Northwestern University

Feinberg School of Medicine

Northwestern Brain Tumor Institute

Lurie Comprehensive Cancer Center

Dysregulated Signaling and Epigenetics in Glioblastoma

Shi-Yuan Cheng Ph.D.

Department of Neurology

Therapeutic targeting cancer

hallmarks

Douglas Hanahan and Robert A. Weinberg. Hallmarks of Cancer: The Next Generation, Cell, March 2011

Non-coding RNAAutophagy

Epigenetics

RTK

Glioblastoma

• Grade IV astrocytomas (Glioblastoma multiforme, GBM).

• Overall survival 14-16 months, 2-year survival rates <10%.

• Most patients develop resistant to radiation, chemotherapy and molecular targeted therapies.

T1 weighted MRI

2005

Stupp R. et al NEJM

2014

Chinot OL et al NEJM

Genetic Pathways to Primary and Secondary Glioblastoma

Ohgaki H , and Kleihues P Clin Cancer Res 2013; 19:764-772

The Cancer Genome Atlas Research Network Nature , 2008

Oncogenic Signaling Pathways Are Activated in GBMs

PDGFR Signaling Promotes GBM Growth and Invasion

Liu, KW, 2011, JCI; Feng, H. et al, 2011, JCI,

2012, Oncogene, 2015, Neuro-Onc

TK

Ig

Ig

Ig

Ig

TK

Ig

JM

pTyrs

pTyrs

Glioma

CellsPDGFPDGF

PDGFR

Rac1

Dock1

Cell

Migration

ELMO1

Src

CrkIIPI3K

SHP-2

Cell

Growth

Dyn2

PKA

Andrae, J., Gallini, R. & Betsholts, C., 2008,

Genes & Dev

Kun-wei Liu, PhD Haizhong Feng,

PhD

EGFR Signaling Promotes Glioma Growth and Invasion

TK

Ig

Ig

Ig

Ig

TK

Ig

JM

pTyrs

pTyrs

Glioma

CellsEGF

EGF

EGFR

Rac1

Cell

Migration

Survival

ELMO1

Src PKA

Dock1

Feng, H. et al, 2012, PNAS;

2013, Oncogene

Haizhong Feng,

PhD

EGFR Phosphorylation of DCBLD2 Recruits TRAF6 andStimulates Akt-promoted Tumorigenesis

DCBLD2

DCBLD2 DCBLD2

EGFR

EGFR EGFR

EGF EGF

TRAF6

TRAF6 TRAF6

Akt

AktAkt Akt

Akt

EGF

P

DCBLD2 EGFR

EGF

TRAF6Akt

P

P

P

P

T308

S473

Ub

UbUb

UbUb

Feng, H. et al., JCI , 2014

Haizhong Feng,

PhD

GBM Subtypes/Heterogeneity

Verhaak, R.G.W., et al., 2010, Cancer Cell, 17:98-110

Other papers

Targeting Mature GBM Cells and Glioma Stem Cells

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19 20

Nduom, E.K-

E., et al,

2012, The

Cancer JBiochem Pharmacol. 2010 80(5): 654–6Cancer Cell 21, 2012, SnapShot

Two Distinct Subtypes of Glioma Stem Cells (GSC)

Derived from Clinical Glioma Tumor Tissues

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19 20

Mao, P. et al, 2013, PNAS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19 20

Gene Profile Analyses Identify GBM Subtypes of GSCs

Mao, P. et al, 2013, PNAS

MicroRNA (miR) Biogenesis

Tianzhi Huang,

PhD

Differentially Expressed miRs Classify PN and MES GBM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19 20

PNMES

miR-

Top 20 miRNAs

22a

let-7g126b

let-7dlet-7f

116130

99a354b138b

31

193a-5p

112

3

83

52

8

81

6

84

AC

20

17

71

8

1303711

193a-3p

630

517b

Rank in Ordered Dataset

0.8

0.6

0.4

0.2

0

0.25

0

0.50 100 200 300 400 500

“MES” (negatively

correlated)

Ran

k lis

t M

etr

icE

nri

chm

en

t S

core

Enrichment plot: Top 20 PN Upegulated

P=0.03808

TCGA dataset

Re

lative

exp

ressio

n le

ve

l

MES PN

0

4

6

8

2

112

38

33

02

02

81

11

71

92

33

415

78

4

**

miR-125b

50

25

151050

11

23

83

30

20

267

302

17

19

23

34

157

84

11

MES PN

**

miR-20b

Re

lative

exp

ressio

n le

ve

lHuang, TZ et al, 2016,

Nat. Comms.

miRs Regulate GBM Tumorigenicity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19 20

