1. Basi molecolari delle terapie mirate nel carcinoma del rene metastatico Nicola Tinari Oncologia Medica Università G. D’Annunzio Chieti

2. Targeted therapies as a paradigm shift in the treatment of metastatic kidney cancer Historically considered as an “orphan” disease for its limited response to conventional chemotherapeutics agents (ORR 2.4%) “First generation” immunotherapy (IFN, high dose IL-2) associated with an ORR of 12.4% and a median OS of 1 yr The development of targeted agents has completely changed the therapeutic landscape of mRCC Starting with sorafenib in 2005, seven different agents have been sequentially approved targeting VEGF/VEGFR or mTOR Rini. JCO 2009; 27:3225-3234 Coppin et al. Cochrane Database Syst Rev 2005; CD001425

3. Molecularly Targeted agents currently approved by EMA in mRCC TYROSINE KINASE INHIBITORS SUNITINIBinhibits VEGFR, PDGFR, FLT-3 and c-Kit PAZOPANIBinhibits VEGFR, PDGFR, c-Kit SORAFENIBInhibits VEGFR, PDGFR, FLT-3 and Raf AXITINIBInhibits VEGFR, PDGF, c-Kit MONOCLONAL ANTIBODIES BEVACIZUMABAnti-VEGF antibody SERINE-THREONINE KINASE INHIBITORS TEMSIROLIMUS Inhibits mTOR EVEROLIMUSInhibits mTOR

4. The story behind the use of targeted therapies in mRCC

5. History of research on the von Hippel-Lindau disease Latif et al. Science 1993; 260:1317-1320 Gossage et al. Nature Rev Cancer 2015; 15:55-64 CNS Hemangioblastomas, pheochromocytoma, papillary cystoadenomas, endocrine pancreatic tumors and clear-cell carcinoma of the kidney Germline heterozygous inactivation of the tumor-suppressor gene VHL (3p25), followed by somatic inactivation or loss of the second wild-type allele VHL encodes for pVHL

6. Somatic biallelic inactivation of VHL gene in sporadic mRCC It is the main pathogenetic event Consistent with the Knudson “two-hits” model of tumorigenesis Prevalence in sporadic RCC: 50% to 90% of cases VHL inactivation may occur through: - mutations - promoter hypermethylation - chromosomal loss Restoration of pVHL function suppresses tumor formation in vivo and restores the ability to enter G0 in low serum Iliopoulos et al. Nat Med 1995; 1: 822-826Kondo et al. Genes Chromosomes Cancer 2002; 34: 58-68 Pause et al. PNAS 1998; 95: 993-998Nickerson et al. Clin Cancer Res 2008; 14: 4726-4734, 2008 Zimmer et al. Mol Cancer Res 2004; 2: 89-95

7. pVHL is a component of a E3-ubiquitin ligase Stebbins et al. Science 1999; 284: 455-461Iwai et al. PNAS 1999; 96:12436–12441 Lonergan et al. Mol Cell Biol 1998; 18: 732-741Maxwell et al. Nature 1999; 399: 271-275 Kamura et al. Science 1999; 284: 657-661 Cullin-2 Elongin C Elongin B Rbx-1 VHL E2 VHL: the substrate-recognition component (the “receptor”) Elongin B/C: the connection between VHL and Cul2 Rbx-1: E2 enzyme recruiter Cullin-2: the rigid scaffold E2:ubiquitinase The specific substrate of the VBC-CR complex is HIF-α HIF-a α-domain β-domain

8. HIF (hypoxia-inducible factor) Belongs to the PAS (Per-arylhydrocarbon receptor nuclear translocator, ARNT) family of basic helix-loop-helix transcription factors Three isoforms: HIF1, HIF2 and HIF3 Binds hypoxia-response elements (HRE) on DNA as a heterodimer: α-subunit (oxygen-sensitive, interacts with pVHL) β-subunit (constitutively expressed,) More than 800 genes controlled by HIF Among them: VEGF, PDGF, TGF-α, EPO, Glut-1 HIF2-α is the key driver of RCC Semenza. Trends Pharmacol Sci 2012; 33: 207-214 Keith et al. Nature Rev Cancer 2012; 12: 9-22 Gordan et al. Cancer Cell 2009; 14: 435-446 β β α α

