cancer stem cells and therapeutic targeting via ccr5 · •founder and cso of aaa phoenix and...
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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
1/b-
cate
nin
Year of Publication by Pestell Lab p
21
Cip
1
20
01
No
tch
NFk
B
Pp
arg
Dac
h1
20
03
2005
20
07
20
09
2011
20
13
20
15
20
17
Cyc
lin D
1
C-J
un
Cyc
lin D
1
• 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
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
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 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
acute GVHD are 38.5% for grade II to IV disease
and 21.9% for grade III or IV disease.
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
acute GVHD are 38.5% for grade II to IV disease
and 21.9% for grade III or IV disease.
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
acute GVHD are 38.5% for grade II to IV disease
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
acute GVHD are 38.5% for grade II to IV disease
and 21.9% for grade III or IV disease.
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
acute GVHD are 38.5% for grade II to IV disease
and 21.9% for grade III or IV disease.
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%
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