SUMMARY: Cyclin-Dependent Kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50=5nM) that has broad anti-cancer activity in-vitro and is effective in in-vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Rationale: Shifts in the gene expression of nuclear protein in chronic obstructive pulmonary disease (COPD), a progressive disease that is characterized by extensive lung inflammation and apoptosis, are common; however, the extent of the elevation of the core histones, which are the major components of nuclear proteins and their consequences in COPD, has not been characterized, which is important because extracellular histones are cytotoxic to endothelial and airway epithelial cells. Objectives: To investigate the role of extracellular histones in COPD disease progression. Methods: We analyzed the nuclear lung proteomes of ex-smokers with and without the disease. Further studies on the consequences of H3.3 were also performed. Measurements and Main Results: A striking finding was a COPDspecific eightfold increase of hyperacetylated histone H3.3. The hyperacetylation renders H3.3 resistant to proteasomal degradation despite ubiquitination; when combined with the reduction in proteasome activity that is known for COPD, this resistance helps account for the increased levels of H3.3. Using anti-H3 antibodies, we found H3.3 in the airway lumen, alveolar fluid, and plasma of COPD samples. H3.3 was cytotoxic to lung structural cells via a mechanism that involves the perturbation of Ca 21 homeostasis and mitochondrial toxicity. We used the primary human airway epithelial cells and found that the antibodies to either the C or N terminus of H3 could partially reverse H3.3 toxicity. Conclusions: Our data indicate that there is an uncontrolled positive feedback loop in which the damaged cells release acetylated H3.3, which causes more damage, adds H3.3 release, and contributes toward the disease progression.Keywords: chronic obstructive pulmonary disease; histone H3.3; acetylation; cytotoxicity; proteomicsThe prevalence of chronic obstructive pulmonary disease (COPD) is increasing in industrialized countries (1, 2). COPD is the third leading cause of death worldwide (3). In 2011, the CDC estimated that 15 million adults in the United States had COPD, which contributed to $49 billion in direct and indirect healthcare costs (4, 5). Disease management can relieve symptoms and prolong life, although there are no treatments to stop the disease progression, which ultimately results in death.Although the molecular pathophysiology that underlies COPD has not been established, it is known that the expression of proinflammatory molecules, lung cell death, and tissue remodeling play critical roles (2, 6-8). Shifts in transcription factor activation and epigenetic markers, such as altered histone acetylation, are associated with changes in signaling proteins upstream of regulatory genes and in the assembly of nuclear chromatin complexes (9, 10). Core histones H2A, H2B, H3, H4 and their variants are key components of nuclear chromatin, the scaffolding that controls interactions between DNA with transcription factors and RNA polymerase, thereby regulating gene expression (11). Post-translational modification of histones causes chromatin ...

Polyamines are low molecular weight, positively charged compounds that are ubiquitous in all living cells. They play a crucial role in many biochemical processes including regulation of transcription and translation, modulation of enzyme activities, regulation of ion channels and apoptosis. A strict balance between synthesis, catabolism and excretion tightly controls the cellular concentration of polyamines. The concentrations of rate-limiting enzymes in the polyamine synthesis and degradation pathways are regulated at different levels, including transcription, translation and degradation. Polyamines can modulate the translation of most of the enzymes required for their synthesis and catabolism through feedback mechanisms that are unique for each enzyme. Translational control is associated with cis-acting and trans-acting factors that can be influenced by the concentration of polyamines through mechanisms that are not completely understood. In this review, we present an overview of the translational control mechanisms of the proteins in the polyamine pathway, including ornithine decarboxylase (ODC), ODC antizyme, S-adenosylmethionine decarboxylase and spermidine/spermine N(1) acetyltransferase, highlighting the areas where more research is needed. A better understanding of the translational control of these enzymes would offer the possibility of a novel pharmacological intervention against cancer and other diseases.

Rapid synthesis of the polyamine catabolic enzyme spermidine/spermine-N 1 -acetyltransferase (SSAT) in response to increased polyamines is an important polyamine homeostatic mechanism. Indirect evidence has suggested that there is an important control mechanism involving the release of a translational repressor protein that allows the immediate initiation of SSAT protein synthesis without RNA transcription, maturation, or translocation. To identify a repressor protein, we used a mass spectroscopy-based RNA-protein interaction system and found six proteins that bind to the coding region of SSAT mRNA. Individual small interfering RNA (siRNA) experiments showed that nucleolin knockdown enhances SSAT translation. Nucleolin exists in several isoforms, and we report that the isoform that binds to SSAT mRNA undergoes autocatalysis in the presence of polyamines, a result suggesting that there is a negative feedback system that helps control the cellular content of polyamines. Preliminary molecular interaction data show that a nucleolin isoform binds to a 5= stem-loop of the coding region of SSAT mRNA. The glycine/arginine-rich C terminus of nucleolin is required for binding, and the four RNA recognition motif domains are included in the isoform that blocks SSAT translation. Understanding SSAT translational control mechanisms has the potential for the development of therapeutic strategies against cancer and obesity. P olyamines are small positively charged molecules present in all cells. The common polyamines putrescine, spermidine, and spermine are essential for cell growth. The rate of synthesis and content both increase with increased cell proliferation (47). Polyamine functions include stabilization of polynucleotides, regulation of transcription and translation, control of enzyme activities, modulation of ion channels, and response to oxidative stress (42, 61). Because so many processes are affected, levels are maintained within a relatively narrow range by shifts in anabolism/catabolism and import/export (1, 46). Manipulation of polyamine metabolism has been an anticancer strategy, with pool depletion in tumor cells used as a surrogate marker of efficacy (21,34,38,48).Many mechanisms contribute to control of eukaryotic polyamine metabolic enzymes (15,41,43,50). Ornithine decarboxylase (ODC), the rate-limiting anabolic enzyme, is regulated allosterically, at transcription, at translation, and by "ODC antizyme," a protein that binds to ODC monomers, thereby blocking homodimerization required for activity and accelerating monomer degradation (41). Antizyme itself is regulated by a translation control mechanism involving polyamine-induced ribosomal frameshifting (15, 35). Spermidine/spermine-N 1 -acetyltransferase (SSAT) is the principal catabolic regulator. SSAT acetylates spermidine and spermine using acetyl-coenzyme A (CoA), thereby altering their charge and facilitating excretion. SSAT basal activity is very low but increases quickly when polyamines are in excess (11). There is evidence for transcription, translati...

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