EGFR-mutant Lung Adenocarcinoma Cell Research Articles

NEAT1_1 confers gefitinib resistance in lung adenocarcinoma through promoting AKR1C1-mediated ferroptosis defence

Gefitinib is one of the most extensively utilized epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) for treating advanced lung adenocarcinoma (LUAD) patients harboring EGFR mutation. However, the emergence of drug resistance significantly compromised the clinical efficacy of EGFR-TKIs. Gaining further insights into the molecular mechanisms underlying gefitinib resistance holds promise for developing novel strategies to overcome the resistance and improve the prognosis in LUAD patients. Here, we identified that the inhibitory efficacy of gefitinib on EGFR-mutated LUAD cells was partially dependent on the induction of ferroptosis, and ferroptosis protection resulted in gefitinib resistance. Among the ferroptosis suppressors, aldo-keto reductase family 1 member C1 (AKR1C1) exhibited significant upregulation in gefitinib-resistant strains of LUAD cells and predicted poor progression-free survival (PFS) and overall survival (OS) of LUAD patients who received first-generation EGFR-TKI treatment. Knockdown of AKR1C1 partially reversed drug resistance by re-sensitizing the LUAD cells to gefitinib-mediated ferroptosis. The decreased expression of miR-338-3p contributed to the aberrant upregulation of AKR1C1 in gefitinib-resistant LUAD cells. Furthermore, upregulated long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1_1 (NEAT1_1) sponged miR-338-3p to neutralize its suppression on AKR1C1. Dual-luciferase reporter assay and miRNA rescue experiment confirmed the NEAT1_1/miR-338-3p/AKR1C1 axis in EGFR-mutated LUAD cells. Gain- and loss-of-function assays demonstrated that the NEAT1_1/miR-338-3p/AKR1C1 axis promoted gefitinib resistance, proliferation, migration, and invasion in LUAD cells. This study reveals the effects of NEAT1_1/miR-338-3p/AKR1C1 axis-mediated ferroptosis defence in gefitinib resistance in LUAD. Thus, targeting NEAT1_1/miR-338-3p/AKR1C1 axis might be a novel strategy for overcoming gefitinib resistance in LUAD harboring EGFR mutation.

Induction of Multiple Alternative Mitogenic Signaling Pathways Accompanies the Emergence of Drug-Tolerant Cancer Cells.

Drug resistance can evolve from a subpopulation of cancer cells that initially survive drug treatment and then gradually form a pool of drug-tolerant cells. Several studies have pinpointed the activation of a specific bypass pathway that appears to provide the critical therapeutic target for preventing drug tolerance. Here, we take a systems-biology approach, using proteomics and genomics to examine the development of drug tolerance to EGFR inhibitors in EGFR-mutant lung adenocarcinoma cells and BRAF inhibitors in BRAF-mutant melanoma cells. We found that there are numerous alternative mitogenic pathways that become activated in both cases, including YAP, STAT3, IGFR1, and phospholipase C (PLC)/protein kinase C (PKC) pathways. Our results suggest that an effective therapeutic strategy to prevent drug tolerance will need to take multiple alternative mitogenic pathways into account rather than focusing on one specific pathway.

Novel CDK12/13 Inhibitors AU-15506 and AU-16770 Are Potent Anti-Cancer Agents in EGFR Mutant Lung Adenocarcinoma with and without Osimertinib Resistance.

Osimertinib is a third-generation epidermal growth factor receptor and tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of lung adenocarcinoma patients harboring EGFR mutations. However, acquired resistance to this targeted therapy is inevitable, leading to disease relapse within a few years. Therefore, understanding the molecular mechanisms of osimertinib resistance and identifying novel targets to overcome such resistance are unmet needs of cancer patients. Here, we investigated the efficacy of two novel CDK12/13 inhibitors, AU-15506 and AU-16770, in osimertinib-resistant EGFR mutant lung adenocarcinoma cells in culture and xenograft models in vivo. We demonstrate that these drugs, either alone or in combination with osimertinib, are potent inhibitors of osimertinib-resistant as well as -sensitive lung adenocarcinoma cells in culture. Interestingly, only the CDK12/13 inhibitor in combination with osimertinib, although not as monotherapy, suppresses the growth of resistant tumors in xenograft models in vivo. Taken together, the results of this study suggest that inhibition of CDK12/13 in combination with osimertinib has the potential to overcome osimertinib resistance in EGFR mutant lung adenocarcinoma patients.

