Single and multiple-editing vectors were constructed to target MAPK and PI3K pathways
In order to obtain an effective dual-inhibition of MAPK and PI3K pathways, four important components of MAPK and PI3K pathways (KRAS, MEK1, PIK3CA, and MTOR) were selected for depletion (Figure 1A). The most efficient single guide RNAs (sgRNAs) of CRISPR/SaCas9 system for these four genes were selected by using Benchling tool (https://benchling.com). Moreover, the vectors required for single and multiple depletions were respectively constructed through Golden Gate ligation of corresponding sgRNA cassettes. In addition to single-editing vectors, the multiple-editing vectors included double-depletion of KRAS and MEK1 (KM-KO), and PIK3CA and MTOR (PM-KO), as well as quadruple-depletion of the four genes (KMPM-KO) were constructed (Figure 1B). According to the results of T7E1 assay of HEK293T cells, the vectors of selected single gRNAs (labeled as #1 in Figure 1C-1F) could edit the corresponding gene sites more efficiently compared with those carrying non-overlapping gRNAs targeting the same genes (labeled as #2 in Additional File 3: Figure S2). Moreover, all the vectors with double and quadruple gRNAs could efficiently edit the corresponding targets, with efficiencies ranging from 44.7% to 56.6% (Figure 1G-1I). The expression levels of four gRNAs and the corresponding mutation statuses of KRAS, MEK1, PIK3CA, and MTOR were respectively confirmed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Sanger sequencing in HEK293T cells with quadruple-depletion (KMPM-KO). The abundances of the four gRNAs were almost comparable at the mRNA level, and those could efficiently induce on-target mutations of the corresponding genes (Additional File 4: Figure S3 and Figure 1J). Therefore, the single- and multiple-editing vectors targeting MAPK and PI3K pathways were successfully constructed, and they could be used to investigate the editing effects of dual-pathway on CRC cells.
Quadruple-editing of KRAS, MEK1, PIK3CA, and MTOR efficiently inhibited MAPK and PI3K pathways in CRC cells with oncogenic mutations of KRAS and PIK3CA
About 50% of CRC cells with oncogenic mutation of KRAS simultaneously carried the activating-mutation of PIK3CA . The oncogenic mutations of KRAS (G12D/V/C/S/A/R, G13D/C) and PIK3CA (E545K and H1047R) could induce the aberrant activation of MAPK and PI3K pathways and progression of CRC cells . Thus, we first detected the effects of single- and multiple-editing of KRAS, MEK1, PIK3CA, and MTOR on HCT116 cells with oncogenic mutations of KRAS (G13D) and PIK3CA (H1047R), comparing with the effects of inhibitors of MAPK and PI3K pathways. The half maximal inhibitory concentration (IC50) values of MEK inhibitor (AZD6244) and PI3K/MTOR inhibitor (BEZ235) in HCT116 cells were respectively 1.0 mM and 2.0 mM (Figure 2A-2B). According to the results of Western blotting on HCT116 cells with single- and multiple-editing, the protein levels of the four target genes were significantly down-regulated by the corresponding editing vectors (Figure 2C-2E). Single- and double-depletion of KRAS and MEK1 (KRAS-KO, MEK1-KO, and KM-KO), and the treatment of AZD6244 down-regulated the phosphorylation level of extracellular signal-regulated kinase (ERK), a downstream component of MAPK pathway, while did not affect the phosphorylation level of AKT, an important component of PI3K pathway. The down-regulation of p-ERK by KM-KO was accompanied with effects similar to those of AZD6244 treatment, and was more significantly noticeable than the effects of KRAS-KO and MEK1-KO (Figure 2C). In contrast, single- and double-depletion of PIK3CA and MTOR (PIK3CA-KO, MTOR-KO, and PM-KO), and the treatment of BEZ235 down-regulated the phosphorylation levels of AKT and S6K, two important components of PI3K pathway, whereas did not influence the phosphorylation level of ERK. The down-regulation of p-AKT and p-S6K by PM-KO showed effects similar to those of BEZ235 treatment, and were more markedly noticeable than the effects of PIK3CA-KO and MTOR-KO (Figure 2D). Furthermore, quadruple-depletion of the four target genes (KMPM-KO) significantly down-regulated the phosphorylation levels of ERK, AKT, and S6K, which were similar to the effects of combining treatment of AZD6244 and BEZ235 (Figure 2E). These results revealed that inhibitory effect of gene editing on the corresponding signaling pathway was specific. The inhibitory effects of double-editing were more noticeable than those of single-editing when a pathway was targeted. Quadruple-editing of KRAS, MEK1, PIK3CA, and MTOR could efficiently and specifically inactivate both MAPK and PI3K pathways. Correspondingly, KMPM-KO also significantly inhibited the proliferation, migration, and invasion of HCT116 cells, with a greater inhibitory effect than single- and double-depletion (Figure 2F-2G). Therefore, quadruple-editing of MAPK and PI3K pathways effectively inhibited the malignant phenotypes of CRC cells with oncogenic mutations of KRAS and PIK3CA.
