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Aim This study aimed to investigate the contamination levels of antibiotics and antibiotic resistance genes (ARGs) in the landfill leachates and their correlations with the bacteria. Methods and results Using HPLC-MS, quantitative PCR and high-throughput sequencing, we measured the pollution levels of 14 antibiotics and 10 ARGs in the leachates of the landfill in Taiyuan, China, and analysed changes in the bacterial community and the correlations of bacteria with antibiotics and ARGs. The main results showed high levels of antibiotics (like enrofloxacin, pefloxacin and oxytetracycline) and ARGs (like sulfonamides, tetracycline, macrolides, quinolones and β-lactam-resistance genes) in the landfill leachates, along with higher diversity and richness of the bacteria. Some types of antibiotics had positive correlations with their corresponding ARGs. The dominant bacteria in the landfill leachates were Pseudomonas, Defluviitoga and Sulfurimonas, which correlated with the antibiotics and ARGs and might have potential effects on degrading them. Conclusions Antibiotics and ARG pollution existed in the landfill leachates, while bacteria were closely associated with them. Significance and impact of the study It will provide helpful information for the potential application of the bacteria in antibiotics and ARGs pollution control and landfill leachate management.

Acquired resistance is inevitable in non-small cell lung cancers (NSCLCs) treated with osimertinib (OSI), and the mechanisms are not well defined. The MERTK ligand GAS6 promoted downstream oncogenic signaling in EGFR-mutated (EGFRMT) NSCLC cells treated with OSI, suggesting a role for MERTK activation in OSI resistance. Indeed, treatment with MRX-2843, a first-in-class MERTK kinase inhibitor, resensitized GAS6-treated NSCLC cells to OSI. Both GAS6 and EGF stimulated downstream PI3K/AKT and MAPK/ERK signaling in parental cells, but only GAS6 activated these pathways in OSI-resistant (OSIR) derivative cell lines. Functionally, OSIR cells were more sensitive to MRX-2843 than parental cells, suggesting acquired dependence on MERTK signaling. Furthermore, MERTK and/or its ligands were dramatically upregulated in EGFRMT tumors after treatment with OSI in both xenograft models and patient samples, consistent with induction of autocrine/paracrine MERTK activation. Moreover, treatment with MRX-2843 in combination with OSI, but not OSI alone, provided durable suppression of tumor growth in vivo, even after treatment was stopped. These data identify MERTK as a driver of bypass signaling in treatment-naive and EGFRMT-OSIR NSCLC cells and predict that MRX-2843 and OSI combination therapy will provide clinical benefit in patients with EGFRMT NSCLC.

Osimertinib is currently the preferred treatment for EGFR-mutated non-small cell lung cancer (NSCLC) patients due to its superior therapeutic efficacy and prolonged overall survival compared to earlier generation EGFR tyrosine kinase inhibitors, but durable responses to osimertinib treatment are rare due to acquired drug resistance. Thus, there is an urgent need for novel strategies to treat osimertinib-resistant NSCLC. Recently, we found that treatment with MRX-2843, a novel MERTK-selective kinase inhibitor currently in Phase I clinical trials, resulted in dose-dependent inhibition of cell expansion and colony formation in an osimertinib-resistant (osiR) H4006 derivative cell line. An unbiased screen of 378 kinase inhibitors was carried out to identify compounds that synergized with MRX-2843 to inhibit expansion of an osiR derivative of the EGFR-mutated H4011 cell line. Treatment with 1µM PIM kinase inhibitor SGI-1776 or 100nM MRX-2843 alone reduced cell density by 5±3% and 44±7%, respectively, while treatment with MRX-2843 and SGI-1776 combined mediated an 82±0.4% decrease. Synergy was also observed in H4006 osiR and H1650 osiR derivative cell lines. Furthermore, treatment with PIM447, a structurally distinct PIM kinase inhibitor, and MRX-2843 decreased cell expansion more effectively than either agent alone. Mechanistically, treatment with a PIM kinase inhibitor in combination with MRX-2843 decreased downstream PI3K-AKT and MAPK-ERK signaling more effectively than single agents. Additionally, combined treatment with MRX-2843 and SGI-1776 prevented colony formation, while single agents had limited effect. In all, these data indicate that combining MRX-2843 and a PIM TKI may control osimertinib resistant tumor growth, providing a potential treatment strategy for osimertinib resistant EGFR-mutated NSCLC patients for whom the choices are still limited. Citation Format: Dan Yan, Zikang Tan, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, III, Deborah DeRyckere, Douglas K. Graham. A novel strategy to cope with osimertinib resistance in non-small cell lung cancer by treatment with a PIM kinase inhibitor in combination with a MERTK-selective kinase inhibitor [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 1109.

Osimertinib (OSI) was recently FDA-approved as a front-line agent for newly diagnosed EGFRMT non-small cell lung cancer (NSCLC). However, unmet clinical needs have arisen in conjunction with OSI use, including understanding mechanisms of OSI resistance and developing novel approaches to prevent or reverse resistance and/or enhance OSI efficacy in responsive patients. To address these issues, osimertinib-resistant (osiR) derivatives of five EGFRMT NSCLC cell lines were generated and roles for MERTK, a receptor tyrosine kinase that has been implicated as a potential therapeutic target in NSCLC, were characterized. PI3K-AKT and MAPK-ERK signaling pathways were activated in osiR cells, even when EGFR was not active. Treatment with the MERTK ligands GAS6 or PROS1 stimulated AKT, ERK, and ribosomal S6 phosphorylation in parental cells treated with OSI and in osiR cells, implicating MERTK as a mediator of resistance to OSI. Downstream signaling was responsive to both EGF and GAS6 stimulation in parental cells but was only activated by GAS6 in osiR cells. OSI blocked EGF-dependent signaling through AKT, ERK and S6 in parental cells in the absence of GAS6, but combined treatment with OSI and MRX-2843, a novel MERTK inhibitor currently in Phase I clinical trials, was required to block signaling in the presence of GAS6. However, treatment with MRX-2843 alone had little impact on downstream signaling in the presence of activated EGFR. Thus, MERTK is not the dominant driver of downstream signaling in parental cells. In contrast, treatment with MRX-2843 alone was sufficient to inhibit downstream signaling in osiR cells and osiR cells were also more sensitive to treatment with MRX-2843 in clonogenic assays. Thus, osiR cells have increased dependence on MERTK kinase activity relative to parental cells. Interestingly, EGFR and MERTK co-precipitated from parental cell lysates and GAS6 stimulation enhanced this interaction. In contrast, MERTK and EGFR interaction was not detected in osiR cells, suggesting a more complex interplay between these two receptors. MERTK and the ligand PROS1 were dramatically upregulated in EGFRMT tumors treated with OSI in vivo, consistent with a role for autocrine MERTK activation in osiR tumor growth. Indeed, treatment with OSI alone or in combination with MRX-2843 was sufficient to block tumor growth in vivo, but when treatment was stopped, tumors treated with OSI alone started to grow, while treatment with the combination resulted in durable suppression of tumor growth. Together these data implicate MERTK as a mediator of resistance to OSI and suggest that combining MRX-2843 and OSI therapy will control tumor growth. Citation Format: Dan Yan, Justus Huelse, Rebecca Parker, Zikang Tan, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. MERTK drives residual tumor growth in EGFR-mutated non-small cell lung cancer cells treated with osimertinib [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 1882.