Tyrosyl DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily and hydrolyzes 3′phospho-DNA adducts via two conserved catalytic histidines, one acting as the lead nucleophile and the second as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease SCAN1. We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics and theoretical chemistry. The structures of wild type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observed in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts access of a nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitution with Asn, Gln, Leu, Ala, Ser and Thr all result in severely compromised enzymes and Top1-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate which suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pKa of this histidine is crucially dependent upon the second histidine and the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily.

DNA is subject to a wide range of insults, resulting from endogenous and exogenous sources that need to be metabolized/resolved to maintain genome integrity. Tyrosyl-DNA phosphodiesterase I (Tdp1) is a eukaryotic DNA repair enzyme that catalyzes the removal of covalent 3'-DNA adducts. As a phospholipase D superfamily member Tdp1 utilizes two catalytic histidines each within a His-Lys-Asn motif. Tdp1 was discovered for its ability to hydrolyze the 3'-phospho-tyrosyl that in the cell covalently links DNA Topoisomerase I (Topo1) and DNA. Tdp1's list of substrates has since grown and can be divided into two groups: protein-DNA adducts, such as camptothecin stabilized Topo1-DNA adducts, and modified nucleotides, including oxidized nucleotides and chain terminating nucleoside analogs. Since many of Tdp1's substrates are generated by clinically relevant chemotherapeutics, Tdp1 became a therapeutic target for molecularly targeted small molecules. Tdp1's unique catalytic cycle allows for two different targeting strategies: (1) the intuitive inhibition of Tdp1 catalysis to prevent Tdp1-mediated repair of chemotherapeutically induced DNA adducts, thereby enhancing their toxicity and (2) stabilization of the Tdp1-DNA covalent reaction intermediate, prevents resolution of Tdp1-DNA adduct and increases the half-life of this potentially toxic DNA adduct. This concept is best illustrated by a catalytic Tdp1 mutant that forms the molecular basis of the autosomal recessive neurodegenerative disease spinocerebellar ataxia with axonal neuropathy, and results in an increased stability of its Tdp1-DNA reaction intermediate. Here, we will discuss Tdp1 catalysis from a structure-function perspective, Tdp1 substrates and Tdp1 potential as a therapeutic target.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. The highest rates of treatment failure occur in specific genetic subsets of ALL, including hypodiploid B-cell ALL (B-ALL), for which effective alternative therapies to current intensive chemotherapy treatments have yet to be developed. Here, we integrated biochemical and genomic profiling with functional drug assays to select effective agents with therapeutic potential against hypodiploid B-ALL. ABT-199, a selective Bcl-2 inhibitor, was effective in reducing leukemic burden in vitro and in vivo in patientderived xenograft models of hypodiploid B-ALL. Daily oral treatment with ABT-199 significantly increased survival in xenografted mice. The unexpected efficacy of observed in hypodiploid leukemias lacking BIM expression (the major reported mediator of ABT-199-induced apoptosis) led us to investigate the mechanism of action of ABT-199 in the absence of BIM. Treatment with ABT-199 elicited responses in a dose-dependent manner, from cell-cycle arrest at low nanomolar concentrations to cell death at concentrations above 100 nmol/L. Collectively, these results demonstrate the efficacy of Bcl-2 inhibition and potential therapeutic strategy in hypodiploid B-ALL.Significance: These results demonstrate the efficacy of ABT-199 in vivo and provide encouraging preclinical data of Bcl-2 as a potential target for the treatment of hypodiploid B-ALL.

Acute lymphoblastic leukemia (ALL) is an aggressive neoplasm of B- or T-lymphoid progenitors and is the commonest childhood tumor. ALL comprises multiple subtypes characterized by distinct genetic alterations, with stereotyped patterns of aneuploidy present in many cases. Although alterations of are common in many tumors, they are infrequent in ALL, with the exception of two ALL subtypes associated with poor outcome: relapsed disease and ALL with hypodiploidy. alterations are present in almost all cases of ALL with low hypodiploidy and are associated with alterations of the lymphoid transcription factor and the tumor-suppressor gene loci and Remarkably, more than half of mutations in low-hypodiploid ALL in children are present in nontumor cells, indicating that low-hypodiploid ALL is a manifestation of Li-Fraumeni syndrome. These findings have profound implications for our understanding of the genetic pathogenesis of hypodiploid ALL, suggesting that alteration of TP53 function may promote the distinctive aneuploidy characteristic of hypodiploid ALL. Moreover, the identification of hypodiploidy mandates offering testing for mutational status to patients and their relatives, with appropriate counseling and disease surveillance.

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