Effective eradication of cancer requires treatment directed against multiple targets. The p53 and nuclear factor κB (NF-κB) pathways are dysregulated in nearly all tumors, making them attractive targets for therapeutic activation and inhibition, respectively. We have isolated and structurally optimized small molecules, curaxins, that simultaneously activate p53 and inhibit NF-κB without causing detectable genotoxicity. Curaxins demonstrated anticancer activity against all tested human tumor xenografts grown in mice. We report here that the effects of curaxins on p53 and NF-κB, as well as their toxicity to cancer cells, result from “chromatin trapping” of the FACT (facilitates chromatin transcription) complex. This FACT inaccessibility leads to phosphorylation of the p53 Ser392 by casein kinase 2 and inhibition of NF-κB–dependent transcription, which requires FACT activity at the elongation stage. These results identify FACT as a prospective anticancer target enabling simultaneous modulation of several pathways frequently dysregulated in cancer without induction of DNA damage. Curaxins have the potential to be developed into effective and safe anticancer drugs.

Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. We used a MYC target gene signature that predicts poor neuroblastoma prognosis to identify the histone chaperone, FAcilitates Chromatin Transcription (FACT), as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small molecule curaxin compound, CBL0137, markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with standard chemotherapy by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells and suggesting a treatment strategy for MYCN-driven neuroblastoma.

Two major stable oxidation products of 2'-deoxycytidine are 2'-deoxy-5-hydroxycytidine (5-OHdC) and 2'-deoxy-5-hydroxyuridine (5-OHdU). In order to study the in vitro incorporation of 5-OHdC and 5-OHdU into DNA by DNA polymerase, and to check the base pairing specificity of these modified bases, 5-OHdCTP and 5-OHdUTP were synthesized. Incorporation studies showed that 5-OHdCTP can replace dCTP, and to a much lesser extent dTTP, as a substrate for Escherichia coli DNA polymerase I Klenow fragment (exonuclease free). However, 5-OHdUTP can only be incorporated into DNA in place of dTTP. To study the specificity of nucleotide incorporation opposite 5-hydroxypyrimidines in template DNA, 18- and 45-member oligodeoxyribonucleotides, containing an internal 5-OHdC or 5-OHdU in two different sequence contexts, were used. Translesion synthesis past 5-OHdC and 5-OHdU in both oligonucleotides occurred, but pauses both opposite, and one nucleotide prior to, the modified base in the template were observed. The specificity of nucleotide incorporation opposite 5-OHdC and 5-OHdU in the template was sequence context dependent. In one sequence context, dG was the predominant nucleotide incorporated opposite 5-OHdC with dA incorporation also observed; in this sequence context, dA was the principal nucleotide incorporated opposite 5-OHdU. However in a second sequence context, dC was the predominant base incorporated opposite 5-OHdC. In that same sequence context, dC was also the predominant nucleotide incorporated opposite 5-OHdU. These data suggest that the 5-hydroxypyrimidines have the potential to be premutagenic lesions leading to C-->T transitions and C-->G transversions.

No abstract

The multidrug resistance-associated protein 1 (MRP1) has been closely linked to poor treatment response in several cancers, most notably neuroblastoma. Homozygous deletion of the MRP1 gene in primary murine neuroblastoma tumors resulted in increased sensitivity to MRP1 substrate drugs (vincristine, etoposide, and doxorubicin) compared with tumors containing both copies of wild-type MRP1, indicating that MRP1 plays a significant role in the drug resistance in this tumor type and defining this multidrug transporter as a target for pharmacologic suppression. A cell-based readout system was created to functionally determine intracellular accumulation of MRP1 substrates using a p53-responsive reporter as an indicator of drug-induced DNA damage. Screening of small-molecule libraries in this readout system revealed pyrazolopyrimidines as a prominent structural class of potent MRP1 inhibitors. Reversan, the lead compound of this class, increased the efficacy of both vincristine and etoposide in murine models of neuroblastoma (syngeneic and human xenografts). As opposed to the majority of inhibitors of multidrug transporters, Reversan was not toxic by itself nor did it increase the toxicity of chemotherapeutic drug exposure in mice. Therefore, Reversan represents a new class of nontoxic MRP1 inhibitor, which may be clinically useful for the treatment of neuroblastoma and other MRP1-overexpressing drug-refractory tumors by increasing their sensitivity to conventional chemotherapy. [Cancer Res 2009;69(16):6573-80]