Targeting the ERG oncogene with splice-switching oligonucleotides as a novel therapeutic strategy in prostate cancer.

BACKGROUND: The ERG oncogene, a member of the ETS family of transcription factor encoding genes, is a genetic driver of prostate cancer. It is activated through a fusion with the androgen-responsive TMPRSS2 promoter in 50% of cases. There is therefore significant interest in developing novel therapeutic agents that target ERG. We have taken an antisense approach and designed morpholino-based oligonucleotides that target ERG by inducing skipping of its constitutive exon 4. METHODS: We designed antisense morpholino oligonucleotides (splice-switching oligonucleotides, SSOs) that target both the 5' and 3' splice sites of ERG's exon 4. We tested their efficacy in terms of inducing exon 4 skipping in two ERG-positive cell lines, VCaP prostate cancer cells and MG63 osteosarcoma cells. We measured their effect on cell proliferation, migration and apoptosis. We also tested their effect on xenograft tumour growth in mice and on ERG protein expression in a human prostate cancer radical prostatectomy sample ex vivo. RESULTS: In VCaP cells, both SSOs were effective at inducing exon 4 skipping, which resulted in a reduction of overall ERG protein levels up to 96 h following a single transfection. SSO-induced ERG reduction decreased cell proliferation, cell migration and significantly increased apoptosis. We observed a concomitant reduction in protein levels for cyclin D1, c-Myc and the Wnt signalling pathway member ß-catenin as well as a marker of activated Wnt signalling, p-LRP6. We tested the 3' splice site SSO in MG63 xenografts in mice and observed a reduction in tumour growth. We also demonstrated that the 3' splice site SSO caused a reduction in ERG expression in a patient-derived prostate tumour tissue cultured ex vivo. CONCLUSIONS: We have successfully designed and tested morpholino-based SSOs that cause a marked reduction in ERG expression, resulting in decreased cell proliferation, a reduced migratory phenotype and increased apoptosis. Our initial tests on mouse xenografts and a human prostate cancer radical prostatectomy specimen indicate that SSOs can be effective for oncogene targeting in vivo. As such, this study encourages further in vivo therapeutic studies using SSOs targeting the ERG oncogene.

Pharmacology of Modulators of Alternative Splicing.

More than 95% of genes in the human genome are alternatively spliced to form multiple transcripts, often encoding proteins with differing or opposing function. The control of alternative splicing is now being elucidated, and with this comes the opportunity to develop modulators of alternative splicing that can control cellular function. A number of approaches have been taken to develop compounds that can experimentally, and sometimes clinically, affect splicing control, resulting in potential novel therapeutics. Here we develop the concepts that targeting alternative splicing can result in relatively specific pathway inhibitors/activators that result in dampening down of physiologic or pathologic processes, from changes in muscle physiology to altering angiogenesis or pain. The targets and pharmacology of some of the current inhibitors/activators of alternative splicing are demonstrated and future directions discussed.

Targeting Angiogenesis in Prostate Cancer.

Prostate cancer is the most commonly diagnosed cancer among men in the Western world. Although localized disease can be effectively treated with established surgical and radiopharmaceutical treatments options, the prognosis of castration-resistant advanced prostate cancer is still disappointing. The objective of this study was to review the role of angiogenesis in prostate cancer and to investigate the effectiveness of anti-angiogenic therapies. A literature search of clinical trials testing the efficacy of anti-angiogenic therapy in prostate cancer was performed using Pubmed. Surrogate markers of angiogenic activity (microvessel density and vascular endothelial growth factor A (VEGF-A) expression) were found to be associated with tumor grade, metastasis, and prognosis. Six randomizedstudies were included in this review: two phase II trials on localized and hormone-sensitive disease (n = 60 and 99 patients) and four phase III trials on castration-resistant refractory disease (n = 873 to 1224 patients). Although the phase II trials showed improved relapse-free survival and stabilisation of the disease, the phase III trials found increased toxicity and no significant improvement in overall survival. Although angiogenesis appears to have an important role in prostate cancer, the results of anti-angiogenic therapy in castration-resistant refractory disease have hitherto been disappointing. There are various possible explanations for this lack of efficacy in castration-resistant refractory disease: redundancy of angiogenic pathways, molecular heterogeneity of the disease, loss of tumor suppressor protein phosphatase and tensin homolog (PTEN) expression as well as various VEGF-A splicing isoforms with pro- and anti-angiogenic activity. A better understanding of the molecular mechanisms of angiogenesis may help to develop effective anti-angiogenic therapy in prostate cancer.

Alternative Splicing in Angiogenesis.

Alternative splicing of pre-mRNA allows the generation of multiple splice isoforms from a given gene, which can have distinct functions. In fact, splice isoforms can have opposing functions and there are many instances whereby a splice isoform acts as an inhibitor of canonical isoform function, thereby adding an additional layer of regulation to important processes. Angiogenesis is an important process that is governed by alternative splicing mechanisms. This review focuses on the alternative spliced isoforms of key genes that are involved in the angiogenesis process; VEGF-A, VEGFR1, VEGFR2, NRP-1, FGFRs, Vasohibin-1, Vasohibin-2, HIF-1α, Angiopoietin-1 and Angiopoietin-2.

The many faces of SRPK1.

Serine-arginine protein kinase 1 (SRPK1) phosphorylates proteins involved in the regulation of several mRNA-processing pathways, including alternative splicing. SRPK1 has been recently reported to be overexpressed in multiple cancers, including prostate cancer, breast cancer, lung cancer, and glioma. Several studies have shown that inhibition of SRPK1 has anti-tumoural effects, and SRPK1 has therefore become a new candidate for targeted therapies. Interestingly, in terms of molecular mechanism, SRPK1 seems to act heterogeneously, and has been reported to affect several processes in different cancers, e.g. angiogenesis in prostate and colon cancer, apoptosis in breast and colon cancer, and migration in breast cancer. A recent report adds to this puzzle, showing that the main effect of SRPK1 overexpression in non-small-cell lung carcinoma is to stimulate a stem cell-like phenotype. This pleiotropy might be related to preferential activation of different downstream signalling pathways by SRPK1 in various cancers. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Differential Expression of VEGF-Axxx Isoforms Is Critical for Development of Pulmonary Fibrosis.

RATIONALE: Fibrosis after lung injury is related to poor outcome, and idiopathic pulmonary fibrosis (IPF) can be regarded as an exemplar. Vascular endothelial growth factor (VEGF)-A has been implicated in this context, but there are conflicting reports as to whether it is a contributory or protective factor. Differential splicing of the VEGF-A gene produces multiple functional isoforms including VEGF-A165a and VEGF-A165b, a member of the inhibitory family. To date there is no clear information on the role of VEGF-A in IPF. OBJECTIVES: To establish VEGF-A isoform expression and functional effects in IPF. METHODS: We used tissue sections, plasma, and lung fibroblasts from patients with IPF and control subjects. In a bleomycin-induced lung fibrosis model we used wild-type MMTV mice and a triple transgenic mouse SPC-rtTA+/-TetoCre+/-LoxP-VEGF-A+/+ to conditionally induce VEGF-A isoform deletion specifically in the alveolar type II (ATII) cells of adult mice. MEASUREMENTS AND MAIN RESULTS: IPF and normal lung fibroblasts differentially expressed and responded to VEGF-A165a and VEGF-A165b in terms of proliferation and matrix expression. Increased VEGF-A165b was detected in plasma of progressing patients with IPF. In a mouse model of pulmonary fibrosis, ATII-specific deficiency of VEGF-A or constitutive overexpression of VEGF-A165b inhibited the development of pulmonary fibrosis, as did treatment with intraperitoneal delivery of VEGF-A165b to wild-type mice. CONCLUSIONS: These results indicate that changes in the bioavailability of VEGF-A sourced from ATII cells, namely the ratio of VEGF-Axxxa to VEGF-Axxxb, are critical in development of pulmonary fibrosis and may be a paradigm for the regulation of tissue repair.

VEGF-A165 b protects against proteinuria in a mouse model with progressive depletion of all endogenous VEGF-A splice isoforms from the kidney.

KEY POINTS: Progressive depletion of all vascular endothelial growth factor A (VEGF-A) splice isoforms from the kidney results in proteinuria and increased glomerular water permeability, which are both rescued by over-expression of VEGF-A165 b only. VEGF-A165 b rescues the increase in glomerular basement membrane and podocyte slit width, as well as the decrease in sub-podocyte space coverage, produced by VEGF-A depletion. VEGF-A165 b restores the expression of platelet endothelial cell adhesion molecule in glomerular endothelial cells and glomerular capillary circumference. VEGF-A165 b has opposite effects to VEGF-A165 on the expression of genes involved in endothelial cell migration and proliferation. ABSTRACT: Chronic kidney disease is strongly associated with a decrease in the expression of vascular endothelial growth factor A (VEGF-A). However, little is known about the contribution of VEGF-A splice isoforms to kidney physiology and pathology. Previous studies suggest that the splice isoform VEGF-A165 b (resulting from alternative usage of a 3' splice site in the terminal exon) is protective for kidney function. In the present study, we show, in a quad-transgenic model, that over-expression of VEGF-A165 b alone is sufficient to rescue the increase in proteinuria, as well as glomerular water permeability, in the context of progressive depletion of all VEGF-A isoforms from the podocytes. Ultrastructural studies show that the glomerular basement membrane is thickened, podocyte slit width is increased and sub-podocyte space coverage is reduced when VEGF-A is depleted, all of which are rescued in VEGF-A165 b over-expressors. VEGF-A165 b restores the expression of platelet endothelial cell adhesion molecule-1 in glomerular endothelial cells and glomerular capillary circumference. Mechanistically, it increases VEGF receptor 2 expression both in vivo and in vitro and down-regulates genes involved in migration and proliferation of endothelial cells, otherwise up-regulated by the canonical isoform VEGF-A165 . The results of the present study indicate that manipulation of VEGF-A splice isoforms could be a novel therapeutic avenue in chronic glomerular disease.

Alternative Splicing in CKD.

Alternative splicing (AS) has emerged in the postgenomic era as one of the main drivers of proteome diversity, with ≥94% of multiexon genes alternatively spliced in humans. AS is therefore one of the main control mechanisms for cell phenotype, and is a process deregulated in disease. Numerous reports describe pathogenic mutations in splice factors, splice sites, or regulatory sequences. Additionally, compared with the physiologic state, disease often associates with an abnormal proportion of splice isoforms (or novel isoforms), without an apparent driver mutation. It is therefore essential to study how AS is regulated in physiology, how it contributes to pathogenesis, and whether we can manipulate faulty splicing for therapeutic advantage. Although the disease most commonly linked to deregulation of AS in several genes is cancer, many reports detail pathogenic splice variants in diseases ranging from neuromuscular disorders to diabetes or cardiomyopathies. A plethora of splice variants have been implicated in CKDs as well. In this review, we describe examples of these CKD-associated splice variants and ideas on how to manipulate them for therapeutic benefit.

SRPK1 inhibition in prostate cancer: A novel anti-angiogenic treatment through modulation of VEGF alternative splicing.

Prostate cancer remains one of the leading causes of cancer death in men around the world, regardless of intense research and development of novel therapies in the last 10 years. One of the new avenues that has been tested - inhibition of angiogenesis - has been disappointing so far in clinical studies in spite of strong evidence that determinants of angiogenesis (e.g. vascular endothelial growth factor) are strongly associated with disease progression. One of the reasons for these outcomes may be our poor understanding of the biology of angiogenesis in prostate cancer (and probably other cancers as well) resulting in inhibition of both detrimental and favourable molecules. We discuss here novel targeted and more specific approaches to inhibit angiogenesis in prostate cancer as well as a completely new therapeutic modality to do this - modulation of alternative splicing - that may be applicable to other molecules/biological processes as well.

Vascular Endothelial Growth Factor-A165b Is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy.

