CSL324, a granulocyte colony-stimulating factor receptor antagonist, blocks neutrophil migration markers that are upregulated in hidradenitis suppurativa.

BACKGROUND: Neutrophils have been shown to contribute to the pathophysiology of hidradenitis suppurativa (HS), a chronic, painful and debilitating inflammatory skin disease, yet their exact role remains to be fully defined. Granulocyte colony-stimulating factor (G-CSF), a major regulator of neutrophil development and survival, can be blocked by the novel, fully human anti-G-CSF receptor (G-CSFR) monoclonal antibody CSL324. OBJECTIVES: We investigated the activation and migration of neutrophils in HS and the impact of blocking G-CSFR with CSL324. METHODS: Biopsy and peripheral blood samples were taken from participants of two studies: 2018.206, a noninterventional research study of systemic and dermal neutrophils and inflammatory markers in patients with neutrophilic skin diseases, and CSL324_1001 (ACTRN12616000846426), a single-dose ascending and repeated dose, randomized, double-blind, placebo-controlled study to assess the safety, pharmacokinetics and pharmacodynamics of CSL324 in healthy adult subjects. Ex vivo experiments were performed, including neutrophil enumeration and immunophenotyping, migration, receptor occupancy and transcriptome analysis. RESULTS: The number of cells positive for the neutrophil markers myeloperoxidase (MPO) and neutrophil elastase (NE) was significantly higher in HS lesions compared with biopsies from healthy donors (HDs) (P < 0.0001 and P = 0.0223, respectively). In peripheral blood samples, mean neutrophil counts were significantly higher in patients with HS than in HDs (2.98 vs. 1.60 × 109 L-1, respectively; P = 8.8 × 10-4). Neutrophil migration pathways in peripheral blood were increased in patients with HS and their neutrophils demonstrated an increased migration phenotype, with higher mean CXCR1 on the surface of neutrophils in patients with HS (24453.20 vs. 20798.47 for HD; P = 0.03). G-CSF was a key driver of the transcriptomic changes in the peripheral blood of patients with HS and was elevated in serum from patients with HS compared with HDs (mean 6.61 vs. 3.84 pg mL-1, respectively; P = 0.013). Administration of CSL324 inhibited G-CSF-induced transcriptional changes in HDs, similar to those observed in the HS cohort, as highlighted by expression changes in genes related to neutrophil migratory capacity. CONCLUSIONS: Data suggest that neutrophils contribute to HS pathophysiology and that neutrophils are increased in lesions due to an increase in G-CSF-driven migration. CSL324 counteracted G-CSF-induced transcriptomic changes and blocked neutrophil migration by reducing cell-surface levels of chemokine receptors.

Activated MLKL attenuates autophagy following its translocation to intracellular membranes.

Necroptosis is an inflammatory form of programmed cell death mediated by the pseudokinase mixed-lineage kinase domain-like protein (MLKL). Upon phosphorylation by receptor-interacting protein kinase-3 (RIPK3), MLKL oligomerizes, and translocates to and disrupts the plasma membrane, thereby causing necroptotic cell lysis. Herein, we show that activation of necroptosis in mouse dermal fibroblasts (MDFs) and HT-29 human colorectal cancer cells results in accumulation of the autophagic marker, lipidated LC3B (also known as MAP1LC3B), in an MLKL-dependent manner. Unexpectedly, the necroptosis-induced increase in lipidated LC3B was due to inhibition of autophagic flux, not the activation of autophagy. Inhibition of autophagy by MLKL correlated with a decrease in autophagosome and/or autolysosome function, and required the association of activated MLKL with intracellular membranes. Collectively, our findings uncover an additional role for the MLKL pseudokinase, namely to inhibit autophagy during necroptosis.

Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner.

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-1ß. 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 MLKL-induced IL-1ß 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-1ß release, was not essential for MLKL-dependent death or IL-1ß 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-1ß 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 MLKL-dependent diseases.

AMP-activated protein kinase selectively inhibited by the type II inhibitor SBI-0206965.

