Fig 2: Spontaneous chromosomal instability in Fancc-/-; Mad2+/- mice. (A) Graphic illustrating the rationale of the quantitative red blood cell (RBC) micronucleation flow cytometry assay. Unlike normal nuclei, micronuclei (MN) arising from mitotic chromosome mis-segregation are not efficiently removed from maturing erythroblasts and therefore, DNA-positive mature RBCs signify abnormal chromosome sorting during erythropoiesis. (B) Example of a micronucleated cell in Fancc-/-; Mad2+/- bone marrow. Red arrows point to micronuclei (MNs). Nuc, nucleus. (C, D) Flow cytometry demonstrating an increased frequency of chromosome mis-segregation (p<0.0001) during Fancc-/-; Mad2+/- erythropoiesis compared to all other mouse genotypes. CD71 was used as a marker for immature peripheral blood red blood cells (RBCs). Frequencies of DNA-containing mature RBCs (CD71-, propidium iodide+) were statistically compared using Fisher’s exact test with n of at least 9 age-matched mice per genotype. (E, F) Karyotype demonstrating genomic instability in non-malignant Fancc-/-;Mad2+/- hematopoietic cells. Normal murine chromosome complement is 40, thus the presence of cells with <40 or >40 chromosomes is suggestive of aneuploidy. Statistical analysis was performed using Fisher’s exact test and Bonferroni post-hoc analysis (n=150 metaphase spreads per genotype, ****p < 0.0001, **p = 0.001).

Fig 3: Lethal hematopoietic malignancy in wt mice transplanted with marrows of moribund Fancc-/-; Mad2+/- mice. (A) Experimental design of competitive hematopoietic cell transplantation. (B) Disease-free survival (p<0.0001) and (C) overall survival (p=0.043) of wt recipients transplanted with bone marrows of indicated genotypes, n =8 recipients of wt donor marrow and n=13 recipients of marrow from leukemic Fancc-/-; Mad2+/- donors. Statistical significance was calculated via log-rank Kaplan-Meier Mantel-Cox tests. (D–G) Comparison of peripheral blood and marrow flow cytometry, bone marrow and liver morphology in a wt mouse, Fancc-/-; Mad2+/- donor and wt recipient of Fancc-/-; Mad2+/- marrow. (D, E) Myeloid marker flow cytometry, (F) evaluation of bone marrow architecture and (G) liver infiltrates demonstrating similar immunophenotype and histopathologic findings in leukemic donor and recipients. (H) Malignant cell infiltration of lymph nodes and spleen in a representative moribund transplant recipient. (I) Survival of three recipient mice transplanted with bone marrow from a Fancc-/-; Mad2+/- donor mouse (p=0.0246). Statistical significance was determined with log-rank Kaplan-Meier Mantel-Cox tests. *p < 0.05, ****p < 0.0001.

Fig 4: Mitotic infidelity and genomic lesions in Fancc-/-; Mad2+/- malignancies. (A) Representative images of abnormal mitotic figures in Fancc-/-; Mad2+/- hematopoietic malignancies in situ and (B) in bone marrow cytospins. Red arrows indicate lagging anaphase chromosomes, multi-nucleation and bi-nucleation in bone marrow cells isolated from Fancc-/-; Mad2+/- AML mice. (C) SKY analysis of Fancc-/-; Mad2+/- leukemia shows clonal hematopoiesis with whole-chromosome gains and losses. (D) Circular synteny map shows mapping of human chromosomes 1, 3, 7 and 21, whose changes had been implicated in FA MDS/AML in previous studies, to mouse chromosomes. Mouse chromosomes containing regions of more than one of the four human chromosomes are highlighted in circles. (E–H) Representative images of Fancc-/-; Mad2+/- malignancies. (I) Whole-exome sequencing of four Fancc-/-; Mad2+/- malignancies revealed copy-number variations of mouse chromosomes 1, 4, 6 and 16. Unique Fancc-/-; Mad2+/- malignancies are represented by letters A-D. Red denotes amplifications and blue represents copy number losses. Additional somatic variants of genes implicated in human cancer and genome stability identified by WES in Fancc-/-; Mad2+/- malignancies are shown in (J). Types of mutations are shown in the visual legend.

Fig 5: Exacerbation of SAC defect but not of DNA cross-linker hypersensitivity in Fancc-/-; Mad2+/- mice. (A) Graphic illustrating assay design for quantifying SAC impairment in primary low-density mononuclear hematopoietic cells (LDMNCs). Live c-kit+ cells were labeled with EdU and treated with 100 ng/mL nocodazole for 12 hours. Immunofluorescence microscopy quantifies cells that progressed through S-phase (EdU+) and escaped nocodazole-mediated SAC arrest to exit mitosis (pH3-) and undergo multi-nucleation (DNA). (B) Representative immunofluorescent images of c-kit+ cells in S/G2 phase (EdU+, pH3-, single nucleus), mitosis (M) (EdU+, pH3+, condensed chromosomes) and after SAC slippage (EdU+, pH3-, multi-nucleated). (C) Percentage of multinucleated Edu+, pH3- cells in wt, Mad2+/- and Fancc-/- and Fancc-/-; Mad2+/- c-kit+ cells, suggesting that SAC deficiency in Fancc-/-; Mad2+/- c-kit+ cells is more severe compared to wt (p<0.0001), Mad2+/- (p<0.001) and Fancc-/- (p<0.01) genotypes. Fancc-/- and Mad2+/- cells demonstrate an intermediate SAC defect. For each genotype, >200 cells were quantified, and results were compared by Fisher’s exact test. (D) Representative time-lapse video frames of wt and Fancc-/-; Mad2+/- BMDMs exposed to 2 µm taxol and imaged on a live imaging microscope at 2-minute intervals for 24 hours. SAC-arrested cells are round. SAC escape is followed by mitotic exit and flattening of multi-nucleated cells. (E) Evaluation of taxol-induced SAC arrest in Fancc-/-; Mad2+/- BMDMs. Time in mitosis (defined as the time from nuclear envelope breakdown to the time the nuclear envelope reforms) was recorded for at least 90 cells/genotype and analyzed by one-way ANOVA and Tukey’s post-hoc multiple comparisons test. The length of taxol-induced arrest in Fancc-/-; Mad2+/- BMDMs was significantly shorter compared to wt (p<0.0001), Mad2+/- (p<0.05) and Fancc-/- (p<0.01) genotypes. (F) Chromosome breaks (red asterisks) in Fancc-/-; Mad2+/- hematopoietic cells exposed to 50 nM MMC. Enlarged examples of fractured chromosomes are shown. (G) Mad2 haploinsufficiency does not affect chromosome breakage in hematopoietic cells exposed to 50 nM MMC. Note that expected high frequency of MMC-induced chromosome breaks in Fancc-/- cells does not differ from Fancc-/-; Mad2+/- cells. Statistical significance was compared using Fisher’s exact test with at least 100 cells per genotype analyzed. ("ns" denotes "not significant"). (H) Images of representative hematopoietic colonies of indicated genotypes in MMC methylcellulose assays. (I) Evaluation of colony forming ability following MMC demonstrates that Mad2 heterozygosity does not affect MMC hypersensitivity of Fancc-/- cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.