Protein folding:

sculpting evolutionary

change, human health,

and disease.

Hemoglobin GFP

25 39 °C

hsp104hsp90hsp70

hsp26

hsp60

Heat shock response

Induced heatand

many other stresses

25 39 °C

hsp104hsp90hsp70

hsp26

hsp60 Master regulatorHSF1

Yeast to man

Hsp90 facilitates evolution

CancerDrug resistance

Hsp90 in yeast

• Essential for life

• Very abundant

• Normally don’t need much

• Need much more under stress

• Acts as buffer

Hsp90 in Mammals (several labs)

• Complexed with inactive hormone

receptors

• Complexed with inactive oncogenic

kinases

• Complexed with inactive hormone

receptors

• Complexed with inactive oncogenic

kinases

Hypothesis: Hsp90 is a repressor

Hypothesis: Hsp90 keeps proteins repressed

Inactive

90 9090

v-src GR

Hypothesis: Hsp90 keeps proteins repressedv-src GR

Active

v-SrcGAL promoter

accumulation

Wt hsp90

activity

Wt hsp90 Wt

20 x exposure

hsp90

c-Src

activity

Normal vs. Oncogenic src Kinase

Self-inhibited c-src

YP mutation

P

P

P

P

P

Active mutant v-src

Normal vs. Oncogenic src Kinase

Self-inhibited wild-type c-src

YP mutation

Misfolded, unstable mutant protein

Hsp90 Saves it

90

Hsp90 Promotes Activation

90

But while bound it is inactive

Hsp90 Promotes Activation

P

P

P

P

P

Hsp90 drug reverses transformation

V-Src

Luke Whitesell

V-Src + GA

PharmaDD Online 2006

Vernalis Novartis*

Conforma Biogen-Idec*

Serenex Pfizer*

Kosan BMS*

Kyowa Hakko Kogyo*

Synta*

Infinity/Medimmune*

Sanofi-Aventis

Merck

Others??

June 2008

Hsp90 Inhibitors:

Rapid Progress in

Clinical Development

* Phase I/II clinical trials in progress

Is any of this relevant to humans?

1. Hsp90 chaperones mutant proteins

Mutations new activities.

But proteins donʼt fold well.

90

P

Activation & repression of gene expression

Development & morphogenesis

Cell cycle progression

Responses to environment

Cell growth

Apoptosis

Activation & repression of gene expression

Development & morphogenesis

Cell cycle progression

Responses to environment

Cell growth

Apoptosis

Many co-chaperones andVariations on the theme

90

90

Signal Transduction Networks and Hsp90

Hanahan and Weinberg, Cell 2000

Drug Resistance in Fungi

ubiquitous

Emergence of drug resistance in Candida albicans

- + Fluconazole

-

+ Radicicol

Evolution in a human host

Resistance of Clinical Isolates

GdA

FL (µg/ml)0 256

Isolates collected by Ted White, Spencer Redding and colleagues.

CAI4

0 256

In v

ivo

sele

ctio

n

CaCi

FL (µg/ml)

39°C

FL (µg/ml)0 256

Mechanism of Hsp90-Dependent Resistance

Ergosterol

Erg3Ergosterol

Erg11

. .WT

Azoles

Mechanism of Hsp90-Dependent Resistance

Toxic sterol

WT

Erg3 Erg11Ergosterol

AzolesMutation

Mechanism of Hsp90-Dependent Resistance

Alternate sterol

WT

Erg3 Erg11Ergosterol

AzolesMutation

. .

HSP90

Calcineurin

Response!

. .

2. Hsp90 chaperones responders.

mutations simultaneously solve & create problems

Aspergillus fumigatus

• Diverged ~ 1 BY

• Major human pathogen

• Mortality rates ~ 90 %

• Already resistant to new chemical entity

Hsp90 Modulates Caspofungin Resistance in A. fumigatus Clinical Isolates

Isolate 1 Isolate 2 Isolate 3

10µM GdA

Cowen LE et al., PNAS 2009

test strip

Informer Screen 25,000 Compounds

Two hundred hits

most not toxic to human cells

RO3 - Screen up to 500,000 new compounds

NIH Comprehensive Screening Center &

Molecular Libraries Probe Production Center Network

That is just one wayone hsp interfaces with

disease and how we might manipulate it.

What about others?

25 39 °C

hsp104hsp90hsp70

hsp26

hsp60

Master regulator

HSF1

Yeast to man

Heat Shock Transription Factor

Heat shock/stress response ---a systematic cellular defense to stresses

Protein homeostasis

Not needed without stress!

Hsf1-/-

Hsf1 enables chemical carcinogenesis

Hsf1+/+

Ras oncogeneHsf1 Reduces Survival

Hsf1 Reduces Survival p53+/R172H

What about humans?

