Patent Application
20240209353
The instant application contains a Sequence Listing which has been submitted in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 6, 2022, is named S171570052WO00-SEQ-JIB and is 4510 bytes in size.
Mammalian cells depend on the inter-conversion of nicotinamide adenine dinucleotide phosphate (NADP) molecules between the oxidized (NADP+) and reduced (NADPH) forms to support reductive biosynthesis and to maintain cellular antioxidant defense. NADP+ and NADPH molecules (also referred to as “NADP(H)”) are unable to cross subcellular membranes. As a result, cellular pools of NADP(H) are compartmentalized. In the cytosol, NADP(H) is derived from nicotinamide adenine dinucleotide [(NAD)H] by NAD kinase 1 (NADK1). Cytosolic NADPH acts as a substrate in fatty acid biosynthesis, and as the reducing equivalent required to regenerate reduced glutathione (GSH) and thioredoxin for antioxidant defense. Mitochondria host a number of biosynthetic activities critical for cellular metabolism but are also major sites for reactive oxygen species (ROS) generation. Mammalian mitochondrial NAD kinase 2 (NADK2) converts NAD(H) to NADP(H) through phosphorylation.
The present disclosure is based on the surprising discovery that mitochondrial nicotinamide adenine dinucleotide phosphate (NADPH) produced by nicotinamide adenine dinucleotide kinase 2 (NADK2) is critical to proline synthesis, protein synthesis, and maintaining cell proliferation in a nutrient-deficient environment. Inhibiting the activity of NADK2 inhibits protein synthesis and cell proliferation in vitro and in vivo. Thus, antagonists of NADK2 may be used to treat diseases or disorders characterized by increased protein synthesis (e.g., fibrosis) and/or increased cell proliferation (e.g., cancer).
In some aspects, the present disclosure provides a method of treating a cancer characterized as having an isocitrate dehydrogenase 2 (IDH2) mutation, the method comprising administering to a subject in need thereof an antagonist of nicotinamide adenine dinucleotide kinase 2 (NADK2) in an amount effective to treat the cancer.
In some aspects, the present disclosure provides a method for inhibiting cancer cell proliferation, the method comprising contacting cancer cells expressing a mutant IDH2 protein with an antagonist of NADK2, wherein the mutant IDH2 protein has neomorphic enzymatic activity.
In further aspects, the present disclosure provides a method for inhibiting cell proliferation comprising: providing a population of cells in a nutrient-deficient environment; and contacting a cell of the population of cells with an antagonist of NADK2, wherein the cell contacted with the antagonist has decreased proliferation compared to a cell not contacted with the antagonist of NADK2.
In further aspects, the present disclosure provides a composition comprising (i) a nutrient-deficient cell culture medium; and (ii) an antagonist of NADK2. In some embodiments, the nutrient-deficient cell culture medium is deficient in one or more amino acids. In some embodiments, the composition further comprises (iii) a population of cells. In some embodiments, the population of cells comprises cancer cells. In some embodiments, the cancer cells express a mutant IDH2 protein. In some embodiments, the nutrient-deficient cell culture medium comprises 10% serum, 100 units/mL penicillin, and/or 100 μg/mL streptomycin.
Accordingly, in some aspects, the present disclosure provides compositions and methods for use in treating a cancer and/or inhibiting proliferation of a cancer cell. In some embodiments, the cancer is characterized as having an isocitrate dehydrogenase 2 (IDH2) mutation. In some embodiments, the cancer is characterized as having increased levels of 2-hydroxyglutrarate (2HG) relative to a known reference value. In some embodiments, the cancer is characterized as having decreased levels of alpha-ketoglutarate (αKG) relative to a known reference value. In some embodiments, the known reference value is from a cell characterized as not having the IDH2 mutation. In some embodiments, the known reference value is from a non-cancerous cell and/or a cell that does not express a mutant IDH2 protein. In some embodiments, the cell is a non-cancerous cell of the subject. In some embodiments, the cancer is characterized as not having an isocitrate dehydrogenase 1 (IDH1) mutation.
In some embodiments, the IDH2 mutation produces a mutant IDH2 protein having a neomorphic enzymatic activity. In some embodiments, the neomorphic enzymatic activity is a reduction of αKG to 2HG. In some embodiments, the IDH2 mutation is selected from R172S, exon 4 mutation, a codon 140 missense mutation, R140Q, a codon 172 missense mutation, R172K, an amplification of IDH2, a loss of IDH2, R172W, R172M, R140W, R172G, V305M, H384Q, T350P. R172T, V355I, K155N, A416V, W21S, X39 splice, R159H, A347T, D390Y, D259N, A370T. A174T, or a combination thereof.
In some embodiments, the mutant IDH2 protein comprises one or more IDH2 mutations selected from R172S, exon 4 mutation, a codon 140 missense mutation, R140Q, a codon 172 missense mutation, R172K, an amplification of IDH2, a loss of IDH2, R172W, R172M, R140W, R172G, V305M, H384Q, T350P, R172T, V355I, K155N, A416V, W21S, X39 splice, R159H, A347T, D390Y, D259N, A370T, and A174T.
In some embodiments, the cancer is an adenocarcinoma. In some embodiments, the adenocarcinoma is selected from colon adenocarcinoma, lung adenocarcinoma, high grade ovarian serous adenocarcinoma, colorectal adenocarcinoma, rectal adenocarcinoma, prostate adenocarcinoma, or a combination thereof.
In some embodiments, the cancer is a carcinoma. In some embodiments, the carcinoma is selected from breast invasive ductal carcinoma, intrahepatic cholangiocarcinoma, endometrial endometrioid carcinoma, bladder urothelial carcinoma, endometrial carcinoma, squamous cell lung carcinoma, or a combination thereof.
In some embodiments, the cancer is selected from acute myeloid leukemia, oligodendroglioma, myelodysplastic syndrome, cutaneous melanoma, gliobastoma multiforme, angioimmunoblastic T-cell lymphoma, acute monoblastic and monocytic leukemia, or a combination thereof.
In further aspects, the present disclosure provides a method of treating a fibrotic disorder, the method comprising administering to a subject in need thereof an antagonist of NADK2 in an amount effective to treat the fibrotic disorder.
In some embodiments, the fibrotic disorder is characterized by increased levels of NADK2 relative to a known reference value. In some embodiments, the fibrotic disorder is characterized by increased levels of pyrroline-5-carboxylate synthase (P5CS) relative to a known reference value. In some embodiments, the known reference value is from a normal cell of the subject.
In some embodiments, the fibrotic disorder is characterized by increased levels of an extracellular matrix protein. In some embodiments, the extracellular matrix protein is collagen, elastin, fibronectin, and/or laminin. In some embodiments, the fibrotic disorder is pulmonary fibrosis or liver fibrosis.
In further aspects, the present disclosure provides a method for inhibiting protein synthesis, the method comprising contacting a cell from a population of cells with an antagonist of NADK2.
In some embodiments, the protein synthesis is decreased as compared to a cell that has not been contacted with the NADK2 antagonist. In some embodiments, the cell that has not been contacted with the antagonist is from the population of cells.
In some embodiments, the cell from the population of cells is contacted with the antagonist in a nutrient-deficient environment. In some embodiments, the nutrient-deficient environment has reduced levels of one or more amino acids compared to a nutrient-replete environment. In some embodiments, the nutrient-deficient environment contains a maximum of 300 μM of proline.
In some embodiments, the cytosolic protein is collagen, elastin, fibronectin, and/or laminin. In some embodiments, the cytosolic protein is collagen, and collagen synthesis is decreased in the cell contacted with the NADK2 antagonist as measured by staining collagen protein. In some embodiments, the collagen protein is stained by Picrosirius red staining.
In some embodiments, proline biosynthesis is decreased in the cell contacted with the NADK2 antagonist as measured by gas chromatography-mass spectrometry (GC-MS) and/or liquid chromatography-mass spectrometry (LC-MS). In some embodiments, proline is labeled with an isotopologue.
In some aspects, the present disclosure provides a method for decreasing protein synthesis, the method comprising: providing a cell expressing nicotinamide adenine dinucleotide kinase 2 (NADK2) in a nutrient-deficient environment; and contacting the cell with an antagonist of NADK2, wherein the cell contacted with the antagonist has decreased protein synthesis compared to a control cell not contacted with the antagonist.
In some embodiments, the protein (e.g., the protein having decreased synthesis) is collagen, elastin, fibronectin, and/or laminin. Accordingly, in some embodiments, the method is a method for decreasing synthesis of collagen, elastin, fibronectin, and/or laminin.
In some embodiments, the nutrient-deficient environment is deficient in one or more amino acids. In some embodiments, the nutrient-deficient environment is in vitro. In some embodiments, the nutrient-deficient environment is in vivo. In some embodiments, the nutrient-deficient environment comprises a subject on a restrictive diet.
In some embodiments, the cell contacted with the antagonist has reduced survival and/or proliferation compared to the control cell not contacted with the antagonist. In some embodiments, the cell contacted with the antagonist expresses pyrroline-5-carboxylate synthase (P5CS). In some embodiments, the cell contacted with the antagonist is associated with a fibrotic disorder. In some embodiments, the cell contacted with the antagonist expresses increased levels of NADK2 compared to a cell not associated with a fibrotic disorder. In some embodiments, the fibrotic disorder is pulmonary fibrosis or liver fibrosis. In some embodiments, the cell contacted with the antagonist expresses increased levels of P5CS compared to a cell not associated with a fibrotic disorder.
The details of certain embodiments of the invention are set forth in the Detailed Description, as described below. Other features, objects, and advantages of the invention will be apparent from the Examples, Drawings, and Claims.
The accompanying drawings, which constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.
