NEK7-induced phosphorylation of EGFR on serine 1070 drives the acquired lenvatinib resistance in hepatocellular carcinoma

Abstract

Background and Aims:

Lenvatinib is recognized as a first-line therapy for inoperable hepatocellular carcinoma (HCC) patients. Growing evidence indicates that lenvatinib resistance can be acquired in HCC cells via kinase rewiring.

Approach and Results:

We established acquired lenvatinib-resistant organoids and HCC cell lines. NIMA-related coiled-coil kinase 7 (NEK7) was identified as an HCC lenvatinib acquired resistance gene by kinase CRISPR–Cas9 genetic screen. Functional analyses demonstrate that NEK7 enhanced lenvatinib resistance in HCC, and NEK7 knockdown or knockout displays the antitumor effects in acquired lenvatinib HCC cells and organoids. Mechanistically, NEK7 binds to the endothelial growth factor receptor (EGFR), leading to the phosphorylation of EGFR specifically at the serine 1070 residue, which contributes to the activation of MAPK (mitogen-activated protein kinase) and PI3K/AKT (phosphoinositide 3-kinase/Akt) signaling pathways. Consistently, designed inhibitory peptides targeting the domain from amino acids 979 to 1099 were proven to inhibit phosphorylation of EGFR S1070 site and therapeutically inhibit antitumor activity of acquired lenvatinib resistance HCC.

Conclusions:

Our results unveil insights into the acquired lenvatinib resistance mechanism that NEK7 phosphorylates EGFR at S1070 to promote acquired lenvatinib resistance in HCC.

INTRODUCTION

Hepatocellular carcinoma (HCC), a prevalent form of liver cancer which is known as the most lethal disease, as it is the sixth frequently occurring cancer and the primary contributor to cancer-related deaths, placing it fourth in mortality statistics in the world.¹ Clinically, HCC patients are often diagnosed at an advanced stage, and a limited array of therapeutic choices is available.² The new advancement in the fields of molecular targeted therapy has emerged, promising development in advanced stages of HCC, including sorafenib, lenvatinib, and PD-1/PD-L1 therapies.^3–5 Following sorafenib,⁶ lenvatinib, the multitargeted tyrosine kinase inhibitor (TKI) which selectively inhibits vascular endothelial growth factor receptor (VEGFR) 1/2/3, fibroblast growth factor receptor (FGFR) 1/2/3/4, platelet-derived growth factor receptor (PDGFR) α, RET, and KIT has been approved by the FDA as the first-line treatment for unresectable advanced HCC patients.^7–9 Furthermore, in lenvatinib-resistant patients, after the initial response to lenvatinib, the continuous presence of lenvatinib exerts a selective pressure on HCC cells, leading to recurrence of HCC.^7,10–13 Consequently, there is an urgent demand for research into the mechanisms underlying the acquired lenvatinib resistance.

Lenvatinib resistance can be acquired in HCC cells via kinase rewiring to compensate for the disrupted signals caused by TKI.^13–15 Consequently, pinpointing the key kinases involved in the reorganization of signaling pathways in acquired lenvatinib resistance is crucial. However, there are challenges in measuring the protein kinase level from a dynamic range, and the drug-resistance models at present are not yet fully capable of representing the proteomics of the human drug-resistance mechanism.^16,17 Patient-derived organoids (PDOs) are an emerging significant preclinical model that preserves the genetic and morphological diversity of tumors,^18,19 and offers exceptional utility in tumorigenesis,²⁰ resistance to drugs,²¹ screening novel pharmaceuticals,²² tailored treatment approaches,²³ and various other research areas.²⁴ Therefore, we established acquired lenvatinib resistance patient-derived organoids-based xenograft (ALR PDOX) via xenotransplantation and maintained continuous treatment with lenvatinib. And we also established the lenvatinib resistance (LR) Hep3B and Huh7 cell models. Loss-of-function screens using an sgRNA library targeting about 482 kinase genes were applied to acquired lenvatinib resistance organoids. NIMA-related coiled-coil kinase 7 (NEK7) was identified as synthetic lethal with lenvatinib selection in vivo and vitro. NEK7 regulates a variety of biological processes, including DNA damage response, telomere integrity,²⁵ mitosis,²⁶ it serves as an essential component of the NLRP3 inflammasome, playing a crucial role in its activation.²⁷ As a serine/threonine kinase, NEK7 exhibits high mRNA and protein levels in breast cancer, colorectal cancer, and lung cancer.²⁸

In this work, we determine that NEK7 promotes the acquired lenvatinib resistance process via directly binding to EGFR and phosphorylating EGFR at serine 1070 residue, leading to the recruitment of the adaptor protein GRB2 (growth factor receptor-bound protein 2) and P85 (phosphoinositide 3-kinase regulatory subunit p85), which activate MAPK (mitogen-activated protein kinase) and PI3K/AKT (phosphoinositide 3-kinase/Akt) signaling. Furthermore, peptides that bind to the EGFR c-tail (979–1099) residue disrupt the NEK7 phosphorylation at EGFR serine 1070, which has been demonstrated to enhance the sensitivity to lenvatinib in animal models and HCC acquired lenvatinib resistance organoids. Collectively, this work uncovers the reconfiguration of kinase networks and identifies novel EGFR phosphorylation sites, shedding light on the mechanisms behind acquired resistance to lenvatinib therapy in HCC.

METHODS

Lenvatinib resistance in the HCC cell model

Huh7 and Hep3B cells were cultured with increasing doses of lenvatinib (Selleck) in varying concentrations of lenvatinib from 3 to 30 μM for 6 months. The clones that had developed resistance to lenvatinib were named Huh7 LR and Hep3B LR, which were cultured in a medium containing 10 μM concentrations of lenvatinib for maintenance.

Clinical specimens and study approval

All research was conducted in accordance with the Declarations of Helsinki and Istanbul. The clinical specimens were promptly immersed in a formalin solution within 30 minutes following the extraction of the tissue, or tissues were placed in liquid nitrogen for immediate preservation, or alternatively, human HCC organoids were generated immediately. All the clinical HCC samples were obtained from the Hepatic Surgery Center, Tongji Hospital of Huazhong University of Science and Technology (Wuhan, China), upon receiving endorsement from the university's ethical review committee for institutional assessment (TJ-IRB20211163). In all, 133 paired clinical HCC samples for tissue microarray and patient-derived specimens and the 25 paired lenvatinib-sensitive patients and the lenvatinib-resistant HCC patients were obtained from patients who received hepatectomy for HCC at Hepatic Surgery Center, Tongji Hospital of Huazhong University of Science and Technology (Wuhan, China). All the lenvatinib-treated patients received preoperative cancer treatment with lenvatinib.