Inhibition of the metabolic regulator AMP-activated protein kinase (AMPK) is increasingly being investigated for its therapeutic potential in diseases where AMPK hyperactivity results in poor prognoses, as in established cancers and neurodegeneration. However, AMPK-inhibitory tool compounds are largely limited to compound C, which has a poor selectivity profile. Here we identify the pyrimidine derivative SBI-0206965 as a direct AMPK inhibitor. SBI-0206965 inhibits AMPK with 40-fold greater potency and markedly lower kinase promiscuity than compound C and inhibits cellular AMPK signaling. Biochemical characterization reveals that SBI-0206965 is a mixed-type inhibitor. A co-crystal structure of the AMPK kinase domain/SBI-0206965 complex shows that the drug occupies a pocket that partially overlaps the ATP active site in a type IIb inhibitor manner. SBI-0206965 has utility as a tool compound for investigating physiological roles for AMPK and provides fresh impetus to small-molecule AMPK inhibitor therapeutic development.

A tumour suppressor network relying on the polyamine-hypusine axis.

Tumour suppressor genes encode a broad class of molecules whose mutational attenuation contributes to malignant progression. In the canonical situation, the tumour suppressor is completely inactivated through a two-hit process involving a point mutation in one allele and chromosomal deletion of the other. Here, to identify tumour suppressor genes in lymphoma, we screen a short hairpin RNA library targeting genes deleted in human lymphomas. We functionally identify those genes whose suppression promotes tumorigenesis in a mouse lymphoma model. Of the nine tumour suppressors we identified, eight correspond to genes occurring in three physically linked 'clusters', suggesting that the common occurrence of large chromosomal deletions in human tumours reflects selective pressure to attenuate multiple genes. Among the new tumour suppressors are adenosylmethionine decarboxylase 1 (AMD1) and eukaryotic translation initiation factor 5A (eIF5A), two genes associated with hypusine, a unique amino acid produced as a product of polyamine metabolism through a highly conserved pathway. Through a secondary screen surveying the impact of all polyamine enzymes on tumorigenesis, we establish the polyamine-hypusine axis as a new tumour suppressor network regulating apoptosis. Unexpectedly, heterozygous deletions encompassing AMD1 and eIF5A often occur together in human lymphomas and co-suppression of both genes promotes lymphomagenesis in mice. Thus, some tumour suppressor functions can be disabled through a two-step process targeting different genes acting in the same pathway.

The molecular relationships between apoptosis, autophagy and necroptosis.

Cells are constantly subjected to a vast range of potentially lethal insults, which may activate specific molecular pathways that have evolved to kill the cell. Cell death pathways are defined partly by their morphology, and more specifically by the molecules that regulate and enact them. As these pathways become more thoroughly characterized, interesting molecular links between them have emerged, some still controversial and others hinting at the physiological and pathophysiological roles these death pathways play. We describe specific molecular programs controlling cell death, with a focus on some of the distinct features of the pathways and the molecular links between them.

Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak.

Antiapoptotic B-cell lymphoma 2 (Bcl-2) family members such as Bcl-2, myeloid cell leukemia 1 (Mcl-1), and B-cell lymphoma-X large (Bcl-xL) are proposed to inhibit autophagy by directly binding to the BH3 domain of Beclin 1/Atg6. However, these Bcl-2 family proteins also block the proapoptotic activity of Bcl-2-associated X (Bax) and Bcl-2 homologous antagonist/killer (Bak), and many inducers of autophagy also cause cell death. Therefore, when the mitochondrial-mediated apoptosis pathway is functional, interpretation of such experiments is complicated. To directly test the impact of the endogenous antiapoptotic Bcl-2 family members on autophagy in the absence of apoptosis, we inhibited their activity in cells lacking the essential cell death mediators Bax and Bak. We also used inducible lentiviral vectors to overexpress Bcl-2, Bcl-xL, or Mcl-1 in cells and subjected them to treatments that promote autophagy. In the absence of Bax and Bak, Bcl-2, Bcl-xL, and Mcl-1 had no detectable effect on autophagy or cell death in myeloid or fibroblast cell lines. On the other hand, when Bax and Bak were present, inhibiting the prosurvival Bcl-2 family members stimulated autophagy, but this correlated with increased cell death. In addition, inhibition of autophagy induced by amino acid starvation, etoposide, or interleukin-3 withdrawal did not affect cell death in the absence of Bax and Bak. These results demonstrate that the antiapoptotic Bcl-2 family members do not directly inhibit components of the autophagic pathway but instead affect autophagy indirectly, owing to their inhibition of Bax and Bak.

