EGFR-mutant Lung Cancer Cell Lines Research Articles

Effect of P. gingivalis on osimertinib resistance in EGFR-mutant lung cancer.

e20511 Background: While most patients with EGFR-mutant lung cancers respond to osimertinib, resistance inevitably develops. Mechanisms of osimertinib resistance remain inadequately understood. Recent studies suggest a potential link between intra-tumoral bacteria and therapy resistance in cancer. Our previous studies found that the pre-conditioned medium (PCM) of certain bacteria in the Bacteroidetes phylum conferred resistance of all EGFR-TKIs tested. In this study, we specifically investigate the effect of Porphyromonas gingivalis, a common component of the human oral microbiome and the main pathogen responsible for periodontal disease, on osimertinib sensitivity in EGFR mutant lung cancer. Methods: P. gingivalis PCM (P-CM) was applied to EGFR-mutant lung cancer cell lines for 3 days and cell viability was measured. Western blot and receptor tyrosine kinase (RTKs) assay were used to analyze the changes of cell signaling pathways, and the contribution of identified signaling pathway to P. gingivalis-mediated resistance to osimertinib were further validated by rescue experiments. To investigate the existence of P. gingivalis in human lung cancer tissues, we used Fluorescence In Situ Hybridization (FISH) probes specific to P. gingivalis and hybridized probes to a tissue microarray (TMA) containing 3 mm punches of lung adenocarcinoma tissue from 30 patients in duplicate. The underlying mechanisms of osimertinib resistance conferred by P. gingivalis were explored by a combination of bacterial genomic and cellular proteomic approaches. Results: P-PCM led to osimertinib resistance in all EGFR-mutant lung cancer cell lines tested. Both Western blot and RTKs assay showed that phosphorylated insulin-like growth factor 1 receptor (pIGF1R) level significantly increased when P-PCM was applied to PC9 EGFR mutant lung cancer cells. We found that both IGF1R inhibitor (linsitinib) and IGF1R knockdown with siRNA eliminated the effect of P-PCM-mediated osimertinib resistance. We found that two cysteine proteases secreted by P. gingivalis, lysine-specific gingipain (Kgp) and arginine-specific gingipain A (RgpA) led to osimertinib, whereas arginine-specific gingipain B (RgpB), which lacks haemagglutinin/adhesin (HA) region of RgpA had no effect on osimertinib response. We also found that gingipains-mediated osimertinib resistance is independent of enzymatic activity. We detected intra-tumoral P. gingivalis in tumor tissue of 9 (30%) patients by using FISH probes specific to P. gingivalis. Conclusions: Our studies demonstrate that P. gingivalis can induce osimertinib resistance in EGFR-mutant lung cancer cells through secreted gingipains that activate the IGF1R pathway through an enzymatic- independent mechanism. P. gingivalis is frequently detected in human lung cancer tissues , suggesting that therapies targeting P. gingivalis may improve EGFR-TKI response in certain patients with EGFR-mutant lung cancer.

Abstract 5826: Comprehensive analysis with single-cell RNA sequence and spatial transcriptomics unravels distinctive expression profile of apoptotic-related genes in EGFR-mutated lung cancer

Abstract Despite an initial marked response to targeted therapy with tyrosine kinase inhibitors (TKIs) in EGFR-mutated lung cancer, the duration of response is limited due to the inevitable development of acquired resistance. Anti- and pro-apoptotic genes play an important role in the survival of tumor cells. Currently, variations in these gene expression levels are not well characterized due to the complex interplay between the inducement of expression changes by EGFR-TKIs and the presence of inter- and intra-tumor heterogeneity. Here, a comprehensive analysis of apoptosis-related genes, including BCL-2 family members and the IAP family members, was conducted via single-cell RNA sequence (scRNA-seq) and spatial transcriptomics. scRNA-seq captured changes in apoptosis-related gene expression after EGFR-TKI treatment using different EGFR-mutated lung cancer cell lines. Notably, BCL2L1 expression increased, whereas MCL1 expression decreased after EGFR-TKI treatment. Other anti- and pro-apoptotic genes did not display consistent trends in the cell lines used. Additionally, scRNA-seq was carried out on samples collected from patients with EGFR-mutated lung cancer after the development of resistance to EGFR-TKIs in order to validate the results obtained from cell line data and to compare gene expression levels between tumor and non-tumor cells. BCL2L1 exhibited high expression specifically in tumor cells; in contrast, MCL1 expression was lower in tumor cells compared to non-tumor cells. Moreover, BAD and XIAP were highly expressed in a tumor-specific manner. To further corroborate the scRNA-seq findings and delineate the spatial heterogeneity in tumors, spatial transcriptomics was performed using EGFR-driven lung cancer transgenic mouse specimens in three conditions: prior to treatment, during therapeutic response after short-term treatment, and following acquired resistance to EGFR-TKIs. Spatial transcriptomics data demonstrated that an increased BCL2L1 expression was observed in tumor tissues and that MCL1 expression was significantly higher in tissue adjacent to tumors than in tumors. Finally, we demonstrated that genetic ablation of BCL2L1 and pharmacological inhibition of BCL2L1 overcame or delayed resistance to EGFR-TKIs in vitro and in vivo. Overall, BCL2L1 is particularly important for tumor cell survival during the emergence of drug tolerance and the development of acquired resistance to EGFR-TKIs. Citation Format: Motohiro Izumi, Masanori Fujii, Vivian Ho, Ikei S. Kobayashi, Susumu S. Kobayashi. Comprehensive analysis with single-cell RNA sequence and spatial transcriptomics unravels distinctive expression profile of apoptotic-related genes in EGFR-mutated lung cancer [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 5826.

