Joyoti Dey, MPH Advisor: Dr. James M. OlsonFred Hutchinson Cancer Research Center &

University of Washington Graduate Program

Investigating two Oncogenic Mutations in Smoothened with

vastly different effects on Cerebellar Development

• Background

- Cerebellar Development & Neuronal Circuitry - Role of Shh in Cerebellar Development- Deregulated Shh signaling: SmoA1 & SmoA2 - Medulloblastoma mouse models

• Characterization of the SmoA2 mouse model

• Future Directions

Outline

• Represents 10% of total brain, yet > 50% of total neurons

• Involved in fine tuning motor movements and balance

• One of the first brain structures to begin differentiation, one of the last to attain maturity

The Cerebellum

www.nih.nlm.org

Cerebellar Neuronal Circuitry

Wang et al. Nature Rev. Neurosci .(2001)

Embryonic Phase of Cerebellar Development• Neurons arise from two germinal matrices -

Ventricular zone & Rhombic Lip

Mouse E13.5 Mouse E17.5

EGL: External Granule Layer GNP: Granule Neuron

Precursor

Ref: Schuller et al. Cancer Cell (2008)

Ref: Hevner R, Reelin Glycoprotein (2008)

Postnatal Cerebellar Development in Mouse

Ref: RIKEN Brain Institute

Molecular Layer

Purkinje

LayerInner Granule Layer

Cellu

lar

Even

tsTim

e S

ch

ed

ule

GNP

Postnatal Cerebellar Development in Mouse

P5 P14 P28

Sonic Hedgehog in Cerebellar Development

Ref: Altaba et al. Nature Rev. Neuroscience (2002)

Mammalian Sonic Hedgehog Pathway

Without Shh

With Shh

Ref: Rubin et al. Nature Reviews: Drug Discovery (2006)

• Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma

- Mutations activating proto-oncogene Smoothened

- Mutations inactivating tumor suppressor Patched

• Medulloblastoma: most common pediatric brain tumor

- Increased activity of Shh pathway due to mutations in 20% cases

- Tumors thought to arise from granule neuron precursors

Ref: Children’s Hospital, Seattle

Shh Pathway Aberrations & Cancer

Oncogenic Smoothened Mutations

• SmoA1 (S537N) : Human Sporadic Basal Cell Carcinoma

• SmoA2 (W539L): Human Medulloblastoma

Ref: Taipale et al.Nature (2000)

Mouse Models with Oncogenic Smoothened MutationsND2 1kb SmoA1* His6/Myc3

P5 P14 P28 Symptomatic Tumor

Hatton et al. Cancer Research (2008)

Tumors serially transplantable

Leptomeningeal Spread

Mouse Models with Oncogenic Smoothened Mutations

ND2 1kb SmoA1* His6/Myc3

P5 P14 P28 Symptomatic Tumor

ND2 1kb SmoA2* HA

P14P5 P28 Symptomatic Tumor

Developmental Defects in SmoA2 Mice

• Disrupted laminar organization

• Disrupted foliation

WT

SmoA2

P5

P5

adult

adult

• Early hyperproliferation

• Ectopic clusters of Purkinje cells

Wildtype

SmoA2

Neurobehavioral Assessment

• Spontaneous Activity• Locomotor Activity• Rotarod Test• Gait Assessment

• Body Position• Tail position• Posture

Neurobehavioral Physical Phenotype Assessment

Assessment of Ataxia: “Footprinting” Assay

No detectable Ataxia in SmoA2 mice

SmoA2

Huntington’s Disease.

Human Cerebellar Dysplasias: Correlation with PNETs

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Figure: Jeffrey A. Golden & Brian N. Harding. (2004). Developmental Neuropathology

Common in apparently normal infant brains

Rorke et al (1968) showed histologically, clusters of mature neurons in white matterfocal or perivascular GNPs, heterotaxias heterotopias

Possible Cause : Inappropriate progenitor cell death and/or neuronal & glial migration

Contributory Role in Medulloblastoma: Dysplasias might be targets for neoplastic transformation (Yachnis et al.(1994), Jay V.(1996)

Bilateral, vertical, orientated folia Enlarged fourth ventricle.

Human Cerebellar Dysplasias

Right cerebellar hypertrophy &

vertical folia

Soto-Ares et al. Am J Neuroradiol (2000)

Abnormal Arborization of White Matter Heterotopia

Normal

SmoA2 & Human CBL Dysplasias: Shared Features

Dysplasia & Contiguous Tumor

Heterotopia &

Heterotaxia

Vertical Foliation

SmoA2

Wildtype

Hyperproliferation in SmoA2 Cerebella

P5 P14 P28

Wildtype

SmoA2

Cyclin

D1

Mouse postnatal timepoints

Rel

ativ

e m

RN

A e

xpre

ssio

n

SmoA2

SmoA2

Expression of Shh Canonical Targets in SmoA2 & SmoA1 cerebella, compared to WT controls

SmoA1

SmoA2 >10 >17

N = 3 WT & 3 Mutant/ timepoint

P5 P14 P21 2m

P5 P14 P21 2m

Purkinje Cell Alignment in SmoA2 Cerebella

P5 P14 P28

Wildtype

SmoA2

Calb

ind

in

Radial Glia Alignment in SmoA2 Cerebella

Wildtype

10X

S100

SmoA2

20X

Wildtype SmoA2

Nov 6, 2008

Abnormal Embryonic Development of SmoA2 Cerebella

Ki67

E15.5 dpc

Conclusions

• Significant difference in cerebellar development due to SmoA1 and SmoA2 oncogenic mutations

• Severe defects in neuronal and glial migration yet no neurobehaviourial abnormalities in the SmoA2 mice

• More robust and earlier activation of Shh pathway due to SmoA2 mutation

• Several shared key features with Human Cerebellar Dyslasias

Future Directions

A connection between Human Cerebellar Dysplasias and the SmoA2 phenotype ?

Human SmoA2

Future Directions

Determine the molecular basis of abnormal neuronal/glial migration

? ?

?

Future DirectionsND2 1kb SmoA1* His6/Myc3

P5 P14 P28 Symptomatic Tumor

ND2 1kb SmoA2* HA

P14P5 P28 Symptomatic Tumor

Investigate Molecular Differences between the SmoA1 and SmoA2 variants

Acknowledgements

Dr. Jim Olson & Our Team !

Acknowledgements

FHCRC Experimental Histopathology Core:

Dr. Julie Randolph Habecker

Dr. Sue Knoblaugh

FHCRC Scientific Imaging Core:

Dr. Julio Vasquez

FHCRC Animal Health Resources

Dr. Jon Cooper

Tapscott Lab: Laurie Snider

Vasioukhin Lab: Dr. Olga Klezovitch