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• W2259502103 abstract "•Aging Fancc−/− mice are prone to hematopoietic malignancies•Chromosomal abnormalities in hematopoietic Fancc−/− cells precede the development of leukemia•Fancc−/− leukemia can be successfully transplanted into healthy wt recipients Fanconi anemia (FA) is an inherited disorder of genomic instability associated with high risk of myelodysplasia and acute myeloid leukemia (AML). Young mice deficient in FA core complex genes do not naturally develop cancer, hampering preclinical studies on malignant hematopoiesis in FA. Here we describe that aging Fancc−/− mice are prone to genomically unstable AML and other hematologic neoplasms. We report that aneuploidy precedes malignant transformation during Fancc−/− hematopoiesis. Our observations reveal that Fancc−/− mice develop hematopoietic chromosomal instability followed by leukemia in an age-dependent manner, recapitulating the clinical phenotype of human FA and providing a proof of concept for future development of preclinical models of FA-associated leukemogenesis. Fanconi anemia (FA) is an inherited disorder of genomic instability associated with high risk of myelodysplasia and acute myeloid leukemia (AML). Young mice deficient in FA core complex genes do not naturally develop cancer, hampering preclinical studies on malignant hematopoiesis in FA. Here we describe that aging Fancc−/− mice are prone to genomically unstable AML and other hematologic neoplasms. We report that aneuploidy precedes malignant transformation during Fancc−/− hematopoiesis. Our observations reveal that Fancc−/− mice develop hematopoietic chromosomal instability followed by leukemia in an age-dependent manner, recapitulating the clinical phenotype of human FA and providing a proof of concept for future development of preclinical models of FA-associated leukemogenesis. The Fanconi anemia (FA) signaling network protects genomic integrity and prevents cancer by facilitating interphase DNA repair and orchestrating cell division [1Kottemann M.C. Smogorzewska A. Fanconi anaemia and the repair of Watson and Crick DNA crosslinks.Nature. 2013; 493: 356-363Crossref PubMed Scopus (436) Google Scholar, 2Nalepa G. Clapp D.W. Fanconi anemia and the cell cycle: New perspectives on aneuploidy.F1000Prime Rep. 2014; 6: 23Crossref PubMed Scopus (23) Google Scholar, 3D’Andrea A.D. Susceptibility pathways in Fanconi's anemia and breast cancer.N Engl J Med. 2010; 362: 1909-1919Crossref PubMed Scopus (273) Google Scholar]. Germline biallelic mutations of any FA genes cause Fanconi anemia, an inherited bone marrow failure syndrome associated with myelodysplasia (MDS) and acute myeloid leukemia (AML). The overall risk of leukemia in FA is increased six hundredfold [4Alter B.P. Fanconi anemia and the development of leukemia.Best Pract Res Clin Haematol. 2014; 27: 214-221Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar]. Young mice deficient in core FA genes do not spontaneously recapitulate clinical hematopoietic manifestations of Fanconi anemia [5Parmar K. D'Andrea A. Niedernhofer L.J. Mouse models of Fanconi anemia.Mutat Res. 2009; 668: 133-140Crossref PubMed Scopus (115) Google Scholar]. Fancc−/− mice demonstrate hypersensitivity to cross-linking agents [6Chen M. Tomkins D.J. Auerbach W. et al.Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia.Nat Genet. 1996; 12: 448-451Crossref PubMed Scopus (220) Google Scholar], decreased hematopoietic stem cell repopulating ability [7Haneline L.S. Gobbett T.A. Ramani R. et al.Loss of FancC function results in decreased hematopoietic stem cell repopulating ability.Blood. 1999; 94: 1-8Crossref PubMed Google Scholar, 8Whitney M.A. Royle G. Low M.J. et al.Germ cell defects and hematopoietic hypersensitivity to gamma-interferon in mice with a targeted disruption of the Fanconi anemia C gene.Blood. 1996; 88: 49-58Crossref PubMed Google Scholar], and hypersensitivity to interferon-γ [8Whitney M.A. Royle G. Low M.J. et al.Germ cell defects and hematopoietic hypersensitivity to gamma-interferon in mice with a targeted disruption of the Fanconi anemia C gene.Blood. 1996; 88: 49-58Crossref PubMed Google Scholar], reflecting disruption of the FA signaling network during hematopoiesis. However, young Fancc−/− mice do not develop spontaneous leukemia or bone marrow failure [6Chen M. Tomkins D.J. Auerbach W. et al.Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia.Nat Genet. 1996; 12: 448-451Crossref PubMed Scopus (220) Google Scholar, 8Whitney M.A. Royle G. Low M.J. et al.Germ cell defects and hematopoietic hypersensitivity to gamma-interferon in mice with a targeted disruption of the Fanconi anemia C gene.Blood. 1996; 88: 49-58Crossref PubMed Google Scholar]. One observation study of a small Fancc−/− mouse cohort (n = 8) did not detect decreased survival [9Pulliam-Leath A.C. Ciccone S.L. Nalepa G. et al.Genetic disruption of both Fancc and Fancg in mice recapitulates the hematopoietic manifestations of Fanconi anemia.Blood. 2010; 116: 2915-2920Crossref PubMed Scopus (40) Google Scholar]. However, FA patients rarely develop AML in their first year of life [10Alter B.P. Giri N. Savage S.A. et al.Malignancies and survival patterns in the National Cancer Institute inherited bone marrow failure syndromes cohort study.Br J Haematol. 2010; 150: 179-188PubMed Google Scholar], and two soft tissue tumors (adenocarcinoma and histiocytic sarcoma) have been reported in >13-month-old Fancc−/− mice [11Carreau M. Not-so-novel phenotypes in the Fanconi anemia group D2 mouse model.Blood. 2004; 103: 2430Crossref PubMed Scopus (24) Google Scholar]. Thus, we hypothesized that aging Fancc−/− mice may be predisposed to hematopoietic malignancies. If the absolute time to the onset of leukemia is similar in FA humans and mice, long-term observation of FA−/− mice may be crucial to detection of cancer predisposition. To address this translationally relevant question, we asked whether Fancc−/− mice develop malignancies as they age. C57Bl/6J Fancc−/− mice were a gift of David W. Clapp (Indiana University). Mice were genotyped by polymerase chain reaction (PCR), as described [9Pulliam-Leath A.C. Ciccone S.L. Nalepa G. et al.Genetic disruption of both Fancc and Fancg in mice recapitulates the hematopoietic manifestations of Fanconi anemia.Blood. 2010; 116: 2915-2920Crossref PubMed Scopus (40) Google Scholar]. B6.SJL-PtprcaPepcb/BoyJ mice were purchased from the Indiana University In Vivo Therapeutics Core. All studies were approved by the Institutional Animal Care and Use Committee at Indiana University. Bone marrow cells were flushed from mouse femurs using a 23-gauge needle/syringe (Becton Dickinson). Light-density mononuclear cells (LDMNCs) were isolated by density gradient using Histopaque-1119 (Sigma) and centrifuging for 30 min at 1,800 rpm with no brake. After centrifugation, LDMNCs were removed from the interface and used for experiments. Cytospins were made by resuspending LDMNCs in phosphate-buffered saline (PBS) and centrifuging onto slides at 450 rpm for 5 min on a Shandon Cytospin 3 Cytocentrifuge (Thermo Scientific). Donor test LDMNCs (1.5 × 106, C57Bl/6J background) and donor competitor BoyJ LDMNCs (1.5 × 106) were transplanted into recipients via tail vein injection. Recipients were 8-week-old female B6.SJL-PtprcaPepcb/BoyJ mice that underwent whole-body split-dose 