Quantification of ongoing APOBEC3A activity in tumor cells by monitoring RNA editing at hotspots

Abstract

APOBEC3A is a cytidine deaminase driving mutagenesis, DNA replication stress and DNA damage in cancer cells. While the APOBEC3A-induced vulnerability of cancers offers an opportunity for therapy, APOBEC3A protein and mRNA are difficult to quantify in tumors due to their low abundance. Here, we describe a quantitative and sensitive assay to measure the ongoing activity of APOBEC3A in tumors. Using hotspot RNA mutations identified from APOBEC3A-positive tumors and droplet digital PCR, we develop an assay to quantify the RNA-editing activity of APOBEC3A. This assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the activity of APOBEC3A on DNA. Importantly, we demonstrate that the RNA mutation-based APOBEC3A assay is applicable to clinical samples from cancer patients. Our study presents a strategy to follow the dysregulation of APOBEC3A in tumors, providing opportunities to investigate the role of APOBEC3A in tumor evolution and to target the APOBEC3A-induced vulnerability in therapy.

Fig. 1. APOBEC3A and APOBEC3B mutation landscape in patients’ tumors.

a, b Whole Genome Sequencing (WGS) of patient tumor samples (from TCGA and other projects, see Supplementary Table 1) were analyzed for their mutation frequency in the TpC motif. Each patient’s tumor samples were plotted by their level of mutations in the TpC motif and their mutation frequency in RTC versus YTC sequences. c, d Patients’ tumor samples were plotted according to their A3A expression level and mutation frequency in YTC sequences. Color-codes indicate patients’ tumor types (a, c) or APOBEC3A mutation frequency in DNA stem-loops (b, d).

Fig. 2. APOBEC3A expression does not correlate with APOBEC activity monitored by DNA deaminase activity assay.

a Analysis of A3A and A3B mRNA expression in a panel of cancer cell lines determined by RT-qPCR. Error bar: S.D. (n = 3). b A3B levels in the indicated cancer cell lines were analyzed by western Blotting. c Schematic representation of DNA oligonucleotides used in this study. polyA-TC: oligonucleotide without any secondary structure (single-stranded DNA). NUP93: natural occurring DNA hairpin in the gene NUP93. d Deamination activity on PolyA-TC and NUP93 substrates from indicated cell extracts (30 μg). e A3A and/or A3B knockdown in BICR6 cells. Deamination activity in BICR6 cell extracts (30 μg) was monitor after indicated knockdown on both PolyA-TC and NUP93 substrates. Source data are provided as a Source Data file.

Fig. 3. APOBEC3A promotes hotspot RNA mutations at specific stem-loop structures.

a Comparison of A3A-dominated tumors (APOBEC3A+) and tumors without APOBEC mutations or activity (APOBEC-) revealed sites undergoing frequent APOBEC-dependent C->U RNA editing. The top 50 most frequently edited sites were aggregated into a logogram showing relative frequencies of the bases at each position. b Classification of sites by hairpin characteristics revealed structural preferences of APOBEC-dependent editing. UpC sites exposed in a hairpin loop are mutated at higher frequencies. The position of the U in the loop affects mutability, with the highest RNA editing frequencies observed for C’s positioned at the 3′-most position in a loop of four nucleotides. c Schematic representation of stem-loop structures in the genes DDOST and CYFIP1, the two most highly APOBEC-edited RNA sites in patient tumor samples. d RNA stem-loop editing levels detected in each patient were superimposed in green on the patients from Fig. 1b for whom RNA-sequencing data was available. e RNA stem-loop editing levels correlate strongly with the amount of A3A expression in patient tumors (ρ = +0.72, p = 3 × 10⁻⁵⁵).

Fig. 4. An RNA-based assay to detect APOBEC3A activity in cells.

a Schematic representation of the droplet digital PCR (ddPCR) strategy to detect RNA-editing by A3A. b Scatter plots of wild type (green) or edited (blue) DDOST amplified by ddPCR after expression of wild-type A3A or a catalytically inactive mutant A3A (A3A^E72A) in U2OS cells. c, d U2OS-derived or RPE1-hTERT-derived cell lines inducibly expressing A3A, A3A^E72A, or A3B were induced with doxycycline (DOX) or left uninduced. The level of edited DDOST^558C>U and CYFIP1^3222C>U in U2OS or RPE1-hTERT cells was quantified by ddPCR assay. Error bar: S.D. (n ≥ 3). e HEK-293T cells were transfected with an increasing amount of vector expressing A3A-Flag/GFP. A3A protein levels in HEK-293T cell extracts were analyzed by western Blotting. E.V. empty vector. f Deamination activity on NUP93 and PolyA-TC substrates was measured following incubation with 0.2 μg or 1 μg of HEK-293T cell extracts, respectively, expressing an increasing level of A3A as shown in e. g Quantification of edited DDOST^558C>U by ddPCR assay tracked closely with monitoring of cleavage of NUP93 DNA by Electrophoretic Mobility Shift Assay as shown in f. Error bar: S.D. (n = 3). For the ddPCR quantification, RNAs were purified from the same pool of HEK-293T cells shown in e and f. Source data are provided as a Source Data file.

Fig. 5. Correlation between APOBEC3A expression and RNA editing activity in a panel of cancer cell lines.

a Analysis of A3A and A3B mRNA expression in a panel of cancer cell lines determined by RT-qPCR. Error bar: S.D. (n = 2). b Quantification of DDOST^558C>U and CYFIP1^3222C>U levels in BICR6 cells by ddPCR assay after A3A knowndown. Error bar: S.D. (n ≥ 3). c Quantification of edited DDOST^558C>T and CYFIP1^3222C>T by ddPCR assay. Error bar: S.D. (n = 3). (*p = 0.0352 for DDOST and p = 0.0111 for CYFIP1 by two-tailed t-test) Source data are provided as a Source Data file.

Fig. 6. RNA mutation-based ddPCR assay is the most accurate method to monitor APOBEC3A level in cancer cells.

a–f Level of A3A was monitored by RT-qPCR, ddPCR, and western blot in BICR6 or PC9 cells following treatment with Gemcitabine (0.5 μM) and Interferon-αA/D (750 U ml⁻¹) for 48 h and release into drug-free media for 24 h, 48 h or 72 h. Error bar: S.D. (n ≥ 3). Source data are provided as a Source Data file.

Fig. 7. APOBEC3A activity in a panel blood tumors.

a Levels of edited DDOST^558C>U were quantified using the ddPCR assay in a panel of patient blood tumor samples. For each sample, independent PCR reactions were performed to determine the level of edited DDOST^558C>U. Error bar: S.D. (n ≥ 3) b, c Levels of A3A mRNA and edited DDOST^558C>U were quantified by RT-qPCR and ddPCR respectively in the indicated patients’ blood tumor samples. For each sample, three or more independent PCR reactions were performed to determine the level of A3A mRNA or DDOST^558C>U. Error bar: S.D. Edited DDOST^558C>U level results showed in c were duplicated from the a of the corresponding patient samples. Source data are provided as a Source Data file.