NAT-A Overcomes EGFR-TKIs Resistance in Non-Small Cell Lung Cancer by Targeting the β-Catenin/PKM2 Axis

NAT-A Overcomes EGFR-TKIs Resistance in Non-Small Cell Lung Cancer by Targeting the β-Catenin/PKM2 Axis | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 28 October 2025 V1 Latest version Share on NAT-A Overcomes EGFR-TKIs Resistance in Non-Small Cell Lung Cancer by Targeting the β-Catenin/PKM2 Axis Authors : Liyun Liu , Xin Wang , Xinkai Wang , Li Guan , Yahui Liao , Qi He , Yanxi Sun , … Show All … , Ying Wu , Xiao Lin , Shuping Wang , Yongjun Zhou , Guohua Zhou , Weihua Jiao , Hou wen Lin , and Fan Sun [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.176164609.95049633/v1 185 views 100 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose: Hyperactivation of the Wnt/ β -catenin pathway is a key driver of EGFR-TKI resistance in non-small cell lung cancer (NSCLC), with pyruvate kinase M2 (PKM2) serving as a critical transcriptional coactivator of β -catenin. Neoantimycin-A (NAT-A) isolated from Streptomyces conglobatus by our group demonstrats excellent anti-cancer activities against various human tumor cells. However, whether NAT-A can overcome acquired EGFR-TKI resistance and its underlying mechanisms remain elusive in NSCLC. Experimental Approach: The anti-tumor effects of NAT-A on EGFR-TKI-resistant NSCLC cells were investigated through luciferase reporter assays, immunoblotting, immunofluorescence, flow cytometry, co-immunoprecipitation, siRNA transfection and plasmid construction assays. In addition, the in vivo efficacy of NAT-A was further evaluated in EGFR-TKI-resistant NSCLC xenograft models. Key Results We demonstrate that PKM2 upregulation drives TKI resistance independently of glycolysis via stabilizing β -catenin and amplifying Wnt/ β -catenin signaling. Here, we identify NAT-A as a novel therapeutic agent capable of overcoming EGFR-TKI resistance. Mechanistically, NAT-A disrupts the interaction between phosphorylated PKM2 (Ser37/Y105) and β -catenin, triggering β -catenin degradation via PKM2-mediated ubiquitination and subsequent suppression of downstream oncogenic signaling. Both in vitro and in vivo studies confirm NAT-A’s anti-tumor efficacy, including inhibition of cell proliferation and induction of apoptosis in EGFR-TKI-resistant models. Conclusion and Implications: NAT-A overcomes EGFR-TKI resistance by disrupting the PKM2/ β -catenin interaction and inducing its degradation, thereby inhibiting tumor growth. Our findings unveil the PKM2/ β -catenin axis as a novel therapeutic target and highlight NAT-A as a unique small molecule disrupting this interaction for treating refractory EGFR-TKI-resistant NSCLC. NAT-A Overcomes EGFR-TKIs Resistance in Non-Small Cell Lung Cancer by Targeting the β -Catenin/PKM2 Axis Liyun Liu a, # , Xin Wang d, # , Xinkai Wang a,e , Li Guan a , Yahui Liao a , Qi He a , Yanxi Sun a , Ying Wu a , Xiao Lin a , Shuping Wang a , Yongjun Zhou a , Guohua Zhou f , Weihua Jiao a,* , Hou wen Lin a,b, c* , Fan Sun, a,* . a Department of Pharmacy, Ren Ji Hospital, College of Clinical Pharmacy, State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China b Integrated Innovation Center for Marine Drugs, Shanghai Jiao Tong University, Shanghai 200240, China c Marine Biomedical Science and Technology Innovation Platform of Lin-gang Special Area, Shanghai 201306, China d Department of Pharmacy, 904th Hospital of Joint Logistics Support Force of PLA, Wuxi 214000, China e School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China f School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China # The first two author contributed equally. * Corresponding author Weihua Jiao, Houwen Lin and Fan Sun, Department of Pharmacy, Ren Ji Hospital, College of Clinical Pharmacy, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China E-mail addresses: [email protected] (Weihua Jiao), [email protected] (Houwen Lin), and [email protected] (Fan Sun). Phone:021-68383339, Fax: +86-21-58732594 Abstract Background and Purpose: Hyperactivation of the Wnt/ β -catenin pathway is a key driver of EGFR-TKI resistance in non-small cell lung cancer (NSCLC), with pyruvate kinase M2 (PKM2) serving as a critical transcriptional coactivator of β -catenin. Neoantimycin-A (NAT-A) isolated from Streptomyces conglobatus by our group demonstrats excellent anti-cancer activities against various human tumor cells. However, whether NAT-A can overcome acquired EGFR-TKI resistance and its underlying mechanisms remain elusive in NSCLC. Experimental Approach: The anti-tumor effects of NAT-A on EGFR-TKI-resistant NSCLC cells were investigated through luciferase reporter assays, immunoblotting, immunofluorescence, flow cytometry, co-immunoprecipitation, siRNA transfection and plasmid construction assays. In addition, the in vivo efficacy of NAT-A was further evaluated in EGFR-TKI-resistant NSCLC xenograft models. Key Results We demonstrate that PKM2 upregulation drives TKI resistance independently of glycolysis via stabilizing β -catenin and amplifying Wnt/ β -catenin signaling. Here, we identify NAT-A as a novel therapeutic agent capable of overcoming EGFR-TKI resistance. Mechanistically, NAT-A disrupts the interaction between phosphorylated PKM2 (Ser37/Y105) and β -catenin, triggering β -catenin degradation via PKM2-mediated ubiquitination and subsequent suppression of downstream oncogenic signaling. Both in vitro and in vivo studies confirm NAT-A’s anti-tumor efficacy, including inhibition of cell proliferation and induction of apoptosis in EGFR-TKI-resistant models. Conclusion and Implications: NAT-A overcomes EGFR-TKI resistance by disrupting the PKM2/ β -catenin interaction and inducing its degradation, thereby inhibiting tumor growth. Our findings unveil the PKM2/ β -catenin axis as a novel therapeutic target and highlight NAT-A as a unique small molecule disrupting this interaction for treating refractory EGFR-TKI-resistant NSCLC. Keywords: Non-Small Cell Lung Cancer (NSCLC), EGFR-TKI resistance, β -catenin/PKM2 Axis, Wnt