1,100-rad irradiation (700 rads/400 rads, 4 hours apart). For chimerism analysis, peripheral blood was collected from lateral tail veins into EDTA-coated tubes, incubated with red blood cell lysis solution (Qiagen) for 10 min at room temperature, washed, stained with anti-Cd45.2-flluorescein isothiocyanate (BD Biosciences) and anti-Cd45.1-phycoerythrin (BD Biosciences), as described [9Pulliam-Leath A.C. Ciccone S.L. Nalepa G. et al.Genetic disruption of both Fancc and Fancg in mice recapitulates the hematopoietic manifestations of Fanconi anemia.Blood. 2010; 116: 2915-2920Crossref PubMed Scopus (40) Google Scholar], and analyzed on a FacsCalibur machine (Becton-Dickinson). At least 10,000 events/sample were acquired and analyzed using FlowJo Software. Bone marrow cells flushed from tibias were cultured in Iscove's modified Dulbecco's medium (IMDM) plus 20% fetal bovine serum, murine stem cell factor (100 ng/mL), and interleukin-6 (200 ng/mL) for 2 days. Cells were then exposed to 0.2 μg/mL colcemid (Life Tech) for 4 hours and pelleted at 800 rpm for 5 min. Cells were resuspended dropwise in prewarmed (37°C) 75 mM KCl and incubated at 37°C for 15 min. After pelleting, cells were resuspended in a 3:1 methanol:glacial acetic acid fixative. Cells were pelleted and resuspended in fixative two additional times before being dropped onto slides and dried overnight. Spreads were stained with Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories). Murine tissues obtained postmortem were fixed in 10% formalin, paraffin-embedded, sectioned (5-μm sections), and stained with hematoxylin and eosin. Peripheral blood smears and bone marrow cytospins were stained with Giemsa, using the automated Siemens Hematek 3000 (Fisher) system. For flow cytometry, peripheral blood cells were incubated in RBC lysis solution, and bone marrow LDMNCS were isolated as described above. Cells were stained with either Gr-1-APC (Ly6G, clone: RB6-8C5) or B220-FITC (clone: RA3-6B2) and analyzed on a FacsCalibur machine, using live gating followed by data quantification with FlowJo software. Leukemia diagnoses were made using criteria established in the Bethesda proposal for classification of nonlymphoid neoplasms in mice [12Kogan S.C. Ward J.M. Anver M.R. et al.Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice.Blood. 2002; 100: 238-245Crossref PubMed Scopus (345) Google Scholar], and were independently validated by a veterinary pathologist at the Indiana University School of Medicine. Images of smears, cytospins, and histologic sections were obtained using a Zeiss Axiolab microscope with a color camera. Metaphase spreads were imaged on a Deltavision personalDx deconvolution microscope (Applied Precision). Image stacks (distance between z-sections: 0.2 μm) were deconvolved using Softworx and analyzed using Imaris software suite (Bitplane). Statistical analysis was performed with GraphPad Prism 6 software (GraphPad Software, La Jolla, CA). We observed cohorts of wild-type (WT; n = 20) and Fancc−/− (n = 18) mice for 24 months and noticed decreased survival of Fancc−/− mice (p = 0.01) (Fig. 1A). Five Fancc−/− mice (27.8%) died between 8 and 24 months of age from leukemia or lymphoma (Fig. 1B–L). Specifically, we diagnosed AML in two moribund Fancc−/− mice with peripheral blasts, predominance of Gr-1+ (Ly-6G) peripheral blood LDMNCs, and myeloid infiltrates around the liver vessels (Fig. 1C–E). One Fancc−/−mouse developed lethal B-cell acute lymphoblastic leukemia (ALL), as evidenced by expansion of B220+ blasts that replaced >90% of bone marrow and infiltrated the liver (Fig. 1F–H). Additionally, two Fancc−/− mice died from metastatic abdominal T-cell lymphoma manifested