Functional characterization of KEAP1 mutations in lung squamous cell carincoma Bridgid Hast Major Lab UNC Chapel Hill

KEAP1/NRF2 regulates intracellular redox homeostasis

CUL3

Risk

for D

isea

se

KEAP1 null

NRF2 null

Effective Prevention

NRF2 mediated transcription

Neurodegeration Cancer

NRF2 activity modulates survival via redox homeostasis

NRF2 target genes -Heme oxygenase 1 (HMOX1) -Glutathione synthesis (GCS) -NADH quinone oxidoreductase 1 (NQO1) -Multidrug resistance proteins (MRP)

*Mitigate acute spikes in ROS *Chemotherapeutic/xenobiotic clearance *Control metabolically-derived ROS

Pathway mutations in KEAP1/NRF2 signaling occur in squamous cell lung carcinoma

-178 total squamous cell lung carcinomas analyzed -Mutations in KEAP1 and NRF2 are mutually exclusive -Primarily in classical subtype -Collectively KEAP1, NRF2, and CUL3 mutations are altered in 34% of total samples The Cancer Genome Atlas Research Network, Nature 2012

KEAP1 mutations exhibit differential suppression of NRF2-mediated transcription

0

0.2

0.4

0.6

0.8

1

1.2

GFP

KE

AP1

R

554Q

R

320Q

W

554C

R

470C

G

423V

N

469f

s G

480W

D

422N

G

333C

L2

31V

P3

18L

G18

6R

S243

C

P318

de

l V

167F

R

71K

V15

5F

Fire

fly/R

en

illa

KEAP1 mutants differentially bind to interacting proteins

KEAP1 mutants differentially bind to interacting proteins

KEAP1 mutants differentially bind to interacting proteins

The KEAP1 mutants cluster into four classes

IVR 180-314

KELCH 315-359

KELCH 361-410

KELCH 412-457

KELCH 459-504

KELCH 506-551

KELCH 553-598

NTR 1-60

CTR 599-624

BTB 61-179

Class I: Strong binders of NRF2 but cannot suppress NRF2-mediated transcription

IVR 180-314

KELCH 315-359

KELCH 361-410

KELCH 412-457

KELCH 459-504

KELCH 506-551

KELCH 553-598

NTR 1-60

CTR 599-624

BTB 61-179

IVR 180-314

KELCH 315-359

KELCH 361-410

KELCH 412-457

KELCH 459-504

KELCH 506-551

KELCH 553-598

NTR 1-60

CTR 599-624

BTB 61-179

IVR 180-314

KELCH 315-359

KELCH 361-410

KELCH 412-457

KELCH 459-504

KELCH 506-551

KELCH 553-598

NTR 1-60

CTR 599-624

BTB 61-179

Class II: Do not bind NRF2 and cannot suppress NRF2

Class III: Weakly bind NRF2 and cannot suppress NRF2

Class IV: Behave like wildtype

R320Q D422N G423V

R470C G186R S243L V155F

W544C R554Q N469fs

G333C P318L G480W

V167F

L231V S224Y P318del R71K

KEAP1 mutants differentially bind to interacting proteins

-“Superbinders” only bind more NRF2

- Cannot suppress NRF2-mediated transcription

- Exhibit increased NRF2 half-life

- Have enhanced cell viability in response to chemotherapeutic insult Mechanism?

0

0.2

0.4

0.6

0.8

1

1.2

GFP

KE

AP1

R

554Q

R

320Q

W

554C

R

470C

G

423V

N

469f

s G

480W

D

422N

G

333C

L2

31V

P3

18L

G18

6R

S243

C

P318

de

l V

167F

R

71K

V15

5F

Fire

fly/R

en

illa

“Superbinders”: slow cyclers or subpar structures?

IVR

KELCH

NRF2

90°

D422 R470

D422 R470

P318 R320

NRF2 DLG and ETGE

-Class I mutants are on “bottom” of KELCH domain, not at KELCH/NRF2 interface -Mutants in IVR and linker between IVR and KELCH are also in Class I. Predicted to be near IVR/BTB interface -More likely that Class I mutants perturb KEAP1 structure than act as “superbinders” 0

0.2

0.4

0.6

0.8

1

1.2

GFP

KEA

P1

R55

4Q

R32

0Q

W55

4C

R47

0C

G42

3V

N46

9fs

G48

0W

D42

2N

G33

3C

L231

V

P318

L

G18

6R

S243

C

P318

de

l

V16

7F

R71

K

V15

5F

Fire

fly/R

en

illa

KEAP1 cysteine residues are stress-specific

-C151 forms adducts with electrophiles -H129, K131, R135, K150, and H154 comprise microenvironment that alters reactivity of C151

-H225/C226 and C613 are reactive to heavy metals -C288 specific reactivity to alkenals

McMahon et al, PNAS 2010

Is cysteine reactivity in KEAP1 altered in cancer?

KEAP1 mutations cluster

Are clustered mutations “pointing” to important regions of KEAP1?

Cys 241, 249, 319, 368, 434, 489 have been shown to react with electrophilic fatty acids as well as sulforaphane

240 320-350 430 Approximate Residue

KEAP1 mutations are hypomorphic and can be further inactivated by interacting proteins

(DPP3)

-Overexpression of the ETGE-containing protein DPP3 further activates NRF2 signaling in a KEAP1 mutant background -DPP3 is overexpressed in tumor verses normal lung squamous cell carcinoma (p=4.6e-14)

Summary

-Mutations in KEAP1 from lung squamous cell carcinoma can be grouped into four phenotypic classes -The “superbinder” class exhibits enhanced NRF2 activity and stability, and is likely a result of structural changes in the KEAP1 homodimer -KEAP1 mutations in cancer cluster around cysteines with reactivity to electrophilic compounds -Overexpression of ETGE-containing proteins can further activate NRF2 activity in a KEAP1 mutant background

Major Lab Ben Major Erica Cloer Kathleen Mulvaney Dennis Goldfarb Priscila Siesser Matt Walker Feng Yan Alex Rabinowitz

Hayes Lab Neil Hayes Matt Wilkerson

TCGA Research Network

Ning Zheng (U. Washington)

Questions?