PIM1 inhibitor synergizes the anti-tumor effect of osimertinib via STAT3 dephosphorylation in EGFR-mutant non-small cell lung cancer

Background An increasing amount of evidence has demonstrated that combined or multiple targeted therapies could bring about more durable clinical outcomes, and it is known that epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance is related to bypass activation. This study aims to explore a specific solution for third-generation EGFR-TKI resistance caused by bypass activation, and to examine the antitumor effects of the combination of a novel inhibitor CX-6258 HCl with osimertinib, along with its underlining mechanisms. Methods A bioinformatics analysis was performed to detect the relations between the provirus integration site for Moloney murine leukemia virus 1 (PIM1) expression and prognosis of lung cancer. The EGFR-mutated lung cancer cell lines were treated with the combination of CX-6258 HCl and osimertinib to analyze cell proliferation using the Cell Counting Kit-8, colony formation, and in vivo experiments. Cell migration was analyzed using wound healing and Transwell assays. The apoptosis level was detected using Annexin V–propidium iodide flow cytometry. The expression levels of EGFR and STAT3 were determined using Western blot analysis. Results High expression level of PIM1 was related to the poor prognosis of non-small cell lung cancer (NSCLC). The combined administration of osimertinib and CX-6258 HCl significantly inhibited cell proliferation and migration and effectively induced apoptosis in lung cancer cells. It was more efficient in suppressing EGFR activation and phosphorylation of STAT3 compared with osimertinib treatment alone. Furthermore, it showed a durable efficacy in a xenograft model. Conclusions This study showed that PIM1 is a poor prognostic factor for NSCLC. CX-6258 HCl is a potential molecular inhibitor to sensitize the antitumor effects of osimertinib through the inhibiting of the phosphorylation of STAT3 in NSCLC.


Introduction
Somatic mutations in the epidermal growth factor receptor (EGFR) gene account for approximately 15% and 40% of non-small cell lung cancer (NSCLC) in Caucasians and East Asians, respectively (1,2). The progression-free survival (PFS) of patients with EGFR-mutant lung cancer using EGFR-tyrosine kinase inhibitors (TKIs) is significantly better than the PFS of patients using traditional chemotherapy (3,4). However, in 10 months, most patients experience therapy failure due to resistance to different drugs. Therefore, an improved targeted therapy that can offer better anti-cancer outcomes needs to be explored.
PIM kinases are a class of serine/threonine kinases, which consist of 3 different isoforms (PIM1, PIM2, and PIM3). PIM1 kinase enhances tumor cell proliferation by suppressing cell cycle suppressor p27 (5). Down regulating the expression of PIM1 inhibits tumor cell proliferation through inducing cell cycle arrest in the G1 phase (6). It can also inhibit apoptosis and induce drug resistance through the phosphorylation of the Bcl-2-associated death promoter (7,8). PIM1 kinase is widely expressed in hematological malignancies and is recognized as effective carcinogenic drivers (9,10). Recent studies have found that PIM1 kinase is overexpressed in solid tumors such as prostate cancer, breast cancer, pancreatic cancer, and salivary adenoid cystic carcinoma (11)(12)(13)(14). In most lung tumor tissues, PIM1 is upregulated, and its expression is closely linked to advanced stage and poor prognosis (15).
PIM kinases are widely overexpressed in different types of tumors and involved in many oncogenic signaling pathways (15,16). A structural analysis of x-rays showed that PIM1 contains a special hinge region used as a target region to produce inhibitors of small molecules (17). Therefore, it is conceivable that PIM1 is an ideal therapeutic target in lung cancer therapy. PIM kinases play an important role in resistance to targeted therapy, chemotherapy, and radiotherapy in many tumors (16,18), and inhibiting the expression of PIM1 can increase the sensitivity of NSCLC cells to cisplatin and gefitinib (16). However, whether the combination of PIM kinase inhibitors and EGFR-TKIs can exert a better antitumor effect compared with osimertinib on EGFR-mutated NSCLCs is uncertain.
The elevated PIM1 expression may be associated to poor lung cancer prognosis. CX-6258 HCl, a pan-PIM kinase inhibitor with robust biochemical efficiency and kinase selectivity (15), and the third-generation EGFR-TKI, osimertinib, were therefore chosen to demonstrate the additive inhibitory effects on tumors. The results could provide useful guides to clinical practice.

