Mechanistic Insights of Aberrant Splicing with Splicing Factor Mutations Found in Myelodysplastic Syndromes

Pre-mRNA splicing is an essential process for gene expression in higher eukaryotes, which requires a high order of accuracy. Mutations in splicing factors or regulatory elements in pre-mRNAs often result in many human diseases. Myelodysplastic syndrome (MDS) is a heterogeneous group of chronic myeloid neoplasms characterized by many symptoms and a high risk of progression to acute myeloid leukemia. Recent findings indicate that mutations in splicing factors represent a novel class of driver mutations in human cancers and affect about 50% of Myelodysplastic syndrome (MDS) patients. Somatic mutations in MDS patients are frequently found in genes SF3B1, SRSF2, U2AF1, and ZRSR2. Interestingly, they are involved in the recognition of 3′ splice sites and exons. It has been reported that mutations in these splicing regulators result in aberrant splicing of many genes. In this review article, we first describe molecular mechanism of pre-mRNA splicing as an introduction and mainly focus on those four splicing factors to describe their mutations and their associated aberrant splicing patterns.     

The Importance of Hypoxia-Inducible Factors (HIF-1 and HIF-2) for the Pathophysiology of Inflammatory Bowel Disease

(1) Background: Hypoxia is a common feature of inflammation when hypoxia inducible factors (HIFs) adapt cells to conditions of low oxygen tension and inflammation. We studied the role of HIF-1 and HIF-2 in cells of the myeloid lineage in a mouse model of acute colitis. (2) Methods: Mice with and without a conditional knockout for either Hif-1a or Hif-2a or Hif-1a and Hif-2a in cells of the myeloid lineage were treated with 2.5% dextran sodium sulfate (DSS) for 6 days to induce an acute colitis. We analyzed the course of inflammation with respect to macroscopic (disease activity index) and microscopic (histology score and immunohistochemical staining of immune cells) parameters and quantified the mRNA expression of cytokines and chemokines in the colon and the mesenteric lymph nodes. (3) Results: A conditional knockout of myeloid Hif-1a ameliorated whereas the knockout of Hif-2a aggravated murine DSS colitis by increased recruitment of neutrophils to deeper layers of the colon. This led to higher expression of Il6, Ifng, Cd11c, Cd4, and Cd8 in the colon but also induced anti-inflammatory mediators such as Foxp3 and Il10. A conditional knockout of Hif-1a and Hif-2a did not show any differences compared to wildtype mice. (4) Conclusions: Myeloid HIF-1α and HIF-2α play opposing roles in acute DSS colitis. Thus, not only a cell type specific, but also the isoform specific modulation of HIFs needs to be addressed in attempts to modify HIF for therapeutic purposes.     

Potential Relationship between Inadequate Response to DNA Damage and Development of Myelodysplastic Syndrome

Hematopoietic stem cells (HSCs) are responsible for the continuous regeneration of all types of blood cells, including themselves. To ensure the functional and genomic integrity of blood tissue, a network of regulatory pathways tightly controls the proliferative status of HSCs. Nevertheless, normal HSC aging is associated with a noticeable decline in regenerative potential and possible changes in other functions. Myelodysplastic syndrome (MDS) is an age-associated hematopoietic malignancy, characterized by abnormal blood cell maturation and a high propensity for leukemic transformation. It is furthermore thought to originate in a HSC and to be associated with the accrual of multiple genetic and epigenetic aberrations. This raises the question whether MDS is, in part, related to an inability to adequately cope with DNA damage. Here we discuss the various components of the cellular response to DNA damage. For each component, we evaluate related studies that may shed light on a potential relationship between MDS development and aberrant DNA damage response/repair.     

Inflammation Regulates Haematopoietic Stem Cells and Their Niche

Haematopoietic stem cells (HSCs) reside in the bone marrow and are supported by the specialised microenvironment, a niche to maintain HSC quiescence. To deal with haematopoietic equilibrium disrupted during inflammation, HSCs are activated from quiescence directly and indirectly to generate more mature immune cells, especially the myeloid lineage cells. In the process of proliferation and differentiation, HSCs gradually lose their self-renewal potential. The extensive inflammation might cause HSC exhaustion/senescence and malignant transformation. Here, we summarise the current understanding of how HSC functions are maintained, damaged, or exhausted during acute, prolonged, and pathological inflammatory conditions. We also highlight the inflammation-altered HSC niche and its impact on escalating the insults on HSCs.     

