Inhibition of E-Selectin or E-selectin together with CXCR4 Re-sensitizes Multiple Myeloma to Treatment Barbara Muz 1 , Ph.D. Henna Bazai 1 , Anita Sekula 1 , William Fogler 2 , Ted Smith 2 , John Magnani 2 and Abdel Kareem Azab 1 1 Department of Radiation Oncology, Cancer Biology Division, Washington University in St. Louis School of Medicine, St. Louis, MO, USA; 2 GlycoMimetics Inc., Rockville, MD, USA AACR Annual Meeting 2017, Washington DC Minisymposium – Microenvironmental Cues in Immune Escape and Therapy Resistance Tuesday, April 4 th 2017
Transcript
Inhibition of E-Selectin or E-selectin together with CXCR4 Re-sensitizes Multiple Myeloma to Treatment
Barbara Muz 1, Ph.D.
Henna Bazai1, Anita Sekula1, William Fogler2 , Ted Smith2, John Magnani2 and Abdel Kareem Azab1 1Department of Radiation Oncology, Cancer Biology Division, Washington University in St. Louis School of Medicine, St. Louis, MO, USA; 2GlycoMimetics Inc., Rockville, MD, USA
AACR Annual Meeting 2017, Washington DC Minisymposium – Microenvironmental Cues in Immune Escape and Therapy Resistance Tuesday, April 4th 2017
Disclosure Information
I have no financial relationships to disclose.
This study was partially supported by GlycoMimetics Inc.
- and -
I will not discuss off label use and/or investigational use in my presentation.
AACR Annual Meeting 2017, Washington DC Minisymposium – Microenvironmental Cues in Immune Escape and Therapy Resistance
Presenter: Barbara Muz, Ph.D.
Multiple myeloma (MM)
Lu et al 2012
Areas of active myeloma (PET scan)
4 April 2017 Barbara Muz
• Plasma cell malignancy localized mainly in the bone marrow
• Characterized by metastasis of MM cells across the skeletal system
• The progression of MM involves a continuous egress (re-circulation) of the tumor cells in the peripheral blood and homing (re-entrance) into the bone marrow
Bone Marrow Primary Site Bone Marrow Secondary Site
Egress Homing
Cellular trafficking in hematologic malignancies
Blood Vessel
The supportive role of the bone marrow microenvironment in MM
Azab AK et al. Blood 2012;119:1468-1478 (Selectins and endothelial cells) Azab AK et al. Blood 2009;113:4341-4351 (Chemokines an stroma) Azab AK et al. Blood 2009;114:619-29 (ECM and stroma) De la Puente P et al. Haematologica 2016 Jul;101(7):e307-11 (MSP-1) De la Puente P et al. Biomaterials 2015 (3DTEBM)
Bone Marrow
Accessory Cells
Extracellular Matrix Soluble factors
4 April 2017 Barbara Muz
Blood vessels
• The interactions of tumor cells with their bone marrow microenvironment facilitate tumor progression, metastasis and drug resistance
Muz B et al. BioMed Res Int 2015; 2015:417586 (P-selectin and PSGL-1) Muz B et al. Mol. Cancer Res 2015 (Cell trafficking) Muz B et al. Blood Cancer J 2015 (Hypoxia) McMillin DW et al. Nat Med 2010;16:483-9 (Stroma) Fulciniti M et al. Clin Cancer Res 2009;15:7144-52 (IL-6)
Cellular compartment:
Non-cellular compartment:
