Abstract

The tumor suppressor gene NF1 encodes neurofibromin, a negative regulator of the RAS/MAPK signaling pathway. Somatic alterations of NF1 have been identified across multiple sporadic cancer types beyond the hereditary context of neurofibromatosis type 1 (MIM#162200). In lung adenocarcinoma, a common malignancy with poor prognosis, the molecular landscape plays a critical role in guiding therapeutic decisions.

My research team has confirmed that somatic NF1 mutations define a specific subtype of bronchial adenocarcinomas. To date, no targeted therapies are specifically approved for patients with this cancer subtype. Therefore, improving patient outcomes requires a deeper understanding of the cellular and signaling mechanisms associated with NF1 loss of function, with the goal of identifying personalized therapeutic strategies.

This doctoral thesis focused on three main objectives, all centered on somatic NF1 loss-of-function mutations in lung adenocarcinoma: (i) evaluating their functional impact and the activation of key signaling pathways, (ii) investigating the immune response associated with these mutations, and (iii) identifying novel synthetic lethal targets. To achieve this, we employed isogenic wild-type or NF1-mutated bronchial epithelial models (HBE4-E6/E7-C1).

We confirmed RAS/MAPK pathway activation in NF1-null cells, with bi-allelic alterations acting as oncogenic drivers that induced a tumor-like phenotype. Analysis of public drug sensitivity datasets revealed increased vulnerability of NF1-mutant lung adenocarcinoma cell lines to RAS/MAPK and PI3K-AKT-mTOR pathway inhibitors. In vitro pharmacological assays demonstrated enhanced sensitivity of NF1 bi-allelic mutant cells to Trametinib (a MEK inhibitor), with a synergistic cytotoxic effect observed in combination with Buparlisib (a PI3K inhibitor), both in vitro and in vivo.

To explore the link between NF1 somatic mutations and potential responsiveness to immune checkpoint blockade, we analyzed publicly available datasets from 4,181 lung adenocarcinoma samples. NF1-mutant tumors exhibited significantly higher tumor mutational burden, overexpression of immune markers including PDL-1, increased CD8+ T-cell and macrophage infiltration, and were enriched among immunologically “hot” tumors, suggesting enhanced sensitivity to immunotherapy.

Finally, we applied a synthetic lethality approach to uncover novel therapeutic vulnerabilities. Two CRISPR-Cas9 negative selection screens were conducted using our isogenic models, allowing us to investigate the impact of NF1 loss in a genetically controlled context. These screens identified 27 candidate synthetic lethal genes, of which five were validated using competitive proliferation assays. Our results underscore the critical dependency of NF1-mutant lung adenocarcinoma cells on the ubiquitin-proteasome system. In particular, we identified two E3 ubiquitin ligases, HUWE1 and FBXW11, as robust synthetic lethal partners. Ongoing pharmacological testing with the HUWE1 inhibitor BI-8622 is underway.

In conclusion, this work provides a comprehensive characterization of NF1 somatic alterations in lung adenocarcinomas, elucidates their functional consequences, and proposes innovative therapeutic avenues, including targeted combinatorial approaches and novel synthetic lethality-based strategies.

Keywords : Non small cell lung cancer; Lung adenocarcinoma; NF1 gene; Neurofibromin; Somatic mutations; RAS/MAPK signaling pathway; Targeted therapy; Immunotherapy; CRISPR-Cas9 genome-wide screen; Synthetic Lethality