Nuove molecole e peptidi quali farmaci regolatori del ciclo cellulare e della risposta a chemioterapici nei tumori epiteliali e cutanei

Alessandro Terrinoni: IDI-IRCCS

PROGRAMMA 3Trasferimento delle conoscenze allo sviluppo di interventi volti a prevenire, diagnosticare e trattare il cancro.

Coordinatore del Progetto: Prof. Gerry Melino

• UO 1: Prof. Gerry Melino (IDI-IRCCS)E.Candi (Universita’ Tor Vergata)

• UO 2: Dott. Giovanni Blandino (Ist. Naz. Tumori Regina Elena)Prof.ssa MG Santoro (Universita’ di Tor Vergata)Dott.ssa L Lanfrancone (Istituto Europeo di Oncologia)

• UO 3: Dott.ssa Stefania D’Atri (IDI-IRCCS)Dott.ssa E Alvino (CNR, Ist. Neurobiologia e Med. Molec.)Dott. U Pfeffer (Ist. Naz. Per la Ricerca sul Cancro)Prof. Gulino (Universita’ della Sapienza)

OBIETTIVO PRINCIPALE del PROGETTO:

Identificare nuove molecole e peptidi quali farmaci regolatori del ciclo cellulare e della risposta a chemioterapici nei tumori epiteliali e cutanei

• Sviluppare nuove molecole inibitori della E3 ubiquitin ligasi ITCH e di validare tali molecole nella via di p63/p73 e di Hedgehog/Gli;

• Studio del potenziale terapeutico di pepidi capaci di disassemblare complessi proteici (mp53/p73) ad attivita’ oncogenica;

• Studio del potenziale terapeutico di inibitori selettivi di NF-kB/IKK e delle cicline-chinasi dipendenti (CDK), come singoli agenti ed in associazione agli inibitori di ITCH e ai chemoterapici;

• Studio dell’espressione della proteina chinasica RaLP nei melanomi e caratterizzazione del pathway di trasduzione del segnale.

OBIETTIVI SPECIFICI:

Development of inhibitorsof the Ub E3 ligase ITCHto regulate the p73 and p63

pathway in Cancer

DNA damage Replicative Stress

Cell Cyle ArrestApoptosis

Sensors

Effectors

Signals

EFFECTS

ALTERATIONS

ATM ATR

p53chk1

chk2

p21gadd45 ….

mdm2

c-abl

bax CD9514-3-3Noxa ….

MRE11rad50

BRCA1NSB1

DNA-PK PARP

cdc25

G1 S G2

Ku70/Ku86

14-3-3cdc25

Cdc2 ….

DNA Repair

HRNHEJ

NERMMRBERp73p63

Incidence of p73 alterations in cancer

0.41% 21.76%

p73 in hepatoma cells

ΔNp73 is a negativePrognostic Factor

p73 as prognostic factor?

Hepathoma patients

N TA PR DBD OD CPR SAM TI

N PR DBD OD CPR SAM TI

TAp73p63 ISOFORM

ΔNp73p63 ISOFORM

ΔNp73 inhibits TAp73 and p53 apoptosis

ΔNp73 is induced by TAp73 and p53

ΔNp73/p63 acts as an oncogenehypothesis

TAp73 induces apoptosis TAp73/p63 acts as a tumour suppressorhypothesis

The ratio TAp73/ΔNp73 dictates the response to chemotherapy

p53

Senescence Apoptosis Cell Cycle Arrest

ONCOGENES

DNA damage

Ub E3 ligase inhib.nutlin

activatorsPRIMA-1

DNA methyltransferase inhibitors ARF

mdm2

Question 1

How are p63/p73 protein levels controlled ?

