How genetic & biochemical alterations in brain tumors contribute to epileptogenesis December 2 nd, 2012 Joon H. Uhm, MD FRCPC Departments of Neurology & Oncology Mayo Clinic, Rochester, MN American Epilepsy Society Annual Meeting
Disclosures Name of Commercial Interests NONE Type of Financial Relationship N/A American Epilepsy Society Annual Meeting 2012
Learning Objectives To gain a better understanding of: The key genetic alterations in gliomas How these genetic alterations lead to production of factors that contribute to epileptogenesis How tumor-derived epileptogenic factors may be targeted for therapeutics American Epilepsy Society Annual Meeting 2012
Molecular basis of tumor-associated epilepsy: Are we there yet? 4
Link between tumor biology & epileptogenesis Tumor biology Epilepsy 5
Start with a clinical case 44 y.o. man, intractable seizures 1998 GTC seizure, surgery - low grade glioma 1999 Radiation, chemo April 2010 intractable seizures (no tumor growth) June 2011 Vagal nerve stimulator Oct 2011 R. sided focal motor seizures every hour No tumor growth Surgery too risky Plan: Try chemo, hope for best Outcome: Sz frequency: once per week Tumor stable What leads to seizures in glioma patients? Tumor-derived factors that alter the microenvironment? (In this pt, tumor did not grow, but +++SZ) Can we target these factors with drugs? 6
Where do we start?... Key components of the glioma engine
Genetic alterations in gliomas 8
TCGA (The Cancer Genome Atlas): Human genome project focused on human glioblastoma What are the key genes that are altered in glioblastoma? First human tumor to be comprehensively evaluated at genetic level 9
Beyond morphology Era of molecular genetics Gene expression profile of human glioblastoma 3 molecular classes of glioblastoma Survival: PN>Prolif>Mes Patient prognosis (and, in future, treatment) based upon molecular data Rich databases to be mined for molecular alterations that contribute to epileptogenesis Phillips/Adape Cancer Cell 2006
Effector functions Gene induction Cell cycling Signal transduction Genetic & biochemical alterations in glioma (Mutation frequencies from: TCGA, Nature 455, 1061-1068, 2008) Ras msos Ras Grb2 SHC Raf-MEK- MAPK G F P P PLC-g Mutation, deletion in 36% DAG PKC EGFR PI3K Mutation/amplification in 45% PTEN mtor Mutation in 87% AKT Survival T p53 Protease Proteolysis Invasion RNA DNA Y Integrin Y Motility 11 TF Altered in 78% of gliomas - Gear box of tumor engine M G1 - Alteration shifts Cell Cycle vegf from STOP to G2 S GO G1 CDK4 STOP T Rb P16 S Proliferation Blood vessels G1/S Checkpoint GO Rb-P Angiogenesis
12
How do these genetic alterations then lead to epilepsy? 13
Mechanism #1: Tumor growth leads to mass effect on adjacent brain seizure Molecular alterations tumor proliferation mass effect seizure Reduction in tumor mass (surgery, radiation, chemo) improvement in Sz control But.. Can t be due to mass effect or growth rate alone
Low grade tumors are more likely to cause seizures TUMOR TYPE Dysembryonic neuroepithelial tumor 100% Ganglioglioma 80-90% Low grade astrocytoma 75% Meningioma 29-60% Glioblastoma 29-49% Metastases 20-35% Primary CNS lymphoma 10% SEIZURE FREQUENCY (adapted from: Van Breemen, Wilms, Vecht. Lancet Neurol 6:421-430, 2007) Effect of tumor type & location? (TL location of ganglioglioma) Effect of survival time? (Low grade tumor pts live longer) 15
Mechanism #2: Infiltration of adjacent neural tissue leads to Sz 16
Infiltration: 1) Degrade 2) Move 3) Bring in supplies Ras msos Ras Grb2 SHC Raf-MEK- MAPK GFR G F P P PLC-g DAG PKC PI3K PTEN mtor AKT Survival T p53 Protease Proteolysis Invasion RNA DNA Y Integrin vegf Y Motility 17 TF M G2 G1 Cell Cycle S G1 CDK4 STOP T Rb P16 S Proliferation G1/S Checkpoint GO Rb-P Blood vessels Angiogenesis
Proteases in tumor invasiveness & epileptogenesis: Matrix metalloproteinase-9 (MMP9) Mizoguchi H et al, J Neurosci 31:12963 (2011) Glioma invasiveness Abundantly expressed by gliomas Mediates glioma cell invasiveness Epileptogenesis JH Uhm et al.; JS Rao, Univ of Illinois - Peoria Increased levels in SE and febrile seizures Kindled seizure model increased MMP9 If knock out MMP9 expression no kindling Mechanism? 18
19
MMP9 Pro-BDNF BDNF Kindling/Epilepsy Protease activity & Kindling 20
MMPs as therapeutic targets? MMP inhibitors evaluated in glioma clinical studies (Marimastat, AG3340) No impact on patient survival Could it impact upon Sz control? Issue of adequate CNS delivery (poor BBB penetration) Tetracyclines, minocyclines Area of further research & development 21
Infiltration: 1) Degrade 2) Move 3) Bring in supplies Ras msos Ras Grb2 SHC Raf-MEK- MAPK GFR G F P P PLC-g DAG PKC PI3K PTEN mtor AKT Survival T p53 Protease Proteolysis Invasion RNA DNA Y Integrin vegf Y Motility 22 TF M G2 G1 Cell Cycle S G1 CDK4 STOP T Rb P16 S Proliferation G1/S Checkpoint GO Rb-P Blood vessels Angiogenesis
Need blood supply to grow beyond size of head of pin Angiogenesis
Vascular endothelial growth factor (vegf) is a key angiogenic protein secreted by gliomas 24
The simplified view of vegf Bevacizumab (BEV) Y vegfr Blood vessels Blood vessels
Bevacizumab is very effective in reducing tumor size, edema, and steroid requirement. 26
vegf its inhibitors a more realistic (& complicated) view vegf double-edged sword Croll SD et al. Adv Exp Med Biol 2004 vegfr Y Inflammatory cytokines ICAM-1, Mip-1 BEV Sz Y vegfr Y Infiltration of brain vegfr Blood vessels Blood vessels Tumor moves to blood vessel 27
Cancer Cell 15, 220-231 (2009) Cancer Cell 15, 232-239 (2009)
Increased invasiveness as mech of resistance 1-07 9-07 29
Mechanism #3: Tumors secrete excitotoxic factors that ultimately lead to seizures The glutamate story 30
Glu release Sz (mouse model) Buckingham SC et al. (Sontheimer H), Nat Med 2011 Human glioma implanted into mouse brain Maintains gene expression and invasive growth characteristics of original tumor. Giannini et al. Neurooncol 7, 164, 2005. ~40% experience Sz Increase Glu release by tumor slices Previous in vitro studies Glu release by glioma cells mediated by Xc- cystine/glutamate transporter SAS (sulfasalazine), which blocks Xc- transporter, was able to inhibit glu release and the hyperexcitability 31
Glu release by glioma induces epileptic activity Buckingham SC et al. (Sontheimer H), Nat Med 2011 32
Glu release Sz (human data) Yuen TI et al. Neurology 2012; 79:883 Compare glioma specimens from pts with tumor-associated seizurs (TAS) vs those who did not have TAS In TAS samples: Increased glu Decreased expression of EAAT2 (excitatory amno acid transporter-2; function is to clear glu via reuptake Increased expression of Xc- transporter (directs glu from intra- to extra-cellular compartment) Net effect: increase extracellular Glu 33
Clinical implications Glu Sz Glu increases glioma growth & invasiveness Could targeting Glu pathway be antitumorgenic and antiepileptic? 34
GluR inhibitor (Talampanel) in glioma clinical study Phase II study (Grossman SA et al., J Clin Oncol 27:4155-4161. 2009) Well tolerated in combination w/ radiation/chemotherapy regimen Pending randomized study 35
Is Xc- transporter a viable target? Phase I/2 study of sulfasalazine (SAS) in recurrent glioma pts (10 pts) Rationale: SAS inhibits NF-kappaB and Xc- transporter Outcome: No tumor shrinkage; stable disease in 1 pt 50% experienced increased cerebral edema; 2 experienced increased seizures (cerebral edema, reduced AED levels) Early termination of study (Robe PA et al., BMC Cancer 2009, 9:372) 36
Mechanism #4: Altered bioenergetics in gliomas lead to the production of a metabolite that is epileptogenic Discovery of the IDH1 mutation
Parsons DW et al., Science 2008, 321:1807 38 Identification of a Kreb s cycle enzyme that is mutated in human gliomas: IDH1
IDH1 mutation 39
Mutant IDH1 generates 2HG Dang L et al., Nature 2009, 462: 739 IDH1 (wt) IDH1 (mutant) D-2-hydroxyglutarate (2HG) Generalized Sz in pt w/ D-2-hydroxyglutaric aciduria D-2HG is NMDA receptor agonist IDH1 mutation seen predominantly in low grade gliomas; rarely in primary glioblastoma Sz frequency is higher in low grade gliomas 40
IDH mutation occurs early in glioma development 41
Mutant IDH1 as a potential therapeutic target Would inhibition of mutant IDH1 lead to better seizure control? Would inhibition of mutant IDH1 inhibit a low grade glioma from transforming to a higher grade tumor? No current drugs to target mutant IDH1, but intense area of research 42
In summary 43
Epilepsy 44 DNA Y VEGF Integrin Protease RNA P P PTEN AKT mtor Survival p53 T PI3K PKC DAG TF PLC-g STOP GO CDK4 T P16 Rb Rb-P G1 S G1/S Checkpoint Y Blood vessels G1 S G2 M Cell Cycle Proliferation Angiogenesis Motility Proteolysis Invasion EGFR G F Ras SHC Grb2 msos Raf-MEK- MAPK Ras 44 Proteases Angiogenic factors Glutamate 2HG (IDH1)
Proteases Infiltration, BDNF Angiogenic Infiltration factors Inflammatory Glutamate cytokines Excitotoxic Tumor-derived pollutants promote epileptogenesis And they may be amenable to therapeutics 45 2HG (IDH1)
In closing The identification & understanding of key pathways that drive glioma growth leads not only to more effective antineoplastic therapies, but potentially more effective antiepileptic strategies. 46
From the Mayo team, Thank you for your attention! 47