Quadruple-editing of KRAS, MEK1, PIK3CA, and MTOR effectively blocked the compensated PI3K activation in KRAS-mutated CRC cells with MAPK suppression
A previous research showed that RTK-dependent activation of PI3K pathway was a resistant mechanism against the suppression of MAPK pathway in CRC cells with KRAS mutation and wild-type PIK3CA (Figure 1A) . In order to indicate whether the gene editing of MAPK pathway can induce compensated PI3K activation, we first determined the IC50 value of AZD6244 in SW620 cells with KRAS G12V mutation and wild-type PIK3CA. The IC50 value of AZD6244 in SW620 cells was 1.0 mM (Figure 3A). Then, the activation status of MAPK and PI3K pathways in SW620 cells with AZD6244 treatment and single- and double-depletion of MAPK pathway (KRAS-KO, MEK1-KO, and KM-KO) was detected by Western blotting. As shown in Figure 3B and 3C, similar to AZD6244 treatment, the single- and double-depletion significantly reduced the phosphorylation level of ERK and enhanced the phosphorylation level of AKT. The up-regulation of p-AKT by KM-KO was more noticeable than that by KRAS-KO and MEK1-KO (Figure 3C). The above-mentioned findings suggested that the gene editing of MAPK pathway also induced compensated PI3K activation in KRAS-mutated CRC cells. In contrast, single- and double-depletion of PIK3CA and MTOR (PIK3CA-KO, MTOR-KO, and PM-KO) remarkably down-regulated the expression levels of p-AKT and p-S6K, while did not affect the expression level of p-ERK. The down-regulated expression levels of p-AKT and p-S6K by PM-KO were more notable than those by PIK3CA-KO and MTOR-KO (Figure 3D). Furthermore, quadruple-depletion of the four target genes (KMPM-KO) significantly down-regulated the expression levels of p-ERK, p-AKT and p-S6K (Figure 3E). Thus, quadruple-editing of KRAS, MEK1, PIK3CA, and MTOR could efficiently block the compensated activation of PI3K pathway under MAPK suppression. Correspondingly, KMPM-KO also markedly inhibited the proliferation, migration, and invasion of SW620 cells, with a greater inhibitory effect than single- and double-depletion of either MAPK or PI3K pathway (Figure 3F-3G). Therefore, compared with only suppressing MAPK pathway, quadruple-editing of MAPK and PI3K pathways could enhance the anti-tumor effects on KRAS-mutated CRC cells through inhibiting the compensated PI3K activation.
In summary, the tumor-suppressive effects of quadruple-editing of KRAS, MEK1, PIK3CA, and MTOR were superior to those of single- and double-editing of MAPK or PI3K pathway in CRC cells with oncogenic mutations of KRAS and PIK3CA or with KRAS mutation and compensated PI3K activation.
