Tuberous Sclerosis Complex: From Genes to Therapeutics

Orrin Devinsky, MDNYU Tuberous Sclerosis & Epilepsy

Centers

TSC Facts

~1 in 6,000 live births ~1 in 9,000 in the general population

~50,000 Americans have TSC >1 million worldwide Neurological manifestations often most devastating – affecting almost all individuals with TSC in some manner, from mild to severe

Remarkable Progress

TSC1 and TSC2 genes identified -1990’s

TSC1/2 complex in cell growth, cell size and proliferation discovered - 2002

Uncovering role of TSC1/2 complex in regulating mTOR

First clinical trial in 2005 Approval of first treatment specifically for TSC in 2010 (Everolimus (Affinitor))

Data from TS Alliance, CDMRP and NIH

0

50

100

150

200

250

300

350

Number

1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010

Year

TSC Publications

TSC

Data derived from PubMed at http://www.ncbi.nlm.nih.gov/pubmed/

TSC and mTOR Publications

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200

400

600

800

1000

1200

1400

1600

1800

1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

Years

TSC

mTOR

Data derived from PubMed at http://www.ncbi.nlm.nih.gov/pubmed

NIH Funding for mTOR Research

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100

200

300

400

500

600

2000 2010

Number of Projects

Number of Projects

0

50000000

100000000

150000000

200000000

250000000

2000 2010

Total Funding

Total Funding

Where are we now?

From bedside to bench and back to bedside All components of TSC research are still critical to progress Basic research – signaling pathway, roles of mTORC1 and mTORC2, how specific gene mutations lead to specific manifestations of disease

Translational research – identifying new drug targets, testing new compounds, drugs and combination therapies in animal models of TSC

Clinical research – moving basic and translational research findings into clinical studies and clinical trials

Genetics of TSC

Mutations in TSC1 and TSC2 genes found in ~85% of individuals with clinical diagnosis of TSC

No mutation identified in 10% to 20% of individuals with TSC ?New gene ?Regulatory DNA outside of exome

Mosaicism is not the answer

Function of TSC1/2 Genes

Drosophila (Fruit Fly) provided first clues to function of TSC1/2 in regulation of cell growth and cell size

Pointed to involvement in regulation of mTOR

TSC1

mTOR

mTOR Signaling Pathway

mTOR = mammalian Target of Rapamycin

TSC2

mTOR

mTOR Signaling Pathway

Rheb

TSC2TSC1

mTORC1

mTOR Signaling Pathway

Rheb

mTORC2

Sirolimus (Rapamycin)Temsirolimus (CCI-779)Everolimus (Afinitor®)

TSC1 TSC2

mTOR Pathway

RTK

Rictor

4EBP

GβL

TSC2

Raptor

AMPK

DGKξ

PI3K

Redd1

eIF4B

eIF3

PIP2PIP3

PKC

p70S6KmTOR

AMP

eIF4GeIF4A

eIF4ERheb

eIF3

eIF4B

eIF4GeIF4A

eIF4EeIF4B

GβLmTOR

eIF4E 4EBPVEGF

eIF4GeIF4AeIF4E

Growth Factors

Insulin Insulin ReceptorReceptorInsulin Insulin

ReceptorReceptor

Energy Energy StressStressSerumSerum NutrientNutrient

HypoxiaHypoxia

Actin Organization

P

PDK-1

mTORC1mTORC1

mTORC2mTORC2

Translation off

40SRPS6

40S

Scanning

Akt/PKB

Translation On

SecondaryStructure

Unwinding

TSC1

PP2A

FKBP12

Rho

HIF1α

VEGFPathway

VEGFPathway

Akt SignalingAkt Signaling

PKC

Rac

PMA

PLD

PKCα

PC

LKB1

Akt

PA

DAG

AUG

Ras

RSK

AUG

MAPK

IRS1

InsulinInsulin 2009ProteinLounge.com 2009ProteinLounge.com

C

Rapamycin Prevents and Treats Epilepsy in a Mouse Model of Tuberous Sclerosis Complex

Early treatment with rapamycin prevented the development of epilepsy and premature death Tsc1(GFAP)CKO mice.

