cdk8 inhibitor sel120-34a has therapeu?c efficacy in ......0.008 µm 0.04 µm 0.2 µm 1 µm 5 µm...
TRANSCRIPT
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CDK8InhibitorSEL120-34AHasTherapeu?cEfficacyinMurineandHumanAMLModels
Disclosures:Mazan,Majewska,Wiklik,Combik,Masiejczyk,Fiedor,Obacz,Bialas,Chesy,Gabor-WorwaSelvitaS.A.:Employment.Brzózka:SelvitaS.A.:Employment,EquityOwnership,Membershiponanen?ty'sBoardofDirectorsoradvisorycommiXees.Rzymski:SelvitaS.A.:Employment,EquityOwnership.Fioretos:Cantargia:EquityOwnership,Membershiponanen?ty'sBoardofDirectorsoradvisorycommiXees.Flygare:LUHolding:Patents
&Royal?es:Patent.Otherauthorshavenorela?onshipstodisclose.
Background• Cyclin-dependentkinase8(CDK)inhibitorshavean:-leukemic
ac:vityinhumanAMLcelllines.• Efficacyhasbeenassociatedwithac:va:onofsuperenhancer
regions(Pelishetal,Nature,2015).• SEL120-34A,aCDK8/CDK19inhibitor,hasan:-leukemiceffectin
apanelofAMLcelllines,associatedwithareduc:onofSTAT5S726phosphoryla:on(Rzymskietal,Oncotarget,2017).
M.Chapellier1,J.Chen2,C.Sandén1,M.Mazan3,E.Majewska3,K.Wiklik3,M.Combik3,M.Masiejczyk3,E.Fiedor3,M.Obacz3,A.Polak4,A.Białas3,P.Chęsy3,E.Gabor-Worwa3,A.Grochowska5,U.Kuklińska5,Z.Sandowska-Markiewicz5,M.Statkiewicz5,M.Kopczynski5,M.Cybulska5,M.Mikula5,P.Juszczynski4,K.Brzózka3,T.Rzymski3,T.Fioretos1,J.Flygare2*andM.Järås1*
1DivisionofClinicalGene?cs,DepartmentofLaboratoryMedicine,LundUniversity,Lund,Sweden;2DivisionofMolecularMedicineandGeneTherapy,LundStemCellCenter,LundUniversity,Lund,Sweden;3R&Ddepartment,SelvitaS.A.,Kraków,Poland;4Ins?tuteofHematologyandTransfusionMedicine,Warsaw,Poland;5DepartmentofGene?cs,MariaSklodowska-CurieMemorialCancerCenterandIns?tuteofOncology,Warsaw,Poland;*Equalcontribu?on
Travelgrant
1.TEXcellsaresensi?vetoCDK8inhibi?on
2.SEL120-34AinhibitsmurineMLL-AF9AMLcells
4.SEL120-34AreducesleukemiaburdeninamurineAMLmodel
5.SEL120-34AinhibitsAMLpa?entcellsinvitro
6.SEL120-34Ashowedstrongan?-leukemiceffectinanAMLPDXmousemodel
Conclusion• CDK8inhibi:onresultedinreducedAMLcellgrowthin
humanandmurineAMLcellsinvitro.• TreatmentofleukemicmicewithSEL120-34Aresultedin
reducedleukemiaburdeninbonemarrowandblood.• SEL120-34AtreatmentofmicetransplantedwithAMLPDX
cellsresultedinstrongan:-leukemicac:vity.
AMLpa:entderivedxenogra[s(PDX)cellsweretreatedex-vivofor3to7dayswithCDK8 inhibitors. Viable cell count revealed a significant an:-leukemic ac:vity ofSEL120-34Ain5outofthe10samplestested.
c o n t r o l
S E L 1 2 0 - 3 4 A 5 u M
S E L 1 2 0 - 3 4 A 1 , 6 7 u M
S E L 1 2 0 - 3 4 A 0 , 5 6 u M
S E L 1 2 0 - 3 4 A 0 , 1 8 u M
S E L 1 2 0 - 3 4 A 0 , 0 6 u M
S E L 1 2 0 - 3 4 A 0 , 0 2 u M
S E L 1 2 0 - 3 4 A 0 ,0 0 7 u M
0 5 1 0 1 5
0
1 1́ 0 6
2 1́ 0 6
3 1́ 0 6
4 1́ 0 6
T E X , S E L 1 2 0 - 3 4 A
d a y s
the
ore
tic
al
ce
ll n
um
be
r
0 5 1 0 1 5
0
1 1́ 0 6
2 1́ 0 6
3 1́ 0 6
4 1́ 0 6
T E X , S e n e x i n B
d a y s
the
ore
tic
al
ce
ll n
um
be
r
c o n t r o l
S e n e x i n B 5 u M
S e n e x i n B 1 , 6 7 u M
S e n e x i n B 0 , 5 6 u M
S e n e x i n B 0 , 1 8 u M
S e n e x i n B 0 , 0 6 u M
S e n e x i n B 0 , 0 2 u M
S e n e x i n B 0 , 0 0 7 u M
STAT1pS727
STAT5pS726
Ac,n
STAT5tot
0510.50.10.050
STAT5pY694
GAPDH
SEL120-34A[uM]
STAT1pY701
STAT1tot
SEL120-34AtreatmentofTEXcells,whichisaleukemiastemcell(LSC)-likecellline (Warner et al, leukemia, 2005), resulted in reduced cell growth andinhibi:on of STAT1 and STAT5 phosphoryla:on. Gene expression analysis ofSEL120-34A treated cells revealed enrichment of gene sets downregulated inLSCsandlinkedtodifferen:a:on.SenexinB,anotherCDK8inhibitor,wasusedascontrol.
