Hester Liu

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.

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 RNA expression and deep sequencing analyses of the nucleolus reveal the presence of nucleolus‐associated microRNAs

Micro RNAs (miRNA) are non‐coding RNAs expressed in the cytoplasm as their mature, 21–22‐nucleotide short forms. More recently, mature miRNAs have also been detected in the nucleus, raising the possibility that their spatial distribution may be more complex than anticipated. Here we undertook comprehensive systematic analyses of miRNA distribution in several subcellular compartments of human cancer cells. In particular, we focused on the potential presence of miRNAs in the nucleolus, which contains an abundance of small non‐coding RNAs. We employed two miRNA expression array platforms and small RNA deep sequencing of small RNAs isolated from cells, nuclei, cytoplasm and the nucleoli. We developed an assay to compare RNAs of isolated nucleoli before and after denaturation and used Northern hybridization to verify the presence of miRNAs in the subcellular compartments. Consistently, we found more than 10 miRNAs associated with the nucleolar preparations. Several miRNAs had greater relative abundance in the nucleolus compared to the other compartments. The nucleolar presence of miRNAs was independent of Dicer and the main activity of the nucleolus, RNA polymerase I transcription, but was dependent on CRM1 previously associated with nucleolar trafficking of small nucleolar RNAs. These results highlight the complexity of miRNA spatial arrangement and regulation.

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.

Differential epithelium DNA damage response to ATM and DNA-PK pathway inhibition in human prostate tissue culture

The ability of cells to respond and repair DNA damage is fundamental for the maintenance of genomic integrity. Ex vivo culturing of surgery-derived human tissues has provided a significant advancement to assess DNA damage response (DDR) in the context of normal cytoarchitecture in a non-proliferating tissue. Here, we assess the dependency of prostate epithelium DDR on ATM and DNA-PKcs, the major kinases responsible for damage detection and repair by nonhomologous end-joining (NHEJ), respectively. DNA damage was caused by ionizing radiation (IR) and cytotoxic drugs, cultured tissues were treated with ATM and DNA-PK inhibitors, and DDR was assessed by phosphorylation of ATM and its targets H2AX and KAP1, a heterochromatin binding protein. Phosphorylation of H2AX and KAP1 was fast, transient and fully dependent on ATM, but these responses were moderate in luminal cells. In contrast, DNA-PKcs was phosphorylated in both luminal and basal cells, suggesting that DNA-PK-dependent repair was also activated in the luminal cells despite the diminished H2AX and KAP1 responses. These results indicate that prostate epithelial cell types have constitutively dissimilar responses to DNA damage. We correlate the altered damage response to the differential chromatin state of the cells. These findings are relevant in understanding how the epithelium senses and responds to DNA damage.

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.

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.

Novel Assay to Detect RNA Polymerase I Activity In Vivo

This report develops an analytically validated chromogenic in situ hybridization (CISH) assay using branched DNA signal amplification (RNAscope) for detecting the expression of the 5′ external transcribed spacer (ETS) of the 45S ribosomal (r) RNA precursor in formalin-fixed and paraffin-embedded (FFPE) human tissues. 5′ETS/45S CISH was performed on standard clinical specimens and tissue microarrays (TMA) from untreated prostate carcinomas, high-grade prostatic intraepithelial neoplasia (PIN), and matched benign prostatic tissues. Signals were quantified using image analysis software. The 5′ETS rRNA signal was restricted to the nucleolus. The signal was markedly attenuated in cell lines and in prostate tissue slices after pharmacologic inhibition of RNA polymerase I (Pol I) using BMH-21 or actinomycin D, and by RNAi depletion of Pol I, demonstrating validity as a measure of Pol I activity. Clinical human prostate FFPE tissue sections and TMAs showed a marked increase in the signal in the presumptive precursor lesion (high-grade PIN) and invasive adenocarcinoma lesions (P = 0.0001 and P = 0.0001, respectively) compared with non-neoplastic luminal epithelium. The increase in 5′ETS rRNA signal was present throughout all Gleason scores and pathologic stages at radical prostatectomy, with no marked difference among these. This precursor rRNA assay has potential utility for detection of increased rRNA production in various tumor types and as a novel companion diagnostic for clinical trials involving Pol I inhibition. Implications: Increased rRNA production, a possible therapeutic target for multiple cancers, can be detected with a new, validated assay that also serves as a pharmacodynamic marker for Pol I inhibitors. Mol Cancer Res; 15(5); 577–84. ©2017 AACR.

