Loss of NF2 drives malignant transformation of human pancreatic acinar cells and enhances cell fitness under nutrient deprivation and therapeutical stress [CRISPR_screen]
Pancreatic ductal adenocarcinoma (PDAC) occurs as a complex, multifaceted event driven by the interplay of tumor permissive genetic mutations, nature of cellular origin and microenvironmental stress. In this study, we present a novel model to screen previously underappreciated tumor suppressor genes in human pancreatic acinar-derived PDAC and probe their implications under nutrient deprived environmental context. Primary human pancreatic acinar 3D organoids were engineered to harbor triple PDAC driver mutations—KRAS G12V, TP53 inactivation, and CDKN2A deletion (designated as KPT organoids). Using a pooled CRISPR knockout library targeting 199 potential tumor suppressors curated from recurrent mutations in clinical PDAC samples, we performed in vivo and in vitro screening with KPT cells and revealed significant enrichment of a list of candidate tumor suppressors, with NF2 emerging as the top target. Functional validation confirmed that loss of NF2 promotes the transition of PDAC from a non-invasive to an invasive state, potentially through extracellular matrix (ECM) modulation. Additionally, we found that the fibroblast heterogeneity in these organoids-derived tumors correlates with the cancer progression, suggesting the important roles of cancer-stroma communications in tumor evolution. Strikingly, NF2 inactivation was found to enhance PDAC cell fitness under nutrient starvation, a condition reflective of the harsh tumor microenvironment. This adaptation not only reinforces the oncogenic state but also confers therapeutical resistance. These findings establish NF2 as a critical tumor suppressor in PDAC and uncover its role in mediating nutrient adaptation and drug resistance. Importantly, this study provides new insights into drug resistance mechanisms and potential therapeutic targets in PDAC. Overall design: In order to identify potential driver mutations in PDAC, we curated a list of 199 potential tumor suppressors associated with recurrent loss of function mutations in clinical PDAC samples. We then isolated primary acinar cells from healthy human pancreatic tissues which were maintained as 3D organoid culture. The organoids were engineered to carry three key PDAC driver mutations: oncogenic KRAS G12V, CRISPR-mediated knockout of TP53 and CDKN2A (designated as KPT organoids). A pooled CRISPR knockout library targeting the curated 199 genes was introduced into KPT organoids by lentivirus infection. The transduced cells were expanded for one passage, a fraction of cells was collected to serve as a reference control. For the in vivo screen, the expanded cells were subcutaneously transplanted into the hind flank of NSG (NOD/SCID/gamma) mice. The xenograft tumor tissues were collected after 8 weeks followed by DNA extraction. For the in vitro screen, the expanded cells were continuously passaged for 8 weeks (7-9 passages), followed by extraction of cellular DNA. The screen was performed using 4 independently established cultures, for each, one control sample, one organoid sample and two tumor samples were collected.