Epidermal Growth Factor Receptor-mutant Cell Research Articles

Abstract B099: Targeting Non Small Cell Lung Cancer EGFR-Mutant and EGFR-Inhibitor resistant cell lines by ferroptosis induction: A potential therapeutic approach

Abstract The epidermal growth factor receptor (EGFR) is a crucial signaling protein involved in regulating cellular growth, survival, and proliferation. EGFR mutations, particularly in non-small cell lung cancer (NSCLC), have been extensively studied due to their role in driving tumor progression and resistance to conventional therapies. While EGFR-mutant tumors initially respond well to EGFR inhibitors, emergence of resistance remains a significant challenge. Ferroptosis is a recently discovered form of regulated cell death characterized by accumulation of lipid peroxides and oxidative stress. It has gained attention as a potential therapeutic strategy in combating drug resistance. However, the vulnerability of various EGFR-mutant and resistant NSCLC cells to ferroptosis induction remains poorly understood. To bridge the existing knowledge gap, we investigated the responsiveness of both EGFR-mutant and their resistant counterpart NSCLC cell lines to ferroptosis induction. Interestingly, NSCLC cell lines and patients’ tumors which harbor EGFR mutations are distinct from those carrying STK11 and KEAP1 mutations, which are known to contribute to resistance to ferroptosis. This observation prompted us to test whether ferroptosis inducers could effectively target this specific subset of NSCLC. In our experimental approach, we first utilized a panel of commercially available EGFR mutant cell lines (n=3), including one harboring T790M resistance mutation, which is a common mechanism of acquired resistance to first-generation inhibitors. We subjected the EGFR-mutant cell lines in vitro to a process of acquiring resistance against three generations of EGFR inhibitors (n=4). Subsequently, upon confirmation of resistance, we evaluated their susceptibility to our proprietary ferroptosis inducer. Exome sequencing data of EGFR-inhibitor resistant cell lines and their age-matched control cell lines revealed that the former acquired genetic alterations that have previously been found to confer resistance in EGFR-inhibitor resistant patients’ tumors, suggesting that our approach is valid for modeling relevant resistance mechanisms. Our results indicate that both EGFR-mutant and resistant cell lines exhibit sensitivity to ferroptosis induction. Furthermore, we observed that EGFR-mutant patient-derived cells, which are refractory to EGFR inhibitors of several generations, were strongly cytostatic to our ferroptosis inducer. These findings provide valuable insights into the interplay between EGFR mutations, drug resistance, and ferroptosis. Exploiting the vulnerability of EGFR-inhibitor resistant NSCLC cells to ferroptosis could offer novel therapeutic options for overcoming drug resistance in EGFR-driven cancers. Further investigations into the underlying molecular mechanisms of ferroptosis inducers in NSCLC are warranted for the development of effective therapies. Citation Format: Taronish Dubash, Maria Cristina Munteanu, Cristian Nunez, Shrouq Farah, Laurence Jadin, Branko Radetich, Francesco M Marincola, Darby Schmidt, Nadia Gurvich. Targeting Non Small Cell Lung Cancer EGFR-Mutant and EGFR-Inhibitor resistant cell lines by ferroptosis induction: A potential therapeutic approach [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B099.

D-mannose induces TFE3-dependent lysosomal degradation of EGFR and inhibits the progression of NSCLC.

In non-small cell lung cancer (NSCLC), the overexpression or abnormal activation of epidermal growth factor receptor (EGFR) is associated with tumor progression and drug resistance. EGFR tyrosine kinase inhibitors (TKIs) are currently the first-line treatment of NSCLC. However, patients inevitably acquired EGFR TKIs resistance mutations, which led to disease progression, so it is urgent to find new treatment. Here, we report that D-mannose up-regulates lysosomal activity by enhancing TFE3-mediated lysosomal biogenesis, thereby increasing the degradation of EGFR and significantly down-regulating its protein level. Therefore, D-mannose significantly inhibited the proliferation, migration and invasion of wild-type EGFR (WT-EGFR) and EGFR mutant cells (E746-A750 deletion, L858R and T790M mutations) in vitro. Oral administration of D-mannose strongly inhibited tumor growth in mice, showing similar effects with osimertinib. Taken together, these data suggest that D-mannose may represent a new strategy for clinical treatment of NSCLC.

Abstract 3499: Inhibition of SOS1 by MRTX0902 augments the anti-tumor response of the targeted EGFR inhibitor osimertinib in NSCLC

