Relevant Disclosures. Targeting VHL Tumors with RTK Inhibitors. VHL Gene and Protein. VHL Mutation Replicates the Hypoxic State. HIF Non HIF Proteins

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1 HIF Non HIF Proteins HIF Targets Targeting Tumors with RTK Inhibitors Relevant Disclosures Consultant: Peloton, Pfizer, Novartis Research Funding: Pfizer, Novartis Eric Jonasch, MD UT MD Anderson Cancer Center Houston Texas Gene and Protein Mutation Replicates the Hypoxic State On chromosome 3p amino acid protein Binds to Elongin C/B Forms VBC complex Elongin C (15kDa) Elongin B (18kDa) Transcription of: Other angiogenic factors HIF HIF Nucleus Cul 2 Modified from Stebbins and Pavletich, Science, Vol 284, 16 April 1999 = vascular endothelial growth factor; HIF = hypoxia inducible factor. 1

2 Loss of Mediated HIF Regulation Drives Angiogenesis HIF Non HIF Proteins HIF Targets R PDGFR Stromal cells Tumor cells / / / EGFR If HIF Regulation is the Main Function of, is Blockade of Downstream Consequences of HIF Upregulation Sufficient to Normalize Phenotype? R = vascular endothelial growth factor receptor; EGFR = endothelial growth factor receptor; PDGFR = platelet derived growth factor receptor. Blocking Agents Response to Sunitinib Roma and Zonegel Fam Cancer

3 One Case Report of Hemangioblastoma Response to Sunitinib 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 Reyes Bottaro and Sanson J Neurooncol 2012 Jonasch and Matin, Annals of Oncology 2011 *(P=0.014) 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 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 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) Jonasch and Matin, Annals of Oncology 2011 *(P=0.014) 3

4 Pancreatic NETs Responded Renal Masses Responded Baseline 12 Weeks 24 Weeks Jonasch and Matin, Annals of Oncology 2011 Hemangioblastomas Did Not Respond Comparison of Hemangioblastoma and Renal Cell Carcinoma Tissue Blood Vessels. 4

5 Related Tumors Are Endothelial Cells in RCC and Hemangioblastomas Driven by Same Growth Factors? R R Stromal cells Stromal cells -/- -/- Tumor cells / -/- PDGFR R EGFR Tumor cells / -/- PDGFR R EGFR Hemangioblastomas Densely packed, seemingly normal blood vessel channels of varying sizes, separated by stromal cells. 20 Hemangioblastomas Evaluate Status of Different Receptor Types in Blood Vessels Using Laser Scanning Cytometry Determine Differences Between Hb and RCC 20 Renal Cell Carcinomas Jonasch et al Annals of Oncology

6 log(hb) log(rcc) t-test Wilcoxon s rank test N mean SD N mean SD p-value p-value pr2 in CD31 Cells tr in CD31 Cell ppdgfr in CD31 Cells tpdgfr in CD31 Cells R.ratio PDGFR.ratio Bottom line: log(hb) log(rcc) t-test Wilcoxon s rank test N mean SD N mean SD p-value p-value Tie2 in CD31 Positive Cells Tie2 in Tumor Tissue FGFR3 in CD31 Positive Cells FGFR3 in Tumor Tissue pfrs2 in CD31 Positive Cells pfrs2 in Tumor Tissue receptor activation state is higher in kidney tumors compared to hemangioblastomas. Bottom line: pfrs2 (a marker for FGF receptor activation) is higher in hemangioblastomas compared to kidney tumors Summary Next Steps: Decreased activation state of pr in hemangioblastoma vs RCC tissue. Increased activation state of pfrs2 in hemangioblastomas vs RCC tissue. 1. To explore FGF receptor blockade in patients with hemangioblastomas 2. To further develop the concept of antiangiogenic drugs in patients with RCC and NETs. 6

7 Two Trials Moved Ahead Dovitinib 1. Single center pilot study testing dovitinib in patients with hemangioblastomas NCT Oral anti-fgf receptor inhibitor. Also blocks R, c-kit and Flt3. 2. Multicenter 40 patient pazopanib trial for patients with RCC NCT Study Design Pazopanib in Disease 14 patient, single arm study with option to increase sample size if response is seen in hemangioblastomas. To be eligible, all patients had hemangioblastomas Study stopped after six patients- stable disease, but unacceptable skin toxicity Considering other agents with greater FGFR specificity and lower toxicity Kim Jonasch and McCutcheon Targ Oncol

