Hematology Department
Cancer Research Center
Universit
Univ
y Hospital Salamanca
University of Salamanca
Reversibility of the Resistance
to Lenalidomide & Pomalidomide and
a
Absence of Cross-Resistance
in a Murine Model of MM
Enrique M. Ocio1,2, Diego Fernandez-Lazaro2, Laura San-Segundo2, Lorena Gonzalez-Mendez2,
Montserrat Martin-Sanchez2, Mercedes Garayoa2, Teresa Paino2, Antonio Garcia-Gomez2, Norma
Gutierrez1,2, Manuel Delgado2, Juan C Montero2, Nuria Quintana3, Jose L. Garcia3, Atanasio
Pandiella2, Jesus F. San Miguel1,2
1. Hospital Universitario de Salamanca. Spain
2. Cancer Research Center (CSIC-IBMCC). University of Salamanca. Spain
3. Celgene Corporation. Madrid. Spain
Activity of IMiDs + Dex in R/R MM
Lenalidomide
(RevlimidTM CC-5013)
O
O
H
N
2 Phase III2 (n=702)
Thalidomide
N
O
60% PR
O
O
H
TTP: 13.4 months
N
NH2
N
O
Pomalidomide
O
(ActimidTM CC-4047)
Metaanalysis1 (n=451)
O
O
H
46% PR
N
Phase II3 (n=60)
N
O
EFS: 8 months
63% PR
PFS: 11.6 months
O
NH2
1. von Lilienfeld-Toal M, Eur J Haematol. 2008
2. Weber D, NEJM 2007, Dimopoulos M, NEJM 2007. Dimopoulos M, Leukemia 2009
3. Lacy, JCO 2009
1
Is Retreatment with IMiDs feasible?
Salvage IMiD
Thalidomide
Lenalidomide
1st Line IMiD (n)
Thal (23)
Len (11)
Thal (58)
Len (48)
Response Rate ( PR)
Overall (n=140)
30%
20%
48%
54%
Pts <PR with 1st IMiD (n=28)
0%
0%
64%
33%
Pts that discontinued 1st IMiD due to PD (n=17)
0%
25%
50%
25%
Thalidomide is not able to rescue previous resistance to IMiDs
Lenalidomide rescues from Thal resistance and retreatment is possible
Madan. Blood 2011
Activity of Pomalidomide + Dex in Len refr. pts
n
Population
Dose
PR
PFS/TTP/DOR
Lacy1
34
Len refr
2 mg (1-28)
32 %
PFS 4.8 m
4 mg (1-21)
35 %
PFS 6.3 m
Leleu2
84
Len & Btz refr
4 mg (1-28)
34 %
PFS 6.3 m
2 mg (1-28)
26 %
DOR 12 m
Lacy3
70
Len & Btz refr
4 mg (1-28)
26 %
DOR NR
Dex 40 mg weekly
1. Lacy. Leukemia. 2010 Nov;24(11):1934-9
2. Leleu ASH 2011. Abstract 812
3. Lacy ASH 2010 . Abstract 863
2
Murine Model of Resistance to IMiDs
·
Human subcutaneous plasmacytoma model in mice
CB17-SCID mice implanted sc with 3 x106 MM1S cells
·
Once tumors became palpable, mice were randomized to:
·
Vehicle (control)
·
LD: Lenalidomide 25 mg/Kg x 5/w + Dex 1 mg/Kg x 2/w
·
PD: Pomalidomide 7 mg/Kg x 5/w + Dex 1 mg/Kg x 2/w
Tumor volume
· Monitorization of
Weight
three times / week
Clinical features
· Mice were sacrificed when tumor diameter reached 2 cm
Develop MM cells in vivo resistant to IMIDs
3500
Control
3000
Cont LD
Cont PD
2500
(mm3) 2000
th
Grow 1500
mor
Tu 1000
500 mm3
500
Limit for Resistance
0
0
1020304050
607080
Days of treatment
After 30 days of sensitivity tumors develop resistance to LD & PD
3
Develop MM cells in vivo resistant to IMIDs
TT500
Tumor growth after 500 mm3
p
1.0
C vs LD
0.01
4500
C vs PD
0.001
4000
0.8
LD vs PD
<0.05
3500
aliv
3000
0.6
(mm3)
Surv
2500
Control
lume
Cum.0.4
o 2000
V
Cont LD
1500
mor
Cont PD
0.2
Tu 1000
500
0.0 0
10
20
30
40
50
60
0
Days
0
1020
3040
50
Days of treatment
TT500 Median (range)
Contro
8 (0-27)
,... once they reach 500 mm3 they
l
become completely resistant to
LD
42 (35-48)
the administered treatment.
