Identification of novel therapeutic targets in lung squamous cell carcinoma by CRISPR/Cas9

Resumen

NRF2 is the master regulator of the antioxidant response, whose activity is regulated by KEAP1. Several reports have related this transcription factor to the development of cancer. In fact, the overactivation of this signaling pathway has been found in several tumor types including lung adenocarcinoma (LUAD). However, less is known about its role in lung squamous cell carcinoma (LUSC) and the targeted therapies available. In this sense, some studies indicated that this pathway was mainly altered in LUSC tumours (~30-40%), suggesting an important role for its development. All these findings together prompted the study of NRF2/KEAP1 in LUSC in more detail, including the consequences of its interference as well as the study of targetable downstream genes related with this signaling pathway. In this work it was found that NRF2/KEAP1 pathway was overactivated in LUSC samples harboring NFE2L2/KEAP1 alterations. This fact makes the active NRF2 LUSC behaves as resistant to cell death by oxidative stress (OS) challenge in comparison to WT NRF2 tumors. With the hypothesis in mind of such tumors been addicted to NRF2, the consequences of NFE2L2 silencing were studied in vitro, by using stable Tet-On shRNA NFE2L2 clones of LUSC cell lines. Cell viability was drastically reduced upon induction of OS in the active NRF2 cell line after NFE2L2 silencing. In the case of in vivo experiments, the silencing of NFE2L2 in a xenograft model with the active NRF2 LUSC cell line, reduced tumor growth, suggesting a potential role of NRF2 as a tumor driver in LUSC. Confirmation of NRF2-dependence for LUSC samples with active NRF2 aimed the study of potential downstream genes of this pathway. These were studied by using the CRISPR/Cas9 activation (a) technology. It was used the CRISPRa/dCas9 SAM system, whose efficiency was tested by confirming the increase in gene expression using a sgRNA for CXCR4. The use of the sgRNA library in the active NRF2 LUSC cells subjected to NRF2 depletion and OS, permitted a partial survival recovery. This suggests that some sgRNAs from the library induced the increase expression of survival genes. Further NGS analyses and validation indicated three possible genes as downstream survival gene candidates. After several validations, one of them stands out as a potential novel target gene in LUSC harboring NFE2L2/KEAP1 alterations, opening a new field of study for future targeted therapies