nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer.

The pleiotropic roles of nSMase2-generated ceramide include regulation of intracellular ceramide signaling and exosome biogenesis. We investigated the effects of eliminating nSMase2 on early and advanced PDA, including its influence on the microenvironment. Employing the KPC mouse model of pancreatic cancer, we demonstrate that pancreatic epithelial nSMase2 ablation reduces neoplasia and promotes a PDA subtype switch from aggressive basal-like to classical.

NF-κB inhibitor alpha controls SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing.

Focal adhesion kinase-YAP signaling axis drives drug-tolerant persister cells and residual disease in lung cancer.

Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies.

Cutting Edge: Induced Loss of Rasgrp1 in Peripheral CD4+ T Cells of Conditional Rasgrp1-Deficient Mice Reveals an Essential Role for Rasgrp1 in TCR/CD28-Induced Ras-MAPK Signaling.

Ras guanine nucleotide-releasing protein 1 (Rasgrp1) is a Ras guanine nucleotide exchange factor that participates in the activation of the Ras-ERK signaling pathway in developing T cells and is required for efficient thymic T cell positive selection. However, the role of Rasgrp1 in mature peripheral T cells has not been definitively addressed, in part because peripheral T cells from constitutive Rasgrp1-deficient mice show an abnormal activated phenotype. In this study, we generated an inducible Rasgrp1-deficient mouse model to allow acute disruption of Rasgrp1 in peripheral CD4+ T cells in the context of normal T cell development.

Transcriptional-translational conflict is a barrier to cellular transformation and cancer progression.

We uncover a tumor-suppressive process in urothelium called transcriptional-translational conflict caused by deregulation of the central chromatin remodeling component ARID1A. Loss of Arid1a triggers an increase in a nexus of pro-proliferation transcripts, but a simultaneous inhibition of the eukaryotic elongation factor 2 (eEF2), which results in tumor suppression. Resolution of this conflict through enhancing translation elongation speed enables the efficient and precise synthesis of a network of poised mRNAs resulting in uncontrolled proliferation, clonogenic growth, and bladder cancer progression.

NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing.

Cellular architecture of human brain metastases.

Brain metastasis (BrM) is the most common form of brain cancer, characterized by neurologic disability and an abysmal prognosis. Unfortunately, our understanding of the biology underlying human BrMs remains rudimentary. Here, we present an integrative analysis of >100,000 malignant and non-malignant cells from 15 human parenchymal BrMs, generated by single-cell transcriptomics, mass cytometry, and complemented with mouse model- and in silico approaches.

SARS-CoV-2 infection studies in lung organoids identify TSPAN8 as novel mediator.

SARS coronavirus-2 (SARS-CoV-2) is causing a global pandemic with large variation in COVID-19 disease spectrum. SARS-CoV-2 infection requires host receptor ACE2 on lung epithelium, but epithelial underpinnings of variation are largely unknown. We capitalized on comprehensive organoid assays to report remarkable variation in SARS-CoV-2 infection rates of lung organoids from different subjects.

Regulation of the Small GTPase Ras and Its Relevance to Human Disease.

Ras research has experienced a considerable boost in recent years, not least prompted by the Ras initiative launched by the NCI in 2013 ( https://www.cancer.gov/research/key-initiatives/ras ), accompanied and conditioned by a strongly reinvigorated determination within the Ras community to develop therapeutics attacking directly the Ras oncoproteins.

T cells: a dedicated effector kinase pathways for every trait?

Signaling pathways play critical roles in regulating the activation of T cells. Recognition of foreign peptide presented by MHC to the T cell receptor (TCR) triggers a signaling cascade of proximal kinases and adapter molecules that lead to the activation of Effector kinase pathways. These effector kinase pathways play pivotal roles in T cell activation, differentiation, and proliferation.

Bioinformatic Approaches to Validation and Functional Analysis of 3D Lung Cancer Models.

3D models of cancer have the potential to improve basic, translational, and clinical studies. Patient-derived xenografts, spheroids, and organoids are broad categories of 3D models of cancer, and to date, these 3D models of cancer have been established for a variety of cancer types. In lung cancer, for example, 3D models offer a promising new avenue to gain novel insights into lung tumor biology and improve outcomes for patients afflicted with the number one cancer killer worldwide.

Investigating increased hematopoietic stem cell fitness in a novel mouse model.

T-cell acute lymphoblastic leukaemia (T-ALL) is a bone marrow (BM) malignancy affecting children and adults. Typically treated with chemotherapy, leukaemia remains a major death cause in people under 20 years old. Understanding molecularly altered pathways in T-ALL may lead to new therapeutic avenues in the future.

Nras Q61R/+ and Kras-/- cooperate to downregulate Rasgrp1 and promote lympho-myeloid leukemia in early T-cell precursors.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of T-cell ALL. Although genetic mutations hyperactivating cytokine receptor/Ras signaling are prevalent in ETP-ALL, it remains unknown how activated Ras signaling contributes to ETP-ALL. Here, we find that in addition to the frequent oncogenic RAS mutations, wild-type (WT) KRAS transcript level was significantly downregulated in human ETP-ALL cells.

Protocol for Barcoding T Cells Combined with Timed Stimulations.

Stimulation of naive T lymphocytes via the T cell receptor (TCR) induces distinct phosphorylation patterns that can be used to explore various signaling pathways within the cell. This protocol can be used to characterize different perturbations to the signaling pathways and the variations in time of stimulation. Here, we provide a method of barcoding and consolidating a maximum of 24 different sample conditions using two florescent dyes.

Protocol for Comprehensive Synthetic Lethality Screens.

Here, we provide a detailed protocol for synthetic lethality screens in a Jurkat T cell leukemia line using cell death as the readout measuring the combinatorial effect of a pan-PI3K inhibitor (GDC0941) with specific gene depletion by shRNA. We describe the use of an ultra-complex shRNA library, coverage considerations, time frames, protocol details, and bottlenecks with images to facilitate similar approaches. We discuss how this protocol resource can be readily adapted by investigators.

Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo.

Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6.