Karita Peltonen

Nucleolar protein NPM interacts with HDM2 and protects tumor suppressor protein p53 from HDM2-mediated degradation.

Nucleophosmin (NPM, B23) is an abundant nucleolar phosphoprotein involved in ribosome biogenesis, and interacts with tumor suppressor proteins p53 and Rb. Here we show that NPM is a UV damage response protein that undergoes nucleoplasmic redistribution and regulates p53 and HDM2 levels and their interaction. By utilizing RNAi approaches and analyses of endogenous and ectopically expressed proteins, we demonstrate that NPM binds HDM2 and acts as a negative regulator of p53-HDM2 interaction. Viral stress, enforced by expression of Kaposis sarcoma virus K cyclin, causes NPM redistribution, K cyclin-NPM association, and p53 stabilization by dissociation of HDM2-p53 complexes. The results demonstrate novel associations of HDM2 and K cyclin with NPM and implicate NPM as a crucial controller of p53 through inhibition of HDM2.

A Targeting Modality for Destruction of RNA Polymerase I that Possesses Anticancer Activity

We define the activity and mechanisms of action of a small molecule lead compound for cancer targeting. We show that the compound, BMH-21, has wide and potent antitumorigenic activity across NCI60 cancer cell lines and represses tumor growth in vivo. BMH-21 binds GC-rich sequences, which are present at a high frequency in ribosomal DNA genes, and potently and rapidly represses RNA polymerase I (Pol I) transcription. Strikingly, we find that BMH-21 causes proteasome-dependent destruction of RPA194, the large catalytic subunit protein of Pol I holocomplex, and this correlates with cancer cell killing. Our results show that Pol I activity is under proteasome-mediated control, which reveals an unexpected therapeutic opportunity.

Human prostate epithelium lacks Wee1A-mediated DNA damage-induced checkpoint enforcement

Cellular DNA damage triggers the DNA damage response pathway and leads to enforcement of cell cycle checkpoints, which are essential for the maintenance of genomic integrity and are activated in early stages of tumorigenesis. A special feature of prostate cancer is its high incidence and multifocality. To address the functionality of DNA damage checkpoints in the prostate, we analyzed the responses of human primary prostate epithelial cells (HPECs) and freshly isolated human prostate tissues to γ-irradiation. We find that γ-irradiation activates the ataxia telangiectasia mutated-associated DNA damage response pathway in the HPECs but that the clearance of phosphorylated histone H2AX (γH2AX) foci is delayed. Surprisingly, γ-irradiated HPECs were unable to enforce cell cycle checkpoint arrest and had sustained cyclin-dependent kinase 2 (Cdk2)-associated kinase activity because of a lack of inhibitory Cdk phosphorylation by Wee1A tyrosine kinase. We further show that HPECs express low levels of Wee1A and that ectopic Wee1A efficiently rescues the checkpoints. We recapitulate the absence of checkpoint responses in epithelium of ex vivo irradiated human prostate tissue despite robust induction of γH2AX. The findings show that prostate epithelium has a surprising inability to control checkpoint arrest, the lack of which may predispose to accrual of DNA lesions.

Nucleophosmin, HDM2 and p53: Players in UV Damage Incited Nucleolar Stress Response

p53 tumor suppressor protein acts as a critical monitor preventing survival of cells with irreparable genetic damage. Its levels are tightly controlled by its negative regulator HDM2, and are allowed to rise only during cellular stress. In our recent paper (Kurki, et al. Cancer Cell 2004; 5:465-75) we identify a novel mechanism leading to p53 stabilization following UV damage of the cells. This involves UV damage provoked nucleoplasmic relocalization of a nucleolar protein, nucleophosmin (NPM, B23) and its rapid and transient interactions with both p53 and HDM2. We discuss here implications of recent findings that several p53 pathway proteins interact with NPM and find that its participation in cellular damage responses is limited to transcriptional stress but absent in direct ds DNA breaks. These findings suggest divergence in the routes provoking p53 stability and implicate the nucleolus as a central site participating in transcriptional stress responses.

Identification of Novel p53 Pathway Activating Small-Molecule Compounds Reveals Unexpected Similarities with Known Therapeutic Agents

Manipulation of the activity of the p53 tumor suppressor pathway has demonstrated potential benefit in preclinical mouse tumor models and has entered human clinical trials. We describe here an improved, extensive small-molecule chemical compound library screen for p53 pathway activation in a human cancer cell line devised to identify hits with potent antitumor activity. We uncover six novel small-molecule lead compounds, which activate p53 and repress the growth of human cancer cells. Two tested compounds suppress in vivo tumor growth in an orthotopic mouse model of human B-cell lymphoma. All compounds interact with DNA, and two activate p53 pathway in a DNA damage signaling-dependent manner. A further screen of a drug library of approved drugs for medicinal uses and analysis of gene-expression signatures of the novel compounds revealed similarities to known DNA intercalating and topoisomerase interfering agents and unexpected connectivities to known drugs without previously demonstrated anticancer activities. These included several neuroleptics, glycosides, antihistamines and adrenoreceptor antagonists. This unbiased screen pinpoints interference with the DNA topology as the predominant mean of pharmacological activation of the p53 pathway and identifies potential novel antitumor agents.

