THE DNA DSB RESPONSE : The. Role in IgH Class Switching and Suppression of Genomic Instability and Cancer

Transcription

1 THE DNA DSB RESPONSE : The Role in IgH Class Switching and Suppression of Genomic Instability and Cancer

2 GENERAL DSB REPAIR Via Non-Homologous End-Joining (NHEJ) DSBs IR, etc. DSB NHEJ NHEJ KNOWN NHEJ FACTORS: KU70, KU80, DNAPKcs, ARTEMIS, XRCC4, LIGASE 4

3 NHEJ-Deficient Mice Severe combined immune deficiency Radiosensitivity Variable additional phenotypes (apoptosis of newly generated neurons) Modest cancer predisposition (G1 Checkpoint elimination of cells with unrepaired DSBs)

4 Percent survival NHEJ -/- p53 -/- mice die early from RAG-dependent Pro-B Lymphomas /12 weeks NHEJ +/- p53-/- NHEJ -/- p53-/ Age (days)

5 NHEJ/p53 deficiency Predisposes Mice to Pro-B Lymphomas IgH Gene Rearrangement B220 + CD43 + IgL Gene Rearrangement Pro B V-J Feedback & k/l D-J H V-DJ H Expansion pre-bcr B220 + CD43 - Pre B Receptor Editing BCR Immature B B220 + IgM + Mature B Proliferation and CSR B220 + Ig + Pro-B Tumor (12/15 translocation (non-reciprocal) which fuses IgH far downstream of c-myc leading to amplification via BFB)

6 NHEJ/p53-DEFICIENT MATURE B CELLS: LYMPHOMAS WITH IgH/c-MYC TRANSLOCATIONS VIA ABERRANT IgH CSR

7 (n= ) V H 1 V H n D H (1-12) J H Em Constant Regions DNAPKcs Ku70, Ku80 Artemis XRCC4/LigaseIV V(D)J Sm RAG1/RAG2 NHEJ Cm Cd V(D)J Recombination Cg3 Cg1 Cg2b Cg2a Ce Ca Se 3 RR V regions Transcription AID DNA repair IgM SHM Class Switch Recombination Ce Ca S regions Transcription AID DNA repair Recombined C H Locus + IgE Excised Circle

8 CONDITIONAL INACTIVATION OF XRCC4 IN P53-DEFICIENT MATURE B CELLS: NOVEL MATURE B-CELL LYMPHOMA 100 % Survival CD21creXc/- p53-/- (N=19) CD21creXc/-p53+/- (N=18) CD21cre p53-/- (N=10) CD21creXc/- (N=20) Days

9 CD21cre/XC/p53N (CXP) Tumor Summary Tumor # Rearrangements C-myc Translocations Break points IgH IgL (k/l) Rearranged Ex1 & Ex2 Amplified CXP35 Sa +/+ no *N-myc+ 3 IgH+ t(12;12;16), t(6;16), t(3;y),t(11:x) nd CXP62 + +/+ + - t(16;15), t(15;16), t(12;14) cloned CXP68 Sa +/+ + - t(12;15), t(15; 12) cloned Sg3 t(12;16) CXP153 Sg1 +/+ + - t(12;15), t(15; 12), nonclonal Chr 16 translocations cloned CXP162 Sg 2b +/+ + - t(12;15), t(15;12); t(6;16), t(6;16;4) nd CXP163 +;Su +/+ + - t(12;15), t(15; 12), t(12;16); t(16;12;16) cloned CXP221 Sa +/+ + - t(12;15), t(15; 12), t(15;7) nd

10 CXP 162 SKY t(12;15) t(15;12)* SKY FISH FISH Chr IgH Chr. 15 Chr. 12 Chr. 15 Chr. 12 C-Myc Chr. 15 Chr IgH *not detectable by SKY

11 Translocations Between 12 and 15 Result from Aberrant CSR IgHRR Ca S S S S Sg1 Sg3 Sm Em Ce Cg2a Cg2b Cg1 Cg3 Cd Cm Im JH4 JH3 JH2 JH1 DD V VV 3 1' 2' Chr 12 Centromere IgH Locus Telomere exon 1 exon 2 exon 3 Chr ' 2' c-myc 1'

12 Rearrangements involving IgH sequences directly into the c-myc locus in CXP NHEJ/p53 Deficient Mature B cell Lymphomas CXP153 t(12;15) Sg1 (breakpoint ~500bp upstream of Exon1) exon 1 exon 2 exon 3 t(15;12) (breakpoint 1.1kb downstream of Exon1) exon 1 I m Em CXP163 t(12;15) Im (breakpoint 445bp upstream of Exon1) exon 1 exon 2 exon 3 t(15;12) 5 Exon of 1 J H3 (breakpoint 900bp upstream of Exon1) CXP68 t(15;12) 5 Exon 1 Sg3 (breakpoint 2.1kb upstream of Exon1)

13 Different IgH/c-Myc Translocations in NHEJ/p53 Deficient Pro-B versus Mature B-Cell Lymphomas Far 3 of c-myc with amplification (pro-b) versus into c- Myc with ectopic activation (mature B). --Differential stage-specific activities of IgH enhancers? --RAG versus AID-mediated mechanism? --Stage-specific translocation acceptor sites (e.g., fragile sites; non-standard DNA )? --Proximal localization?

14 XRCC4/p53-Deficient Mature B Cells: a model for Mature B Lymphomas --Translocations of IgH locus to exon 1 region of c-myc gene (similar to Human BL). --Reciprocal Translocations (like human BL but not mouse pro-b tumors). --Chromosome 12 translocations appear to involve aberrant CSR. --Most tumors have second translocation involving chromosome 16.

15 Recurrent Chromosome 16 translocations Bcl6 Igl germline locus V2 Vx J2 C2 yj4 C4 V1 J3 C3 J1 C1 Chr. 16 BAC P9 BAC E14 BAC P12-147kb t(15;16) SKY FISH Chr. 15 Chr. 16 Chr. 15 Igl BAC P9 Chr. 16 Chr. 15 Igl BAC E14 Chr. 16 Chr. 15 Igl BAC P12 Chr. 16 CXP 62 t(16;15) SKY FIS H Chr. 16 Chr. 16 Chr Chr. 16 Chr. 15 Igl BAC P9 Chr. 15 Igl BAC E14 Chr. 15 Igl BAC P12 Chr. 15

16 Translocations Involving Chr16 in CXP Tumors 1 Bcl11b cyclink IgH Chr12 D12Ertd771e BAC Centromere 1 4 Telomere 3' IgL Enh Cl1 Jl1 Cl3 Jl3 Vl1 Bcl6 Chr16 4' 4 lambda Chr15 4' 1.2kb exon 1 exon 2 exon 3 c-myc

17 Translocations involving Chr16 in CXP Tumors CXP163 T(16;12;16) 3' IgL Enh Cl1 Jl1 Bcl11b?? Bcl6 D12Ertd771e BAC CXP62 T(16;15) 3' IgL Enh Cl1 Jl1 exon 1 exon 2 exon 3 c-myc CXP62 T(15;16) Chr 15 Bcl6

18 Deletion of Igk and Aberrant Igl Rearrangements : Editing in the Absence of NHEJ? Chr. 16 V1/2 J3 C3 J1 C1 CXP35 (4/4) V2Æ1kb upstream J1 (J3D?) Nonproductive CXP62 (5/5) V1 Æ within J1 exon (bp -2) Nonproductive CXP153 (4/6) V1 Æ within J1 exon (bp -15) Nonproductive CXP162 (cloned) 5 J3 Æ3 V1 Æ3 J1 Nonproductive CXP162 (4/4) V1 Æ within J1 exon (bp -57) Nonproductive CXP163 (6/7) V1/2Æ15bp downstream J1 (bp -70) Nonproductive

19 IGl LOCUS REARRANGEMENTS AND TRANSLOCATIONS IN NHEJ/p53-DEFICIENT B-CELL LYMPHOMAS --Igk deletion and non-productive/aberrant Igl rearrangements in most tumors --Igl translocations in most tumors (along with 12/15 translocations) --Reciprocal 16/15 and 15/16 juxtapose Igl1 and c- Myc --Non-reciprocal translocations of 16 juxtapose Igl into various chromosomes (activation/inactivation of target genes?)

