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Despite recent advances in EGFR-tyrosine kinase inhibitor (TKI) drugs for glioblastoma multiforme (GBM), intrinsic EGFR alterations in GBM have resulted in drug resistance and unsatisfactory clinical development of EGFR-TKIs. Determining the unknown mechanisms underlying EGFR-TKI drug resistance is an urgent, but unmet, medical need for GBM. Although several m6A RNA methylation regulators, such as reader YTHDF1/2, were recently predicted to be related to GBM recurrence, none was associated with resistance to the 3rd generation EGFR-TKI osimertinib.BACKGROUND/AIMDespite recent advances in EGFR-tyrosine kinase inhibitor (TKI) drugs for glioblastoma multiforme (GBM), intrinsic EGFR alterations in GBM have resulted in drug resistance and unsatisfactory clinical development of EGFR-TKIs. Determining the unknown mechanisms underlying EGFR-TKI drug resistance is an urgent, but unmet, medical need for GBM. Although several m6A RNA methylation regulators, such as reader YTHDF1/2, were recently predicted to be related to GBM recurrence, none was associated with resistance to the 3rd generation EGFR-TKI osimertinib.Osimertinib-resistant GBM cells (U87OSR) were established to ascertain the correlation between m6A expression and osimertinib resistance, prior to systemic analyses on m6A writers, erasers, and readers. YTHDF3-silencing was employed to reveal changes in IC50, cellular migration, cancer stemness, and p21-guided senescence in U87OSR cells. Signaling pathways and an in vivo xenograft model of U87OSR cells were investigated to delineate the influence of osimertinib-resistance and elevated YTHDF3 expression.MATERIALS AND METHODSOsimertinib-resistant GBM cells (U87OSR) were established to ascertain the correlation between m6A expression and osimertinib resistance, prior to systemic analyses on m6A writers, erasers, and readers. YTHDF3-silencing was employed to reveal changes in IC50, cellular migration, cancer stemness, and p21-guided senescence in U87OSR cells. Signaling pathways and an in vivo xenograft model of U87OSR cells were investigated to delineate the influence of osimertinib-resistance and elevated YTHDF3 expression.YTHDF3 played a crucial role in inducing cellular proliferation, migration, and stemness in U87OSR GBM cells. Importantly, silencing of YTHDF3 markedly reduced the activation of certain signaling pathways, including EGFR- or ITGA7- AKT, and ERK in U87OSR cells. Our study also revealed the oncogenic function of YTHDF3 in inducing senescence escape via p21 down-regulation. In contrast, silencing of YTHDF3 resulted in increased p21 expression, senescence, and suppressed tumor growth in our osimertinib-resistant preclinical model.RESULTSYTHDF3 played a crucial role in inducing cellular proliferation, migration, and stemness in U87OSR GBM cells. Importantly, silencing of YTHDF3 markedly reduced the activation of certain signaling pathways, including EGFR- or ITGA7- AKT, and ERK in U87OSR cells. Our study also revealed the oncogenic function of YTHDF3 in inducing senescence escape via p21 down-regulation. In contrast, silencing of YTHDF3 resulted in increased p21 expression, senescence, and suppressed tumor growth in our osimertinib-resistant preclinical model.Overall, our research underscores the novel potential of YTHDF3 as a new pharmacological target in GBM treatment, specifically for patients with osimertinib-resistant or refractory tumors.CONCLUSIONOverall, our research underscores the novel potential of YTHDF3 as a new pharmacological target in GBM treatment, specifically for patients with osimertinib-resistant or refractory tumors.