Small molecule BMH-compounds that inhibit RNA polymerase I and cause nucleolar stress.

Activation of the p53 pathway has been considered a therapeutic strategy to target cancers. We have previously identified several p53-activating small molecules in a cell-based screen. Two of the compounds activated p53 by causing DNA damage, but this modality was absent in the other four. We recently showed that one of these, BMH-21, inhibits RNA polymerase I (Pol I) transcription, causes the degradation of Pol I catalytic subunit RPA194, and has potent anticancer activity. We show here that three remaining compounds in this screen, BMH-9, BMH-22, and BMH-23, cause reorganization of nucleolar marker proteins consistent with segregation of the nucleolus, a hallmark of Pol I transcription stress. Further, the compounds destabilize RPA194 in a proteasome-dependent manner and inhibit nascent rRNA synthesis and expression of the 45S rRNA precursor. BMH-9– and BMH-22–mediated nucleolar stress was detected in ex vivo–cultured human prostate tissues indicating good tissue bioactivity. Testing of closely related analogues showed that their activities were chemically constrained. Viability screen for BMH-9, BMH-22, and BMH-23 in the NCI60 cancer cell lines showed potent anticancer activity across many tumor types. Finally, we show that the Pol I transcription stress by BMH-9, BMH-22, and BMH-23 is independent of p53 function. These results highlight the dominant impact of Pol I transcription stress on p53 pathway activation and bring forward chemically novel lead molecules for Pol I inhibition, and, potentially, cancer targeting. Mol Cancer Ther; 13(11); 2537–46. ©2014 AACR.

Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication

Kaposi’s sarcoma herpesvirus (KSHV) causes Kaposi’s sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication.

KIT overexpression induces proliferation in astrocytes in an imatinib-responsive manner and associates with proliferation index in gliomas

Activating gene mutations, gene amplifications and overexpressed proteins may be useful as targets for novel therapies. Alterations at chromosome locus 4q12 are associated with gliomas and the region harbors the receptor tyrosine kinase gene KIT, which is frequently amplified in gliomas, and also overexpressed in a subset of gliomas. KIT and its ligand stem cell factor are widely expressed in embryonic and adult mouse brain, and they play a role in many signal transduction pathways involved in cellular proliferation, differentiation and cancer cell metastasis. However, the function of KIT in gliomagenesis or disease progression remains unresolved as well as its role in neural and brain tumor development. In this study, we utilized lentivirus‐mediated gene transfer to deliver the KIT gene into mouse astrocytes. The growth properties of KIT overexpressing cells were analyzed using several in vitro functional assays. The effect of receptor tyrosine kinase inhibitor imatinib on astrocyte growth was also investigated. Our results indicate that overexpression of KIT in mouse astrocytes promotes cell proliferation, and the increased proliferation is partly inhibited by imatinib treatment. Furthermore, KIT overexpression induces phenotypic changes in the cells suggesting that KIT may play a role in astrocyte growth regulation.

Design, synthesis, and structure-activity relationships of pyridoquinazolinecarboxamides as RNA polymerase i inhibitors

RNA polymerase I (Pol I) is a dedicated polymerase that transcribes the 45S ribosomal (r) RNA precursor. The 45S rRNA precursor is subsequently processed into the mature 5.8S, 18S, and 28S rRNAs and assembled into ribosomes in the nucleolus. Pol I activity is commonly deregulated in human cancers. On the basis of the discovery of lead molecule BMH-21, a series of pyridoquinazolinecarboxamides have been evaluated as inhibitors of Pol I and activators of the destruction of RPA194, the Pol I large catalytic subunit protein. Structure–activity relationships in assays of nucleolar stress and cell viability demonstrate key pharmacophores and their physicochemical properties required for potent activation of Pol I stress and cytotoxicity. This work identifies a set of bioactive compounds that potently cause RPA194 degradation that function in a tightly constrained chemical space. This work has yielded novel derivatives that contribute to the development of Pol I inhibitory cancer therapeutic strategies.

Proteasome Activity Influences UV-Mediated Subnuclear Localization Changes of NPM

UV damage activates cellular stress signaling pathways, causes DNA helix distortions and inhibits transcription by RNA polymerases I and II. In particular, the nucleolus, which is the site of RNA polymerase I transcription and ribosome biogenesis, disintegrates following UV damage. The disintegration is characterized by reorganization of the subnucleolar structures and change of localization of many nucleolar proteins. Here we have queried the basis of localization change of nucleophosmin (NPM), a nucleolar granular component protein, which is increasingly detected in the nucleoplasm following UV radiation. Using photobleaching experiments of NPM-fluorescent fusion protein in live human cells we show that NPM mobility increases after UV damage. However, we show that the increase in NPM nucleoplasmic abundance after UV is independent of UV-activated cellular stress and DNA damage signaling pathways. Unexpectedly, we find that proteasome activity affects NPM redistribution. NPM nucleolar expression was maintained when the UV-treated cells were exposed to proteasome inhibitors or when the expression of proteasome subunits was inhibited using RNAi. However, there was no evidence of increased NPM turnover in the UV damaged cells, or that ubiquitin or ubiquitin recycling affected NPM localization. These findings suggest that proteasome activity couples to nucleolar protein localizations in UV damage stress.