EGFR-TKI Resistance In Non-small Cell Lung Cancer Research Articles

HDAC1 and FOXK1 mediate EGFR-TKI resistance of non-small cell lung cancer through miR-33a silencing

BackgroundThe development of acquired EGFR-TKI treatment resistance is still a major clinical challenge in the treatment of non-small cell lung cancer (NSCLC). This study aimed to investigate the role of HDAC1/FOXK1/miR-33a signaling in EGFR-TKI resistance.MethodsThe expression levels of miR‐33a, HDAC1, and FOXK1 were examined using quantitative polymerase chain reaction (PCR) and bioinformatics analysis. Cell proliferation, migration, and apoptosis were explored by cell number assay, Transwell, and flow cytometry assays, respectively. After overexpression or knockdown of HDAC1, miR-33a expression in the cells, cell functions were tested. Immunoprecipitation and correlation analyses were used to evaluate the interaction between HDAC1 and FOXK1 protein. The tumor-suppressive role of miR-33a was investigated by animal experiments.ResultsThe suppression of miR-33a increased TKI resistance by affecting cell proliferation, migration, and apoptosis in gefitinib-resistant cells. HDAC1 is the key upstream molecule that inhibits miR-33 expression. HDAC1 upregulation increased gefitinib resistance by its binding to FOXK1 in cells to silence miR-33a expression. MiR-33a overexpression exerts tumor-suppressive effects by negatively regulating ABCB7 and p70S6K1 expression. Moreover, overexpression of miR-33a inhibited tumor growth in a xenograft nude mouse model.ConclusionsHDAC1/FOXK1 upregulation and miR-33a silencing are new mechanisms of EGFR-TKI resistance in NSCLC.

PrPC controls epithelial-to-mesenchymal transition in EGFR-mutated NSCLC: implications for TKI resistance and patient follow-up

Patients with EGFR-mutated non-small cell lung cancer (NSCLC) benefit from treatment with tyrosine kinase inhibitors (TKI) targeting EGFR. Despite improvements in patient care, especially with the 3rd generation TKI osimertinib, disease relapse is observed in all patients. Among the various processes involved in TKI resistance, epithelial-to-mesenchymal transition (EMT) is far from being fully characterized. We hypothesized that the cellular prion protein PrPC could be involved in EMT and EGFR-TKI resistance in NSCLC. Using 5 independent lung adenocarcinoma datasets, including our own cohort, we document that the expression of the PRNP gene encoding PrPC is associated with EMT. By manipulating the levels of PrPC in different EGFR-mutated NSCLC cell lines, we firmly establish that the expression of PrPC is mandatory for cells to maintain or acquire a mesenchymal phenotype. Mechanistically, we show that PrPC operates through an ILK-RBPJ cascade, which also controls the expression of EGFR. Our data further demonstrate that PrPC levels are elevated in EGFR-mutated versus wild-type tumours or upon EGFR activation in vitro. In addition, we provide evidence that PRNP levels increase with TKI resistance and that reducing PRNP expression sensitizes cells to osimertinib. Finally, we found that plasma PrPC levels are increased in EGFR-mutated NSCLC patients from 2 independent cohorts and that their longitudinal evolution mirrors that of disease. Altogether, these findings define PrPC as a candidate driver of EMT-dependent resistance to EGFR-TKI in NSCLC. They further suggest that monitoring plasma PrPC levels may represent a valuable non-invasive strategy for patient follow-up and warrant considering PrPC-targeted therapies for EGFR-mutated NSCLC patients with TKI failure.

Clinical-genomic characteristics in patients with EGFR-mutated non-small cell lung cancer harboring MET copy number gains after progression on EGFR-TKI: A report from LC-SCRUM-TRY.

8628 Background: MET amplification (amp) account for 10-15% of resistance mechanism in EGFR-mutated non-small cell lung cancer (NSCLC) to EGFR-tyrosine kinase inhibitor (TKI) treatment. MET copy number (CN) gains consists of MET focal amp and polysomy (poly), but the clinical characteristics and outcome of EGFR-TKI resistant NSCLC patients harboring MET amp, poly remains unclear. Methods: The clinical-genomic characteristics and treatment outcome ofEGFR-TKI resistant EGFR-mutated NSCLC registered in LC-SCRUM-TRY (UMIN000041957) were analyzed. Genetic resistant alterations were screened using next-generation sequencing (NGS) (Tissue; Oncomine Precision Assay [OPA] or Plasma; Guardant360 [G360]), and MET-fluorescence in-situ hybridization (FISH). MET amp was defined as MET CN ≥4 (OPA), MET CN ≥2.12 (G360) or MET/CEP7 ratio ≥2 (FISH). MET poly was defined as MET CN≥5 and MET/CEP7 ratio <2 by FISH. Results: A total of 1559 patients had been enrolled in LC-SCRUM-TRY between Sep 2020 and Jan 2024. Of 1537 analyzed samples, 666 (43%) were EGFR-TKI resistant samples. Of these, 92 (14%), 239 (36%) and 335 (50%) had MET amp, poly, and no CN gain (no CNG), respectively. Patient characteristics and treatment outcome with 1st line EGFR-TKI and platinum-based chemotherapy (PBC) post EGFR-TKI is summarized (Table 1). The overall response rate (ORR) of PBC post EGFR-TKI in the patients with MET amp, poly and no CNG was 41%, 29% and 31%, respectively (p=0.34), with median progression-free survival (mPFS) being 5.7 months, 6.8 months, and 6.7 months (p=0.02). In addition, 12 and 11 of patients with MET amp and poly were enrolled into clinical trial of MET-targeted therapy (MET-TKI, n=2; MET-antibody-drug conjugate [ADC], n=4; EGFR/MET-antibody, n=2), and MET non-targeted therapy (EGFR-TKI, n=5; other ADCs, n=10: immunotherapy; n=1). The ORR of MET-targeted therapy/MET non-targeted therapy were 60/0% for MET amp, and 0/33% for MET poly, respectively. Conclusions: Patients with shorter PFS with prior 1st line EGFR-TKI are more likely to have MET amp. The efficacy of standard PBC post EGFR-TKI in patients with MET CNG is limited. Current MET-targeted therapy may not be effective for EGFR-mutated NSCLC harboring MET poly after progression on EGFR-TKI. [Table: see text]

Abstract 7206: Inhibition of CAD, an enzyme of de novo pyrimidine synthesis, to overcome EGFR-TKI resistance in NSCLC

Abstract Given the fast-growing cancer cells, there is an increased demand for large amounts of energy and nucleotides for DNA and RNA synthesis. Thus, targeting nucleotide metabolism, specifically de novo nucleotide biosynthesis, is a promising strategy for cancer treatment. Hyperactivation of Carbamoyl-phosphate synthetase, aspartate transcarbamylase (CAD), a key enzyme catalyzing the first three steps of de novo pyrimidine synthesis, is a common event in tumors and has been associated with chemoresistance in tumors. Our analysis, using TCGA data, revealed that increased expression of CAD is associated with poor prognosis in non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have demonstrated significant benefits to patients with NSCLC harboring EGFR-activating mutations; however, acquired resistance limits their long-term efficacy. Therefore, it remains urgent to discover the underlying mechanisms for discovering novel therapeutic strategies for overcoming TKI resistance. Our previous findings demonstrated that blockade of S6K1 overcomes acquired resistance to EGFR-TKIs in NSCLC. It was reported that S6K1 is involved in de novo pyrimidine biosynthesis. In this study, we aimed to investigate whether CAD mediates S6K1-induced EGFR-TKI resistance in NSCLC. Immunohistochemistry analysis on a tissue microarray of lung adenocarcinoma tissues indicated that increased co-expression of p-CAD and S6K1 proteins is associated with clinicopathological parameters and poor survival outcomes. Our in vitro results showed an increased expression level of CAD and p-CAD proteins in TKI-resistant cells compared with parental cells upon TKI treatment. Knockdown of CAD by siRNA led to sensitizing TKI-resistant cells to osimertinib, as indicated by BrdU cell proliferation assay. Inhibition of S6K1 by a siRNA or specific inhibitors PF-470867 and LY2584702 effectively reduced the phosphorylation levels of CAD protein in TKI-resistant cells, suggesting that S6K1 may serve as an upstream regulator of CAD for TKI resistance. The String database predicted a protein-protein interaction between S6K1 and CAD. Indeed, our co-immunoprecipitation (co-IP) results revealed a direct association between S6K1 and CAD proteins. Importantly, TKI treatment enhanced the binding between S6K1 and CAD. Taken together, these findings provide evidence that CAD may mediate S6K1-mediated EGFR-TKI resistance in NSCLC. Our ongoing and future work will assess whether inhibition of CAD sensitizes resistant cells to TKI in vivo and elucidate the associated mechanism. Citation Format: Leili Saeednejad Zanjani, Lei Zhao, Shaomin Wang, Margot Federoff, Xin Sun, Jun He. Inhibition of CAD, an enzyme of de novo pyrimidine synthesis, to overcome EGFR-TKI resistance in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7206.

