CDK8 Inhibitor SEL120-34A Has Therapeu?c Efficacy in ......0.008 µM 0.04 µM 0.2 µM 1 µM 5 µM...
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CDK8 Inhibitor SEL120-34A Has Therapeu?c Efficacy in Murine and Human AML Models Disclosures : Mazan, Majewska, Wiklik, Combik, Masiejczyk, Fiedor, Obacz, Bialas, Chesy, Gabor-Worwa Selvita S.A.: Employment. Brzózka: Selvita S.A.: Employment, Equity Ownership, Membership on an en?ty's Board of Directors or advisory commiXees. Rzymski: Selvita S.A.: Employment, Equity Ownership. Fioretos: Cantargia: Equity Ownership, Membership on an en?ty's Board of Directors or advisory commiXees. Flygare: LU Holding: Patents & Royal?es: Patent. Other authors have no rela?onships to disclose. Background • Cyclin-dependent kinase 8 (CDK) inhibitors have an:-leukemic ac:vity in human AML cell lines. • Efficacy has been associated with ac:va:on of super enhancer regions (Pelish et al, Nature, 2015). • SEL120-34A, a CDK8/CDK19 inhibitor, has an:-leukemic effect in a panel of AML cell lines, associated with a reduc:on of STAT5 S726 phosphoryla:on (Rzymski et al, Oncotarget, 2017). M. Chapellier 1 , J. Chen 2 , C. Sandén 1 , M. Mazan 3 , E. Majewska 3 , K. Wiklik 3 , M. Combik 3 , M. Masiejczyk 3 , E. Fiedor 3 , M. Obacz 3 , A. Polak 4 , A. Białas 3 , P. Chęsy 3 , E. Gabor-Worwa 3 , A. Grochowska 5 , U. Kuklińska 5 , Z. Sandowska-Markiewicz 5 , M. Statkiewicz 5 , M. Kopczynski 5 , M. Cybulska 5 , M. Mikula 5 , P. Juszczynski 4 , K. Brzózka 3 , T. Rzymski 3 , T. Fioretos 1 , J. Flygare 2* and M. Järås 1* 1 Division of Clinical Gene?cs, Department of Laboratory Medicine, Lund University, Lund, Sweden; 2 Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden; 3 R&D department, Selvita S.A., Kraków, Poland; 4 Ins?tute of Hematology and Transfusion Medicine, Warsaw, Poland; 5 Department of Gene?cs, Maria Sklodowska-Curie Memorial Cancer Center and Ins?tute of Oncology, Warsaw, Poland; *Equal contribu?on Travel grant 1. TEX cells are sensi?ve to CDK8 inhibi?on 2. SEL120-34A inhibits murine MLL-AF9 AML cells 4. SEL120-34A reduces leukemia burden in a murine AML model 5. SEL120-34A inhibits AML pa?ent cells in vitro 6. SEL120-34A showed strong an?-leukemic effect in an AML PDX mouse model Conclusion • CDK8 inhibi:on resulted in reduced AML cell growth in human and murine AML cells in vitro. • Treatment of leukemic mice with SEL120-34A resulted in reduced leukemia burden in bone marrow and blood. • SEL120-34A treatment of mice transplanted with AML PDX cells resulted in strong an:-leukemic ac:vity. AML pa:ent derived xenogra[s (PDX) cells were treated ex-vivo for 3 to 7 days with CDK8 inhibitors. Viable cell count revealed a significant an:-leukemic ac:vity of SEL120-34A in 5 out of the 10 samples tested. SEL120-34A 5uM SEL120-34A 1,67 uM SEL120-34A 0,56 uM SEL120-34A 0,18 uM SEL120-34A 0,06 uM SEL120-34A 0,02 uM SEL120-34A 0,007 uM 0 5 10 15 0 1 ´ 10 6 2 ´ 10 6 3 ´ 10 6 days theoretical cell number 0 5 10 15 0 1 ´ 10 6 2 ´ 10 6 3 ´ 10 6 days theoretical cell number Senexin B 5uM Senexin B 1,67 uM Senexin B 0,56 uM Senexin B 0,18 uM Senexin B 0,06 uM Senexin B 0,02 uM Senexin B 0,007 uM STAT1 pS727 STAT5 pS726 Ac,n STAT5 tot 0 5 1 0.5 0.1 0.05 0 STAT5 pY694 GAPDH SEL120-34A [uM] STAT1 pY701 STAT1 tot SEL120-34A treatment of TEX cells, which is a leukemia stem cell (LSC)-like cell line (Warner et al, leukemia, 2005), resulted in reduced cell growth and inhibi:on of STAT1 and STAT5 phosphoryla:on. Gene expression analysis of SEL120-34A treated cells revealed enrichment of gene sets downregulated in LSCs and linked to differen:a:on. Senexin