EGFR Mutant Lung Adenocarcinoma Showed Reduced Radiosensitivity after Acquired Resistance to EGFR TKIs

<h3>Purpose/Objective(s)</h3> Though earlier application of local radiotherapy for the primary lesion may improve the survival of patients, the timing of radiotherapy intervention for advanced EGFR mutant lung adenocarcinoma is still controversial. This study aimed to detect the difference of radiosensitivity before and after resistance and explore the related mechanism by constructing EGFR mutant lung adenocarcinoma cell lines with acquired resistance to gefitinib in vitro, so as to provide theoretical basis for determining the optimal intervention timing of radiotherapy. <h3>Materials/Methods</h3> Two lung adenocarcinoma cell lines harboring EGFR mutation-PC9 and HCC827, were used for establishment of acquired resistant cells to gefitinib. The radiosensitivity was detected using a cell counting kit. Flow cytometry was introduced to explore the cell cycle distribution and Western blot was applied to further test cell cycle and DNA repair related proteins. Soft agar assay and Western blot were performed to determine the cancer stem cell (CSC) characteristics. RT-PCR detected the expression of CSC markers. Finally, the relevant signal pathways were explored. <h3>Results</h3> The cell counting kit showed that the inhibitory activity of cells with irradiation after gefitinib resistance is weaker than that before resistance. Flow cytometry revealed that the G2/M phase proportion of PC9 and HCC827 were significantly higher than that of PC9/GR and HCC827/GR under the same irradiation dose, which is consistent with the results of western blot that the increased expression of G2/M regulatory protein, including p21, Cyclin B1, CyclinA2, pCDC2 and Myt1 were more significant increased in parental cells than resistant cells. The phosphorylated level of RAD17, ATM and CHK2 which were DNA damage repair protein were more upregulated in PC9GR and HCC827GR cells. Furthermore, GR had strong capability of sphere-forming and EMT phenotype. RT-PCR showed that the expression of many genes regulating CSC characteristics increased obviously after gefitinib resistance, including SOX2, OCT4, Nanog, Notch1, Notch3, ALDH1A1, ALDH1A3 and BMI. But the up-regulated genes in the two drug-resistant cells were not completely same. Western blot and RT-PCR showed that BMP/SMAD signal pathway was more activated in both resistant cells than that in parental cells. <h3>Conclusion</h3> Lung adenocarcinoma with EGFR mutations showed reduced radiosensitivity after acquired resistance to EGFR TKIs through obtaining CSC characteristics, which may be mediated by BMP/SMAD signal pathway.

ARID1A deficiency reverses the response to anti-PD(L)1 therapy in EGFR-mutant lung adenocarcinoma by enhancing autophagy-inhibited type I interferon production

IntroductionEGFR mutations in non-small cell lung cancer (NSCLC) are associated with a poor response to immune checkpoint inhibitors (ICIs), and only 20% of NSCLC patients harboring EGFR mutations benefit from immunotherapy. Novel biomarkers or therapeutics are needed to predict NSCLC prognosis and enhance the efficacy of ICIs in NSCLC patients harboring EGFR mutations, especially lung adenocarcinoma (LUAD) patients, who account for approximately 40–50% of all NSCLC cases.MethodsAn ARID1A-knockdown (ARID1A-KD) EGFR-mutant LUAD cell line was constructed using lentivirus. RNA-seq and mass spectrometry were performed. Western blotting and IHC were used for protein expression evaluation. Effects of 3-MA and rapamycin on cells were explored. Immunofluorescence assays were used for immune cell infiltration examination.ResultsARID1A expression was negatively associated with immune cell infiltration and immune scores for ICIs in LUAD with EGFR mutations. In vitro experiments suggested that ARID1A-KD activates the EGFR/PI3K/Akt/mTOR pathway and inhibits autophagy, which attenuates the inhibition of Rig-I-like receptor pathway activity and type I interferon production in EGFR-mutant LUAD cells. In addition, 3-MA upregulated production of type I interferon in EGFR-mutant LUAD cells, with an similar effect to ARID1A-KD. On the other hand, rapamycin attenuated the enhanced production of type I interferon in ARID1A-KD EGFR-mutant LUAD cells. ARID1A function appears to influence the tumor immune microenvironment and response to ICIs.ConclusionARID1A deficiency reverses response to ICIs in EGFR-mutant LUAD by enhancing autophagy-inhibited type I interferon production.4p38v18Tik7_Gskdth_SehVideo

The Interaction of the IFNγ/JAK/STAT1 and JAK/STAT3 Signalling Pathways in EGFR-Mutated Lung Adenocarcinoma Cells.

