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Lipid uptake occurs through caveolae, plasma membrane invaginations formed by caveolins (CAV) and caveolae-associated protein 1 (CAVIN1). Genetic alterations of CAV1N1 and CAV1 modify lipid metabolism and underpin lipodystrophy syndromes. Lipids contribute to tumorigenesis by providing fuel to cancer metabolism and supporting growth and signaling. Tumor stroma promotes tumor proliferation, invasion, and metastasis, but how stromal lipids influence these processes remain to be defined. Here, we show that stromal CAVIN1 regulates lipid abundance in the prostate cancer microenvironment and suppresses metastasis. We show that depletion of CAVIN1 in prostate stromal cells markedly reduces their lipid droplet accumulation and increases inflammation. Stromal cells lacking CAVIN1 enhance prostate cancer cell migration and invasion. Remarkably, they increase lipid uptake and M2 inflammatory macrophage infiltration in the primary tumors and metastasis to distant sites. Our data support the concept that stromal cells contribute to prostate cancer aggressiveness by modulating lipid content and inflammation in the tumor microenvironment.Implications:This study showed that stromal CAVIN1 suppresses prostate cancer metastasis by modulating tumor microenvironment, lipid content, and inflammatory response.

Figure S1 shows cell growth and lipid distribution data. Figure S2 shows lipid distribution and invasion data. Figure S3 shows additional transcriptomic data. Figure S4 and S5 shows additional orthotopic data.

Supplementary table ("clinicopathological features of cases on the TMAs") and figures (1; "increased 5'ETS/45S CISH signal in cancer glands", 2; "scatterplot showing correlation between 5ʹETS/45S CISH levels by quantitative image analysis and quantitative real time PCR levels from the experiments shown in Figure 2E") S1. Increased 5ʹETS/45S CISH signals in cancer glands. S2. Scatterplot showing correlation between 5ʹETS/45S CISH levels by quantitative image analysis and quantitative real time PCR levels from the experiments shown in Figure 2E.

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.

Supplementary Figures S1-S3. Supplementary Figure 1. BMH-9, BMH-22 and BMH-23 cause a reduction in RPA194 half life. Supplementary Figure 2. BMH-9, BMH-22 and BMH-23 do not activate the DNA damage response. Supplementary Figure 3. p53 activation by Nutlin-3 does not affect the integrity of the nucleolus or rRNA synthesis.

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.

Cancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and represses cancer cell growth. Several recent studies have uncovered key mechanisms by which BMH-21 inhibits ribosome biosynthesis but the selectivity of BMH-21 for Pol I has not been directly measured. Here, we quantify the effects of BMH-21 on Pol I, RNA polymerase II (Pol II), and RNA polymerase III (Pol III) in vitro using purified components. We found that BMH-21 directly impairs nucleotide addition by Pol I, with no or modest effect on Pols II and III, respectively. Additionally, we found that BMH-21 does not affect the stability of any of the Pols’ elongation complexes. These data demonstrate that BMH-21 directly exploits unique vulnerabilities of Pol I.

Memory CD8(+) T cells are characterized by their ability to persist long after the initial antigen encounter and their capacity to generate a rapid recall response. Recent studies have identified a role for metabolic reprogramming and mitochondrial function in promoting the longevity of memory T cells. However, detailed mechanisms involved in promoting their rapid recall response are incompletely understood. Here, we identify a role for the initial and continued activation of the trifunctional rate-limiting enzyme of the de novo pyrimidine synthesis pathway CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) as critical in promoting the rapid recall response of previously-activated CD8(+) T cells. We found that CAD was rapidly phosphorylated upon naïve T cell activation in an mTORC1-dependent manner, yet remained phosphorylated long after initial activation. Previously-activated CD8(+) T cells displayed continued de novo pyrimidine synthesis in the absence of mitogenic signals, and interfering with this pathway diminished the speed and magnitude of cytokine production upon rechallenge. Inhibition of CAD did not affect cytokine transcript levels, but diminished available pre-rRNA, the polycistronic rRNA precursor whose synthesis is the rate-limiting step in ribosomal biogenesis. CAD inhibition additionally decreased levels of detectable ribosomal proteins in previously-activated CD8(+) T cells. Conversely, overexpression of CAD improved both the cytokine response and proliferation of memory T cells. Overall, our studies reveal a critical role for CAD-induced pyrimidine synthesis and ribosomal biogenesis in promoting the rapid recall response characteristic of memory T cells.

Polymorphism drives survival under stress and provides adaptability. Genetic polymorphism of ribosomal RNA (rRNA) genes derives from internal repeat variation of this multicopy gene, and from interindividual variation. A considerable amount of rRNA sequence heterogeneity has been proposed but has been challenging to estimate given the scarcity of accurate reference sequences. We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling and used high-coverage whole-genome sequencing (WGS) data from the 1000 Genomes Project to analyze variants in 2504 individuals from 26 populations. We identified a total of 3791 variant positions. The variants positioned nonrandomly on the rRNA gene. Invariant regions included the promoter, early 5′ ETS, most of 18S, 5.8S, ITS1, and large areas of the intragenic spacer. A total of 470 variant positions were observed on 28S rRNA. The majority of the 28S rRNA variants were located on highly flexible human-expanded rRNA helical folds ES7L and ES27L, suggesting that these represent positions of diversity and are potentially under continuous evolution. Several variants were validated based on RNA-seq analyses. Population analyses showed remarkable ancestry-linked genetic variance and the presence of both high penetrance and frequent variants in the 5′ ETS, ITS2, and 28S regions segregating according to the continental populations. These findings provide a genetic view of rRNA gene array heterogeneity and raise the need to functionally assess how the 28S rRNA variants affect ribosome functions.

Lipid uptake occurs through caveolae, plasma membrane invaginations formed by caveolins (CAV) and caveolae-associated protein 1 (CAVIN1). Genetic alterations of CAV1N1 and CAV1 modify lipid metabolism and underpin lipodystrophy syndromes. Lipids contribute to tumorigenesis by providing fuel to cancer metabolism and supporting growth and signaling. Tumor stroma promotes tumor proliferation, invasion, and metastasis, but how stromal lipids influence these processes remain to be defined. Here, we show that stromal CAVIN1 regulates lipid abundance in the prostate cancer microenvironment and suppresses metastasis. We show that depletion of CAVIN1 in prostate stromal cells markedly reduces their lipid droplet accumulation and increases inflammation. Stromal cells lacking CAVIN1 enhance prostate cancer cell migration and invasion. Remarkably, they increase lipid uptake and M2 inflammatory macrophage infiltration in the primary tumors and metastasis to distant sites. Our data support the concept that stromal cells contribute to prostate cancer aggressiveness by modulating lipid content and inflammation in the tumor microenvironment. Implications: This study showed that stromal CAVIN1 suppresses prostate cancer metastasis by modulating tumor microenvironment, lipid content, and inflammatory response.

[Image: see text] RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) into the 47S ribosomal RNA (rRNA) precursor. Further processing produces the 28S, 5.8S, and 18S rRNAs that are assembled into mature ribosomes. Many cancers exhibit higher Pol I transcriptional activity, reflecting a need for increased ribosome biogenesis and protein synthesis and making the inhibition of this process an attractive therapeutic strategy. Lead molecule BMH-21 (1) has been established as a Pol I inhibitor by affecting the destruction of RPA194, the Pol I large catalytic subunit. A previous structure–activity relationship (SAR) study uncovered key pharmacophores, but activity was constrained within a tight chemical space. This work details further SAR efforts that have yielded new scaffolds and improved off-target activity while retaining the desired RPA194 degradation potency. Pharmacokinetic profiling was obtained and provides a starting point for further optimization. New compounds present additional opportunities for the development of Pol I inhibitory cancer therapies.

The discovery of small, “cave-like” invaginations at the plasma membrane, called caveola, has opened up a new and exciting research area in health and diseases revolving around this cellular ultrastructure. Caveolae are rich in cholesterol and orchestrate cellular signaling events. Within caveola, the caveola-associated proteins, caveolins and cavins, are critical components for the formation of these lipid rafts, their dynamics, and cellular pathophysiology. Their alterations underlie human diseases such as lipodystrophy, muscular dystrophy, cardiovascular disease, and diabetes. The expression of caveolins and cavins is modulated in tumors and in tumor stroma, and their alterations are connected with cancer progression and treatment resistance. To date, although substantial breakthroughs in cancer drug development have been made, drug resistance remains a problem leading to treatment failures and challenging translation and bench-to-bedside research. Here, we summarize the current progress in understanding cancer drug resistance in the context of caveola-associated molecules and tumor stroma and discuss how we can potentially design therapeutic avenues to target these molecules in order to overcome treatment resistance.

