JI017 Sensitizes EGFR-T790M NSCLC to Erlotinib by Extinguishing the STAT3

Abstract

Introduction

First-generation EGFR-TKIs in NSCLC frequently lose efficacy as a result of the secondary EGFR-T790M mutation and a “persistent-STAT3” prosurvival pathway that sustains STAT3–survivin signaling in the face of EGFR inhibition. Preclinical evidence demonstrates that phytochemicals in the JI017 herbal formulation (2:1:1, Angelica gigas: processed Aconitum carmichaeli: Zingiber officinale) can reinstate erlotinib sensitivity in T790M-positive NSCLC, and relevant molecular mechanisms have been examined.

Methods

Anti-proliferative activities were evaluated in A549 (EGFR-WT), HCC827 (EGFR Δ19), and H1975 (EGFR L858R/T790M) cells utilizing MTT assays, colony formation, and Annexin V/7-AAD flow cytometry. Mechanistic analyses included immunoblotting for p-EGFR (Tyr1068/1173), p-JAK2, p-STAT3 (Tyr705), PARP, Bcl-2, survivin, and AXL, complemented by RT-qPCR for BIRC5 and AXL transcripts. Drug interaction effects were determined using the Chou–Talalay combination index (CompuSyn). Anti-tumor efficacy was assessed in H1975 xenografts treated for 14 days with vehicle, JI017, erlotinib, or combination therapy; tumors underwent H&E staining and IHC for p-STAT3, survivin, and Ki-67.

Results

JI017 inhibited proliferation in all NSCLC cell lines tested, showing greatest effectiveness in H1975, where it triggered PARP cleavage and suppression of Bcl-2 and survivin expression. In H1975, co-treatment with JI017 and erlotinib led to synergistic growth inhibition, eradicated colony growth, and significantly elevated apoptotic cell populations compared to single treatments. While erlotinib alone reduced p-EGFR and p-JAK2, it left p-STAT3 largely unaltered, reflecting persistent-STAT3 activity. The combination regimen abrogated p-STAT3, further lowered p-EGFR and p-JAK2 levels, diminished BIRC5 mRNA, and decreased both AXL protein and transcript levels. In vivo, the drug combination achieved sustained tumor stasis relative to controls or monotherapy; combination group tumors displayed widespread necrosis and substantial decreases in p-STAT3, survivin, and Ki-67.

Discussion

These data support the suppression of STAT3–survivin as the primary mechanism by which JI017 sensitizes EGFR-T790M models to erlotinib. The consistent down-regulation of AXL indicates the inhibition of an AXL-mediated bypass that may maintain STAT3 signaling during EGFR blockade, although causality has yet to be confirmed. The marked in-vivo tumor inhibition without observable toxicity underscores the translational promise as a low-toxicity therapeutic adjunct.

Conclusions

JI017 restores erlotinib sensitivity in EGFR-T790M NSCLC by inhibiting STAT3–survivin signaling and possibly reducing AXL-mediated resistance, resulting in durable antitumor effects both in vitro and in vivo. Additional preclinical studies and early-phase clinical assessment of JI017 in combination with erlotinib are justified.

Keywords

JI017 herbal formulation erlotinib sensitization STAT3 survivin (BIRC5)