Purpose It was reported that the EGFR (epidermal growth factor receptor) mutation status was related to primary immune resistance in NSCLC (non-small-cell lung cancer). ICIs (immune checkpoint inhibitors) have poor efficacy and large side effects for people with EGFR mutation. EGFR mutation was considered as a sign of immune therapeutic resistance, but its underlying mechanism is difficult to be determined. Combined with our research basis, we tried to explore the possible mechanism of primary drug resistance in EFGR mutant lung adenocarcinoma through the interaction between the JAK/STAT1 and JAK/STAT3 pathway. Materials and Methods Cell apoptosis and viability test were used to study the role of the JAK/STAT signalling pathway in lung adenocarcinoma cell survival. Western blot, RT-PCR, and flow cytometry were employed to explore the changes of expression in JAK1/2, STAT1/3, PD-L1, and related signal molecules in the case of activation or inhibition of the JAK/STAT3 signalling pathway. Results With inhibition of inhibiting the JAK/STAT3 signalling pathway by STAT3 inhibitors, we found IFNγ-JAK-STAT1 pathway activation by IFNγ could further keep lung adenocarcinoma cells from proliferation and promote its apoptosis. The inhibition of the JAK/STAT3 pathway results in the upregulation of JAK1/2, STAT1, IRF1, IRF9, and PD-L1 and downregulation of STAT3 and SOCS1. Conclusions The absence of the IFNγ-JAK-STAT1 signal pathway is one of the main mechanisms for the ICI endogenous resistance. The abnormal activation of the downstream JAK/STAT3 pathway in cells with EGFR mutation may have antagonistic effects on the STAT1 induced antitumor immune response, which may cause the IFNγ-JAK-STAT1 pathway to lose its function. The mechanism may result in production of the immune tolerance of the EGFR mutant, which promotes immune escape.

SRC Family Kinase Inhibition Targets YES1 and YAP1 as Primary Drivers of Lung Cancer and as Mediators of Acquired Resistance to ALK and Epidermal Growth Factor Receptor Inhibitors.

PURPOSEThe identification of novel oncogenic driver alterations and novel mechanisms of acquired resistance (AR) is the key for further development of personalized therapy. The current study investigates the potential role of YES1 amplification as a primary driver of tumorigenesis and of YES1/YAP1 amplifications as mediators of AR to ALK and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs).METHODSModels of ectopic expression were established and characterized for YES1 and YAP1 in human bronchial epithelial cells and ALK fusion–positive (ALK+) and EGFR-mutant lung adenocarcinoma cell lines. MSK-IMPACT data for all lung adenocarcinoma cases and for ALK and EGFR TKI AR cases were surveyed for YES1 and YAP1 amplification.RESULTSWe report response to SRC family kinase (SFK) inhibition in a patient whose lung cancer exhibited YES1 amplification, without any well-established primary driver alteration, suggesting that YES1 amplification can also function as a primary oncogenic driver. To investigate the possibility of YES1 as a primary driver in tumorigenesis, we established preclinical models of YES1 overexpression using human bronchial epithelial cells and normal human breast epithelial cells. We showed that YES1 overexpression conferred sensitivity to SFK TKIs and promoted EGF-independent growth in a YAP1-dependent manner. Analysis of clinical genomic sequencing data from cases of AR to EGFR and ALK inhibitors revealed acquired amplification of YAP1 in four cases. EGFR-mutant and ALK fusion–positive cells overexpressing YES1 or YAP1 were resistant to EGFR and ALK TKIs, respectively, but were sensitive to dual inhibition of the primary driver and YES1.CONCLUSIONOur results demonstrate the therapeutic potential of SFK inhibition in primary tumorigenesis and AR driven by YES1/YAP1 signaling.

BIX01294 inhibits EGFR signaling in EGFR-mutant lung adenocarcinoma cells through a BCKDHA-mediated reduction in the EGFR level

BIX01294 (BIX), an inhibitor of the G9a histone methyltransferase, has been reported to have antitumor activity against a variety of cancers. However, the molecular mechanisms underlying its anticancer effects, particularly those against lung cancer, remain unclear. Here, we report that BIX induces apoptotic cell death in EGFR-mutant non-small cell lung cancer (NSCLC) cells but not in their wild-type counterparts. Treatment with BIX resulted in a significant reduction in the EGFR level and inhibition of EGFR signaling only in EGFR-mutant NSCLC cells, leading to apoptosis. BIX also inhibited mitochondrial metabolic function and decreased the cellular energy levels that are critical for maintaining the EGFR level. Furthermore, BIX transcriptionally downregulated the transcription of branched-chain α-keto acid dehydrogenase (BCKDHA), which is essential for fueling the tricarboxylic acid (TCA) cycle. Interestingly, this BCKDHA downregulation was due to inhibition of Jumanji-domain histone demethylases but not the G9a histone methyltransferase. We observed that KDM3A, a Jumonji histone demethylase, epigenetically regulates BCKDHA expression by binding to the BCKDHA gene promoter. BIX exposure also led to a significant decrease in the EGFR level, causing apoptosis in EGFR-TKI (tyrosine kinase inhibitor)-resistant cell lines, which are dependent on EGFR signaling for survival. Taken together, our current data suggest that BIX triggers apoptosis only in EGFR-mutant NSCLC cells via inhibition of BCKDHA-mediated mitochondrial metabolic function.

