Flipping the Switch: Can You Turn Genes "On" and "Off?”

Craig J. Kutz, 1,2 Steven L. Holshouser,1 Robert A. Casero, Jr.,3 Donald R. Menick2 and Patrick M. Woster1

1Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, 70 President St., Charleston, SC 29425

2Department of Medicine, Medical University of South Carolina, 141 Ashley Ave., Charleston, SC 29425

3Sidney Kimmel Comprehensive Cancer Institute, Johns Hopkins School of Medicine, 1650 Orleans St., Baltimore, MD 21231

Lou Hawthorne, the Missyplicity Project and

Genetic Savings and Clone

Lou Hawthorne and Missy Rainbow Copycat

Allie and Copycat Rainbow and Copycat

Epigenetics and ChromatinOrganization

The problem: How

to fit 1.8m DNA into

a nucleus of 2 um?

DNA Methylation and Histone Modifications:

Compartmentalization of the genome into domains of

different transcriptional potentials (important for

development and differentiation).

hypoacetylated histones

Dense DNA methylation

Histone H3-K9 methylation

Histone H3-K27 methylation

hyperacetylated histones

Low DNA methylation

Histone H3-K4 methylation

From P. Vertino

Epigenetics

• Heritable traits that do not involve changes to the underlying DNA sequence.

• Epigenetic changes can lead to gene silencing or gene activation, depending on the chromatin mark involved.

• Regulated by changes in DNA CpG methylation and histone protein modification.

Control of Gene Expression Through Post-Translational

Modification of Histones

Histone PTMs are coordinated with methylation of

CPG Islands at DNA promoter sites.

LYSorARG

NH2

LYSorARG

HN

HistoneTail

HistoneTail

HistoneTail

HistoneTail

histone acetyltransferasehistone lysine methyltransferase

protein arginine methyltransferase

histone deacetylasehistone demethylase

AlteredGene

Expression

Post-translationalModification

epigenetic writers

epigenetic erasers

epigenetic readers

Components of Cellular Epigenetic Modulation

Via Histone Post Translational Modifications

Epigenetic Writers, Erasers and Readers

Epigenetic Writers

Histone acetyltransferases (HATs)

GNAT family (Gcn5, PCAF and ELP3)

p300/CBP family (p300 and cyclic AMP-responsive element

binding protein)

MYST family (Tip60 and MYST 1-4)

Histone lysine methyltransferases

Protein lysine methyltransferases (KMTs) – SET1, SET2,

SUV39, EZH1,EZH2, PRDM, other SET, non-SET

All but non-SET contain SU(VAR)3–9, enhancer-of-Zeste, Trihorax)

Protein arginine methyltransferases (PRMTs)

PRMT Type I, PRMT Type II

Histone lysine phosphorylases (H3 Thr3, Ser10, Thr11, and Ser28)

Epigenetic erasers

Histone Deacetylases (HDAC 1-11)

Histone lysine demethylases

KDM1 (FAD dependent)

KDM2 – KDM6 (Fe(II)/2-oxoglutarate-dependent)

Varier, R.A.; Timmers, H.T.M.: Bioch. Bioph. Acta 2011, 1815, 75-89

Epigenetic Readers

Musselman, C. A.; Lalonde, M. E.; Cote, J.; Kutateladze, T. G.

Nat Struct Mol Biol 2012, 19 (12), 1218-1227.

Transcriptional control via histone lysine methylation

Methylation of specific lysine residues on histone tails can lead to either transcriptional activation or

repression

17 lysine residues and 7 arginine residues have been shown to undergo methylation/demethylation

10 lysine methyltransferases and nine arginine methyltransferases are known

Lysine demethylases:

lysine-specific demethylase 1 (LSD1) bound to CoREST complex – specific for

H3K4me1 and H3K4me2 (activating chromatin mark)

lysine-specific demethylase 2 (LSD2) - specific for H3K4me1 and H3K4me2 but not bound

to CoREST or another protein complex

LSD1 bound to androgen receptor – specific for H3K9me1 and H3K9me2 (a deactivating

chromatin mark)

Jumonji (JmjC)-domain containing demethylases

JHDM1A – specific for H3K6me1 and H3K6me2

JHDM2A – H3K9me1 and H3K9me2

Other Jumonji demethylases specific for trimethylated lysines

Epigenetics and Cancer

• DNA methylation and histone modifications contribute to aberrant gene

silencing.

• A functional link of aberrant epigenetic gene silencing to the pathophysiology

of cancer has been established.

