thioguanine-anhydrous and Melanoma

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carmustine; Clinical Trials as Topic; Drug Evaluation; Female; Humans; Male; Melanoma; Middle Aged; Neoplasm Metastasis; Thioguanine

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).

Topics: Animals; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Immune Checkpoint Inhibitors; Melanoma; Mice; Mutation; Thioguanine; Tumor Microenvironment

In vivo hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient T cells (MT) from melanoma patients are enriched for T cells with in vivo clonal amplifications that traffic between blood and tumor tissues. Melanoma is thus a model cancer to test the hypothesis that in vivo MT from cancer patients can be used as immunological probes for immunogenic tumor antigens. MT were obtained by 6-thioguanine (TG) selection of lymphocytes from peripheral blood and tumor tissues, and wild-type T cells (WT) were obtained analogously without TG selection. cDNA sequences of the T cell receptor beta chains (TRB) were used as unambiguous biomarkers of in vivo clonality and as indicators of T cell specificity. Public TRB were identified in MT from the blood and tumor of different melanoma patients. Such public TRB were not found in normal control MT or WT. As an indicator of T cell specificity for melanoma, the >2600 MT and WT TRB, including the public TRB from melanoma patients, were compared to a literature-derived empirical database of >1270 TRB from melanoma-reactive T cells. Various degrees of similarity, ranging from 100% conservation to 3-amino acid motifs (3-mer), were found between both melanoma patient MT and WT TRBs and the empirical database. The frequency of 3-mer and 4-mer TRB matching to the empirical database was significantly higher in MT compared with WT in the tumor (p=0.0285 and p=0.006, respectively). In summary, in vivo MT from melanoma patients contain public TRB as well as T cells with specificity for characterized melanoma antigens. We conclude that in vivo MT merit study as novel probes for uncharacterized immunogenic antigens in melanoma and other malignancies.

Topics: Adult; Aged; Amino Acid Motifs; Amino Acid Sequence; Antigens, Neoplasm; Antineoplastic Agents; Biomarkers, Tumor; Case-Control Studies; Cells, Cultured; Drug Resistance, Neoplasm; Female; Genes, T-Cell Receptor beta; Humans; Hypoxanthine Phosphoribosyltransferase; Lymphocyte Activation; Male; Melanoma; Melanoma-Specific Antigens; Middle Aged; Molecular Sequence Data; Mutation; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes; Thioguanine

O(6)-methylguanine-DNA methyltransferase (MGMT), is a DNA repair enzyme that recognizes O(6)-alkylated guanine, a base analog resulting from treatment with alkylating agents. O(6)-6-thioguanine (6-TG) is used clinically to treat malignant as well as inflammatory diseases. Although MGMT participates in resistance to alkylating agents, it has not been shown to be involved in resistance of tumors to 6-TG. In this study we used a human melanoma cell line (GA) and its selected 6-TG drug resistant variant (GA-6-TG) to investigate whether MGMT plays a role in determining the drug resistant phenotype of GA-6-TG cells. We showed that GA-6-TG resistant cells express about three fold more MGMT protein and mRNA than GA cells. Treatment with 6-TG diminishes significantly MGMT amounts in both cell lines. Increased amounts of MGMT in resistant cells, are consistent with hypermethylation of the MGMT gene coding-region. Pretreatment of cells with the MGMT inhibitor O6 benzyl guanine, resulted in sensitization of GA-6-TG cells to 6-TG. Taken together, our data suggests that MGMT is associated with 6-TG drug resistance. In analogy to patients treated with alkylating agents, patients with tumors containing increased MGMT amounts, may be more resistant to 6-TG and therefore may benefit from treatment with MGMT inhibitors.

