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.

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.

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.

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.

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.

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.

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.

Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing.

Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3-dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3-dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3-dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3ß phosphorylated these proteins in vitro RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing.

Autophagy is a signature of a signaling network that maintains hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are able to self-renew and to differentiate into all blood cells. HSCs reside in a low-perfusion niche and depend on local signals to survive and to maintain the capacity for self-renewal. HSCs removed from the niche are unable to survive without addition of hematopoietic cytokines and rapidly lose their ability to self-renew. We reported previously that inhibition of both GSK-3 and mTORC1 is essential to maintain long-term HSCs ex vivo. Although Wnt/ß-catenin signaling downstream of GSK-3 is required for this response, the downstream effectors of mTORC1 remain undefined. We now report that HSCs express a pro-autophagic gene signature and accumulate LC3 puncta only when both mTORC1 and GSK-3 are inhibited, identifying autophagy as a signature for a signaling network that maintains HSCs ex vivo. In addition, these conditions sustain HSC repopulating function despite an increased rate of global translation. Together, these findings provide new insight into the relative contributions of various mTORC1 outputs toward the maintenance of HSC function and build upon the growing body of literature implicating autophagy and tightly controlled protein synthesis as important modulators of diverse stem cell populations.

CKAP4 is identified as a receptor for Dickkopf in cancer cells.

The secretory protein Dickkopf-1 (DKK-1) is a known Wnt antagonist and has been shown to suppress tumorigenesis in some cancer cells; however, it is also upregulated in many types of cancer and associated with poor prognosis. Wnt-independent mechanisms by which DKK-1 promotes cancer cell proliferation are not well understood. In this issue of the JCI, Kimura and colleagues demonstrate that DKK-1 interacts with cytoskeleton-associated protein 4 (CKAP4) to promote activation of AKT. They show that both DKK-1 and CKAP4 are frequently upregulated in pancreatic and lung cancers. Importantly, targeting this interaction with an anti-CKAP4 antibody prevented tumor formation in murine xenograft models. These results identify a previously unrecognized DKK-1-mediated pathway and suggest CKAP4 as a potential therapeutic target for certain cancers.

Collagenoma in an African Clawed Frog (Xenopus laevis).

A 3-y-old female Xenopus laevis was reported for a gray mass on the abdomen. The frog was used for egg collection and was otherwise experimentally naïve. On physical exam, the frog was bright and active and had a firm, gray, lobulated mass (1.5 cm × 0.5 cm × 0.5 cm) in the cutaneous tissue of the left lateral abdomen. An excisional biopsy was performed under anesthesia, and the entire mass was removed and processed for histopathology. Microscopically, the dermis was greatly expanded by connective tissue with a marked decrease in the number of glands, and occasional degenerative glands were present. When stained with Masson trichrome, the excessive connective tissue stained blue, indicating that it was composed of collagen. With Verhoeff-van Gieson staining, the connective tissue stained bright red with an absence of black-staining material, demonstrating the presence of collagen and ruling out elastic fibers. In light of the morphology of the mass and the results of the special stains, the mass was diagnosed as a collagenoma. To our knowledge, this report is the first description of a collagenoma in X. laevis.

Wnt Signaling in Normal and Malignant Stem Cells.

Wnt signaling plays important roles in stem cell self-renewal and differentiation in adults as well as in embryonic development. Mutations that activate canonical Wnt/ß-catenin signaling also initiate and maintain several cancer states, including colorectal cancer and leukemia, and hence Wnt inhibitors are currently being explored as therapeutic options. In this review, we summarize previous studies and update recent findings on canonical Wnt signaling and its components, as well as their roles in somatic stem cell homeostasis and maintenance of cancer initiating cells.

The Msi Family of RNA-Binding Proteins Function Redundantly as Intestinal Oncoproteins.

Members of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers.

Evaluation of Presurgical Skin Preparation Agents in African Clawed Frogs (Xenopus laevis).

Despite the routine collection of oocytes from African clawed frogs (Xenopus laevis) for use in research, few studies have evaluated methods for preparing their skin for surgery. We evaluated 3 skin preparatory agents by examining their antibacterial efficacy and the gross and microscopic appearance of Xenopus skin after exposure. Frogs (n = 14) were sedated and treated (contact time, 10 min) with 0.9% sterile NaCl on one-half of the ventrum and with 0.5% povidone-iodine or 0.75% chlorhexidine on the other half. Bacterial cultures were obtained before and after skin treatment; bacteria were identified by mass spectrometry. To assess inflammation and degenerative changes, the incision sites were photographed and biopsied at 0, 1, and 7 d after surgery. We isolated at least 22 genera of bacteria from the skin of our frog population (mean ± SE, 5.21 ± 0.82 genera per frog). Iodine (2.00 ± 0.44 genera) and chlorhexidine (0.29 ± 0.76 genera) both had greater antimicrobial activity than did saline. Skin erythema did not correlate with treatment group. Histologic evidence of epidermal degeneration and necrosis was greater on days 1 and 7 after chlorhexidine treatment than after iodine or saline. In addition, frogs treated with chlorhexidine had a higher incidence of clinical illness associated with the exposure site. In summary, although chlorhexidine has adequate antimicrobial activity against organisms on X. laevis skin, it leads to skin damage and subsequent clinical complications. We therefore do not recommend chlorhexidine as a preoperative preparation agent in Xenopus.