTOR target RPS6K upon (a) gabapentin treatment and (b BCAT1 knockdown in the cell lines U-87MG, U-373MG and Hs683.
BCAT1 knockdown causes apoptosis in a glioblastoma spheroi primary culture. (a) Reduction of proliferation induced b lentiviral transduction of two shRNA constructs (n=3, *** P 0.0001). (b) Cell cycle analysis showing strong increase of the subG1 fraction following BCAT1 knockdown (n=3). The Western blot at the top shows the increased presence of the G1-arrest marker CDKN1B in the knockdown cells. (c AnnexinV/7AAD assay confirming apoptotic death of spheroi cells with BCAT1 knockdown.
See Example 2 for experimental details.
See Example 3 for experimental details
Thus, the present invention relates to a compound capable of reducing or inhibiting (a) the biological activity o branched-chain-aminotransferase-1 (BCAT1) or (b) the expression of the gene encoding BCAT1 for use in a method of treating a neoplasia.
“Neoplasm” is an abnormal mass of tissue as a result of neoplasia. “Neoplasia” is the abnormal proliferation of cells The growth of the cells exceeds and is uncoordinated with respect to the normal tissues around it. The growth persistant in the same excessive manner, even after cessation of the stimuli. It usually causes a lump or tumor. Neoplasms may be benign, pre-malignant (carcinoma-in-situ) or malignant (cancer). The neoplasms to be treated according to the present invention are those which (over)express BCAT1. Thus, the determination of BCAT1 in a neoplasm is an indication to start with a BCAT inhibiting therapy. Neoplasms to be treated ar brain tumors, particularly an astrocytic brain tumor, glioma or glioblastoma, in particular those expressing IDH1 wildtype.
The branched-chain amino acids (BCAAs) valine, leucine an isoleucine are essential amino acids that escape live catabolism and are available in the general circulation. BCA metabolism provides an important transport system to move nitrogen throughout the body for the synthesis of non-essential amino acids, including the neurotransmitte glutamate in the central nervous system. Deregulation of the BCAA catabolic pathways frequently results in neura dysfunction. The first step of BCAA catabolism involves the transfer of the alpha-amino group to alpha-ketoglutarat (alpha-KG) by the cytosolic branched-chain amino acid transaminase 1 (BCAT1) or the mitochondrial BCAT2 isoenzyme with glutamate and the respective branched chain ketoaci (BCKA) as products (Ichihara et al., J. Biochem. 59, pp. 160 169, (1966); Taylor et al., J. Biol. Chem. 241, pp. 4396-4405 (1966)). Expression of BCAT2 is ubiquitous, whereas expression of BCAT1 is restricted to a small number of tissues including brain, where BCAAs are a major source of nitrogen for the synthesis of the neurotransmitter glutamate. Following transamination, BCKAs are catabolized further to acetyl-Co and succinyl-CoA, which enter the tricarboxylic acid (TCA cycle (
Mutations in IDH1 (isocitrat dehydrogenase 1), originally detected in a fraction of glioblastomas are present in the great majority of World Health Organization (WHO) grade II an III gliomas and secondary glioblastomas, but are rare in primary glioblastomas (Bales et al., Acta Neuropath. 116, pp 597-602 (2008)). Mutations in IDH2 also have been detected albeit at a lower frequency of 5-10% (Bales, Bales et al. Acta Neuropath. 116, pp. 597-602 (2008); Yan et al., N. Eng J. Med. 360, pp. 765-773 (2009)). IDH mutations play a central role in glioma pathogenesis (Parsons et al, Science 321, pp 1807-1812 (2008)) and have been shown to constitute a ke classifier distinguishing two major glioma subgroups that were identified initially based on RNA expression and DN methylation patterns. It has been revealed that brain tumor showing an IDH1 mutation (IDHmut) have a better prognosis that those where IDH1 is not mutated (IDHwt) (Hartmann et al., Act Neuropathol. 120, pp. 707-718 (2010)). The inventors found out that one difference between IDHm (having IDH1-R132H mutation) and IDHwt brain tumors is that BCAT1 overexpression is a highly specific feature of IDH glioblastomas, the most common and most aggressive adult brain tumor. The observed specific methylation of the BCAT1 promoted in IDHmut tumors, but not in IDHwt tumors and normal brain strongly show that low BCAT1 expression in IDHmut tumors is consequence of IDH1 mutation-associated DNA methylation, which is thought to be mediated through inhibition of histon demethylases and the TET family of 5-methylcytosin hydroxylases by the product of mutant IDH1 and IDH2 enzymes the oncometabolite 2-hydroxyglutarate. Interestingly regulation of BCAT1 mRNA expression originating from promote appears to be achieved by an IDH-independent epigeneti mechanism, through methylation of a bindung site for the HEY transcriptional repressor in IDHwt but not IDHmut tumors. These diametrically opposed patterns of DNA methylation in the two promoters make clear that BCAT1 suppression does not occur a a mere byproduct of IDH1 mutation through “passenger methylation, but rather that the differential regulation of cell metabolism in IDHwt and IDHmut tumors requires tight control of BCAT1 expression in each group.
Similar to the commonly used diagnostic IDH1-R132H staining BCAT1 staining may help to distinguish IDHwt from IDHm gliomas; however, BCAT1 staining offers the added advantage c also distinguishing IDHwt primary glioblastoma from the clos to 10% of IDHmut astrocytomas with non-IDH1-R132H mutations Most importantly, the present invention shows that BCAT1 an BCAA metabolism provide the basis for the development of nove metabolism-based approaches in glioma therapy.
This means that suppression of BCAT1 in IDHwt glioblastomas has the potential to significantly impede tumor growth as well a the excretion of glutamate by the tumor cells, which frequently causes neurotoxicity to surrounding brain tissue and leads to tumor-associated epilepsy in brain tumor patients.
In addition, the data of the present invention show that availability of large amounts of glucose and glutamine, the two nutrients considered to be most important for supporting malignant cell growth, is not sufficient to support sustained fast growth of IDHwt glioblastoma.
