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Metabolic Targets in Renal Cell Carcinoma Brian Shuch, MD Assistant Professor of Urology and Radiology Cancer Genetics and Prevention Program Yale Cancer Center Brian Shuch, MD Disclosures 1. Company Relationship: honoraria, speakers’ bureau, grants, research support, stock, consultant, et cetera Pfizer Consultant Overview • Review the molecular genetics of RCC • Discuss biology and therapeutic targets in HLRCC • Discuss biology and therapeutic targets in ccRCC Overview of Kidney Cancer 2013 (ISUP Vancouver Meeting) Clear cell RCC Multilocular clear cell RCC Papillary I and II RCC Chromophobe Hybrid oncocytic/chromophobe Collecting duct Renal medullary carcinoma Neuroblastoma associated Mucinous tubular & spindle cell Tubulocystic RCC Acquired cystic disease RCC Clear cell papillary RCC Unclassified RCC MiT family translocation HLRCC • Clear cell kidney cancer represents 75% of RCC • All the rest lumped into a basket considered “non-clear cell RCC” Shuch, B., et al. (2014). Understanding Pathologic Variants of Renal Cell Carcinoma: Distilling Therapeutic Opportunities from Biologic Complexity. Eur Urol. “Non-Clear Cell RCC”- Descriptive?? Phone Market Sneaker Market Lumping rare kidney cancer variants into a “non-clear cell” basket term has led to trials not focusing on unique biology . “The Genetic Basis of Hereditary RCC” Clear Cell Papillary Type 1 VHL MET Papillary Type 2 FH Chromophobe Oncocytoma FLCN • Different germline alterations lead to different forms of kidney cancer • Much of our understanding of sporadic RCC comes from hereditary RCC • However, sporadic forms significantly more complex than previously thought Genomic Alterations Differ by Subtype: Pan-RCC Analysis • Distinct pattern of genetic mutations in kidney cancer • However, several themes emerge common to several forms of kidney cancer Chen, F., et al. (2016). Multilevel Genomics-Based Taxonomy of Renal Cell Carcinoma. Cell Reports. 2016 Genomic Alterations and Dysregulated Metabolism in RCC Ricketts, C. J., et al. (2016). SnapShot: Renal Cell Carcinoma. Cancer Cell, 29(4), 610–610.e1. Kidney Cancer Metabolic Targets • Shared mutations lead to activated metabolic pathways • Overlapping gene alterations in the hypoxia pathway, PI3K/mTOR pathway, chromatin regulation, cell cycle control, and antioxidant response Durinck, S., et al. (2015). Spectrum of diverse genomic alterations define non-clear cell renal carcinoma subtypes. Nat Genet, 47(1), 13–21. Kidney Cancer Metabolic Targets • Glycolysis and the Krebs cycle central to energy processing • Dysregulated glucose metabolism common in RCC • Understanding the metabolic and genetic basis of RCC will provide the foundation for the further development of effective forms of therapy High grade Clear cell/Chromophobe &Oncocytoma FH/SDH Kidney Cancer and high grade pRCC Kidney Cancer- Metabolic Targets Hexokinase Inhibitors (2DG) PKM2 Inhibitors • Obligate metabolism may lead to exquisite sensitivity to inhibition of specific pathways • Carbon metabolism is central to generation of ATP and lipids • Several enzymes have been postulated as therapeutic targets in RCC Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) • Estimated incidence is 1 in 50,000-100,000 • Incidence likely significantly higher from our group’s estimates from EXAC/1000 genomes (~1/1500) • Autosomal dominant inheritance pattern • Linked to 1q42 and found to be Fumarate hydratase (FH) in 2002 • Behaves like classic tumor suppressor gene with LOH found in tumors • Responsible for Krebs cycle enzyme that is responsible for oxidation of fumarate to malate Tomlinson, I. P., Alam, N. A., Rowan, A. J. et al.: Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet, 30: 406, 2002 Cutaneous Leiomyomas In HLRCC Occur in 75% of individuals, generally in clusters. HLRCC Kidney Cancer • 15-30% of patients with HLRCC • Tumors are very aggressive/infiltrative • Can metastasize even when tumors 1-2 cm • ~70% of patients present with Stage III/IV disease • Over 50% of individuals with HLRCC kidney cancer had died of disease • Patients with limited treatment options and those with metastatic disease classically have lived ~12 months • Resistant to classic forms