Murine c-Kit+MLL-AF9 leukemic cells (Miller et al, Cancer Cell, 2013) weretreatedex-vivowithCDK8inhibitors.Cellcountreadouta[er3,7and10daysrevealedstronginhibi:onofcellgrowth.
VolcanoplotLogra-o
-log10p-ad
j
GSEA
GSEAsummaryplotfortop50C2genesetswithFDR<0.5andsortedaccordingto|NES|
Gene Log2foldchangeLILRB2 -2.14MEIS1 -1.37CD38 3.92CD36 5.18TNF 5.85IL-12 8.56
3.CDK8inhibi?onincreasesapoptosisofmurineleukemiacells
Murine c-Kit+ MLL-AF9 leukemic cells were transplanted into sublethallyirradiatedC57/bl6recipientmice.Miceweredividedinto3groups:Vehicle,20mg/kgof SEL-120, and40mg/kgof SEL-120 . Treatmentwas started10daysa[er the injec:on of leukemia cells. SEL120-34A treated animals showed adose-dependent selec:ve an:-leukemic ac:vity in peripheral blood andbonemarrowatendpointanalysis(12daysoftreatment),associatedwithreducedwhitebloodcellcount(WBC)andsmallerspleensize.Treatmentalsoresultedinstronginhibi:onofSTAT1andSTAT3biomarkers.
c-Kit+ dsRed MLL-AF9 cells
Sublethally irradiated C57bl6 recipient mice
10 days latency
Vehicle / SEL-120 20mg/kg SEL-120 40mg/kg
Daily treatment 12 days
Leukemia burden analysis
/
Vehicle 20 mg/kg 40 mg/kg0
5
10
15
WB
C 1
0^9/
L
White blood cell count
SEL-120
Vehicle 20 mg/kg 40 mg/kg0
10
20
30
40
50
% o
f dsR
ed+
cells
in P
B
Circulating leukemia cells
SEL-120
Vehicle 20 mg/kg 40 mg/kg0
20
40
60
WB
C 1
0^9/
L
White blood cell count
SEL-120
Vehicle 20 mg/kg 40 mg/kg50
60
70
80
90
100
% o
f dsR
ed+
Circulating leukemia cells
*
SEL-120
7daysoftreatment 12daysoftreatment
Vehicle 20 mg/kg 40 mg/kg0
100
200
300
400
500
wei
ght m
g
spleen weight**
SEL-120
Vehicle 20 mg/kg 40 mg/kg85
90
95
100
% o
f dsR
ed+
Leukemic cells in bone marrow
**
SEL-120
12daysoftreatment
Murine c-Kit+ MLL-AF9 leukemic cells were treated ex-vivo with the CDK8inhibitors SEL120-34A or Senexin B. CDK8 inhibi:on resulted in reducednumbersofac:velycyclingcellsandanincreaseinapopto:ccells.
SLV-
1103
-12
Sene
xin B
0.0
5.0×103
1.0×104
1.5×104
2.0×104
cell
num
ber
Day 3
5 uM1 uM0.2 uM0.04 uM0.008 uM0 uM
SLV-
1103
-12
Sene
xin B
0
1×105
2×105
3×105
4×105
5×105
theo
ritic
al c
ell n
umbe
r
Day 7
5 uM1 uM0.2 uM0.04 uM0.008 uM0 uM
day 0
day 3
day 7
day 1
00.0
5.0×105
1.0×106
1.5×106
theo
ritic
al c
ell n
umbe
r
SLV-1103-12
0 uM0.008 uM0.04 uM0.2 uM1 uM5 uM
day 0
day 3
day 7
day 1
00.0
5.0×105
1.0×106
1.5×106
theo
ritic
al c
ell n
umbe
r
Senexin B
0 uM0.008 uM0.04 uM0.2 uM1 uM5 uM
input cell number 10 000 cells
SLV-
1103
-12
Sene
xin B
0.0
5.0×105
1.0×106
1.5×106
theo
ritic
al c
ell n
umbe
r
Day 10
5 uM1 uM0.2 uM0.04 uM0.008 uM0 uM
SEL-120-34A SenexinB
AMLPDXcellswere transplanted intoNSGmice. Fromday10,mice receiveddailytreatmentswithvehicleorSEL120-34Aat45mg/kg.Leukemiaburdenwasanalyzeda[er28daysoftreatment.AnalysisofhumanCD45andCD34expressionrevealedinvivoan:-leukemicac:vityofSEL120-34Aonprimi:veAMLcells.