Tet methylcytosine dioxygenase 2 (TET2) deficiency elicits EGFR-TKI (tyrosine kinase inhibitors) resistance in non-small cell lung cancer

Despite epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have shown remarkable efficacy in patients with EGFR-mutant non-small cell lung cancer (NSCLC), acquired resistance inevitably develops, limiting clinical efficacy. We found that TET2 was poly-ubiquitinated by E3 ligase CUL7FBXW11 and degraded in EGFR-TKI resistant NSCLC cells. Genetic perturbation of TET2 rendered parental cells more tolerant to TKI treatment. TET2 was stabilized by MEK1 phosphorylation at Ser 1107, while MEK1 inactivation promoted its proteasome degradation by enhancing the recruitment of CUL7FBXW11. Loss of TET2 resulted in the upregulation of TNF/NF-κB signaling that confers the EGFR-TKI resistance. Genetic or pharmacological inhibition of NF-κB attenuate the TKI resistance both in vitro and in vivo. Our findings exemplified how a cell growth controlling kinase MEK1 leveraged the epigenetic homeostasis by regulating TET2, and demonstrated an alternative path of non-mutational acquired EGFR-TKI resistance modulated by TET2 deficiency. Therefore, combined strategy exploiting EGFR-TKI and inhibitors of TET2/NF-κB axis holds therapeutic potential for treating NSCLC patients who suffered from this resistance.

Recent Developments in Tyrosine Kinase Inhibitor-based Nanotherapeutics for EGFR-resistant Non-small Cell Lung Cancer.

The advent of drug resistance in response to epidermal growth factor receptor (EGFR)- tyrosine kinase inhibitor (TKI) targeted therapy represents a serious challenge in the management of non-small cell lung cancer (NSCLC). These acquired resistance mutations, attributed to several advanced EGFR mutations and, necessitated the development of new-generation TKIs. Nanomedicine approaches provide a plausible way to address these problems by providing targeted delivery and sustained release, which have demonstrated success in preclinical trials. This review article provides a summary of nano-formulations designed for EGFR-TKI-resistant NSCLC, highlighting their efficacy in both in vitro and in vivo models. These findings reveal insights into the design of nanoparticles and multifunctional nanosystems, offering a potential avenue for efficacious treatment of EGFR-TKIresistant NSCLC.

Targeting HER3 to overcome EGFR TKI resistance in NSCLC.

Receptor tyrosine kinases (RTKs) play a crucial role in cellular signaling and oncogenic progression. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have become the standard treatment for advanced non-small cell lung cancer (NSCLC) patients with EGFR-sensitizing mutations, but resistance frequently emerges between 10 to 14 months. A significant factor in this resistance is the role of human EGFR 3 (HER3), an EGFR family member. Despite its significance, effective targeting of HER3 is still developing. This review aims to bridge this gap by deeply examining HER3's pivotal contribution to EGFR TKI resistance and spotlighting emerging HER3-centered therapeutic avenues, including monoclonal antibodies (mAbs), TKIs, and antibody-drug conjugates (ADCs). Preliminary results indicate combining HER3-specific treatments with EGFR TKIs enhances antitumor effects, leading to an increased objective response rate (ORR) and prolonged overall survival (OS) in resistant cases. Embracing HER3-targeting therapies represents a transformative approach against EGFR TKI resistance and emphasizes the importance of further research to optimize patient stratification and understand resistance mechanisms.

PTPN23 ubiquitination by WDR4 suppresses EGFR and c-MET degradation to define a lung cancer therapeutic target

Aberrant overexpression or activation of EGFR drives the development of non-small cell lung cancer (NSCLC) and acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) by secondary EGFR mutations or c-MET amplification/activation remains as a major hurdle for NSCLC treatment. We previously identified WDR4 as a substrate adaptor of Cullin 4 ubiquitin ligase and an association of WDR4 high expression with poor prognosis of lung cancer. Here, using an unbiased ubiquitylome analysis, we uncover PTPN23, a component of the ESCRT complex, as a substrate of WDR4-based ubiquitin ligase. WDR4-mediated PTPN23 ubiquitination leads to its proteasomal degradation, thereby suppressing lysosome trafficking and degradation of wild type EGFR, EGFR mutant, and c-MET. Through this mechanism, WDR4 sustains EGFR and c-MET signaling to promote NSCLC proliferation, migration, invasion, stemness, and metastasis. Clinically, PTPN23 is downregulated in lung cancer and its low expression correlates with WDR4 high expression and poor prognosis. Targeting WDR4-mediated PTPN23 ubiquitination by a peptide that competes with PTPN23 for binding WDR4 promotes EGFR and c-MET degradation to block the growth and progression of EGFR TKI-resistant NSCLC. These findings identify a central role of WDR4/PTPN23 axis in EGFR and c-MET trafficking and a potential therapeutic target for treating EGFR TKI-resistant NSCLC.