B, another CDK8 inhibitor, was used as control. Murine c-Kit + MLL-AF9 leukemic cells (Miller et al, Cancer Cell, 2013) were treated ex-vivo with CDK8 inhibitors. Cell count readout a[er 3, 7 and 10 days revealed strong inhibi:on of cell growth. Volcano plot Log ra-o -log10 p-adj GSEA GSEA summary plot for top 50 C2 gene sets with FDR < 0.5 and sorted according to |NES| Gene Log2 fold change LILRB2 -2.14 MEIS1 -1.37 CD38 3.92 CD36 5.18 TNF 5.85 IL-12 8.56 3. CDK8 inhibi?on increases apoptosis of murine leukemia cells Murine c-Kit + MLL-AF9 leukemic cells were transplanted into sublethally irradiated C57/bl6 recipient mice. Mice were divided into 3 groups: Vehicle, 20 mg/kg of SEL-120, and 40 mg/kg of SEL-120 . Treatment was started 10 days a[er the injec:on of leukemia cells. SEL120-34A treated animals showed a dose-dependent selec:ve an:-leukemic ac:vity in peripheral blood and bone marrow at end point analysis (12 days of treatment), associated with reduced white blood cell count (WBC) and smaller spleen size. Treatment also resulted in strong inhibi:on of STAT1 and STAT3 biomarkers. 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/kg 0 5 10 15 WBC 10^9/L White blood cell count SEL-120 Vehicle 20 mg/kg 40 mg/kg 0 10 20 30 40 50 % of dsRed+ cells in PB Circulating leukemia cells SEL-120 Vehicle 20 mg/kg 40 mg/kg 0 20 40 60 WBC 10^9/L White blood cell count SEL-120 Vehicle 20 mg/kg 40 mg/kg 50 60 70 80 90 100 % of dsRed+ Circulating leukemia cells * SEL-120 7 days of treatment 12 days of treatment Vehicle 20 mg/kg 40 mg/kg 0 100 200 300 400 500 weight mg spleen weight ** SEL-120 Vehicle 20 mg/kg 40 mg/kg 85 90 95 100 % of dsRed+ Leukemic cells in bone marrow * * SEL-120 12 days of treatment Murine c-Kit + MLL-AF9 leukemic cells were treated ex-vivo with the CDK8 inhibitors SEL120-34A or Senexin B. CDK8 inhibi:on resulted in reduced numbers of ac:vely cycling cells and an increase in apopto:c cells. 1.5×10 6 1.5×10 6 SEL-120-34A Senexin B AML PDX cells were transplanted into NSG mice. From day 10, mice received daily treatments with vehicle or SEL120-34A at 45 mg/kg. Leukemia burden was analyzed a[er 28 days of treatment. Analysis of human CD45 and CD34 expression revealed in vivo an:-leukemic ac:vity of SEL120-34A on primi:ve AML cells. Control 200 nM 500 nM 200 nM 500 nM 0 500 1000 1500 Ki67 (gMFI) 24h * Senexin B SEL-120 Control 200 nM 500 nM 200 nM 500 nM 0 1000 2000 3000 4000 Ki67 (gMFI) ** * * 48h Senexin B SEL-120 Control 200 nM 500 nM 200 nM 500 nM 0 500 1000 1500 2000 Ki67 (gMFI) Senexin B SEL-120 72h Control 200 nM 500 nM 200 nM 500 nM 0 2000 4000 6000 8000 Annexin V (gMFI) 24h * * * * Senexin B SEL-120 Control 200 nM 500 nM 200 nM 500 nM 0 5000 10000 15000 20000 Annexin V (gMFI) 48h * ** * * Senexin B SEL-120 Control 200 nM 500 nM 200 nM 500 nM 0 5000 10000 15000 20000 Annexin V (gMFI) 72h ** * *** * Senexin B SEL-120 Cell cycle Apoptosis day 0 day 3 day 7 day 10 0 1×10 5 2×10 5 3×10 5 4×10 5 cell number SEL120-34A 0 μM 0.008 μM 0.04 μM 0.2 μM 1 μM 5 μM day 0 day 3 day 7 day 10 0 1×10 5 2×10 5 3×10 5 4×10 5 cell number Senexin B 0 μM 0.008 μM 0.04 μM 0.2 μM 1 μM 5 μM Vehicle SEL-120 0 20 40 60 80 100 %hCD45+ hCD34+ cells Circulating leukemia cells ** Vehicle SEL-120 0 50 100 %hCD34+ cells %hCD34+ cells in bone marrow ** Vehicle SEL-120 0 100 200 300 spleen weight (mg) spleen weight ** PDX cells Sublethally irradiated NSG mice 10 days latency Vehicle / SEL-120 45mg/kg Daily treatment 28 days Leukemia burden analysis patient 2 0 20000 40000 60000 cell number ** patient 1 0 100000 200000 300000 cell number * * * patient 6 0 2000 4000 6000 8000 cell number patient 10 0 1000 2000 3000 4000 cell number patient 4 0 200000 400000 600000 800000 1000000 cell number patient 9 0 5000 10000 15000 20000 cell number * *** *** patient 7 0 20000 40000 60000 80000 cell number * * * 7 days control Senexin B 0.2 uM Senexin B 1 uM SEL120 0.2 uM SEL 120 1 uM patient 8 0 5000 10000 15000 cell number patient 5 0 50 100 150 200 cell number patient 3 0 500 1000 1500 2000 2500 cell number *** *** *** *** 3 days control Senexin B 0.2 uM Senexin B 1 uM SEL120 0.2 uM SEL 120 1 uM
