KCNQ1/Kv7, a low-voltage-gated K+ channel, regulates cardiac rhythm and glucose homeostasis. While KCNQ1 mutations are associated with long-QT syndrome and type2 diabetes, its function in human pancreatic cells remains controversial. We identified a homozygous KCNQ1 mutation (R397W) in an individual with permanent neonatal diabetes melitus (PNDM) without cardiovascular symptoms. To decipher the potential mechanism(s), we introduced the mutation into human embryonic stem cells and generated islet-like organoids (SC-islets) using CRISPR-mediated homology-repair. The mutation did not affect pancreatic differentiation, but affected channel function by increasing spike frequency and Ca2+ flux, leading to insulin hypersecretion. With prolonged culturing, the mutant islets decreased their secretion and gradually deteriorated, modeling a diabetic state, which accelerated by high glucose levels. The molecular basis was the downregulated expression of voltage-activated Ca2+ channels and oxidative phosphorylation. Our study provides a better understanding of the role of KCNQ1 in regulating insulin secretion and β-cell survival in hereditary diabetes pathology.© 2024 The Authors.

Readily accessible human pancreatic beta cells that are functionally close to primary adult beta cells are a crucial model to better understand human beta cell physiology and develop new treatments for diabetes. We here report the characterization of EndoC-βH5 cells, the latest in the EndoC-βH cell family.
EndoC-βH5 cells were generated by integrative gene transfer of immortalizing transgenes hTERT and SV40 large T along with Herpes Simplex Virus-1 thymidine kinase into human fetal pancreas. Immortalizing transgenes were removed after amplification using CRE activation and remaining non-excized cells eliminated using ganciclovir. Resulting cells were distributed as ready to use EndoC-βH5 cells. We performed transcriptome, immunological and extensive functional assays.
Ready to use EndoC-βH5 cells display highly efficient glucose dependent insulin secretion. A robust 10-fold insulin secretion index was observed and reproduced in four independent laboratories across Europe. EndoC-βH5 cells secrete insulin in a dynamic manner in response to glucose and secretion is further potentiated by GIP and GLP-1 analogs. RNA-seq confirmed abundant expression of beta cell transcription factors and functional markers, including incretin receptors. Cytokines induce a gene expression signature of inflammatory pathways and antigen processing and presentation. Finally, modified HLA-A2 expressing EndoC-βH5 cells elicit specific A2-alloreactive CD8 T cell activation.
EndoC-βH5 cells represent a unique storable and ready to use human pancreatic beta cell model with highly robust and reproducible features. Such cells are thus relevant for the study of beta cell function, screening and validation of new drugs, and development of disease models.
Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.

The HNF1αp291fsinsC truncation is the most common mutation associated with maturity-onset diabetes of the young 3 (MODY3). Although shown to impair HNF1α signaling, the mechanism by which HNF1αp291fsinsC causes MODY3 is not fully understood. Here we use MODY3 patient and CRISPR/Cas9-engineered human induced pluripotent stem cells (hiPSCs) grown as 3D organoids to investigate how HNF1αp291fsinsC affects hiPSC differentiation during pancreatic development. HNF1αp291fsinsC hiPSCs shows reduced pancreatic progenitor and β cell differentiation. Mechanistically, HNF1αp291fsinsC interacts with HNF1β and inhibits its function, and disrupting this interaction partially rescues HNF1β-dependent transcription. HNF1β overexpression in the HNF1αp291fsinsC patient organoid line increases PDX1+ progenitors, while HNF1β overexpression in the HNF1αp291fsinsC patient iPSC line partially rescues β cell differentiation. Our study highlights the capability of pancreas progenitor-derived organoids to model disease in vitro. Additionally, it uncovers an HNF1β-mediated mechanism linked to HNF1α truncation that affects progenitor differentiation and could explain the clinical heterogeneity observed in MODY3 patients.Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

Combining immune checkpoint therapy (ICT) and targeted therapy holds great promises for broad and long-lasting anti-cancer therapies. However, combining ICT with anti-PI3K inhibitors have been challenging because the multifaceted effects of PI3K on both cancer cells and immune cells within the tumor microenvironment. Here we find that intermittent but not daily dosing of a PI3Kα/β/δ inhibitor, BAY1082439, on Pten-null prostate cancer models could overcome ICT resistance and unleash CD8+ T cell-dependent anti-tumor immunity in vivo. Mechanistically, BAY1082439 converts cancer cell-intrinsic immune-suppression to immune-stimulation by promoting IFNα/IFNγ pathway activation, β2-microglubin expression and CXCL10/CCL5 secretion. With its preferential regulatory T cell inhibition activity, BAY1082439 promotes clonal expansion of tumor-associated CD8+ T cells, most likely via tertiary lymphoid structures. Once primed, tumors remain T cell-inflamed, become responsive to anti-PD-1 therapy and have durable therapeutic effect. Our data suggest that intermittent PI3K inhibition can alleviate Pten-null cancer cell-intrinsic immunosuppressive activity and turn "cold" tumors into T cell-inflamed ones, paving the way for successful ICT.
© 2022. The Author(s).

h4>Summary/h4> Prostate cancers generally lack T cell infiltration and display resistance to immune checkpoint therapies (ICT). We found that intermittent but not daily dosing of PI3Kα/β/δ inhibitor BAY1082439 on a Pten -null spontaneous prostate cancer model could overcome ICT resistance and unleash CD8 + T cell-dependent anti-tumor immunity in vivo . Mechanistically, BAY1082439 converts Pten -null cancer cell-intrinsic immune-suppression to immune-stimulation by promoting IFNα/γ pathway activation, β2-microglubin expression and CXCL10/CCL5 secretion. Together with its preferential Treg inhibition activity, BAY1082439 promotes clonal expansion of tumor-associated CD8 + T cells. Once primed, tumors remain as T cell-inflamed and become responsive to anti-PD-1 therapy. Our data suggest that intermittent PI3K inhibition can alleviate Pten -null cancer cell-intrinsic immunosuppressive activity and turn “cold” tumors into T cell-inflamed ones, paving the way for successful ICT. h4>Significance/h4> The combination of ICT and targeted therapies holds great promises for broad and long-lasting therapeutic effects for cancers. However, combining ICT with anti-PI3K inhibitors have been difficult because the multifaceted effects of PI3K on both cancer cells and immune cells within the tumor microenvironment. Here we show a carefully designed anti-PI3K treatment, both in its specificity and dosing schedule, to inhibit cancer cell growth while promoting anti-tumor immunity, is critically important for successful ICT. Since the PI3K pathway is one of the most frequently altered signaling pathways in human cancers, our work may shed light on treating those cancers with PI3K activation and overcome resistance to ICT. h4>Highlights/h4> Intermittent PI3Kα/β/δ inhibitor BAY1082439 treatment overcomes ICT resistance BAY1082439 turns Pten -null prostate cancer from “cold” to T cell-inflamed BAY1082439 inhibits cancer cell-intrinsic immunosuppressive activity and Treg BAY1082439 promotes clonal expansion and immunity of tumor-associated CD8 + T cells