Sclerostin Depletion Induces Inflammation in the Bone Marrow of Mice

Romosozumab, a humanized monoclonal antibody specific for sclerostin (SOST), has been approved for treatment of postmenopausal women with osteoporosis at a high risk for fracture. Previous work in sclerostin global knockout (Sost^−/−) mice indicated alterations in immune cell development in the bone marrow (BM), which could be a possible side effect in romosozumab-treated patients. Here, we examined the effects of short-term sclerostin depletion in the BM on hematopoiesis in young mice receiving sclerostin antibody (Scl-Ab) treatment for 6 weeks, and the effects of long-term Sost deficiency on wild-type (WT) long-term hematopoietic stem cells transplanted into older cohorts of Sost^−/− mice. Our analyses revealed an increased frequency of granulocytes in the BM of Scl-Ab-treated mice and WT→Sost^−/− chimeras, indicating myeloid-biased differentiation in Sost-deficient BM microenvironments. This myeloid bias extended to extramedullary hematopoiesis in the spleen and was correlated with an increase in inflammatory cytokines TNFα, IL-1α, and MCP-1 in Sost^−/− BM serum. Additionally, we observed alterations in erythrocyte differentiation in the BM and spleen of Sost^−/− mice. Taken together, our current study indicates novel roles for Sost in the regulation of myelopoiesis and control of inflammation in the BM.     

Uncovering a Hub Signaling Pathway of Antimicrobial-Antifungal-Anticancer Peptides’ Axis on Short Cationic Peptides via Network Pharmacology Study

Short cationic peptides (SCPs) with therapeutic efficacy of antimicrobial peptides (AMPs), antifungal peptides (AFPs), and anticancer peptides (ACPs) are known as an enhancement of the host defense system. Here, we investigated the uppermost peptide(s), hub signaling pathway(s), and their associated target(s) through network pharmacology. Firstly, we selected SCPs with positive amino acid residues on N- and C- terminals under 500 Dalton via RStudio. Secondly, the overlapping targets between the bacteria-responsive targets (TTD and OMIM) and AMPs’ targets were visualized by VENNY 2.1. Thirdly, the overlapping targets between AFPs’ targets and fungal-responsive targets were exhibited by VENNY 2.1. Fourthly, the overlapping targets between cancer-related targets (TTD and OMIM) and fungal-responsive targets were displayed by VENNY 2.1. Finally, a molecular docking study (MDS) was carried out to discover the most potent peptides on a hub signaling pathway. A total of 1833 SCPs were identified, and AMPs’, AFPs’, and ACPs’ filtration suggested that 197 peptides (30 targets), 81 peptides (6 targets), and 59 peptides (4 targets) were connected, respectively. The AMPs―AFPs―ACPs’ axis indicated that 27 peptides (2 targets) were associated. Each hub signaling pathway for the enhancement of the host defense system was “Inactivation of Rap1 signaling pathway on AMPs”, “Activation of Notch signaling pathway on AMPs―AFPs’ axis”, and “Inactivation of HIF-1 signaling pathway on AMPs―AFPs―ACPs’ axis”. The most potent peptides were assessed via MDS; finally, HPIK on STAT3 and HVTK on NOS2 and on HIF-1 signaling pathway were the most stable complexes. Furthermore, the two peptides had better affinity scores than standard inhibitors (Stattic, 1400 W). Overall, the most potent SCPs for the human defense system were HPIK on STAT3 and HVTK on NOS2, which might inactivate the HIF-1 signaling pathway.     

Targeting Casein Kinase 1 (CK1) in Hematological Cancers

The casein kinase 1 enzymes (CK1) form a family of serine/threonine kinases with seven CK1 isoforms identified in humans. The most important substrates of CK1 kinases are proteins that act in the regulatory nodes essential for tumorigenesis of hematological malignancies. Among those, the most important are the functions of CK1s in the regulation of Wnt pathways, cell proliferation, apoptosis and autophagy. In this review we summarize the recent developments in the understanding of biology and therapeutic potential of the inhibition of CK1 isoforms in the pathogenesis of chronic lymphocytic leukemia (CLL), other non-Hodgkin lymphomas (NHL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and multiple myeloma (MM). CK1δ/ε inhibitors block CLL development in preclinical models via inhibition of WNT-5A/ROR1-driven non-canonical Wnt pathway. While no selective CK1 inhibitors have reached clinical stage to date, one dual PI3Kδ and CK1ε inhibitor, umbralisib, is currently in clinical trials for CLL and NHL patients. In MDS, AML and MM, inhibition of CK1α, acting via activation of p53 pathway, showed promising preclinical activities and the first CK1α inhibitor has now entered the clinical trials.     