Memory CD8+ T cells are characterized by their ability to persist long after the initial antigen encounter and their ability to generate a rapid recall response. Recent studies have identified a role for metabolic reprogramming and mitochondrial function in promoting the longevity of memory T cells. However, detailed mechanisms involved in promoting the rapid recall response are incompletely understood. Here we identify a novel role for the initial and continued activation of the trifunctional rate-limiting enzyme of the de novo pyrimidine synthesis pathway CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) as critical in promoting the rapid recall response of previously-activated CD8+ T cells. CAD is rapidly phosphorylated upon T cell activation in an mTORC1-dependent manner yet remains phosphorylated long after initial activation. Previously-activated CD8+ T cells display continued de novo pyrimidine synthesis in the absence of mitogenic signals and interfering with this pathway diminishes the speed and magnitude of cytokine production upon rechallenge. Inhibition of CAD does not affect cytokine transcript levels, but diminishes available pre-rRNA, the polycistronic rRNA precursor whose synthesis is the rate-limiting step in ribosomal biogenesis. CAD inhibition additionally decreases levels of detectable ribosomal proteins in previously-activated CD8+ T cells. Overexpression of CAD improves both the cytokine response and proliferation of memory T cells. Overall, our studies reveal a novel and critical role for CAD-induced pyrimidine synthesis and ribosomal biogenesis in promoting the rapid recall response characteristic of memory T cells.One Sentence SummaryPyrimidine synthesis fuels ribosomal biogenesis to facilitate rapid recall responses in CD8+ T cells

Publisher Copyright: © 2022 The Authors RNA Polymerase I (Pol I) synthesizes rRNA, which is the first and rate-limiting step in ribosome biogenesis. Factors governing the stability of the polymerase complex are not known. Previous studies characterizing Pol I inhibitor BMH-21 revealed a transcriptional stress-dependent pathway for degradation of the largest subunit of Pol I, RPA194. To identify the E3 ligase(s) involved, we conducted a cell-based RNAi screen for ubiquitin pathway genes. We establish Skp–Cullin–F-box protein complex F-box protein FBXL14 as an E3 ligase for RPA194. We show that FBXL14 binds to RPA194 and mediates RPA194 ubiquitination and degradation in cancer cells treated with BMH-21. Mutation analysis in yeast identified lysines 1150, 1153, and 1156 on Rpa190 relevant for the protein degradation. These results reveal the regulated turnover of Pol I, showing that the stability of the catalytic subunit is controlled by the F-box protein FBXL14 in response to transcription stress.

RNA polymerase I is a highly processive enzyme with fast initiation and elongation rates. The structure of Pol I, with its in-built RNA cleavage ability and incorporation of subunits homologous to transcription factors, enables it to quickly and efficiently synthesize the enormous amount of rRNA required for ribosome biogenesis. Each step of Pol I transcription is carefully controlled. However, cancers have highjacked these control points to switch the enzyme, and its transcription, on permanently. While this provides an exceptional benefit to cancer cells, it also creates a potential cancer therapeutic vulnerability. We review the current research on the regulation of Pol I transcription, and we discuss chemical biology efforts to develop new targeted agents against this process. Lastly, we highlight challenges that have arisen from the introduction of agents with promiscuous mechanisms of action and provide examples of agents with specificity and selectivity against Pol I.

Cancer cells are dependent upon an abundance of ribosomes to maintain rapid cell growth and proliferation. The rate-limiting step of ribosome biogenesis is ribosomal RNA (rRNA) synthesis by RNA polymerase I (Pol I). Therefore, a goal of the cancer therapeutic field is to develop and characterize Pol I inhibitors. Here, we elucidate the mechanism of Pol I inhibition by a first-in-class small molecule, BMH-21. To characterize the effects of BMH-21 on Pol I transcription, we leveraged high-resolution in vitro transcription assays and in vivo native elongating transcript sequencing (NET-seq). We find that Pol I transcription initiation, promoter-escape, and elongation are all inhibited by BMH-21 in vitro. In particular, the transcription elongation phase is highly sensitive to BMH-21 treatment, as it causes a decrease in transcription elongation rate and an increase in paused Pols on the ribosomal DNA (rDNA) template. In vivo NET-seq experiments complement these findings by revealing a reduction in Pol I occupancy on the template and an increase in sequence-specific pausing upstream of G-rich rDNA sequences after BMH-21 treatment. Collectively, these data reveal the mechanism of action of BMH-21, which is a critical step forward in the development of this compound and its derivatives for clinical use.

Polymorphism drives survival under stress and provides adaptability. Genetic polymorphism of ribosomal RNA (rRNA) genes derives from internal repeat variation of this multicopy gene, and from interindividual variation. A considerable amount of rRNA sequence heterogeneity has been proposed but has been challenging to estimate given the scarcity of accurate reference sequences. We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. Pairwise alignment of the rRNA coding sequences of these copies showed differences in sequence and length. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling. We employed whole genome sequencing (WGS) data from the 1000 Genomes Project phase 3 and analyzed variants in 2,504 individuals from 26 populations. Using the pipeline, we identified a total of 3,790 variant positions. The variants positioned non-randomly on the rRNA gene. Invariant regions included the promoter, early 5’ ETS, 5.8S, ITS1 and certain regions of the 28S rRNA, and large areas of the intragenic spacer. 18S rRNA coding region had very few variants, while a total of 470 variant positions were observed on 28S rRNA. The majority of the 28S rRNA variants located on highly flexible human-expanded rRNA helical folds ES7L and ES27L, suggesting that these represent positions of diversity and are potentially under continuous evolution. These findings provide a genetic view for rRNA heterogeneity and raise the need to functional assess how the 28S rRNA variants affect ribosome functions.