TP53 gain-of-function mutations promote osimertinib resistance via TNF-α–NF-κB signaling in EGFR-mutated lung cancer

EGFR tyrosine kinase inhibitors (TKIs) are effective against EGFR-mutated lung cancer, but tumors eventually develop resistance to these drugs. Although TP53 gain-of-function (GOF) mutations promote carcinogenesis, their effect on EGFR-TKI efficacy has remained unclear. We here established EGFR-mutated lung cancer cell lines that express wild-type (WT) or various mutant p53 proteins with CRISPR-Cas9 technology and found that TP53-GOF mutations promote early development of resistance to the EGFR-TKI osimertinib associated with sustained activation of ERK and expression of c-Myc. Gene expression analysis revealed that osimertinib activates TNF-α–NF-κB signaling specifically in TP53-GOF mutant cells. In such cells, osimertinib promoted interaction of p53 with the NF-κB subunit p65, translocation of the resulting complex to the nucleus and its binding to the TNF promoter, and TNF-α production. Concurrent treatment of TP53-GOF mutant cells with the TNF-α inhibitor infliximab suppressed acquisition of osimertinib resistance as well as restored osimertinib sensitivity in resistant cells in association with attenuation of ERK activation and c-Myc expression. Our findings indicate that induction of TNF-α expression by osimertinib in TP53-GOF mutant cells contributes to the early development of osimertinib resistance, and that TNF-α inhibition may therefore be an effective strategy to overcome such resistance in EGFR-mutant lung cancer with TP53-GOF mutations.

SLC12A8 mediates TKI resistance in EGFR-mutant lung cancer via PDK1/AKT axis.

Epidermal growth factor receptor (EGFR) mutation is a prominent driver of lung cancer. Tyrosine kinase inhibitors (TKIs) have shown efficacy in treating EGFR-mutant lung cancer, but the emergence of drug resistance poses a significant challenge. Recent research has highlighted solute carrier family 12 member 8 (SLC12A8) as one of the highly upregulated genes in various cancer types. However, its oncogenic function remains largely unexplored. 343 consecutive lung cancer patients were prospectively recruited and were followed for over 10years. SLC12A8 expression in lung cancer tissues was measured by qPCR and was associated with patient survival. The association of SLC12A8 with TKI resistance was studied in in vitro EGFR-mutant lung cancer cell line as well as in in vivo xenograft tumor model. High-throughput kinome screening was employed to investigate SLC12A8-mediated oncogenic signaling pathway in lung cancer. SLC12A8 is a predictive biomarker of poor prognosis in lung cancer, particularly in patients with EGFR mutations. SLC12A8 overexpression diminishes the effectiveness of TKIs in EGFR-mutant lung cancer, resulting in treatment failure and disease progression. More importantly, SLC12A8-induced TKI resistance is mediated by the PDK1/AKT signaling axis, while silencing SLC12A8 expression inhibits oncogenic PDK1/AKT signaling, restoring TKI sensitivity in lung cancer cells. SLC12A8 mediates TKI resistance in EGFR-mutant lung cancer via PDK1/AKT axis. These findings not only advance our understanding of the molecular mechanisms driving TKI resistance, but also offer novel alternative strategies for the treatment of lung cancer.

Abstract 2859: High-resolution lineage tracing for the study of cancer drug persistence at the single-cell level

Abstract Cancer drug persistence allows small fractions of otherwise drug-sensitive populations of cancer cells to survive treatment with anti-cancer drugs. Unlike drug resistance, persistence mechanisms are not driven by genetic mutations, and are thus considered reversible after a ‘drug holiday’ period. While most persistent cells remain in cell cycle arrest under drug treatment, recent work with EGFR-mutant Non-Small Cell Lung Cancer (NSCLC) cell lines has demonstrated that a small fraction of persister cells continue to undergo cell divisions under treatment with EGFR inhibitors. Thus, persister cells have a two-fold potential clinical significance: (i) they can serve as an intermediate state, allowing a non-resistant cancer cell to survive long enough and continue to divide under drug treatment to acquire a resistance mutation; and (ii) cycling persister cells might directly contribute to relapse, without a secondary resistance-driving mutation. Despite this, little is known about the underlying mechanisms by which affect the potential of a given cell to persist, or about the epigenetic traits that govern the distribution of persistence potential in a cell population. In this work, we use high-resolution lineage tracing to study lineages of drug persistent cells within a cancer cell population. Using the PC9 NSCLC cell line as a model, we demonstrate that while most lineages within the PC9 cell population can generate persisters, certain branches within the lineage tree have significantly higher persistence potentials than other branches, suggesting that inherited events determine the probability of a cell to persist under treatment. Moreover, we analyze the rate of change of the potential to persist within a single-cell-derived lineage of NSCLC cells and assess which branches within the lineage tree generate cycling persisters. Furthermore, we couple single-cell RNAseq data with lineage tracing information to identify gene expression patterns that correlate with the probability of drug-naïve cells to persist under drug treatment, allowing for insight into the cellular and molecular mechanisms underlying the continuum of the probability to persist. Finally, we demonstrate that while persistent-derived PC9 cells are indeed resensitized after a drug holiday, they give rise to a significantly higher proportion of cycling persister cells upon resumption of drug treatment, allowing for a rapid repopulation of the cell culture by cycling persisters. These results suggest that lineage tracing is a powerful tool to study drug-induced persistence and resistance and we will apply it to multiple other cell lines and tumor types. Citation Format: Benny Zhitomirsky, Jideofor Ezike, Elizabeth Hopkins, Brian P. Danysh, Laxmi Parida, Yaara Oren, Gad Getz. High-resolution lineage tracing for the study of cancer drug persistence at the single-cell level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2859.