ITCH E3 ligase regulates p73 stability

Itch

Ubiquitin E3 ligase (NEDD4 family)

18H Mice natural Knockout (immune defects)

Interacts with Atrophin

Degrades (transcription factor):Jun-B, c-Jun, Notch, FLIP

HECTcWW WWC2Ca2+ dependent lipids interaction

protein-protein interacting domain

catalytic domain transfering ubiquitin to substrate

CN

Phage Display

TA DNA Binding Domain OD SAM PR PRCN TAp73

UbiquitylationBinding

ITCH wtITCH mut

p73 p53

IP: p73/p53 WB: HA(Ub)

Ubiquitylatedp73

pcDNA Cyclo-hexamide (h) 0 1 2 4 6 8

< p73

< tubulinpcDNA

ITCH

< p73

< tubulin

Degraded p73

Degradation

ITCH wtITCH mut

p73 p53pcDNA

IP: Itch WB: p73/p53

p73 bound

Rossi et al. EmboJ 2005 25:836

ITCH regulates endogenous p73 protein levels

siRNA ITCH-/- reconstitutionITCH-/-

ITCH degrades p63

Itch interacts with p63 Itch ubiquitilates p63

shRNA inhibitionof ITCH

Itch can regulate cell death by doxocycline

ITCH reintroductionIn -/- cells

Hansen et al. BBRC 2007. 361,1:33

Question 2

What do we know on ITCH E3 activity ?

Itch interaction with UbcH7

ring of negatively charged aa on Itch’s HECT surfaceUbcH7 ring of positively charged aa

Itch interaction with UbcH7

HECTresidues

UbcH7residues

E193E193D191D172D191

R5K9K9K64R6

Itch interaction with UbcH7

HECTresidues

UbcH7residues

R5

K64

K9

E193

D172

E193

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

5

5.2

Column B

time (ps)

Dis

tanc

e E

193

- R5

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

22.5

33.5

44.5

55.5

66.5

77.5

88.5

9

Main Title

Column B

time (ps)

Dis

tenc

e D

172

- K

64

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

Main title

Column B

time (ps)

Dis

tanc

e E

193

- K

9

time

time

time

500

500

500

1000

1000

1000

1500 ps

1500 ps

1500 ps

Itch interaction with UbcH7

504

Itch interacts with p63 Y504

Itch interacts with p73 Y487

Question 3

Is ITCH E3 activity regulated ?

A model for Itch-mediated regulation of p63/p73 function

Tumor cell

p73Ub

p73ItchItch

Itch

Itch

Tumor cell

p73Ub

p73ItchItch

Itch

DNA damage

ApoptosisApoptosis

Itch

N4BP1

p73 p73

p73puma, noxa,CD95,

p63ITCH Ub, degradation

N4BP1

p73

Question 4

Can we inhibit ITCH E3 activity ?

ELISA configuration for Itch autoubiquitination

SCREENING STRATEGY

0.0

0.4

0.8

1.2

1.6

2.0+FLAG UbNo Ub

-0.5 0.0 0.5 1.0 1.5 2.00.0

0.5

1.0

1.5

2.0

ItchMutant

EC50 5ng/well

r2 = 0.99

Log (ng/well itch)

Titration of Itch activity in ELISAunder optimised conditions

The assay requires E1, E2, ubiquitin and wild type E3

Electrochemiluminescence assay configuration for Itch autoubiquitination

SCREENING STRATEGY

-1.0 -0.5 0.0 0.5 1.0 1.50

50000

100000

150000ItchItch mutant

r2 = 0.96

EC50 3ng/well

Log (ng/well E3)

Titration of Itch in electrochemiluminescence assay

FRET configuration for Itch autoubiquitination

Can we control p63/p73 protein levels?

HTP screening

Open question

Itch inhibitors search by HighThroughPut Screening

++ Hits cut off < 50%+++ Hits cut off < 70%

COMPOUND PCT-Activity PCT-Activity PCT-Activity PCT-ActivityScreen Validation1 Validation2 MEAN

#2 44.25 76.5 82.7 67.8# 3 26.52 86.5 85.1 66.0# 5 49.71 79.1 82.2 70.4# 7 35.58 75.5 84.9 65.4# 8 45.12 79.9 74.9 67.2# 10 37.35 84.5 78.3 66.7# 12 47.23 83.9 82.7 71.3

SCREENING RESULTS

% inhibition

SCREENING RESULTS

% inhibition

compounds manual

-2 -1 0 1 2 3 40.0

0.2

0.4

0.6

0.8

1.0

1.2 339863427633224174341376416121

log µM

321

87

11

Compound 8:

• Sensitizes cells to chemotherapeutics at 10µM• Shows specificity towards Hect rather than RING ligases• Analogues shows in vitro activity against Itch/p73 and sensitize cells to chemotherapy

Compound 8 itself may or may not be a starting point for drug development but there is potential to find other starting points from further screens

DMSOComp.8E2

E3

substrate

Effect of compound nEffect of compound nºº 8 8 on E2 chargeon E2 charge

Ubch7 -

Effect of compound nEffect of compound nºº 8 8 on E3 chargeon E3 charge

Senescence ??? Apoptosis Cell Cycle Arrest

DNA damage

Ub E3 ligase inhib.Compound 8 ??

