Construction of 74,389 EGFR Variants
EGFR pathogenic variants, including substitutions, deletions, and exon 20 insertions [29], are mainly located in exons 18–21 (residues 688–875). To cover all the major types of mutations, two EGFR libraries were constructed using the MITE method [11]. Parallel synthesized oligonucleotides were used to construct a saturation EGFR Sub-Del library containing 3,572 (theoretical number) single AA substitution (Sub) variants, 188 (theoretical number) single AA deletion (Del-1) variants, and 182 (theoretical number, for cloning reason, six deletion variants were not covered) two consecutive AA deletion (Del-2) variants. All Sub-Del library variants were located at AA positions 688–875. Similarly, multiple degenerate primers were used to construct a saturation EGFR Ins library containing 126,300 (theoretical number) in-frame insertion variants. All Ins library variants were located in the EGFR insertion hotspot (AA position, 762–776) and consisted of one, two, or three tandem NNS codons (N = adenine, cytosine, guanine, or thymine; S = cytosine or guanine) inserted between adjacent AAs, with each variant containing one insertion in each of the AA positions. The Ins library was composed of three subtype libraries: Ins-1 library (300 variants, each with a single-AA inserted), Ins-2 library (6,000 variants, each with a double-AA inserted), and Ins-3 library (120,000 variants, each with a triple-AA inserted). Variants of Sub-Del and Ins libraries were cloned into lentiviral vectors, and the uniformity and completeness of each library were determined by high-throughput sequencing of the plasmids. The Sub-Del library showed decent uniformity (Additional file 2: Figure S2a), and 99.3% of the designed variants were detected more than 20 times (>20 reads) (Additional file 3: Figure S3a). Similarly, variants of the Ins library also showed high uniformity (Additional file 2: Figure S2b). Because the Ins library was derived from mixing the Ins-1, Ins-2, and Ins-3 libraries at a mass ratio of 1:5:5, the ratio of the average proportion of each variant from Ins-1, Ins-2, and Ins-3 libraries was 80:20:1. Not surprisingly, most of the designed variants in the Ins-1 (99.7%) and Ins-2 (93.6%) libraries were detected (>20 reads for each variant). However, only 53.8% of the designed Ins-3 library variants were detected (>20 reads for each variant) (Additional file 3: Figure S3b). Overall, the coverage and uniformity of Sub-Del and Ins libraries were as expected, and the total number of detected variants was 74,389.
Deletion of Endogenous EGFR in the PC-9 Cell Model
The human lung adenocarcinoma cell line, PC9 (contains EGFR exon 19 deletions), was chosen as the model. The cell line identity was confirmed by STR profiling and genomic mutations were further reviewed through next-generation sequencing. A Glu746–Ala750 deletion was confirmed in EGFR exon 19 and a p.Arg248Gln mutation was detected in TP53. The p.Arg248Gln variant is a gain-of-function mutation that promotes tumorigenesis [30]. These results indicate that the PC9 cell line is a genetically clear cell model for evaluating the sensitivity of EGFR variants to TKIs.
To eliminate the endogenous EGFR, we separately introduced Cas9 and the sgRNA-EGFR variant (codon-optimized) into PC9 cells using two lentiviral vectors. Firstly, Cas9 was introduced into PC9 cells to obtain the PC9-Cas9 cell line, stably expressing Cas9. Subsequently, the sgRNA-EGFR variant was introduced into the PC9-Cas9 cells. The endogenous EGFR was knocked out using the CRISPR/Cas9 system and replaced with the exogenous EGFR variant in approximately 10 days. Because direct knockout of EGFR may seriously affect the viability of PC9 cells [31], we tested the CRISPR/Cas9 gene-editing efficiency in A549 cells (with a wildtype EGFR and mutated KRAS background). Considering that KRAS locates downstream of the EGFR signaling pathway, EGFR can be knocked out without seriously affecting the viability of A549 cells. FACS and western blotting analyses confirmed that the endogenous EGFR had been efficiently knocked out by CRISPR/Cas9 (Additional file 4-5, 11: Figure S4-S5, Table S2). We predicted that these validated CRISPR/Cas9 sgRNAs would also efficiently knockout the endogenous EGFR in PC9-Cas9 cells.
EGFR-TKI Cytotoxicity Screening
To systematically evaluate the sensitivity of EGFR variants to different TKIs, we separately introduced Sub-Del and Ins variants into the PC9-Cas9 cells and treated them with TKIs. Expression of exogenous EGFR variants was checked through FACS analysis and found to be slightly higher than that of EGFR in PC9 cells (Additional file 6: Figure S6). The concentrations of EGFR-TKIs were set mainly referring to the clinical plasma concentration of each TKI (Additional file 12: Table S3). Specifically, the concentrations of reversible TKIs (erlotinib, gefitinib, and icotinib) were set to 600 nM, whereas those of irreversible TKIs were set at two levels. The afatinib clinical plasma concentration is much lower than 600 nM [32]; we designed two assays for 50 and 600 nM afatinib. Because incubation with 600 nM osimertinib showed strong cytotoxicity to cells with Sub-Del variants in vitro, we designed two assays for 200 and 600 nM osimertinib. The TKI treatments lasted 2 weeks and cells were counted every 2 days. The number of cells containing the Sub-Del variants in the erlotinib, gefitinib, icotinib, afatinib (50 nM), and osimertinib (200 nM) assays decreased substantially at days 5 and 7, reached the minimum at day 9, and then began to increase. The cell numbers in the afatinib (600 nM) and osimertinib (600 nM) assays reached the minimum at days 11 and 15, respectively (Fig. 2a). For cells containing the Ins variants, the minimum cell number in the afatinib (600 nM) and osimertinib (600 nM) assays was reached at days 9 and 13, respectively, whereas the cell number in other TKI assays was not decreased during 2-week of TKI treatment (Fig. 2b).
Osimertinib Inhibited Almost all Types of Variants
To investigate the enrichment of variants after incubation with TKIs, we harvested cells from each TKI assay for gDNA extraction, mutation region amplification, and high-throughput sequencing. The relative drug sensitivity for each variant was defined as the fold change of a variant’s theoretical cell number post-TKI screening (time 1) to that at the early time point (time 0). The theoretical cell number for each variant was calculated using the read proportion of a variant among the total valid reads multiplied by the final cell number in each assay. Compared with reversible TKIs (erlotinib, gefitinib, and icotinib), irreversible TKIs (afatinib 50 nM and osimertinib 200 nM) showed stronger inhibition for Sub-Del variants (enrichment score mean: afatinib, 0.331; erlotinib, 0.793; gefitinib, 1.73; icotinib, 4.44; osimertinib, 0.388; false discovery rate (FDR)-adjusted P values <0.0001) (Fig. 2c). For the Ins variants, osimertinib (200 nM) had a greater inhibition than the other TKIs (enrichment score mean: afatinib, 113; erlotinib, 89.3; gefitinib, 111; icotinib, 176; osimertinib, 4.46; FDR-adjusted P values <0.0001) (Fig. 2d). Overall, these results indicated that the Ins library variants were more resistant to TKI treatment than the Sub-Del library variants, and osimertinib (200 nM) showed stronger inhibition for variants in both the libraries.
To investigate whether distinct EGFR variant subtypes present different sensitivity against the same EGFR-TKI, we separately analyzed relative drug sensitivity of the Sub, Del-1, and Del-2 variants in the Sub-Del library and the Ins-1, Ins-2, and Ins-3 variants in the Ins library. No significant difference in the inhibition of Sub, Del-1, and Del-2 variants to any of the five TKIs was observed (Fig. 2e, FDR-adjusted P values >0.05). In contrast, the sensitivity of Ins-1, Ins-2, and Ins-3 variants to each of the five TKIs was significantly different (Fig. 2f, FDR-adjusted P values < 0.05). However, the difference in enrichment scores (log2) cannot be entirely attributed to TKI treatment because differences were also observed for Ins-1, Ins-2, and Ins-3 variants in TKI-free assay (control).
