We used patient-derived tumor organoids to study the molecular factors underlying heterogeneous chemotherapy responses in colorectal cancers. We identified an epithelial interferon-stimulated gene (ISG) program, linked to high JAK–STAT signaling, that was associated with reduced sensitivity to standard-of-care chemotherapy. JAK–STAT signaling was further modulated by neoadjuvant chemotherapy, with EPSTI1 emerging as a key effector. Loss-of-function experiments demonstrated that EPSTI1 depletion reduced cancer-cell viability and increased chemotherapy sensitivity. These findings nominate the JAK–STAT–EPSTI1 axis as a potential therapeutic target and demonstrate the value of patient-derived tumor organoids for uncovering tumor-intrinsic mechanisms that shape treatment response in colorectal cancer