Mi-63, a small molecule inhibitor of MDM2-p53 interaction, has significant in vitro activity in Multiple Myeloma
Shaji Kumar, MD1, Michael Kline, PhD1, Terry Kimlinger, BA1, Michael Timm1, Jessica Haug1, Dajun Yang, MD,PhD2 and S. Vincent Rajkumar, MD1. 1Hematology, Mayo Clinic, Rochester, MN, United States, 55905 and 2Ascenta
Therapeutics, San Diego, CA, United States.
Background
Results
0 hr
6 hrs
12 hrs
18 hrs
24 hrs
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· Multiple myeloma (MM) is a clonal plasma cell disorder that
MM1.
MM1 S
characterized by low proliferative and apoptotic rates compared to other
120
RPMI-8226
120
MM1
MM R
OPM2
LR5
LR
malignancies.
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Dox
100
l)
l
100
ro
tron
nt
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o
·The tumor suppressor gene p53, responsible for induction of cellular
80
c
fc
80
fc
80
o
of%
apoptosis in response to genotoxic stimuli, is relatively intact in most
(%
(
yit 60
60
il
lity
b
lbi
cases of myeloma. However, p53 mutations or deletion can occur late in
iaV
iaV
40
40
the course of disease.
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20
·Here we evaluate a novel small molecule inhibitor (Mi63) of the
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0
interaction between p53 and its negative regulator, MDM2, in the setting
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2.5
2
5
7.5
7
10
15
20
25
0
2.5
5
7.5
10
15
20
25
Drug
Dru Dose
o
(uMo
(uM l
o )
l
Dru
Dr g Dose (u
( Mol)
l
of myeloma.
BrdU
Mi63 was cytotoxic to several myeloma cell lines, including those resistant to conventional drugs as
Mi-63 inhibits proliferation of myeloma cells (RPMI top row and MM1.S
measured by MTT assay after 48 hours of incubation.
Results
bottom row) measured by BrdU incorporation
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1
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70
·Mi-63 was cytotoxic to several different myeloma cell lines with a median
90
90
80
80
effect observed at approximately 2.5 µM in cell lines including MM1.S that
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express wild type p53 and between 10-15 µM in cells with mutated p53 as
9.2%
8.8%
8.
50
50
40
50
40
measured using an MTT cell viability assay. Additionally, Mi63 induced
30
30
20
2.8%
3.7%
3.
20
bcl xl%
cytotoxicity in myeloma cell lines resistant to conventional agents such as
40
10
10
2.7
bcl2%
po 0
0
A
APO%
O
Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R),
010
01
25
2
50
5
010
25
2
50
ing
mcl1
l %
s 70
100
s
30
e
indicating non-overlapping mechanisms.
bax %
pr
90
60
ex
80
lls 50
70
ce
20
%
60
·To evaluate the ability of the drug to induce cell death in the tumor
40
50
7.7%
10.7%
10.
30
40
microenvironment, MM cells were co-cultured with marrow stromal cells
10
20
30
20
20
or in the presence of VEGF or IL-6, two cytokines known to be important
10
10
4.6%
23.7%
23.
0
PI
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0
for myeloma growth and survival. Mi63 was cytotoxic to myeloma cells
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25
010
25
0
2 hr
h s4
r
hr
h s8
r
hr
h s
r
none
ke
ke
k
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ke
k
ke
under these conditions as well, at doses similar to those seen with myeloma
Annexin V
cells alone.
Mi-63 induces time dependent apoptosis of
Mi-63 treatment leads to changes in anti and pro-apoptotic
Mi-63 induces apoptosis of primary patient cells as measured
myeloma cell lines measured by Annexin/PI
molecule expression measured by flow cytometry
by Apo 2.7 positive cells
·Mi63 was able to inhibit proliferation and induce apoptosis in myeloma
cells in a dose- and time-dependent fashion, as demonstrated by flow
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2 hrs
4 hrs
8 hrs
Conclusions
cytometry using Annexin/PI staining as well as cell cycle studies.
·Treatment of myeloma cells with Mi63 was associated with early
Mi-63 has significant activity in vitro in the setting of myeloma as demonstrated
mitochondrial membrane depolarization, inversion of Bax/Bcl-2 ratio, and
by its effect on myeloma cell lines and primary patient cells. It clearly induces
apoptosis in myeloma cells, with higher activity seen in cells with wild type
down
regulation
of
Mcl-1,
indicating
induction
of
mitochondrial
12.1%
7.6%
7.6
16.8%
42.6%
p53. Given the lack of p53 abnormalities in most of the patients with myeloma,
mechanisms of cell death.
this drug alone or in combination is likely to have significant clinical activity.
red
·Mi63 was also cytotoxic to freshly isolated primary patient myeloma cells,
Studies combining this with various DNA damaging drugs are in progress.
green
These studies will eventually form the framework for future clinical studies.
inducing apoptosis in a dose-dependent manner. In the patient cells the
Mi-63 treatment leads to mitochondrial membrane depolarization measured by Mitocapture® assay
drug appears to have a differential effect on the CD45 + and - cells.
Disclosures: DY is an employee of Ascenta Pharmaceuticals, other authors have no disclosures

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