BLOOD VESSEL
ENDOTHELIUM
Rolling
Adhesion
Extravasation
Stromal cells
BONE MARROW Homing
STROMA
Signaling Pathways
Eph-B2 FGFR3
VLA4
Fibronectin VCAM
E-selectin : CLA
CXCR4 : SDF-1
Targeting cell trafficking as a strategy to sensitize MM cells
Selectins
Chemokines
Integrins
RTK’s
Extracellular Matrix
To test the role of E-selectin (GMI-1271) and E-selectin/CXCR4 (GMI-1359)
antagonists on MM cell trafficking in vitro and in vivo as a potential approach to
overcome bone marrow microenvironment-induced drug resistance
Aim
4 April 2017 Barbara Muz
4 April 2017 Barbara Muz
0
5
10
15
20
25
E-selectin CLA CXCR4
Pro
tein
Exp
ress
ion
(
MFI
tar
get/
MFI
iso
typ
e)
MM1s
H929
RPMI8226
0
5
10
15
20
25
E-selectin CLA CXCR4
Pro
tein
Exp
ress
ion
(
MFI
tar
get/
MFI
iso
typ
e)
HUVECs
MSP1
HS5
Expression of cell surface molecules in endothelial, stromal and myeloma cells
MSP1 – MM-derived stromal cell line (de la Puente P et al. Haematologica 2016 Jul;101(7):e307-11) CLA - Cutaneous Lymphocyte Antigen (Rossiter H et al. European J Immunol 1994;24(1): 205-10) HUVECs – human umbilical vascular endothelial cells SDF-1 – stromal-derived growth factor-1 HS5 – stromal cell line (normal)
0
20
40
60
80
100
120
untr GMI-1271 GMI-1359
# o
f M
M.1
S ce
lls m
igra
ted
(R
ela
tive
to
un
tre
ate
d)
HUVECs media
SDF1 50nM
HS5 media
MSP1 media
4 April 2017 Barbara Muz
(20mM) (20mM)
In the presence of SDF-1, GMI-1359 inhibits MM cell chemotaxis more effectively than GMI-1271
Boyden Chamber
METHOD
Conditioned media: HUVECs
HS5 MSP1
MM.1S cells pre-treated with GMI-1271 or GMI-1359 for 1hr
6hrs
HUVECs – human umbilical vascular endothelial cells SDF1 – stromal-derived growth factor-1 HS5 – stromal cell line (normal) MSP1 – MM-derived stromal cell line (de la Puente P et al. Haematologica. 2016 Jul;101(7):e307-11)
HUVECs MSP1
Normoxic RPMI8226
METHOD
0
50
100
150
200
250
untr GMI-1271 GMI-1359
Tran
s-En
do
the
lial M
igra
tio
n
(% o
f u
ntr
eat
ed
no
rmo
xic
MM
ce
lls)
Normoxia
Hypoxia
Hypoxic RPMI8226
4 April 2017 Barbara Muz
(20mM) (20mM)
GMI-1359 inhibits MM cell trans-endothelial migration more effectively than GMI-1271, especially under hypoxic conditions
Labeled MM.cells
Cultured in Normoxia or Hypoxia (1%O2) for 24hrs
Pre-treated with GMI-1271 or GMI-1359 for 1hr
6hrs
0
50
100
150
200
250
Untreated GMI-1271 GMI-1359
Nu
mb
er
of
circ
ula
tin
g M
M c
ells
(
in 1
0,0
00
MN
Cs)
MM Untreated
(Calcein Violet)
MM GMI-1271
(Calcein Orange)
MM GMI-1359
(Calcein Green)
Mix
IV injection
Sacrifice at 90 mins Collect Blood
Analyze by flow cytometry
4 April 2017 Barbara Muz
(20mM) (20mM)
GMI-1359 inhibits extravasation of MM cells to the bone marrow in vivo
METHOD
0
5000
10000
15000
20000
25000
30000
0 7 14 21
Tum
or
gro
wth
(R
OI n
orm
aliz
ed
to
Day
0)
Days post-treatment initiation
vehicle
GMI-1271 (40mg/kg)
Lenali (25mg/kg)
Combo
*
In vivo - Human Xenograft Disseminated Mouse Model
4 April 2017 Barbara Muz