EGFR-TKI targeted therapy is one of the most effective treatments for lung cancer patients harboring EGFR activating mutations. However, inhibition response is easily attenuated by drug resistance, which is mainly due to bypass activation or downstream activation. Herein, we established osimertinib-resistant cells by stepwise dose-escalation in vitro and an osimertinib-resistant patient-derived xenograft model through persistent treatment in vivo. Phosphorylated proteomics identified that MEK1 and AKT1/2 were abnormally activated in resistant cells compared with parental cells. Likewise, EGFR inhibition by osimertinib induced activation of MEK1 and AKT1/2, which weakened osimertinib sensitivity in NSCLC cells. Consequently, this study aimed to identify a novel inhibitor which could suppress resistant cell growth by dual targeting of MEK1 and AKT1/2. Based on computational screening, we identified that costunolide could interact with MEK1 and AKT1/2. Further exploration using in vitro kinase assays validated that costunolide inhibited the kinase activity of MEK1 and AKT1/2, which restrained downstream ERK-RSK2 and GSK3β signal transduction and significantly induced cell apoptosis. Remarkably, the combination of osimertinib and costunolide showed synergistic or additive inhibitory effects on tumor growth in osimertinib-resistant cell lines and PDX model. Hence, this study highlights a potential therapeutic strategy for osimertinib-resistant patients through targeting of MEK1 and AKT1/2 by costunolide.

Background: Osimertinib-based therapy effectively improves the prognosis of lung adenocarcinoma (LUAD) patients with epidermal growth factor receptor mutations. However, patients will have cancer progression after approximately one year due to the occurrence of drug resistance. Extensive evidence has revealed that lipid metabolism and tumor-associated macrophage (TAM) are associated with drug resistance, which deserves further exploration. Methods: An osimertinib resistance index (ORi) was built to investigate the link between lipid metabolism and osimertinib resistance. The ORi was constructed and validated using TCGA and GEO data, and the relationship between ORi and immune infiltration was discussed. Weighted gene co-expression network analysis based on the M2/M1 macrophage ratio determined the hub gene TIAM2 and the biological function of TIAM2 in LUAD was verified in vitro. Results: ORi based on nine lipid metabolism-related genes was successfully constructed, which could accurately reflect the resistance of LUAD patients to osimertinib, predict the prognosis, and correlate with M2-like TAM. Additionally, TIAM2 was found to increase osimertinib tolerance, enhance cell motility, and promote M2-like TAM polarization in LUAD. Conclusions: The lipid metabolism gene is strongly connected with osimertinib resistance. TIAM2 contributes to osimertinib resistance, enhances cell motility, and induces M2-like TAM polarization in LUAD.

Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI); it has achieved favorable progression-free survival (PFS) in non-small cell lung cancer (NSCLC) patients with EGFR mutation, however, the resistance occurs in most patients, and the underlying mechanism remain to be elucidated. Cancer-associated fibroblasts (CAFs) are major stromal cells in tumor microenvironment. Despite accumulating evidence suggests that CAFs contribute to drug resistance, the role of CAFs in osimertinib resistance in NSCLC is not fully understood. Here, we reported that CAFs promoted the resistance of NSCLC cells to osimertinib through enhancing stemness of NSCLC cells and reducing apoptosis induced by osimertinib. CAFs possessed a high level of Neuregulin-1 (NRG1), and CAFs-secreted NRG1 mediated the promoting effect of CAFs on osimertinib resistance, demonstrated by applying recombinant human NRG1 (rhNRG1) and NRG1 knockdown. We also found that osimertinib stimulated NRG1 secretion by CAFs, which may further enhance osimertinib resistance. Further study revealed that CAFs promoted the resistance of NSCLC cells to osimertinib via NRG1-mediated HER3/AKT/NF-κB pathway. Moreover, the mouse xenograft study demonstrated that CAFs enhanced osimertinib-treated tumor growth . Our finding highlights the potential value of CAFs-derived NRG1 as a novel therapeutic target for osimertinib resistance in lung cancer.