Introduction

According to the latest GLOBOCAN estimates, lung cancer continues to be the leading cause of cancer-related mortality on a global scale. In 2020, there were approximately 2.2 million new lung cancer cases (constituting 11.4 % of all cancers) and nearly 1.8 million deaths (accounting for 18.0 % of all cancer deaths).¹ Lung cancer is usually classified into non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), with NSCLC representing approximately 80–85 % of all cases.² Activating mutations within the epidermal growth factor receptor (EGFR) gene are among the most frequent genetic events in NSCLC and appear with notably high prevalence in Asian populations.^3-5 First-generation EGFR tyrosine-kinase inhibitors (EGFR-TKIs), including gefitinib and erlotinib, produce objective response rates of roughly 70 % in EGFR-mutant, advanced NSCLC and have thus been established as standard first-line therapy.^6-8 However, almost all patients who initially respond eventually experience disease progression within 9–12 months due to acquired resistance, with secondary gate-keeper EGFR-T790M mutations responsible for 50–60 % of these cases.^9,10 Although third-generation EGFR-TKIs (for example, Osimertinib) were engineered to target T790M, resistance continues to arise and is frequently associated with adverse effects or economic barriers.¹¹ Furthermore, a portion of patients presents with intrinsic resistance to EGFR-TKIs, which is driven by EGFR-independent mechanisms such as activation of the STAT3–survivin pathway or AXL over-expression.^12,13Consequently, combination strategies have attracted increasing interest as an effective approach to address both intrinsic and acquired EGFR-TKI resistance. The co-administration of anti-VEGF agents (bevacizumab or ramucirumab) substantially extends progression-free survival (PFS) compared to EGFR-TKI monotherapy,¹⁴ and the use of AXL inhibitors in combination regimens has also demonstrated promising clinical outcomes.¹⁵ Targeting the STAT3–survivin axis is an emerging strategy, as IL-6/JAK1/2–STAT3 signaling contributes to de-novo EGFR-TKI resistance through up-regulation of survivin; furthermore, pre-clinical studies indicate that combining the STAT3 inhibitor OPB-111077 or the survivin inhibitor YM155 with EGFR-TKIs produces distinct synergistic effects.^16-18 Collectively, these findings underscore VEGF, AXL, and the STAT3–survivin axis as actionable targets for the development of effective combination therapies against EGFR-mutant NSCLC.

JI017 is a 2:1:1 herbal formulation consisting of Angelica gigas, Aconitum carmichaeli, and Zingiber officinale, which has exhibited broad-spectrum anti-tumor effects in breast, ovarian, prostate, and lung cancers.^19-22 JI017 initiates endoplasmic reticulum (ER) stress-mediated cell death and, in lung-cancer cells, promotes the generation of reactive oxygen species (ROS), triggering both autophagy and apoptosis. The two principal marker compounds in JI017, decursin and 6-gingerol, have demonstrated the ability to eliminate TKI-resistant cancer cells through inhibition of the STAT3–survivin pathway.^23-25 Due to its multitargeted actions, JI017 shows promise as an adjunct to EGFR-TKIs in T790M-positive NSCLC.

In this study, we assess whether JI017 acts synergistically with erlotinib to counteract T790M-mediated resistance. We quantify the degree of apoptotic synergy produced by the JI017 and erlotinib combination, elucidate the roles of STAT3–survivin pathway inhibition and AXL/EGFR bypass blockade as critical mechanisms, and provide evidence of therapeutic benefit in an EGFR-T790M xenograft model. These results support the potential of a low-toxicity, natural product-based strategy as a clinically relevant treatment option for patients with resistance to first or second generation EGFR-TKIs.

Materials & Methods

Preparation for JI017 and Erlotinib

JI017 is a botanical formulation consisting of Angelica gigas (Ag), Zingiber officinale Roscoe (Zo), and processed Aconitum carmichaeli (Ac) in a 2:1:1 proportion. The powdered dried extract was provided by Hanpoong Pharmaceutical Co., Ltd. (Jeonju, Republic of Korea) and dissolved in DMSO to generate concentrated stock solutions. Stock solutions were aliquoted and stored at −20 °C, protected from light. Erlotinib (#S7786; SelleckChem, Houston, TX, USA) was prepared as a 10 mM DMSO stock, aliquoted, and stored at −20 °C.

Cell Culture

The human NSCLC cell lines A549 (EGFR-WT; ATCC, CCL-185; RRID: CVCL_0023), HCC827 (EGFR exon 19 deletion; KCLB, 70827; RRID: CVCL_2063), and H1975 (EGFR L858R/T790M; ATCC, CRL-5908; RRID: CVCL_1511) were maintained in RPMI-1640 medium (Welgene, Gyeongsan, Republic of Korea) supplemented with 10% fetal bovine serum (FBS; #16000-044, GIBCO) and 1% antibiotic-antimycotic solution (#LS203-01, WELGENE). Cells were cultured at 37°C in a humidified incubator with 5% CO₂.

MTT Assay

Cells were plated in 96 well plates (A549 and H1975, 4 × 10³ cells/well; HCC827, 6 × 10³ cells/well) and treated the following day with JI017, erlotinib, or their combination for 24, 48, or 72 h. Aqueous MTT reagent (5 mg/ml, Sigma-Aldrich) was subsequently added to each well, and plates were incubated at 37°C for 2 h. After removing the supernatant, formazan crystals were solubilized in 100μl of DMSO (Duksan, Seoul, Republic of Korea), and absorbance was measured at 570 nm using a microplate reader (Synergy H1; BioTek, USA). IC₅₀ values were determined with GraphPad Prism 8 software. Drug interactions were evaluated with the Chou–Talalay method using CompuSyn. The combination index (CI) was calculated for JI017 and erlotinib, with CI < 1 indicating synergy, CI = 1 additivity, and CI > 1 antagonism.