Bioinformatics analysis
PIM1 association result was downloaded from the Linked Omics database (http://www.linkedomics.org). This result was analyzed using the Pearson correlation test. The biological process, cellular component, and molecular function that positively correlated with PIM1, were analyzed using enrichment analysis. Oncomine (https:// www.oncomine.org) was used to determine the expression level of candidate genes in lung cancer, and PROGgeneV2 (http://genomics.jefferson.edu/proggene/) was used to determine the prognostic significance of PIM1.

Cell lines and reagents
Osimertinib and CX-6258 HCl were purchased from Selleck Chemicals (TX, USA). EGF was obtained from Sigma-Aldrich (St. Louis, MO, USA). Human NSCLC cell lines NCI-H1975 (amplified EGFR-T790M/del19) and PC9 (amplified EGFR-del19) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in Roswell Park Memorial Institute-1640 Medium (RPMI-1640, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were kept in a humidified incubator at 37 ℃ in the presence of 5% CO 2 .

Cell proliferation assay
The proliferation of NSCLC cells was measured using a Cell Counting Kit-8 (CCK-8; Dojindo, Japan) following the manufacturer's protocol. Briefly, the cells were plated at a density of 3×10 3 cells per well in 96-well plates. The cells were treated with vehicle, 2.5 or 5 μM osimertinib, 2.5 or 5 μM CX-6258 HCl, or a combination of both (2.5 or 5 μM) in H1975 and PC9 cells for 24 or 48 h after attachment to the wall overnight. Then, the culture media was changed to 10% CCK-8 solution, and the cells were incubated for 3 h at 37 ℃. The cell numbers were estimated using a spectrophotometer at 450 nm.

Wound-healing assay
The cells were plated in six-well plates and allowed to cover the well overnight. The wounds were scratched in the cell monolayer using a 10-µL micropipette tip. The floating cells and debris were rinsed with PBS. To test the effects of drugs on the migration of NSCLCs, H1975, and PC9 cells were seeded in serum-free RPMI-1640 medium with the vehicle, 5 μM CX-6258 HCl, 5 μM osimertinib, or a combination of both (5 μM). Wound healing was recorded using a microscope at 0 and 48 h.

Transwell assay
The cells were plated at a density of 5×10 4 cells in a serumfree medium in the top chamber of a Transwell (Corning, NY, USA). The lower chambers were filled with 600 μL of the complete medium with the vehicle, osimertinib, CX-6258 HCl, or a combination. The medium was refreshed 6 h later. The cells were incubated for 24 h, washed with PBS twice, fixed in methanol, and stained with 0.4% crystal violet. The average number of migrating cells was determined in 6 random high-power fields.

Apoptosis assay
Apoptosis assay was performed using an Annexin V-FITC/ propidium iodide (PI) apoptosis detection kit (Vazyme Biotech Co., Ltd, Nanjing, China) according to the manufacturer's protocols. Briefly, the cells were plated in six-well plates overnight and treated with vehicle, 5 μM CX-6258 HCl, 5 μM osimertinib, or a combination of both (5 μM). After 24 h of treatment, the cells were collected and washed with PBS twice. They were stained with Annexin V-FITC and PI for 15 min. The cell apoptosis was analyzed using flow cytometry (BD Biosciences, FACS Calibur).

Western blot analysis
The cells were plated in six-well plates until 80% density was achieved. Osimertinib or CX-6258 HCl was then added for 6 h, following which the cells were stimulated for 15 min with 10 ng/mL EGF ( The protein bands were visualized using the enhanced chemiluminescence (ECL) detection system (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocols.

Xenograft model
Six-week-old female BALB/c nu/nu mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). They were subcutaneously inoculated into the right flank with 5×10 6 NCI-H1975 cells (Day 1). After the tumor had grown to around 200-300 mm 3 of tumor volume (TV), the mice were randomly allocated into 4 groups: control, osimertinib, CX-6258 HCl, and osimertinib combined with CX-6258 HCl. Osimertinib (1 mg/kg in 1% DMSO) and CX-6258 HCl (25 mg/kg in 1% DMSO) were given daily by oral gavage. The tumors were measured using a Vernier caliper 3 times a week. The antitumor activity was evaluated using TV, which was estimated from the following equation: TV = AB 2 /2 (where A and B are tumor length and width, respectively). The mice were euthanized by cervical dislocation at the end of the experiments. All experiments involving animals were performed following ARRIVE guidelines and after the approval of the local ethical committee of Hunan Cancer Hospital.
The degree of immunostaining was evaluated using the proportion of positively stained tumor cells and staining intensities. This project has been reviewed and approved by Hunan Cancer Hospital IRB Committee (Approval number: 201902651).

