PC-9 Xenograft Models Research Articles

Discovery of a Novel Benzimidazole Derivative Targeting Histone Deacetylase to Induce Ferroptosis and Trigger Immunogenic Cell Death.

Ferroptosis is a unique type of cell death, characterized by its reliance on iron dependency and lipid peroxidation (LPO). Consequently, small-molecule ferroptosis modulators have garnered substantial interest as a promising avenue for cancer therapy. Herein, we explored the ferroptosis sensitivity of epigenetic modulators and found that the antiproliferative effects of class I histone deacetylase (HDAC) inhibitors are significantly reliant on ferroptosis. Subsequently, we developed a novel series of HDAC inhibitors, identifying HL-5s with robust inhibitory activity against class I HDACs, particularly HDAC1. Notably, HL-5s induces ferroptosis by augmenting LPO production. Mechanistically, HL-5s increased the YB-1 acetylation and inhibited the Nrf2/HO-1 signaling pathway. Furthermore, HL-5s not only significantly suppresses tumor growth in the PC-9 xenograft model but also remodels the tumor microenvironment in the LLC allograft model. Our study has unveiled that class I HDAC inhibitors can exert antitumor effects by triggering ferroptosis, and HL-5s may serve as a promising candidate for future cancer treatment.

Abstract 656: DNA repair-interfering molecule AsiDNA® overcomes resistance to tyrosine kinase inhibitors in lung cancer

Abstract Purpose: Drug-tolerance has emerged as one of the major non-genetic mechanism that drives resistance to targeted therapies in lung cancers. Identification of drug-tolerant cells (DTC) vulnerabilities thus represents an attractive strategy to prevent relapse in patients. We observed that EGFR-TKI-derived DTC displayed an increased level of DNA Double Strand Breaks (DSB), raising the hypothesis that genomic instability in these cells could favor the onset of resistance mechanisms. We tested AsiDNA, a small molecule that interferes with the DNA repair machinery, to prevent the emergence of resistances Experimental design: AsiDNA is a double stranded DNA molecule (decoy oligonucleotide) that mimics DNA DSB to interfere with DNA repair, by over-activating a false signaling of DNA damage through DNA-PK and PARP enzymes. We used EGFR-mutated (PC9 and HCC827) or ALK-translocated (H3122) lung tumor cells as well as a PC9 xenograft model, to assess whether a long-term combination of AsiDNA and TKI (erlotinib or osimertinib for EGFR-mutated and alectinib for ALK-translocated cells) could prevent the emergence of resistances arising from DTC. We also performed RNAseq to identify molecular mechanisms underlying AsiDNA effect on TKI-derived DTC. Results:Concomitant treatment of TKI with AsiDNA strongly prevented the emergence of TKI-resistant clones in the three cell lines tested, whereas AsiDNA alone did not display cytotoxic activity. Interestingly, AsiDNA didn’t increase the apoptotic activity of TKI but rather prevented the re-entry of drug-tolerant “dormant” cells into a proliferative state, ultimately resulting in DTC death. RNAseq data revealed a strong downregulation of gene signatures associated with cell cycle, DNA repair but also several pathways known to promote DTC survival such as Epithelial-to-Mesenchymal Transition (EMT) or Interferon pathway in AsiDNA+osimertinib vs osimertinib alone. In vivo, AsiDNA strongly interfered with the emergence of resistances in a PC9 xenograft model treated with erlotinib, resulting in a delayed relapse for the majority of tumors and a complete tumor regression for 20% of them, with no evidence of toxicity for mice. Conclusion: Inhibition of the DNA repair machinery using AsiDNA efficiently prevented the emergence of resistances to tyrosine kinase inhibitors and point to the therapeutic opportunity of combining AsiDNA and TKI to overcome resistance in clinical situation. These findings should have immediate implications to design drug combination strategies to prevent TKI-induced resistance in lung cancers. Citation Format: Sarah Figarol, Sandra Pagano, Wael Jdey, Hugo Guillaume, Gilles Favre, Olivier Calvayrac. DNA repair-interfering molecule AsiDNA® overcomes resistance to tyrosine kinase inhibitors in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 656.

