The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient and unresponsive to immunotherapy, while MMR-deficient (MMRd) tumors often respond to immune-checkpoint-blockade. We previously reported that treatment of CRC preclinical models with temozolomide (TMZ) leads to MMR-deficiency, increased tumor mutational burden (TMB) and sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-Methylguanine-DNA-methyltransferase (MGMT) deficient, MMR-proficient, RAS mutant mCRC patients received priming therapy with TMZ. Analysis of tissue biopsies and circulating tumor DNA (ctDNA) revealed the emergence of a distinct mutational signature and increased TMB after TMZ treatment. Multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes and the p.T1219I MSH6 variant was detected in ctDNA and tissue of 94% (16/17) of the cases. A patient's subset whose tumors displayed the MSH6 mutation, the TMZ mutational signature and increased TMB, achieved disease stabilization upon pembrolizumab treatment.