10

20

30

40

23.5

6.0

22.5

5.5

****

Ctrl + - + -miR - + - +

83 1123

Fre

qu

en

cy o

f sp

here

form

ing

cells

(%

)

23.2

7.5

21.8

6.60

10

20

30

40 ****

+ - + - - + - +

1000 cells/well 500 cells/well 200 cells/well

100 cells/well 50 cells/well

83 1123 125b 20b

MES

83

11

23

Ctrl 125b

**

T

um

or

siz

e (

mm

3)

0

10

20

30

Ctrl + - + -125b - + - +

83 1123

**

MES

MES

Fre

que

ncy o

f sp

he

refo

rmin

g c

ells

(%

)

0

20

40

8.5

12.0

10

14.9

**

0

10

20

30

8.5 10 1012.3

**

1000 cells/well 500 cells/well 200 cells/well

100 cells/well 50 cells/well

Ctrl + - + -Zip - + - +

157 84

+ - + - - + - +

157 84 125b 20b

PN

0

10

20

30

T

um

or

siz

e (

mm

3)

Ctrl + - + -125bZip - + - +

157 84

** **

PN

15

78

4

Ctrl 125b Zip

PN

Huang, TZ et al, 2016,

Nat Comms.

Canonical Wnt Signaling Pathway: Off and On

Anastas, JN and Moon, RT, Nat Rev Cancer, 13, 11-26 (2013)

miR-Wnt Regulates GBM Phenotypes through FDZ6

+ - + -- + - +

83

FZD6

Ctrl 20b

APC

β-actin

1123

+ - -- + - +

157-Zip+

FZD6

Ctrl 20b

APC

β-actin

+ - + -- + - +

83 1123Ctrl

125b

+ - -- + - +

84-Zip+ Ctrl

125b

MES

PN

FZD6 mRNA

miR-20b

5' ...UAUUGUGAUAUUUUAGCACUUUG...3'

GAUGGACGUGAUACUCGUGAAAC

FZD6 mRNA

miR-125b

CTTGTTCACCCGGTTTCAGGAG...3'

AGUGUUCAAUCCCAGAGUCCCU

5' ...

0

20

40

60

Ctrl 20b Zip 125b Zip

0

5

10

15

Rela

tive e

xp

ressio

n level

FZD6PN84PN157

****

* ***

**

* *APC FZD6APC

831123

sh-Ctrl sh-FZD6

MES

APCAxin

GSK3bpβ-catenin

β-catenin miR-20b miR-125b

Nucleus

Self-renewalCell growth

PN GBM

Positivefeedback loop

PP2A

Destruction complex

ALDH1A3

FZD6

cell growth

self-renewal

TCF4 LEF

APC

Re

lative

lu

cife

rase

activity

T F T F T F

Ctrl

0

1

2

3

4

** ***

**

Ctrl20b125b20b+125b

ABC

Ctrl 125b

20b

125b

+20b

β-actin

TCF4

BIOWnt3A

Huang, TZ et al, 2016,

Nat. Comms

miR-Wnt Regulates GBM Phenotypes through FDZ6

FZD6β-actin

23Ctrl

FZD6 - + TCF4ABC

p-Stat3Stat3

p65p-p65

17+ -- +

TAK1p-TAK1

NLK

Ctrl FZD6

*

**

Rela

tive e

xpre

ssio

n level

**

+ -

12

8

4

0

ALD

H1A

3

CD44

Olig

2

Sox

2

1.2

0.8

0.4

0

PNPN23

Rela

tive

expre

ssio

n leve

l

Ctrl

KN93

5z-7

-oxo

sh-N

LK

FZD6

KN93

+FZD

6

5z-7

-oxo

+FZD

6

sh-N

LK+F

ZD6

3

2

1

0

20b 125b

***

*

LEF1

*

*

ID2

*

*

*

0

1

2

3

4

01

2

3

4

Ctrl

FZD6

KN93

5z-7

-oxo

+FZD6

sh-N

LK+F

ZD6

KN93

+FZD

6

5z-7

-oxo

sh-N

LK Ctrl

FZD6

KN93

5z-7

-oxo

+FZD

6

sh-N

LK+F

ZD6

KN93

+FZD

6

5z-7

-oxo

sh-N

LK

Rela

tive e

xpre

ssio

n levelPN23 PN23

Ctr

lF

ZD

6

PN23

FZD6

KN93CaMKII

TAK1

NLK

Nucleus

Self-

5Z-7-oxo

MES GBM

miR-20b miR-125b x

NF-kB

Stat3

b-catenin

TCF4

cell growth

self-renewal

LEF

Huang, TZ et al, 2016,

Nat Comms.