RNA polymerase I inhibitors represent a new class of cancer therapeutics with potential application in many cancer types. By curbing the transcription of ribosomal RNAs they block the rate-limiting step in ribosome biogenesis and protein synthesis. We have recently identified specific and selective first-in-class inhibitors of Pol I. This class of Pol I inhibitors, represented by small-molecule BMH-21, blocks transcription initiation and elongation and causes the destruction of the enzyme catalytic subunit. BMH-21 activity is genetically dependent on Pol I in cancer cells and in yeast confirming the targeting specificity. Our earlier mechanistic and efficacy studies with BMH-21 have demonstrated the utility of the approach in targeting colon and prostate cancers and melanoma. The goal of this study was to pinpoint cancer types with specific sensitivity to these inhibitors and to identify markers of this sensitivity. To do so, we undertook unbiased large-scale cancer cell line screens with our Pol I inhibitors and conducted correlative analyses using open source DepMap and Achilles databases to identify cancer types and features that sensitize cancer cells to these inhibitors. The most sensitive lineages were colorectal, endometrial, esophageal, leukemia, myeloma, and ovarian. Comparison of the cancer cell line responses to the Achilles gene essentiality database showed high correlation to genetic knock-out of three Pol I subunits. This unbiased finding strongly supports identification of Pol I as the target. Further, we identified two ribosomal proteins, RPL22 and RPL22L1 as the top genetic and expression markers denoting sensitivity, and MDM4, a p53 transcriptional repressor, as the top protein marker. Together, these three markers conform to a new p53 regulatory pathway. These findings were validated using genetic and mechanistic analyses. We show that Pol I inhibition alters the MDM4 splicing and decreases the expression of MDM4 isoform that represses p53, and that this activity is dependent on the RPL22/RPL22L1 proteins. Notably, perturbation of the biomarkers is strongly associated with mismatch repair defective (MMRd) cancers. We show the efficacy of the Pol I inhibitors in xenograft and patient-derived MMRd models. Our data suggest that RPL22mutation could serve as tumor-agnostic biomarker for targeting cancers with the Pol I inhibitors. Furthermore, this work identifies a new Pol I transcription-dependent ribosomal protein-governed pathway that converges on the regulation of p53. Citation Format: Wenjun Fan, Hester Liu, Asma Begum, Stephanie Pitts, Brittany Ford, Tony Dorado, Pablo de Leon, Rajeshkumar NV, James C. Barrow, Marikki Laiho. Therapeutic inhibition of RNA polymerase I reveals vulnerability of mismatch repair defective cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2644.

The dose-limiting factor in radiation therapy (RT) is damage to healthy tissues, which presents as radiation-induced lung toxicity (RILT). Acute RILT, pneumonitis, is an early inflammatory-driven toxicity that develops within weeks to months following RT. Purified poloxamer 188 (PP188) is a triblock copolymer that associates with and protects cell membranes from oxidative insult, minimizing secondary inflammatory damage. This study aimed to investigate the effects of PP188 to mitigate the onset of acute radiation pneumonitis in a rat model of RILT, and to test the effect of PP188 on the efficacy of RT on tumor growth of a syngeneic CT26.WT murine colon carcinoma in Balb/c mice. To evaluate the efficacy of PP188 to mitigate RILT in healthy tissues, Sprague Dawley rats were given 200mg/kg PP188 or vehicle via IV 1hr prior to a single dose of partial volume (6mm) 20Gy X-irradiation to the right lung, and then were followed for 6 weeks. To validate PP188 treatment does not interfere with RT efficacy to reduce tumor volume, Balb/c mice were implanted with CT26.WT murine colon adenocarcinoma in the high axilla space, given 5Gy SARRP irradiation, and tumor volume was monitored. Rats were evaluated for histopathological changes indicative of acute RILT, including leakage of proteins into alveolar space, thickening of alveolar septa, and alteration of bronchial epithelium and capillaries; for presence of inflammatory infiltrate (Hanker-Yates Peroxidase Leukocyte kit); and for immunohistochemical expression of alpha-smooth muscle actin (SMA). Without PP188, significant inflammatory infiltrate was observed in all lungs bilaterally (p Citation Format: Sarah G. Dennis-Little, Wilfred Ngwa, Marikki Laiho, Graham Warren, Michael J. Yost. Purified poloxamer 188 mitigates radiation-induced lung toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3319.

