APOBEC3 activity promotes the survival and evolution of drug-tolerant persister cells during acquired resistance to EGFR inhibitors in lung cancer

Abstract

APOBEC mutagenesis is one of the most common endogenous sources of mutations in human cancer and is a major source of genetic intratumor heterogeneity. High levels of APOBEC mutagenesis are associated with poor prognosis and aggressive disease across diverse cancers, but the mechanistic and functional impacts of APOBEC mutagenesis on tumor evolution and therapy resistance remain relatively unexplored. To address this, we investigated the contribution of APOBEC mutagenesis to acquired therapy resistance in a model of EGFR-mutant non-small cell lung cancer. We find that inhibition of EGFR in lung cancer cells leads to a rapid and pronounced induction of APOBEC3 expression and activity. Functionally, APOBEC expression promotes the survival of drug-tolerant persister cells (DTPs) following EGFR inhibition. Constitutive expression of APOBEC3B alters the evolutionary trajectory of acquired resistance to the EGFR inhibitor gefitinib, making it more likely that resistance arises through de novo acquisition of the T790M gatekeeper mutation and squamous transdifferentiation during the DTP state. APOBEC3B expression is associated with increased expression of the squamous cell transcription factor deltaNp63 and squamous cell transdifferentiation in gefitinib-resistant cells. Knockout of deltaNp63 in gefitinib- resistant cells reduces the expression of the p63 target genes IL1alpha/beta and sensitizes these cells to the third- generation EGFR inhibitor osimertinib. These results suggest that APOBEC activity promotes acquired resistance by facilitating evolution and transdifferentiation in DTPs, and suggest that approaches to target deltaNp63 in gefitinib-resistant lung cancers may have therapeutic benefit.

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

“…Our work expands upon prior studies suggesting a potential association between APOBEC-mediated mutagenesis and acquisition of putative resistance mutations in the APOBEC-preferred context during the treatment of EGFR-driven lung cancers 74,75 . Our data suggest that inhibition of APOBEC3 family members could suppress the emergence of one pathway to resistance and thereby improve response to targeted therapy, consistent with the work of others in the field that suggests that multiple APOBEC3 family members including A3B contribute to targeted therapy resistance 5,32 , with both A3A and A3B shown to be contributors of mutagenesis 6,32,36,76 . The role of A3B in promoting resistance to TKI is likely multifaceted, and our data do not discount the contribution of other possible parallel cytosine deaminase-independent mechanisms, such as induced CIN 4,77 , regulation of cell cycle 22 and regulation of the DNA damage repair pathway 78,79 .…”

Induction of Multiple Alternative Mitogenic Signaling Pathways Accompanies the Emergence of Drug-Tolerant Cancer Cells

“…Given the observed high levels of reactive oxygen species (ROS) in persister cells, we further explored the expression patterns of these genes. Moreover, given the observed elevation of repair enzymes and error-prone DNA polymerases in persister cells [ 24 , 25 , 26 , 27 ], we included an examination of these enzymes in our analysis. Notably, among the scrutinized genes, GAS6, GSTA4, and GSTM3 exhibited significant up-regulation in both lung and melanoma persister cells (refer to Table 1 ).…”

APOBEC Mutagenesis in Cancer Development and Susceptibility

“…However, the precise contributions of APOBEC deaminases to cancer evolution remains less well understood. While APOBEC enzymes may contribute to cancer evolution through non-mutagenic mechanisms [25,36,37], mutagenesis by these enzymes appears to have a more widespread impact on cancer [20,[38][39][40][41][42][43][44]. APOBEC mutagenesis endures in vitro in human cancer cell lines [45], and its signatures often appear in the subclonal phylogenetic branches of primary tumors and metastatic cancers, with incidental observations of driver mutations in APOBEC-associated sequence contexts [46][47][48][49][50][51][52].…”

“…APOBEC mutagenesis endures in vitro in human cancer cell lines [45], and its signatures often appear in the subclonal phylogenetic branches of primary tumors and metastatic cancers, with incidental observations of driver mutations in APOBEC-associated sequence contexts [46][47][48][49][50][51][52]. APOBEC3A and APOBEC3B have been linked to persistent cell evolution and therapy resistance in lung cancers [41,43,44], and APOBEC3B has been linked to resistance against androgen receptor (AR)-targeted therapy and Tamoxifen in prostate and estrogen receptor-positive (ER+) breast cancers [40,53]. Furthermore, in vivo studies suggest that APOBEC mutagenesis can promote tumor heterogeneity [20,[54][55][56].…”