A mitochondrial surveillance mechanism activated by SRSF2 mutations in hematologic malignancies.

Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.

A mitochondrial surveillance mechanism activated by SRSF2 mutations in hematologic malignancies.

Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.

Expansion of human hematopoietic stem cells by inhibiting translation.

Hematopoietic stem cell (HSC) transplantation using umbilical cord blood (UCB) is a potentially life-saving treatment for leukemia and bone marrow failure but is limited by the low number of HSCs in UCB. The loss of HSCs after ex vivo manipulation is also a major obstacle to gene editing for inherited blood disorders. HSCs require a low rate of translation to maintain their capacity for self-renewal, but hematopoietic cytokines used to expand HSCs stimulate protein synthesis and impair long-term self-renewal. We previously described cytokine-free conditions that maintain but do not expand human and mouse HSCs ex vivo. Here we performed a high throughput screen and identified translation inhibitors that allow ex vivo expansion of human HSCs while minimizing cytokine exposure. Transplantation assays show a ~5-fold expansion of long-term HSCs from UCB after one week of culture in low cytokine conditions. Single cell transcriptomic analysis demonstrates maintenance of HSCs expressing mediators of the unfolded protein stress response, further supporting the importance of regulated proteostasis in HSC maintenance and expansion. This expansion method maintains and expands human HSCs after CRISPR/Cas9 editing of the BCL11A+58 enhancer, overcoming a major obstacle to ex vivo gene correction for human hemoglobinopathies.

When You Come to a Fork in the Road, Take It: Wnt Signaling Activates Multiple Pathways through the APC/Axin/GSK-3 Complex.

The Wnt signaling pathway is a highly conserved regulator of metazoan development and stem cell maintenance. Activation of Wnt signaling is an early step in diverse malignancies. Work over the past four decades has defined a "canonical" Wnt pathway that is initiated by Wnt proteins, secreted glycoproteins that bind to a surface receptor complex and activate intracellular signal transduction by inhibiting a catalytic complex composed of the classical tumor suppressor Adenomatous Polyposis Coli (APC), Axin, and Glycogen Synthase Kinase-3 (GSK-3). The best characterized effector of this complex is ß-catenin, which is stabilized by inhibition of GSK-3, allowing ß-catenin entrance to the nucleus and activation of Wnt target gene transcription, leading to multiple cancers when inappropriately activated. However, canonical Wnt signaling through the APC/Axin/GSK-3 complex impinges on other effectors, independently of ß-catenin, including the mechanistic Target of Rapamycin (mTOR), regulators of protein stability, mitotic spindle orientation, and Hippo signaling. This review focuses on these alternative effectors of the canonical Wnt pathway and how they may contribute to cancers.

The Tumor Suppressor Adenomatous Polyposis Coli (apc) Is Required for Neural Crest-Dependent Craniofacial Development in Zebrafish.

Neural crest (NC) is a unique vertebrate cell type arising from the border of the neural plate and epidermis that gives rise to diverse tissues along the entire body axis. Roberto Mayor and colleagues have made major contributions to our understanding of NC induction, delamination, and migration. We report that a truncating mutation of the classical tumor suppressor Adenomatous Polyposis Coli (apc) disrupts craniofacial development in zebrafish larvae, with a marked reduction in the cranial neural crest (CNC) cells that contribute to mandibular and hyoid pharyngeal arches. While the mechanism is not yet clear, the altered expression of signaling molecules that guide CNC migration could underlie this phenotype. For example, apcmcr/mcr larvae express substantially higher levels of complement c3, which Mayor and colleagues showed impairs CNC cell migration when overexpressed. However, we also observe reduction in stroma-derived factor 1 (sdf1/cxcl12), which is required for CNC migration into the head. Consistent with our previous work showing that APC directly enhances the activity of glycogen synthase kinase 3 (GSK-3) and, independently, that GSK-3 phosphorylates multiple core mRNA splicing factors, we identify 340 mRNA splicing variations in apc mutant zebrafish, including a splice variant that deletes a conserved domain in semaphorin 3f (sema3f), an axonal guidance molecule and a known regulator of CNC migration. Here, we discuss potential roles for apc in CNC development in the context of some of the seminal findings of Mayor and colleagues.