RNA polymerase I (Pol I) is responsible for the transcription of ribosomal DNA (rDNA), the first and rate-limiting step of ribosome biogenesis. Pol I is the most active RNA polymerase, and it comprises over 60% of transcriptional activity in cancer cells. The robust transcriptional activity, coupled with the high density of elongating Pol I transcription complexes on the rDNA, renders Pol I vulnerable to transcriptional blocks. In healthy cells, this type of transcriptional stress activates inhibitory downstream cascades such as the p53 pathway. However, cancer cells elude this regulatory mechanism to sustain high levels of growth. Further, oncogenic drivers upregulate Pol I activity and achieve abnormally high rates of ribosome biogenesis. This makes Pol I a highly relevant target for cancer therapeutics. Yet little is known about the stability and regulation of the enzyme complex and whether this knowledge can be used for therapeutic purposes. Our lab recently discovered a first-in-class small molecule, BMH-21, that inhibits Pol I transcription and induces the degradation of the catalytic subunit of Pol I, RPA194. We showed that the inducible degradation of RPA194 is cancer cell-specific, correlated with cell death, and is mediated by the proteasome system. A detailed understanding of the degradation process will provide essential knowledge about the stability of the Pol I complex and benefit therapeutic implementation of Pol I inhibitory strategies. To identify the E3 ligases involved, we conducted a cell-based RNAi screen for ubiquitin pathway genes associated with RPA194 degradation. This screen identified a Skp-Cullin-F-box (SCF) complex as a promising candidate E3 ligase. We focused on validating the role of the identified F-box protein, FBXL14, in RPA194 degradation. To do this, we conducted knockout, knockdown, and overexpression analyses of FBXL14 in melanoma, osteosarcoma, breast cancer and chronic myelogenous leukemia cancer cell lines. We show here that both the basal expression of RPA194 and the BMH-21-induced degradation depend on FBXL14. The expression of FBXL14 significantly affected the half-life of RPA194. Knockdown of FBXL14 increases RPA194 turnover upon BMH-21 treatment, while its overexpression reduces RPA194 half-life. Using co-immunoprecipitation analyses, we demonstrate that FBXL14 interacts with RPA194 both in cell lines and in vitro. To investigate the correlation between RPA194 degradation and cell death, we analyzed how the expression of FBXL14 affects sensitivity to BMH-21 using cell growth, viability, and colony formation assays. We found that FBXL14 overexpression increased the sensitivity to the drug. Our results have revealed novel regulatory aspects of Pol I, showing that the stability of the catalytic subunit is controlled by the F-box protein FBXL14. We discuss the implications of these findings on Pol I transcription activity and its regulation. Citation Format: Stephanie Pitts, Hester Liu, Wenjun Fan, Brittany Ford, Ronald Hay, Marikki Laiho. Identification of an E3 ligase regulating the catalytic subunit of RNA polymerase I [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 678.

Increased ribosome biogenesis is a hallmark of cancer and targeting this process with RNA polymerase I (Pol I) inhibitors is a promising strategy for cancer therapy. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and induces degradation of the enzyme. This approach is effective across many cancer types. However, a heterogeneous response was observed in cancer cell lines emphasizing the need to increase knowledge of factors that underlie the response to this therapeutic strategy. To identify genes that modulate the response of cancer cells to BMH-21, we performed genome-wide CRISPR-Cas9-based positive selection screens in human colorectal carcinoma cells. These screens identified high-confidence hits accounting for BMH-21 drug resistance that included all key positive regulators of the mTORC1 complex. Given that p53 has been identified as a downstream effector of Pol I transcription stress, we conducted the screens in TP53 isogenic cells. Notably, the mTORC1 pathway hits were identified in all screens indicating that the resistance was p53 independent. These findings are particularly striking given that mTOR is a major driver of ribosome biogenesis and cellular translational programs. The findings were validated using chemical and genetic approaches. Torin-1, a catalytic mTOR inhibitor, was found to cause resistance to BMH-21. mTOR signaling pathway knockout and rescue cell lines were generated and tested for changes in the drug responses by growth, viability, colony formation and GFP competition assays. In each case, compromised mTOR activity led to resistance to BMH-21. However, compromised mTOR activity did not abrogate Pol I transcription inhibition by BMH-21. To assess impact on protein translation, we used polysome profiling. BMH-21 treatment was found to cause a severe ribosome biogenesis defect. Surprisingly, mTOR inactivation partially rescued the translation ability under the drug treatment suggesting that this translation is pivotal for cell survival. To uncover factors critical for the survival, we performed Ribo-seq and RNA-seq in the drug-treated BMH-21 sensitive and resistant cells. The profiling results revealed that mTOR inactivation led to elevated translation efficiency of mRNAs encoding ribosomal proteins. These findings suggest that mTOR inactivation evokes compensatory selective translation of ribosomal proteins under severe ribosome biogenesis defect caused by the Pol I inhibition. The findings indicate that mTOR inactivation regulates selective translation as means to bypass Pol I inhibition, and more generally, that maintenance of translational capacity strongly contributes to treatment resistance. These findings reveal an unexpected complication by mTOR inhibitory strategies as well as have implications on exploring drug combinations in cancer. Citation Format: Wenjun Fan, Hester Liu, Stephanie Pitts, Brittany Ford, Rajeshkumar NV, Marikki Laiho. Functional CRISPR-Cas9 screens identify master regulators of resistance to chemical targeting of RNA polymerase I [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3247.

