Non-small Cell Lung Cancer Xenograft Research Articles

Abrogation of STAT3 activation cascade by Ginkgolide C mitigates tumourigenesis in lung cancer preclinical model.

Ginkgolide C (GGC) isolated from Ginkgo biloba (Ginkgoaceae) leaf can demonstrate pleiotropic pharmacological actions. However, its anti-oncogenic impact in non-small cell lung cancer (NSCLC) model has not been reconnoitered. As signal transducer and activator of transcription 3 (STAT3) cascade can promote tumour growth and survival, we contemplated that GGC may interrupt this signalling cascade to expend its anti-cancer actions in NSCLC. The effect of GGC on STAT3 activation, associated protein kinases, STAT3-regulated gene products, cellular proliferation and apoptosis was examined. The in-vivo effect of GGC on the growth of human NSCLC xenograft tumours in athymic nu/nu female mice was also investigated. GGC attenuated the phosphorylation of STAT3 and STAT3 upstream kinases effectively. Exposure to pervanadate modulated GGC-induced down-regulation of STAT3 activation and promoted an elevation in the level of PTPε protein. Indeed, silencing of the PTPε gene reversed the GGC-promoted abrogation of STAT3 activation and apoptosis. Moreover, GGC exposure significantly reduced NSCLC tumour growth without demonstrating significant adverse effects via decreasing levels of p-STAT3 in mice tissues. Overall, the findings support that GGC may exhibit anti-neoplastic actions by mitigation of STAT3 signalling cascade in NSCLC.

Circ_PIP5K1A regulates cisplatin resistance and malignant progression in non-small cell lung cancer cells and xenograft murine model via depending on miR-493-5p/ROCK1 axis

BackgroundChemoresistance limits the therapeutic effect of cisplatin (DDP) on non-small cell lung cancer (NSCLC). Circular RNAs (circRNAs) function as important regulators in chemoresistance. This study aimed to explore the regulation of circRNA Phosphatidylinositol-4-Phosphate 5-Kinase Type 1 Alpha (circ_PIP5K1A) in DDP resistance.MethodsThe expression analysis of circ_PIP5K1A, micoRNA-493-5p (miR-493-5p) and Rho Associated Coiled-Coil Containing Protein Kinase 1 (ROCK1) was conducted through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell sensitivity was determined using 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell proliferation and cell viability were evaluated by colony formation assay and MTT assay, respectively. Cell cycle and apoptosis detection was performed via flow cytometry. Cell motility was examined by transwell migration or invasion assay. Dual-luciferase reporter assay was applied to confirm the target binding. ROCK1 protein level was assayed via Western blot. In vivo assay was carried out using xenograft model in mice.ResultsCirc_PIP5K1A level was abnormally increased in DDP-resistant NSCLC tissues and cells. Silencing circ_PIP5K1A reduced DDP resistance, proliferation, cell cycle progression and cell motility in DDP-resistant NSCLC cells. Circ_PIP5K1A directly interacted with miR-493-5p in NSCLC cells. The function of circ_PIP5K1A was dependent on the negative regulation of miR-493-5p. MiR-493-5p directly targeted ROCK1 and circ_PIP5K1A regulated the ROCK1 level via acting as a sponge of miR-493-5p. Overexpression of miR-493-5p inhibited chemoresistance and cancer progression by downregulating ROCK1 expression in DDP-resistant NSCLC cells. Circ_PIP5K1A regulated DDP sensitivity in vivo via the miR-493-5p/ROCK1 axis.ConclusionThese findings suggested that circ_PIP5K1A upregulated the ROCK1 expression to promote DDP resistance and cancer progression in NSCLC by sponging miR-493-5p.

