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1.
Mol Cancer Ther ; 2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38684230

ABSTRACT

Advances in linker payload technology and target selection have been at the forefront of recent improvements in antibody-drug conjugate (ADC) design, leading to several approvals over the last decade. In contrast, the potential of novel ADC technologies to enhance payload delivery to tumors is relatively underexplored. We demonstrate that incorporation of pH-dependent binding in the antibody component of a cMET targeting ADC (MYTX-011) can overcome the requirement for high cMET expression on tumors, an innovation that has the potential to benefit a broader population of patients with lower cMET levels. MYTX-011 drove four-fold higher net internalization than a non-pH engineered parent ADC in non-small cell lung cancer (NSCLC) cells and showed increased cytotoxicity against a panel of cell lines from various solid tumors. A single dose of MYTX-011 showed at least three-fold higher efficacy than a benchmark ADC in mouse xenograft models of NSCLC ranging from low to high cMET expression. Moreover, MYTX-011 showed improved pharmacokinetics over parent and benchmark ADCs. In a repeat dose toxicology study, MYTX-011 exhibited a toxicity profile similar to other MMAE-based ADCs. These results highlight the potential of MYTX-011 for treating a broader range of NSCLC patients with cMET expression than other cMET targeting ADCs. A first in human study is ongoing to determine the safety, tolerability, and preliminary efficacy of MYTX-011 in patients with NSCLC (NCT05652868).

2.
Nat Metab ; 1(9): 861-867, 2019 09.
Article in English | MEDLINE | ID: mdl-31598584

ABSTRACT

The de novo serine synthesis pathway is upregulated in many cancers. However, even cancer cells with increased serine synthesis take up large amounts of serine from the environment1 and we confirm that exogenous serine is needed for maximal proliferation of these cells. Here we show that even when enzymes in the serine synthesis pathway are genetically upregulated, the demand for oxidized NAD+ constrains serine synthesis, rendering serine-deprived cells sensitive to conditions that decrease the cellular NAD+/NADH ratio. Further, purine depletion is a major consequence of reduced intracellular serine availability, particularly when NAD+ regeneration is impaired. Thus, cells rely on exogenous serine consumption to maintain purine biosynthesis. In support of this explanation, providing exogenous purine nucleobases, or increasing NAD+ availability to facilitate de novo serine and purine synthesis, both rescue maximal proliferation even in the absence of extracellular serine. Together, these data indicate that NAD+ is an endogenous limitation for cancer cells to synthesize the serine needed for purine production to support rapid proliferation.


Subject(s)
Cell Proliferation , Neoplasms/metabolism , Nucleotides/biosynthesis , Serine/biosynthesis , Humans , Neoplasms/pathology , Nucleotides/metabolism , Oxidation-Reduction , Serine/metabolism
3.
BMC Cancer ; 19(1): 723, 2019 Jul 22.
Article in English | MEDLINE | ID: mdl-31331318

ABSTRACT

BACKGROUND: Copy number gain of the D-3-phosphoglycerate dehydrogenase (PHGDH) gene, which encodes the first enzyme in serine biosynthesis, is found in some human cancers including a subset of melanomas. METHODS: In order to study the effect of increased PHGDH expression in tissues in vivo, we generated mice harboring a PHGDHtetO allele that allows tissue-specific, doxycycline-inducible PHGDH expression, and we analyzed the phenotype of mice with a ubiquitous increase in PHGDH expression. RESULTS: Tissues and cells derived from PHGDHtetO mice exhibit increased serine biosynthesis. Histological examination of skin tissue from PHGDHtetO mice reveals the presence of melanin granules in early anagen hair follicles, despite the fact that melanin synthesis is closely coupled to the hair follicle cycle and does not normally begin until later in the cycle. This phenotype occurs in the absence of any global change in hair follicle cycle timing. The aberrant presence of melanin early in the hair follicle cycle following PHGDH expression is also accompanied by increased melanocyte abundance in early anagen skin. CONCLUSIONS: These data suggest increased PHGDH expression impacts normal melanocyte biology, but PHGDH expression alone is not sufficient to cause cancer.