GMI-1271 in combination with lenalidomide overcomes stroma-induced drug resistance in vitro and inhibits tumor growth in vivo
0
20
40
60
80
100
120
vehicle GMI-1271 Lenali Combo
Surv
ival
(%
of
veh
icle
)
In vitro – MTT assay
**
MM.1S with stroma
MM.1S alone
PO QDx20
IP QDx20
Treatment N MST
(days) P vs
saline P vs CFZ
saline 10 36.5 --
GMI-127140 mg/kg IP QDx14
10 36.5 0.5212
CFZ 3 mg/kg IV QDx2
10 40.0 0.0274 --
GMI-1271 + CFZ 10 49.5 0.0001 0.0006
In vivo - Syngeneic 5TGM1 Disseminated Mouse Model
10 20 30 40 50 60
0
20
40
60
80
100
Days Post Tumor Injection
Perc
ent
Surv
ival
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0
20
40
60
80
100
120
vehicle GMI-1271 CFZ Combo
Surv
ival
(%
of v
eh
icle
)
GMI-1271 in combination with carfilzomib (CFZ) overcomes stroma-induced drug resistance in vitro and prolongs mice survival
*
In vitro – MTT assay
MM.1S with stroma
MM.1S alone
Treatment N MST
(days) P vs
saline P vs CFZ
saline 10 32.5 --
GMI-1359 40 mg/kg IP QDx14
10 34 0.5215
CFZ 3 mg/kg IV QDx2
10 38.5 0.0002 --
GMI-1359 + CFZ 10 49 0.0001 0.014
In vivo - Syngeneic 5TGM Disseminated Mouse Model
20 25 30 35 40 45 50 55 60 65 70
0
20
40
60
80
100
Days Post Tumor Implants
% S
urv
ivin
g
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0
20
40
60
80
100
120
vehicle GMI-1359 CFZ Combo
Surv
ival
(%
of
veh
icle
)
GMI-1359 in combination with carfilzomib (CFZ) overcomes stroma-induced drug resistance in vitro and prolongs mice survival
*
In vitro – MTT assay
MM.1S with stroma
MM.1S alone
Summary
0
5
10
15
20
25
MM1s
0
5
10
15
20
25HUVECs
MSP1• Endothelial cells (HUVECs) and stromal cells (MSP-1 and HS5) express high levels of E-selectin
• CXCR4 is highly expressed on MM cell lines; CLA is highly expressed in RPMI8226
• In vitro, MM cell adhesion, chemotaxis, and trans-endothelial migration is decreased by GMI-1271 and even further by GMI-1359, in the presence of SDF-1
0
20
40
60
80
100
120
0 10 20 50
RPMI8226
0
50
100
150
200
250
Untreated GMI-1271 GMI-1359
Nu
mb
er o
f ci
rcu
lati
ng
MM
ce
lls (
in 1
0,00
0 M
NC
s) • In vivo, GMI-1359 significantly inhibits extravasation of MM cells to the bone marrow
0
20
40
60
80
100
120
vehicle GMI-1271 CFZ Combo
Surv
ival
(% o
f ve
hic
le)
H929 alone H929 with MSP1
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• In vitro, GMI-1271 and GMI-1359 combined with either lenalidomide or carfilzomib overcome stroma-mediated drug resistance
• In vivo, GMI-1271 in combination with lenalidomide reduces tumor growth
• Mice survival is prolonged by GMI-1271 combined with carfilzomib, and even further by GMI-1359 combined with carfilzomib
20 25 30 35 40 45 50 55 60 65 70 0
20
40
60
80
100
Days Post Tumor Implants
% S
urv
ivin
g
0
10000
20000
30000
0 7 14 21
vehicle
GMI-1271 (40mg/kg)
Lenali (25mg/kg)
Combo
Acknowledgements
Kareem Azab Henna Bazai Anita Sekula Feda Azab Pilar de la Puente Cinzia Federico Micah Luderer Hubert Kusdono
