Posttranslational Regulation of the Fanconi Anemia Cancer Susceptibility Pathway

Hyungjin Kim, Ph.D.

Stony Brook University

Department of Pharmacological Sciences

Assistant professor

DNA repair interest group 04/21/15

DNA Repair System Preserves Genome Stability

Uracil Abasic site

8-Oxiguanine Single-strand break

(6-4)PP Bulky adduct

CPD

Interstrand cross-link Double-strand break

Base pair mismatch Insertion/deletion

Base- excision repair

(BER)

Nucleotide- excision repair

(NER)

Fanconi anemia (FA) pathway HR/NHEJ

Mismatch repair

DNA REPAIR

X-rays Oxygen radicals Alkylating agents

Spontaneous reactions

UV light Polycyclic aromatic

hydrocarbons

X-rays Anti-tumor agents (cisplatin, MMC)

Replication errors

DNA DAMAGE

Cell-cycle arrest

Cell death

DNA DAMAGE

RESPONSE

Cancer •  Mutations •  Chromosome aberrations

DNA REPAIR

DEFECT

Adapted from Hoeijmakers (2001) Nature

Environmental Mutagen Oncogenic Stress

Checkpoint, DNA repair

DNA damage response

Tumorigenesis Genome instability

•  Hereditary cancers: mutations in DNA repair genes (BRCA1, BRCA2, MSH2, etc)

•  Sporadic cancers: - Oncogene-induced replication stress - Inaccurate DNA repair caused by selection of somatic mutations in DNA repair genes

DNA Repair Mechanism as a Tumor Suppressor Network Cancer is a disease of DNA repair

TCGA network, (2011) Nature

Germ-line and Somatic Disruption of DNA Repair Is Prevalent in Cancer

The Cancer Genome Atlas (TCGA): Genomic characterization and sequence analysis of various tumors from patient samples

Clinical and Cellular Features of Fanconi Anemia (FA)

Fanconi Anemia

Chromosome instability syndrome

Mutation in 17 genes that cooperate in DNA interstrand

cross-link (ICL) repair i.e. the FA pathway

Disease of defective DNA repair

Developmental defect Bone marrow failure Increased cancer risks

AML

Model for cancer pathogenesis

FANCA

FANCB

FANCC

BRCA2/FANCD1

FANCD2 FANCE

FANCF

FANCG

FANCI

BRIP1/FANCJ FANCL

FANCM PALB2/FANCN

RAD51C/FANCO

SLX4/FANCP

Frequencies of FA complementation group mutations!

60-70%!

The Seventeen Fanconi Anemia Genes in the FA/BRCA Pathway

XPF/FANCQ

FANCA FANCB FANCC FANCD1 FANCD2 FANCE FANCF FANCG FANCI FANCJ FANCL FANCM FANCN FANCO FANCP FANCQ FANCS

17 FANC genes!(Complementation group)!

BRCA1/FANCS

FA core

D2

I

The FA Pathway Regulates Interstrand Cross-link (ICL) Repair

ICL

Nucleolytic Incision

Translesion DNA synthesis (TLS)

TLS Pol. (REV1, Pol ζ)

O HR J

N D1

D2

I

NER RAD51

D2 I USP1

UAF1

Kim & D’Andrea (2012) Genes & Dev

SLX4 (P) MUS81

EME1

XPF (Q) ERCC1 Ub D2

I Ub

D2

I

A

A

S

Regulation of FANCD2 Mono- and De-Ubiquitination

+ damage

α FANCD2 IF

Garcia-Higuera et al., (2001) Mol. Cell Kim et al., (2009) Dev. Cell

FANCD2-L: FANCD2-Ub

I

D2 Ub FA

Core USP1: Deubiquitinating enzyme

Fanconi Anemia

Ubiquitin Signaling

DNA Repair Process Cancer Pathogenesis

Bioinformatic Search of UBZ4-Containing Proteins

QILTCPVCFRAQGCISLEALNKHVDECLDGPS

UBZ4 domain from TLS polymerase κ

Hidden Markov Model (HMM)

Polκ-1

RAD18SLX4-1SLX4-2FAN1

SNM1AUBZ4 #1

Polκ-2

RAP80

UBZ4 #2UBZ4 #3

Target

Ub

UBD

UBD: Ubiquitin Binding Domain

UBZ4 (Ub-Binding Zn-finger 4)

Ub

Predominantly found in DNA repair factors

FAAP20

FAAP20 Regulates FANCD2 Activation in the FA Pathway

180 a.a.