Abstract Osimertinib is a third generation, irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor recommended as a first line therapy in patients with advanced non-small lung cancer (NSCLC) characterized by EGFR activating mutations (exon 19 deletions and exon 21 L858R) and the resistance associated exon 20 T790M mutation, which is present in approximately 40-55% of patients following treatment with first generation EGFR-targeting inhibitors. Unfortunately, osimertinib monotherapy inevitably leads to both EGFR-dependent and EGFR-independent resistance mechanisms that reactivate RTK/MAPK pathway signaling. Thus, exploration of new combination strategies that target RTK/MAPK-mediated bypass of EGFR dependence may circumvent or delay therapeutic resistance are a key research focus. Preclinically, it has been shown that EGFR-mutant NSCLC cell lines are sensitive to inhibition of an upstream node of the MAPK pathway, the Son of Sevenless homolog 1 (SOS1) protein, which functions as a guanine nucleotide exchange factor (GEF) for the RAS subfamily of small GTPases. MRTX0902 is a selective and potent SOS1 inhibitor currently in Phase 1 clinical trials and functions by disrupting the KRAS/SOS1 protein-protein interaction to prevent SOS1-mediated nucleotide exchange on KRAS, ultimately leading to downregulation of KRAS-MAPK pathway signaling. Here we demonstrate that dual inhibition of SOS1 and mutant EGFR by osimertinib results in greater anchorage-independent (3D) cell death and further downregulation of MAPK and PI3K/AKT pathway signaling compared to single agent osimertinib treatment. In our current study, we characterized the anti-tumor effects of SOS1 inhibition by MRTX0902 as a monotherapy and in combination with osimertinib in EGFR mutant models of NSCLC. While single agent MRTX0902 treatment effectively inhibited EGFR mutant cell survival, combination treatment of MRTX0902 with osimertinib resulted in (1) deeper and sustained inhibition of MAPK and PI3K/AKT pathway signaling, (2) enhanced inhibition of 3D cell viability and increased apoptosis, and (3) increased anti-tumor efficacy compared to osimertinib monotherapy in EGFR mutant CDX models in vivo. These studies uncover the potential clinical application of combined vertical inhibition of RTK/MAPK pathway signaling by osimertinib and MRTX0902 and ultimately aide in the understanding of EGFR and RAS biology in targeted cancer therapy. Citation Format: Shilpi Khare, Niranjan Sudhakar, Jade Laguer, David M. Briere, Larry Yan, Allan Hebbert, Andrew Calinisan, Lars D. Engstrom, Fadia Qiryaqos, Peter Olson, James G. Christensen, Jacob R. Haling. Inhibition of SOS1 by MRTX0902 augments the anti-tumor response of the targeted EGFR inhibitor osimertinib in NSCLC [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 3499.

Abstract 6141: Mechanisms by which Mito-lonidamine inhibits EGFR mutant lung cancer

Abstract Lung cancer remains the leading cause of cancer mortality. Efficacious targeted therapy options are urgently needed for lung cancer prevention and treatment. Epidermal growth factor receptor (EGFR) mutant lung cancer represents about 15% of lung cancer cases generally occurring in patients with minimal to no smoking history and with higher prevalence in females. Linkages to environmental exposures have been postulated. Our group synthesized Mito-lonidamine (Mito-LND), a mitochondrial targeted analog of lonidamine which inhibits aerobic glycolysis in cancer cells. We previously identified that Mito-LND induces mitophagy in KRAS mutant lung cells and inhibits lung tumorigenesis and brain metastasis in vivo. Yet, the cancer inhibitory capacity of Mito-LND in EGFR mutant lung cancer has not been investigated. Viability staining assessed Mito-LND induced cell death in EGFR mutant cell lines PC9 (parent) and PC9BrM3 (brain metastatic). Transcriptomic profiling was conducted following Mito-LND treatment and results analyzed in Metacore to assess molecular changes. Deconvolution analysis interrogated alterations in immune cell populations. To discern cell death mechanisms, we evaluated FDA-approved inhibitors for autophagy (chloroquine, CQ), mitophagy/apoptosis (cyclosporin A, CsA) and necrosis (Ponatinib) in EGFR mutant cell lines using viability assays with Mito-LND treatment. In PC9 cells, the IC50 for Mito-LND at 24h and 48h was 1.5µM and 0.5µM, respectively and in PC9BrM3 cells at 24h and 48h was 1.5µM and 0.7µM, respectively. These data support that EGFR mutant cell lines are >2-fold more sensitive to Mito-LND induced cell death compared to KRAS mutant lung cell lines H2030 and H2030BrM3. Transcriptomic profiling results show that Mito-LND treatment upregulated pathway maps linked to heat shock protein and HIF-1A signaling in both PC9 and PC9BrM3 cells. Process networks linked to unfolded protein response, response to hypoxia and oxidative stress, apoptosis and the immune response were upregulated with Mito-LND treatment. Deconvolution analysis revealed monocytes and macrophages were upregulated in Mito-LND treated EGFR mutant cell lines, with CD8+ T cells increased in PC9BrM3 cells and regulatory T cells increased in PC9 cells. Last, CsA significantly inhibited Mito-LND induced cell death in both cell lines, while CQ had no effect suggesting blocking apoptosis, but not late autophagy inhibits cell death induction in EGFR mutant cell lines. Further, necrosis inhibition with Ponatinib synergistically increased Mito-LND induced cell death in EGFR mutant cell lines. These results begin to define the cancer inhibitory mechanisms of Mito-LND in EGFR mutant lung cancer cells which appear to differ from those observed in KRAS mutant lung cancer cells. Future directions include dissecting apoptotic and necrosis driven Mito-LND induced cell death using additional pharmacological inhibitors and genetic approaches. Citation Format: Katherine M. Weh, Connor Howard, Yun Zhang, Jean-Jack Riethoven, Jennifer Clarke, Gang Cheng, Balaraman Kalyanaraman, Ming You, Laura Kresty. Mechanisms by which Mito-lonidamine inhibits EGFR mutant lung cancer [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 6141.

