New Horizons on bone targeted therapies Daniele Santini CBM - - PowerPoint PPT Presentation

Daniele Santini

CBM University Rome

New Horizons on bone targeted therapies

…we have specific in vitro models of bone cells

IN VITRO MODELS OF BONE CELLS

DIFFERENTIATION (TRAP ASSAY) ACTIVITY (OSTEOASSAY)

PBMCs ISOLATION CD14 + CELLS LABELING WITH MICROBEADS MAGNETIC ISOLATION SEEDING

UNDIFFERENTIATED UNDIFFERENTIATED DIFFERENTIATED DIFFERENTIATED

RESORBED AREA BONE LAYER

PRIMARY HUMAN OSTEOCLASTS male healthy donors Cultured for 12 days with M-CSF and RANKL

IN VITRO MODELS OF BONE CELLS

CHEMICAL DIGESTION SEEDING CELLULAR OUTGROWN

PRIMARY HUMAN OSTEOBLASTS DIFFERENTIATION (ALP ASSAY) ACTIVITY (ALIZARIN RED ASSAY)

UNDIFFERENTIATED UNDIFFERENTIATED DIFFERENTIATED DIFFERENTIATED

From human mesenchymal stem cells (hMSCs) obtained from bone fragments of non-oncological orthopaedic surgery patients

OBL differentiation was monitored by alkaline phosphatase (ALP) staining, and bone matrix deposition as a marker of OBL activity by Alizarin Red staining

Abiraterone and bone microenvironment

Starting from preclinical and clinical evidence we designed an in vitro/translational study in order to investigate a potential direct effect

• f Abiraterone in our models of primary human OCLs/OBLs

“ New therapeutic tar argets s in in the he bo bone mic icroenvironment for

• r treatment of
• f bo

bone metastasi sis”

Abir iraterone In Inhib ibit its Androgen Bio

iosynthesis is Th

Through CY CYP17

• Androgens produced at

t 3 criti critical l sit ites es:

• Tes

estes

• Adr

Adrenal gland

• Pros
• state tu

tumor

• r cell

ells

• Abiraterone in

inhibits biosynthes esis is of

• f

androgens that stimula late tu tumor cell gr growth

1.

• 1. Attard G

G et al. J J Clin in Onc ncol

• l. 20

2008 08;26:4563-4571; 2.

• 2. Attard G

G et al. J J Clin in Onc ncol

• l. 20

2009 09;27:3742-3748; 3.

• 3. Reid

id AH H et t al. J Clin in Onc

• ncol. 20

2010 10;28:1489- 14 1495 95; 4.

• 4. Ryan CJ et

t al. J Clin in Onc

• ncol. 20

2010 10;28:1481-1488; 5.

• 5. Dan

anila ila DC et al. J Clin in Onc

• ncol. 20

2010 10;28:1496-1501; 6.

• 6. de

de Bon

• no JS et

t al. Ann nn Onc ncol. . 20 2010 10;21(suppl 8) 8): Abstract LB LBA5.

RE REAL TIME TIME PCR CR

CYP17A1 is expressed in primary human osteoclast/osteoblast

Mol

• lecular ch

characterization of

• f ab

abir iraterone tar argets in in human prim rimary os

• steocla

last/osteobla last

Os Osteocla lasts Os Osteobla lasts

Abiraterone treatment inhibits osteoclast differentiation and activity both in presence and absence of steroids

Steroids NO Steroids

* P < 0.05 ** P < 0.01 *** P < 0.001 **** P < 0.0001

Abiraterone treatment increases osteoblast differentiation and activity both in presence and absence of steroids

Steroids NO Steroids

* P < 0.05

Abiraterone down-modulates osteoclasts marker genes (at gene and protein level)

Osteoclastic gene markers: TRAP Cathepsin K (Cath-k) Metalloproteinase-9 (MMP-9) Osteoblastic gene markers: ALP Osteocalcin (OCN) Runx2

Abiraterone up-regulates osteoblasts marker genes (at gene and protein level)