Cell culture

The human embryonic kidney 293 cell line (HEK293T), as well as HCC cell lines including Hep3B, Huh7, were all procured from the China Center for Type Culture Collection (CCTCC), located in Wuhan, China. Human cancer cells were cultivated in the Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The incubation conditions were maintained at a temperature of 37°C in an environment containing 5% carbon dioxide.

Statistical analysis

The statistical significance of the results, including cell viability assay, migration and invasion assay, immunohistochemical staining, tumor weight and volume, was statistically analyzed using either the t test or the Mann–Whitney U test as suitable using GraphPad Prism (GraphPad Software). The significance was determined with a 95% CI for all the tests (ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001).

RESULTS

Identification of NEK7 as the acquired lenvatinib resistance gene in HCC

To establish the ALR PDOX models in vivo, we first developed HCC organoids from lenvatinib-untreated HCC patients. We divided the lenvatinib-sensitive patients from IC50 values to identify the congenital lenvatinib resistance organoids and lenvatinib-sensitive organoids (Supplemental Figure S1A, http://links.lww.com/HEP/J882). Then, 6 cases of lenvatinib-sensitive organoids with the lowest IC50 values were subcutaneously injected into immunodeficient mice with 30 mg/kg lenvatinib treatment to build up ALR PDOXs; the tumor sizes were measured during the whole xenotransplantation (Figure 1A). The tumor size line chart demonstrated whether ALR tumors were filtered out, and we chose ALR tumors that rebuilt ALR PDOXs (Supplemental Figures S1B–D, http://links.lww.com/HEP/J882). Compared with the parent strains, ALR patient-derived organoids (ALR PDOs) elevated IC50 values from 5.43 to 64.31 μM (Figure 1B). Lenvatinib resistance mechanisms have demonstrated that kinases are reconfigured to compensate for the loss of signaling in response to TKIs.¹⁵ Based on the ALR PDOXs model, we conducted the human kinase domain-focused CRISPR knockout library using a sgRNA library targeting about 482 kinase genes (Supplemental Table 3, http://links.lww.com/HEP/J882) (Figures 1C, D). Following CRISPR knockout library transduction, we injected the ALR PDOXs subcutaneously into immunodeficient mice and treated them with lenvatinib. After 15 days of treatment, the tumors were harvested and analyzed (Figures 1E, F). Synthetic lethal with lenvatinib genes in vivo were listed in the Top negative genes (Supplemental Table 4, http://links.lww.com/HEP/J882) (Figure 1G and Supplemental Figure S1F, http://links.lww.com/HEP/J882).

FIGURE 1.

Identification of NEK7 as the acquired lenvatinib resistance gene in HCC. (A) Schematic diagram of the ALR PDOXs selection. (B) The corresponding lenvatinib-sensitive organoids and ALR PDOs IC50 values were measured. (C) Schematic diagram of the synthetic lethal screen in vivo. The tumors were harvested and mixed in the same group. (D) Western blot of the protein expression of Cas9 to validate the successful transduction of the sgRNA library. (E) Subcutaneous xenograft model transplanted with ALR PDOs transduced with sgRNA library, followed by treatment with lenvatinib (30 mg/kg/d) when the tumor reached a volume of ~150 mm³ in size (n=5, scale bar: 1 cm). (F) Quantification of tumor weight (n=5). (G) Presentation of the comparative frequencies of gRNA sequences in the ALR PDOXs model. (H) Schematic diagram of the synthetic lethal screen in Hep3B LR cell lines. (I) Presentation of the comparative frequencies of gRNA sequences in Hep3B LR model. (J) Schematic diagram of the acquired lenvatinib resistance Trp53^KO/MYC^OE HCC mouse model. (K) DIA analysis was conducted to evaluate the protein expression profiles between the acquired lenvatinib resistance Trp53^KO/MYC^OE HCC mouse model and the Trp53^KO/MYC^OE HCC mouse model. Abbreviations: ALR PDOs, acquired lenvatinib resistance patient-derived organoids; ALR PDOXs, acquired lenvatinib resistance patient-derived organoids-based xenograft; DIA, data-independent acquisition; NEK7, NIMA-related coiled-coil kinase 7.

Hep3B and Huh7 cell lines were recognized as lenvatinib-sensitive cell lines.¹⁵ We treated Hep3B and Huh7 cell lines in varying concentrations of lenvatinib from 3 to 30 μM for 6 months to establish the lenvatinib resistance (LR) cell models. Successful development of resistance to lenvatinib was confirmed, as Hep3B LR cell lines and Huh7 LR cell lines have 10.9 times and 20.2 times higher IC50 in lenvatinib-resistant cells compared with control Hep3B and Huh7 cell lines, respectively (Supplemental Figure S1E, http://links.lww.com/HEP/J882). The CRISPR knockout library was also transduced in Hep3B LR cell lines following the 30 μM lenvatinib treatment to further validate the synthetic lethality with lenvatinib genes in vitro (Supplemental Table 5, http://links.lww.com/HEP/J882) (Figures 1H, I and Supplemental Figure S1G, http://links.lww.com/HEP/J882).

Acquired lenvatinib resistance models in immunocompetent mice have been reported to be constructed by the Trp53^KO/MYC^OE HCC mouse model.¹³ In wild-type C57BL/6 J mice, we induced HCC by the overexpression of the activated forms of c-myc-IRES-Luc, along with PX330-sg-p53 and Sleeping Beauty transposase, through hydrodynamic injection. Fourteen days post-injection of the plasmids, the mice were divided into 2 groups with administered placebo or lenvatinib at a dosage of 30 mg/kg for a period of 35 days to replicate HCC patients who have no response to lenvatinib treatment (Figure 1J). The initial decrease followed by an increase in the level of bioluminescent signal as an indicator of successful drug-resistance model establishment, and tumor growth accelerated more rapidly in the lenvatinib-resistance group compared with the placebo group (Supplemental Figures S1H, I, http://links.lww.com/HEP/J882). In addition to investigating the kinase regulation, data-independent acquisition (DIA) demonstrated that NEK7 was upregulated in the acquired lenvatinib resistance HCC immunocompetent mouse model (Figure 1K). Western blot assay demonstrated NEK7 was upregulated in both Hep3B LR cell lines and Huh7 LR cell lines (Supplemental Figure S1J, http://links.lww.com/HEP/J882).