20 XRCC4/p53-DEFICIENT MATURE B-CELL LYMPHOMAS Finding --Harbor independent translocations involving aberrant CSR and aberrant V(D)J recombination (editing) in a mature B cell IMPLICATIONS --Defective NHEJ can predispose to oncogenic translocations in mature B cells --A potential stage in mature B cells in which CSR and V(D)J recombination are activated (revision?)

21 Question: What other mutations may synergize with NHEJ/p53 deficiency to promote genomic instability? Observation: NHEJ potently inhibits translocations by strongly favoring re-ligation of DNA ends within a DSB as opposed to joining ends of separate DSBs. Hypothesis: Chromatin-Associated Proteins Sense and Facilitate Proper Repair of DSBs via NHEJ Through Alteration of Chromatin Structure.

22 Role of DSB Response Factors (H2AX, 53BP1, MDC-1) in DSBR, CSR, and Suppression of Genomic Instability

23 Histone H2AX H2A variant with C-terminal SQE motif that is rapidly phosphorylated by ATM, ATR, DNA-PKcs along Mb domains flanking DSBs (leading to co-localized repair and checkpoint protein foci) (Bonner). H2AX deficiency or haploinsuffiency leads to genomic instability and (in p53-deficient background) lymphomas and other cancers. AID-dependent g-h2ax foci at the IgH locus during CSR (Nussenzweig). H2AX-haploinsufficiency (on a p53-deficient background) leads to Mature B cell lymphomas with S-region translocations.

24 g-h2ax FUNCTIONS AS A SCAFFOLD FOR THE ASSEMBLY OF MULTIPLE PROTEIN/DNA COMPLEXES H2AX Double-Strand Break (ATM, ATR, DNA-PK) 53BP1 MDC1 MRN g-h2ax

25 g-h2ax May Function as Anchor to Hold Broken Chromosomal DNA Ends in Close Proximity Normal Cell V General DSB g-h2ax.. MRN.. 53BP NHEJ MDC1 v Repair H2AX-Deficient Cell Two DSBs Aberrant Repair Translocation

26 H2AX-, 53BP1-, and MDC-1-deficient Mice Have Overlapping Phenotpes Viable, smaller, and IR-sensitive Similar tumor onset and spectrum in 53BP1/p53- and H2AX/p53-deficient Mice. Modestly impaired lymphocyte development but normal V(D)J recombination levels. Impaired IgH class switch recombination (53BP1 > H2AX > MDC1) with normal SHM ---What is role of proteins in CSR?

27 Normal Mature B Cell Sm H2AX-deficient (or 53BP1- or MDC-deficient?) Mature B Cell Sm AID-initiated DSBs Sg1 AID-initiated DSBs Sg1 DSB response Factors stabilize ends No Change in Chromatin Structure DSBs Repaired Accurately by NHEJ DSBs Not held Together for repair

28 Prediction of Model --ENDS SHOULD NOT BE HELD PROPERLY DURING CSR IN H2AX-, P53BP1- OR MDC-1-DEFICIENT B CELLS --LEADING TO LARGE NUMBERS OF BROKEN ENDS WITHIN THE IgH REGION IN DSBR FACTOR-DEFICIENT B CELLS ACTIVATED FOR CSR.