Control 200 nM 500 nM 200 nM 500 nM0
500
1000
1500
Ki6
7 (g
MFI
)
24h
*
Senexin B SEL-120
Control 200 nM 500 nM 200 nM 500 nM0
1000
2000
3000
4000
Ki6
7 (g
MFI
)
***
*
48h
Senexin B SEL-120
Control 200 nM 500 nM 200 nM 500 nM0
500
1000
1500
2000
Ki6
7 (g
MFI
)
Senexin B SEL-120
72h
Control 200 nM 500 nM 200 nM 500 nM0
2000
4000
6000
8000
Ann
exin
V (g
MFI
)
24h
*
*
**
Senexin B SEL-120
Control 200 nM 500 nM 200 nM 500 nM0
5000
10000
15000
20000
Ann
exin
V (g
MFI
)
48h
*
**
**
Senexin B SEL-120
Control 200 nM 500 nM 200 nM 500 nM0
5000
10000
15000
20000
Ann
exin
V (g
MFI
)
72h
**
*
****
Senexin B SEL-120
Cellcycle
Apop
tosis
day 0 day 3 day 7 day 100
1×105
2×105
3×105
4×105
cell
num
ber
SEL120-34A
0 µM0.008 µM0.04 µM0.2 µM1 µM5 µM
day 0 day 3 day 7 day 100
1×105
2×105
3×105
4×105
cell
num
ber
Senexin B
0 µM0.008 µM0.04 µM0.2 µM1 µM5 µM
Vehicle SEL-1200
20
40
60
80
100
%hC
D45
+ hC
D34
+ ce
lls
Circulating leukemia cells
**
Vehicle SEL-1200
50
100
%hC
D34
+ ce
lls
%hCD34+ cells in bone marrow
**
Vehicle SEL-1200
100
200
300
sple
en w
eigh
t (m
g)
spleen weight
**
PDX cells Sublethally irradiated NSG mice
10 days latency
Vehicle / SEL-120 45mg/kg
Daily treatment 28 days
Leukemia burden analysis
patient 10
100000
200000
300000
cell
num
ber
**
*
patient 60
2000
4000
6000
8000
cell
num
ber
patient 20
20000
40000
60000
cell
num
ber
**
patient 70
20000
40000
60000
80000
cell
num
ber
**
*
patient 80
500
1000
1500
cell
num
ber
patient 40
200000
400000
600000
800000
1000000
cell
num
ber
patient 90
5000
10000
15000
20000
cell
num
ber
****
***
patient 100
2000
4000
6000
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 10
100000
200000
300000
cell
num
ber
**
*
patient 60
2000
4000
6000
8000
cell
num
ber
patient 20
20000
40000
60000
cell
num
ber
**
patient 70
20000
40000
60000
80000
cell
num
ber
**
*
patient 80
500
1000
1500
cell
num
ber
patient 40
200000
400000
600000
800000
1000000
cell
num
ber
patient 90
5000
10000
15000
20000
cell
num
ber
****
***
patient 100
2000
4000
6000
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 10
100000
200000
300000
cell
num
ber
**
*
patient 60
2000
4000
6000
8000
cell
num
ber
patient 20
20000
40000
60000
cell
num
ber
**
patient 70
20000
40000
60000
80000
cell
num
ber
**
*
patient 80
500
1000
1500
cell
num
ber
patient 40
200000
400000
600000
800000
1000000
cell
num
ber
patient 90
5000
10000
15000
20000
cell
num
ber
****
***
patient 100
2000
4000
6000
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 10
20000
40000
60000
80000
100000
cell
num
ber
*
patient 60
1000
2000
3000
4000
5000
cell
num
ber
****
***
patient 20
20000
40000
60000
80000
100000
cell
num
ber
****
*
patient 70
10000
20000
30000
40000
50000
cell
num
ber
patient 30
500
1000
1500
2000
2500
cell
num
ber
******
***
***
patient 80
5000
10000
15000
cell
num
ber
patient 40
20000
40000
60000
cell
num
ber
****
patient 90
2000
4000
6000
8000
10000
cell
num
ber
**
patient 50
50
100
150
200
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 100
1000
2000
3000
4000
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 10
100000
200000
300000
cell
num
ber
**
*
patient 60
2000
4000
6000
8000
cell
num
ber
patient 20
20000
40000
60000
cell
num
ber
**
patient 70
20000
40000
60000
80000
cell
num
ber
**
*
patient 80
500
1000
1500
cell
num
ber
patient 40
200000
400000
600000
800000
1000000
cell
num
ber
patient 90
5000
10000
15000
20000
cell
num
ber
****
***
patient 100
2000
4000
6000
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 10
100000
200000
300000
cell
num
ber
**
*
patient 60
2000
4000
6000
8000
cell
num
ber
patient 20
20000
40000
60000
cell
num
ber
**
patient 70
20000
40000
60000
80000
cell
