RFX6 haploinsufficiency predisposes to diabetes through impaired beta cell function.

AIMS/HYPOTHESIS: Regulatory factor X 6 (RFX6) is crucial for pancreatic endocrine development and differentiation. The RFX6 variant p.His293LeufsTer7 is significantly enriched in the Finnish population, with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 and gestational diabetes. However, the precise mechanism of this predisposition remains unknown. METHODS: To understand the role of this variant in beta cell development and function, we used CRISPR technology to generate allelic series of pluripotent stem cells. We created two isogenic stem cell models: a human embryonic stem cell model; and a patient-derived stem cell model. Both were differentiated into pancreatic islet lineages (stem-cell-derived islets, SC-islets), followed by implantation in immunocompromised NOD-SCID-Gamma mice. RESULTS: Stem cell models of the homozygous variant RFX6-/- predictably failed to generate insulin-secreting pancreatic beta cells, mirroring the phenotype observed in Mitchell-Riley syndrome. Notably, at the pancreatic endocrine stage, there was an upregulation of precursor markers NEUROG3 and SOX9, accompanied by increased apoptosis. Intriguingly, heterozygous RFX6+/- SC-islets exhibited RFX6 haploinsufficiency (54.2% reduction in protein expression), associated with reduced beta cell maturation markers, altered calcium signalling and impaired insulin secretion (62% and 54% reduction in basal and high glucose conditions, respectively). However, RFX6 haploinsufficiency did not have an impact on beta cell number or insulin content. The reduced insulin secretion persisted after in vivo implantation in mice, aligning with the increased risk of variant carriers to develop diabetes. CONCLUSIONS/INTERPRETATION: Our allelic series isogenic SC-islet models represent a powerful tool to elucidate specific aetiologies of diabetes in humans, enabling the sensitive detection of aberrations in both beta cell development and function. We highlight the critical role of RFX6 in augmenting and maintaining the pancreatic progenitor pool, with an endocrine roadblock and increased cell death upon its loss. We demonstrate that RFX6 haploinsufficiency does not affect beta cell number or insulin content but does impair function, predisposing heterozygous carriers of loss-of-function variants to diabetes. DATA AVAILABILITY: Ultra-deep bulk RNA-seq data for pancreatic differentiation stages 3, 5 and 7 of H1 RFX6 genotypes are deposited in the Gene Expression Omnibus database with accession code GSE234289. Original western blot images are deposited at Mendeley ( https://data.mendeley.com/datasets/g75drr3mgw/2 ).

Alternative splicing encodes functional intracellular CD59 isoforms that mediate insulin secretion and are down-regulated in diabetic islets.

Human pancreatic islets highly express CD59, which is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein and is required for insulin secretion. How cell-surface CD59 could interact with intracellular exocytotic machinery has so far not been described. We now demonstrate the existence of CD59 splice variants in human pancreatic islets, which have unique C-terminal domains replacing the GPI-anchoring signal sequence. These isoforms are found in the cytosol of ß-cells, interact with SNARE proteins VAMP2 and SNAP25, colocalize with insulin granules, and rescue insulin secretion in CD59-knockout (KO) cells. We therefore named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Antibodies raised against each isoform revealed that expression of both IRIS-1 and IRIS-2 is significantly lower in islets isolated from human type 2 diabetes (T2D) patients, as compared to healthy controls. Further, glucotoxicity induced in primary, healthy human islets led to a significant decrease of IRIS-1 expression, suggesting that hyperglycemia (raised glucose levels) and subsequent decreased IRIS-1 expression may contribute to relative insulin deficiency in T2D patients. Similar isoforms were also identified in the mouse CD59B gene, and targeted CRISPR/Cas9-mediated knockout showed that these intracellular isoforms, but not canonical CD59B, are involved in insulin secretion from mouse ß-cells. Mouse IRIS-2 is also down-regulated in diabetic db/db mouse islets. These findings establish the endogenous existence of previously undescribed non­GPI-anchored intracellular isoforms of human CD59 and mouse CD59B, which are required for normal insulin secretion.

Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells.

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.

Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetes.

Glucagon is released from pancreatic α-cells to activate pathways that raise blood glucose. Its secretion is regulated by α-cell-intrinsic glucose sensing and paracrine control through insulin and somatostatin. To understand the inadequately high glucagon levels that contribute to hyperglycemia in type-2 diabetes (T2D), we analyzed granule behavior, exocytosis and membrane excitability in α-cells of 68 non-diabetic and 21 T2D human donors. We report that exocytosis is moderately reduced in α-cells of T2D donors, without changes in voltage-dependent ion currents or granule trafficking. Dispersed α-cells have a non-physiological V-shaped dose response to glucose, with maximal exocytosis at hyperglycemia. Within intact islets, hyperglycemia instead inhibits α-cell exocytosis, but not in T2D or when paracrine inhibition by insulin or somatostatin is blocked. Surface expression of somatostatin-receptor-2 is reduced in T2D, suggesting a mechanism for the observed somatostatin resistance. Thus, elevated glucagon in human T2D may reflect α-cell insensitivity to paracrine inhibition at hyperglycemia.

Biaryl sulfonamide motifs up- or down-regulate ion channel activity by activating voltage sensors.

Voltage-gated ion channels are key molecules for the generation of cellular electrical excitability. Many pharmaceutical drugs target these channels by blocking their ion-conducting pore, but in many cases, channel-opening compounds would be more beneficial. Here, to search for new channel-opening compounds, we screen 18,000 compounds with high-throughput patch-clamp technology and find several potassium-channel openers that share a distinct biaryl-sulfonamide motif. Our data suggest that the negatively charged variants of these compounds bind to the top of the voltage-sensor domain, between transmembrane segments 3 and 4, to open the channel. Although we show here that biaryl-sulfonamide compounds open a potassium channel, they have also been reported to block sodium and calcium channels. However, because they inactivate voltage-gated sodium channels by promoting activation of one voltage sensor, we suggest that, despite different effects on the channel gates, the biaryl-sulfonamide motif is a general ion-channel activator motif. Because these compounds block action potential-generating sodium and calcium channels and open an action potential-dampening potassium channel, they should have a high propensity to reduce excitability. This opens up the possibility to build new excitability-reducing pharmaceutical drugs from the biaryl-sulfonamide scaffold.

A deep conical agarose microwell array for adhesion independent three-dimensional cell culture and dynamic volume measurement.

Multicellular spheroids represent a well-established 3D model to study healthy and diseased cells in vitro. The use of conventional 3D cell culture platforms for the generation of multicellular spheroids is limited to cell types that easily self-assemble into spheroids because less adhesive cells fail to form stable aggregates. A high-precision micromoulding technique developed in our laboratory produces deep conical agarose microwell arrays that allow the cultivation of uniform multicellular aggregates, irrespective of the spheroid formation capacity of the cells. Such hydrogel arrays warrant a steady nutrient supply for several weeks, permit live volumetric measurements to monitor cell growth, enable immunohistochemical staining, fluorescence-based microscopy, and facilitate immediate harvesting of cell aggregates. This system also allows co-cultures of two distinct cell types either in direct cell-cell contact or at a distance as the hydrogel permits diffusion of soluble compounds. Notably, we show that co-culture of a breast cancer cell line with bone marrow stromal cells enhances 3D growth of the cancer cells in this system.

Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes.

