Henna M. Moore

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.

Proteasome inhibitors induce nucleolar aggregation of proteasome target proteins and polyadenylated RNA by altering ubiquitin availability

The ubiquitin–proteasome pathway is essential for most cellular processes, including protein quality control, cell cycle, transcription, signaling, protein transport, DNA repair and stress responses. Hampered proteasome activity leads to the accumulation of polyubiquitylated proteins, endoplastic reticulum (ER) stress and even cell death. The ability of chemical proteasome inhibitors (PIs) to induce apoptosis is utilized in cancer therapy. During PI treatment, misfolded proteins accrue to cytoplasmic aggresomes. The formation of aggresome-like structures in the nucleus has remained obscure. We identify here a nucleolus-associated RNA-protein aggregate (NoA) formed by the inhibition of proteasome activity in mammalian cells. The aggregate forms within the nucleolus and is dependent on nucleolar integrity, yet is a separate structure, lacking nucleolar marker proteins, ribosomal RNA (rRNA) and rRNA synthesis activity. The NoAs contain polyadenylated RNA, conjugated ubiquitin and numerous nucleoplasmic proteasome target proteins. Several of these are key factors in oncogenesis, including transcription factors p53 and retinoblastoma protein (Rb), several cell cycle-regulating cyclins and cyclin-dependent kinases (CDKs), and stress response kinases ataxia-telangiectasia mutated (ATM) and Chk1. The aggregate formation depends on ubiquitin availability, as shown by modulating the levels of ubiquitin and deubiquitinases. Furthermore, inhibition of chromosome region maintenance 1 protein homolog (CRM1) export pathway aggravates the formation of NoAs. Taken together, we identify here a novel nuclear stress body, which forms upon proteasome inactivity within the nucleolus and is detectable in mammalian cell lines and in human tissue. These findings show that the nucleolus controls protein and RNA surveillance and export by the ubiquitin pathway in a previously unidentified manner, and provide mechanistic insight into the cellular effects of PIs.

Quantitative Proteomics and Dynamic Imaging of the Nucleolus Reveal Distinct Responses to UV and Ionizing Radiation

The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.

Nucleophosmin Phosphorylation by v-Cyclin-CDK6 Controls KSHV Latency

Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposis sarcoma herpesvirus (KSHV) is the etiological agent of Kaposis sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA.

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.

CRM1 and its ribosome export adaptor NMD3 localize to the nucleolus and affect rRNA synthesis

CRM1 is an export factor that together with its adaptor NMD3 transports numerous cargo molecules from the nucleus to cytoplasm through the nuclear pore. Previous studies have suggested that CRM1 and NMD3 are detected in the nucleolus. However, their localization with subnucleolar domains or participation in the activities of the nucleolus are unclear. We demonstrate here biochemically and using imaging analyses that CRM1 and NMD3 co-localize with nucleolar marker proteins in the nucleolus. In particular, their nucleolar localization is markedly increased by inhibition of RNA polymerase I (Pol I) transcription by actinomycin D or by silencing Pol I catalytic subunit, RPA194. We show that CRM1 nucleolar localization is dependent on its activity and the expression of NMD3, whereas NMD3 nucleolar localization is independent of CRM1. This suggests that NMD3 provides nucleolar tethering of CRM1. While inhibition of CRM1 by leptomycin B inhibited processing of 28S ribosomal (r) RNA, depletion of NMD3 did not, suggesting that their effects on 28S rRNA processing are distinct. Markedly, depletion of NMD3 and inhibition of CRM1 reduced the rate of pre-47S rRNA synthesis. However, their inactivation did not lead to nucleolar disintegration, a hallmark of Pol I transcription stress, suggesting that they do not directly regulate transcription. These results indicate that CRM1 and NMD3 have complex functions in pathways that couple rRNA synthetic and processing engines and that the rRNA synthesis rate may be adjusted according to proficiency in rRNA processing and export.

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.

Cysteine-rich protein 1 is regulated by transforming growth factor-β1 and expressed in lung fibrosis

Transforming growth factor‐β (TGF‐β) is a diverse cytokine regulating growth, apoptosis, differentiation, adhesion, invasion, and extracellular matrix production. Dysregulation of TGF‐β is associated with fibrotic disorders and epithelial‐mesenchymal transition, and has been linked with idiopathic pulmonary fibrosis (IPF). Cysteine‐rich protein 1 (CRP1) is a small LIM‐domain containing protein involved in smooth muscle differentiation. Here, we show that TGF‐β1 increases the expression of CRP1 protein and that CRP1 levels increase in a biphasic fashion. A rapid transient (15–45 min) increase in CRP1 is followed by a subsequent, sustained increase in CRP1 a few hours afterwards that lasts several days. We find that TGF‐β1 regulates the expression of CRP1 through Smad and non‐conventional p38 MAPK signaling pathways in a transcription‐independent manner and that the induction occurs concomitant with an increase in myofibroblast differentiation. Using CRP1 silencing by shRNA, we identify CRP1 as a novel factor mediating cell contractility. Furthermore, we localize CRP1 to fibroblastic foci in IPF lungs and find that CRP1 is significantly more expressed in IPF as compared to control lung tissue. The results show that CRP1 is a novel TGF‐β1 regulated protein that is expressed in fibrotic lesions and may be relevant in the IPF disease. J. Cell. Physiol. 227: 2605–2612, 2012.

Ultraviolet B radiation regulates cysteine-rich protein 1 in human keratinocytes

Background: Cysteine‐rich protein 1 (CRP1) is a growth‐inhibitory cytoskeletal protein that is induced by ultraviolet (UV) C radiation radiation in fibroblasts. Our aim was to investigate the effects of UV radiation on CRP1 in keratinocytes, the main cell type subjected to UV radiation in the human body.