MG53 suppresses tumor progression and stress granule formation by modulating G3BP2 activity in non-small cell lung cancer

BackgroundCancer cells develop resistance to chemotherapeutic intervention by excessive formation of stress granules (SGs), which are modulated by an oncogenic protein G3BP2. Selective control of G3BP2/SG signaling is a potential means to treat non-small cell lung cancer (NSCLC).MethodsCo-immunoprecipitation was conducted to identify the interaction of MG53 and G3BP2. Immunohistochemistry and live cell imaging were performed to visualize the subcellular expression or co-localization. We used shRNA to knock-down the expression MG53 or G3BP2 to test the cell migration and colony formation. The expression level of MG53 and G3BP2 in human NSCLC tissues was tested by western blot analysis. The ATO-induced oxidative stress model was used to examine the effect of rhMG53 on SG formation. Moue NSCLC allograft experiments were performed on wild type and transgenic mice with either knockout of MG53, or overexpression of MG53. Human NSCLC xenograft model in mice was used to evaluate the effect of MG53 overexpression on tumorigenesis.ResultsWe show that MG53, a member of the TRIM protein family (TRIM72), modulates G3BP2 activity to control lung cancer progression. Loss of MG53 results in the progressive development of lung cancer in mg53-/- mice. Transgenic mice with sustained elevation of MG53 in the bloodstream demonstrate reduced tumor growth following allograft transplantation of mouse NSCLC cells. Biochemical assay reveals physical interaction between G3BP2 and MG53 through the TRIM domain of MG53. Knockdown of MG53 enhances proliferation and migration of NSCLC cells, whereas reduced tumorigenicity is seen in NSCLC cells with knockdown of G3BP2 expression. The recombinant human MG53 (rhMG53) protein can enter the NSCLC cells to induce nuclear translation of G3BP2 and block arsenic trioxide-induced SG formation. The anti-proliferative effect of rhMG53 on NSCLC cells was abolished with knockout of G3BP2. rhMG53 can enhance sensitivity of NSCLC cells to undergo cell death upon treatment with cisplatin. Tailored induction of MG53 expression in NSCLC cells suppresses lung cancer growth via reduced SG formation in a xenograft model.ConclusionOverall, these findings support the notion that MG53 functions as a tumor suppressor by targeting G3BP2/SG activity in NSCLCs.

Tumor-associated macrophages (TAMs)-derived osteopontin (OPN) upregulates PD-L1 expression and predicts poor prognosis in non-small cell lung cancer (NSCLC).

BackgroundProgrammed cell death ligand 1 (PD‐L1) is widely known as an immune checkpoint molecule in tumor cells. Osteopontin (OPN) is expressed by both tumor cells and tumor‐associated macrophages (TAMs), and both autocrine and paracrine of OPN are considered to be involved in tumor metastasis, proliferation and immunosuppression. However, little is known about the relationship between OPN expressed in TAMs (TOPN) and PD‐L1 in non‐small cell lung cancer (NSCLC).MethodsTissue microarray was used to detect the expression of TOPN, TAMs and PD‐L1 by multiple quantitative fluorescence staining in 509 NSCLC patients undergoing complete pulmonary resection. The correlations between TOPN, PD‐L1 and clinicopathological data were analyzed. An in vitro coculture system was established to investigate the crosstalk between TOPN and neoplastic PD‐L1. In vivo, the intrinsic features of PD‐L1 in NSCLC xenografts were evaluated after being coinjected with OPN‐positive TAMs, and a series of key cytokines and chemokines were detected in the tumor microenvironment.ResultsA positive association between the TOPN and PD‐L1 expression in tumor tissues from 509 patients with NSCLC was verified. In addition, TOPN and PD‐L1 were independent prognostic factors for overall survival (OS) and disease‐free survival (DFS) of NSCLC patients. Moreover, TOPN upregulated PD‐L1 expression in NSCLC cells through the nuclear factor‐κB (NF‐κB) pathway in vitro TOPN induced the PD‐L1 expression promoted the tumor growth in tumor‐bearing mice, altering immune‐related cytokines and chemokines.ConclusionsTOPN regulates PD‐L1 expression through the NF‐κB pathway in NSCLS, which is a potential independent biomarker and target for prognosis as well as immunotherapy.