Quadruple-editing inhibited the survival of primary CRC cells with diverse mutations of KRAS and PIK3CA
To further confirm the anti-tumor effects of quadruple-editing on CRC cells, the cells of primary tumors and patient-derived xenografts (PDXs) were isolated. Their mutation statuses at hot spots of KRAS and PIK3CA genes, including exon-2 of KRAS, as well as exon-10 and exon-21 of PIK3CA, were detected by Sanger sequencing. Four primary tumor cells and three PDX tumor cells with different types of KRAS mutations or oncogenic mutations of KRAS/PIK3CA were studied. The pathological and mutation data of the seven CRC cases were summarized in Additional File 1: Table S3. These tumor cells were respectively infected with the lentivirus of quadruple-editing, MEK inhibitor (AZD6244), and PI3K/MTOR inhibitor (BEZ235). The inhibition rates of cell survival under various treatments compared with non-treated controls were presented in Figure 4A-4C and 4E-4H. In all the seven cases, quadruple-editing led to a more remarkable inhibition of cell survival than a single-treatment with AZD6244 or BEZ235, and showed similar or more significant inhibitory effects to those of combined therapies. Furthermore, Western blotting results indicated that quadruple-editing of KRAS, MEK1, PIK3CA, and MTOR could efficiently inactivate MAPK and PI3K pathways in primary CRC cells carrying KRAS/PIK3CA double mutations (CRC-P01) or KRAS single mutation (CRC-PDX01). The inhibitory effects of quadruple-editing were more noticeable than those of single-treatment with AZD6244 and BEZ235, and were similar or a little superior to those of combined therapies (Figure 4D and 4I). The above-mentioned results suggested that quadruple-editing had prevalent anti-tumor effects on KRAS-mutated CRC cells.
The complex combining ADV5 and engineered proteins intravenously delivered CRISPR system to CRC with high efficiency and specificity
The ADV5 is extensively utilized in gene therapy of cancer. However, the current administration of ADV5 is only local injection because of its off-target tissue tropism under a systemic delivery. Thus, two proteins, an adaptor and a protector, were engineered to make ADV5 as a proper vector for the intravenous delivery of CRISPR system to CRC. ADV5 infected host cells through a high-affinity interaction between the knob domain of the viral fiber proteins and coxsackievirus and adenovirus receptor (CXADR) displayed on the target cell surface . Therefore, the adaptor protein was composed of the ectodomain of ADV receptor CXADR of cells (ECXADR), and a humanized single-chain variable fragment (scFv) recognizing a certain surface marker on CRC cells, which could be linked by a phage T4 fibritin polypeptide. Adaptor protein could interact with the knob protein of ADV5 fiber through ECXADR domain, and retarget ADV5 to CRC cells through scFv (Figure 5A) . In addition, a scFv antibody against the hexon protein on ADV5 surface was fused to a phage T4 fibritin polypeptide to form a protector protein, which could cover ADV5 to reduce the tissue off-targeting (Figure 5A) . The fibritin induced the trimerization of adaptor and protector proteins, and up-regulated their affinities with ADV5 (Figure 5A) [19, 20].
EpCAM is a transmembrane glycoprotein mediating Ca2+-independent homotypic cell–cell adhesion in epithelia. It has an extremely high rate of over-expression (≥80%) in CRC cells [23, 24]. The results of RT-qPCR showed that compared with 293T cells, EpCAM was over-expressed in 4 human CRC cell lines, including HCT116, LOVO, SW480, and SW620, and in seven types of CRC cells isolated from primary and PDX tumors (Figure 5B-5C). In contrast, Mucin 1, cell surface associated (MUC1), showed lower expression levels in CRC cell lines and primary tumor cells compared with EpCAM (Figure 5B-5C). Therefore, an adaptor protein targeting EpCAM was constructed to enhance the infection efficiency of ADV5 to CRC cells, and a MUC1 adaptor was also developed as a control to confirm the function of the adaptor protein.