Background Recent evidence has demonstrated that abnormal expression and regulation of circular RNA (circRNAs) are involved in the occurrence and development of a variety of tumors. The aim of this study was to investigate the effects of circ_PPAPDC1A in Osimertinib resistance in NSCLC. Methods Human circRNAs microarray analysis was conducted to identify differentially expressed (DE) circRNAs in Osimertinib-acquired resistance tissues of NSCLC. The effect of circ_PPAPDC1A on cell proliferation, invasion, migration, and apoptosis was assessed in both in vitro and in vivo. Dual-luciferase reporter assay, RT-qPCR, Western-blot, and rescue assay were employed to confirm the interaction between circ_PPAPDC1A/miR-30a-3p/IGF1R axis. Results The results revealed that circ_PPAPDC1A was significantly upregulated in Osimertinib acquired resistance tissues of NSCLC. circ_PPAPDC1A reduced the sensitivity of PC9 and HCC827 cells to Osimertinib and promoted cell proliferation, invasion, migration, while inhibiting apoptosis in Osimertinib-resistant PC9/OR and HCC829/OR cells, both in vitro and in vivo. Silencing circ_PPAPDC1A partially reversed Osimertinib resistance. Additionally, circ_PPAPDC1A acted as a competing endogenous RNA (ceRNA) by targeting miR-30a-3p, and Insulin-like Growth Factor 1 Receptor (IGF1R) was identified as a functional gene for miR-30a-3p in NSCLC. Furthermore, the results confirmed that circ_PPAPDC1A/miR-30a-3p/IGF1R axis plays a role in activating the PI3K/AKT/mTOR signaling pathway in NSCLC with Osimertinib resistance. Conclusions Therefore, for the first time we identified that circ_PPAPDC1A was significantly upregulated and exerts an oncogenic role in NSCLC with Osimertinib resistance by sponging miR-30a-3p to active IGF1R/PI3K/AKT/mTOR pathway. circ_PPAPDC1A may serve as a novel diagnostic biomarker and therapeutic target for NSCLC patients with Osimertinib resistance. Graphical Abstract

Osimertinib is a third‐generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR‐TKI) for lung adenocarcinoma (LUAD) harboring activating mutations, but patients ultimately develop acquired resistance. Circular RNAs are involved in EGFR‐TKI resistance, while the role of hsa_circ_0005576 in the osimertinib resistance of LUAD remains unknown. In this study, we demonstrated that hsa_circ_0005576 could facilitate osimertinib‐resistant LUAD cells. Briefly, knockdown of hsa_circ_0005576 not only suppressed the proliferation and promoted the apoptosis of resistant LUAD cells, but also increased their sensitivity to osimertinib. Mechanistically, hsa_circ_0005576, serving as an miRNA sponge, could directly interact with miR‐512‐5p and subsequently upregulate the miR‐512‐5p‐targeted insulin‐like growth factor 1 receptor. Rescue assays indicated that miR‐512‐5p inhibition could reverse the effects of hsa_circ_0005576 knockdown in LUAD cells resistant to osimertinib. Overall, our study revealed that hsa_circ_0005576 regulates proliferation and apoptosis through miR‐512‐5p/IGF1R signaling, which contributes further to the resistance of LUAD cells to osimertinib. In addition, this study provides a novel insight into the mechanisms underlying osimertinib resistance of LUAD. This study revealed that hsa_circ_0005576 regulates proliferation and apoptosis through miR‐512‐5p/IGF1R signaling, which contributes further to the resistance of LUAD cells to osimertinib. In addition, it provides a novel insight into the mechanism underlying osimertinib resistance of LUAD.

Osimertinib (AZD9291) has been widely used for the treatment of EGFR mutant non-small cell lung cancer. However, resistance to osimertinib is inevitable. In this study we elucidated the molecular mechanisms of resistance in osimertinib-resistant NCI-H1975/OSIR cells. We showed that NCI-H1975/OSIR cells underwent epithelial–mesenchymal transition (EMT), which conferred sensitivity to the GPX4 inhibitor 1 S , 3 R -RSL3 to induce ferroptotic cell death. The EMT occurrence resulted from osimertinib-induced upregulation of TGFβ2 that activated SMAD2. On the other hand, we revealed that NCI-H1975/OSIR cells were highly dependent on NF-κB pathway for survival, since treatment with the NF-κB pathway inhibitor BAY 11–7082 or genetic silence of p65 caused much greater cell death as compared with the parental NCI-H1975 cells. In NCI-H1975 cells, osimertinib activated NF-κB pathway, evidenced by the increased p65 nuclear translocation, which was abolished by knockdown of TGFβ2. In the cancer genome atlas lung adenocarcinoma data, TGFB2 transcript abundance significantly correlated with EMT-associated genes and NF-κB pathway. In addition, coexistence of EMT and activation of NF-κB pathway was observed in several NCI-H1975/OSIR clones. These findings shed new light on distinct roles of TGFβ2 in osimertinib-resistant cells and provide new strategies for treatment of this resistant status.

With the wide clinical use of the third‐generation epidermal growth factor receptor (EGFR) inhibitor osimertinib for the treatment of EGFR‐mutated non–small cell lung cancer (NSCLC), acquired resistance caused by EGFR C797S tertiary mutation has become a concern. Therefore, fourth‐generation EGFR inhibitors that could overcome this mutation have gained increasing attention in recent years. Here, we identified LS‐106 as a novel EGFR inhibitor against C797S mutation and evaluated its antitumor activity both in vitro and in vivo. In cell‐free assay, LS‐106 potently inhibited the kinase activities of EGFR19del/T790M/C797S and EGFRL858R/T790M/C797S with IC50 values of 2.4 nmol/L and 3.1 nmol/L, respectively, which was more potent than osimertinib. Meanwhile, LS‐106 exhibited comparable kinase inhibitory effect to osimertinib on EGFRL858R/T790M and wild‐type EGFR. Results from cellular experiments demonstrated that LS‐106 potently blocked the phosphorylation of EGFR C797S triple mutations in the constructed BaF3 cells that highly expressed EGFR19del/T790M/C797S or EGFRL858R/T790M/C797S, and thus inhibited the proliferation of these cells. We also constructed tumor cells harboring EGFR19del/T790M/C797S (named PC‐9‐OR cells) using the CRISPR/Cas9 system and found that LS‐106 markedly suppressed the activation of EGFR19del/T790M/C797S and the proliferation of PC‐9‐OR cells. Moreover, cells harboring EGFR19del/T790M/C797S underwent remarkable apoptosis upon LS‐106 treatment. In vivo experiments further demonstrated that oral administration of LS‐106 caused significant tumor regression in a PC‐9‐OR xenograft model, with a tumor growth inhibition rate (TGI) of 83.5% and 136.6% at doses of 30 and 60 mg/kg, respectively. Taken together, we identified LS‐106 as a novel fourth‐generation EGFR inhibitor against C797S mutation and confirmed its preclinical antitumor effects in C797S–triple‐mutant tumor models. In this study, we identified LS‐106 as a novel inhibitor against C797S–triple‐mutant epidermal growth factor receptor (EGFR) (EGFR19del/T790M/C797S and EGFRL858R/T790M/C797S), which showed great in vitro and in vivo antitumor activity in EGFR‐C797S–triple‐mutant osimertinib‐resistant tumor models.

Background Osimertinib resistance limits the treatment of epidermal growth factor receptor-(EGFR)-mutated non-small-cell lung carcinoma (NSCLC). The mechanisms of osimertinib resistance need to be elucidated to determine alternative treatment strategies. This study explores the role of M2 type tumor-associated macrophage (TAM)-derived exosomal MSTRG.292666.16 in osimertinib resistance, and its related competing endogenous RNA (ceRNA) mechanism. Methods M2 type TAMs were induced with 200 ng/mL phorbol 12-myristate 13-acetate, 20 ng/mL IL-4 and IL-13, and M2 type macrophage markers were measured by RT-qPCR. Next, the exosomes were isolated and characterized. Tumor formation in nude mice was conducted using H1975 cells under different treatment conditions. Small RNA sequencing was performed on exosomes derived from sensitive and resistant plasma, and ceRNA networks were constructed. Fluorescence in situ hybridization was used to observe the localization of MSTRG.292666.16, and a ceRNA network (MSTRG.292666.16-miR-6836-5p-MAPK8IP3) was selected for further validation. Results M2 type TAMs, and M2 type TAM-derived exosomes were successfully induced and isolated. Nude mice results showed that M2 type TAM-derived exosomes and MSTRG.292666.16 overexpression significantly increased tumor volume after administration of osimertinib for 4 weeks. M2 type TAMs were found in the resistant plasma, and MSTRG.292666.16 localized in the cytoplasm of H1975 cells. In addition, the genes in the ceRNA networks were significantly enriched in eight GO terms and seven KEGG pathways, including the MAPK signaling pathway. Subsequently, the levels of MSTRG.292666.16 and MAPK8IP3 significantly increased in both resistant plasma-derived exosomes and M2 type TAM-derived exosomes, while miR-6836-5p levels were significantly reduced. Finally, MSTRG.292666.16, miR-6836-5p, and MAPK8IP3 were part of the same network. Conclusions M2 type TAM-derived exosomes promoted osimertinib resistance in NSCLC by regulating the MSTRG.292666.16/miR-6386-5p/MAPK8IP3 axis.

Background Overcoming resistance to Osimertinib in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) is clinically challenging because the underlying mechanisms are not fully understood. The murine double minute 2 (MDM2) has been extensively described as a tumor promotor in various malignancies, mainly through a negative regulatory machinery on the p53 tumor suppressor. However, the significance of MDM2 on the sensitivity to Osimertinib has not been described. Methods Osimertinib resistant cells were generated by standard dose escalation strategy and individual resistant clones were isolated for MDM2 testing. The MDM2 and its mutant constructs (ΔPBD, ΔRING, C464A) were introduced into PC-9, HCC827 and H1975 cells and evaluated for the sensitivity to Osimertinib by MTT assay, colony formation, EdU assay and TUNEL assay. MDM2 expression in resistant cells was manipulated by pharmacological and molecular approaches, respectively. Proteins that were implicated in PI3K/Akt, MAPK/Erk and apoptosis signaling were measured by Western blot analysis. Candidate proteins that interacted with MDM2 were captured by immunoprecipitation and probed with indicated antibodies. Results In comparison with parental PC-9 cells, the PC-9 OR resistant cells expressed high level of MDM2. Ectopic expression of MDM2 in PC-9, HCC827 and H1975 sensitive cells generated an Osimertinib resistant phenotype, regardless of p53 status. MDM2 promoted resistance to Osimertinib through a PI3K/Akt and MAPK/Erk-independent machinery, in contrast, MDM2 selectively stabilized MCL-1 protein to arrest Osimertinib-induced cancer cell apoptosis. Mechanistically, MDM2 acted as a E3 ligase to ubiquitinate FBW7, a well-established E3 ligase for MCL-1, at Lys412 residue, which resulted in FBW7 destruction and MCL-1 stabilization. Targeting MDM2 to augment MCL-1 protein breakdown overcame resistance to Osimertinib in vitro and in vivo. Finally, the clinical relevance of MDM2-FBW7-MCL-1 regulatory axis was validated in mouse xenograft tumor model and in NSCLC specimen. Conclusion Overexpression of MDM2 is a novel resistant mechanism to Osimertinib in EGFR mutant NSCLC. MDM2 utilizes its E3 ligase activity to provoke FBW7 destruction and sequentially leads to MCL-1 stabilization. Cancer cells with aberrant MDM2 state are refractory to apoptosis induction and elicit a resistant phenotype to Osimertinib. Therefore, targeting MDM2 would be a feasible approach to overcome resistance to Osimertinib in EGFR mutant NSCLC.