In other words, in the present invention it has been shown that BCAT1 overexpression is a highly characteristic feature of glioblastoma, in particular IDHwt glioblastoma, and essential for their aggressive clinical behavior. Thus, BCAT1 expression and BCAA catabolism are promising markers for the diagnostic and prognostic assessment of gliomas and serve a novel therapeutic targets. Furthermore, the present invention represents the first example of silencing of a metabolic gen that is central to glioma pathomechanism by IDH1 mutation associated aberrant DNA methylation. Silencing of BCAT1 earl in tumor development will prevent IDHmut gliomas from utilizing BCAAs as a metabolic resource and offers an explanation for the less malignant growth behaviour of IDHmut gliomas relative to the BCAT1-dependent IDHwt glioblastomas.
The reduction, silencing or inhibition of the biological activity can be effected by direct interaction or binding of compound to BCAT1 or by indirect interaction, e.g., b interacting with a compound that is associated with the biological activity of BCAT1. The reduction or inhibition of the biological activity can also be achieved by the application of altered, e.g., inactive forms of BCAT1 preferably in excess.
Examples of suitable compounds reducing, silencing o inhibiting the biological activity of BCAT1 or the expression of the gene encoding BCAT1 with the aim to get a therapeutic effect are:
(a) Plasmids, vectors or natural/synthetic/mutated viruses oligonucleotides of various types of modification (e.g. PTO LNA, 2′F-ANA, protein-nucleotide complexes, RNAi, siRNA o mikromiRNA, shRNA, Methylmethoxy-, Phosphoroamidates, PNA Morpholino, Phosphoramidate, Cyclohexen (CeNA), gap-meres ribozymes, aptamers, CpG-oligos, DNA-zymes, riboswitches, o lipids or lipid containing molecules;
(b) peptides, peptide complexes, including all types of linkers,
(c) small molecules;
(d) antibodies and their derivatives, especially chimeras Fab-fragments, Fc-fragments, or
(e) carriers, liposomes, nanoparticles, complexes, or an other delivery systems containing the above named constructs,
(f) oxidizing agents or sulfhydryl (SH groups) modifying agents.
Further compounds suitable for the purposes of the present invention and methods how to identify/select such compound are in more detail described below.
Preferably, in a pharmaceutical composition, such compounds as described above are combined with a pharmaceuticall acceptable carrier. “Pharmaceutically acceptable” is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the activ ingredient and that is not toxic to the host to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered salin solutions, water, emulsions, such as oil/water emulsions various types of wetting agents, sterile solutions etc. Suc carriers can be formulated by conventional methods and the active compound can be administered to the subject at an effective dose.
An “effective dose” refers to an amount of the activ ingredient that is sufficient to affect the course and the severity of the neoplasia, leading to the reduction of remission of such a pathology. An “effective dose” useful for treating and/or preventing neoplasias may be determined using methods known to one skilled in the art (see for example Fingl et al., The Pharmocological Basis of Therapeutics Goodman and Gilman, eds. Macmillan Publishing Co., New York pp. 1-46 ((1975)).
Administration of the suitable compositions may be effected b different ways, e.g. by intravenous, intraperitoneal subcutaneous, intramuscular, topical or intraderma administration. The route of administration, of course depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, th particular compound to be administered, time and route o administration, the kind of therapy, general health and of the drugs being administered concurrently.
The person skilled in the art can easily identify or generate compounds useful for the treatments of the present invention based on the knowledge of the amino acid sequence of BCAT1 and the nucleotide sequence of the gene encoding this protein Respective sequences are found in the UniProtKB/Swiss-Pro database (P54687; BCAT1 HUMAN), in Genbank (NCBI Reference Sequence: NM—005504) and the Human Genome Organization Gen Nomenclature Committee (HGNC) database (HGNC ID: 976).
In a further preferred embodiment of the present invention the compound useful for reducing or inhibiting the expression of the gene encoding BCAT1 is an antisense oligonucleotide shRNA or siRNA specific for BCAT1. Preferably, the compound useful for silencing the BCAT1 expression are independent Mission® shRNA constructs targeting different regions of th human BCAT1 (BCAT1 shRNAI NM—005504.3-1064s1c1 and BCAT shRNAII NM—005504.3-751s1c1) and human IDH1 (IDH1 shRNA NM—005896.2-1363s1c1 and IDH1 shRNAII NM—005896.2-292s1c1 mRNA transcripts.
The generation of suitable antisense oligonucleotides include determination of a site or sites within the BCAT1 encoding gene for the antisense interaction to occur such that the desired effect, e.g., inhibition of the expression of the protein, will result. A preferred intragenic site is (a) the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene or (b) region of the mRNA which is a “loop” or “bulge”, i.e., no part of a secondary structure. If one or more target site have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridiz sufficiently well and with sufficient specificity, to give the desired effect. In the context of this invention “hybridization” means hydrogen bonding, which may be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleoside or nucleotide bases
“Complementary” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if nucleotide at a certain position of an oligonucleotide i capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can make hydrogen bonds with each other Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable an specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound does not need to be 100 complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound i specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-targe sequences under conditions in which specific binding i desired, i.e., in the case of therapeutic treatment.
The skilled person can generate antisense compounds and siRNA or shRNAs according to the present invention on the basis of the known DNA sequence for BCAT1.
“Oligonucleotide” refers to an oligomer or polymer o ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) o mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well a oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhance cellular uptake, enhanced affinity for nucleic acid target an increased stability in the presence of nucleases. While antisense oligonucleotides are a preferred form of the antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention compris from about 8 to about 50 nucleobases (i.e. from about 8 t about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferable those comprising from about 15 to about 25 nucleobases Antisense compounds include ribozymes, external guide sequences (EGS), oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and inhibit it expression.