pathway, Neoantimycin-A CCK-8, Cell Counting Kit-8; CHX, cycloheximide; Co-IP, Co-immunoprecipitation; EGFR-TKI, Epidermal growth factor receptor tyrosine kinase inhibitor; DAPI, 4,6-diamino-2-phenylindole; H&E, hematoxylin and eosin; NSCLC, Non-Small Cell Lung Cancer; WB, Western Blot; IC 50 , half maximal inhibitory concentration. NATs, Neoantimycins; NAT-A, Neoantimycin-A; PKM2, The pyruvate kinase M2; HCC827-GR, HCC827- gefitinib -resistant; HCC827-OR, HCC827-osimertinib-resistant; PC9-GR, PC9- gefitinib -resistant; PC9-OR, PC9-osimertinib-resistant. Introduction Epidermal growth factor receptor (EGFR) mutations drive oncogenesis in approximately 80% of non-small cell lung cancer (NSCLC) cases. Despite therapeutic advances with EGFR-tyrosine kinase inhibitors (TKIs) such as gefitinib and osimertinib, the 5-year survival rate remains below 21% [Soria et al., 2018; Siegel et al., 2025]. A major obstacle to sustained treatment efficacy is the inevitable development of acquired resistance to successive generations of EGFR-TKIs, highlighting an urgent need for novel strategies targeting resistance mechanisms [Shah, R et al., 2020; Piper-Vallillo et al., 2020]. The Wnt/ β -catenin pathway, a critical regulator of cancer stemness and therapeutic resistance, has emerged as a pivotal contributor to EGFR-TKI resistance [Liu et al., 2020]. Conventional Wnt inhibitors like XAV939 have shown limited efficacy, failing to effectively downregulate β -catenin and reverse resistance [Huang et al., 2023]. Recent investigations demonstrate that transcriptional co-regulators DDX17 and Notch3 directly bind cytoplasmic β -catenin, preventing its proteasomal degradation and enhancing its nuclear translocation to amplify transcriptional activation of oncogenic targets [Li et al., 2017; Arasada et al., 2018]. Notably, the circFBXW7-encoded 185AA peptide suppresses Wnt signaling by enhancing β -catenin ubiquitination, effectively restoring EGFR-TKI sensitivity in resistant models [Li et al., 2023], reinforcing β -catenin degradation as a viable therapeutic avenue. The pyruvate kinase M2 (PKM2), a pivotal regulator of aerobic glycolysis, is aberrantly overexpressed across human malignancies where it drives tumor progression [Wang et al., 2020] and correlates with poor clinical outcomes [Chang et al., 2021]. As a β -catenin transcriptional coactivator, nuclear PKM2 directly binds β -catenin to form a functional complex that drives β -catenin-dependent transcription and cell proliferation [Yang et al., 2011]. This PKM2/ β -catenin axis exacerbates chemoresistance via HIF-1α-mediated multidrug resistance (MDR) protein upregulation, while cooperative interactions with regulators like Gankyrin amplify resistance through β -catenin/c-Myc/PKM2 metabolic reprogramming in hepatocellular carcinoma [Liu et al., 2019]. Pharmacological disruption of this axis reverses chemoresistance in diverse malignancies: it suppresses metabolic adaptation in head and neck cancer and overcomes gemcitabine resistance in cholangiocarcinoma by blocking β -catenin signaling [Gupta et al., 2018]. A study further highlights cryptotanshinone, a herbal inhibitor of the PKM2/ β -catenin pathway, as a promising agent for improving survival and overcoming drug resistance in breast cancer [Zhou et al., 2020]. Nonetheless, the role of PKM2 in regulating β -catenin-mediated EGFR-TKI resistance remains uncharacterized, and the therapeutic potential of targeting PKM2/ β -catenin interactions awaits further exploration. Our group isolated Neoantimycins (NATs) from Streptomyces conglobatus , and these compounds exhibit excellent anti - cancer activities against various human tumor cells [Zhou et al., 2018; Lin et al., 2019]. Subsequent research demonstrated that NAT - F, a new NAT member, can trigger mitochondrion - mediated apoptotic pathways in NSCLC [Liu et al., 2019]. Notably, our recent progress has shown that NAT - A and its derivatives have significant antitumor activity against chemotherapeutic - resistant human gastric and colon cancer [Lin et al., 2019; Zhu et al., 2024]. However, it is still unclear whether NATs can overcome acquired resistance to EGFR - TKIs and what the underlying mechanisms might be. In this study, we demonstrate that the natural compound NAT-A suppresses Wnt/ β -catenin signaling in gefitinib- and osimertinib resistant NSCLC cells. We confirmed that PKM2 stabilized β -catenin to activate Wnt/ β -catenin signaling independently of glycolysis, which is effectively abrogated by NAT-A. Specifically, NAT-A disrupts the phosphorylation-dependent interaction between PKM2 (Ser37/Y105) and β -catenin, inducing PKM2-dependent β -catenin ubiquitination and subsequent proteasomal degradation. This molecular intervention translates to robust anti-cancer efficacy, as evidenced by in vitro dose-response studies and in vivo xenograft experiments. Our findings establish NAT-A as a novel PKM2/ β -catenin axis-targeting agent and propose a therapeutic strategy for refractory EGFR-TKI-resistant NSCLC. Materials and methods Experimental materials NAT-A (Figure S1A) was isolated from Streptomyces conglobatus by our group [Zhou et al., 2018] . Gefitinib , Osimertinib , MG-132 was purchased from Sigma-Aldrich (SML1135, Missouri, USA). CHIR-99021(S1263), Cycloheximide (CHX) (S7418) and Shikonin (S8279) were purchased from Selleckchem (Houston, TX, USA). Cell lines and cell culture PC9 and HCC827 cell lines were exposed to increasing concentrations of osimertinib for 6 months to establish osimertinib-resistant sublines (PC9-OR and HCC827-OR). All cell lines were cultured in RPMI 1640 containing 10% FBS at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air. The HEK293, HCT116, SW480, HCC827-GR, PC9-GR, PC9 and HCC827 cell lines and their culture conditions were the same as described previously [Liu et al., 2020; Zhu et al.,2024]. Cell proliferation assays For CCK-8 assays, cells were seeded into 96-well plates at a density of 3000 cells per well followed by treatment with the tested agents at