Splicing targeting drugs highlight intron retention as an actionable vulnerability in advanced prostate cancer.

BACKGROUND: Advanced prostate cancer (PC) is characterized by insensitivity to androgen deprivation therapy and chemotherapy, resulting in poor outcome for most patients. Thus, advanced PC urgently needs novel therapeutic strategies. Mounting evidence points to splicing dysregulation as a hallmark of advanced PC. Moreover, pharmacologic inhibition of the splicing process is emerging as a promising option for this disease. METHOD: By using a representative androgen-insensitive PC cell line (22Rv1), we have investigated the genome-wide transcriptomic effects underlying the cytotoxic effects exerted by three splicing-targeting drugs: Pladienolide B, indisulam and THZ531. Bioinformatic analyses were performed to uncover the gene structural features underlying sensitivity to transcriptional and splicing regulation by these treatments. Biological pathways altered by these treatments were annotated by gene ontology analyses and validated by functional experiments in cell models. RESULTS: Although eliciting similar cytotoxic effects on advanced PC cells, Pladienolide B, indisulam and THZ531 modulate specific transcriptional and splicing signatures. Drug sensitivity is associated with distinct gene structural features, expression levels and cis-acting sequence elements in the regulated exons and introns. Importantly, we identified PC-relevant genes (i.e. EZH2, MDM4) whose drug-induced splicing alteration exerts an impact on cell survival. Moreover, computational analyses uncovered a widespread impact of splicing-targeting drugs on intron retention, with enrichment in genes implicated in pre-mRNA 3'-end processing (i.e. CSTF3, PCF11). Coherently, advanced PC cells displayed high sensitivity to a specific inhibitor of the cleavage and polyadenylation complex, which enhances the effects of chemotherapeutic drugs that are already in use for this cancer. CONCLUSIONS: Our study uncovers intron retention as an actionable vulnerability for advanced PC, which may be exploited to improve therapeutic management of this currently incurable disease.

Dual inhibition of CDK12 and CDK13 uncovers actionable vulnerabilities in patient-derived ovarian cancer organoids.

BACKGROUND: High grade serous ovarian cancer (HGSOC) is highly lethal, partly due to chemotherapy resistance and limited availability of targeted approaches. Cyclin dependent kinases 12 and 13 (CDK12/13) are promising therapeutic targets in human cancers, including HGSOC. Nevertheless, the effects of their inhibition in HGSOC and the potential synergy with other drugs are poorly known. METHODS: We analyzed the effects of the CDK12/13 inhibitor THZ531 in HGSOC cells and patient-derived organoids (PDOs). RNA sequencing and quantitative PCR analyses were performed to identify the genome-wide effects of short-term CDK12/13 inhibition on the transcriptome of HGSOC cells. Viability assays with HGSOC cells and PDOs were performed to assess the efficacy of THZ531 as single agent or in combination with clinically relevant drugs. RESULTS: The CDK12 and CDK13 genes are deregulated in HGSOC and their concomitant up-regulation with the oncogene MYC predicts poor prognosis. HGSOC cells and PDOs display high sensitivity to CDK12/13 inhibition, which synergizes with drugs in clinical use for HGSOC. Transcriptome analyses revealed cancer-relevant genes whose expression is repressed by dual CDK12/13 inhibition through impaired splicing. Combined treatment with THZ531 and inhibitors of pathways regulated by these cancer relevant genes (EGFR, RPTOR, ATRIP) exerted synergic effects on HGSOC PDO viability. CONCLUSIONS: CDK12 and CDK13 represent valuable therapeutic targets for HGSOC. We uncovered a wide spectrum of CDK12/13 targets as potential therapeutic vulnerabilities for HGSOC. Moreover, our study indicates that CDK12/13 inhibition enhances the efficacy of approved drugs that are already in use for HGSOC or other human cancers.

The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer.

Alternative polyadenylation (APA) yields transcripts differing in their 3'-end, and its regulation is altered in cancer, including prostate cancer. Here we have uncovered a mechanism of APA regulation impinging on the interaction between the exonuclease XRN2 and the RNA-binding protein Sam68, whose increased expression in prostate cancer is promoted by the transcription factor MYC. Genome-wide transcriptome profiling revealed a widespread impact of the Sam68/XRN2 complex on APA. XRN2 promotes recruitment of Sam68 to its target transcripts, where it competes with the cleavage and polyadenylation specificity factor for binding to strong polyadenylation signals at distal ends of genes, thus promoting usage of suboptimal proximal polyadenylation signals. This mechanism leads to 3' untranslated region shortening and translation of transcripts encoding proteins involved in G1/S progression and proliferation. Thus, our findings indicate that the APA program driven by Sam68/XRN2 promotes cell cycle progression and may represent an actionable target for therapeutic intervention.