Non

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.

In two brothers born to consanguineous parents, we identified an unusual neurological disease that manifested with ataxia, psychomotor retardation, cerebellar and cerebral atrophy, and leukodystrophy. Via linkage analysis and exome sequencing, we identified homozygous c.2801C>T (p.(Ser934Leu)) in POLR1A (encoding RPA194, largest subunit of RNA polymerase I) and c.511C>T (p.(Arg171Trp)) in OSBPL11 (encoding oxysterol-binding protein-like protein 11). Although in silico analysis, histopathologic evidence and functional verification indicated that both variants were deleterious, segregation with the patient phenotype established that the POLR1A defect underlies the disease, as a clinically unaffected sister also was homozygous for the OSBPL11 variant. Decreased nucleolar RPA194 was observed in the skin fibroblasts of only the affected brothers, whereas intracellular cholesterol accumulation was observed in the skin biopsies of the patients and the sister homozygous for the OSBPL11 variant. Our findings provide the first report showing a complex leukodystrophy associated with POLR1A. Variants in three other RNA polymerase subunits, POLR1C, POLR3A and POLR3B, are known to cause recessive leukodystrophy similar to the disease afflicting the present family but with a later onset. Of those, POLR1C is also implicated in a mandibulofacial dysostosis syndrome without leukodystrophy as POLR1A is. This syndrome is absent in the family we present.

Background Advanced prostate cancers depend on protein synthesis for continued survival and accelerated rates of metabolism for growth. RNA polymerase I (Pol I) is the enzyme responsible for ribosomal RNA (rRNA) transcription and a rate-limiting step for ribosome biogenesis. We have shown using a specific and sensitive RNA probe for the 45S rRNA precursor that rRNA synthesis is increased in prostate adenocarcinoma compared to nonmalignant epithelium. We have introduced a first-in-class Pol I inhibitor, BMH-21, that targets cancer cells of multiple origins, and holds potential for clinical translation. Methods The effect of BMH-21 was tested in prostate cancer cell lines and in prostate cancer xenograft and mouse genetic models. Results We show that BMH-21 inhibits Pol I transcription in metastatic, castration-resistant, and enzalutamide treatment-resistant prostate cancer cell lines. The genetic abrogation of Pol I effectively blocks the growth of prostate cancer cells. Silencing of p53, a pathway activated downstream of Pol I, does not diminish this effect. We find that BMH-21 significantly inhibited tumor growth and reduced the Ki67 proliferation index in an enzalutamide-resistant xenograft tumor model. A decrease in 45S rRNA synthesis demonstrated on-target activity. Furthermore, the Pol I inhibitor significantly inhibited tumor growth and pathology in an aggressive genetically modified Hoxb13-MYC|Hoxb13-Cre|Ptenfl/fl (BMPC) mouse prostate cancer model. Conclusion Taken together, BMH-21 is a novel promising molecule for the treatment of castration-resistant prostate cancer.