various concentrations for indicated time points. Cell viability was then assessed with CCK-8 reagents as described previously [Liu et al., 2019]. For colony formation assays, cells were cultivated in six-well plates at a density of 1000 cells per well and incubated for 24h. Followed by NAT-A treatment for indicated time, cells were maintained at 37 °C with 5% CO 2 for 8 ~12 days. At the end of the experiment, colonies formed were washed with PBS, fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 30min. Colonies and the images were recorded under the light microscope. Flow cytometry assays The resistant cell lines were plated in 6-well plates (1× 10 5 cells per well) for routine culture for 24 h. Cells were then treated with indicated concentrations of NAT-A for 48h. After treatment, cells were harvested and subjected to an Annexin V-PI staining as previously described [Liu et al., 2019]. Immunofluorescence assay The cells were washed with ice-cold PBS and fixed for 30min in 4% paraformaldehyde at room temperature. Following 1 h blocking in 5% BSA at room temperature, the specified primary antibodies incubation proceeded overnight at 4°C, followed by 1h secondary antibody (Cell Signaling Technology, Danvers, USA) incubation at room temperature. After DAPI nuclear staining (5 min), images were captured with a Leica SP8 confocal system Antibody against p-PKM2 (Tyr105) (Cat# PA5-101033, RRID: AB_2815862) and p-PKM2 (Ser37) (Cat# PA5-37684, RRID: AB_2554292) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The β -catenin antibody was purchased from Abcam (Cambridge, MA, USA). Real-time quantitative PCR (RT-qPCR) Total mRNA from each sample was extracted using RNA simple Total RNA Kit (Tiangen Biotech, Beijing, China). First-strand cDNA was synthesized with PrimeScript™ RT reagent Kit (Perfect Real Time) (Takara, Japan) according to the manufacturer’s instructions. RT-qPCR analysis was performed by SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Japan) using Light Cycler 480 System (Roche, Switzerland). The primer sequences for cyclind1 and c-myc were as previously reported [Liu et al., 2020]. Luciferase reporter assay β -catenin transcriptional activity was analyzed using TOP/FOP flash luciferase reporter as described previously [Liu et al., 2020]. Briefly, cells were seeded in 24-well plates for 24 h before co-transfection with wild-type or mutant TCF/LEF reporter and Renilla luciferase plasmids via X-tremeGENE HP (Roche, Germany). Cells were treated with either vehicle control or NATs, followed by lysis in passive buffer. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, WI, USA), with Wnt/ β -catenin signaling transcriptional activity expressed as the TOP/FOP ratio. siRNA transfection The interfering small RNA (siPKM2) and control siRNA were provided by OBiO Technology (Shanghai, China) Corp., Ltd., and their sequences were listed in Table 1. The β -catenin siRNAs and control siRNA, as well as the transfection procedure, were the same as described in our previous study [Liu et al., 2020]. Briefly, cells were plated in 6-well plates at a density of 2 × 10 5 cells/mL and cultured for 24 h. Gene-specific siRNAs and corresponding control siRNA were transfected into cells using siRNA-Mate plus according to the manufacturer’s protocol (GenePharma, China). Table 1. The sequences of si PKM2 and negative control used in siRNA. si PKM2 #1 CCUGGACAUUGAUUCACCATT UGGUGAAUCAAUGUCCAGGTT si PKM2 #2 GCCAUCUACCACUUGCAAUTT AUUGCAAGUGGUAGAUGGCTT si PKM2 #3 GGCUGGACUACAAGAACAUTT AUGUUCUUGUAGUCCAGCCTT control siRNA UUCUUCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT Plasmid construction Vectors encoding the Flag-tagged PKM2, Flag-Ub, HA-Ub and HA-tagged β -catenin were constructed by GenePharma (Shanghai, China). Cells were seeded in 6-well plates. The next day, they were transfected with 2μg of Flag-tagged PKM2 or HA-tagged β -catenin DNA using 6 μL Lipofectamine TM LTX Reagent (Invitrogen) and 2.5 μL PLUS TM Reagent (Invitrogen, USA) in 200 μL serum-free Opti-MEM (Life Technologies, USA). After 6 h, the culture medium was replaced and 24 h later, cells were treated with NAT-A for indicated time points. Co-immunoprecipitation (Co-IP) assays Immunoprecipitation was performed with the lysates from indicated cultured cells and followed by immunoblotting with corresponding antibodies. Briefly, the cells were collected and washed three times with cold PBS. Following 30-min lysis in ice-cold buffer at 4°C, protein concentrations were quantified. Equal amounts of cell lysates were then subjected to immunoprecipitation by overnight rotation with indicated antibodies at 4°C. Pierce™ Protein A/G Magnetic Beads (Thermo Fisher, #88802; 20 μL/sample) were added, and incubated with rotation for 2 h at 4°C. Beads were washed three times with PBS buffer, and immunocomplexes were collected by centrifugation at 1,000 g and resolved on SDS-PAGE gel and subsequently transferred to PVDF membranes (Millipore) for subsequent western blot analysis. Antibody against PKM2 (Cat# ab131021, AB_11156038) was purchased from Abcam (Cambridge, MA, USA). Antibody against Flag (Cat# F3165, AB_259529) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Antibodies against HA (Cat# ab9110, AB_307019) and Ubiquitin (Cat# ab134953, AB_2801561) were purchased from Abcam (Cambridge, MA, USA). Western blot analysis Total cellular proteins, along with nuclear and cytosolic fractions, were extracted from cancer cells according to established protocols, followed by western blot analysis as previously described [Liu et al.,2019]. Briefly, the cells were lysed in RIPA buffer on ice for 30 min, followed by centrifugation (12,000rpm, 10 min, 4°C) to collect supernatants. A BCA protein assay (Beyotime Biotechnology, Shanghai, China) was employed to determine protein concentrations. Equal amounts of protein were separated by 10%～15% SDS-PAGE and electro transferred to PVDF membranes (Millipore, MA, USA). Following 1 h room temperature blocking, membranes were incubated overnight at 4°C with specific primary antibodies, then with HRP-conjugated secondary antibodies. The immunoblots were profiled by using the ECL system (LI-COR Biosciences). Antibodies against PKM1(Cat#7067, AB_2715534), PKM2(Cat #4053, AB_1904096), p-PKM2(Tyr105) (Cat #3827, AB_1950369), EGFR (Cat #4267, AB_2895042), p-EGFR (Cat #3777, AB_2277657), Akt (Cat #9272, AB_39825), and p-Akt (#Ser37) (Cat#4060, AB_2315049) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against active- β -catenin (Cat# 05-665, AB_309887) was obtained from Millipore (Merck, Darmstadt, Germany). The remaining antibodies and reagents used in this study were described previously [Liu et al., 2019; Liu et al.,2020]. In vivo xenograft assay Four-to-five-week- old male BALB/c nude mice (20∼22 g) were purchased from Shanghai Laboratory Animal Center (Shanghai, China).To establish tumor xenografts, 1×10⁶ EGFR-TKI-resistant or parental cells in 100 μL PBS/Matrigel (1:1; BD Biosciences) were subcutaneously injected into the right flank (n=8 per group).When tumor volumes reached 80～100 mm³, mice were randomized to vehicle control (i.p.), gefitinib(i.p.), Osimertinib (i.p.), or NAT-A(i.p.) every other day at designated doses (n=8 per group). Tumor growth was monitored by measuring tumor size with calipers twice per week. Tumor volume (TV) was calculated as V = (length × width 2 ) /2. At the experimental endpoint, mice were sacrificed and tumors were collected for immediate imaging and weight measurement. Tissue samples were either fixed in 4% neutral buffered formalin or flash-frozen in liquid nitrogen for preservation at -80°C until analysis. All animal experiments were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Ren Ji Hospital, Shanghai, China. Statistical analysis The data from at least three independent experiments are presented as mean ± standard deviation (SD) analyzed by GraphPad Prism8.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was performed using Student’s t-test for paired samples, while comparisons involving multiple groups were assessed through one-way ANOVA followed by Dunnett’s post-test. Values of * p < 0.05, ** p < 0.01, and *** p < 0.001 were regarded as statistically significant. Results NAT-A inhibits aberrant Wnt/β-catenin signaling in EGFR-TKI-resistant NSCLC Building upon our observation of Wnt/ β -catenin pathway activation in gefitinib [Liu et al., 2020] and osimertinib-resistant NSCLC cells (Fig. S1B), we systematically evaluated NAT analogs derived from Streptomyces conglobatus by our group for Wnt modulatory activity [Zhou et al., 2018]. Initial screening revealed NAT-A and NAT-F, particularly NAT-A at 1 μM, significantly suppressed Wnt3a-CM and CHIR-99021-induced β -catenin transcriptional activity in HEK293 cells (Fig. 1A), a phenomenon consistently observed in Wnt/ β -catenin hyperactivated cell models (Fig. S1.C). Dose-response analyses further revealed that NAT-A treatment reduced β -catenin transcriptional activity by approximately 50% in these cells (Fig1.B). Complementary western blot validation confirmed that NAT-A decreased total β -catenin, its active form, and Wnt target protein Cyclin D1 and C-myc (Fig. 1C, and Fig. S1D), collectively demonstrating NAT-A’s potent Wnt pathway inhibition. Notably, NAT-A treatment dramatically inhibited TOPFlash/FOPFlash reporter activity across four EGFR-TKI-resistant cell lines (Fig1.D), and reduced cyclinD1 and c-myc mRNA levels in responsive cells (Fig. S1E). Furthermore, NAT-A consistently reduced the protein expression levels of β -catenin and Wnt target proteins in these cell lines (Fig. 1E, Fig. S1F), extending to both nuclear and cytoplasmic β -catenin as confirmed by nuclear-cytoplasmic fractionation and immunofluorescence (Fig1F, G, and Fig1 S1G, H). A significant reduction in β -catenin protein levels was observed upon co-treatment with NAT-A and the protein synthesis inhibitor CHX (Fig. 1H, Fig.S1I). Furthermore, MG-132 partially restored the levels of β -catenin in NAT-A treated EGFR-TKI-resistant cells (Fig. 1I, S1J), suggesting that NAT-A promotes the proteasomal degradation of β -catenin. Consistently, NAT-A-mediated β -catenin decrease occurred via polyubiquitin-dependent degradation in the resistant cells (Fig. 1J) and HA- β -catenin/Flag-Ub-transfected HEK293 cells (Fig1. S1K). Functionally, NAT-A exhibited potent anti-proliferative effects (IC 50 : 0.42 ~1.16 μM) (Fig. 1K), suppressed colony formation by 21.1 %-33.8 % (Fig. 1L), and induced apoptosis (3.5 ~ 5.0-fold increase in Annexin V+ cells) (Fig.1M). These data indicate that NAT-A suppresses hyperactivated Wnt signaling through ubiquitination-mediated degradation of β -catenin to overcome EGFR-TKI resistance in NSCLC. Fig1 . NAT-A suppresses hyperactivated Wnt signaling and inhibits the growth of EGFR-TKI-resistant NSCLC cells . ( A ) NATs (A, F, D, J, K) inhibit TOPFlash reporter activity in HEK293 cells. Cells transfected with the TOPFlash plasmid were pretreated with DMSO or NATs for 2 h, then exposed to Ctrl-CM, Wnt3a- CM, or CHIR-99021 for 17h. Luciferase activity was then measured. ( B ) NAT-A dose-dependently suppresses TOPFlash reporter activity in Wnt/ β -catenin hyperactivated cell models (HEK293, HCT116, SW480) following 12h treatment. ( C ) Western blot analysis of β -catenin expression in HEK293 cells following NAT-A treatment for 12 h. ( D ) NAT-A suppressed TOPFlash reporter activity in EGFR-TKI-resistant cell lines following 24h treatment. ( E, F ) Effects of NAT-A on β -catenin signaling in gefitinib- and osimertinib resistant PC9 cells. Cells were analyzed by immunoblotting, assessing protein levels, downstream targets, and nuclear-cytoplasmic distribution. ( G ) Representative immunofluorescence images and quantifications of β -catenin in PC9-GR cells treated with 1 µM NAT-A for 24 h. Scale bar=25μm. ( H-J ) Immunoblotting assessed the stability and degradation of β -catenin in EGFR-TKI-resistant cells treated with CHX or MG-132 containing NAT-A or not at specified times. ( K-M) Inhibitory effects of