* P < 0.05 ** P < 0.01

A significant decrease of CTX values and an increase of ALP was found in serum of 49 mCRPC patients treated with Abiraterone post-docetaxel

“ New therapeutic tar argets s in in the he bo bone mic icroenvironment for

• r treatment of
• f bo

bone metastasi sis”

Abiraterone: TUMOR TARGET AND BONE TARGET THERAPY

Cabozantinib and bone microenvironment

Starting from preclinical and clinical evidence we designed an in vitro/translational study in order to investigate a direct effect of CBZ in

• ur models of primary human OCLs/OBLs

“ New therapeutic tar argets s in in the he bo bone mic icroenvironment for

• r treatment of
• f bo

bone metastasi sis”

Cabozantin inib ib (XL184): Target Profil ile

Kinase IC50, nM MET 1.8 VEGFR2 0.035 RET 5.2 KIT 4.6 AXL 7.0 TIE2 14 FLT3 14 S/T Ks (47) >200

Data courtesy of Ron Weitzman and Dana Aftab.

ATP competitive, reversible

RTK Cellular IC50, nM, Autophosphorylation MET 8 VEGFR2 4 Cabozantinib, mg/kg

pMET MET

V 3 10 30 100

VEGFR2 pVEGFR2

H441 tumors* Mouse lung†

*No growth factor stimulation.

†VEGF-A administered 30 min prior to harvest.

• 12
• 10
• 8
• 6
• 4
• 2

2 4 6 7 14 21

mRNA FOLD CHANGE vs GUSβ TIME (days)

MET VEGFR2

• 14
• 12
• 10
• 8
• 6
• 4
• 2

2 4 6 8 10 12 14

mRNA FOLD CHANGE vs GUSβ TIME (days)

MET VEGFR2

* * * * *

A B

3dd 6dd 12dd c-MET β-actin 7dd 14dd 21dd c-MET β-actin

D C

Osteoclasti Osteblasti Under Review on Scientific Reports

Mol

• lecular ch

characterization of

• f cab

abozantin inib tar argets in in human pri rimary ry os

• steocla

last/osteobla last

Cabozantinib inhibits osteoclast differentiation and activity

Under Review on Scientific Reports

* P < 0.05

Under Review on Scientific Reports

Cabozantinib does not affect osteoblast differentiation and activity

Cabozantinib treatment up-regulates OPG gene/protein sectretion and down- modulates RANKL gene/protein secretion altering RANKL/OPG balance

Under Review on Scientific Reports

* P < 0.05

mRNA Proteins

Cabozantinib pre-treated osteoblasts influence osteoclasts differentiation?

OBLs CTRL CBZ PRE-TREATED OBLs

RANKL/OPG RANKL/OPG

COCOLTURE OSTEOBLAST/OSTEOCLAST “CELL-TO-CELL CONTACT”

Experim imental me method

• dology:
• Osteoblasts were differentiated in presence/absence of CABO ZANTINIB
• Osteoclasts were seeded and differentiated directly on osteoblast layer (un/treated with CABO) without exogenous RANKL supplement
• At day 12 the number of cathepsin k positive cells (identified as osteoclasts) was evaluated

Cabozantinib pre-treated osteoblasts reduced osteoclast differentiation compared to untreated osteoblast

Under Review on Scientific Reports

OBLs mono-colture OBLs CTRL + OCLs OBLs pre-treated CBZ (3μM) + OCLs OBLs pre-treated CBZ (5μM) + OCLs

Trap assay

* P < 0.05

Enzalutamide and bone microenvironment

Starting from preclinical and clinical evidence we designed an in vitro/translational study in order to investigate a direct effect of ENZA in our models of primary human OCLs/OBLs

“ New therapeutic tar argets s in in the he bo bone mic icroenvironment for

• r treatment of
• f bo

bone metastasi sis”

