Abstract 5544: Identification, isolation and molecular characterization of drug-resistant sub-populations of pancreatic cancer cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, since most PDAC tumors develop resistance to the standard of care (SoC) treatments like chemotherapy, radiation, and targeted therapies. All tumors consist of multiple, genetically related subpopulations of cancer cells that evolve in parallel and display heterogeneity at genomic, epigenetic, or phenotypic levels. As cancer develops, some subpopulations of cancer cells may show faster growth, increased metastatic potential, and resistance toward SoC treatment. A key challenge in PDAC management is understanding chemoresistance driven by cancer subpopulation dynamics. This would broaden our understanding of tumor adaptation to treatments and guide targeting resistant subpopulations to improve therapeutic outcomes. In this study, we identified PDAC cell subpopulations resistant to chemotherapy using our DNA barcoding system B-GLI (barcode-guide lineage isolation). The B-GLI system leverages a highly complex DNA barcode library and CRISPR activation (CRISPRa) to trace and isolate sub-lineages within heterogeneous cell populations by their DNA barcodes. PDAC cell lines PANC-1 and Mia-PaCa-2 were barcoded with the B-GLI barcode library, which consist of two optimized guide RNA binding sites in each barcode. After DNA barcoding, we performed treatments with two SoC chemotherapeutics, gemcitabine + paclitaxel and FOLFIRINOX, to induce a selection pressure in the PDAC cell lines, and then identified the resistant subpopulations through differentially represented barcodes and sequencing. Furthermore, we designed barcode-specific single guide RNAs (sgRNAs) targeting the resistant subpopulations and activated the expression of the puromycin resistance gene by CRISPRa. This allowed us to enrich and isolate chemoresistant subpopulations by puromycin treatment. After having isolated the resistant PDAC subpopulations, we performed ATAC-seq and RNA-seq for molecular characterization of the resistant and parental cell populations, along with phenotypic drug screening with 384 compounds to identify novel treatment vulnerabilities. The compound testing confirmed that the resistant cells were less sensitive to the two SoC treatments, as well as identified selective sensitivity to specific compounds. ATAC-seq and RNA-seq data will allow us to decode whether and how chromatin structure and transcriptomic changes are associated with the treatment resistance, and the confirmation screen of the drug sensitivities will identify potential compounds for future pre-clinical and clinical testing in PDAC models and patients with resistant disease. In summary, this study will identify targeted treatment alternatives, accompanied by molecular biomarkers for chemotherapy resistant PDAC. Citation Format: Subhendu Roy Choudhury, Shixiong Wang, Yevhen Akimov, Katarina Willoch, Biswajyoti Sahu, Alfonso Urbanucci, Thomas Fleischer, Tero Aittokallio. Identification, isolation and molecular characterization of drug-resistant sub-populations of pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5544.