NAT-A on EGFR-TKIs resistant cell lines were assayed by comparative heatmap of IC 50 values by CCK-8 assay (K), colony formation analysis (L) and apoptosis quantification by flow cytometry (M). Data were represented as mean ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to control. NAT-A promotes β-catenin degradation via PKM2-mediated ubiquitination Emerging evidence highlights PKM2’s critical role in stabilizing oncoproteins such as β -catenin [Yang et al, et al., 2011], with its isoform-specific overexpression strongly linked to therapeutic resistance in lung cancers [Wang et al 2021]. Analysis of GEPIA database revealed a positive correlation of pkm2 with CTNNB1 transcript in NSCLC patient samples (Fig.2A). EGFR-TKI-resistant cell lines exhibited significantly elevated PKM2 expression compared to their parental counterparts, while PKM1 levels remained unchanged (Fig.S2A). This observation was further supported by a strong positive correlation between PKM2 and β -catenin protein levels in resistant cells (Pearson correlation, r =0.6588, p = 0.0198) (Fig.2B). The PKM2-selective enzymatic inhibitor shikonin did not alter the inhibitory effect of NAT-A on resistant cells (Fig. 2C), whereas PKM2 siRNA-mediated knockdown partly abolished its effects (Fig. 2D, Fig.S2B), demonstrating that NAT-A’s inhibitory effects depend on PKM2 protein expression. Subsequently, we found that PKM2 silencing reduced both active and total β -catenin levels, concurrently downregulating the drug resistance marker ABCG2 (Fig.2E, Fig.S2C). Furthermore, overexpression of wild-type PKM2 in parental cells markedly increased β -catenin expression and activated its downstream signaling (Fig.2F), an effect maintained even with the catalytically inactive K367M mutant (Fig2. S2D, E), confirming PKM2’ s β -catenin regulation is independent of its metabolic function. Notably, β -catenin silencing left PKM2 levels unaffected, demonstrating that PKM2 regulation occurs independently of β -catenin signaling and suggesting that NAT-A may mediate its effects on β -catenin through a PKM2-dependent mechanism (Fig.2G). Indeed, NAT-A treatment effectively reversed PKM2-induced β -catenin hyperactivation, suppressing β -catenin and its effectors c-Myc/cyclin D1 without altering PKM2 stability (Fig.2H, Fig.S2F). To elucidate whether NAT-A disrupts PKM2-mediated β -catenin stabilization, overexpression of either wild-type PKM2 or its catalytically inactive mutant (K367M) demonstrated that NAT-A treatment consistently promoted β -catenin degradation while simultaneously increasing polyubiquitination levels (Fig.2I), while PKM2 knockdown partially rescued NAT-A-induced β -catenin destabilization (Fig.2J). These findings definitively establish that NAT-A promotes β -catenin degradation through PKM2-dependent ubiquitination. Fig2 . NAT-A triggers PKM2-dependent ubiquitination to drive β -catenin degradation . ( A ) GEPIA database result indicated a positive correlation between CTNNB1 and pkm2 mRNA expression in NSCLC human samples. ( B ) Pearson correlation analysis of protein expression of β -catenin and PKM2 in four EGFR-TKIs resistant cell lines (r =0.6588, p = 0.0198, n=12). ( C, D ) CCK-8 assay assessed cell viability in PC9-GR and PC9-OR cells treated with NAT-A following PKM2 inhibitor shikonin treatment (C) or knockdown(D). ( E, F ) Western blot analyses revealed that PKM2 knockdown reduced β -catenin and its active-form protein levels (E), while PKM2 overexpression significantly active β -catenin signaling (F). ( G ) Immunoblot analyses of the expression of PKM2 protein remained unchanged upon β -catenin knockdown. ( H ) Immunoblotting assessed the β -catenin and its downstream targets in PKM2-overexpressing parent cells upon NAT-A treatment. ( I, J) Effect of NAT-A on β -catenin degradation. PC9 cells expressing wild-type or K367M mutant PKM2 (I) or PK9-OR cells with PKM2 knockdown and transfected HA-Ub plasmid (J), were treated with NAT-A and analyzed by immunoblotting. Data were represented as mean ± S.D. of three independent experiments. *** p < 0.001, compared to control. NAT-A disrupts cytoplasmic β-catenin-PKM2 interaction via suppression of PKM2 phosphorylation at Ser37/Y105 Enhanced PKM2- β -catenin binding in EGFR-TKI-resistant cells was confirmed through co-immunoprecipitation (Co-IP) assays, aligning with the established role of nuclear PKM2/β-catenin complexes in oncogenic growth regulation [Yang et al., 2011] (Fig. S3A). NAT-A treatment significantly attenuated endogenous PKM2- β -catenin interactions in resistant cell lines (Fig. 3A), as demonstrated by dose-dependent reductions in β -catenin binding to both Flag-tagged PKM2 and endogenous PKM2 in transfected parental cells (Fig. 3B) (Fig. S3B). This inhibitory effect was further validated in HEK293 cells expressing exogenous PKM2 and β -catenin (Fig.S3C). Notably, PKM2 phosphorylation at Ser37 [Niu et al., 2020] and Tyr105 [Liu et al., 2025] induce its dimerization of PKM2 and nuclear translocation, thereby facilitating PKM2-mediated transcriptional regulation of tumor-associated genes through β -catenin collaboration. Indeed, elevated levels of pSer37/Y105-PKM2 were observed in resistant cells (Fig. S3D). NAT-A reversed PKM2 overexpression-induced hyperphosphorylation at these sites and disrupted the interaction between phosphorylated PKM2 and β -catenin, as evidenced by Co-IP assays (Fig. 3C and D). Subcellular fractionation immunoblotting revealed enhanced cytoplasmic PKM2 accumulation in the resistant versus parent cells (Fig.3E). Cytoplasmic-localized NES-tagged PKM2 was overexpressed in parental cells, where NAT-A treatment effectively reduced β -catenin-PKM2 binding, confirming its action in the cytoplasmic compartment (Fig. 3F). Notably, STAT3 - a known PKM2 substrate [Chen et al., 2020; Nagasawa et al., 2020] and resistance mediator-retained its interaction with PKM2 despite NAT-A treatment (Fig. S3E), correlating with unaltered STAT3 activation in resistant models (Fig. S3F). Furthermore, NAT-A potently suppressed hyperactivated phosphorylation of EGFR, AKT, and ERK pathways in both resistant cells and PKM2-overexpressing parental lines (Fig3G and H, Fig. S3G). Collectively, our findings demonstrate that NAT-A specifically disrupts cytoplasmic β -catenin-PKM2 axis while sparing STAT3-related signaling, ultimately reversing resistance-associated pathway activation. Fig3 . NAT-A selectively abrogates the cytoplasmic interaction between β -catenin and PKM2 in EGFR-TKI-resistant NSCLC cells. (A) Co-IP analysis of endogenous interaction between PKM2 and β -catenin in PC9-GR and PC9-OR cells. (B) In Flag-PKM2-expressing PC9 and HCC827 cells treated with NAT-A (0.2 and1 μM) for 24 h, Co-IP with anti-Flag antibody showed dose-dependent disruption of PKM2- β -catenin binding, as detected by immunoblotting for PKM2 and β -catenin. (C) Western blot analysis of β -catenin and pSer37/Y105-PKM2 in PKM2-overexpressing PC9 and HCC827 cells treated with 0.2 μM NAT-A for 24h. ( D) Co-IP analysis in Flag-PKM2-transfected PC9 cells showed that 0.2 μM NAT-A for 24 h disrupted pPKM2(S37/Y105)- β -catenin binding, as verified by immunoblotting. ( E ) Western blot analysis of the β -catenin in cytoplasmic and nuclear fractionation of parent (PC9) and resistant (PC9-GR and PC9-OR) cell lines. LMNA and GAPDH were considered as nuclear and cytosolic loading control, respectively. ( F) NES-PKM2-expressing PC9 and HCC827 cells treated with 0.2 μM NAT-A for 24 h were subjected to Flag- immunoprecipitation, with PKM2 and β -catenin interactions analyzed by immunoblotting. ( G, H). Western blot analysis of total and phosphorylated EGFR, AKT, and ERK in the resistant and PKM2-overexpressing parent cells upon NAT-A treatment. NAT-A overcomes EGFR-TKI resistance by targeting PKM2-dependent Wnt/β-catenin signaling In EGFR-TKI-resistant cells, NAT-A’s disruption of the β -catenin-PKM2 complex was modulated by PKM2 expression levels, with overexpression reducing TOPFlash suppression by ~60% and genetic ablation completely abolishing the effect (Fig. 4A, Fig. S4A). Similarly, overexpression of PKM2 in parental cells did not alter NAT-A’s potency relative to controls (IC 50 : 0.78 ± 0.11 vs 0.61 ± 0.08 μM, p > 0.05) (Fig. 4B), whereas PKM2 knockdown in resistant cells significantly reduced sensitivity (IC 50 increased from 0.67 ± 0.12 to 2.23 ± 0.58 μM, p < 0.01) (Fig. 4B and Fig. S4B). Consistently, NAT-A suppressed colony formation in PKM2-overexpressing sensitive cells but lost efficacy upon PKM2 depletion (Fig. 4C, D and Fig. S4C). Furthermore, NAT-A specifically induced apoptosis in PKM2-overexpressing cells, with PKM2 depletion substantially attenuating this pro-apoptotic effect (Fig. 4E and Fig. S4D). Mechanistically, NAT-A treatment triggered a coordinated shift in apoptotic regulators-elevating pro-apoptotic Bax while suppressing anti-apoptotic Bcl-2 (Fig. 4F and Fig. S4E). These data establish that NAT-A overcomes EGFR-TKI resistance by suppressing the PKM2-driven Wnt/ β -catenin signaling, thereby restoring apoptotic sensitivity through Bax/Bcl-2 modulation. Fig4 . NAT-A overcomes EGFR-TKI resistance by suppressing PKM2-dependent Wnt signaling pathway. ( A-D ) Effects of NAT-A inhibition in PKM2-overexpressing and PKM2-knockdown cells were assessed by TOPFlash luciferase reporter assays for β -catenin transcriptional activity (A), CCK-8 assays for IC 50 values (B), and colony formation assays for cell proliferation (C, D). ( E, F ) The effect of NAT-A on apoptosis in PKM2-knockdown and PKM2-overexpressing cells was determined by flow cytometry analysis and western blotting of Bax and Bcl-2 protein levels following 48 h exposure. Quantified colony numbers and apoptotic fractions (mean ± SD) shown. NAT-A suppresses EGFR-TKIs resistant NSCLC progression via PKM2/β-catenin axis inactivation in xenograft models To assess therapeutic potential in vivo , we established osimertinib-resistant PC9-OR xenograft models, in which osimertinib (5, 10 mg/kg) significantly inhibited PC9 tumor growth (T/C ratios: 66.67 ± 4.48% to 56.98 ± 5.23%) but failed to suppress PC9-OR tumors (Fig. S5A-D). Subsequently, NAT-A exhibited induced dose-dependent tumor weight reductions of 54% and 60% (0.025 and 0.05 mg/kg) in PC9-OR xenografts (Fig. 5A-C, S5E). In PC9-GR xenografts, T/C ratios decreased from 56.98% to 16.04% (0.01-0.05 mg/kg), while gefitinib remained ineffective (Fig. 5A-C, S5E). Histopathological analysis revealed increased necrosis in both PC9-GR and PC9-OR xenografts by H&E staining, with corresponding antiproliferative effects as evidenced by 73.29 and 83.33% reduction in Ki67-positive cells and 5.50 and 4.20 -fold elevation in TUNEL-positive apoptotic cells, respectively (Fig. 5D and E). Critically, NAT-A maintained an excellent safety profile, with no significant body weight loss or histopathological toxicity in major organs (Fig S5 Fand G). These data confirm NAT-A’s ability to selectively eradicate resistant tumors through coordinated suppression of proliferation and induction of apoptotic cell death. Consistent with in vitro findings, IHC analysis confirmed NAT-A-mediated β -catenin degradation in resistant xenografts, with 57.57 and 63.34% reduction in nuclear β -catenin intensity compared to controls (Fig. 5F). Immunofluorescence validation revealed that NAT-A treatment substantially inhibited p-PKM2 phosphorylation, with 79.34 -79.46% reduction at Ser37 and a 43.60-64.41% reduction at Tyr105 (Fig. 5G and Fig. S5H). The concomitant dephosphorylation of PKM2 and destabilization of β -catenin through disruption of PKM2/ β -catenin complex formation across both mouse xenografts underscore NAT-A’s efficacy in overcoming TKI resistance. Fig5 . NAT-A inhibits in vivo progression of EGFR-TKI-resistant NSCLC xenografts. ( A-C) . Tumor photographs (A), weight change (B), and growth kinetics (C) of PC9-GR and PC9-OR-CDX models treated with varying NAT-A doses or gefitinib (10 mg/kg). n = 6 mice per group. ( D ) Representative images of H&E and the proliferation marker Ki67 staining on tumor tissues excised from different groups in mice. Scale bars, 50μM. ( E ) Representative fluorescence images of tumors sections were taken were acquired following TUNEL staining, with green and blue represent TUNEL signal and nuclear stain, respectively. Scale bar, 50μm. Quantitative image analysis was conducted using ImageJ software, with data presented as mean ± SD (n = 6 mice/group). Discussion Despite significant advances in molecularly targeted therapies, NSCLC treatment continues to confront formidable clinical challenges, primarily due to high relapse rates and mortality driven by EGFR-TKI resistance-a dominant therapeutic barrier [Wang et al., 2021]. The refractory phenotypes associated with EGFR-TKI resistance underscore an urgent need to elucidate underlying molecular mechanisms and develop novel therapeutic strategies to improve patient outcomes. Emerging evidence positions the β -catenin-PKM2 signaling axis as a pivotal regulator of tumor aggressiveness, metabolic reprogramming, and drug resistance across malignancies, including lung cancer [Li et al., 2017; Tripathi et al., 2021]. While PKM2’s canonical glycolytic role in chemoresistance is well-characterized, its non-metabolic functions-particularly in stabilizing β -catenin and sustaining EGFR-TKI resistance-remain poorly understood, highlighting a critical knowledge gap in therapeutic targeting. NATs, a class of depsipeptide natural products, exhibit broad-spectrum anticancer activity through diverse mechanisms [Salim et al., 2014]. Salim et al. demonstrated that NAT-A and its derivatives (F, G, H) exhibited cytotoxicity against colon cancer via K-Ras plasma membrane localization disruption [Salim et al., 2014]. Our prior work identified a series of NATs from Streptomyces conglobatus , including NAT-A, which demonstrated potent anticancer activity (IC 50 = 2.9 nM ~7.3 μM) surpassing cisplatin across multiple cell lines [Zhou et al.,2018]. Notably, NAT derivatives such as prunustatin A, JBIR-04 and JBIR-05 function as GRP-78 suppressors [Umeda et al., 2005; Izumikawa et al., 2007]- a key associated molecular chaperone- while retaining anticancer efficacy against colorectal cancer through evasion of P-gp-mediated drug efflux [Salim et al., 2014], highlighting their potential to overcome multidrug resistance. Herein, our screening of five NATs derived from Streptomyces conglobatus identified NAT-A as the most potent inhibitor of Wnt/ β -catenin signaling. NAT-A effectively inhibited four EGFR-TKI-resistant cell lines (IC 50 = 0.42～1.16 μM), demonstrating its potential to overcome EGFR-TKI resistance. Building on our previous finding that NAT-A derivative NAT-F promotes mitochondria-related apoptosis in NSCLCs [Liu et al., 2019], we further show here that NAT-A specifically disrupts cytoplasmic β- catenin-PKM2 interactions, induces β -catenin degradation, and thereby triggers apoptosis in resistant cells. Accumulating evidences highlights the Wnt/ β -catenin pathway as a key regulator of stem-like properties and EMT driving EGFR-TKI resistance in NSCLC. Our prior studies validated its critical role in maintaining cancer stemness and conferring resistance to gefitinib in NSCLC stem cell models [Liu et al., 2020]. Recent studies demonstrate that oncoproteins SRPK1 or PAK2 promote β -catenin nuclear translocation and Wnt activation to sustain osimertinib and gefitinib resistance in NSCLC [Huang et al., 2023; Yi et al.,2022]. Pharmacological inhibition of aberrant Wnt/ β -catenin signaling has emerged as a synergistic strategy to enhance irreversible EGFR-TKI efficacy [Li et al., 2017; Arasada et al., 2018], though existing approaches face limitations. The Wnt/ β -catenin pathway inhibitor XAV939 failed to overcome EGFR-TKI resistance due to impaired β -catenin degradation [Huang et al., 2023]. Recent advances discovered that the circFBXW7-encoded 185AA peptide that induces β -catenin ubiquitination [Li et al., 2023], reinforce the critical role of β- catenin degradation as a pivotal therapeutic strategy. IMU1003, a compound derived from filamentous fungi , overcomes osimertinib resistance by reducing β -catenin localization in resistant lung cancer cells, but its effect on β -catenin degradation remains undetermined [Katagiri et al., 2023]. Herein, we identified NAT-A as a novel Wnt/ β -catenin signaling inhibitor overcomes these limitations. In both vitro and in vivo EGFR-TKI resistance models, NAT-A effectively mediated β -catenin ubiquitination and degradation, overcomes these limitations. Collectively, our finding provides a mechanistically distinct approach to reverse TKI resistance, aligning with emerging paradigms prioritizing protein degradation in refractory NSCLC therapy. PKM2, a pivotal glycolytic enzyme in tumor cells, aberrantly expressed across multiple cancers and correlates with aggressive tumor behavior and poor clinical outcomes. Emerging evidence implicates PKM2 overexpression in chemotherapeutic resistance via canonical glucose metabolic reprogramming [Wang et al., 2021; Chen et al., 2021]. In cisplatin-resistant NSCLC cells, PKM2 upregulation has been established as a biomarker and therapeutic target [Wang et al., 2021; Sun et al., 2020], while our findings reveal its specific cytoplasmic overexpression in EGFR-TKI-resistant cells compared to parental counterparts, with unchanged PKM1 levels-strongly suggesting PKM2’s non-metabolic role in TKI resistance. Chen et al . observed elevated PKM2 in erlotinib-resistant NSCLC, where it interacts with lncRNA H19 to activate Akt and confer resistance [Chen et al., 2020]. Further mechanistic studies demonstrate PKM2’s direct binding to mutant EGFR and HSP90, stabilizing EGFR-T790M in gefitinib-resistant cells via HSP90-cochaperone interactions [Chang et al., 2021], strengthening the evidence of cytoplasmic PKM2’s importance in non-metabolic functions. PKM2 acts as a multifunctional scaffold, promoting β-catenin-mediated malignancy progression, metastasis, and drug resistance through non-enzymatic mechanisms [Lin et al., 2018]. While nuclear PKM2-β-catenin interactions activate oncogenes like cyclin D1 and c-Myc in glioblastoma [Gupta et al., 2018], its role in β -catenin-driven EGFR-TKI resistance remained unexplored. PKM2 undergoes critical post-translational modifications, particularly phosphorylation at Ser37 and Tyr105, which regulate its nuclear translocation and oncogenic functions. Phosphorylation at Tyr105 disrupts PKM2’s tetrameric active form by interfering with fructose-1,6-bisphosphate binding, thereby reducing glycolytic activity while enhancing non-canonical roles in transcriptional regulation [Hitosugi et al., 2009]. Concurrently, Ser37 phosphorylation facilitates PKM2 nuclear entry and β -catenin-mediated transcriptional activation of tumor-associated genes [Niu et al., 2020; Liu et al., 2025]. In EGFR-TKI-resistant NSCLC models, hyperphosphorylation at Ser37/Y105 correlates with hyperactivation of Wnt/ β -catenin, AKT, and ERK pathways, underscoring the dominance of PKM2’s non-glycolytic functions in driving therapeutic resistance. Supporting this, Wang et al. identified PKM2 Y105 phosphorylation as a critical regulator of cancer stemness and drug resistance in NSCLC [Wang et al 2025], while lambertianic acid-a natural compound targeting p-PKM2 (Tyr105)-demonstrates efficacy in disrupting PKM2/ β -catenin interactions and inducing apoptosis in prostate cancer [Pak et al., 2023]. These findings collectively position PKM2 phosphorylation as a druggable node in resistance mechanisms. NAT-A emerges as a unique inhibitor of PKM2/ β -catenin signaling by selectively targeting phosphorylation-dependent interactions. Both in vitro and in viv o studies demonstrate that NAT-A reduces Ser37/Y105 phosphorylation, thereby blocking PKM2- β -catenin binding and abrogating downstream oncogenic transcription. Unlike sanguinarine, which disrupts PKM2/ β -catenin complexes alongside metabolic reprogramming in HCC [Kong et al., 2024], NAT-A specifically destabilizes cytoplasmic β -catenin without affecting PKM2-STAT3 interactions-a distinction from the dual-target inhibitor NPD10084 [Nagasawa et al., 2024]. This selectivity, combined with NAT-A’s concurrent suppression of AKT and EGFR pathways, highlights its capacity to disrupt multiple resistance-driving axes. Although the structural basis of NAT-A’s interaction with phosphorylated PKM2 remains to be elucidated, its ability to degrade β- catenin and reverse TKI resistance positions it as both a therapeutic candidate and a chemical probe for studying PKM2’s non-glycolytic functions. Given the scarcity of agents targeting PKM2’s non-glycolytic functions, NAT-A provides a strategic tool to dissect phosphorylation-dependent protein interactions and refine resistance-overcoming strategies in NSCLC. Conclusions Our study identifys NAT-A as a novel small molecule that selectively disrupts β -catenin-PKM2 interactions, triggering proteasomal degradation of β -catenin and effectively reversing EGFR-TKI resistance. These findings not only redefine PKM2’s role as a cytosolic β -catenin stabilizer independent of its glycolytic activity but also position NAT-A as a mechanistically distinct therapeutic candidate for EGFR-TKI-resistant NSCLC. Conflict of interest The authors declare that there are no conflicts of interest associated with this manuscript. Funding The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the National Key Research and Development Program of China (2021YFF0502400 and 2022YFC2804100), National Natural Science Foundation of China (82104033, 82304371, 22137006, 82073713 and 82473795). Authorship contribution statement Fan Sun , Houwen Lin , and Weihua Jiao led the project’s conceptualization, experimental design, coordination, and manuscript development. Liyun Liu and Xin Wang designed and conducted the experiments, performed data analysis, and authored the manuscript. Xinkai Wang performed EGFR-TKIs resistant cell lines assays. Li Guan , and Yahui Liao were primarily responsible for performing the in vivo experiments. Qi He , Yanxi Sun and Ying Wu contributed to cell culture maintenance and participated in manuscript proofreading. Xiao Lin and Yongjun Zhou isolated NATs from Streptomyces conglobatus and contributed to compound synthesis. Shuping Wang and Guohua Zhou contributed to manuscript refinement. 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Information & Authors Information Version history V1 Version 1 28 October 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Liyun Liu Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Xin Wang Central Military Commission Joint Logistics Support Force 904th Hospital View all articles by this author Xinkai Wang Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Li Guan Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Yahui Liao Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Qi He Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Yanxi Sun Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Ying Wu Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Xiao Lin Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Shuping Wang Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Yongjun Zhou Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Guohua Zhou China Pharmaceutical University College of Life Science and Technology View all articles by this author Weihua Jiao Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Hou wen Lin Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Fan Sun [email protected] Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital View all articles by this author Metrics & Citations Metrics Article Usage 185 views 100 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Liyun Liu, Xin Wang, Xinkai Wang, et al. NAT-A Overcomes EGFR-TKIs Resistance in Non-Small Cell Lung Cancer by Targeting the β-Catenin/PKM2 Axis. Authorea . 28 October 2025. DOI: https://doi.org/10.22541/au.176164609.95049633/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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