Long non-coding RNAs in non-small cell lung cancer: implications for EGFR-TKI resistance.

Non-small cell lung cancer (NSCLC) is one of the most common types of malignant tumors as well as the leading cause of cancer-related deaths in the world. The application of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) has dramatically improved the prognosis of NSCLC patients who harbor EGFR mutations. However, despite an excellent initial response, NSCLC inevitably becomes resistant to EGFR-TKIs, leading to irreversible disease progression. Hence, it is of great significance to shed light on the molecular mechanisms underlying the EGFR-TKI resistance in NSCLC. Long non-coding RNAs (lncRNAs) are critical gene modulators that are able to act as oncogenes or tumor suppressors that modulate tumorigenesis, invasion, and metastasis. Recently, extensive evidence demonstrates that lncRNAs also have a significant function in modulating EGFR-TKI resistance in NSCLC. In this review, we present a comprehensive summary of the lncRNAs involved in EGFR-TKI resistance in NSCLC and focus on their detailed mechanisms of action, including activation of alternative bypass signaling pathways, phenotypic transformation, intercellular communication in the tumor microenvironment, competing endogenous RNAs (ceRNAs) networks, and epigenetic modifications. In addition, we briefly discuss the limitations and the clinical implications of current lncRNAs research in this field.

HER3-targeted therapeutic antibodies and antibody–drug conjugates in non-small cell lung cancer refractory to EGFR-tyrosine kinase inhibitors

Human epidermal growth factor receptor 3 (HER3) is a unique member of the human epidermal growth factor receptor (HER/EGFR) family, since it has negligible kinase activity. Therefore, HER3 must interact with a kinase-proficient receptor to form a heterodimer, leading to the activation of signaling cascades. Overexpression of HER3 is observed in various human cancers, including non-small cell lung cancer (NSCLC), and correlates with poor clinical outcomes in patients. Studies on the underlying mechanism demonstrate that HER3-initiated signaling promotes tumor metastasis and causes treatment failure in human cancers. Upregulation of HER3 is frequently observed in EGFR-mutant NSCLC treated with EGFR-tyrosine kinase inhibitors (TKIs). Increased expression of HER3 triggers the so-called EGFR-independent mechanism via interactions with other receptors to activate “bypass signaling pathways”, thereby resulting in resistance to EGFR-TKIs. To date, no HER3-targeted therapy has been approved for cancer treatment. In both preclinical and clinical studies, targeting HER3 with a blocking antibody (Ab) is the only strategy being examined. Recent evaluations of an anti-HER3 Ab-drug conjugate (ADC) show promising results in patients with EGFR-TKI-resistant NSCLC. Herein, we summarize our understanding of the unique biology of HER3 in NSCLC refractory to EGFR-TKIs, with a focus on its dimerization partners and subsequent activation of signaling pathways. We also discuss the latest development of the therapeutic Abs and ADCs targeting HER3 to abrogate EGFR-TKI resistance in NSCLC.

AZ12756122, a novel fatty acid synthase inhibitor, decreases resistance features in EGFR-TKI resistant EGFR-mutated NSCLC cell models

Different EGFR tyrosine kinase inhibitors (TKIs) have been developed for the treatment of non-small cell lung cancer (NSCLC) patients harboring sensitizing mutations in the EGFR gene. Apart from acquired secondary mutations, multiple resistance mechanisms have been reported, such as the overexpression of fatty acid synthase (FASN), a multi-functional enzyme essential for the de novo lipogenesis, or the increase of cancer stem cells, a small subpopulation within the tumor responsible for relapse, metastasis, and resistance to therapies. Hence, the purpose of this work is to evaluate the novel FASN inhibitor AZ12756122, both alone and in combination with gefitinib and osimertinib, in EGFR-mutated (EGFRm) lung adenocarcinoma cell models sensitive and resistant to EGFR-TKIs. The molecular effect of AZ12756122 (alone and in combination with EGFR-TKI) on FASN, EGFR/STAT3, Akt/mTOR, and MAPK signaling pathways was analyzed using RT-qPCR and Western blot. FASN expression was also evaluated in samples from patients with EGFRm NSCLC through immunohistochemistry. Our findings revealed that AZ12756122 caused cytotoxic effects inducing apoptosis, downregulated FASN expression and activity, decreased the activation of EGFR and Akt/mTOR pathway, and reduced cancer stem-like cells. Furthermore, the combination of AZ12756122 and osimertinib sensitized cells to EGFR-TKI, showing a synergistic effect that resulted in a reduction in the activation of EGFR, Akt/mTOR, and MAPK signaling pathways. Our study also showed that FASN+ EGFRm NSCLC patients exhibited a longer mPFS in patients who responded to EGFR-TKI treatment. In conclusion, FASN inhibition should be further studied for the treatment, alone or in combination with EGFR-TKIs, for EGFRm NSCLC patients.

Targeted Co-Delivery of Gefitinib and Rapamycin by Aptamer-Modified Nanoparticles Overcomes EGFR-TKI Resistance in NSCLC via Promoting Autophagy

Acquired drug resistance decreases the efficacy of gefitinib after approximately 1 year of treatment in non-small-cell lung cancer (NSCLC). Autophagy is a process that could lead to cell death when it is prolonged. Thus, we investigated a drug combination therapy of gefitinib with rapamycin—a cell autophagy activator—in gefitinib-resistant NSCLC cell line H1975 to improve the therapeutic efficacy of gefitinib in advanced NSCLC cells through acute cell autophagy induction. Cell viability and tumor formation assays indicated that rapamycin is strongly synergistic with gefitinib inhibition, both in vitro and in vivo. Mechanistic studies demonstrated that EGFR expression and cell autophagy decreased under gefitinib treatment and were restored after the drug combination therapy, indicating a potential cell autophagy–EGFR positive feedback regulation. To further optimize the delivery efficiency of the combinational agents, we constructed an anti-EGFR aptamer-functionalized nanoparticle (NP-Apt) carrier system. The microscopic observation and cell proliferation assays suggested that NP-Apt achieved remarkably targeted delivery and cytotoxicity in the cancer cells. Taken together, our results suggest that combining rapamycin and gefitinib can be an efficacious therapy to overcome gefitinib resistance in NSCLC, and targeted delivery of the drugs using the aptamer-nanoparticle carrier system further enhances the therapeutic efficacy of gefitinib.

Abstract 548: EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC

Abstract The leading cause of cancer-related deaths in the US is non-small cell lung cancer (NSCLC), and about 10-15% of NSCLC cases harbor EGFR activating mutations. While these patients are initially responsive to EGFR tyrosine kinase inhibitors (TKIs), they eventually experience progression and develop acquired TKI resistance. Currently, there are no approved therapies for NSCLC patients with acquired resistance to the third-generation EGFR TKI osimertinib (OSI). Moreover, EGFR-TKI resistant NSCLCs are refractory to immune checkpoint inhibitors. Therefore, novel treatment strategies are urgently needed. Chimeric antigen receptors (CARs) have been used to enhance the anti-tumor activity of immune effector cells. Here, we developed CAR-based therapies targeting EGFR as a novel strategy to overcome immune suppression and target EGFR TKI-resistant NSCLC. We designed multi-generational EGFR CAR constructs and transduced them into T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. We then evaluated their activity against NSCLC cell lines in vitro using firefly luciferase assays. EGFR CAR-T and CAR-NK cells showed potent cytotoxicity against NSCLC cells expressing EGFR including EGFR wild-type and mutant NSCLCs, as well as NSCLC cells with acquired resistance to OSI. However, EGFR CAR-T cells showed decreased killing activity against OSI-resistant NSCLC cells compared to their parental cells. In contrast, EGFR CAR-NK cells showed stronger cytotoxicity against NSCLC with acquired OSI resistance as compared to parental cells. Transcriptomic analysis revealed that NSCLC cells with acquired resistance to TKI had undergone epithelial-mesenchymal transition and had downregulated expression of genes related to antigen presentation and upregulated expression of B7-H6 (NCR3LG1), which have been reported to be associated with increased responsiveness to NK cells. Moreover, treatment of TKI resistant cells with OSI resulted in upregulation of cell surface EGFR and potentiated CAR-NK cells-mediated killing. TGF-β is a critical immune-suppressive cytokine. We found that EGFR-TKI resistant NSCLC cells elaborated expression of TGF-β as compared to parental cells. Blockade of the TGF-β pathway using galunisertib significantly enhanced the activity of EGFR CAR-NK cells against OSI-resistant NSCLC cells. Next, we engineered the dominant-negative TGF-β receptor II (DNTGFBRII) into our EGFR CAR constructs. The expression of DNTGFBRII inhibited the phosphorylation of SMAD2 and the downregulation of granzyme B and perforin induced by TGF-β. DNTGFBRII-CAR-NK cells showed higher killing activity against OSI-resistant NSCLC cells and were resistant to TGF-β-mediated immunosuppression. In conclusion, EGFR CAR-NK cells show significant anti-tumor activity to EGFR TKI-resistant NSCLC cells, and the cytotoxicity is enhanced in combination with OSI treatment and blockade of the TGF-β pathway. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Jacqulyne Robichaux, Alissa Poteete, Xiaoxing Yu, Fahao Zhang, Simon Heeke, Yu Qian, Junqin He, John Heymach. EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 548.

Combining EGFR-TKI With SAHA Overcomes EGFR-TKI-Acquired Resistance by Reducing the Protective Autophagy in Non-Small Cell Lung Cancer.

Nowadays, lung cancer has the highest mortality worldwide. The emergence of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has greatly improved the survival of patients with non-small cell lung cancer (NSCLC) having EGFR-TKI-sensitive mutations. Unfortunately, acquired resistance happens for most patients. In the present research, we found that EGFR-TKIs (such as gefitinib and osimertinib) can induce autophagy in NSCLC cell lines. Compared with parental sensitive cells, drug-resistant cells have higher autophagy activity. The use of an autophagy inhibitor could enhance the toxicity of gefitinib and osimertinib, which indicates that the enhancement of protective autophagy might be one of the mechanisms of EGFR-TKI resistance in NSCLC. In addition, increased autophagy activity is associated with decreased enhancer of zeste homolog 2 (EZH2) expression. Knockdown of EZH2 or EZH2 inhibitor treatment could lead to increased autophagy in NSCLC cells, indicating that EZH2 is a negative regulator of autophagy. We revealed that the increase in autophagy caused by the reduction of EZH2 was reversed in vitro and in vivo when combining gefitinib or osimertinib with suberoylanilide hydroxamic acid (SAHA), a broad-spectrum histone deacetylase inhibitor (HDACi). In conclusion, our results indicated that the combination of EGFR-TKIs and SAHA may be a new strategy to overcome EGFR-TKIs acquired resistance.

Pedigree analysis of the EGFR p.V1010M germline mutation in a family with a family history of non-small-cell lung cancer.

BackgroundTumors can be caused by genetic or environmental factors, but previous studies have shown that genetic factors contribute less to lung cancer than environmental factors. The epidermal growth factor receptor (EGFR) is the most common driver gene in non-small-cell lung cancer (NSCLC), but most variations are somatic. In this study, we reported on the pedigree of the EGFR p.V1010M germline mutation for the first time, and explored the correlation between the V1010M and the occurrence of NSCLC. Further, the effect of the V1010M on the treatment of the EGFR-tyrosine kinase inhibitors (TKIs) was investigated through the treatment of the proband with the simultaneous somatic mutation of the EGFR p.L858R.MethodsThe family members were screened using next-generation sequencing (NGS) and Sanger sequencing, and the pedigree was analyzed to examine the relationship between the EGFR p.V1010M and the occurrence of NSCLC. Schrodinger software was used to predict the structural function of the mutant amino acid sequence proteins.ResultsA total of 10 blood samples were collected from 4 generations of family members, many of whom had suffered from lung cancer. Six carriers of the EGFR p.V1010M were detected. The pedigree analysis showed that there was still no evidence of a correlation between the EGFR p.V1010M and disease occurrence. Additionally, the proband had the EGFR p.L858R somatic mutation, and the response after the treatment of gifitinib was stable disease (SD), which turned to progressive disease (PD) some 4 months later. Schrodinger software showed that the 1010th amino acid valine was located near the C terminal, and the variation to methionine had little effect on the structure of the EGFR dimer.ConclusionsThis study is the first report on pedigree with the EGFR p.V1010M germline mutation. Further research needs to be conducted to determine whether this mutation is pathogenic, but it is likely related to EGFR-TKI resistance in NSCLC.

Patritumab Deruxtecan: Paving the Way for EGFR-TKI–Resistant NSCLC

HER3 is ubiquitously expressed in EGFR-mutant non-small cell lung cancer (NSCLC) irrespective of resistant mechanisms to EGFR tyrosine kinase inhibitors, thus garnering attention as a valuable therapeutic target. In this issue of Cancer Discovery, Jänne and colleagues highlight early clinical data supporting patritumab deruxtecan as a potentially appreciable agent for previously treated EGFR-mutant NSCLC.See related article by Jänne et al., p. 74.

P71.04 MiR-1243 Increases Radiosensitivity in EGFR-TKI Resistant Non-Small Cell Lung Cancer Cells

Acquired resistance is an important issue which limits the application of EGFR-TKI in advanced non-small cell lung cancer (NSCLC). Our data showed nearly half of EGFR-mutant patients develop local relapse or limited metastases at disease progression. Previous studies have confirmed the value of radiotherapy in advanced lung cancer patients. EGFR-TKI resistant cells with T790M exhibited enhanced sensitivity to radiation. Studies have shown that microRNAs are involved in DNA damage repair mechanisms and thus affect the sensitivity of radiotherapy. Therefore, it is important to explore the detailed mechanism on miRNAs regulating radiosensitivity in EGFR-TKI resistant NSCLC. We performed miRNA microarray to screen miRNAs differentially expressed in the paired gefitinib-sensitivity PC-9 cells and gefitinib-resistant PC-9-GR cells (T790M mutation). Furthermore, we use bioinformatics to screen out target proteins and verify the expression differences of these target proteins. The proliferation of lung cancer cells was determined by CCK-8 assay. Proteins and the phosphorylation of proteins were evaluated by western blot assay. Colony formation assay showed PC-9-GR cells were more sensitive to radiation than PC-9 cells. MiRNA microarray showed 156 differentially expressed miRNAs between PC-9 and PC-9-GR cells. miR-1243 was in the top10 differentially expressed miRNAs and it was up-regulated in PC-9-GR cells. Stable knock-down miR-1243 PC-9-GR cells were further constructed using, termed as PC-9-GR-miR1243KD. CCK-8 assay showed similar proliferation kinetics between PC-9-GR and PC-9-GR-miR1243KD. It was found that PC-9-GR-miR1243KD cells showed a significant reduction in clone formation compared with PC-9-GR and PC-9-GR-NC cells after radiation, indicating higher radiosensitivity. Bioinformatics showed miR-1243 may function as a tumor oncogene to regulate apoptosis of gefitinib resistant cell with T790M mutation. SPOCK1 was one of its targeting gene. Western blots showed SPOCK1 was high expression in PC-9-GR-miR1243KD cells. This study found novel molecular mechanism might correlate with increase of radiosensitivity in EGFR-TKI resistant NSCLC cells. It could be a new therapeutic strategy for patients in advanced NSCLC to aid expansion of the effectiveness of TKI treatment through radiotherapy.

Reduced PHLPP Expression Leads to EGFR-TKI Resistance in Lung Cancer by Activating PI3K-AKT and MAPK-ERK Dual Signaling.

BackgroundEpidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in advanced EGFR-mutation non-small cell lung cancer (NSCLC) but the magnitude of tumor regression varies, and drug resistance is unavoidable. The pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) levels are reduced or lost and acts as a tumor suppressor in many cancers. Here, we hypothesized that PHLPP is a key regulator of EGFR-TKI sensitivity and a potential treatment target for overcoming resistance to EGFR-TKI in lung cancer.MethodsCell proliferation and growth inhibition were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assay. PHLPP- knockdown stable cell lines were generated by lentivirus-mediated delivery of PHLPP shRNAs. The expression of PHLPP mRNA and protein levels was detected by real-time quantitative polymerase chain reaction (qPCR) and Western blotting. Immunohistochemical (IHC) staining was performed to detect the PHLPP expression in clinical patient tissue samples. A transcriptomic assay of genome-wide RNA expressions of PHLPP in NSCLC cell lines according to gefitinib sensitivity was obtained from Gene Expression Omnibus (GEO) database. Murine xenograft model was established to verify the function of PHLPP in gefitinib resistance in vivo.ResultsPHLPP highly expressed in gefitinib-sensitive NSCLC cell lines than gefitinib-resistant NSCLC cell lines. In gefitinib-acquired resistance cell line HCC827-GR, PHLPP expression even dramatically reduced. Knockdown of PHLPP in NSCLC cells decreased cell death induced by the EGFR-TKI, while overexpression PHLPP in gefitinib-resistance NSCLC cells can enhance or restore EGFR-TKIs sensitivity. Mechanism study indicated that PHLPP downregulation attenuates the effect of EGFR-TKI on the both AKT and ERK pathway, thereby decreasing the cell death sensitivity to EGFR inhibitors. In xenograft mice, knockdown of PHLPP decreased tumor response to gefitinib and advanced tumor cells re-growth after gefitinib treatment. In clinical, PHLPP expression were reduced in the post-relapse tumor compared to that of pre-treatment, and lower pre-treatment PHLPP levels were significantly correlated with shorter progression-free survival (PFS) in patients with EGFR-mutant lung adenocarcinoma whom treated with EGFR-TKI.ConclusionsOur data strongly demonstrated that loss of PHLPP function was a key factor of EGFR-TKI resistance in NSCLC. Downregulated PHLPP expression activated PI3K-AKT and MAPK-ERK pathway which strengthened cell survival to EGFR-TKI. Therefore, PHLPP expression level was not only a potential biomarker to predict EGFR-TKIs sensitivity but also as a therapeutic target in EGFR-TKIs therapy, enhancing PHLPP expression may be a valuable strategy for delaying or overcoming EGFR-TKIs drug resistance.

Effects of hyperinsulinemia on acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitor via the PI3K/AKT pathway in non-small cell lung cancer cells in vitro.

Patients with lung cancer harboring activating epidermal growth factor (EGFR) mutations and pre-existing diabetes have been demonstrated to exhibit poor responses to first-line EGFR-tyrosine kinase inhibitor (TKI) therapy. Strategies for the management of acquired resistance to EGFR-TKIs in patients with advanced non-small cell lung cancer (NSCLC) are urgently required. Only a limited number of studies have been published to date on the effects of insulin on EGFR-TKI resistance in NSCLC. Hence, the aim of the present study was to investigate the roles of hyperinsulinemia and hyperglycemia in mediating gefitinib resistance in NSCLC cells with activating EGFR mutations. In the present study, the HCC4006 cell line, which harbors EGFR mutations, was co-treated with gefitinib and long-acting insulin glargine. Whether hyperinsulinemia is able to mediate EGFR-TKI resistance in the NSCLC cell line harboring activating EGFR mutations was also investigated, and the possible underlying mechanisms responsible for these actions were explored. The inhibition of cell proliferation, and the potential mechanism of gefitinib resistance, were examined using an MTS proliferation assay and western blot analysis, and through the transfection of siRNAs. Whether the inhibition of AKT is able to overcome EGFR-TKI resistance induced by long-acting insulin was also investigated. The results obtained suggested that hyperinsulinemia induced by glargine upregulated NSCLC cell proliferation and survival, and induced gefitinib resistance. By contrast, the morphology and proliferation of the cells in a medium containing a 2-fold concentration of glucose were not significantly affected. Gefitinib resistance induced by hyperinsulinemia may have been mediated via the phosphoinositide 3-kinase (PI3K)/AKT pathway rather than the mitogen-activated protein kinase extracellular signal regulated kinase (MAPK/ERK) pathway. AKT serine/threonine kinase 1 knockdown by siRNA rescued the gefitinib resistance that was induced by hyperinsulinemia. In conclusion, hyperinsulinemia, but not hyperglycemia, was identified to cause the development of gefitinib resistance in NSCLC cells with activating EGFR mutations. However, additional studies are required to investigate strategies, such as co targeting hyperinsulinemia and the PI3K/AKT pathway, for overcoming EGFR-TKI resistance in patients with NSCLC.

1366P Efficacy and safety of apatinib plus EGFR-TKI in advanced non-small cell lung cancer with EGFR-TKI resistance (date updated)

Treatment failure frequently occurs in patients with EGFR-mutant non-small cell lung cancer (NSCLC) who respond to EGFR-TKI initially. Previous studies have shown that apatinib (a TKI against VEGFR-2) combined with EGFR-TKI might prevent progression of the disease. We conducted this trial to investigate the efficacy and safety of apatinib (a TKI against VEGFR-2) plus EGFR-TKIs (including erlotinib, gefitinib, icotinib, afatinib and osimertinib) compared with chemotherapy for EGFR-TKI resistant NSCLC patients. From Mar 2017 to Nov 2019, this study enrolled 42 advanced NSCLC patients who acquired resistance to the EGFR-TKI therapy. 27 patients received apatinib plus EGFR-TKI (apatinib in start dose of 250 mg plus original EGFR-TKI dose), 15 patients received chemotherapy (pemetrexed or vinorelbine with platinum). In the apatinib group, 24/27 patients were available evaluated. The objective response rate was 21% (5/24) and the disease control rate was 96% (24/27). The most common adverse events in the apatinib group were diarrhea (63%, 17/27), hypertension (63%, 17/27) and hand-foot syndrome (41%, 11/27). The most common grade 3 or 4 toxicity was proteinuria (11%, 3/27). Most of adverse events were grade 1 or 2, as well as controllable and tolerable. Six patients with brain metastases in the apatinib group got long median progressive-free survival (PFS) of 15.7 months. In the chemotherapy group, 12/15 patients were available evaluated. The objective response rate was 25% (3/12) and the disease control rate was 92% (11/12). No new adverse event occured. The median PFS of apatinib group was 10.5 months, and chemotherapy group was 4.5 months. The longest PFS in the apatinib group was 29.8 months. Apatinib plus EGFR-TKI shown a good clinical efficacy in patients with acquired EGFR-TKI (1st, 2nd or 3rd generation) resisitance. Patient's quality of life and the compliance of therapy had been increased by oral drugs. Besides, we found that patients’ PFS tended to be prolonged who with EGFR 21 mutation (15.7 months), or male (17 months).