Clonogenic Assay

H1975 and HCC827 cells (200 cells/well) were seeded in 6 well plates and, after 24h, subjected to treatment with JI017, erlotinib, or their combination for 12 days. Colonies were stained with 0.5% crystal violet (#V5265, Sigma-Aldrich, St. Louis, MO, USA), rinsed, imaged, and subsequently dissolved in extract buffer. Absorbance results were normalized to the vehicle control. The absorbance was quantified at 590nm using a microplate reader.

Annexin V/7-AAD Apoptosis Analysis

Following 24 h of treatment with JI017, erlotinib, or the combination, cells were harvested, washed with PBS, and stained with Annexin V (BD Biosciences, NJ, USA) and 7-AAD (Abcam, Cambridge, UK) for 15 min at room temperature. Acquisition was performed using CytoFLEX flow cytometry (Beckman Coulter, CA, USA), and subsequent analysis was conducted with FlowJo software (Treestar, OR, USA).

Western Blotting

Cells were washed twice with PBS, lysed using RIPA buffer (Biosesang, Yongin, Republic of Korea), and maintained on ice for 30 min. Cellular debris was eliminated by centrifugation at 13,000 rpm for 30 min at 4°C. Protein concentrations were assessed using a Bio-Rad protein assay dye (Bio-Rad Laboratories, Hercules, CA, USA). Equal quantities of protein were separated on SDS-PAGE gels and transferred to nitrocellulose(NC) membranes (Cytiva, Little Chalfont, Buckinghamshire, UK) at 100 volts for 2h. Blots were incubated with primary antibodies overnight at 4°C, washed with PBS-T (0.1% Tween-20 in PBS), and visualized using an ECL detection kit (DoGEN, Seoul, Korea). The following primary antibodies were applied: PARP (#9542; RRID: AB_2160739), Bcl-2 (#15071; RRID:AB_2744528), Survivin (#2808; RRID: AB_2063948), PI3K (#42574257; RRID: AB_2281020), p-PI3K (#4228; RRID: AB_659940), AKT (#9272; RRID: AB_329827), p-AKT (#9271; RRID: AB_329825), mTOR (#2972; RRID: AB_330978), p-mTOR (#2974; RRID: AB_2262884), EGFR (#2232; RRID: AB_331707), p-EGFR (Tyr1068; #3777; RRID: AB_2096270), p-EGFR (Tyr1173; #4407; RRID: AB_331795), AXL (#8661; RRID: AB_11217435), JAK2 (#3230; RRID: AB_2128522), p-JAK2 (Tyr1007; #4406; RRID: AB_10706164), STAT3 (#4904; RRID: AB_331269), p-STAT3 (Tyr705; #9145; RRID: AB_2491009) and GAPDH(#5174; RRID: AB_10622025; Cell Signaling Technology, MA, USA).

RNA Isolation and Real-Time PCR

Cells were collected and total RNA was isolated using the easy-BLUE Total RNA Extraction Kit (#17061; iNtRON Biotech, Republic of Korea) following the manufacturer’s instructions. RNA purity and concentration were determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). cDNA was synthesized from 2 µg total RNA using the cDNA Synthesis Kit (#6110B; TaKaRa, Otsu, Shinga, Japan). Real-time PCR was conducted on a Light Cycler 96 system (Roche) using the SensiFAST™ SYBR® Hi-ROX kit (#BIO-92020; Meridian Bioscience, OH, USA). Expression levels were calculated by the 2-ΔΔCt method with GAPDH serving as a reference gene. Primer sequences are provided in Table 1.

Table 1.