by massive mesenteric lymph node conglomerates (Fig. 1I–J) and accompanied by Cd3+ liver infiltrates (Fig. 1K–L) in the absence of bone marrow or peripheral blood abnormalities. After 24 months of observation, all surviving Fancc−/− and WT mice were sacrificed and examined by necropsy. Four of 13 (30.8%) 2-year-old Fancc−/− animals had hematopoietic solid tumors and/or peripheral blasts, consistent with leukemia/lymphoma. Serial blood counts did not reveal progressive pancytopenia in aging Fancc−/− mice, suggesting that the development of leukemia may not be preceded by bone marrow failure in this animal model of FA. Nine of 18 Fancc−/− mice developed hematopoietic malignancies by 2 years of age (including 5 animals that died prematurely from disease), compared with zero of 20 control WT mice (p = 0.0003) (Fig. 1B). Thus, aging Fancc−/− mice are prone to hematopoietic neoplasms, reflecting the age-dependent risk of leukemia in FA patients [4Alter B.P. Fanconi anemia and the development of leukemia.Best Pract Res Clin Haematol. 2014; 27: 214-221Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 10Alter B.P. Giri N. Savage S.A. et al.Malignancies and survival patterns in the National Cancer Institute inherited bone marrow failure syndromes cohort study.Br J Haematol. 2010; 150: 179-188PubMed Google Scholar, 13Kutler D.I. Singh B. Satagopan J. et al.A 20-year perspective on the International Fanconi Anemia Registry (IFAR).Blood. 2003; 101: 1249-1256Crossref PubMed Scopus (564) Google Scholar]. We next asked whether Fancc−/− AML can be propagated in WT mice via competitive stem cell transplantation. We mixed donor Fancc−/− Cd45.2+ LDMNCs isolated from a moribund AML Fancc−/− mouse (Fig. 1C–E) with WT Cd45.1+ competitor LDMNCs at a 1:1 ratio and transplanted the mixed cells into three lethally irradiated WT recipients. Three WT recipients of age-matched WT Cd45.2+ LDMNCs mixed with WT Cd45.1+ LDMNCs served as controls (Fig. 2A). By 50 days posttransplantation, all recipients of Fancc−/− LDMNCs had died of AML, whereas control recipients of WT LDMNCs remained healthy (Fig. 2B). The diagnosis of AML was confirmed in all recipients by flow cytometry, peripheral blood smears (Fig. 2C), and splenomegaly (p = 0.0216) (Fig. 2D). Peripheral blood flow cytometry revealed increased Cd45.2+ chimerism (p = 0.0436) in recipients of leukemic Fancc−/− LDMNCs compared with controls at 1 month posttransplant (Fig. 2E, F), highlighting the malignant potential of leukemic Fancc−/− LDMNCs to outcompete WT hematopoietic cells in the host marrow. The FA signaling network maintains genomic integrity during Fancc−/− hematopoiesis in vivo [14Abdul-Sater Z. Cerabona D. Sierra Potchanant E. et al.FANCA safeguards interphase and mitosis during hematopoiesis in vivo.Exp Hematol. 2015; 43: 1031-1046Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar], and genomic instability promotes cancer [15Gordon D.J. Resio B. Pellman D. Causes and consequences of aneuploidy in cancer.Nat Rev Genet. 2012; 13: 189-203PubMed Google Scholar]. Thus, we asked whether leukemic Fancc−/− mice exhibit increased chromosomal instability and whether chromosomal instability precedes the onset of leukemia during Fancc−/− hematopoiesis. We compared karyotypes of LDMNCs isolated from leukemic Fancc−/− with those of age-matched WT and healthy Fancc−/− marrows. Bone marrow cells isolated from healthy Fancc−/− mice had a higher incidence of aneuploidy and an increased frequency of abnormal mitotic figures compared with WT LDMNCs (Fig. 3A–D), indicating that Fancc−/− hematopoietic cells become chromosomally unstable before overt leukemogenesis occurs. Similarly, FA patients develop hematopoietic chromosomal and nuclear abnormalities prior to the onset of leukemia [14Abdul-Sater Z. Cerabona D. Sierra Potchanant E. et al.FANCA safeguards interphase and mitosis during hematopoiesis in vivo.Exp Hematol. 