• Tumor-suppressor genes are frequently inactivated in association with

promoter CpG island methylation.

• Aberrant DNA methylation and histone modifications have been shown to

have potential in risk-assessment, early detection, disease classification

and prognosis prediction in a variety of cancers.

• DNA-methyltransferase inhibitors reactivate functional expression of tumor-

suppressor genes silenced in cancer.

Baylin et al. Nature Reviews Cancer 6, 107–116 ,

2006

Klose and Zhang, Nat Rev. Molec. Cell Biol, 2007 8, 307-318

a | The LSD1 reaction mechanism detailing the removal

of a mono-methyl group. LSD1 is proposed to mediate

demethylation of mono- and di-methylated lysine residues

through an amine oxidation reaction using FAD as a

cofactor. Loss of the methyl group from mono-methyl lysine

occurs through an imine intermediate (1), which is

hydrolysed to form formaldehyde by a non-enzymatic

process (2). b | A polypeptide backbone cartoon structure

of LSD1 bound to Co-REST and the cofactor FAD. The

two-lobed amine oxidase (AO) domain is shown in orange

and yellow. The Tower domain is in green and the SWIRM

domain in blue. The Co-REST linker region (pink)

associates with the LSD1 Tower domain and the SANT

domain (red) situated at the top of the Tower domain.

c | Depiction of the potential association of LSD1–Co-REST

with nucleosomal DNA. The bottom half shows a

nucleosome with the core histone octamer in the centre

and the associated DNA double helix in blue. The LSD1–

Co-REST complex modelled onto a nucleosome indicates

that the SANT domain of Co-REST (red) could interact with

nucleosomal DNA, whereas LSD1 targets the histone H3

tail where it protrudes from the DNA gyres (shown by the

arrow). d | LSD1 as part of the Co-REST complexes

contributes to repression of neuronal genes in non-neuronal

cells. LSD1 contributes to repression by removing H3K4

methylation. e | When bound to the androgen receptor (AR),

LSD1 is converted from a transcriptional repressor to an

activator by changing the substrate specificity of LSD1 so

that it catalyses the removal of H3K9 methylation.

Polyamine-related compounds that inhibit amine oxidases

Differential Inhibition of SMO and APAO by Polyamino(bis)guanidines and Polyaminobiguanides

1g

1g

2a

2a

Polyamino(bis)guanidines and Polyaminobiguanides Inhibit Purified LSD1

0

20000

40000

60000

80000

100000

1a

1c 1f

1d

1e

2a

2e 2f

2c

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1b

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Untr

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ted

(pm

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“You can observe a lot by just watching.”-Yogi Berra

Inhibition of LSD1 Activity by Oligoamine Analogues in HCT116 Human Colon Adenocarcinoma Cells In Vitro

H3K4me2

PCNA

Un

treate

d

0.2

5 m

M

0.5

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1 m

M

5 m

M

10 m

M

1cA

H3K4me3

H3K9me2

H3K4me1

B

1c (mM)

0123456789

10

0 0.25 0.5 1 5 10

Rel

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H3K4me1

H3K4me2

Un

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0.5

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1 m

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5 m

M

10 m

M

2d

H3K4me2

PCNA

H3K4me3

H3K9me2

H3K4me1

0

2

4

6

8

10

12

14

0 0.25 0.5 1 5 10

Rel

ativ

e q

uan

tity

2d (mM)

H3K4me1

H3K4me2

Polyamino(bis)guanidines and Polyaminobiguanides are Non-Competitive Inhibitors of LSD1

-100

-50

0

50

100

150

-0.3 -0.2 -0.1 0 0.1 0.2 0.3

1/H3K4me2 (mM)

1/V

0 mM

0.25 mM

0.5 mM

1 mM

2.5 mM

-100

-50

0

50

100

150

-0.3 -0.2 -0.1 0 0.1 0.2 0.3

1/H3K4me2 (mM)

1/V

0 mM

0.25 mM

0.5 mM

1 mM

2.5 mM

Compound 1c

Compound 2d

GAPDH

SFRP5

SFRP4

Co

ntr

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1c

(5m

M)

2d

(5m

M)

DC

A (

1m

M)

TS

A (

30

0n

M)

1d

(5m

M)

2b

(5m

M)