DNA-PK inhibition by M3814 enhances chemosensitivity in non-small cell lung cancer

A significant proportion of non-small cell lung cancer (NSCLC) patients experience accumulating chemotherapy-related adverse events, motivating the design of chemosensitizating strategies. The main cytotoxic damage induced by chemotherapeutic agents is DNA double-strand breaks (DSB). It is thus conceivable that DNA-dependent protein kinase (DNA-PK) inhibitors which attenuate DNA repair would enhance the anti-tumor effect of chemotherapy. The present study aims to systematically evaluate the efficacy and safety of a novel DNA-PK inhibitor M3814 in synergy with chemotherapies on NSCLC. We identified increased expression of DNA-PK in human NSCLC tissues which was associated with poor prognosis. M3814 potentiated the anti-tumor effect of paclitaxel and etoposide in A549, H460 and H1703 NSCLC cell lines. In the four combinations based on two NSCLC xenograft models and two chemotherapy, we also observed tumor regression at tolerated doses in vivo. Moreover, we identified a P53-dependent accelerated senescence response by M3814 following treatment with paclitaxel/etoposide. The present study provides a theoretical basis for the use of M3814 in combination with paclitaxel and etoposide in clinical practice, with hope to aid the optimization of NSCLC treatment.

The requirement of mitochondrial RNA polymerase for non-small cell lung cancer cell growth

POLRMT (RNA polymerase mitochondrial) is responsible for the transcription of mitochondrial genome encoding key components of oxidative phosphorylation. This process is important for cancer cell growth. The current study tested expression and potential functions of POLRMT in non-small cell lung cancer (NSCLC). TCGA cohorts and the results from the local lung cancer tissues showed that POLRMT is overexpressed in human lung cancer tissues. In both primary human NSCLC cells and A549 cells, POLRMT silencing (by targeted lentiviral shRNAs) or knockout (through CRSIPR/Cas9 gene editing method) potently inhibited cell viability, proliferation, migration, and invasion, and induced apoptosis activation. On the contrast, ectopic overexpression of POLRMT using a lentiviral construct accelerated cell proliferation and migration in NSCLC cells. The mtDNA contents, mRNA levels of mitochondrial transcripts, and subunits of respiratory chain complexes, as well as S6 phosphorylation, were decreased in POLRMT-silenced or -knockout NSCLC cells, but increased after ectopic POLRMT overexpression. In vivo, intratumoral injection of POLRMT shRNA adeno-associated virus (AAV) potently inhibited NSCLC xenograft growth in severe combined immune deficiency mice. The mtDNA contents, mRNA levels of mitochondria respiratory chain complex subunits, and S6 phosphorylation were decreased in POLRMT shRNA AAV-injected NSCLC xenograft tissues. These results show that POLRMT is a novel and important oncogene required for NSCLC cell growth in vitro and in vivo.

Epigenetic inhibition assisted chemotherapeutic treatment of lung cancer based on artificial exosomes

We adopted a novel strategy by combining histone deacetylase (HDAC) inhibitors with traditional chemotherapeutics to treat solid tumors. However, chemotherapeutics often have a narrow therapeutic index and need multiple administrations with undesired side effects that lead to the intolerance. To reduce the non-specificity of chemotherapeutics, targeted therapy was introduced to restrict such agents in the tumor with minimum effects on other tissues. We developed bioinspired artificial exosomes (AE), which enabled to deliver chemotherapeutics to the tumors effectively after systemic administration. AE were produced by incorporating membrane proteins from cancer cells into phospholipid liposomes that mimicked the plasma membrane. The synthesized AE were used for the delivery of broad-spectrum chemotherapeutic doxorubicin (DOX) and vorinostat (SAHA), an epigenetic inhibitor. The combination of DOX and SAHA showed synergistic effects on suppressing non-small cell lung cancer cells and xenograft tumors without apparent adverse effects. AE facilitated the delivery of drugs to tumor tissue and extended the retention time of drugs within tumors. Taken together, these studies suggest that the bioengineered artificial exosomes may serve as novel delivery strategy for chemotherapeutics to treat non-small cell lung cancer.

Targeting c-Myc to Overcome Acquired Resistance of EGFR Mutant NSCLC Cells to the Third-Generation EGFR Tyrosine Kinase Inhibitor, Osimertinib.