Diabetic nephropathy is the leading cause of ESRD in high-income countries and a growing problem across the world. Vascular endothelial growth factor-A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, yet VEGF-A knockout and overexpression of angiogenic VEGF-A isoforms each worsen diabetic nephropathy. We examined the vasculoprotective effects of the VEGF-A isoform VEGF-A165b in diabetic nephropathy. Renal expression of VEGF-A165b mRNA was upregulated in diabetic individuals with well preserved kidney function, but not in those with progressive disease. Reproducing this VEGF-A165b upregulation in mouse podocytes in vivo prevented functional and histologic abnormalities in diabetic nephropathy. Biweekly systemic injections of recombinant human VEGF-A165b reduced features of diabetic nephropathy when initiated during early or advanced nephropathy in a model of type 1 diabetes and when initiated during early nephropathy in a model of type 2 diabetes. VEGF-A165b normalized glomerular permeability through phosphorylation of VEGF receptor 2 in glomerular endothelial cells, and reversed diabetes-induced damage to the glomerular endothelial glycocalyx. VEGF-A165b also improved the permeability function of isolated diabetic human glomeruli. These results show that VEGF-A165b acts via the endothelium to protect blood vessels and ameliorate diabetic nephropathy.

Regulation of Epithelial-Mesenchymal Transitions by Alternative Splicing: Potential New Area for Cancer Therapeutics.

The epithelial-mesenchymal transition (EMT) is a complicated biological process in which cells with epithelial phenotype are transformed into mesenchymal cells with loss of cell polarity and cell-cell adhesion and gain of the ability to migrate. EMT and the reverse mesenchymal-epithelial transitions (METs) are present during cancer progression and metastasis. Using the dynamic switch between EMT and MET, tumour cells can migrate to neighbouring organs or metastasize in the distance and develop resistance to traditional chemotherapy and targeted drug treatments. Growing evidence shows that reversing or inhibiting EMT may be an advantageous approach for suppressing the migration of tumour cells or distant metastasis. Among different levels of modulation of EMT, alternative splicing (AS) plays an important role. An in-depth understanding of the role of AS and EMT in cancer is not only helpful to better understand the occurrence and regulation of EMT in cancer progression, but also may provide new therapeutic strategies. This review will present and discuss various splice variants and splicing factors that have been shown to play a crucial role in EMT.

Detection of genomic mutations in blood and urine free circulating tumour DNA in patients with inoperable and metastatic lung adenocarcinoma harbouring an EGFR mutation in tissue: a UK pilot study.

The development of methodologies to analyse circulating tumour DNA (ctDNA) in the blood or urine of cancer patients provides an invaluable resource that can be used for diagnosis and prognosis and to evaluate response to treatments. Lung cancer has seen in the last years a revolution in treatment strategy with the use of several classes of EGFR inhibitors. However, almost invariably, resistance to such therapies appears. In this paper, we describe a pilot, longitudinal study with 20 patients with confirmed EGFR mutations in tissue biopsy for lung cancer. The objective of the study was to determine whether ctDNA from plasma and/or urine could be used to monitor the EGFR mutational status of patients with confirmed EGFR mutation-positive non-small cell lung cancer (NSCLC) during treatment with EGFR inhibitors. Blood and urine were collected monthly over periods ranging from 6 to 16 months. CtDNA was analysed in each patient for the presence of several known mutations that predispose to resistance to EGFR inhibitors. We have proven that serial monitoring of ctDNA from both plasma and urine is feasible and that patients are willing to participate in this process. We have also shown that longitudinal ctDNA monitoring may detect resistance mutations before the development of radiological and clinical disease progression.

VEGF165b overexpression restores normal glomerular water permeability in VEGF164-overexpressing adult mice.

