1. Optimizing Renal Cell Cancer Treatment: Molecular Rationale and Evidence of Sequencing TKI-mTOR Ignacio Duran, MD PhD UGC Oncología Medica y Radioterapica Hospital Universitario Virgen del Rocío Sevilla

2. Outline Molecular Basis of Renal Cell Cancer “classical view” New Inputs from the Atlas Genome Project Clinical evidence of sequencing TKI-mTOR Conclusions Q&A

3. 3 Introduction HIF VHL ub HIF Proteosome Degradation E3 ubiquitin ligase VEGF PDGF TGFa Tumor Growth and Angiogenesis RECEPTORS Clear Cell Renal Cell Carcinoma PI3K mTor c-Met Papillary Type I Renal Carcinoma Prolyl- Hydoxylase O2O2 Fumarate Hydratase Fumarate Malate Papillary Type II Renal Carcinoma 2-oxoglutarate Adapted from: Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell Review 2007. 12(1): 9-22; Suarez et al. Molecular basis for the treatment of renal cell carcinoma. Clin Transl Oncol (2010) 12:15-21. FNIP1 FLCN FNIP2 Cromophobe Renal Carcinoma

4. 4 Treatment Development: Rationale HIF VHL ub HIF Proteosome Degradation E3 ubiquitin ligase VEGF PDGF TGFa Tumor Growth and Angiogenesis RECEPTORS Clear Cell Renal Cell Carcinoma PI3K mTor c-Met Papillary Type I Renal Carcinoma Prolyl- Hydoxylase O2O2 Fumarate Hydratase Fumarate Malate Papillary Type II Renal Carcinoma 2-oxoglutarate Adapted from: Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell Review 2007. 12(1): 9-22; Suarez et al. Molecular basis for the treatment of renal cell carcinoma. Clin Transl Oncol (2010) 12:15-21. mTOR Inhibit C-Met Inhibit TKIs mAb FNIP1 FLCN FNIP2

5. Introduction PAZOPANIB/SUNITINIB/BEVA-INF TEMSIROLIMUS [Only for poor prognosis] Thus, for the majority of patients with mRCC, there is little controversy around the use of a VEGF-targeted agent as 1st line First Line Options mRCC Bellmunt J et al. Clin Transl Oncol. 2014 Dec;16(12):1043-50

6. Resistencia intrínseca Resistencia evasiva Rini BI, and Flaherty K, Urol Oncol 2008 Refractario primario (2–3 meses de tratamiento) Progresión temprana (6–12 meses de tratamiento) Progresión tardía Variación de las medidas tumorales (%) PATIENTS DEVELOP TREATMENT RESISTANCE

7. Key Question So, my patient has been treated with a TKI and has progressed to treatment…. How effective could be a similar strategy and how a major switch in the therapeutic approach? Let’s review the “molecular evidence” of resistance to make a rationale approach

8. Why patients progress? Modes of resistance to VEGF pathway inhibitors include upregulation of alternative pro-angiogenic factors, downregulation of angiostatic factors, recruitment of bone marrow-derived cells for the development of new blood vessels, and invasion without angiogenesis. Ravaud A, Gross-Goupil M. Overcoming resistance to tyrosine kinase inhibitors in renal cell carcinoma. Cancer Treat Rev 2012.

9. Why patients progress? Moreover, tumor cells may reduce their dependence on angiogenesis and evade by up-regulating other pathways involved in tumor survival, invasiveness, and metastasis. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646–74.

10. Resistance Mechanisms Two major mechanisms have been proposed: – 1.- A rapid scape to anti-VEGF mechanisms through alternative intracellular via – 2.- The capacity to survive in a microenvironment intrinsically resistant to anti-VEGF therapy.

11. A rapid scape to anti-VEGF mechanisms IL- 8 ( pro-angiogenic cytokine) CD11b+Gr1+ cells (MDSCs) Basic fibroblast growth factor( bFGF) Ephitelial to mesenquimal transition (EMT)

12. The capacity to survive in a microenvironment intrinsically resistant to anti-VEGF therapy

13. Tumor Stromal Architecture Consideration of the tumor stromal architecture may be an important determinant of whether tumors will be susceptible to treatment with VEGFi or other vascular modulating agents Neil RS et al. Clin Cancer Res; 19(24);6943–56. 2013 Two dominant morphologies were identified: – Tumor vessel phenotype Tumor structure with vessels embedded through out the tumor cells – Stromal vessel phenotype Pattern of tumor cell nests surrounded by well developed stromal structures containing the majoritly of vessels

14. Introduction Would make sense at this point a different approach? ….Probably so

15. Molecular Basis Targeting the PI3K-AKT-mTOR pathway may be crucial as its activation leads to constitutive HIF-1α expression The development of mTOR inhibitors have been supported by their potential to inhibit both tumor cell proliferation and angiogenesis Posadas EM, Limvorasak S, Sharma S, Figlin RA. Targeting angiogenesis in renal cell carcinoma. Expert Opin Pharmacother. 2013 Nov; 14(16): 2221-36.

16. mTOR Inhibition The mechanism of anti-tumor activity of mTOR inhibitors differs from that of VEGF inhibitors. Targeted agents that inhibit mTOR signaling block the tumor cell cycle at the G1 phase, resulting in decreased tumor growth and proliferation, cellular metabolism, and angiogenesis

17. One Step Beyond: Why this pathway? In a comprehensive molecular characterization of 400 RCC tumor samples using different genomic platforms, 19 significantly mutated genes were identified The PI3K/Akt pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target Cancer Genome Atlas Research Network. Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature. 2013 Jul 4; 499(7456): 43-9.

18. One Step Beyond Cancer Genome Atlas Research Network. Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature. 2013 Jul 4; 499(7456): 43-9. Inputs from the Cancer Genome Atlas suggest that PI3K/AKT pathway might be critical in RCC pathogenesis

19. PI3K/AKT/mTOR In the PI3K/Akt/mTOR pathway (altered in ~28% of tumors), the MEMo algorithm identified a pattern of mutually exclusive gene alterations (somatic mutations, copy alterations, and aberrant mRNA expression)targeting multiple components, including 2 genes from the recurrent amplicon on 5q35.3.

20. The Pathway So it seems obvious that this pathway is relevant in RCC biology and resistance Let’s try to understand how it works and how we target it

22. mTOR Inhibition after VEGFR-TKI failure –This strategy has been reported to provide median progression-free survival (PFS) or time to progression (TTP) ranging from 4.9 to 9.7 months in prospective studies and from 1.4 to 5.5 months in retrospective studies of patients with mRCC whose disease progressed after initial VEGFR- TKI therapy Gerullis H, Bergmann L, Maute L, et al. Med Oncol 2010;27:373–8. Bergmann L, Kube U, Kindler M, et al. J Clin Oncol 2011;29(Suppl.) [abstr 4552]. Roca S, Quivy A, Gross-Goupil M, Bernhard JC, De Clermont H, Ravaud A.. Acta Oncol 2011;50:1135–6. Gruenwald V, Seidel C, Busch J, Fenner M, Weikert S. J Clin Oncol 2011;29(Suppl.) [abstr e15028]. Weikert S, Kempkensteffen C, Busch J, et al. World J Urol 2011 Grunwald V, Weikert S, Seidel C, et al. Onkologie 2011;34:310–4. Mackenzie MJ, Rini BI, Elson P, et al. Ann Oncol 2011;22:145–8. Feinberg BA, Jolly P, Wang ST, et al.. Med Oncol 2011. Wood L, Bukowski RN, Dreicer R, et al. ASCO Genitourinary Cancers Symposium; Feb 14–16, 2008; San Francisco, CA, USA; abstract 353.

23. Upon Disease Progression Safety Interim Analysis 416 patients randomized between December 2006 and November 2007 Analysis cut-off: Feb-28-08, based on 266 PFS events 2nd interim analysis based on cut-off:15-Oct-07, efficacy boundary crossed with 410 patients/191 PFS events (Motzer et al. Lancet. 2008;372:449–456), complete study unblinded on 28-Feb-08 2nd Interim Analysis Data Cut-Off: 15-Oct- 07, N = 410 End of Double- Blind Analysis Data Cut-Off: 28- Feb-08 Survival Follow-Up: 15-Nov-08 Study Unblinded N = 416 Stratification Prior VEGFr-TKI: 1 or 2 MSKCC risk group: favorable, intermediate, or poor Everolimus 10mg/day + BSC (n = 277) Placebo + BSC (n = 139) RANDOMISATION (2:1) Double-Blind Previous Beva, IL2 or INF-alpha allowed RECORD-1Study: design and conduct

24. Motzer R et al August 9, 2008

25. RECORD-1: Motzer RJ, Escudier B, Oudard S, et al. Phase 3 trial of everolimus for metastatic renal cell carcinoma: final results and analysis of prognostic factors. Cancer 2010;116:4256–65.

26. RECORD1: Sub-group Analysis Sub-group of one previous VEGFR-TKI (n=308) – PFS: 5.4 mos Vs 1.9 HR, 0.32; 95% CI, 0.24–0.43; P < 0.001) Sub-group of two previous TKIs (n=108) – 4 mos Vs 1.8 HR, 0.32; 95% CI, 0.19–0.54; P < 0.001 Sub-group of previous only Sunitinib treated (n=56) – 43 Everolimus -13 Placebo – 4.6 mos Vs 1.8 HR, 0.22; 95% CI, 0.09–0.55; P < 0.001)

27. Other mTOR inhibitors Hutson T et al. J Clin Oncol 2013. 32:760-767 Some unanswered questions: Was the OS benefit driven only by the study drugs? What were the treatments received upon progression? 94% of missing data in that topic

28. Some relevant studies just completed METEOR – Cabozantinib Vs Everolimus [after TKI] – PFS CHECKMATE-025 – Anti-PD1 (Nivo) Vs Everolimus [2nd/3rdline] – OS These studies wont completely answer the sequencing question

29. Sequencing Therapies SWITCH (Two TKIs): Weak conclussions; many loss RECORD-3: mTOR-TKI Vs TKI-mTOR – Non-inferiority Phase II design – Better results from TKI-mTOR Others ongoing: – SWITCH 2 – START (MD Anderson): 6 Sequences

30. Conclusions There is a strong molecular rationale to consider the PI3K-AKT-mTOR pathway as a key player in mRCC and treatment resistance The sequence TKI-mTOR inhibitor seems logical from a mechanistic stand-point Despite this, there is still room for improvement in the clinical arena being the data modest in this setting

31. Conclusions Better patient selection will most likely improve treatment outcomes in the near future Continuous effort is needed in this research field

32. Muchas Gracias!!!

33. These processes lead to production of HIF 1α and cell-cycle regulators: c-myc and cyclin D1 In fact mTOR inhibitors are considered both ANTIANGIOGENICS and CELL CYCLE INHIBITORS

34. 34 Treatment Development: Rationale HIF VHL ub HIF Proteosome Degradation E3 ubiquitin ligase VEGF PDGF TGFa Tumor Growth and Angiogenesis RECEPTORS Clear Cell Renal Cell Carcinoma PI3K mTor c-Met Papillary Type I Renal Carcinoma Prolyl- Hydoxylase O2O2 Fumarate Hydratase Fumarate Malate Papillary Type II Renal Carcinoma 2-oxoglutarate Adapted from: Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell Review 2007. 12(1): 9-22; Suarez et al. Molecular basis for the treatment of renal cell carcinoma. Clin Transl Oncol (2010) 12:15-21. FNIP1 FLCN FNIP2 Cromophobe Renal Carcinoma

35. 35 Treatment Development: Rationale HIF VHL ub HIF Proteosome Degradation E3 ubiquitin ligase VEGF PDGF TGFa Tumor Growth and Angiogenesis RECEPTORS Clear Cell Renal Cell Carcinoma PI3K mTor c-Met Papillary Type I Renal Carcinoma Prolyl- Hydoxylase O2O2 Fumarate Hydratase Fumarate Malate Papillary Type II Renal Carcinoma 2-oxoglutarate Adapted from: Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell Review 2007. 12(1): 9-22; Suarez et al. Molecular basis for the treatment of renal cell carcinoma. Clin Transl Oncol (2010) 12:15-21. mTOR Inhibit C-Met Inhibit TKIs mAb FNIP1 FLCN FNIP2

36. RCC treatment outcomes evolution There has been a significant improvement in PFS compared with the cytokine era

37. RCC treatment outcomes evolution There has been a significant improvement in OS compared with the cytokine era

38. Upon Disease Progression Safety Interim Analysis 416 patients randomized between December 2006 and November 2007 Analysis cut-off: Feb-28-08, based on 266 PFS events 2nd interim analysis based on cut-off:15-Oct-07, efficacy boundary crossed with 410 patients/191 PFS events (Motzer et al. Lancet. 2008;372:449–456), complete study unblinded on 28-Feb-08 2nd Interim Analysis Data Cut-Off: 15-Oct- 07, N = 410 End of Double- Blind Analysis Data Cut-Off: 28- Feb-08 Survival Follow-Up: 15-Nov-08 Study Unblinded N = 416 Stratification Prior VEGFr-TKI: 1 or 2 MSKCC risk group: favorable, intermediate, or poor Everolimus 10mg/day + BSC (n = 277) Placebo + BSC (n = 139) RANDOMISATION (2:1) Double-Blind Previous Beva, IL2 or INF-alpha allowed RECORD-1Study: design and conduct

39. RECORD-1:

40. RECORD1: Sub-group Analysis Sub-group of one previous VEGFR-TKI (n=308) – PFS: 5.4 mos Vs 1.9 HR, 0.32; 95% CI, 0.24–0.43; P < 0.001) Sub-group of two previous TKIs (n=108) – 4 mos Vs 1.8 HR, 0.32; 95% CI, 0.19–0.54; P < 0.001 Sub-group of previous only Sunitinib treated (n=56) – 43 Everolimus -13 Placebo – 4.6 mos Vs 1.8 HR, 0.22; 95% CI, 0.09–0.55; P < 0.001)

41. Conclusion When compared to placebo mTOR inhibition seems to be a good strategy when failure to TKI is observed in patients with advanced mRCC Solid data from Randomized Control Trial although a heterogenous population Everolimus is a valid option for the treatment of 2 nd line mRCC

42. Literature Evidence

43. Evidence

46. Optimizar

47. Optimizar: “Buscar la mejor manera de hacer una actividad”

48. “Results have been universally poor. At this time, there appears to be no single agent, hormonal manipulation, or combination drug regimen which is useful in controlling disseminated renal cancer” Quoted from Alan Yagoda 1984 Most of RCC patients dont do very well and a few do very poorly Renal Cell Carcinoma: The XX Century Slide modified from Motzer RJ (ASCO 2010)

49. 2014 …Currently there are data to support that RCC can be approached from different perspectives Multiple drugs have shown activity in this setting

50. Optimizing: Renal Cell Cancer:

51. Optimizing: Renal Cell Cancer

52. Molecular Biology: Summary HIF VHL ub HIF Proteosome Degradation E3 ubiquitin ligase VEGF PDGF TGFa Tumor Growth and Angiogenesis RECEPTORS Clear Cell Renal Cell Carcinoma PI3K mTor c-Met Papillary Type I Renal Carcinoma Prolyl- Hydoxylase O2O2 Fumarate Hydratase Fumarate Malate Papillary Type II Renal Carcinoma 2-oxoglutarate Adapted from: Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell Review 2007. 12(1): 9-22; Suarez et al. Molecular basis for the treatment of renal cell carcinoma. Clin Transl Oncol (2010) 12:15-21. FNIP1 FLCN FNIP2 Cromophobe Renal Carcinoma

53. Treatment Development: Rationale HIF VHL ub HIF Proteosome Degradation E3 ubiquitin ligase VEGF PDGF TGFa Tumor Growth and Angiogenesis RECEPTORS Clear Cell Renal Cell Carcinoma PI3K mTor c-Met Papillary Type I Renal Carcinoma Prolyl- Hydoxylase O2O2 Fumarate Hydratase Fumarate Malate Papillary Type II Renal Carcinoma 2-oxoglutarate Adapted from: Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell Review 2007. 12(1): 9-22; Suarez et al. Molecular basis for the treatment of renal cell carcinoma. Clin Transl Oncol (2010) 12:15-21. mTOR Inhibit C-Met Inhibit TKIs mAb FNIP1 FLCN FNIP2

54. Molecular Biology: Summary HIF VHL ub HIF Proteosome Degradation E3 ubiquitin ligase VEGF PDGF TGFa Tumor Growth and Angiogenesis RECEPTORS Clear Cell Renal Cell Carcinoma PI3K mTor c-Met Papillary Type I Renal Carcinoma Prolyl- Hydoxylase O2O2 Fumarate Hydratase Fumarate Malate Papillary Type II Renal Carcinoma 2-oxoglutarate Adapted from: Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell Review 2007. 12(1): 9-22; Suarez et al. Molecular basis for the treatment of renal cell carcinoma. Clin Transl Oncol (2010) 12:15-21. FNIP1 FLCN FNIP2 Cromophobe Renal Carcinoma mTOR Inhibit C-Met Inhibit TKIs mAb

55. Molecular Biology: Summary HIF VHL ub HIF Proteosome Degradation E3 ubiquitin ligase VEGF PDGF TGFa Tumor Growth and Angiogenesis RECEPTORS Clear Cell Renal Cell Carcinoma PI3K mTor c-Met Papillary Type I Renal Carcinoma Prolyl- Hydoxylase O2O2 Fumarate Hydratase Fumarate Malate Papillary Type II Renal Carcinoma 2-oxoglutarate Adapted from: Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell Review 2007. 12(1): 9-22; Suarez et al. Molecular basis for the treatment of renal cell carcinoma. Clin Transl Oncol (2010) 12:15-21. FNIP1 FLCN FNIP2 Cromophobe Renal Carcinoma

56. Treatment Development: Rationale HIF VHL ub HIF Proteosome Degradation E3 ubiquitin ligase VEGF PDGF TGFa Tumor Growth and Angiogenesis RECEPTORS Clear Cell Renal Cell Carcinoma PI3K mTor c-Met Papillary Type I Renal Carcinoma Prolyl- Hydoxylase O2O2 Fumarate Hydratase Fumarate Malate Papillary Type II Renal Carcinoma 2-oxoglutarate Adapted from: Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell Review 2007. 12(1): 9-22; Suarez et al. Molecular basis for the treatment of renal cell carcinoma. Clin Transl Oncol (2010) 12:15-21. mTOR Inhibit C-Met Inhibit TKIs mAb FNIP1 FLCN FNIP2

57. There are two different mTOR complexes, mTORC1 and mTORC2 Both of them consist of: mTOR, DEP-domain-containing mTOR-interacting protein (Deptor) and mammalian lethal with Sec13 protein 8 (mLST8)

58. mTORC1 mTORC1 comprises 2 distinct components: -Regulatory associated protein of mTOR (Raptor) - Proline-rich AKT substrate 40 kDa (PRAS40);

59. mTORC2 mTORC2 consists of 3 other proteins: mammalian stress- activated protein kinase interacting protein (mSIN1) Rapamycin insensitive companion of tor (RICTOR) and protein observed with Rictor-1 (Protor-1)

60. It is also useful to distinguish two basic signal cascades leading to mTOR1 activation: The first one occurs via the insulin - and the second one via Ras – pathway

61. The first stage of the insulin signaling cascade involves binding of insulin to its receptor which displays tyrosine kinase activity towards insulin receptor substrate 1 (IRS1) When IRS1 is recruited and activated, the signal is transduced via the activation of phosphatidylinositide 3-kinase (PI3K), which subsequently activates phosphoinositide- dependent kinase-1 (PDK1) and then Akt

62. The second pathway, which starts from Ras activation, involves signal transduction via Raf andthen MEK 1/2 to mitogen-activated protein kinase (MAPK) and ribosomal s6 kinases (RSKs)

63. The stimulation of these two pathways by growth factors increases the phosphorylation of tuberin (TSC2) and this way inactivate hamartin-tuberin complex.

64. In the insulin pathway phosphorylation is driven by AKT [23] In the RASpathway signal transduction is mediated by MAPK also known as extracellular signal-regulated kinase 1/2 (ERK1/2) [24].

65. More precisely, mTORC1 is under control of TSC1/2 complex [25] through its GTPase- activating protein activity towards the G- protein Ras homologue enriched in brain (Rheb) [25]. When TSC1 is inactivated, Rheb level is increased and activation of mTOR pathway occurs

66. Regardless this core pathway, the AKT is able to activate mTORC1 in a TSC1/2 nondependent manner. The mechanism involves prolinerich Akt substrate 40 (PRAS40), which regulates mTORC1 by functioning as a direct inhibitor of substrate binding [30]

67. There are two main downstream targets of mTORC1 activity: – the eukaryotic initiation factor 4E (eIF4E)- bindingprotein 1 (4E-BP1) – p70 ribosomal S6 kinase 1 (S6K1) The phosphorylation of 4E-BP1 prevents its binding to eIF4E, enabling eIF4E to promote cap-dependent translation [30]. Translation is also stimulated by S6K1