CDK9 inhibition activates innate immune response through viral mimicry.

Cancer cells frequently exhibit hyperactivation of transcription, which can lead to increased sensitivity to compounds targeting the transcriptional kinases, in particular CDK9. However, mechanistic details of CDK9 inhibition-induced cancer cell-selective anti-proliferative effects remain largely unknown. Here, we discover that CDK9 inhibition activates the innate immune response through viral mimicry in cancer cells. In MYC over-expressing prostate cancer cells, CDK9 inhibition leads to the gross accumulation of mis-spliced RNA. Double-stranded RNA (dsRNA)-activated kinase can recognize these mis-spliced RNAs, and we show that the activity of this kinase is required for the CDK9 inhibitor-induced anti-proliferative effects. Using time-resolved transcriptional profiling (SLAM-seq), targeted proteomics, and ChIP-seq, we show that, similar to viral infection, CDK9 inhibition significantly suppresses transcription of most genes but allows selective transcription and translation of cytokines related to the innate immune response. In particular, CDK9 inhibition activates NFκB-driven cytokine signaling at the transcriptional and secretome levels. The transcriptional signature induced by CDK9 inhibition identifies prostate cancers with a high level of genome instability. We propose that it is possible to induce similar effects in patients using CDK9 inhibition, which, we show, causes DNA damage in vitro. In the future, it is important to establish whether CDK9 inhibitors can potentiate the effects of immunotherapy against late-stage prostate cancer, a currently lethal disease.

Spatial transcriptomic analysis of virtual prostate biopsy reveals confounding effect of tissue heterogeneity on genomic signatures.

Genetic signatures have added a molecular dimension to prognostics and therapeutic decision-making. However, tumour heterogeneity in prostate cancer and current sampling methods could confound accurate assessment. Based on previously published spatial transcriptomic data from multifocal prostate cancer, we created virtual biopsy models that mimic conventional biopsy placement and core size. We then analysed the gene expression of different prognostic signatures (OncotypeDx®, Decipher®, Prostadiag®) using a step-wise approach with increasing resolution from pseudo-bulk analysis of the whole biopsy, to differentiation by tissue subtype (benign, stroma, tumour), followed by distinct tumour grade and finally clonal resolution. The gene expression profile of virtual tumour biopsies revealed clear differences between grade groups and tumour clones, compared to a benign control, which were not reflected in bulk analyses. This suggests that bulk analyses of whole biopsies or tumour-only areas, as used in clinical practice, may provide an inaccurate assessment of gene profiles. The type of tissue, the grade of the tumour and the clonal composition all influence the gene expression in a biopsy. Clinical decision making based on biopsy genomics should be made with caution while we await more precise targeting and cost-effective spatial analyses.

O-GlcNAc transferase maintains metabolic homeostasis in response to CDK9 inhibition.

Co-targeting of O-GlcNAc transferase (OGT) and the transcriptional kinase cyclin-dependent kinase 9 (CDK9) is toxic to prostate cancer cells. As OGT is an essential glycosyltransferase, identifying an alternative target showing similar effects is of great interest. Here, we used a multiomics approach (transcriptomics, metabolomics, and proteomics) to better understand the mechanistic basis of the combinatorial lethality between OGT and CDK9 inhibition. CDK9 inhibition preferentially affected transcription. In contrast, depletion of OGT activity predominantly remodeled the metabolome. Using an unbiased systems biology approach (weighted gene correlation network analysis), we discovered that CDK9 inhibition alters mitochondrial activity/flux, and high OGT activity is essential to maintain mitochondrial respiration when CDK9 activity is depleted. Our metabolite profiling data revealed that pantothenic acid (vitamin B5) is the metabolite that is most robustly induced by both OGT and OGT+CDK9 inhibitor treatments but not by CDK9 inhibition alone. Finally, supplementing prostate cancer cell lines with vitamin B5 in the presence of CDK9 inhibitor mimics the effects of co-targeting OGT and CDK9.

