Background <p>Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by desmoplastic stroma, immunosuppressive tumor microenvironment (TME), and resistance to standard therapies. Natural killer (NK) cell-based immunotherapies have shown limited efficacy due to impaired persistence, infiltration, and function in PDAC.</p> Methods <p>We established a direct reprogramming strategy to generate cytotoxic NK cells (1&#xa0;F-NKs) by targeting <i>BCL11B</i>, a transcription factor essential for T cell lineage commitment, using shRNA or CRISPR/Cas9 in peripheral blood mononuclear cells (PBMCs). A genome-wide CRISPR/Cas9 screen identified tumor-intrinsic modulators of NK resistance. Functional and in vivo studies assesses the efficacy of 1&#xa0;F-NKs alone and in combination with mesothelin (MSLN)-CAR engineering and PKMYT1 inhibition.</p> Results <p>BCL11B depletion enabled the generation of CD56^brightCD16^bright 1&#xa0;F-NKs with potent cytotoxicity and elevated NKG2D and CX3CR1 expression. Site-specific integration of a mesothelin (MSLN)-CAR into <i>BCL11B</i> locus generated MSLN-1&#xa0;F-NKs with stable antigen specific activity. A genome-wide screen identified <i>PKMYT1</i> as a modulator of tumor resistance to NK cell-mediated killing; its inhibition by RP6306 upregulated NKG2D ligands (MICA/B) and CX3CL1, sensitizing PDACs to 1&#xa0;F-NK cytotoxicity. In PDAC xenograft models, 1&#xa0;F-NKs alone or combined with CAR engineering and RP6306 significantly reduced tumor growth and prolonged survival. Notably, this triple combination elicited a synergistic antitumor effect, outperforming each monotherapy or dual combination.</p> Conclusions <p>This study presents a synergistic immunotherapy platform that integrates NK reprogramming, CAR engineering, and tumor sensitization. The combinatorial approach significantly enhances antitumor efficacy in PDAC and offers a promising strategy for overcoming immune resistance in solid tumors.</p>