No abstract

The atypical bacterial pathogen Mycoplasma pneumoniae is a leading etiological agent of community-acquired pneumonia in humans; infections are often recalcitrant, recurrent and resistant to antibiotic treatment. These characteristics suggest a mechanism that facilitates long-term colonization in hosts. In an in vitro setting, M. pneumoniae forms biofilms that are unusual in that motility plays no more than a very limited role in their formation and development. Given the unusual nature of M. pneumoniae biofilms, open questions remain concerning phenotypes associated with persistence, such as what properties might favour the bacteria while minimizing host damage. M. pneumoniae also produces several cytotoxic molecules including community-acquired respiratory distress syndrome (CARDS) toxin, H2S and H2O2, but how it deploys these agents during growth is unknown. Whereas several biochemical techniques for biofilm disruption were ineffective, sonication was required for disruption of M. pneumoniae biofilms to generate individual cells for comparative studies, suggesting unusual physical properties likely related to the atypical cell envelope. Nonetheless, like for other bacteria, biofilms were less susceptible to antibiotic inhibition and complement killing than dispersed cells, with resistance increasing as the biofilms matured. CARDS toxin levels and enzymatic activities associated with H2S and H2O2 production were highest during early biofilm formation and decreased over time, suggesting attenuation of virulence in connection with chronic infection. Collectively, these findings result in a model of how M. pneumoniae biofilms contribute to both the establishment and propagation of M. pneumoniae infections, and how both biofilm towers and individual cells participate in persistence and chronic disease.

Mycoplasma genitalium is an important etiologic agent of non-gonococcal urethritis (NGU), known for chronicity and multidrug resistance, in which biofilms may play an integral role. In some bacterial species capable of forming biofilms, extracellular polymeric substances (EPS) composed of poly-N-acetylglucosamine (PNAG) are a crucial component of the matrix. Monosaccharide analysis of M. genitalium strains revealed high abundance of GlcNAc, suggesting a biofilm-specific EPS. Chromatograms also showed high concentrations of galactose and glucose as observed in other mycoplasma species. Fluorescence microscopy of M. genitalium biofilms utilizing fluorcoupled lectins revealed differential staining of biofilm structures. Scanning electron microscopy (SEM) showed increasing maturation over time of bacterial "towers" seen in biofilm development. As seen with Mycoplasma pneumoniae, organisms within fully mature M. genitalium biofilms exhibited loss of cell polarization. Bacteria associated with disrupted biofilms exhibited decreased dose-dependent viability after treatment with antibiotics compared to bacteria with intact biofilms. In addition, growth index analysis demonstrated decreases in metabolism in cultures with disrupted biofilms with antibiotic treatment. Taken together, these data suggest that M. genitalium biofilms are a contributing factor in antibiotic resistance.

MET exon 14 skipping alteration (METΔ14Ex) is an actionable oncogenic driver that occurs in 2% to 4% of non–small cell lung cancer (NSCLC) cases. The precise role of METΔ14Ex in tumor progression of NSCLC is poorly understood. Using multiple isogenic METΔ14Ex cell models established with CRISPR editing, we demonstrate that METΔ14Ex expression increases receptor kinase activity and downstream signaling by impairing receptor internalization and endocytic degradation, significantly boosting cell scatter, migration, and invasion capacity in vitro as well as metastasis in vivo. RNA sequencing analysis revealed that METΔ14Ex preferentially activates biological processes associated with cell movement, providing novel insights into its unique molecular mechanism of action. Activation of PI3K/Akt/Rac1 signaling and upregulation of multiple matrix metallopeptidases (MMP) by METΔ14Ex induced cytoskeleton remodeling and extracellular matrix disassembly, which are critical functional pathways that facilitate cell invasion and metastasis. Therapeutically, MET inhibitors dramatically repressed METΔ14Ex-mediated tumor growth and metastasis in vivo, indicating potential therapeutic options for METΔ14Ex-altered NSCLC patients. These mechanistic insights into METΔ14Ex-mediated invasion and metastasis provide a deeper understanding of the role of METΔ14Ex in NSCLC. Significance: These findings reveal the mechanistic function of METΔ14Ex alteration in driving metastasis and define novel metastasis-related pathways that could be targeted for more effective treatment of lung cancer with METΔ14Ex alterations.