Inhibition of GBM Subtypes by Pathway-Specific Inhibitors

Ctrl

LGK974

BAY-117082

MES83

BAY-117082 LGK974Ctrl

Tu

mo

r vo

lum

es (

mm

3)

0

400

800

1200

0 5 8 12 14 17 19

CtrlLGK974BAY-117082

***

Days

BAY-117082

Enriched in

MES GBM

Ctrl

LGK974

BAY-117082

PN23

BAY-117082LGK974Ctrl

Tu

mo

r vo

lum

es (

mm

3)

Days

0

400

800

1200 BAY-117082LGK974Ctrl

40 50 60 70

***

0

LGK974

Enriched in

PN GBM

Huang, TZ et al, 2016,

Nat Comms.

miRs-Wnt Signaling establish Regulatory Circuits

Controlling GBM Phenotypes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19 20APC

AxinGSK3βp

β-catenin

β-catenin miR-20b miR-125b

NucleusSelf-renewal

KN93/KDCaMKII

TAK1

NLK

Nucleus

Self-

5Z-7-oxo/KD

Cell growth

MES GBMPN GBM

miR-20b miR-125b

Positive

feedback loop

PP2A

ALDH1A3

FZD6

x

Wnt/β-Catenin

NF-kB

Stat3

β-catenin

cell growth

self-renewal

TCF4 LEF TCF4

APC

cell growth

self-renewal

miR-125b/20b

FZD6

LEF

KD

FZD6

PORC

LGK974

Wnt

Wnt

CBP

ICG-001

Indo/KD

KD

LRP6

FZD

Huang, TZ et al, 2016,

Nat. Comms

Jones, A.P.. et al., 2012, Nat Rev Genet.

CpG Sites in Chromatin and Their Roles in Gene Expression

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19 20

DNA Methylation Regulates Gene Expression

Pangeni RP. et al, 2016,

in submission

CCCGTTTCGTAGAGCGCGTGT

CH3

GCCCGTTTCGTAGAGCGCGTGT

TTTGCGCGGGCGTGGCCTTTCGTAGAGTTCGTGG

CH3 CH3 CH3

GeneNOTexpressedPromoter

TTTGCGCGGGCGTGGCCTTTCGTAGAGTTCGTGG

CH3 CH3

Normalcell

Cancercell

Promoter

Geneexpressed

mRNA Protein

TSS

CpGisland

CpGisland

Intragenicregion

Intragenicregion

Unmethylated Methylated

Hypermethylated Hypomethylated(demethylated)

(Genesilencing) (Genomicdysregula on

A)

B)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19 20

Epigenetic Determinant for GBM Phenotypes

Pangeni RP. et al, 2016,

in submission

Rajendra Pangeni,

PhD

DNA Methylation Profiling Identifies Subtype-

associated Methylation landscapes in GBM and GSCs

GSC GBM GSC GBM

MES N CL PN PN U1 U2

Hypermethylated

Hypomethylated

0

25

50

0

3

6

0

0.6

1.2

0

2

4

MES

0

8

16

0

10

20

0

12

24

0

12

24

No of genes (x100) No of genes (x100)

603

16

34

528

84

157

19

67

92

46

608

1123

83

30

600

17

83

586

42

44

59

543

576

PN MESU2 U1

Pangeni RP. et al, 2016,

in submission

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19 20

Subtypes-specific Methylation Signatures in

GBM and GSCs

Pangeni RP. et al, 2016,

in submission

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19 20

Epigenetic Dysregulation Correlate to Patients Prognosis

PathwaysassociatedwithMES-methylatedsignaturegenesinGSCs

PathwaysassociatedwithPN-methylatedsignaturegenesinGSCs

High, n=271Low, n=271CFLAR

GBM (TCGA)

Months

p=0.009

0.4

0.8

0.0

6633 99 132

PrognosisofPN-methylatedgenes

High, n=103Low, n=103TMCC1

Glioma (TCGA)

Months

0.4

0.8

0.0

6633 99 132 198165

P=0.003

PrognosisofMES-methylatedgenes

Pangeni RP. et al, 2016,

in submission

Cheng Laboratory At Northwestern Univ.

