The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.</p

Roles of the APOBECs in the generation of cellular heterogeneity

AID/APOBEC proteins are able to deaminate cytosines in the context of single strand nuclei acids. Their physiological role is involved in innate and adaptive immune system. However, their mutagenic activity is responsible for somatic mutations and chromosomal alterations involved in human cancers. Recent studies have reported that APOBEC3 proteins can target not only chromosomal DNA, but also RNA and extrachromosomal circular DNAs (eccDNA). However, the impact of the significance of these two APOBEC-mediated processes in the context of cancer were not explored yet. In addition, the mechanisms that regulate the expression of APOBEC3 genes and APOBEC-mediated mutagenesis are not fully understood. In the first part of these thesis, I investigate the role of AID/APOBECs in the circularization of DNA fragments DNAs by assaying their effect on CRISPR-C system, a cellular model for extrachromosomal circular DNA formation. Here I show evidence that APOBEC-mediated deamination is necessary for DNA circularization. In addition, I have characterized eccDNAs from normal and cancer cells using Nanopore technology and showed that in presence of APOBEC3 proteins, eccDNAs derive mainly from genomic regions target by the APOBECs. We are currently investigating the molecular mechanisms through which these deaminases affect eccDNA biogenesis, either by facilitating circularisation of the eccDNA or increasing their stability Taking advantage of the CRISPR-C system I investigated the molecular mechanisms involved in eccDNA formation and clearance. I applied a genome wide CRISPR screening to identify genes that may modulate the circularisation of DNA fragments after the introduction of DNA cleavage. I identified several genes whose absence causes high levels of CRISPR-induced eccDNA. To validate the results from our CRISPR library screening, I generated HEK293T single knockout clones which have shown a similar increase in eccDNA levels. I am currently investigating how these genes mediate circularization of DNA fragments and whether the same genes also affect the level of endogenous eccDNA in the context of cancer. 7 As eccDNA affects the evolution of cancer, investigating circularisation of linear chromosomal DNA is a key step to understand how eccDNAs can affect cancer progression and whether those factors/processes involved in its biogenesis can be used as a biomarker. In the second part of this thesis, I have investigated the mechanism that triggers APOBEC3 genes expression in the context of bladder. Here, I show that metabolic activation of Benzo[a]pyrene through CYP450 enzymes and following DNA damage result to induction of APOBEC3A and APOBEC3B genes. Conversely, enzymatic inhibition of CYP450s abolished the induction of APOBEC3A and APOBEC3B. In the final part of this thesis, I have investigated the role of APOBEC3A-mediated RNA editing of DDOST in the cellular biology. Here I show that editing of DDOST mRNA results in a lower amount of protein. Based on this data we hypothesized that APOBEC3A may be able to modulate N-glycosylation in in processes in which a rapid shutdown of the ER might be beneficial

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy