Abstract The tumor mutational burden affects immune surveillance and is associated with response to immune checkpoint blockade. We recently reported that inactivation of the DNA mismatch repair (MMR) pathway in cancer cells increases the mutational burden and modifies the neoantigen landscapes of cancer cells leading to their increased recognition by the immune system. We designed a pharmacologic screening to identify FDA-approved drugs capable of differentially affecting cancer cells MMR proficient and deficient. MMR-deficienT-cells displayed lower sensitivity to the alkylating agent Temozolomide (TMZ) and to the antimetabolite 6-Thioguanine (6-TG). Cells lacking key elements of the MMR pathway such as MutL homolog1, MutS homolog2 (MSH2) or MutS homolog 6 (MSH6), displayed an increased resistance to both TMZ and 6-TG. Next we treated two mouse colorectal cancer cell lines (MC38 and CT26) with TMZ until resistant populations emerged. MC38 cells acquired TMZ resistance through inactivation of the MMR pathway. Bioinformatic analysis revealed that these cells had higher numbers of neoantigens compared to parental cells. Importantly, when MC38 MMRd cells were injected in syngeneic mice, they were unable to form tumors. On the contrary, CT26 cells that acquired TMZ-resistance through other mechanisms, efficiently formed tumors in mice. Therefore, TMZ-induced MMR inactivation, and not TMZ treatment per se, triggered immune surveillance. To assess whether results obtained in mouse cancer models might translate to human disease, we tested TMZ in 47 molecularly annotated colorectal cancer (CRC) cancer cell lines. Only MMR-proficienT-cells and cells in which O6-methylguanine-DNA- methyltransferase (MGMT, the enzyme responsible for repairing the DNA adducts formed by TMZ) was not expressed were sensible to TMZ. Ten sensitive cell lines were chronically treated with TMZ until resistant populations emerged; we found that MGMT re-expression and loss of MMR genes were the main mechanisms of acquired resistance. In agreement with in vitro observations, analysis of biopsies from eight patients relapsing upon TMZ-based therapeutic regimens revealed MGMT re-expression (5 patients) and MMR genes mutations (i.e., MSH2 or MSH6) as main resistance mechanism. In both cell lines and biopsies, MMR inactivation led to increased mutational load and, consequently, to higher levels of predicted neo-antigens, suggesting an augmented immunogenicity. These preclinical data led to the clinical trial Arethusa (NCT03519412; https://clinicaltrials.gov/ct2/show/NCT03519412). Within Arethusa MMR-proficient patients will be tested for (MGMT) expression (IHC) and then for MGMT promoter methylation. MGMT negative patients will be treated with temozolomide (TMZ). Patients progressing under temozolomide will be tested for tumor mutational burden (TMB) and proceed to pembrolizumab if TMB is > 20 mutations/Mb. The primary study hypothesis is that tumors with acquired resistance to temozolomide might become hypermutated and could be sensitive to the anti PD-1 antibody, pembrolizumab. Citation Format: Vito Amodio, Giovanni Grmano, Ludovic Barault, Simona Lamba, Giuseppe Rospo, Alessandro Magrì, Federica Maione, Giovanni Crisafulli, Carlotta Cancelliere, Giulia Lerda, Alice Bartolini, Giulia Siravegna, Benedetta Mussolin, Roberta Frappolli, Monica Montone, Giovanni Randon, Filippo de Braud, Nabil Amirouchene Angelozzi, Silvia Marsoni, Maurizio D'Incalci, Armando Orlandi, Enrico Giraudo, Andrea Satore-Bianchi, Salvatore Siena, Filippo Pietrantonio, Federica Di Nicolantonio, Alberto Bardelli. Temozolomide drives mismatch repair deficiency and fosters neoantigen generation in tumor cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B069.