Targeting 1 Integrins
Integrins in
Multiple Myeloma
Lori Hazlehurst
Hazlehurst
H. Lee Moffitt Cancer Center and
Research Institute
Bone Marrow Microenvironment Contributes
to de-novo Resistance
Hematopoietic cell
VLA-4,
LFA-1
VLA-5
VLA-6
VLA-1

v 3
NCAM VLA-4
NCAM VCAMICAM
FN
CO LM VN
Bone Marrow
Marr
Stro
Str ma
Adhesion to FN protects myeloma patient
specimens from melphalan
pp
induced apo
p ptosis
p
100
90
Sus
80
FN
osis 70
60
Apopt 50
ific 40
30
Spec% 20
10
0
12
345
678
9
10
Melphalan [200 M]
Targeting Beta 1 Integrin
Integrin Signaling
FN
A. HYD1

Integrins
Src
ILK
Paxillin
P
P
FAK/Pyk2
p130cas
P
?
Bim
Topo II
Drug Resistance
HYD1 peptide
·
D-amino acid peptide
·
Combinatorial peptide chemistry- One bead one peptide technology
·
Functional cell binding assay to the bead.
·
Secondary screen was use of functional integrin blocking antibody
·
HYD1 inhibited beta 1 integrin mediated adhesion of prostate cells
to ECM proteins including laminin 1, FN, laminin 5 and collagen IV
·
HYD1S is a scrambled peptide derivative of HYD1 that does not
i h
n ibit
hibit dh
a
esion.
·
Work done by our collaborator Anne Cress, Ph.D.
HYD1 but not the scrambled peptide (HYD1S) inhibits cell
adhesion of H929 myeloma cells to FN
0.4
FN
BSA
0.3
OD 0.2
0.1
0 12.5 25 50 100 12.5 25 50 100 VC a4b1 a5b1
HYD1S [ug/ml]
HYD1 [ug/ml]
HYD1 but not the scrambled peptide (HYD1S) inhibits cell
adhesion of 8226 myeloma cells to FN.
0.5
0.4
FN
BSA
03
0.3
DO
0.2
0.1
0
12.5
25
50
100
12.5
25
50
100
HYD1S [ug/ml]
HYD1 [ug/ml]
HYD1 inhibits adhesion of 8226 cells to the BMS HS-5 cell line.
1800
1600
s
1400
HS-5
Unit
BSA
ce1200
n1000
800
luoresceF 600
400
200
0 12.5 25 50 100 12.5 25 50 100 VC I IgG
HYD1S [ug/ml]
HYD1 [ug/ml]
HYD1 does not inhibit adhesion of H929 cells to the BMS HS-5 cell line.
3500
HS-5
3000
BSA
s 2500
Unit
ce 2000
n
1500
luoresce 1000
F
500
0 12.5 25 50 100 12.5 25 50 100 VC
I
IgG
HYD1S [ug/ml]
HYD1 [ug/ml]
HYD1 reduces resistance associated with the bone marrow
stroma co-culture model of drug resistance in H929 cells
70
*
Suspension
*
h
60
Co-culture
deat
*
50
cell
40
specific
n
30
20
elphalaM
%
10
0
no peptide
HYD1 [50ug/ml]
HYD1S [50
[50ug/ l
m ]
l]
HYD1 reverses resistance associated with the bone marrow
stroma co-culture model of drug resistance in 8226 cells
*
Suspension
eath 40
Co-culture
ed 35
*
cellfic 30
25
speci
lan 20
15
Melpha 10
% 5
0
no peptide
HYD1 [50ug/ml]
HYD1S [50ug/ml]
[50ug/ml]
HYD1 induces cell death in suspension cultures
80
70
death
H929
60
cell
8226
50
cifice 40
sp
30
YD1H
% 20
10
0
12.5 ug/ml
25 ug/ml
50 ug/ml
100 ug/ml
-10
[HYD1]
[]
HYD1 induces minimal caspase 3 activation
s
70000
6 hr
60000
Unit
50000
ce
cells 40000
n 6 30000
10 20000
Per
0
luoresce
100
25
50
100
12.5
25
50
F
HYD1S
HYD1 [ug/ml]
Melphalan [uM]
24 hr
its
24 hr
110000
1
Un
90000
cells 70000
ence 6
c
0
c
50000
1
30000
Per 10000
Fluores
-10000
100
25
50
100
12.5
25
50
HYD1S
HYD1 [ug/ml]
Melphalan [uM]
HYD1 does not activate caspase 8
6000
6 hr
5000
Units
4000
e
ells
c
c6 3000
10 2000
rescen
er 1000
o
P
0
Flu
-1000
100
25
50
100
Trail 50 nM
6000
HYD1S
24 hr
HYD1 [ug/ml]
5000
Units
4000
ce
cells
n 6
3000
10
2000
1000