Alternatively, the compound of the invention is a vector allowing to transcribe an antisense oligonucleotide of the invention, e.g., in a mammalian host. Preferably, such vector is a vector useful for gene therapy. Preferred vector useful for gene therapy are viral vectors, e.g. adenovirus herpes virus, vaccinia, or, more preferably, an RNA virus suc as a retrovirus. Even more preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples o such retroviral vectors which can be used in the presen invention are: Moloney murine leukemia virus (MoMuLV), Harve murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV) and Rous sarcoma virus (RSV). Most preferably, a non-human primate retroviral vector is employed, such as th gibbon ape leukemia virus (GaLV), providing a broader hos range compared to murine vectors. Since recombinan retroviruses are defective, assistance is required in order to produce infectious particles. Such assistance can be provided e.g., by using helper cell lines that contain plasmid encoding all of the structural genes of the retrovirus unde the control of regulatory sequences within the LTR. Suitable helper cell lines are well known to those skilled in the art Said vectors can additionally contain a gene encoding selectable marker so that the transduced cells can b identified. Moreover, the retroviral vectors can be modified in such a way that they become target specific. This can be achieved, e.g., by inserting a polynucleotide encoding sugar, a glycolipid, or a protein, preferably an antibody Those skilled in the art know additional methods for generating target specific vectors. Further suitable vector and methods for in vitro- or in vivo-gene therapy ar described in the literature and are known to the person skilled in the art; see, e.g., WO 94/29469 or WO 97/00957.
In order to achieve expression only in the target organ, e.g. a brain tumor, the DNA sequences for transcription of the antisense oligonucleotides can be linked to a tissue specific promoter and used for gene therapy. Such promoters are well known to those skilled in the art (see e.g. Zimmermann et al. (1994) Neuron 12, 11-24; Vidal et al.; (1990) EMBO J. 9, 833 840; Mayford et al., (1995), Cell 81, 891-904; Pinkert et al. (1987) Genes & Dev. 1, 268-76).
Within an oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbon of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Specific examples of preferred antisense compounds useful in the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotide backbones which can result in increased stability are known to the person skilled in the art preferably such modification is a phosphorothioate linkage.
A preferred oligonucleotide mimetic is an oligonucleotide mimetic that has been shown to have excellent hybridizatio properties, and is referred to as a peptide nucleic aci (PNA). In PNA compounds, the sugar-backbone of a oligonucleotide is replaced with an amide containing backbone in particular an aminoethylglycine backbone. The nucleobase are retained and are bound directly or indirectly to az nitrogen atoms of the amide portion of the backbone (see e.g., Nielsen et al., Science 254 (1991), 1497-1500.)
Modified oligonucleotides may also contain one or more substituted or modified sugar moieties. Preferred oligonucleotides comprise one of the following at the 2 position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O- S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 t C10 alkyl or C2 to C10 alkenyl and alkynyl. A particularly preferred modified sugar moiety is a 2′-O-methoxyethyl suga moiety.
Oligonucleotides of the invention may also include nucleobas modifications or substitutions. Modified nucleobases include other synthetic and natural nucleobases such as 5 methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine hypoxanthine, 2-aminoadenine, 6-methyl and other alky derivatives of adenine and guanine, 2-propyl and other alky derivatives of adenine and guanine, 2-thiouracil, 2 thiothymine and 2-thiocytosine etc., with 5-methylcytosin substitutions being preferred since these modifications have been shown to increase nucleic acid duplex stability.
Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include lipid moieties such as a cholesterol moiety cholic acid, a thioether, a thiocholesterol, an aliphati chain, e.g., dodecandiol or undecyl residues, a phospholipid a polyamine or a polyethylene glycol chain, or adamantan acetic acid, a palmityl moiety, or an octadecylamine o hexylamino-carbonyl-oxycholesterol moiety.
The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds o “chimeras,” in the context of this invention, are antisens compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide i modified so as to confer upon the oligonucleotide increase resistance to nuclease degradation, increased cellular uptake and/or increased binding affinity for the target nucleic acid An additional region of the oligonucleotide may serve as substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonucleas which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotide when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleoside and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids o gapmers.
In a further preferred embodiment of the present invention the compounds for use in a method of treating a neoplasia are compounds reducing or inhibiting the biological activity o BCAT1.
Such compounds are described in the art for medica indications differing from the indication of the present invention and, preferably, comprise compounds like 1 (aminomethyl)cyclohexaneacetic acid (gabapentin) or a compound described in Goto et al., The Journal of Biological Chemistry 280(44) (2005), 37246-56, Hu et al., Bioorganic & Medicinal Chemistry Letters 16 (2006), 2337-40, Caballero et al. Molecular Diversity 13 (2009), 493-500, U.S. Pat. No. 6,632,831, U.S. Pat. No. 6,809,119, and EP-B1 1 157 000.
Further examples of compounds capable of reducing o inhibiting the biological activity of BCAT1 are (neutralizing antibodies directed against BCAT1 or fragments thereof having substantially the same binding specificity. The term “antibody”, preferably, relates to antibodies which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations. Monoclonal antibodies are made from a antigen containing, e.g., a fragment of BCAT1 by methods well known to those skilled in the art (see, e.g., Köhler et al. Nature 256 (1975), 495). As used herein, the term “antibody (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example Fab and F(ab′).2 fragments) which are capable of specifically binding to protein. Fab and F(ab′)2 fragments lack the F fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. (Wahl et al., J. Nucl. Med. 24: 316 325 (1983)). Thus, these fragments are preferred, as well a the products of a FAB or other immunoglobulin expression library. Moreover, antibodies useful for the purposes of the present invention include chimerical, single chain, an humanized antibodies.
Alternatively, preferred compounds for the purpose of the invention are inactive versions of BCAT1 or nucleic acid sequences encoding inactive versions of BCAT1 that can be introduced according to the approaches/vectors describe above. Such inactive versions can be generated according to well known methods of mutagenesis. Such compounds can have therapeutic effect in the human body by displacing their functionally active counterpart, in particular when applied i excess. Analyses of potentially inactive versions of BCAT1 can be carried out by assaying the (reversible) transamination o branched-chain L-amino acids to branched-chain alpha-ket acids, e.g., by determining the production of glutamate Suitable assays are described in the literature and, e.g., i Example 39 of U.S. Pat. No. 6,809,119, Hu et al. (2006), an EP-B1 1 157 000.
In a preferred embodiment of the present invention the compound described in detail above is used in a method o treating an astrocytic brain tumor or glioblastoma.
The present invention also relates to a method for identifying a compound reducing or inhibiting the biological activity o BCAT1 and/or its expression, comprising the steps of:
-
- (a) incubating a candidate compound with a test system comprising BCAT1 or its gene; and
- (b) assaying a biological activity of BCAT1;
- wherein an inhibition or loss of a biological activity o BCAT1, preferably compared to a test system in the absence of said test compound, is indicative of the presence of candidate compound having the desired property.