Sequences of Primers Used for Real-Time PCR

Primer type	Primer name	Primer sequence
Human	AXL	F: 5′-GTT TGG AGC TGT GAT GGA AGG C-3′
	AXL	R: 5′-CGC TTC ACT CAG GAA ATC CTC C-3′
	BIRC5	F: 5′-CCA CTG AGA ACG AGC CAG ACT T-3′
	BIRC5	R: 5′-GTA TTA CAG GCG TAA GCC ACC G-3′
	GAPDH	F: 5′-GTC TCC TCT GAC TTC AAC AGC G-3′
	GAPDH	R: 5′-ACC ACC CTG TTG CTG TAG CCA A-3′

Animal Studies

BALB/c Nude mice (5 weeks old, male) were obtained from Nara Biotech (Seoul, Korea). The animals underwent an acclimatization period of one week prior to experimentation. H1975 cells (2.5 x 107) were injected subcutaneously in a 1:2 mixture (100 μL) of Matrigel and PBS. Once tumors were established after 7 days, the mice were randomly assigned to four groups (n = 6) and administered JI017 and erlotinib orally at the specified doses and time points for 2 weeks (Figure 4A). Tumor volume was assessed three times per week using the formula (length x width2/2). All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Kyung Hee University (KHU; KHSASP-25-068), and all mice were maintained under specific pathogen-free conditions with a 12/12 h light/dark cycle.

JI017 powder was dissolved in sterile distilled water to obtain non-cytotoxic working concentrations, using a 2:1:1 ratio of Angelica gigas: Zingiber officinale: Aconitum carmichaeli. Erlotinib was prepared and diluted following the manufacturer’s instructions.

Histology and Immunohistochemistry (IHC)

Tumor tissues fixed in formaldehyde and paraffin-embedded (FFPE) were sectioned at 7 μm and stained with hematoxylin and eosin (H&E). For IHC analysis, FFPE tumors were sectioned at 7 μm, deparaffinized, and rehydrated. Antigen retrieval was performed in sodium citrate buffer (pH 6.0) for 10 min using a microwave. Following cooling, endogenous peroxidase activity was blocked with hydrogen peroxide, and tissues were incubated in blocking solution for 1 h. Primary antibodies were applied overnight at 4 °C. Afterward, sections were incubated with biotinylated secondary antibodies (Vectastain ABC-AP staining kit, Vector Laboratories, CA USA) for 1 h and developed with 3,3′-Diaminobenzidine (DAB). Slides were examined using a microscope (Carl Zeiss, Germany). Antibodies used were: p-STAT3 (Tyr705, #9145; Cell Signaling Technology, MA, USA), Survivin (#2808; Cell Signaling Technology, MA, USA), and Ki67 (#ab16667; Abcam, Cambridge, UK).

Statistical Analysis

Quantitative results are reported as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). Statistical significance was analyzed via one-way analysis of variance (ANOVA) or two-way ANOVA based on experimental requirements. All statistical evaluations were conducted with GraphPad Prism (version 8.0. GraphPad Software Inc., USA). A P-value less than 0.05 was considered statistically significant. Statistically significant group differences are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

Results

JI017 Potently Inhibits Cell Proliferation and Induces Apoptosis in EGFR-T790M NSCLC Cell Lines

To determine the anti-proliferative effects of JI017, MTT assays were conducted in A549 (EGFR Wild type), HCC827 (EGFR Δ19), and H1975 (EGFR L858R/T790M) cell lines after exposure to a range of JI017 concentrations for 24, 48, and 72 h.

As illustrated in Figure 1A and B, JI017 decreased cell viability in a manner dependent on both dose and time in all three cell lines, with H1975 cells being the most sensitive, relative to HCC827 and A549. At 48 h, treatment with 100 µg/mL JI017 reduced H1975 cell viability to below 50% compared with untreated controls, whereas HCC827 and A549 cells exhibited more modest reductions.The IC50 values of JI017 at 48 h were 67.8 µg/mL for H1975 cells, 230.6 µg/mL for HCC827 cells, and 158.3 µg/mL for A549 cells. Long-term colony formation assays corroborated these observations: H1975 cells exposed to JI017 (50–200 µg/mL) for 10 days showed a dose-dependent reduction in clonogenic survival, reaching statistical significance at 200 µg/mL (p = 0.0045 vs. untreated control), while HCC827 cells displayed only minimal and statistically insignificant changes under comparable conditions (Figure 1C). To assess whether the growth suppression observed was attributed to apoptosis, we subsequently examined principal apoptotic markers by Western blot. In H1975 cells, JI017 triggered PARP cleavage and decreased expression of anti-apoptotic proteins Bcl-2 and survivin in a concentration-dependent manner; in contrast, these proteins remained essentially stable in HCC827 cells (Figure 1D). In agreement with these molecular findings, flow cytometric analysis using Annexin V/7-AAD staining showed a substantial elevation in apoptotic H1975 cells at 200 µg/mL compared with controls, whereas HCC827 cells exhibited only a slight increase in apoptosis with the same treatment (Figure 1E). This marked difference indicates that JI017 preferentially induces apoptosis in EGFR-T790M mutant, erlotinib-resistant H1975 cells rather than in EGFR-TKI–sensitive HCC827 cells.