UBZ4 1

FAAP20 (C1orf86): Fanconi anemia-associated protein 20 kD

* *

FANCD2 DAPI

si Control

si C1orf86

HU

FA Core

I

D2 Ub

E3 ligase

FAAP20

FANCA: FAAP20

The FAAP20-FANCA Interaction Is Required for Maintaining FANCA Level

pre-IP α-­Flag�  IP

Flag-­FAAP20

FANCA

Flag-­FAAP20Flag-­FAAP20�  ΔC

IgL

- wt ΔN - wt

25-­

150-­

kD

20-­

1 2 3 4 5 6 7 8

Flag-­FAAP20�  ΔN

ΔC ΔNΔC

15-­

UBZ4

ΔN ΔC wt

Flag-FAAP20

β-actin37-

siRNA

kD

150-

Ctrl

F20

#3

F20

#3

FANCA

25-

20-

ΔC

F20

#3

F20

#3

wt ΔN Flag-FAAP20

Flag-FAAP20Flag-FAAP20 ΔCFlag-FAAP20 ΔN

- -

siRNA FAAP20 + siRNA resistant cDNA

FANCA interaction

FAAP20 Connects the FA Pathway with Translesion DNA Synthesis

Kim et al., (2012) Nat. Struc. Mol. Biol.

FANCP

Ub D2/I

Ub

REV1

FA Core

Pol ζ

FAAP20 D2 Monoubiquitination

Nucleolytic Incision

Rev1 Recruitment

Nucleotide adduct bypass by TLS

Replication-associated HR

ICL Recognition

UBZ4 (C) N

1) Nucleolytic Incision

2) TLS

3) HR

FA Core complex FAAP20

A

Ub

Solution Structure of the FAAP20-Ubiquitin Complex

FAAP20 UBZ

C147

C150

H166

C170

The disordered C-terminal tail of FAAP20 is involved in ubiquitin binding

Wojtaszek et al., (2014) Nucleic Acids Res.

Tail

The Very C-terminal Tryptophan Is Required for Ubiquitin Binding

Isothermal Titration Calorimetry (ITC)

The Very C-terminal Tryptophan Is Required for DNA ICL Repair

CA: C147A & C150A WA: W180A

* * * * *

* *

Ubiquitin Recognition of FAAP20 Is Distinct from Canonical UBZ Module

Unconventional FAAP20 UBZ motif

E3 ligase

FAAP20 FANCA

Ø  Mechanism of maintaining FANCA stability by FAAP20?

Ø  Mechanism of FANCA degradation and its implication in tumorigenesis?

Regulation of the FA Pathway by the FANCA-FAAP20 Axis

FA Core E3 ligase

A 20

150 kD

Characterization of a FA-like Breast Cancer Patient

Ø  33 year-old triple negative breast cancer (TNBC) patient (not a FA patient per se)

Ø  Features of FA (i.e. short stature, café au la spot, mild macrocytic anemia)

DF2231

DF2231

20 ng/mL MMC

Quadriradial chromosome

Characterization of a FA-like Breast Cancer Patient

0

0.2

0.4

0.6

0.8

1

1.2

0 12.5 25

GM0357 FA-I DF2231

MMC (nM)

Sur

viva

l

FANCD2FANCD2+ DAPI

MMC

GM

0637

DF2

231

Upstream defect

GM0637

2816T>G; Ile939Ser

The I939S Point Mutation in Patient FANCA Destabilizes FANCA

kD

150-

150-

50-

FANCA

Tubulin

FANCD2-UbFANCD2

DF2231

GM0637

MMC (nM)

Sur

viva

l

0

0.2

0.4

0.6

0.8

1

1.2

0 12.5 25

DF2231+Vector DF2231+FANCA GM0637

2840C>G; Ser947stop

FAAP20

1st allele 2nd allele

FANCA gene

(Savoia et al. 1996, Levran et al. 1997, Savino et al. 2003)