Statistical analysis
Data were expressed as mean ± standard deviation (SD). Statistical analysis was conducted using SPSS 16.0 software. P values were calculated using the unpaired two-tailed Student t-test or one-way ANOVA test. The differences were considered significant at P values less than 0.05.

High expression of PIM1 was related to the poor prognosis of lung cancer
Increasing evidence has shown that PIM1 kinase is an effective carcinogenic driver of different malignant tumors (17,19). The Oncomine database showed that the expression level of the PIM1 gene was higher in different pathological types of lung cancer compared with normal tissues ( Figure 1A). The PIM1 association result was analyzed using the Pearson correlation test on the Linked Omics database ( Figure 1B). Meanwhile, the survival analysis of PIM1 from the GSE30219 and GSE 3141 datasets was performed in the PROGgeneV2 database, revealing that high the expression of the PIM1 gene had a significantly negative correlation with overall survival ( Figure 1C,D). Furthermore, we found that cell proliferation and cell cycle were positively correlated with PIM1. This suggests that PIM1 might promote cell proliferation via modulating cell cycle ( Figures S1,S2). These findings indicate that PIM1 is a potential oncogenic biomarker in lung cancer and targeting PIM1 might be an ideal approach in the targeted therapy of lung cancer.

CX-6258 HCl combined with osimertinib inhibited the proliferation of H1975 and PC9 cells
The effects of the combination of osimertinib with CX-6258 HCl on the proliferation of H1975 (EGFR-T790M) and PC9 (exon 19 deletions) lung cancer cells were investigated. As shown in Figure 2A,B, both H1975 and PC9 cells were more sensitive to osimertinib than to CX-6258 HCl. The administration of osimertinib combined with CX-6258 HCl was more effective in suppressing cell viability in a dose-and time-dependent manner (Figure 2A,B,C,D). The colony formation assay showed that the combined treatment exerted an additive inhibitory effect on colony formation compared with osimertinib alone ( Figure 2E,F).
The colonies in the combined-treatment group were much smaller and fewer compared with the colonies in the control group ( Figure 2G,H). These findings showed that CX-6258 HCl, combined with osimertinib, had a better growthinhibitory effect compared with either inhibitor used alone.

CX-6258 HCl combined with osimertinib inhibited cell migration in H1975 and PC9 cells
The wound-healing assay was used to detect the effects of combined administration on cell migration. Treatment with the combination of osimertinib and CX-6258 HCl for 48 h significantly increased the open space compared with single administration ( Figure 3A,B). Results presented in Figure 3C,D showed that the combination inhibited cell migration in H1975 and PC9 cells more effectively in quantification. In the transwell assay, the combination elicited a strong inhibitory effect on tumor cell migration ( Figure 3E,F). Quantification is shown in Figure 3G,H. Taken together, the results indicate that the combination of osimertinib and CX-6258 HCl can exert additively inhibitory effects on cell migration.

CX-6258 HCl combined with osimertinib induced apoptosis
An Annexin V-FITC/PI apoptosis detection kit was used to analyze the effect of the combination of osimertinib and CX-6258 HCl on apoptosis. Most cells in the control group remained intact. However, apoptosis was induced in the osimertinib-treated H1975 cells from 5.1%±0.1% to 11.2%±0.9% (P<0.001, Figure 4A,B), and in the osimertinib-treated PC9 cells from 6.9%±0.4% to 11.5%±1.2% (P<0.001, Figure 4C,D). The apoptotic rate increased to 16.0%±0.3% and 16.2%±0.5%, respectively, in the CX-6258 HCl-treated H1975 and PC9 cells compared with the osimertinib-treated cells. The combined treatment produced the most significant effect on apoptosis, with the apoptotic rate 20.6%±0.7% in H1975 cells and 24.4%±0.5% in PC9 cells (P<0.001, Figure 4B,D). These data showed that CX-6258 HCl alone not only induced the apoptosis of EGFR-mutated NSCLC cell lines but also significantly increased the apoptotic rate when combined with osimertinib. Furthermore, the expression levels of cleaved PARP and cleaved caspase-3 were significantly higher in the combined-treatment group compared with the untreated and single-treatment groups ( Figure 5). These findings confirm that CX-6258 HCl has great potential in combination with other drugs to promote apoptosis.