Taken together, by intersecting the synthetic lethal genes with lenvatinib from the 2 CRISPR–Cas9 sequencing datasets and the DIA analysis of the Trp53^KO/MYC^OE HCC lenvatinib-resistance mouse model, we identified NEK7 as a key kinase involved in the acquired lenvatinib resistance in the treatment of HCC.

Knockdown of NEK7 promotes the sensitivity of HCC to lenvatinib

Given that NEK7 was critical in acquired lenvatinib resistance, we focused on sensitizing acquired lenvatinib resistance HCC cells to lenvatinib treatment via suppressing NEK7. In Hep3B LR cells, the IC50 value of lenvatinib significantly decreased from 60.50 to 22.16 and 24.05 μM, and in Huh7 LR cells IC50 value of lenvatinib decreased from 66.32 to 23.68 and 21.95 μM after knockdown NEK7 (Figures 2A, B and Supplemental Figures S2A, B, http://links.lww.com/HEP/J882). We found knockdown of NEK7 in ALR PDOs reduced resistance to lenvatinib at various doses (20 and 40 μmol/L) (Figures 2C, D). Furthermore, we constructed Nek7^flox/flox (Nek7-WT) mice and Nek7^ΔHep (Nek7-CKO) mice in a liver fibrosis-induced liver cancer model (Supplemental Figure S2C, http://links.lww.com/HEP/J882). The results showed Nek7^ΔHep mice had lower tumor number and max tumor volume compared with Nek7^flox/flox mice (Supplemental Figures S2D, E, http://links.lww.com/HEP/J882).

FIGURE 2.

Knockdown of NEK7 promotes the sensitivity of HCC to lenvatinib. (A) The IC50 values were measured and compared in shNC and shNEK7-1 and shNEK7-2 in Hep3B LR cells. (B) Long-term colony formation assay of WT, LR shNC, and LR shNEK7 in Hep3B LR cells. (C) Representative images of ALR PDOs response to lenvatinib at concentrations of 20 or 40 μM for 6 days after NEK7 knockdown (Scale bar: 100 μm). (D) Relative cell viability to DMSO (fold change) is presented. (E) Schematic plot about the establishment of Nek7^flox/flox mice and Nek7^ΔHep mice in acquired lenvatinib resistance Trp53^KO/MYC^OE HCC mouse model with placebo or lenvatinib treatment. (F) The level of bioluminescent signal serves as a marker for HCC development. (G) Representative images of HCC tumors and luciferase activity images from Nek7^flox/flox and Nek7^ΔHep groups at the endpoint with placebo or lenvatinib treatment. (H) Quantification of signal intensity of the livers in Nek7^flox/flox and Nek7^ΔHep groups with placebo or lenvatinib treatment (n=5). (I) Quantification of liver/body weight ratio (n=5). (J) Subcutaneous xenograft model transplanted with Hep3B or Hep3B LR cells followed by treatment with lenvatinib (30 mg/kg/d) when the tumor reached a volume of ~150 mm³ in size (n=6, scale bar: 1 cm). (K) Quantification of tumor weight (n=6). (L) Tumor volume monitored and the growth curves measured for 20 days (n=6). Abbreviations: Abbreviations: ALR PDOs, acquired lenvatinib resistance patient-derived organoids; LR, lenvatinib resistance; NEK7, NIMA-related coiled-coil kinase 7; WT, wild type.

To further investigate the role of NEK7 in lenvatinib resistance, we utilized Nek7^flox/flox mice or Nek7^ΔHep mice and validated in the lenvatinib resistance Trp53^KO/MYC^OE HCC mouse model (Figure 2E). The results showed that, in the placebo groups, Nek7^ΔHep mice led to a decreased tumor burden compared with the Nek7^flox/flox mice. In lenvatinib treatment groups, there was an initial decrease followed by an increase in the level of bioluminescent signal after lenvatinib treatment, which indicates that the lenvatinib-resistant model is successfully established in Nek7^flox/flox mice. In contrast, in Nek7^ΔHep mice, the level of bioluminescent signal continued to decrease with sustained lenvatinib administration (Figure 2F). Compared with Nek7^flox/flox mice, Nek7^ΔHep mice exhibited a lower level of bioluminescent signal and a reduced ratio of liver weight to body weight (Figures 2G–I). In addition, Ki-67 staining was downregulated, and cleaved caspase-3 staining was increased in the Nek7^ΔHep lenvatinib treatment group (Supplemental Figures S2F, G, http://links.lww.com/HEP/J882). These findings indicate that specifically knocking out Nek7 in hepatocytes significantly inhibited the acquired lenvatinib resistance in the Trp53^KO/MYC^OE model.

Consistent with our finding, in a subcutaneously implanted tumor model, knockdown of NEK7 inhibited tumor progression by enhancing the efficacy of lenvatinib (Figures 2J–L). Moreover, the Ki-67 staining was downregulated, and cleaved caspase-3 staining was increased in the shNEK7 group (Supplemental Figures S2H, I, http://links.lww.com/HEP/J882). These findings indicate that knockdown or deletion of NEK7 restores the efficacy of lenvatinib and inhibits acquired lenvatinib resistance progression in vitro and vivo.

