EGFR Tyrosine Kinase Inhibitor Resistant Cell Research Articles

Efficacy of the CDK7 Inhibitor on EMT-Associated Resistance to 3rd Generation EGFR-TKIs in Non-Small Cell Lung Cancer Cell Lines.

Epithelial to mesenchymal transition (EMT) is associated with resistance during EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy. Here, we investigated whether EMT is associated with acquired resistance to 3rd generation EGFR-TKIs, and we explored the effects of cyclin-dependent kinase 7 (CDK7) inhibitors on EMT-mediated EGFR-TKIs resistance in non-small cell lung cancer (NSCLC). We established 3rd generation EGFR-TKI resistant cell lines (H1975/WR and H1975/OR) via repeated exposure to WZ4002 and osimertinib. The two resistant cell lines showed phenotypic changes to a spindle-cell shape, had a reduction of epithelial marker proteins, an induction of vimentin expression, and enhanced cellular mobility. The EMT-related resistant cells had higher sensitivity to THZ1 than the parental cells, although THZ1 treatment did not inhibit EGFR activity. This phenomenon was also observed in TGF-β1 induced EMT cell lines. THZ1 treatment induced G2/M cell cycle arrest and apoptosis in all of the cell lines. In addition, THZ1 treatment led to drug-tolerant, EMT-related resistant cells, and these THZ1-tolerant cells partially recovered their sensitivity to 3rd generation EGFR-TKIs. Taken together, EMT was associated with acquired resistance to 3rd generation EGFR-TKIs, and CDK7 inhibitors could potentially be used as a therapeutic strategy to overcome EMT associated EGFR-TKI resistance in NSCLC.

Abstract 4543: Proteomic characterization of AXL kinase inhibitors and signaling pathways

Abstract AXL is an attractive drug target because of its role in EMT-mediated resistance to EGFR tyrosine kinase inhibitor (TKI) in lung cancer (LC). Lack of genetic alterations and the role of stroma-mediated AXL activation in cancer cells, underscore the need to better characterize AXL TKIs, understand their effects on signaling and phenotype of cells, and develop assays to visualize active AXL signaling complexes. For this, 25 LC cells were analyzed for total (t) and phosphorylated (p) AXL expression. AXL TKIs, RXDX106, R428 and Cabozantinib, were profiled using western blotting (WB), viability assay and activity-based protein profiling (ABPP). Phosphoproteins (pSTY) altered by RXDX106 were identified using mass spectrometry. Effects of RXDX106 on signaling, viability and migration of LC cells were also evaluated. Cell line models of EMT-mediated acquired drug resistance, treated with a combination of AXL and EGFR TKIs, were analyzed for changes in signaling, cell viability and EMT. Immunoprecipitation (IP) identified adaptors of AXL signaling, and Proximity Ligation Assays (PLA) were developed to detect these active complexes in situ. H1299 cells, expressing highest levels of p and t AXL among the LC lines screened, was used in this study. RXDX106 and Cabozantinib potently inhibited pAXL in H1299 cells, but did not affect cell viability at these doses. R428 reduced cell viability at doses that did not efficiently inhibit pAXL, suggesting AXL independent phenotypic effects. Our ABPP data shows that apart from AXL, these TKIs target other overlapping and distinct subsets of proteins. R428 has the highest number of off targets and its unique ability to inhibit the FoxO pathway may explain the AXL independent phenotypic effects of R428. The pSTY data shows that RXDX106 deregulates phosphorylation of proteins involved in PI3K signaling, receptor endocytosis and cell migration pathways in H1299 cells. WB and phenotypic assays support these results by showing that RXDX106 inhibits pAXL, downstream pAKT but not pERK, and migration/invasion in these cells. In EGFR TKI resistant cells, EGFR and AXL TKI combination fails to alter downstream signaling, cell viability or EMT. Consistent with the WB and pSTY analyses, IP identifies PI3KR1 as an AXL interactor. PLAs to detect active AXL:PI3KR1 and AXL:pY100 signaling complexes show high basal PLA foci in H1299 and Calu1 cells that are abrogated by AXL TKI. HCC827 cells, which lack ligand independent pAXL, do not show significant labeling by either PLA. Overall, we demonstrate that different AXL TKIs have distinct target profiles and that inhibition of AXL suppresses downstream PI3K/AKT signaling and migration/ invasion of LC cells. We also show that AXL TKI fails to suppress downstream signaling, cell viability or EMT in EGFR TKI resistant cell lines. We have also established a PLA to annotate AXL adaptor foci that could be developed as a tool to measure drug-targetable active AXL complexes in patient tissues. Citation Format: Anurima Majumder, Guolin Zhang, Emma Adhikari, Bin Fang, Eric A. Welsh, John M. Koomen, Eric B. Haura. Proteomic characterization of AXL kinase inhibitors and signaling pathways [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 4543.