Novel third-generation pyrimidines-based EGFR tyrosine kinase inhibitors targeting EGFR T790M mutation in advanced non-small cell lung cancer

The critical T790M secondary mutation in epidermal growth-factor receptor (EGFR) mediates resistance to first- and second-generation EGFR tyrosine kinase inhibitors. Herein, we identified 12 new 2,4-diaryl pyrimidine derivatives containing thiophene fragments as new selective third-generation EGFR inhibitors. Among them, Compound 6a showed good inhibitory activity against EGFR mutant cells with an IC50 value of 0.0022 ± 0.001 μM and was approximately 1730-fold less potent against EGFR WT cells (IC50: 4.499 ± 0.057 μM). Moreover, it strongly affected EGFR-mediated signaling pathways, attenuated tumor proliferation via the intrinsic mitochondrial apoptotic pathway, arrested the cell cycle at G0/G1 phase, and induced apoptosis in H1975 cells. It also displayed appropriate pharmacokinetic (PK) parameters with an oral bioavailability value of 33.57%. Additionally, in vivo studies confirmed that 6a suppressed tumor growth in an H1975 xenograft model (25 mg/kg/d, TGI: 90.24%). Overall, these results suggest that 6a could be a promising lead compound for overcoming the clinical EGFR T790M resistance of patients with non-small-cell lung cancers (NSCLCs).

Abstract 1466: ORIC-114, a brain penetrant, orally bioavailable, irreversible inhibitor selectively targets EGFR and HER2 exon20 insertion mutants and regresses intracranial NSCLC xenograft tumors

Abstract Genomic insertions within exon20 of both epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are oncogenic drivers most commonly found in non-small cell lung cancer (NSCLC) but also occurring in multiple other tumor types. Exon20 insertions render the receptors resistant to currently approved inhibitors, giving patients with tumors harboring such insertions a worse prognosis than with other activating EGFR mutations. Moreover, approximately one-third of patients with exon20 insertion mutations may develop central nervous system (CNS) metastases over the course of their disease. To address this unmet medical need, ORIC-114, a brain penetrant, orally bioavailable, irreversible small molecule inhibitor was designed to target exon20 insertions in EGFR and HER2. ORIC-114 is highly selective to the EGFR family of receptors, showing superior kinome selectivity compared with other reported exon20 inhibitors. In biochemical assays ORIC-114 displays low nanomolar potency, and importantly has enhanced potency on the EGFR exon20 NPG insertion relative to wildtype EGFR protein. ORIC-114 also demonstrates low nanomolar potency across exon20 insertion variants using cell-based assays measuring EGFR phosphorylation, downstream signaling and cell viability. ORIC-114 is readily brain available across multiple preclinical species hence studies were undertaken to investigate activity in both subcutaneous and intracranial tumor models. Herein, we explored the in vivo activity of ORIC-114. Consistent with our in vitro findings, robust activity was observed in EGFR exon20 patient-derived xenograft models using once daily oral administration, with greater than 90% tumor growth inhibition in the absence of body weight loss. Moreover, these significant antitumor effects correlate with decreased pharmacodynamic response as measured by phosphorylated EGFR in terminal tumors. To investigate whether the brain-penetrant attributes of ORIC-114 translate into therapeutic CNS activity, we utilized an intracranial luciferase-labeled EGFR mutant cell line model. Once daily oral administration of ORIC-114 significantly regressed established intracranial NSCLC tumors, demonstrating greater efficacy than TAK-788, commensurate with the superior brain to plasma exposure of ORIC-114. Taken together, these data establish ORIC-114 as a selective, irreversible, and brain penetrant EGFR inhibitor, making it a promising therapeutic candidate for development in patients with tumors harboring EGFR and HER2 exon20 insertions, including those with CNS metastases. ORIC-114 is anticipated to enter a global Phase 1/2 tumor-agnostic trial in genetically defined cancers in the second half of 2021. Citation Format: Melissa R. Junttila, Sunghwan Kim, Younho Lee, Hwan Kim, Juhee Kang, Jiyoon Seok, Jihye Yoo, Youngyi Lee, Dong-Hyuk Seo, Jung Beom Son, Daekwon Kim, Hwan Geun Choi, Nam Doo Kim, Akash Das, Dena Sutimantanapi, Tatiana Zavorotinskaya, Chelsea Chen, Jae Chang, Matthew Panuwat, Lori Friedman. ORIC-114, a brain penetrant, orally bioavailable, irreversible inhibitor selectively targets EGFR and HER2 exon20 insertion mutants and regresses intracranial NSCLC xenograft tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1466.

Apoptotic activities of brusatol in human non-small cell lung cancer cells: Involvement of ROS-mediated mitochondrial-dependent pathway and inhibition of Nrf2-mediated antioxidant response

Brusatol occurs as a characteristic bioactive principle of Brucea javanica (L.) Merr., a traditional medicinal herb frequently employed to tackle cancer in China. This work endeavored to unravel the potential anti-cancer activity and action mechanism of brusatol against non-small cell lung cancer (NSCLC) cell lines. The findings indicated that brusatol remarkably inhibited the growth of wild-type NSCLC cell lines (A549 and H1650) and epidermal growth factor receptor-mutant cell lines (PC9 and HCC827) in a dose- and time-related fashion, and profoundly inhibited the clonogenic capability and migratory capacity of PC9 cells. Treatment with brusatol resulted in significant apoptosis in PC9 cells, as evidenced by Hoechst 33342 staining and flow cytometric analysis. The apoptotic effect was closely related to induction of G0-G1 cell cycle arrest, stimulation of reactive oxygen species (ROS) and malondialdehyde, decrease of glutathione levels and disruption of mitochondrial membrane potential. Furthermore, pretreatment with N-acetylcysteine, a typical ROS scavenger, markedly ameliorated the brusatol-induced inhibition of PC9 cells. Western blotting assay indicated that brusatol pronouncedly suppressed the expression levels of mitochondrial apoptotic pathway-associated proteins Bcl-2 and Bcl-xl, accentuated the expression of Bax and Bak, and upregulated the protein expression of XIAP, cleaved caspase-3/pro caspase-3, cleaved caspase-8/pro caspase-8, and cleaved PARP/total PARP. In addition, brusatol significantly suppressed the expression of Nrf2 and HO-1, and abrogated tBHQ-induced Nrf2 activation. Combinational administration of brusatol with four chemotherapeutic agents exhibited marked synergetic effect on PC9 cells. Together, the inhibition of PC9 cells proliferation by brusatol might be intimately associated with the modulation of ROS-mediated mitochondrial-dependent pathway and inhibition of Nrf2-mediated antioxidant response. This novel insight might provide further evidence to buttress the antineoplastic efficacy of B. javanica, and support a role for brusatol as a promising anti-cancer candidate or adjuvant to current chemotherapeutic medication in the therapy of EGFR-mutant NSCLC.

ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation.

Rationale: Immune checkpoint inhibitors (ICIs) against the PD-1/PD-L1 pathway showed limited success in non-small cell lung cancer (NSCLC) patients, especially in those with activating epidermal growth factor receptor (EGFR) mutations. Elucidation of the mechanisms underlying EGFR-mediated tumor immune escape and the development of effective immune therapeutics are urgently needed. Immunoglobulin-like transcript (ILT) 4, a crucial immunosuppressive molecule initially identified in myeloid cells, is enriched in solid tumor cells and promotes the malignant behavior of NSCLC. However, the upstream regulation of ILT4 overexpression and its function in tumor immunity of NSCLC with EGFR activation remains unclear.Methods: ILT4 expression and EGFR phosphorylation in human NSCLC tissues and cell lines were analyzed using immunohistochemistry (IHC), real-time PCR, Western blotting, immunofluorescence, and flow cytometry. The molecular signaling for EGFR-regulated ILT4 expression was investigated using mRNA microarray and The Cancer Genome Atlas (TCGA) database analyses and then confirmed by Western blotting. The regulation of tumor cell proliferation and apoptosis by ILT4 was examined by CCK8 proliferation and apoptosis assays. The impact of ILT4 and PD-L1 on tumor-associated macrophage (TAM) recruitment and polarization was evaluated using Transwell migration assay, flow cytometry, enzyme linked immunosorbent assay (ELISA) and real-time PCR, while their impact on T cell survival and cytotoxicity was analyzed by CFSE proliferation assay, apoptotic assay, flow cytometry, ELISA and cytolytic assay. Tumor immunotherapy models targeting at paired Ig-like receptor B (PIR-B, an ortholog of ILT4 in mouse)/ILT4 and/or PD-L1 were established in C57BL/6 mice inoculated with stable EGFR- overexpressing Lewis lung carcinoma (LLC) cells and in humanized NSG mice inoculated with EGFR mutant, gefitinib-resistant PC9 (PC9-GR) or EGFR-overexpressing wild type H1299 cells. PIR-B and ILT4 inhibition was implemented by infection of specific knockdown lentivirus and PD-L1 was blocked using human/mouse neutralizing antibodies. The tumor growth model was established in NSG mice injected with PIR-B-downregulated LLC cells to evaluate the effect of PIR-B on tumor proliferation. The frequencies and phenotypes of macrophages and T cells in mouse spleens and blood were detected by flow cytometry while those in tumor tissues were determined by IHC and immunofluorescence.Results: We found that ILT4 expression in tumor cells was positively correlated with EGFR phosphorylation in human NSCLC tissues. Using NSCLC cell lines, we demonstrated that ILT4 was upregulated by both tyrosine kinase mutation-induced and epidermal growth factor (EGF)-dependent EGFR activation and subsequent AKT/ERK1/2 phosphorylation. Overexpressed ILT4 in EGFR-activated tumor cells induced TAM recruitment and M2-like polarization, which impaired T cell function. ILT4 also directly inhibited T cell proliferation, cytotoxicity, and IFN-γ expression and secretion. In EGFR-activated cell lines in vitro and in wild-type EGFR-activated C57BL/6 and humanized NSG immunotherapy models in vivo, either ILT4 (PIR-B) or PD-L1 inhibition enhanced anti-tumor immunity and suppressed tumor progression by counteracting TAM- and dysfunctional T cell- induced immuno-suppressive TME; the combined inhibition of both molecules showed the most dramatic tumor retraction. Surprisingly, in EGFR mutant, TKI resistant humanized NSG immunotherapy model, ILT4 inhibition alone rather than in combination with a PD-L1 inhibitor suppressed tumor growth and immune evasion.Conclusions: ILT4 was induced by activation of EGFR-AKT and ERK1/2 signaling in NSCLC cells. Overexpressed ILT4 suppressed tumor immunity by recruiting M2-like TAMs and impairing T cell response, while ILT4 inhibition prevented immunosuppression and tumor promotion. Furthermore, ILT4 inhibition enhanced the efficacy of PD-L1 inhibitor in EGFR wild-type but not in EGFR mutant NSCLC. Our study identified novel mechanisms for EGFR-mediated tumor immune escape, and provided promising immunotherapeutic strategies for patients with EGFR-activated NSCLC.

Abstract 5066: Investigating the role of IL-6 to modulate the immunosuppressive tumor microenvironment of EGFR mutant NSCLC

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death with a less than 15% five-year survival. Approximately 15% of NSCLC cases in the US harbor epidermal growth factor receptor (EGFR) activating mutations and are initially highly sensitive to EGFR tyrosine kinase inhibitors (TKI). Unfortunately, the vast majority of tumors acquire EGFR TKI resistance, and treating resistant disease remains a clinical challenge. Additional therapeutic modalities, such as immunotherapy, are needed to improve clinical outcome. While immunotherapy has shown clinical activity in multiple cancer types including NSCLC, a lack of efficacy has been observed in EGFR mutant NSCLC patients. The biological mechanisms driving immunotherapy resistance in EGFR mutant NSCLC are not well understood, and additional EGFR mutant NSCLC patients. We previously reported that IL-6 is upregulated in NSCLC cells with acquired resistance to EGFR TKIs. Given the role of IL-6 as an immunomodulatory cytokine, we evaluated the effects of IL-6 signaling on immunosuppression in genetically engineered mouse models (GEMMs) of EGFR mutant NSCLC. IL-6 knockout (EGFRL858R/IL-6KO) resulted in decreased PD-L1 expression on the surface of EGFR mutant tumor cells compared to tumors in IL-6 proficient mice. Additionally, we observed a modest increase in PD-1 expression on CD8+ T cells in EGFRL858R/IL-6KO mice compared to EGFRL858R mice, suggesting that IL-6 acts on both tumor and immune populations to facilitate a “cold” immunosuppressive tumor microenvironment. We next analyzed immune populations in EGFR mutant tumors from mice with or without knockout of IL-6 by single cell RNA sequencing and validated these findings by immunohistochemistry. Tumors in EGFRL858R/IL-6KO mice displayed decreased B-cell (CD20) populations, increased macrophages (F480), and increased natural killer cells (NKp46) compared to tumors from IL-6 expressing EGFRL858R mice. Our data suggests that the increased expression of IL-6 in EGFR TKI resistant NSCLC may alter immune cell populations within the tumor microenvironment and impact immunosuppression, and that therapeutic approach targeting IL-6 merit further investigation for EGFR-mutant NSCLC. Citation Format: Sonia A. Patel, Monique Nilsson, Sungnam Cho, Xiuning Le, John Heymach. Investigating the role of IL-6 to modulate the immunosuppressive tumor microenvironment of EGFR mutant NSCLC [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 5066.

SRC-homology 2 domain-containing phosphatase 2 (SHP2) in epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD).

9540 Background: Epidermal growth factor (EGFR)-mutant lung adenocarcinomas (LUADs) display impaired phosphorylation of extracellular signal-regulated kinase (ERK) and SRC-homology 2 domain-containing phosphatase 2 (SHP2) in comparison with EGFR wild-type LUADs. However, the function of SHP2 in early EGFR-mutant LUADs and EGFR wild-type LUADs has not been reported. We posit that SHP2 mRNA expression could be a predictive marker in resected EGFR-mutant LUADs versus EGFR wild-type patients (pts). Methods: We examined 267 resected LUADs from Japan and Spain. mRNA expression levels of AXL, MET, CDCP1, STAT3, YAP1 and SHP2 were analyzed by quantitative reverse transcriptase polymerase chain reaction (PCR). EGFR mutant cell lines were investigated for their activity of SHP2. Results: Among the 267 enrolled pts, 100 (37.3%) were EGFR-mutant LUADs. Five-year recurrence-free survival (RFS) and overall survival (OS) were lower for EGFR-mutant LUADs with high SHP2 mRNA levels (hazard ratio = 1.83 and 2.28, respectively. p = 0.03 and p = 0.04). However, SHP2 was not associated with RFS nor OS in the 167 wild-type EGFR LUADs. In EGFR-mutant cells, RMC-4550 (SHP2 inhibitor) plus erlotinib showed synergism via inhibition of AKT (S473) and ERK1/2 (T202/Y204). While erlotinib translocates SHP2 (Y542) into the nucleus, either RMC-4550 alone, or in combination with erlotinib, relocalizes SHP2 into the cytoplasm membrane, limiting AKT and ERK activation. Conclusions: High SHP2 mRNA is related to shorter RFS and OS in EGFR-mutant LUADs, but not in EGFR wild-type LUADs. The findings indicate that the addition of SHP2 inhibitors could improve adjuvant therapy in EGFR-mutant LUADs.