In order to validate the function of adaptor protein in vitro, the green fluorescent protein (GFP)-expressing ADV5 was pre-incubated with various amounts of EpCAM adaptor protein, or a truncated adaptor protein losing an anti-EpCAM scFv fragment and remaining only ECXADR domain as well as fibritin, and then, infected SW620 cells. The fluorescence-activated cell sorting (FACS) analysis revealed that EpCAM adaptor protein gradually up-regulated the infection efficiency of ADV5 in SW620 cells when increasing amount of protein was pre-incubated with ADV5. However, the truncated adaptor protein gradually down-regulated the infection efficiency of ADV5 in SW620 cells when the amount of protein increased (Additional File 5: Figure S4). The results indicated that EpCAM adaptor protein blocked the interaction between ADV5 and its receptor CXADR through ECXADR domain, and retargeted the virus to EpCAM protein on CRC cells by anti-EpCAM scFv. Afterwards, the GFP-expressing ADV5 was respectively pre-incubated with various amounts of EpCAM and MUC1 adaptor proteins, and infected SW620 cells. As displayed in Figure 5D, these two adaptor proteins could dramatically enhance the infection efficiency of ADV5 in SW620 cells. The infection efficiency up-regulated by EpCAM adaptor was more remarkable than that by MUC1 adaptor, which could be correlated with the difference of their expression levels in SW620 cells (Figure 5B). The results indicated that the infection efficiency of the complex combining ADV5 and adaptor depended on expression level of a cell surface marker targeted by adaptor. Since EpCAM was extensively over-expressed in CRC cells, EpCAM adaptor might up-regulate the infection efficiency of ADV5 in the majority of CRC cases.
Furthermore, the functions of engineered proteins were validated in vivo. The ADV5 expressing SaCas9 (Additional File 2: Figure S1B) was intratumorally or intravenously injected into nude mice with SW620 cells-derived xenografts (SW620 CDX model), individually or via combination of EpCAM adaptor and protector proteins. According to the expression levels of SaCas9 in tissues collected 48 h after administration, the intratumorally delivered ADV5 was mainly enriched in tumor tissue. The intravenously delivered ADV5 had diverse tissue tropisms, especially towards liver. Adaptor protein significantly enhanced the tumor tropism of ADV5, and reduced the off-target tropisms towards the majority of organs except for colon where EpCAM was also over-expressed (Figure 5E). The addition of protector protein significantly reduced the off-targeting tropisms in a variety of organs including colon, and enhanced the tumor tropism compared with adaptor only (Figure 5E). Therefore, the combination of adaptor and protector could retarget ADV5 to CRC cells over-expressing EpCAM, and reduce its tissue off-targeting in vivo. The complex combining ADV5 and the two engineered proteins might be a proper vector to deliver CRISPR system intravenously.
Quadruple-editing of KRAS, MEK1, PIK3CA, and MTOR blocked the progression of KRAS-mutated CRC cells in vivo
To evaluate the therapeutic potential of quadruple-editing of MAPK and PI3K pathways mediated by the complex combining ADV5 and engineered proteins, the ADV5 vectors depleting KRAS, MEK1, PIK3CA, and MTOR (KMPM-KO) and non-targeting controls (NT) were pre-incubated with adaptor and protector proteins, and then, were intravenously injected into CDX model of HCT116 (KRAS G13D and PIK3CA H1047R) and PDX model of CRC-PDX01 (KRAS G12V), respectively. The ADV-protein complex (APC) was injected for 2 rounds on Day-0 and Day-12, in which the number of viral particles was 7.0×109 and the amount of protein was 1.0×10-7 pmol. The viral distributions in CDX and PDX mice injected with NT and KMPM-KO were checked on Day-2 post-administration (Figure 6A). According to the expression level of SaCas9 in different tissues, NT and KMPM-KO mainly infected the tumor tissues of CDX and PDX mice, indicating the infection specificity of APC. Similar expression level of SaCas9 in the tumor tissues of mice infected with NT