[Abstract unavailable

Disablement of cell death programs in cancer cells contributes to drug resistance and in some cases has been associated with altered translational control. As eukaryotic translation initiation factor 4E (eIF4E) cooperates with c-Myc during lymphomagenesis, induces drug resistance, and is a genetic modifier of the rapamycin response, we have investigated the effect of dysregulation of the ribosome recruitment phase of translation initiation on tumor progression and chemosensitivity. eIF4E is a subunit of eIF4F, a complex that stimulates ribosome recruitment during translation initiation by delivering the DEAD-box RNA helicase eIF4A to the 5′ end of mRNAs. eIF4A is thought to prepare a ribosome landing pad on mRNA templates for incoming 40S ribosomes (and associated factors). Using small molecule screening, we found that cyclopenta[b]benzofuran flavaglines, a class of natural products, modulate eIF4A activity and inhibit translation initiation. One member of this class of compounds, silvestrol, was able to enhance chemosensitivity in a mouse lymphoma model in which carcinogenesis is driven by phosphatase and tensin homolog (PTEN) inactivation or elevated eIF4E levels. These results establish that targeting translation initiation can restore drug sensitivity in vivo and provide an approach to modulating chemosensitivity.

RNA helicases are molecular motors that are involved in virtually all aspects of RNA metabolism. Eukaryotic initiation factor (eIF) 4A is the prototypical member of the DEAD-box family of RNA helicases. It is thought to use energy from ATP hydrolysis to unwind mRNA structure and, in conjunction with other translation factors, it prepares mRNA templates for ribosome recruitment during translation initiation. In screening marine extracts for new eukaryotic translation initiation inhibitors, we identified the natural product hippuristanol. We show here that this compound is a selective and potent inhibitor of eIF4A RNA-binding activity that can be used to distinguish between eIF4A-dependent and -independent modes of translation initiation in vitro and in vivo. We also show that poliovirus replication is delayed when infected cells are exposed to hippuristanol. Our study demonstrates the feasibility of selectively targeting members of the DEAD-box helicase family with small-molecule inhibitors.

Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1β. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKLinduced IL-1β secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1β release, was not essential for MLKL-dependent death or IL-1β secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1β cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKLdependent diseases.aspase-dependent apoptotic cell death is required for mammalian development and the prevention of autoimmune and neoplastic diseases. Programmed cell death can also act to eliminate pathogen-infected cells, with recent studies highlighting how targeted apoptosis-inducing anticancer compounds can treat viral and intracellular bacterial infections (1, 2). On the other hand, the recently characterized caspase-independent necroptotic cell death pathway is dispensable for organism development but, like apoptosis, can be triggered to kill cells harboring pathogenic microbes (3). A number of studies have also reported how pathological activation of necroptotic signaling may contribute to diverse disease states, such as ischemia-reperfusion injury, atherosclerosis, and liver disease, presumably through cell death and the release of proinflammatory mediators (4).The execution of necroptosis is dependent on receptor interacting serine-threonine protein kinase 3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), and MLKL's association with, and disruption of, plasma membrane integrity (5).In the absence of caspase activit...

RNA helicases are the largest group of enzymes in eukaryotic RNA metabolism. The DEXD͞H-box putative RNA helicases form the helicase superfamily II, whose members are defined by seven highly conserved amino acid motifs, making specific targeting of selected members a challenging pharmacological problem. The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase that works in conjunction with eIF4B and eIF4H and as a subunit of eIF4F to prepare the mRNA template for ribosome binding, possibly by unwinding the secondary structure proximal to the 5 m 7 GpppN cap structure. We report the identification and characterization of a small molecule inhibitor of eukaryotic translation initiation that acts in an unusual manner by stimulating eIF4A-associated activities. Our results suggest that proper control of eIF4A helicase activity is necessary for efficient ribosome binding and demonstrate the feasibility of selectively targeting DEADbox RNA helicases with small molecules.chemical biology ͉ DEAD-box helicase ͉ pateamine T he ribosome recruitment step of translation initiation is ratelimiting and an important regulatory point whereby cellular environmental cues (e.g., amino acid starvation, mitogenic stimulation, and hypoxia) are linked to the process of translation (1). Two distinct pathways exist for recruitment of the ribosome to the mRNA template. One mechanism is cap-dependent and is facilitated by the presence of the 5Ј cap structure (m 7 GpppN, where N is any nucleotide) on the mRNA. It is catalyzed by the eIF4 class of translation initiation factors and involves the recruitment of ribosomes near the 5Ј end of the mRNA template (1). The second mode involves ribosome recruitment in a cap-independent fashion to an internal ribosome entry site (IRES). Initiation factor requirement for internal ribosome binding varies among IRESes, with some not requiring any factors (2).Preparation of the mRNA template for cap-dependent ribosome recruitment is achieved by eIF4F, eIF4A, eIF4B, eIF4H, and ATP hydrolysis (1). The eIF4F complex is comprised of three subunits: (i) eIF4E, which binds the mRNA cap structure in an ATP-independent fashion; (ii) eIF4A, an RNA helicase that exhibits RNA-dependent ATPase activity and ATPstimulated RNA binding activity (3); and (iii) eIF4G, a modular scaffold that mediates mRNA binding of the 43S preinitiation complex through interactions with eIF3. eIF4B, and eIF4H cooperate with eIF4A to make its helicase activity more processive (4, 5). eIF4A exists as a free form (referred to herein as eIF4A f ) and as a subunit of eIF4F (eIF4A c ) and is thought to cycle through the eIF4F complex during initiation (6-8). When localized in the eIF4F complex, eIF4A c is Ϸ20-fold more efficient as an RNA helicase than when found alone (4, 9), leading to the proposal that eIF4A c is the functional helicase for translation initiation (10). The helicase activity of eIF4F (via eIF4A c ) is thought to unwind local secondary structure in the 5Ј UTR of mRNAs to facilitate cap-dependent ribosome...

Highlights d MCL-1 and BCL-XL prevent BAX/BAK-induced IL-1b activation and death in macrophages d BAX/BAK-induced IL-1b requires the downstream effector caspases, caspase-3 and -7 d Caspase-3/-7 cause potassium efflux to activate the NLRP3 inflammasome and IL-1b d BAX/BAK-induced caspase-3/-7 and IAP loss also trigger caspase-8 to cleave IL-1b

RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved.

It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence. However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function. To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds. We found that knockdown of Zmat3, Ctsf and Cav1, promoted lymphoma/leukemia development only when PUMA and p21, the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence, were also absent. Notably, loss of the DNA repair gene Mlh1 caused lymphoma in a wild-type background, and its enforced expression was able to delay tumor development driven by loss of p53. Further examination of direct p53 target genes implicated in DNA repair showed that knockdown of Mlh1, Msh2, Rnf144b, Cav1 and Ddit4 accelerated MYC-driven lymphoma development to a similar extent as knockdown of p53. Collectively, these findings demonstrate that extensive functional overlap of several p53-regulated processes safeguards against cancer and that coordination of DNA repair appears to be an important process by which p53 suppresses tumor development.