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APOBEC3A drives deaminase domain-independent chromosomal instability to promote pancreatic cancer metastasis

Nature Cancer volume 2, pages 1338–1356 (2021)Cite this article

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Abstract

Despite efforts in understanding its underlying mechanisms, the etiology of chromosomal instability (CIN) remains unclear for many tumor types. Here, we identify CIN initiation as a previously undescribed function for APOBEC3A (A3A), a cytidine deaminase upregulated across cancer types. Using genetic mouse models of pancreatic ductal adenocarcinoma (PDA) and genomics analyses in human tumor cells we show that A3A-induced CIN leads to aggressive tumors characterized by enhanced early dissemination and metastasis in a STING-dependent manner and independently of the canonical deaminase functions of A3A. We show that A3A upregulation recapitulates numerous copy number alterations commonly observed in patients with PDA, including co-deletions in DNA repair pathway genes, which in turn render these tumors susceptible to poly (ADP-ribose) polymerase inhibition. Overall, our results demonstrate that A3A plays an unexpected role in PDA as a specific driver of CIN, with significant effects on disease progression and treatment.

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Fig. 1: APOBEC3A expression, not deaminase activity, is associated with survival in human PDA.
Fig. 2: APOBEC3A drives widespread deaminase-independent CIN in PDA.
Fig. 3: APOBEC3A enhances metastatic colonization.
Fig. 4: A3A-mediated CIN drives metastatic seeding through cGAS–STING signaling in PDA.
Fig. 5: A3A expression accelerates tumor formation in an autochthonous model of PDA independent of deaminase function.
Fig. 6: Ectopic expression of deaminase-deficient A3A in murine tumors recapitulates copy number changes of human PDAs.
Fig. 7: Identification of a A3A high, CIN high PDA cohort with acquired deficiencies in DNA damage repair.

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Data availability

The datasets generated and/or analyzed during the current study are available through ArrayExpress (CU44, E-MTAB-9843 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-9843; CU37, E-MTAB-9972 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-9972); CU42, E-MTAB-10115 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-10115); RS8, E-MTAB-9966 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-9966); RS10, E-MTAB-10116 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-10116); Sting OE orthotopic Tumors, E-MTAB-10722 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-10722) and EGA at https://ega-archive.org/studies/EGAS00001002543 or ICGC at https://dcc.icgc.org/releases/release_20/ Projects/PACA-CA. Source data for Figs. 1–7 and Extended Data Fig. 1–10 are provided with the paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

The codes supporting the current study are available from the corresponding author on request.

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Acknowledgements

We thank members of the Rhim laboratory for critical reading of the manuscript. S.M.W. is supported by the German Cancer Aid, Mildred-Scheel-Postdoctoral Fellow program. A.D.R. is supported by the V Foundation (V Scholar Grant), Doris Duke Foundation (Clinical Scholar Grant), Andrew Sabin Family Foundation, Cockrell Family Foundation, NCI (MD Anderson SPORE), MD Anderson Cancer Center (Physician Scientist Program, Pancreatic Cancer Moonshot) and CPRIT (Rising Stars Award). S.R.R. was supported by NIA (R21AG047114). Cancer studies in the Reuben Harris (R.S.H.) lab are supported by NCI P01 CA234228. R.L.K. received salary support from NIH T32 HL007062 and F32 CA232458. R.L.K. is an Awardee of the Weizmann Institute of Science–National Postdoctoral Award Program for Advancing Women in Science. R.S.H. is the Margaret Harvey Schering Land Grant Chair for Cancer Research, a Distinguished University McKnight Professor and an Investigator of the Howard Hughes Medical Institute. M.P.K. is supported by the NIH/NCI K08CA218690. Genomic instability analysis was done in the Cytogenetics and Cell Authentication Core (CCAC) at MD Anderson Cancer Center.

