The natural product berberine synergizes with osimertinib preferentially against MET-amplified osimertinib-resistant lung cancer via direct MET inhibition.

Berberine is a natural product that has long been used in traditional Chinese medicine due to its antimicrobial, anti-inflammatory and metabolism-regulatory properties. Osimertinib is the first third-generation EGFR-tyrosine kinase inhibitor (TKI) approved for the treatment of non-small cell lung cancer (NSCLC) with activating EGFR mutations and those resistant to earlier generation EGFR-TKIs due to a T790M mutation. However, emergence of acquired resistance to osimertinib limits its long-term efficacy in the clinic. One known mechanism of acquired resistance to osimertinib and other EGFR-TKIs is MET (c-MET) gene amplification. Here, we report that berberine, when combined with osimertinib, synergistically and selectively decreased the survival of several MET-amplified osimertinib-resistant EGFR mutant NSCLC cell lines with enhanced induction of apoptosis likely through Bim elevation and Mcl-1 reduction. Importantly, this combination effectively enhanced suppressive effect on the growth of MET-amplified osimertinib-resistant xenografts in nude mice and was well tolerated. Molecular modeling showed that berberine was able to bind to the kinase domain of non-phosphorylated MET, occupy the front of the binding pocket, and interact with the activation loop, in a similar way as other known MET inhibitors do. MET kinase assay showed clear concentration-dependent inhibitory effects of berberine against MET activity, confirming its kinase inhibitory activity. These findings collectively suggest that berberine can act as a naturally-existing MET inhibitor to synergize with osimertinib in overcoming osimertinib acquired resistance caused by MET amplification.

Chen Z ，Vallega KA ，Chen H ，Zhou J ，Ramalingam SS ，Sun SY ... -  《-》

Trametinib overcomes KRAS-G12V-induced osimertinib resistance in a leptomeningeal carcinomatosis model of EGFR-mutant lung cancer.

Leptomeningeal carcinomatosis (LMC) occurs frequently in non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations and is associated with acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, the mechanism by which LMC acquires resistance to osimertinib, a third-generation EGFR-TKI, is unclear. In this study, we elucidated the resistance mechanism and searched for a novel therapeutic strategy. We induced osimertinib resistance in a mouse model of LMC using an EGFR-mutant NSCLC cell line (PC9) via continuous oral osimertinib treatment and administration of established resistant cells and examined the resistance mechanism using next-generation sequencing. We detected the Kirsten rat sarcoma (KRAS)-G12V mutation in resistant cells, which retained the EGFR exon 19 deletion. Experiments involving KRAS knockdown in resistant cells and KRAS-G12V overexpression in parental cells revealed the involvement of KRAS-G12V in osimertinib resistance. Cotreatment with trametinib (a MEK inhibitor) and osimertinib resensitized the cells to osimertinib. Furthermore, in the mouse model of LMC with resistant cells, combined osimertinib and trametinib treatment successfully controlled LMC progression. These findings suggest a potential novel therapy against KRAS-G12V-harboring osimertinib-resistant LMC in EGFR-mutant NSCLC.

Fukuda K ，Otani S ，Takeuchi S ，Arai S ，Nanjo S ，Tanimoto A ，Nishiyama A ，Naoki K ，Yano S ... -  《-》

ERK inhibition effectively overcomes acquired resistance of epidermal growth factor receptor-mutant non-small cell lung cancer cells to osimertinib.

Osimertinib (AZD9291), a third-generation, mutation-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR-TKI), is an approved drug for patients who have non-small cell lung cancer (NSCLC) with activating EGFR mutations or those harboring a resistant T790M mutation. Unfortunately, all patients eventually relapse and develop resistance to osimertinib. The current study addressed whether ERK inhibition exerts effects similar to those produced by MEK inhibition in overcoming acquired resistance to osimertinib. Drug effects on cell and tumor growth were assessed by measuring cell number alterations and colony formation in vitro and with xenografts in nude mice in vivo. Apoptosis was assessed with annexin V/flow cytometry and protein cleavage. Protein alterations in cells were detected with Western blot analysis. Gene overexpression and knockout were achieved with lentiviral infection and CRISPR/Cas9, respectively. The combination of osimertinib with an ERK inhibitor synergistically decreased the survival of osimertinib-resistant cell lines with enhanced induction of apoptosis and effectively inhibited the growth of osimertinib-resistant xenografts in nude mice. Moreover, the combination of an MEK or ERK inhibitor with a first-generation (eg, erlotinib) or second-generation (eg, afatinib) EGFR-TKI also very effectively inhibited the growth of osimertinib-resistant cells in vitro and of tumors in vivo, although these cell lines were cross-resistant to first-generation or second-generation EGFR-TKIs. The current findings emphasize the importance of targeting MEK/ERK signaling in maintaining the long-term benefit of osimertinib through overcoming acquired resistance to osimertinib, warranting further investigation of this therapeutic strategy to improve the therapeutic efficacy of osimertinib in the clinic.

