Myc regulation of histone Myc regulation of histone acetylationacetylation

TCA cycle

[U-13C] Glucose

[U-13C]-Pyruvate 13C-FA, 13C-acetate and 13C-acetoacetate

13C-Ac13C-Ac

GCN5

[U-13C]-Acetyl-CoA

[U-13C]-Citrate

Nuclear histones

Analysis of Analysis of 1313C acetyl group C acetyl group transfer to Histone H4transfer to Histone H4

Incubate myc-/- and myc+/+ cells in [U-13C] glucose

Acid extract and RP-HPLC purify histones

Identify fractions enriched with H4 by LC-MS

Detect 12C, 13C, and deuterium labeled K16-H4 by ESI-MS/MS

In vivo label with deuterium acetic anhydride

Histone H4 acetylation Histone H4 acetylation analysisanalysis

Acetylated peptides

Myc increases Myc increases 1313C labeling of C labeling of acetyl groups on Histone H4 acetyl groups on Histone H4

20.6%

12.7%

Morrish et al. J. Biol Chem 2010

Myc regulates the supply of Myc regulates the supply of metabolic substrates for histone metabolic substrates for histone

acetylationacetylation

Rapid modification of chromatinRapid modification of chromatin

Increased gene transcriptionIncreased gene transcription

Induce rapid cellular re-programmingInduce rapid cellular re-programming

Regulation of metabolism Regulation of metabolism during tumor progression during tumor progression

and regressionand regression

Induction/regression of islet tumors

21 day +Tamoxifen

pInsMyc/Bcl-XL

Bcl-XL

8 day no tamoxifen

Islet tumor

Glutamate Glucose

KetonesLactatePyruvate Plasma

Acetyl CoA HMG CoA

Cholesterol

SphingolipidsKetones.Isoleucine, leucine and valine

CellSlc1a1 Slc2a5 Slc16a1

Slc25a1Palmitoyl CoA

TCA cycle

Glud1

Eno1Fbp1G6p

IdhCS

Fasn

HmgcrMvdMvkFdpsDhcr7Sqle

Ppap2aDegs2Sgpl1

Oxct1

Oxct1AcadmBcat2BckdhaAox1Aldh2Aldh7a1

Genes for substrate transport and pathways Genes for substrate transport and pathways for conversion to lipids induced by Myc in for conversion to lipids induced by Myc in

cellscells

1

211

21

TCA

Data compiled from results in Lawlor et al 2006

Time-course analysis for Time-course analysis for neoplasia induction and neoplasia induction and

regressionregression

48 hrs 16 days 21 days

6 days

Regression

Induction of neoplasia

•Each time point performed 5 times with 5 MycER/Bcl-XL mice and 5 Bcl-Xl mice

Daily tamoxifen injections

1313C labeling of islets C labeling of islets 1,000 islets from MycER/Bcl-XL and Bcl-XL mice

Incubate RPMI media

O/N

Pre-incubate in Krebs Ringer buffer

Incubate in 20 mM 13C glucose

1 hr

2 hr

Quench in -700 methanol

Extract and derivatize

Stearate

Summary of changes in Summary of changes in de novode novo metabolismmetabolism

Summary of events Summary of events Glucose

Pyruvate

Succinate

Lactate

Alanine

Glucose

Pyruvate

Malate

Lactate

Alanine

2 days 16 days

Glutamate

Glutamine

Citrate PalmitateStearate

Pyruvate

Succinate

Lactate

Alanine

Glutamate

Glutamine

Citrate PalmitateStearate

Glucose

Pyruvate

Succinate

Lactate

Alanine

Glutamate

Glutamine

Citrate PalmitateStearate

Glucose

Glutamate

Citrate PalmitateStearate

Glutamine

21 days 8 day regression

Lipid metabolism on Lipid metabolism on regressionregression

18:2n-6

18:3 n6

20:3 n-6

20:4 n-6

C20:2 n-6

Elongation

Delta6-desaturationFADS2

Phospholipids

*

* *

*

**

• Suppression of miR34a• Esr1

Lipotoxicity protection mechanisms

*

**

Switching off Myc alters Switching off Myc alters cellular FAO capacity and ATP cellular FAO capacity and ATP

productionproductionATP generation with palmitateOxygen consumption with palmitate

ConclusionsConclusions Myc is a dual regulator of cell cycle Myc is a dual regulator of cell cycle

genes and metabolism genes and metabolism Myc regulates anabolic and catabolic Myc regulates anabolic and catabolic

metabolism via the TCA cyclemetabolism via the TCA cycle Myc links metabolome and proteome Myc links metabolome and proteome

via protein post-translational via protein post-translational modificationmodification

Myc-induces metabolic switching Myc-induces metabolic switching providing metabolic flexibility for providing metabolic flexibility for tumor progression tumor progression

Myc-induced lipid production Myc-induced lipid production provides an “Achilles Heel” for provides an “Achilles Heel” for targeted drug therapytargeted drug therapy

AcknowledgementsAcknowledgementsFHCRCDavid HockenberyJhoanna Noonan

Marc VanGilst Carissa Perez

Phillip Gafken

University of Washington DERCIan Sweet Ben ReedChemistry Mass SpectrometryMartin Sadelik

PNNLNancy Isern

UT South WesternMark Jeffrey

MITJoanne Kelleher

Funding sources: NIH/NCI

UCSFGerard Evan

Myc temporally regulates genes Myc temporally regulates genes related to acetyl-CoA metabolismrelated to acetyl-CoA metabolism

Sirt3 Acat1

Myc regulation of the histone Myc regulation of the histone acetyltransferase GCN5acetyltransferase GCN5

GCN5

Myc increases Myc increases 1313C labeling of C labeling of acetyl groups on Histone H4acetyl groups on Histone H4

FM012908_021308_TGR #4508-4758 RT: 21.24-22.15 AV: 84 NL: 6.00E5F: ITMS + c NSI d Full ms2 722.91@cid35.00 [185.00-1460.00]

524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540

m/z

0

20

40

60

80

100

Re

lativ

e A

bu

nd

an

ce

533.39

530.36 532.40

534.40

531.39535.41

536.42 539.46537.40529.41 538.49526.30524.37 528.40527.41525.35

FM030308_031908_KO #4149-4398 RT: 19.75-20.66 AV: 84 NL: 1.15E6F: ITMS + c NSI d Full ms2 722.91@cid35.00 [185.00-1460.00]

524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540

m/z

0

20

40

60

80

100

Re

lativ

e A

bu

nd

an

ce

533.45

530.42534.45

532.52531.46

535.48 536.48 539.43537.46 538.48525.33 528.42526.35524.43 527.42 529.28

MS2 Analysis y5 fragment (K16)(provides % acetylation occupancy at specific lysines )

TGR

KO

0 Ac

1 Ac 1 Ac (2 x 13C)

0 Ac

1 Ac

1 Ac (2 x 13C)

20.6%

12.7%

Myc+/+

Myc-/-

Morrish et al J. Biol Chem 2010

Function of metabolic and Function of metabolic and bioenergetic cell cycle bioenergetic cell cycle

checkpointscheckpoints

Quiescent cell

Stimulus

Nutritional status?

Good

Inadequate

No response to stimulus

Cell division