Topics: Antineoplastic Agents, Alkylating; Cell Line, Tumor; DNA Methylation; Dose-Response Relationship, Drug; Guanine; Humans; Melanoma; O(6)-Methylguanine-DNA Methyltransferase; Thioguanine

The identification of specific lymphocyte populations that mediate tumor immune responses is required for elucidating the mechanisms underlying these responses and facilitating therapeutic interventions in humans with cancer. To this end, mutant hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficient (HPRT-) T-cells were used as probes to detect T-cell clonal amplifications and trafficking in vivo in patients with advanced melanoma. Mutant T-cells from peripheral blood were obtained as clonal isolates or in mass cultures in the presence of 6-thioguanine (TG) selection and from tumor-bearing lymph nodes (LNs) or metastatic melanoma tissues by TG-selected mass cultures. Nonmutant (wild-type) cells were obtained from all sites by analogous means, but without TG selection. cDNA sequences of the T-cell receptor (TCR) beta chains (TCR-beta), determined directly (clonal isolates) or following insertion into plasmids (mass cultures), were used as unambiguous biomarkers of in vivo clonality of mature T-cell clones. Clonal amplifications, identified as repetitive TCR-beta V-region, complementarity determining region 3 (CDR3), and J-region gene sequences, were demonstrated at all sites studied, that is, peripheral blood, LNs, and metastatic tumors. Amplifications were significantly enriched among the mutant compared with the wild-type T-cell fractions. Importantly, T-cell trafficking was manifested by identical TCR-beta cDNA sequences, including the hypervariable CDR3 motifs, being found in both blood and tissues in individual patients. The findings described herein indicate that the mutant T-cell fractions from melanoma patients are enriched for proliferating T-cells that infiltrate the tumor, making them candidates for investigations of potentially protective immunological responses.

Topics: Cell Proliferation; Clone Cells; Drug Resistance; Humans; Hypoxanthine Phosphoribosyltransferase; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Melanoma; Mutation; Neoplasms; Receptors, Antigen, T-Cell; T-Lymphocytes; Thioguanine

The various members of the Trk tyrosine kinase family and p75 neurotrophin receptor (p75(NTR)) have been identified as signaling receptors for the structurally related members of the neurotrophins (NT) family. We have previously reported that NT treatment of murine and human brain-metastatic melanoma cells affects their invasive capacities and increases the production of extracellular-matrix degradative enzymes. These cells express aberrant levels of functional p75(NTR) and TrkC, the putative high-affinity receptor for the neurotrophin NT-3. Here we demonstrate that, by using sensitive immune-complex kinase assays in human brain-metastatic (70W) melanoma cells, TrkC receptors associate with a kinase activity exhibiting a dose-dependent susceptibility to inhibition by the purine-analogs 6-thioguanine and 2-aminopurine. The activity of this purine-analog-sensitive kinase (PASK) was induced by NT-3 in a time-dependent fashion, phosphorylating exogenous myelin basic protein (MBP) but not denatured enolase. It is similar to the one reported to relate with p75(NTR) and TrkA receptors and stimulated by the prototypic NT, nerve growth factor. Thus, PASKs may represent unique signaling components common to NT receptors that could engage joint downstream signaling effectors in brain-metastatic melanoma.

Topics: 2-Aminopurine; Brain Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Melanoma; Neoplasm Metastasis; Neurotrophin 3; Protein Kinases; Protein Serine-Threonine Kinases; Receptor, Nerve Growth Factor; Receptor, trkC; Receptors, Nerve Growth Factor; Signal Transduction; Thioguanine; Time Factors

T-cell activation by malignant melanoma would be anticipated to stimulate T-cell proliferation, which in turn has been associated with increasing the likelihood of somatic gene mutation. The purpose of this study was to test the hypothesis that in vivo hypoxanthine guanine phosphoribosyltransferase (hprt) mutant frequencies (MFs) are increased in peripheral blood T-cells from melanoma patients compared to normal controls. Assays were made of 48 peripheral blood samples from melanoma patients with stage 3 (13 patients) and stage 4 (35 patients) disease, 38 normal controls, and of nine tumor bearing lymph nodes. The mean hprt log(10)(MF) in patient peripheral blood was -4.77 (geometric mean hprt MF=17.0x10(-6)) compared to a mean hprt log(10)(MF) of -4.87 (geometric mean hprt MF=13.5x10(-6)) in controls. Although modest, this difference is statistically significant both by t-test (P=0.049) and after adjustment for covariates of age, gender, and cigarette smoking by regression analysis (P=0.001). Among the melanoma patients, the mean log(10)(MF) for the 17 patients who had received potentially genotoxic therapies was not significantly different from the mean log(10)(MF) for the 31 patients not receiving such therapies. The hprt MFs in the nine tumor bearing nodes were compared with MFs in peripheral blood from the same patients and revealed a non-significant (P=0.07) trend for increasing MFs in blood. Furthermore, analyses of T-cell receptor gene rearrangement patterns revealed hprt mutants originating from the same in vivo clone in both peripheral blood and a tumor-bearing node. The finding of elevated hprt MFs not entirely explained by genotoxic therapies in patients compared to controls can be explained either by hypermutability or in vivo T-cell activation. The similar MFs in peripheral blood and tumor bearing lymph nodes, as well as the finding of mutant representatives of the same in vivo T-cell clone in both locations, support monitoring peripheral blood to detect events in the nodes. If in vivo proliferation accounts for the current findings, the hprt deficient (hprt-) mutant fraction in blood may be enriched for T-cells that mediate the host immune response against malignant melanoma. Further studies will characterize the functional reactivity of hprt mutant isolates against melanoma-related antigens.