A Bispecific Inhibitor of the EGFR/ADAM17 Axis Decreases Cell Proliferation and Migration of EGFR-Dependent Cancer Cells

Dysregulated epidermal growth factor receptor (EGFR) is an oncogenic driver of many human cancers, promoting aberrant cell proliferation, migration, and survival. Pharmacological targeting of EGFR is often challenged by acquired mechanisms of resistance. Ligand-dependent mechanisms in EGFR wild-type cells rely on ligand or receptor overexpression, allowing cells to outcompete inhibitors and perpetuate signaling in an autocrine manner. Importantly, EGFR ligands are synthesized as membrane-bound precursors that must be solubilized to enable receptor-ligand interactions. The A disintegrin and metalloproteinase 17 (ADAM17) is considered the main sheddase of several EGFR ligands, and a potential pharmacological target. However, its broad substrate range and ubiquitous expression complicate its therapeutic targeting. Here, we present a novel bispecific fusion protein construct consisting of the inhibitory prodomain of ADAM17 (TPD), fused to an EGFR-targeting designed ankyrin repeat protein (DARPin). TPD is a natural inhibitor of ADAM17, maintaining the protease in a zymogen-like form. Meanwhile, the high affinity anti-EGFR DARPin E01 binds to EGFR and inhibits ligand binding. The resulting fusion protein E01-GS-TPD retained binding ability to both molecular targets EGFR and ADAM17. The large difference in affinity for each target resulted in enrichment of the fusion protein in EGFR-positive cells compared to EGFR-negative cells, suggesting a possible application in autocrine signaling inhibition. Accordingly, E01-GS-TPD decreased migration and proliferation of EGFR-dependent cell lines with no significant increase in apoptotic cell death. Finally, inhibition of proliferation was observed through EGFR ligand-dependent mechanisms as growth inhibition was not observed in EGFR mutant or KRAS mutant cell lines. The use of bispecific proteins targeting the EGFR/ADAM17 axis could be an innovative strategy for the treatment of EGFR-dependent cancers.

EGFR-TKI resistance and MAP17 are associated with cancer stem cell like properties.

Patients with non-small-cell lung cancer (NSCLC) with sensitive epidermal growth factor receptor (EGFR) mutations generally react well to tyrosine kinase inhibitors (TKIs). However acquired resistance eventually occurs. Several mechanisms contribute to the resistance including T790M mutation, c-Met amplification and PIK3CA mutation. In recent years, cancer stem cells (CSCs) have been suggested to be involved in TKI resistance. MAP17 is aberrantly overexpressed in a number of malignancies. However, the expression pattern and function of MAP17 in CSCs are still unclear. The aim the present study was to illustrate the effect of CSC-like cells on the resistance to TKIs in EGFR mutant NSCLC cells and explore the possible role of MAP17 in CSCs. The EGFR mutant cell line PC9 was cultured under serum-deprived undifferentiated conditions. The CSC properties including expression of stem cell markers CD133, CD44, Oct-4 and ABCG2, ability of self-renewal, invasion, proliferation and tumorigenesis were examined. The expression of MAP17 was compared in sphere and parent cells. Sphere cells displayed stem cells phenotypes and were resistant to erlotinib. Sphere cells expressed higher levels of MAP17, and MAP17 was associated with self-renewal and TKI resistance. The function of MAP17 demonstrated to be partially dependent on Na-dependent glucose transporter 1. Collectively these findings suggest that MAP17 serves a role in TKI resistance through regulation of CSCs in lung cancer.

Abstract 2061: Non-covalent EGFR T790M targeting TKIs inhibit AZD9291 resistant EGFR C797S mutants

Abstract Approximately 10-15% of non-small-cell lung cancers (NSCLC) have epidermal growth factor receptor (EGFR) mutations resulting in increased sensitivity to 1st generation tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. For common mutations in EGFR, treatment with 1st generation TKIs results in approximately 70% overall response rate, increased progression free survival, and increased quality of life compared to chemotherapy alone. However, resistance to 1st generation TKIs typically develops within ~12 months, and approximately 55% of patients acquire a secondary mutation in EGFR Exon 20, T790M. To overcome resistance, 2nd and 3rd generation covalently binding TKIs targeting T790M mutations have been developed. Recent studies show that approximately 40% of acquired resistance to 2nd and 3rd generation TKIs can also occur via a third acquired EGFR mutation at the site of covalent binding, C797S. To date, there are no standard approved targeted therapies for treating EGFR C797S mutant NSCLC. Moreover, with proposed increased use of covalent inhibitors in the first line setting, C797S mutations are expected to become more prevalent and new strategies to overcome therapeutic resistance will be required. To this end, we have generated stable Ba/F3 and HCC827 NSCLC cell lines expressing C797S mutant EGF receptors with common mutations in EGFR including, L858R/T790M/C797S, and Ex19del/T790M/C797S. EGFR mutant cell lines expressing C797S were screened against 1st, 2nd, and 3rd generation EGFR TKIs and cell viability was determined using Cell Titer Glo. Triple mutant cell lines containing T790M and C797S mutations were not sensitive to any 1st, 2nd, or 3rd generation inhibitors with IC50 values of >10µM, 7.0µM, and 7.6µM, respectively. However Ba/F3 and HCC827 cell lines transfected with EGFR triple mutants were inhibited by non-covalent inhibitors: CUDC-101, an EGFR, HER2 and HDAC inhibitor, and PKC412, a FLT3 inhibitor. In triple mutant Ba/F3 cells, CUDC-101 and PKC412 had IC50 values of 470nM and 250nM in Ex19del/T790M/C797S EGFR mutants, respectively, and IC50 values of 690nM and 270nM in L858R/T790M/C797S EGFR mutants. In HCC827 cells, PKC412 had IC50 values of 610nM and 520nM in L858R/T790M/C797S and Ex19del/T790M/C797S triple mutants, respectively. In addition, western blot analysis of triple mutant Ba/F3 cells showed decreased phosphorylation of EGFR in presence of 50nM PKC412 and 500nM of CUDC-101. Moreover, PKC412 partially decreased p-EGFR expression at 50nM in HCC827 triple mutant cell lines. In conclusion, unlike other EGFR TKIs, the non-covalent EGFR inhibitors CUDC-101 and PKC412 inhibit both T790M and C797S EGFR mutants at low concentrations. Currently, we are establishing additional osimertinib resistant HCC827, H1975, PC9 and H4006 cell lines for further in vitro and in vivo studies. Citation Format: Jacqulyne P. Robichaux, Monique Nilsson, John V. Heymach. Non-covalent EGFR T790M targeting TKIs inhibit AZD9291 resistant EGFR C797S mutants [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 2061. doi:10.1158/1538-7445.AM2017-2061