DNA Damage Regulates the Functions of the RNA Binding Protein Sam68 through ATM-Dependent Phosphorylation.

Cancer cells frequently exhibit dysregulation of the DNA damage response (DDR), genomic instability, and altered RNA metabolism. Recent genome-wide studies have strongly suggested an interaction between the pathways involved in the cellular response to DDR and in the regulation of RNA metabolism, but the molecular mechanism(s) involved in this crosstalk are largely unknown. Herein, we found that activation of the DDR kinase ATM promotes its interaction with Sam68, leading to phosphorylation of this multifunctional RNA binding protein (RBP) on three residues: threonine 61, serine 388 and serine 390. Moreover, we demonstrate that ATM-dependent phosphorylation of threonine 61 promotes the function of Sam68 in the DDR pathway and enhances its RNA processing activity. Importantly, ATM-mediated phosphorylation of Sam68 in prostate cancer cells modulates alternative polyadenylation of transcripts that are targets of Sam68, supporting the notion that the ATM-Sam68 axis exerts a multifaceted role in the response to DNA damage. Thus, our work validates Sam68 as an ATM kinase substrate and uncovers an unexpected bidirectional interplay between ATM and Sam68, which couples the DDR pathway to modulation of RNA metabolism in response to genotoxic stress.

The androgen receptor couples promoter recruitment of RNA processing factors to regulation of alternative polyadenylation at the 3' end of transcripts.

Prostate cancer (PC) relies on androgen receptor (AR) signaling. While hormonal therapy (HT) is efficacious, most patients evolve to an incurable castration-resistant stage (CRPC). To date, most proposed mechanisms of acquired resistance to HT have focused on AR transcriptional activity. Herein, we uncover a new role for the AR in alternative cleavage and polyadenylation (APA). Inhibition of the AR by Enzalutamide globally regulates APA in PC cells, with specific enrichment in genes related to transcription and DNA topology, suggesting their involvement in transcriptome reprogramming. AR inhibition selects promoter-distal polyadenylation sites (pAs) enriched in cis-elements recognized by the cleavage and polyadenylation specificity factor (CPSF) complex. Conversely, promoter-proximal intronic pAs relying on the cleavage stimulation factor (CSTF) complex are repressed. Mechanistically, Enzalutamide induces rearrangement of APA subcomplexes and impairs the interaction between CPSF and CSTF. AR inhibition also induces co-transcriptional CPSF recruitment to gene promoters, predisposing the selection of pAs depending on this complex. Importantly, the scaffold CPSF160 protein is up-regulated in CRPC cells and its depletion represses HT-induced APA patterns. These findings uncover an unexpected role for the AR in APA regulation and suggest that APA-mediated transcriptome reprogramming represents an adaptive response of PC cells to HT.

Functional Interaction Between the Oncogenic Kinase NEK2 and Sam68 Promotes a Splicing Program Involved in Migration and Invasion in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents the most aggressive breast cancer subtype. Poor prognosis in TNBC is partly due to lack of efficacious targeted therapy and high propensity to metastasize. Dysregulation of alternative splicing has recently emerged as a trait of TNBC, suggesting that unveiling the molecular mechanisms underlying its regulation could uncover new druggable cancer vulnerabilities. The oncogenic kinase NEK2 is significantly upregulated in TNBC and contributes to shaping their unique splicing profile. Herein, we found that NEK2 interacts with the RNA binding protein Sam68 in TNBC cells and that NEK2-mediated phosphorylation of Sam68 enhances its splicing activity. Genome-wide transcriptome analyses identified the splicing targets of Sam68 in TNBC cells and revealed a common set of exons that are co-regulated by NEK2. Functional annotation of splicing-regulated genes highlighted cell migration and spreading as biological processes regulated by Sam68. Accordingly, Sam68 depletion reduces TNBC cell migration and invasion, and these effects are potentiated by the concomitant inhibition of NEK2 activity. Our findings indicate that Sam68 and NEK2 functionally cooperate in the regulation of a splicing program that sustains the pro-metastatic features of TNBC cells.