Effects of lithium administration on vertebral bone disease in mucopolysaccharidosis I dogs.

Mucopolysaccharidosis (MPS) I is a lysosomal storage disease characterized by deficient activity of the enzyme alpha-L-iduronidase, leading to abnormal accumulation of heparan and dermatan sulfate glycosaminoglycans in cells and tissues. Patients commonly exhibit progressive skeletal abnormalities, in part due to failures of endochondral ossification during postnatal growth. Previously, using the naturally-occurring canine model, we showed that bone and cartilage cells in MPS I exhibit elevated lysosomal storage from an early age and that animals subsequently exhibit significantly diminished vertebral trabecular bone formation. Wnts are critical regulators of endochondral ossification that depend on glycosaminoglycans for signaling. The objective of this study was to examine whether lithium, a glycogen synthase kinase-3 inhibitor and stimulator of Wnt/beta-catenin signaling, administered during postnatal growth could attenuate progression of vertebral trabecular bone disease in MPS I. MPS I dogs were treated orally with therapeutic levels of lithium carbonate from 14 days to 6 months-of-age. Untreated heterozygous and MPS I dogs served as controls. Serum was collected at 3 and 6 months for assessment of bone turnover markers. At the study end point, thoracic vertebrae were excised and assessed using microcomputed tomography and histology. Lithium-treated animals exhibited significantly improved trabecular spacing, number and connectivity density, and serum bone-specific alkaline phosphatase levels compared to untreated animals. Growth plates from lithium-treated animals exhibited increased numbers of hypertrophic chondrocytes relative to both untreated MPS I and heterozygous animals. These findings suggest that bone and cartilage cells in MPS I are still capable of responding to exogenous osteogenic signals even in the presence of significant lysosomal storage, and that targeted osteogenic therapies may represent a promising approach for attenuating bone disease progression in MPS I.

Targeting the coronavirus nucleocapsid protein through GSK-3 inhibition.

The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors, including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR-tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35-0.74], P = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type-dependent manner. Targeting GSK-3 may therefore provide an approach to treat COVID-19 and future coronavirus outbreaks.

Restoring two tumor suppressor pathways with one PAWI.

In this issue of Cell Chemical Biology, Cheng et al. (2021) identify a class of drugs that activate a mitotic stress-dependent signaling cascade, which culminates in p53 activation and Wnt pathway inhibition (PAWI). PAWI compounds may therefore be effective in cancers associated with loss of p53 and activation of Wnt signaling.

Targeting the Coronavirus Nucleocapsid Protein through GSK-3 Inhibition.

The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome (MERS-CoV), and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35 - 0.74], p = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type dependent manner. Targeting GSK-3 may therefore provide a new approach to treat COVID-19 and future coronavirus outbreaks. SIGNIFICANCE: COVID-19 is taking a major toll on personal health, healthcare systems, and the global economy. With three betacoronavirus epidemics in less than 20 years, there is an urgent need for therapies to combat new and existing coronavirus outbreaks. Our analysis of clinical data from over 300,000 patients in three major health systems demonstrates a 50% reduced risk of COVID-19 in patients taking lithium, a direct inhibitor of glycogen synthase kinase-3 (GSK-3). We further show that GSK-3 is essential for phosphorylation of the SARS-CoV-2 nucleocapsid protein and that GSK-3 inhibition blocks SARS-CoV-2 infection in human lung epithelial cells. These findings suggest an antiviral strategy for COVID-19 and new coronaviruses that may arise in the future.

Lithium and Therapeutic Targeting of GSK-3.

