Fig 2: Analysis of A3A and A3B mutational signatures in TCGA HNSCCTumors were classified by HPV status and substrate specificity for A3A and A3B. Xt[c]a > t/g polymorphisms were considered APOBEC related. Mutational signatures from individual tumors with >=50% A/Gt[c]a > t/g mutations were considered A3B like; and conversely tumors with >50% T/Ct[c]a > t/g designated A3A like, see also STAR Methods.(A) APOBEC mutations per HNSCC tumor in the TCGA, stratified by HPV status and A3A vs. A3B mutational signature. Log10(n APOBEC mutations + 1) is plotted for clarity. Wilcoxon rank sum test. *** p-value < 5*10ˆ-3.(B and C) Progression-free Survival of HPV-negative (B) and HPV-positive (C) HNSCC by APOBEC mutation profile. HPV+ HNSCC included oropharyngeal cancers in patients with up to 30 pack year tobacco smoke exposure, see STAR Methods.

Fig 3: Preclinical and clinical trial specimens showing 5-AzaC upregulates A3B in HPV+ HNSCC(A) A3B is upregulated in HPV+, but not in HPV- head and neck cancer cells after 5-AzaC treatment. Relative to GPDH mRNA, levels of A3B in cells after 5-AzaC treatment.(B) A3B protein levels in HPV+ UMSCC47 and UMSCC47 A3B CRISPR cells (UMSCC47 A3B-7) after 5-AzaC treatment, GPDH shown as loading control.(C) Relative mRNA levels of A3B in tumors before and after 5 or 7 days of 5-AzaC treatment from HNSCC patients enrolled in a window clinical trial. 1–5 represent tumors from five different HPV HNSCC patients; “post” indicates a tumor sample after 5-AzaC treatment.(D) Clonogenic survival after 5-AzaC treatment of HPV+ UMSCC47 and APOBEC3B CRISPR (A3B-7) cells. Data are represented as mean ± SD.

Fig 4: Effect of ssDNA Primary Sequence on A3B-FL Cytidine Deaminase Activity(A) Effect of the identity of the nucleotide in the -2 position on the rate of A3B cytidine deaminase activity.(B) Effect of the identity of the nucleotide in the -1 position on the rate of A3B cytidine deaminase activity. Constructs with a pyrimidine in the -1 position are deaminated more completely than those with a purine in the -1 position. Constructs with a thymine in the -1 position undergo complete product formation.(C) Effect of the identity of the nucleotide in the +1 position on the rate of A3B deaminase activity.(D) Effect of the identity of the nucleotide in the +2 position on the rate of A3B deaminase activity. Data are represented as mean ± SD.

Fig 5: Endogenous A3B from HNSCC cell lines confirms preference for purine at -2 position(A) Whole cell lysates from five out of six HNSCC cell lines prefer a guanine at -2 position over a thymine, except for SCC35 cell lysate, whose cytidine deaminase activity is undetectable. Nucleotides at -2 position and the cytidine that is deaminated are indicated by underscore. Red box designates substrate (S) and blue box product (P).(B) Western blot showing the expression level of A3B in all six HNSCC cell lines. A3B expression in SCC35 cells is below the detection limit, and HPV-positive cells (UMSCC47, UDSCC2, and YSCC94) have higher A3B expression compared to HPV-negative cells (SCC35, UNC521, and WSCC283).(C) Correlation of APOBEC3B expression with mutational profiles in HNSCC tumors. Tumors were classified by HPV status and substrate specificity for A3A and A3B. Xt[c]a > t/g polymorphisms were considered APOBEC related. Mutational signatures from individual tumors with >=50% A/Gt[c]a > t/g mutations were considered A3B like; and conversely tumors with >50% T/Ct[c]a > t/g designated A3A like, see also STAR Methods. Wilcoxon rank sum test. ns – not significant. *** p-value < 5*10ˆ-3. TPM – transcripts per million.(D) Western blot showing A3B expression in A3B CRISPR and A3B siRNA clones. A3A expression is not significantly altered. C = siRNA control.(E) A3B knockout and knockdown decrease the cytidine deaminase activity to below the detection level with the DNA substrate having a guanine at -2 position. DNA substrates (S) and products (P) are indicated in red and blue boxes, respectively. C = siRNA control.(F) A3B knockout and knockdown also decrease the turnover of the DNA substrate with a thymine at -2 position to below the detection level. DNA substrates (S) and products (P) are indicated in red and blue boxes, respectively. C, siRNA control.