29 H2AX -/- B cells show increased genomic instability aberrations per metaphase (%) H2AX+/+ H2AX+/- H2AX-/- DAPI LPS CD40L/ IL-4 Con A 100 LPS CD40L/IL-4 Chromosome breaks Chromatid breaks Fragments Other aberrations aberrations DAPI + (TTAGGG) 3 aberrations per metaphase (%)

30 Increased IgH locus breaks in H2AX -/- B cells Telomere PNA FISH/3 IgH FISH TELOMERE V D J Em C 3 RR CENTROMERE (TTAGGG) 3 BAC#199 number of breaks LPS number of breaks CD40L/Il-4 (TTAGGG) 3 2 broken chromatids 1 broken chromatid 2 broken chromatids 1 broken chromatid number of breaks concanavalin A BAC199 hybridizes at the breakpoint BAC199 does not hybridize at the breakpoint 0 BAC 199 (IgH 3 RR) 2 broken chromatids 1 broken chromatid

31 Combined 5 IgH and 3 IgH two-color FISH TELOMERE V D J Em C 3 RR CENTROMERE BAC#207 BAC#199 LPS -CD40/ IL-4 -CD40 Con A H2AX +/ H2AX +/ H2AX -/ H2AX -/ % IgH locus breaks Time course Day 3 Day 4 H2AX -/- Mouse 1 H2AX -/- Mouse 2 H2AX +/- H2AX -/- H2AX -/- DAPI BAC199 (3 IgH) BAC207 (5 IgH)

32 Igh Locus Breaks and General Breaks in Switching H2AX -/- B Cells Are Mechanistically Distinct IgH Locus Breaks vs General Breaks 25 CD40L 80 CD40L IgH locus breaks (%) general breaks (%) LPS a-cd40/ IL-4 a-cd40 Con A 0 LPS a-cd40/ IL-4 a-cd40 Con A

33 End-dissociation of S Regions Underlies the Switch Defect of H2AX-, ATM-, MDC1-, and 53bp1-deficient B Cells -inverse correlation between CSR efficiency and frequency of IgH locus breaks- MDC1 -/- H2AX -/- 53BP1 -/- CSR efficiency Mildly reduced Moderately reduced Severely reduced Frequency 10 of IgH locus 5 0 breaks (%) 6.0± ± ±4.2 +/+ -/- +/+ -/- -/- MDC1 H2AX 53BP1/p53

34 53BP1 -/- B cells A Model to Dissociate General and IgH-Locus Genomic Instability ATM -/- H2AX -/- MDC1 -/- 53BP1 -/- General chromosome breaks IgH locus breaks % 20 0 LPS CD40L/Il-4 All chromosome breaks IgH locus breaks CD40L

35 End-dissociation of S Regions Generates Translocation Acceptors H2AX -/- MDC1 -/- 53BP1 -/- ATM -/- 53BP1>>H2AXªATM>>MDC1 In the first mitosis after switching Non-clonal Centric 12 Acentric 12 Free ends a fragment Translocated onto: a chromatid break an acentric Broken free ends Dicentric with the other centric 12 Dicentric with a non-12 centric Translocated onto a centric

36 Conclusions H2AX, MDC1, 53BP1 and ATM are required to maintain general genomic integrity in B and T cells 53BP1-, H2AX-, ATM- or MDC1- deficient mature B Cell Sm Sg1 In switching B cells, lack of H2AX, ATM, MDC1 or 53BP1 results in failure to rejoin AID-induced S region DSBs in one or the two IgH loci. The frequency of IgH-locus breaks correlates inversely with the switch deficit, suggesting that this is the underlying molecular lesion 53BP1>>H2AXªATM >> MDC1 IgH locus breaks always involve both chromatids, implying that AID-induced DSBs are introduced in prereplicative DNA Replicated IgH locus breaks are often involved in chromosomal translocations, which could contribute to lymphomas AID-initiated DSBs No Change in Chromatin Structure (foci formation, recruitment of NHEJ) DSBs is replicated Loss of genetic material Substrate for translocations

37 Acknowledgments Alt Lab Sonia Franco Catherine Yan Shan Zha Monica Gostissa Michael Murphy Jing Wang Abishek Datta Joanne Sekiguchi Craig Bassing Collaborators Klaus Rajewsky Phil Carpenter Julio Morales John Manis Junjie Chen