CX-6258 HCl combined with osimertinib blocked the phosphorylation of EGFR and STAT3
To elucidate the role of CX-6258 HCl and osimertinib in H1975 and PC9 cells, the expression levels of key proteins related to STAT and EGFR pathways were assessed. CX-6258 HCl decreased the expression level of PIM1 and phosphorylation levels of STAT3. The combination resulted in a more visible suppression compared with single administration ( Figure 5). The expression of EGFR and its downstream molecules AKT and ERK was detected.
Osimertinib effectively inhibited the phosphorylation of p-EGFR, p-ERK, and p-AKT, but did not alter the total protein levels of EGFR, ERK, and AKT in H1975 and PC9 cell lines. However, the combination of CX-6258   The experiment was repeated 3 times. The Student's t-test was used between the osimertinib group and the combination group.  HCl and osimertinib blocked the phosphorylation of EGFR, ERK, and AKT and caused the weaker expression of p-EGFR compared with osimertinib alone ( Figure 5). These results showed that the combination suppressed the phosphorylation of EGFR.

CX-6258 HCl combined with osimertinib inhibited tumor growth in a xenograft model
The NCI-H1975 xenograft model was used to evaluate whether the combination had an antitumor effect in vivo. H1975 cells were implanted in the flanks of BALB/c nu/nu mice, and the animals were randomized into 4 groups until tumors grew to around 200-300 mm 3 of TV. The results showed that the combined administration did not gain satisfactory achievements in the first week. However, it did eventually suppress the growth of the H1975 subcutaneous tumor ( Figure 6A,B). Meanwhile, the safety of the combined administration was evaluated by taking the weight of mice.
The combined administration did not significantly influence body weight (P>0.05, Figure 6C). Isolated tumors from each treatment group were presented in Figure 6D. Next, mice were euthanized, and the tumors were excised. The expression of PIM1, p-EGFR, and caspase-3 was detected in tumor tissues using immunohistochemistry. The number of caspase 3+ tumor cells increased with each single drug administration. As expected, it was further increased in the combined administration group (Figure 7). The results revealed a strong expression of p-EGFR in tumor cells of the control group. CX-6258 HCl slightly decreased the expression level of p-EGFR. However, both osimertinib and combined administration further decreased the expression of p-EGFR (Figure 7). The expression level of PIM1 significantly decreased in tumor cells treated with CX-6258 HCl (Figure 7). These data were consistent with the in vitro data.