VPRBP Functions Downstream of the Androgen Receptor and OGT to Restrict p53 Activation in Prostate Cancer.

Androgen receptor (AR) is a major driver of prostate cancer initiation and progression. O-GlcNAc transferase (OGT), the enzyme that catalyzes the covalent addition of UDP-N-acetylglucosamine (UDP-GlcNAc) to serine and threonine residues of proteins, is often highly expressed in prostate cancer with its expression correlated with high Gleason score. In this study, we have identified an AR and OGT coregulated factor, Vpr (HIV-1) binding protein (VPRBP) also known as DDB1 and CUL4 Associated Factor 1 (DCAF1). We show that VPRBP is regulated by the AR at the transcript level, and stabilized by OGT at the protein level. VPRBP knockdown in prostate cancer cells led to a significant decrease in cell proliferation, p53 stabilization, nucleolar fragmentation, and increased p53 recruitment to the chromatin. In human prostate tumor samples, VPRBP protein overexpression correlated with AR amplification, OGT overexpression, a shorter time to postoperative biochemical progression and poor clinical outcome. In clinical transcriptomic data, VPRBP expression was positively correlated with the AR and also with AR activity gene signatures. IMPLICATIONS: In conclusion, we have shown that VPRBP/DCAF1 promotes prostate cancer cell proliferation by restraining p53 activation under the influence of the AR and OGT.

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.

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.

CDK9 Inhibition Induces a Metabolic Switch that Renders Prostate Cancer Cells Dependent on Fatty Acid Oxidation.

Cyclin-dependent kinase 9 (CDK9), a key regulator of RNA-polymerase II, is a candidate drug target for cancers driven by transcriptional deregulation. Here we report a multi-omics-profiling of prostate cancer cell responses to CDK9 inhibition to identify synthetic lethal interactions. These interactions were validated using live-cell imaging, mitochondrial flux-, viability- and cell death activation assays. We show that CDK9 inhibition induces acute metabolic stress in prostate cancer cells. This is manifested by a drastic down-regulation of mitochondrial oxidative phosphorylation, ATP depletion and induction of a rapid and sustained phosphorylation of AMP-activated protein kinase (AMPK), the key sensor of cellular energy homeostasis. We used metabolomics to demonstrate that inhibition of CDK9 leads to accumulation of acyl-carnitines, metabolic intermediates in fatty acid oxidation (FAO). Acyl-carnitines are produced by carnitine palmitoyltransferase enzymes 1 and 2 (CPT), and we used both genetic and pharmacological tools to show that inhibition of CPT-activity is synthetically lethal with CDK9 inhibition. To our knowledge this is the first report to show that CDK9 inhibition dramatically alters cancer cell metabolism.

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.

High fructose-mediated attenuation of insulin receptor signaling does not affect PDGF-induced proliferative signaling in vascular smooth muscle cells.

Insulin resistance is associated with accelerated atherosclerosis. Although high fructose is known to induce insulin resistance, it remains unclear as to how fructose regulates insulin receptor signaling and proliferative phenotype in vascular smooth muscle cells (VSMCs), which play a major role in atherosclerosis. Using human aortic VSMCs, we investigated the effects of high fructose treatment on insulin receptor substrate-1 (IRS-1) serine phosphorylation, insulin versus platelet-derived growth factor (PDGF)-induced phosphorylation of Akt, S6 ribosomal protein, and extracellular signal-regulated kinase (ERK), and cell cycle proteins. In comparison with PDGF (a potent mitogen), neither fructose nor insulin enhanced VSMC proliferation and cyclin D1 expression. d-[14C(U)]fructose uptake studies revealed a progressive increase in fructose uptake in a time-dependent manner. Concentration-dependent studies with high fructose (5-25mM) showed marked increases in IRS-1 serine phosphorylation, a key adapter protein in insulin receptor signaling. Accordingly, high fructose treatment led to significant diminutions in insulin-induced phosphorylation of downstream signaling components including Akt and S6. In addition, high fructose significantly diminished insulin-induced ERK phosphorylation. Nevertheless, high fructose did not affect PDGF-induced key proliferative signaling events including phosphorylation of Akt, S6, and ERK and expression of cyclin D1 protein. Together, high fructose dysregulates IRS-1 phosphorylation state and proximal insulin receptor signaling in VSMCs, but does not affect PDGF-induced proliferative signaling. These findings suggest that systemic insulin resistance rather than VSMC-specific dysregulation of insulin receptor signaling by high fructose may play a major role in enhancing atherosclerosis and neointimal hyperplasia.