(Mouse ≠ human)

What about maintenance?

HSF1 requirement correlates with malignant state

normal tumorigenic(primary) (hTERT) (hTERT, LTA and HRAS)

HSF1 maintains the growth and survival of human breast cancer cells

HSF1 maintains the growth and survival of human cancer cells withdiverse histopathological origins

(Fibroblasts) (Cervical) (Prostate) (Kidney) (MPNST)

Hsf1

What aboutneurodegeneration?

Neurodegenerative Diseases

Adapted from Forman, Trojanowski, and Lee 2004

Alzheimer FTDP-17 Parkinson

Huntington ALS CJD

Yeast cells as living test tubes

Parkinsonʼs Disease

• Second most common neurodegenerative disorder.

• Loss of dopamine-producing neurons from substantia nigra. Severe motor defects…...

• Bewildering environmental influences.

• Mostly sporadic but some genetic forms.

• Hallmark of the disease inclusions of aSynuclein

α-Synuclein Localization

α-synuclein GFPGal

one copy two copies

glucose

Identify genes that make cells

better or worse

αSyn-GFP

Library of ~5,000 yeast genes

. . . . . .gene1, gene2, gene3, gene4…….gene5000

galactose

better

worse

No effect

Screened 5,000 ORFs~ 60 modifiers

empty well

• Galactose induction• Protein trafficking• Manganese transport• Nitrosative & Oxidative stress• Kinases and Phosphatases• Protein quality control• Unknown Function

Genetic hit list

• Galactose induction• Protein trafficking• Manganese transport• Nitrosative & Oxidative stress• Kinases and Phosphatases• Protein quality control• Unknown Function

Genetic hit list

ER l Golgi Enhancers

ER Golgi Suppressors

Primary Neurons

Rat embryonic midbrain cultures(Chris Rochet)

Drosophila PD model(Nancy Bonini)

C. Elegans PD model(Guy Caldwell)

Hits from yeast screen

Rab1 rescues α-Syn-induced loss in primary rat midbrain cultures

With Chris Rochet (Purdue)

P < 0.01

Several others, diverse

functionsas well!

Enhancers and Suppressors tested in rat primary neurons

Suppressors• Rab3A* & Rab8A*• Pde2/PDE9A*• Cdc5/PLK2*• Hrd1/SYVN1• Ubp3/USP10

Not confirmed• Yck3/CSNK1G3

*also effective in nematode model

Enhancers and Suppressors tested in rat primary neurons

Suppressors• Rab3A* & Rab8A*• Pde2/PDE9A*• Cdc5/PLK2*• Hrd1/SYVN1• Ubp3/USP10

Not confirmed• Yck3/CSNK1G3

*also effective in nematode modelYeast homolog rescues cell from aSyn

tip

iceberg

Huntingtin “OFF” Huntingtin “ON”

25Q

47Q

72Q

103Q

0Q

PolyQ length-dependent toxicity serial dilution

Virtually everythingfrom high throughputscreens is different.

Except: The Heat Shock Response

Hsf1 knockouts die faster from prion infection (intracranial)

Hard Place

Rock

There is still great hope!

Short term therapy for cancer?Blood brain barrier?

HSF potentiators for ND rather than inducers?

Specific arms of the response?

Breaking the feedback loop?

Finding the right balance?

Human Glioma Progenitor Cells – Orthotopic Transplantation Model

Keith Ligon and Santosh Kesari/Stiles Lab - DFCI

!

Katherine BorkovichDebbie Nathan

Didier PicardKeith Yamamoto

Xang XuMike Singer

AcknowledgementsLeah Cowen

--------

Theodore White (Seattle Biomedical Ins.)James Anderson (University of Toronto)

Thanks!

Aaron GitlerTiago Outiero Anil Cashikar

Julie Su, Esti Yeger Lotem, Ernest Fraenkel

Esti Yeger-Lotem

Ernest Fraenkel

Linhui Julie Su

Smitha Jagadesh

Sylvia KrobitschMartin DuennwaldSmitha Jagadish

Antony Cooper Paul Muchowski Chris Rochet

Nancy Bonini Guy Caldwell Charles Barlowe

Bhupinder Bhullar Josh LaBaer

Harvard Institute of Proteomics

Harvard Institute Chemical &Cell Biology at Longwood Caroline Shamu

Chengkai DaiLuke Whitesell

Sandro Santagata

Leslie Gunatilaka-Natural Products Chemistry

University of Arizona

Dana Farber Cancer InstituteSantosh Kesari/Stiles LabTan Ince

Hsf-/- micegenerous gift ofIvor Benjamin

Chief of CardiologyUniversity of Utah

and Xianzhong Xiao

Tan InceDepartment of

Pathology

Brigham & WomensHospital