NEK7 promotes the acquired lenvatinib resistance through the EGFR signaling pathway

NEK7 is a serine/threonine kinase that plays a crucial role by adding phosphate groups to specific serine or threonine residues on target proteins.^25,29 We employed quantitative phosphoproteomics in Hep3B LR shNC and shNEK7 cells. The results demonstrated that the EGFR signaling pathway and its downstream signaling of MAPK and PI3K/AKT signaling pathways were affected by NEK7 (Figure 3A). We next focused on whether NEK7 could regulate these signaling pathways. Consequently, we determined the levels of EGF secretion in the culture medium of Huh7/Huh7 LR and Hep3B/Hep3B LR cell lines by ELISA, and observed EGF secretion upregulated in both Huh7 LR and Hep3B LR cell lines (Supplemental Figure S3A, http://links.lww.com/HEP/J882). To exclude insulin affected PI3K/AKT signaling activation, we treated cells with insulin (100 ng/mL) in a time-dependent manner in NEK7 knockdown or NEK7 overexpression cell lines, under the same stimulation time (5, 10​​​​, and 15 min), the expression of p-AKT did not significantly change between the shNC and shNEK7 groups, or the Vector and NEK7 groups (Supplemental Figure S3B, http://links.lww.com/HEP/J882). Western blot assay demonstrated that after being treated with human EGF (100 ng/mL) in a time-dependent manner, the level of p-ERK and p-AKT, which represented the activation of MAPK and PI3K/AKT signaling pathway, was decreased after knockdown of NEK7 (Figures 3B, C). Furthermore, the level of p-ERK and p-AKT was increased in NEK7 overexpression cells (Supplemental Figure S3C, http://links.lww.com/HEP/J882). However, under the same stimulation time (5, 10, and 15 min), the WB assay showed that the classical EGFR phosphorylation sites EGFR (Y1068) and EGFR (Y1173) had no significant change in both NEK7 knockdown or overexpression cell lines (Figures 3B, C and Supplemental Figure S3C, http://links.lww.com/HEP/J882). To further investigate the relationship between NEK7 and EGFR, we constructed EGFR KO cell lines in Hep3B LR and Huh7 LR cells using CRISPR/Cas9 (Supplemental Figure S3D, http://links.lww.com/HEP/J882) and subsequently stably overexpressed NEK7. We observed that EGFR KO significantly inhibited the proliferation of Hep3B LR and Huh7 LR cells, which had a lower IC50 compared with the LR Vector group upon lenvatinib treatment. Meanwhile, overexpression of NEK7 promoted the proliferation of Hep3B LR and Huh7 LR cells; however, it could not rescue the inhibition level following EGFR KO upon lenvatinib treatment (Figures 3D, E and Supplemental Figures S3E1, E2, http://links.lww.com/HEP/J882). Consistently, WB assay demonstrated that overexpression of NEK7 could not rescue the EGFR downstream inhibition after EGFR KO (Supplemental Figure S3F, http://links.lww.com/HEP/J882). In vivo experiments demonstrated that overexpression of NEK7 could not rescue the growth of xenografts following EGFR KO upon lenvatinib treatment (Figures 3F–H). Furthermore, EGFR KO led to lower Ki-67 and higher cleaved caspase-3 upon lenvatinib treatment, and this effect could not be rescued by overexpression of NEK7 (Figure 3I and Supplemental Figure S3G, http://links.lww.com/HEP/J882). Overall, these findings implied NEK7 promotes the acquired lenvatinib resistance of HCC in an EGFR-dependent manner.

FIGURE 3.

NEK7 modulates the acquired lenvatinib resistance progression through the MAPK and PI3K/AKT signaling pathway. (A) Gene Ontology biological process (Environmental Information Processing) analysis of proteins identified in quantitative phosphorylation MS via the Database for Annotation, Visualization, and Integrated Discovery. And volcano plot shows the EGFR signaling gene in quantitative phosphorylation MS in Hep3B LR (shNC vs. shNEK7). (B, C) Western blot of the protein expression of EGFR signaling downstream MAPK and PI3K/AKT signaling pathway for the activation of p-EGFR (Y1068), EGFR (Y1173), p-AKT (T308), p-AKT (S473), p-ERK in Hep3B LR and Huh7 LR shNC or shNEK7 cell lines, treated with EGF (50 ng/mL) in a time-dependent manner. (D, E) Long-term colony formation assay of Vector, NEK7 overexpression, with sgNC or sgEGFR in Hep3B LR cells, the corresponding IC50 values were measured and compared. (F) Subcutaneous xenograft model transplanted with Vector, NEK7 overexpression, with sgNC or sgEGFR in Hep3B LR cells, followed by treatment with lenvatinib (30 mg/kg/d) when the tumor reached a volume of ~150 mm³ in size (n=6, scale bar: 1 cm). (G) Quantification of tumor weight (n=6). (H) Tumor volume monitored and the growth curves measured for 20 days (n=6). (I) Representative images of immunohistochemical staining of HE, Ki-67, and cleaved caspase-3 (n=6, scale bar: 20 μm). Abbreviations: EGFR, endothelial growth factor receptor; LR, lenvatinib resistance; MAPK, mitogen-activated protein kinase; NEK7, NIMA-related coiled-coil kinase 7; PI3K/AKT, phosphoinositide 3-kinase/Akt.

EGFR C-terminal tail residue S1070 is phosphorylated by NEK7

To elucidate the mechanisms by which NEK7 activates the EGFR signaling pathway. The interaction between NEK7 and EGFR was confirmed by exogenous and endogenous co-IP assay, respectively (Figures 4A, B). Furthermore, immunofluorescent colocalization of NEK7 and EGFR on the cytomembrane was observed through confocal microscopy (Figure 4C). We then conducted molecular docking simulations based on the protein structure of NEK7 and EGFR. We observed the interacting ability between NEK7 and EGFR (Supplemental Figure S4A, http://links.lww.com/HEP/J882). To identify the interacting regions of EGFR that interact with NEK7, several truncated forms of EGFR were established. Full-length EGFR and other truncated forms interacted with NEK7 (Figure 4D). As EGFR is a transmembrane protein, purified proteins were constructed separately as the outer segment of the cell membrane, His-EGFR (25–645) and the inner segment of the cell membrane, His-EGFR (669–1210). In vitro GST pull-down assay further demonstrated the direct binding between EGFR (669–1210) and NEK7 (Figure 4E and Supplemental Figure S4B, http://links.lww.com/HEP/J882). Based on NEK7 being a serine–threonine kinase, we hypothesized that NEK7 should phosphorylate EGFR at the inner segment of the cell membrane (669–1210). We firstly constructed NEK7 kinase-dead mutant (lysins 63/64 at the ATP-binding site are replaced with methionine, K63/64M) and NEK7 kinase-active mutant (Tyr residue at the auto-inhibitory domain was mutated to an Ala, thus lacking auto-inhibitory function, Y97A) according to previous study.²⁵ The WB assay demonstrated the pan-Phospho-(Ser/Thr) level was significantly enhanced after NEK7 (Y97A) overexpression but not NEK7 (K63/64M) compared with NEK7 (WT) overexpression (Figure 4F). Furthermore, to determine the specific phosphorylation site on EGFR, we pulled down EGFR proteins with or without NEK7 overexpression, then sent them for phosphorylation mass spectrometry analysis to identify the potential phosphorylation site on EGFR activated by NEK7 (Figure 4G). The phosphorylation mass spectrum showed that 2 phosphorylation sites, S1026 and S1070, were identified in a set of differences (Figure 4H). Subsequently, the quantification co-IP assay showed that the S1070 rather than S1026 was essential to the pan-Phospho-(Ser/Thr) level of EGFR induced by NEK7 overexpression (Figure 4I). Furthermore, we generated EGFR S1070-specific mono-phosphorylation antibody (p-EGFR S1070), which specifically recognized the EGFR S1070 site to validate NEK7 activation on EGFR. Results showed that only HA-EGFR WT could be catalyzed on the S1070 site by NEK7; activation of the site HA-EGFR S1070D and HA-EGFR S1070E sustained activation on the EGFR S1070 site, simultaneously, inactivation of the site HA-EGFR S1070A sustained inactivation on the EGFR S1070 site. Meanwhile, ectopically expressing NEK7 had no effect on p-EGFR Y1068 activation (Figure 4J). Of note, the EGFR S1070 site was highly conserved among various species (Figure 4K). Next, we employed an in vitro phosphorylation assay to confirm that NEK7 could phosphorylate EGFR on the S1070 site (Figure 4L). Taken together, our data provide significant evidence to support that NEK7 induced phosphorylation on the EGFR c-tail residue S1070 site (Supplemental Table 1, http://links.lww.com/HEP/J882).