Alterations in the Global Proteome and Phosphoproteome in Third Generation EGFR TKI Resistance Reveal Drug Targets to Circumvent Resistance.

Lung cancer is the leading cause of cancer mortality worldwide. The treatment of patients with lung cancer harboring mutant EGFR with orally administered EGFR tyrosine kinase inhibitors (TKI) has been a paradigm shift. Osimertinib and rociletinib are third-generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard of care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here we characterized the proteome and phosphoproteome of a series of isogenic EGFR-mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs, comprising the most comprehensive proteomic dataset resource to date to investigate third generation EGFR TKI resistance in lung adenocarcinoma. Unbiased global quantitative mass spectrometry uncovered alterations in signaling pathways, revealed a proteomic signature of epithelial-mesenchymal transition, and identified kinases and phosphatases with altered expression and phosphorylation in TKI-resistant cells. Decreased tyrosine phosphorylation of key sites in the phosphatase SHP2 suggests its inhibition, resulting in subsequent inhibition of RAS/MAPK and activation of PI3K/AKT pathways. Anticorrelation analyses of this phosphoproteomic dataset with published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures program predicted drugs with the potential to overcome EGFR TKI resistance. The PI3K/MTOR inhibitor dactolisib in combination with osimertinib overcame resistance both in vitro and in vivo. Taken together, this study reveals global proteomic alterations upon third generation EGFR TKI resistance and highlights potential novel approaches to overcome resistance. SIGNIFICANCE: Global quantitative proteomics reveals changes in the proteome and phosphoproteome in lung cancer cells resistant to third generation EGFR TKIs, identifying the PI3K/mTOR inhibitor dactolisib as a potential approach to overcome resistance.

YES1 amplification as a primary driver of lung tumorigenesis and YES1/YAP1 amplifications as mediators of acquired resistance (AR) to ALK and EGFR tyrosine kinase inhibitors (TKIs).

e21591 Background: Our previous work identified YES1 amplification as a mechanism of AR to EGFR TKIs. The current study investigates the potential role of YES1 amplification as a primary driver of tumorigenesis and of YES1/YAP1 amplifications as mediators of AR to ALK and EGFR TKIs. Methods: Models of ectopic expression were established and characterized for YES1 and YAP1 in human bronchial epithelial cells (HBECs) and ALK fusion-positive ( ALK+) and EGFR-mutant ( EGFRm) lung adenocarcinoma (LUAD) cell lines. MSK-IMPACT data for all LUAD cases and for ALK and EGFR TKI AR cases were surveyed for YES1 and YAP1 amplification. Results: YES1 was found to be amplified in 0.5% of all LUAD cases currently in the MSK-IMPACT database. To demonstrate its potential as a primary driver of tumorigenesis, YES1 was inducibly expressed in HBECs and shown to promote EGF-independent growth, focus formation, and increases in proliferation rate and sensitivity to Src family kinase (SFK) TKIs. Consistent with these findings, a partial response by RECIST criteria to third-line dasatinib was observed for a patient with stage IV LUAD in which MSK-IMPACT detected YES1 amplification and no established primary driver alteration. In models of AR, overexpression of either YES1 or YAP1 in multiple ALK+ and EGFRm cell lines conferred resistance to ALK and EGFR TKIs. Sensitivity to ALK or EGFR TKIs was partially recovered in YES1-overexpressing cells lines by siRNA-mediated gene knockdown of YAP1, and was restored in YAP1-overexpressing cells lines by knockdown of YES1 or mutation of the YES1-phosphorylation site on YAP1. ALK + or EGFR m cells overexpressing either YES1 or YAP1 were sensitive to dual inhibition of the primary driver and SFKs with single-agent repotrectinib or dasatinib, respectively. In contrast, treatment of YAP1-overexpressing ALK+ H3122 cells with alectinib induced changes in morphology and gene expression consistent with epithelial-to-mesenchymal transition (EMT) and rendered the cells insensitive to repotrectinib. Similar resistance to dual inhibition was seen in other YAP1-overexpressing cell lines after treatment with only ALK or EGFR blockade. To date, one LUAD case each of EGFR and ALK TKI AR has been found through MSK-IMPACT™ to be associated with YAP1 amplification. Conclusions: SFK inhibition can potentially be exploited to therapeutically target YES1 amplification as a primary driver in tumorigenesis and YES1/YAP1 amplifications as mediators of AR to ALK and EGFR TKIs.