Alteration of cytokine profile following hemorrhagic shock.

Hemorrhage is one of the leading causes of death in patients with trauma. We recently demonstrated that resveratrol can improve cardiac function and prolong life following severe hemorrhagic injury (HI) in a rat model. The present work is focused on determining changes in NF-κB dependent gene expression in the heart and the systemic cytokine milieu following HI and the effect of resveratrol treatment. The results indicate an increase in phosphorylated NF-κB in the heart with a concomitant increase in the expression of NF-κB dependent genes following HI. There was also a significant increase of systemic cytokine levels, both pro and anti-inflammatory, following HI and resolution when treated with resveratrol. This study demonstrates the potential role NF-κB has in the physiological response to HI and the effectiveness of resveratrol in reducing immune activation.

Sirtuin regulation in aging and injury.

Sirtuins or Sir2 family of proteins are a class of NAD(+) dependent protein deacetylases which are evolutionarily conserved from bacteria to humans. Some sirtuins also exhibit mono-ADP ribosyl transferase, demalonylation and desuccinylation activities. Originally identified in the yeast, these proteins regulate key cellular processes like cell cycle, apoptosis, metabolic regulation and inflammation. Humans encode seven sirtuin isoforms SIRT1-SIRT7 with varying intracellular distribution. Apart from their classic role as histone deacetylases regulating transcription, a number of cytoplasmic and mitochondrial targets of sirtuins have also been identified. Sirtuins have been implicated in longevity and accumulating evidence indicate their role in a spectrum of diseases like cancer, diabetes, obesity and neurodegenerative diseases. A number of studies have reported profound changes in SIRT1 expression and activity linked to mitochondrial functional alterations following hypoxic-ischemic conditions and following reoxygenation injury. The SIRT1 mediated deacetylation of targets such as PGC-1α, FOXO3, p53 and NF-κb has profound effect on mitochondrial function, apoptosis and inflammation. These biological processes and functions are critical in life-span determination and outcome following injury. Aging is reported to be characterized by declining SIRT1 activity, and its increased expression or activation demonstrated prolonged life-span in lower forms of animals. A pseudohypoxic state due to declining NAD(+) has also been implicated in aging. In this review we provide an overview of studies on the role of sirtuins in aging and injury.

Resveratrol Improves Survival and Prolongs Life Following Hemorrhagic Shock.

Resveratrol has been shown to potentiate mitochondrial function and extend longevity; however, there is no evidence to support whether resveratrol can improve survival or prolong life following hemorrhagic shock. We sought to determine whether (a) resveratrol can improve survival following hemorrhage and resuscitation and (b) prolong life in the absence of resuscitation. Using a hemorrhagic injury (HI) model in the rat, we describe for the first time that the naturally occurring small molecule, resveratrol, may be an effective adjunct to resuscitation fluid. In a series of three sets of experiments we show that resveratrol administration during resuscitation improves survival following HI (p < 0.05), resveratrol and its synthetic mimic SRT1720 can significantly prolong life in the absence of resuscitation fluid (<30 min versus up to 4 h; p < 0.05), and resveratrol as well as SRT1720 restores left ventricular function following HI. We also found significant changes in the expression level of mitochondria-related transcription factors Ppar-α and Tfam, as well as Pgc-1α in the left ventricular tissues of rats subjected to HI and treated with resveratrol. The results indicate that resveratrol is a strong candidate adjunct to resuscitation following severe hemorrhage.

