Journal of Carcinogenesis & Mutagenesis

Presumptive Evidence for ADAR1 A-to-I Deamination at WA-sites as the Mutagenic Genomic Driver in Hepatocellular and Related ADAR1-Hi Cancers

Robyn A. Lindley^1,2 and Edward J. Steele^{3,4* 1}Department of Clinical Pathology Faculty of Medicine, Dentistry & Health Sciences, University of Melbourne, Australia
²Scientific Officer and Director of GMDx Group Ltd, Melbourne, Australia
³Centre for Astrobiology, University of Ruhuna, Matara, Sri Lanka
⁴C Y O’Connor, ERADE Village, Foundation, Piara Waters, Perth 6112 WA, Australia
^*Correspondence: Edward J. Steele, Centre for Astrobiology, University of Ruhuna, Matara, Sri Lanka, Tel: +61 (0) 420 863 551, Email:

Received: 10-Nov-2020 Published: 30-Nov-2020, DOI: 10.35248/2157-2518.20.S14.002

Introduction

For a number of years we have been marshalling evidence in support of the reverse transcriptase mechanism of somatic hypermutation (SHM) in rearranged immunoglobulin (Ig) genes [1-4] and in non-Ig genes undergoing “dysregulated” SHM Ig-like responses in non-lymphoid cancer genomes [3,4-9]. The most compelling data from cancer genomes are from those mutagenic processes targeting the DNA binding region of the TP53 gene in a variety of common cancers including those of breast, lung, bladder, ovary curated in the IARC TP53 database [6,7].

The main components of the reverse transcriptase mechanism following initiation by AID of cytosine to uracil (C-to-U) DNA deamination lesions in genomic DNA [10,11] are:

1). DNA polymerase- ƞ is the sole error-prone DNA polymerase targeting A:T base pairs at WA-sites during SHM in vivo [12,13] and also an efficient cellular reverse transcriptase (RT) together with potential back-up RT activity from Y family relative DNA polymerase-ҡ[1,4,14,15].

ii). The signature for ADAR deaminase A-to-I(G) by the RNA editor ADAR1, also targeting WA-sites, where the edits appear in pre-mRNA co-transcriptionally [16,17]. Indeed, the analyses show that ADAR1 introduces A-to-I(G) modifications in the nascent pre-mRNA in post-transcriptional DNA [2,6]. Also recently, such editing events have been identified in the RNA and DNA moieties of RNA: DNA hybrids in vitro [18], implying that adenines in both RNA and DNA moieties of RNA:DNA hybrids are A-to-I (G) edited at stalled transcription bubbles in vivo [3].

These findings together infer a transcription-linked model and evoking a RNA to DNA copying process back into genomic DNA. That is, involving an implied mRNA intermediary by a reverse transcription (RT) step at the genomic target site.

Consistent this conclusion it has been found that amongst several strand-biased in vivo mutation signatures observed in Ig SHM [19] and in Ig SHM-like patterns in cancer genomes [5-7] the somatic mutations are found at WA motifs. These are always strand biased with A-to-G mutations exceeding T-to-C mutations by 2 to 3 fold (mutations read by convention 5’ to 3’ on the nontranscribed strand). A-to-C and A-to-T transversion mutations also exceed their T-to-G and T-to-A Watson-Crick pyrimidine complements. More recently, genome-wide analyses have also confirmed wide-spread WA-site/DNA Polymerase-ƞ associated mutation signatures in pan-cancer genomes [20,21].

We ask: Does evidence already exist for a strong signal of ADAR1 A-to-I(G) RNA editing in the DNA of ADAR1 overexpressing hepatocellular carcinomas? In these (and some related) cancers it has been established that ADAR1 overexpression is the cause of significantly elevated A-to-I RNA editing and ADAR1 acts as an oncogene promoting cancer progression causing increased risk of liver cirrhosis and postoperative recurrence with poor prognoses [22]. The same appears to be happening in gastric cancer [23]. The cause of this over expression could be ADAR1 gene amplification at 1q21 as demonstrated in other cancers [24].

A striking feature of genome-wide sequencing of cancer genomes [25] are the single base substitution (SBS) hepatocellular mutations annotated as signatures 12 and 16 which exhibit an extremely strong transcriptional strand bias with A-to-G mutations greatly exceeding T-to-C mutations mainly at WA motifs, about 5 fold higher in the case of signature 16 (see the updates in [26] and at the COSMIC Note that SBS signatures 12 and 16 have also been identified in some other cancers (Kidney-RCC, Biliary-AdenoCA, Prost-AdenoCA, Head -SCC). A similar A:T focused yet now reversed orientation strand-biased signature in SBS 21 is evident in stomach cancers [25], suggesting to us the interpretation [3] that A-to-I deamination has occurred directly on the transcribed DNA strand at the annealed RNA:DNA hybrid in these cancers. However with respect to the striking adenine-targeted strand bias on the nontranscribed strand in HCC Alexandrov et al [25] discuss the striking strand biases as follows:

“On the assumption that the transcriptional strand biases in signatures 12 and 16 are introduced by transcription-coupled NER, these currently unexplained signatures may be the result of bulky DNA helix-distorting adducts on adenine. However, there is no previous basis for invoking transcription-coupled NER in the genesis of these signatures and other causes of transcriptional strand bias may exist”.

This is a key qualification. The formal explanations at the COSMIC website are (author addition and emphasis in italics):

• For signature 12 (SBS12): “ Transcriptional strand-bias with more mutations of A than T on the untranscribed strands of genes consistent with damage to adenine and repair by transcription-coupled nucleotide excision repair” [27] and

• For signature 16 (SBS16): “ Extremely strong transcriptional strand bias with more mutations of A than T (by five fold and mainly A-to-G exceeding T-to-C) on the untranscribed strands of genes consistent with damage to adenine and differential repair of the transcribed strand by transcription coupled nucleotide excision repair [27] . There is also evidence for SBS16 of transcription coupled DNA damage, with more damage to A on untranscribed than on transcribed strands (in addition to transcription coupled nucleotide excision repair).” The latter conclusion is to a type of mechanism we infer in the later work of Haradhvala et al., [28].

It needs to be noted here that there is a preference in both signatures for A-to-G mutations to occur at WA-sites where the 5’ nucleotide is A or T i.e. consistent with the well-known ADAR signature motif WA, where W=T or A).

Thus, Haradhvala et al., have asserted that adenines are somehow specifically targeted for damage during transcription by a process that they refer to as “Transcriptional Coupled Damage” (TCD) [28]. They elucidate further by adding “we propose that this is due to two separate processes operating on different strands-TCD and TCR (transcription-coupled repair). This explains the extreme transcriptional asymmetry of liver A/G compared to other signatures these results suggest that the A/G signature is caused by a mutational process distinct from typical bulky-adduct damage. Finally, we noticed that one colorectal patient (‘‘CRC-8’’) from an earlier study of nine colorectal whole genomes showed the same signature of TCD. Thus, this phenomenon may be enriched in liver but not exclusive to it.”

Thus, their explanation is that the extreme bias of A-to-G over Tto- C mutations in hepatocellular cancer is caused by some unspecified damage to adenine on the non-transcribed strand (NTS) coupled to transcription itself. This targeting is over and above any differential Transcription Coupled Repair that preferentially clears bulky adducts [27] of the template or transcribed strand (TS) leaving an excess of unrepaired A-to-G mutations on the displaced coding or NTS. They call this Transcription Coupled Damage (restricted to adenines) but suggest no plausible molecular mechanism to explain the data as it is assumed to be a part of the TCD signature.

We find that our own data analyses are consistent with the Alexandrov et al., and Haradhvala et al., empirical observations [25,28]. However, we now offer a molecular explanation for the strong strand biases reported. From our perspective, the strand biased signatures at WA-sites, where mutations of A-to-G far exceed mutations of T-to-C by some factors of 4-fold or more, are expected and readily explained by the ADAR1/Pol-ƞ coupled target-site reverse transcriptase (TSRT) mechanism premised on a DNA-to-RNA-to-DNA transcription linked process [1-4]. Given that several other cancers also display ADAR1 over-expression e.g. gastric cancers [23] and lung adenocarcinomas [24, 29], this new interpretation of the causative molecular mechanisms in ADAR1-over expressing cancers is worth investigating from the point of view of therapeutic targeting of the presumptive causative deaminase, ADAR1. Our explanation is also consistent in part with the recent work by Tasakis et al., for ADAR1 driving mutagenic progression in multiple myeloma at larger R-loops across the cancer genome [30]. In this case the A-to-I(G) editing on the transcript appears also to generate A-to-I (G) mutations in the transcribed (-) DNA strand, identifying as T-to-C mutations on the reference (+) strand [28].

Conclusion

In conclusion we believe that our interpretation is also consistent with, and is an extension of, the DNA damage and repair mechanisms proposed to explain the observed mutational strand asymmetries of APOBEC signatures in cancer genomes.

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