Cytotoxicity Screening Results were Consistent with Known Clinical Annotations
Next, we evaluated the consistency of TKI screening results with known clinical annotations. For this, we compiled a list of EGFR mutations from the Clinical Interpretation of Variants in Cancer (CIViC) knowledgebase and compared the clinical annotations (resistant or sensitive) of mutations with the enrichment scores (log2) obtained in cytotoxicity assays by plotting those mutations on a scale bar comprising all the results (Additional file 7: Figure S7). TKI-sensitive variants (such as p.Leu858Arg and p.Gly719Ala/Ser) were located in the middle or lower half of the bar, whereas TKI-resistant variants (such as p.Thr790Met) were located in the middle or upper half of the bar (Additional file 7: Figure 7a). Similarly, the annotated Ins variants were distributed at expected positions in the bars (Additional file 7: Figure 7b). However, some annotated variants, such as p.Cys797Ser (0.53) and p.Ser768Ile (1.19), had contradictory enrichment scores in the osimertinib assay. These discrepancies could be attributed to the presence of additional mutations. For example, EGFR mutation combinations in cis (on the same allele) or trans (on different alleles) might lead to altered drug response outcomes compared with those for independent mutations [33-36]. Indeed, p.Cys797Ser has been detected as a secondary mutation coexisting with p.Thr790Met in cis and results in resistance to osimertinib [33-35]. In addition, another variant, p.Gly724Ser, has been associated with increased sensitivity to osimertinib when combined in trans with p.Leu858Arg than with exon 19 deletions [36]. In general, our TKI sensitivity evaluation results are consistent with the known annotation.
Cytotoxicity Screening Identifies Potentially Drug Resistant Variants
The variant enrichment scores (log2) in each TKI assay for each variant were presented as heatmaps (Fig. 3, 4, Additional file 8-9: Figure S8-S9). For Sub-Del variants, 280 (7.2%) variants in afatinib assay (50 nM), 841 (21.5%) variants in erlotinib assay, 1,802 (46.2%) variants in gefitinib assay, 3,440 (88.0%) variants in icotinib assay, and 271 (7.2%) variants in osimertinib assay (200 nM) of all variants had a positive enrichment score (log2), indicating that the number of those variants were increased after 2 weeks of TKI incubation. We divided all substitution variants into two categories, the complex and simple variants. The complex variants can only be achieved through two or three point mutations in the corresponding codon whereas the simple variants can be obtained through one point mutation. Under normal circumstances, the probability of multiple independent point mutations occurring in the same codon is relatively low, and therefore, simple variants are more likely to be encountered in the clinic. Enriched variants from all three reversible TKI assays have 813 variants (complex variants = 571, simple variants = 242) in common (Fig. 5 a, c, e). The variants enriched in the two irreversible TKI assays have 51 variants (complex variants = 36, simple variants =15) in common (Fig. 5 b, d, f). Overall, in our systematic cytotoxicity screening, we found 813 and 51 substitution variants that potentially mediate resistance to EGFR reversible and irreversible TKIs, respectively.
The Top 5% Enriched Insertions Showed Insertional Position and AA Type Preference
For Ins variants, the proportion of variants with positive enrichment scores (log2) was much larger than of their Sub-Del counterparts: 95% for afatinib (50 nM) assay, 93% for erlotinib assay, 94% for gefitinib assay, 97% for icotinib assay, and 57% for osimertinib (200 nM) assay. To study whether the insertional position and the AA type affect the sensitivity of exon 20 insertions to different TKIs, we selected the top 5% enriched insertions from each TKI assay and determined the cumulative frequency of the inserted AA at each AA position. The top enriched exon 20 insertions shared many common features (Fig. 6 a-f): (1) the highly enriched insertion variants showed preference for insertion at AA positions 770–774; (2) the highly enriched insertion variants showed preference of glycine and serine. To evaluate the sensitivity of the clinically detected exon 20 insertions to TKIs, we determined the cumulative frequency of insertional AA in variants from the Chinese population [37] and COSMIC database (Fig. 6 g-i). The detected rare exon 20 insertions were mainly concentrated within AA positions 770–774. Our results explain the resistance of these clinically identified exon 20 insertions to TKIs. Together, these results reveal that insertion in the EGFR AA positions 770–774 might induce a stronger resistance to TKIs, especially when the insertions contain glycine or serine.