Total synthesis of a biotinylated rocaglate: Selective targeting of the translation factors eIF4AI/II.

The total synthesis of a biotinylated derivative of methyl rocaglate is described. This compound was accessed from synthetic methyl rocaglate (2) via formation of the propargyl amide and subsequent click reaction with a biotin azide. Affinity purification revealed that biotinylated rocaglate (8) and methyl rocaglate (2) bind with high specificity to translation factors eIF4AI/II. This remarkable selectivity is in line with that found for the more complex rocaglate silvestrol (3).

The eukaryotic initiation factor eIF4H facilitates loop-binding, repetitive RNA unwinding by the eIF4A DEAD-box helicase.

Eukaryotic translation initiation is a highly regulated process in protein synthesis. The principal translation initiation factor eIF4AI displays helicase activity, unwinding secondary structures in the mRNAs 5'-UTR. Single molecule fluorescence resonance energy transfer (sm-FRET) is applied here to directly observe and quantify the helicase activity of eIF4AI in the presence of the ancillary RNA-binding factor eIF4H. Results show that eIF4H can significantly enhance the helicase activity of eIF4AI by strongly binding both to loop structures within the RNA transcript as well as to eIF4AI. In the presence of ATP, the eIF4AI/eIF4H complex exhibits persistent rapid and repetitive cycles of unwinding and re-annealing. ATP titration assays suggest that this process consumes a single ATP molecule per cycle. In contrast, helicase unwinding activity does not occur in the presence of the non-hydrolysable analog ATP-γS. Based on our sm-FRET results, we propose an unwinding mechanism where eIF4AI/eIF4H can bind directly to loop structures to destabilize duplexes. Since eIF4AI is the prototypical example of a DEA(D/H)-box RNA helicase, it is highly likely that this unwinding mechanism is applicable to a myriad of DEAD-box helicases employed in RNA metabolism.

Inhibition of translation by cytotrienin A--a member of the ansamycin family.

The ansamycins are a diverse and often physiologically active group of compounds that include geldanamycin and rifamycin, inhibitors of heat shock protein 90 and prokaryotic DNA-dependent RNA synthesis, respectively. Cytotrienin A is an ansamycin-type small molecule with potent antiproliferative and proapoptotic properties. Here, we report that this compound inhibits eukaryotic protein synthesis by targeting translation elongation and interfering with eukaryotic elongation factor 1A function. We also find that cytotrienin A prevents HUVEC tube formation and diminishes microvessel formation in the chorioallantoic membrane assay. These results provide a molecular understanding into cytotrienin A's previously reported properties as an anticancer apoptosis-inducing drug.

Total synthesis of 2''',5'''-diepisilvestrol and its C1''' epimer: key structure activity relationships at C1''' and C2'''.

The first total synthesis of the low-abundance natural product 2''',5'''-diepisilvestrol (4) is described. The key step involved a Mitsunobu coupling between cyclopenta[b]benzofuran phenol 7 and dioxane lactol 6. Deprotection then gave a 1:2.6 ratio of natural product 2''',5'''-diepisilvestrol (4) and its C1 epimer 1''',2''',5'''-triepisilvestrol (15) in 50% overall yield. An in vitro protein translation inhibition assay showed that 2''',5'''-diepisilvestrol (4) was considerably less active than episilvestrol (2), while the unnatural isomer 1''',2''',5'''-triepisilvestrol (15) was essentially inactive, showing that the configuration at C1''' and C2''' has a large effect on the biological activity.

The Impact of Hospital Level of Care on the Management of Acute Cholecystitis: a Population-Based Study.

BACKGROUND: The organization of healthcare could have an impact on the outcome of patients treated for acute cholecystitis (AC). The aim of this study was to analyze the way in which patients with AC are managed relative to the level of care by the treating hospital. METHODS: Data were collected from the Swedish Register for Gallstone Surgery and ERCP (GallRiks). Cholecystectomies between 2010 and 2019 were included. The inclusion criterion was acute cholecystectomy in patients with AC operated at either tertiary referral centers (TRCs) or regional hospitals. RESULTS: A total of 24,194 cholecystectomies with AC met the inclusion criterion. The time between admission and acute surgery was significantly elongated at TRCs compared with regional hospitals (2.2 ± 1.7 days vs. 1.6 ± 1.4 days, mean ± SD; p < 0.0001). Patients with a history of AC were more frequent at TRC (10.1% vs. 8.9%, p < 0.0056) and had a higher adverse event rate compared with those at regional hospitals (OR 1.61; CI 1.40-1.84, p < 0.0001). Surprisingly, an increased number of hospital beds correlated slightly with an increased number of days between admission and surgery (R2 = 0.132; p = 0.0075). CONCLUSION: Compared with regional hospitals, patients with AC had to wait longer at TRCs before surgery. A history of AC significantly increased the risk of adverse events. These findings indicate that logistic and organizational aspects of hospital care may affect the management of patients with AC. However, whether these findings can be generalized to healthcare organizations outside Sweden requires further investigation.