Together, these findings indicate that JI017 produces selective cytotoxic and pro-apoptotic responses in EGFR-T790M mutant NSCLC cells, underscoring its potential to counteract resistance mediated by this mutation.

JI017 Synergizes With Erlotinib to Overcome Intrinsic Resistance and Inhibit Proliferation in EGFR-T790M NSCLC Cells

To investigate whether JI017 can enhance the antiproliferative activity of erlotinib, H1975 cells were treated for 48 h with both erlotinib and JI017. As shown in Figure 2A, the combination of 5 µM erlotinib and 80 µg/mL JI017 resulted in significant inhibition of cell viability, yielding a combination index (CI) of 0.57, indicative of synergism. This synergy was corroborated by colony formation assays. H1975 cells received 12-day treatments with no drug, JI017 (80 µg/mL), erlotinib (5 µM), or the two in combination. The combination significantly suppressed colony growth compared to single-agent treatments (Figure 2B). To clarify the underlying mechanisms of this synergy, we evaluated alterations in critical survival signaling molecules (PI3K/AKT/mTOR) after 24 h of exposure (Figure 2C). Compared with untreated controls, the combination treatment significantly reduced phosphorylation of PI3K (p < 0.05) and mTOR (p < 0.01), whereas phosphorylation of AKT was not significantly altered. Quantitative analysis normalized to GAPDH further confirmed that the suppression of mTOR phosphorylation by the combination was significantly greater than that observed with either JI017 or erlotinib alone (p < 0.05; Supplementary Figure 3B).

Combination of JI017 With Erlotinib Abolishes Canonical and Bypass Survival Pathways in EGFR-T790M NSCLC

We next examined whether the increased apoptosis contributes to the observed growth inhibition. Western blot analysis performed after a 24 h treatment in H1975 cells showed that the combination of JI017 and erlotinib significantly enhanced PARP cleavage (p < 0.05 vs. erlotinib; p < 0.01 vs. JI017) and resulted in a more pronounced downregulation of the anti-apoptotic proteins Bcl-2 (p < 0.05 vs. control) and survivin(p < 0.0001 vs. control), compared to either agent alone (Figure 3A). In alignment with these molecular alterations, flow cytometric analysis using Annexin V/7-AAD staining indicated that the combination of JI017 and erlotinib induced greater cell apoptosis compared to monotherapy (Figure 3B).

Furthermore, to understand the mechanism responsible for the synergistic cytotoxicity of JI017 and erlotinib, we analyzed key survival pathways in H1975 cells after a 2 h treatment with Control (No-treat), JI017, erlotinib, or their combination. As indicated in Figure 3C, erlotinib alone reduced levels of p-EGFR (Tyr1068, Tyr1173) and p-JAK2(p < 0.05 vs. control for p-JAK2), but did not suppress p-STAT3 (Tyr705). This finding is consistent with the “persistent-STAT3” activity described for first-generation TKIs.¹⁷ Monotherapy with JI017 only modestly reduced p-STAT3 levels. Conversely, the combination significantly reduced p-STAT3 levels (p < 0.05 vs. control and JI017), concurrently down-regulated total AXL protein(p < 0.05 vs. control and JI017), and further inhibited both p-EGFR and p-JAK2, implying that JI017 disrupts the AXL-mediated bypass that maintains STAT3 activity under EGFR inhibition. Mirroring these protein-level effects, qPCR analyses demonstrated that the combined treatment led to reduced expression of BIRC5 (survivin) and AXL (Figure 3D). Notably, BIRC5 encodes survivin, and the corresponding changes at the protein level are shown in Figure 3A.