0

5

10

15

20

25

30

35

40

Control 1c 2d TSA 1d 2b

% o

f D

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in

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SFRP5

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Scramble RNAi

Rela

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0

20

40

60

80

100

120

100

15

Scramble RNAi

Rela

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0

20

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60

80

100

120

100

15

B

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Mo

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Sc

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Mo

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Sc

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Ai

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SFRP1

SFRP4

SFRP5

GATA5

Input No antibody -H3K4me2

C

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Sc

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Ai

Mo

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Sc

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SFRP5

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Input No antibody -H3K4me2

SFRP1

SFRP4

SFRP5

GATA5

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Mo

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1c2d

Re-expression of SFRP1, SFRP4, SFRP5 and GATA5 by Compounds 1c and 2d

SFRP4

SFRP5 GATA5

The Polyaminobiguanide Verlindamycin is a Potent Epigenetic Modulator

-Acts as a non-competitive inhibitor of recombinant LSD1/CoREST (KI = 6.7 mM)

-Promotes a 6.5-fold increase in global H3K4me2 in HCT116 cells in vitro; no change in methylation levels at H3K9 or H3K27

-Causes significant re-expression of aberrantly silenced tumor suppressor proteins SFRP1, 4 and 5 and GATA 5.

Huang, Y. et al.: Proc. Nat. Acad. Sci. USA 2007, 104,

8023-8028.

Huang, Y. et al.: Clin. Cancer Res. 2009, 15,

7217-7228

In vivo effects of compound 2d in the presence and absence of 5-azacytidineVerlindamycin (2d) Is Effective In Vivo in Combination with 5-Azacytidine

Epigenetics in the Heart

Epigenetics refers to alterations in gene expression independent of the genetic code.

HDACs have been extensively studied in cardiovascular disease

HDAC inhibitors shown to be cardioprotective in both ischemia reperfusion injury and heart failure

Recent evidence implies a crosstalk, or even an interdependency, of HDACs with histone demethylases

Chandrasekaran, S. et al.: Histone deacetylases facilitate sodium/calcium exchanger up-regulation in adult cardiomyocytes. FASEB J. 2009, 23(11), 3851-3864.

HDAC activity causes

histone methylation

Assessment of Drug Effects in the Langendorff Heart Model

Normal rabbit heart Heart after ischemia reperfusion injury

Left Ventricular

HCT 116 human colorectal tumor xenograft in Balb/c mice

0 10 20 30 40 50 60 70 80 90 1000

50

100

150

200

Time(mins)

mm

Hg

Developed Pressure

Vehicle (n=6)

2d (n=5)

No IR (n=1)

ReperfusionNo-Flow

Ischemia

**** *** **

****p<0.0001; ***p<0.001; **p<0.01; *p<0.05

In vivo effects of compound 2d in the presence and absence of 5-azacytidine

HCT 116 human colorectal tumor xenograft in Balb/c mice

0 10 20 30 40 50 60 70 80 90 1000

20

40

60

80

Time(mins)

mm

Hg

End Diastolic Pressure

No IR (n=1)ReperfusionNo-Flow Ischemia **** ***

****p<0.0001; ***p<0.001; **p<0.01; *p<0.05

Vehicle (n=6)

2d (n=5)

Left Ventricular

IR Injury Verlindamycin

MDL 72527

TCP

!

-- -- -- --

+ -- -- --

+ + -- --

+ -- + --

+ -- -- +

IR Injury Verlindamycin

MDL 72527

TCP

!

-- -- -- --

+ -- -- --

+ + -- --

+ -- + --

+ -- -- +

IR Injury Verlindamycin

MDL 72527 TCP

!

-- -- -- --

+ -- -- --

+ + -- --

+ -- + --

+ -- -- +

IR Injury Verlindamycin

MDL 72527

TCP

!

-- -- -- --

+ -- -- --

+ + -- --

+ -- + --

+ -- -- +

IR Injury

Verlindamycin

Tranylcypromine

Hearts were stained a with triphenyl tetrazolium chloride (TTC) after treatment. TTC turns red in live cells, and infarcted areas appear in white. Infarct area was determined by ImageJ. Each image is the average of data from 3 hearts.

