DYRK1B blockade promotes tumoricidal macrophage activity in pancreatic cancer.

OBJECTIVE: Highly malignant pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant immunosuppressive and fibrotic tumour microenvironment (TME). Future therapeutic attempts will therefore demand the targeting of tumours and stromal compartments in order to be effective. Here we investigate whether dual specificity and tyrosine phosphorylation-regulated kinase 1B (DYRK1B) fulfil these criteria and represent a promising anticancer target in PDAC. DESIGN: We used transplantation and autochthonous mouse models of PDAC with either genetic Dyrk1b loss or pharmacological DYRK1B inhibition, respectively. Mechanistic interactions between tumour cells and macrophages were studied in direct or indirect co-culture experiments. Histological analyses used tissue microarrays from patients with PDAC. Additional methodological approaches included bulk mRNA sequencing (transcriptomics) and proteomics (secretomics). RESULTS: We found that DYRK1B is mainly expressed by pancreatic epithelial cancer cells and modulates the influx and activity of TME-associated macrophages through effects on the cancer cells themselves as well as through the tumour secretome. Mechanistically, genetic ablation or pharmacological inhibition of DYRK1B strongly attracts tumoricidal macrophages and, in addition, downregulates the phagocytosis checkpoint and 'don't eat me' signal CD24 on cancer cells, resulting in enhanced tumour cell phagocytosis. Consequently, tumour cells lacking DYRK1B hardly expand in transplantation experiments, despite their rapid growth in culture. Furthermore, combining a small-molecule DYRK1B-directed therapy with mammalian target of rapamycin inhibition and conventional chemotherapy stalls the growth of established tumours and results in a significant extension of life span in a highly aggressive autochthonous model of PDAC. CONCLUSION: In light of DYRK inhibitors currently entering clinical phase testing, our data thus provide a novel and clinically translatable approach targeting both the cancer cell compartment and its microenvironment.

PPARß/δ recruits NCOR and regulates transcription reinitiation of ANGPTL4.

In the absence of ligands, the nuclear receptor PPARß/δ recruits the NCOR and SMRT corepressors, which form complexes with HDAC3, to canonical target genes. Agonistic ligands cause dissociation of corepressors and enable enhanced transcription. Vice versa, synthetic inverse agonists augment corepressor recruitment and repression. Both basal repression of the target gene ANGPTL4 and reinforced repression elicited by inverse agonists are partially insensitive to HDAC inhibition. This raises the question how PPARß/δ represses transcription mechanistically. We show that the PPARß/δ inverse agonist PT-S264 impairs transcription initiation by decreasing recruitment of activating Mediator subunits, RNA polymerase II, and TFIIB, but not of TFIIA, to the ANGPTL4 promoter. Mass spectrometry identifies NCOR as the main PT-S264-dependent interactor of PPARß/δ. Reconstitution of knockout cells with PPARß/δ mutants deficient in basal repression results in diminished recruitment of NCOR, SMRT, and HDAC3 to PPAR target genes, while occupancy by RNA polymerase II is increased. PT-S264 restores binding of NCOR, SMRT, and HDAC3 to the mutants, resulting in reduced polymerase II occupancy. Our findings corroborate deacetylase-dependent and -independent repressive functions of HDAC3-containing complexes, which act in parallel to downregulate transcription.

Tumor-suppressive disruption of cancer subtype-associated super enhancer circuits by small molecule treatment.

Transcriptional cancer subtypes which correlate with traits such as tumor growth, drug sensitivity or the chances of relapse and metastasis, have been described for several malignancies. The core regulatory circuits (CRCs) defining these subtypes are established by chromatin super enhancers (SEs) driving key transcription factors (TFs) specific for the particular cell state. In neuroblastoma (NB), one of the most frequent solid pediatric cancer entities, two major SE-directed molecular subtypes have been described: A more lineage-committed adrenergic (ADRN) and a mesenchymal (MES) subtype. Here, we found that a small isoxazole molecule (ISX), a frequently used pro-neural drug, reprogrammed SE activity and switched NB cells from an ADRN subtype towards a growth-retarded MES-like state. The MES-like state shared strong transcriptional overlap with ganglioneuroma (GN), a benign and highly differentiated tumor of the neural crest. Mechanistically, ISX suppressed chromatin binding of N-MYC, a CRC-amplifying transcription factor, resulting in loss of key ADRN subtype-enriched components such as N-MYC itself, PHOX2B and ALK, while concomitently, MES subtype markers were induced. Globally, ISX treatment installed a chromatin accessibility landscape typically associated with low risk NB. In summary, we provide evidence that CRCs and cancer subtype reprogramming might be amenable to future therapeutic targeting.