of kidney cancer therapy Grubb et al. Hereditary leiomyomatosis and renal cell cancer: a syndrome associated with an aggressive form of inherited renal cancer. J Urol vol. 177 (6) pp. 2074-9 Renal Manifestations: 46 year old man with hematuria HLRCC and Exquisite Sensitivity to Withdrawal of Glucose HLRCC Sporadic Papillary Type II Relative Cell Growth (% of growth with normal glucose) 120% 100% 80% 60% 40% 20% 0% UOK262 LABAZ1 HRC86T2 MDACC-55 Glucose • Cell lines incredibly sensitive to glucose withdrawal (12 hours) • Blocking glucose uptake postulated to be therapeutic strategy Shuch, B. et al In Preparation • Knock down of FH similarly leads to HIF accumulation • Addition of fumarate/succinate lead to HIF accumulation • HIF1a known to lead to GLUT-1 upregulation • Targeting downstream pathways on HLRCC proven modestly effective with bevacizumab/erlotinib combination • Single arm phase II study – VEGF tx targeting angiogenesis and EGFR perhaps impairing glucose uptake • Arm 1: HLRCC -overall response rate was 65% (all PR) -Median PFS was 24.2 months -some with durable response for >3 years • Arm 2: Sporadic pRCC -29% response rate (all PR) -Median PFS of 7.4 months • NRF2 can protect cancer cells from oxidative stress and promote cell proliferation. • Recent studies reveal that activation of the Nrf2 pathway is critical for resistance to chemotherapeutic agents • Small molecule inhibitors of NRF2 have been recently described • Treatment with NRF2 inhibitors has been shown to re-sensitize nonsmall cell lung ca to platinum based therapy, may Fumarate levels cause cysteine residues to succination alters Keap1 And prevents NRF2 binding and ubiquitination • C13 Radiolabeled Glucose assessed in cells with altered Krebs cycle/ETC, most citrate contained no C13 (m+0), indicating suppressed entry of glucose into Krebs Cycle • Reductive Glutamine metabolism allows generation of AKG and the Krebs Cycle can reverse to allow citrate generation • Galactose oxidation produces no ATP- common in glycolytic cells • Glutamine in FH (-/-) cell line cells cannot grow without carbon source LDH catalyzes the reversible reduction of lactate to pyruvate. LDH-A is upregulated in solid tumors reliant on glycolysis, inhibition of this enzyme has been shown to inhibit growth both in vitro and in vivo Metabolic Targets in HLRCC/HLRCC-like Kidney Cancer Summary of the Mechanism of Tumorigenesis in HLRCC Ub Decreased HR repair αKG-dependent dioxygenase(s) Fumarate HPH X HIFα C TeT proteins KEAP1 NRF2 5hmC 5mC αKG succinate Regulation of DNA methylation Loss of miR200EMT C S C KEAP1 C HIFα HIFα HIFα HIFα HIFα S NRF2 Anti-Oxidant Response Angiogenesis Proliferation Glucose Transport Principle Component Analysis: Comparing Papillary I/II RCC to HLRCC HLRCC Pap I Pap II Published CEL files assessing mRNA using Affymetric HG 133 U Plus 2 Shuch, B ASCO GU 2012 Altered Metabolism of HLRCC 140 Oxygen Consumption Extracellular Acidification OCR (O2/min/ug of prot) ECAR (mpH/min/ug of prot) 180 120 100 80 60 40 20 160 140 120 100 80 60 40 20 0 LABAZ HRC86T2 MDA55 UOK262 UOK262 had significantly high amounts of extracellular acidification/glycolysis Shuch, B ASCO GU 2012 0 LABAZ HRC86T2 MDA55 UOK262 The sporadic papillary lines have normal oxygen consumption while UOK262 has none Dysregulated Glucose Metabolism Gene Expression ***Gene expression for Glycolysis, Krebs Cycle, PPP, glycogen, pyruvate pathways Shuch, B ASCO GU 2012 Applicability to Sporadic Papillary RCC Identification of CIMP Cluster/Warburg Class of Tumors Similar to HLRCC • CpG Island Methylated Phenotype cluster: • Germline FH alterations and other mechanisms such as CDKN2A loss, and alterations in NRF2/KEAP1 pathway • Awful prognosis • CIMP phenotype may be interesting population to target with inhibitors of DNA methylation, CDK4/6 inhibitors, or glycolytic inhibitors (Warburg effect) Altered Metabolism in the HLRCC-like CIMP cohort Aggressive type 2 pRCC can have a similar metabolic phenotype to HLRCC How common this is in advanced pRCC is unknown, but will be investigated in S1500 SWOG trial, n=180 metastatic pRCC patients Clear Cell Kidney Cancer • VHL loss (mutation/hypermethylation) in >80% of clear cell RCC • 95% of clear cell RCC demonstrate 