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

Abstract Purpose: Drug-tolerant “dormant” cells (DTC) have emerged as one of the major non-genetic mechanisms driving resistance to targeted therapy in lung cancer, although the sequence of events leading to entry and exit from dormancy remain poorly described. Here, we provide a step-by-step phenotypic and molecular characterization of the different processes involved during the adaptive response to osimertinib using several EGFR-mutated lung cancer models. This strategy led to the identification of a common vulnerability of drug-tolerant cells which could be efficiently and safely targeted by a clinical stage drug. Experimental design: We used the FUCCI (fluorescence ubiquitination cell cycle indicator) system to determine the cell cycle dynamics in real time during the adaptive response to osimertinib in a panel of EGFR-mutated lung cancer cell lines. We performed scRNAseq on untreated and osimertinib-treated G1 and S/G2 sorted cells during early relapse to determine the molecular mechanisms underlying entry and exit from dormancy. We validated our observations in several in vitro and in vivo models as well as in publicly available patient data. Results: FUCCI labelling allowed the identification of a rare population of S/G2 cycling cells (referred to as early escapers) that emerged in the first hours of treatment amongst a majority of stably arrested and progressively dying G1 cells. scRNAseq data revealed that early escapers emerged from a differentiated alveolar type 1 (AT1) phenotype which was invariably associated with an increase in contractile-related gene signatures, F-actin polymerization and Rho/ROCK pathway activation. Using a screen of Rho-pathway inhibitors, we found that tipifarnib, a farnesyltransferase inhibitor (FTi), induced a complete clearance of DTC in vitro. Co-treatment with tipifarnib, a clinically active FTi, safely and durably prevented relapse to osimertinib in a PC9-xenograft model as well as in a PDX model of EGFRL858R/T790M lung cancer for up to 6 months with no evidence of toxicity. Several farnesylated targets were identified in both G1 and S/G2 treated cells, which could explain the high efficiency of tipifarnib in preventing the adaptive response to osimertinib. Finally, we observed that osimertinib + tipifarnib co-treatment completely suppressed the emergence of the AT1 phenotype, prevented mitosis of S/G2-treated cells and increased the apoptotic response through activation of the unfolded protein response (UPR) pathway. Conclusion: Our data strongly support the use of tipifarnib in combination with osimertinib in the clinic to effectively, durably and safely prevent relapse. Citation Format: Sarah Figarol, Célia Delahaye, Rémi Gence, Raghda Asslan, Sandra Pagano, Jacques Colinge, Jean-Philippe Villemin, Antonio Maraver, Isabelle Lajoie-Mazenc, Estelle Clermont, Anne Casanova, Anne Pradines, Julien Mazières, Olivier Calvayrac, Gilles Favre. Tipifarnib prevents emergence of resistance to osimertinib in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB080.

Fucoxanthin extracted from Laminaria Japonica inhibits metastasis and enhances the sensitivity of lung cancer to Gefitinib

Ethnopharmacological relevanceLaminaria japonica, a brown seaweed, has been used in Traditional Chinese Medicine (TCM) to treat a variety of diseases including lung cancer. Aim of the studyTo demonstrate the effects of Fucoxanthin (FX), a major active component extracted from Laminaria japonica on metastasis and Gefitinib (Gef) sensitivity in human lung cancer cells both in vitro and in vivo. Materials and methodsInvasion and migration of lung cancer cells were detected using the wound healing assay and transwell assay. Epithelial-to-mesenchymal transition (EMT) factors and PI3K/AKT/NF-κB pathways were analyzed by western blotting. RNA interference (RNAi) technology was used to silence TIMP-2 gene expression in A549 cells. The anti-metastatic effect of FX was evaluated in vivo in an experimental lung metastatic tumor model. On the other hand, cell counting kit-8 assay was used to study the cell viability of human lung cancer PC9 cells and Gef resistant PC9 cells (PC9/G) after Gef, FX or FX combined with Gef treatment. PC9 xenograft model was established to explore the anti-tumor effect of FX or combined with Gef. Immunohistochemistry staining assay and immunofluorescence staining assay were used to reveal the effects of FX on lung cancer cell proliferation and apoptosis. ResultsFX was able to significantly inhibit lung cancer cells migration and invasion in vitro. FX suppressed the expressions of Snail, Twist, Fibronectin, N-cadherin, MMP-2, PI3K, p-AKT and NF-κB, and increased the expression of TIMP-2. Furthermore, knockdown of TIMP-2 attenuated FX-mediated invasion inhibition. Additionally, we demonstrated that FX inhibited lung cancer cells metastasis in vivo. The anti-metastatic effects of FX on lung cancer cells might be attributed to inhibition of EMT and PI3K/AKT/NF-κB pathway. We further demonstrated that the anti-tumor activity of FX was not only limited to the drug sensitive cell lines, but also prominent on lung cancer cells with Gef resistant phenotype. Furthermore, in vivo xenograft assay confirmed that FX inhibited tumor growth and enhanced the sensitivity of lung cancer cells to Gef and this effect may be due to inhibition of tumor cell proliferation and activation of apoptosis. ConclusionCollectively, our findings suggested that FX suppresses metastasis of lung cancer cells and overcomes EGFR TKIs resistance. Thus, FX is worthy of further investigation as a drug candidate for the treatment of lung cancer.