Loss of first-phase insulin secretion is an early sign of developing type 2 diabetes (T2D). Ca2+ entry through voltage-gated L-type Ca2+ channels triggers exocytosis of insulin-containing granules in pancreatic ß cells and is required for the postprandial spike in insulin secretion. Using high-resolution microscopy, we have identified a subset of docked insulin granules in human ß cells and rat-derived clonal insulin 1 (INS1) cells for which localized Ca2+ influx triggers exocytosis with high probability and minimal latency. This immediately releasable pool (IRP) of granules, identified both structurally and functionally, was absent in ß cells from human T2D donors and in INS1 cells cultured in fatty acids that mimic the diabetic state. Upon arrival at the plasma membrane, IRP granules slowly associated with 15 to 20 L-type channels. We determined that recruitment depended on a direct interaction with the synaptic protein Munc13, because expression of the II-III loop of the channel, the C2 domain of Munc13-1, or of Munc13-1 with a mutated C2 domain all disrupted L-type channel clustering at granules and ablated fast exocytosis. Thus, rapid insulin secretion requires Munc13-mediated recruitment of L-type Ca2+ channels in close proximity to insulin granules. Loss of this organization underlies disturbed insulin secretion kinetics in T2D.

Resin-acid derivatives as potent electrostatic openers of voltage-gated K channels and suppressors of neuronal excitability.

Voltage-gated ion channels generate cellular excitability, cause diseases when mutated, and act as drug targets in hyperexcitability diseases, such as epilepsy, cardiac arrhythmia and pain. Unfortunately, many patients do not satisfactorily respond to the present-day drugs. We found that the naturally occurring resin acid dehydroabietic acid (DHAA) is a potent opener of a voltage-gated K channel and thereby a potential suppressor of cellular excitability. DHAA acts via a non-traditional mechanism, by electrostatically activating the voltage-sensor domain, rather than directly targeting the ion-conducting pore domain. By systematic iterative modifications of DHAA we synthesized 71 derivatives and found 32 compounds more potent than DHAA. The most potent compound, Compound 77, is 240 times more efficient than DHAA in opening a K channel. This and other potent compounds reduced excitability in dorsal root ganglion neurons, suggesting that resin-acid derivatives can become the first members of a new family of drugs with the potential for treatment of hyperexcitability diseases.

Potency optimization of Huwentoxin-IV on hNav1.7: a neurotoxin TTX-S sodium-channel antagonist from the venom of the Chinese bird-eating spider Selenocosmia huwena.

The spider venom peptide Huwentoxin-IV (HwTx-IV) 1 is a potent antagonist of hNav1.7 (IC50 determined herein as 17 ± 2 nM). Nav1.7 is a voltage-gated sodium channel involved in the generation and conduction of neuropathic and nociceptive pain signals. We prepared a number of HwTx-IV analogs as part of a structure-function study into Nav1.7 antagonism. The inhibitory potency of these analogs was determined by automated electrophysiology and is reported herein. In particular, the native residues Glu(1), Glu(4), Phe(6) and Tyr(33) were revealed as important activity modulators and several peptides bearing mutations in these positions showed significantly increased potency on hNav1.7 while maintaining the original selectivity profile of the wild-type peptide 1 on hNav1.5. Peptide 47 (Gly(1), Gly(4), Trp(33)-HwTx) demonstrated the largest potency increase on hNav1.7 (IC50 0.4 ± 0.1 nM).

In vivo and ex vivo inhibition of spinal nerve ligation-induced ectopic activity by sodium channel blockers correlate to in vitro inhibition of NaV1.7 and clinical efficacy: a pharmacokinetic-pharmacodynamic translational approach.

PURPOSE: In vivo and ex vivo inhibition of ectopic activity of clinically used and newly developed sodium channel (NaV) blockers were quantified in the rat spinal nerve ligation (SNL) model using a pharmacokinetic-pharmacodynamic (PKPD) approach and correlated to in vitro NaV1.7 channel inhibition and clinical effective concentrations. METHODS: In vivo, drug exposure and inhibition of ectopic activity were assessed in anaesthetized SNL rats at two dose levels. Ex vivo, compounds were applied at increasing concentrations to dorsal root ganglias isolated from SNL rats. The inhibitory potency (IC 50 ) was estimated using PKPD analysis. In vitro IC 50 was estimated using an electrophysiology-based assay using recombinant rat and human NaV1.7 expressing HEK293 cells. RESULTS: In vivo and ex vivo inhibition of ectopic activity correlated well with the in vitro inhibition on the rat NaV1.7 channel. The estimated IC 50s for inhibition of ectopic activity in the SNL model occurred at similar unbound concentrations as clinical effective concentrations in humans. CONCLUSIONS: Inhibition of ectopic activity in the SNL model could be useful in predicting clinical effective concentrations for novel sodium channel blockers. In addition, in vitro potency could be used for screening, characterization and selection of compounds, thereby reducing the need for in vivo testing.