Mitochondrial DNA (mtDNA) mutagenesis and nuclear DNA repair defects are considered cellular mechanisms of ageing. mtDNA mutator mice with increased mtDNA mutagenesis show signs of premature ageing. However, why patients with mitochondrial diseases, or mice with other forms of mitochondrial dysfunction, do not age prematurely remains unknown. Here, we show that cells from mutator mice display challenged nuclear genome maintenance similar to that observed in progeric cells with defects in nuclear DNA repair. Cells from mutator mice show slow nuclear DNA replication fork progression, cell cycle stalling and chronic DNA replication stress, leading to double-strand DNA breaks in proliferating progenitor or stem cells. The underlying mechanism involves increased mtDNA replication frequency, sequestering of nucleotides to mitochondria, depletion of total cellular nucleotide pools, decreased deoxynucleoside 5'-triphosphate (dNTP) availability for nuclear genome replication and compromised nuclear genome maintenance. Our data indicate that defects in mtDNA replication can challenge nuclear genome stability. We suggest that defects in nuclear genome maintenance, particularly in the stem cell compartment, represent a unified mechanism for mouse progerias. Therefore, through their destabilizing effects on the nuclear genome, mtDNA mutations are indirect contributors to organismal ageing, suggesting that the direct role of mtDNA mutations in driving ageing-like symptoms might need to be revisited.

Objective While compelling data suggest a cancer-induced autoimmunity model in scleroderma patients with anti-RNA polymerase III large subunit (anti-RPC155) antibodies, ~85% of these patients do not manifest cancer. This study was undertaken to determine whether additional autoantigens are targeted in anti-RPC155-positive scleroderma patients without detectable cancer. Methods The study included 168 scleroderma patients with anti-RPC155 antibodies (80 with a history of cancer and 88 with no cancer diagnosis after >5 years of follow-up). Thirty-five sera (17 from patients with cancer and 18 from patients without cancer) were randomly selected for autoantibody discovery using immunoprecipitation (IP). An ~194-kd band was enriched in the subgroup without cancer; this was identified as RNA polymerase I large subunit (RPA194). Results RPA194 generated by in vitro transcription/translation was used for IPs performed on the entire cohort to test whether anti-RPA194 was enriched among anti-RPC155-positive patients without cancer. Anti-RPA194 antibodies were significantly more common in the group without cancer (16 [18.2%] of 88) than in the group with cancer (3 [3.8%] of 80) (P = 0.003). Patients with both anti-RPA194 and anti-RPC155 were significantly less likely to have severe gastrointestinal disease than patients with anti-RPC155 only (26.3% versus 51.0%; P = 0.043). Conclusion Anti-RPA194 antibodies are enriched in anti-RPC155-positive scleroderma patients without cancer. Since somatic mutations in the gene encoding RPC155 in cancer in scleroderma patients appears to play a role in immune response initiation against RPC155 in those patients, these data raise the possibility that the development of immune responses to both RPC155 and RPA194 may influence clinical cancer emergence. Further study is required to define whether different autoantibody combinations have utility as tools for cancer risk stratification in scleroderma.

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.

DNA damage responses are relevant to prostate cancer initiation, progression and treatment. Few models of the normal and malignant human prostate that maintain stromal-epithelial interactions in vivo exist in which to study DNA damage responses. We evaluated the feasibility of maintaining tissue slice grafts at subcutaneous vs subrenal capsular sites in RAG2(-/-)γC(-/-) mice to study the DNA damage responses of normal and malignant glands.We compared the take rate and histology of tissue slice grafts from fresh, precision cut surgical specimens that were maintained for 1 to 4 weeks in subcutaneous vs subrenal capsular sites. Induction of γH2AX, p53, ATM and apoptosis was evaluated as a measure of the DNA damage response after irradiation.The take rate of subcutaneous tissue slice grafts was higher than typically reported but lower than at the subrenal capsular site. Subcutaneous tissue slice grafts frequently showed basal cell hyperplasia, squamous metaplasia and cystic atrophy, and cancer did not survive. In contrast, normal and malignant histology was well maintained in subrenal capsular tissue slice grafts. Regardless of implantation site the induction of γH2AX and ATM occurred in tissue slice graft epithelium 1 hour after irradiation and decreased to basal level by 24 hours, indicating DNA damage recognition and repair. As observed previously in prostatic ex vivo models, p53 was not activated. Notably, tumor but not normal cells responded to irradiation by undergoing apoptosis.To our knowledge this is the first study of DNA damage responses in a patient derived prostate tissue graft model. The subrenal capsular site of RAG2(-/-)γC(-/-) mice optimally maintains normal and malignant histology and function, permitting novel studies of DNA damage responses in a physiological context.