2015; 43: 1031-1046Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 16Mehta P.A. Harris R.E. Davies S.M. et al.Numerical chromosomal changes and risk of development of myelodysplastic syndrome: Acute myeloid leukemia in patients with Fanconi anemia.Cancer Genet Cytogenet. 2010; 203: 180-186Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 17Barton J.C. Parmley R.T. Carroll A.J. et al.Preleukemia in Fanconi's anemia: Hematopoietic cell multinuclearity, membrane duplication, and dysgranulogenesis.J Submicrosc Cytol. 1987; 19: 355-364PubMed Google Scholar]. Leukemic Fancc−/− bone marrows were more aneuploid (Fig. 3B) with higher mitotic index compared with both age-matched WT (p = 0.001) and Fancc−/− nonleukemic (p < 0.0001) (Fig. 3D) marrows. This observation is consistent with further exacerbation of genomic instability and acquisition of bizarre karyotypic abnormalities reported in human FA-associated AML [18Woo H.I. Kim H.J. Lee S.H. Yoo K.H. Koo H.H. Kim S.H. Acute myeloid leukemia with complex hypodiploidy and loss of heterozygosity of 17p in a boy with Fanconi anemia.Ann Clin Lab Sci. 2011; 41: 66-70PubMed Google Scholar, 19Quentin S. Cuccuini W. Ceccaldi R. et al.Myelodysplasia and leukemia of Fanconi anemia are associated with a specific pattern of genomic abnormalities that includes cryptic RUNX1/AML1 lesions.Blood. 2011; 117: e161-e170Crossref PubMed Scopus (129) Google Scholar]. Fancc−/− mice develop chromosomally unstable hematopoietic malignancies as they age, recapitulating clinical and genomic abnormalities seen in patients with Fanconi anemia (Figure 1, Figure 3). Interestingly, a similar incidence of tumors has been reported in old mice deficient in another FA core gene, Fanca, although that observation has not reached statistical significance because of small sample sizes [20Wong J.C. Alon N. McKerlie C. Huang J.R. Meyn M.S. Buchwald M. Targeted disruption of exons 1 to 6 of the Fanconi anemia group A gene leads to growth retardation, strain-specific microphthalmia, meiotic defects and primordial germ cell hypoplasia.Hum Mol Genet. 2003; 12: 2063-2076Crossref PubMed Scopus (133) Google Scholar]. Thus, late-onset carcinogenesis may be a common phenotype of murine FA core gene knockouts. AML arising in Fancc−/− mice can be propagated via hematopoietic stem cell transplant and can produce rapid onset of lethal leukemia in WT transplant recipients (Fig. 2). As large-scale cohorts of leukemic mice are essential for preclinical drug testing, our observations may facilitate the development of future preclinical models of FA−/− AML. We thank the following funding sources: NIH K12 Pediatric Scientist Award, the Barth Syndrome/Bone Marrow Failure Research Fund at Riley Children's Foundation, the Heroes Foundation, and NIH T32 HL007910 “Basic Science Studies on Gene Therapy of Blood Diseases” grant. We thank Dr. George Sandusky for his pathology expertise, the Indiana University In Vivo Therapeutics Core for use of their irradiation facility, and Li Jiang for transplantation assistance. We acknowledge the work of our colleagues who we were unable to cite here because of space limitations. No financial interests/relationships with financial interest relating to the topic of this article have been declared." @default.
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• W2259502103 title "Leukemia and chromosomal instability in aged Fancc−/− mice" @default.
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