OH

F

CN

Cl O Cl

CN

O Cl

NH2

S

SNNC

O

Cl

HNN

S

NC

NH2H2N

EtOH

microwave

90oC, 10min

ether

microwave,

40oC, 5 min

DMSO, K2CO3

microwave, 190oC

6 min

+

LiAlH4

ether, 0oC, 24 h

CH3

CH3

H3C

O

Cl

HNH

N

NN

H2N

X

HNN

N

NH

H2N

General Structure

Scheme 1

21 22 2324

266

25R1

R2

R3

R4

Cl

In Silico Model of Compound C1 Bound to LSD1/CoRest

Substituted Triazoles Selectively Inhibit LSD1 with Minimal Cytotoxicity

Cellular Effects of C1 and C15 in the Calu6 Lung Adenocarcinoma Cell Line

Pe

rce

nta

ge

of

Rela

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e C

ou

nt

Freq. Distribution H3K4me2

Vehicle

30µM TCP

1µM 6 10µM 7

1µM 6 10µM 7

DAPI F-Actin H3K4me2

***

***

***

10000 20000 300000

50

100 Vehicle

6 (10µM)6 (1µM)

10000 20000 300000

50

100 Vehicle

TCP (30mM)

10000 20000 300000

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100

Average Intensity (RFU)

Vehicle

7 (10µM)

7 (1µM)

Vehicle

30µM TCP

1µM 6 10µM 6

1µM 7 10µM 7

DAPI F-Actin H3K4me2

A B

!C D

! E

!

Figure S3. Comparison of the cytotoxicity of compounds 6 and 7 to known agents verlindamycin 2 and TCP in 5 cell lines in vitro using a standard MTS

reduction assay. Panel A: CA46 Burlitt’s Lymphoma cell line; Panel B: PC3 human prostate cancer cell line; Panel C: PANC-1 human pancreatic cancer cell

line; Panel D: MDA-MB-231 estrogen receptor negative breast cancer cell line; Panel E: MCF-10A human breast epithelial cell line. In Panels B and C,

verlindamycin 2 was run at 8 mM as a positive control, while in Panels A, D and E a dose-response curve was generated for 2. Each data point is the average of 3

determinations + standard error.

0 10 20 30 40 50 60 70 80 90 1000

50

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Time(mins)

Develo

ped

Pre

ssu

re (

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)Left Ventricular Developed Pressure

(1-hr pretreatment)

Vehicle (n=6)ReperfusionNo-Flow

Ischemia C1 (n=3)

Verlindamycin (n=3)

No IR (n=2)

0 10 20 30 40 50 60 70 80 90 1000

20

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60

80

Time(mins)

En

d D

iasto

lic P

ressu

re (

mm

HG

)Left Ventricular End Diastolic Pressure

(1-hr pretreatment)No IR (n=2)

ReperfusionNo-Flow

Ischemia C1 (n=3)

Verlindamycin (n=3)

Vehicle (n=6)

Vehicle Verlindamycin C1

0

20

40

60In

farc

t A

rea (

% o

f to

tal LV

are

a)

***

* p-value < 0.05

** p-value < 0.01

n = 3 n = 3 n = 3

Effect of Verlindamycin and C1 on Infarct Area Following Ischemia Reperfusion

Figure 1. LSD1/HDAC/CoREST corepressor complex. LSD1 inhibitors or HDAC

inhibitors (HDACi) can independently re-express silenced promoters through post-translational histone modifications.

Is the Major Effect of Inhibitors of Chromatin Remodeling Enzymes Mediated at the Epigenetic Complex?

Primary feline cardiomyocytes were treated for 3 h with 5 mM verlindamycin (V), 1 mM C1 or 2 mM C15. The co-repressor HDAC:CoREST:LSD1 complex was initially pulled down with an antibody for HDAC1 and a Western blot for CoREST was performed. The figure shows that verlindamycin and C1 disrupted the interaction between HDAC1 and CoREST, indicating that LSD1 inhibition may cause disruption of the entire co-repressor complex.

Additional experiments were performed with a pull-down with LSD1 antibody, showing that C1 and verlindamycin disrupted LSD1:HDAC1 interaction as well.

Pull-down Experiment for HDAC1/CoREST/LSD1 Complex

Wb: CoREST!

NC ! Veh! 2d! C1! C15!

IP: HDAC1!

IgG Veh V C1 C15

CoREST

Non-specific

Acknowledgements

MUSC Johns Hopkins University University of Pretoria

Dr. Donald R. Menick Robert A. Casero Lyn-Marie Birkholtz

Isuru Kumarasinghe Tracey Murray-Stewart Bianca Verlinden

Sun Choi Shannon Nowatarski Jandeli Niemand

Steven Holshouser Valentina Battaglia

Melissa Sokolosky Christina Destefano-Shields Jawarhal Nehru University

Craig Kutz Christin Hanigan Rentala Madhubala

Youxuan Li

Hereward “Cliff” Wimborne

Benefactors

NIH grant RO1 CA149095-01