3p loss, leading to LOH • VHL dysregulation central to hypoxia pathway • Hypoxia adaptive changes include upregulation of glycolysis • Accounts for 90% of metastatic RCC • Loss of VHL and resultant downsteam changes including upregulation of the VEGF pathway have led to our current systemic therapy approaches to treatment • Over 100 other downstream targets of HIF which may also be therapeutic targets in ccRCC • VHL also has HIF independent functions and loss has not been a focus on research Shuch, B., et al. (2014). Understanding Pathologic Variants of Renal Cell Carcinoma: Distilling Therapeutic Opportunities from Biologic Complexity. Eur Urol. • Around time of discovery of VHL, team from JHU discovered that HIF1 was a major regulator of glycolytic enzymes • HIF1a responsible for transcriptional regulation of most of the enzymes regulating glycolytic flux • HIF1a and HIF2a with similar actions bind to HIF1b to form a heterodimer and bind to same HRE • Inducible HIF1a/HIF2a Lead to transcription of many of the same genes • However they play non-redundant roles, and activation of each one results in a distinctly different phenotype. • HIF2 DOESN’T REGULATE GLYCOLYSIS • Two “flavors” of VHL (-/-) kidney cancer, HIF1/2 vs HIF2 • Metabolism very different, HIF1/2 tumors with high expression of glycolysis genes • Perhaps different therapeutic response to anti-metabolic therapy • Oxygen is the ultimate electron acceptor for mitochondrial respiration, a process catalyzed by cytochrome c oxidase (COX), the terminal enzyme on ETC • HIF1a leads to downregulation of mitochondrial respiration via COX regulation ultimately suppressing the Krebs cycle/oxidative phosphorylation • ccRCC has fundamental shift towards Warburg biology • Hypoxia/HIF1 upregulation leads to glycolysis • C13 analysis demonstrates reductive carboxylation in hypoxic cells resulting in shuttling of glutamine into Krebs cycle for carbon intermediates • VHL (-/-) lines in normoxic conditions share a similar phenotype due to HIF dysregulation. • HIF1a K.D. reverses phenotype • Metabolic findings supported in ccRCC • Suppression of Krebs cycle enzymes observed in the TCGA ccRCC analysis of 412 tumors • Evidence of other dysfunctional pathways including upregulation of Oxidative Pentose Phosphate Pathway and Fatty Acid Synthesis Cancer Genome Atlas Research Network. (2013). Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature, 499(7456), 43–49. • Later MSKCC group matched tumor/normal kidney pairs (n=138) and performed mass spec for metabolites, RNAseq, and pathway analysis • Other dysregulated pathways included PPP and lipid metabolism • Tumor progression associated with metabolite increases in glutathione and cysteine/methionine metabolism Hakimi, A. A., Reznik, E., Lee, C.-H., Creighton, C. J., Brannon, A. R., Luna, A., et al. (2016). An Integrated Metabolic Atlas of Clear Cell Renal Cell Carcinoma. Cancer Cell, 29(1), 104–116. Targeting Lipid Metabolism in ccRCC • Clear cell is “clear” due to lipid accumulation • Reductive carboxylation through glutaminase for citrate source for lipogenesis • ccRCC with overexpression of FASN have significantly worse outcome Albiges, L., et al. (2016). Body Mass Index and Metastatic Renal Cell Carcinoma: Clinical and Biological Correlations. JCO. 34, 30 (October 2016) 3655-3663. • • FASN inhibitor C75 was studied in ccRCC models Lead to growth inhibition in cell culture and xenografts. • FASN inhibitors have made it into clinical development for cancer • ccRCC may be an interesting tumor to study similar agents due to role of FASN/lipid metabolism Conclusions • Renal cell carcinoma (RCC) is a heterogenous group of cancers however common dysregulated pathways lead to similar metabolic phenotype • HLRCC and related tumors have an extreme Warburg phenotype, reliance on glutamine metabolism, and are driven through dysregulation of metabolites (oncometabolites). • ccRCC, due to VHL loss have similar metabolic properties including upregulation of glycolysis, Krebs cycle depression, and reductive carboxylation. • Complete reliance on these pathways may make them exquisitely vulnerable to novel metabolic strategies currently in early clinical development. Questions? [email protected]