Activators ??

DNA methyltransferase Inhibitors ?? N4BP1

ITCH

p73/p63

???ONCOGENES

p53

ARF

mdm2

- p63/p73 are involved in DNA repair/Cancer - p63/p73 regulate chemosensitivity in cancer - p63/p73 are transcriptionally regulated by PML- p63/p73 are Ub & degraded by Itch- Itch is regulated by N4BP1- low MW Itch inhibitors are under development

ITCH is a candidatetherapeutical target

(to regulate p63/p73)

perspective

CONCLUSIONS conclusion

E1= 10

E2= 100

E3= 1000

Proteasome= 1

human

PLC-g1, phospholipase C-g1; PKC-t, protein kinase t, c-FLIP, cellular FLICE-inhibitory protein; CXCR4, chemokine receptor 4

ITCH SUBSTRATES

ITCH REGULATORS

JNK, Jun _N-terminal kinase; N4BP1, Nedd 4 binding partner 1, Ndfip1, Nedd 4 family interacting protein 1

Melino G, De Laurenzi V, Vousden KH p73: friend or foe in tumorigenesis. Nature Review Cancer. (2002) 2: 605-615

evolutionof thep53 family

20.0

Method: UPGMA; Best Tree;tie breaking = SystematicDistance: Absolute (# differences)Gaps distributed proportionally

DNADAMAGE

DifferentiationSignals ?

MLH1c-Abl

ATMchk2

?

Viral proteins

p53

p73

Development

DNAdamage

?

?

p63

PARTICIPANT UNIT 2Scientific Responsible: Dr. Giovanni Blandino

Translational Oncogenomic UnitRegina Elena Cancer Institute, Rome-Italy

Mutant p53/p73 protein complex : a new molecular target for novel anticancer therapeutic approaches?

Short Interfering peptides disrupt the protein complex mutant p53/p73 and increase selectively the chemosensitivity of mutant p53

tumor cells

p53 status and DNA damage

p53 wild type

Bax

p21p53AIP1

MDM214-3-3σ

p63p73 p53

growth arrestapoptosis

p53 null mutant p53

DNA DAMAGE

p63p73

growth arrestapoptosis

X

Bax

p21p53AIP1

MDM214-3-3σ

X

p63

mp53

p73

mp53

growth arrestapoptosis

XX

Bax

p21p53AIP1

MDM214-3-3σ

mp53

?

EGR1NFkB2

Fas/cd95

Chemoresistence

MSP/MST-1Others

p73core

mp53

DBD

p73DBD

p73

core

mp53peptides

Apoptosis

BAX

p53AIP1

PUMA

p21

Cyclin G

Mdm2

p73

p73

p73

p73

p73

Hypotesis: By competition with p73α in the binding to mutant p53, peptides might be capable to restore p73 transcriptional and apoptotic activities

Peptides designed on DBD of human p73α

FQQSSTAKSA TWTYSPLLKK LYCQIAKTCP IQIKVSTPPP PGTAIRAMPV

YKKAEHVTDV VKRCPNHELG RDFNEGQSAP ASHLIRVEGN NLSQYVDDPV

TGRQSVVVPY EPPQVGTEFT TILYNFMCNS SCVGGMNRRP ILIIITLEMR

DGQVLGRRSF EGRICACPGR DRKADEDHYR

131 141 171151 161

SIMP1SIMP5SIMP6

ChIP (Chromatin Immunoprecipitation assay)

- - --+ + ++No Ab

contr contr

Input

αp73αH4Ac

SKBR3 SW480

BAX

ADR 3μM

SIMP5,6 SIMP5,6

p21

No Ab

Input

No Ab

Input

PIG3

No Ab

Input

PUMA1

αp73αH4Ac

αp73αH4Ac

αp73αH4Ac

Synthetic peptides promote the activation of p73apoptotic target genesin response to DNA damage