Small-Molecule Targeting of RNA Polymerase I Activates a Conserved Transcription Elongation Checkpoint

SUMMARY Inhibition of RNA polymerase I (Pol I) is a promising strategy for modern cancer therapy. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and induces degradation of the enzyme, but how this exceptional response is enforced is not known. Here, we define key elements requisite for the response. We show that Pol I preinitiation factors and polymerase subunits (e.g., RPA135) are required for BMH-21-mediated degradation of RPA194. We further find that Pol I inhibition and induced degradation by BMH-21 are conserved in yeast. Genetic analyses demonstrate that mutations that induce transcription elongation defects in Pol I result in hypersensitivity to BMH-21. Using a fully reconstituted Pol I transcription assay, we show that BMH-21 directly impairs transcription elongation by Pol I, resulting in long-lived polymerase pausing. These studies define a conserved regulatory checkpoint that monitors Pol I transcription and is activated by therapeutic intervention.

Abstract LB-186: Development and validation of a novel RNA in situ hybridization assay to detect RNA polymerase I activity in vivo

Increased ribosome biogenesis is a critical process requisite for the cancer cell phenotype as it is required to doulbe protein levels for cell replication. Ribosome synthesis is a complex process that is stimulated by a number of well-studied oncogenic pathways (MYC, mTOR, RAS-RAF-ERK) and repressed by tumor suppressors (pRb, p53, PTEN, p14/ARF). A key first step in ribosome biogenesis is the increased synthesis by RNA Polymerase I (Pol I) of the precursor rRNA, referred to as 45S rRNA, that is later processed into the 28S, 5.8S and 18S mature rRNA forms. This process is compartmentalized into the nucleolus. As such, the pharmacological inhibition of RNA Pol I is becoming a potentially important therapeutic strategy in cancer (PMIDs 20055700, 23953479, 24434211). A validated assay that can reflect relative levels of Pol I activity in routinely obtained tissue specimens would be a useful tool. For example it would allow the pretreatment screening (as a predictive biomarker) for those tumors likely to respond to Pol I inhibition, and, could also serve as a pharmacodynamic biomarker of Pol I inhibition. The 5’ external transcribed spacer (5’ETS) of the nascent precursor 45S transcript is rapidly removed and degraded; thus, its levels can be used as a surrogate for new rRNA transcription. We employed branched DNA amplified RNA in situ hybridization using probes developed with Advanced Cellular Diagnostics (ACD RNAscope 2.0). The 5’ ETS/45S CISH signal was restricted to nucleoli. The signal was markedly attenuated in cell lines and in formalin fixed paraffin embedded (FFPE) prostate tissue slices after pharmacological inhibition of RNA Pol I using BMH-21 or actinomycin D, demonstrating validity as a measure of RNA Pol I transcriptional activity. In clinical human prostate FFPE tissue sections and TMAs there was a marked increase in the signal in the presumptive precursor lesion (high grade prostatic intraepithelial neoplasia/HGPIN, n = 25) and in invasive adenocarcinoma lesions (n = 72, Kruskal-Wallis, p = 0.0001 and p = 0.0001, respectively) compared with normal luminal epithelium. These results are consistent with prior work using RT-PCR showing an increase in 5’ ETS/45S rRNA levels in prostate cancer tissues. In adenocarcinomas, the increase in 5’ ETS/45S signal was present throughout all Gleason scores and pathological stages at radical prostatectomy, with no marked difference among these. We developed and analytically validated a chromogenic in situ hybridization (CISH) protocol using RNAscope for detecting the 5’ external transcribed spacer (ETS) of the precursor 45S ribosomal RNA in FFPE human tissues. This novel assay was used to show increased rRNA transcription in human clinical samples of PIN and carcinoma, and should prove useful for detection of increased rRNA production in various tumor types and as a novel pharmacodynamic biomarker in clinical trials of RNA Pol I inhibitors. Citation Format: Gunes Guner, Paul Sirajuddin, Qizhi Zheng, Hester Liu, Ibrahim Kulac, Marikki K. Laiho, Angelo M. De Marzo. Development and validation of a novel RNA in situ hybridization assay to detect RNA polymerase I activity in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-186.