Abstract 7121: CRISPR druggable-genome screen identifies BRD4 as a synergistic vulnerability with the natural product cerberin in pancreatic cancer organoids

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) acquires resistance to systemic therapy, requiring novel therapeutic combinations. Our prior work demonstrated that cardiac glycosides including cerberin can inhibit PI3K/AKT/mTOR signaling yielding low nanomolar potency in PDAC viability. We recently optimized pooled CRISPR-Cas9 “druggable-genome” screening in 3D PDAC patient-derived organoids (PDOs). Here, we used this platform to define genetic dependencies under cerberin pressure and to nominate synthetic lethal combinations. Methods: The KRASG12D-mutant PDO line Pan21 was transduced with a lentiviral CRISPR-Cas9 library targeting 2,312 druggable genes (∼10,000 sgRNAs; Millipore-Sigma) using optimized viral particle: cell ratios and 6-day puromycin selection. Baseline samples were collected following. PDOs were expanded using media control and cerberin 10nM for 6 days, followed by 2 days of recovery. Guide representation was assessed by next-generation sequencing, and gene-level dropout was analyzed with MAGeCK robust rank aggregation (RRA). For functional validation, dose-response combination matrices were generated for cerberin or digoxin in combination with the BET inhibitor ZEN-3694 in Pan21 PDOs. Viability was measured at 144h using 3D CellTiter-Glo (CTG, 50% v/v), and synergy was quantified in SynergyFinder 3.0. Results: Library QC confirmed stable Cas9 expression, recovery of more than 95% of sgRNAs, and similar log2 read-count profiles across baseline, control, and cerberin-treated samples. Cerberin treatment generated a consistent dropout pattern enriched for signaling and metabolic regulators; among the highest-ranked negative-selection hits were CYGB, ANXA2, NR1I2, PIK3R1, TWF2, ENPP2, FDX1, PRG2, and the epigenetic reader BRD4. Although this pilot was limited to stringent FDR thresholds, these genes showed low RRA scores at nominal p-values 0.005. BRD4 was selected among the top 10 depleted genes (RRA score ∼6 × 10-4; nominal p ≈ 0.003) due to availability for synthetic inhibitors from NCI’s Cancer Therapeutics Evaluation Program. In Pan21 PDOs, dose-response combinations of cerberin with ZEN-3694 produced robust synergy (HSA 26.2, Bliss 23.3, ZIP 23.6), with values in a range generally interpreted as strong positive drug-drug interaction and clear synergy “hot spots” at submaximal concentrations of both agents. Similarly, digoxin with ZEN-3694 exhibited a synergistic landscape (HSA 25.3, Bliss 24.7), supporting a cardenolide class effect. Conclusions: Adopting a CRISPR/Cas9 druggable screen in 3D PDAC PDO models, we provide proof-of-concept for synergy between cardiac glycosides and BRD4/BET inhibitors. This work links a PDO-based CRISPR platform with natural-product pharmacology to provide a framework for mechanistic partners to advance the use of cardiac glycosides for clinical translation. Citation Format: Md Shahadat Hossan, Lauryn E. Flannagan, Michela Cadarso, Molly A. Nellen, Ethan S. Lin, Austin Stram, Dustin Rubinstein, Derek Pavelec, Mark E. Berres, Sean Ronnekleiv-Kelly, Jeremy D. Kratz. CRISPR druggable-genome screen identifies BRD4 as a synergistic vulnerability with the natural product cerberin in pancreatic cancer organoids abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7121.