Role of EZH2 Inhibitor Combined with Gefitinib in EGFR-TKIs Resistant Lung Cancer Cells

背景与目的肺癌是危害身体健康的常见恶性肿瘤之一。随着表观遗传学的发展，研究者发现Zeste基因增强子同源物基因2（enhancer of Zeste homolog 2, EZH2）在肺癌中高表达且其表达量与预后息息相关，与此同时EZH2抑制剂也被发现能够提升肿瘤细胞对多种抗肿瘤药物的敏感性。本研究的目的在于探讨EZH2抑制剂与吉非替尼（gefitinib）联合应用，对耐吉非替尼细胞的增殖、凋亡及迁移的影响。方法以PC9吉非替尼敏感细胞和PC9/AB2吉非替尼耐药细胞为研究对象，应用CCK-8及EdU实验检测联合用药对于细胞活性及增殖的影响；通过划痕及Transwell小室检测联合用药对于细胞迁移能力的影响；并应用流式细胞学检测联合用药对于细胞凋亡的影响；并通过Western blot观察联合用药对EZH2及表皮生长因子（epidermal growth factor receptor, EGFR）信号通路相关蛋白的作用。结果在吉非替尼耐药细胞PC9/AB2细胞中，联合EZH2抑制剂GSK343与吉非替尼能够显著抑制耐药细胞的活性，降低细胞的迁移能力并诱导耐药细胞的凋亡。同时，联合用药也能够显著抑制EZH2表达及EGFR蛋白的磷酸化。结论EZH2抑制剂GSK343与吉非替尼联合应用能够克服PC9/AB2对吉非替尼的耐药作用，此研究也提示协同治疗在肺癌EGFR-TKIs耐药逆转中具有一定的作用。

MTORC2 contributes to the metabolic reprogramming in EGFR tyrosine-kinase inhibitor resistant cells in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations are often successfully treated with EGFR tyrosine kinase inhibitor (TKI) such as erlotinib; however, treatment resistance inevitably occurs. Given tumor metabolism of glucose and therapeutic response are intimately linked, we explored the metabolic differences between isogenic erlotinib-sensitive and -resistant NSCLC cell lines. We discovered that the growth of erlotinib-resistant cells is more sensitive to glucose deprivation. Seahorse metabolic assay revealed erlotinib-resistant cells have lower spare respiratory capacity (SRC), an indicator of metabolic flexibility, compared to erlotinib-sensitive cells. Additionally, we found downstream components of mTORC2 signaling to be phosphorylated in erlotinib-resistant cells. Knockdown of an mTORC2 component, Rictor, enhanced the SRC and rescued the growth rate of erlotinib-resistant cells during glucose deprivation. Among NSCLCs with activating EGFR mutations, gene sets involved in glucose metabolism were enriched in patients with high expression of p-NDGR1, a readout of mTORC2 activity. Furthermore, overall survival was negatively correlated with p-NDRG1. Our work uncovers a link between mTORC2 and metabolic reprogramming in EGFR TKI-resistant cells and highlights the significance of mTORC2 in the progression of EGFR-mutated NSCLC.

