Malignant and fibroblast programs in aggressive PDAC molecular subtypes

Pancreatic Ductal Adenocarcinoma (PDAC) is the deadliest cancer worldwide. Late diagnosis and a complex biology make this a difficult-to-treat disease. PDAC displays extensive heterogeneity both in the malignant and non-malignant compartments. Of the non-malignant cells, cancer-associated fibroblasts (CAFs) are the most abundant cell type which critically influences tumour biology and response to treatments. The different PDAC cell states are shaped by the integration of cell intrinsic and cell extrinsic inputs. It is now well established that the transcriptional cell state (basal-like/squamous) displaying suppression of pancreatic endodermal gene programs has the most aggressive biological behaviour. Whether and how basal-like/squamous cells instruct the CAFs differently from the less aggressive classical phenotype remains to be elucidated. In this thesis work, we sought to disclose novel determinants of cell lineage specification as well as to identify subtype-specific stromal phenotypes. Receptor-Tyrosine Kinases (RTKs) are a large family of metazoan-specific plasma-membrane receptors that control several cellular processes including cell fate determination. Therefore, we focused on the potential role of RTKs in defining or sustaining aggressive molecular phenotypes of neoplastic cells. We found that the expression of FGFR4 was significantly elevated in the classical PDAC subtype and associated with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligned with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlated with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably led to the enrichment of basal-like/squamous gene programs and associated with either partial or full switch of phenotype. Whole-genome sequencing of advanced PDAC tissues and functional interrogation of organoid-based xenotransplantation have suggested RAS hyperactivation as a determinant of the basal-like/squamous subtype. Here, we used pathway mapping analysis and context-dependent pathway response signatures to infer MAPK activity and dependency in heterogeneous expression data from models and patients' samples. In situ expression analyses complemented this approach, which undisclosed an important role for the MAPK signalling pathway in the definition of PDAC CAFs phenotypes. We found that the epithelial activity of MAPK did not discriminate basal-like from classical tumours. Conversely, hyperactivation of MAPK signalling occurred in myCAFs populating basal-like/squamous tumour niches. Short-term inhibition of MAPK was invariably associated with a dramatic change of the myCAFs/iCAFs ratio in mouse PDAC tissue due to a unique dependency of myCAF on a proficient MAPK signalling. Gene expression signatures of MAPKhigh CAFs (sMEK) from mouse tumours suggested metabolic rewiring and immunoregulatory function. Finally, we found that the sMEK signature correlated with poor prognosis in several cancer conditions, including PDAC, and with reduced response to immunecheckpoint inhibition in bladder cancers