State of the Art of Therapeutic Approaches for VHL Disease. VHL Gene and Protein

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1 State of the Art of Therapeutic Approaches for VHL Disease Eric Jonasch, MD UT MD Anderson Cancer Center Houston Texas Coming Up With A Cure: Many Layers of Knowledge are Needed! Identification of the VHL Gene Description of VHL Protein Function Identifying and Characterizing Additional Genes Disrupted in VHL Disease Therapeutic Avenues? Development of Relevant Model Systems VHL Gene and Protein HIF Upregulation Alters Regulation of Reductive Carboxylation On chromosome 3p amino acid protein Binds to Elongin C/B Forms VBC complex Elongin C (15kDa) Elongin B (18kDa) Cul 2 Modified from Stebbins and Pavletich, Science, Vol 284, 16 April 1999 Gameiro PA et al, Molecular Cell 2013 ILIOPOULOS LAB, MGH Cancer Center/Harvard Medical School Courtesy of Othon Iliopoulos 1

2 VHL- A Regulatory Hub DNA The VHL Life Cycle Elongin C Elongin B Cul 2 Chaperones TRIC Elongin C p53 * Extracellular Matrix Control p53 Regulation? Primary Cilium Function Angiogenesis Protein Production Mature VHL Complex Ohh et al, Mol Cell, Vol 1, , 1998 Kurban et al, Cancer Res 2006; 66: (3). Roe and Youn Mol Cell May 2006 Thoma et al Nature Cell Biology Aug 2009 Kuehn et al Ca Res May 15, 2007 Pugh et al Narture Medicine 2003 Kerbel NEJM May 2008 * Roe and Youn Mol Cell 2006 DNA Mutated VHL May Fail to Bind TRiC Protein Folding Mutated VHL May Fail to Release from TRiC if VBC Complex not Formed DNA Chaperones TRIC Chaperones TRIC Inability to Bind Elongin C HSP90 Protein Production Proteasomal Degradation Protein Production Proteasomal Degradation 2

3 Mutated VHL May Fail to Bind to Other Substrates Over One Third of Mutations are Missense (Hereditary and Sporadic) TRIC Collagen IV AURKA HIF Others What this means is you have a full sized protein, that can possibly be fixed Zbar et al: Human Mut 1996;8(4): Point Mutations Destabilize VHL But May Retain Functionality C77A W117A L118P F148A VHL-mm C162F R167Q WT VHL / And It s Not Only VHL That is Mutated! Renal Cell Carcinoma: SETD2, PBRM1, BAP1 VHL30 VHL19 Hemangioblastomas: HNF1B C terminal Venus Tagged Proteins HIF2 Knowing how these genes interact will be critical to fully understanding VHL disease, develop relevant model systems and discover treatments 3

4 Currently Evolving Treatment Paradigms Currently Evolving Treatment Paradigms Targeting HIF Dependent Downstream Consequences of VHL Loss HIF, HAF, VEGF Modulating Agents and Metabolism Modifiers Targeting HIF Dependent Consequences of VHL Loss HIF, HAF, VEGF Modulating Agents and Metabolism Modifiers Restabilizing/Refunctionalizin g Mutated VHL Modulators of VHL Proteostasis Restabilizing/Refunctionalizin g Mutated VHL Developing Synthetic Lethal Approaches that Target the Tumor Cell Modulators of Autophagy, or of co Mutated Genes Developing Synthetic Lethal Approaches that Target the Tumor Cell Targeting the Immune Microenvironment Immune Checkpoint Inhibitors Targeting the Immune Microenvironment Targeting Downstream Consequences of HIF Dysregulation VEGF VEGF VEGFR PDGFR Stromal cells Tumor cells VHL / VHL / VEGF VHL / EGFR VEGFR = vascular endothelial growth factor receptor; EGFR = endothelial growth factor receptor; PDGFR = platelet derived growth factor receptor. 4

5 15 Patient Sunitinib Study Lesion site Number of PR (%) SD (%) PD (%) Lesions Hemangioblastoma* (91) 2(9) Renal cell carcinoma* 18 6 (33) 10(67) 2(10) Renal cyst (100) 0 Retinal angiomas (100) 0 Pancreatic NET (100) 0 Pancreatic cyst (100) 0 Nine out of 15 patients completed study most came off study due to poor tolerability Jonasch and Matin, Annals of Oncology 2011 *(P=0.014) Pazopanib in VHL Disease 37 patient study: One RECIST PR, some shrinkage in a number of patients. Ram Srinivasan Kim Jonasch and McCutcheon Targ Oncol

6 Ongoing Study NCT , a Phase II Study of Pazopanib in VHL Patients. Must have at least one measurable lesion. Targeting HIF So far 23 out of 40 patients enrolled. Geldanamycin, an HSP90 Inhibitor, Destabilizes HIF Low Oxygen Or Mutation HIF HIF Generates VEGF Other angiogenic factors VHL Nucleus Ram Srinivasan 6

7 NIH Geldanamycin Study HAF Differentially Regulates HIF1a and HIF2a, and is Potentially Targetable Geldamycin administered to 8 patients with VHL disease and measurable RCC lesions. Stable disease seen in all patients. Trial closed due to slow accrual. Koh and Powis TIBS 2012 HIF2a inhibitors decreases hypoxia induced erythropoiesis and angiogenesis A 0.01% DMSO DMOG + DMSO DMOG + 10nM 76 Othon Iliopoulos: VHL Models and Novel Therapeutics Massachusetts General Hospital, Boston MA B C 0.01% DMSO DMOG + DMSO DMOG + 10nM 76 Total Intensity from pixelmachine learning DMSO 76 compound DMOG * * + + 1nM 10nM Ana Metelo et al ILIOPOULOS LAB, MGH Cancer Center Harvard Medical School Zebrafish are tiny fish that can be genetically modified VHL mutation in zebrafish can represent aspects of human biology Dr. Iliopoulos will use zebrafish to discover new drugs that may rescue consequences of VHL mutation. Courtesy of Othon Iliopoulos 7

8 Loss of PGC 1α Induces the Clear Cell Phenotype in Renal Proximal tubule Cells Metabolomics PAS (Glycogen)Oil Red O (Lipids) HK2 Cells shgfp shpgc-1 #1 shpgc-1 #2 Genetic suppression of PGC 1α results in lipid and glycogen accumulation, consistent with clear cell phenotype of renal cell carcinoma. Mechanism of lipid accumulation unclear Increased lipid synthesis or uptake driving lipid accumulation? Decreased fatty acid oxidation? Courtesy of Amato Giaccia Rel Abs. Units/mg Protein shgfp Oil Red O Quantification * * shpgc-1 #1 shpgc-1 #2 PGC 1α Expression Impedes Tumor Growth Summary In vivo In vitro NOD/SCID mice were injected subcutaneously with 5x10⁶ 786 O cells transduced with control or PGC 1α retrovirus (n=8 per group) Ectopic expression of PGC 1α impairs 786 O tumor growth Does PGC 1α effect any metabolic features of ccrcc in vivo? Courtesy of Amato Giaccia HIF suppresses PGC 1α via activation of Dec1 Loss of PGC 1α results in decreased OXPHOS activity and promotes clear cell phenotype Induction of PGC 1α induces oxidative stress, reverts metabolic phenotypes, and suppresses tumor growth Courtesy of Amato Giaccia 8

9 Regulation of reductive carboxylation by HIF Loss of VHL renders RCC cells/tumors sensitive to glutaminase inhibitors in vivo Tumor size (mm 3 ) Control Day 0Day 2Day 4Day 6Day 8 * BPTES 0.7 * 0.6 * 0.5 * * 0.4 Day 10 Day 12 Day 14 Tumor weight (g) Control BPTES Gameiro PA et al, Molecular Cell 2013 ILIOPOULOS LAB, MGH Cancer Center/Harvard Medical School Courtesy of Othon Iliopoulos Gameiro PA et al, Molecular Cell 2013 ILIOPOULOS LAB, MGH Cancer Center/Harvard Medical School Courtesy of Othon Iliopoulos Phase I Study Currently Evolving Treatment Paradigms NCT A Phase 1 Study of the Safety, Pharmacokinetics, and Pharmacodynamics of Escalating Oral Doses of the Glutaminase Inhibitor CB 839 in Patients with Advanced and/or Treatment Refractory Solid Tumors. Trial allows recruitment of patients with metastatic RCC. Targeting HIF Dependent Downstream Consequences of VHL Loss Restabilizing/Refunctionalizin g Mutated VHL Developing Synthetic Lethal Approaches that Target the Tumor Cell Targeting the Immune Microenvironment HIF, HAF, VEGF Modulating Agents and Metabolism Modifiers Modulators of VHL Proteostasis Modulators of Autophagy, or of co Mutated Genes Immune Checkpoint Inhibitors 9

10 Genetic Titration of Mutant VHL Levels Alters Growth in Xenograft Models Pharmacological Modulation of VHL Levels Can Augment Function All VHL isoforms in this figure are untagged, with lower basal levels Cell Line Ongoing Trial NCT : Carfilzomib in patients with refractory RCC. Trial on hold after 12 patients, with tissue specific analysis to test genotype phenotype link. 10

11 Preclinical Study: Daniel Segal Fixing Broken VHL Tel Aviv University, Tel Aviv Currently Evolving Treatment Paradigms Targeting HIF Dependent Consequences of VHL Loss HIF, HAF, VEGF Modulating Agents and Metabolism Modifiers Dr. Segal will study a candidate substance, D Arginine, which appears to stabilize mutant VHL protein. Restabilizing/Refunctionalizin g Mutated VHL Developing Synthetic Lethal Approaches that Target the Tumor Cell Modulators of VHL Proteostasis Modulators of Autophagy, or of co Mutated Genes Targeting the Immune Microenvironment Immune Checkpoint Inhibitors Currently Evolving Treatment Paradigms Targeting HIF Dependent Consequences of VHL Loss HIF, HAF, VEGF Modulating Agents and Metabolism Modifiers Restabilizing/Refunctionalizin g Mutated VHL Modulators of VHL Proteostasis Treatment with STF Developing Synthetic Lethal Approaches that Target the Tumor Cell Modulators of Autophagy, or of co Mutated Genes Targeting the Immune Microenvironment Immune Checkpoint Inhibitors 11

12 Tumor Cell or Antigen Presenting Cell Immune Checkpoints Signal 2 T cell But what if all you see below is immunogenic? We need to proceed, but with caution B7.1/2 CD28 CTLA 4 B7 H1 (PD L1) HLA Class II MHC Signal 1 antigen T Cell Receptor LAG 3 PD 1 C. Drake Past Present and Future 3p Shows Multiple Hits in RCC Identification of the VHL Gene Q arm P arm Targeting VHL Gene Deficiency PBRM1 3p VHL 3p25 1 BAP1 3p SETD2 3p Identifying and Characterizing Additional Genes Disrupted in VHL Disease 1: Latif et al Science1993 May 28;260(5112): : Dalgliesh and Futreal, Nature Jan 21;463(7279): : Varela and Futreal, Nature 469 (7331): , Pena Llopis and Brugarolas, Nature Genetics,

13 Focal loss of chromosome 17q12,32 in hemangioblastomas Case L2 Hydroxyglutarate Dehydrogenase (on 14q) Suppresses In Vitro Tumor Phenotypes A CV (+) L2HGDH (+) Days A498 CV CV cell count (cells/ 6 well) cell count (cells/ 6 well) RXF393 CV (+) L2HGDH (+) Days RXF393 L2HGDH L2HGDH Mianen Sun Courtesy of Sunil Sudarshan Summary Current therapies that can round off the edges of biology arising from VHL deficiency, and may in some cases be very helpful. Only by characterizing the additional genomic drivers of RCC, hemangioblastoma, pheo and NETs will we be able to devise appropriate model systems that can help develop curative therapy. 13