Therapeutic suppression of translation initiation modulates chemosensitivity in a mouse lymphoma model.

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.

Synthesis of the antiproliferative agent hippuristanol and its analogues via Suárez cyclizations and Hg(II)-catalyzed spiroketalizations.

A full account of the synthesis of hippuristanol and its analogues is described. Hecogenin acetate was identified as a suitable and economical starting material for this work, and substrate-controlled stereoselection was obtained throughout the construction of the key spiroketal unit. Suárez cyclization was first used, but Hg(II)-catalyzed spiroketalization of the 3-alkyne-1,7-diol motif was finally identified as the most convenient strategy.

Regional variations in the treatment of gallstone disease may affect patient outcome: a large, population-based register study in sweden.

BACKGROUND: The lack of studies showing benefit from surgery in patients with symptoms of gallstone disease has led to a divergence in local practices and standards of care. This study aimed to explore regional differences in management and complications in Sweden. Furthermore, to study whether population density had an impact on management. METHODS: Data were collected from the Swedish National Register for Gallstone Surgery and Endoscopic Retrograde Cholangiopancreatography (GallRiks). Cholecystectomies undertaken for gallstone disease between January 2006 and December 2017 were included. Age, sex, American Society of Anesthesiologists (ASA) classification, intra- and post-operative complications, and the proportion of patients with acute cholecystitis who underwent surgery within 2 days of hospital admission were analyzed. The 21 different geographical regions in Sweden were compared, and each variable was analyzed according to population density. RESULTS: A total of 139,444 cholecystectomies cases were included in this study. There were large differences between regions regarding indications for surgery and intra- and post-operative complications. In the analyses, there were greater divergences than would be expected by chance for most of the variables analyzed. Age of the cholecystectomized patients correlated with population density of the regions (R2 = 0.310; p = 0.0088). CONCLUSIONS: There are major differences between the different regions in Sweden in terms of the treatment of gallstone disease and outcome, but these did not correlate to population density, suggesting that local routines are more likely to have an impact on treatment strategies rather than demographic factors. These differences need further investigation to reveal the underlying causes.

Cap-dependent eukaryotic initiation factor-mRNA interactions probed by cross-linking.

Cap-dependent ribosome recruitment to eukaryotic mRNAs during translation initiation is stimulated by the eukaryotic initiation factor (eIF) 4F complex and eIF4B. eIF4F is a heterotrimeric complex composed of three subunits: eIF4E, a 7-methyl guanosine cap binding protein; eIF4A, a DEAD-box RNA helicase; and eIF4G. The interactions of eIF4E, eIF4A, and eIF4B with mRNA have previously been monitored by chemical- and UV-based cross-linking approaches aimed at characterizing the initial protein/mRNA interactions that lead to ribosome recruitment. These studies have led to a model whereby eIF4E interacts with the 7-methyl guanosine cap structure in an ATP-independent manner, followed by an ATP-dependent interaction of eIF4A and eIF4B. Herein, we apply a splint-ligation-mediated approach to generate 4-thiouridine-containing mRNA adjacent to a radiolabel group that we utilize to monitor cap-dependent cross-linking of proteins adjacent to, and downstream from, the cap structure. Using this approach, we demonstrate interactions between eIF4G, eIF4H, and eIF3 subunits with the mRNA during the cap recognition process.

High-throughput assays probing protein-RNA interactions of eukaryotic translation initiation factors.