Activation of signal transducer and activator of transcription 3 (STAT3) signaling in EGFR mutant non-small-cell lung cancer (NSCLC).

Gefitinib, erlotinib or afatinib are the current treatment for non-small-cell lung cancer (NSCLC) harboring an activating mutation of the epidermal growth factor receptor (EGFR), but less than 5% of patients achieve a complete response and the median progression-free survival is no longer than 12 months. Early adaptive resistance can occur as soon as two hours after starting treatment by activating signal transducer and activation of transcription 3 (STAT3) signaling. We investigated the activation of STAT3 in a panel of gefitinib-sensitive EGFR mutant cell lines, and gefitinib-resistant PC9 cell lines developed in our laboratory. Afatinib has great activity in gefitinib-sensitive as well as in gefitinib-resistant EGFR mutant NSCLC cell lines. However, afatinib therapy causes phosphorylation of STAT3 tyrosine 705 (pSTAT3Tyr705) and elevation of STAT3 and RANTES mRNA levels. The combination of afatinib with TPCA-1 (a STAT3 inhibitor) ablated pSTAT3Tyr705 and down-regulated STAT3 and RANTES mRNA levels with significant growth inhibitory effect in both gefitinib-sensitive and gefitinib-resistant EGFR mutant NSCLC cell lines. Aldehyde dehydrogenase positive (ALDH+) cells were still observed with the combination at the time that Hairy and Enhancer of Split 1 (HES1) mRNA expression was elevated following therapy. Although the combination of afatinib with STAT3 inhibition cannot eliminate the potential problem of a remnant cancer stem cell population, it represents a substantial advantage and opportunity to further prolong progression free survival and probably could increase the response rate in comparison to the current standard of single therapy.

The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation

Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR.In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance.

Epidermal growth factor signals regulate dihydropyrimidine dehydrogenase expression in EGFR-mutated non-small-cell lung cancer.

BackgroundIt has been shown that epidermal growth factor receptor (EGFR) mutation status is associated with 5-fluorouracil (5-FU) sensitivity in non-small-cell lung cancer (NSCLC). However, the relationship between EGFR mutation status and dihydropyrimidine dehydrogenase (DPD), a 5-FU degrading enzyme, is unknown.MethodsWe elucidated the crosstalk among the EGFR signal cascade, the DPD gene (DPYD), and DPD protein expression via the transcription factor Sp1 and the effect of EGFR mutation status on the crosstalk.ResultsIn the PC9 (exon19 E746-A750) study, EGF treatment induced up-regulation of both Sp1 and DPD; gefitinib, an EGFR-tyrosine kinase inhibitor (EGFR-TKI), and mithramycin A, a specific Sp-1 inhibitor, suppressed them. Among EGFR-mutated (PC9, HCC827; exon19 E746-A750 and H1975; exon21 L858R, T790M, gefitinib resistant) and -non-mutated (H1437, H1299) cell lines, EGF administration increased DPYD mRNA expression only in mutated cells (p < 0.05). Accordingly, gefitinib inhibited DPD protein expression only in PC9 and HCC827 cells, and mithramycin A inhibited it in EGFR-mutated cell lines, but not in wild-type. FU treatment decreased the level of cell viability more in gefitinib-treated EGFR-TKI sensitive cell lines. Further, combination treatment of FU and mithramycin A suppressed cell viability even in a gefitinib resistant cell line.ConclusionsThe EGFR signal cascade regulates DPD expression via Sp1 in EGFR mutant cells. These results might be a step towards new therapies targeting Sp1 and DPD in NSCLC with different EGFR mutant status.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2392-0) contains supplementary material, which is available to authorized users.

Glycoproteomic Approach Identifies KRAS as a Positive Regulator of CREG1 in Non-small Cell Lung Cancer Cells.

