LB-209

Hek 293 + Wnt1 + SEN461 700

700

RLU (% vs CTR)

600

RLU (% vs CTR)

LB-209

Hek 293 + Wnt1 + SEN461

600

Identification and characterization of small molecule inhibitor of Wnt canonical pathway in a glioma setting De Rosa

1 0 Antonella 100

200

, Giordano Cinzia,1 Varrone Maurizio,1 Nencini Arianna1, Pratelli Carmela1, Bakker Annette and Salerno Massimiliano 1 100

0

Biotech S.p.A., Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy, e-mail: [email protected]

CTR SiRNA 100pmoles 200pmoles

-

+

+

+

+

+

0

30

100

300

1000

wnt3a

SEN-A (nM)

-

+

+

+

+

+

0

30

100

300

1000

Hek293 + LRP6 + Wnt3a + SEN461

Hek293 + Wnt3a RLU (% vs CTR)

700

Figure 1: -catenin siRNA induced a less proliferative and less tumorigenic phenotype in GBM cells. Ability to grow in anchorage independent fashion is strongly reduced after β-catenin KO (A). β-catenin KO induced a G0/G1 cell cycle arrest and strong S phase reduction (B).

600 500 400

300 200

800 700 600 +500 SEN461 400 300 200 100 0

100 0

+ + 0

800 700 600 500 400 300+ 200 + 100 1000 0

RLU (% vs CTR)

B)

+ + 3000

3 +

3 +

10 +

10 M SEN-B M SEN-A + DMSO

SEN-A

50 IC50IC(IC M)( M) 50 ( M)

Axin1

80

80

% Activity

100

% Activity

100

60 40

TCF-Luciferase

20 0

1

40

TCF-Luciferase

20

TA-Renilla

TA-Renilla

0

( M) 10 Conc (uM)

1030 10 820 8 6 66 4 44 2 22 0 00

60

10 1 ( M) Conc (uM)

SEN-A SEN-A SEN-A

+

Wnt3a CM

+

SEN-A -catenin Axin

GAPDH

1.2E-02 1.0E-02 8.0E-03 6.0E-03 4.0E-03 2.0E-03 0.0E-00

IWR2

IWR2

IWR2 XAV939

SEN461 IWR2

PARP1

PARP2

PARP3

PARP6

18

2.9

>10

>10

>10

>10

0.01

0.01

0.12

0.05

>10

>10

Soft SoftAgar AgarSensitivity Sensitivity Soft Agar Sensitivity

+ -

+ + -

+ + +

DMSO Wnt3A CM SEN-A

Figure 4: effect of SEN-A on Wnt molecular markers in HEK293 cells. SEN-A (10µM) reversed the effects of Wnt3a inducing a reduction of total β-catenin and an increased of Axin1 levels (A). Activation of the Wnt/β-catenin target gene Axin2 by Wnt3a CM was inhibited by SEN-A (B).

and

• SEN-A showed a weak effect on Tankyrases stabilization and auto-parsylation compared to the Tankyrase inhibitor XAV939.

233

• SEN-A treatment confirmed in-vitro antitumoral activity in a panel of commercially available and patient derived GBMs.

122

• SEN-A treatment showed in-vivo antitumoral activity in a GBM xenograft model Figure 10: in vitro response to SEN-A in a panel of GBM cells. The halfmaximal inhibitory concentration (IC50) for 11 GBM cancer cell lines, determined from the soft agar assay ordered from lowest to highest. (* = Primary patient derived GBMs)

IP: Axin2

• All data suggest Wnt inhibition mediated by SEN-A treatment as a potential therapeutic approach in glioblastoma.

Vehicle 10 mL/kg Vehicle 30mpk30 BID, 14 days SEN461 mg/Kg/BIDx14 100mpk, 14 days SEN461 100QD, mg/Kg/day 500 mpk, 2 wks SEN461 500Qwk, mg/Kg/day

1200

Axin2h h Axin2 Tubulin Axin2 Tubulin Tubulin

Axin2 qPCR

• SEN-A prevented Axin2 ubiquitination subsequent proteasomal degradation.

1400

A A h Tubulin

B) Normalized Relative Expression

-

+ -

TNKS2

MEAN and SEM

h Axin2

-

• SEN-A increased the amount of cytosolic phosphorylated β-catenin, decreased total β-catenin and stabilized Axin at protein level but decreased Axin2 at transcriptional level.

SEN-B SEN-B SEN-B

A

HEK293

TNKS1

• SEN-A inhibited either Wnt1 or Wnt3a mediated transcriptional activity in a concentration-dependent manner in HEK293 cells.

00

Figure 6: SEN-A activity on Wnt pathway components at the protein level in DBTRG cells. SEN-A treatment stabilized Axins, increased phosphorylated βcatenin (Ser33/37/Thr41) with a concomitant decrease of total β-catenin.

