Metabolomics Analysis Reveals Differential T-Cell Serine Metabolism As a Target in Autoimmunity

Metabolomics Analysis Reveals Differential T-Cell Serine Metabolism as a Target in Autoimmunity

Gabriela Andrejeva Jeffrey Rathmell lab Vanderbilt University Medical Center [email protected]

2018 UK Metabolism Symposium Monday, 30th July 2018 Outline 2

dUMP dTMP

Betaine aldehyde

Choline

TYMS

CHDH

Folate Se-Adenosylselenomethionine S-Adenosyl-L-methionine Dihydropteroate Dihydrofolate

Cob(II)alamin 10-Formyltetrahydrofolylpolyglutamate10-Formyltetrahydrofolyl Betaine L-glutamate MAT2B MAT1A DHFR MAT2A Tetrahydrofolyl-[Glu](n)

Formate Poly-L-glutamate ALDH1L2 MTR 3-Phospho-D-glycerate BHMT2 ALDH1L1 BHMT FPGS S-Adenosyl-L-homocysteine MTHFD1L GGH PHGDH MTHFD1 ATIC AHCYL1 L-Methionine MTHFD2 10-Formyltetrahydrofolate L-Homocysteine MTFMT "N,N-Dimethylglycine" Tetrahydrofolate 3-Phosphonooxypyruvate PDPR AHCY 5-Methyltetrahydrofolate Selenohomocysteine MTHFR GART "5,10-Methylenetetrahydrofolate" "5,10-Methenyltetrahydrofolate" DMGDH SHMT2 PSAT1O-Phospho-4-hydroxy-L-threonine IL4I1 MTHFS Cystathionine AMT Sarcosine SARDH SHMT1 5-Formyltetrahydrofolate O-Phospho-L-serine CBS FTCD N-Formimino-L-glutamate PSPH 2-Oxo-3-hydroxy-4-phosphobutanoate NAGS GNMT NH3 L-Serine

L-Pipecolate PIPOX 5-Formiminotetrahydrofolate PPIG L-Cystathionine Thiocysteine Glycine Background and hypothesis GCAT Oxaloacetate L-AlanineL-Glutamate CTH 2-Oxobutanoate L-2-Amino-3-oxobutanoic acid L-Cysteine AGXT Hydroxypyruvate SDS ANPEP 2-Aminoacrylate ALAS2 AGXT2 S-Aminomethyldihydrolipoylprotein GLDC L-Cystine ALAS1 CP GOT1 GPT GPT2 GOT2 GSS ADC CO2 5-Aminolevulinate PPCS Pyruvate CDO1 Succinyl-CoA 2-Oxoglutarate GAD2 Mercaptopyruvate GATM GAD1 Glutathione 3-Sulfino-L-alanine L-Arginine 4-AminobutanoateL-Cysteate Hypotaurine

DLD Taurine 4-Guanidinobutanoate CSAD beta-Alanine MPST

Guanidinoacetate Global metabolomics and proteomics studies of CD4 T cells Acetyl-CoA 5,10-Methylenetetrahydrofolate" CoA

Cyanide Glucose Extracelular

GAMT Serine MITOCHONDRION Serine Serine Creatine

Methylene-THF METHIONINE Methylene-THF Serine pathway in CD4 T cell subsets MTHFD2 GSH NADPH NADPH/ GSH PURINES NADH Formate Formate Serine pathway and MTHFD2 in EAE and multiple sclerosis CYTOSOL

Th1 Summary and future directions Th17

Treg

Outline 3

Background and hypothesis Global metabolomics and proteomics studies of CD4 T cells Serine pathway in CD4 T cell subsets Serine pathway and MTHFD2 in EAE and multiple sclerosis Summary and future directions

CD4 T Cells and Metabolism 4

TCR

Antigen Recognition & Co-Stimulation Naïve

POLARIZING TGFβ TGFβ IL-12 IL-6 MILEU IL-2 IFN� IL-4 IL-23

Treg Th1 Th2 Th17 FoxP3 T-bet GATA3 Ror�t

EFFECTOR IL-10 IFN�, TNF IL-4, IL-5, IL-17A, IL-17F, MEDIATORS IL-13 IL-22

Regulation, Macrophage EFFECTOR Allergic and Inflammation suppression of activation, helminth FUNCTIONS inflammatory inflammation responses responses