Identification of novel NaV1.7 antagonists using high throughput screening platforms.

Congenital Insensitivity to Pain (CIP) is a loss of function mutation resulting in a truncated NaV1.7 protein, suggesting a pivotal role in pain signaling and rendering it an important pharmaceutical target for multiple pain conditions. The structural homology in the NaV-channel family makes it challenging to design effective analgesic compounds without inducing for example cardiotoxicity or seizure liabilities. An additional approach to structural isoform selectivity is to identify compounds with use- or state-dependent profiles, i.e. inhibition efficacy based on the gating of the ion channel. In general nerve cells in damaged or inflamed tissue are more depolarized and electrically active compared to healthy nerve cells in for instance the heart. This observation has led to the design of two types of screening protocols emulating the voltage condition of peripheral neurons or cardiac tissue. The two voltage protocols have been developed to identify both use- and state-dependent antagonists. In this paper we describe an attempt to merge the two different protocols into one to increase screening efficacy, while retaining relevant state- and use-dependent pharmacology. The new protocol is constructed of two stimulation pulses and a slow voltage ramp for simultaneous assessment of resting and state-dependent block. By comparing all protocols we show that the new protocol indeed filter compounds for state-dependence and increase the prediction power of selecting use-dependent compounds.

Phenethyl nicotinamides, a novel class of Na(V)1.7 channel blockers: structure and activity relationship.

The Na(V)1.7 ion channel is an attractive target for development of potential analgesic drugs based on strong genetic links between mutations in the gene coding for the channel protein and inheritable pain conditions. The (S)-N-chroman-3-ylcarboxamide series, exemplified by 1, was used as a starting point for development of new channel blockers, resulting in the phenethyl nicotinamide series. The structure and activity relationship for this series was established and the metabolic issues of early analogues were addressed by appropriate substitutions. Compound 33 displayed acceptable overall in vitro properties and in vivo rat PK profile.

Structure and activity relationship in the (S)-N-chroman-3-ylcarboxamide series of voltage-gated sodium channel blockers.

Recent findings showing a relation between mutations in the Na(V)1.7 channel in humans and altered pain sensation has contributed to increase the attractiveness of this ion channel as target for development of potential analgesics. Amido chromanes 1 and 2 were identified as blockers of the Na(V)1.7 channel and analogues with modifications of the 5-substituent and the carboxamide part of the molecule were prepared to establish the structure-activity relationship. Compounds 13 and 29 with good overall in vitro and in vivo rat PK profile were identified. Furthermore, 29 showed in vivo efficacy in a nociceptive pain model.

3-Oxoisoindoline-1-carboxamides: potent, state-dependent blockers of voltage-gated sodium channel Na(V)1.7 with efficacy in rat pain models.

The voltage-gated sodium channel Na(V)1.7 is believed to be a critical mediator of pain sensation based on clinical genetic studies and pharmacological results. Clinical utility of nonselective sodium channel blockers is limited due to serious adverse drug effects. Here, we present the optimization, structure-activity relationships, and in vitro and in vivo characterization of a novel series of Na(V)1.7 inhibitors based on the oxoisoindoline core. Extensive studies with focus on optimization of Na(V)1.7 potency, selectivity over Na(V)1.5, and metabolic stability properties produced several interesting oxoisoindoline carboxamides (16A, 26B, 28, 51, 60, and 62) that were further characterized. The oxoisoindoline carboxamides interacted with the local anesthetics binding site. In spite of this, several compounds showed functional selectivity versus Na(V)1.5 of more than 100-fold. This appeared to be a combination of subtype and state-dependent selectivity. Compound 28 showed concentration-dependent inhibition of nerve injury-induced ectopic in an ex vivo DRG preparation from SNL rats. Compounds 16A and 26B demonstrated concentration-dependent efficacy in preclinical behavioral pain models. The oxoisoindoline carboxamides series described here may be valuable for further investigations for pain therapeutics.