IL-22 Confers EGFR-TKI Resistance in NSCLC via the AKT and ERK Signaling Pathways

Background: The efficacy of an EGFR-targeted treatment strategy for non-small cell lung cancer (NSCLC) is reduced by drug resistance. IL-22 enhances tumor growth and induces chemotherapy resistance in human lung cancer cells. The present study elucidated the IL-22-induced mechanism underlying EGFR-tyrosine kinase inhibitor (TKI) resistance in NSCLC.Methods: The plasma and tissues of patients who received EGFR-TKIs were utilized to determine the association between IL-22 expression and gefitinib efficacy. The IL-22 effect on the EGFR/ERK/AKT pathways in NSCLC HCC827 and PC-9 cells was determined using the CCK-8 assay, western blot, and flow cytometric analysis. A PC-9 xenograft model of IL-22 exposure was established. Gefitinib was administered to mice in combination with IL-22 or vehicle.Results: We showed that IL-22 expression was higher in the EGFR-TKI-resistant group compared to EGFR-TKI-sensitive group. IL-22 expression was associated with EGFR-TKI efficacy in plasma. Additional treatment of IL-22 induced gefitinib resistance and reduced apoptosis in PC-9 and HCC827 cell lines. Furthermore, we showed that the effects of IL-22 attributed to p-ERK, p-EGFR, and p-AKT up-regulation. IL-22 neutralizing antibody completely abrogated the effects of IL-22 on apoptosis and AKT/EGFR/ERK signaling. Finally, we showed that IL-22 enhanced tumor growth and induced gefitinib resistance in the PC-9 xenograft model. Moreover, compared with gefitinib alone, the combination of IL-22 and gefitinib led to an increase in Ki67-positive staining and a reduction in TUNEL staining.Conclusions: Our findings indicate that IL-22 plays a role in tumor progression and EGFR-TKI resistance in NSCLC. Thus, IL-22 might serve as a novel biomarker to overcome resistance of EGFR-TKI.

EP1.03-27 The Anti-Migration and Anti-Invation Mechanisms of Capilliposide C from Lysimachia Capillipes on Lung Cancer

Approximately 50% of lung cancer patients had distant metastases when initial diagnosed. Lysimachia capillipes is one of traditional medicine in China. It is proved to be safety in clinical use. Several studies have showed the antitumor effects of Capilliposide from Lysimachia capillipes (LC) in vitro. Our preliminary data showed LC could inhibit the migration and invasion of lung cancer. This study aims to explore the detailed mechanisms of LC as well as its extracts on lung cancer. Four non-small cell lines were selected. The invasive response of lung cancer cells was determined by Wound Healing assay. Proteins and the phosphorylation of proteins were evaluated by iTRAQ-based proteomics. The phosphorylation chip was used to evaluate the phosphorylation effect. PC-9 xenografts were used to evaluate the antitumor of LC-C in vivo. Immunohistochemistry (IHC) was used to evaluate the antitumor mechanisms in vivo. The migration capa city of lung cancer cells was significantly reduced after treatment of LC-C. Proteomics showed there were 364 differentially expressed proteins and 456 differentially expressed phosphorylated proteins in both LC-C treated PC-9 and H1975 cells. Differentially expressed proteins were enriched in EGF receptor signaling pathway, Wnt signaling pathway, Cadherin signaling pathway, Notch signaling pathway, TGF-beta signaling pathway, and p38 MAPK pathway by bioinformatic analysis. Phosphorylation chip showed LC reduced the phosphorylation of AKT, WNK1 and PRAS40, but not EGFR. Western blot showed the phosphorylation of mTOR, AKT and PRAS40 were significantly inhibited after LC-C treatment; besides, the inhibitory effects of AKT and mTOR were dose-dependent. While the total proteins of AKT and mTOR were not changed. Western blot showed the phosphorylation of Smad2 and Smad3 were significantly inhibited after LC-C treatment. Western blot showed E-Cadherin was up-regulated and N-Cadherin was down-regulated after LC-C treatment; while other EMT related proteins including ZO-1, Snail and Vimentin were not changed. Nor did cell adhesion related protein Claudin-1. PC-9 xenograft model showed the tumor growth inhibitory rates were 114.4% in 7 days after LC-C administration. IHC showed the phosphorylation of AKT was down-regulation after LC-C administration, Ki-67 was also down-regulation, while cleaved caspase-3 was not changed. The study showed LC-C inhibit the growth and the capacity of invasion and migration of lung cancer cells. The detailed mechanisms might crosstalk with several critical pathway such as AKT pathway, TGF-β pathway and EMT.

P2.13-29 IL-22 Confers Resistance To EGFR-TKIs In Non-Small Cell Lung Cancer Bearing EGFR Gene Mutation and Amplification

The effect of epidermal growth factor receptor(EGFR)-targeted strategy is hindered by drug resistance. IL-22 can promote tumor growth and induce resistance to chemotherapy in human lung cancer cells. The aim of our study was to investigate the mechanism of IL-22-induced resistance to EGFR-tyrosine kinase inhibitors(EGFR-TKIs)in the NSCLC. The tissues and plasma of patients taking EGFR-TKIs were used to examine the correlation between the drug’s efficacy and IL-22 level. We identified IL-22-induced EGFR-TKIs resistance and the effect of IL-22 on EGFR/AKT/ERK pathways in NSCLC PC-9 and HCC827 cells by CCK-8 assay, flow cytometric analysis, and western blotting. Then, we established the PC-9 xenograft model with IL-22 exposure and used gefitinib combined with vehicle or IL-22 to treat mice. We confirmed that IL-22 can inhibit the effect of gefitinib on NSCLC cells and determined the effects of IL-22 on EGFR/AKT/ERK pathways. Then, we showed that IL-22 exposures could promote tumor growth and induce resistance to gefitinib in the PC-9 xenograft model. These results suggest that IL-22 contributes to tumor progression and EGFR-TKIs resistance in NSCLC. Therefore, IL-22 is a potential therapeutic target for EGFR-TKIs resistance.

Synergistic Inhibition of Thalidomide and Icotinib on Human Non-Small Cell Lung Carcinomas Through ERK and AKT Signaling.

BackgroundEpidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been widely used in the treatment of non-small cell lung cancer (NSCLC) patients with sensitive EGFR mutations. However, the survival of patients with EGFR-TKI administration is limited by the inevitable development of acquired drug resistance. Recently, multi-targeted drugs combination has been shown to be a promising strategy to improve the efficacy of EGFR-TKI treatment and enable the reduction of drug resistance in NSCLC.Material/MethodsHumanized NSCLC cell lines PC9 and A549 were co-cultured with thalidomide and/or icotinib to test for anti-tumor efficiency. Cell proliferation was measured by MTT assay, cell apoptosis by flow cytometry and cell migration by wound healing assay. Western blot was performed to determine the expression of caspase-3, -8, -9, Bax, EGFR, VEGF-R, AKT, ERK, MMP2, MMP9, and NF-κB. The xenograft mouse model was used to explore the effects of thalidomide and icotinib in vivo. Immunohistochemical testing was used to determine the expression of Ki-67 and TUNEL staining in tumor tissues.ResultsTreatments of thalidomide and/or icotinib reduced cell viability, induced apoptosis, and suppressed migration. Attenuation of pEGFR and pVEGF-R resulted in deactivation of ERK and AKT pathways, which eventually increased the anti-proliferative response. In PC9 xenograft model, combined administration of thalidomide and icotinib restrained tumor growth with remarkable reduced Ki-67 index and increased TUNEL positive cells.ConclusionsThalidomide sensitizes icotinib to increase apoptosis and prevent migration, and it may be a potentially promising anti-tumor drug in lung cancer multi-modality therapy.

Abstract 4119: The development of EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism

Abstract EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism. Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 15% of lung adenocarcinomas. This leads to constitutive activation of EGFR, which triggers multiple downstream survival and proliferation pathways. Currently, EGFR tyrosine kinase inhibitors (TKIs) are first line therapy for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. Despite initial significant response to TKIs, most tumors develop resistance. The main mechanism of resistance detected in 50-60% of cases is a cytosine to thymine (C>T) single nucleotide transition mutation at position 2369. This causes a threonine to methionine amino acid change at position 790 (i.e. T790M). Our data suggests that the C>T mutation is an acquired event secondary to cytosine deamination by Activation Induced Cytosine Deamination enzyme (AICDA). Results: Single cell clones of lung adenocarcinoma cell line, PC9, were treated with EGFR TKI. At baseline, these clones have EGFR exon 19 deletion but no evidence of T790M mutation by digital droplet PCR (ddPCR). However, after treatment with a serial increasing dose of EGFR TKI, T790M mutation was detected by ddPCR. Assessing whether cytosine deamination enzymes were altered by this treatment, a significant increase in AICDA expression was seen. Furthermore, recombinant AICDA protein could deaminate cytosine at position 2369 in vitro. In addition, using mass spectrometry and methylation specific primers, we determined that cytosine at position 2369 is in fact methylated. This further supports our hypothesis since 5-methyl cytosine is deaminated into thymine directly. Since in germinal center B-lymphocytes, AICDA is activated through a non-canonical NFkB mechanism, we assessed NFkB pathway in PC9 cell line. RelB and p52 expression were significantly increased after TKI treatment. In addition direct interaction between RelB and AICDA promoter was confirmed by ChIP Assay. These findings were also seen in a mouse PC9 xenograft model. Daily oral gavage of EGFR TKI caused significant increase in the expression of RelB as well as AICDA. Adding NFkB inhibitor twice weekly inhibited the expression of RelB and AICDA. Finally, knocking down AICDA by shRNA prevented the development of T790M mutation in PC9 cell lines after TKI exposure. Conclusion: Our data suggest that the T790M mutation could be actively acquired after TKI treatment through a cytosine deamination process by AICDA. This would have significant implications for treatment with targeted therapy. In fact, Imatinib resistance in CML and GIST tumors have a similar C>T single nucleotide transition mutation. Citation Format: Khaled Hassan, Najwa El Kadi, April Davis, Gregory Kalemkerian, Luo Wang, Hasan Korkaya. The development of EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism [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 4119. doi:10.1158/1538-7445.AM2017-4119

Combination of Gefitinib and Pemetrexed Prevents the Acquisition of TKI Resistance in NSCLC Cell Lines Carrying EGFR-Activating Mutation

IntroductionDevelopment of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors is a clinical issue in patients with epidermal growth factor receptor gene (EGFR)-mutated non–small cell lung cancer (NSCLC). The aim of this study was to investigate the potential of combining gefitinib and pemetrexed in preventing the acquisition of resistance to EGFR tyrosine kinase inhibitors in NSCLC cell lines harboring EGFR exon 19 deletion. MethodsThe effect of different combinatorial schedules of gefitinib and pemetrexed on cell proliferation, cell cycle, apoptosis, and acquisition of gefitinib resistance in PC9 and HCC827 NSCLC cell lines and in PC9 xenograft models was investigated. ResultsSimultaneous treatment with gefitinib and pemetrexed enhanced cell growth inhibition and cell death and prevented the appearance of gefitinib resistance mediated by T790M mutation or epithelial-to-mesenchymal transition (EMT) in PC9 and HCC827 cells, respectively. In PC9 cells and in PC9 xenografts the combination of gefitinib and pemetrexed, with different schedules, prevented gefitinib resistance only when pemetrexed was the first treatment, given alone or together with gefitinib. Conversely, when gefitinib alone was administered first and pemetrexed sequentially alternated, a negative interaction was observed and no prevention of gefitinib resistance was documented. The mechanisms of resistance that developed in vivo included T790M mutation and EMT. The induction of EMT was a feature of tumors treated with gefitinib when given before pemetrexed, whereas T790M was recorded only in tumors treated with gefitinib alone. ConclusionsThe combination of gefitinib and pemetrexed is effective in preventing gefitinib resistance; the application of intermittent treatments requires that gefitinib not be administered before pemetrexed.

Nicotine induces resistance to erlotinib via cross-talk between α 1 nAChR and EGFR in the non-small cell lung cancer xenograft model.