EGFR-independent EGFR-mutant lung adenocarcinoma cells depend on Bcl-xL and MCL1 for survival

Although most EGFR-mutant lung adenocarcinomas initially respond to EGFR inhibitors, disease progression almost inevitably occurs. We previously reported that two EGFR-mutant lung adenocarcinoma cell lines, HCC827 and H1975, contain subpopulations of cells that display an epithelial-to-mesenchymal phenotype and can thrive independently of EGFR signaling. In this study, we explored to what extent these two sublines, HCC827 GR2 and H1975 WR7, depended on the anti-apoptotic BCL2 family members, Bcl-xL and/or MCL1, for survival. Although HCC827 GR2 cells were hardly affected by Bcl-xL or MCL1 knockdown alone, dual inhibition of Bcl-xL and MCL1 caused the cells to undergo apoptosis, resulting in decreased viability. In H1975 WR7 cells, not only dual inhibition, but also MCL1 silencing alone, induced the cells to undergo apoptosis. Interestingly, the two sublines markedly declined in number when autophagy flux was suppressed, because they depend, in part, on active autophagy for survival. However, autophagy inhibition was inferior to dual inhibition of Bcl-xL plus MCL1 for GR2 cells, or MCL1 inhibition alone, for decreasing the viability of WR7 cells. Collectively, these findings suggest that inhibiting Bcl-xL plus MCL1, or MCL1 alone, may represent a new approach to treat EGFR-independent EGFR-mutant cancer cells.

The influence of tumor heterogeneity on sensitivity of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs

Background: Studies have shown that extensive genetic or spatial heterogeneity is present within tumors. The present study explored the influence of heterogeneous cells in the tumor microenvironment (TME) on the sensitivity of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs and further investigated associated molecular mechanisms. Methods: Tumor heterogeneity was simulated using transwell co-culture technique with H1975, A549 or MRC-5 cells grown in the upper chambers and PC-9 cells cultured in the bottom chamber. Each co-culture system was grouped based on different proportions of cells in the upper and lower chambers (from 1% to 300%), and each group was subdivided into erlotinib treatment group (erlotinib+) and erlotinib non-treatment group (erlotinib−). The viability of PC-9 cells was analyzed by CCK-8; HGF, IGF-1 and TGF-α were determined by ELISA; MET amplification was detected by qRT-PCR, and the relevant proteins were detected by Western blot. Results: The viability of PC-9 cells increased with increased proportion of A549/PC-9 or MRC-5/PC-9 cells from 1% to 300%. HGF increased with increased proportion of H1975 cells from 1% to 300%. In all three co-culture systems, TGF-α production in the erlotinib+ subgroup was significantly lower than that in erlotinib- subgroup, and phosphorylated AKT protein showed an ascending tendency with the increased proportion of upper chamber cells relative to PC-9 cells from 1% to 300%. H1975 cells could induce MET amplification of PC-9 cells. MRC5 cells inhibited MET amplification. Conclusions: Tumor heterogeneity partially accounts for the resistance of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs. The possible mechanisms may be related to AKT signaling pathways activated by growth factors, which are secreted by heterogeneous cells in the TME under the pressure of EGFR-TKIs.

The influence of tumor heterogeneity on sensitivity of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs.

BackgroundStudies have shown that extensive genetic or spatial heterogeneity is present within tumors. The present study explored the influence of heterogeneous cells in the tumor microenvironment (TME) on the sensitivity of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs and further investigated associated molecular mechanisms.MethodsTumor heterogeneity was simulated using transwell co-culture technique with H1975, A549 or MRC-5 cells grown in the upper chambers and PC-9 cells cultured in the bottom chamber. Each co-culture system was grouped based on different proportions of cells in the upper and lower chambers (from 1% to 300%), and each group was subdivided into erlotinib treatment group (erlotinib+) and erlotinib non-treatment group (erlotinib−). The viability of PC-9 cells was analyzed by CCK-8; HGF, IGF-1 and TGF-α were determined by ELISA; MET amplification was detected by qRT-PCR, and the relevant proteins were detected by Western blot.ResultsThe viability of PC-9 cells increased with increased proportion of A549/PC-9 or MRC-5/PC-9 cells from 1% to 300%. HGF increased with increased proportion of H1975 cells from 1% to 300%. In all three co-culture systems, TGF-α production in the erlotinib+ subgroup was significantly lower than that in erlotinib- subgroup, and phosphorylated AKT protein showed an ascending tendency with the increased proportion of upper chamber cells relative to PC-9 cells from 1% to 300%. H1975 cells could induce MET amplification of PC-9 cells. MRC5 cells inhibited MET amplification.ConclusionsTumor heterogeneity partially accounts for the resistance of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs. The possible mechanisms may be related to AKT signaling pathways activated by growth factors, which are secreted by heterogeneous cells in the TME under the pressure of EGFR-TKIs.