Transformation-dependent silencing of tumor-selective apoptosis-inducing TRAIL by DNA hypermethylation is antagonized by decitabine.

TNF-related apoptosis-inducing ligand (TRAIL) kills tumor cells selectively. We asked how emerging tumor cells escape elimination by TRAIL and how tumor-specific killing by TRAIL could then be restored. We found that TRAIL expression is consistently downregulated in HRAS(G12V)-transformed cells in stepwise tumorigenesis models derived from four different tissues due to DNA hypermethylation of CpG clusters within the TRAIL promoter. Decitabine de-silenced TRAIL, which remained inducible by interferon, while induction of TRAIL by blocking the HRAS(G12V)-activated mitogen-activated protein kinase pathway was subordinated to epigenetic silencing. Decitabine induced apoptosis through upregulation of endogenous TRAIL in cooperation with favorable regulation of key players acting in TRAIL-mediated apoptosis. Apoptosis induction by exogenously added TRAIL was largely increased by decitabine. In vivo treatment of xenografted human HRAS(G12V)-transformed human epithelial kidney or syngenic mice tumors by decitabine blocked tumor growth induced TRAIL expression and apoptosis. Our results emphasize the potential of decitabine to enhance TRAIL-induced apoptosis in tumors and thus provide a rationale for combination therapies with decitabine to increase tumor-selective apoptosis.

Phenyl isoxazole voltage-gated sodium channel blockers: structure and activity relationship.

Blocking of certain sodium channels is considered to be an attractive mechanism to treat chronic pain conditions. Phenyl isoxazole carbamate 1 was identified as a potent and selective Na(V)1.7 blocker. Structural analogues of 1, both carbamates, ureas and amides, were proven to be useful in establishing the structure-activity relationship and improving ADME related properties. Amide 24 showed a good overall in vitro profile, that translated well to rat in vivo PK.

Identification of Y-box binding protein 1 as a core regulator of MEK/ERK pathway-dependent gene signatures in colorectal cancer cells.

Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties.

Down-regulation of HLA Class I and NKG2D ligands through a concerted action of MAPK and DNA methyltransferases in colorectal cancer cells.

Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. It is still unclear, to what extend the escape of emerging cancer cells from recognition and elimination by the immune system is determined by similar mechanisms. We compared the transcriptomes of HCT116 colorectal cancer cells deficient in DNA methyltransferases (DNMTs) and of cells, in which the RAS pathway as the major growth-promoting signaling system is blocked by inhibition of MAPK. We identified the MHC Class I genes HLA-A1/A2 and the ULBP2 gene encoding 1 of the 8 known ligands of the activating NK receptor NKG2D among a cluster of immune genes up-regulated under the conditions of both DNMT-deficiency and MEK-inhibition. Bisulphite sequencing analyses of HCT116 with DNMT deficiency or after MEK-inhibition showed that de-methylation of the ULPB2 promoter correlated with its enhanced surface expression. The HLA-A promoters were not methylated indicating that components of the HLA assembly machinery were also suppressed in DNMT-deficient and MEK-inhibited cells. Increased HLA-A2 surface expression was correlated with enhanced recognition and lysis by A2-specific CTL. On the contrary, elevated ULBP2 expression was not reflected by enhanced recognition and lysis by NK cells. Cosuppression of HLA Class I and NKG2D ligands and genes encoding peptide transporters or proteasomal genes mediates a strong functional link between RAS activation, DNMT activity and disruption of the antigen presenting system controlling immune recognition in colorectal cancer cells.