ABSTRACT Despite the progress in molecular therapy and immunotherapy, multiple underlying cellular mechanisms cause resistance to cancer therapy. There are urgent needs to develop innovative approaches to meet these challenges. The proposed study is to develop subcellular enzyme-instructed self-assembly (sEISA), which includes mitochondrial EISA (mitoEISA) and cytoplasmic EISA (cytoEISA), for generating molecular nanofibers to overcome drug resistance and immunosuppression in cancer therapy. Our preliminary studies have shown that sEISA selectively targets the mitochondria of cancer cells and minimizes drug resistance. Most importantly, our preliminary study shows that sEISA inhibits the growth of immunosuppressive tumors in vivo. Thus, we propose to further develop sEISA against drug resistant cancer cells and tumors. The proposed research has three specific aims: Aim 1, developing mitoEISA for selectively targeting cancer cells; Aim 2, developing cytoEISA for minimizing drug resistance and immunosuppression; and Aim 3, evaluating sEISA in ovarian cancer xenograft murine models. The central hypothesis is that sEISA spatiotemporally generates molecular nanofibers, which interact with multiple cellular proteins and interrupt multiple cellular processes inside cancer cells to minimize drug resistance. Our preliminary results support the central hypothesis. The innovation is that the mechanisms of the action of the molecular nanofibers significantly depart from the ligand-receptor dogma of the current anticancer drugs. The long-term goal of the proposed work is to develop sEISA to generate molecular nanofibers for overcoming resistance in cancer therapy. We anticipate that this research will provide innovative anticancer approaches to address the problems of drug resistance and immunosuppression in cancer therapy, thus ultimately will improve the survivorship of cancer patients.