No abstract

EGFR-mutated lung cancer accounts for a significant proportion of lung cancer cases worldwide. For these cases, osimertinib, a thirdgeneration EGFR tyrosine kinase inhibitor, is extensively used as a first-line or second-line treatment. However, lung cancer cells acquire resistance to osimertinib in 1 to 2 years. Thus, a thorough clarification of resistance mechanisms to osimertinib is highly anticipated. Recent next-generation sequencing (NGS) of lung cancer samples identified several genetically defined resistance mechanisms to osimertinib, such as EGFR C797S or MET amplification. However, nongenetically defined mechanisms are not well evaluated. For a thorough clarification of osimertinib resistance, both genetic and nongenetic mechanisms are essential. By using our comprehensive protein phosphorylation array, we detected IGF1R bypass pathway activation after EGFR abolishment. Both of our established lung cancer cells and patient-derived lung cancer cells demonstrated IGF2 autocrine-mediated IGF1R pathway activation as a mechanism of osimertinib resistance. Notably, this resistance mechanism was not detected by a previously performed NGS, highlighting the essential roles of living cancer cells for a thorough clarification of resistance mechanisms. Interestingly, the immunohistochemical analysis confirmed the increased IGF2 expression in lung cancer patients who were treated with osimertinib and met the established clinical definition of acquired resistance. The findings highlight the crucial roles of cell-autonomous ligand expression in osimertinib resistance. Here, we report for the first time the IGF2 autocrine-mediated IGF1R activation as a nongenetic mechanism of osimertinib resistance in lung cancer at a clinically relevant level. Implications: Using comprehensive protein phosphorylation array and patient-derived lung cancer cells, we found that IGF2 autocrinemediated IGF1R pathway activation is a clinically relevant and common mechanism of acquired resistance to osimertinib.

Although the main cellular target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be alveolar cells, the absence of their tractable culture system has precluded the development of a clinically-relevant SARS-CoV-2 infection model. Here, we established an efficient human alveolosphere culture method and sphere-based drug testing platform for SARS-CoV-2. Alveolospheres exhibited indolent growth in a Wnt and R-spondin dependent manner. Gene expression, immunofluorescence and electron microscopy analyses revealed the presence of alveolar cells in alveolospheres. Alveolospheres expressed ACE2 and allowed SARS-CoV-2 to propagate nearly 100,000- fold in three days of infection. While lopinavir and nelfinavir, protease inhibitors used for the treatment of HIV infection, had a modest anti-viral effect on SARS-CoV-2, remdesivir, a nucleotide prodrug, showed anti-viral effect at the concentration comparable to the circulating drug level. These results demonstrated the validity of alveolosphere culture system for the development of therapeutic agents to combat SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.

No abstract