FIGURE 4.

EGFR C-terminal tail residue S1070 is phosphorylated by NEK7. (A) The interaction between exogenous Flag-NEK7 and HA-EGFR was confirmed in HEK293T cells. (B) The interaction between endogenous NEK7 and EGFR was confirmed in Hep3B LR cells. (C) Confocal immunofluorescence analysis revealed the colocalization of NEK7 and EGFR on the cell membrane. (D) Mapping EGFR regions involved in NEK7 binding; schematic diagram of EGFR truncation tagged with HA. Western blot confirmed the interaction between NEK7 and different functional regions of EGFR. (E) GST pull-down assay indicating direct interaction between NEK7 and EGFR (669–1210). The input was tested by silver staining (left). (F) pan-Phospho-(Ser/Thr) level changes after transfected with NEK7-WT, NEK7-Y97A, and NEK7-K63/64M. (G) The schematic model of detecting the phosphorylation site of EGFR activated by NEK7. (H) Secondary phosphorylation mass spectrometry results of EGFR phosphorylation site in HA-EGFR co-IP by HA (up) and HA-EGFR and Flag-NEK7 co-IP by HA (bottom). (I) HEK293T cells transfected with HA-EGFR or HA-EGFR mutant plasmids as indicated, WCLs were collected for IP with HA antibody, followed by Immunoblots (IB) analysis. (J) HEK293T cells transfected with HA-EGFR or HA-EGFR S1070A, S1070D or S1070E mutant plasmids, WCEs were collected for IP with HA antibody, followed by IB analysis. (K) The EGFR S1070 site amino acids in different species. (L) In vitro kinase assay was performed, purified GST-NEK7 incubated with His-EGFR (669–1210) in the adenosine 5’-triphosphate, followed by IB analysis for p-EGFR S1070 level using p-EGFR S1070-specific phosphorylation antibody (right). The input was tested by silver staining (left). Abbreviations: co-IP, co-immunoprecipitation; EGFR, endothelial growth factor receptor; GST, glutathione S-transferase; HA, hemagglutinin; HEK293T, human embryonic kidney 293 cell line; IP, immunoprecipitation; LR, lenvatinib resistance; NEK7, NIMA-related coiled-coil kinase 7; Ser, serine; Thr, threonine; WCEs, whole-cell extracts; WCLs, whole-cell lysates; WT, wild type.

The phosphorylation of EGFR C-terminal tails residue S1070 promotes the interaction between EGFR, P85, and GRB2

EGFR signals play a crucial role in regulating the MAPK and PI3K/AKT signaling pathways.^30,31 After the phosphorylation on the C-terminal tails, GRB2 can bind via its SH2 domain, which subsequently recruits SOS and activates the MAPK signaling pathway.^32,33 The SH2 domain of P85 (a regulatory subunit of PI3K) is reported to bind to EGFR complex and relieves P110 (a catalytic subunit of PI3K) with inhibitory contacts, the catalytic positions in P110 can access substrate and receive further inputs in activating PI3K/AKT signaling pathway.^34,35 We generated ectopically stably expressed EGFR WT and EGFR S1070A in Hep3B KO EGFR LR cells and Huh7 KO EGFR LR cells to investigate the potential function of EGFR residue S1070. We observed a significant decrease in MAPK and PI3K/AKT signaling in KO EGFR cells and EGFR S1070A cells by detecting p-ERK and p-AKT levels (Figure 5A and Supplemental Figure S5A, http://links.lww.com/HEP/J882). We employed exogenous HA-EGFR and HA-EGFR S1070A for overexpression. We further proved that P85 and GRB2 interacting with EGFR were reduced with EGFR S1070A (Figure 5B). Then, in the endogenous protein quantification co-IP assay, the interacting ability of P85 and GRB2 with EGFR was reduced with EGFR S1070A (Figure 5C and Supplemental Figure S5B, http://links.lww.com/HEP/J882). We then determined the role of NEK7 in P85–EGFR and GRB2–EGFR interactions. WB assay results showed that in Huh7 LR cells, overexpression of NEK7 enhanced P85 and GRB2 interaction with EGFR after EGF stimulation for 10 minutes. In Hep3B LR cells, knockdown of NEK7 attenuated P85 and GRB2 interacting with EGFR after EGF stimulation for 10 minutes. EGF activated phosphorylation of EGFR firstly occurs at the plasma membrane.³⁶ We further determined whether NEK7 impacted on P85 and GRB2 membrane recruitment. Data demonstrated that NEK7 overexpression led to P85 and GRB2 accumulation on the membrane after EGF stimulation (Figure 5F). Knockdown of NEK7 impaired EGF stimulation of P85 and GRB2 membrane recruitment (Figure 5G). To further confirm EGFR S1070A impaired the P85 and GRB2 membrane recruitment, live cells transfected with mCherry-P85 and pEGFP-GRB2 were observed by immunofluorescent cytochemistry and an IF assay was employed. We observed P85 and GRB2 accumulated on the membrane in EGFR WT cells more than in EGFR S1070A cells, suggesting EGFR S1070A limited the P85–EGFR and GRB2–EGFR interactions (Figure 5H and Supplemental Figure S5C, http://links.lww.com/HEP/J882). Furthermore, we reintroduced EGFR WT, EGFR S1070A, and NEK7 in EGFR KO Hep3B LR cell lines to investigate whether the phosphorylation of EGFR at S1070 is crucial for NEK7-mediated lenvatinib resistance. We found that when EGFR was KO while NEK7 was overexpressed, the cells lost their resistance to lenvatinib. However, when EGFR WT was reintroduced along with NEK7, the resistance was restored. In contrast, when EGFR S1070A was reintroduced with NEK7, the resistance was not significantly recovered (Supplemental Figures S5D, E, http://links.lww.com/HEP/J882). Overall, these findings suggested that phosphorylation of the EGFR S1070 site was essential in NEK7-mediated lenvatinib resistance through EGFR–P85 and EGFR–GRB2 interactions.

