cancer stem cells and therapeutic targeting via ccr5 · cancer stem cells and therapeutic targeting...

Cancer stem cells and therapeutic

targeting via CCR5

11th Annual Conference on Stem Cell and

Regenerative Medicine

October 15

Richard G Pestell

President Pennsylvania Cancer and Regenerative

Medicine Center

Acknowledgements

Pennsylvania Cancer and Regenerative Medicine Center

X. Jiao Xiaoming Ju

DISCLOSURE

• The flying FINNS

• Hannes Kolehmainen

• Paavo Nurmi

• Lasse Viren

• Ville Ritola

DISCLOSURE

• Supported from NIH (R01CA70896, R01CA75503, R01CA107382,

R01CA8607 P30CA56036)

• Supported from Breast Cancer Research Foundation

• Supported from Marian Falk Foundation

• Supported from State of Pennsylvania Department of Health

• Founder and CSO of AAA Phoenix and ProstaGene

• CMO CytoDyn

• External Advisor to 7 NCI cancer centers

• President PCARM • Blumberg Institute,

• European Cancer Stem Cell Institute,

• Weizmann Institute

• University of Stuttgart

• Karolinska Institute

• University of Helsinki

• 2015 Precision Medicine Initiative

• Public trust

• Accountability

• Data sharing, quality integrity

Precise Medicine

Precise Medicine

Targeting Cancer Stem Cells

Breast CSC markers

Genetic Determinants of Mammary Cancer Stem Cell

20

00

Cyc

lin D

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Year of Publication by Pestell Lab p

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Dac

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2005

20

07

20

09

2011

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20

15

20

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• Liu ..Pestell,RG., 2009, Proc Natl Acad Sci USA 106:55, p19035

• Genander, M., ..Pestell,RG.,Frisen J. 2009, Cell, 139 (4), p679-92

• Liu .. Pestell,RG., 2010 The canonical NF-κB pathway governs mammary

tumorigenesis in transgenic mice via tumor stem cell expansion Cancer

Res.70(24) p10464-10473.

p21CIP1 Determines Mammary Cancer Stem Cell

EMT ↑Stem cells ↑

C

c-MycRas

p21

Twist

SOX2

VSNL1

CER1

NFE2L3

INDO

ADAM23

SFRP1

BUB3

WDHD1

MTHFD2

KRT8

TERF1

CACHD1

MSH2

DLG7

KIF2C

CRABP1

PTTG1

MCM6

MFGE8

ST6GAL1

Myc

Ras

A

p21+/+

p21-/-

p21+/+

p21-/-

• Liu ..Pestell,RG., 2009, Proc Natl Acad Sci USA 106:55, p19035

Dach1 Governs Stem Cell

100 m 20 m

; Green, CK8; Blue, DAPI

(Dach

1+

/+)

(Da

ch

1-/

- )

Red, CK5 (basal)

Green, CK8 (luminal) Cancer stem cell

Mammary stem cell

Dach1

1. Wu K, . Pestell RG. Cell fate determination factor Dachshund reprograms

breast cancer stem cell function. J Biol Chem. 2011 Jan 21;286(3):2132-42.

2. Jiao, .. Pestell R. Cell Stem Cell Reports, 2018

https://www.ncbi.nlm.nih.gov/pubmed/20937839

Therapies Targeting CSC

Cancer Stem Cell Niche

CCL3 MIP1a

CCL4 MIP1b

CCL5 RANTES

CCL8

CCL11 Eotaxin1

CCL13

CCL14 HCC-1

CCL16 LEC, MTN-1

CCL3, CCL4, CCL5, CCL8, CCL11, CCL13, CCL14, CCL16

CCR5 Signaling and Cancer

The HIV CCR5 Receptor Signaling and Function

Breast oncogene induction of CCR5 Receptor

Signaling and Invasion

Inv

asion

MCF-10A:Vector Neu-T Ras Src

P<0.05 P<0.05

P>0.05

P>0.05

Con

trol

CCL5

CCR5 Receptor expression in Breast Cancer

A

Cancer Stem Cell Characteristics

CCR5 - Minor population in Breast Cancer

- Associated with Relapse

20151050

1.0

0.8

0.6

0.4

0.2

0.0Low CCR5 (N=226)High CCR5 (N=323)

Logrank p=0.002

Node-negative Breast Cancer

Time (Years)