Figure 3: effect of SEN-A and the inactive analog SEN-B on Wnt transcriptional activity (A) and anchorage independent growth (B) in DBTRG cells.

A)

cells.

TNKS2 TNKS2

344

h Axin2h Axin2 Tubulin Tubulin

+IP: Axin2 + + DMSO B M + SEN461 10 M) + ++ MG-132 DMSO + +0 0.5 2 4 6 16 (CHX + SEN-A) IP: MG-132 + Axin2 + + B (CHX 100 M + SEN461 10 M) + SEN-A SEN461 IP: Axin2 DMSO + + + 0.5 M22+ SEN461 44 661016 0 B (CHX M) Hours 0 100 0.5 16 MG-132 DMSO +- + ++ 0 0.5 2 M) 4 6 16 Axin2 MG-132 -- +- + (CHX 100 SEN461 0 0.5 2 4 6 16 - +IB: Ub SEN461 WB: Ub Tubulin

A) A) (CHX 100

B)

B) 1000

TUMOR MASS (mm )

120

transcriptional growth in GBM

3

SEN-A

TNKS2 TNKS2 • SEN-A inhibited Wnt mediated TNKS2 TNKS2TNKS2 TNKS2 activity and anchorage independent TNKS1 TNKS1 TNKS1 TNKS1TNKS1 TNKS1

GBM Soft Agar Sensity DBTRG GBMSoft Soft Agar Agar Sensitivity Sensity

( M) IC50IC(50M) IC50 ( M)

SEN0082461:695/014/01

TNKS1 TNKS1

011

GAPDH

B)

KO induced a less proliferative and less tumorigenic phenotype suggesting a key role of Wnt DMSO pathway activity in GBM.

TNKS2 DMSO +DMSOTNKS2

10 45

Axin2

SEN0082973:682/018/01 SEN-B % Activity 120

TNKS1 TNKS1 TNKS1 TNKS1 Tubulin Tubulin TNKS2 TNKS2 TNKS2 TNKS2

20 5

β-catenin

Figure 2: DBTRG cells showed to be responsive to positive and negative modulators of the Wnt pathway. Exogenous expression of positive (A) and negative (B) Wnt pathway modulators.

TNKS2

TNKS1 TNKS1 - M SEN-A M SEN-A

Figure 9: biochemical assay to evaluate TNKS1, TNKS2 and PARP activities after SEN-A and XAV939 treatment.

P-β-catenin

A)

TNKS2

- 10 -30 10 10030- 100- + + ++ ++ ++ ++ +

IC50( M)

XAV939

DBTRG +

Axin2 Axin2 TNKS1 TNKS1 Axin2 Axin2

10 ++- +- +-+++- +- ++-+1+ +-10 ++ + 1+ +DMSO ++ M XAV939 + +DMSOM XAV939 +DMSO

Figure 8: effects of different compounds on Axins and Tankyrases protein levels. Axins and Tankyrase stabilization in DBTRG cells (A). Concentration dependent stabilization of TNKS1 and TNKS2 in DBTRG cells (B).


A)

Axin2

Tubulin Tubulin Tubulin Tubulin

Figure 5: effect of SEN-A on Wnt ligands mediated transcription in HEK293 cells. SEN-A inhibited either Wnt1 or Wnt3a-mediated luciferase activity in a concentration-dependent manner, without affecting the Wnt-independent TARenilla activity . 800 700 600 500 400 300 200 100 0

Axin1

Axin2


-

wnt3a LRP6 SEN-A (nM)

IWR2 XAV939 XAV939

Axin1

100 0

+-

NCI-N87

0

M1=G0/G1=83% M2=S=5% M3=G2/M=7%

wnt1 SEN-A (nM)

Axin1Axin1 Axin1Axin1

DBTRG

M1=G0/G1=59% M2=S=16% M3=G2/M=23%

0

300 200

DBTRG NCI-N87

20

100

400

CONCLUSIONS

B) B) B)10 M XAV939 - - 1 - 101 M XAV939 - - -10 M XAV939 M XAV939 - - - - - - 1 - 10 1 B) - B) β-Catenin 10- 3010 100 30 100 -10 DBTRG M•SEN461 -M- SEN461- -M SEN461- M SEN461 - 30 10100 30 - 100- NCI-N87

β-catenin siRNA

Scramble siRNA

200

A)

10 M o/n

B)

DBTRG

40

100 0

A)

A)10 M o/n

DBTRG NCI-N87

60

300

300 200

A)

A)

600 500

B)

10 MDBTRG o/n10 M o/n

NCI-N87

GAPDH

400

400

700

A) CTR CTRL

80

500

500

Luciferase (RLU, CTR)%) RLU (% vs

100

600

A)

Hek293 + Wnt3a + SEN461

RLU (% vs CTR)

β-catenin

RLU (% vs CTR)

-catenin

700

vs CTR) RLU (% (RLU, %) activity Luciferase

Scramble

120

Colonies (% of control)

