The Novel Chemical Entity YTR107 Inhibits Recruitment of Nucleophosmin to Sites of DNA Damage, Suppressing Repair of DNA Double-Strand Breaks and Enhancing Radiosensitization

Abstract

Purpose Radiation therapy continues to be an important therapeutic strategy for providing definitive local/regional control of human cancer. However, oncogenes that harbor driver mutations and/or amplifications can compromise therapeutic efficacy. Thus, there is a need for novel approaches that enhance the DNA damage produced by ionizing radiation. Experimental Design A forward chemical genetic approach coupled with cell-based phenotypic screening of several tumor cell lines was used to identify a novel chemical entity (NCE) that functioned as a radiation sensitizer. Proteomics, comet assays, confocal microscopy and immunoblotting were used to identify the biological target. Results The screening process identified a 5-((N-benzyl-1H-indol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)trione as a NCE that radiosensitized cancer cells expressing amplified and/or mutated RAS, ErbB, PIK3CA, and/or BRAF oncogenes. Affinity-based solid phase resin capture followed by LC/MS/MS analysis identified the chaperone nucleophosmin as the NCE target. SiRNA suppression of nucleophosmin abrogated radiosensitization by the NCE. Confocal microscopy demonstrated that the NCE inhibited nucleophosmin shuttling to radiation-induced DNA damage repair foci and analysis of comet assays indicated a diminished rate of DNA double strand break repair. Conclusion These data support the hypothesis that inhibition of DNA repair due to inhibition of nucleophosmin shuttling increases the efficacy of DNA damaging therapeutic strategies.

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

“…4). In agreement with this view, many reports point to the involvement of NPM1 in different aspects of the DDR, yet, the exact contribution(s) of this protein to the stress response is currently elusive (31,79,127). It is worth pointing out that the lack of NPM1 has been proved to sensitize cells to genotoxins that elicit a BER response and that APE1 catalytic activity is impaired in NPM1 knock out cells (150).…”

Nucleolin and nucleophosmin: nucleolar proteins with multiple functions in DNA repair

“…Tumor suppressor activities have not been reported for NCL so far; D r a f t 33 however, overexpression of NCL has been linked to the development, progression and malignancy of a wide variety of solid and hematological tumors, identifying this protein as a major oncogene in numerous cellular systems (Berger et al 2015). This diversity of pathogenic roles for NPM1 and NCL, and their contribution to numerous essential proliferative pathways, make these two proteins exciting future targets for the development of multi-active chemotherapeutic agents (Poletto et al 2015;Sekhar et al 2011). such domains have been reported; dotted brackets for NCL indicate that a region which is not absolutely required for the interaction but enhances the affinity of NCL for the target.…”

Molecules that target nucleophosmin for cancer treatment: an update

“…Sekhar and colleagues [129], through affinity-based solid-phase resin capture and liquid chromatography/tandem mass spectrometry (LC/MS-MS), have identified NPM1 as the biological target of YTR107, a potent radiosensitizing compound previously identified [130]. YTR107 ((Z) −5 - ((N-benzyl-1H-indol-3-yl) methylene) pyrimidine-2, 4, 6 (1H, 3H, 5H) trione) (Figure 2I) interferes with DNA damage repair mechanisms and is therefore capable of sensitizing to radiation different tumour cell lines, including HT29 colorectal adenocarcinoma cells, D54 glioblastoma cells, PANC1 pancreatic cancer cells, different breast cancer cell models and NSCLC cell lines (Table 1) [127].…”