Integrated CRISPR screening and drug profiling identifies combination opportunities for EGFR, ALK, and BRAF/MEK inhibitors

Anti-tumor efficacy of targeted therapies is variable across patients and cancer types. Even in patients with initial deep response, tumors are typically not eradicated and eventually relapse. To address these challenges, we present a systematic screen for targets that limit the anti-tumor efficacy of EGFR and ALK inhibitors in non-small cell lung cancer and BRAF/MEK inhibitors in colorectal cancer. Our approach includes genome-wide CRISPR screens with or without drugs targeting the oncogenic driver ("anchor therapy"), and large-scale pairwise combination screens of anchor therapies with 351 other drugs. Interestingly, targeting of a small number of genes, including MCL1, BCL2L1, and YAP1, sensitizes multiple cell lines to the respective anchor therapy. Data from drug combination screens with EGF816 and ceritinib indicate that dasatinib and agents disrupting microtubules act synergistically across many cell lines. Finally, we show that a higher-order-combination screen with 26 selected drugs in two resistant EGFR-mutant lung cancer cell lines identified active triplet combinations.

Chlorpromazine cooperatively induces apoptosis with tyrosine kinase inhibitors in EGFR-mutated lung cancer cell lines and restores the sensitivity to gefitinib in T790M-harboring resistant cells

We previously reported that the antipsychotic drug chlorpromazine (CPZ), which inhibits the formation of clathrin-coated vesicles (CCVs) essential for endocytosis and intracellular transport of receptor tyrosine kinase (RTK), inhibits the growth/survival of acute myeloid leukemia cells with mutated RTK (KIT D816V or FLT3-ITD) by perturbing the intracellular localization of these molecules. Here, we examined whether these findings are applicable to epidermal growth factor receptor (EGFR). CPZ dose-dependently inhibited the growth/survival of the non-small cell lung cancer (NSCLC) cell line, PC9 harboring an EGFR-activating (EGFR exon 19 deletion). In addition, CPZ not only suppressed the growth/survival of gefitinib (GEF)-resistant PC9ZD cells harboring T790 M, but also restored their sensitivities to GEF. Furthermore, CPZ overcame GEF resistance caused by Met amplification in HCC827GR cells. As for the mechanism of CPZ-induced growth suppression, we found that although CPZ hardly influenced the phosphorylation of EGFR, it effectively reduced the phosphorylation of ERK and AKT. When utilized in combination with trametinib (a MEK inhibitor), dabrafenib (an RAF inhibitor), and everolimus (an mTOR inhibitor), CPZ suppressed the growth of PC9ZD cells cooperatively with everolimus but not with trametinib or dabrafenib. Immunofluorescent staining revealed that EGFR shows a perinuclear pattern and was intensely colocalized with the late endosome marker, Rab11. However, after CPZ treatment, EGFR was unevenly distributed in the cells, and colocalization with the early endosome marker Rab5 and EEA1 became more apparent, indicating that CPZ disrupted the intracellular transport of EGFR. These results suggest that CPZ has therapeutic potential for NSCLC with mutated EGFR by a novel mechanism different from conventional TKIs alone or in combination with other agents.

Abstract LB080: Tipifarnib prevents emergence of resistance to osimertinib in EGFR-mutant NSCLC

