DNA mismatch repair enzymes (e.g., MSH2) maintain genomic integrity, and their deficiency predisposes to several human cancers and to drug resistance. We found that leukemia cells from a substantial proportion of patients (~11%) with newly diagnosed acute lymphoblastic leukemia (ALL) have low or undetectable MSH2 protein levels (MSH2-L), despite abundant wild-type MSH2 mRNA. MSH2-L leukemia cells contained partial or complete somatic deletions of 1–4 genes that regulate MSH2 degradation (FRAP1, HERC1, PRKCZ, PIK3C2B); these deletions were also found in adult ALL (16%) and sporadic colorectal cancer (13.5%). Knockdown of these genes in human leukemia cells recapitulated the MSH2 protein deficiency by enhancing MSH2-degradation, leading to significant reduction in DNA mismatch repair (MMR) and increased resistance to thiopurines. These findings reveal a previously unrecognized mechanism whereby somatic deletions of genes regulating MSH2 degradation result in undetectable levels of MSH2 protein in leukemia cells, MMR deficiency and drug resistance.

Mercaptopurine and thioguanine, two of the most widely used antileukemic agents, exert their cytotoxic, therapeutic effects by being incorporated into DNA as deoxy-6-thioguanosine. However, the molecular mechanism(s) by which incorporation of these thiopurines into DNA translates into cytotoxicity is unknown. The solution structure of thioguanine-modified duplex DNA presented here shows that the effects of the modification on DNA structure were subtle and localized to the modified base pair. Specifically, thioguanine existed in the keto form, formed weakened Watson-Crick hydrogen bonds with cytosine and caused a modest ϳ10°o pening of the modified base pair toward the major groove. In contrast, thioguanine significantly altered base pair dynamics, causing an ϳ80-fold decrease in the base pair lifetime with cytosine compared with normal guanine. This perturbation was consistent with the ϳ6°C decrease in DNA melting temperature of the modified oligonucleotide, the 1.13 ppm upfield shift of the thioguanine imino proton resonance, and the large increase in the exchange rate of the thioguanine imino proton with water. Our studies provide new mechanistic insight into the effects of thioguanine incorporation into DNA at the level of DNA structure and dynamics, provide explanations for the effects of thioguanine incorporation on the activity of DNA-processing enzymes, and provide a molecular basis for the specific recognition of thioguanine-substituted sites by proteins. These combined effects likely cooperate to produce the cellular responses that underlie the therapeutic effects of thiopurines.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with glycolytic and nonglycolytic functions, including pro-apoptotic activity. GAPDH accumulates in the nucleus after cells are treated with genotoxic drugs, and it is present in a protein complex that binds DNA modified by thioguanine incorporation. We identified a novel CRM1-dependent nuclear export signal (NES) comprising 13 amino acids (KKVVKQASEGPLK) in the C-terminal domain of GAPDH, truncation or mutation of which abrogated CRM1 binding and caused nuclear accumulation of GAPDH. Alanine scanning of the sequence encompassing the putative NES demonstrated at least two regions important for nuclear export. Site mutagenesis of Lys 259 did not affect oligomerization but impaired nuclear efflux of GAPDH, indicating that this amino acid residue is essential for proper functioning of this NES. This novel NES does not contain multiple leucine residues unlike other CRM1-interacting NES, is conserved in GAPDH from multiple species, and has sequence similarities to the export signal found in feline immunodeficiency virus Rev protein. Similar sequences (KKVV*7-13PLK) were found in two other human proteins, U5 small nuclear ribonucleoprotein, and transcription factor BT3.

The thiopurine drugs are purine antimetabolites widely used in the treatment of acute lymphoblastic leukemia, autoimmune disorders (e.g. Crohn’s disease and rheumatoid arthritis) and of organ transplant recipients. As inactive prodrugs, 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and azathioprine require intracellular activation, catalyzed by multiple enzymes, to exert cytotoxicity. The first step in azathioprine activation is the release of 6-MP, which involves glutathione transferases and nonenzy-matic conversion. Transport of 6-MP into the cell involves SLC28A2, SLC28A3, SLC29A1 and SLC29A2. After the uptake, 6-MP is converted into thioinosine mono-phosphate by hypoxanthine guanine phosphoribosyl transferase, with 5-phospho-D-ribose-1-pyrophosphate as the phosphoribosyl donor. In a similar way, 6-TG can be converted into thioguanosine monophosphate (TGMP). Thioinosine monophosphate can be converted into TGMP in two steps: first, thioxanthosine monophosphate is formed by inositol monophosphate dehydrogenase; second, TGMP is formed by guanosine monophosphate synthetase. Subsequently, TGMP can be converted into TG nucleotide diphosphates and triphosphates. Cyto-toxic effects of thiopurine drugs are achieved through incorporation of thio-deoxyguanosine triphosphate into DNA and of thioguanosine triphosphate into RNA, by inhibition of de novo purine synthesis by methylmercap-topurine nucleotides and by inhibition of Rac1 (this inhibition induces apoptosis in activated T-cells). The thio-deoxyguanosine triphosphate incorporation inhibits the function of several enzymes involved in DNA replication and repair, and induces DNA damage such as single strand-breaks, DNA–protein cross-links and chromatid exchanges. The pathway that leads to synthesis of active metabolites is in competition with inactivation pathways catalyzed by xanthine oxidase or the polymorphic thiopurine methyltransferase

The identification of new molecular components of the DNA damage signaling cascade opens novel avenues to enhance the efficacy of chemotherapeutic drugs. High-mobility group protein 1 (HMGB1) is a DNA damage sensor responsive to the incorporation of nonnatural nucleosides into DNA; several nuclear and cytosolic proteins are functionally integrated with HMGB1 in the context of DNA damage response. The functional role of HMGB1 and HMGB1-associated proteins (high-mobility group protein B2, HMGB2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; protein disulfide isomerase family A member 3, PDIA3; and heat shock 70 kDa protein 8, HSPA8) in DNA damage response was assessed in human carcinoma cells A549 and UO31 by transient knockdown with short interfering RNAs. Using the cell proliferation assay, we found that knockdown of HMGB1-associated proteins resulted in 8-fold to 50-fold decreased chemosensitivity of A549 cells to cytarabine. Western blot analysis and immunofluorescent microscopy were used to evaluate genotoxic stress markers in knocked-down cancer cells after 24 to 72 hours of incubation with 1 μmol/L of cytarabine. Our results dissect the roles of HMGB1-associated proteins in DNA damage response: HMGB1 and HMGB2 facilitate p53 phosphorylation after exposure to genotoxic stress, and PDIA3 has been found essential for H2AX phosphorylation (no γ-H2AX accumulated after 24-72 hours of incubation with 1 μmol/L of cytarabine in PDIA3 knockdown cells). We conclude that phosphorylation of p53 and phosphorylation of H2AX occur in two distinct branches of the DNA damage response. These findings identify new molecular components of the DNA damage signaling cascade and provide novel promising targets for chemotherapeutic intervention.