ABSTRACT
An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc, we found that KO of Alk, Bclaf3, or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non-Lgr5-expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3ß, while Bclaf3 and Prkra suppress regenerating islet-derived (Reg) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation.
Subject(s)
Adult Stem Cells/metabolism , Anaplastic Lymphoma Kinase/genetics , Epithelial Cells/metabolism , Gene Editing/methods , Organoids/metabolism , RNA-Binding Proteins/genetics , Wnt Signaling Pathway/genetics , Adenomatous Polyposis Coli Protein/genetics , Adenomatous Polyposis Coli Protein/metabolism , Adult Stem Cells/cytology , Anaplastic Lymphoma Kinase/metabolism , Animals , CRISPR-Cas Systems , Cell Proliferation , Epithelial Cells/cytology , Gastric Mucosa/cytology , Gastric Mucosa/metabolism , Gene Expression Regulation , Gene Library , Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , HEK293 Cells , Humans , Interleukins/genetics , Interleukins/metabolism , Mice , Organoids/cytology , RNA-Binding Proteins/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Stomach/cytologyABSTRACT
Recent strategies for treating CML patients have focused on investigating new combinations of tyrosine kinase inhibitors (TKIs) as well as identifying novel translational research agents that can eradicate CML leukemia-initiating cells (CML-LICs). However, little is known about the therapeutic benefits such CML-LIC targeting therapies might bring to CML patients. In this study, we investigated the therapeutic potential of EW-7197, an orally bioavailable transforming growth factor-ß signaling inhibitor which has recently been approved as an Investigational New Drug (NIH, USA), to suppress CML-LICs in vivo. Compared to TKI treatment alone, administration of TKI plus EW-7197 to CML-affected mice significantly delayed disease relapse and prolonged survival. Notably, combined treatment with EW-7197 plus TKI was effective in eliminating CML-LICs even if they expressed the TKI-resistant T315I mutant BCR-ABL1 oncogene. Collectively, these results indicate that EW-7197 may be a promising candidate for a new therapeutic that can greatly benefit CML patients by working in combination with TKIs to eradicate CML-LICs.
Subject(s)
Aniline Compounds/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Triazoles/pharmacology , Animals , Cell Proliferation/drug effects , Disease Models, Animal , Humans , Imidazoles/administration & dosage , Mice , Mice, Inbred C57BL , Protein Kinase Inhibitors/administration & dosage , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridazines/administration & dosage , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Transfection , Transforming Growth Factor beta/antagonists & inhibitorsABSTRACT
Understanding the specific survival of the rare chronic myelogenous leukaemia (CML) stem cell population could provide a target for therapeutics aimed at eradicating these cells. However, little is known about how survival signalling is regulated in CML stem cells. In this study, we survey global metabolic differences between murine normal haematopoietic stem cells (HSCs) and CML stem cells using metabolomics techniques. Strikingly, we show that CML stem cells accumulate significantly higher levels of certain dipeptide species than normal HSCs. Once internalized, these dipeptide species activate amino-acid signalling via a pathway involving p38MAPK and the stemness transcription factor Smad3, which promotes CML stem cell maintenance. Importantly, pharmacological inhibition of dipeptide uptake inhibits CML stem cell activity in vivo. Our results demonstrate that dipeptide species support CML stem cell maintenance by activating p38MAPK-Smad3 signalling in vivo, and thus point towards a potential therapeutic target for CML treatment.