Osimertinib (AZD9291 or TAGRISSO) is a promising and approved third-generation EGFR tyrosine kinase inhibitor (TKI) for treating patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR-activating mutations or the resistant T790M mutation. However, the inevitable emergence of acquired resistance limits its long-term efficacy. A fuller understanding of the mechanism of action of osimertinib and its linkage to acquired resistance will enable the development of more efficacious therapeutic strategies. Consequently, we have identified a novel connection between osimertinib or other EGFR-TKIs and c-Myc. Osimertinib rapidly and sustainably decreased c-Myc levels primarily via enhancing protein degradation in EGFR-mutant (EGFRm) NSCLC cell lines and xenograft tumors. c-Myc levels were substantially elevated in different EGFRm NSCLC cell lines with acquired resistance to osimertinib in comparison with their corresponding parental cell lines and could not be reduced any further by osimertinib. Consistently, c-Myc levels were elevated in the majority of EGFRm NSCLC tissues relapsed from EGFR-TKI treatment compared with their corresponding untreated baseline c-Myc levels. Suppression of c-Myc through knockdown or pharmacologic targeting with BET inhibitors restored the response of resistant cell lines to osimertinib. These findings indicate that c-Myc modulation mediates the therapeutic efficacy of osimertinib and the development of osimertinib acquired resistance. Furthermore, they establish c-Myc as a potential therapeutic target and warrant clinical testing of BET inhibition as a potential strategy to overcome acquired resistance to osimertinib or other EGFR inhibitors. SIGNIFICANCE: This study demonstrates a critical role of c-Myc modulation in mediating therapeutic efficacy of osimertinib including osimertinib acquired resistance and suggests targeting c-Myc as a potential strategy to overcome osimertinib acquired resistance.

Abstract LB234: The Neuropilin-2 targeting antibody ATYR2810 inhibits non-small cell lung cancer tumor growth in monotherapy and combination therapy

Abstract Lung cancer remains one of the leading cancer types and is the foremost cause of cancer-related deaths, yet treatment options for advanced lung cancer remain limited. Novel therapies targeting factors critical to the progression of lung cancer are essential. Neuropilin-2 (NRP2) acts as a co-receptor for VEGF and has been correlated with aggressive cancer growth and metastasis. The expression of NRP2 has been shown to be upregulated in several cancers, including non-small cell lung cancer (NSCLC), and the expression of NRP2 in NSCLC is associated with increased invasive tumor growth in vitro as well as a significant decrease in patient survival. As shown previously, aTyr has developed ATYR2810, a domain-specific anti-human monoclonal antibody to NRP2 which blocks its binding to VEGF. Importantly, ATYR2810 blocks VEGF binding in a highly specific manner and does not affect the binding of Semaphorin-3F or subsequent Semaphorin-3F-induced dimerization of NRP2 and PlexinA1. In this study, we have used ATYR2810 to examine the effects of blocking VEGF-mediated NRP2 signaling both as monotherapy and in combination therapy. Using in vitro 3D colony formation assays, we found that ATYR2810 used as monotherapy led to a significant reduction in colony formation in the A549 NSCLC cell line. The effects of ATYR2810 were also examined in NSCLC xenografts, where the use of ATYR2810 as a single agent was found to result in the inhibition of tumor growth. Interestingly, the tumor growth inhibition observed with ATYR2810 as monotherapy was comparable to that of cisplatin monotherapy, suggesting that ATYR2810 may be a potent inhibitor of tumor growth in the A549 xenograft model. The effects of ATYR2810 combination therapy in NSCLC xenografts was also examined, and we found that the use of ATYR2810 in combination with either 5-FU or cisplatin enhanced tumor growth inhibition as compared to the use of either chemotherapeutic agent alone. Taken together, these results suggest that NRP2 may be an important target in aggressive NSCLC and that the use of our novel ATYR2810 antibody to block VEGF-mediated NRP2 signaling may serve as a valuable therapeutic agent both as monotherapy as well as in combination therapy. Citation Format: Alison G. Barber, Zhiwen Xu, Justin Rahman, Hira Lal Goel, Arthur M. Mercurio, Christoph Burkart, Leslie A. Nangle. The Neuropilin-2 targeting antibody ATYR2810 inhibits non-small cell lung cancer tumor growth in monotherapy and combination therapy [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 LB234.

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.