Introduction. Infections with the respiratory pathogen Mycoplasma pneumoniae are often chronic, recurrent and resistant, persisting after antibiotic treatment. M. pneumoniae grown on glass forms protective biofilms, consistent with a role for biofilms in persistence. These biofilms consist of towers of bacteria interspersed with individual adherent cells. Hypothesis/Gap Statement. A tissue culture model for M. pneumoniae biofilms has not been described or evaluated to address whether growth, development and resistance properties are consistent with persistence in the host. Moreover, it is unclear whether the M. pneumoniae cells in the biofilm towers and individual bacterial cells have distinct roles in disease. Aim. We evaluated the properties of biofilms of M. pneumoniae grown on the immortalized human bronchial epithelial cell line BEAS-2B in relation to persistence in the host. We observed nucleation of biofilm towers and the disposition of individual cells in culture, leading to a model of how tower and individual cells contribute to infection and disease. Methodology. With submerged BEAS-2B cells as a substrate, we evaluated growth and development of M. pneumoniae biofilms using scanning electron microscopy and confocal laser scanning microscopy. We characterized resistance to erythromycin and complement using minimum inhibitory concentration assays and quantification of colony forming units. We monitored biofilm tower formation using time-lapse microscopic analysis of host-cell-free M. pneumoniae cultures. Results. Bacteria grown on host cells underwent similar development to those grown without host cells, including tower formation, rounding and incidence of individual cells outside towers. Erythromycin and complement significantly reduced growth of M. pneumoniae . Towers formed exclusively from pre-existing aggregates of bacteria. We discuss a model of the M. pneumoniae biofilm life cycle in which protective towers derive from pre-existing aggregates, and generate individual cytotoxic cells. Conclusion . M. pneumoniae can form protective biofilms in a tissue culture model, implicating biofilms in chronic infections, with aggregates of M. pneumoniae cells being important for establishing infections.

Supplementary Table from Functional Analysis of <i>MET</i> Exon 14 Skipping Alteration in Cancer Invasion and Metastatic Dissemination

<div>Abstract<p><i>MET</i> exon 14 skipping alteration (<i>METΔ14Ex</i>) is an actionable oncogenic driver that occurs in 2% to 4% of non–small cell lung cancer (NSCLC) cases. The precise role of <i>METΔ14Ex</i> in tumor progression of NSCLC is poorly understood. Using multiple isogenic <i>METΔ14Ex</i> cell models established with CRISPR editing, we demonstrate that METΔ14Ex expression increases receptor kinase activity and downstream signaling by impairing receptor internalization and endocytic degradation, significantly boosting cell scatter, migration, and invasion capacity <i>in vitro</i> as well as metastasis <i>in vivo</i>. RNA sequencing analysis revealed that <i>METΔ14Ex</i> preferentially activates biological processes associated with cell movement, providing novel insights into its unique molecular mechanism of action. Activation of PI3K/Akt/Rac1 signaling and upregulation of multiple matrix metallopeptidases (MMP) by METΔ14Ex induced cytoskeleton remodeling and extracellular matrix disassembly, which are critical functional pathways that facilitate cell invasion and metastasis. Therapeutically, MET inhibitors dramatically repressed <i>METΔ14Ex</i>-mediated tumor growth and metastasis <i>in vivo</i>, indicating potential therapeutic options for <i>METΔ14Ex</i>-altered NSCLC patients. These mechanistic insights into <i>METΔ14Ex</i>-mediated invasion and metastasis provide a deeper understanding of the role of <i>METΔ14Ex</i> in NSCLC.</p>Significance:<p>These findings reveal the mechanistic function of <i>METΔ14Ex</i> alteration in driving metastasis and define novel metastasis-related pathways that could be targeted for more effective treatment of lung cancer with <i>METΔ14Ex</i> alterations.</p></div>

3597 Background: Approximately 20% to 50% of patients with advanced lung cancer develop brain metastases, which are associated with debilitating neurologic impairment and a dismal prognosis. There have been very limited studies investigating the genomics of brain metastases in lung cancer. Methods: We comprehensively investigated the frequency of PI3K/AKT/RICTOR/mTOR pathway aberrations in primary and metastatic sites using an extensive database of 11845 cases of lung adenocarcinoma by NGS (FoundationOne). The potential roles of RICTOR amplification in the development of brain metastases were studied both in vitro and in vivo in orthotopic mouse models. Results: Compared to the primary tumor, PI3K/AKT/mTOR gene alterations were more frequent in metastatic sites, with particular enrichment noted in brain metastases. RICTOR amplification alone accounted for the observed higher frequency both in brain metastases (brain vs. primary: 9.73% vs 3.50%, P = 2.6E-14; brain vs. other mets: 9.73% vs. 7.3%, P = 0.03) and other metastatic sites (other mets vs. primary: 7.3% vs.3.5%, P = 10E-15), whereas the frequency of PTEN, AKT1, PK3CA or mTOR genetic alterations was not different in the primary tumor, brain and other metastatic sites. In vitro, inducible RICTOR knockdown in H23 lung cancer cells (parental line with RICTOR amplification) was associated with reduced cell migration and invasion, whereas upregulation of RICTOR in HCC827 lung cancer cells (parental line with normal RICTOR copy numbers) was associated with an increase of both processes. These results were confirmed with pharmacological inhibition using mTOR1/2 inhibitors with known CNS penetration. In vivo, both inducible ablation of RICTOR and the mTOR1/2 inhibitor TAK228 (Sapanisertinib) significantly inhibited lung cancer H23-R4-Luc tumor growth in the brain, including a number of near complete responses. Mechanistic studies suggest that RICTOR may regulate the brain metastasis process through AKT and CXCL12 chemokine-CXCR4 axis. Conclusions: RICTOR amplification is the first identified actionable target that is markedly enriched in brain metastases. Our study provides a strong rationale for the development of RICTOR-targeted therapeutic strategies for the treatment and/or prevention of these major causes of lung cancer morbidity and mortality.