9. Cullin-2 Elongin C Elongin B Rbx-1 VHL E2 OH NORMAL CELL NORMOXIA HYPOXIA Pro 402 & 564 Hydroxilation (PHD) NO hydroxilation HIF-α OH- HIF-α OH- HIF-α nucleus Cullin-2 Elongin C Elongin B Rbx-1 VHL E2 VEGF PDGF TGF-α Epo HIF-α OH- PROTEASOME Poly-Ub HIF-αHIF-β CANCER CELL (VHL defective) Cullin-2 Elongin C Elongin B Rbx-1 E2 NORMOXIA/HYPOXIA HIF-α OH- HIF-α OH- nucleus HIF-β Pro 402 & 564 Hydroxilation (PHD) VHL VEGF PDGF TGF-α Epo

10. mTOR signaling Activated by growth factors (via Pi3K or MAPK pathways), nutrients, stress Often induced by mutations of PTEN or TSC1/TSC2 Temsirolimus and Everolimus mainly inhibit mTORC1 The mTORC1 activate a negative feedback loop on mTORC2 HIF1-α is induced by mTORC1 HIF2-α is induced by mTORC2

11. Cellular pathways implicated in clear cell RCC Shuch et al. Eur Urol 2015; 67: 85–97

12. Chromatin remodelling genes as potential therapeutic targets in ccRCC Cancer Genome Atlas Research Network. Nature 2013; 499: 43-49

13. Renal cancers were not created equal… Renal cell tumors listed in the International Society of Urological Pathology Vancouver Modification Srigley et al. Mod Pathol 2009;22(Suppl 2):S2–3.

14. Papillary renal-cell carcinoma Second most common subtype of RCC, occurring in approximately 10-15% of cases Two microscopic pattern designated papillary type 1 and 2, sporadic or hereditary, with different molecular profile Hereditary papillary renal cancer syndrome (HPRC): -risk of bilateral, multifocal pRCC type 1 -activating mutations of MET (more often chromosome 7 polysomy in the sporadic form) Hereditary leiomyomatosis RCC (HLRCC) - cutaneous and uterine leyomiomatosis, pRCC type 2 - inactivating mutations of FH (fumarate hydratase)

15. MET signaling pathway Cabozantinib Foretinib

16. Pathways activated by loss of fumarate hydratase (FH) in the familiar form of pRCC type II Srinivasan et al. Clin Cancer Res 2015;21:10-17

17. Chromophobe renal cell carcinoma Third most common subtype of RCC accounting for 5-7% of case Prognosis is generally excellent exept when sarcomatoid transformation is present Sporadic and hereditary forms Limited genomic characterization is currently available Birt-Hogg-Dubé syndrome: benign cutaneous tumors, pulmunary cysts and chromophobe RCC Mutations of FLCN gene, encoding for Fulliculin, in 90% of the affected families.

18. Bukowski, Figlin, Motzer (Eds). Renal cell carcinoma. Molecular targets and clinical applications. 3 Ed. Springer Science+Business Media New York (2015) Fulliculin pathway

19. All roads lead to HIF!! Linehan et al. Nature Rev Urol 2010; 7: 277-285

20. Alterated signaling converge on HIF, regardless of the underlying molecular defect Targets of TKIs are only a small portion of the downstream genes regulated by HIF mTOR inhibitors only marginally affect HIF2-α which is induced by mTORC2 Inhibition of HIF2-α may represent a more potent therapeutic strategy More targets to be exploited Remarks

22. Alterated signaling converge on HIF, regardless of the underlying molecular defect Targets of TKIs are only a small portion of the downstream genes regulated by HIF mTOR inhibitors only marginally affect HIF2-α which is induced by mTORC2 Inhibition of HIF2-α may represent a more potent therapeutic strategy A number of HIF2-α inhibitors are being tested, but clinical grade agents have yet to be developed More targets to be exploited Remarks

23. Impact on survival of targeted therapies Targeted therapies clinical trials showed ORR of 30% and median OS exceeding 2 years Patient risk and agents account for variations among studies Impromements might be less pronounced in the heterogeneous, “real world” population of mRCC Coppin et al. Cochrane Database Syst 2005; CD001425 Albiges et al. Eur Urol 2015; 67: 100-110 MacLeod et al. Urology 2015; 86: 262-268