Given our previously published study, α 1 nicotinic acetylcholine receptor (nAChR) plays an essential role in nicotine-induced cell signaling and nicotine-induced resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) in non-small cell lung cancer (NSCLC) PC9 cells. The aim of this study was to investigate the potential mechanism between nAChR and EGFR for nicotine-induced resistance to EGFR-TKI erlotinib in the NSCLC xenograft model. We identified the role of nicotine to EGFR/AKT/ERK pathways and to erlotinib-resistance in NSCLC PC9 and HCC827 cells by MTS assay and western blot. Then, we established the PC9 xenograft model with nicotine exposure and treated mice with erlotinib combined with vehicle or nicotine. We confirmed the effects of nicotine on EGFR/AKT/ERK pathways and determined nicotine's potential in preventing from the effect of erlotinib on NSCLC cells. Then, we showed that nicotine exposures can promote tumor growth and induce resistance to erlotinib in the PC9 xenograft model. Our results also indicated that chronic oral administration of nicotine can cause more significant erlotinib-resistance compared with acute i.v. injection of nicotine through activating α 1 nAChR and EGFR pathways. These results suggest that nicotine contributes to the progression and erlotinib-resistance of the NSCLC xenograft model via the cooperation between nAChR and EGFR.

Antagonism of adenosine A2A receptor expressed by lung adenocarcinoma tumor cells and cancer associated fibroblasts inhibits their growth

Recently it has become clear that the cost associated with the Warburg effect, which is inefficient production of ATP, is offset by selective advantages that are produced by resultant intracellular metabolic alterations. In fact tumors may be addicted to the Warburg effect. In addition these alterations result in changes in the extracellular tumor microenvironment that can also produce selective advantages for tumor cell growth and survival. One such extracellular alteration is increased adenosine concentrations that have been shown to impair T cell mediated rejection and support angiogenesis. The expression of the A2A receptor in non-small cell cancer (NSCLC) tissues, cell lines and cancer associated fibroblasts (CAF) was determined by performing immunohistrochemistry and immunoblot analysis. The efficacy of the A2A receptor antagonists in vivo was evaluated in a PC9 xenograft model. To determine the mode of cell death induced by A2A receptor antagonists flow cytometry, immunoblot, and cytotoxic analysis were performed. We found that a significant number of lung adenocarcinomas express adenosine A2A receptors. Antagonism of these receptors impaired CAF and tumor cell growth in vitro and inhibited human tumor xenograft growth in mice. These observations add to the rationale for testing adenosine A2A receptor antagonists as anticancer therapeutics. Not only could there be prevention of negative signaling in T cells within the tumor microenvironment and inhibition of angiogenesis, but also an inhibitory effect on tumor-promoting, immunosuppressive CAFs and a direct inhibitory effect on the tumor cells themselves.

SKLB1206, a Novel Orally Available Multikinase Inhibitor Targeting EGFR Activating and T790M Mutants, ErbB2, ErbB4, and VEGFR2, Displays Potent Antitumor Activity Both In Vitro and In Vivo

Anti-epidermal growth factor receptor (EGFR) treatment has been successfully applied in clinical cancer therapy. However, the clinical efficacy of first-generation reversible EGFR inhibitors, such as gefitinib and erlotinib, is limited by the development of drug-resistant mutations, including the gatekeeper T790M mutation and upregulation of alternative signaling pathways. Second-generation irreversible EGFR inhibitors that were designed to overcome the drug resistance due to the T790M mutation have thus far had limited success. Here, we report a novel reversible EGFR inhibitor, SKLB1206, which has potent activity against EGFR with gefitinib-sensitive and -resistant (T790M) mutations. In addition, SKLB1206 has also considerable inhibition potency against some other related oncokinases, including ErbB2, ErbB4, and VEGF receptor 2 (VEGFR2). SKLB1206 exhibited highly antiproliferative activity against a range of EGFR-mutant cell lines, including gefitinib-sensitive and -resistant cell lines, and EGFR or ErbB2-overexpressing cell lines. SKLB1206 also showed a potent antiangiogenesis effect in vitro, in a zebrafish embryonic angiogenesis assay, and in an alginate-encapsulate tumor cell assay. In vivo, oral administration of SKLB1206 showed complete tumor regression in gefitinib-sensitive HCC827 and PC-9 xenograft models and showed a considerable antitumor effect on the gefitinib-resistant H1975 model as well as other EGFR/ErbB2-overexpressing or -dependent tumor models including A431, LoVo, and N87 established in athymic mice. SKLB1206 also showed a very good oral bioavailability (50.1%). Collectively, these preclinical evaluations may support clinical development of SKLB1206 for cancers with EGFR-activating/resistance mutations or EGFR/ErbB2 overexpressed.