Aspects of the present disclosure relate to the discovery that NADPH produced by NADK2 is required for proline biosynthesis, cytosolic protein synthesis, and cell proliferation in a nutrient-deficient environment. Cells contacted with an antagonist of NADK2 in a nutrient-deficient environment will have reduced proliferation due to decreased proline biosynthesis. Thus, methods and compositions provided herein may be used to treat disorders (e.g., cancer, fibrotic disorder) by inhibiting cell proliferation and cytosolic protein synthesis.
In some aspects, methods provided in the present disclosure are drawn to treating a disease or disorder by administering to a subject in need thereof an antagonist of nicotinamide adenine dinucleotide kinase 2.
In some aspects, methods and compositions provided in the present disclosure comprise an antagonist of nicotinamide adenine dinucleotide kinase 2 (NADK2). NADK2 is a mitochondrial enzyme that phosphorylates nicotinamide adenine dinucleotide (NAD+) to produce NADP+. NAD+ and NADH and NADP+ and NADPH may be used interchangeably herein. Because NADP+ is membrane impermeable, mitochondrial NADP+ is separate from cytosolic NADP+ produced by nicotinamide adenine dinucleotide kinase 1 (NADK1). As demonstrated herein, NADP+ produced from NADK2 is required for cell proliferation, proline biosynthesis, and cytosolic protein synthesis. Thus, antagonizing the activity of NADK2 (e.g., with an NADK2 antagonist) is an effective strategy for inhibiting cell proliferation, proline biosynthesis, and cytosolic protein synthesis.
NADK2 herein may be NADK2 expressed in any organism known in the art. NADK2 is conserved in human (Gene ID: 133686), mouse (Gene ID: 68646), rat (Gene ID: 365699), frog (Gene ID: 780144), non-human primates (Gene IDs: 704285, 461919), cow (Gene ID: 506968), zebrafish (Gene ID: 445071), chicken (Gene ID: 417438), dog (Gene ID: 612569), hamster (Gene ID: 101837077), horse (Gene ID: 100067696) and fish (Gene IDs: 108279376, 108900730, 109868343). In some embodiments, NADK2 is human NADK2.
Human NADK2 may be any human NADK2 sequence known in the art. Human NADK2 is alternatively spliced to produce 3 different isoforms. Human NADK2 isoform 1 (Q4G0N4-1) is 442 amino acids in length and is considered full-length. Human NADK2 isoform 2 (Q4G0N4-2) is 410 amino acids in length and is missing amino acids 288-319 from the NADK2 isoform 1 sequence. Human NADK2 isoform 3 (Q4G0N4-3) is 279 amino acids in length and is missing amino acids 1-163 from the NADK2 isoform 1 sequence.
In some embodiments, an antagonist of NADK2 is administered to a subject in need thereof. An antagonist is a compound or molecule that inhibits the activity of a protein. An antagonist of NADK2 may decrease NADK2 activity by 10%-100%, 20%-90%, 30%-80%, 40%-70%, or 50%-60%. In some embodiments, an antagonist of NADK2 may decrease NADK2 activity by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
An antagonist of NADK2 inhibits the activity of NADK2 directly or indirectly. A direct antagonist of NADK2 binds to NADK2 protein and inhibits its catalytic activity (e.g., by blocking the enzyme active site). An indirect antagonist of NADK2 inhibits the production of NADK2 protein (e.g., NADK2 transcription, NADK2 translation).
An antagonist of NADK2 may be any NADK2 antagonist known in the art (see, e.g., WO 2016/170348). Non-limiting examples of potential NADK2 antagonists include small organic compounds having a molecular weight of less than about 1,000 g/mol; nucleotide compounds including a guide RNA used in a clustered regularly interspaced short palindromic repeats (CRISPR/Cas) genome editing system, an antisense oligonucleotide, a ribozyme, a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), a short (or small) activating RNA (saRNA) or a combination thereof; an anti-NADK2 antibody; and an anti-NADK2 nucleic acid aptamer.
In some embodiments, an antagonist of NADK2 is a guide RNA (gRNA) used in a CRISPR/Cas genome editing system. CRISPR/Cas genome editing is well-known in the art (see, e.g., Wang et al., Ann. Rev. Biochem., 2016, 85: 227-264; Pickar-Oliver and Gersbach, Nature Reviews Molecular Cellular Biology, 2019, 20: 490-507; Aldi, Nature Communications, 2018, 9: 1911). In some embodiments, a gRNA antagonist of NADK2 knocks out (removes) NADK2 from the genome, decreases expression of NADK2 from the gnome, decreases NADK2 enzyme activity, or a combination thereof. A gRNA antagonist of NADK2 may be 1-10, 2-9, 3-8, 4-7, or 5-6 gRNAs. In some embodiments, a gRNA antagonist of NADK2 may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more gRNAs.
A subject in need thereof may be administered one antagonist of NADK2 or multiple antagonists of NADK2. When multiple antagonists of NADK2 are administered, the multiple antagonists may have the same mechanism of action (e.g., inhibiting NADK2 expression, inhibiting NADK2 enzymatic activity), different mechanisms of action, or a combination thereof. In some embodiments, 1-10, 2-9, 3-8, 4-7, or 5-6 antagonists of NADK2 are administered to a subject in need thereof. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more antagonists of NADK2 are administered to a subject in need thereof. When multiple antagonists of NADK2 are administered to a subject, they may be administered in the same administration or in multiple administrations.
In some aspects, the present disclosure provides a method of treating a cancer. Treating a cancer may be killing cancer cells, inhibiting the proliferation of cancer cells, inhibiting the growth of cancer cells, inhibiting the metastasis of cancer cells, or any other measure of treating cancer known in the art. A cancer treated with a method provided herein may be a primary cancer or a secondary cancer. A primary cancer is a cancer that is confined to the original location where the cancer began (e.g., breast, colon, etc.), and a secondary cancer is a cancer that originated in a different location and metastasized. A cancer treated with a method provided herein may be a first occurrence of the cancer or may be a subsequent occurrence of the cancer (relapsed or recurrent cancer).
In some embodiments, a method provided herein includes treating a cancer characterized as having an isocitrate dehydrogenase 2 (IDH2) mutation. Characterized as having means that a mutation (e.g., IDH2 mutation) has been detected in the cancer. IDH2 is a mitochondrial enzyme produced by expression of the IDH2 gene. IDH2 catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate (αKG, also known as 2-oxoglutarate) as part of the tricarboxylic acid (TCA) cycle that produces energy in the form of adenine trinucleotide phosphate (ATP). Because αKG is membrane impermeable, mitochondrial αKG is separate from cytosolic αKG produced by isocitrate dehydrogenase 1 (IDH1).
IDH2 herein may be IDH2 from any organism known in the art. IDH2 is expressed in human (Gene ID: 3418), mouse (Gene ID: 269951), rat (Gene ID: 361596), pig (Gene ID: 397603), frog (Gene ID: 448026), non-human primates (Gene IDs: 701480, 453645), cow (Gene ID: 327669), zebrafish (Gene ID: 386951), chicken (Gene ID: 431056), dog (Gene ID: 479043), and fish (Gene IDs: 100194639, 100304677, 105025672). In some embodiments, IDH2 is human IDH2.
Human IDH2 may be any human IDH2 sequence known in the art. Human IDH2 is alternatively spliced to produce 2 different isoforms. Human IDH2 isoform 1 (P48735-1) is 452 amino acids in length and is considered full-length. Human IDH2 isoform 2 (P48735-2) is 400 amino acids in length and is missing amino acids 1-52 from the IDH2 isoform 1 sequence.
An IDH2 mutation may be any mutation known in the art that is associated with cancer. Associated with cancer means that an IDH2 mutation has been detected in a cancer cell. IDH2 is mutated in 1.39% of all cancers, with acute myeloid leukemia, breast invasive ductal carcinoma, colon adenocarcinoma, lung adenocarcinoma, and oligodendroglioma having the greatest prevalence of IDH2 mutations (31).
An IDH2 mutation may be a gain-of-function mutation or a loss-of-function mutation. A gain-of-function IDH2 mutation is a mutation that confers a stronger (e.g., higher activity, more constitutive activity, etc.) enzymatic function or an additional enzymatic function to an IDH2 protein compared to wild-type IDH2. A loss-of-function IDH2 mutation is a mutation that confers a weaker (e.g., lower activity, less constitutive activity, etc.) enzymatic activity or losing an enzymatic function that is expressed compared to wild-type IDH2.
An IDH2 mutation may be any mutation known in the art. Non-limiting examples of IDH2 mutations include R172S, exon 4 mutation, a codon 140 missense mutation, R140Q, a codon 172 missense mutation, R172K, an amplification of IDH2, a loss of IDH2, R172W, R172M, R140W. R172G, V305M, H384Q, T350P, R172T, V355I, K155N, A416V, W21S, X39 splice, R159H, A347T, D390Y, D259N, A370T, and A174T.
In some embodiments, a cancer characterized as having an IDH2 mutation has a combination of IDH2 mutations known in the art. In some embodiments, a cancer characterized as having an IDH2 mutation has 1-10, 2-9, 3-8, 4-7, or 5-6 mutations. In some embodiments, a cancer characterized as having an IDH2 mutation has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations.
In some embodiments, an IDH2 mutation produces a mutant IDH2 protein having a neomorphic activity. A neomorphic activity is an enzymatic function that the mutant IDH2 protein possesses and does not normally have or has at a higher level than a wild-type protein. Mutations in IDH2 may contribute to cancer through production of 2-hydroxyglutarate (2HG) from αKG. Thus, in some embodiments, mutations in IDH2 that confer a neomorphic (e.g., gain-of-function) activity to the IDH2 enzyme produce increased levels of 2HG compared to wild-type IDH2 enzyme (32). Therefore, in some embodiments, a cancer that has an IDH2 mutation has increased levels of 2HG relative to a reference value. In some embodiments, a cancer that has an IDH2 mutation has decreased levels of αKG relative to a reference value. Levels of 2HG and αKG may be measured by any method known in the art. Non-limiting examples of methods for measuring levels of 2HG and αKG include: gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), colorimetric assay, and fluorometric assays.