num
ber
**
*
patient 80
500
1000
1500
cell
num
ber
patient 40
200000
400000
600000
800000
1000000
cell
num
ber
patient 90
5000
10000
15000
20000
cell
num
ber
****
***
patient 100
2000
4000
6000
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 10
100000
200000
300000
cell
num
ber
**
*
patient 60
2000
4000
6000
8000
cell
num
ber
patient 20
20000
40000
60000
cell
num
ber
**
patient 70
20000
40000
60000
80000
cell
num
ber
**
*
patient 80
500
1000
1500
cell
num
ber
patient 40
200000
400000
600000
800000
1000000
cell
num
ber
patient 90
5000
10000
15000
20000
cell
num
ber
****
***
patient 100
2000
4000
6000
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
7dayspatient 1
0
20000
40000
60000
80000
100000
cell
num
ber
*
patient 60
1000
2000
3000
4000
5000
cell
num
ber
****
***
patient 20
20000
40000
60000
80000
100000
cell
num
ber
****
*
patient 70
10000
20000
30000
40000
50000
cell
num
ber
patient 30
500
1000
1500
2000
2500
cell
num
ber
******
***
***
patient 80
5000
10000
15000
cell
num
ber
patient 40
20000
40000
60000
cell
num
ber
****
patient 90
2000
4000
6000
8000
10000
cell
num
ber
**
patient 50
50
100
150
200
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 100
1000
2000
3000
4000
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 10
20000
40000
60000
80000
100000
cell
num
ber
*
patient 60
1000
2000
3000
4000
5000
cell
num
ber
****
***
patient 20
20000
40000
60000
80000
100000
cell
num
ber
****
*
patient 70
10000
20000
30000
40000
50000
cell
num
ber
patient 30
500
1000
1500
2000
2500
cell
num
ber
******
***
***
patient 80
5000
10000
15000
cell
num
ber
patient 40
20000
40000
60000
cell
num
ber
****
patient 90
2000
4000
6000
8000
10000
cell
num
ber
**
patient 50
50
100
150
200
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 100
1000
2000
3000
4000
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 10
20000
40000
60000
80000
100000
cell
num
ber
*
patient 60
1000
2000
3000
4000
5000
cell
num
ber
****
***
patient 20
20000
40000
60000
80000
100000
cell
num
ber
****
*
patient 70
10000
20000
30000
40000
50000
cell
num
ber
patient 30
500
1000
1500
2000
2500
cell
num
ber
******
***
***
patient 80
5000
10000
15000
cell
num
ber
patient 40
20000
40000
60000
cell
num
ber
****
patient 90
2000
4000
6000
8000
10000
cell
num
ber
**
patient 50
50
100
150
200
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 100
1000
2000
3000
4000
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 10
20000
40000
60000
80000
100000
cell
num
ber
*
patient 60
1000
2000
3000
4000
5000
cell
num
ber
****
***
patient 20
20000
40000
60000
80000
100000
cell
num
ber
****
*
patient 70
10000
20000
30000
40000
50000
cell
num
ber
patient 30
500
1000
1500
2000
2500
cell
num
ber
******
***
***
patient 80
5000
10000
15000
cell
num
ber
patient 40
20000
40000
60000
cell
num
ber
****
patient 90
2000
4000
6000
8000
10000
cell
num
ber
**
patient 50
50
100
150
200
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 100
1000
2000
3000
4000
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
3days
patient 10
20000
40000
60000
80000
100000
cell
num
ber
*
patient 60
1000
2000
3000
4000
5000
cell
num
ber
****
***
patient 20
20000
40000
60000
80000
100000
cell
num
ber
****
*
patient 70
10000
20000
30000
40000
50000
cell
num
ber
patient 30
500
1000
1500
2000
2500
cell
num
ber
******
***
***
patient 80
5000
10000
15000
cell
num
ber
patient 40
20000
40000
60000
cell
num
ber
****
patient 90
2000
4000
6000
8000
10000
cell
num
ber
**
patient 50
50
100
150
200
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
patient 100
1000
2000
3000
4000
cell
num
ber
control
Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM