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 ΔNp63 and squamous cell transdifferentiation in gefitinib-resistant cells. Knockout of p63 in gefitinib-resistant cells reduces the expression of the ΔNp63 target genes IL1α/β 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 ΔNp63 in gefitinib-resistant lung cancers may have therapeutic benefit.

Keywords: APOBEC; APOBEC3B; lung cancer; mutational signatures; p63; squamous cell; transdifferentiation.

Figure 1.. APOBEC3 activity is induced following EGFR inhibition.

A. qRT-PCR analysis of a representative experiment showing A3A and A3B expression in PC9 cells following treatment with gefitinib or osimertinib for 24 hours. Error bars represent SEM of two biological replicates. Significance relative to the vehicle was determined using unpaired t-test. *** indicates p<0.0005 and **** indicates p<0.0001. B. Western blot for A3B protein in PC9 cells following treatment with gefitinib or osimertinib for 24 hours. BT474 cells are shown as a positive control. Short and long exposures for A3B are shown. C. In vitro deaminase activity of PC9 cells treated with gefitinib or osimertinib for 24 hours. BT474 and SKBR3 cells are shown as positive and negative controls, respectively. % deamination was calculated as described in Methods. D. Quantification of % deamination shown in C. Error bars represent SEM of two technical replicates. Error bars represent SEM of two technical replicates. Significance relative to the vehicle was determined using unpaired t-test. *** indicates p<0.0005. E. Western blot for A3B protein in PC9 cells expressing non-targeting gRNAs (NT) or gRNAs targeting both A3A and A3B (A3A/A3B KO). A pooled population is shown for NT cells, and two clones are shown for A3A/A3B KO cells. Cells were treated with gefitinib or osimertinib for 24 hours. F. In vitro deaminase activity of PC9 cells expressing non-targeting gRNAs (NT) or gRNAs targeting both A3A and A3B (A3A/A3B KO). A pooled population is shown for NT cells, and two clones are shown A3A/A3B KO cells. Cells were treated with gefitinib or osimertinib for 24 hours and % deamination was calculated as described in Methods. G. Quantification of % deamination shown in F. Error bars represent SEM of two technical replicates. Significance relative to the NT vehicle was determined using unpaired t-test. * indicates p<0.05 and ** indicates p<0.005. H. Schematic of competition assay via flow cytometry. PC9 cells expressing non-targeting gRNAs (NT) or gRNAs targeting both A3A and A3B (A3A/A3B KO) were used for the experiment. I. Quantification of % live cells, either GFP-negative or -positive, after 7 or 14 days of EGFR inhibition. A pooled population is shown for NT cells and two clones are shown for A3A/A3B KO cells. Error bars represent SEM of two biological replicates. Unpaired t-test was used to determine statistical significance of GFP-positive cells in treated condition relative to vehicle. * indicates p<0.05, ** indicates p<0.005 and *** indicates p<0.0005.

Figure 2.. Engineering an inducible system for APOBEC3B expression in PC9 cells.

A. qRT-PCR analysis showing A3A and A3B expression in PC9 cells following treatment with gefitinib or osimertinib over the course of 14 days. Error bars represent SEM of three technical replicates. B. Schematic of Cre-inducible APOBEC3B expression. C. qRT-PCR analysis showing A3B expression in PC9 cells following infection with Cre recombinase. Error bars represent SEM of three technical replicates. * indicates p<0.05. BT474 and SKBR3 cells are shown as controls. D. Western blot showing protein expression of HA-tagged A3B in PC9 cells following infection with Cre recombinase. E. In vitro deaminase activity assay in PC9 cells following infection with Cre recombinase. BT474 and SKBR3 cells are shown as controls. % deamination was calculated as described in Methods. F. Quantification of % deamination in two replicates of control PC9 cells (−Cre) and five replicates of A3B-expressing PC9 (+Cre). % deamination was calculated as described in Methods. Unpaired t-test was performed to determine statistical significance. ** indicates p<0.005. G. Growth curves for PC9 cells expressing A3B (+Cre) or control cells (−Cre). Error bars represent SEM of two biological replicates. Two-way ANOVA was performed to determine statistical significance. ns = not significant

Figure 3.. APOBEC3B expression alters the evolutionary trajectory of acquired resistance to EGFR inhibitors.

A. Kinetics of evolution of gefitinib resistance in PC9 cells with or without A3B expression. B. Representative dose-response curves to gefitinib for sensitive and resistant A3B-off and A3B-on PC9 cells. C. Quantification of area under the curve (AUC) of dose-response curves to gefitinib for sensitive and resistant A3B-off and A3B-on PC9 cells. Unpaired t-test was performed to determine statistical significance. ** indicates p<0.05. *** indicates p<0.0005. D. Frequency of EGFR T790M mutation as determined by ddPCR in sensitive and gefitinib resistant A3B-off and A3B-on PC9 cells. % EGFR T790M was calculated as described in Methods. E. Contingency table summarizing the relationship between resistance kinetics and EGFR mutation status in A3B-off and A3B-on PC9 cells.

Figure 4.. Integrated genomic analysis of gefitinib-resistant PC9 cells

A. Quantification of mutations per exome in A3B off and A3B on gefitinib-resistant cells. Mutations were called as described in Methods. B. Principle component analysis from RNA sequencing data. C. Gene set enrichment analysis comparing A3B-on and A3B-off sensitive PC9 cells.

Figure 5.. APOBEC3B-expressing PC9 cells show evidence of squamous cell transdifferentiation during acquired gefitinib resistance.

A. Normalized gene counts for TP63 from RNA sequencing in sensitive and gefitinib-resistant PC9 cells, with or without A3B expression. B. qRT-PCR analysis showing ΔNp63 and total p63 mRNA expression in sensitive and gefitinib-resistant PC9 A3B-on cells. Parental PC9 are shown as a control. Error bars represent SEM of 3 technical replicates. Unpaired t-test was performed to determine statistical significance. *** indicates p<0.0005 and **** indicates p<0.0001. C. Normalized gene counts for MUC5B, IL1A, IL1B, and KRT6A from RNA sequencing in sensitive and gefitinib-resistant PC9 cells expressing A3B. D. Immunohistochemistry images showing ΔNp63 protein expression in sensitive and resistant PC9 A3B-on cells. Insets are shown at 20X magnification. E. Quantification of ΔNp63-positive cells for sensitive and resistant PC9 A3B-on cells shown in D. Each point represents a field of view at 20X magnification. Error bars represent SEM of five fields of view. Unpaired t-test was performed to determine statistical significance. *** indicates p<0.0005 and **** indicates p<0.0001.

Figure 6.. p63 knockout reduces inflammatory gene expression and sensitizes PC9 GR cells to EGFR inhibition.

A. Dose-response curve to osimertinib in sensitive and gefitinib-resistant PC9 cells expressing A3B. Viability wasmeasured using CellTiter Glo following 3 days of drug treatment. Two-way ANOVA with Tukey’s multiple comparisons test was used to determine statistical significance at each dose. **** indicates p<0.0001 when comparing PC9 A3B-on GRF and GRE cells at the indicated dose. B. Western blot for pEGFR (Y1068) and total EGFR in sensitive and gefitinib-resistant PC9 cells expressing A3B.Cells were treated with osimertinib for 24 hours. C. Western blot for p63α in PC9 A3B-on GRF cells expressing an empty vector or gRNAs targeting p63 (sg1 and sg3). PC9 A3B-on F cells are shown as a control. D. qRT-PCR analysis showing IL1A and IL1B expression in PC9 A3B-on GRF cells expressing an empty vector orgRNAs targeting p63 (sg1 and sg3). PC9 A3B-on F cells are shown as a control. Error bars represent SEM of three technical replicates. Unpaired t-test was performed to determine statistical significance. ** inidicates p<0.005 and **** indicates p<0.0001. E. In vitro growth curves of PC9 A3B-on GRF cells with or without p63 knockout. Two-way ANOVA was performed todetermine statistical significance. ns = not significant. F. Dose-response curve to osimertinib in PC9 A3B-on GRF cells expressing an empty vector or gRNAs targeting p63(sg1 and sg3). Viability was measured using CellTiter Glo following 3 days of drug treatment. Two-way ANOVA was performed and determined a significant p63 status x dose interaction with a p value of 0.02. G. Viability of PC9 A3B-on GRF cells expressing an empty vector or gRNAs targeting p63 (sg1 and sg3) followingtreatment with osimertinib for 6 days. A two-way ANOVA with Dunnett’s test was performed to determine statistical significance. **** indicates p<0.00001.