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

New Horizons on bone targeted therapies Daniele Santini CBM University Rome

…we have specific in vitro models of bone cells

IN VITRO MODELS OF BONE CELLS PRIMARY HUMAN OSTEOCLASTS CD14 + CELLS LABELING WITH MICROBEAD S PBMCs ISOLATION MAGNETIC ISOLATION SEEDING male healthy donors DIFFERENTIATION (TRAP ASSAY) ACTIVITY (OSTEOASSAY) UNDIFFERENTIATED DIFFERENTIATED UNDIFFERENTIATED DIFFERENTIATED Cultured for 12 days with M-CSF and RANKL BONE LAYER RESORBED AREA

IN VITRO MODELS OF BONE CELLS PRIMARY HUMAN OSTEOBLASTS CHEMICAL DIGESTION CELLULAR SEEDING OUTGROWN From human mesenchymal stem cells (hMSCs) obtained from bone fragments of non-oncological orthopaedic surgery patients DIFFERENTIATION (ALP ASSAY) ACTIVITY (ALIZARIN RED ASSAY) UNDIFFERENTIATED DIFFERENTIATED UNDIFFERENTIATED DIFFERENTIATED 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 of Abiraterone in our models of primary human OCLs/OBLs

its Androgen Bio iosynthesis is Th Abir iraterone In Inhib ibit Through CY CYP17 “ New therapeutic tar argets s in in the he bo bone mic icroenvironment for or treatment of of bo bone metastasi sis ” • Androgens produced at t 3 criti critical l sit ites es: • Tes estes • Adr Adrenal gland • Pros ostate tu tumor or cell ells • Abiraterone in inhibits biosynthes esis is of of androgens that stimula late tu tumor cell gr growth 1. 1. Attard G G et al. J J Clin in Onc ncol ol. 20 2008 08;26:4563-4571; 2. 2. Attard G G et al. J J Clin in Onc ncol ol. 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 ono JS et t al. Ann nn Onc ncol. . 20 2010 10;21(suppl 8) 8): Abstract LB LBA5.

Mol olecular ch characterization of of ab abir iraterone tar argets in in human prim rimary os osteocla last/osteobla last RE REAL TIME TIME PCR CR Os Osteobla lasts Os Osteocla lasts CYP17A1 is expressed in primary human osteoclast/osteoblast

Abiraterone treatment inhibits osteoclast differentiation and activity both in presence and absence of steroids * P < 0.05 ** P < 0.01 *** P < 0.001 **** P < 0.0001 Steroids NO Steroids

Abiraterone treatment increases osteoblast differentiation and activity both in presence and absence of steroids Steroids * P < 0.05 NO Steroids

Abiraterone down-modulates osteoclasts marker genes (at gene and protein level) * P < 0.05 ** P < 0.01 Osteoclastic gene markers: TRAP Cathepsin K (Cath-k) Metalloproteinase-9 (MMP-9) Abiraterone up-regulates osteoblasts marker genes (at gene and protein level) Osteoblastic gene markers: ALP Osteocalcin (OCN) Runx2

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

Abiraterone: TUMOR TARGET AND BONE TARGET THERAPY “ New therapeutic tar argets s in in the he bo bone mic icroenvironment for or treatment of of bo bone metastasi sis ”

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 our models of primary human OCLs/OBLs

Cabozantin inib ib (XL184): Target Profil ile “ New therapeutic tar argets s in in the he bo bone mic icroenvironment for or treatment of of bo bone metastasi sis ” Kinase IC 50 , nM RTK Cellular IC 50 , nM, Autophosphorylation MET 1.8 MET 8 VEGFR2 0.035 VEGFR2 4 RET 5.2 Cabozantinib, mg/kg KIT 4.6 V 3 10 30 100 AXL 7.0 H441 pMET TIE2 14 tumors* FLT3 14 MET S/T Ks (47) >200 pVEGFR2 Mouse ATP competitive, reversible lung † VEGFR2 *No growth factor stimulation. † VEGF-A administered 30 min prior to harvest. Data courtesy of Ron Weitzman and Dana Aftab.

Mol olecular ch characterization of of cab abozantin inib tar argets in in human pri rimary ry os osteocla last/osteobla last A B Osteoclasti Osteblasti 0 6 mRNA FOLD CHANGE vs GUS β -2 mRNA FOLD CHANGE vs GUS β 4 * 2 -4 * 0 * -6 -2 -8 -4 -6 -10 -8 * -12 * -10 -14 -12 0 2 4 6 8 10 12 14 0 7 14 21 TIME (days) TIME (days ) MET VEGFR2 MET VEGFR2 C D 3dd 6dd 12dd 7dd 14dd 21dd c-MET c-MET β -actin β -actin Under Review on Scientific Reports

Cabozantinib inhibits osteoclast differentiation and activity * P < 0.05 Under Review on Scientific Reports

Cabozantinib does not affect osteoblast differentiation and activity Under Review on Scientific Reports

Cabozantinib treatment up-regulates OPG gene/protein sectretion and down- modulates RANKL gene/protein secretion altering RANKL/OPG balance * P < 0.05 mRNA Proteins Under Review on Scientific Reports

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 odology: • 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 OBLs mono-colture OBLs CTRL + OCLs * P < 0.05 Trap assay OBLs pre-treated CBZ (3 μ M) OBLs pre-treated CBZ (5 μ M) + OCLs + OCLs Under Review on Scientific Reports

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

Enzalu lutamid ide: : mechanis ism of f actio tion “ New therapeutic tar argets s in in the he bo bone mic icroenvironment for or treatment of of bo bone metastasi sis ” 3. Blocks DNA binding 2. Impairs nuclear translocation 1. Blocks AR binding and activation Cytoplasm Nucleus DHT AR Enzalutamide Enzalutamide Enzalutamide 2 – 3 fold lower affinity than DHT

Mol olecular ch characterization of of enzalutamid ide tar argets s in in human prim rimary ry osteoclast/osteobla os last RE REAL TIME TIME PCR CR Os Osteocla lasts Os Osteoblasts AR is expressed in primary human osteoclast/osteoblast

Enzalutamide does not affect osteoclast differentiation and activity 500 Trap+ Osteoclasts 400 Trap assay 300 200 100 0 ENZ 5 μ M ENZ 10 μ M CTRL Osteoassay 120 Bone resorption area (%) 100 80 60 Enzalutamide Control 40 20 0 ENZ 5 μ M ENZ 10 μ M CTRL Unpublished data

Enzalutamide does not affect osteoblast differentiation and activity 1.000 900 800 Signal intensity (arbitrary unit) Alp assay 700 600 500 400 300 200 100 0 DMSO ENZA 5 μ M ENZA 10 μ M Alizarin red 1600 assay 1400 1200 Signal intensity (arbitrary unit ) 1000 800 Control Enzalutamide 600 400 200 0 CTRL ENZ 5 μ M ENZ 10 μ M Unpublished data

Enzalutamide treatment up-regulates some osteoblastic marker genes: CXCL12, OSTERIX and RUNX2 7 * P < 0.05 * 6 5 * 4 3 * 2 1 mRNA 0 CTRL ENZALUTAMIDE Unpublished data

Enzalutamide treatment up-regulates pro-inflammatory genes IL-6, IL-8, IL-12 * P < 0.05 4,5 * 4 3,5 FOLD CHANGE 3 * 2,5 2 * 1,5 1 mRNA 0,5 0 IL-6 IL-8 IL-11 IL-12 TNF IL-10 CTRL ENZALUTAMIDE Unpublished data

Enzalutamide treatment up-regulates RANKL gene expression * P < 0.05 * 2 1,8 1,6 1,4 FOLD CHANGE 1,2 1 0,8 0,6 mRNA 0,4 0,2 0 OPG RANKL CTRL ENZALUTAMIDE Unpublished data

Enzalutamide treatment up-regulates RANKL protein secretion * P < 0.05 * 2500 1200 2000 1000 RANKL (pg/ml ) OPG (pg/ml ) 800 1500 600 1000 400 500 200 0 0 CTRL ENZ 5 μ M ENZ 10 μ M CTRL ENZ 5 μ M ENZ 10 μ M Proteins Proteins Unpublished data

Future perspectives: Abiraterone and Enzalutamide  To investigate the “indirect” osteoblast -mediated effects of Abiraterone and Enzalutamide on 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 on the androgen receptor activation of CRPC exposed to pre-treated OBL conditioned media .