Current Funding:NIH CA158911, NS095634, NS093843 & CA209345;

James McDonnell Foundation (PI: Hu); Northwestern Brain Tumor Institute

Zell Scholar Fund & NU Department of Neurology

AcknowledgementUSA and Europe

Webster Cavenee, Frank Furnari, Ludwig Inst for Cancer Res & UCSD, CA

Hai Yan, Duke Univ., NC; Jenny Grandis, UCSF, CA

Philip Auron, Duquesne Univ. Pittsburgh, PA

Kristiina Vuori, Sanford Burnham Med Res Institute, La Jolla CA

Xinghau Lu, R. Hamilton, Univ. of Pittsburgh, PA

Ichiro Nakano, Univ. of Alabama, AL

Wei Zhang, C. David James, Craig Horbinsky, Jeffery Raizer, John Kessler,

Northwestern Univ. Chicago, IL

Hui-Kuan Lin, Wake Forest Univ. NC

Susan Keezer, Cell Signaling Technology, Danvers, MA

Cameron Brenann, Sloan Kettering Cancer Center, NY

Erik Sulman, MD Anderson Cancer Center, Houston, TX

Gaetano Finocchiaro, Istituto Neurologico Besta, Via Celoria, Italy

Ming Tan, Robert Sobol, Univ of South Alabama, AL,Jan Sarkaria, Mayo Clinic, MN

JapanRyo Nishikawa, Saitama Medical Univ.Motoo Nagane, Kyorin Univ.

ChinaHaizhong, Feng, Wei-Qiang Gao, Shanghai Jiao Tong Univ.& Ren Ji Stem Cell CtrTao Cheng, Yanxin Li, Chinese Academy of Med Sci, Tainjin

Wnt Signaling is active in PN GBM but not in MES GBM

TCF4

ABCβ-actin

APC

FZD6

19

23

84

15

71

73

03

26

83

11

23

PN MES

Re

lative

TO

P/F

OP

expre

ssio

n

83

11

23

17

84

15

7

PN MES

0

5

10

15

*

**

*

Huang, TZ et al, 2016,

Nat Comm in revision

Wnt Signaling Induces miRs in GBM

Huang, TZ et al, 2016,

Nat Comm in revision

Correlative expressions of miRs, TCF-4 and FDZ6 in GBM

0

0.5

1

1.5

2

T1

T2

T3

T4

T5

T6

T7

T8

T9

T10

T11

T12

T13

T14

T15

T16

T17

T18

T19

T20

T21

T22

T23

T24

T25

T26

T27

T28

T29

T30

T31

T32

T33

T34

T35

T36

T37

T38

T39

20b 125b

Re

lative

exp

ressi

on

leve

l

TCF4FZD6

b-actin

100

50

00 20 40 P

erc

en

t su

rviv

al

Months

20b high 20b low P=0.0407

TCF4 highTCF4 low

100

50

00 40 80

Months

Pe

rce

nt

surv

iva

l

FZD6 highFZD6 low

P=0.0047 P=0.0013

100

50

0

125b high125b low P=0.0348

Pe

rce

nt su

rviv

al

0 20 40

Months

0 40 80Months

Pe

rce

nt

surv

iva

l

100

50

0

TCF4 high/FZD6 low (n=8)

TCF4 low/FZD6 high (n=15)

P=0.0057

100

50

00 20 40 60 80

months

Pe

rce

nt

su

rviv

al

Huang, TZ et al, 2016,

Nat Comm in revision

PDGFR Signaling Promotes Glioma Cell Growth and Invasion

TK

Ig

Ig

Ig

Ig

TK

Ig

JM

pTyrs

pTyrs

Glioma

CellsPDGF

PDGF

PDGFR

PI3K

SHP-2

Cell

Growth

Dyn2

Liu, K. et al., 2011, JCI; Feng, H. et al, 2011: JCI, 2012:

Oncogene; 2014: Neuro-Oncol.

Rac1

Dock1

Cell

Migration

ELMO1

Src

CrkII

PKA

Haizhong Feng,

PhD

Unconventional Guanine Nucleotide

Exchange Factors (GEFs)

DHR-1 DHR-2

ELMO1 PIP3 Rac1 CrkII

SH3 PxxP

MicroRNAs Targeting the Hallmarks of Cancer

Iorio, M.V. and Croce, C.M., 2012, EMBO Mol Med

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19 20

Methylation Profiling (MIDN) in PN and Mes GSC

GSC GBMA

B C

0

0.4

0.8

MU

β-

valu

e

MES PN

0

0.4

0.8

MES PN

MU

PN MES

15

7

U C

Sa

m D

NA

U C

84

U C

19

U C

17

U C

54

3

U C

16

U C

34

U C

83

U C

30

U C60

0U C

11

23

U C

** *

*

*

52

8

U C

5 1084

30

83

1123

14

PN

ME

S

CpG1 CpG2 CpG3 CpG4

* Methylated, U: Uncut control, C: Cut by enzyme

UnmethylatedMethylated

Pangeni RP. et al, 2016,

in submission