Molecular mechanism of acquired drug resistance in the EGFR-TKI resistant cell line HCC827-TR.

The first-line standard treatment of non-small cell lung cancer (NSCLC) with EGFR mutation is EGFR-tyrosine kinase inhibitors (TKIs). However, most patients will develop acquired resistance after 9-13 months. This study investigated novel molecular mechanisms of acquired resistance to EGFR-TKIs to identify a potential new treatment for EGFR-TKI resistant NSCLC patients. We established an EGFR-TKI resistant cell line (HCC827-TR) by culturing the HCC827-P cell line through continuous erlotinib culture. We used Sanger sequencing, RT-PCR, and western blot to rule out known resistance mechanisms in HCC827-TR cells, including EGFR-T790M and MET, PTEN, or EGFR expression changes. Next-generation sequencing was performed and identified differentially expressed genes between two cell lines and examined the genes with GO and KEGG pathway database analyses. We also examined the molecular alterations in COSMIC and GDSC databases and performed hazard predictions using SIFT, PolyPhen-2, Mutation Taster, and CADD. Our results identified FGF2 as a differentially expressed gene with a G101T point mutation in HCC827-TR cells that showed high mutation frequency and hazard score. HCC827-TR cells showed elevated FGF2 compared to parental cells. It is noteworthy that treatment with the FGFR inhibitor AZD4547 could restore the sensitivity of HCC872-TR cells to erlotinib. An erlotinib-resistant cell line HCC827-TR was successfully constructed and we identified the EGFR-TKI resistance mechanism involving the FGF2 gene mutation. Targeted inhibition of the FGF2/FGFR signaling pathway may effectively restore the sensitivity of the resistant cells to erlotinib. These results suggest a novel treatment strategy for EGFR-TKI resistant NSCLC patients. Significant findings of the study: Identifies a novel molecular mechanism for EGFR-TKI acquired resistance. A potential novel strategy for the treatment of EGFR-TKI resistant NSCLC patients.

Yu T ，Xia Q ，Gong T ，Wang J ，Zhong D ... -  《-》

LMNA Reduced Acquired Resistance to Erlotinib in NSCLC by Reversing the Epithelial-Mesenchymal Transition via the FGFR/MAPK/c-fos Signaling Pathway.

Hu C ，Zhou A ，Hu X ，Xiang Y ，Huang M ，Huang J ，Yang D ，Tang Y ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Anlotinib can overcome acquired resistance to EGFR-TKIs via FGFR1 signaling in non-small cell lung cancer without harboring EGFR T790M mutation.

Although many studies have defined mechanisms of resistance to EGFR-TKIs, acquired resistance remains the major limitation of monotherapy with EGFR-TKIs. Cell viability was analyzed using a Cell Counting Kit-8 (CCK-8) assay. EGFR T790M mutation was sequenced on a HiSeq 4000 platform. mRNAs from HCC827 and HCC827 gefitinib-resistant (GR) cells were analyzed by genome analyzer-based deep sequencing. The effect of anlotinib on apoptosis and cell cycle arrest of HCC827 GR was detected by fluorescence-activated cell sorting (FACS) analysis. A mouse xenograft model was used to assess the effect of anlotinib on HCC827 GR cells. The T790M mutation was found in the PC-9 GR cell line but not in the HCC827 GR cell line. Anlotinib could suppress the growth of HCC827 GR cells by inhibiting FGFR1 in vitro and in a mouse xenograft model. Moreover, FGFR1 was overexpressed in HCC827 GR cells, and the knockdown of FGFR1 reversed gefitinib resistance in HCC827 GR cells. Furthermore, anlotinib induced apoptosis and cell cycle arrest in HCC827 GR cells by increasing the activity of Caspase-3. FGFR1 overexpression could be the mechanism of EGFR-TKI acquired resistance and anlotinib can suppresse the growth of EGFR-TKI-resistant NSCLC cells without T790M mutation.

Lian Z ，Du W ，Zhang Y ，Fu Y ，Liu T ，Wang A ，Cai T ，Zhu J ，Zeng Y ，Liu Z ，Huang JA ... -  《-》

Enhanced Sensitivity of Nonsmall Cell Lung Cancer with Acquired Resistance to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors to Phenformin: The Roles of a Metabolic Shift to Oxidative Phosphorylation and Redox Balance.

Although the efficacy of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR- TKI) therapy has been proven in non-small cell lung cancer (NSCLC) patients, acquired resistance to EGFR-TKIs presents a serious clinical problem. Hence, the identification of new therapeutic strategy is needed to treat EGFR-TKI-resistant NSCLC. Acquired EGFR-TKI-resistant lung cancer cell lines (HCC827, H1993, and H292 cells with acquired resistance to gefitinib or erlotinib) were used for cell-based studies. IncuCyte live cell analysis system and XFp analyzer were used for the determination of cell proliferation and energy metabolism, respectively. In vivo anticancer effect of phenformin was assessed in xenografts implanting HCC827 and gefitinib-resistant HCC827 (HCC827 GR) cells. HCC827 GR and erlotinib-resistant H1993 (H1993 ER) cells exhibited different metabolic properties compared with their respective parental cells, HCC827, and H1993. In EGFR-TKI-resistant NSCLC cells, glycolysis markers including the glucose consumption rate, intracellular lactate level, and extracellular acidification rate were decreased; however, mitochondrial oxidative phosphorylation (OXPHOS) markers including mitochondria-driven ATP production, mitochondrial membrane potential, and maximal OXPHOS capacity were increased. Cell proliferation and tumor growth were strongly inhibited by biguanide phenformin via targeting of mitochondrial OXPHOS complex 1 in EGFR-TKI-resistant NSCLC cells. Inhibition of OXPHOS resulted in a reduced NAD+/NADH ratio and intracellular aspartate levels. Recovery of glycolysis by hexokinase 2 overexpression in erlotinib-resistant H292 (H292 ER) cells significantly reduced the anticancer effects of phenformin. Long-term treatment with EGFR-TKIs causes reactivation of mitochondrial metabolism, resulting in vulnerability to OXPHOS inhibitor such as phenformin. We propose a new therapeutic option for NSCLC with acquired EGFR-TKI resistance that focuses on cancer metabolism.

Kim S ，Im JH ，Kim WK ，Choi YJ ，Lee JY ，Kim SK ，Kim SJ ，Kwon SW ，Kang KW ... -  《-》

AXL/MET dual inhibitor, CB469, has activity in non-small cell lung cancer with acquired resistance to EGFR TKI with AXL or MET activation.

In non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations, acquired resistance to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) may occur via bypass signals such as AXL or MET activation. We investigated the antitumor activity of CB469, a newly developed drug that targets both AXL and MET, in EGFR TKI-resistant NSCLC cells. We generated EGFR TKI-resistant NSCLC cell lines with acquired resistance to erlotinib, gefitinib, and osimertinib (PC9/ER, HCC827/GR and HCC827/OR, respectively). We characterized the mechanisms of CB469 action in resistant cells and investigated the antitumor efficacy of CB469 both in vitro and in vivo. Resistant cells showed activation of phosphorylated EGFR, as well as AXL and MET activation and phosphorylation. The combination of CB469 and EGFR TKIs synergistically inhibited cell proliferation and colony formation rates in resistant cell lines. The combination of CB469 and erlotinib induced apoptosis of PC9/ER cells. Mechanistically, resistant cells showed an interaction of AXL and MET. CB469 and EGFR TKI also demonstrated antitumor activity by reducing phosphorylated AXL and MET in mouse xenograft models with HCC827/GR cells. The combination of CB469 and EGFR TKI can overcome the acquired resistance to EGFR TKI mediated by AXL and MET activation. We anticipate that the dual inhibitory actions of CB469 will assist with the development of targeted therapy for EGFR-mutant NSCLC patients who fail initial EGFR TKI therapy.

Yang YM ，Jang Y ，Lee SH ，Kang B ，Lim SM ... -  《-》