SKBR3 SAOS

BAX

0 1 3 0 1 3 0 1 3 0 1 3

contr contr

p21

GAPDH

SIMP5,6 SIMP5,6

aldolase

p53AIP1

Killer/DR5

μM ADR

RT-PCR

αtubulin

contr SIMP5,6

αBAX

αp21

SKBR3

μM ADR 0 01 13 35 5

contr SIMP5,60 01 13 35 5

SAOS

Western Blotting

PARTICIPANT UNIT 3Scientific Responsible: Stefania D’Atri, IDI-IRCCS, Rome

1. Istituto Dermopatico dell’Immacolata-IRCCS, Rome (Stefania D’Atri)2. Institute of Neurobiology and Molecular Medicine, Rome (Ester Alvino)3. National Institute for Cancer Research, Genoa (Ulrich Pfeffer)4. Department of Experimental Medicine, University of Rome “La Sapienza”, Rome

(Alberto Gulino)

Evaluation of the therapeutic potential of two new inhibitors of CDKs, either alone or in combination with chemotherapeutic agents or Itch inhibitors, in melanoma (RG1, RG2, RG3)

Identification of inhibitors of the Hedgehog/Gli pathway and evaluation of their therapeutic potential in brain tumors (RG4)

Research Groups

Objectives

PARTICIPANT UNIT 3

Seven melanoma cell lines, sensitive or resistant to temozolomide (TMZ), showed a marked susceptibility to the growth inhibitory effects of the panCDK-I and the CDK2-I under investigation. For each cell line, the IC50 values of panCDK-I and CDK2-I were below the peak plasma concentrations described for the two drugs in the ongoing phase I/II clinical studies

Sensitivity of melanoma cells to the CDK2-I showed a good correlation with the expression of CDK2, and was mainly dependent on drug-induced G1 arrest. No apoptosis was found at CDK2-I concentrations up to those corresponding to the peak plasma concentration

Additive or synergistic growth suppressive effects were observed in melanoma cells exposed to TMZ followed by the CDK2-I

Evaluation of the therapeutic potential of two new inhibitors of CDKs, either alone or in combination with chemotherapeutic agents or Itch inhibitors, in melanoma (RG1, RG2, RG3)

Results

PubblicazioniBelardo G, Piva R, Santoro MG (submitted) Hyperthermia inhibits constitutive NF-kB activity in human malignant B-cells and triggers apoptosis by impairing NF-kB survival signaling Leukemia.

Bernassola F, Karin M, Ciechanover A, Melino G (2008) The HECT family of E3 ubiquitin ligases: multiple players in cancer development. Cancer Cell 14:10-21.

Di Agostino S, Cortese G, Monti O, Dell'orso S, Sacchi A, Eisenstein M, et al. (2008) The disruption of the protein complex mutantp53/p73 increases selectively the response of tumor cells to anticancer drugs. Cell Cycle 7.

Ferretti E, De Smaele E, Miele E, Laneve P, Po A, Pelloni M, et al. (2008) Concerted microRNA control of Hedgehog signalling in cerebellar neuronal progenitor and tumour cells. EMBO J 27:2616-2627.

Ferretti E, De Smaele E, Po A, Di Marcotullio L, Tosi E, Espinola MSB, et al. (in press) microRNA profiling in human medulloblastoma. Int J Cancer

Hansen TM, Rossi M, Roperch JP, Ansell K, Simpson K, Taylor D, et al. (2007) Itch inhibition regulates chemosensitivity in vitro. Biochem Biophys Res Commun 361:33-36.

Melino G, Gallagher E, Aqeilan RI, Knight R, Peschiaroli A, Rossi M, et al. (2008) Itch: a HECT-type E3 ligase regulating immunity, skin and cancer. Cell Death Differ 15:1103-1112.

Raimondo D, Giorgetti A, Bernassola F, Melino G, Tramontano A (2008) Modelling and molecular dynamics of the interaction between the E3 ubiquitin ligase Itch and the E2 UbcH7. Biochem Pharmacol.

Scialpi F, Malatesta M, Peschiaroli A, Rossi M, Melino G, Bernassola F (2008) Itch self-polyubiquitylation occurs through lysine-63 linkages. Biochem Pharmacol.