A reference value may be from a cell characterized as not having an IDH2 mutation, a non-cancerous cell, or a cell that is not contacted with an antagonist of NADK2. A non-cancerous cell is a cell that does not possess a mutation associated with cancer. A mutation associated with cancer may be any mutation known in the art to occur in cancer cells.
In some embodiments, a cancer provided herein is characterized as not having an isocitrate dehydrogenase (IDH1) mutation. IDH1 catalyzes the oxidative decarboxylation of isocitrate to αKG in the cytosol of a cell as part of the TCA cycle that produces energy in the form of ATP.
In some embodiments, a cancer treated with a method provided herein is an adenocarcinoma. An adenocarcinoma is a cancer that forms in epithelial cells that produce fluids or mucus. An adenocarcinoma may be any adenocarcinoma known in the art. Non-limiting examples of adenocarcinomas include colon adenocarcinoma, lung adenocarcinoma, high grade ovarian serous adenocarcinoma, colorectal adenocarcinoma, rectal adenocarcinoma, prostate adenocarcinoma, breast adenocarcinoma, or a combination thereof.
In some embodiments, a cancer treated with a method provided herein is a carcinoma. Carcinoma is the most common type of cancer and is formed by epithelial cells. A carcinoma may be any carcinoma known in the art. Non-limiting examples of carcinoma include: breast invasive ductal carcinoma, intrahepatic cholangiocarcinoma, endometrial endometrioid carcinoma, bladder urothelial carcinoma, endometrial carcinoma, squamous cell lung carcinoma, or a combination thereof.
In some embodiments, a cancer is selected from acute myeloid leukemia, oligodendroglioma, myelodysplastic syndrome, cutaneous melanoma, glioblastoma multiforme, angioimmunoblastic T-cell lymphoma, acute monoblastic and monocytic leukemia, or a combination thereof.
In some aspects, the present disclosure provides a method of treating a fibrotic disorder by administering to a subject in need thereof an antagonist of NADK2 in an amount effective to treat the fibrotic disorder. A fibrotic disorder is a disorder in which extracellular matrix molecules uncontrollably and progressively accumulate in affected tissues and organs, causing their ultimate failure. Fibrosis is a predominant feature of the pathology of a wide range of diseases across numerous organ systems, and fibrotic disorders are estimated to contribute to up to 45% of all-cause mortality in the United States. Despite this prevalence of fibrotic disorders, effective therapies are limited.
In some embodiments, a fibrotic disorder that is treated with a method provided herein is characterized by increased levels of an extracellular matrix (ECM) protein. An ECM protein is a protein in a three-dimensional network of extracellular macromolecules and minerals that exists between cells. An ECM protein herein may be any ECM protein known the in art. Non-limiting examples of ECM proteins include: collagen, elastin, fibronectin, and laminin. More than one ECM protein may also have increased levels in a fibrotic disorder treated herein. In some embodiments, a fibrotic disorder is characterized by increased levels of 1-10, 2-9, 3-8, 4-7, or 5-6 ECM proteins. In some embodiments, a fibrotic disorder is characterized by increased levels of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more ECM proteins.
In some embodiments, a fibrotic disorder that is treated with a method provided herein is characterized by increased levels of a collagen protein. Collagens are the most abundant protein in the ECM and the human body. Collagen is produced in cells and exocytosed in precursor form (procollagen) which is then cleaved and assembled into mature collagen extracellular. Collagen proteins may be divided into several families based on the types of structures that they form, including, but not limited to: fibrillar (Types I, II, III, V, and XI collagens), facit (Types IX, XII, and XIV collagens), short chain (Types VIII and X collagens), basement membrane (Type IV), and other structures (Types VI, VII, and XIII).
Extracellular matrix proteins require amino acids, such as proline, that confer structural rigidity to fold into and maintain the proper architecture. In addition to its role in promoting cell proliferation discussed above, NADP+ produced by NADK2 is also required for proline biosynthesis in a nutrient-deficient environment. A nutrient-deficient environment lacks sufficient levels of one or more nutrients to allow cellular processes (e.g., cell proliferation, protein synthesis, proline biosynthesis). Proline is produced by the conversion of glutamate to pyrroline-5-carboxylate (P5C) by pyrroline-5-carboxylate synthase (P5CS), which requires NADPH produced by NADK2. P5C is further reduced to proline by mitochondrial pyrroline-5-carobxylate reductases (PYCR1 and PYCR2). Thus, contacting NADK2 with an antagonist reduces proline biosynthesis in a nutrient-deficient environment by inhibiting the conversion of glutamate to P5C.
As described above, NADK2 and P5CS are required for proline biosynthesis and fibrosis in a nutrient-deficient environment. Thus, in some embodiments, a fibrotic disorder treated with a method provided herein is characterized by increased levels of NADK2, increased levels of P5CS, or increased levels of NADK2 and increased levels of P5CS relative to a known reference value.
A reference value may be a normal cell, a cell that is not contacted with an antagonist of NADK2, or a cell in a nutrient-replete environment. A normal cell is a cell that is not associated with fibrosis and does not have an increased level of NADK2, P5CS, or NADK2 and P5CS. A nutrient-replete environment has sufficient levels of one or more nutrients to allow cellular processes (e.g., cell proliferation, protein synthesis, proline biosynthesis).
A fibrotic disorder may be any fibrotic disorder known in the art. Non-limiting examples of fibrotic disorders include: idiopathic pulmonary fibrosis (IPF), hepatic fibrosis, systemic sclerosis, sclerodermatous graft vs. host disease, nephrogenic systemic fibrosis, radiation-induced fibrosis, cardiac fibrosis, kidney fibrosis, or a combination thereof. Treating a fibrotic disorder may mean decreased proline synthesis, decreased synthesis of ECM proteins, decreased deposition of ECM proteins, reduction of existing depositions of ECM proteins, or a combination thereof.
Proline synthesis may be measured by any method known in the art including, but not limited to: isotopologue labeling followed by GC-MS quantification, isotopologue labeling following by LC-MS quantification, ninhydrin staining, and colorimetric assays. Any isotopologue known in the art may be used in methods of quantifying proline, including but not limited to: [13C], [16O], [17O], [18O], [2H], [15N], [2,3,3-2H3]serine, [U-13C], [U-16O], [U-17O], [U-18O], [U-2H], and [U-15N].
Extracellular matrix protein may be measured by any method known in the art including, but not limited to: protein staining, isobaric demethylated leucine (DiLeu) labeling and quantification, mass spectrometry, reversed phase liquid chromatography, second harmonic generation (SHG) microscopy, and strong cation exchange chromatography. In some embodiments, ECM proteins are measured by protein staining. Non-limiting examples of protein staining of ECM proteins include: Picrosirius Red staining. Masson's Trichrome staining, and hematoxylin and eosin staining.
Methods provided herein may be used to treat a subject in need thereof. A subject in need thereof may have any disease or disorder provided herein including, but not limited to, a cancer (e.g., adenocarcinoma, carcinoma, leukemia, glioma) and a fibrotic disease (e.g., pulmonary fibrosis, liver fibrosis, kidney fibrosis). A subject may have one or more diseases or disorders provided herein. In some embodiments, a subject has 1-10 diseases or disorders, 2-9 diseases or disorders, 3-8 diseases or disorders, 4-7 diseases or disorders, or 5-6 diseases or disorders. In some embodiments, a subject has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more diseases or disorders provided herein.
In some embodiments, a subject is administered an effective amount of an antagonist of NADK2 to treat a disease or disorder. An effective amount of an antagonist of NADK2 is any amount that decreases cell proliferation, decreases cell survival, decreases protein synthesis, decreases proline biosynthesis, decreases ECM protein deposition, decreases fibrosis, or a combination thereof.
An effective amount of an antagonist of NADK2 will vary based on factors that are known to a person skilled in the art, including, but not limited to: age of a subject, height of a subject, weight of a subject, pre-existing conditions, stage of a disease or disorder, other treatments or medications that a subject is being administered, or a combination thereof. In some embodiments, an effective amount of an antagonist of NADK2 is 1 μg/kg-1,000 mg/kg, 10 μg/kg-100 mg/kg, 100 μg/kg-10 mg/kg, or 500 μg/kg-1 mg/kg. In some embodiments, an effective amount of an antagonist of NADK2 is 1 μg/kg, 10 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg, 200 μg/kg, 250 μg/kg, 300 μg/kg, 350 μg/kg, 400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg, 600 μg/kg, 650 μg/kg, 700 μg/kg, 750 μg/kg, 800 μg/kg, 850 μg/kg, 900 μg/kg, 950 μg/kg, 1 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 μg mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, or 1,000 mg/kg.
In some embodiments, a subject is a vertebrate. A vertebrate may be any vertebrate known in the art including, but not limited to: a human, a rodent (e.g., mouse, rat, hamster), a non-human primate (e.g., Rhesus monkey, chimpanzee, orangutan), a pet (e.g., dog, cat, ferret), a livestock animal (e.g., pig, cow, sheep, chicken), or a fish (zebrafish, catfish, perch).
An antagonist of NADK2 may be administered to a subject by any method known in the art. Non-limiting examples of methods for administering an antagonist of NADK2 include: injection (e.g., intravenous, intramuscular, intraarterial), inhalation (e.g., by nebulizer, by inhaler), ingestion (e.g., oral, rectal, vaginal), sublingual or buccal dissolution, ocular placement, otic placement, and absorbed through skin (e.g., cutaneously, transdermally).
Methods provided herein may be used in vitro (e.g., in a cultured cell) or in vivo (e.g., in a subject) to antagonize NADK2. Because NADK2 is required for proline biosynthesis, cytosolic protein synthesis, and cell proliferation in a nutrient-deficient environment, methods provided herein may be used to inhibit protein synthesis and cell proliferation in vitro or in vivo.
