PAX Genes in Cardiovascular Development.

The mammalian heart is a four-chambered organ with systemic and pulmonary circulations to deliver oxygenated blood to the body, and a tightly regulated genetic network exists to shape normal development of the heart and its associated major arteries. A key process during cardiovascular morphogenesis is the septation of the outflow tract which initially forms as a single vessel before separating into the aorta and pulmonary trunk. The outflow tract connects to the aortic arch arteries which are derived from the pharyngeal arch arteries. Congenital heart defects are a major cause of death and morbidity and are frequently associated with a failure to deliver oxygenated blood to the body. The Pax transcription factor family is characterised through their highly conserved paired box and DNA binding domains and are crucial in organogenesis, regulating the development of a wide range of cells, organs and tissues including the cardiovascular system. Studies altering the expression of these genes in murine models, notably Pax3 and Pax9, have found a range of cardiovascular patterning abnormalities such as interruption of the aortic arch and common arterial trunk. This suggests that these Pax genes play a crucial role in the regulatory networks governing cardiovascular development.

Inhibition of CDK9 activity compromises global splicing in prostate cancer cells.

Cyclin-dependent kinase 9 (CDK9) phosphorylates RNA polymerase II to promote productive transcription elongation. Here we show that short-term CDK9 inhibition affects the splicing of thousands of mRNAs. CDK9 inhibition impairs global splicing and there is no evidence for a coordinated response between the alternative splicing and the overall transcriptome. Alternative splicing is a feature of aggressive prostate cancer (CRPC) and enables the generation of the anti-androgen resistant version of the ligand-independent androgen receptor, AR-v7. We show that CDK9 inhibition results in the loss of AR and AR-v7 expression due to the defects in splicing, which sensitizes CRPC cells to androgen deprivation. Finally, we demonstrate that CDK9 expression increases as PC cells develop CRPC-phenotype both in vitro and also in patient samples. To conclude, here we show that CDK9 inhibition compromises splicing in PC cells, which can be capitalized on by targeting the PC-specific addiction androgen receptor.

Modulating the unfolded protein response with ONC201 to impact on radiation response in prostate cancer cells.

Prostate cancer (PCa) is the most common non-cutaneous cancer in men and a notable cause of cancer mortality when it metastasises. The unfolded protein response (UPR) can be cytoprotective but when acutely activated can lead to cell death. In this study, we sought to enhance the acute activation of the UPR using radiation and ONC201, an UPR activator. Treating PCa cells with ONC201 quickly increased the expression of all the key regulators of the UPR and reduced the oxidative phosphorylation, with cell death occurring 72 h later. We exploited this time lag to sensitize prostate cancer cells to radiation through short-term treatment with ONC201. To understand how priming occurred, we performed RNA-Seq analysis and found that ONC201 suppressed the expression of cell cycle and DNA repair factors. In conclusion, we have shown that ONC201 can prime enhanced radiation response.

Propagation of human prostate tissue from induced pluripotent stem cells.

Primary culture of human prostate organoids and patient-derived xenografts is inefficient and has limited access to clinical tissues. This hampers their use for translational study to identify new treatments. To overcome this, we established a complementary approach where rapidly proliferating and easily handled induced pluripotent stem cells enabled the generation of human prostate tissue in vivo and in vitro. By using a coculture technique with inductive urogenital sinus mesenchyme, we comprehensively recapitulated in situ 3D prostate histology, and overcame limitations in the primary culture of human prostate stem, luminal and neuroendocrine cells, as well as the stromal microenvironment. This model now unlocks new opportunities to undertake translational studies of benign and malignant prostate disease.

Inhibition of O-GlcNAc Transferase Renders Prostate Cancer Cells Dependent on CDK9.

O-GlcNAc transferase (OGT) is a nutrient-sensitive glycosyltransferase that is overexpressed in prostate cancer, the most common cancer in males. We recently developed a specific and potent inhibitor targeting this enzyme, and here, we report a synthetic lethality screen using this compound. Our screen identified pan-cyclin-dependent kinase (CDK) inhibitor AT7519 as lethal in combination with OGT inhibition. Follow-up chemical and genetic approaches identified CDK9 as the major target for synthetic lethality with OGT inhibition in prostate cancer cells. OGT expression is regulated through retention of the fourth intron in the gene and CDK9 inhibition blunted this regulatory mechanism. CDK9 phosphorylates carboxy-terminal domain (CTD) of RNA Polymerase II to promote transcription elongation. We show that OGT inhibition augments effects of CDK9 inhibitors on CTD phosphorylation and general transcription. Finally, the combined inhibition of both OGT and CDK9 blocked growth of organoids derived from patients with metastatic prostate cancer, but had minimal effects on normal prostate spheroids. We report a novel synthetic lethal interaction between inhibitors of OGT and CDK9 that specifically kills prostate cancer cells, but not normal cells. Our study highlights the potential of combining OGT inhibitors with other treatments to exploit cancer-specific vulnerabilities. IMPLICATIONS: The primary contribution of OGT to cell proliferation is unknown, and in this study, we used a compound screen to indicate that OGT and CDK9 collaborate to sustain a cancer cell-specific pro-proliferative program. A better understanding of how OGT and CDK9 cross-talk will refine our understanding of this novel synthetic lethal interaction.

