Myc regulation of histone acetylation
[U-13C] Glucose
[U-13C]-Pyruvate
13C-FA, 13C-acetate
and 13C-acetoacetate
[U-13C]-Acetyl-CoA
TCA cycle
GCN5
[U-13C]-Citrate
13C-Ac
Nuclear histones
13C-Ac
Analysis of
13C
acetyl group transfer
to Histone H4
Incubate myc-/- and myc+/+ cells in [U-13C] glucose
Acid extract and RP-HPLC purify histones
In vivo label with deuterium acetic anhydride
Identify fractions enriched with H4 by LC-MS
Detect 12C, 13C, and deuterium labeled K16-H4 by ESI-MS/MS
Histone H4 acetylation analysis
Acetylated peptides
Myc increases 13C labeling of acetyl
groups on Histone H4
12.7%
20.6%
Morrish et al. J. Biol Chem 2010
Myc regulates the supply of metabolic
substrates for histone acetylation

Rapid modification of chromatin

Increased gene transcription

Induce rapid cellular re-programming
Regulation of metabolism during
tumor progression and regression
Induction/regression of islet tumors
21 day +Tamoxifen
Islet tumor
pInsMyc/
Bcl-XL
Bcl-XL
8 day no tamoxifen
Genes for substrate transport and pathways for
conversion to lipids induced by Myc in b cells
Glutamate
Ketones
Lactate
Pyruvate
Glucose
Plasma
1
Slc1a1
Glud1
Ketones.
Isoleucine,
leucine and
valine
Oxct1
Acadm
Bcat2
Bckdha
Aox1
Aldh2
Aldh7a1
Slc2a5
Eno1
Fbp1
G6p
Slc16a1
1
Cell
21
Oxct1
21
Sphingolipids
TCA
cycle
TCA
Idh
CS
Slc25a1
Ppap2a
Degs2
Sgpl1
Palmitoyl CoA
Fasn
Acetyl CoA
HMG CoA
Cholesterol
Hmgcr
Mvd
Mvk
Fdps
Dhcr7
Sqle
Data compiled from results in Lawlor et al 2006
Time-course analysis for neoplasia
induction and regression
Induction of neoplasia
Daily tamoxifen injections
48 hrs
16 days
21 days
Regression
6 days
•Each time point performed 5 times with 5 MycER/Bcl-XL mice and 5 Bcl-Xl mice
13C
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
1 hr
Incubate in 20 mM 13C glucose
2 hr
Quench in -700 methanol
Extract and derivatize
Summary of changes in de novo metabolism
Stearate
Summary of events
2 days
Glucose
16 days
Glucose
Lactate
Lactate
Pyruvate
Pyruvate
Palmitate
Stearate
Citrate
Alanine
Citrate
Alanine
Malate
Succinate
Glutamate
Glutamate
Glutamine
Glutamine
Glucose
Palmitate
Stearate
Glucose
Lactate
Lactate
Pyruvate
Pyruvate
Palmitate
Stearate
Citrate
Alanine
Succinate
Alanine
Citrate
Palmitate
Stearate
Succinate
Glutamate
Glutamine
21 days
Glutamate
Glutamine
8 day regression
Lipid metabolism on regression
*
*
*
**
*
*
*
*
18:2n-6
Lipotoxicity protection mechanisms
• Suppression of miR34a
• Esr1
Elongation
18:3 n6
C20:2 n-6
20:3 n-6
Delta6-desaturation
FADS2
20:4 n-6
Phospholipids
Switching off Myc alters cellular
FAO capacity and ATP production
Oxygen consumption with palmitate
ATP generation with palmitate
Conclusions





Myc is a dual regulator of cell cycle genes
and metabolism
Myc regulates anabolic and catabolic
metabolism via the TCA cycle
Myc links metabolome and proteome via
protein post-translational modification
Myc-induces metabolic switching providing
metabolic flexibility for tumor progression
Myc-induced lipid production provides an
“Achilles Heel” for targeted drug therapy
Acknowledgements
FHCRC
David Hockenbery
Jhoanna Noonan
Marc VanGilst
Carissa Perez
Phillip Gafken
PNNL
Nancy Isern
MIT
Joanne Kelleher
UCSF
Gerard Evan
University of Washington
DERC
Ian Sweet
Ben Reed
Chemistry Mass Spectrometry
Martin Sadelik
UT South Western
Mark Jeffrey
Funding sources: NIH/NCI
Myc temporally regulates genes related to
acetyl-CoA metabolism
Sirt3
Acat1
Myc regulation of the histone
acetyltransferase GCN5
GCN5
Myc increases 13C labeling of acetyl
groups on Histone H4
MS2 Analysis y5 fragment (K16)
(provides % acetylation occupancy at specific lysines )
FM012908_021308_TGR #4508-4758 RT: 21.24-22.15 AV: 84 NL: 6.00E5
F: ITMS + c NSI d Full ms2 722.91@cid35.00 [185.00-1460.00]
0 Ac
20.6%
100
TGR
Myc+/+
533.39
80
60
1 Ac
1 Ac (2 x 13C)
40
530.36
532.40
534.40
20
531.39
524.37
0
524
525.35
525
526.30
526
527.41
527
528.40
528
535.41
529.41
529
530
531
532
533
534
535
536.42
536
537.40
537
538.49
538
539.46
539
540
m/z
FM030308_031908_KO #4149-4398 RT: 19.75-20.66 AV: 84 NL: 1.15E6
F: ITMS + c NSI d Full ms2 722.91@cid35.00 [185.00-1460.00]
0 Ac
100
12.7%
80
60
40
1 Ac
530.42
20
524.43 525.33 526.35
527.42 528.42 529.28
0
524
525
526
527
528
529
530
Morrish et al J. Biol Chem 2010
Myc-/KO
533.45
1 Ac (2 x 13C)
534.45
532.52
531.46
531
535.48
532
m/z
533
534
535
536.48
536
537.46
537
538.48
538
539.43
539
540
Function of metabolic and
bioenergetic cell cycle checkpoints
Stimulus
Cell division
Nutritional status?
Good
Quiescent cell
Inadequate
No response to stimulus