As described above, NADK2 is required for proline biosynthesis in nutrient-deficient environments. Proline that is produced in mitochondria is utilized in protein synthesis, particularly for proteins that require structural rigidity and specific conformations (e.g., ECM proteins). Thus, in some aspects, methods provided herein may be used to inhibit protein synthesis. These methods may be used to inhibit protein synthesis in vitro (e.g., in cell culture) or in vivo (e.g., in a subject).
When methods provided herein for inhibiting protein synthesis are in vivo in a subject in need thereof, they may be used to treat a disease or disorder associated with increased or aberrant protein synthesis. Aberrant protein synthesis may be synthesis of mutant protein, synthesis of a pathologic protein, or a combination thereof. A pathologic protein may be a protein that malfunctioned protein folding (compared to its wild-type counterpart).
In some embodiments, when methods provided herein for inhibiting protein synthesis are in vivo in a subject in need thereof, the subject is on a restrictive diet. A restrictive diet decreases and/or increases the consumption of specific foods or limits nutrient intake to a certain number of calories (also known as kilocalories). Non-limiting examples of foods that may be decreased on a restrictive diet include refined grains (e.g., fried rice, granola, biscuits, sweet rolls, muffins, scones, coffee bread, doughnuts, cheese bread), sweets (e.g., cookies, cakes, candy, ice cream), snacks (e.g., chips, pretzels, crackers), certain proteins (e.g., duck, goose, bacon, sausage, hot dogs, cold cuts, nuts, nut butters), dairy (e.g., whole milk, cream, whole milk yogurt, whole milk cheese), beverages (e.g., alcohol, carbonated beverages with sugar, juices with added sugar), or any combination thereof. Non-limiting examples of foods that may be increased on a restrictive diet include fruits (e.g., berries, apples, citrus), vegetables (e.g., green beans, peas, carrots, lettuce, cabbage), whole grains (e.g., rice, popcorn, bread, pasta, cereal), natural sweeteners (e.g., honey, agave syrup, maple syrup), lean proteins (e.g., chicken, turkey, fish, beans, beans, legumes, eggs), dairy (e.g., reduced fat or non-fat milk, reduced fat or non-fat cheese, reduced fat or non-fat yogurt), beverages (e.g., coffee, tea, water), or some combination thereof. Non-limiting examples of certain numbers of calories that may be consumed daily on a restrictive diet include: 800 calories-1900 calories, 900 calories-1800 calories, 1000 calories-1700 calories, 1100 calories-1600 calories, 1200 calories-1500 calories, 1300 calories-1400 calories. A restrictive diet may be any restrictive diet known in the art including, but not limited to: 5:2 diet, Body for Life, cookie diet, The Hacker's Diet, Nurtisystem® diet, Weight Watchers® diet, inedia, KE diet, Atkins® diet, Dukan diet, South Beach Diet®, Stillman diet, Beverly Hills® diet, cabbage soup diet, grapefruit diet, monotrophic diet, Subway® diet, juice fasting, Master Cleanse®, DASH diet, diabetic diet, elemental diet, ketogenic diet, liquid diet, low-FODMAP diet, vegetarian diet, pescatarian diet, vegan diet, and soft diet.
Any disease or disorder associated with increased or aberrant protein synthesis known in the art may be treated with methods provided herein. Non-limited examples of diseases or disorders associated with increased or aberrant protein synthesis include: fibrosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, cystic fibrosis, Gaucher's disease, amyloidosis, multiple system atrophy, and prion diseases (e.g., kuru, fatal familial insomnia, Creutzfeldt-Jakob Disease (CJD), variant Creutzfeldt-Jakob Disease (vCJD)).
Cellular protein synthesis may be measured by any method known in the art. Non-limiting examples of measuring protein synthesis include: radioactive isotope labeling (e.g., 3H-phenylalanine, 35S-methionine), stable isotope labeling (e.g., 15N-lysine, 13C-leucine, ring-13C6-phenylalanine), puromycin Surface Sensing of Translation (SUnSET) labeling, Western blot, GC-MS, LC-MS, and protein staining.
As described above, NADK2 is required for cell proliferation in a nutrient-deficient environment (e.g., nutrient-deficient cell culture media). Thus, in some aspects, methods provided herein may be used to inhibit cell proliferation. These methods may be used to inhibit cell proliferation in vitro (e.g., in cell culture) or in vivo (e.g., in a subject).
When methods provided herein for inhibiting cell proliferation are in vivo in a subject in need thereof, they may be used to treat a disease or disorder associated with increased cell proliferation. Any disease or disorder associated with increased cell proliferation known in the art may be treated with methods provided herein. Non-limiting examples of diseases or disorders associated with increased cell proliferation include: cancer, ataxia telangiectasia, xeroderma pigmentosum, autoimmune lymphoproliferative syndrome (types I and II), systemic lupus erythematosus, polycythemia vera, familial hemophagocytic lymphohistiocytosis, Niemann-Pick disease, osteoporosis, adenovirus infection, baculovirus infection, Epstein-Barr virus infection, Herpes virus infection, poxvirus infection, Down's syndrome, progeria, and atherosclerosis.
Cell proliferation may be an increase in cell metabolites or an increase in cell numbers. Cell proliferation may be measured or monitored by any method known in the art. Non-limiting methods of cell proliferation include: bromodeoxyuridine (BrdU) incorporation, 5-Ethynyl-2′-deoxyuridine (EdU) incorporation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide (MTT) salt cleavage, (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) (XTT) salt cleavage, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) salt cleavage, (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) (WST-8) salt cleavage, and Ki67 nuclear protein antibody labeling.
The present disclosure demonstrates that NADK2 is required for proline biosynthesis and cell proliferation in a nutrient-deficient environment, including a nutrient-deficient cell culture medium. Cells contacted with an antagonist of NADK2 in nutrient-deficient cell culture medium will have reduced proliferation due to decreased proline biosynthesis. Thus, in some aspects, the present disclosure provides a composition comprising (i) nutrient-deficient cell culture medium; and (ii) an antagonist of NADK2. This composition may be used in methods of treating a subject having a disease or disorder (e.g., cancer, fibrotic disorder).
Nutrient-deficient cell culture medium is cell culture medium deficient in one or more nutrients required for cellular processes, including but not limited to: amino acids, vitamins, and ions. Deficient in one or more amino acids means that the cell culture medium does not contain sufficient levels of one or more amino acids to support cellular processes. The cellular processes that are not supported in nutrient-deficient cell culture medium may be cell proliferation, survival, proline biosynthesis, ECM protein, ECM deposition, or a combination thereof.
Nutrient-deficient cell culture medium may be deficient in any amino acid including, but not limited to, arginine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or any combination thereof. In some embodiments, nutrient-deficient cell culture medium is deficient in 1-20, 2-19, 3-18, 4-17, 5-16, 6-15, 7-14, 8-13, 9-12, or 10-11 amino acids. In some embodiments, nutrient-deficient cell culture medium is deficient in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, nutrient-deficient cell culture medium is deficient in proline.
In some embodiments, a composition provided herein further comprises a population of cells. A population of cells may be a homogeneous population composed of the same cell type or a heterogenous population composed of a mixture of cell types. A population of cells may be in vitro (e.g., in cell culture medium) or in vivo (e.g., in a subject). In some embodiments, a population of cells is obtained from a subject and maintained in vitro (e.g., in cell culture medium).
A population of cells may contain any number of cells including, but not limited to: 5 cells-100 cells, 50 cells-500 cells, 250 cells-1,000 cells, 500 cells-10,000 cells, 5,000 cells-100,000 cells, 50,000 cells-1,000,000 cells, 500,000 cells-10,000,000 cells, 1,000,000-1,000,000,000 cells, 5,000,000 cells-10,000,000,000 cells or more.
In some embodiments, the population of cells comprises cancer cells. The cancer cells may be derived from any cancer provided herein or a combination of cancers provided herein. In some embodiments, a population of cancer cells express a mutant IDH2 protein. A mutant IDH2 protein may be any mutant IDH2 protein provided herein.
In some embodiments, a mutant IDH2 protein in a cancer cell population provided herein has a neomorphic enzymatic activity. In some embodiments, the neomorphic enzymatic activity is a reduction of αKG to 2HG. Thus, in some embodiments, a cancer cell population expressing a mutant IDH2 protein having a neomorphic activity contains increased levels of 2HG relative to a known reference value. In some embodiments, a cancer cell population expressing a mutant IDH2 protein having a neomorphic activity contains reduced levels of 2HG relative to a known reference value.
A nutrient-deficient cell culture medium provided herein may contain one or more additives. Additives are exogenous compounds that are added to a nutrient-deficient medium. An additive may be any compound known in the art to be added to cell medium. Non-limiting examples of classes of compounds that are added to cell medium include: antibiotics (e.g., streptomycin, penicillin, ampicillin, kanamycin), serum (e.g., bovine serum albumin, human serum albumin, fetal bovine serum), amino acids (e.g., arginine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine), inorganic salt (e.g., ammonium molybdate, ammonium metavandate, calcium chloride, cupric sulfate, ferric nitrate, ferrous sulfate, manganese sulfate, magnesium chloride, magnesium sulfate, nickel chloride, potassium chloride, sodium metasilicate, sodium selenite, sodium phosphate dibasic, sodium phosphate monobasic, stannous chloride, zinc sulfate), vitamins (e.g., biotin, choline chloride, folic acid, myo-inositol, niacinamide, pantothenic acid, pyridoxal, pyridoxine, riboflavin, thiamine, vitamin B12), buffers (e.g., glucose, HEPES, hypoxanthine, linoleic acid, Phenol Red, putrescine, pyruvic acid, thioctic acid, thymidine, sodium bicarbonate).