Step (b) can be carried out by assaying the (reversible transamination of branched-chain L-amino acids to branched chain alpha-keto acids using an assay as described above.
Examples of such candidate molecules include antibodies oligonucleotides, proteins, or small molecules. Such molecule can be rationally designed using known techniques.
Preferably, said test system used for screening comprise substances of similar chemical and/or physical properties most preferably said substances are almost identical. The compounds which can be prepared and identified according to use of the present invention may be expression libraries e.g., cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, ligands, hormones peptidomimetics, PNAs or the like.
WO 98/25146 describes further methods for screening libraries of complexes for compounds having a desired property especially, the capacity to agonize, bind to, or antagonize polypeptide or its cellular receptor. The complexes in such libraries comprise a compound under test, a tag recording a least one step in synthesis of the compound, and a tethe susceptible to modification by a reporter molecule Modification of the tether is used to signify that a comple contains a compound having a desired property. The tag can be decoded to reveal at least one step in the synthesis of such compound. Other methods for identifying compounds which interac with BCAT1 or nucleic acid molecules encoding such molecule are, for example, the in vitro screening with the phage display system as well as filter binding assays or “real time” measuring of interaction.
It is also well known to the person skilled in the art, that it is possible to design, synthesize and evaluate mimetics o small organic compounds that, for example, can act as substrate or ligand to BCAT1. For example, it has been described that D-glucose mimetics of hapalosin exhibited similar efficiency as hapalosin in antagonizing multidru resistance assistance-associated protein in cytotoxicity; se Dinh, J. Med. Chem. 41 (1998), 981-987.
All these methods can be used in accordance with the present invention to identify a compound reducing or inhibiting the biological activity of BCAT1 or its expression.
The gene encoding BCAT1 can also serve as a target for the screening of inhibitors. Inhibitors may comprise, for example proteins that bind to the mRNA of the genes encoding BCAT1 thereby destabilizing the native conformation of the mRNA an hampering transcription and/or translation. Furthermore methods are described in the literature for identifying nucleic acid molecules such as a RNA fragment that mimics the structure of a defined or undefined target RNA molecule t which a compound binds inside of a cell resulting in the retardation of the cell growth or cell death; see, e.g., W 98/18947 and references cited therein. These nucleic acid molecules can be used for identifying unknown compounds o pharmaceutical interest, and for identifying unknown RNA targets for use in treating a disease. These methods an compositions can be used for identifying compounds useful to reduce expression levels of BCAT1.
The compounds which can be tested and identified according to the method of the invention may be expression libraries, e.g. cDNA expression libraries, peptides, proteins, nucleic acids antibodies, small organic compounds, hormones peptidomimetics, PNAs or the like (Milner, Nature Medicine (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 7 (1994), 193-198 and references cited supra). Furthermore genes encoding a putative regulator of BCAT1 and/or which exert their effects up- or downstream of BCAT1 may be identified using, for example, insertion mutagenesis using for example, gene targeting vectors known in the art. Sai compounds can also be functional derivatives or analogues o known inhibitors. Such useful compounds can be for example transacting factors which bind to BCAT1 or regulator sequences of the gene encoding it. Identification o transacting factors can be carried out using standard method in the art. To determine whether a protein binds to the protein itself or regulatory sequences, standard native gel shift analyses can be carried out. In order to identify transacting factor which binds to the protein or regulator sequence, the protein or regulatory sequence can be used as a affinity reagent in standard protein purification methods, o as a probe for screening an expression library. Th identification of nucleic acid molecules which encod polypeptides which interact with BCAT1 can also be achieved, for example, as described in Scofield (Science 274 (1996), 2063 2065) by use of the so-called yeast “two-hybrid system”. In this system BCAT1 is linked to the DNA-binding domain of the GAL transcription factor. A yeast strain expressing this fusio polypeptide and comprising a lacZ reporter gene driven by a appropriate promoter, which is recognized by the GAL transcription factor, is transformed with a library of cDNA which will express plant proteins or peptides thereof fused t an activation domain. Thus, if a peptide encoded by one of the cDNAs is able to interact with the fusion peptide comprising peptide of BCAT1, the complex is able to direct expression o the reporter gene. In this way, BCAT1 and the gene encoding BCAT1 can be used to identify peptides and proteins interacting with BCAT1. It is apparent to the person skilled in the art that this and similar systems may then further be exploited for the identification of inhibitors.
The below example explains the invention in more detail bu are not construed as a limitation of the invention.
Example 1 General Methods(a) RNA Isolation and Quantitative Real-Time PCR (qRT-PCR)
Total RNA was extracted using the AllPrep DNA/RNA/Protein Min Kit (Qiagen) according to the manufacturer's instructions FirstChoice® Human Brain Reference Total RNA from Ambio served as normal brain RNA pool (n=23 donors). Total RNA (50 ng) was reverse-transcribed using random primers an superscript II (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. Each complimentary DNA sample was analysed in triplicate with the Applied Biosystems Pris 7900HT Fast Real-Time PCR System (Applied Biosystems, Foste City, Calif., USA) using Absolute SYBR Green ROX Mix (ABgene Epsom, UK). The relative amount of specific mRNA was normalised to ARF, B2M and TBP. Primer sequences are given in the following Table 1A.
(b) Western Blot Analysis
All experiments involving the use of human tissues are carried out in line with the guidelines of the institutional revie board of the Medical Faculty at Heinrich Heine University Düsseldorf. Fresh frozen human tumor tissue samples and a non-neoplastic brain tissue sample were lysed in guanidin isothiocyanate solution (4M) using an ULTRA-TURRAX (IKA Staufen, Germany) and subjected to CsCl-ultracentrifugation Diafiltration of the obtained protein fractions was performed using the Amicon Ultra-0.5 centrifugal filter device (Millipore, Schwalbach, Germany; 3 kDa cut-off) essentially a described45. Total protein of cell lines was extracted using the AllPrep DNA/RNA/Protein Mini kit (Qiagen). 10 μg o protein were separated by 10% SDS-PAGE and transferred to PVD membranes (Millipore). The membrane was blocked in blocking solution (5% BSA in TBS, 0.1% Tween 20) and incubated with primary antibodies overnight at 4° C. Horseradish peroxidas (HRP)-conjugated secondary antibodies were incubated for hour at room temperature prior to the chemiluminescent detection of protein (ECL kit; GE Healthcare, Little Chalfont UK).