Combination of JI017 With Erlotinib Suppresses Tumor Growth and Survival Signaling in an EGFR-T790M Xenograft Model

Building on the in vitro synergy and mechanistic data described above, we next evaluated the in vivo efficacy of the combination regimen. One week after subcutaneous inoculation of H1975 (L858R/T790M) cells, the combination treatment was administered over a 2-week period as outlined in Figure 4A. Monotherapy with either JI017 or erlotinib resulted in only a modest delay in tumor progression, while the combined regimen induced sustained tumor stasis throughout the 14-day treatment period. Notably, upon cessation of dosing, tumors from the control and erlotinib groups exhibited rapid regrowth, mirroring the pre-clinical counterpart of the clinical flare phenomenon,^9,26 whereas those receiving the combination showed limited regrowth throughout the observation window (Figure 4B). Macroscopically excised tumors in the combination group were substantially smaller compared to control and single-agent groups, confirming the superior antitumor efficacy of the combination (Figure 4C). In addition, no significant body weight changes were noted in any treatment group, indicating the absence of systemic toxicity (Figure 4D). These results suggest that, as an adjunct, JI017 can maintain suppression of tumor proliferation and mitigate the withdrawal flare commonly observed after discontinuation of first-generation EGFR TKIs, thereby preventing rapid disease progression and providing a pharmacologic bridge pending initiation of subsequent therapy. H&E staining demonstrated densely packed, viable cells in control tumors, with focal necrosis after each monotherapy and extensive necrotic regions in combination-treated lesions (Figure 5A). Immunohistochemical analysis revealed that combination therapy nearly abolished the proliferation marker Ki-67 and reduced p-STAT3 and survivin expression in tumor tissues, whereas monotherapies achieved only partial inhibition (Figure 5B).

Taken together with the previous in vitro findings, these in vivo results demonstrate that JI017 enhances erlotinib activity at the tumor site, translating molecular inhibition of the EGFR–JAK2 pathway and the compensatory AXL–STAT3–survivin signaling into prolonged tumor growth arrest and increased apoptosis in EGFR-T790M NSCLC.

Discussion

In this study, we demonstrate that the herbal formulation JI017 overcomes erlotinib resistance in EGFR-T790M non-small cell lung cancer (NSCLC) by inhibiting the STAT3/survivin axis and down modulating AXL. JI017 as a single agent exhibited limited cytotoxicity in EGFR wild type A549 and exon 19 deleted HCC827 cells, but significantly suppressed proliferation and induced apoptosis in T790M mutant H1975 cells, highlighting its selective antitumor activity against the inherently resistant genotype. When combined, erlotinib and JI017 displayed in vitro synergy, and the combination treatment resulted in sustained tumor stasis in the H1975 xenograft model, preventing the rapid tumor regrowth observed after discontinuation of erlotinib and thereby supporting its chemosensitizing potential. In this context, the present study extends previous observations of EGFR-TKI resistance by demonstrating that suppression of the STAT3–survivin axis can be functionally linked to both enhanced erlotinib sensitivity and sustained tumor growth control in an EGFR-T790M setting. Unlike prior studies that primarily described persistent STAT3 signaling as a resistance-associated phenotype, our findings provide experimental evidence that pharmacologic disruption of this pathway can translate into durable antitumor effects in vivo, particularly following treatment withdrawal.

Our molecular findings provide insight into the complementary effects of JI017 and erlotinib. At the molecular level, erlotinib monotherapy reduced p-EGFR and downstream p-JAK2, but had little impact on p-STAT3,^27-29 which is consistent with a “persistent-STAT3” phenotype widely implicated as a central mechanism of TKI resistance.^30-32 The addition of JI017 concurrently reduced total AXL, ablated p-STAT3, and suppressed survivin while further diminishing p-EGFR/p-JAK2. Since AXL over-expression is a well-characterized bypass pathway supporting STAT3 activation in TKI-treated tumors,^33-35 these results indicate that JI017 acts as a STAT3-survivin inhibitor that closes a vulnerability left by first generation TKIs.

TKI cessation in patients who are awaiting re-biopsy or transitioning to a third generation agent frequently triggers a disease flare, manifesting as rapid tumor progression and clinical decline.^9,26,36 In our xenograft model, tumors in the vehicle and erlotinib groups exhibited immediate growth resurgence following treatment cessation. Conversely, tumors treated with JI017 and erlotinib showed only minimal regrowth. The observation that JI017 mitigates the withdrawal flare highlights its potential as a low toxicity bridging therapy during molecular confirmation, or in settings where osimertinib is inaccessible or associated with prohibitive costs. Furthermore, as JI017 reduced p-STAT3 and survivin, which are markers related to de-novo resistance and poor prognosis, it may offer clinical benefit for the 10∼20 % of EGFR mutant patients who fail to respond to TKIs.^30,31,37,38 With respect to mechanism, several studies have established that JI017, as well as its constituents decursin, benzoyl aconitine, and 6-gingerol, enhance intracellular reactive oxygen species (ROS) levels, thereby activating PERK/eIF2α dependent endoplasmic reticulum (ER) stress and subsequent antitumor apoptosis.^19-22,39-43 The current study proposes that the ROS/ER stress signaling cascade may function as an upstream regulator of AXL degradation, STAT3 dephosphorylation, and survivin destabilization. Nevertheless, additional investigation is necessary to elucidate the precise molecular targets.

Current strategies to overcome EGFR-TKI resistance have expanded beyond AXL blockade and STAT3/survivin inhibition to encompass mutant-selective fourth generation EGFR inhibitors, MET-targeted bispecific antibodies or TKIs, and HER3-specific antibody drug conjugates.^44-46 Nonetheless, epithelial to mesenchymal transition via AXL and activation of the persistent STAT3/survivin signaling circuit remain the most commonly triggered adaptive pathways during first and second generation TKI failure.^33,47 JI017 inhibits the PI3K/AKT/mTOR pathway and disrupts the STAT3/survivin survival circuit, thereby enhancing erlotinib-induced cytotoxicity and facilitating apoptotic cell death. This concurrent inhibition of primary growth pathways and compensatory escape mechanisms is rarely accomplished by a single small-molecule compound, potentially delaying the development of multiclonal resistance that often limits narrowly targeted therapies. Importantly, unlike existing small molecule AXL inhibitors, JI017 was not associated with weight loss or organ toxicity in our xenograft studies, supporting its potential as a low-toxicity adjuvant or maintenance approach.^48,49

This study is limited by the use of a single T790M cell line and a fixed dosing regimen. Validation in osimertinib-resistant, C797S-positive, and MET-amplified models, as well as in patient-derived xenografts and immune-competent systems, will be necessary to generalize these results and to assess potential immunomodulatory effects. Chemoproteomic pulldown assays and genome-wide CRISPR screens are needed to determine whether JI017 exerts direct interaction with the AXL–STAT3 complex or primarily modulates upstream redox signaling. Moreover, comprehensive pharmacokinetic analysis and formulation development are critical for future clinical application. Finally, factorial studies combining JI017 with fourth-generation EGFR-TKIs, MET inhibitors, or HER3-directed antibody drug conjugates will help to clarify whether its multitarget actions yield additive or synergistic efficacy within dynamic treatment paradigms.

Conclusion

JI017 restores erlotinib sensitivity in EGFR-T790M NSCLC through inhibition of the STAT3/survivin axis and downregulation of AXL, thus suppressing both canonical and compensatory survival pathways. This dual targeting results in sustained antitumor efficacy in vivo and prevents the TKI-withdrawal flare, without inducing detectable toxicity. Overall, these results support further pre-clinical evaluation and early-phase clinical investigation of JI017 combined with erlotinib as a low-toxicity regimen for patients with intrinsic or acquired resistance to first- or second-generation EGFR-TKIs.

Supplemental Material

Supplemental Material - JI017 Sensitizes EGFR-T790M NSCLC to Erlotinib by Extinguishing the STAT3–Survivin Axis

Supplemental Material for JI017 Sensitizes EGFR-T790M NSCLC to Erlotinib by Extinguishing the STAT3–Survivin Axis by Daeun Kim, Young-Ha Kim, Gaeun Choi, Chunhoo Cheon, Seong-Gyu Ko in Integrative Cancer Therapies

Footnotes

ORCID iDs

Chunhoo Cheon

Seong-Gyu Ko

Ethical Considerations

The animal study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Kyung Hee University (Seoul, Republic of Korea) under approval number KHSASP-25-068.

Author Contributions

KDE designed and conceptualized the study, performed data acquisition and analysis, and prepared the manuscript. KYH contributed to manuscript review and editing. CGE conducted experiments and assisted with data collection. CHC reviewed the manuscript. KSG participated in study conceptualization, provided oversight, and critically reviewed the manuscript. All authors reviewed and approved the final version of the manuscript.

Funding

This research was supported by the National Research Foundation of Korea (NRF) grant, funded by the Korean government (MSIT) (No. RS-2020-NR049559).

Declaration of Conflicting Interests

The authors state that they have no competing interests.

Data Availability Statement

All data produced or examined in this study are provided within this published article.*

Supplemental Material

Supplemental material for this article is available online.

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