In some embodiments, nutrient-deficient cell culture medium contains serum, penicillin, and streptomycin. The concentration of serum, penicillin, and streptomycin may be any concentration in cell culture medium known in the art. In some embodiments, nutrient-deficient cell culture medium contains 1%-30%, 2%-29%, 3%-28%, 4%-27%, 5%-26%, 6%-25%, 7%-24%, 8%-23%, 9%-22%, 10%-21%, 11%-20%, 12%-19%, 13%-18%, 14%-17%, or 15%-16% serum. In some embodiments, nutrient-deficient cell culture medium contains 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% serum. In some embodiments, nutrient-deficient cell culture medium contains 10 units/mL-150 units/mL, 20 units/mL-140 units/mL, 30 units/mL-130 units/mL, 40 units/mL-120 units/mL, 50 units/mL-110 units/mL, 60 units/mL-100 units/mL, or 70 units/mL-90 units/mL penicillin. In some embodiments, nutrient-deficient cell culture medium contains 10 units/mL, 20 units/mL, 30 units/mL, 40 units/mL, 50 units/mL, 60 units/mL, 70 units/mL, 80 units/mL, 90 units/mL, 100 units/mL, 110 units/mL, 120 units/mL, 130 units/mL, 140 units/mL, or 150 units/mL penicillin. In some embodiments, nutrient-deficient cell culture medium contains 10 μg/mL-150 μg/mL, 20 μg/mL 140 μg/mL, 30 μg/mL-130 μg/mL, 40 μg/mL-120 μg/mL, 50 μg/mL-110 μg/mL, 60 μg/mL-100 μg/mL, or 70 μg/mL-90 μg/mL streptomycin. In some embodiments, nutrient-deficient cell culture medium contains 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, 100 μg/mL, 110 μg/mL, 120 μg/mL, 130 μg/mL, 140 μg/mL, or 150 μg/mL streptomycin.
The data in this Example demonstrates that NADK2 is required to maintain mitochondrial NADPH and mitochondrial 2-hydroxyglutrate (2-HG) in cells expressing mutant IDH2.
Mammalian cells depend on the inter-conversion of nicotinamide adenine dinucleotide phosphate (NADP) molecules between the oxidized (NADP+) and reduced (NADPH) forms to support reductive biosynthesis and to maintain cellular antioxidant defense. NADP+ and NADPH molecules (also referred to as “NADP(H)”) are unable to cross subcellular membranes (1, 2). As a result, cellular pools of NADP(H) are compartmentalized. In the cytosol, NADP(H) is derived from nicotinamide adenine dinucleotide [(NAD)H] by NAD kinase 1 (NADK1). Cytosolic NADPH acts as a substrate in fatty acid biosynthesis, and as the reducing equivalent required to regenerate reduced glutathione (GSH) and thioredoxin for antioxidant defense. Mitochondria host a number of biosynthetic activities critical for cellular metabolism but are also major sites for reactive oxygen species (ROS) generation. Mammalian mitochondrial NAD kinase 2 (NADK2) converts NAD(H) to NADP(H) through phosphorylation (3).
Using subcellular fractionation, it was confirmed that NADK2 purified in the membrane-associated fraction in cultured human cell lines (
1Detected in whole cell samples, but not in Mito-IP samples
2Detected in Mito-IP samples, and sgCtrl OMP25HA Mito-IP signal is more than 1.5-fold higher than OMP25Myc Mito-IP signal
3Detected in Mito-IP samples, and sgCtrl OMP25HA Mito-IP signal is less than 1.5-fold higher than OMP25Myc Mito-IP signal
Oncogenic mutant forms of isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) require cytosolic and mitochondrial NADPH, respectively, to produce 2-hydroxyglutarate (2HG) from α-ketoglutarate (αKG) (8) (
Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and MTHFD2-like (MTHFD2L) use either NAD+ or NADP+as electron acceptors in the mitochondrial folate pathway. Using [2,3,3-2H3]serine isotope tracing, cells lacking MTHFD2 or serine hydroxymethyltransferase 2 (SHMT2) both displayed an increase in doubly-labeled thymidine triphosphate (TTP M+2) when compared to control cells (
Isotope tracing experiments were performed with uniformly labeled [U-13C]glucose or [U-13C]glutamine comparing control and NADK2-deleted cells to analyze tricarboxylic acid (TCA) cycle activity. Consistent changes were not observed in the TCA cycle intermediates derived from either glucose or glutamine (
Mitochondria are major sites of reactive oxygen species (ROS) generation in cells (11), and depletion of mitochondrial NADP(H) is thought to lead to oxidative stress. However, in all cell types that were tested, cells lacking NADK2 did not display increased cellular ROS or mitochondrial superoxide (MitoSox) abundance (
Hyper-oxidation of peroxiredoxins (PRXs-SO3) indicates oxidative stress of the cellular thioredoxin system. Similar amounts of mitochondrial peroxiredoxin (PRX3) were observed, as well as cytosolic (PRX1) and nuclear (PRX2) peroxiredoxin oxidation, when comparing cells lacking NADK2 with control cells (
Proliferation of cells lacking NADK2 was not perturbed compared to that of control cells when cultured in a nutrient rich medium (DMEM/F12) (
The data in this Example demonstrates that NADK2 is required to maintain mitochondrial proline biosynthesis. NADK2 knock-out cells were shown to have decreased mitochondrial proline biosynthesis and decreased collagen production and deposition.
Growth of cells lacking NADK2 was restored in DMEM by supplementing non-essential amino acids (NEAAs), but not by other nutrients present in DMEM/F12 media (
Metabolite profiling was performed on cells lacking NADK2 cultured in DMEM, and confirmed the depletion of intracellular proline, while amounts of many other amino acids were slightly increased (
Proline biosynthesis takes place in the mitochondria, where glutamine-derived glutamate is converted to pyrroline-5-carboxylate (P5C) by pyrroline-5-carboxylate synthase (P5CS). P5C is further reduced to proline by mitochondrial pyrroline-5-carboxylate reductases (PYCR1 and PYCR2) (
P5CS is an NADPH-dependent enzyme, whereas PYCR1 and PYCR2 have higher affinity for NADH than for NADPH (19-21). To test if loss of NADK2 impairs conversion of glutamate to P5C by P5CS, the fact that cellular P5C is in equilibrium with glutamate-5-semialdhyde (GSA), which can be diverted to produce ornithine for polyamine biosynthesis was exploited (
Incorporation of the proline pyrrolidine ring slows protein translation (22, 23), but endows proline-containing polypeptides with conformational rigidity. As a result, proline and its post-translationally modified form, hydroxyproline, are abundant in collagen proteins (24), so a consequence of decreased mitochondrial NADP(H) generation could be impaired collagen production. Cultured MEFs lacking NADK2 had decreased expression of collagen when grown in DMEM (
Additionally, PC5S deletion diminished expression of collagen protein both in untreated and TGFβ-treated cells, which was restored by addition of proline to the culture medium (
To test whether P5CS expression could also be relevant for fibrotic diseases that are characterized by excessive collagen deposition in idiopathic pulmonary fibrosis (IPF), PC5S expression was analyzed in publicly available gene expression datasets from lungs of mice treated with bleomycin to induce pulmonary fibrosis or from lungs of IPF patients. P5CS was significantly upregulated in the bleomycin mouse model of pulmonary fibrosis (
Next, the role of fibroblast pyruvate carboxylase (PC) and glutamine synthetase (GluI) in maintaining collagen levels and tumor growth in vivo was investigated. Pyruvate carboxylase converts pyruvate to oxaloacetate, a tricarboxylic acid cycle intermediate that is required to produce isocitrate, which is converted to alpha ketoglutarate (αKG) in mitochondria by IDH2. Glutamine synthetase converts glutamate to αKG in mitochondria. Low numbers of pancreatic ductal adenocarcinoma (KPC) cells were injected subcutaneously into the flanks of nude mice, cither alone or with pancreatic stellate cells (PSCs) expressing either a control, PC, or GluI single guide RNA (sgControl, sgPC, sgGluI) (
The ability of fibroblast pyruvate carboxylase (PC) to regulate tumor growth and collagen content was also investigated. Specifically, the possibility that the growth of DB7 murine mammary tumors could be supported by matrix proteins such as collagen secreted by primary mammary fibroblasts (MFB) was tested. Consistent with this, co-injection of MFBs substantially increased the collagen content of DB7 allograft tumors after engraftment, as measured by the levels of hydroxyproline in tumor acid hydrosylates and by Western blot (
These findings provide insights into the regulation of intracellular metabolism. In endosymbiosis with the host cell, mitochondria produce NADP(H) that supplies biosynthetic precursors to their host and appear not to use the NADP(H) for antioxidant defense in support of their own homeostasis. Compartmentalization of cellular metabolism thus has important roles in cukaryotic cells beyond the well-known collaborative production of ATP.
Antibodies (commercial source, catalog number, detected molecular weight) used in this study were: Tubulin (Sigma, T9026, 50 kD), CS (Cell Signaling Technology, 14309, 45 kD), NADK2 (Abcam, ab181028, 45 kD), COX IV (Cell Signaling Technology, 4850T, 17 kD), Lamin A/C (Cell Signaling Technology, 4777, 75 kD and 65 kD), H3 (Abcam, ab1791, 17 kD), Vinculin (Sigma, V9131, 120 kD), CAT (Cell Signaling Technology, 12980, 60 kD), GOLGA1 (Cell Signaling Technology, 13192, 100 kD), CALR (Cell Signaling Technology, 12238, 55 kD), LAMP2 (Santa Cruz Biotechnology, sc-18822, 120 kD), CTSC (Santa Cruz Biotechnology, sc-74590, 25 kD), PRX-SO3 (Abcam, ab16830, PRX3-SO3 at 25 kD and PRX1/2-SO3 at 22 kD), PRX3 (Abcam, ab73349, 25 kD), Collagen I (Abcam, ab21286, 120 kD and 160 kD), ATF4 (Cell Signaling Technology, 11815, 47 kD), PYCRL (Thermo Fisher, MA5-25335, 30 kD), Collagen IV (Proteintech Group Inc., 55131-1-AP, 190 kD), MTHFD2 (Proteintech Group Inc., 12270-1-AP, 35 kD), SHMT2 (Cell Signaling Technology, 12762, 50 kD), Flag (Sigma, F1804, POS5-Flag at 49 kD. TPNOX-Flag at 52 kD), GCLC (Santa Cruz Biotechnology, sc-390811, 70 kD), GCLM (Proteintech Group Inc., 14241-1-AP, 30 kD), xCT (Cell Signaling Technology, 12691, 38 kD), SOD2 (Proteintech Group Inc., 24127-1-AP, 25 kD), GSR (Santa Cruz Biotechnology, sc-133245, 50 kD), NADK1 (Cell Signaling Technology, 55948, 48 kD), Cyclin D1 (Cell Signaling Technology, 55506, 35 kD).
