Mattia Poletto

Critical lysine residues within the overlooked N-terminal domain of human APE1 regulate its biological functions

Apurinic/apyrimidinic endonuclease 1 (APE1), an essential protein in mammals, is involved in base excision DNA repair (BER) and in regulation of gene expression, acting as a redox co-activator of several transcription factors. Recent findings highlight a novel role for APE1 in RNA metabolism, which is modulated by nucleophosmin (NPM1). The results reported in this article show that five lysine residues (K24, K25, K27, K31 and K32), located in the APE1 N-terminal unstructured domain, are involved in the interaction of APE1 with both RNA and NPM1, thus supporting a competitive binding mechanism. Data from kinetic experiments demonstrate that the APE1 N-terminal domain also serves as a device for fine regulation of protein catalytic activity on abasic DNA. Interestingly, some of these critical lysine residues undergo acetylation in vivo. These results suggest that protein–protein interactions and/or post-translational modifications involving APE1 N-terminal domain may play important in vivo roles, in better coordinating and fine-tuning protein BER activity and function on RNA metabolism.

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

SIGNIFICANCE An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. RECENT ADVANCES After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. CRITICAL ISSUES A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. FUTURE DIRECTIONS A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world.

Nucleolar accumulation of APE1 depends on charged Lysine residues that undergo acetylation upon genotoxic stress and modulate its BER activity in cells

The functional importance of APE1 nucleolar accumulation is described. It is shown that acetylation of Lys27–35, affecting local conformation, regulates APE1 function by 1) controlling its interaction with NPM1 and rRNA and its nucleolar accumulation, 2) modulating K6/K7 acetylation status, and 3) promoting APE1 BER activity in cells.

G-quadruplex DNA recognition by nucleophosmin: New insights from protein dissection

BACKGROUND Nucleophosmin (NPM1, B23) is a multifunctional protein that is involved in a variety of fundamental biological processes. NPM1/B23 deregulation is implicated in the pathogenesis of several human malignancies. This protein exerts its functions through the interaction with a multiplicity of biological partners. Very recently it is has been shown that NPM1/B23 specifically recognizes DNA G-quadruplexes through its C-terminal region. METHODS Through a rational dissection approach of protein here we show that the intrinsically unfolded regions of NPM1/B23 significantly contribute to the binding of c-MYC G-quadruplex motif. Interestingly, the analysis of the ability of distinct NPM1/B23 fragments to bind this quadruplex led to the identifications of distinct NPM1/B23-based peptides that individually present a high affinity for this motif. RESULTS These results suggest that the tight binding of NPM1/B23 to the G-quadruplex is achieved through the cooperation of both folded and unfolded regions that are individually able to bind it. The dissection of NPM1/B23 also unveils that its H1 helix is intrinsically endowed with an unusual thermal stability. CONCLUSIONS These findings have implications for the unfolding mechanism of NPM1/B23, for the G-quadruplex affinity of the different NPM1/B23 isoforms and for the design of peptide-based molecules able to interact with this DNA motif. GENERAL OBSERVATION This study sheds new light in the molecular mechanism of the complex NPM1/G-quadruplex involved in acute myeloid leukemia (AML) disease.

Nucleophosmin modulates stability, activity, and nucleolar accumulation of base excision repair proteins

NPM1 is a novel modulator of the BER pathway. NPM1 depletion results in BER protein up-regulation; NPM1 has a stimulatory effect on BER capacity and promotes accumulation of BER proteins within nucleoli. Cisplatin leads to redistribution of NPM1 and BER proteins from nucleoli.

Acetylation on critical lysine residues of Apurinic/apyrimidinic endonuclease 1 (APE1) in triple negative breast cancers.