8 Ongoing Study NCT , a Phase II Study of Pazopanib in Patients. Must have at least one measurable lesion. 08 C 0020: Phase II Study of ZD6474 (Vandetanib) in Patients with von Hippel Lindau () Disease and Renal Tumors So far 31 out of 40 patients enrolled. Courtesy of Ram Srinivasan Objectives Primary Overall response rate in patients with renal tumors Secondary Safety and tolerability in patients Progression free survival Effect of ZD6474 on non renal tumors Study Design Single arm, open label phase 2 study ZD6474 oral Continuous daily dosing 300mg/day Simon optimal two stage design Initial stage: 12 patients If 1 or more of initial 12 respond, maximum of 37 patients will be enrolled Assess response by RECIST q 12 weeks 8

9 Eligibility Criteria Demographics Clinical diagnosis of One or more measurable tumors Adequate organ function ECOG 2 Response 9

10 A Regulatory Hub Antiangiogenic Agents Summary Response in RCC and in NETs. Long term tolerability an issue. Less successful in hemangioblastomas reason unclear at moment. Require additional study to identify vulnerabilities in Hb Consider more potent FGFR inhibitors. Li and Kim J Cell Mol Med 2011 HAF Differentially Regulates HIF1a and HIF2a, and is Potentially Targetable HIF Non HIF Proteins HIF Targets HIF Non HIF Proteins HIF Targets Koh and Powis TIBS

11 HIF Non HIF Proteins HIF2a Inhibitor HIF Targets Peloton pharmaceuticals HIF2 inhibitor in clinical trials. May be a good agent to test in patients with disease. HIF Non HIF Proteins HIF Targets Point Mutations Destabilize But May Retain Functionality Over One Third of Mutations are Missense (Hereditary and Sporadic) C77A W117A L118P F148A -mm C162F R167Q WT / HIF2 C terminal Venus Tagged Proteins Can we rescue these mutant proteins? Zhiyong Ding Zbar et al: Human Mut 1996;8(4):

12 Raising Level of Rescuable Mutant R167Q Isoform Abrogated Tumor Growth Group A: parental line Peter German Name That Band Group B: with L117A mutation and a C terminal Venus tag Zi Group C: with R167Q mutation and a C terminal Venus tag Group D: with R167Q mutation and no tag 30 Other Band 19 Zhiyong Ding Ding and Jonasch, Ca Research 2014 Researchers have long noticed a second band below 30, which seems to vary from blot to blot. We observed that proteasome inhibition enhanced this band. Proteasome inhibition enhances lower band in a time dependent manner. Edman degradation identified lower band as arising from N terminal cleavage at AA 23 and is dependent on protein synthesis. 12

13 A CK2 inhibitor, CX4945 (silmitasertib) markedly reduced accumulation of the lower band after proteasome inhibition A CK2 inhibitor, CX4945 (silmitasertib) markedly reduced accumulation of the lower band after proteasome inhibition Alanine substitution of the CK2 phosphorylation sites (aa 33, 38 and 43) ablated presence of lower band. Alanine substitution of the CK2 phosphorylation sites (aa 33, 38 and 43) ablated presence of lower band. A chymotrypsin inhibitor, AEBSF, markedly reduced accumulation of the lower band after proteasome inhibition CK2 blockade enhances R82P mutant s ability to regulate HI2 levels in RCC cell lines. 13

14 CK2 effect is dependent. Therapeutic Opportunities Therapeutic Opportunities 14

15 HIF Non HIF Proteins HIF Targets Evolution of Treatment Approaches Silmitasertib Identify Gene AKA CX4945 Highly specific CK2 inhibitor In clinical trials for other diseases Relatively well tolerated Targeting downstream consequences of deficiency Trial design in discussion with Senhwa Biosciences Move upstream to more proximal targets of intervention Summary Antiangiogenic RTK therapy can round off the edges of phenotype. We need to perform additional detailed analysis of disease related endothelial biology to refine RTK approach. Future efforts should focus on upstream stabilization of defect. NANOMEDICINE TEAM Wah Chiu Judith Frydman Bill Mobley NIH ROADMAP TEAM Richard Fischer Jeffery Schloss COLLABORATORS Shuxing Zhang Peter Davies Kenneth Scott Amato Giaccia CENTER Surena Matin Steven Waguespack Ian McCutcheon Dan Gombos LAB Zhiyong Ding Lijun Zhou Xiande Liu Mianen Sun Peter German Shanshan Bai Xuesong Zhang 15