Subject(s)
Gene Expression , Melanins/metabolism , Phosphoglycerate Dehydrogenase/genetics , Alleles , Animals , Cell Line , Doxycycline/pharmacology , Hair Follicle/physiology , Humans , Melanocytes/metabolism , Melanoma/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phenotype , Serine/biosynthesis , Skin/metabolism , Skin Neoplasms/metabolism
5.
Nat Chem Biol ; 12(6): 452-8, 2016 06.
Article in English | MEDLINE | ID: mdl-27110680

ABSTRACT

Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.


Subject(s)
Carbon/metabolism , Enzyme Inhibitors/pharmacology , Phosphoglycerate Dehydrogenase/antagonists & inhibitors , Serine/biosynthesis , Small Molecule Libraries/pharmacology , Animals , Carbon/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Female , Glycolysis/drug effects , Humans , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/enzymology , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice , Molecular Structure , Phosphoglycerate Dehydrogenase/metabolism , Purines/biosynthesis , Serine/chemistry , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Thymidine/biosynthesis , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
6.
Mol Cell ; 59(5): 850-7, 2015 Sep 03.
Article in English | MEDLINE | ID: mdl-26300261

ABSTRACT

The role of pyruvate kinase M2 (PKM2) in cell proliferation is controversial. A unique function of PKM2 proposed to be important for the proliferation of some cancer cells involves the direct activity of this enzyme as a protein kinase; however, a detailed biochemical characterization of this activity is lacking. Using [(32)P]-phosphoenolpyruvate (PEP) we examine the direct substrates of PKM2 using recombinant enzyme and in vitro systems where PKM2 is genetically deleted. Labeling of some protein species from [(32)P]-PEP can be observed; however, most were dependent on the presence of ADP, and none were dependent on the presence of PKM2. In addition, we also failed to observe PKM2-dependent transfer of phosphate from ATP directly to protein. These findings argue against a role for PKM2 as a protein kinase.


Subject(s)
Carrier Proteins/metabolism , Membrane Proteins/metabolism , Protein Kinases/metabolism , Pyruvate Kinase/metabolism , Thyroid Hormones/metabolism , Adenosine Triphosphate/metabolism , Animals , Carrier Proteins/genetics , Cell Line, Tumor , Cell Proliferation/physiology , Cells, Cultured , Gene Deletion , Glycolysis , Humans , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Phosphoenolpyruvate/metabolism , Phosphorylation , Protein Kinases/deficiency , Protein Kinases/genetics , Pyruvate Kinase/deficiency , Pyruvate Kinase/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Thyroid Hormones/deficiency , Thyroid Hormones/genetics , Thyroid Hormone-Binding Proteins
7.
Cancer Metab ; 3: 5, 2015.
Article in English | MEDLINE | ID: mdl-25926973

