DNA repair interest group 04/21/15 Posttranslational Regulation of the Fanconi Anemia Cancer Susceptibility Pathway 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 I 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 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 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 Ub 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 .