Per
uoresce
0
Fl
-1000
100
25
50
100
Trail 50 nM
HYD1S
HYD1 [ug/ml]
Z-VAD-FMK does not inhibit HYD1 induced cell death
15 uM melp VC
h l
VC
a an
75 ug/ml HYD1
104
.13%
104
3.08%104
0.14%
0.72%
9.32%
38.4%
103
103
103
102
102
PI
102
PI
90.7%
PI
59.6%
40.8%
101
101
101
6.05%
100
30.9%
20.2%
100
100
10 1
10 2
10 3
104
100
100
101
102
Annexin-FITC
103
10 4
100
101
102
103
104
Annexin-FITC
Annexin-FITC
VC + 200 uM Z-VAD
15 uM melphalan + Z-VAD
VA
75 ug/ml HYD1 + Z-VAD
VA
104
0.15%
104
104
0.25%
6.19%
0.9%
11.9%
41.2%
103
103
103
102
102
PI
PI
86.5%
102
PI
78.2%
43.1%
101
101
101
7.15%
9.66%
14.7%
100
100
100
100
101
102
103
104
100
101
102
103
10 4
100
10 1
10 2
10 3
104
Annexin-FITC
Annexin-FITC
Annexin-FITC
Caspase Independent
Independent Cell Death
· Autophagy-Bulk degradation of
of proteins
proteins and
organelles
­ Protective mechanism/Nutrient deprivation
­ Other stimuli-can lead to cell death.
· Formation of a double membrane (phagophore)
that expands and sequesters cytosolic and
organelles (autophagasome) fuses with a
lysosome (autophagolysosome)
lti
resu ng in th
the
eventual degradation of contents.
HYD1 treatment increases the number and size of
acidic vesicles in H929 cells
6 hr treatment
VC
100 ug/ml HYD1S
VC
100 ug/ml
15 uM melphalan
25 ug/ml HYD1
50 ug/ml HYD1
100 ug/ml HYD1
HYD1 treatment increases the number and size of acidic
vesicles in 8226 cells
6h
6 hr t
t
rea
t
men
VC
100 ug/ml
g
HYD1S
20 uM melphalan
25 ug/ml HYD1
50 ug/ml HYD1
100 ug/ml HYD1
HYD1 induces formation of double membrane
vacuoles
HYD1S treatment 4 hours
HYD1
4 hr treatment H929
autophagolysosome
Target Validation-MOA
· Partial agonist-Clustering but in the absence of adhesion
inappropri t
a /
e i
/i
l
ncomp t
e e i
s
li
gna ng occurs
lti
resu ng
l
ce lll
death
· Inhibitory peptide- Myeloma cells have basal activation
f
o b t
e a 1 it
integrin
d
me itd
diated i
s
l
gna iling in
i
suspens on
cultures.
8226
H929
2 hr
6 hr
2 hr
6 hr
1S
1
1S
1
1S
1
1S
1
D
D
D
D
D
D
D
D
HY
HY
HY
HY
HY
HY
HY
HY
P-Pyk2
Pyk2
Treatment Schedule for SCID-hu Model
Day 28: ELISA
HYD12
HYD1 2
/
mg k
/kg D i
a lily I.P. 14d
14 days
Day 29: Melphalan 1.5 mg/kg
Day 33: Melphalan 1.5 mg/kg
Day 1:Tumor cell inj
yjection
Day
Da 35
35 + 42 ELISA
ELIS
6 weeks
4 Weeks
Day 49 ELISA
Implant
remove bones
3
VC
HYD1
melphalan
levels
2.5
HYD1/melphalan
da
lamb
2
28y
1.5
levels/Da
tlambda
1
n
Curre
0.5
30
40
days post tumor cell inoculation (50,000 8226 cells)
Summary
· HYD1 has activity as a single agent in vitro and
in-vivo.
· Reduces resistance associated with BMS co-
culture model.
· HYD1 induced cell death is
is independent
independent of
capase activity.
· HYD1 results in increase size and number of
acidic vesicles.
· EM indicates that HYD1 induces large double
membrane vesicle suggestive of autophagy
gy.
­ Continuation of further preclinical development and
delineation of the MOA of HYD1
Acknowledgements
Moffitt Cancer Center
William Dalton
Michael Emmons
Melissa Alsina
Mohamad Hussein
Raul Argilagos
Hong Gong Wang
Daniel Sullivan
Bill Kerr
Supported by
U of A
Multiple Myeloma Research Foundation
Anne Cress
Bankhead-Coley Florida State Grant