Adapted from Swain et al., Nat Rev Immunol, 2012 CD4 T Cells and Metabolism 5

TCR

Antigen Recognition & Co-Stimulation Naïve

POLARIZING TGFβ TGFβ IL-12 IL-6 MILEU IL-2 IFN� IL-4 IL-23

Treg Th1 Th2 Th17 FoxP3 T-bet GATA3 Ror�t

EFFECTOR IL-10 IFN�, TNF IL-4, IL-5, IL-17A, IL-17F, MEDIATORS IL-13 IL-22

Regulation, Macrophage EFFECTOR Allergic and Inflammation suppression of activation, helminth FUNCTIONS inflammatory inflammation responses responses

Adapted from Swain et al., Nat Rev Immunol, 2012 Sugiura & Rathmell., J Immunol, 2018 CD4 T Cells and the Immunological Balance 6

Th1 Th1 Th2 Treg Th1 Th17 Treg Th17 Th2 Th17 Treg Th2

Immunosuppression Tolerance Inflammation Cancer Normal Immunity Autoimmunity Hypothesis 7

Cytokine signaling during CD4 T cell development induces differential metabolic rewiring to fulfill biosynthetic, signaling and epigenetic roles. These are selectively essential for the development and function of different CD4 T cell subsets.

Outline 8

Experimental System for Metabolomics and 9 Proteomics

Isolated naïve Cytokines added Outcome mouse TGFβ Treg CD4+ T cell TGFβ + IL-2 Treg proliferative IL-12 Th1 CD3 CD28 SIGNAL 1 IL-12 + IL-2 Th1 24h, 48h, 72h Immune Characterization SIGNAL 2 TGFβ + IL-6 Th17 • Transcription Factor Expression TCR anti-CD3 anti-CD28 TGFβ + IL-6 + IL-23 More pathogenic Th17 • Cytokine Secretion TGFβ + IL-6 + IL-23 Pathogenic Th17 anti-CD3 and anti-CD28 + IL-1β coated plate (5ug/ml each) Metabolic Characterization Nothing Th0 • Intracellular Metabolite MS • Medium Analysis by NMR • Proteomics Isolated Cytokines added Outcome naïve mouse IL-7 Naïve T cell CD4+ T cell Outline 10

Serine/Glycine/One-Carbon Metabolism 11

Glucose Extracellular Serine MITOCHONDRION Glycine Serine Serine

Glycine Methylene-THF METHIONINE Methylene-THF

GSH NADPH NADPH/ GSH PURINES NADH Formate Formate

CYTOSOL Serine/Glycine/One-Carbon Metabolism 12

Glucose Extracellular Serine MITOCHONDRION Glycine Serine Serine

Glycine Methylene-THF METHIONINE Methylene-THF MTHFD2

GSH NADPH NADPH/ GSH PURINES NADH Formate Formate

CYTOSOL Serine/Glycine/One-Carbon Metabolism 13

Glucose Extracellular Serine MITOCHONDRION Glycine Serine Serine

Glycine Methylene-THF METHIONINE Methylene-THF ? Th1 MTHFD2 Th17 TReg GSH NADPH NADPH/ GSH PURINES NADH Formate Formate

CYTOSOL Possible Mechanisms of MTHFD2 Inhibitor Action

Nucleotide Synthesis Reactive Oxygen Species The Model of MTHFD2 Inhibitor Action 15

Glucose Extracellular Serine MITOCHONDRION Glycine Serine Serine

Glycine Methylene-THF METHIONINE Methylene-THF Th1 MTHFD2 Th17 TReg GSH NADPH NADPH/ GSH PURINES AICAR NADH

Formate Formate AMPK CYTOSOL mTORC1 Outline 16

Outline 17

Summary and Future Directions 18

dUMP dTMP

Betaine aldehyde

Choline

TYMS

CHDH

Folate Se-Adenosylselenomethionine S-Adenosyl-L-methionine Dihydropteroate Dihydrofolate