Aging and injury: alterations in cellular energetics and organ function.

Aging is characterized by increased oxidative stress, heightened inflammatory response, accelerated cellular senescence and progressive organ dysfunction. The homeostatic imbalance with aging significantly alters cellular responses to injury. Though it is unclear whether cellular energetic imbalance is a cause or effect of the aging process, preservation of mitochondrial function has been reported to be important in organ function restoration following severe injury. Unintentional injuries are ranked among the top 10 causes of death in adults of both sexes, 65 years and older. Aging associated decline in mitochondrial function has been shown to enhance the vulnerability of heart, lung, liver and kidney to ischemia/reperfusion injury. Studies have identified alterations in the level or activity of factors such as SIRT1, PGC-1α, HIF-1α and c-MYC involved in key regulatory processes in the maintenance of mitochondrial structural integrity, biogenesis and function. Studies using experimental models of hemorrhagic injury and burn have demonstrated significant influence of aging in metabolic regulation and organ function. Understanding the age-associated molecular mechanisms regulating mitochondrial dysfunction following injury is important towards identifying novel targets and therapeutic strategies to improve the outcome after injury in the elderly.

Expression of conventional and novel glucose transporters, GLUT1, -9, -10, and -12, in vascular smooth muscle cells.

Intimal hyperplasia is characterized by exaggerated proliferation of vascular smooth muscle cells (VSMCs). Enhanced VSMC growth is dependent on increased glucose uptake and metabolism. Facilitative glucose transporters (GLUTs) are comprised of conventional GLUT isoforms (GLUT1-5) and novel GLUT isoforms (GLUT6-14). Previous studies demonstrate that GLUT1 overexpression or GLUT10 downregulation contribute to phenotypic changes in VSMCs. To date, the expression profile of all 14 GLUT isoforms has not been fully examined in VSMCs. Using the proliferative and differentiated phenotypes of human aortic VSMCs, the present study has determined the relative abundance of GLUT1-14 mRNAs by quantitative real-time PCR analysis. Twelve GLUT mRNAs excluding GLUT7 and GLUT14 were detectable in VSMCs. In the proliferative phenotype, the relative abundance of key GLUT mRNAs was GLUT1 (∼43%)>GLUT10 (∼26%)>GLUT9 (∼13%)>GLUT12 (∼4%), whereas in the differentiated phenotype the relative abundance was GLUT10 (∼28%)>GLUT1 (∼25%)>GLUT12 (∼20%)>GLUT9 (∼14%), together constituting 86-87% of total GLUT transcripts. To confirm the expression of key GLUT proteins, immunoblot and immunocytochemical analyses were performed using GLUT isoform-specific primary antibodies. The protein bands characteristic of GLUT1, -9, -10, and -12 were detected in VSMCs in parallel with respective positive controls. In particular, GLUT1 protein expression showed different molecular forms representative of altered glycosylation. While GLUT1 protein displayed a predominant distribution in the plasma membrane, GLUT9, -10, and -12 proteins were mostly distributed in the intracellular compartments. The present study provides the first direct evidence for GLUT9 and GLUT12 expression in VSMCs in conjunction with the previously identified GLUT1 and GLUT10.

Vanillin induces adipocyte differentiation in 3T3-L1 cells by activating extracellular signal regulated kinase 42/44.