PD
53 (45-60)
Although treated tumors have longer TT500, ...
Ex vivo sensitivity of resistant cells
Cells excised from control & resistant tumors were ex vivo treated with LD & PD
Doses: Pomalidomide 10 µM, Lenalidomide 10 µM & Dex 10 nM for 5 days
LD
PD
100
100
80
80
60
60
40
40
20
20
Control
Res LD
Control
Res PD
n=3
n=3
n=3
n=3
In vivo resistance confers resistance to ex vivo treatment
4
LD & PD do not present Cross-Resistance
Tumors that had developed resistance to LD & reached 1.700 mm3,
where switched to receive PD
4000
3500
3000
2500
(mm3)
Cont. LD
2000
lume
LD - PD
ov
1500
mor
Change to PD
Tu
1000
500
0
-61
-54
-47
-40
-33
-26
-19
-12
-5
1
8
15
21
28
Days from treatment change
PD overcomes resistance to LD
LD & PD do not present Cross-Resistance
Tumors that had developed resistance to PD & reached 1.700 mm3,
where switched to receive LD
4000
3500
3000
2500
(mm3)
Cont. PD
2000
lume
PD - LD
ov
1500
Change to LD
mor
Tu
1000
500
0
-61
-54
-47
-40
-33
-26
-19
-12
-5
1
8
15
21
28
Days from treatment change
LD overcomes resistance to PD
5
PD is more potent than LD in reverting Resistance
Tumor growth
Maximal Response
3000
LD
2500
p=0.005
PD
(mm3) 2000
100
1500
lume
)
ov 1000
75
mor
olume)
500
Tu
esponse
V
R
ial 50
0
-61
-54
-47
-40
-33
-26
-19
-12
-5
1
8
15
21
28
init
Days from treatment change
of
Maximal
(% 25
1.0
TTP
n=10
n=10
0
LD
PD
0.8
aliv
TTP
Median (range)
0.6
Surv
LD
18 (0-28)
0.4
PD
27 (19-52)
Cum.
p=0.004
0.2
0.0 0
10
20
30
40
50
60
Days
What about tumors that develop resistance to
the 1st IMID and, later on, to the 2nd one
These mice were again treated with the 1st IMID to whom they had initially
developed resistance, and, surprisingly, they respond again
Previous LD resistance
Previous PD
resistance
3000
3000
2500
2500
(mm3) 2000
(mm3) 2000
lume 1500
lume 1500
o
o
V
V
1000
1000
mor
mor
u
U
T 500
500
T
0
0
-19 -17 -15 -12 -10 -8 -5 -3 -1 1 3 5 8 10 12
-18 -17 -15 -12 -10 -8 -5 -3 -1 1
3
5
8 1012151719 21232528303235
Days from treatment change
Days from treatment change
LD - PD
PD - LD
Change to LD
Change to PD
LD - PD - LD
PD - LD - PD
Resistance to PD & LD is reversible after an intermediate treatment with
the alternative IMID
6
Mechanisms of Resistance to IMIDs + Dex
Untreated (Control)
Fresh cells from tumors
Treated & responding (Sensitive)
Treated & non responding (Resistant)
were extracted and changes in Gene Expression Profile were
analyzed by Microarrays.
Control
Control
Sensitive LD
Resistant LD
Sensitive PD
Resistant PD
GEP of cells untreated, sensitive & resistant to IMiDs + Dex
GEP of cells excised from control, sensitive & resistant tumors was analyzed
Lenalidomide + Dex
Pomalidomide + Dex
Sensitivity
Resistance
Sensitivity
Resistance
SLD vs Control
RLD vs Control
SPD vs Control
RPD vs Control
0
22
9
2
194
30
CS
S S
CC R R R
Control
RPD
SPD
The genomic deregulation induced by IMIDs + Dex reverts to the basal
situation in the resistant cells
SAM algorithm (FDR < 0.1%)/Hierarchical Clustering (Cluster and TreeView software)
7
GEP of cells untreated, sensitive & resistant to PD
GEP of cells excised from control, sensitive & resistant tumors to PD was analyzed
CELLULAR MACROMOLECULE
MEMBRANE
SIGNAL TRANSDUCTION
METABOLIC PROCESS
SPD Ctrl RPD
SPD Ctrl RPD
SPD Ctrl RPD
Among the genes deregulated in the sensitive and Resistant cells several
components of the MEK/ERK pathway
Gene Set Enrichment Analysis (GSEA)
Role of MEK/ERK pathway in IMIDs' activity & resistance
Fresh cells from untreated, sensitive and resistant tumors to LD & PD were extracted and
different signaling pathways were analyzed by WB
Control
LD
PD
Sensitive
Resistant
Sensitive
Resistant
RAS
p-RAF
RAF
74 KDa
p-MEK-1/2
MEK-1,2
60KDa
MEK 1/2 inh.