and KMPM-KO indicated equal infection efficiencies of these two types of virus (Figure 6B, 6G). The expression level of SaCas9 in the tumor tissues of CRC-PDX01 mice was higher than that in HCT116-CDX mice, which might result from the higher expression level of EpCAM in CRC-PDX01 than that in HCT116 cells (Figure 5B-5C, 6B, 6G). The tumor growth in CDX and PDX mice that received KMPM-KO was significantly blocked compared with those that were given NT (Figure 6C, 6H). Besides, the final tumor volume in CDX and PDX mice that received KMPM-KO was markedly reduced compared with those that were given NT (Figure 6D, 6I). Furthermore, H&E staining of different tissues in CDX and PDX mice showed that the tumor tissues injected with KMPM-KO had a more significant cell necrosis compared with those that received NT (Figure 6E, 6J). These results strongly suggested that quadruple-editing of MAPK and PI3K pathways blocked the progression of KRAS-mutated CRC cells in vivo. In addition, no injuries were observed by H&E staining in liver, lung, kidney, and colon of CDX and PDX mice that were given NT and KMPM-KO (Figure 6E, 6J). No significant difference was noted in body weight of CDX and PDX mice that received NT and KMPM-KO APCs (Figure 6F, 6K). Therefore, the EpCAM-targeting APC could be a specific and safe vector for intravenous delivery of CRISPR system to CRC.
Quadruple-editing blocked MAPK and PI3K pathways in KRAS-mutated CRC cells in vivo
The results of T7E1 assay in the tumor tissues of CDX and PDX mice injected with NT and KMPM-KO showed that compared with NT samples, the genomic regions of the four target genes, including KRAS, MEK1, PIK3CA, and MTOR, were all efficiently edited in KMPM-KO samples (Figure 7A, 7D). According to the results of immunohistochemistry (IHC) and Western blotting, the expression levels of the four target genes were significantly down-regulated in KMPM-KO samples compared with those in NT samples (Figure 7B-7C, 7E-7F). The Ki67 staining indicated that the proliferation of tumor cells after quadruple-depletion was notably down-regulated compared with that in control cells (Figure 7B, 7E). Furthermore, the phosphorylation levels of components of MAPK and PI3K pathways, such as ERK, AKT, and S6K, were remarkably down-regulated in KMPM-KO samples compared with those in NT samples (Figure 7C, 7F). The above-mentioned findings indicated that quadruple-editing of the four target genes mediated by EpCAM-targeting APC efficiently blocked MAPK and PI3K pathways in KRAS-mutated CRC cells in vivo.
The status of on-target and off-target mutations in tumor tissues induced by quadruple-editing
Whole-exome sequencing (WES) was performed in the tumor tissues of HCT116 CDX mice that received NT and KMPM-KO to detect the mutation status induced by quadruple-editing. The sequencing depth was over 100×. Totally, seven types of on-target deletions were detected at genomic regions of the four target genes (KRAS, MEK1, PIK3CA, and MTOR). The total mutation frequencies of the four target genes were 34.9%, 80%, 40.4%, and 73.3%, respectively (Figure 8A). Among 167 potential off-target loci of these genes predicted by Benchling tool, only one locus of MTOR was detected with a real off-target single-nucleotide variant (SNV) (Chr16, 28603692 C>T), whose mutation frequency was 1.68% (Figure 8B). Furthermore, the total number of variants detected in NT and KMPM-KO samples was almost equal (24420 versus 24539). The number of insertion/deletions (INDELs) and single nucleotide variations (SNVs) detected at different genomic regions was also similar in NT and KMPM-KO samples (Figure 8C-8D). Additionally, the majority of variants detected at different chromosomes were common ones shared by NT and KMPM-KO samples (Figure 8E). These results strongly suggested the minor effect of quadruple-editing on the mutation status of CRC.
Altogether, the quadruple-depletion of KRAS, MEK1, PIK3CA, and MTOR intravenously delivered by the EpCAM-targeting APC blocked MAPK and PI3K pathways and the progression of KRAS-mutated CRC cells with high efficiency and specificity.