FIGURE 5.

The phosphorylation of EGFR C-terminal tails residue S1070 promotes the interaction between EGFR, P85, and GRB2. (A) Hep3B LR knockout EGFR cells were transfected with EGFR or EGFR S1070A, then treated with EGF (50 ng/mL) in a time-dependent manner, WCE were collected for IB analysis. (B) HEK293T cells were transfected with HA-EGFR or HA-EGFR S1070A, then transfected with Flag-NEK7, WCE were collected for IP with HA antibody, followed by IB analysis. (C) Hep3B LR knockout EGFR cells overexpression of EGFR or EGFR S1070A, WCE were collected for IP with HA antibody, followed by IB analysis. (D, E) Huh7 LR overexpression NEK7 cells and Hep3B LR knocking down NEK7 cells, treated with EGF (50 ng/mL) in a time-dependent manner, WCE were collected for IP with HA antibody, followed by IB analysis. (F, G) The membrane and cytosolic fractions from Huh7 LR and Hep3B LR Vector or overexpression NEK7 cells, treated with EGF (50 ng/mL) for 10 minutes, WCE were collected, followed by IB analysis. (H) Hep3B LR sgEGFR cells stably transfected with EGFR WT or EGFR S1070A and treated with EGF (50 ng/mL), IF showing the localization of P85 and GRB2. Abbreviations: EGFR, endothelial growth factor receptor; GRB2, growth factor receptor-bound protein 2; HA, hemagglutinin; HEK293T, human embryonic kidney 293 cell line; IB, immunoblotting; IF, immunofluorescence; IP, immunoprecipitation; LR, lenvatinib resistance; NEK7, NIMA-related kinase 7; P85, phosphoinositide 3-kinase regulatory subunit p85; WCE, whole-cell extract; WT, wild type.

The phosphorylation of EGFR C-terminal tails residue S1070 induced by NEK7 promotes acquired resistance to lenvatinib therapy

To further validate the relationship between EGFR C-terminal tails residue S1070 and NEK7 in the acquired lenvatinib resistance, we stably overexpressed EGFR (Hep3B LR^EGFR) and EGFR S1070A (Hep3B LR^{EGFR S1070A}) in Hep3B LR KO EGFR cells. The IC50 value of lenvatinib in EGFR S1070A significantly decreased 1.64 times and 1.69 times compared with EGFR in Hep3B KO EGFR cells and Huh7 KO EGFR cells, respectively (Figures 6A, B and Supplemental Figure S6A, B, http://links.lww.com/HEP/J882). Consistently, in vivo xenograft tumors showed that the tumor growth of Hep3B LR^{EGFR S1070A} cells obviously decreased compared with Hep3B LR^WT cells under placebo treatment and Hep3B LR^WT cells and Hep3B LR^EGFR cells under lenvatinib treatment (Figures 6C, D and Supplemental Figure S6C, http://links.lww.com/HEP/J882). Consistently, Hep3B LR^{EGFR S1070A} tumor exhibited a decrease in Ki-67 positive rates and a higher cleaved caspase-3 positive rate (Supplemental Figure S6D–F, http://links.lww.com/HEP/J882). These data suggested that EGFR S1070A was essential for the efficacy of lenvatinib therapy. We then examined the strong correlation of NEK7 or EGFR and p-EGFR S1070 by the WB assay (Figure 6E and Supplemental Figure S6G, http://links.lww.com/HEP/J882). Using clinical data, we found that the expression level of p-EGFR S1070 was related to worse OS in HCC patients (Figure 6F and Supplemental Figure S6H, http://links.lww.com/HEP/J882). Consistently, we analyzed clinical data of patients who received lenvatinib therapy. A higher expression level of NEK7 was observed from HCC tissue microarray in a lenvatinib resistance patient (Tongji cohort 1) (Figures 6G, H). Next, we examined 119 paired samples from the HCC tissue microarray, which confirmed the upregulation of NEK7 in tumor samples and related to a worse OS (Figures 6I, J). Notably, NEK7 levels correlated with differentiation, AFP, and tumor size (Supplemental Table 2, http://links.lww.com/HEP/J882) (Supplemental Figure S6I, http://links.lww.com/HEP/J882). To further validate these results, a large-scale data mining analysis of 89 cohorts of HCC patients was presented. NEK7 mRNA was increased in 67 datasets, and 52 datasets had a significant increase of NEK7 mRNA in HCC cohorts (Figure 6K). Collectively, these findings suggest that the level of EGFR C-terminal tails residue S1070 and NEK7 is closely related to the efficacy of lenvatinib therapy and the malignant progression of HCC. EGFR S1070 phosphorylation site could function as a predictive marker for acquired lenvatinib patients.

FIGURE 6.