Transcript of CDK8 Inhibitor SEL120-34A Has Therapeu?c Efficacy in ......0.008 µM 0.04 µM 0.2 µM 1 µM 5 µM...
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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
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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
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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
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Vehicle 20 mg/kg 40 mg/kg0
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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
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theo
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day 0
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1.5×106
theo
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al c
ell n
umbe
r
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0 uM0.008 uM0.04 uM0.2 uM1 uM5 uM
input cell number 10 000 cells
SLV-
1103
-12
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xin B
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1.5×106
theo
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ell n
umbe
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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
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Ki6
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MFI
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Control 200 nM 500 nM 200 nM 500 nM0
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****
Senexin B SEL-120
Cellcycle
Apop
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day 0 day 3 day 7 day 100
1×105
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day 0 day 3 day 7 day 100
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0 µM0.008 µM0.04 µM0.2 µM1 µM5 µM
Vehicle SEL-1200
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%hC
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+ hC
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+ ce
lls
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D34
+ ce
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%hCD34+ cells in bone marrow
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Vehicle SEL-1200
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PDX cells Sublethally irradiated NSG mice
10 days latency
Vehicle / SEL-120 45mg/kg
Daily treatment 28 days
Leukemia burden analysis
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SEL 120 1 uM
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Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
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Senexin B 0.2 uM
Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
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***
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Senexin B 1 uM
SEL120 0.2 uM
SEL 120 1 uM
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SEL120 0.2 uM
SEL 120 1 uM
patient 10
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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