Lithium salts have been in the therapeutic toolbox for better or worse since the 19th century, with purported benefit in gout, hangover, insomnia, and early suggestions that lithium improved psychiatric disorders. However, the remarkable effects of lithium reported by John Cade and subsequently by Mogens Schou revolutionized the treatment of bipolar disorder. The known molecular targets of lithium are surprisingly few and include the signaling kinase glycogen synthase kinase-3 (GSK-3), a group of structurally related phosphomonoesterases that includes inositol monophosphatases, and phosphoglucomutase. Here we present a brief history of the therapeutic uses of lithium and then focus on GSK-3 as a therapeutic target in diverse diseases, including bipolar disorder, cancer, and coronavirus infections.

Leukemic Stem Cell Culture in Cytokine-Free Medium.

Leukemia-initiating cells, also known as leukemic stem cells (LSCs), are experimentally defined by their ability to engraft immunocompromised mice and are believed to be a major cause of relapse in acute myeloid leukemia (AML). Despite the aggressive characteristics of acute leukemia, AML blasts are difficult to culture once removed from the patient, and LSCs, which are a minor fraction of the blast population, are especially difficult to transplant after culture. This impedes development of new therapies for AML that target LSCs. Here, we present a simple strategy to culture LSCs in cytokine-free medium and to perform flow cytometric analysis of the resulting cell population for the characterization of LSCs maintenance and differentiation.

Chromatin accessibility and histone acetylation in the regulation of competence in early development.

As development proceeds, inductive cues are interpreted by competent tissues in a spatially and temporally restricted manner. While key inductive signaling pathways within competent cells are well-described at a molecular level, the mechanisms by which tissues lose responsiveness to inductive signals are not well understood. Localized activation of Wnt signaling before zygotic gene activation in Xenopus laevis leads to dorsal development, but competence to induce dorsal genes in response to Wnts is lost by the late blastula stage. We hypothesize that loss of competence is mediated by changes in histone modifications leading to a loss of chromatin accessibility at the promoters of Wnt target genes. We use ATAC-seq to evaluate genome-wide changes in chromatin accessibility across several developmental stages. Based on overlap with p300 binding, we identify thousands of putative cis-regulatory elements at the gastrula stage, including sites that lose accessibility by the end of gastrulation and are enriched for pluripotency factor binding motifs. Dorsal Wnt target gene promoters are not accessible after the loss of competence in the early gastrula while genes involved in mesoderm and neural crest development maintain accessibility at their promoters. Inhibition of histone deacetylases increases acetylation at the promoters of dorsal Wnt target genes and extends competence for dorsal gene induction by Wnt signaling. Histone deacetylase inhibition, however, is not sufficient to extend competence for mesoderm or neural crest induction. These data suggest that chromatin state regulates the loss of competence to inductive signals in a context-dependent manner.

Correction: Transient stabilization, rather than inhibition, of MYC amplifies extrinsic apoptosis and therapeutic responses in refractory B-cell lymphoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single Cell Proteomics by Data-Independent Acquisition To Study Embryonic Asymmetry in Xenopus laevis.

Techniques that allow single cell analysis are gaining widespread attention, and most of these studies utilize genomics-based approaches. While nanofluidic technologies have enabled mass spectrometric analysis of single cells, these measurements have been limited to metabolomics and lipidomic studies. Single cell proteomics has the potential to improve our understanding of intercellular heterogeneity. However, this approach has faced challenges including limited sample availability, as well as a requirement of highly sensitive methods for sample collection, cleanup, and detection. We present a technique to overcome these limitations by combining a micropipette (pulled glass capillary) based sample collection strategy with offline sample preparation and nanoLC-MS/MS to analyze proteins through a bottom-up proteomic strategy. This study explores two types of proteomics data acquisition strategies namely data-dependent (DDA) and data-independent acquisition (DIA). Results from the study indicate DIA to be more sensitive enabling analysis of >1600 proteins from ∼130 µm Xenopus laevis embryonic cells containing <6 nL of cytoplasm. The method was found to be robust in obtaining reproducible protein quantifications from single cells spanning the 1-128-cell stages of development. Furthermore, we used micropipette sampling to study intercellular heterogeneity within cells in a single embryo and investigated embryonic asymmetry along both animal-vegetal and dorsal-ventral axes during early stages of development. Investigation of the animal-vegetal axis led to discovery of various asymmetrically distributed proteins along the animal-vegetal axis. We have further compared the hits found from our proteomic data sets with other studies and validated a few hits using an orthogonal imaging technique. This study forms the first report of vegetal enrichment of the germ plasm associated protein DDX4/VASA in Xenopus embyos. Overall, the method and data presented here holds promise to enable important leads in developmental biology.