p-ERK-1/2
ERK-1,2
AZD-6244
42,44 KDa
ARRY 142886
Selumetinib
ERK-(total)
42 KDa
Tumor
Growth
differentiation
& R
Treatment
esistance
with IMIDs + Dex induces a decrease of pERK whereas
resistance to PD is associated with a significant activation of this pathway
8
Ex vivo combination of MEK 1/ 2 inhibitor with PD
Cells from tumors resistant to PD were excised and ex vivo treated with PD +/- AZD 6244
Doses: Pomalidomide 10 µM, Dex 10 nM & AZD-6244 100 nM (2 pulses) for 5 days
Cells from control tumors
Cells from RPD tumors
100
100
80
80
cells
cells
+V
+
60
V
60
nnexin 40
nnexin
40
A
A
%
%
20
20
0
0
Ex vivo Treatment
PD
AZD
PD + AZD
PD
AZD
PD + AZD
The addition of a MEK 1/2 inhibitor completely overcomes the resistance
to PD ex vivo
In vivo treatment with MEK 1/2 inhibitor AZD6244
In two mice with tumors resistant to PD a MEK 1/2 inhibitor was added
AZD-6244 10 mg/Kg /12 h
2000
1750
RPD
1500
AZD-6244
1250
RPD + AZD
(mm3)
1000
lumeoV
750
mor
Tu 500
250
0
1
8
15
21
29
36
42
49
56
63
70
77
84
91
94
Days
In vivo inhibition of MEK ½ partially abrogates PD resistance
9
Expression of CRBN & IRF-4 in sens. & res. tumors
Cells from tumors sensitive & resistant to LD & PD were excised and CRBN & IRD-4
expression were analyzed by PCR
CRBN
IRF-4
1.8
1.6
1.6
1.4
ion 1.4
s
on
s
1.2
1.2
prex
1.0
E 1.0
e
0.8
*
tiv 0.8
la
ativeExpressi 0.6
Re 0.6
4RelF- 0.4
0.4
IR
CRBN
n=3 mice per group
0.2
0.2
* p<0.05 as
0.0
0.0
compared to
control
Treated tumors tend to have higher
Treatment decreases the expression
expression CRBN while it tends to
of IRF-4, mostly in PD, but it is
decrease in the resistant tumors
restored in the resistant setting
Summary
·
We have developed an in vivo model of acquired resistance to IMiDs + Dex & in this
model:
·
Lenalidomide + Dex & Pomalidomide + Dex do not present cross resistance,
probably indicating different mechanisms of resistance. Pomalidomide was
more potent.
·
Resistance to IMiDs + Dex is reversible after a period without the treatment
·
Cells that develop resistance to IMiDs + Dex turn-off most the GEP changes
induced after treatment with these drugs
·
The upregulation of the MEK/ERK pathway seems to be an important
mechanism in the acquired resistance to Pomalidomide + Dex.
·
Treated tumors tend to have higher expression CRBN while it decreases in the
resistant tumors. Treatment decreases the expression of IRF-4 but it is restored
in the resistant setting in the case of PD
10
Acknowledgments
Cancer Research Center
Universit
Univ
y Hospital
Laboratory 15
Laboratory 12
Department of Hematology
Atanasio Pandiella
Enrique M. Ocio
Mª Victoria Mateos
Juan Carlos Montero
Teresa Paíno
Ramón García-Sanz
Stela Álvarez-Hernández
Mercedes Garayoa
Norma C. Gutiérrez
Xi Chen
Patricia Maiso
Bruno Paiva
Elena Díaz
Laura San Segundo
Luis Corchete
Diego Fernández-Lázaro
Antonio García
Montserrat Martín
Jesús F. San Miguel
Lorena González
University of Salamanca - Spain
S
11