Single base pair mismatches, and indels of between 1-4 base pair mismatches, occur during DNA replication when DNA polymerase?s proofreading abilities are compromised. These errors are repaired via an intact mismatch repair (MMR) pathway. Deficiencies in DNA MMR are associated with somatic cancers, including endometrial (30%) and colorectal cancers (15-17%), and in various germline diseases that predispose individuals to cancer development. MMR defect (MMRD) endometrial and colorectal cancers are highly resistant to conventional adjuvant chemotherapy and thus novel therapies are urgently needed for the treatment of MMRD cancers. To this end, we have generated a robust gene signature that can faithfully predict MMRD cancer, and based on this signature, we further identified MLN4924 as an effective drug that diminishes cell viability of MMRD cells in vitro and in vivo. MLN4924 is a NEDD8 Activating Enzyme E1 Subunit 1 (NAE1) inhibitor, which prevents ned-dylation of tumor suppressors. Similar to ubiquitination, the addition of a NEDD8 moiety to protein substrates targets them for degradation via the proteasome. Intriguingly, we found that global neddylation increases in MMRD versus MMR intact endometrioid and colon carcinoma cell lines, which is suppressed with MLN4924 treatment. These data suggest that a high mutational burden in MMRD cancer cells may lead to the excessive aggregation of misfolded mutant proteins and elevated proteotoxic stress levels in MMRD cancers; MLN4924 further increases proteotoxic stress and kills MMRD cancer cells. Moreover, because proteotoxic stress induces pro-inflammatory responses, MLN4924 may enhance the immune checkpoint therapies that have been heavily investigated for the treatment of MMRD colorectal cancer. All of these promising findings strongly support the hypothesis that MLN4924 can specifically target MMRD cancers by increasing proteotoxic stress, thereby potentiating the pro-inflammatory response and enhancing the effects of PD-1 immunotherapy. These hypotheses will be tested via three specific aims: (1) to determine if MLN4924 treatment will inhibit the tumorigenicity of MMRD patient-derived xenografts (PDX) in colon and endometrioid carcinomas. We will establish both subcutaneous and orthotopic endometrioid and colon PDX models to critically evaluate the therapeutic effect of MLN4924 on MMRD cancers; (2) to determine the underlying mechanisms for the therapeutic efficacy of MLN4924 inhibition in MMRD cancers. We will investigate how MLN4924 mechanistically kills MMRD cancer cells; and (3) to determine if MLN4924 will potentiate the effects of PD-1 immunotherapy in MMRD tumors. We will establish syngeneic endometrial and colon mouse models to evaluate the monotherapies and combination therapies using MLN4924 and an anti-mouse PD-1 antibody. All of the proposed studies will lead to the development of novel and effective MLN4924-based mono- and combination therapies to effectively treat MMRD cancers.