The human DEK oncogene: metabolic reprogramming of engineered epidermis

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1 The human DEK oncogene: metabolic reprogramming of engineered epidermis Markey Cancer Center, CESB, University of Kentucky 218 Metabolomics Symposium NMR Based Metabolomics Core CESB Co-director, UKY Marion Brusadelli Lindsey Romick- Rosendale Miki Watanabe Sara Vicente Munoz Andrew Lane

2 Human epidermis our first line of defense Outermost layer of human skin and mucosa Physical barrier that separates us from a most hostile environment Regulatory, sensory, metabolic, infectious, mechanical roles Tissue of origin for SCC, composed largely of keratinocytes

3 Wells Lab Interests Biology and engineering of human epidermis Tissue response & susceptibility to exogenous stress Viral infection HPV DNA repair and human disease Cancer development and progression (Human DEK oncogene)

4 A role for the human DEK oncogene in keratinocyte metabolism DEK is a human oncogene nuclear protein supporting a multitude of processes overexpressed in SCC (cervix, head and neck, skin) not an enzyme, not yet targetable drives tumor initiation, promotion and progression

5 Organotypic epithelial rafts mimic early phenotypes in human SCC development Human Keratinocytes DEK overexpression promotes proliferation/hyperplasia in 3D epidermis (but not in cultured cells) In cultured cells, DEK OE stimulates ECAR and OCR Drives metabolic enzyme expression and the accumulation of key metabolites Hypothesis: DEK overexpression reprograms cellular metabolism to promote sustained SCC development. Where?

6 A subcellular fraction of DEK is localized at mitochondria A. B. DEK MT Dendra-2 DAPI Hypothesis: DEK reprograms cellular metabolism as a nuclear or mitochondrial protein. How?

7 Stable Isotope-Resolved Metabolomics (SIRM) approach Workflow of SIRM metabolomic studies by NMR 1 Tracer 1 Rafts/ Media Polar/ Non-polar 6 5 C6-Glc C5, 15 N2-Gln Tracer experiment Sample collection Grinding/ Quenching Metabolite Extraction NMR sample preparation NMR Spectroscopy

8 Experimental Design and Sample Collection R78-NIKs Day Raft generation 1 Day C-Glc or 12 C-Glc Raft collection at 24h C 6 -Glc RAFTS 12 C 6 -Glc DEK-NIKs Metabolite extraction (CH 3 OH:H 2 O:CHCl 3 ) Polar/Lipid phase C 6 -Glc add DSS 12 C 6 -Glc NMR analysis of Polar phase 2x spectra acquired t=h t=2h t=4h t=8h t=12h t=24h MEDIA 5 ul 5 ul 5 ul 5 ul Acetone extraction 5 ul 5 ul add DSS NMR analysis of Polar phase 2x spectra acquired 8

9 NMR Spectroscopy 1) 1D 1 H-PRESAT SPECTRUM - Depicts all non exchangeable 1 H present in molecules - Arrows: C satellites of Lac and Ala 2) 1D 1 H- C-HSQC SPECTRUM - ONLY depicts 1 H atoms attached to C C 6 -Glucose Unlabeled C 6 -Glucose Unlabeled ( C: 1.1% Nat.Abundance) DSS: 17.5 nmoles DSS: 8.75 nmoles 9

10 1 H NMR ASSIGNMENTS IN RAFTS 1D 1 H-PRESAT SPECTRUM 12 C-N2-NAD+ C 12 C-N2-NADP+ 12 C-N6-NAD+ 12 C-N4-NAD+ 12 C-8-ATP 12 C-8-AMP 12 C-8-ADP 12 C-Formate C-2-AXP C-N5-NAD+ 12 C-A2-NAD+ 12 C-3,5-Phe 12 C-6-UXP 12 C-4-Phe 12 C-5-His 12 C-2,6-Phe B 12 C-GSSG 12 C-GSH 12 C-1-βGlucose 12 C-2,6-Tyr 12 C-6-His 12 C-3,5-Tyr C-1- βglucose 12 C-2,3-Fumarate 12 C-2-Lac C-1 -AXP C-N1 -NAD+ 12 C-1 -AXP 12 C-2-MyoIno 12 C-1 -UXP 12 C-P-Creatine 12 C-Creatine 12 C-5-UXP C-1- αglucose C-aGlycogen 12 C-Glycogen C-bGlycogen 12 C-2-Gly 12 C-1,3-MyoIno 12 C-1- αglucose 12 C-NMe-PCholine C-1-αGlucose 12 C-P-Creatine 12 C-Creatine 12 C-Cys-3-GSH A 12 C-3-Asp 12 C-GSH-GSSG-4 12 C-4-Gln 12 C-3-Gln 12 C-4-Glu 12 C-4-Glu+GSH+GSSG 12 C-3-Glu 12 C-2-Acetate C3-1,2,3-Ala 12 C-3-Ala C3-1,2,3-Lac C3-1,2,3-Ala 12 C-3-Thr C3-1,2,3-Lac 12 C-3-Lac 12 C-6-Ile 12 C-5-Ile 12 C-4-Val 12 C-5-Val 1

