Thiopurine 6-thioguanine Research Articles

Thiopurine 6TG treatment increases tumor immunogenicity and response to immune checkpoint blockade

ABSTRACT Immune-checkpoint inhibitors (ICI) are highly effective in reinvigorating T cells to attack cancer. Nevertheless, a large subset of patients fails to benefit from ICI, partly due to lack of the cancer neoepitopes necessary to trigger an immune response. In this study, we used the thiopurine 6-thioguanine (6TG) to induce random mutations and thus increase the level of neoepitopes presented by tumor cells. Thiopurines are prodrugs which are converted into thioguanine nucleotides that are incorporated into DNA (DNA-TG), where they can induce mutation through single nucleotide mismatching. In a pre-clinical mouse model of a mutation-low melanoma cell line, we demonstrated that 6TG induced clinical-grade DNA-TG integration resulting in an improved tumor control that was strongly T cell dependent. 6TG exposure increased the tumor mutational burden, without affecting tumor cell proliferation and cell death. Moreover, 6TG treatment re-shaped the tumor microenvironment by increasing T and NK immune cells, making the tumors more responsive to immune-checkpoint blockade. We further validated that 6TG exposure improved tumor control in additional mouse models of melanoma. These findings have paved the way for a phase I/II clinical trial that explores whether treatment with thiopurines can increase the proportion of otherwise treatment-resistant cancer patients who may benefit from ICI therapy (NCT05276284).

Liquid chromatography–tandem mass spectrometry quantification of 6-thioguanine in DNA using endogenous guanine as internal standard

Thiopurines are S-substituted antimetabolites that are widely used in the treatment of hematological malignancies and as immunosuppressants. Because of extensive inter-individual variation in drug disposition and the significant toxicity associated with thiopurine therapy, there is a need for improved individualized treatment. We here present a fast and sensitive method for quantifying the pharmacological end-point of thiopurines, 6-thioguanine (TG) in chromosomal DNA. Purine nucleobases are released from DNA, etheno-derivatized with chloroacetaldehyde, separated by HILIC and quantified by tandem mass spectrometry using endogenous chromosomal guanine as internal standard. The method is linear up to at least 10 pmol TG/μg DNA and the limit of detection and quantification are 4.2 and 14.1 fmol TG/μg DNA, respectively. The matrix (DNA) had no effect upon quantification of TG. SPE recovery was estimated at 63% (RSD 26%), which is corrected for by the internal standard resulting in stable quantification. The TG levels found were above the LOQ in 18 out of 18 childhood leukemia patients on 6-mercaptopurine/methotrexate maintenance therapy (median 377, range 45–1190 fmol/μg DNA) with intra- and inter-day RSDs of less than 11%. The method uses 2 μg DNA/sample, which can easily be obtained from these patients.

Ambiguous coding is required for the lethal interaction between methylated DNA bases and DNA mismatch repair

The thiopurine 6-thioguanine (S 6G) is used to treat acute leukaemia. Its cytotoxic effect requires an active DNA mismatch repair (MMR) system. S 6G is incorporated into DNA where a small fraction undergoes in situ conversion to S 6-thiomethylguanine (S 6meG). After replication, S 6meG-containing base pairs interact with MMR. This interaction is ultimately lethal and MMR-defective cells are resistant to S 6G. Here, we report that growing human cells extensively incorporate the thiopyrimidine nucleoside 4-thiothymidine (S 4TdR) into their DNA. The incorporated thiopyrimidine (S 4T) can also undergo facile S-methylation to 4-thiomethylthymine (S 4meT). The rate of methylation of S 4TdR in model substrates is similar to that for the conversion of S 6G to S 6meG indicating that the DNA of cells grown in S 4TdR will contain significant levels of S 4meT. Despite this, S 4TdR is not associated with MMR-related cell death. We demonstrate that, in contrast to S 6meG, neither DNA S 4T nor S 4meT codes ambiguously. S 4T retains the coding properties of unmodified T, whereas S 4meT behaves like a normal cytosine and exclusively directs the incorporation of guanine. The preferred S 4meT:G base pair is also a poor substrate for binding by the hMutSα mismatch recognition factor. We suggest that the ability of S 4meT to produce a structurally acceptable base pair during replication underlies the absence of MMR-related death in cells treated with S 4TdR.