Ove

rall

Su

rviv

al

CCR5 activates calcium In Breast Cancer

CCL5 FBS

MDA-MB-231

RFI

Single cell sequencing of CCR5+ cells

Increased “volatility” of gene expression

C D

0

8 -

6 -

4 -

2 -

0 -

-2 -

-4 -

-6 -

-8 -

Lo

g2

ge

ne

exp

ressio

n

- - -

Significant genes

-

503010 70

SUM-159 cells

CCR5+ cells

CCR5- cells

1050-5PC1

PC

2

5

0

-5

-

-

-

- - - -

CCR5+ cells CCR5- cells

Single cell sequencing of CCR5+ cells

Increased “volatility” of gene expression

AB CCR5+ cells CCR5- cells

Fold Change (Log2 )

P V

alu

e

1050 15-10 -5-15

EFNA5FGF8

STFD2KAT7

RAD54L2TNRC6B CNNM2

ANK3 RP1-59D14.9

RP11-294N21.2

SNHG3IGHV1-30-34RP11-118A1.2

0.0

03

0.0

30

.01

0.0

5

acute GVHD are 38.5% for grade II to IV disease

and 21.9% for grade III or IV disease.

CCR5+ cells give rise to mammospheres

P<0.05

CC

R5+

CC

R5-

Ma

mm

osp

he

res/1

000

ce

lls

APC-aCCR5

Count

11.4%

0

20

40

60

CC

R5+

CC

R5-

Ma

mm

osp

he

res/1

000

ce

lls

P<0.05

0

20

40

60

80

0

20

40

60

80

SUM149

CC

R5+

CC

R5-

Ma

mm

osp

he

res/1

000

ce

lls

FC-IBC-02

P<0.05

SUM159



CCR5+ cells give rise to tumors

CCR5+

CCR5-

Phot

on f

lux (

x10

9p/

sec/

cm2/s

r)

0.4

0.6

0.8

1.0

0.2 0 4 0 4 months

CCR5+ CCR5-

Ph

oto

n flu

x

(x1

08

p/s

ec/c

m2/s

r)

0

1

2

3

4

P<0.05

P<0.05

P>0.05

P<0.05



SUM159-CCR5

SUM159-Vector

Ph

oto

n flu

x

(x1

09

p/s

ec/c

m2/s

r)

P<0.05

CCR5Vector0

3

2

1

4

Reintroduction of CCR5 into CCR5- BCa cells

give rise to tumors in vivo

CCR5 Receptor Expression Promotes Breast

Cancer Cell invasion

CCR5+ CCR5-0

10

20

30

40

50

60

70 P=0.004

Ce

ll n

um

be

r a

t 24

0m

m

FBS

FBS

CCR5+

CCR5-

A

Control Maraviroc 100 nM Vicriviroc 100nm

CCR5 Receptor Inhibition blocks

Breast Cancer cellular invasion

Time (weeks)1 2 3 4

Co

ntr

ol

Mar

avir

oc

CCR5 antagonists block Breast Cancer

metastasis



0 100 200 0 100 200

Vector CCR5

SUM-159

p-gH2AX

Vinculin

Doxorubicin (nM)

DMSO

Co

ntr

ol

CC

L5

Ma

raviro

c

Vic

riviro

c

Doxorubicin (100 nM)

Co

ntr

ol

CC

L5

Ma

raviro

c

Vic

riviro

c

SUM-159

p-gH2AX

Vinculin

CCR5 restrains DNA damage response in

breast tumors

Pe

rce

nta

ge

of

GF

P+

ce

ll

no

rma

lize

d w

ith

tra

ns

fec

tio

n e

ffic

ien

cy

0

2

4

6

CCR5- CCR5+

P<0.01

DR-GFP

0

1

2

3

4

DR-GFP SA-GFP

Vector CCR5

Pe

rce

nta

ge

of

GF

P+

ce

ll

no

rma

lize

d w

ith

tra

ns

fec

tio

n e

ffic

ien

cy

SceGFP iGFP

I-SceI site Bcgl

GFP+

Bcgl Bcgl

iGFPI-SceI

HDR

GFP+

I-SceI site

5’GFP Sce3’GFPI-SceI

SSA

SA-GFP

CCR5 Receptor induces DNA damage repair


and 21.9% for grade III or IV disease.0.039 0.078 0.156 0.312 0.625 1.25 2.5 5 10 (nM)

HCC70

HCC1395

HCC1569

HCC1937

MDA-MB-231

MDA-MB-436

SUM-149

SUM-159

Doxorubicin

Maraviroc

DoxorubicinPARP inhibitors

CCR5 inhibitors increase breast cancer cell

killing



CCR5 governs DNA damage repair and breast cancer stem

cell expansion. Cancer Res. Pestell et al 2018 78:7, 1657

Chemotherapy induces CCR5

CCR5 function in Breast cancer

Metastasis

• CCR5 signaling induced in human basal breast cancer

• Oncogenes induce CCR5 signaling

• CCR5 overexpressed in basal breast epithelial cells

• CCR5 induces invasion and metastasis

• CCR5 inhibition blocks basal breast cancer metastasis in

pre-clinical models

• CCR5/CCL axis induced in >50% of human BCa

– Cancer.