Li Y ，Zang H ，Qian G ，Owonikoko TK ，Ramalingam SR ，Sun SY ... -  《-》

Dual MET and ERBB inhibition overcomes intratumor plasticity in osimertinib-resistant-advanced non-small-cell lung cancer (NSCLC).

Third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as osimertinib are the last line of targeted treatment of metastatic non-small-cell lung cancer (NSCLC) EGFR-mutant harboring T790M. Different mechanisms of acquired resistance to third-generation EGFR-TKIs have been proposed. It is therefore crucial to identify new and effective strategies to overcome successive acquired mechanisms of resistance. For Amplicon-seq analysis, samples from the index patient (primary and metastasis lesions at different timepoints) as well as the patient-derived orthotopic xenograft tumors corresponding to the different treatment arms were used. All samples were formalin-fixed paraffin-embedded, selected and evaluated by a pathologist. For droplet digital PCR, 20 patients diagnosed with NSCLC at baseline or progression to different lines of TKI therapies were selected. Formalin-fixed paraffin-embedded blocks corresponding to either primary tumor or metastasis specimens were used for analysis. For single-cell analysis, orthotopically grown metastases were dissected from the brain of an athymic nu/nu mouse and cryopreserved at -80°C. In a brain metastasis lesion from a NSCLC patient presenting an EGFR T790M mutation, we detected MET gene amplification after prolonged treatment with osimertinib. Importantly, the combination of capmatinib (c-MET inhibitor) and afatinib (ErbB-1/2/4 inhibitor) completely suppressed tumor growth in mice orthotopically injected with cells derived from this brain metastasis. In those mice treated with capmatinib or afatinib as monotherapy, we observed the emergence of KRAS G12C clones. Single-cell gene expression analyses also revealed intratumor heterogeneity, indicating the presence of a KRAS-driven subclone. We also detected low-frequent KRAS G12C alleles in patients treated with various EGFR-TKIs. Acquired resistance to subsequent EGFR-TKI treatment lines in EGFR-mutant lung cancer patients may induce genetic plasticity. We assess the biological insights of tumor heterogeneity in an osimertinib-resistant tumor with acquired MET-amplification and propose new treatment strategies in this situation.

Martinez-Marti A ，Felip E ，Matito J ，Mereu E ，Navarro A ，Cedrés S ，Pardo N ，Martinez de Castro A ，Remon J ，Miquel JM ，Guillaumet-Adkins A ，Nadal E ，Rodriguez-Esteban G ，Arqués O ，Fasani R ，Nuciforo P ，Heyn H ，Villanueva A ，Palmer HG ，Vivancos A ... -  《-》

Overcoming acquired resistance of epidermal growth factor receptor-mutant non-small cell lung cancer cells to osimertinib by combining osimertinib with the histone deacetylase inhibitor panobinostat (LBH589).

The major clinical obstacle that limits the long-term benefits of treatment with osimertinib (AZD9291) in patients with epidermal growth factor receptor-mutant non-small cell lung cancer is the development of acquired resistance. Therefore, effective strategies that can overcome acquired resistance to osimertinib are urgently needed. The authors' current efforts in this direction have identified LBH589 (panobinostat), a clinically used histone deacetylase inhibitor, as a potential agent in overcoming osimertinib resistance. Cell growth and apoptosis in vitro were evaluated by measuring cell numbers and colony formation and by detecting annexin V-positive cells and protein cleavage, respectively. Drug effects on tumor growth in vivo were assessed with xenografts in nude mice. Alterations of tested proteins in cells were monitored with Western blot analysis. Gene knockout was achieved using the CRISPR/Cas9 technique. The combination of LBH589 and osimertinib synergistically decreased the survival of different osimertinib-resistant cell lines, including those harboring C797S mutations, with greater inhibition of cell colony formation and growth. The combination enhanced the induction of apoptosis in osimertinib-resistant cells. Importantly, the combination effectively inhibited the growth of osimertinib-resistant xenograft tumors in nude mice. Mechanistically, the combination of LBH589 and osimertinib enhanced the elevation of Bim in osimertinib-resistant cells. Knockout of Bim in osimertinib-resistant cells substantially attenuated or abolished apoptosis enhanced by the LBH589 and osimertinib combination. These results collectively support a critical role of Bim elevation in the induction of apoptosis of osimertinib-resistant cells for this combination. The current findings provide strong preclinical evidence in support of the potential for LBH589 to overcome osimertinib resistance in the clinic.

Zang H ，Qian G ，Zong D ，Fan S ，Owonikoko TK ，Ramalingam SS ，Sun SY ... -  《-》