Mechanism of
of the
the anti-myeloma
-
activity of PU-H71, a novel purine
scaffold HSP90 inhibitor
inhibitor
Saad Z. Usmani1, Robert D. Bona1,
2
1
Gabriela Chiosis2, Zihai Li
1.
Division of Hematology-Oncology, Lea's Center for Hematologic Disorders/ Neag
Comprehensive Cancer Center, University of Connecticut Health Center,
Farmington, CT
2.
Program in Molecular Pharmacology and Chemistry, Department of Medicine,
Memorial Sloan Kettering Cancer Center, New York, NY
Session: Lymphoma and Plasma Cell Disorders
Type: General Poster Session
Time: Saturday June 5, 8:00 AM to 12:00 PM
Location: S Hall A2
Learning Obj
gjectives
After reading and reviewing this material, the participant
should be
be better able to
to understand:
· In vitro anti-myeloma activity
yy of PU-H71, a novel HSP90
inhibitor.
· Mechanism of anti-myeloma activity of PU-H71
­ Activation of the unfolded protein response
­ Effects via potential inhibition of HSP90B1(a.k.a, gp96, grp94,
endoplasmin)
Outline Slide
· Role of unfolded protein response in plasma cell
dl
development
· Introduction to HSP90B1(a.k.a, gp96, grp94,
endl
dopl
i
asm n)
· In vitro anti-myeloma activity data on PU-H71, a
novel purine scaff
scaf old
f
inhibitor
inhibitor
Unfolded Protein Response:
Role in plasma cell development
·
Plasma cell differentiation requires the transcription factor XBP-1
Reinmold AM et al Nature 2001
·
XBP1 is required for plasma cell differentiation and the unfolded
protein response Iwakoshi et al Immunol Rev 2003; van Anken et al Immunity 2003
·
XBP1 is upregulated in MM primary plasma cells & its excess leads
to MM in a transgenic mouse model Carassco et al. Cancer Cell 2007
·
XBP1 regulates signal transduction, transcription factors and bone
marrow colonization in B cells Hu AC et al EMBO 2009
·
XBP-1-Deficient plasmablasts show normal protein folding but
altered gly
gycosylation and lipid sy
pynthesis McGehee AM et al J Immunol 2009
HSP90B1 (gp96), the endoplasmic
paralogue of cytosolic HSP90
HSP90
· HSP90 family
family member that resides predominantly in the ER
ER
· gp96, a.k.a. grp94, HSP90b1, Endoplasmin
· Implicated in B-cell function (physiological & pathological)
· Client proteins
­ Toll-like receptors (TLR): TLR1, TLR2, TLR4, TLR6, TLR7,
TLR9
­ 18 of 26 Integrins (4, L, V, 3, 7)
· Role in lymphopoeisis
· Mediates ER unfolded protein response (UPR)
Y Yang & Z Li. Mol Cell 2005; Y Yang et al. Immunity.2007;
B Lui & Z Li. Blood.2008; Staron et al. Blood 2009
PU-H71, a novel purine scaffold HSP90
inhibitor
·
Efficacy of the Novel Non-Quinone Based HSP-90 Inhibitor PU-H71
in JAK2V617F and MPLW515L-Induced Murine Models of
Myeloproliferative Neoplasms
Neoplasms . ASH 2009
·
A purine scaffold Hsp90 inhibitor destabilizes BCL-6 and has
specific antitumor activity in BCL-6-dependent B cell lymphomas.
Nat Med. 2009 Dec;15(12):1369-76.
·
Tt
Targe i
ting h
t
ea shock prot i
e n 90
90
i
w th
ith
ii
non-quinone i h
n ibit
hibitors: a novel
chemotherapeutic approach in human hepatocellular carcinoma.
Hepatology. 2009 Jul;50(1):102-12.
·
Hsp90 inhibitor PU-H71, a multimodal inhibitor of malignancy,
induces complete responses in triple-negative breast cancer
models. Proc Natl Acad Sci U S A. 2009 May 19;106(20):8368 73
-
Figure 1.
1. Dose-
Dose response curves
curves of human myeloma
cell lines exposed to PU-H71 or control for 24 hours.
(A) Drug sensitive cell lines; IC50 ranging from 100 nM to 300 nM.
Figure 1.
1. Dose-
Dose response curves
curves of human myeloma
cell lines exposed to PU-H71 or control for 24 hours.
(B) Drug resistant cell lines; IC50 ranging from 100 nM to 300 nM.
A
B
C
Figure 2. Dose-response of PU-H71 on the unfolded
protein response
response, apoptosis
apoptosis and cell cycle.
(A) Increased protein levels of IRE1a, XBP1 and grp78 (B)
Upregulation of phospho-eIF2. (C) Increased cleavage of caspase
3 and PARP
D
E
Cyclin D1
100
80
60
Max
of 40
%
20
0
100
101
102
103
104
F
D
E
Figure 2. Dose-response of PU-H71 on the unfolded
F
protein response
response, apoptosis
apoptosis and cell cycle.
(D) DNA content by PI staining showing decrease in cells in S/G2
phase. (E) Decline in Cyclin D1 after 24 hour drug exposure (F)
Increased protein levels of p27kip1 after 24 hour drug exposure
A
B
C
E
Figure 3. Generation of HSP90B1 (gp96) knockdown
F
human myeloma cell line.
.(A) Relative surface expression of selected integrins and gp96 in
human myeloma cell lines. (B) Schema representing gp96
dependent and independent integrins (C) gp96 knockdown, integrin
expression on RPMI-8226 cell line.
AB
CD
E
Figure 4. Dose-response of anti-myeloma drugs on
F
RPMI-8226 empty vector and gp96 knockdown
knockdown cells
(A)PU-H71 (B) 17-AAG (C) 17-DMAG (D) Dexamethasone
EF
GH
E
Figure 4. Dose-response of anti-myeloma drugs on
F
RPMI-8226 empty vector and gp96 knockdown
knockdown cells
(E) Melphalan (F) Thalidomide (G) Doxorubicin (H) Bortezomib
Summary
PU-H71 has potent in vitro anti-myeloma activity
PU H71
-
i d
n uces
l
mye oma
ll
ce
f
un l
o d
lded
t
pro i
e n
response and apoptosis via caspase dependent
pathway.
py
It works in part by inhibition of HSP90B1 (a.k.a. gp96,
grp4, endoplasmin, etc.) as observed by decreased anti-
myeloma activity
activity in
in gp96 knockdown cells
cells.
HSP90B1 or gp96 knockdown cells appear more
susceptible
p
to most anti-myeloma drug
yg classes.
Future Directions
Compare unfolded protein response and apoptosis in
gp96 knockdown cells
cells and empty vector
vector cells
cells
In vivo studies in SCID/NOD mice
THIS WORK IS SUPPORTED BY THE UNIVERSITY OF
CONNECTICUT HEALTH CENTER MULTIPLE MYELOMA
SEED GRANT 2009 AND THE LEA'S FOUNDATION FOR
LEUKEMIA RESEARCH