Conclusions
Like other targeted therapies, resistance to EGFR-TKI is inevitable. Many studies have discovered the molecular mechanism underlying acquired drug resistance to EGFR inhibitors. However, little is known about the ways to delay or overcome this resistance. Increasing evidence has demonstrated that combined or multiple targeted therapies can help overcome EGFR-TKI resistance. This novel study showed that the combination of CX-6258 HCl and osimertinib exerted a synergetic anti-tumor effect in vivo and in vitro; the JAK/STAT3 pathway was also suppressed ( Figure 8).
PIM kinases are responsible for the improvement in sensitivity to tumor inducers (9). The expression level of PIM1 in different pathologic types of lung cancer was higher than that in normal lung tissue. Moreover, the high expression of PIM1 might correlate with the poor prognosis of NSCLC. These findings may offer clinical support for subsequent research. CX-6258 HCl is a pan-PIM kinase inhibitor that selectively inhibits all 3 PIM kinases (PIM1, PIM2, and PIM3) (15); it has the most substantial inhibitory effect on PIM1.
Sustaining proliferative signaling and promoting invasion and metastasis are 2 significant features of tumors (20). In the present study, the proliferation and migration of EGFR-mutated cells were significantly repressed with the combined administration of 2 inhibitors compared with the control or single administration. Such results were linked to the combination of osimertinib and CX-6258 HCl for inhibition of EGFR activation. EGFR is well known as a receptor for the epithelial growth factor in cell proliferation, invasion, and metastasis as well as signal transduction. This study demonstrated that the combination of osimertinib and CX-6258 HCl significantly inhibited the expression of p-EGFR compared with osimertinib alone. Additionally, the expression of p-EGFR was detected in the tumor tissue from xenograft models using immunohistochemistry, which was consistent with the results in vitro. These results are related to the fact that PIM kinases promoted tumor cell proliferation by regulating cell cycle suppressors p27 and p21 (21,22) and regulated migration through inhibiting the JAK/STAT pathway (23,24). Also, the combined treatment markedly reduced the tumor size in an H1975 xenograft model. Meanwhile, minimal bodyweight loss or toxicity was seen in the combined treatment group, suggesting that the simultaneous inhibition of PIM kinases led to more durable antitumor effects.
Programmed cell death by apoptosis is an important mechanism of homeostatic balance. Impaired apoptosis plays a central role in cancer development (25)(26)(27). PIM kinases are known to prevent apoptosis through the direct phosphorylation of BAD and the release of anti-apoptotic Bcl-2 protein (28,29). In this study, Annexin V-FITC/ PI double staining results revealed that the combination effectively induced apoptosis in H1975 and PC9 cells. As the main effector caspase, caspase-3 is a common downstream effector of multiple apoptotic pathways and occupies a core position in the process of apoptosis. Once activated, caspase-3 has a high proteolytic ability to degrade intracellular target substances on a large scale and eventually cause irreversible cell death (30). The activation of caspase-3 depends heavily on the release of cyt-c (31,32). However, PIM kinases increase the release of Bcl-2 (33,34). The overexpressed Bcl-2 forms a heterodimer with BAX, which closes the mitochondrial permeability transition pore and inhibits the delivery of cyt-c, thereby hindering the activation of caspase-3 (35,36). The combination of osimertinib and CX-6258 HCl caused the increased expression of cleaved caspase-3 and cleaved PARP, further proving that CX-6258HCl exerted a robust synergistic action by promoting apoptosis. In line with the in vitro experiment, the expression of activated caspase-3 was upregulated.
The third-generation EGFR-TKIs bind covalently to a cysteine on codon 797, which not only works on the original mutation but also targets the most common mode  (37,38). In this study, two NSCLC cell lines with different mutations were selected to observe the efficacy of the combination of osimertinib and CX-6258 HCl. Acquired resistance of EGFR-TKIs leads to the failure of clinical treatment in EGFR-mutated NSCLC. Hata et al. found that the inhibitory effect of the third-generation irreversible EGFR inhibitor was stronger on early-resistant PC9-GR2 cells than on lateresistant PC9-GR3 cells, which was attributed to the downregulation of Bcl-2 interacting mediator of cell death (BIM) in late-resistant PC9-GR3 cells (39). A significant feature of drug-resistant cells is the evasion of drug-induced apoptosis, especially in late-resistant cells. Thus, antiapoptotic inhibitors can restore the sensitivity of resistant clones to drugs. Also, MacKeigan found that STAT3 was overexpressed in EGFR-mutant NSCLC cells, but EGFR-TKI did not inhibit the activation of STAT3. The inhibitors of JAK2 not only blocked the activation of STAT3 but also significantly enhanced the effect of erlotinib to repress the formation of clones and the growth of xenografts in vivo (40,41). As a highly selective pan-PIM inhibitor, CX-6258 HCl possesses the capability of inducing apoptosis, besides directly binding to STAT3 and reducing its phosphorylation (42). Therefore, the expression of STAT3 and p-STAT3 was evaluated in the present study. The combination of CX-6258 HCl and osimertinib significantly downregulated the expression of p-STAT3, suggesting that the pan-PIM kinase inhibitor with EGFR-TKI suppressed tumor growth via targeting the STAT3 pathway in NSCLC. Nevertheless, it was not possible to verify the hypothesis that the joint method could reverse osimertinib resistance because the development of the osimertinib-resistant cell lines and animal models was not very successful.
In conclusion, this study was novel in reporting the antitumor effect of the combination of pan-PIM inhibitor and osimertinib on EGFR-mutated NSCLC cell lines. The interventions designed in this study not only more efficiently inhibited the phosphorylation of EGFR but also restored drug-induced apoptosis and repressed other alternative pathways. Therefore, CX-6258 HCl can act synergistically with other targeted drugs to exert more powerful anti-tumor effects. Immunohistochemistry images for PIM1, p-EGFR, and caspase-3 staining of tumor tissue in BALB/c nu/nu mice. All images were 20× magnification; bar =50 μm.