Author information

Authors and Affiliations

  1. Ahmed Cancer Center for Pancreatic Cancer Research, MD Anderson Cancer Center, University of Texas, Houston, TX, USA

    Sonja M. Wörmann, Fredrik I. Thege, Robert W. Cowan, Dhwani N. Rupani, Runsheng Wang, Sara L. Manning, Kimal I. Rajapakshe, Anirban Maitra & Andrew D. Rhim

  2. Department of Translational Molecular Pathology, MD Anderson Cancer Center, University of Texas, Houston, TX, USA

    Sonja M. Wörmann, Fredrik I. Thege & Anirban Maitra

  3. Ontario Institute for Cancer Research, Toronto, Ontario, Canada

    Amy Zhang, Steven Gallinger & Faiyaz Notta

  4. Department of Gastroenterology, Hepatology & Nutrition, MD Anderson Cancer Center, University of Texas, Houston, TX, USA

    Robert W. Cowan, Dhwani N. Rupani, Runsheng Wang, Sara L. Manning & Andrew D. Rhim

  5. BRCF Bioinformatics Core, University of Michigan, School of Medicine, Ann Arbor, MI, USA

    Chris Gates & Weisheng Wu

  6. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA

    Rena Levin-Klein & Reuben S. Harris

  7. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA

    Rena Levin-Klein & Reuben S. Harris

  8. Institute for Molecular Virology, University of Minnesota, Minneapolis, MN, USA

    Rena Levin-Klein & Reuben S. Harris

  9. Department of Experimental Radiation Oncology, MD Anderson Cancer Center, University of Texas, Houston, TX, USA

    Meifang Yu & Cullen M. Taniguchi

  10. Department of Genetics, MD Anderson Cancer Center, University of Texas, Houston, TX, USA

    Asha S. Multani

  11. Department of Surgical Oncology, MD Anderson Cancer Center, University of Texas, Houston, TX, USA

    Ya’an Kang, Matthew H. G. Katz & Michael P. Kim

  12. Department of Cancer Biology, Division of Basic Science Research, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Katharina Schlacher

  13. University of Minnesota Medical Center, Schulze Diabetes Institute, Minneapolis, MN, USA

    Melena D. Bellin

  14. Department of Gastrointestinal Oncology, H. Lee Moffitt Cancer Center, Tampa, FL, USA

    Jason B. Fleming

  15. Department of Internal Medicine and Hamon Center for Therapeutic Oncology Research and Children’s Research Institute, UT Southwestern Medical Center, Dallas, TX, USA

    Ravikanth Maddipati

  16. Howard Hughes Medical Institute, University of Minnesota, Minneapolis, Minnesota, USA

    Reuben S. Harris

  17. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Susan R. Ross

  18. Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA

    Susan R. Ross

Authors
  1. Sonja M. Wörmann
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  2. Amy Zhang
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  27. Andrew D. Rhim
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Contributions

A.D.R. conceived of the project. S.M.W. designed and performed experiments and analyzed the data. F.I.T., R.W.C., D.R., R.W., S.L.M. and M.Y. performed experiments. S.R.R. provided the A3A mouse model. R.L.K. and R.S.H. provided the deaminase assay, A3B antibody and optimized protocols. A.S.M. performed cytogenetic analyses. A.Z., F.N., C.G., W.W. and K.I.R, performed bioinformatic analyses. Y.K., M.H.G.K., M.P.K, and J.B.F. provided the hPDX (MDA cohort) data set. M.D.B. provided the human pancreatitis data set. A.Z., F.N. and S.G. provided the resected and advanced PDA Toronto datasets. A.M., S.R.R., K.S., R.M., F.I.T. and C.M.T. revised and edited the manuscript. A.D.R. designed experiments and analyzed data. A.D.R. and S.M.W. wrote and revised the manuscript.

Corresponding authors

Correspondence to Sonja M. Wörmann or Andrew D. Rhim.

Ethics declarations

Competing interests

R.S.H. is a co-founder, shareholder and consultant of ApoGen Biotechnologies Inc. The other authors declare no competing interests.

Additional information

Peer review information Nature Cancer thanks Samuel Bakhoum, Francisco X. Real and Fran Supek for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 A3A expression, not deaminase activity, is associated with tumor burden in PDA.