Abstract 1263: Dual RAF/MEK inhibitor VS-6766 enhances antitumor efficacy of KRAS-G12C inhibitors through a vertical pathway inhibition strategy

Abstract KRAS is one of the most frequently mutated oncogenes in cancer, stimulating tumor growth through activation of the RAS/RAF/MEK/ERK pathway. G12C inhibitors (G12Ci), sotorasib (AMG 510) and adagrasib (MTRX849), have demonstrated promising antitumor activity in patients with KRAS G12C mutant (mt) non-small cell lung cancer (NSCLC). Several recent studies have shown that simultaneous targeting of multiple nodes in the RAS/RAF/MEK/ERK pathway may be optimal for durable pathway inhibition and response. VS-6766 is a unique dual RAF/MEK inhibitor. In contrast to other MEK inhibitors, VS-6766 is a potent allosteric inhibitor of MEK kinase activity which promotes a dominant negative RAF/MEK complex preventing phosphorylation of MEK by wildtype BRAF, BRAF V600E mt and CRAF. This unique mechanism allows VS-6766 to block MEK signaling without the compensatory activation of MEK that appears to limit the efficacy of other inhibitors. Clinical studies are ongoing evaluating VS-6766 alone or in combination with the focal adhesion kinase (FAK) inhibitor defactinib for the treatment of various solid tumors. Here, we tested the hypothesis that vertical pharmacological blockade of RAS, RAF and MEK with G12Ci in combination with VS-6766 might yield superior pathway blockade and antitumor efficacy. In 3D proliferation assays in vitro, VS-6766 was synergistic with both sotorasib and adagrasib in reducing viability of a panel of KRAS G12C mt NSCLC and colorectal cancer cell lines. Accordingly, VS-6766 effectively suppressed RAS pathway signaling (pMEK, pERK, p-p90RSK) across KRAS G12C mt NSCLC cell lines as a single agent, and the combination of VS-6766 + G12Ci showed improved depth and duration of RAS pathway signaling relative to G12Ci alone. Similarly, in the H2122 KRAS G12C mt NSCLC xenograft model, VS-6766 combination with sotorasib showed improved inhibition of pMEK and pERK in tumors relative to sotorasib alone. In KRAS G12C mt NSCLC xenograft models in vivo, combination with VS-6766 (0.3 mg/kg QD) augmented tumor growth inhibition by sotorasib (30 mg/kg QD) in the H2122 KRAS G12C mt NSCLC model, whereas trametinib (0.3 mg/kg QD) was much less effective in augmenting sotorasib efficacy. Sotorasib monotherapy induced >20% tumor regression in 0/10 mice, whereas combination of VS-6766, trametinib or a FAK inhibitor with sotorasib induced >20% tumor regression in 5/10, 1/10 and 4/10 mice, respectively, following 10 days of treatment. Strikingly, triple combination of VS-6766, sotorasib and a FAK inhibitor conferred tumor reductions of ≥35% in 10/10 mice. Similarly, in the H358 KRAS G12C mt NSCLC model, triple combination of VS-6766, sotorasib and a FAK inhibitor induced >35% tumor regression in 10/10 mice following 21 days of treatment. These results support the clinical evaluation of VS-6766 ± defactinib in combination with a G12C inhibitor for treatment of KRAS G12C mt solid tumors. Citation Format: Silvia Coma, Sanjib Chowdhury, Jonathan A. Pachter. Dual RAF/MEK inhibitor VS-6766 enhances antitumor efficacy of KRAS-G12C inhibitors through a vertical pathway inhibition strategy [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 1263.

Abstract 2423: Discovery of ZN-e4, an irreversible EGFR-TKI with potent anti-tumor activity in EGFR mutant non-small-cell lung cancer