Chemicals (commercial source, catalog number) used in this study were: Erastin (Med Chem Express, HY-15763), RSL3 (Cayman, 1219810-16-8), H2O2 (Sigma, H1009), MitoParaquat (Cayman, 18808), FK866 (Sigma, F8557), Buthionine sulfoximine (Cayman, 14484), L-alanine (Sigma, A7627), L-aspartate (Sigma, A8949), L-asparagine (Sigma, A0884), L-glutamate (Sigma, G1251), L-proline (Sigma, P0380), [U-13C]L-glutamine (Cambridge Isotope Laboratories, CLM-1822-H-0.25), [U-13C]L-arginine (Cambridge Isotope Laboratories, CLM-2265-H-0.1), [U-13C]glucose (Cambridge Isotope Laboratories, CLM-1396-5), [2,3,3-2H3]serine (Cambridge Isotope Laboratories, DLM-582-0.1), Lipoic acid (Sigma, T1395), Pyruvate (Life Technologies, 11360070), Biotin (Sigma, B4639), Vitamin B12 (Sigma, V6629).
The HEK293T cell line, the cancer cell lines U2OS, DLD1, T47D and Saos2, the non-malignant cell lines HaCaT and MCF10A, and the NIH-3T3 cell line were obtained from the American Type Culture Collection (ATCC). The chondrosarcoma cell lines JJ012 with an endogenous IDH1 R132G mutation and CS1 with an endogenous IDH2 R172S mutation were previously validated by sequencing the IDH1 and IDH2 genes as described (26, 27). The MEF cell line was derived by SV40 large T antigen immortalization. The MCF10A cell line was maintained in DMEM/F12 (Thermo Fisher 11320) based medium supplemented with 5% horse serum (Thermo Fisher 16050122), 20 ng/mL EGF (Peprotech, AF-100-15), 0.5 mg/mL hydrocortisone (Sigma, H0888), 100 ng/mL cholera toxin (Sigma, C8052), 10 μg/mL insulin (Sigma, 10516), and 100 unit/mL penicillin and 100 μg/mL streptomycin. Other cell lines were maintained in DMEM/F12 based medium supplemented with 10% FBS (Gemini) and 100 unit/mL penicillin and 100 μg/mL streptomycin. All cell lines were cultured in a 37° C. incubator at 20% oxygen, and were routinely verified to be mycoplasma-free by MycoAlert Mycoplasma Detection Kit (Lonza).
Contact-inhibition of MEFs was induced by seeding 125,000 cells per well in 0.1% gelatin-coated 24-well plates. Complete confluency was observed after 48 hours, and the cells were maintained for additional 96 hours, with medium change every 24 hours, before the downstream analyses.
For experiments involving nutrient and medium component manipulation, and stable isotope tracing, the denoted medium was supplemented with 10% dialyzed FBS (Gemini) and 100 unit/mL penicillin and 100 μg/mL streptomycin. The level of nutrient supplementation was determined by the amount present in DMEM/F12 medium unless otherwise specified.
CRISPR-Cas9 mediated gene knockout was achieved using the lentiCRISPR v2 system (Addgene 52961 and 98292), and polyclonal cell populations were used for the experiments. The human control sgRNA (sgCtrl) is targeting the silent gene PRM1 in order to achieve genome cutting, but at a non-expressed gene. Similarly, the mouse control sgRNA is targeting the ROSA26 locus. cDNA for NADK2 was obtained from Origene (RC214247), and was mutagenized to prevent targeting by guide RNA but preserve the wild-type protein sequence. cDNA for POS5 synthesized at GENEWIZ was codon optimized (see Table 2 for codon optimized POS5 cDNA) for mammalian cell expression.
A FLAG tag was further fused to the C-terminus of the POS5 protein to allow antibody detection. cDNA for FLAG-tagged cytoTPNOX and mitoTPNOX were obtained from Addgene (87853 and 87854). Ectopic gene expression of cytoTPNOX and mitoTPNOX in U2OS cells was achieved through the pINDUCER20 (Addgene, 44012) tet-on viral expression system. All the other ectopic gene expression described in this study (including cytoTPNOX and mitoTPNOX in MEFs) was achieved through the pTURN-hygro-rtTA retroviral tet-on expression system. Doxycycline was used at 100 ng/mL for gene induction. The Mito-Grx1-roGFP2 and Mito-Orp1-roGFP2 constructs were obtained from Addgene (64977 and 64991). Complete antibiotic selection was applied to all genetically modified cells before proceeding to experiments. sgRNA sequences used in this study are shown in Table 3.
Cells were lysed in RIPA lysis buffer (Millipore 20-188) supplemented with protease inhibitors (Thermo Fisher, 78428). Protein concentration was determined by BCA protein assay (Thermo Fisher, 23228), following which equal amount of protein was loaded and separated in polyacrylamide gels. Protein was then transferred to nitrocellulose membrane for immunoblotting.
Subcellular fraction was performed as previously described (28). Briefly, cells were washed, pelleted and lysed in cytosol extraction buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 M hexylene glycol, 100 μM digitonin) for 10 minutes on ice. Lysates were centrifuged at 500 g for 5 min at 4° C., and supernatants were collected (cytosolic fraction) while pellets were further lysed in membrane extraction buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 M hexylene glycol, 1% IGEPAL) and incubated at 4° C. for 10 min. Samples were then centrifuged at 3,000 g for 5 min at 4° C., and supernatants were collected (membrane fraction). Remaining pellets were resuspended in RIPA lysis buffer and incubated for 30 min at 4° C. Samples were centrifuged at 16,000 g for 15 min at 4° C., and supernatants were collected as the nuclear fraction. The same volume of extraction buffer was used for each subcellular fraction and for the whole cell lysate, such that each fraction can be compared by Western blot on the basis of equal cell number.
Rapid immunopurification of mitochondria was performed following the published methodology (4). In brief, cells with control or NADK2 knockout were engineered to express the HA-tagged OMP25 protein (Addgene, 83356); or in the case of
NAD(H) and NADP(H) measurements were performed using colorimetric quantification assays (Sigma, MAK037 and MAK038, respectively), with modifications as described in (6). Briefly, metabolites from whole cells or Mito-IP samples were extracted with 80:20 methanol:water. Supernatant of the extracted metabolites was dried down in a vacuum evaporator (Gene Vac EZ-2 Elite) for 2 hours. Metabolites were then resuspended in the manufacture's NADH or NADPH extraction buffer and centrifuged for 2 min at 3000 g. The supernatant was then split in half. One half was subjected to 60° C. incubation for 30 min to decompose NAD+ or NADP+. The other half was kept on ice for 30 min. 50 μL of each half of the supernatant was then transferred to a clear-bottom 96-well plate. For each assay, a series of NADH standards of 0, 1.25, 2.5, 5, 10, 20, 40, and 80 pmol/well, or NADPH standards of 0, 1.25, 2.5, 5, 10, 20, 40, and 80 pmol/well were included. 100 μL of NAD cycling buffer and enzyme mix, or NADP cycling buffer and enzyme mix (98 μL cycling buffer and 2 μL cycling enzyme mix from the manufacture) was added to each sample and incubated for 5 min to convert all NAD+ to NADH, or NADP+ to NADPH, respectively. 10 μL of manufacturer's NADH or NADPH developer was added into each well. Values were recorded with a plate reader at 450 nm at 2 hours. The amount of NADH or NADPH was calculated from the corresponding standard curves. The ice-incubated sample indicated the total abundance of NAD(H) or NADP(H), whereas the 60° C.-incubated sample indicated only the NADH or NADPH species.
Measurement of whole cell or mitochondrial GSH abundance or GSH/GSSG ratio was performed using GSH/GSSG-Glo assay (Promega, V6611) following the manufacture's protocol. In brief, whole cell samples were cultured in duplicate sets or Mito-IP samples were split in half following immunopurification and KPBS washes. 50 μL of total glutathione lysis reagent or oxidized glutathione lysis reagent (from the manufacture) was added to the whole cell samples, or was used to elute the Mito-IP samples. 50 μL of total glutathione lysis reagent was also added to a series of 0, 0.125, 0.25, 0.5, 1, 2, 4, and 8 μM GSH standards. After 5 min incubation at room temperature, 50 μL of luciferin generation reagent (from the manufacture) was added to each sample and incubated at room temperature for 30 min. 100 μL of luciferin detection reagent was then added to each sample. After 15 min incubation, luminescence values were measured using a Cytation 3 imaging reader. The total glutathione lysis reagent sample indicated the total abundance of GSH (both GSH and GSSG species), whereas the corresponding oxidized glutathione lysis reagent sample indicated the GSSG species.