Protein acetylation plays many roles within living cells, modulating metabolism, signaling and cell response to environmental stimuli, as well as having an impact on pathological conditions, such as cancer pathogenesis and progression. The Apurinic/apyrimidinic endonuclease APE1 is a vital protein that exerts many functions in mammalian cells, acting as a pivotal enzyme in the base excision repair (BER) pathway of DNA lesions, as transcriptional modulator and being also involved in RNA metabolism. As an eclectic and abundant protein, APE1 is extensively modulated through post-translational modifications, including acetylation. Many findings have linked APE1 to cancer development and onset of chemo- and radio-resistance. Here, we focus on APE1 acetylation pattern in triple negative breast cancer (TNBC). We describe the validation and characterization of a polyclonal antibody that is specific for the acetylation on lysine 35 of the protein. Finally, we use the new antibody to analyze the APE1 acetylation pattern on a cohort of TNBC specimens, exploiting immunohistochemistry. Our findings reveal a profound deregulation of APE1 acetylation status in TNBC, opening new perspectives for future improvements on treatment and prognosis of this molecular subtype of breast carcinomas.

Inhibitors of the apurinic/apyrimidinic endonuclease 1 (APE1)/nucleophosmin (NPM1) interaction that display anti-tumor properties

The apurinic/apyrimidinic endonuclease 1 (APE1) is a protein central to the base excision DNA repair pathway and operates in the modulation of gene expression through redox‐dependent and independent mechanisms. Aberrant expression and localization of APE1 in tumors are recurrent hallmarks of aggressiveness and resistance to therapy. We identified and characterized the molecular association between APE1 and nucleophosmin (NPM1), a multifunctional protein involved in the preservation of genome stability and rRNA maturation. This protein–protein interaction modulates subcellular localization and endonuclease activity of APE1. Moreover, we reported a correlation between APE1 and NPM1 expression levels in ovarian cancer, with NPM1 overexpression being a marker of poor prognosis. These observations suggest that tumors that display an augmented APE1/NPM1 association may exhibit increased aggressiveness and resistance. Therefore, targeting the APE1/NPM1 interaction might represent an innovative strategy for the development of anticancer drugs, as tumor cells relying on higher levels of APE1 and NPM1 for proliferation and survival may be more sensitive than untransformed cells. We set up a chemiluminescence‐based high‐throughput screening assay in order to find small molecules able to interfere with the APE1/NPM1 interaction. This screening led to the identification of a set of bioactive compounds that impair the APE1/NPM1 association in living cells. Interestingly, some of these molecules display anti‐proliferative activity and sensitize cells to therapeutically relevant genotoxins. Given the prognostic significance of APE1 and NPM1, these compounds might prove effective in the treatment of tumors that show abundant levels of both proteins, such as ovarian or hepatic carcinomas.

Expression and Prognostic Significance of APE1/Ref-1 and NPM1 Proteins in High-Grade Ovarian Serous Cancer

OBJECTIVES To correlate the expression profile of human apurinic endonuclease/redox factor 1 (APE1/Ref-1) with that of nucleolar/nucleoplasmic protein nucleophosmin 1 (NPM1) in association with the aggressiveness and progression of high-grade ovarian serous cancer. METHODS Retrospective study analyzing a tissue microarray of 73 women affected by high-grade ovarian serous cancer. Protein expression was assessed by immunohistochemistry on primary tumor masses and synchronous peritoneal metastases if present. RESULTS APE1/Ref-1 and NPM1 showed a significant correlation in ovarian serous cancer. Patients with a poorer outcome showed a significant overexpression of nuclear NPM1 protein. A Cox proportional hazards multivariate regression model revealed NPM1 expression to be independently significant for overall survival in high-grade ovarian serous cancers after correcting for stage, age, cytoreduction completeness, and platinum resistance. CONCLUSIONS APE1/Ref-1 interacts with NPM1 to control the DNA damage repair system, and it is likely that this interaction plays a defining role in high-grade ovarian serous carcinoma. A high NPM1 immunohistochemical expression was independently correlated with a shorter survival period and thus appears to be an important prognostic factor.

Role of mutual interactions in the chemical and thermal stability of nucleophosmin NPM1 domains.