Which Properties Allow Ligands to Open and Bind to the Transient Binding Pocket of Human Aldose Reductase?

The transient specificity pocket of aldose reductase only opens in response to specific ligands. This pocket may offer an advantage for the development of novel, more selective ligands for proteins with similar topology that lack such an adaptive pocket. Our aim was to elucidate which properties allow an inhibitor to bind in the specificity pocket. A series of inhibitors that share the same parent scaffold but differ in their attached aromatic substituents were screened using ITC and X-ray crystallography for their ability to occupy the pocket. Additionally, we investigated the electrostatic potentials and charge distribution across the attached terminal aromatic groups with respect to their potential to bind to the transient pocket of the enzyme using ESP calculations. These methods allowed us to confirm the previously established hypothesis that an electron-deficient aromatic group is an important prerequisite for opening and occupying the specificity pocket. We also demonstrated from our crystal structures that a pH shift between 5 and 8 does not affect the binding position of the ligand in the specificity pocket. This allows for a comparison between thermodynamic and crystallographic data collected at different pH values.

Mitochondrial rescue prevents glutathione peroxidase-dependent ferroptosis.

Research into oxidative cell death is producing exciting new mechanisms, such as ferroptosis, in the neuropathologies of cerebral ischemia and hemorrhagic brain insults. Ferroptosis is an oxidative form of regulated necrotic cell death featuring glutathione (GSH) depletion, disrupted glutathione peroxidase-4 (GPX4) redox defense and detrimental lipid reactive oxygen species (ROS) formation. Further, our recent findings identified mitochondrial damage in models of oxidative glutamate toxicity, glutathione peroxidase depletion, and ferroptosis. Despite knowledge on the signaling pathways of ferroptosis increasing, the particular role of mitochondrial damage requires more in depth investigation in order to achieve effective treatment options targeting mitochondria. In the present study, we applied RSL3 to induce ferroptosis in neuronal HT22 cells and mouse embryonic fibroblasts. In both cell types, RSL3 mediated concentration-dependent inhibition of GPX4, lipid peroxidation, enhanced mitochondrial fragmentation, loss of mitochondrial membrane potential, and reduced mitochondrial respiration. Ferroptosis inhibitors, such as deferoxamine, ferrostatin-1 and liproxstatin-1, but also CRISPR/Cas9 Bid knockout and the BID inhibitor BI-6c9 protected against RSL3 toxicity. We found compelling new information that the mitochondria-targeted ROS scavenger mitoquinone (MitoQ) preserved mitochondrial integrity and function, and cell viability despite significant loss of GPX4 expression and associated increases in general lipid peroxidation after exposure to RSL3. Our data demonstrate that rescuing mitochondrial integrity and function through the inhibition of BID or by the mitochondria-targeted ROS scavenger MitoQ serves as a most effective strategy in the prevention of ferroptosis in different cell types. These findings expose mitochondria as promising targets for novel therapeutic intervention strategies in oxidative cell death.

A Small-Molecule Inhibitor of Bax and Bak Oligomerization Prevents Genotoxic Cell Death and Promotes Neuroprotection.

Aberrant apoptosis can lead to acute or chronic degenerative diseases. Mitochondrial outer membrane permeabilization (MOMP) triggered by the oligomerization of the Bcl-2 family proteins Bax/Bak is an irreversible step leading to execution of apoptosis. Here, we describe the discovery of small-molecule inhibitors of Bax/Bak oligomerization that prevent MOMP. We demonstrate that these molecules disrupt multiple, but not all, interactions between Bax dimer interfaces thereby interfering with the formation of higher-order oligomers in the MOM, but not recruitment of Bax to the MOM. Small-molecule inhibition of Bax/Bak oligomerization allowed cells to evade apoptotic stimuli and rescued neurons from death after excitotoxicity, demonstrating that oligomerization of Bax is essential for MOMP. Our discovery of small-molecule Bax/Bak inhibitors provides novel tools for the investigation of the mechanisms leading to MOMP and will ultimately facilitate development of compounds inhibiting Bax/Bak in acute and chronic degenerative diseases.