AIMS: To investigate the effect of vanillin, a dietary component, on adipocyte differentiation and the mechanism involved in the process using 3T3-L1 murine preadipocytes. MAIN METHODS: The effect of vanillin on adipocyte differentiation was detected by Oil Red O analysis. The activation of extracellular signal regulated kinase 42/44 (ERK 42/44), Akt, expression of the key regulator of adipocyte differentiation peroxisome proliferators-activated receptor (PPARγ) and its target gene glucose transporter 4 (GLUT4) were detected by western blotting. Glucose uptake assay was used to determine the insulin sensitivity of adipocytes differentiated by vanillin treatment. To confirm the role of ERK 42/44 and Akt, Oil Red O analysis was performed with cells differentiated in the presence or absence of ERK inhibitor U0126 or Akt kinase 1/2 inhibitor. KEY FINDINGS: Vanillin induced adipocyte differentiation in 3T3-L1 cells in a dose dependent manner and also increased the expression levels of PPARγ and its target gene GLUT4. The adipocytes differentiated by vanillin exhibited insulin sensitivity as demonstrated by a significant increase in glucose uptake. Vanillin treatment activated the phosphorylation of ERK 42/44 during the initial phase of adipocyte differentiation but there was no significant change in the Akt phosphorylation status. SIGNIFICANCE: The data show that vanillin induces adipocyte differentiation in 3T3-L1 cells by activating ERK42/44 and these adipocytes are insulin sensitive in nature.

Molecular dynamics simulation studies of GLUT4: substrate-free and substrate-induced dynamics and ATP-mediated glucose transport inhibition.

BACKGROUND: Glucose transporter 4 (GLUT4) is an insulin facilitated glucose transporter that plays an important role in maintaining blood glucose homeostasis. GLUT4 is sequestered into intracellular vesicles in unstimulated cells and translocated to the plasma membrane by various stimuli. Understanding the structural details of GLUT4 will provide insights into the mechanism of glucose transport and its regulation. To date, a crystal structure for GLUT4 is not available. However, earlier work from our laboratory proposed a well validated homology model for GLUT4 based on the experimental data available on GLUT1 and the crystal structure data obtained from the glycerol 3-phosphate transporter. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, the dynamic behavior of GLUT4 in a membrane environment was analyzed using three forms of GLUT4 (apo, substrate and ATP-substrate bound states). Apo form simulation analysis revealed an extracellular open conformation of GLUT4 in the membrane favoring easy exofacial binding of substrate. Simulation studies with the substrate bound form proposed a stable state of GLUT4 with glucose, which can be a substrate-occluded state of the transporter. Principal component analysis suggested a clockwise movement for the domains in the apo form, whereas ATP substrate-bound form induced an anti-clockwise rotation. Simulation studies suggested distinct conformational changes for the GLUT4 domains in the ATP substrate-bound form and favor a constricted behavior for the transport channel. Various inter-domain hydrogen bonds and switching of a salt-bridge network from E345-R350-E409 to E345-R169-E409 contributed to this ATP-mediated channel constriction favoring substrate occlusion and prevention of its release into cytoplasm. These data are consistent with the biochemical studies, suggesting an inhibitory role for ATP in GLUT-mediated glucose transport. CONCLUSIONS/SIGNIFICANCE: In the absence of a crystal structure for any glucose transporter, this study provides mechanistic details of the conformational changes in GLUT4 induced by substrate and its regulator.

Homology modeling of GLUT4, an insulin regulated facilitated glucose transporter and docking studies with ATP and its inhibitors.

GLUT4 is a 12 transmembrane (TM) protein belonging to the Class I facilitated glucose transporter family that transports glucose into the cells in an insulin regulated manner. GLUT4 plays a key role in the maintenance of blood glucose homeostasis and inhibition of glucose transporter activity may lead to insulin resistance, hallmark of type 2 diabetes. No crystal structure data is available for any members of the facilitated glucose transporter family. Here, in this paper, we have generated a homology model of GLUT4 based on experimental data available on GLUT1, a Class I facilitated glucose transporter and the crystal structure data obtained from the Glycerol 3-phosphate transporter. The model identified regions in GLUT4 that form a channel for the transport of glucose along with the substrate interacting residues. Docking and electrostatic potential data analysis of GLUT4 model has mapped an ATP binding region close to the binding site of cytochalasin B and genistein, two GLUT4 inhibitors, and this may explain the mechanism by which these inhibitors could potentially affect the GLUT4 function.