Vascular endothelial growth factor (VEGF)-A, a family of differentially spliced proteins produced by glomerular podocytes, maintains glomerular filtration barrier function. The expression of VEGF molecules is altered in human nephropathy. We aimed to determine the roles of the angiogenic VEGF(164) isoform, and the antiangiogenic VEGF(165)b isoform in mature, adult glomeruli in vivo using conditional, inducible transgenic overexpression systems in mice. Podocyte-specific VEGF(164) overexpression (up to 100 days) was induced by oral administration of doxycycline to adult podocin-rtTA/TetO-VEGF(164) double transgenic mice. The consequences of simultaneous overexpression of VEGF(164) and VEGF(165)b were assessed in triple-transgenic podocin-rtTA/TetO-VEGF(164)/nephrin-VEGF(165)b mice. Persistent VEGF(164) overexpression did not cause proteinuria but did increase glomerular ultrafiltration coefficient between days 3 and 7. Despite persistently increased VEGF(164) levels, glomerular ultrafiltration coefficient normalized by day 14 and remained normal up to 100 days. Decreased subpodocyte space (SPS) coverage of the glomerular capillary wall accompanied increased glomerular hydraulic conductivity in VEGF(164)-overexpressing mice. The changes in glomerular ultrafiltration coefficient and SPS coverage induced by 7 days of overexpression of VEGF(164) were not present in triple transgenic VEGF(164) and VEGF(165)b overexpressing mice. These results indicate that 1) the adult mouse glomerulus is relatively resistant to induced VEGF(164) overexpression. VEGF(164) overexpression altered glomerular permeability but did not cause proteinuria in these mature, adult animals; 2) the SPS is a dynamic VEGF-responsive modulator of glomerular function; and 3) the balance of VEGF isoforms plays a critical role in the regulation of glomerular permeability. VEGF(165)b is capable of preventing VEGF(164)-induced changes in glomerular permeability and ultrastructure in vivo.

SRPK1 inhibition in vivo: modulation of VEGF splicing and potential treatment for multiple diseases.

SRPK1 (serine-arginine protein kinase 1) is a protein kinase that specifically phosphorylates proteins containing serine-arginine-rich domains. Its substrates include a family of SR proteins that are key regulators of mRNA AS (alternative splicing). VEGF (vascular endothelial growth factor), a principal angiogenesis factor contains an alternative 3' splice site in the terminal exon that defines a family of isoforms with a different amino acid sequence at the C-terminal end, resulting in anti-angiogenic activity in the context of VEGF165-driven neovascularization. It has been shown recently in our laboratories that SRPK1 regulates the choice of this splice site through phosphorylation of the splicing factor SRSF1 (serine/arginine-rich splicing factor 1). The present review summarizes progress that has been made to understand how SRPK1 inhibition may be used to manipulate the balance of pro- and anti-angiogenic VEGF isoforms in animal models in vivo and therefore control abnormal angiogenesis and other pathophysiological processes in multiple disease states.

A repositioning screen using an FGFR2 splicing reporter reveals compounds that regulate epithelial-mesenchymal transitions and inhibit growth of prostate cancer xenografts.

Research in the area of hallmarks of cancer has opened the possibility of designing new therapies based on modulating these cancer properties. We present here a screen designed to find chemicals that modulate epithelial-mesenchymal transitions (EMTs) in prostate cancer. For screening, we used a repurposing library and, as a readout, an FGFR2-based splicing reporter, which has been shown previously to be a sensor for EMTs. Various properties of cancer cells were assessed, signaling pathways investigated, and in vivo experiments in nude mice xenografts performed. The screen yielded three hit compounds (a T-type Ca channel inhibitor, an L-type Ca channel inhibitor, and an opioid antagonist) that switch FGFR2 splicing and induce an epithelial phenotype in prostate cancer cells. The compounds affected differently various properties of cancer cells, but all of them decreased cell migration, which is in line with modulating EMTs. We further present mechanistic insights into one of the compounds, nemadipine-A. The administration of nemadipine-A intraperitoneally in a nude mouse xenograft model of prostate cancer slowed tumor growth. To conclude, we show that knowledge of the molecular mechanisms that connect alternative splicing and various cancer properties may be used as a platform for drug development.

The epithelial splicing regulator ESRP2 is epigenetically repressed by DNA hypermethylation in Wilms tumour and acts as a tumour suppressor.

Wilms tumour (WT), an embryonal kidney cancer, has been extensively characterised for genetic and epigenetic alterations, but a proportion of WTs still lack identifiable abnormalities. To uncover DNA methylation changes critical for WT pathogenesis, we compared the epigenome of foetal kidney with two WT cell lines, filtering our results to remove common cancer-associated epigenetic changes and to enrich for genes involved in early kidney development. This identified four hypermethylated genes, of which ESRP2 (epithelial splicing regulatory protein 2) was the most promising for further study. ESRP2 was commonly repressed by DNA methylation in WT, and this occurred early in WT development (in nephrogenic rests). ESRP2 expression was reactivated by DNA methyltransferase inhibition in WT cell lines. When ESRP2 was overexpressed in WT cell lines, it inhibited cellular proliferation in vitro, and in vivo it suppressed tumour growth of orthotopic xenografts in nude mice. RNA-seq of the ESRP2-expressing WT cell lines identified several novel splicing targets. We propose a model in which epigenetic inactivation of ESRP2 disrupts the mesenchymal to epithelial transition in early kidney development to generate WT.