Late treatment with rapamycin suppressed seizures and prolonged survival in Tsc1(GFAP)CKO mice that had already developed epilepsy.

Rapamycin inhibited the abnormal activation of the mammalian target of rapamycin pathway, astrogliosis, and neuronal disorganization, and increased brain size in Tsc1(GFAP)CKO mice.

Rapamycin has strong efficacy for preventing seizures and prolonging survival in Tsc1(GFAP)CKO mice.

International, multicenter Phase 3 clinical trial being planned by Novartis Oncology.

Zeng, Xu, Gutmann, Wong (2008) Ann Neurol63:444–453

Anti-epileptogenesis in TSC Referral of all infants with cardiac rhabdomyomas or

TSC at birth Monthly EEG and monitoring Treat with vigabatrin when first identify abnormal

EEG (prior to first clinical seizure) Significantly reduced incidence of infantile spasms Seizures that do occur are not refractory Significantly improved cognitive outcomes Do we need to shift our mindset to preventing

seizures rather than trying to treat them once they manifest? In other words, should all individuals with TSC receive an EEG at diagnosis and at follow-up? Should we treat the EEG and not wait for clinical seizures to appear?

NINDS to issue RFP for anti-epileptogenesis “Center Without Walls” in 2012

Unpublished observations, Sergiusz Jozwiak, MD, PhD, Warsaw, Poland

20 100

Global Intellectual Ability (‘IQ’) in TSC

Normal Distribution (ND) TSC phenotype

Profound (P) TSC phenotype

70

Normal distribution in the general population

Prather & de Vries, JCN 2004, de Vries & Prather, NEJM, 2007

Distribution of IQ in TSC

Reversal of Learning Deficits in a Tsc2+/– Mouse Model of Tuberous Sclerosis

Deficits observed in learning in mouse model of TSC.

These deficits included impairments in two spatial learning tasks and in contextual discrimination.

Brief treatment with the mTOR inhibitor rapamycin in adult mice rescues the behavioral deficits in this animal model of TSC.

Results reveal a biological basis for some of the cognitive deficits associated with TSC, and treatment with mTOR inhibitors ameliorates cognitive dysfunction in a mouse model of TSC.

Neurocognitive clinical trial starting to recruit study participants in Boston (and Cincinnati once IRB approved); PIs: Mustafa Sahin, MD, PhD, Petrus de Vries, MD, PhD, and David Franz, MD; funded by Novartis Oncology, TS Alliance and Autism Speaks.

Ehninger, Han, Shilyansky, Zhou, Li, Kwiatkowski, Ramesh, Silva (2008) Nature Medicine 14(8):843-8

Afinitor Approved to Treat SEGA Associated with TSC

First medication approved specifically to treat individuals with TSC.

Afinitor received conditional approval by FDA on October 29, 2010 for treatment of SEGA associated with TSC

At least 30% reduction in size of SEGA in 21 patients (75%)

At least 50% reduction in size of SEGA in 9 patients (32%)

Marked reductions were seen within 3 months and were sustained

In 16 patients who had 24-hour video EEG, seizure frequency decreased in 9 patients, did not change in 6, and increased in 1 patient

Krueger et al (2010) Everolimus for subependymal giant-cell astrocytomas in tuberous sclerosis. N Engl J Med 363(19):1801-11

Axial T1 Axial T1 +C+C

Axial T1 +CAxial T1 +C

MRI after treatmentMRI after treatmentMRI prior to treatmentMRI prior to treatment

Rapamycin Afinitor®

O O OH

OOO

N

OO

O O

OH

OH

O

O O OH

OOO

N

OO

O O

O

OH

O

OH

Chemical StructureRapamycin and Everolimus (Afinitor®)

VEGF-D Assay

Vascular endothelial growth factor D (VEGF-D) promotes the growth of blood vessels and lymphatic vessels in the body

Elevated in blood of ~70% of women with sporadic lymphangioleiomyomatosis (LAM) and almost all women with TSC + LAM*

Used as a diagnostic test for LAM in women with cystic lung disease

Increased levels may also correlate with growing renal AMLs in individuals with TSC (and sporadic-LAM??)

Further research needed before understand utility in individuals with TSC

*Young et al, Chest 2010; 138:674-681

Additional Clinical Trials Topical Rapamycin to Treat Facial

Angiofibromas; PI: Mary Kay Koenig, MD, University of Texas- Houston; funded by TSCRP

Treatment of Renal AMLs and LAM Using Ultrasound-Guided MRI; PI: John Bissler, MD, Cincinnati Children’s Hospital; funded by TS Alliance Rothberg Courage Award

VEGF-D as a Biomarker for TSC and LAM

TSC Drug Screening Program

Goal: To screen potential new compounds and FDA-approved drugs for treatment of TSC

Awarded two grants in 2010 Program to be continued and expanded Use to leverage funding for translational studies at NIH and/or clinical trials

TSC Natural History Database Project

1,050 study participants now enrolled at 15 sites in the USA and 1 in Belgium

First publication including data from database published in 2010

Several additional studies underway Utilized to identify study participants for clinical trials

NYU will be analyzing seizure data

TSC Research at NYU

25 peer-reviewed papers Pioneered multistage surgery in TSC Recruitment of Delia Talos, MD from Harvard to expand research on human tissue and examine animal models Studies of tuber v. perituberal tissue Studies of neuroinflammation, mTOR pathway, excitatory neurotransmitters

Animal models of epilepsy & SUDEP in TSC

High field (7T) imaging

Pros High resolution images (excellent

T1 contrast) T2* (T2 star)- excellent

delineation of venous structures, calcifications, blood products

Neg Field inhomogeneities Sensitivity to air/artifacts Long sequences for high

resolution Research magnet, no implants

Human Studies – NYU TSC Ctr

Biopsy tissue from epilepsy surgery cases Immunocytochemical and western blot

analysis (neuroinflammation markers and synaptic proteins)

Standard extracellular and whole-cell patch clamp techniques (LTP/LTD, AMPA/NMDA)

Ex vivo pharmacologic studies

Vimentin/DAPI Vimentin/DAPIVeronique RuppePelin Dilsiz

Cellular Abnormalities in Human TSC

TUBER PERI-TUBER

Altered mTORC1 signaling in human cortical tubers

Veronique RuppePelin Dilsiz

SMI 311/pS6/DAPI Vimentin/pS6/DAPI

Altered mTORC1 signaling in human peri-tuberal cortex

Veronique RuppePelin Dilsiz

SMI 311/pS6/DAPI Vimentin/pS6/DAPI

Animal Model Studies – NYU TSC Ctr

Time course experiments Early life inflammatory /seizure challenge Treatment efficacy

Seizures

Natural history of epilepsy in TSC mouse model

P7 P14

1 month 3 monthsP21

Brain collection

Epidural electrode implantation

Long term video EEG monitoringVideo EEG monitoring at intervals

Repeated subcutaneous electrode placements

Brain collection

Brain collection

Brain collection

Seizures

Early treatment

P7 P14

1 month 3 monthsP21

Epidural electrode implantation

Long term video EEG monitoring

Brain collection

Brain collection

Brain collection

Daily anti-inflammatory drug treatment

Seizures

Late treatments of epilepsy

P14

1 month 2 monthsP21

Epidural electrode implantation

Long term video EEG monitoring

Brain collection

Brain collection

2. Anti-inflammatory drug

1.5 month

1. Conventional AED

3. Combination treatment

Conclusions New research on

genetics of TSC Important to support

basic, translational and clinical research

Bedside to bench to bedside important!

TSC Natural History Database Project

TSC Drug Screening Program

Expanded research and clinical program for TSC at NYU

Dr. Jim Riviello to become new NYU TSC Director

Questions?