ABSTRACT

BACKGROUND: The gene encoding the serine biosynthesis pathway enzyme PHGDH is located in a region of focal genomic copy number gain in human cancers. Cells with PHGDH amplification are dependent on enzyme expression for proliferation. However, dependence on increased PHGDH expression extends beyond production of serine alone, and further studies of PHGDH function are necessary to elucidate its role in cancer cells. These studies will require a physiologically relevant form of the enzyme for experiments using engineered cell lines and recombinant protein. RESULTS: The addition of an N-terminal epitope tag to PHGDH abolished the ability to support proliferation of PHGDH-amplified cells despite retention of some activity to convert 3-PG to PHP. Introducing an R236E mutation into PHGDH eliminates enzyme activity, and this catalytically inactive enzyme cannot support proliferation of PHGDH-dependent cells, arguing that canonical enzyme activity is required. Tagged and untagged PHGDH exhibit the same intracellular localization and ability to produce D-2-hydroxyglutarate (D-2HG), an error product of PHGDH, arguing that neither mislocalization nor loss of D-2HG production explains the inability of epitope-tagged PHGDH to support proliferation. To enable studies of PHGDH function, we report a method to purify recombinant PHGDH and found that untagged enzyme activity was greater than N-terminally tagged enzyme. Analysis of tagged and untagged PHGDH using size exclusion chromatography and electron microscopy found that an N-terminal epitope tag alters enzyme structure. CONCLUSIONS: Purification of untagged recombinant PHGDH eliminates the need to use an epitope tag for enzyme studies. Furthermore, while tagged PHGDH retains some ability to convert 3PG to PHP, the structural alterations caused by including an epitope tag disrupts the ability of PHGDH to sustain cancer cell proliferation.

8.
Nature ; 520(7547): 363-7, 2015 Apr 16.
Article in English | MEDLINE | ID: mdl-25855294

ABSTRACT

Cancer cells adapt their metabolic processes to support rapid proliferation, but less is known about how cancer cells alter metabolism to promote cell survival in a poorly vascularized tumour microenvironment. Here we identify a key role for serine and glycine metabolism in the survival of brain cancer cells within the ischaemic zones of gliomas. In human glioblastoma multiforme, mitochondrial serine hydroxymethyltransferase (SHMT2) and glycine decarboxylase (GLDC) are highly expressed in the pseudopalisading cells that surround necrotic foci. We find that SHMT2 activity limits that of pyruvate kinase (PKM2) and reduces oxygen consumption, eliciting a metabolic state that confers a profound survival advantage to cells in poorly vascularized tumour regions. GLDC inhibition impairs cells with high SHMT2 levels as the excess glycine not metabolized by GLDC can be converted to the toxic molecules aminoacetone and methylglyoxal. Thus, SHMT2 is required for cancer cells to adapt to the tumour environment, but also renders these cells sensitive to glycine cleavage system inhibition.


Subject(s)
Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Glioblastoma/metabolism , Glioblastoma/pathology , Glycine Hydroxymethyltransferase/metabolism , Glycine/metabolism , Ischemia/metabolism , Acetone/analogs & derivatives , Acetone/metabolism , Acetone/toxicity , Animals , Brain Neoplasms/blood supply , Brain Neoplasms/enzymology , Cell Hypoxia , Cell Line, Tumor , Cell Survival , Female , Glioblastoma/blood supply , Glioblastoma/enzymology , Glycine Dehydrogenase (Decarboxylating)/antagonists & inhibitors , Glycine Dehydrogenase (Decarboxylating)/metabolism , Humans , Ischemia/enzymology , Ischemia/pathology , Mice , Necrosis , Oxygen Consumption , Pyruvaldehyde/metabolism , Pyruvaldehyde/toxicity , Pyruvate Kinase/metabolism , Tumor Microenvironment , Xenograft Model Antitumor Assays
9.
Nat Med ; 20(10): 1193-1198, 2014 10.
Article in English | MEDLINE | ID: mdl-25261994

ABSTRACT

Most patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed with advanced disease and survive less than 12 months. PDAC has been linked with obesity and glucose intolerance, but whether changes in circulating metabolites are associated with early cancer progression is unknown. To better understand metabolic derangements associated with early disease, we profiled metabolites in prediagnostic plasma from individuals with pancreatic cancer (cases) and matched controls from four prospective cohort studies. We find that elevated plasma levels of branched-chain amino acids (BCAAs) are associated with a greater than twofold increased risk of future pancreatic cancer diagnosis. This elevated risk was independent of known predisposing factors, with the strongest association observed among subjects with samples collected 2 to 5 years before diagnosis, when occult disease is probably present. We show that plasma BCAAs are also elevated in mice with early-stage pancreatic cancers driven by mutant Kras expression but not in mice with Kras-driven tumors in other tissues, and that breakdown of tissue protein accounts for the increase in plasma BCAAs that accompanies early-stage disease. Together, these findings suggest that increased whole-body protein breakdown is an early event in development of PDAC.


