Análisis genómico funcional de la resistencia a las terapias anti-egfr asociada al fenotipo mesenquimal en el cáncer pulmonar

Resumen

Lung cancer is the leading cause of cancer death in western countries. It is highly resistant to the conventional therapy, metastatic and with a 5-years survival rate of 15% for the most predominant histological subtype, non-small cell lung cancer (NSCLC). The improvements of genetic diagnosis, along with the latest advances in the design of chemicals inhibiting molecules targets (usually protein kinases), have provided a significant clinical benefit for a small subsets of patients whose tumors have activating mutations in genes encoding target proteins (called oncogenic drivers) critical for tumor progression such as ALK, MET, and EGFR in NSCLC. Specific inhibitors targeting tyrosine kinase domain (TKIs) of EGFR, such as first and second generation TKIs erlotinib, gefitinib and afatinib, have shown an unprecedented clinical efficacy for the treatment of EGFR-mutant NSCLC. However, the clinical benefit is usually limited to a fraction of patients, and those showing an initial response inevitably suffer a relapse by the emergence of resistance through multiple molecular mechanisms. Some tumors acquire new mutations in EGFR (T790M, 60% of the cases), but osimertinib, a third-generation TKI, overcomes the resistance, although eventually these patients will also relapse. Other patients show evidences of epithelial-mesenchymal transition (EMT) activation, a transcriptional embryonic program characterized by the fibroblast-like morphology, the repression of epithelial cadherin (CDH1) and the expression of mesenchymal markers (such as vimentin, N-cadherin) and transcription factors ZEB1, SNAIL or TWIST). EMT is also involved in the acquisition of aggressive and metastatic phenotype of carcinomas and the genesis of cancer stem cells. While literature suggests a strong link between drug resistance and EMT in various cancer types, no effective therapies are available for this malignant phenotype which lack druggable targets. EMT mediates dynamic and reversible transitions between multiple phenotypic states as a consequence of the deep reprogramming of the transcriptome by complex regulatory mechanisms, which cause large changes not only in the proteome but also in the metabolome. However, in the context of EGFR-mutant NSCLC with acquired resistance to TKI, there are not comprehensive and global studies regarding such reprogramming in EMT-driven TKI-resistant NSCLC cell models. The overall objective of this work is to achieve a deeper understanding of molecular mechanisms controlling EMT process. We postulate that identifying an EMT-selective druggable vulnerability target would solve resistance to TKIs in NSCLC containing mutant-EGFR, an approach that could be extended to other genetic backgrounds such as EMT-positive KRAS tumors. In order to generate NSCLC cell models resistant to EGFR TKIs, HCC827, HCC4006 and H1975 cells were exposed to increasing concentrations of erlotinib, gefitinib and osimertinb respectively over six months. Western blot using antibodies against canonical epithelial and mesenchymal markers revealed that these TKI-resistant cells underwent EMT. Using a multi-omic approach (transcriptomics, epigenomics and proteomics) on these models, we identified NNMT as one of the genes that is more constitutively and robustly hypomethylated and overexpressed. Functional studies using RNAi tools and lentiviral gene transduction also showed that NNMT overexpression leads and maintains the mesenchymal TKI-resistant phenotype and its repression reverses cells to an epithelial-like state more sensitive to TKI. Thus, pharmacological NNMT inhibition appears to be a feasible strategy for reversing EGFR TKI resistance with EMT in NSCLC. Furthermore, targeted metabolomics also showed that NNMT overexpressing cells shunt nicotinamide to 1-methylnicotinamide production instead of NAD+ synthesis. Then, inhibition of NAD+ salvage pathway leads to a selective cell death of EMT-driven TKI resistant EGFR-mutant cells as well as EMT-positive KRAS-mutant, a new cancer vulnerability of cells with EMT phenotype that could be used as a new therapeutic target using NAD+ depleting agents such as daporinad, a NAMPT inhibitor in phase II clinical trials for the treatment of B-cell chronic lymphocytic leukemia, cutaneous T-cell lymphoma and melanoma. Additionally, the quantitative measurement of 1-methylnicotinamide, the by-product of NNMT activity in liquid biopsies is correlated with poor prognosis of TKI-treated patients, and emerges as a potential biomarker of drug response and metastatic progression in NSCLC patients.