After unresolved endoplasmic reticulum stress, recovery of protein synthesis including increased expression of ribosomal components and translation factors may induce cell death. Using a mouse model of moderate contusive spinal cord injury (SCI) at the T9 level, upregulation of ribosomal biogenesis was observed in the injury epicenter at 24 h after trauma. Such upregulation coincided with endoplasmic reticulum stress response as previously reported in this model. It was also accompanied by changes in expression of many other genes associated with translational regulation. Systemic treatment with a pharmacological inhibitor of RNA-Polymerase-1, BMH-21 reduced rRNA transcription in the spinal cord. Moreover, in the injury epicenter, treatment with BMH-21 increased expression of oligodendrocyte-specific transcripts including Mbp and Cldn11 at 3 days post injury. Although such findings may suggest at least transient reduction of oligodendrocyte death, locomotor outcome was mostly unaffected except slightly accelerated recovery of hindlimb function at week 2 post-injury. Therefore, at least in mice, RNA-Polymerase-1 does not appear to be a robust target for therapies to protect spinal cord tissue after contusion. However, these findings raise an interesting possibility that altered rate of ribosomal biogenesis contributes to the apparent translational reprogramming after contusive SCI. Such a reprogramming could be a major regulator of SCI-induced gene expression.

The nucleolus is a subcellular compartment with a key essential function in ribosome biogenesis. The nucleolus is rich in noncoding RNAs, mostly the ribosomal RNAs and small nucleolar RNAs. Surprisingly, also several miRNAs have been detected in the nucleolus, raising the question as to whether other small RNA species are present and functional in the nucleolus. We have developed a strategy for stepwise enrichment of nucleolar small RNAs from the total nucleolar RNA extracts and subsequent construction of nucleolar small RNA libraries which are suitable for deep sequencing. Our method successfully isolates the small RNA population from total RNAs and monitors the RNA quality in each step to ensure that small RNAs recovered represent the actual small RNA population in the nucleolus and not degradation products from larger RNAs. We have further applied this approach to characterize the distribution of small RNAs in different cellular compartments.

Small RNAs (size 20-30 nt) of various types have been actively investigated in recent years, and their subcellular compartmentalization and relative concentrations are likely to be of importance to their cellular and physiological functions. Comprehensive data on this subset of the transcriptome can only be obtained by application of high-throughput sequencing, which yields data that are inherently complex and multidimensional, as sequence composition, length, and abundance will all inform to the small RNA function. Subsequent data analysis, hypothesis testing, and presentation/visualization of the results are correspondingly challenging. We have constructed small RNA libraries derived from different cellular compartments, including the nucleolus, and asked whether small RNAs exist in the nucleolus and whether they are distinct from cytoplasmic and nuclear small RNAs, the miRNAs. Here, we present a workflow for analysis of small RNA sequencing data generated by the Ion Torrent PGM sequencer from samples derived from different cellular compartments.

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 (TMAs) 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 pharmacological 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 prostatic intraepithelial neoplasia/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 pathological 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.

The nucleolus is a subcellular compartment with a key essential function in ribosome biogenesis. The nucleolus is rich in noncoding RNAs, mostly the ribosomal RNAs and small nucleolar RNAs. Surprisingly, also several miRNAs have been detected in the nucleolus, raising the question as to whether other small RNA species are present and functional in the nucleolus. We have developed a strategy for stepwise enrichment of nucleolar small RNAs from the total nucleolar RNA extracts and subsequent construction of nucleolar small RNA libraries which are suitable for deep sequencing. Our method successfully isolates the small RNA population from total RNAs and monitors the RNA quality in each step to ensure that small RNAs recovered represent the actual small RNA population in the nucleolus and not degradation products from larger RNAs. We have further applied this approach to characterize the distribution of small RNAs in different cellular compartments.

Small RNAs (size 20-30 nt) of various types have been actively investigated in recent years, and their subcellular compartmentalization and relative concentrations are likely to be of importance to their cellular and physiological functions. Comprehensive data on this subset of the transcriptome can only be obtained by application of high-throughput sequencing, which yields data that are inherently complex and multidimensional, as sequence composition, length, and abundance will all inform to the small RNA function. Subsequent data analysis, hypothesis testing, and presentation/visualization of the results are correspondingly challenging. We have constructed small RNA libraries derived from different cellular compartments, including the nucleolus, and asked whether small RNAs exist in the nucleolus and whether they are distinct from cytoplasmic and nuclear small RNAs, the miRNAs. Here, we present a workflow for analysis of small RNA sequencing data generated by the Ion Torrent PGM sequencer from samples derived from different cellular compartments.

Efficient extraction and accurate quantification of nucleolar macromolecules are critical for in vitro analysis, especially for studying RNA, DNA, and protein dynamics under identical conditions. There is presently no single method that efficiently and simultaneously isolates these three macromolecular constituents from purified nucleoli. We have developed an optimized method, which without evident loss, extracts, and solubilizes protein recovered from a single sample following TRIzol isolation of RNA and DNA. The solubilized protein can be accurately quantified by protein bicinchoninic acid assay and assessed by polyacrylamide gel electrophoresis. We have successfully applied this approach to extract and quantify all three nucleolar components, and to study nucleolar protein responses after actinomycin D treatment.