Abstract Purpose: Drug-tolerant “dormant” cells (DTC) have emerged as one of the major non-genetic mechanisms driving resistance to targeted therapy in lung cancer, although the sequence of events leading to entry and exit from dormancy remain poorly described. Here, we provide a step-by-step phenotypic and molecular characterization of the different processes involved during the adaptive response to osimertinib using several EGFR-mutated lung cancer models. This strategy led to the identification of a common vulnerability of drug-tolerant cells which could be efficiently and safely targeted by a clinical stage drug. Experimental design: We used the FUCCI (fluorescence ubiquitination cell cycle indicator) system to determine the cell cycle dynamics in real time during the adaptive response to osimertinib in a panel of EGFR-mutated lung cancer cell lines. We performed scRNAseq on untreated and osimertinib-treated G1 and S/G2 sorted cells during early relapse to determine the molecular mechanisms underlying entry and exit from dormancy. We validated our observations in several in vitro and in vivo models as well as in publicly available patient data. Results: FUCCI labelling allowed the identification of a rare population of S/G2 cycling cells (referred to as early escapers) that emerged in the first hours of treatment amongst a majority of stably arrested and progressively dying G1 cells. scRNAseq data revealed that early escapers emerged from a differentiated alveolar type 1 (AT1) phenotype which was invariably associated with an increase in contractile-related gene signatures, F-actin polymerization and Rho/ROCK pathway activation. Using a screen of Rho-pathway inhibitors, we found that tipifarnib, a farnesyltransferase inhibitor (FTi), induced a complete clearance of DTC in vitro. Co-treatment with tipifarnib, a clinically active FTi, safely and durably prevented relapse to osimertinib in a PC9-xenograft model as well as in a PDX model of EGFRL858R/T790M lung cancer for up to 6 months with no evidence of toxicity. Several farnesylated targets were identified in both G1 and S/G2 treated cells, which could explain the high efficiency of tipifarnib in preventing the adaptive response to osimertinib. Finally, we observed that osimertinib + tipifarnib co-treatment completely suppressed the emergence of the AT1 phenotype, prevented mitosis of S/G2-treated cells and increased the apoptotic response through activation of the unfolded protein response (UPR) pathway. Conclusion: Our data strongly support the use of tipifarnib in combination with osimertinib in the clinic to effectively, durably and safely prevent relapse. Citation Format: Sarah Figarol, Célia Delahaye, Rémi Gence, Raghda Asslan, Sandra Pagano, Jacques Colinge, Jean-Philippe Villemin, Antonio Maraver, Isabelle Lajoie-Mazenc, Estelle Clermont, Anne Casanova, Anne Pradines, Julien Mazières, Olivier Calvayrac, Gilles Favre. Tipifarnib prevents emergence of resistance to osimertinib in EGFR-mutant 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 LB080.

Early On-Treatment Prediction of the Mechanisms of Acquired Resistance to EGFR Tyrosine Kinase Inhibitors.

Simple SummaryEGFR T790M-positive clones expand in treated by EGFR inhibitors. C-MET amplified clones expand in HCC827 lung cancer cells treated by EGFR inhibitors. Early on-treatment kinetics of the resistance-related gene predict the resistance mechanism.Background: Prediction of resistance mechanisms for epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) remains challenging. Thus, we investigated whether resistant cancer cells that expand shortly after EGFR-TKI treatment would eventually cause the resistant phenotype. Methods: We generated two EGFR-mutant lung cancer cell lines resistant to gefitinib (PC9GR and HCC827GR). The parent cell lines were exposed to short-term treatment with gefitinib or paclitaxel and then were assessed for EGFR T790M mutation and C-MET expression. These experiments were repeated in vivo and in clinically relevant patient-derived cell (PDC) models. For validation in clinical cases, we measured these gene alterations in plasma circulating tumor DNA (ctDNA) before and 8 weeks after starting EGFR-TKIs in four patients with EGFR-mutant lung cancer. Results: T790M mutation was only detected in the PC9GR cells, whereas C-MET amplification was detected in the HCC827GR cells. The T790M mutation level significantly increased in PC9 cells after short-term treatment with gefitinib but not in the paclitaxel. C-MET mRNA expression was only significantly increased in gefitinib-treated HCC827 cells. We confirmed that the C-MET copy number in HCC827 cells that survived after short-term gefitinib treatment was significantly higher than that in dead HCC827 cells. These findings were reproduced in the in vivo and PDC models. An early on-treatment increase in the plasma ctDNA level of these gene alterations was correlated with the corresponding resistance mechanism to EGFR-TKIs, a finding that was confirmed in post-treatment tumor tissues. Conclusions: Early on-treatment kinetics in resistance-related gene alterations may predict the final mechanism of EGFR-TKI resistance.

Beneficial effect of erlotinib and trastuzumab emtansine combination in lung tumors harboring EGFR mutations

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is the standard therapy for non-small cell lung cancer (NSCLC) harboring EGFR mutations, but the resistance is inevitable. The drug-tolerant persister cancer cells are thought to be involved in the resistance. We recently reported that HER2 expression had a negative impact on time-to-treatment-failure in patients with EGFR mutant NSCLC. In this study, we hypothesized that HER2 might be a potential target for alternative combination therapy in NSCLC harboring EGFR mutations. In vitro study showed that the level of HER2 expression had no correlation with the sensitivity to EGFR-TKI, erlotinib but showed some correlation with HER2-inhibitor, ado-trastuzumab emtansine (T-DM1) in multiple EGFR-mutant lung cancer cell lines. In addition, HER2 expression was increased in persister cancer cells in 11–18 cell line harboring EGFR L858R or HCC827 cell line harboring EGFR exon 19 deletion after the exposure to erlotinib in vitro and in vivo. The combination of erlotinib and T-DM1 showed a superior inhibitory effect on cell proliferation compared with those of the erlotinib or T-DM1 alone in either 11–18 or HCC827 cells in vitro. The combination therapy also induced a significantly greater inhibitory effect on tumor growth in xenograft model in mice transplanted with either 11–18 or HCC827 cells compared with erlotinib alone or T-DM1 alone. No body weight loss was observed in these mice. These results suggested that the combination therapy with EGFR-TKI and T-DM1 might be a potentially promising strategy for treating lung cancer harboring EGFR mutations.