The phosphorylation of EGFR C-terminal tails residue S1070 phosphorylated by NEK7 modulates the efficacy of acquired resistance to lenvatinib therapy. (A, B) Long-term colony formation assay of Hep3B LR sgNC or sgEGFR cells stably overexpressing EGFR WT or EGFR S1070A, the corresponding IC50 values were measured and compared. (C) Subcutaneous xenograft model transplanted with Hep3B LR sgEGFR cells stably overexpressing EGFR WT or EGFR S1070A, followed by treatment with lenvatinib (30 mg/kg/d) when the tumor reached a volume of ~150 mm³ in size (n=6, scale bar: 1 cm). (D) Quantification of tumor weight (n=6). (E) The Pearson correlation analysis of the NEK7 and p-EGFR S1070, EGFR, and p-EGFR S1070 protein levels in the HCC cohort as determined by western blot. (F) Kaplan–Meier curves of the OS with differential p-EGFR S1070 expression in Tongji cohort 2. (G) Representative immunohistochemistry images of NEK7 expression in sensitive and resistant HCC patients. (H) The immunohistochemistry scores demonstrated NEK7 expression was high in lenvatinib-resistant HCC patients (n=25). (I) Representative immunohistochemistry images of NEK7 expression in adjacent tissues and cancerous tissues from Tongji cohort 2. (J) The immunohistochemistry scores demonstrated NEK7 expression was high in cancer tissues (left) and Kaplan–Meier curves of the OS with differential NEK7 expression in Tongji cohort 2 (right) (n=119). (K) Large-scale data mining techniques were employed to analyze the differential expression of NEK7 mRNA between HCC tissues and adjacent non-tumorous liver tissues. Abbreviations: EGFR, endothelial growth factor receptor; IHC, immunohistochemistry; LR, lenvatinib resistance; NEK7, NIMA-related coiled-coil kinase 7; WT, wild type.

Pharmacological blocking of EGFR S1070 attenuates acquired lenvatinib resistance of HCC

Based on the fact that NEK7 phosphorylated EGFR S1070 site is essential for MAPK and PI3K/AKT signaling pathway activation, as well as its role in acquired resistance to lenvatinib, we explored the potential therapeutic targeting of the EGFR S1070 site. We utilized the Alphafold2 database to obtain the EGFR C-terminal tails (979–1099) structure and further designed 2 peptides with cell-penetrating property of the TAT-peptide, which scored highest in the EGFR interface analyzer result (Figure 7A and Supplemental Figure S7A, http://links.lww.com/HEP/J882). To assess whether the TAT-peptides could impact EGFR S1070 phosphorylation and its downstream pathways, data demonstrated that TAT-pep10 significantly inhibited activation of MAPK and PI3K/AKT signaling pathways in Hep3B LR cells (Figure 7B). In vitro antitumor effects, TAT-pep10 significantly inhibited the invasion, migration, and tumor cell proliferation of Hep3B LR cells (Supplemental Figures S7B, C, http://links.lww.com/HEP/J882). And TAT-pep10 showed a synergistic effect when combined with lenvatinib (Supplemental Figure S7D, http://links.lww.com/HEP/J882). Furthermore, after treating the cells with TAT-pep10, the quantification co-IP assay demonstrated that the interaction of EGFR with NEK7, P85 and GRB2 was inhibited in a concentration-dependent manner in 293T cells (Figure 7C). Importantly, TAT-pep10 inhibited phosphorylation of EGFR S1070 and its downstream pathways in a concentration-dependent manner in Hep3B LR cells (Figure 7D). Next, surface plasmon resonance (SPR) detection assays showed that TAT-pep10 could interact with the EGFR inner segment of cell membrane residue (669–1210) (Figure 7E). Furthermore, TAT-pep10 reduces the size and growth rate of acquired lenvatinib resistance organoids in a concentration-dependent manner (10, 20, and 40 μM), and the combination of TAT-pep10 and lenvatinib effectively inhibited the size and growth rate of acquired lenvatinib resistance organoids (Figures 7F, G). We verify the therapeutic efficacy for the combination of TAT-pep10 via tail vein (1 mg/mL, 100 μL) and lenvatinib via oral gavage (10 mg/kg, 1/3 dose used alone). In the subcutaneous xenograft model, the combination treatment groups demonstrated significant growth inhibition compared with the placebo and lenvatinib groups (Figures 7H–J). Consistently, in lenvatinib-resistance Trp53^KO/MYC^OE HCC model (Supplemental Figure S7E, http://links.lww.com/HEP/J882), the combination treatment groups exhibited lower signal of luciferase intensity and liver weight to body weight compared with the placebo and lenvatinib groups (Figures 7K–M). The Ki-67 positive rates were lower in combination groups, while cleaved caspase-3 positive rates were significantly higher compared with the placebo and lenvatinib groups in Hep3B LR subcutaneous xenograft model (Supplemental Figures S7H, I2, http://links.lww.com/HEP/J882) and lenvatinib-resistance Trp53^KO/MYC^OE HCC model (Supplemental Figures S7J, K2, http://links.lww.com/HEP/J882). In the course of this study, the animals receiving the combined therapy exhibited no significant differences in mouse body weight (Supplemental Figure S7G, http://links.lww.com/HEP/J882). Collectively, our data revealed the significance of NEK7-mediated EGFR S1070 phosphorylation in acquired lenvatinib resistance; targeting EGFR C-terminal tails residue S1070 may be a possible therapeutic strategy for acquired LR treatment.

FIGURE 7.

Pharmacological blocking of EGFR S1070 inhibits MAPK and PI3K/AKT signaling pathways activation and the development of acquired lenvatinib resistance in HCC. (A) Three-dimensional (3D) binding model of Pep1 and Pep10 with EGFR (669–1210) domains. (B) Effect of TAT-NC, TAT-pep1, and TAT-pep10 (10 μM) in Hep3B LR cells for 48 hours, WCE was collected, followed by IB analysis. (C) Effect of TAT-NC, TAT-pep10 (10, 20​​​​, and 40 μM) on EGFR and NEK7, P85, GRB2 interaction in HEK293T cells. (D) Effect of TAT-NC, TAT-pep10 (10, 20​​​​, and 40 μM) in Hep3B LR cells for 48 hours, WCE was collected, followed by IB analysis. (E) SPR analysis confirmed the binding of TAT-pep10 and EGFR (669–1210) domains. (F) Representative images of ALR HCC organoids of TAT-pep10 (10, 20​​​​, and 40 μM) or TAT-pep10 (10 μM), lenvatinib (30 μM), or combination (TAT-pep10 3 μM, lenvatinib 10 μM) for 5 days. (G) Relative cell viability to placebo (fold change) is presented on day 5. (H) Subcutaneous xenograft model transplanted with Hep3B LR cells followed by treatment with lenvatinib (30 mg/kg/d), TAT-pep10 tail vein (3 mg/mL, 100 μL) and combination oral gavage lenvatinib (10 mg/kg, 1/3 dose used alone), and TAT-pep10 tail vein (1 mg/mL, 100 μL) when the tumor reached a volume of ~150 mm³ in size (n=6, scale bar: 1 cm). (I) Tumor volume was monitored, and the growth curves were measured for 20 days (n=6). (J) Quantification of tumor weight (n=6). (K) Representative images of HCC tumors and luciferase activity images from 4 groups at the endpoint. (L) Quantification of signal intensity of the livers in 3 groups (n=5). (M) Quantification of liver/body weight ratio (n=5). Abbreviations: ALR, acquired lenvatinib resistance; EGFR, endothelial growth factor receptor; GRB2, growth factor receptor-bound protein 2; HEK293T, human embryonic kidney 293 cell line; IB, immunoblotting; LR, lenvatinib resistance; MAPK, mitogen-activated protein kinase; NEK7, NIMA-related coiled-coil kinase 7; PI3K/AKT, phosphoinositide 3-kinase/Akt; SPR, surface plasmon resonance; WCE, whole-cell extract.