Secretion of high amounts of hepatocyte growth factor is a characteristic feature of cancer-associated fibroblasts with EGFR-TKI resistance-promoting phenotype: A study of 18 cases of cancer-associated fibroblasts.

Humoral factors from cancer-associated fibroblasts (CAFs) reportedly affect epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance in cancer cells with EGFR mutations. The aim of this study was to identify the robust humoral factors secreted from CAFs that induce the primary resistance to EGFR-TKI. We evaluated the EGFR-TKI sensitivity of EGFR-mutant lung adenocarcinoma cell line (PC-9) treated with condition media (CM) from 18 cases of CAFs and matched non-cancerous-tissue-associated fibroblasts (NCAFs). We measured the expression levels of hepatocyte growth factor (HGF), interleukin-6, fibroblast growth factor-2, insulin-like growth factor-1, and vascular endothelial growth factor-A in CAFs and NCAFs. We examined whether HGF neutralizing antibody could annul the EGFR-TKI resistance induced by CM from CAFs. Compared to CM from NCAFs, CM from CAFs increased the resistance of PC-9 cells to EGFR-TKI in five out of 18 cases. Relative expression ratio of HGF messenger RNA was significantly higher in these five CAFs compared to others (P = 0.0013), whereas other cytokines were not. In four of these five cases, the addition of HGF neutralizing antibody significantly decreased the survival ratio of PC-9 cells. This study suggests that the secretion of higher amounts of HGF is the robust feature of EGFR-TKI resistance-promoting CAFs.

Clinical importance of long noncoding RNA LINC00460 expression in plasma cell-free tumor RNA in EGFR-mutant adenocarcinoma.

e20529 Background: Long noncoding RNA (lncRNA) is longer than 200 nucleotides and does not encode proteins. The IncRNA LINC00460 was reported to be involved in physiological and pathological processes such as proliferation, invasion, metastasis, and epithelial–mesenchymal transition (EMT). However, its role in epidermal growth factor receptor ( EGFR)-mutant lung adenocarcinoma is unclear. Methods: Non–small cell lung cancer (NSCLC) cells with knockout of LINC00460 were generated with a CRISPR-Cas9 system and used to investigate the effect of LINC00460 on gefitinib-induced cell death in an MTT assay. Western blot was used to observe change in EMT-related proteins, and changes in expression of EMT-related proteins were detected with an immunofluorescence assay. In addition, among 38 EGFR mutant lung adenocarcinomas treated by EGFR-TKI, cell-free tumor RNA extracted from blood plasma samples was analyzed for LINC00460 by using a real-time PCR assay. In addition, we assessed the association between LINC00460 expression and progression-free survival (PFS) after EGFR-TKI therapy. Results: LINC00460 was up-regulated by EGFR activation. LINC00460 expressions were significantly greater in NSCLC tumors than in adjacent normal tissues (p &lt; 0.01). H1299 cells with LINC00460 knockout were more sensitive to gefitinib. LINC00460 was induced by transcription factors such as FOS and NF-κB in EGFR-mutant lung adenocarcinoma and gefitinib-resistant cells. LINC00460 overexpression was involved in gefitinib resistance. The MTT assay showed that LINC00460 expression was greater in cells with acquired resistance to EGFR-TKIs (gefitinib, erlotinib, and osimertinib). Western blot and immunofluorescence revealed that LINC00460 expression was associated with changes in the expression of EMT-related proteins. Patients with high LINC00460 expression in plasma cell-free tumor RNA had significantly shorter PFS after EGFR-TKI therapy than did those with low expression (median PFS: 688 vs 1089 days, respectively; p = 0.024). Conclusions: LINC00460 appears to be a novel factor involved in malignant transformation of EGFR mutant–positive lung adenocarcinoma and increased EGFR-TKI resistance. LINC00460 expression in plasma cell-free tumor RNA in EGFR-mutant adenocarcinoma was associated with shorter PFS after EGFR-TKI therapy.

MCL1 inhibition enhances the therapeutic effect of MEK inhibitors in KRAS-mutant lung adenocarcinoma cells

ObjectivesMCL1 is an anti-apoptotic BCL2 family member that is highly expressed in various malignant tumors. However, little is known about the role of MCL1 in KRAS-mutant lung adenocarcinomas. In this study, we aimed to clarify whether MCL1 could be a therapeutic target in KRAS-mutant lung adenocarcinomas for which no effective molecular targeted drugs are available. Materials and methodsWe examined to what extent MCL1 knockdown either alone or in combination with MEK inhibitor trametinib suppressed growth or induced apoptosis in the KRAS-mutant lung adenocarcinoma cell line H441 and EGFR-mutant lung adenocarcinoma cell line H1975. Furthermore, we investigated the therapeutic effects of dual inhibition of MCL1 and Bcl-xL, another anti-apoptotic BCL2 family member, in these two cell lines. ResultsMCL1 knockdown alone did not induce apoptosis in H441 or H1975 cells. However, MCL1-depleted H441 and H1975 cells underwent apoptosis and decreased in number in the presence of trametinib. We also confirmed that combined therapy by MCL1 knockdown and trametinib almost completely suppressed the growth of H441 cells in vivo. Moreover, dual knockdown of MCL1 and Bcl-xL induced extensive apoptosis in H441 and H1975 cells. ConclusionThese findings suggest that combined treatments of MCL1 knockdown and trametinib or dual inhibition of MCL1 and Bcl-xL would be effective therapies for lung adenocarcinomas including the KRAS-mutant subtype.