a, Survival of patients after PDA resection from a, b, TCGA, c, resectable Toronto, and d, MD Anderson (MDA) according to either A3A or A3B expression level in the resected tumors: a, b, TCGA A3A/A3B high, (ms 518 d and 596 d, respectively), medium (ms 598 d and 607 d) and low (ms 661 d and 592 d); c, d, resectable Toronto (A3A) and MDA (A3B) high (ms 579 d and 490 d, respectively), medium (ms 647 d and 679 d) and low (ms 608 d and 951 d); e, Time to recurrence of hPDAs (MDA) with A3A high (n = 16) and low (n = 14) expression. *P = 0.0488. f, Time to recurrence of hPDAs (MDA) with A3B high (n = 16) and low (n = 14) expression. P = NS. e, f, Data are presented as mean values + /- s.d. Two-sided unpaired Student’s t test. Total SNV count of g, A3A high (n = 79) and low (n = 80) resected A3A and h, high (n = 84) and low (n = 85) advanced hPDAs (Toronto). Two-sided Wilcoxon. Boxplot center line represents median, lower and upper box limits represent Q1 and Q3 respectively, whiskers represent Q1 - 1.5 IQR and Q3 + 1.5 IQR. i, Total number of mutations called in A3A high (n = 20) and low (n = 20) hPDAs (MDA). Data are presented as mean values + /- s.d. Two-sided unpaired Student’s t test, P = N.S. j, Pearson correlation of total number of mutations with A3A expression in hPDAs (n = 40) (MDA). k, Total number of mutations contributed by APOBEC signature in different tumors. l, Trinucleotide context-dependent SNV frequencies in A3A high (n = 20) and A3A low (n = 20) hPDAs (MDA). m, Total number of mutations in A3B high (n = 20) versus A3B low (n = 20) hPDAs (MDA). Data are presented as mean values + /- s.d. Two-sided unpaired Student’s t test. P = NS. n, Pearson correlation of the number of mutations with A3B expression in hPDAs (n = 40) (MDA). P = NS. o, Trinucleotide context-dependent SNV frequencies in A3B high and A3B low hPDAs (MDA). a-o, n=biologically independent hPDAs.

Source data

Extended Data Fig. 2 A3A expression correlates with APOBEC mutagenesis in PDA.

a, b, APOBEC specific motif enrichment in a, CG-coordinated and b, restricted CG-coordinated clusters with variants restricted to C > G, C > T in A3A high (n = 20) versus A3A low (n = 20) hPDAs (MDA). c, tCa versus tCw and d, ytCa versus rtCa motif enrichment in CG-coordinated clusters (restricted) in A3A high hPDAs (n = 20) (MDA). e, f, APOBEC specific motif enrichment in e, CG-coordinated and f, restricted CG-coordinated clusters with variants restricted to C > G, C > T in A3B high (n = 20) versus A3B low (n = 20) hPDAs (MDA). g, tCa versus tCw and h, ytCa versus rtCa motif enrichment in CG-coordinated clusters (restricted) in A3B high hPDAs (n = 20) (MDA). i, Venn diagram for the number and % of genes carrying mutations and uniquely found in A3A low (n = 20), in A3A high (n = 20) or in both hPDAs (MDA); Pie chart for uniquely mutated genes in A3A low, in A3A high hPDAs and found in up to 6 hPDAs respectively. j, Heatmap showing the % of mutations in A3A high (n = 20) versus A3A low (n = 20) hPDAs (MDA) belonging to a certain functional class (SNPEFF_FUNCTIONAL CLASS). a-j, n=biologically independent hPDAs. k, Allelic frequency average (AF) in % of somatic variants found in A3A high hPDAs (MDA).

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Extended Data Fig. 3 A3A drives widespread deaminase-independent CIN in PDA.

a, mRNA expression (2-ddCT) of A3A in MiaPaca-2 and Panc1 cells expressing A3AWTLV196 treated + /- Dox (n = 2 each). b, mRNA expression (2-ddCT) of A3A in MiaPaca-2 and Panc1 cells expressing inducible shRNA A3A and treated + /- Dox (n = 2 each). a, b, n=technical replicates. c, d, γH2AX staining and quantitation in MiaPaca-2 and Panc1 cells expressing A3AWTLV196 + /-Dox (n = 3 each) or inducible shRNA A3A + /- Dox (MiaPaca + /- Dox n = 3; Panc1 n = 3 (-Dox), n = 4 (+Dox); n=technical replicates. Data are represented as mean. e-g, % γH2AX positive cells within e-f, PKCY cells expressing indicated constructs of A3A (PKCY control (n = 5) versus A3AC161Y (n = 3) P = NS., control versus A3AE72A (n = 4) *P = 0.0159, control versus A3ATR (n = 3) *P = 0.0357, A3AWTLV196 + Dox versus -Dox (n = 3 each) P = NS. and within g, MiaPaca-2 +Dox versus -Dox (n = 2 each) and Panc1 +Dox versus -Dox (n = 2 each) cells expressing A3AWTLV196 or shRNA to A3A. n=biologically independent cells lines; e-f, Two-sided Mann–Whitney-U test. h-p, Giemsa staining with analysis of 35 or more metaphases of PKCY tumor cells expressing A3AWTLV196 + /- Dox (n = 4 each). q-y, Giemsa staining with analysis of 35 or more metaphases of PKCY tumor cells expressing A3AC161Y and control vector (n = 4 each). z-hh, Giemsa staining with analysis of 35 or more metaphases of PKCY tumor cells expressing A3AE72A and control vector (n = 3 each). ii-qq Giemsa staining with analysis of 35 or more metaphases of PKCY tumor cells expressing A3ATR and control vector (n = 2 each). rr, Representative images of Giemsa staining of primary A3A high and A3A low hPDX-derived cells. Scale bars = 100 μm. ss-zz, Giemsa staining with analysis of 35 or more metaphases of primary A3A high and A3A low hPDX-derived cell lines (n = 4 each); ss-zz: ***P < 0.0001; Chi-square test w/o Yate’s correction. Scale bars = 100 μm. h-zz, n=biologically independent cell lines. All insets at x80.