Investigating Radiotherapy Response in a Novel Syngeneic Model of Prostate Cancer.

The prostate cancer (PCa) field lacks clinically relevant, syngeneic mouse models which retain the tumour microenvironment observed in PCa patients. This study establishes a cell line from prostate tumour tissue derived from the Pten-/-/trp53-/- mouse, termed DVL3 which when subcutaneously implanted in immunocompetent C57BL/6 mice, forms tumours with distinct glandular morphology, strong cytokeratin 8 and androgen receptor expression, recapitulating high-risk localised human PCa. Compared to the commonly used TRAMP C1 model, generated with SV40 large T-antigen, DVL3 tumours are immunologically cold, with a lower proportion of CD8+ T-cells, and high proportion of immunosuppressive myeloid derived suppressor cells (MDSCs), thus resembling high-risk PCa. Furthermore, DVL3 tumours are responsive to fractionated RT, a standard treatment for localised and metastatic PCa, compared to the TRAMP C1 model. RNA-sequencing of irradiated DVL3 tumours identified upregulation of type-1 interferon and STING pathways, as well as transcripts associated with MDSCs. Upregulation of STING expression in tumour epithelium and the recruitment of MDSCs following irradiation was confirmed by immunohistochemistry. The DVL3 syngeneic model represents substantial progress in preclinical PCa modelling, displaying pathological, micro-environmental and treatment responses observed in molecular high-risk disease. Our study supports using this model for development and validation of treatments targeting PCa, especially novel immune therapeutic agents.

The impact of transcription on metabolism in prostate and breast cancers.

Metabolic dysregulation is regarded as an important driver in cancer development and progression. The impact of transcriptional changes on metabolism has been intensively studied in hormone-dependent cancers, and in particular, in prostate and breast cancer. These cancers have strong similarities in the function of important transcriptional drivers, such as the oestrogen and androgen receptors, at the level of dietary risk and epidemiology, genetics and therapeutically. In this review, we will focus on the function of these nuclear hormone receptors and their downstream impact on metabolism, with a particular focus on lipid metabolism. We go on to discuss how lipid metabolism remains dysregulated as the cancers progress. We conclude by discussing the opportunities that this presents for drug repurposing, imaging and the development and testing of new therapeutics and treatment combinations.

Silencing Mist1 Gene Expression Is Essential for Recovery from Acute Pancreatitis.

Acinar cells of the exocrine pancreas are tasked with synthesizing, packaging and secreting vast quantities of pro-digestive enzymes to maintain proper metabolic homeostasis for the organism. Because the synthesis of high levels of hydrolases is potentially dangerous, the pancreas is prone to acute pancreatitis (AP), a disease that targets acinar cells, leading to acinar-ductal metaplasia (ADM), inflammation and fibrosis-events that can transition into the earliest stages of pancreatic ductal adenocarcinoma. Despite a wealth of information concerning the broad phenotype associated with pancreatitis, little is understood regarding specific transcriptional regulatory networks that are susceptible to AP and the role these networks play in acinar cell and exocrine pancreas responses. In this study, we examined the importance of the acinar-specific maturation transcription factor MIST1 to AP damage and organ recovery. Analysis of wild-type and Mist1 conditional null mice revealed that Mist1 gene transcription and protein accumulation were dramatically reduced as acinar cells underwent ADM alterations during AP episodes. To test if loss of MIST1 function was primarily responsible for the damaged status of the organ, mice harboring a Cre-inducible Mist1 transgene (iMist1) were utilized to determine if sustained MIST1 activity could alleviate AP damage responses. Unexpectedly, constitutive iMist1 expression during AP led to a dramatic increase in organ damage followed by acinar cell death. We conclude that the transient silencing of Mist1 expression is critical for acinar cells to survive an AP episode, providing cells an opportunity to suppress their secretory function and regenerate damaged cells. The importance of MIST1 to these events suggests that modulating key pancreas transcription networks could ease clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer.