cdk8 inhibitor sel120-34a has therapeu?c efficacy in ......0.008 µm 0.04 µm 0.2 µm 1 µm 5 µm...

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