Abstract LB-385: miR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein

Abstract Bromodomain Containing 4 (BRD4) mediates transcriptional elongation of the oncogene MYC by binding to acetylated histones. BRD4 has been shown to play a critical role in tumorigenesis in several cancers, and the BRD4-NUT fusion gene is a driver of NUT midline carcinoma (NMC), a rare but highly lethal cancer. microRNAs (miRNAs) are endogenous small non-coding RNAs that suppress target gene expression by binding to complementary mRNA sequences. Here, we show that miR-3140, which was identified as a novel tumor suppressive miRNA by function-based screening of a library containing 1090 miRNA mimics, directly suppressed BRD4 by binding to its coding sequence (CDS). miR-3140 concurrently downregulated BRD3 by bind to its CDS as well as CDK2 and EGFR by binding to their 3' untranslated regions. miR-3140 inhibited tumor cell growth in vitro in various cancer cell lines, including EGFR tyrosine kinase inhibitor-resistant cells. Interestingly, we found that miR-3140 downregulated the BRD4-NUT fusion protein and suppressed in vitro tumor cell growth in a NMC cell line, Ty-82 cells. Furthermore, administration of miR-3140 suppressed in vivo tumor growth in a xenograft mouse model. Our results suggest that miR-3140 is a candidate for the development of miRNA-based cancer therapeutics. Citation Format: Tomoki Muramatsu, Erina Tonouchi, Yasuyuki Gen, Hidekazu Hiramoto, Kousuke Tanimoto, Jun Inoue, Johji Inazawa. miR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-385.

MiR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein

Bromodomain Containing 4 (BRD4) mediates transcriptional elongation of the oncogene MYC by binding to acetylated histones. BRD4 has been shown to play a critical role in tumorigenesis in several cancers, and the BRD4-NUT fusion gene is a driver of NUT midline carcinoma (NMC), a rare but highly lethal cancer. microRNAs (miRNAs) are endogenous small non-coding RNAs that suppress target gene expression by binding to complementary mRNA sequences. Here, we show that miR-3140, which was identified as a novel tumor suppressive miRNA by function-based screening of a library containing 1090 miRNA mimics, directly suppressed BRD4 by binding to its coding sequence (CDS). miR-3140 concurrently downregulated BRD3 by bind to its CDS as well as CDK2 and EGFR by binding to their 3’ untranslated regions. miR-3140 inhibited tumor cell growth in vitro in various cancer cell lines, including EGFR tyrosine kinase inhibitor-resistant cells. Interestingly, we found that miR-3140 downregulated the BRD4-NUT fusion protein and suppressed in vitro tumor cell growth in a NMC cell line, Ty-82 cells. Furthermore, administration of miR-3140 suppressed in vivo tumor growth in a xenograft mouse model. Our results suggest that miR-3140 is a candidate for the development of miRNA-based cancer therapeutics.

Abstract 5861: Enhanced radiosenistivity of EGFR-TKI sensitive and resistant NSCLC cells by abemaciclib is mediated by altered DNA repair and metabolic pathways

Abstract Dysregulation of the p16(INK4a)-CyclinD-CDK4/6-Rb pathways in patients with NSCLC (Non-Small Cell Lung Cancer) is a rational therapeutic target. The current study investigated the radiosensitizing potential of a novel CDK4/6 inhibitor, LY2835219 (LY, Abemaciclib) in NSCLC cell lines with varied genomic context to identify genomic and metabolic biomarkers that are predictive of a response over conventional EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy. NSCLC cell lines were exposed to LY (0-10uM) for 24 hr immediately after 0-10 Gy radiation. Cell survival was assessed by clonogenic assay and cell cycle distribution was quantified by flow cytometry. Dose modifying factors (DMF) were calculated at 10% survival from radiation survival curves. Altered DNA repair pathways and metabolic profiling of cells post LY treatment was assessed by immunoblot and LC/MS mass spectrometry analysis. LY treatment enhanced radiosensitivity of EGFR-TKI sensitive (HCC827, PC9) and EGFR-TKI resistant (H820 and H1975) cell lines with DMF of 1.3 (±0.06), 1.4 (±0.30), 1.5 (±0.51) and 1.3 (±0.02), respectively. Values in the parenthesis indicates standard deviation. Wild type EGFR expressing cells (A549 and H460) also showed enhanced radiosensitivity by LY with DMF of 1.6 (±0.09) and 1.75(±0.15), respectively. Interestingly, no radiation enhancement by LY was observed for cells deficient in functional PTEN (H1650), Rb (H82) and p53 (H460 DNp53 and H1299) protein. Radiosensitization was also observed for cells made resistant to third generation EGFR-TKI, AZD9291. Flow cytometry analysis of majority of cell types exposed to LY exhibited 55% to 94% G1 arrest (depending on cell type). Mechanistically, the combinatorial treatment in radiosensitive cells showed elevated phosphorylated-γH2Ax. Combination treatment also reduced expression of ATR, ATM, DNA-PK, Rad51 and Chk-2 suggestive of reduced DNA repair compared to radiation alone. LY treatment brought major changes in the glycolysis/TCA/ total amino acids. LY increased significantly Acetyl-CoA, fumarate and malate, indicating enhanced oxidative phosphorylation. LY significantly elevated Uric acid levels suggestive of oxidative stress and elevated nucleotide degradation. Finally, administration of 100mg/kg LY2835219 for five days in combination with fractionated dose of radiation (3 Gy) significantly delayed tumor regrowth in H460 xenograft (p< 0.014). Collectively, our pre-clinical data indicates altered Rb, p53 and PTEN status are distinct predictive biomarkers of response for LY mediated radiosensitivity and provides an alternative therapeutic option in overcoming EGFR-TKI resistance in NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Sarwat Naz, William DeGraff, Anastasia Sowers, Rajani Choudhuri, Maria Wissler, Janet Gamson, John Cook, James B. Mitchell. Enhanced radiosenistivity of EGFR-TKI sensitive and resistant NSCLC cells by abemaciclib is mediated by altered DNA repair and metabolic pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5861. doi:10.1158/1538-7445.AM2017-5861