TRIM59 promotes gefitinib resistance in EGFR mutant lung adenocarcinoma cells

AimsThe relationship between TRIM59 and drug resistance is elusive despite of its multiple uncovered roles in human cancers. Here we aimed to characterize the expression status of TRIM59 in gefitinib-resistant EGFR mutant lung adenocarcinoma cells and elucidate its mechanism underlying the drug resistance. Main methodsGefitinib-resistant cell lines were established by progressive dosage. Relative expression of TRIM59 was determined by both real-time PCR and Western blot. Target gene knockdown was achieved by specific shRNAs. Cell viability was measured by MTT assay. Cell apoptosis was analyzed by flow cytometry with Annexin V/7-AAD double staining. Cell proliferation was determined by clonogenic formation assay. Migration and invasion capacities were detected using transwell chamber assay. Direct interaction between TRIM59 and STAT3 was analyzed by co-immunoprecipitation assay. Key findingsWe first observed overexpression of TRIM59 in gefitinib-resistant EGFR mutant lung adenocarcinoma cells. ShRNA-mediated knockdown of TRIM59 significantly inhibited cell viability and stimulated apoptosis. Meanwhile, TRIM59-deficiency suppressed cell migration and invasion. We further identified the interaction between TRIM59 and STAT3. TRIM59-deficiency remarkably impaired the activation of STAT3 signaling. STAT3-specific shRNAs significantly re-sensitized TRIM59-proficient EGFR mutant lung adenocarcinoma cells to gefitinib. SignificanceOur data characterized aberrant TRIM59 overexpression in gefitinib-resistance EGFR mutant lung adenocarcinoma cells, and indicated the potential involvement of TRIM59-STAT3 signaling in the occurrence of gefitinib-resistance.

Aurora kinase A drives the evolution of resistance to third-generation EGFR inhibitors in lung cancer.

Although targeted therapies often elicit profound initial patient responses, these effects are transient due to residual disease leading to acquired resistance. How tumors transition between drug responsiveness, tolerance and resistance, especially in the absence of pre-existing subclones, remains unclear. In EGFR-mutant lung adenocarcinoma cells, we demonstrate that residual disease and acquired resistance in response to EGFR inhibitors requires AURKA activity. Non-genetic resistance through the activation of AURKA by its co-activator TPX2 emerges in response to chronic EGFR inhibition where it mitigates drug-induced apoptosis. Aurora kinase inhibitors suppress this adaptive survival program, increasing the magnitude and duration of EGFR inhibitor response in pre-clinical models. Treatment induced activation of AURKA was associated with resistance to EGFR inhibitors in-vitro, in-vivo and in individuals with EGFR-mutant lung adenocarcinoma. These findings delineate a path whereby drug resistance emerges from drug-tolerant cells and unveils a synthetic lethal strategy for enhancing responses to EGFR inhibitors by suppressing AURKA driven residual disease and acquired resistance.

Abstract B24: Differential sensitivity to Aurora kinase inhibition in RIT1- and KRAS-mutant lung adenocarcinoma