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Extended Data Fig. 4 A3A enhances metastatic colonization after hemi-splenic injection.

a-c, IB for A3AC-Terminal of PKCY tumor cells transfected with A3AWTLV196 and treated + /-Dox. HSP90 and β-actin served as control. d, IB for Myc-tag of PKCY tumor cells transfected with A3AC161Y and controls. HSP90 served as control. e-g, IB for Myc-tag of PKCY tumor cells transfected with A3AE72A and controls. HSP90 served as control. h, IB for Myc-tag of PKCY tumor cells transfected with A3ATR. HSP90 served as control. i, Quantitation of tumor cell area in the liver 3d and at euthanasia after hemi-splenic injections. PKCY tumor cells transfected with A3AWTLV196 and treated +Dox (d3 n = 3; survival n = 4) or -Dox (d3 n = 3; survival n = 4); d3 P = NS.; survival *P = 0.0286; A3AC16Y (d3 n = 6; survival n = 5) and control (d3 n = 5; survival n = 5); d3 **P = 0.0043; survival **P = 0.0079; A3AE72A (d3 n = 3; survival n = 3) and control vector (d3 n = 3; survival n = 4); P = NS. (j) Quantitation of tumor cell proliferation of cells seeded into liver after 1 d, 3 d and at euthanasia after hemi-splenic injections. PKCY tumor cells transfected with A3AWTLV196 and treated +Dox (d1 n = 5, d3 n = 4, survival n = 4) or -Dox (d1 n = 4, d3 n = 3, survival n = 4); P = NS.; P = NS.; *P = 0.0286; A3AC16Y (d1 n = 3, d3 n = 4, survival n = 4) and control (d1 n = 3, d3 n = 4, survival n = 4); P = NS.; P = NS.; *P = 0.0286; A3AE72A (d1 n = 3, d3 n = 4, survival n = 3) and control (d1 n = 4, d3 n = 3, survival n = 3); P = NS.; A3ATR(n = 3) and control (n = 4); P = NS. i, j, n=biologically independent tumors. Data are presented as mean values + /- s.d. Two- sided Mann-Whitney-U test.

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Extended Data Fig. 5 A3A enhances migration and tumor cell growth.

a, Relative wound density in % for PKCY A3AWTLV196 tumor cells + /-Dox; 4093 A3AWTLV196#2 +Dox (n = 8), 4093 A3AWTLV196 #2 -Dox (n = 4); 4093 A3AWTLV196 #3 +Dox (n = 7), 4093 A3AWTLV196#3 -Dox (n = 4). b, Confluency in % of PKCY A3AWTLV196 tumor cell + /-Dox; 4093 A3AWTLV196#2 +Dox (n = 4), -Dox (n = 5); 4093 A3AWTLV19#3 +Dox (n = 3), -Dox (n = 2). c, Relative wound density, PKCY A3AC161Y tumor cells or control (n = 2 each). d, Confluency, PKCY A3AC161Y tumor cells or control. 4093 A3AC161Y#5 (n = 5), 4093 GFP#1 (n = 5); 5699 A3AC161Y#2 (n = 6), 5699 GFP#2 (n = 6); 6178 A3AC161Y#2 (n = 5), 6178 GPF#2 (n = 7); 4093 A3AC161Y#2 (n = 7), 4093 GFP#2 (n = 4). e, Relative wound density, PKCY A3AE72A tumor cells or control; 4093 A3AE72A#9 (n = 4), 4093 control#1 (n = 4), 4093 A3AE72A#5 (n = 3), 4093 control#3 (n = 3). f, Confluency, PKCY A3AE72A tumor cells or control; 4093 A3AE72A#9 (n = 5), 4093 control#1 (n = 6), 4093 A3AE72A#5 (n = 5), 4093 control#3 (n = 5). g, Relative wound density, PKCY A3ATR tumor cells or control; 6178 A3ATR#105 (n = 5), 6178 GFP#2 (n = 7); 4093 A3ATR#132 (n = 5), 4093 GFP#1 (n = 3), 4093 A3ATR#131 (n = 8), 4093 GFP#2 (n = 5). h, Confluency, PKCY A3ATR tumor cells or control; 4093 A3ATR#131 (n = 6), 4093 GFP#2 (n = 3), 4093 A3ATR#132 (n = 3), 4093 GFP#1 (n = 3); 9308 A3ATR#136 (n = 4), 9308 GFP#2 (n = 3). i, Confluency, MiaPaca-2 A3AWTLV196 tumor cells + /-Dox; MiaPaca A3AWTLV196 + Dox (n = 4), MiaPaca A3AWTLV196 -Dox (n = 2). j, Confluency, of Panc1 A3AWTLV196 tumor cells + /-Dox; Panc1 A3AWTLV196 + Dox (n = 6), Panc1 A3AWTLV196 -Dox (n = 2); a-j, n=technical replicates. Data presented as mean. k-m, % of cells in each cell cycle phase 12 h after double thymidine block release. k, l, PKCY cells expressing indicated constructs of A3A; for all cells in G1 and S: P = NS.; G2: PKCY control (n = 5) versus A3AC161Y (n = 3) *P = 0.0357; PKCY control versus A3AE72A (n = 4) G2: *P = 0.0159; PKCY control versus A3ATR (n = 3) G2: *P = 0.0357; A3AWTLV196 + Dox versus -Dox (n = 3 each) P = NS. and m, MiaPaca-2 and Panc1 cell expressing A3AWTLV196 + /- Dox (n = 2 each). n=biologically independent cells lines. k, l, Two-sided Mann-Whitney-U test.

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Extended Data Fig. 6 A3A-mediated CIN drives metastatic seeding through cGAS-STING signaling in PDA.

a, GSEA enrichment plot of A3A high (n = 20) versus A3A low (n = 20) hPDAs (MDA). b, IB analysis for p65, pp65, p100, p52, pp100, Relb, pStat3, Stat3 in cytoplasmic, soluble nuclear and chromatin bound nuclear subcellular fractions of PKCY tumor cells transfected with either vector control (control) or A3AC161Y together with shcontrol or shSting. HSP90, HDAC1 and Histone H3 served as controls. The experiment was repeated twice with similar results. c, d, GSEA enrichment plots of A3A high (n = 20) versus A3A low (n = 20) hPDA (MDA). a, c, d, n=biologically independent tumors. e, RNA seq and comprehensive GSEA analysis of various RNA seq data sets as indicated. FDR < 0.25 considered significant. Significance levels and enrichments scores are displayed by color (POS = positive enriched FDR<0.25; POS_NS = positive enriched FDR>0.25), NES scores by size. f, g, i, hemi-splenic injections: f, IF staining and g, quantitation for GPF, ki67 and DAPI of indicated tumor cells seeded into liver after 1d, 3d and at euthanasia after hemi-splenic injections. Right: PKCY tumor cells transfected with A3AC161Y and shSting (d3 n = 5; survival n = 6) or shcontrol (d3 n = 4; survival n = 6); d3 *P = 0.0159; survival **P = 0.0043. Left: PKCY primary tumor cells transfected with Sting (OE Sting) (survival n = 3) or vector control (survival n = 4); survival P = NS. h, orthotopic transplantations: Metastatic rates. i, hemi-splenic injections: Tumor cell proliferation. Right: Primary PKCY tumor cells transfected with A3AC161Y and shSting (d1 n = 4; d3 n = 4; survival n = 4) or shcontrol (d1 n = 4; d3 n = 5; survival n = 6); d1 P = NS.; d3 *P = 0.0159; survival **P = 0.0095. Left: PKCY primary tumor cells transfected with STING (OE STING) (d1 n = 4; survival n = 3) or control (d1 n = 3; survival n = 4); d1 P = NS. For g, i, n=biologically independent mice. Mean values + /- SD. Two-sided Mann-Whitney-U test. j-l, orthotopic transplantations: j, Metastatic rates. k, l Kaplan-Meier plot with log-rank test (Mantel–Cox) for k, survival of A3AC161Y shSting (ms 34 d) and A3AC161Y shcontrol (ms 28 d) and for l, PKCY OE Sting (ms 23.5 d) and control (ms 30 d). m, IB for Sting and A3AN-Terminal of indicated tumor cells. n, IB for Sting. n, m, HSP90 served as control. o, p, IHC of PDA tissues obtained from orthotopic transplantations with indicated cells and for indicated marker. o-p, At least, 3 biologically independent tumor tissues were stained for each marker and each group. Scale bars = 100 μm. All insets at x80.