Effect of AZD1480 in an epidermal growth factor receptor-driven lung cancer model

ObjectiveSTAT3 plays a vital role in inducing and maintaining a pro-carcinogenic inflammatory microenvironment and is reported to be a critical mediator of the oncogenic effects of EGFR mutations. STAT3 activation is mediated through JAK family kinases. We investigated the effect of the JAK1/2 inhibitor AZD1480 on lung tumors induced by an activating EGFR mutation. Materials and methodsThree EGFR tyrosine kinase inhibitor-resistant cell lines (RPC-9, PC-9/Van-R and PC-9/ER3) established from PC-9 harboring an EGFR exon19 deletion mutation were used. Growth inhibition was measured using an MTT assay. Effects of AZD1480 were also evaluated in the xenograft model and in the EGFR transgenic mice model. Protein expressions were assessed by immunoblotting and immunohistochemistry. Group differences were compared using Student's t-test. To evaluate the efficacy of AZD1480 on survival, AZD1480 or vehicle was administered orally from 7 weeks of age of the transgenic mice. Overall survival curves were calculated using the Kaplan–Meier method. ResultsThe sensitivities of resistant and parent cells to AZD1480 were similar in vitro. AZD1480 (30 or 50mg/kg/day, per os) reduced angiogenesis and revealed significant tumor regression in a mouse xenograft model. Subsequently, the transgenic mice were treated with AZD1480 (30mg/kg/day) or vehicle alone. The numbers of lung tumors (long axis exceeding 1mm) in the AZD1480-treated group and control group were 0.37±0.18 and 2.25±0.53 (p<0.001), respectively. AZD1480 treatment suppressed pSTAT3, pJAK1, pJAK2 and angiogenesis. The median survival time in the AZD1480-treated group (217 days) was significantly greater than that in the control group (106 days) (log-rank test, p<0.0001). ConclusionAZD1480 may be effective against lung tumors driven by an activating EGFR mutation.

Abstract 3994: Expression levels of EGFR-ligands are up-regulated in EGFR tyrosine kinase inhibitor-resistant cell line

Abstract Epidermal growth factor receptor (EGFR) is often highly expressed in many solid tumors and EGFR tyrosine kinase inhibitor (EGFR-TKI) demonstrated the therapeutic benefit for non small cell lung cancer in clinical setting. We previously established the EGFR-TKI resistant cell lines derived from highly parental sensitive cell line with constitutively active EGFR mutation, PC-9. EGFR-TKI-resistant cell line shows about 1000 fold resistant for EGFR-TKI compared with parental PC-9 cells. We performed the microarray and exon array analysis for these cell lines and identified that 30 genes were altered the gene expressions over 5 fold between two cell lines. There were no gene expression changes in EGFR, HER4, AKT and adaptor molecules, whereas HER2 and HER3 were down-regulated and MET were up-regulated in resistant cells. Interestingly, several ligands of EGFR such as TGF alpha, epiregulin, amphiregulin and HB-EGF were up-regulated in EGFR-TKI resistant cell lines, suggesting that the ligand expression change are associated with the phenotype of drug resistant and may be useful molecular biomarkers for EGFR-TKI treatment. We are now investigating the functional analysis for these identified genes and analysing the result of exon array. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3994.