Topics: Adult; Aged; Case-Control Studies; Drug Resistance; Female; Humans; Hypoxanthine Phosphoribosyltransferase; In Vitro Techniques; Lymphatic Metastasis; Lymphocyte Activation; Male; Melanoma; Middle Aged; Mutation; T-Lymphocytes; Thioguanine

A human melanoma cell line (MM96L) had a spontaneous mutation rate at the HGPRT locus of approx. 7 times normal. The cells had elevated dATP and dGTP pools, lacked purine nucleoside phosphorylase (PNP) and were sensitive to killing by deoxyadenosine, deoxyinosine and related purines but not to inosine or hypoxanthine. Four other melanoma cell lines exhibited a range of nucleoside sensitivities and dNTP pool sizes. Failure of intact MM96L cells to degrade exogenous deoxyadenosine and deoxyinosine to hypoxanthine was confirmed by NMR of culture medium. Normal melanocytes were PNP+ and were insensitive to deoxyinosine. Comparison of the metabolites of [14C]deoxyinosine from MM96L and a PNP+ cell line of similar doubling time (HeLa) showed that both cell types produced 14C-labelled guanine and adenine nucleotides, with [14C]dATP and [14C]dADP being found in MM96L. This indicates that human sAMP synthetase or a similar enzyme catalyses the conversion of dIMP to dAMP, the resultant elevation of dATP causing base misincorporation and a mutator phenotype.

Topics: Alanine; Antibiotics, Antineoplastic; Antineoplastic Agents; Cell Survival; Chromatography, High Pressure Liquid; Deoxyadenosines; Deoxyribonucleotides; HeLa Cells; Humans; Hypoxanthine Phosphoribosyltransferase; Inosine; Magnetic Resonance Spectroscopy; Melanoma; Mutation; Ribavirin; Thioguanine; Tumor Cells, Cultured

Methotrexate (MTX), 6-thioguanine (6-TG) and cytosine arabinoside (ara-C) inhibited the replication of adenovirus (viral capacity) more in drug-sensitive than in resistant human melanoma cell lines. By comparison, inhibition of cellular DNA and RNA synthesis after short treatment periods (less than 48 hr) was not a good predictor of cellular sensitivity. MTX, an inhibitor of de novo nucleotide synthesis, was most effective when added to cells just before infection with virus and inhibited viral capacity at doses 10-1000-fold lower than those required to affect cell survival. The MTX-sensitive cell lines, members of a DNA repair deficient group sensitive also to killing by methylating agents (the Mer- phenotype), were not deficient in dihydrofolate reductase but exhibited DNA fragmentation after treatment with MTX for 48 hr. 6-TG and ara-C, inhibitors of purine and pyrimidine salvage, were most inhibitory to viral capacity when added greater than 36 hr before virus infection and were less effective than MTX (doses 5-7-fold and 4-24-fold higher than for cell survival respectively). No correlation was found between MTX sensitivity and sensitivity to 6-TG or ara-C. These results indicate that (i) inhibition of viral capacity is a more comprehensive test of antimetabolite cytotoxicity than inhibition of cellular DNA or RNA synthesis; (ii) the viral capacity assay correctly predicts cellular sensitivity to MTX, 6-TG and ara-C and therefore has potential for application to primary cultures of human tumours; and (iii) MTX-sensitive cell lines and adenovirus replication rely heavily on de novo nucleotide synthesis, which in Mer- cells appears to be linked to a DNA repair defect as yet undefined.