Abstract 1874: Anti-VEGF therapy sensitizes EGFR/MET co-altered NSCLC cells to TKIs via inhibiting the VEGF/ERK/MET pathway

Abstract Mesenchymal epithelial transition factor (MET) activation has been considered to mediate primary and acquired resistance to EGFR tyrosine kinase inhibitors (TKI) in epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC), but the mechanisms are not wholly clear. Meanwhile, the effective therapeutic strategy for these patients remains controversial. We previously presented a lung adenocarcinoma patient with dual EGFR-MET alterations benefited from bevacizumab + erlotinib as first-line treatment, achieving 13 months partial response (PR). PFS of another phase reached to 9 months with the combination of osimertinib + crizotinib + bevacizumab when disease proceeded due to increased MET copy number. Persistent PFS benefit was confirmed when the patient was administrated with different TKIs treatment in the presence of bevacizumab. However, the underlying mechanism remains elusive. Thus, we assumed that VEGF might be involved in the regulation of the MET signaling pathway and the addition of anti-VEGF therapy might be promising. In this study, we firstly confirmed whether MET overexpression alone might causally responsible for resistance to gefitinib treatment. Thus, we conducted in vitro forward genetic experiments, we took advantage of the EGFR-mutant lung cancer cell line PC-9, which had been shown to be sensitive to gefitinib. Ectopic overexpression of MET in PC-9 parental cells (PC-9/MET) was able to confer resistance to gefitinib by activating AKT and ERK signaling and sustaining proliferation.Meanwhile we got a gefitinib resistant EGFR mutant NSCLC cell line, HCC827 (HCC827-GR), which showing acquired MET amplification. Interestingly, we found that VEGF promoted cell survival and elicited adaptive resistance to gefitinib and crizotinib alone or in combination in MET transduced PC9-MET cells. This phenomenon also existed in HCC827-GR which harboring MET amplification. Importantly, addition of the VEGF inhibitor bevacizumab was able to restore partial sensitivity to gefitinib in both PC-9/MET and HCC827-GR cells while triple inhibition of EGFR, MET, and VEGF could suppresses the cell proliferation more remarkable. Moreover, we observed that VEGF could facilitate MET phosphorylation and activate several intracellular signaling pathways, including ERK, AKT, and Stat3. Then we pretreated cells with several specific kinase inhibitors, followed by treatment with VEGF. We found that VEGF-mediated up-regulation of MET depended on the ERK pathway and was independent of AKT and Stat3 pathways. These findings were further confirmed in human NSCLC Xenograft models. Addition of bevacizumab to TKIs resulted in stronger inhibition of tumor growth than TKI alone or TKIs combination treatment, by blocking both EGFR and MET signal transduction. In conclusion, bevacizumab + TKIs enhanced antitumor activity in NSCLC with EGFR/MET co-alteration through inhibition of the VEGF/ ERK/MET signaling axis. Citation Format: Qian Chu, Shanshan Huang, Yuan Gao, Jie Lin, Yuan Chen, Bo Zhu, Kongming Wu. Anti-VEGF therapy sensitizes EGFR/MET co-altered NSCLC cells to TKIs via inhibiting the VEGF/ERK/MET pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1874.

PIM1 inhibitor synergizes the anti-tumor effect of osimertinib via STAT3 dephosphorylation in EGFR-mutant non-small cell lung cancer.

BackgroundAn increasing amount of evidence has demonstrated that combined or multiple targeted therapies could bring about more durable clinical outcomes, and it is known that epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance is related to bypass activation. This study aims to explore a specific solution for third-generation EGFR-TKI resistance caused by bypass activation, and to examine the antitumor effects of the combination of a novel inhibitor CX-6258 HCl with osimertinib, along with its underlining mechanisms.MethodsA bioinformatics analysis was performed to detect the relations between the provirus integration site for Moloney murine leukemia virus 1 (PIM1) expression and prognosis of lung cancer. The EGFR-mutated lung cancer cell lines were treated with the combination of CX-6258 HCl and osimertinib to analyze cell proliferation using the Cell Counting Kit-8, colony formation, and in vivo experiments. Cell migration was analyzed using wound healing and Transwell assays. The apoptosis level was detected using Annexin V–propidium iodide flow cytometry. The expression levels of EGFR and STAT3 were determined using Western blot analysis.ResultsHigh expression level of PIM1 was related to the poor prognosis of non-small cell lung cancer (NSCLC). The combined administration of osimertinib and CX-6258 HCl significantly inhibited cell proliferation and migration and effectively induced apoptosis in lung cancer cells. It was more efficient in suppressing EGFR activation and phosphorylation of STAT3 compared with osimertinib treatment alone. Furthermore, it showed a durable efficacy in a xenograft model.ConclusionsThis study showed that PIM1 is a poor prognostic factor for NSCLC. CX-6258 HCl is a potential molecular inhibitor to sensitize the antitumor effects of osimertinib through the inhibiting of the phosphorylation of STAT3 in NSCLC.

Reciprocal regulation of miR-206 and IL-6/STAT3 pathway mediates IL6-induced gefitinib resistance in EGFR-mutant lung cancer cells.

Persistently activated IL‐6/STAT3 pathway promotes acquired resistance to targeted therapy with epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs) in non–small‐cell lung cancer (NSCLC) treatment. miR‐206 has been verified to be dysregulated and plays as a negative regulator in lung cancer. However, whether miR‐206 may overcome IL6‐induced gefitinib resistance in EGFR‐mutant lung cancer remains elusive. In this study, we investigated the role of miR‐206 in IL6‐induced gefitinib‐resistant EGFR‐mutated lung cancer cell lines. We showed that forced miR‐206 expression restored gefitinib sensitivity in IL6‐induced gefitinib‐resistant EGFR‐mutant lung cancer cells by inhibiting IL6/JAK1/STAT3 pathway. Specifically, mechanistic investigations revealed that miR‐206 blocked IL‐6/STAT3 signalling via directly targeting the 3'‐UTR of intracellular IL‐6 messenger RNA. Moreover, IL‐6 induced miR‐206 down‐regulation by reducing the cropping process of primary miR‐206 (pri‐miR‐206) into the Drosha/DGCR8 complex. Taken together, our findings reveal a direct role of miR‐206 in regulating IL‐6/STAT3 pathway and contrarily activated IL‐6/STAT3 signalling mediates the miR‐206 maturation process in gefitinib‐resistant EGFR‐mutant lung cancer cells.

Abstract 1273: Molecular dynamics prediction of the mechanism of acquired resistance to EGFR inhibitors in EGFR-mutant lung cancer

Abstract Background Acquired drug resistance is the most challenging problem in treating nonsmall cell lung cancer (NSCLC) patients with EGFR tyrosine kinase inhibitor (TKI). We evaluated whether resistance cell subclones significantly increased shortly after treatment with EGFR-TKIs could determine the final resistance phenotype for EGFR-TKIs in the tumor with EGFR mutations. Methods We generated 2 EGFR-mutant lung cancer cell lines (PC9GR and HCC827GR) resistant to gefitinib and evaluated the resistance mechanisms in each cell line. In parent cell lines (PC9 and HCC827) the amounts of EGFR T790M mutation and C-MET and VIMENTIN mRNA expression were measured before and after 48 hours treatment with gefitinib or paclitaxel. Using a cell line derived directly from tumors of treatment-naïve NSCLC patient with EGFR mutation, the above tests were repeated. We prospectively collected the bloods from 4 NSCLC patients with EGFR mutations at baseline and eight weeks after EGFR-TKI treatment and measured the level of T790M mutation in circulating tumor DNAs (ctDNA) from their bloods by colourimetric assay. Results EGFR T790M mutation was detected in PC9GR cells whereas C-MET amplification in HCC827GR cells. The proportion of EGFR T790M mutation significantly increased in the PC9 cells treated with gefitinib whereas not in the same cells treated with paclitaxel or not in the HCC827 treated with gefitinib or paclitaxel. The mRNA expression of C-MET was significantly increased in the HCC827 treated with gefitinib. The VIMENTIN mRNA expression was not changed in both cell lines. When we treated the patient-derived cells with gefitnib, the frequency of T790M significantly increased whereas C-MET mRNA expression decreased and VIMENTIN mRNA expression was not changed. In the corresponding resistance tumor tissue, only T790M mutation was detected without C-MET amplification and VIMENTIN overexpression. Two cases whose T790M mutation level in their ctDNAs increased at 8 weeks by more than 50% revealed T790M mutation in their final resistance tumor tissue. But, in other cases without an early surge of T790M mutation in their ctDNAs, T790M mutation was not detected in posttreatment biopsy sample. Conclusions Early-on-treatment quantitative change of resistance-related gene alterations may predict the final mechanism of resistance to EGFR-TKI. Citation Format: Youngjoo Lee, Yu-Ra Choi. Molecular dynamics prediction of the mechanism of acquired resistance to EGFR inhibitors in EGFR-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1273.

Acquired BRAF Rearrangements Induce Secondary Resistance to EGFR therapy in EGFR-Mutated Lung Cancers

IntroductionMultiple genetic mechanisms have been identified in EGFR-mutant lung cancers as mediators of acquired resistance (AR) to EGFR tyrosine kinase inhibitors (TKIs), but many cases still lack a known mechanism. MethodsTo identify novel mechanisms of AR, we performed targeted large panel sequencing of samples from 374 consecutive patients with metastatic EGFR-mutant lung cancer, including 174 post-TKI samples, of which 38 also had a matched pre-TKI sample. Alterations hypothesized to confer AR were introduced into drug-sensitive EGFR-mutant lung cancer cell lines (H1975, HCC827, and PC9) by using clustered regularly interspaced short palindromic repeats/Cas9 genome editing. MSK-LX138cl, a cell line with EGFR exon 19 deletion (ex19del) and praja ring finger ubiquitin ligase 2 gene (PJA2)/BRAF fusion, was generated from an EGFR TKI–resistant patient sample. ResultsWe identified four patients (2.3%) with a BRAF fusion (three with acylglycerol kinase gene (AGK)/BRAF and one with PJA2/BRAF) in samples obtained at AR to EGFR TKI therapy (two posterlotinib samples and two posterlotinib and postosimertinib samples). Pre-TKI samples were available for two of four patients and both were negative for BRAF fusion. Induction of AGK/BRAF fusion in H1975 (L858R + T790M), PC9 (ex19del) and HCC827 (ex19del) cells increased phosphorylation of BRAF, MEK1/2, ERK1/2, and signal transducer and activator of transcription 3 and conferred resistance to growth inhibition by osimertinib. MEK inhibition with trametinib synergized with osimertinib to block growth. Alternately, a pan-RAF inhibitor as a single agent blocked growth of all cell lines with mutant EGFR and BRAF fusion. ConclusionBRAF fusion is a mechanism of AR to EGFR TKI therapy in approximately 2% of patients. Combined inhibition of EGFR and MEK (with osimertinib and trametinib) or BRAF (with a pan-RAF inhibitor) are potential therapeutic strategies that should be explored.

The role of histone lysine demethylases in cancer cells' resistance to tyrosine kinase inhibitors.

Current cancer therapies are often associated with treatment failure and reduced patients’ survival due to drug resistance. There are various mechanisms involved in the acquisition of cancer drug resistance, including the selection of advantageous mutations, overexpression of transporter proteins and epigenetic alterations. In this context, epigenetic alterations refer to chromatin-mediated regulation of gene expression that results in heritable changes in the cellular phenotype. There is an ever-growing body of evidence suggesting that epigenetic mechanisms play an important role in bringing about drug resistance in cancer cells. While the relationship between chemotherapy and epigenetics has been widely discussed, emerging evidence indicates that specific epigenetic effectors are also crucial for the development of resistance to tyrosine kinase inhibitors (TKIs). One particular gene that encodes the histone lysine demethylase KDM5A is overexpressed in several cancers. In breast cancer tissues, cells with KDM5A gene amplification were found to be more resistant to erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), when compared to cells without the same amplification. KDM5A was also shown to mediate resistance to a second EGFR inhibitor called gefitinib, in EGFR-mutant lung cancer cell lines. This evidence indicates that KDM5A could activate alternative survival pathways involved in overcoming EGFR inhibition. In line with these results, another histone demethylase (i.e., KDM1A) promotes liver cancer cells’ resistance to the TKI sorafenib. Current evidence provides a suitable rationale to consider the use of specific KDMs inhibitors to sensitize cells to tyrosine kinase targeted therapies and thus, presents an opportunity to prevent the further development of drug resistance. This review discusses the involvement of histone lysine demethylases in the development of resistance to TKI and highlights the importance to develop new cancer treatment regimens to counteract this phenomenon.

Increased Synthesis of MCL-1 Protein Underlies Initial Survival of EGFR-Mutant Lung Cancer to EGFR Inhibitors and Provides a Novel Drug Target.

Purpose: EGFR inhibitors (EGFRi) are effective against EGFR-mutant lung cancers. The efficacy of these drugs, however, is mitigated by the outgrowth of resistant cells, most often driven by a secondary acquired mutation in EGFR, T790M We recently demonstrated that T790M can arise de novo during treatment; it follows that one potential therapeutic strategy to thwart resistance would be identifying and eliminating these cells [referred to as drug-tolerant cells (DTC)] prior to acquiring secondary mutations like T790M Experimental Design: We have developed DTCs to EGFRi in EGFR-mutant lung cancer cell lines. Subsequent analyses of DTCs included RNA-seq, high-content microscopy, and protein translational assays. Based on these results, we tested the ability of MCL-1 BH3 mimetics to combine with EGFR inhibitors to eliminate DTCs and shrink EGFR-mutant lung cancer tumors in vivo Results: We demonstrate surviving EGFR-mutant lung cancer cells upregulate the antiapoptotic protein MCL-1 in response to short-term EGFRi treatment. Mechanistically, DTCs undergo a protein biosynthesis enrichment resulting in increased mTORC1-mediated mRNA translation of MCL-1, revealing a novel mechanism in which lung cancer cells adapt to short-term pressures of apoptosis-inducing kinase inhibitors. Moreover, MCL-1 is a key molecule governing the emergence of early EGFR-mutant DTCs to EGFRi, and we demonstrate it can be effectively cotargeted with clinically emerging MCL-1 inhibitors both in vitro and in vivo Conclusions: Altogether, these data reveal that this novel therapeutic combination may delay the acquisition of secondary mutations, therefore prolonging therapy efficacy. Clin Cancer Res; 24(22); 5658-72. ©2018 AACR.

Abstract PR13: MET copy number gain is associated with gefitinib resistance in leptomeningeal carcinomatosis of EGFR-mutant lung cancer

Abstract Leptomeningeal carcinomatosis occurs frequently in EGFR-mutant lung cancer, and develops acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). This study aimed to clarify the mechanism of EGFR-TKI resistance in leptomeningeal carcinomatosis and seek a novel therapeutic strategy. We examined EGFR mutations, including the T790M gatekeeper mutation, in 32 re-biopsy specimens from 12 leptomeningeal carcinomatosis and 20 extracranial lesions of EGFR-mutant lung cancer patients who became refractory to EGFR-TKI treatment. All 32 specimens had the same baseline EGFR mutations, but the T790M mutation was less frequent in leptomeningeal carcinomatosis specimens than in extracranial specimens (8% vs. 55%, P < 0.01). To study molecular mechanisms of acquired EGFR-TKI resistance in leptomeningeal carcinomatosis, we utilized our previously developed mouse model of leptomeningeal carcinomatosis with the EGFR-mutant lung cancer cell line PC-9/ffluc cells, in which acquired resistance to gefitinib was induced by continuous oral treatment. Compared with subcutaneously inoculated gefitinib-resistant tumors, the T790M mutation was less frequent in leptomeningeal carcinomatosis that acquired resistance to gefitinib. PC-9/LMC-GR cells were established from the gefitinib-resistant leptomeningeal carcinomatosis model, and they were found to be intermediately resistant to gefitinib and osimertinib (third-generation EGFR-TKI). Although EGFR-T790M was negative, gefitinib resistance of PC-9/LMC-GR cells was related to MET copy number gain with MET activation. Moreover, combined use of EGFR-TKI and crizotinib, a MET inhibitor, dramatically regressed leptomeningeal carcinomatosis with acquired resistance to gefitinib or osimertinib. These findings suggest that combination therapy with MET inhibitors may be promising for controlling leptomeningeal carcinomatosis that acquires resistance to EGFR-TKIs. This abstract is also being presented as Poster B29. Citation Format: Shigeki Nanjo, Sachiko Arai, Wei Wang, Akito Hata, Nobuyuki Katakami, Seiji Yano. MET copy number gain is associated with gefitinib resistance in leptomeningeal carcinomatosis of EGFR-mutant lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR13.

Loss of EGFR confers acquired resistance to AZD9291 in an EGFR-mutant non-small cell lung cancer cell line with an epithelial-mesenchymal transition phenotype.

AZD9291 is an irreversible, small-molecule inhibitor which has potency against mutant EGFR- and T790M-resistant mutation. Despite the encouraging efficacy in clinical, the acquired resistance will finally occur. Further study will need to be done to identify the acquired resistance mechanisms and determine the next treatment. We established an AZD9291-resistant cell line (HCC827/AZDR) from parental HCC827 cell line through stepwise pulsed selection of AZD9291. The expression of EGFR and its downstream pathways were determined by western blot analysis or immunofluorescence assay. The sensitivity to indicated agents were evaluated by MTS. Compared with parental HCC827 cells, the HCC827/AZDR cells showed high resistance to AZD9291 and other EGFR-TKIs, and exhibited a mesenchymal-like phenotype. Almost complete loss of EGFR expression was observed in HCC827/AZDR cells. But the activation of downstream pathway, MAPK signaling, was found in HCC827/AZDR cells even in the presence of AZD9291. Inhibition of MAPK signaling had no effect on cell viability of HCC827/AZDR and could not reverse AZD9291 resistance because of the subsequent activation of AKT signaling. When treated with the combination of AKT and MAPK inhibitor, HCC827/AZDR showed remarkable growth inhibition. Loss of EGFR could be proposed as a potential acquired resistance mechanism of AZD9291 in EGFR-mutant NSCLC cells with an EMT phenotype. Despite the loss of EGFR, the activation of MAPK pathway which had crosstalk with AKT pathway could maintain the proliferation and survival of resistant cells. Blocking MAPK and AKT signaling may be a potential therapeutic strategy following AZD9291 resistance.

Antitumor effects of histone deacetylase inhibitor suberoylanilide hydroxamic acid in epidermal growth factor receptor-mutant non-small-cell lung cancer lines in vitro and in vivo.

Histone acetylation is one of the most abundant post-translational modifications in eukaryotic cells; aberrant histone acetylation is related to a range of cancer types because of the dysregulation of histone deacetylases (HDACs). Inhibition of HDACs leads to suppression of tumor growth in multiple cancers, whereas the inhibitory effects of HDAC inhibitors remain incompletely understood in epidermal growth factor receptor (EGFR)-mutant lung cancers. In this study, the antitumor effects of HDACs inhibitor suberoylanilide hydroxamic acid (SAHA, vorinostat) were examined in EGFR-mutant lung cancer cell lines. The results of the present work showed that SAHA markedly inhibited cell viability and proliferation, induced cell apoptosis by arresting the cell cycle in the G2/M phase, and significantly reduced tumor growth in a xenograft model. Further study confirmed that the suppression function of SAHA might be mediated by regulating the ERK-dependent and/or the AKT-dependent pathway; meanwhile, angiogenesis abrogation induced by SAHA exerted effects on tumor regression in vivo. Taken together, our results identify the antitumor effects of HDACs inhibitor SAHA as an alternative therapeutic application for the epigenetic treatment of EGFR-mutant non-small-cell lung cancer.