Adult hippocampal neurogenesis is not necessary for the response to lithium in the forced swim test.

Chronic lithium treatment stimulates adult hippocampal neurogenesis, but whether increased neurogenesis contributes to its therapeutic mechanism remains unclear. We use a genetic model of neural progenitor cell (NPC) ablation to test whether a lithium-sensitive behavior requires hippocampal neurogenesis. NPC-ablated mice were treated with lithium and assessed in the forced swim test (FST). Lithium reduced time immobile in the FST in NPC-ablated and control mice but had no effect on activity in the open field, a control for the locomotion-based FST. These findings show that hippocampal NPCs that proliferate in response to chronic lithium are not necessary for the behavioral response to lithium in the FST. We further show that 4-6 week old immature hippocampal neurons are not required for this response. These data suggest that increased hippocampal neurogenesis does not contribute to the response to lithium in the forced swim test and may not be an essential component of its therapeutic mechanism.

Transient stabilization, rather than inhibition, of MYC amplifies extrinsic apoptosis and therapeutic responses in refractory B-cell lymphoma.

Therapeutic targeting of initiating oncogenes is the mainstay of precision medicine. Considerable efforts have been expended toward silencing MYC, which drives many human cancers including Burkitt lymphomas (BL). Yet, the effects of MYC silencing on standard-of-care therapies are poorly understood. Here we found that inhibition of MYC transcription renders B-lymphoblastoid cells refractory to chemotherapeutic agents. This suggested that in the context of chemotherapy, stabilization of Myc protein could be more beneficial than its inactivation. We tested this hypothesis by pharmacologically inhibiting glycogen synthase kinase 3ß (GSK-3ß), which normally targets Myc for proteasomal degradation. We discovered that chemorefractory BL cell lines responded better to doxorubicin and other anti-cancer drugs when Myc was transiently stabilized. In vivo, GSK3 inhibitors (GSK3i) enhanced doxorubicin-induced apoptosis in BL patient-derived xenografts (BL-PDX), as well as in murine MYC-driven lymphoma allografts. This enhancement was accompanied by and required deregulation of several key genes acting in the extrinsic, death-receptor-mediated apoptotic pathway. Consistent with this mechanism of action, GSK3i also facilitated lymphoma cell killing by a death ligand TRAIL and by a death receptor agonist mapatumumab. Thus, GSK3i synergizes with both standard chemotherapeutics and direct engagers of death receptors and could improve outcomes in patients with refractory lymphomas.

Novel functions of the ubiquitin-independent proteasome system in regulating Xenopus germline development.

In most species, early germline development occurs in the absence of transcription with germline determinants subject to complex translational and post-translational regulations. Here, we report for the first time that early germline development is influenced by dynamic regulation of the proteasome system, previously thought to be ubiquitously expressed and to serve 'housekeeping' roles in controlling protein homeostasis. We show that proteasomes are present in a gradient with the highest levels in the animal hemisphere and extending into the vegetal hemisphere of Xenopus oocytes. This distribution changes dramatically during the oocyte-to-embryo transition, with proteasomes becoming enriched in and restricted to the animal hemisphere and therefore separated from vegetally localized germline determinants. We identify Dead-end1 (Dnd1), a master regulator of vertebrate germline development, as a novel substrate of the ubiquitin-independent proteasomes. In the oocyte, ubiquitin-independent proteasomal degradation acts together with translational repression to prevent premature accumulation of Dnd1 protein. In the embryo, artificially increasing ubiquitin-independent proteasomal degradation in the vegetal pole interferes with germline development. Our work thus reveals novel inhibitory functions and spatial regulation of the ubiquitin-independent proteasome during vertebrate germline development.

Non-acylated Wnts Can Promote Signaling.

Wnts are a family of 19 extracellular ligands that regulate cell fate, proliferation, and migration during metazoan embryogenesis and throughout adulthood. Wnts are acylated post-translationally at a conserved serine and bind the extracellular cysteine-rich domain (CRD) of Frizzled (FZD) seven-pass transmembrane receptors. Although crystal structures suggest that acylation is essential for Wnt binding to FZDs, we show here that several Wnts can promote signaling in Xenopus laevis and Danio rerio embryos, as well as in an in vitro cell culture model, without acylation. The non-acylated Wnts are expressed at levels similar to wild-type counterparts and retain CRD binding. By contrast, we find that certain other Wnts do require acylation for biological activity in Xenopus embryos, although not necessarily for FZD binding. Our data argue that acylation dependence of Wnt activity is context specific. They further suggest that acylation may underlie aspects of ligand-receptor selectivity and/or control other aspects of Wnt function.

Training the physician-scientist: views from program directors and aspiring young investigators.

There is growing concern that the physician-scientist is endangered due to a leaky training pipeline and prolonged time to scientific independence (1). The NIH Physician-Scientist Workforce Working Group has concluded that as many as 1,000 individuals will need to enter the pipeline each year to sustain the workforce (2). Moreover, surveys of postgraduate training programs document considerable variability in disposition and infrastructure (3). Programs can be broadly grouped into two classes: physician-scientist training programs (PSTPs) that span residency and fellowship training, and research-in-residency programs (RiRs), which are limited to residency but trainees are able to match into PSTPs upon transitioning to fellowship (Figure 1). Funding sources for RiRs and PSTPs are varied and include NIH KL2 and T32 awards, charitable foundations, philanthropy, and institutional support. Furthermore, standards for research training and tools for evaluating programmatic success are lacking. Here, we share consensus generated from iterative workshops hosted by the Alliance of Academic Internal Medicine (AAIM) and the student-led American Physician Scientists Association (APSA).

Glycogen synthase kinase-3 and alternative splicing.

Glycogen synthase kinase-3 (GSK-3) is a highly conserved negative regulator of receptor tyrosine kinase, cytokine, and Wnt signaling pathways. Stimulation of these pathways inhibits GSK-3 to modulate diverse downstream effectors that include transcription factors, nutrient sensors, glycogen synthesis, mitochondrial function, circadian rhythm, and cell fate. GSK-3 also regulates alternative splicing in response to T-cell receptor activation, and recent phosphoproteomic studies have revealed that multiple splicing factors and regulators of RNA biosynthesis are phosphorylated in a GSK-3-dependent manner. Furthermore, inhibition of GSK-3 alters the splicing of hundreds of mRNAs, indicating a broad role for GSK-3 in the regulation of RNA processing. GSK-3-regulated phosphoproteins include SF3B1, SRSF2, PSF, RBM8A, nucleophosmin 1 (NPM1), and PHF6, many of which are mutated in leukemia and myelodysplasia. As GSK-3 is inhibited by pathways that are pathologically activated in leukemia and loss of Gsk3 in hematopoietic cells causes a severe myelodysplastic neoplasm in mice, these findings strongly implicate GSK-3 as a critical regulator of mRNA processing in normal and malignant hematopoiesis. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.