Abstract Mutations in Ras-family genes Kirsten rat sarcoma viral oncogene (KRAS) and Ras-like in all tissues (RIT1) occur in 30% and 2% of lung adenocarcinomas, respectively. Targeted therapies have shown promise in lung adenocarcinomas and other cancers; however, there are currently no FDA-approved drugs that directly target RIT1 or KRAS. To identify critical effector pathways and/or genetic dependencies that might be targeted for clinical benefit in these cancers, we undertook complementary proteomic and small-molecule screens in multiple human lung cancer models. To better understand the signaling networks modulated by RIT1 and KRAS, we performed deep characterization of total protein and phosphoproteins in non-transformed human lung epithelial cells engineered to express mutant or wild-type RIT1 or KRAS. 10,131 proteins were detected across all samples, 9,001 of which were detected in every sample. 29,172 phosphorylated sites were detected with 14,769 of these identified in every sample. Gene set enrichment analysis (GSEA) identified pathways significantly modulated by RIT1 M90I, KRAS G12V, or KRAS Q61H mutations. Whereas some pathways, such as MYC, were commonly activated by both mutant RIT1 and KRAS, others, such as Aurora kinase signaling, were specifically perturbed by RIT1. Previously, we found that mutant RIT1 and KRAS can transform human lung epithelial cells and confer resistance to EGFR blockade in EGFR-dependent lung adenocarcinoma. To identify opportunities for therapeutic intervention of RIT1- and KRAS-mutant tumors, we screened a collection of 160 small molecules for their ability to block RIT1/KRAS-induced drug resistance in PC9 cells, an EGFR-mutant lung adenocarcinoma cell line. Interestingly, RIT1 M90I-expressing cells were selectively killed as a result of treatment with the Aurora kinase inhibitors, alisertib and barasertib, while KRAS G12V cells were resistant to these inhibitors. The specific effect of Aurora kinase inhibition on RIT1 mutant cells was also confirmed in a fibroblast transformation assay and could not be attributed to proliferation rate, which was similar between RIT1- and KRAS-expressing cells. Moreover, Aurora kinase B (AURKB) was among the most differentially expressed transcripts when comparing RIT1- or KRAS-expressing cells, suggesting differential effects of RIT1 and KRAS on Aurora kinase activity. These findings nominate Aurora kinase inhibition as a potential therapeutic strategy in RIT1- , but not KRAS-mutant, lung cancer. Citation Format: Kristin D. Holmes, Filip Mundt, Phillip Mertins, Steve Carr, Alice Berger. Differential sensitivity to Aurora kinase inhibition in RIT1- and KRAS-mutant lung adenocarcinoma [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 B24.

Abstract PR08: Functional characterization and evolutionary reconstruction of small cell lung cancer transformation of EGFR-mutant lung adenocarcinomas

Abstract Purpose: Recently, we performed a whole-genome sequencing (WGS) study of serially acquired tumors and demonstrated that an early, complete inactivation of RB1 and TP53 is common among patients with EGFR-mutant lung adenocarcinoma (LADC) whose disease was transformed into small cell lung cancer (SCLC). To further understand this phenomenon, here we functionally characterized a patient-derived cell line of transformed SCLC and related in vitro models. We also deepened our genome analysis focusing on the complex rearrangements to probe their evolutionary paths in detail. Method: mRNA sequencing was performed for SNU-2962A cells and their transcriptome was analyzed with published mRNA sequencing datasets of primary LADCs (n = 87), and SCLCs (n = 81). A cell viability screening of SNU-2962A cells was performed to understand their therapeutic vulnerability using a kinome-targeting siRNA library. We also characterized isogenic RB1/TP53-knockout LADC cell lines with EGFR mutation (PC9, HCC-827 and HCC-4006). In addition, we reconstructed complex genomic rearrangements and analyzed them in relationship with whole-genome duplication to investigate their time points during clonal evolution. Results: The SNU-2962A cell line was established from the pleural effusion of the patient LC1. These cells exhibited adherent morphology in vitro and strongly expressed neuroendocrine markers. A hierarchical clustering of SNU-2962A cells with the published LADCs and SCLCs showed complete clustering with SCLCs. This cell line strongly expressed NEUROD1 and MYC, which was consistent with the variant-type SCLC. Kinome siRNA library screening showed a significantly reduced viability of SNU-2962A cells with knockdown of PI3K-MTOR pathway genes (PIK3CA or MTOR). Pharmacologic inhibition of this pathway using PI3K or AKT inhibitors showed moderate sensitivity. We tried to induce transformation into SCLCs using three different EGFR-mutant LADC cell lines by knockout of both RB1 and TP53 as well as knockin of PIK3CA E545K mutations using CRISPR, but we did not observe any meaningful evidence of neuroendocrine differentiation. We further analyzed our WGS data of 11 tumors from five patients, and found that many large-scaled genomic events such as whole-genome duplication, chromothripsis, or kataegis are frequently shared by LADCs and subsequent SCLCs, indicating their early occurrence. MYC amplification is frequently selected in transformed SCLCs. We also found a somatic deletion of BIM gene in LC3 patient, and this deletion was shared by the early LADC and the late SCLC. This may confer a dysfunctional apoptosis upon EGFR inhibition, which could predispose the early emergence of resistant tumor in this patient. Conclusion: Although a complete inactivation of both RB1 and TP53 is necessary for transformation from LADC into SCLC, it is not sufficient. Phylogenic reconstruction of large structural variations in our WGS dataset further clarified the mutational complexity of the common ancestor clones. This abstract is also being presented as Poster A23. Citation Format: June-Koo Lee, Seongyeol Park, Joon Kim, Tae Min Kim, Young Seok Ju. Functional characterization and evolutionary reconstruction of small cell lung cancer transformation of EGFR-mutant lung adenocarcinomas [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 PR08.