Cob(II)alamin 10-Formyltetrahydrofolylpolyglutamate10-Formyltetrahydrofolyl Betaine L-glutamate MAT2B MAT1A DHFR MAT2A Tetrahydrofolyl-[Glu](n)

Formate Poly-L-glutamate ALDH1L2 MTR 3-Phospho-D-glycerate BHMT2 ALDH1L1 BHMT FPGS S-Adenosyl-L-homocysteine MTHFD1L GGH PHGDH MTHFD1 ATIC AHCYL1 L-Methionine MTHFD2 10-Formyltetrahydrofolate L-Homocysteine MTFMT "N,N-Dimethylglycine" Tetrahydrofolate 3-Phosphonooxypyruvate PDPR AHCY 5-Methyltetrahydrofolate Selenohomocysteine MTHFR GART "5,10-Methylenetetrahydrofolate" "5,10-Methenyltetrahydrofolate" DMGDH SHMT2 PSAT1O-Phospho-4-hydroxy-L-threonine IL4I1 MTHFS Cystathionine AMT Sarcosine SARDH SHMT1 5-Formyltetrahydrofolate O-Phospho-L-serine CBS Global metabolomics analysis can reveal pathways that are FTCD N-Formimino-L-glutamate PSPH 2-Oxo-3-hydroxy-4-phosphobutanoate NAGS GNMT NH3 L-Serine

L-Pipecolate PIPOX 5-Formiminotetrahydrofolate PPIG L-Cystathionine Thiocysteine Glycine GCAT Oxaloacetate

L-AlanineL-Glutamate CTH 2-Oxobutanoate L-2-Amino-3-oxobutanoic acid L-Cysteine AGXT Hydroxypyruvate SDS ANPEP 2-Aminoacrylate ALAS2 AGXT2 S-Aminomethyldihydrolipoylprotein GLDC L-Cystine important in CD4 T cell activation and subset function ALAS1 CP GOT1 GPT GPT2 GOT2 GSS ADC CO2 5-Aminolevulinate PPCS Pyruvate CDO1 Succinyl-CoA 2-Oxoglutarate GAD2 Mercaptopyruvate GATM GAD1 Glutathione 3-Sulfino-L-alanine L-Arginine 4-AminobutanoateL-Cysteate Hypotaurine

DLD Taurine 4-Guanidinobutanoate CSAD beta-Alanine MPST

Guanidinoacetate Acetyl-CoA 5,10-Methylenetetrahydrofolate"

CoA

Cyanide Glucose Extracelular Naïve CD4 T cell differentiation with lineage-specific cytokines GAMT Serine MITOCHONDRION Serine Serine Creatine

Methylene-THF results in significant alterations of serine metabolism METHIONINE Methylene-THF MTHFD2

GSH NADPH NADPH/ GSH PURINES NADH Formate Formate Targeting MTHFD2 might be a viable strategy to shift the CYTOSOL balance between Th1/Th17/Treg in human inflammatory disease

Th1 Th17 Treg In vivo: Does targeting MTHFD2 suppress autoimmunity and promote Treg stability?

In humans: Is MTHFD2 expression increased in human multiple sclerosis patient brain lesions?

Acknowledgements 19

Rathmells’ labs Young lab • Jeffrey Rathmell • Jamey Young • Kimryn Rathmell • Irina Trenary • Kathryn Beckermann • Ali McAtee • Anna Chytil • Diana Contreras Vanderbilt NMR • Aguirre De cubas cores • Stephanie Dudzinski • Donald Stec • Marc Johnson • Markus Voehler • Aaron Lim

• Matthew Madden • Damian Maseda Sriram lab • Frank Mason • Subramaniam Sriram • Stephen Norris • Bradley Reinfeld McLean lab Vanderbilt MS core Norris lab Aune lab • Ayaka Sugiura • Stacy Sherrod • Kevin Schey • Jeremy Norris • Thomas Aune • Esteban Terzo • Alexandra Rutledge • Wade Calcutt • Danielle Guttierez • Jamie Weyandt • Simona Codreanu • Emilio Rivera • Kristie Lindsey Rose • Melissa Wolf • Kirsten Young