Nucleophosmin (NPM1) is a key factor involved in fundamental biological processes. Mutations involving the NPM1 gene are the most frequent molecular alterations in acute myeloid leukemia. Here we report a biophysical characterization of NPM1 and of its domains in order to gain insights into the role that inter-domain interactions plays in the protein stabilization. Thermal denaturation analyses show that the N-terminal domain is endowed with an exceptional thermal stability, as it does not unfold in the investigated temperature range (20-105°C). This finding is corroborated by chemical denaturation experiments showing that this domain is not significantly affected by the addition of 8M urea. These results are consistent with the chaperone function of NPM1. In line with literature data, the other folded domain of the NPM1, a 3-helix bundle domain located at the C-terminus, shows a lower stability. Interestingly, the chemical and thermal stability of this latter domain, which embeds natural mutations related to acute myeloid leukemia, is influenced by the presence of other regions of the protein. Small but significant stabilizations of the C-terminal 3-helix bundle are provided by the adjacent unfolded fragment as well as by the rest of the protein.

Abstract 3337: Modulating the APE1/NPM1 interaction in cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC DNA repair pathways protect the genome from endogenous and environmental DNA damage. Such defensive mechanisms, however, enable tumor cells to survive DNA damage caused by chemo- and radio-therapy. Moreover, alterations of DNA repair pathways that may occur during tumorigenesis can make cancer cells reliant on a subset of repair enzymes for survival. Hence, knowledge of the mechanisms involved in the control of DNA repair proteins is of great interest for cancer diagnosis and treatment. The Apurinic/apyrimidinc endonuclease 1 (APE1) is an essential protein in eukaryotic cells. As the main AP-endonuclease in mammals, this protein is central to the base excision DNA repair pathway (BER). Beside its role in the maintenance of genomic stability, APE1 modulates gene expression by tuning the redox status of several transcription factors. Moreover, recent reports suggest that APE1 may take part in RNA processing and degradation, thus controlling the transcriptional output of the cell. Overexpression and aberrant localization of APE1 are recurrent phenotypes in tumors. Both situations are prognostic indicators of aggressiveness and onset of resistance. Interestingly, downregulation or inhibition of APE1 sensitizes cells to pharmacologically relevant gentoxins. We identified and characterized the molecular association between APE1 and Nucleophosmin (NPM1), a multifunctional protein involved in the preservation of genome stability, rRNA maturation and chromatin remodeling. The association with NPM1 modulates subcellular localization and endonuclease activity of APE1. Accordingly, NPM1−/− cells show lower BER capacity, and expression of an APE1 mutant unable to interact with NPM1 severely impairs cell proliferation. We recently reported a correlation between APE1 and NPM1 expression levels in ovarian cancer, along with a poorer prognosis for patients having higher NPM1 levels. These observations suggest that tumors that display an augmented APE1/NPM1 association may exhibit increased aggressiveness and resistance. Targeting the APE1/NPM1 interaction therefore represents a novel strategy for the development of anticancer drugs, as tumor cells which rely on higher levels of APE1 and NPM1 for proliferation and survival may be more sensitive than normal cells. We exploited the AlphaScreen technology to screen for small molecules that act as inhibitors of the APE1/NPM1 interaction. Our approach detected a set of compounds able to interfere with the APE1/NPM1 association in vivo. Interestingly, some of these molecules display anti-proliferative activity and sensitize cells to genotoxins. To the best of our knowledge, this is the first attempt to target the APE1 interactome. Given the prognostic significance of the APE1 and NPM1 overexpression, these compounds might prove particularly effective in the treatment of tumors that show abundant levels of both proteins, such as ovarian or hepatic carcinomas. Citation Format: Mattia Poletto, Dorjbal Dorjsuren, Lisa Lirussi, Carlo Vascotto, Pasqualina L. Scognamiglio, Daniela Marasco, Ajit Jadhav, David J. Maloney, Anton Simeonov, David M. Wilson, Gianluca Tell. Modulating the APE1/NPM1 interaction in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3337. doi:10.1158/1538-7445.AM2013-3337