The following antibodies were used: monoclonal mouse antibod against α-tubulin (1:2000, #T9026, Sigma-Aldrich, St. Louis Mo. 63178, USA), monoclonal mouse antibody against BCAT (1:2000, ECA39, clone 51, #611270, BD Biosciences, San Jose Calif., USA), monoclonal rat antiserum against IDH1 (1:1, provided by A. von Deimling, DKFZ, Heidelberg, Germany; commercially available from Dianova, Hamburg, Germany), monoclonal mous antibody against HADHSC (1:500, #H00003033-M01, Abnova) monoclonal rabbit antibody against CDKN1B (1:1000, p27 Kip1 #3686, Cell Signaling Technology), monoclonal rabbit antibod against Akt (1:1000, #9272, Cell Signaling Technology) monoclonal rabbit antibody against phospho-Akt (Ser473 (1:1000, #193H12, Cell Signaling Technology), polyclona rabbit antibody against p70 S6 kinase (1:1000, #9202, Cell Signaling Technology), polyclonal rabbit antibody against phospho-p70 S6 kinase (Thr389) (1:1000, #9205, Cell Signaling Technology), HRP-conjugated anti-rat IgG (1:10000, provided by A. von Deimling, DKFZ, Heidelberg, Germany), HRP-conjugated horse anti-mouse IgG (1:5000, #7076, Cell Signaling Technology), HRP-conjugated goat anti-rabbit IgG (1:2000 #7074, Cell Signaling Technology).
(c) Immunohistochemical Staining
Tumor sections were deparaffinised using xylol and rehydrated in decreasing concentrations of ethanol. Antigen retrieval was performed by heating for 40 min in a steamer in 10 mM sodium citrate buffer (pH 6.0). Endogenous peroxidase was inactivate by incubating the tumor sections in 3% hydrogen peroxide Sections were incubated overnight with primary anti-BCAT (ECA39) monoclonal mouse antibody, clone 51 (BD Biosciences San Jose, Calif.) diluted 1:500 or mouse anti-human IDH1 R132 antibody (Dianova, Hamburg, Germany) diluted 1:20 in Dak REAL™ antibody diluent (Dako, Glostrup, Denmark). Staining for detection of bound antibody was performed according to standard protocols using the EnVision™ detection system (peroxidase/DAB+, rabbit/mouse) (Dako, Glostrup, Denmark) subsequent counterstaining was done using hematoxylin.
(d) DNA Methylation Analysis
DNA methylation was analyzed by MassARRAY technique. Briefly 500 ng genomic DNA was chemically modified with sodium bisulfite using the EZ methylation kit (Zymo Research according to the manufacturer's instructions. The bisulfite treated DNA was PCR-amplified with primers generating fou amplicons (A1-A4) from −990 bp to +612 bp around TSS of BCAT transcript 1 (T1, ENST00000261192), and three amplicons (A5 A7) of the promoter of BCAT1 transcript 4 (T4 ENST00000539282) and 6 (T6, ENST00000538118) from −198 bp t +63 bp. The amplicons were transcribed by T7 polymerase followed by T specific-RNAaseA cleavage. The digested fragments were quantified by mass-spectrometry. The prime sequences are given in Table 1B. DNA methylation standard (0%, 20%, 40%, 60%, 80%, and 100% methylated genomic DNA) were used to confirm the unbiased amplification of the amplicons.
(e) Cell Culture
The human glioma cell lines U-87MG (HTB-14), LN-229 (CRL 2611), Hs683 (HTB-138) and U-373MG (HTB-17) were cultured i Dulbecco's Minimal Essential Medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin-mix. Cel lines were authenticated by short tandem repeat (STR analysis. The brain cancer stem-like cells NCH421k were established from primary glioblastoma patients undergoing surgical resection according to the research proposal approved by the institutional review board at the Medica Faculty, University of Heidelberg. They were characterized genotypically and phenotypically in a previous study (Campos B., et al. Differentiation therapy exerts antitumor effects o stem-like glioma cells. Clin Cancer Res 16, 2715-2728 (2010)) NCH421k cells were grown as floating aggregates (neurospheres on uncoated tissue culture dishes in DMEM/F-12 mediu containing 20% BIT serum-free supplement, basic fibroblas growth factor and epidermal growth factor at a concentration of 20 ng/ml each (all from Provitro, Berlin, Germany). HEK293 and HEK293 cells were maintained as monolayer cultures in DME containing 10% fetal calf serum without antibiotics.
All cell lines were cultivated at 37° C. in a humidified incubator with 5% CO2. For hypoxia experiments, cells were cultured at 1% O2 in a nitrogen-supplied C16 hypoxia incubato (Labotect, Goettingen, Germany).
(f) Chromatin Immunoprecipitation (ChIP)
ChIP was performed on HEK293 cells overexpressing flag-tagged HEY1, since the currently available antibodies against HEY were not specific for ChIP assays. Constructs for HEY overexpression were prepared using gateway compatible vector tagged with FLAG; pDest26 (C-terminal tag) and pDest11 (N terminal tag). 1 μg of either the control vectors or HEY expression vectors were transfected into 2.5×10̂6 HEK293 cell per 15 cm plate using TransIT®-LT1 (Mirus Bio LLC, Madison, W 53711, USA) according to slightly modified manufacturer' instructions. The cells from two plates were harvested 4 hours after transfection. Chromatin was prepared using non ionic shearing buffers with Covaris S2, according to the manufacturer's protocol (Covaris Inc.). ChIP was performed using anti-FLAG antibody (Cell signaling #2368). The DNA recovered after ChIP was used for qRT-PCR with input chromati and mock immunoprecipitation (anti-IgG antibody, Diagenode Kch-803-015) serving as controls. qRT-PCR was performed i triplicate with SYBR green detection using primers listed i Table 2. Ratios of bound to input signals are reported.