Overexpression of VEGF165b in podocytes reduces glomerular permeability.

The observation that therapeutic agents targeting vascular endothelial growth factor-A (VEGF-A) associate with renal toxicity suggests that VEGF plays a role in the maintenance of the glomerular filtration barrier. Alternative mRNA splicing produces the VEGF(xxx)b family, which consists of antiangiogenic peptides that reduce permeability and inhibit tumor growth; the contribution of these peptides to normal glomerular function is unknown. Here, we established and characterized heterozygous and homozygous transgenic mice that overexpress VEGF(165)b specifically in podocytes. We confirmed excess production of glomerular VEGF(165)b by reverse transcriptase-PCR, immunohistochemistry, and ELISA in both heterozygous and homozygous animals. Macroscopically, the mice seemed normal up to 18 months of age, unlike the phenotype of transgenic podocyte-specific VEGF(164)-overexpressing mice. Animals overexpressing VEGF(165)b, however, had a significantly reduced normalized glomerular ultrafiltration fraction with accompanying changes in ultrastructure of the glomerular filtration barrier on the vascular side of the glomerular basement membrane. These data highlight the contrasting properties of VEGF splice variants and their impact on glomerular function and phenotype.

CDC2-like (CLK) protein kinase inhibition as a novel targeted therapeutic strategy in prostate cancer.

Dysregulation of alternative splicing is a feature of cancer, both in aetiology and progression. It occurs because of mutations in splice sites or sites that regulate splicing, or because of the altered expression and activity of splice factors and of splice factor kinases that regulate splice factor activity. Recently the CDC2-like kinases (CLKs) have attracted attention due to their increasing involvement in cancer. We measured the effect of the CLK inhibitor, the benzothiazole TG003, on two prostate cancer cell lines. TG003 reduced cell proliferation and increased apoptosis in PC3 and DU145 cells. Conversely, the overexpression of CLK1 in PC3 cells prevented TG003 from reducing cell proliferation. TG003 slowed scratch closure and reduced cell migration and invasion in a transwell assay. TG003 decisively inhibited the growth of a PC3 cell line xenograft in nude mice. We performed a transcriptomic analysis of cells treated with TG003. We report widespread and consistent changes in alternative splicing of cancer-associated genes including CENPE, ESCO2, CKAP2, MELK, ASPH and CD164 in both HeLa and PC3 cells. Together these findings suggest that targeting CLKs will provide novel therapeutic opportunities in prostate cancer.

A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties.

Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3' splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A's terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3' splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.

A Runaway PRH/HHEX-Notch3-Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition.

Aberrant Notch and Wnt signaling are known drivers of cholangiocarcinoma (CCA), but the underlying factors that initiate and maintain these pathways are not known. Here, we show that the proline-rich homeodomain protein/hematopoietically expressed homeobox (PRH/HHEX) transcription factor forms a positive transcriptional feedback loop with Notch3 that is critical in CCA. PRH/HHEX expression is elevated in CCA, and depletion of PRH reduces CCA tumor growth in a xenograft model. Overexpression of PRH in primary human biliary epithelial cells is sufficient to increase cell proliferation and produce an invasive phenotype. Interrogation of the gene networks regulated by PRH and Notch3 reveals that unlike Notch3, PRH directly activates canonical Wnt signaling. These data indicate that hyperactivation of Notch and Wnt signaling is independent of the underlying mutational landscape and has a common origin in dysregulation of PRH. Moreover, they suggest new therapeutic options based on the dependence of specific Wnt, Notch, and CDK4/6 inhibitors on PRH activity. SIGNIFICANCE: The PRH/HHEX transcription factor is an oncogenic driver in cholangiocarcinoma that confers sensitivity to CDK4/6 inhibitors.