Posttranslational Regulation of the Fanconi Anemia Cancer Susceptibility Pathway

DNA repair interest group 04/21/15

Assistant professor Department of Pharmacological Sciences Stony Brook University Hyungjin Kim, Ph.D. DNA Repair System Preserves Genome Stability

DNA DAMAGE X-rays DNA DAMAGE Oxygen radicals UV light X-rays RESPONSE Alkylating agents Polycyclic aromatic Anti-tumor agents Replication Spontaneous reactions hydrocarbons (cisplatin, MMC) errors Cell-cycle arrest

Cell death

Uracil DNA REPAIR Abasic site (6-4)PP Interstrand cross-link Base pair mismatch DEFECT 8-Oxiguanine Bulky adduct Double-strand break Insertion/deletion Single-strand break CPD Cancer • Mutations • Chromosome Base- Nucleotide- Fanconi anemia (FA) aberrations Mismatch excision repair excision repair pathway repair (BER) (NER) HR/NHEJ

DNA REPAIR

Adapted from Hoeijmakers (2001) Nature DNA Repair Mechanism as a Tumor Suppressor Network Cancer is a disease of DNA repair

Environmental Mutagen Oncogenic Stress • Hereditary cancers: mutations in DNA repair genes (BRCA1, BRCA2, MSH2, etc) DNA damage response

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

TCGA network, (2011) Nature Clinical and Cellular Features of Fanconi Anemia (FA)

Fanconi Anemia

Chromosome instability syndrome

Mutation in 17 genes that Disease of defective DNA repair cooperate in DNA interstrand cross-link (ICL) repair Model for cancer pathogenesis i.e. the FA pathway

Developmental defect Bone marrow failure Increased cancer risks

AML The Seventeen Fanconi Anemia Genes in the FA/BRCA Pathway

17 FANC genes! (Complementation group)! FANCA Frequencies of FA complementation group mutations! FANCB FANCC PALB2/ RAD51C/ FANCM FANCD1 BRIP1/ FANCN FANCO FANCD2 FANCJ FANCL SLX4/FANCP FANCI FANCE FANCF XPF/FANCQ BRCA1/FANCS FANCF FANCE FANCG FANCG FANCD2

FANCI BRCA2/ FANCJ FANCD1 FANCL FANCC FANCM 60-70%! FANCN FANCO FANCA FANCP FANCB FANCQ FANCS

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

D2 FA A core I D2

HR J ICL O D1 S N

Ub A I XPF (Q) RAD51 D2 Ub ERCC1 NER SLX4 (P) MUS81 EME1

Nucleolytic Incision

USP1 I UAF1 D2

I D2

TLS Pol. Translesion DNA synthesis (REV1, Pol ζ) (TLS) Kim & D’Andrea (2012) Genes & Dev Regulation of FANCD2 Mono- and De-Ubiquitination

Ub FA D2 Core I USP1: Deubiquitinating enzyme

FANCD2-L: FANCD2-Ub

+ damage

α FANCD2 IF

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

Fanconi Anemia

DNA Repair Process Cancer Pathogenesis Bioinformatic Search of UBZ4-Containing Proteins

UBD: Ubiquitin Binding Domain

UBZ4 domain from TLS polymerase κ Ub Target QILTCPVCFRAQGCISLEALNKHVDECLDGPS UBD

Hidden Markov Model (HMM)

Polκ-1 UBZ4 (Ub-Binding Zn-finger 4) Polκ-2 RAD18 SLX4-1 SLX4-2 FAN1 RAP80 SNM1A Ub UBZ4 #1 UBZ4 #2 UBZ4 #3 FAAP20

Predominantly found in DNA repair factors FAAP20 Regulates FANCD2 Activation in the FA Pathway

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

1 180 a.a. E3 ligase UBZ4 Ub FA D2 Core I * * FANCA: FAAP20

HU FANCD2 DAPI

si Control

si C1orf86 The FAAP20-FANCA Interaction Is Required for Maintaining FANCA Level

Flag-FAAP20 UBZ4 wt ΔC ΔN siRNA FAAP20 + siRNA resistant cDNA

FANCA interaction siRNA Ctrl F20 #3 F20 #3 F20 #3 F20 #3 pre-IP α-Flag IP - - wt ΔC ΔN Flag-FAAP20 kD - wt ΔC ΔN - wt ΔC ΔN Flag-FAAP20 kD 150- FANCA 150- FANCA β-actin 37- IgL 25- 25- Flag-FAAP20 Flag-FAAP20 Flag-FAAP20 ΔC 20- Flag-FAAP20 ΔC 20- Flag-FAAP20 ΔN 15- Flag-FAAP20 ΔN 1 2 3 4 5 6 7 8 FAAP20 Connects the FA Pathway with Translesion DNA Synthesis