For [U-13C]glutamine and [U-13C]glucose tracing studies, cells were seeded in 6-well plates, and after 40 hours transferred into medium containing 2 mM [U-13C]glutamine or 25 mM [U-13C]glucose, supplemented with 10% dialyzed FBS, and cultured for 6 hours. For other cell-based GC-MS studies, cells were seeded in 6-well plates and incubated as described in the figure legends. Metabolism was quenched by the addition of 1 mL of 80:20 methanol:water and stored at −80° C., overnight. For metabolite measurements from spent culture medium, 30 μL of cell-conditioned medium was extracted by the addition of 1 mL of 80:20 methanol:water and stored at −80° C., overnight. 30 μL of blank medium incubated for the same amount of experimental time was processed in parallel and used as a reference to determine metabolite secretion or consumption. Measured metabolite abundances were converted to approximate concentrations using the media formulation values as a reference.
The methanol-extracted metabolites were cleared by centrifugation and supernatant was dried in a vacuum evaporator (Genevac EZ-2 Elite) for 5 hours. Dried metabolites were dissolved in 40 mg/mL methoxyamine hydrochloride (Sigma, 226904) in pyridine (Thermo Fisher, TS-27530) for 90 min at 30° C., and derivatized with MSTFA with 1% TMCS (Thermo Fisher, TS-48915) for 30 min at 37° ° C. Samples were analyzed using an Agilent 7890A GC connected to an Agilent 5975C Mass Selective Detector with electron impact ionization. The GC was operated in splitless mode with constant helium gas flow at 1 mL/min. 1 μL of derivatized metabolites was injected onto an HP-5MS column, the inlet temperature was 250° C., and the GC oven temperature was ramped from 60 to 290° C., over 25 min. Peak ion chromatograms for metabolites of interest were recorded and extracted at their specific m/z with MassHunter Quantitative Analysis software v10.0 (Agilent Technologies). Ions used for quantification of metabolite levels are as follows: α-ketoglutarate m/z 304; citrate m/z 465; fumarate m/z 245; malate m/z 335; aspartate m/z 232; alanine m/z 218; glutamate m/z 363; glycine m/z 276; isoleucine m/z 260; leucine m/z 260; proline m/z 216; serine m/z 306; threonine m/z 320; tryptophan m/z 202; tyrosine m/z 354; valine m/z 218; methionine m/z 293; glutamine m/z 246; phenylalanine m/z 294; 2-hydroxyglutarate m/z 349. All peaks were manually inspected and verified relative to known spectra for each metabolite. Natural isotope abundance correction was performed using IsoCor (https://isocor.readthedocs.io/en/latest/index.html). For relative quantification, integrated peak areas were normalized to the packed cell volume of each sample.
For [U-13C]glutamine, [U-13C]arginine and [2,3,3-2H]serine tracing studies, cells were seeded in 6-well plates in DMEM with 150 μM proline. Cells were cultured for 40 hours and then transferred into DMEM containing 2 mM [U-13C]glutamine, 400 μM [U-13C]arginine or 400 μM [2,3,3-2H]serine, 10% dialyzed FBS, 100 unit/mL penicillin and 100 μg/mL streptomycin. Proline (150 μM) was also supplemented for [2,3,3-2H]serine tracing experiments. After 8 hours, metabolism was quenched and metabolites were extracted by aspirating medium and adding 1 mL of 80:20 methanol:water previously kept at −80° C. After overnight incubation at −80° C., cells were collected and centrifuged at 20,000 g for 20 min at 4° C. The supernatants were dried in a vacuum evaporator (Genevac EZ-2 Elite) for 3 hours. Dried extracts were resuspended in 60 μL of 60% acetonitrile in water. Samples were vortexed, incubated on ice for 20 min, and clarified by centrifugation at 20,000 g for 20 min at 4° C.
LC-MS analysis was performed with a 6545 Q-TOF mass spectrometer with Dual JetStream source (Agilent) operating in either positive or negative ionization. For positive ionization mode liquid chromatography separation was achieved on a Acquity UPLC BEH Amide column (150 mm×2.1 mm, 1.7 μm particle size, Waters). Mobile phase A was 10 mM ammonium acetate in 10:90 acetonitrile:water with 0.2% acetic acid, pH 4 and mobile phase B was 10 mM ammonium acetate in 90:10 acetonitrile:water with 0.2% acetic acid, pH 4. The gradient was 0 min, 95% B; 9 min, 70% B; 13 min, 30% B; 14 min, 30% B; 14.5 min, 95% B; 15 min, 95% B, 20 min, 95% B; 2 mins posttime. Other LC parameters were: flow rate: 400 μL/min; column temperature: 40° C., and the injection volume was 5 μL. MS parameters were: gas temp: 300° C.; gas flow: 10 L/min; nebulizer pressure: 35 psig; sheath gas temp: 350° C.; sheath gas flow: 12 L/min; VCap: 4,000 V; fragmentor: 125 V.
For negative ionization mode liquid chromatography separation was achieved on an iHILIC-(P) Classic column (100 mm×2.1 mm, 5 μm particle size, HILICON). Mobile phase A was 10 mM ammonium bicarbonate in 10:90 acetonitrile:water with 5 μM medronic acid, pH 9.4 and mobile phase B was 10 mM ammonium bicarbonate in 90:10 acetonitrile:water with 5 μM medronic acid, pH 9.4). The gradient was 0 min, 95% B; 15 min, 50% B; 18 min, 50% B; 19 min, 95% B; 19.10 min, 95% B; 25.5 min, 95% B; 2 mins posttime. Other LC parameters were: flow rate: 200 μL/min; column temperature: 40° C., and injection volume was 2 μL. MS parameters were: gas temp: 300° C.; gas flow: 10 L/min; nebulizer pressure: 40 psig; sheath gas temp: 350° C.; sheath gas flow: 12 L/min; VCap: 3,000 V; fragmentor: 125 V. Data were acquired from m/z 50-1700 with active reference masses correction (m/z: 121.05087 and 922.00980 (positive mode) or m/z: 119.03632 and 980.01638 (negative mode). Peak identification and integration were done based on in-house exact mass and retention time library built from commercial standards. Data analysis and natural isotope abundance correction were performed using MassHunter Profinder software v10.0 (Agilent Technologies).
For TTP measurements only, MS detection was performed using an Agilent 6470 triple quadrupole mass spectrometer operated in negative ionization and MRM mode. Liquid chromatography separation was using the iHILIC-(P) Classic negative method described above. MS parameters were: gas temperature 300° C.; gas flow: 10 L/min; sheath gas temperature: 350° C.; sheath gas flow: 12 L/min; VCap: 3,000 V; fragmentor: 125 V. Individual mass transitions monitored and collision energies (CE) were: TTP M+0: m/z 481.0→158.9; TTP M+1: m/z 482.0→158.9; TTP M+2: m/z 483.0→158.9. For all transitions, collision energy was 32 V, cell accelerator voltage is 4 V. Potentially confounding signals from UTP and CTP were also monitored and chromatographic separation confirmed so they did not interfere with TTP measurements. Data analysis was using MassHunter Quantitative Analysis software v10.0 (Agilent Technologies) and natural isotope abundance correction was performed using IsoCorrectoR (https://github.com/chkohler/IsoCorrectoR).
For metabolomic profiling of the Mito-IP samples, dried extracts were resuspended in 30 μL of 60:40 acetonitrile:water and an additional 7.5 μL of 100% methanol added to prevent phase separation. Samples were vortexed, incubated on ice for 20 min, and clarified by centrifugation at 20,000 g for 20 min at 4° C. LC-MS analysis was using the iHILIC-(P) Classic negative method described with 6545 Q-TOF mass spectrometer (Agilent Technologies). Whole cell extracts were analyzed in parallel and data analysis was performed using MassHunter Profinder v10.0 software (Agilent Technologies). Metabolite identifications reported were based on either (a) exact mass and retention times matched to authentic standards (denoted as RT in Tables 1A-1G) or (b) exact mass and MS2 spectra match using SIRIUS software (denoted as MS2 in Tables 1A-1G) (https://bio.informatik.uni-jena.de/software/sirius/). Metabolites were considered to be mitochondrial if the average peak area measured in anti-HA Mito-IPs from HA-tagged OMP25 cells was at least 1.5-fold more than in anti-HA Mito-IPs from the control cell expressing Myc-tagged OMP25 (scc Tables 1A-1G; FC>1.5 for [OMP25HA sgCtrl MitoIP vs. OMP25Myc MitoIP]). Outlier identification and exclusion were performed with Grubbs' test (α=0.01) for data shown in
Oxygen consumption rate (OCR) was measured using a XFe96 Extracellular Flux Analyzer (Agilent). Cells were plated in Seahorse microplates (Agilent) at appropriate densities (10,000 cells/well for DLD1 and HaCaT cells, or 6,000 cells/well for MEFs), and were allowed to adhere overnight. Cell culture media were then removed and replaced with Seahorse media (DMEM containing 10 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate). OCR analysis was performed at basal level and after subsequent injections of oligomycin (2 μM), FCCP (0.5 μM), and rotenone plus antimycin mix (both 0.5 μM) according to the manufacturer's instructions. Immediately after OCR measurement, cell number and volume were determined using a Multisizer 3 Coulter Counter (Beckman). OCR results were analyzed using the Wave software (Agilent) under default settings and were normalized to packed cell volume.
Cellular ROS levels were measured by the CM-H2DCFDA oxidative stress indicator (Thermo Fisher, C6827) following the recommended manuals. Briefly, cells were incubated with 1 μM CM-H2DCFDA at 37° ° C. for 30 minutes. Cells were then harvested, and fluorescence signals were determined by flow cytometry.
Cells were seeded in 96-well plates at appropriate cell densities (DLD1: 10000 cells/well, T47D: 15000 cells/well), and incubated overnight at 37° C. containing 5% CO2. Contact-inhibited MEFs were seeded in 24-well plates and incubated as described above. Cell were then subjected to treatments as described in figures. Cells were stained with Hoechst 33342 (0.1 μg/ml) to monitor total cell number, and with Sytox Green (5 nM) to monitor cell death. Culture plates were read by Cytation 5 at indicated time point. Percentage of cell death was calculated as Sytox Green-positive cell number over total cell number.