The Amino Acid 913-1095 Region of FANCA Is Required for FAAP20 Interaction

Myc

Flag

WT-

FAN

CA

WT-

FAN

CA

I939

S-F

AN

CA

Flag IP

WT-

FAN

CA

WT-

FAN

CA

I939

S-F

AN

CA

Flag-FAAP20 Flag-FAAP20

WCE

myc-FANCA

FANCA-I939S Mutation Disrupts FAAP20 Binding

I939

FANCA

FAAP20

Identification of SUMOylation Site of FANCA Near the FAAP20 Binding Region

Exon 28 Exon 29

Human Mouse Rat Chicken

I939 K921

Regulation of Protein Stability by Coupled SUMO-Ubiquitin Signaling

RNF4

STUbL (SUMO-Targeted Ubiquitin Ligase)

target S S S

S Ub

Ub Ub

SIM: SUMO-interacting motif

190 1

SIM RING RNF4

1)  RNF4 is a Ubiquitin E3 ligase which regulates the DNA damage response (Galanty, Y et al, Genes Dev 26, 2012; Yin, Y, et al, Genes Dev 26, 2012)

2) RNF4 is the human ortholog of the yeast proteins, Slx5 and Slx8 (discovered in the same screen which identified Slx4; SLX4/FANCP)

FANCA from the Patient Shows Increased SUMOylation and Polyubiquitination

FANCA SUMOylation at K921 Is Required for Polyubiquitination of FANCA

UBC9 and PIAS1 Are Required for FANCA Sumoylation

FANCA-K921R SUMO-Defective Mutant Exhibits Increased Half-life

Cycloheximide blocking

RNF4 Regulates FANCA Stability

FANCA I939S + siRNF4

RNF4 Is a New Player in the FA Pathway

Ub USP1 UAF1

Regulation of DNA ICL Repair by Integrated Ubiquitin-SUMO Signaling

I

D2 Ub

A 20 S

Ub

A

20

S S

Ub Ub

RNF4 A

20

I

D2

M

ü  FANCD2 deubiquitination (USP1)

ü  FANCM release

ü  FANCA degradation

PIAS1

Regulation of DNA ICL Repair by Integrated Ubiquitin-SUMO Signaling

S A

20

S S RNF4 RNF4 loss

Aberrant FANCA accumulation

Delay of replication restart

Ub

The FANC Proteins Are Predicted to be Extensively SUMOylated

Mutational Hotspot of FANCA Corresponds to the FAAP20 Interacting Region

FANCA gene mutations from 97 FA patients

Levran et al., (1997) PNAS

cBioPortal for Cancer Genomics

221 FANCA mutations from 89 studies including TCGA

FANCA Mutations From FA and/or Cancer Patients Disrupt FAAP20 Interaction

FANCA null FA patient cells + FANCA variants

Conclusions

1. DNA repair mechanisms preserve genome integrity and their deregulation contributes to tumorigenesis

2. The FA pathway removes DNA ICL encountered during S phase, and its defect leads to cancer-prone disease, Fanconi anemia

3. Complex interplay of post-translational modification network including ubiquitination/SUMOylation controls DNA repair pathways

- Disruption of the DNA repair activity (germ-line or somatic) could be a major driving force for tumorigenesis

- FAAP20 ubiquitin binding in regulating ICL repair

- FANCA degradation by Ubiquitin plus SUMO

4. Compound heterozygosity of FANCA leads to (or contribute to) TNBC

Alan D’Andrea Lab Kailin Yang, Ph.D. - bioinformatics

Donniphat Dejsuphong, M.D./Ph.D. - DT40

Lisa Moreau – chromosome assay

Leukemia & Lymphoma Postdoctoral Fellowship

Miltenyi Biotech GmhH, Germany

Shunichi Takeda, Ph.D. - DT40 Kyoto University, Japan

Graham C. Walker, Ph.D. - Rev1 structure MIT

Pei Zhou, Ph.D. - Rev1 structure Duke University Medical Center

Jenny Xie, Ph.D. - FANCA patient

Collaborators and Reagents

Raphael Ceccaldi, Ph.D. – TCGA analysis

DFCI

Funding

Judy Garber, M.D. - FANCA patient

Shannon Puhalla, M.D. - FANCA patient U. of Pittsburgh

Acknowledgements

Hyungjin Kim Lab - Pharmacology Ukhyun Jo, Ph.D.

Jingming Wang

Soyoung Joo

Start-up funds - Stony Brook Cancer Center

Kay Hofmann, Ph.D. - Bioinformatics

U. of Washington Ning Zheng, Ph.D. – DUB structure and function Winson Cai