Spatiotemporal regulation of gene expression governs cellular development and malignant transformation. Compared to the understanding of cis-regulatory elements on the linear chromatin, our knowledge about the three-dimensional (3D) organization of the human genome is still limited. Recent advances in chromatin conformation capture techniques coupled with high-throughput sequencing and fluorescence in situ hybridization combined with high-content microscopy greatly advanced the mapping of 3D genome at kilo-base resolutions. However, the mechanisms of the establishment and maintenance of genome folding and the implications of their disruption in cancer are largely unexplored. Moreover, besides a few architectural proteins, the roles of other transcription factors in chromatin topology remain elusive. Here, I used three cancer types with oncogenic mutations in the signaling-dependent developmental transcription factor NOTCH1 as models to probe the contributions of genome misfolding to oncogenesis and anti-cancer therapy resistance. By subjecting triple-negative breast cancer and mantle cell lymphoma cells to short-term Notch inhibition and reactivation, I discovered that beyond its known role in activating distal enhancers, Notch can dynamically reposition distal enhancers to the promoters of pro-survival genes such as MYC but has limited impact on higher-order chromatin structures including topologically associated domains (TADs) and compartments. Interestingly, in T-cell acute lymphoblastic leukemia (T-ALL), short-term Notch inhibition only diminishes MYC enhancer activity but not looping to the promoter, suggesting that Notch mediates chromatin loops in a lineage- and locus-specific manner. In contrast to short-term treatment, I identified widespread refolding of compartments, TADs and loops in T-ALL cells that acquire resistance to long-term Notch inhibition. These events closely coincide with redistribution of chromatin activity and architectural protein and are reversible when Notch inhibitor is removed. Finally, using a combination of sequencing, imaging and genetics approaches, I provided direct evidence that the B-cell lineage determining factor EBF1 is repositioned from the transcriptionally repressive nuclear lamina to the interior during long-term Notch inhibition. Activated EBF1 thus instructs reorganization of the linear and 3D genome to promote therapy resistance. My studies in Notch-mutated cancers advanced the mechanistical understanding of non-architectural transcription factors in cancer genome folding, which can potentially provide insights into their functional roles during normal development.