DISCUSSION

NEK7 was recognized to be involved in early mortality²⁶ and the process of mitosis.³⁷ Subsequently, NEK7 was reported as a component of NLRP3 inflammasome.^27,29,38 The effect and molecular mechanism of NEK7 in tumor cells are poorly understood. Previous studies have demonstrated lenvatinib-resistance Trp53^KO/MYC^OE HCC mouse model,¹³ and the studies on lenvatinib resistance demonstrated that kinase rewiring is the key point in lenvatinib resistance mechanism,^14,15 the kinase CRISPR–Cas9 genetic screen in vivo and vitro and DIA for lenvatinib-resistance model identified NEK7 as a significant kinase gene in acquired lenvatinib resistance. Our study sheds light on the mechanism underlying the role of NEK7 in malignancy progression and acquired resistance to lenvatinib. Inhibition of NEK7 suppressed the progression of acquired lenvatinib resistance in cells and the xenograft model. Furthermore, individuals exhibiting elevated NEK7 levels tend to have poorer prognoses and are more likely to develop resistance to lenvatinib. Therefore, NEK7 could potentially act as a prognostic biomarker and a target for treatment in HCC.

Previous studies have demonstrated the role of EGFR in lenvatinib resistance.^10,14,15 Moreover, integrin subunit beta 8 (ITGB8),³⁹ cyclin-dependent kinase 6 (CDK6),¹³ dual specificity phosphatase 9 (DUSP9)⁴⁰ have been identified as genes associated with lenvatinib resistance. These genes are known to activate MAPK or PI3K/AKT signaling pathways, contributing to the progression of lenvatinib resistance in HCC. Consistent with these findings, our study uncovered that elevated NEK7 expression enhanced the activation of MAPK and PI3K/AKT signaling pathways. The effect of NEK7 on the activation of MAPK and PI3K/AKT signaling pathways via EGFR was confirmed by multiple analyses. Our study elaborated the relationship between NEK7 and EGFR by Co-IP, immunofluorescence, and phosphorylation mass spectrum. Intriguingly, we found that NEK7 phosphorylation of EGFR at the S1070 residue within the C-terminal tail significantly boosted the activation of both the MAPK and PI3K/AKT signaling pathways. Several studies have shown that phosphorylation on the C-terminal tails can activate EGFR downstream signaling pathways by interacting with GRB2 and P85, which releases the catalytic positions.^32–35 In our research, immunofluorescence and membrane fractionation studies provided both visual and quantitative insights into the effects of NEK7 and the EGFR S1070A mutation on GRB2 and P85 membrane activation. This study uncovers a novel phosphorylation site on EGFR at S1070, mediated by NEK7, which significantly contributes to the activation of EGFR's downstream signaling pathways, thereby inducing acquired resistance to lenvatinib.

Pharmacologically targeting EGFR was thought to be a potential therapeutic target for lenvatinib resistance. Regarding EGFR inhibition, a clinical study showed that EGFR inhibitor erlotinib combined with sorafenib has a modest benefit in patients in HCC (NCT0901901).^41,42 And combination of lenvatinib plus gefitinib has resulted in significant clinical responses in previously unresponsive to lenvatinib treatment patients (NCT04642547).¹⁵ Moreover, EGFR inhibitors were shown to inhibit cancer progression in non–small-cell lung cancer,^43,44 pancreatic cancer,⁴⁵ and HCC.¹⁴ In our study, we introduced a novel therapeutic TAT-peptide to target EGFR C-terminal tails residue S1070 phosphorylation. TAT-pep10 is capable of interacting with the C-terminal tails of EGFR, thereby preventing the formation of the EGFR/NEK7 complex and further inhibiting the stimulation of the EGFR/P85/GRB2 complex on the membrane. This interaction also suppresses the activation of the MAPK and PI3K/AKT signaling pathways. Importantly, gefitinib inhibited the EGFR-tyrosine kinase by binding to the ATP-binding (kinase domain) to inhibit EGFR C-terminal tails phosphorylation by the EGFR heterodimer.⁴⁶ Furthermore, NEK7 phosphorylates the S1070 site on the C-terminal tails of EGFR, rather than the classical sites in EGFR phosphorylation. In contrast to conventional EGFR inhibitors, TAT-pep10 demonstrated efficacy in countering NEK7-driven EGFR phosphorylation, which plays a key role in the development of acquired lenvatinib resistance in HCC.

In summary, we identify a novel EGFR phosphorylation site S1070. Our findings demonstrate that NEK7 plays a crucial role in the development of acquired lenvatinib resistance. Moreover, NEK7-mediated phosphorylation at the S1070 site on EGFR is essential for the stimulation of GRB2 and P85 on the membrane, which are regulatory subunits of the MAPK and PI3K/AKT signaling pathways. Importantly, NEK7-mediated EGFR S1070 phosphorylation not only contributed to acquired resistance to lenvatinib but also showed a positive correlation with poor overall survival. Moreover, pharmacologically inhibiting EGFR C-terminal tails enhanced the effectiveness of lenvatinib therapy and inhibited HCC progression. Collectively, our studies expanded the understanding of NEK7-mediated EGFR phosphorylation in acquired lenvatinib resistance in HCC and highlight the therapeutic potential of EGFR C-terminal tails as a target for acquired lenvatinib resistance treatment.

Supplementary Material

hep-84-378-s001.docx ^{(3.1MB, docx)}