Analysis of Secreted Proteins from Prepubertal Ovarian Tissues Exposed In Vitro to Cisplatin and LH.

It is well known that secreted and exosomal proteins are associated with a broad range of physiological processes involving tissue homeostasis and differentiation. In the present paper, our purpose was to characterize the proteome of the culture medium in which the oocytes within the primordial/primary follicles underwent apoptosis induced by cisplatin (CIS) or were, for the most part, protected by LH against the drug. To this aim, prepubertal ovarian tissues were cultured under control and in the presence of CIS, LH, and CIS + LH. The culture media were harvested after 2, 12, and 24 h from chemotherapeutic drug treatment and analyzed by liquid chromatography-mass spectrometry (LC-MS). We found that apoptotic conditions generated by CIS in the cultured ovarian tissues and/or oocytes are reflected in distinct changes in the extracellular microenvironment in which they were cultured. These changes became evident mainly from 12 h onwards and were characterized by the inhibition or decreased release of a variety of compounds, such as the proteases Htra1 and Prss23, the antioxidants Prdx2 and Hbat1, the metabolic regulators Ldha and Pkm, and regulators of apoptotic pathways such as Tmsb4x. Altogether, these results confirm the biological relevance of the LH action on prepuberal ovaries and provide novel information about the proteins released by the ovarian tissues exposed to CIS and LH in the surrounding microenvironment. These data might represent a valuable resource for future studies aimed to clarify the effects and identify biomarkers of these compounds' action on the developing ovary.

Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways.

Despite germ cell tumors (GCTs) responding to cisplatin-based chemotherapy at a high rate, a subset of patients does not respond to treatment and have significantly worse prognosis. The biological mechanisms underlying the resistance remain unknown. In this study, by using two TGCT cell lines that have acquired cisplatin resistance after chronic exposure to the drug, we identified some key proteins and mechanisms of acquired resistance. We show that cisplatin-resistant cell lines had a non-homologous end-joining (NHEJ)-less phenotype. This correlated with a reduced basal expression of TP53-binding protein 1 (53BP1) and DNA-dependent protein kinase (DNA-PKcs) proteins and reduced formation of 53BP1 foci after cisplatin treatment. Consistent with these observations, modulation of 53BP1 protein expression altered the cell line's resistance to cisplatin, and inhibition of DNA-PKcs activity antagonized cisplatin cytotoxicity. Dampening of NHEJ was accompanied by a functional increase in the repair of DNA double-strand breaks (DSBs) by the homologous recombination repair pathway. As a result, cisplatin-resistant cells were more resistant to PARP inhibitor (PARPi) monotherapy. Moreover, when PARPi was given in combination with cisplatin, it exerted an additive/synergistic effect, and reduced the cisplatin dose for cytotoxicity. These results suggest that treatment of cisplatin-refractory patients may benefit from low-dose cisplatin therapy combined with PARPi.

Assessing Homologous Recombination and Interstrand Cross-Link Repair in Embryonal Carcinoma Testicular Germ Cell Tumor Cell Lines.

Testicular germ cell tumors (TGCTs) are typically exquisitely sensitive to DNA interstrand cross-link (ICLs) agents. ICLs covalently link both strands of the DNA duplex, impeding fundamental cellular processes like DNA replication to cause cell death. A leading drug used for the treatment of TGCTs is cisplatin, which introduces ICLs and leads to formation of double strand breaks (DSBs), a DNA lesion that can be repaired in the S/G2 phases of the cell cycle by homologous recombination (HR, also termed homology-direct repair). Although most TGCTs respond to cisplatin-induced ICLs, a fraction is resistant to treatment. One proposed mechanism of TGCT resistance to cisplatin is an enhanced ability to repair DSBs by HR. Other than HR, repair of the ICL-lesions requires additional DNA repair mechanisms, whose action might also be implemented in therapy-resistant cells. This chapter describes GFP assays to measure (a) HR proficiency following formation of a DSB by the endonuclease I-SceI, and (b) HR repair induced by site-specific ICL formation involving psoralen. These experimental approaches can be used to determine the proficiency of TGCT cell lines in DSB repair by HR in comparison to HR repair of ICLs, providing tools to better characterize their recombination profile. Protocols of these assays have been adapted for use in Embryonal Carcinoma (EC) TGCT cell lines. Assays only require transient introduction of plasmids within cells, affording the advantage of testing multiple cell lines in a relatively short time.

Sempervirine inhibits RNA polymerase I transcription independently from p53 in tumor cells.

