(A) Oncoprint plot of genetic alterations in ESCO2 and other SCC-associated genes in TCGA dataset. N = 10950. Number of samples with each mutation: N_(ESCO2) = 404, N_(ESCO1) = 252, N_(SMC3) = 216, N_(SMC1A) = 322, N_(NIPBL) = 743, N_(STAG1) = 429, N_(STAG2) = 374, N_(SGO1) = 165, N_(CDCA5) = 159, N_(WAPL) = 248, N_(PDS5A) = 227 and N_(PDS5B) = 375. P-values shown in the figure indicate significance of co-occurrence with ESCO2 from one-sided Fisher Exact test. (B) Kaplan-Meier survival analysis of patients with ESCO2 deficient (N = 1529) versus ESCO2 WT (N = 8317) tumors within TCGA dataset samples. P-value was determined by log-rank test. (C) Stacked bar plot indicating the percentage of patients with an ESCO2 deletion (blue), amplification (red) or mutation (green) and multiple alterations (grey) in uterine corpus endometrioid (UCEC), prostate (PRAD), colorectal (COADREAD), ovarian (OV), bladder (BLCA), liver (LIHC), lung adenocarcinoma (LUAD), lung squamous (LUSC), and breast cancer (BRCA) in the TCGA dataset. Cancer type is ordered based on the genetic alteration frequency. Number of samples with mutation/ total samples in each cancer type: 45/529 UCEC, 37/494 PRAD, 43/594 COADREAD, 41/584 OV, 26/411 BLCA, 23/372 LIHC, 33/566 LUAD, 23/487 LUSC, 50/1084 BRCA. The frequency of deletion in 100 kbp windows throughout Chromosome 8 in 1,111 pan-cancer type (Figure D, N = 11203) and prostate adenocarcinoma (PRAD, Figure E, N = 502) cancer patients. A region is considered as deleted if Log² (Copy Number/2) < -1. Blue line shows smoothed deletion frequency. Red vertical line indicates the locus of ESCO2 gene.

(A) Zebrafish Kaplan-Meier curves for tumor-free survival for wild-type, esco2^2865/+, p53^J19/+, esco2^2865/+; p53^J19/+, p53^J19/J19, and esco2^2865/+; p53^J19/J19 cohorts. Compared cohorts were established by natural single pair breeding of esco2^m/+ x AB (wild-type strain), esco2^m/+; p53^m/m x AB, or esco2^m/+; p53^m/m x p53^m/m parents (all cohorts were n = 96). The p-value = <0.0001 when comparing p53^J19/+ with esco2^2865/+; p53^J19/+ cohorts based on Log-rank (Mantel-Cox) test. (B) Mouse Kaplan-Meier curves for tumor-free survival for wild-type, Esco2 ^+/-, p53 ^+/-, Esco2 ^+/-; p53 ^+/- and p53 ^-/- cohorts (cohorts with p53^+/+ and p53^+/- background were n>60 and cohorts with p53^-/- background were n>6). The p-value = <0.05 when comparing p53^+/- with esco2^+/-; p53^+/- curves based on Log-rank (Mantel-Cox) test. (C) Frequency of p53 wild-type loss of heterozygosity (LOH) and esco2 wild-type LOH in zebrafish tumors; as well as the frequency of tumors being Malignant Peripheral Nerve sheath tumors (MPNST). (D) Pie charts showing tumor spectrum in Esco2^+/+; p53^+/- (left panel) and Esco2^+/-; p53^+/- (right panel) mice. No statistically significant in tumor spectrum with Chi-square test. (E) Percentage of patients with LOH on TP53 in TCGA cancer samples with or w/o ESCO2 mutation/deletion. The number of WT and deficient samples in each tumor were indicated. BLCA: 393 WT + 13 Deficient; BRCA: 935 WT + 29 Deficient; COADREAD: 482 WT + 38 Deficient; LIHC: 333 WT + 21 Deficient; LUND: 495 WT + 10 Deficient; LUSC: 478 WT + 8 Deficient; OV: 383 WT + 46 Deficient; PRAD: 481 WT + 6 Deficient; UCEC: 479 WT + 43 Deficient. * indicates p<0.05 from Fisher’s exact test. (F) Percentage of patients with LOH covering in tumor suppressor gene PTEN, BRCA1, BRCA2, RB1, NF1 and APC in the TCGA dataset. N = 4,126 for ESCO2-WT cohort and N = 193 for ESCO2-deficiency cohort. ** and *** indicate p<0.01 and p<0.001 with Fisher’s Exact test. (G) The number of LOH events per tumor sample in TCGA cancer samples with or w/o ESCO2 mutation/deletion. The number of WT and deficient samples in each tumor were indicated: PRAD: 481 WT + 6 Deficient; UCEC: 479 WT + 43 Deficient; OV: 383 WT + 46 Deficient; LUAD: 495 WT + 10 Deficient; COADREAD: 482 WT + 38 Deficient; LUSC: 478 WT + 8 Deficient; BRCA: 935 WT + 29 Deficient; BLCA: 393 WT + 13 Deficient. * and ** indicate p<0.05 and p<0.01 from Mann-Whitney test.

(A) Representative images of “paired” and “railroad” (RR) metaphase spreads. Inset shows zoomed-in view of each phenotype. (B) The percentage of metaphase spreads in esco2^+/+ and esco2^2865/+ showing paired or RR phenotypes. Two pools of embryos were used for esco2^+/+ and esco2^2865/+ spreads from two independent experiments. A total of 92 esco2^+/+ and 119 esco2^2865/+ spreads were tallied. (C) The percentage of spreads in sgo1^+/+ and sgo1^+/- showing paired or RR phenotypes. Three pools of embryos were used for sgo1^+/+ and sgo1^m/+ spreads. A total of 131 sgo1^+/+ and 142 sgo1^+/- spreads were tallied. ** p-value < 0.01 and *** p-value < 0.001 by paired t-test. (D) Kaplan-Meier curves for tumor-free survival for p53^+/+, sgo1^+/-; p53^+/+, p53^+/- and sgo1^+/-; p53^+/- cohorts. Cohorts were established by natural single-pair breeding of sgo1^+/- X p53^+2/+ parents (all cohorts were n = 96). P-value = <0.0001 when comparing p53^+/- with sgo1^+/-; p53^+/- curves based on Log-rank (Mantel-Cox) test.

(A) in-vivo confocal imaging of H2A.F/Z-EGFP and CaaX-mCherry mRNA injected embryos at 24 hours post-fertilization (hpf) for two hours. Representative images of normal and defective mitoses in esco2^+/+ and esco2^m/+. Arrow in anaphase bridge time-lapse points towards the anaphase bridge formed. Dotted circular in tri-polar time-lapse represents the three future nuclei that will occur. CaaX-mCherry was removed in tri-polar time-lapse for better visualization. t = time in minutes. (B) Division time calculated for each division in six esco2^+/+ and ten esco2^m/+embryos using two-hour imaging time-lapse data from each embryo. The percentage of cells was calculated for each bin category. N = 73 for esco2^+/+ and N = 132 for esco2^m/+. (C) Table representing mitotic defects and the associated mitotic timing and cell fate observed in six esco2^+/+ and ten esco2^m/+embryos.

(A) Micronuclei (MN) during interphase were counted in six esco2^+/+ and ten esco2^2865/+ H2A.F/Z-EGFP mRNA injected embryos at 24hpf using two-hour live imaging time-lapse data from each embryo. Each dot represents an embryo measured. Percentage MN/embryo was calculated based on the total number of micronuclei per total nuclei present in the time lapse at t = 0. Total nuclei are indicated in parenthesis. Mean ± SD, ** p-value < 0.01 based on unpaired t-test. Representative black and white images of micronuclei in each genotype are shown below. (B) Proposed model where the proportion of cells with MN would correlate with p53 LOH and timing of tumor formation. (C) Experimental workflow to establish high and low MN cohorts, using confocal living-imaging of 3-dpf p53^+2/+; TG^PhOTO-N/+ embryos. Figure B and C were created with BioRender.com. (D) Dot-blot of Micronuclei frequency in individual 3-dpf p53^+2/+; TG^PhOTO-N/+ embryos. Individuals deemed part of the high MN cohort were highlighted with red (MN%>0.9%) and individuals in the low MN cohort were highlighted with green (MN% = 0). (E) Zebrafish Kaplan-Meier curves for tumor-free survival for p53^+2/+; TG^PhOTO-N/+ unsorted (n = 90), p53^+2/+; TG^PhOTO-N/+ with high NM ratio(n = 54) and p53^+2/+; TG^PhOTO-N/+ with high NM ratio (n = 58). P-value is not significance when comparing each other based on Log-rank (Mantel-Cox) test.

(A) Schematic of determining LOH mechanisms/types in p53 heterozygous tumors. The haplotype of nearby p53 marker in tumor and normal paired samples, cooccurring with p53 wild-type or mutant gene are labeled (left). (B) A table showing the percentage of mitotic recombination (MR) in esco2^+/+; p53^zy7/+ and esco2^hi2865/+; p53^zy7/+ tumors. (C) Schematic of determining the mitotic recombination with TCGA database. Figure A and C were created with BioRender.com. (D) The number of mitotic recombination in TCGA cancer samples with or w/o ESCO2 mutation/deletion. Genome-wide data was downloaded from NCI Genomic Data Commons. The number of WT and deficient samples in each tumor were indicated: PRAD: 481 WT + 6 Deficient; UCEC: 479 WT + 43 Deficient; OV: 383 WT + 46 Deficient; LUAD: 495 WT + 10 Deficient; COADREAD: 482 WT + 38 Deficient; LUSC: 478 WT + 8 Deficient; BRCA: 935 WT + 29 Deficient and BLCA: 393 WT + 13 Deficient. *, ** and *** indicate p<0.05, p<0.01 and p<0.001 from Mann-Whitney test.