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Extended Data Fig. 7 A3A expression accelerates tumor formation and progression in an autochthonous model of PDA independent of deaminase function.

a, Oxygen consumption rate (OCR) (pmol/min) in A3AE PKCY (5687 (n = 4)); 7554 (n = 3)) versus PKCY (5417 (n = 3); 4099 (n = 4)) tumor cells. n=technical replicates. Data are represented as mean. b, IB for A3AN-terminal and A3AC-terminal of primary A3AE PKCY and PKCY tumor cells. PKCY tumor cells transfected with A3AC161Y and HSP90 served as control. At least 3 biologically independent cell lines were included in each comparison group with similar results. c, Survival of A3AE PKCY mice with A3A High (ms 108d) versus A3A Low (ms140d) mRNA expression (2-dCT) within the PDA tissue; Kaplan-Meier plot with log-rank test (Mantel–Cox). d, Quantitation of ki67 positive cells in A3AE PKCY (n = 5) versus PKCY (n = 5) PDAs. *P = 0.0317. e, Quantitation of Cleaved Caspase 3 (CC3) positive cells in A3AE PKCY (n = 6) versus PKCY (n = 5) PDAs. **P = 0.0087. (d, e) n=biologically independent tumors. Two-sided Mann-Whitney-U test; Data are represented as mean ± s.e.m. f, Relative wound density in % of A3AE PKCY and PKCY tumor cells; n = 2 technical replicates. g, Growth assay. Confluency in % of A3AE PKCY and PKCY and tumor cells; (7853 (n = 6), 7860 (n = 3), 4971 (n = 3), 7680 (n = 5), 4099 (n = 4), 5789 (n = 7), 4093 (n = 4), 5488 (n = 5), 6178 (n = 4); n=technical replicates. f, g, Data are presented as mean. h, Top, % of Sirius Red stained area in A3AE PKCY (n = 4) versus PKCY (n = 4) PDAs. *P = 0.0286. Bottom, representative aSMA and Sirius Red staining for A3AE PKCY and PKCY PDAs. i, Quantitation of lesions in 7 wks old A3AE PKCY (n = 3) versus PKCY (n = 3) mice. Reactive ducts (RD); P = NS. Bottom, representative H&E staining of 10 wks old A3AE PKCY versus PKCY mice. (h, i) n=biologically independent tissues. Two-sided Mann-Whitney-U test; Data are represented as mean ± s.e.m. j, Survival of A3AE KCY mice (ms 497 d) versus KCY (ms 532 d) mice. k, Top, Metastatic rate of A3AE KCY (n = 7) versus KCY (n = 23) mice; Bottom, table detailing the % of metastatic sites in each group. n=biologically independent mice.Scale bars = 100 μm. Insets for aSMA at x80, for Sirius Red at x20. Scale bars = 100 μm.

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Extended Data Fig. 8 Deaminase deficient A3A drives CIN and cGAS/Sting activation in a mouse model for PDA.

a-h, WES (CU37) of A3AE PKCY (n = 6) and PKCY (n = 4) pancreatic tissues. n=biologically independent tumors. a, Total number of mutations in A3AE PKCY and PKCY pancreatic tissues. Mean values + /- s.d. Two-sided unpaired Student’s t-test. P = NS. b, Trinucleotide context-dependent SNV frequencies. c-d, APOBEC3 specific motif enrichment in c, CG-coordinated clusters and d, CG-coordinated clusters (restricted). Stars indicated the motif enrichment in CG-coordinated clusters (restricted) of hPDA (MDA; red=A3A high; black =A3A low). e, Unique mutations: Venn diagram for the number and % of mutations uniquely found in either A3AE PKCY, PKCY mice or in both. Bar graphs for the % of mutations uniquely found per mouse in either A3AE PKCY or in PKCY and found in up to 3 mice respectively. f, Unique genes: Venn diagram for the number and % of genes carrying mutations and uniquely found in A3AE PKCY, in PKCY mice or in both. g, Heatmaps depicting the % of mutations having a specific impact (SNPEFF_IMPACT) h, Allelic frequency average (AF) in % of variants of A3AE PKCY mouse tissues. i-p, Giemsa staining with quantification (>35 metaphases) of A3AE PCKY (n = 15) and PKCY (n = 17) tumor cells. n=biologically independent cell lines. i, % samples with 15% or more of metaphases with breaks. j, % samples with ≥25% metaphases with fusions. k, % samples with ≥92% metaphases with fragments. l, % samples with an average dicentric count ≥2%. m, % samples with an average ring count ≥5%. n, % samples with an average fusion count ≥60%. o, % samples with an average break count ≥15%. i-o,***p < 0.0001. p, % samples with an average triradial count ≥2%. P = NS. For i-p, Two-sided Chi-square test w/o Yate’s correction. q, Quantification of anaphase bridges in A3AE PKCY (n = 5) versus PKCY (n = 5) tumor cells. *P = 0.0317. n=biologically independent cell lines. Two-sided Mann-Whitney-U test; Mean ± s.e.m. r, Giemsa staining depicting anaphases and anaphase bridges in A3AE PCKY tumor cells. s, Representative IB analysis for indicated antibodies showing cytoplasmic separation from nuclear and mitochondrial compartments in two biologically independent A3AE PKCY primary tumor cells. t, IF staining for Sting, cGas or Irf3 and DAPI in indicated tumor cells. s, t, At least, 3 biologically independent cells were included in each comparison group with similar results. Scale bars = 100 μm. All insets at x80.

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Extended Data Fig. 9 Ectopic expression of deaminase deficient A3A in murine tumors recapitulates copy number changes of human PDA’s.

a, Total SV count per chromosome in A3AE PKCY (n = 5) versus PKCY (n = 4) YFP+ sorted tumor cells. Two-sided Mann-Whitney-U test; Chr2 *P = 0.0159; Chr9 *P = 0.0159; Chr10 *P = 0.0159; Chr15 **P = 0.0079; Chr17 *P = 0.0159; Chr18 *P = 0.0357; all other Chr P = NS.; Data are represented as mean ± s.e.m. b, Top, Venn diagram for the count and percentage of genes with SV in A3AE PKCY, in PKCY or in both tumor cells. Bottom, Pie chart for the % of genes with Sversus found uniquely and in 50% or more of A3AE PKCY, or PKCY tumor cells. c, qPCR for Cdkn2a mRNA expression in A3AE PKCY (n = 13) and PKCY tumor cells (n = 11). n=biologically independent cell lines. Two-sided Mann-Whitney-U test; **P = 0.0043 for PKCY Loss versus Neutral. d, High magnification map for CN status (loss = blue; neutral =yellow) in Chromosome 4 in A3AE PKCY versus PKCY tumor cells. e, Map with unsupervised hierarchical clustering for LOH status of 139 homologous genes in advanced hPDAs (n = 169) (Toronto). All 139 were found primarily located in 3 distinct genomic loci. PDAs were separated by LOH status (blue) in either 2 or more loci (2 + LOH; orange/red square; n = 64) or less than 2 loci (<2 LOH; black/grey square; n = 105). f, A3A expression in advanced hPDA (Toronto) with LOH in <2 loci (n = 105) and 2 + (n = 64). Two-sided Wilcoxon. Boxplot center line represents median, lower and upper box limits represent Q1 and Q3 respectively, whiskers represent Q1 – 1.5 IQR and Q3 + 1.5 IQR, and dots represent outliers. e,f, n=biologically independent hPDAs. g, Survival of advanced hPDAs (Toronto) with 2+LOH (ms 249 d), and <2LOH (ms 258 d); Log-rank test (Mantel–Cox). h, Oncoplot of 144 homologous genes (uniquely found in A3AE PKCY tumor cells) (149 homologous genes thereof 144 found in UTSW data set) reveals a subgroup of 20 (18.3%) patients which had >70% of the same genes co-deleted (CN -1; -2) (subgroup: group1). i, 109 human PDA patients separated into two groups based on the percentage of deletions found per 144 genes. All A3AE PKCY (n = 5), PKCY (n = 4) were FACS sorted for YFP+. All n for A3AE PKCY equals 5, all n for PKCY equals 4 unless noted and represent biologically independent tumor cells.

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Extended Data Fig. 10 Identification of a A3A high, CIN high PDA cohort with acquired deficiencies in DNA damage repair.

a, b, % γH2AX positive cells in each cell cycle phase of a, PKCY cells expressing A3AWTLV196 + /-Dox (n = 3); Two-sided Mann-Whitney-U test. P = NS. and b, MiaPaca-2 and Panc1 cell expressing A3AWTLV196 + /-Dox (n = 2 each). n=biologically independent tumor cells. c, Map for LOH status of HRR genes (111) with a minimum fold change of 1 in advanced PDAs (Toronto) in 2+LOH (n = 64) versus <2LOH (n = 105) cohorts. d, Number of HRR genes with LOH e, Percentage of samples with LOH in each HRR gene in advanced PDA (Toronto) 2+LOH (n = 64) and <2LOH (n = 105) cohorts. c-e, n=biologically independent hPDAs. d, e, Two-sided Wilcoxon. Boxplot center line represents median, lower and upper box limits represent Q1 and Q3 respectively, whiskers represent Q1 - 1.5 IQR and Q3 + 1.5 IQR, and dots represent outliers. f-i, IF for RAD51 and DAPI in indicated tumor cells. At least 2 biologically independent cell lines were included in each comparison group with similar results. j, Distribution of deletions by size found in advanced hPDAs (Toronto) with A3A high (n = 84) versus low (n = 85) expression. <1 kb **P = 0.00915.; 1-10 kb P = NS.; 10-100 kb **P = 0.00195; 100-1 mb ***P = 0.00032; 1-10 mb ***P = 0.000225; >10 mb P = NS.; Two-sided Wilcoxon. k-m, Length of microhomology at deletions in A3A high (n = 79) versus low (n = 80) resected hPDAs, A3A high (n = 84) versus low (n = 85) advanced hPDAs (Toronto cohorts) and in A3AE PKCY (n = 5) versus PKCY (n = 5) mice. Two-sided Wilcoxon. P = NS. n-p, Foldback inversions in human and murine PDA cohorts. n, Toronto resected A3A high (n = 79) versus low (n = 80), o, Toronto advanced A3A high (n = 84) versus low (n = 85), p, A3AE PKCY (n = 5) versus PKCY (n = 4). Two-sided Wilcoxon. P = NS. k-p, Boxplot center line represents median, lower and upper box limits represent Q1 and Q3 respectively, whiskers represent Q1 - 1.5 IQR and Q3 + 1.5 IQR, and dots represent outliers. q, r, number of PDA samples with HRD signature in A3A high (n = 79) versus low (n = 80) resected and A3A high (n = 84) versus low (n = 85) advanced hPDAs. ChiSquare without Yate’s correction. P = NS. j-r, n=biologically independent tumors. s, t, Tumor cells treated with Oxaliplatin [0-1 mM]; Gemcitabine [0-4μM] for 96 h; s, A3AE PKCY (n = 7) versus PKCY (n = 6): *P = 0.0328. A3AE72A (n = 3) versus control (n = 3): ***P = 0.0007; A3AWTLV196 + Dox (n = 3) versus -Dox (n = 3): *P = 0.0496; hPDX A3A high (n = 5) versus low (n = 5): P = NS. t, A3AE PKCY (n = 7) versus PKCY (n = 6): P = NS. A3AE72A (n = 3) versus control (n = 3): P = NS; A3AWTLV196 + Dox (n = 3) versus -Dox (n = 3): P = NS.; hPDX A3A High (n = 5) versus Low (n = 5): P = NS. Two-sided unpaired Student’s t test. u, v MiaPaca-2 and Panc1 tumor cells transfected with shA3A + /-Dox, treated with Gemcitabine [0-1 μM]; Oxaliplatin [0-250 μM]; for 96 h. (n = 2). s-v, n=biologically independent cell lines. Scale bars = 100 μm. All insets at x80.

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Wörmann, S.M., Zhang, A., Thege, F.I. et al. APOBEC3A drives deaminase domain-independent chromosomal instability to promote pancreatic cancer metastasis. Nat Cancer 2, 1338–1356 (2021). https://doi.org/10.1038/s43018-021-00268-8

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