In the search of small molecules that can target MDM2/p53 pathway in testicular germ cell tumors (TGCTs), we identified sempervirine (2,3,4,13-tetrahydro-1H-benz[g]indolo[2,3-a]quinolizin-6-ium), an alkaloid of Gelsemium sempervirens, that has been previously proposed as an inhibitor of MDM2 that targets p53-wildtype (wt) tumor cells. We found that sempervirine not only affects cell growth of p53-wt cancer cells, but it is also active in p53-mutated and p53-null cells by triggering p53-dependent and independent pathways without affecting non-transformed cells. To understand which mechanism/s could be activated both in p53-wt and -null cells, we found that sempervirine induced nucleolar remodeling and nucleolar stress by reducing protein stability of RPA194, the catalytic subunit of RNA polymerase I, that led to rRNA synthesis inhibition and to MDM2 block. As shown for other cancer cell models, MDM2 inhibition by nucleolar stress downregulated E2F1 protein levels both in p53-wt and p53-null TGCT cells with the concomitant upregulation of unphosphorylated pRb. Finally, we show that sempervirine is able to enter the nucleus and accumulates within the nucleolus where it binds rRNA without causing DNA damage. Our results identify semperivirine as a novel rRNA synthesis inhibitor and indicate this drug as a non-genotoxic anticancer small molecule.

Splicing Dysregulation as Oncogenic Driver and Passenger Factor in Brain Tumors.

Brain tumors are a heterogeneous group of neoplasms ranging from almost benign to highly aggressive phenotypes. The malignancy of these tumors mostly relies on gene expression reprogramming, which is frequently accompanied by the aberrant regulation of RNA processing mechanisms. In brain tumors, defects in alternative splicing result either from the dysregulation of expression and activity of splicing factors, or from mutations in the genes encoding splicing machinery components. Aberrant splicing regulation can generate dysfunctional proteins that lead to modification of fundamental physiological cellular processes, thus contributing to the development or progression of brain tumors. Herein, we summarize the current knowledge on splicing abnormalities in brain tumors and how these alterations contribute to the disease by sustaining proliferative signaling, escaping growth suppressors, or establishing a tumor microenvironment that fosters angiogenesis and intercellular communications. Lastly, we review recent efforts aimed at developing novel splicing-targeted cancer therapies, which employ oligonucleotide-based approaches or chemical modulators of alternative splicing that elicit an impact on brain tumor biology.

c-MYC empowers transcription and productive splicing of the oncogenic splicing factor Sam68 in cancer.

The splicing factor Sam68 is upregulated in many human cancers, including prostate cancer (PCa) where it promotes cell proliferation and survival. Nevertheless, in spite of its frequent upregulation in cancer, the mechanism(s) underlying its expression are largely unknown. Herein, bioinformatics analyses identified the promoter region of the Sam68 gene (KHDRBS1) and the proto-oncogenic transcription factor c-MYC as a key regulator of Sam68 expression. Upregulation of Sam68 and c-MYC correlate in PCa patients. c-MYC directly binds to and activates the Sam68 promoter. Furthermore, c-MYC affects productive splicing of the nascent Sam68 transcript by modulating the transcriptional elongation rate within the gene. Importantly, c-MYC-dependent expression of Sam68 is under the tight control of external cues, such as androgens and/or mitogens. These findings uncover an unexpected coordination of transcription and splicing of Sam68 by c-MYC, which may represent a key step in PCa tumorigenesis.

H2AFX and MDC1 promote maintenance of genomic integrity in male germ cells.

In somatic cells, H2afx and Mdc1 are close functional partners in DNA repair and damage response. However, it is not known whether they are also involved in the maintenance of genome integrity in meiosis. By analyzing chromosome dynamics in H2afx-/- spermatocytes, we found that the synapsis of autosomes and X-Y chromosomes was impaired in a fraction of cells. Such defects correlated with an abnormal recombination profile. Conversely, Mdc1 was dispensable for the synapsis of the autosomes and played only a minor role in X-Y synapsis, compared with the action of H2afx This suggested that those genes have non-overlapping functions in chromosome synapsis. However, we observed that both genes play a similar role in the assembly of MLH3 onto chromosomes, a key step in crossover formation. Moreover, we show that H2afx and Mdc1 cooperate in promoting the activation of the recombination-dependent checkpoint, a mechanism that restrains the differentiation of cells with unrepaired DSBs. This occurs by a mechanism that involves P53. Overall, our data show that, in male germ cells, H2afx and Mdc1 promote the maintenance of genome integrity.This article has an associated First Person interview with the first author of the paper.

