Identification of small molecules that mitigate vincristine-induced neurotoxicity while sensitizing leukemia cells to vincristine.

Vincristine (VCR) is one of the most widely prescribed medications for treating solid tumors and acute lymphoblastic leukemia (ALL) in children and adults. However, its major dose-limiting toxicity is peripheral neuropathy that can disrupt curative therapy. Peripheral neuropathy can also persist into adulthood, compromising quality of life of childhood cancer survivors. Reducing VCR-induced neurotoxicity without compromising its anticancer effects would be ideal. Here, we show that low expression of NHP2L1 is associated with increased sensitivity of primary leukemia cells to VCR, and that concomitant administration of VCR with inhibitors of NHP2L1 increases VCR cytotoxicity in leukemia cells, prolongs survival of ALL xenograft mice, but decreases VCR effects on human-induced pluripotent stem cell-derived neurons and mitigates neurotoxicity in mice. These findings offer a strategy for increasing VCR's antileukemic effects while reducing peripheral neuropathy in patients treated with this widely prescribed medication.

Population differences in platinum toxicity as a means to identify novel genetic susceptibility variants.

OBJECTIVES: Clinical studies show that Asians (ASN) are more susceptible to toxicities associated with platinum-containing regimens. We hypothesized that studying ASN as an 'enriched phenotype' population could enable the discovery of novel genetic determinants of platinum susceptibility. METHODS: Using well-genotyped lymphoblastoid cell lines from the HapMap, we determined cisplatin and carboplatin cytotoxicity phenotypes (IC50s) for ASN, Caucasians (CEU), and Africans (YRI). IC50s were used in genome-wide association studies. RESULTS: ASN were most sensitive to platinums, corroborating clinical findings. ASN genome-wide association studies produced 479 single-nucleotide polymorphisms (SNPs) associating with cisplatin susceptibility and 199 with carboplatin susceptibility (P<10). Considering only the most significant variants (P<9.99x10), backwards elimination was then used to identify reduced-model SNPs, which robustly described the drug phenotypes within ASN. These SNPs comprised highly descriptive genetic signatures of susceptibility, with 12 SNPs explaining more than 95% of the susceptibility phenotype variation for cisplatin, and eight SNPs approximately 75% for carboplatin. To determine the possible function of these variants in ASN, the SNPs were tested for association with differential expression of target genes. SNPs were highly associated with the expression of multiple target genes, and notably, the histone H3 family was implicated for both drugs, suggesting a platinum-class mechanism. Histone H3 has repeatedly been described as regulating the formation of platinum-DNA adducts, but this is the first evidence that specific genetic variants might mediate these interactions in a pharmacogenetic manner. Finally, to determine whether any ASN-identified SNPs might also be important in other human populations, we interrogated all 479/199 SNPs for association with platinum susceptibility in an independent combined CEU/YRI population. Three unique SNPs for cisplatin and 10 for carboplatin replicated in CEU/YRI. CONCLUSION: Enriched 'platinum susceptible' populations can be used to discover novel genetic determinants governing interindividual platinum chemotherapy susceptibility.

The Werner's syndrome 4330T>C (Cys1367Arg) gene variant does not affect the in vitro cytotoxicity of topoisomerase inhibitors and platinum compounds.

PURPOSE: Werner's syndrome (WS) is a recessive disorder of premature onset of processes associated with aging. Defective DNA repair has been reported after exposure of cells isolated from WS patients to DNA-damaging agents. The germline 4330T>C (Cys1367Arg) variant in the WS gene (WRN) has been associated with protection from age-related diseases, suggesting it has a functional role. We studied whether the 4330T>C variant confers altered drug sensitivity in vitro. METHODS: 4330T>C was genotyped in 372 human lymphoblastoid cell lines (LCLs) from unrelated healthy Caucasian individuals using a TaqMan-based method. The study was powered to detect the effect of the 4330T>C genotypes after exposure to camptothecin (based upon preliminary data). The effect of the 4330T>C variant on the cytotoxicity of etoposide, carboplatin, cisplatin and daunorubicin was also tested. WRN expression in 57 LCLs was measured by microarray. RESULTS: No significant difference between the IC50 of the cells was observed among genotypes (P = 0.46) after exposure to camptothecin. No association was also observed for etoposide, carboplatin, cisplatin, and daunorubicin (ANOVA, P > 0.05). WRN expression also did not vary across genotypes (ANOVA, P = 0.37). CONCLUSION: These results suggest that this nonsynonymous variant has relatively normal function at the cellular level.

Gender-specific differences in expression in human lymphoblastoid cell lines.

Women and men have different risks for certain diseases and they often respond differently to treatment. These differences could be due to the sex-specific differences in the expression of genes related to primary disease susceptibility or pharmacodynamic targets. To evaluate the sex-specific pattern of gene expression, we compared gene expression levels using a publicly available microarray dataset of 233 (115 women and 118 men) lymphoblastoid cell lines. From the 4799 probes meeting a specified minimal level of expression, 10 genes (P<0.005, permutation adjusted false discovery rate less than 50%) located on autosomal chromosomes were identified using a permutation-based approach. These genes were found to be over-represented in certain gene ontology terms of biological process (cell adhesion, apoptosis, transcription and signal transduction), and molecular function (structural molecule activity, zinc ion binding, transcription factor activity and protein binding). A Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that two known pathways are over-represented: adherens junction and cytokine-cytokine receptor interaction.

Inhibition of DNA repair as a means of increasing the antitumor activity of DNA reactive agents.

Chemotherapeutic alkylnitrosoureas (BCNU, CCNU, streptozotocin) and alkyltriazenes (DTIC, temozolomide) produce a cytotoxic lesion at the O(6)-position of guanine. The DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase removes damage from the O(6)-position in a single-step mechanism without co-factors. There is extensive evidence that this protein is one of the most important factors contributing to alkylnitrosourea and alkyltriazene treatment failure. There is an inverse correlation between the level of this protein and the sensitivity of cells to the cytotoxic effects of O(6)-alkylating agents. Attempts have been made to modulate AGT activity using anti-sense technology, methylating agents, O(6)-alkylguanines, and O(6)-benzylguanine analogs. O(6)-Benzylguanine and its analogs are clearly the most potent direct inactivators of the AGT protein. The mechanism involves O(6)-benzylguanine acting as a low-molecular weight substrate with transfer of the benzyl group to the cysteine residue within the active site of the repair protein. Pretreatment of cells with non-toxic doses of O(6)-benzylguanine results in an increase in the sensitivity to O(6)-alkylating agents. Animal studies revealed that the therapeutic index of BCNU increased when administered in combination with O(6)-benzylguanine. This drug is currently in phase I clinical trials. Evidence from animal studies indicates that myelosuppression may be the dose-limiting toxicity, thus, efforts are aimed at improving the therapeutic index by the stable expression of O(6)-benzylguanine-resistant AGT proteins into targeted normal tissue such as bone marrow. The successful modulation of alkyltransferases brings on an exciting new era for alkylnitrosoureas and alkyltriazenes.