Protein-RNA interactions are involved in all facets of RNA biology. The identification of small molecules that selectively block such bimolecular interactions could provide insight into previously unexplored steps of gene regulation. Such is the case for regulation of eukaryotic protein synthesis where interactions between messenger RNA (mRNA) and several eukaryotic initiation factors govern the recruitment of 40S ribosomes (and associated factors) to mRNA templates during the initiation phase. We have designed simple fluorescence polarization-based high-throughput screening assays that query the binding of several translation factors to RNA and found that the mixed inhibitor p-chloromercuribenzoate interferes with poly(A) binding protein-RNA interaction.

By reducing global mRNA translation in several ways, 2-deoxyglucose lowers MCL-1 protein and sensitizes hemopoietic tumor cells to BH3 mimetic ABT737.

Drugs targeting various pro-survival BCL-2 family members (''BH3 mimetics'') have efficacy in hemopoietic malignancies, but the non-targeted pro-survival family members can promote resistance. Pertinently, the sensitivity of some tumor cell lines to BH3 mimetic ABT737, which targets BCL-2, BCL-XL, and BCL-W but not MCL-1, is enhanced by 2-deoxyglucose (2DG). We found that 2DG augmented apoptosis induced by ABT737 in 3 of 8 human hemopoietic tumor cell lines, most strongly in pre-B acute lymphocytic leukemia cell line NALM-6, the focus of our mechanistic studies. Although 2DG can lower MCL-1 translation, how it does so is incompletely understood, in part because 2DG inhibits both glycolysis and protein glycosylation in the endoplasmic reticulum (ER). Its glycolysis inhibition lowered ATP and, through the AMPK/mTORC1 pathway, markedly reduced global protein synthesis, as did an ER integrated stress response. A dual reporter assay revealed that 2DG impeded not only cap-dependent translation but also elongation or cap-independent translation. MCL-1 protein fell markedly, whereas 12 other BCL-2 family members were unaffected. We ascribe the MCL-1 drop to the global fall in translation, exacerbated for mRNAs with a structured 5' untranslated region (5'UTR) containing potential regulatory motifs like those in MCL-1 mRNA and the short half-life of MCL-1 protein. Pertinently, 2DG downregulated two other short-lived oncoproteins, MYC and MDM2. Thus, our results support MCL-1 as a critical 2DG target, but also reveal multiple effects on global translation that may well also affect its promotion of apoptosis.

In the absence of apoptosis, myeloid cells arrest when deprived of growth factor, but remain viable by consuming extracellular glucose.

Withdrawal of the growth factor interleukin-3 (IL-3) from IL-3-dependent myeloid cells causes them to undergo Bax/Bak1-dependent apoptosis, whereas factor-deprived Bax-/-Bak1-/- cells remain viable, but arrest and shrink. It was reported that withdrawal of IL-3 from Bax-/-Bak1-/- cells caused decreased expression of the glucose transporter Glut1, leading to reduced glucose uptake, so that arrested cells required Atg5-dependent autophagy for long-term survival. In other cell types, a decrease in Glut1 is mediated by the thioredoxin-interacting protein (Txnip), which is induced in IL-3-dependent myeloid cells when growth factor is removed. We mutated Atg5 and Txnip by CRISPR/Cas9 and found that Atg5-dependent autophagy was not necessary for the long-term viability of cycling or arrested Bax-/-Bak1-/- cells, and that Txnip was not required for the decrease in Glut1 expression in response to IL-3 withdrawal. Surprisingly, Atg5-deficient Bax/Bak1 double mutant cells survived for several weeks in medium supplemented with 10% fetal bovine serum (FBS), without high concentrations of added glucose or glutamine. When serum was withdrawn, the provision of an equivalent amount of glucose present in 10% FBS (~0.5 mM) was sufficient to support cell survival for more than a week, in the presence or absence of IL-3. Thus, Bax-/-Bak1-/- myeloid cells deprived of growth factor consume extracellular glucose to maintain long-term viability, without a requirement for Atg5-dependent autophagy.

Cross-talk between protein synthesis, energy metabolism and autophagy in cancer.

Translation is a pivotal step in the regulation of gene expression as well as one of the most energy consuming processes in the cell. Dysregulation of translation caused by the aberrant function of upstream signaling pathways and/or perturbations in the expression or function of components of the translation machinery is frequent in cancer. In this review, we discuss emerging findings that highlight hitherto unappreciated aspects of signaling to the translation apparatus with the particular focus on emerging connections between protein synthesis, autophagy and energy homeostasis in cancer.