11 1 H ATTACHED TO C NMR ASSIGNMENTS IN RAFTS 1D 1 H- C-HSQC SPECTRUM C-N2-NAD+ B C-8-ADP C-Formate C-2-AXP C-6-UXP C-Fumarate C-A1 -AXP C-N1 -NAD+ C-A1 -NAD+ C-1-UXP C-1-GTP C-3-Glu + Gln + GSH + GSSG C-2-Gly C-4-Glu + GSH + GSSG C-4-Glu C-4-Gln C-3-Gln C-3-Ala C-3-Glu C-3-Lac A C-UDP-Glucose C-UDP-Glalactose C-Glycogen C-1-αGlucose C-GSSG C-1-βGlucose C-GSH C-2-Lac C-3-Asp

12 1 H-NMR spectra of media reveal Glc consumption and Lac production Media: t=h, t=2h, t=4h, t=8h, t=12h, t=24h C-α-Glc 12 C-α-Glc C-α-Glc C3 -Lac 12 C-3-Lac C3 -Lac 1 CHO 2 HCOH 3 HOCH 4 HCOH 5 HCOH 6 CH 2 OH C 6 -Glc 1 CHO 2 HCOH 3 HOCH 4 HCOH 5 HCOH 6 CH 2 OH C 6 -Glc Glycolysis Rafts (R78/DEK) 2 CO 3 CH 3 C 3 -Pyr LDHA 2 COH 3 CH 3 C 3 -Lac C 3 -Lactate in media therefore represents lactate newly synthesized from the added C 6 -Glucose 1 COO- 1COO- 1 COO- 2 COH 3 CH 3 C 3 -Lac

13 DEK-NIKs vs R78-NIKs rafts: Lactate RAFTS [U- C]-Glucose MEDIA t24-t 1D 1 H-PRESAT SPECTRUM nmoles in polar R78rNIKs DEKrNIKs G6P F6P DHAP GAP Pyruvate Lactate nmoles in polar R78rNIKs C-bGlc (A) t= t=2 t=4 t=8 t=12 t=24 DEKrNIKs 12C-bGlc C-bGlc (B) t= t=2 t=4 t=8 t=12 t=24

14 DEK-NIKs vs R78-NIKs rafts: Alanine [U- C]-Glucose RAFTS R78rNIKs DEKrNIKs GLYCOLYSIS Glucose G6P F6P GAP DHAP nmoles in polar C3-Ala (A) 12C-3-Ala C3-Ala (B) Succinate-2, Fumarate- 2,3 1D 1 H-PRESAT SPECTRUM 3PG ALT Ala PEP Pyruvate PC PDH AcCoA Pyruvate LDH Malate OAA TCA Citrate αkg GLUTAMINOLYSIS Glu Gln Cytosol Lactate Fumarate Succinate

15 DEK-NIKs vs R78-NIKs rafts: Glu/Gln metabolism and redox status GLYCOLYSIS GAP Pyruvate LDH 3PG PEP Lactate [U- C]-Glucose Glucose G6P F6P DHAP nmoles in polar Malate RAFTS Glu-3 Pyruvate PC ALT Fumarate Gln-4 OAA R78rNIKs PDH TCA Ala Succinate DEKrNIKs GSH+GSSG-3 GSH-8 GSSG-24 AcCoA Citrate αkg GLUTAMINOLYSIS Glu GSH Gln GSSG 1D 1 H-PRESAT SPECTRUM Cytosol nmoles in polar MEDIA T24-T R78rNIKs Glu-3 DEKrNIKs Gln-4 1D 1 H-PRESAT SPECTRUM 1D 1 H- C-HSQC SPECTRUM

16 [U- C]-Glucose DEK-NIKs vs R78-NIKs - Glycine RAFTS Glycine Ser Gly MEDIA Intracellular t= t=2 t=4 t=8 t=12 t=24 t= t=2 t=4 t=8 t=12 t=24 GLYCOLYSIS 3 3 3PG Ser Gly Ser biosynthesis Ser Gly Adapted from: Nat Genet. 215 Dec;47(12): D 1 H-PRESAT SPECTRUM 12 C-2-Gly nmoles in polar C-2-Gly 1D 1 H- C-HSQC SPECTRUM C-2-Gly nmoles in polar C-2-Gly 12C-Gly_R78rNIKs 12C-Gly_DEKrNIKs t24-t R78rNIKs DEKrNIKs C-Gly

17 SIRM is feasible for studies of engineered 3D human epidermis Preliminary data: Isogenic DEK status identifies gene-metabolic flux connections De novo synthesis of Gln, AXP, UDP-Glc, UDP-Gal and glycogen from C-Glc DEK OE stimulates de novo synthesis of Ala, Gln, Glu, Gly, but not Lac Hypotheses: DEK OE drives a hyperactive TCA cycle and anaplerosis via targetable nuclear or mitochondrial activities? This may be reflected by excessive succinate and fumarate accumulation opportunities for targeting these oncometabolites? Gly accumulation might promote GSH/GSSG synthesis High GSSG/GSH ratio may reflect oxidative stress opportunities for antioxidant based therapies? Target DEK OE via metabolic interventions

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20 Alanine C-enrichment - R78: 87% - DEK: 86%