Mitochondrial superoxide levels were measured by the MitoSox indicator (Thermo Fisher, M36008) following the recommended manuals. Briefly, mock or rotenone (Cayman, 13995) treated cells were incubated with 2.5 μM MitoSox reagent in HBSS (Thermo Fisher, 24020117) at 37 ºC for 10 minutes. Cells were then harvested, and fluorescence signals were determined by flow cytometry.
Mitochondrial H2O2 and Mitochondrial Glutathione Oxidation Measurement
Cells expressing Mito-Orp1-roGFP2 were treated with vehicle (DMSO) or MitoParaquat (100 μM) (MitoPQ, Cayman, 18808) for 24 hours. Cells expressing Mito-Grx1-roGFP2 were mock treated or treated with H2O2 (100 μM) (Sigma, H1009) for 4 hours. Cells were washed and incubated with 20 mM N-ethylmaleimide (NEM, Sigma, E3876) for 5 min to prevent further probe oxidation. Cells were harvested, fixed with 4% formaldehyde, and analyzed by flow cytometry using a 520/10-nm filter. The ratio of emission after excitation at 405 and 488 nm was calculated as a measure of mitochondrial H2O2 abundance (Mito-Orp1-roGFP2) or glutathione oxidation (Mito-Grx1-roGFP2). The maximal oxidized and reduced form of the probe was determined for each experiment by incubating cells in extra wells with 5 mM H2O2 or 10 mM DTT (Thermo Fisher, R0861) for 5 min before adding NEM. Oxidation status was expressed as percentage of maximal oxidized form of the probe.
Extracellular matrix (ECM) extraction and collagen staining were performed as previously described (24). In brief, confluent MEFs were grown for two days on plates coated with 0.1% gelatin in the presence of 50 μM ascorbate (Sigma, A4034) in the indicated medium. Plates were decellularized with 20 mM ammonium hydroxide/0.5% Triton X-100 for 5 min on a rotating platform. Three times the volume of PBS was added, and ECM was equilibrated overnight at 4° C., followed by four additional PBS washes. To measure collagen abundance, extracted ECM was stained with the Picro Sirius Red Stain Kit (Abcam, ab150681) according to the manufacturer's instructions. The stain was extracted with 0.1 M NaOH, and optical density was measured at 550 nm using a microplate reader. Values were normalized to the packed cell volume of cells grown on a separate plate under the same experimental conditions.
Female nude mice (Mus musculus, Athymic Nude-Foxn1nu, Envigo 069) between the ages of 7 to 9 weeks old were used for the tumor xenograft experiment. 10 mice were randomly assigned into two groups (5 mice per group). 8 million CS1 cells with control or NADK2 knockout were implanted subcutaneously per flank on both flanks of each mouse. Tumor size was measured by calipers every other day starting from Day 7 post implantation. Measurements were taken in two dimensions, and tumor volume was calculated as length×width2×π/6. On Day 15 post implantation, all tumors were collected and snap-frozen in liquid nitrogen. Metabolites from powdered tumors were extracted using 40:40:20 acetonitrile:methanol:water (20 μL/mg of powdered tumor). Samples were sonicated, vortexed, and subjected to 2 freeze-thaw cycles, then centrifuged at 20,000 g for 20 min at 4° C., and an equal volume of supernatant was dried in a vacuum evaporator for 2 hours. At the time of tumor collection, blood was taken from each of the mice by retro-orbital bleeding and was immediately placed in EDTA-tubes. Blood samples were then centrifuged at 850 g for 10 min at 4° C. to separate plasma. 25 μL of plasma from each sample was taken. Metabolism was quenched and metabolites were extracted by addition of 1 mL of 80:20 methanol:water and kept at −80° C., overnight. Extracted metabolites were centrifuged at 20,000 g for 20 min at 4° C., and supernatant was dried in a vacuum evaporator for 2 hours. GC-MS was performed to examine metabolites in tumors and in plasma samples. Animal experiments described adhered to policies and practices approved by the Memorial Sloan Kettering Cancer Center Institutional Biosafety Committee (IBC) and the Institutional Animal Care and Use Committee (IACUC).
Analysis of gene expression and patient data was performed as previously described (24). Briefly, processed gene expression dataset GSE32537 was downloaded from Gene Expression Omnibus (GEO) with GEOquery package and assigned to groups in R studio v3.6.1 (www.r-project.org). Available clinical data for GSE32537 was correlated to NADK2 gene expression using Pearson correlation analysis. Patients were grouped into low- or high-expressers according to the gene expression of P5CS or NADK2 being within the first (low) or forth quartile (high) of the gene expression range. Data was then filtered for values being present in both the P5CShigh and NADK2high group, or the P5CSLOW and NADK2low group.
Spheroids were generated by plating 1×104 KPC cells in ultra-low attachment spheroid microplates (Corning). The next day, spheroids were transferred to 24-well plates containing synthetic ECM or fibroblast-derived ECM using a P1000 pipette at one spheroid per well. Synthetic ECM was generated by gelating different concentrations of high-concentration rat tail collagen I (Corning) and growth-factor reduced Matrigel (Corning) at a final concentration of 20% in a 37° C. incubator for 1 h. Spheroids were cultured on top of ECM in DMEM with 10% FBS and were imaged 2-3 h after transfer on ECM (d0) and the three following days with a Zeiss AxioCam microscope. Spheroid area, including outgrowing cells, was quantified manually in Fiji.
Flash frozen tumors were ground to a powder in a cryocup grinder (BioSpec) cooled with liquid nitrogen. Acid hydrolysates were generated from aliquots of 5-10 mg ground tumor by addition of 6 N HCl (100 μL/mg) and incubation at 95° C. for 16 h. Samples were cooled to room temperature and centrifuged at 20,000 g for 10 min. 100 μL supernatant was dried in a vacuum evaporator (Genevac EZ-2 Elite) for 2 h, and hydroxyproline levels were measured by GC-MS as described below.
GC-MS measurements were performed as described before (30). Ions used for quantification of metabolite levels were as follows: d5-2HG m/z 354; citrate m/z 465; alpha-ketoglutarate m/z 304; succinate m/z 247; fumarate m/z 245; malate m/z 335; aspartate m/z 232; hydroxyproline m/z 332; proline m/z 216; glutamate m/z 246; glutamine m/z 245; lactate m/z 219; pyruvate m/z 174. All peaks were manually inspected and verified relative to known spectra for each metabolite. For relative quantification of cell samples, integrated peak areas were normalized to the internal standard d5-2HG and to the packed cell volume of each sample. Absolute quantification of hydroxyproline in tumor acid hydrolysates was performed against a standard curve of commercial trans-4-hydroxy-L-proline (Sigma). In stable isotope tracing experiments, natural isotope abundance correction was performed with IsoCor software (30). LC-MS measurements were performed as described before (30). Peak identification and integration were done based on exact mass and retention time match to commercial standards. Data analysis and natural isotope abundance correction were performed with MassHunter Profinder software v10.0 (Agilent Technologies).
For the pancreatic ductal adenocarcinoma (PDAC) allograft model, 1×105 KPC cells alone or together with 5×105 PSCs were resuspended in 100 μL PBS and injected subcutaneously into the flanks of 8-10 weeks old female athymic Nude-Foxn1nu mice (Envigo, 069). For the BRCA allograft model, 5×105 DB7 cells alone or together with 5×105 MFBs were resuspended in 100 μL PBS and injected subcutaneously into the flanks of 8-10 weeks old female FVB/N mice (JAX, 001800). In one experiment, 5×105 DB7 cells were injected in 1:1 of 100 μL Matrigel (Corning) and PBS. At the beginning of each experiment, mice were randomly assigned to the different groups. No estimation of sample size was performed before the experiments. Mice were monitored daily and tumor volume was measured by calipers. Measurements were carried out blindly by members of the MSKCC Antitumor Assessment Core and were taken in two dimensions, and tumor volume was calculated as length×width2×π/6. At the end of the experiment, mice were euthanized with CO2, and tumors were collected and aliquoted for 10% formalin fixation and/or snap freezing.
Tissues were fixed overnight in 10% formalin, dehydrated in ethanol, embedded in paraffin, and cut into 5 μm sections. Picrosirius Red staining was performed with the Picro Sirius Red Stain Kit (Abcam) according to the manufacturer's instructions. Masson's trichrome staining was performed with the Masson's Trichrome Stain Kit (Polysciences) according to the manufacturer's instructions. For immunofluorescence staining, sections were de-paraffinized with Histo-Clear II (National Diagnostics) and rehydrated. Antigen retrieval was performed for 40 min in citrate buffer pH 6.0 (Vector Laboratories) in a steamer (IHC World). Sections were blocked in 5% BSA and 5% normal goat serum (Cell Signaling) in TBS containing 0.1% Tween-20, and incubated in primary antibodies at 4° C. in a humidified chamber overnight. Sections were incubated in secondary antibody in blocking solution for 1 h at room temperature and mounted in Vectashield Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories). The following primary antibodies were used: SMA (Millipore, CBL171), CK8 (DSHB, TROMA-I). The following secondary antibodies were used: donkey anti-mouse Alexa-Fluor 488, donkey anti-rat Alexa Fluor 647 (Thermo Scientific).
In the claims articles such as “a.” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of.” “only one of.” or “exactly one of.” “Consisting essentially of.” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B.” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising.” “including,” “carrying.” “having.” “containing.” “involving.” “holding.” “composed of.” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the application describes “a composition comprising A and B.” the application also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”
Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/172,598, filed Apr. 8, 2021, which is hereby incorporated by reference in its entirety.
This invention was made with government support under CA248711 and CA008748 awarded by the National Institutes of Health. The government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/023788 | 4/7/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63172598 | Apr 2021 | US |