ICL Recognition

Ub FA Core complex 1) Nucleolytic FA Core D2/I FAAP20 Incision Ub UBZ4 (C) N A FANCP FAAP20 Ub Rev1 Recruitment D2 Monoubiquitination REV1 3) HR Pol ζ

2) TLS Nucleotide adduct bypass by TLS Nucleolytic Incision

Replication-associated HR

Kim et al., (2012) Nat. Struc. Mol. Biol. Solution Structure of the FAAP20-Ubiquitin Complex

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

FAAP20 UBZ Tail

C150 C170

H166 C147

Wojtaszek et al., (2014) Nucleic Acids Res. 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 Regulation of the FA Pathway by the FANCA-FAAP20 Axis

A 20 FA Core E3 ligase FAAP20 FANCA E3 ligase 150 kD

Ø Mechanism of maintaining FANCA stability by FAAP20?

Ø Mechanism of FANCA degradation and its implication in tumorigenesis? 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

20 ng/mL MMC

Quadriradial chromosome Characterization of a FA-like Breast Cancer Patient

MMC 1.2 FANCD2 1 FANCD2 + DAPI

0.8

0.6 GM0637 Survival 0.4 GM0357GM0637 0.2 FA-I DF2231

0 DF2231 0 12.5 25 MMC (nM) Upstream defect The I939S Point Mutation in Patient FANCA Destabilizes FANCA

FANCA gene

FAAP20

2816T>G; Ile939Ser

2840C>G; Ser947stop (Savoia et al. 1996, Levran et al. 1997, 1st allele 2nd allele Savino et al. 2003)

1.2 DF2231+Vector DF2231+FANCA 1 GM0637 0.8 kD GM0637DF2231 0.6 FANCD2-Ub FANCD2

Survival 150- 0.4 150- FANCA 0.2 50- Tubulin 0 0 12.5 25

MMC (nM) The Amino Acid 913-1095 Region of FANCA Is Required for FAAP20 Interaction FANCA-I939S Mutation Disrupts FAAP20 Binding

I939

FANCA

FAAP20

Flag-FAAP20 Flag-FAAP20

myc-FANCA

I939S-FANCA I939S-FANCA WT-FANCA WT-FANCA WT-FANCA WT-FANCA Myc

Flag WCE Flag IP Identification of SUMOylation Site of FANCA Near the FAAP20 Binding Region

Exon 28 Exon 29

Human Mouse Rat Chicken

K921 I939 Regulation of Protein Stability by Coupled SUMO-Ubiquitin Signaling

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)

STUbL (SUMO-Targeted Ubiquitin Ligase)

S target S S S RNF4 Ub UbUb 1 190 RNF4 SIM RING SIM: SUMO-interacting motif 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

Regulation of DNA ICL Repair by Integrated Ubiquitin-SUMO Signaling

USP1 Ub UAF1

Ub D2 PIAS1 Ub Ub Ub I D2 S RNF4 A S S A I A M 20 20 20

ü FANCD2 deubiquitination (USP1)

ü FANCM release

ü FANCA degradation Regulation of DNA ICL Repair by Integrated Ubiquitin-SUMO Signaling

RNF4 loss S RNF4 S S A Aberrant FANCA 20 accumulation

Delay of replication restart 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

221 FANCA mutations from 89 studies including TCGA

cBioPortal for Cancer Genomics 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

- FAAP20 ubiquitin binding in regulating ICL repair

- FANCA degradation by Ubiquitin plus SUMO

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

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

Collaborators and Reagents DFCI Alan D’Andrea Lab Judy Garber, M.D. - FANCA patient Kailin Yang, Ph.D. - bioinformatics U. of Pittsburgh Donniphat Dejsuphong, M.D./Ph.D. - DT40 Shannon Puhalla, M.D. - FANCA patient Lisa Moreau – chromosome assay MIT Jenny Xie, Ph.D. - FANCA patient Graham C. Walker, Ph.D. - Rev1 structure Raphael Ceccaldi, Ph.D. – TCGA analysis Duke University Medical Center Pei Zhou, Ph.D. - Rev1 structure

Kyoto University, Japan Hyungjin Kim Lab - Pharmacology Shunichi Takeda, Ph.D. - DT40

Ukhyun Jo, Ph.D. Miltenyi Biotech GmhH, Germany Jingming Wang Kay Hofmann, Ph.D. - Bioinformatics Soyoung Joo U. of Washington Winson Cai Ning Zheng, Ph.D. – DUB structure and function

Funding Leukemia & Lymphoma Postdoctoral Fellowship

Start-up funds - Stony Brook Cancer Center