A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons.

Neurodegeneration associated with amyloid ß (Aß) peptide accumulation, synaptic loss, and memory impairment are pathophysiological features of Alzheimer's disease (AD). Numerous microRNAs regulate amyloid precursor protein (APP) expression and metabolism. We previously reported that miR-101 is a negative regulator of APP expression in cultured hippocampal neurons. In this study, a search for predicted APP metabolism-associated miR-101 targets led to the identification of a conserved miR-101 binding site within the 3' untranslated region (UTR) of the mRNA encoding Ran-binding protein 9 (RanBP9). RanBP9 increases APP processing by ß-amyloid converting enzyme 1 (BACE1), secretion of soluble APPß (sAPPß), and generation of Aß. MiR-101 significantly reduced reporter gene expression when co-transfected with a RanBP9 3'-UTR reporter construct, while site-directed mutagenesis of the predicted miR-101 target site eliminated the reporter response. To investigate the effect of stable inhibition of miR-101 both in vitro and in vivo, a microRNA sponge was developed to bind miR-101 and derepress its targets. Four tandem bulged miR-101 responsive elements (REs), located downstream of the enhanced green fluorescence protein (EGFP) open reading frame and driven by the synapsin promoter, were placed in a lentiviral vector to create the pLSyn-miR-101 sponge. Delivery of the sponge to primary hippocampal neurons significantly increased both APP and RanBP9 expression, as well as sAPPß levels in the conditioned medium. Importantly, silencing of endogenous RanBP9 reduced sAPPß levels in miR-101 sponge-containing hippocampal cultures, indicating that miR-101 inhibition may increase amyloidogenic processing of APP by RanBP9. Lastly, the impact of miR-101 on its targets was demonstrated in vivo by intrahippocampal injection of the pLSyn-miR-101 sponge into C57BL6 mice. This study thus provides the basis for studying the consequences of long-term miR-101 inhibition on the pathology of AD.

Drug delivery by polymeric micelles: an in vitro and in vivo study to deliver lipophilic substances to colonocytes and selectively target inflamed colon.

Colitis is the term used for chronic inflammatory bowel diseases at substantially increased risk of developing a form of colorectal cancer (CRC) known as colitis-associated cancer. In our study we synthesized core-shell polymeric micelles obtained by self-assembly of block copolymers for high efficiency delivery of anti-inflammatory and anti-cancer compounds to colonocytes and colon mucosa. We achieved an efficient intracellular delivery of these hydrophobic compounds (prednisone, retinoic acid and doxorubicin) to cultured colonocytes without cellular toxicity. The efficacy of retinoic acid and doxorubicin administration was significantly increased using these nanosized carriers. Moreover, these polymeric micelles have been shown to overcome the multidrug resistance efflux mechanism effectively delivering doxorubicin to multidrug-resistant colon cancer cells. These nanocarriers are also suitable for selective in vivo delivery of lipophilic drugs by enema administration to the inflamed colon tissue, specifically targeting the inflamed mucosa. FROM THE CLINICAL EDITOR: This team of investigators studied polymeric micelles as highly efficient drug delivery systems enabling intracellular delivery of hydrophobic compounds (prednisone, retinoic acid, and doxorubicin) to cultured colonocytes without cellular toxicity, also demonstrating beneficial in vivo effects.

Loss of miR-101 expression promotes Wnt/ß-catenin signalling pathway activation and malignancy in colon cancer cells.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in Western countries. Although the aberrant expression of several microRNAs (oncomiRs) is associated with CRC progression, the molecular mechanisms of this phenomenon are still under investigation. Here we show that miR-101 expression is differentially impaired in CRC specimens, depending on tumour grade. miR-101 re-expression suppresses cell growth in 3D, hypoxic survival and invasive potential in CRC cells showing low levels of miR-101. Additionally, we provide molecular evidence of a bidirectional regulatory mechanism between miR-101 expression and important CRC pro-malignant features, such as inflammation, activation of the Wnt/ß-catenin signalling pathway and epithelial-mesenchymal transition (EMT). We then propose that up-regulated miR-101 may function as a tumour suppressor in CRC and that its pharmacological restoration might hamper the aggressive behaviour of CRC in vivo. MiR-101 expression may also represent a cancer biomarker for CRC diagnosis and prognosis.