Abstract Small molecule tyrosine kinase inhibitors against the epidermal growth factor (EGFR) have made significant impact in the treatment of advanced non-small-cell lung cancer (NSCLC) with activating EGFR mutations. Although the first and second-generation EGFR inhibitors have remarkably improved survival in advanced EGFR mutant NSCLC patients, most of the patients develop acquired resistance. The gatekeeper mutation (T790M) is the most common acquired resistance mechanism to the first and second-generation EGFR inhibitors. To overcome the T790M acquired resistance, third-generation EGFR inhibitors, such as osimertinib, were developed. However, additional mutant selective EGFR inhibitors with improved toxicity profile are still needed as osimertinib treatment produced incidence of adverse events, such as diarrhea and skin rush. Here, we describe the identification and characterization of ZN-e4, an orally bioavailable, selective, irreversible third generation EGFR inhibitor. ZN-e4 selectively inhibited the kinase activity of several mutated forms of EGFR, including L858R, T790M/L858R, and Exon19 del in biochemical assay. In cell-based assays, ZN-e4 demonstrated a 20-40-fold selectivity ratio for mutant EGFR forms over the wild-type form thus minimizing the potential for on target toxicity. ZN-e4 potently inhibited the proliferation of NSCLC cell lines harboring EGFR activating and T790M mutation and induced tumor regression in small and large tumors with activating EGFR mutations in preclinical human NSCLC xenograft models. Taken together, our data suggest that ZN-e4 is a potent and selective third generation EGFR inhibitor. Currently, ZN-e4 is in a Phase I clinical trial in patients with EGFR mutant NSCLC showing clinical activity and encouraging toxicity profile. Citation Format: Jiali Li, Sunny Abraham, Jianhui Ma, Peter Q. Huang, Kevin D. Bunker, Fernando Doñate, Ahmed A. Samatar. Discovery of ZN-e4, an irreversible EGFR-TKI with potent anti-tumor activity in EGFR mutant non-small-cell lung cancer [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 2423.

Additive Antiproliferative and Antiangiogenic Effects of Metformin and Pemetrexed in a Non-Small-Cell Lung Cancer Xenograft Model.

Lung cancer is heterogeneous and challenging to cope with once it has progressed. Chemotherapy is the first step once no active driver mutation has been discovered. Non-antitumor drugs have been found to be beneficial when used as adjuvants to chemotherapy. In this study, the additive effect and mechanism of metformin combined with pemetrexed in non-small-cell lung cancer (NSCLC) cells were elucidated. Three NSCLC cell lines, A549, H1975, and HCC827, were used to analyze tumor cell proliferation, colony formation and the cell cycle in vitro when exposed to metformin alone, pemetrexed alone or their combination. We found that combination treatment in three cell lines exerted antiproliferative effects through cell cycle arrest in the S phase. An ex vivo chicken chorioallantoic membrane (CAM) assay was used to examine the antiangiogenic effect of metformin combined with pemetrexed on vascular structure formation. We further created an A549 orthotopic xenograft model with an in vivo imaging system (IVIS) and explored the associated indicators involved in the tumorigenic process. The in vitro results showed that the combination of metformin and pemetrexed exhibited an antiproliferative effect in reducing cell viability and colony formation, the downregulation of cyclin D1 and A2 and the upregulation of CDKN1B, which are involved in the G1/S phase. For antiangiogenic effects, the combination therapy inhibited the vascular structure, as proven by the CAM assay. We elucidated that combination therapy could target VEGFA and Endoglin by RT-qPCR, ELISA and histopathological findings in an A549 orthotopic NSCLC xenograft model. Our research demonstrated the additive antiproliferative and antiangiogenic effects of the combination of metformin with pemetrexed in NSCLC and could be applied to clinical lung cancer therapy.

SIRT6 through PI3K/Akt/mTOR signaling pathway to enhance radiosensitivity of non-Small cell lung cancer and inhibit tumor progression.

To explore the effect and mechanism of SIRT6 on radiosensitivity and tumor progression of non-small cell lung cancer (NSCLC). qRT-PCR was performed to detect the expressions of SIRT6 in tumor tissues, adjacent normal tissues and NSCLC cell lines of patients with advanced NSCLC before and after radiotherapy. After overexpression or interference with SIRT6 expression in NSCLC cells, the cells were routinely cultured or transfected for 48 h followed by 4Gy radiation for 24 h. Then, check the cell proliferation, migration, apoptosis and cell cycle by MTT, wound healing assay and flow cytometry, while detect the expression of PI3K/Akt/mTOR signaling pathway-related proteins by Western blot. In addition, the effect of SIRT6 expression on NSCLC tumor growth was analyzed by xenograft tumor assay. SIRT6 showed a low expression in NSCLC tumor tissues and cell lines, while SIRT6 was significantly increased in NSCLC tissues after radiation treatment. Overexpression of SIRT6 in A549 and NCI-H23 cells inhibited cell proliferation viability, migration ability and promoted apoptosis. By comparison, after radiation treatment, NSCLC cells with high SIRT6 expression had lower ability of proliferation and migration and higher apoptosis rate. Overexpression of SIRT6 evidently down-regulated the activity of PI3K/Akt/mTOR signaling pathway in NSCLC cells before and after radiation. In addition, H2009 cells exhibited opposite cellular functions after interference with SIRT6 expression. In vivo experiments showed that overexpression of SIRT6 promoted the inhibitory effect of radiation on the growth of NSCLC xenograft tumors in nude mice. SIRT6 can promote the radiosensitivity of NSCLC and inhibit the development of tumor by down-regulating the activity of PI3K/Akt/mTOR signaling pathway.

The Cardenolide Glycoside Acovenoside A Interferes with Epidermal Growth Factor Receptor Trafficking in Non-Small Cell Lung Cancer Cells.

Cardenolide glycosides are natural compounds known to inhibit the ion pumping function of the Na+/K+-ATPase in cellular systems. Interestingly, various cancer cell types are highly susceptible to cardenolide glycosides. Herein, we explore the cardenolide glycoside Acovenoside A (AcoA) with respect to its influences on human A549 non-small cell lung cancer (NSCLC) cells. We found that exposure to AcoA, digoxin and ouabain increases intracellular sodium and ATP levels indicating that the ion pumping function of the transmembrane Na+/K+-ATPase is effectively inhibited. Like digoxin and ouabain, AcoA inhibits transcription factor NF-κB activation and induces apoptotic cell death in NSCLC cells. This was confirmed by a preclinical in vivo model in which AcoA treatment of NSCLC xenografts grown on chick chorioallantoic membranes inhibited the expression of proliferation antigen Ki-67 and induced apoptotic DNA strand breaks. We aimed to elucidate the underlying mechanisms. The Na+/K+-ATPase transmembrane complex contains Src kinase and epidermal growth factor receptor (EGFR). Indeed, we found that AcoA activates Src kinase in A549 cells, but not in a cell-free assay using recombinant Src kinase. Src kinase is a downstream target of EGFR, and correlation analysis using the NCI60 database pointed to a role of EGFR in cardenolide glycoside-induced cancer cell death. Accordingly, NSCLC cells expressing hyperphosphorylated EGFRmut exhibited resistance to AcoA. To investigate the interaction between cardenolide glycosides and EGFR in detail, we performed immunoblotting studies: Whereas ligand binding and EGFR phosphorylation were not significantly affected, ubiquitinated EGFR accumulated after prolonged incubation with AcoA. To visualize EGFR trafficking we used A549 cells transfected with a fluorescent biosensor which binds to activated EGFR. Pretreatment with AcoA and digoxin induced accumulation of EGFR in endosomal compartments thus inhibiting EGF-induced EGFR degradation comparable to the Na+ ionophore monensin, a known inducer of EGFR endosomal arrest. Intracellular Na+ concentrations regulate EGFR trafficking and signaling. Na+ homeostasis is maintained by the Na+/K+-ATPase, which might account for its close interaction with the EGFR. Cardenolide glycosides inhibit the ATP-dependent Na+/K+ exchange through the Na+/K+-ATPase resulting in higher intracellular Na+ levels. Our data provide first evidence that this impedes efficient EGFR trafficking at the endosomal compartment.

Migration and invasion of NSCLC suppressed by the downregulation of Src/focal adhesion kinase using single, double and tetra domain anti- CEACAM6 antibodies

Carcinoembryonic antigen-related cell adhesion molecules 6 (CEACAM6) is a cell adhesion receptor. Expression of CEACAM6 in non-small cell lung cancer (NSCLC) associated with tumor progression and metastatic condition via Src/FAK signaling pathway. We established three anti-CEACAM6 antibodies with valences, which were designed to be monomeric sdAb, bivalent sdAb (2Ab), and tetravalent sdAb (4Ab). The anti-CEACAM6 antibodies can be used to target CEACAM6 overexpressing NSCLC. Anti-CEACAM6 antibodies, sdAb, 2Ab and 4Ab, were modified with different valency via protein engineering. sdAb and multivalent sdAbs (2Ab & 4Ab) were expressed and purified from E.coli and CHO cells, respectively. We compared the effect of anti-CEACAM6 antibodies with doxorubicin in NSCLC cell line both in vitro and in vivo. The 4Ab showed significant effect on cell viability. In addition, A549 cells treated with 2Ab and 4Ab inhibited the invasion and migration. In western blot, the 2Ab and 4Ab showed significant inhibition of phospho FAK domain Ty397 that is essential for activation of Src kinase family. Meanwhile, overall protein analysis revealed that 2Ab and 4Ab potently inhibited the phosphorylation of pSRC, pERK, pFAK, pAKT, MMP-2, MMP-9 and N-cadherin. Anti-tumor effect was observed in an A549 NSCLC xenograft model treated with 2Ab or 4Ab compared with doxorubicin. Confocal analysis showed higher targeting ability of 4Ab than that of 2Ab at 4 h incubation. Our data suggests that 2Ab and 4Ab inhibits EMT-mediated migration and invasion via suppression of Src/FAK signaling, which exhibits therapeutic efficiency for NSCLC treatment.

Silencing SIX1 by miR-7160 inhibits non-small cell lung cancer cell growth.

The homeoprotein SIX1 is upregulated in non-small cell lung cancer (NSCLC) and associated with NSCLC tumorigenesis and progression. We identified microRNA-7160 (miR-7160) as a SIX1-targeting miRNA. RNA immunoprecipitation results confirmed a direct binding between miR-7160 and SIX1 mRNA in NSCLC cells. In the primary and established NSCLC cells, forced overexpression of miR-7160 downregulated SIX1 and inhibited cancer cell growth, proliferation, migration and invasion. Furthermore, miR-7160 overexpression induced apoptosis activation in NSCLC cells. Conversely, miR-7160 inhibition elevated SIX1 expression and enhanced NSCLC cell progression in vitro. Restoring SIX1 expression, by an untranslated region-depleted SIX1 expression construct, reversed miR-7160-induced anti-NSCLC cell activity. CRISPR/Cas9-inudced knockout of SIX1 mimicked miR-7160-induced actions and produced anti-NSCLC cell activity. In vivo, intratumoral injection of miR-7160-expressing lentivirus downregulated SIX1 mRNA and inhibited NSCLC xenograft growth in severe combined immunodeficient mice. Significantly, miR-7160 expression is downregulated in human NSCLC tissues and is correlated with SIX1 mRNA upregulation. Collectively, miR-7160 silenced SIX1 and inhibited NSCLC cell growth in vitro and in vivo.

Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC)

Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+; Trp53R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFRmut). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFRmut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFRmut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFRmut NSCLC.

P71.02 Estrogen Promotes Resistance to Bevacizumab Treatment in Non-Small Cell Lung Cancer (NSCLC) Xenograft Models

P71.02 Estrogen Promotes Resistance to Bevacizumab Treatment in Non-Small Cell Lung Cancer (NSCLC) Xenograft Models

CircRNA ZNF609 Knockdown Represses the Development of Non-Small Cell Lung Cancer via miR-623/FOXM1 Axis.

BackgroundThe dysregulated circular RNAs (circRNAs) are relevant to the development of non-small cell lung cancer (NSCLC). Nevertheless, the function and mechanism of circRNA zinc finger protein 609 (circZNF609) in NSCLC development remain uncertain.MethodsSixty-two NSCLC patients were recruited. circZNF609, microRNA-623 (miR-623) and forkhead box M1 (FOXM1) abundances were measured via quantitative reverse transcription polymerase chain reaction or Western blot. Cell viability, apoptosis, migration and invasion were analyzed via cell counting kit-8 (CCK8), flow cytometry, caspase3 activity, transwell assay and Western blot. The interaction between miR-623 and circZNF609 or FOXM1 was analyzed via dual-luciferase reporter analysis, RNA immunoprecipitation and pull-down. The function of circZNF609 on cell growth in vivo was tested via xenograft model.ResultscircZNF609 abundance was enhanced in NSCLC tissues and cells. High expression of circZNF609 indicated the lower overall survival. circZNF609 interference restrained cell viability, migration and invasion and increased apoptosis. miR-623 was targeted via circZNF609. FOXM1 was targeted via miR-623 and regulated via circZNF609. miR-623 knockdown or FOXM1 overexpression mitigated the role of circZNF609 silence in NSCLC development. circZNF609 knockdown decreased NSCLC xenograft tumor growth.ConclusioncircZNF609 knockdown repressed NSCLC development via regulating miR-623 and FOXM1.