Purpose:O6-methylguanine DNA methyltransferase (MGMT)-silenced tumors reveal sensitivity to temozolomide (TMZ), which may be enhanced by PARP inhibitors. Approximately 40% of colorectal cancer hasMGMTsilencing and we aimed to measure antitumoral and immunomodulatory effects from TMZ and olaparib in colorectal cancer.Experimental Design:Patients with advanced colorectal cancer were screened forMGMTpromoter hypermethylation using methylation-specific PCR of archival tumor. Eligible patients received TMZ 75 mg/m2days 1–7 with olaparib 150 mg twice daily every 21 days. Pretreatment tumor biopsies were collected for whole-exome sequencing (WES), and multiplex quantitative immunofluorescence (QIF) of MGMT protein expression and immune markers.Results:MGMTpromoter hypermethylation was detected in 18/51 (35%) patients, 9 received study treatment with no objective responses, 5/9 had stable disease (SD) and 4/9 had progressive disease as best response. Three patients had clinical benefit: carcinoembryonic antigen reduction, radiographic tumor regression, and prolonged SD. MGMT expression by multiplex QIF revealed prominent tumor MGMT protein from 6/9 patients without benefit, while MGMT protein was lower in 3/9 with benefit. Moreover, benefitting patients had higher baseline CD8+tumor-infiltrating lymphocytes. WES revealed 8/9 patients with MAP kinase variants (7KRASand 1ERBB2). Flow cytometry identified peripheral expansion of effector T cells.Conclusions:Our results indicate discordance betweenMGMTpromoter hypermethylation and MGMT protein expression. Antitumor activity seen in patients with low MGMT protein expression, supports MGMT protein as a predictor of alkylator sensitivity. Increased CD8+TILs and peripheral activated T cells, suggest a role for immunostimulatory combinations.Significance:TMZ and PARP inhibitors synergizein vitroandin vivoin tumors with MGMT silencing. Up to 40% of colorectal cancer is MGMT promoter hypermethylated, and we investigated whether TMZ and olaparib are effective in this population. We also measured MGMT by QIF and observed efficacy only in patients with low MGMT, suggesting quantitative MGMT biomarkers more accurately predict benefit to alkylator combinations.