Breast, Prostate, colorectal

esophageal, kidney,

Leukemia, liver, lymphoma

Myeloma brain, bladder.

CCR5 is expressed in many

different cancer types

Prostate Cancer

• Most common cancer in American men (ACS 2012)(1/6 men)

• 241,740 men will acquire this year

• 28,170 deaths/per year

• 2.5 M current survivors- no reliable predictors of survivors

• Increasing incidence globally

• Death from metastasis (bone, brain)

• No reliable pre-clinical testing models



Con

trol

Mara

viro

cCCR5 inhibitors block PCa metastasis

CCR5 in Prostate Cancer

• CCR5 signaling activated in vivo in immune competent animals

• Reliable metastasis of isogenic prostate cancer lines

• CCR5 inhibitors reduce metastasis in immune-competent mice in

vivo (total body, lung, bone and bone)

• BUT serious adverse events with Maraviroc in HIV patients…-

CCR5 specific monoclonal antibody therapy for

cancer stem cells

• Leronlimab

• Humanized monoclonal antibody

• Used in HIV treatment (Mono and combination therapy)

• No serious adverse events (SAEs)

• >600 patients in 8 clinical trials for HIV

• Once weekly, easy, subcutaneous

– self administration

Con

tro

lPR

O14

0 (

1/10

0)

20 sec beforeadding CCL5

60 sec afteradding CCL5

240 sec afteradding CCL5

60 sec afteradding FBS

0 200 400 600 800 1000

CCL5FBS

0 200 400 600 800 1000

CCL5

FBS

Control PRO140

A

B C

Time (Sec)Time (Sec)

-1

0

1

2

3

-1

0

1

2

3R

ela

tive F

I

of F

luo-4

Rela

tive F

I

of F

luo-4

Leronlimab blocks

Breast Cancer Ca+2 signaling

Leronlimab blocks

Breast Cancer cellular invasion

Control

Control 1/500 1/1000

PRO140 (1/500) Vicriviroc

PRO140

CC

L5

A

C

0

200

400

600

800

1000

Contr

ol

PR

O140

Vic

riviroc

Dis

tance o

f

Invasio

n (m

)

B

CC

L5

0

200

400

600

800

1000

Dis

tance o

f

Invasio

n (m

)

Contr

ol

1/5

00

1/1

000

D

P<0.001

P<0.001

P<0.001

P<0.001

SW480 Human Colon Carcinoma Xenografts in NCr Nude MicePRO 140, 2 mg i.p. twice/week, started day 1, n=16 tumors/group

p=0.014 p=0.272

Tu

mo

r V

olu

me

in

mm

3(m

ea

n ±

SE

)

0.2 mg dosing2 mg dosing

Leronlimab blocks

Colon Cancer tumor growth

advanced-stage metastatic colorectal cancer who

are refractory to standard chemotherapy,

including regorafenib

Tumoral Immune Cell Exploitation in Colorectal

Cancer Metastases Can Be Targeted Effectively

by Anti-CCR5 Therapy in Cancer Patients

Cancer Cell. 2016 587-601

CCR5 inhibitors objective response

Colon Cancer Phase I

1. April 1, 2018, Pfizer, Phase I, Pembrolizumab + Maraviroc + MSS CRC

2. September 2018, Merck, Phase 2, Pembrolizumab + Vicraviroc + MSS CRC

CCR5 inhibitors objective response

Leronlimab Breast Cancer study

November 2018-March 2019

Phase II

Breakthrough (unmet need)

April 2019-July 2021 (Phase III)

Pro-140 525 mg 1sc/weekCarboplatin AUC 2q week

x3 28 days cycle

TNB

C R

x R

efr

ac

tory

CC

R5+

CTC

Endpoints

1. OS

2. PFS’

3. Decreased CTC

CCR5+ cells in Breast Cancer

Stem Cells

• Stem like cells –(mammospheres)

• Give rise to new tumors

• Contribute to therapy resistance

• Promote metastasis (necessary and sufficient)

• Single cell sequencing- volatility

• Enhanced DNA repair

• CCR5 inhibitors increase DDR cell death- >200%

Spend more time with your family and friends, eat your

favorite foods, visit the places you love.

cancer stem cells and therapeutic targeting via ccr5 · cancer stem cells and therapeutic targeting...

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