DHT

• 1. Blocks AR binding
• 3. Blocks DNA binding

and activation

Enzalutamide Enzalutamide

AR

Cytoplasm Nucleus

• 2. Impairs nuclear translocation

Enzalutamide

2–3 fold lower affinity than DHT

Enzalu lutamid ide: : mechanis ism of f actio tion

RE REAL TIME TIME PCR CR

AR is expressed in primary human osteoclast/osteoblast

Mol

• lecular ch

characterization of

• f enzalutamid

ide tar argets s in in human prim rimary ry

• s
• steoclast/osteobla

last

Os Osteocla lasts Os Osteoblasts

Enzalutamide does not affect osteoclast differentiation and activity

Control Enzalutamide Trap assay Osteoassay

100 200 300 400 500 CTRL ENZ 5 μM ENZ 10 μM

Trap+ Osteoclasts

20 40 60 80 100 120 CTRL ENZ 5 μM ENZ 10 μM

Bone resorption area (%)

Unpublished data

100 200 300 400 500 600 700 800 900 1.000 DMSO ENZA 5 μM ENZA 10 μM

Signal intensity (arbitrary unit)

200 400 600 800 1000 1200 1400 1600 CTRL ENZ 5 μM ENZ 10 μM

Signal intensity (arbitrary unit)

Alp assay Alizarin red assay Control Enzalutamide

Enzalutamide does not affect osteoblast differentiation and activity

Unpublished data

1 2 3 4 5 6 7

CTRL ENZALUTAMIDE

* * *

Enzalutamide treatment up-regulates some osteoblastic marker genes: CXCL12, OSTERIX and RUNX2

* P < 0.05

Unpublished data

mRNA

0,5 1 1,5 2 2,5 3 3,5 4 4,5

IL-6 IL-8 IL-11 IL-12 TNF IL-10

FOLD CHANGE

CTRL ENZALUTAMIDE

* * *

Enzalutamide treatment up-regulates pro-inflammatory genes IL-6, IL-8, IL-12

* P < 0.05

Unpublished data

mRNA

*

0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2

OPG RANKL

FOLD CHANGE

CTRL ENZALUTAMIDE

Enzalutamide treatment up-regulates RANKL gene expression

* P < 0.05

Unpublished data

mRNA

Enzalutamide treatment up-regulates RANKL protein secretion

200 400 600 800 1000 1200 CTRL ENZ 5 μM ENZ 10 μM

OPG (pg/ml)

500 1000 1500 2000 2500 CTRL ENZ 5 μM ENZ 10 μM

RANKL (pg/ml)

*

* P < 0.05

Unpublished data

Proteins Proteins

 To investigate the “indirect” osteoblast-mediated effects of Abiraterone and Enzalutamide

• n the modulation of main biological parameters such as proliferation, apoptosis and cell

cycle of CRPC exposed to pre-treated OBL conditioned media  To investigate the “indirect” osteoblast-mediated effects of Abiraterone and Enzalutamide

• n the androgen receptor activation of CRPC exposed to pre-treated OBL conditioned

media .

Future perspectives: Abiraterone and Enzalutamide

Future perspectives: Abiraterone and Enzalutamide

 To investigate the “indirect” osteoblast-mediated effects of Abiraterone and Enzalutamide in terms of gene expression modulation of CRPC exposed to pre-treated OBL conditioned media using an high-throughput approach  To investigate the “indirect” osteoblast-mediated effects of Abiraterone and Enzalutamide

• n the activation of specific signaling pathways in CRPC exposed to pre-treated OBL

conditioned media in order to identify new potential mechanisms of activity and resistance to anti-androgen therapy

Future perspectives: Cabozantinib

 To investigate the AXL and c-MET activation status in osteoblasts/osteoclasts previously treated with sunitinib and the following modulation of cells in terms of gene expression and protein analyses after cabozantinib administration.  To investigate a potential “indirect” antitumor effect of cabozantinib mediated by

• steoblasts on renal carcinoma (RCC) cells evaluating the modulation of main

biological parameters such as proliferation, apoptosis and cell cycle of cancer cells

Osteoblast

Chronic sunitinib

Thank you very much for the Attention

Paolo Manca Michele Iuliani Francesco Pantano Marco Fioramonti Giulia Ribelli