Hek 293 + Wnt1 +600 SEN461

SiRNA

B)

CTR)%) RLU (% vs Luciferase (RLU,

A)

% Activity

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain tumors. The aggressive and highly invasive phenotype of these tumors makes them among the more destructive human cancers with a median survival of less than one year. In the different GBM tumor subtypes several altered genes and multiple pathways cooperate to promote and sustain tumor growth, tumor invasion and tumor recurrence. Although Wnt pathway activation was historically linked to the presence of mutations involving key components of the network (APC, catenin or AXIN proteins), an increasing number of studies suggest that aberrant Wnt signaling can also be initiated by several alternative mechanisms. Autocrine signaling mediated by specific Wnt ligands has been linked to lung, breast and pancreatic tumours, but also malignant melanoma cells spreading. Wnt signals, both positive and negative, form a class of paracrine growth factors that could act to influence multiple myeloma cell growth, metastatic potential and target tissue erosion. Although very well studied in multiple diseases, the role and importance of Wnt signaling pathway has not been extensively described in GBM tumors. After an initial phase where we showed modulation of the Wnt transcriptional activity and the phenotypic consequences of negative Wnt signaling after catenin KO in glioma cells we started a screening campaign to identify small molecules Wnt inhibitors coupling a pathway/phenotypic approach to oncology relevant phenotypic assays. Among the different chemical classes identified, we characterized a selective canonical Wnt signaling inhibitor, which stabilizes Axin (a negative regulator of the Wnt signalling pathway) at the protein level together with a concomitant decrease at the transcriptional level. Due to the central role of Axin in controlling the ratio between the unphosphorylated (the stable form which can then enter the nucleus and thus activate Wnt target genes) and the phosphorylated (labelled for proteasomal degradation) pool of β-catenin we also observed as a consequence an increase in the amount of cytosolic phosphorylated β-catenin (S33/S37/T41) and a decrease of total β-catenin. One possible explanation for the up-regulation of Axin protein level and the concomitant decrease of steady-state Axin mRNA levels after compound treatment could be via a protein stabilization mechanism, as demonstrated by the effects of the small molecule on the half-life of Axin2 in DBTRG cells. Consistent with a protective effect of the molecule against Axin2 proteasomal degradation, co-treatment of the Wnt inhibitor and the reversible proteasome inhibitor MG-132 almost completely blocked the ubiquitination of Axin2. In vivo studies, used to confirm the in vitro observations, showed antitumor activity in a glioma xenograft model.

700

NCI-N87

ABSTRACT

CTR IWR2 XAV939

Hek293 + Wnt3a + SEN461

XAV939 XAV939 SEN461 SEN-A SEN461

1Siena

Tunici

Patrizia1,

300

CTR

Verani

Margherita,1

200

400

SEN461 SEN-A CTR CTRL

Rossi

Marco,1

300

500

NCI-N87

Valensin

Silvia,1

400

NCI-N87

De Robertis

Alessandra,1

500

36% 38%

800

50% 600

400

0 00

(CHX 100 M) (CHX) (CHX2100 4M) 6 16 0.5 WB: Axin2 WB: Ub 0.5 2 4 6 16 Hours 0.5 2 4 6 16 WB: Ub

Axin2 TCL

Axin2 Axin1

TubulinGAPDH

IB: Axin2

Axin2 Axin1 GAPDH

200

68% 0

TCL

WB: Axin2 Figure 7: SEN-A protects Axin from ubiquitination. SEN-A (10µM) affected the WB: Axin2

half-life of Axin2 (A). Co-treatment of SEN-A (10µM) and the proteasome Axin2 inhibitor MG-132 almost completely prevents the ubiquitination of Axin2 (B). Axin2 TCL TCL

Axin1 Axin1 GAPDH GAPDH

28 31

34 37

40

43

48

51 55 58 TIME (Days)

REFERENCES: 62

65

69

72

76 79

Figure 11: in vivo antitumor activity of SEN-A in the DBTRG tumor xenograft model. DBTRG cells were injected s.c. into CD-1 nude mice on day 0 and SENA p.o. dosing started on Day 28. Treatment groups (10 mice per group) were 30 mg/kg/day twice day from Day 28 to 41 (red line), 100 mg/kg/day daily from Day 28 to 41 (blue line) and 500 mg/kg/day once weekly on Days 28 and 34 (green line). Tumour volume was followed over time until day 79 (37 days after the end of the treatment).

Shih-Min A.Huang et al. (2009) Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461(7264):614-20. Baozhi Chen et al. (2009) Small molecule-mediated disruption of Wntdependent signaling in tissue regeneration and cancer. Nature Chemical Biology 5(2):100-107. Hugh K Arnold et al (2009) The Axin1 scaffold protein promotes formation of a degradation complex for c-Myc. EMBO Journal 28:500-512.