Topics: Adenoviridae; Cell Survival; Cytarabine; DNA; Drug Resistance; Humans; Kinetics; Melanoma; Methotrexate; RNA; Tetrahydrofolate Dehydrogenase; Thioguanine; Tumor Cells, Cultured; Virus Replication

Karyotypes of the human C32r16 line and its HPRT-mutant derivatives were compared. All three HPRT-mutant strains (TG-A, TG-E1, and TG-E2) studied were hypotetraploid and arose by genomic duplication. TG-A and TG-E had distinct karyotypes, reflecting their independent clonal origins. Both TG-E1 and TG-E2 were similar karyotypically, except that TG-E2 differed from TG-E1 by the presence of two new sets of balanced translocations and a significantly higher number of new markers, viz., 24 new markers in 14 TG-E2 cells as compared to 2 new markers in 13 TG-E1 cells. Comparison of the hypothetical 2s karyotype of C32r16 with the modal karyotypes of the mutants revealed (1) that chromosome changes occurred more frequently among marker chromosomes (57.1%) than among normal chromosomes (18.1%) and also more frequently among tetrasomic (28.6%) than among disomic (3.3%) normal chromosomes, and (2) that one member of a group of paired marker chromosomes tended to be eliminated very frequently, whereas disomic normal chromosomes retained their disomic condition. Chromosome 7 was involved in the formation of new marker chromosomes twice as much as any other chromosome, and even increased to pentasomy in TG-E1. Chromosome changes associated with 6-thioguanine treatment are briefly discussed.

Topics: Cell Line; Chromosome Banding; Chromosomes, Human; Drug Resistance; Humans; Hypoxanthine Phosphoribosyltransferase; Karyotyping; Melanoma; Mutation; Thioguanine

Antitumor activities of a combination chemotherapy with a water-soluble nitrosourea, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU), and a single dose of 6-thioguanine were studied using three obstinate murine tumor systems, i.e., Lewis lung carcinoma, B16 melanoma, and an advanced stage of L1210 leukemia systems. Therapeutically synergistic effect was observed either definitely against 1- or 2-day-old Lewis lung carcinoma and 6-day-old L1210 leukemia or moderately against 1-day-old B16 melanoma. Single intravenous treatment on day 7 after subcutaneous implantation of Lewis lung carcinoma, when the tumors had already metastasized to the lungs, produced a significant regression of tumor and a significant increment in survival time of tumor-bearing mice. In comparative studies, the combination of ACNU and 6-thioguanine showed a greater and a wider spectrum of antitumor activities against these tumors than those obtained by the combination with ACNU and a single dose of 5-fluorouracil, methotrexate, or 6-mercaptopurine. Increment in lethal toxicity for normal and tumor-bearing mice was not observed by the combination of ACNU and 6-thioguanine in contrast to definite increases in this toxicity by the combination of ACNU and 5-fluorouracil. The present experimental results may suggest the clinical utility of the combination chemotherapy with ACNU and 6-thioguanine in the treatment of several solid tumors as well as acute leukemias.

Topics: Animals; Antimetabolites, Antineoplastic; Body Weight; Drug Synergism; Drug Therapy, Combination; Female; Injections, Intraperitoneal; Injections, Intravenous; Leukemia L1210; Lung Neoplasms; Male; Melanoma; Mercaptopurine; Mice; Neoplasms, Experimental; Nimustine; Nitrosourea Compounds; Thioguanine

Topics: Animals; Antineoplastic Agents; Child; Cyclohexanes; Cyclophosphamide; Cytarabine; Dactinomycin; Drug Resistance; Drug Synergism; Drug Therapy, Combination; Humans; Imidazoles; Leukemia L1210; Lung Neoplasms; Melanoma; Mice; Neoplasm Metastasis; Neoplasms; Neoplasms, Experimental; Nitrogen Mustard Compounds; Nitrosourea Compounds; Thioguanine; Triazenes; Wilms Tumor