Molecular Basis of Targeted Therapies in Metastatic Renal Carcinoma

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 • SUNITINIB inhibits VEGFR, PDGFR, FLT-3 and c-Kit • PAZOPANIB inhibits VEGFR, PDGFR, c-Kit • SORAFENIB Inhibits VEGFR, PDGFR, FLT-3 and Raf • AXITINIB Inhibits VEGFR, PDGF, c-Kit • MONOCLONAL ANTIBODIES • BEVACIZUMAB Anti-VEGF antibody • SERINE-THREONINE KINASE INHIBITORS • TEMSIROLIMUS InhibitsmTOR • EVEROLIMUS InhibitsmTOR

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

5. History of research on the von Hippel-Lindau disease • 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 Latif et al. Science 1993; 260:1317-1320 Gossage et al. Nature Rev Cancer 2015; 15:55-64

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: 822-826 Kondo et al. GenesChromosomesCancer 2002; 34: 58-68 Pause et al. PNAS 1998; 95: 993-998 Nickerson et al. ClinCancer Res 2008; 14: 4726-4734, 2008 Zimmer et al. MolCancer Res 2004; 2: 89-95

7. pVHL is a component of a E3-ubiquitin ligase 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 Cullin-2 Rbx-1 Elongin B Elongin C E2 HIF-a β-domain α-domain VHL The specific substrate of the VBC-CR complex is HIF-α Stebbins et al. Science 1999; 284: 455-461 Iwai et al. PNAS 1999; 96:12436–12441 Lonergan et al. Mol Cell Biol 1998; 18: 732-741 Maxwell et al. Nature 1999; 399: 271-275 Kamura et al. Science 1999; 284: 657-661

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 α β Semenza. Trends PharmacolSci 2012; 33: 207-214 Keith et al. Nature Rev Cancer 2012; 12: 9-22 Gordan et al. Cancer Cell 2009; 14: 435-446

9. Cullin-2 Rbx-1 OH- OH- OH- OH- OH- Elongin B Elongin C E2 Cullin-2 Cullin-2 Rbx-1 Rbx-1 Elongin B Elongin B Elongin C Elongin C HIF-α HIF-α HIF-β HIF-α HIF-α HIF-α HIF-β HIF-α HIF-α E2 E2 VHL VHL OH CANCER CELL (VHL defective) NORMAL CELL HYPOXIA NORMOXIA NORMOXIA/HYPOXIA Pro402&564 Hydroxilation (PHD) Pro402&564 Hydroxilation (PHD) NO hydroxilation Poly-Ub VHL nucleus nucleus VEGF PDGF TGF-α Epo VEGF PDGF TGF-α Epo PROTEASOME

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 • Twomicroscopic pattern designatedpapillarytype 1 and 2, sporadic or hereditary, with differentmolecularprofile • Hereditarypapillaryrenalcancersyndrome (HPRC): • riskof bilateral, multifocalpRCCtype 1 • activatingmutations of MET(more oftenchromosome 7 polysomy in the sporadicform) • Hereditaryleiomyomatosis RCC (HLRCC) • - cutaneous and uterine leyomiomatosis, pRCCtype 2 • - inactivatingmutations of FH (fumaratehydratase)

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

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

20. Remarks • 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

22. Remarks • 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

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