Significant developments in cancer immunotherapy, such as immunocheckpoint inhibitors (ICI), have revolutionized the treatment of metastatic patients. Despite these breakthroughs, a group of patients does not respond favorably to ICI, partly due to a lack of cancer neoepitopes. Mutations acquired during tumorigenesis can increase the number of neoepitopes on tumor cells, eliciting a cytotoxic T-cell response that is then boosted by ICI therapy. We use a compound called thiopurine 6-thioguanine (6TG) to trigger random mutations in the tumor in order to enhance the number of neoepitopes produced by tumor cells. In preclinical models of low-mutational burden melanoma, we demonstrated that 6TG-induced mutations enhanced T cell-mediated tumor suppression and significantly improved responsiveness to anti-PD-1 therapy. However, while this strategy augmented immune activation, it remained insufficient to achieve complete tumor regression (Nazerai et al. 2023).

YUMM1.1 (Yale University Murine Melanoma) cells have been co-cultured with mouse splenocytes; cell viability and flow cytometry analyses have been performed to evaluate T-cell mediated cell death of tumor cells treated or not by 6TG. YUMM1.1-based syngeneic mouse model has been used to test the effect of 6TG with anti-CTLA-4 monotherapy or combined with anti-PD-1. Tumor growth kinetics and immune memory have been assessed.

We explored additional ICI combinations to optimize treatment outcomes, specifically evaluating anti-CTLA-4 alone or in combination with anti-PD-1 in 6TG-treated tumors. Our findings revealed that anti-CTLA-4—particularly when combined with anti-PD-1—achieved complete tumor remission, but only when treatment was initiated at smaller tumor volumes. In contrast, efficacy declined when therapy began at later stages, emphasizing the critical importance of early intervention. Additionally, we found that mice experiencing complete tumor regression after 6TG + ICI treatment developed long-term immune memory. When re-exposed to tumor cells, these mice effectively suppressed tumor growth, suggesting durable protection against recurrence.

These findings (Nazerai et al. 2025) have significant clinical implications, highlighting the potential of 6TG-based combinatorial strategies to extend the benefits of immunotherapy to low-TMB patients and overcome current resistance.