(g) Transient Transfection of siRNA
HEK293T cells were transfected with siRNA duplexes from Ambio (HEY1: s23868-70) or Dharmacon (control non-targeting: D 001810-10) using DhamaFect1 following slight modifications t the manufacturer's instructions (Dharmacon). Briefly, eac siRNA pool was diluted in 1× siRNA buffer (Dharmacon), wa mixed with RPMI, and then distributed into 6 wells of 96 wel plates. 1.2×10̂4 HEK293 or 9×10̂3 HEK293T cells were seeded o top of the siRNA/DharmaFECT mixture (the volume was 15 μl/well and 20 nM of siRNAs in final). After 48 hour incubation at 37° C., RNAs were isolated from the wells fo further analysis.
(h) Virus Production and Transduction
Lentiviral vectors were produced by cotransfection of HEK293 cells with the psPAX2 (Addgene 12260, Didier Trono, packaging vector), pMD2.G (Addgene 12259, Didier Trono, envelop plasmid), and pLKO.1 shRNA constructs (Sigma-Aldrich) Transfections were carried out using TransIT®-LT1 (Mirus Bi LLC) and virus was harvested at 48 and 72 hours after transfection and combined.
Infection of glioma cells with virus at an M.O.I. of 2 was carried out in the presence of 8 μg/ml of polybrene (Chemicon Billerica, Mass., USA). Virus-containing supernatant was removed after 24 hours and cells were split on day 3, day 5 and day after transduction. Two independent Mission® shRNA construct targeting different regions of the human BCATI (BCAT1 shRNA NM—005504.3-1064s1c1 and BCAT1 shRNAII NM—005504.3-751s1c1 and human IDH1 (IDH1 shRNAI NM—005896.2-1363s1c1 and IDH shRNAII NM—005896.2-292s1c1) mRNA transcripts were used Nontargeting shRNA (Mission SHC002) was used as a control. Quantification of BCAT1 and IDH1 knockdown was assessed b quantitative real-time PCR and Western blot.
(i) Treatment with Dimethyl-α-Ketoglutarate and Gabapentin
5×10̂4 cells/well were seeded in 24-well plates in a total volume of 500 μl cell culture medium. The medium was removed 16 hours after seeding the cells, and replaced with 500μ medium containing 5 mM or 10 mM dimethyl-α-ketoglutarat (Dimethyl 2-oxoglutarate; Sigma-Aldrich) or 5 mM, 10 mM or 2 mM gabapentin (1-(Aminomethyl)-cyclohexane; Sigma-Aldrich) Corresponding volumes of 200 mM HEPES buffer (pH 7.4) were added to the respective control wells.
(j) Cell-Cycle Analysis and Detection of Apoptosis
Cell-cycle analysis was performed 20 hours after treatment with gabapentin and 6 days after lentiviral transduction Nicoletti buffer (0.1% sodium citrate, pH7.4, 0.05% Triton X 100, 50 μg ml−1 propidium iodide) was added to the well containing both dead and living cells. After 4 hours in the dark at 4° C., DNA content was analysed by flow cytometry using FACS Canto II (BD Biosciences, San Jose, Calif., USA). FACS Div software was used to quantify the distribution of cells i each cell-cycle phase: sub-G1 (dead cells), G1, S, and G2/M. For investigation of apoptotic activity after lentivira knockdown NCH421k cells were incubated with annexin V-P (Phycoerythrin) and 7-AAD (7-aminoactinomycin D, B Biosciences) for 15 minutes in the dark, immediately followed by flow cytometry.
(k) Proliferation Analysis
To assess the proliferation of glioma cells after treatment with gabapentin or after lentiviral knockdown, the Click-iT EdU cell proliferation assay (Invitrogen, Karlsruhe, Germany was used following the manufacturer's instructions. The cell were incubated with 10 μM of the nucleoside analog EdU (5 ethynyl-2′deoxyuridine) for 16 hours. Quantification of cell that incorporated EdU was performed using FACS Canto II (B Biosciences).
(l) Glutamate Quantification
Glutamate concentration in the supernatant of cells treated with gabapentin was determined using the glutamine/glutamat determination Kit (GLN-1; Sigma-Aldrich) according to the manufacturer's instructions. The reaction volumes were scaled down to 100 μl total volume and absorbance was measured in triplicate in a microplate (Corning® 96 Well Clear Flat Bottom UV-Transparent Microplate) using a Tecan Infinite M200 plat reader (Tecan, Austria). Data were normalised to the number o cells per well.
(m) Immunofluorescence Staining
Cells were plated on glass coverslips five days after lentiviral transduction. Cells were fixed in 4% formaldehyde rinsed twice in 1×PBS, and permeabilized in PBS containing 0.2% Triton. Following rinsing with 1×PBS cells were incubated in 10% goat serum for 30 minutes at room temperature. Samples were incubated 1 hour with the primary antibody (mouse anti α-tubulin antibody, 1:200, #T9026, Sigma Aldrich) and 1 hour at room temperature with the secondary antibody (FITC-conjugated goat anti mouse antibody, 1:100 ab6785, Abcam) following Mounting with DAPI-containing Vectashield mounting medium (Vector Laboratories, Burlingame Calif., USA). For fluorescence imaging, images were taken using 40× objective lense on a Zeiss Axioplan microscope.
(n) 3D Microchannel Migration Assay
Poly(dimethylsiloxane) (PDMS) based microchannel chips were kindly provided by Dr. Ralf Kemkemer (Max Planck Institute for Intelligent Systems, Germany). Microfabricated channe structures with the dimensions of 5×11×300 μm (W×H×L) were bio-functionalized by incubation with a 50 μg/ml fibronecti solution prior to use. The chip was fixed on a Teflon holde and 2×10̂5 cells were seeded on the chip in close proximity t the channels. After cells were attached on the chip, live-cell imaging was performed for 25 hours. During the experiments n chemical gradient or flow inside the channels was applied Phase-contrast time-lapse pictures of multiple positions were captured every 10 minutes with an automated inverted microscope (Zeiss Cell Observer; Carl Zeiss) equipped with a air-humidified and heated chamber. Images were recorded an processed with Zeiss AxioVision and ImageJ software. Cel behaviors were analyzed and categorized as reported previously (Bai, A. H., et al. MicroRNA-182 promotes leptomeningeal sprea of non-sonic hedgehog-medulloblastoma. Acta Neuropatho (2011); Rolli, C. G., Seufferlein, T., Kemkemer, R. & Spatz J. P. Impact of tumor cell cytoskeleton organization o invasiveness and migration: a microchannel-based approach PLoS One 5, e8726 (2010)).