Subject(s)
Amino Acids, Branched-Chain/blood , Carcinoma, Pancreatic Ductal/blood , Pancreatic Neoplasms/blood , Adult , Aged , Aged, 80 and over , Animals , Carcinoma, Pancreatic Ductal/etiology , Case-Control Studies , Cohort Studies , Disease Models, Animal , Disease Progression , Female , Humans , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Pancreatic Neoplasms/etiology , Prospective Studies , Proto-Oncogene Proteins p21(ras)/genetics , Risk Factors , Time Factors
10.
Mol Cell ; 55(2): 253-63, 2014 Jul 17.
Article in English | MEDLINE | ID: mdl-24882210

ABSTRACT

Eukaryotic cells compartmentalize biochemical processes in different organelles, often relying on metabolic cycles to shuttle reducing equivalents across intracellular membranes. NADPH serves as the electron carrier for the maintenance of redox homeostasis and reductive biosynthesis, with separate cytosolic and mitochondrial pools providing reducing power in each respective location. This cellular organization is critical for numerous functions but complicates analysis of metabolic pathways using available methods. Here we develop an approach to resolve NADP(H)-dependent pathways present within both the cytosol and the mitochondria. By tracing hydrogen in compartmentalized reactions that use NADPH as a cofactor, including the production of 2-hydroxyglutarate by mutant isocitrate dehydrogenase enzymes, we can observe metabolic pathway activity in these distinct cellular compartments. Using this system we determine the direction of serine/glycine interconversion within the mitochondria and cytosol, highlighting the ability of this approach to resolve compartmentalized reactions in intact cells.


Subject(s)
Cytosol/metabolism , Mitochondria/metabolism , NADP/metabolism , Cell Line, Tumor , Glucose/metabolism , Glycine/metabolism , Humans , Isocitrate Dehydrogenase/metabolism , Metabolic Flux Analysis , Serine/metabolism
11.
Cell ; 155(2): 397-409, 2013 Oct 10.
Article in English | MEDLINE | ID: mdl-24120138

ABSTRACT

The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.


Subject(s)
Breast Neoplasms/metabolism , Gene Deletion , Mammary Neoplasms, Experimental/metabolism , Pyruvate Kinase/genetics , Pyruvate Kinase/metabolism , Animals , Base Sequence , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Exons , Female , Gene Knockout Techniques , Heterografts , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Sequence Data , Mutagenesis , Mutation , Neoplasm Metastasis , Neoplasm Transplantation , RNA Splicing
12.
Nat Cell Biol ; 14(8): 790-1, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22854814

ABSTRACT

Proliferating cells of the Xenopus laevis retina facultatively use aerobic glycolysis instead of oxidative phosphorylation. This demonstrates that the metabolic rewiring usually associated with the Warburg effect in tumorigenesis may be a more widespread feature of proliferative metabolism than generally appreciated.


Subject(s)
Glucose/metabolism , Glycolysis , Retina/cytology , Retina/embryology , Animals , Cell Proliferation/drug effects , Oxidative Phosphorylation , Oxygen/pharmacology , Retina/drug effects , Xenopus laevis/embryology
13.
Nat Chem Biol ; 8(10): 839-47, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22922757

ABSTRACT

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.


Subject(s)
Biopolymers/metabolism , Cell Transformation, Neoplastic , Enzyme Activators/pharmacology , Pyruvate Kinase/metabolism , Animals , Biopolymers/chemistry , Blotting, Western , Cell Proliferation , Humans , Mice , Neoplasms/enzymology , Neoplasms/metabolism , Neoplasms/pathology , Pyruvate Kinase/chemistry
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