Pooled CRISPR screening in pancreatic cancer cells implicates co-repressor complexes as a cause of multiple drug resistance via regulation of epithelial-to-mesenchymal transition

Abstract

Background Pancreatic ductal adenocarcinoma (PDAC) patients suffer poor outcomes, including a five-year survival of below 10%. Poor outcomes result in part from therapeutic resistance that limits the impact of cytotoxic first-line therapy. Novel therapeutic approaches are needed, but currently no targeted therapies exist to treat PDAC. Methods To assess cellular resistance mechanisms common to four cytotoxic chemotherapies (gemcitabine, 5-fluorouracil, irinotecan, and oxaliplatin) used to treat PDAC patients, we performed four genome-wide CRISPR activation (CRISPRact) and CRISPR knock-out (CRISPRko) screens in two common PDAC cell lines (Panc-1 and BxPC3). We used pathway analysis to identify gene sets enriched among our hits and conducted RNA-sequencing and chromatin immunoprecipitation-sequencing (ChIP-seq) to characterize top hits from our screen. We used scratch assays to assess changes in cellular migration with HDAC1 overexpression. Results Our data revealed activation of ABCG2, a well-described efflux pump, as the most consistent mediator of resistance in each of our screens. CRISPR-mediated activation of genes involved in transcriptional co-repressor complexes also conferred resistance to multiple drugs. Expression of many of these genes, including HDAC1, is associated with reduced survival in PDAC patients. Up-regulation of HDAC1 in vitro increased promoter occupancy and expression of several genes involved in the epithelial-to-mesenchymal transition (EMT). These cells also displayed phenotypic changes in cellular migration consistent with activation of the EMT pathway. The expression changes resulting from HDAC1 activation were also observed with activation of several other co-repressor complex members. Finally, we developed a publicly available analysis tool, PancDS, which integrates gene expression profiles with our screen results to predict drug sensitivity in resected PDAC tumors and cell lines. Conclusion Our results provide a comprehensive resource for identifying cellular mechanisms of drug resistance in PDAC, mechanistically implicate HDAC1, and co-repressor complex members broadly, in multi-drug resistance, and provide an analytical tool for predicting treatment response in PDAC tumors and cell lines.

Genomic analysis reveals HDAC1 regulates clinically relevant transcriptional programs in pancreatic cancer

“…Increased expression of CD44 , a cell surface protein important for cell adhesion and migration, is associated with a more mesenchymal-like phenotype which is characteristic of EMT 20 . Here we have shown that the mesenchymal marker, CD44 transcript and protein are more abundant in cells with HDAC1 overexpression, agreeing with our past work showing that HDAC1 overexpression leads to increased migration 9 .…”

“…HDAC1 expression has been previously linked to increased cellular resistance to cytotoxic chemotherapeutic drugs 9 . To further evaluate the effects of HDAC1 overexpression on chemotherapeutic resistance, we used CRISPRa 17 to generate a stable MIA PaCa-2 cell line (MP2_HDAC1_OE) expressing HDAC1 at ~3 times the levels of the levels of the control line (MP2_NTC) which expresses a non-targeting control guide ( Supplementary Data Fig.…”

“…We previously demonstrated that overexpression of HDAC1 contributes to multidrug resistance in pancreatic cancer cells 9 . While HDACs are canonically members of repressive complexes, binding of HDAC1 has also been associated with transcriptional activation.…”

Forward Genetic Screens as Tools to Investigate Role and Mechanisms of EMT in Cancer

“…Among these genes, HDAC1 overexpression caused an enrichment of certain genes implicated in EMT. Intriguingly, the expression of canonical EMT transcriptional regulators SNAI1, SNAI2 or TWIST1 was not altered, pointing to the involvement of a partial EMT in this phenomenon [ 46 ].…”

Pre-clinical Models of Metastasis in Pancreatic Cancer

“…Using PDAC cell lines and combining the previously mentioned assays with CRISPR-Cas9 technique has revolutionized the field of metastasis research. Currently, there are available genome-wide or custom made sgRNA CRISPR libraries that have helped identify genes promoting migration, chemoresistance or radioresistance such as Histone Deacetylase 1, ATP binding cassette subfamily G member 2, Endoplasmic reticulum-associated protein degradation ( Du et al, 2021 ; Ramaker et al, 2021 ).…”