Protein glycosylation plays a fundamental role in a multitude of biological processes, and the associated aberrant expression of glycoproteins in cancer has made them attractive biomarkers and therapeutic targets. In this study, we examined differentially expressed glycoproteins in cell lines derived from three different states of lung tumorigenesis: an immortalized bronchial epithelial cell (HBE) line, a non-small cell lung cancer (NSCLC) cell line harboring a Kirsten rat sarcoma viral oncogene homolog (KRAS) activation mutation and a NSCLC cell line harboring an epidermal growth factor receptor (EGFR) activation deletion. Using a Triple SILAC proteomic quantification strategy paired with hydrazide chemistry N-linked glycopeptide enrichment, we quantified 118 glycopeptides in the three cell lines derived from 82 glycoproteins. Proteomic profiling revealed 27 glycopeptides overexpressed in both NSCLC cell lines, 6 glycopeptides overexpressed only in the EGFR mutant cells and 19 glycopeptides overexpressed only in the KRAS mutant cells. Further investigation of a panel of NSCLC cell lines found that Cellular repressor of E1A-stimulated genes (CREG1) overexpression was closely correlated with KRAS mutation status in NSCLC cells and could be down-regulated by inhibition of KRAS expression. Our results indicate that CREG1 is a down-stream effector of KRAS in a sub-type of NSCLC cells and a novel candidate biomarker or therapeutic target for KRAS mutant NSCLC.

Comparative proteome analysis across non-small cell lung cancer cell lines

Non-small cell lung cancer (NSCLC) cell lines are widely used model systems to study molecular aspects of lung cancer. Comparative and in-depth proteome expression data across many NSCLC cell lines has not been generated yet, but would be of utility for the investigation of candidate targets and markers in oncogenesis. We employed a SILAC reference approach to perform replicate proteome quantifications across 23 distinct NSCLC cell lines. On average, close to 4000 distinct proteins were identified and quantified per cell line. These included many known targets and diagnostic markers, indicating that our proteome expression data represents a useful resource for NSCLC pre-clinical research. To assess proteome diversity within the NSCLC cell line panel, we performed hierarchical clustering and principal component analysis of proteome expression data. Our results indicate that general proteome diversity among NSCLC cell lines supersedes potential effects common to K-Ras or epidermal growth factor receptor (EGFR) oncoprotein expression. However, we observed partial segregation of EGFR or KRAS mutant cell lines for certain principal components, which reflected biological differences according to gene ontology enrichment analyses. Moreover, statistical analysis revealed several proteins that were significantly overexpressed in KRAS or EGFR mutant cell lines.

Mechanism for activation of mutated epidermal growth factor receptors in lung cancer

The initiation of epidermal growth factor receptor (EGFR) kinase activity proceeds via an asymmetric dimerization mechanism in which a "donor" tyrosine kinase domain (TKD) contacts an "acceptor" TKD, leading to its activation. In the context of a ligand-induced dimer, identical wild-type EGFR TKDs are thought to assume the donor or acceptor roles in a random manner. Here, we present biochemical reconstitution data demonstrating that activated EGFR mutants found in lung cancer preferentially assume the acceptor role when coexpressed with WT EGFR. Mutated EGFRs show enhanced association with WT EGFR, leading to hyperphosphorylation of the WT counterpart. Mutated EGFRs also hyperphosphorylate the related erythroblastic leukemia viral oncogene (ErbB) family member, ErbB-2, in a similar manner. This directional "superacceptor activity" is particularly pronounced in the drug-resistant L834R/T766M mutant. A 4-Å crystal structure of this mutant in the active conformation reveals an asymmetric dimer interface that is essentially the same as that in WT EGFR. Asymmetric dimer formation induces an allosteric conformational change in the acceptor subunit. Thus, superacceptor activity likely arises simply from a lower energetic cost associated with this conformational change in the mutant EGFR compared with WT, rather than from any structural alteration that impairs the donor role of the mutant. Collectively, these findings define a previously unrecognized mode of mutant-specific intermolecular regulation for ErbB receptors, knowledge of which could potentially be exploited for therapeutic benefit.

RBx10080307, a dual EGFR/IGF-1R inhibitor for anticancer therapy

Pharmacological intervention of epidermal growth factor receptor (EGFR) family members by antibodies or small molecule inhibitors has been one of the most successful approaches for anticancer therapy. However this therapy has its own limitations due to the development of resistance, over a period of time. One of the possible causes of the development of resistance to the therapy with EGFR inhibitors could be the simultaneous activation of parallel pathways. Both EGFR and insulin like growth factor-1 receptor (IGF-1R) pathways are reported to act reciprocal to each other and converge into the mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. Inhibiting one pathway alone may therefore not be sufficient and could be a cause of development of resistance. The other cause could be mutations of EGFR which would be less sensitive to the inhibitors. We, therefore, suggest that co-targeting IGF-1R and EGFR kinases by dual inhibitors can lead to improved efficacy and address the problems of resistance.In the present manuscript, we report the identification of a novel, small molecule dual EGFR/IGF-1R inhibitor, RBx10080307 which displayed in vitro activity at the molecular level and oral efficacy in mouse xenograft model. The compound also showed in vitro activity in an EGFR mutant cell line and may thus have the potential to show activity in resistant conditions. Additional efficacy studies are needed in EGFR resistant mouse cancer model and if found efficacious, this can be a major advantage over standalone erlotinib and other existing therapies.