(o) Animal Experiments
Animal work was approved by the governmental authorities (Regierungspraesidium Karlsruhe, Germany) and supervised b institutional animal protection officials in accordance with the National Institutes of Health guidelines Guide for the Care and Use of Laboratory Animals.
(p) Orthotopic Brain Tumor Model
A total of 2×10̂5 U-87MG cells with BCAT1 shRNAI knockdown o nontargeting shRNA were stereotactically implanted into the brain of six 7-9-week-old athymic mice (CD1 nu/nu; Charle River Laboratories, Wilmington, Mass., USA), respectively. Fou weeks after implantation, animals were sacrificed, brain removed and cryosectioned. Brain sections were stained with hematoxylin and eosin and the tumor volume was calculate using ImageJ.
(q) Perchloric Acid Cell Extraction and NMR Spectroscopy
Cells were harvested by trypsinization, washed once with ice cold PBS, and the cell pellet was frozen at −80° C. Typically 1×108 harvested cells were subjected to perchloric acid extraction followed by KOH neutralization according t published protocols. Briefly, 2 mL of ice-cold 1 N HClO4 were added to a frozen pellet of harvested cells. The pellet was disrupted using a Teflon pestle and a glass homogeniser. 1 m of ice-cold water was added to the lysate, vortexed for 90 and centrifuged at 10000×g for 10 min at 4° C. The pellet was re-extracted and the supernatants were combined. The extrac was neutralised with KOH and the pH was adjusted to 6.5-7.0 Precipitated KClO4 was pelleted at 25000×g for 20 min and the supernatant was lyophilised.
The lyophilised extract residues were dissolved in 0.5 ml of D2O buffer (99.9% D) containing 30 mM sodium phosphate and 0. mM sodium azide (pH 7.02). For quantitative analysis reference capillary (1.5 mm OD, 0.99 mm ID) was filled with solution of 11 mM trimethylsilyl-2,2,3,3-tetradeuteropropanoi acid (TSP, sodium salt) in the D2O buffer described above Subsequently, the capillary was sealed in the flame of Bunsen burner. A calibration solution was prepared by placin weighed amounts of glucose and citric acid in the D2O buffer t give the calculated concentrations of 10.49 mM glucose an 4.95 mm glucose. 1H-NMR spectra of the calibration solution capillary were acquired at 600 MHz (Avance AV-600, Bruke BioSpin GmbH) using a standard 5-mm NMR tube and a triple resonance inverse probe under the same conditions to be use for the extracts (20° C., presaturation of residual HDO signal repetition time TR=7 s, 30° flip angle). The signal integrals for citrate and glucose were set to value corresponding to the concentrations given above, and th integral of the TSP methyl signal was found to be equivalen to 4.60±0.10 mM protons. Cell extracts were measured wit the calibrated reference capillary inserted (512 transients i 1 hour), and the 1H-NMR signals for the branched-chain amin acids, various metabolites, and the TSP reference wer integrated. The TSP integral was defined as 4.60 mM so tha the metabolite signal integrals gave directly the metabolit concentrations in mM (μmol/ml) for the 0.5 ml volumes used These data were then converted to femtomol/cell using the cel counts determined prior to extraction.
(r) Preparation of Cell Homogenates
Cell pellets were diluted in 100 μl water and homogenized b. sonification (Ultrasonic device, 3−5×20 cycle, output 80% Branson Sonifier 450, Dietzenbach, Germany). Protein wa determined according to Lowry (Lowry, O. H., Rosebrough, N. J. Farr, A. L. & Randall, R. J. Protein measurement with the Foli phenol reagent. J Biol Chem 193, 265-275 (1951) with th modifications of Helenius and Simons (Helenius, A. & Simons K. The binding of detergents to lipophilic and hydrophili proteins. J Biol Chem 247, 3656-3661 (1972)) using bovine seru albumin as a standard. The final protein concentrations of th homogenates should be in a range of 2-4 mg/ml. These dilution were used for all analyses.
(s) Statistical Analysis
The relationship between IDH1/IDH2 mutation status and BCAT protein expression (
The human glioblastoma cell lines U87-MG (HTB-14), HS683 (HTB 138) and U373-MG (HTB-17; LGC Standards, Teddington TW11 0LY U.K.) were cultured in Dulbecco's Minimal Essential Mediu supplemented with 10% fetal calf serum and 1 penicillin/streptomycin-mix. 5×104 cells/well were seeded in 2 well plates in a total volume of 500 μl cell culture medium The medium was removed 4 hours after seeding the cells, an replaced with 500 μl medium containing 1-(Aminomethyl)cyclohexylessigsäure (Gabapentin, dissolved in 200 mM HEPE buffer at a concentration of 500 mM) at final concentrations o 5 mM, 10 mM and 20 mM, respectively. Corresponding volumes o 200 mM HEPES were added to the respective control wells. hours after exchanging the medium, 5-ethynyl-2′-deoxyuridin (EdU; Carlsbad, Calif. 92008, USA) was added in order to measur cell proliferation using Invitrogens (Carlsbad, Calif. 92008, USA Click iT® proliferation kit. The cells were harvested 16 hour after EdU application and proliferation was measured according to the manufacturer's instructions using a BD Biosciences FAC Canto II flow cytometer (BD Biosciences, Franklin Lakes, N USA 07417). Three replicate measurements were obtained fo each treatment. In all cell lines a concentration-dependen significant reduction of cell proliferation of about 20-55 was observed in the gabapentin treated cells as compared t mock treated cells. The results are shown in
Lentiviral vectors were produced by the cotransfection o HEK293T cells with the psPAX2 (Addgene 12260, Didier Trono packaging vector), pMD2.G (Addgene 12259, Didier Trono envelope plasmid), and pLKO.1 shRNA constructs (Sigma-Aldrich St. Louis, Mo. 63178, USA). Transfections were carried ou using TransIT®-LT1 (Mirus Bio LLC, Madison, Wis. 53711, USA) an virus was harvested at 48 and 72 hours after transfection. Tw independent shRNA constructs targeting different regions o the BCAT1 mRNA transcript were used: MISSION shRN TRCN0000005907 NM—005504.3-1064s1c1 (shRNA1) an TRCN0000005909 NM—005504.3-751s1c1 (shRNA2); all Sigma Aldrich, St. Louis, Mo. 63178, USA. Nontargeting shRNA was use as a control.
The human glioblastoma cell lines U87-MG (HTB-14), HS683 (HTB 138) and U373-MG (HTB-17; LGC Standards, Teddington TW11 0LY U.K.) were seeded in 24 well plates (5×104 cells/well) in total volume of 500 μl cell culture medium. After 24 hour cells were transduced with virus in the presence of 8 μg/ml o polybrene. Virus-containing supernatant was removed after 2 hours and cells were split on day 3 and day 5 afte transduction. Decreased BCAT1 mRNA and protein expression wa detected using quantitative real-time PCR and Western blo (ECA39 antibody, BD Biosciences Pharmingen, San Diego, Calif. 92121, USA). A proliferation assay was carried out on day 6 using the Click-iT® EdU cell proliferation kit after incubating cells with 10 μM EdU for 16 hours. In all of the BCAT1 shRNA transduced cells a reduction of proliferation was observed ranging from 20-80% depending on the cell line. These results are shown in
Prediction analysis of microarrays on gene expression data from astrocytic gliomas of WHO grades II, III and IV identified BCAT1 as the best classifier distinguishing primary glioblastoma from other astrocytoma as can be seen in the following Table 3:
When classifying tumors based on IDH-mutation status, BCAT1 mRNA expression levels were significantly higher for IDHwt gliomas relative to IDHmut gliomas (
BCAT1 was found to be expressed strongly in the glioblastoma cell lines LN-229, U-87MG, U-373MG and to a lesser extent in A172 (
To further elucidate the differential regulation of BCAT1 expression in IDHwt and IDHmut gliomas, transcript expression in patient samples was quantified. Using RT-PCR and sequencing, expression of three protein-coding BCAT1 transcripts listed in the Ensembl database in IDHwt astrocytic primary tumors as well as in a pool of 23 normal brain tissues (
To investigate the possible mechanisms of BCAT1 transcriptional regulation, quantitative DNA methylation analysis on astrocytomas of all grades using MassARRAY analysis of PCR products amplified from bisulfite-treated DNA covering the two alternative promoters (
To gain insight into the BCAT1 functional role in glioblastomas, cell lines U-87MG and U-373MG cell lines were treated with gabapentin, a leucine analog that specifically inhibits BCAT1, but not BCAT2. 1H-NMR spectroscopy of extracts of cells treated with 20 mM gabapentin for 20 hours demonstrated the intracellular accumulation of BCAAs, consistent with BCAT1 inhibition (
To independently confirm these findings, shRNA-mediated BCAT1 knockdown in U-87MG, U-373MG and Hs683 cells using two shRNAs that both target all three BCAT1 transcripts was performed. Tandem-MS analysis of the U-87MG cell culture media after 24 hour incubation revealed that glutamate release, as well as BCAA uptake were reduced in BCAT1 knockdown cells compared to control cells (
BCAT1 knockdown led to strong downregulation of 3-hydroxyacyl CoA dehydrogenase (HADH), an enzyme participating in the catabolism of valine and isoleucine downstream of BCAT1 (
BCAT1 knockdown strongly affected cell morphology, resulting in a rounded, less extended appearance (
To assess the impact of BCAT1 on tumor cell proliferation, inhibition and knockdown experiments were conducted. A concentration-dependent reduction of proliferation up to 56%, estimated based on EdU-incorporation, was observed, upon treatment with the inhibitor gabapentin (
Comparable reductions of cell proliferation were observed upon BCAT1 knockdown in glioblastoma primary spheroid cultures in serum-free media, except that these cells showed a higher rate of apoptosis as determined by Annexin V/7AAD staining (
The effect of BCAT1 knockdown on tumor growth in vivo was evaluated by intracerebral transplantation of U-87MG cells into CD-1 nude mice (
Claims
1.-12. (canceled)
13. A pharmaceutical composition for treating a brain tumor, wherein said pharmaceutical composition comprises wherein the effective dose leads to a reduction or inhibition of the proliferation of brain tumor cells in a subject,
- an effective dose of a compound capable of reducing or inhibiting (a) the biological activity of a branched-chain-aminotransferase-1 (BCAT1) or (b) the expression of the gene encoding BCAT1,
- and a pharmaceutically acceptable carrier.
14. The pharmaceutical composition of claim 13, wherein said compound is an antisense oligonucleotide or siRNA reducing or inhibiting the expression of the gene encoding BCAT1.
15. The pharmaceutical composition of claim 13, wherein said compound is a compound reducing or inhibiting the biological activity of BCAT1.
16. The pharmaceutical composition of claim 15, wherein said compound is 1-(aminomethyl)cyclohexaneacetic acid.
17. The pharmaceutical composition of claim 15, wherein said compound is an antibody directed against BCAT1 or a fragment thereof.
18. The pharmaceutical composition of claim 13, wherein said compound is an inactive version of BCAT1.
19. The pharmaceutical composition of claim 13, wherein said compound is, wherein the neoplasm to be treated shows BCAT1 (over) expression.
20. The pharmaceutical composition of claim 13, wherein said compound is, wherein said brain tumor to be treated is an astrocytic brain tumor, glioma or glioblastoma.
21. The pharmaceutical composition of claim 20, wherein said compound is, wherein the brain tumor is an IDHwt glioblastoma.
Type: Application
Filed: Jun 26, 2015
Publication Date: Dec 31, 2015
Inventors: Bernhard RADLWIMMER (Heidelberg), Martje TOENJES (Heidelberg), Sebastian BARBUS (Heidelberg), Peter LICHTER (Gaiberg)
Application Number: 14/752,280