Aging and Clonal Behavior of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are the only cell population that possesses both a self-renewing capacity and multipotency, and can give rise to all lineages of blood cells throughout an organism’s life. However, the self-renewal capacity of HSCs is not infinite, and cumulative evidence suggests that HSCs alter their function and become less active during organismal aging, leading ultimately to the disruption of hematopoietic homeostasis, such as anemia, perturbed immunity and increased propensity to hematological malignancies. Thus, understanding how HSCs alter their function during aging is a matter of critical importance to prevent or overcome these age-related changes in the blood system. Recent advances in clonal analysis have revealed the functional heterogeneity of murine HSC pools that is established upon development and skewed toward the clonal expansion of functionally poised HSCs during aging. In humans, next-generation sequencing has revealed age-related clonal hematopoiesis that originates from HSC subsets with acquired somatic mutations, and has highlighted it as a significant risk factor for hematological malignancies and cardiovascular diseases. In this review, we summarize the current fate-mapping strategies that are used to track and visualize HSC clonal behavior during development or after stress. We then review the age-related changes in HSCs that can be inherited by daughter cells and act as a cellular memory to form functionally distinct clones. Altogether, we link aging of the hematopoietic system to HSC clonal evolution and discuss how HSC clones with myeloid skewing and low regenerative potential can be expanded during aging.     

β3-Adrenoreceptors as ROS Balancer in Hematopoietic Stem Cell Transplantation

In the last decades, the therapeutic potential of hematopoietic stem cell transplantation (HSCT) has acquired a primary role in the management of a broad spectrum of diseases including cancer, hematologic conditions, immune system dysregulations, and inborn errors of metabolism. The different types of HSCT, autologous and allogeneic, include risks of severe complications including acute and chronic graft-versus-host disease (GvHD) complications, hepatic veno-occlusive disease, lung injury, and infections. Despite being a dangerous procedure, it improved patient survival. Hence, its use was extended to treat autoimmune diseases, metabolic disorders, malignant infantile disorders, and hereditary skeletal dysplasia. HSCT is performed to restore or treat various congenital conditions in which immunologic functions are compromised, for instance, by chemo- and radiotherapy, and involves the administration of hematopoietic stem cells (HSCs) in patients with depleted or dysfunctional bone marrow (BM). Since HSCs biology is tightly regulated by oxidative stress (OS), the control of reactive oxygen species (ROS) levels is important to maintain their self-renewal capacity. In quiescent HSCs, low ROS levels are essential for stemness maintenance; however, physiological ROS levels promote HSC proliferation and differentiation. High ROS levels are mainly involved in short-term repopulation, whereas low ROS levels are associated with long-term repopulating ability. In this review, we aim summarize the current state of knowledge about the role of β3-adrenoreceptors (β3-ARs) in regulating HSCs redox homeostasis. β3-ARs play a major role in regulating stromal cell differentiation, and the antagonist SR59230A promotes differentiation of different progenitor cells in hematopoietic tumors, suggesting that β3-ARs agonism and antagonism could be exploited for clinical benefit.     

NPM1-Mutated Myeloid Neoplasms with <20% Blasts: A Really Distinct Clinico-Pathologic Entity?

Nucleophosmin (NPM1) gene mutations rarely occur in non-acute myeloid neoplasms (MNs) with <20% blasts. Among nearly 10,000 patients investigated so far, molecular analyses documented NPM1 mutations in around 2% of myelodysplastic syndrome (MDS) cases, mainly belonging to MDS with excess of blasts, and 3% of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) cases, prevalently classified as chronic myelomonocytic leukemia. These uncommon malignancies are associated with an aggressive clinical course, relatively rapid progression to overt acute myeloid leukemia (AML) and poor survival outcomes, raising controversies on their classification as distinct clinico-pathologic entities. Furthermore, fit patients with NPM1-mutated MNs with <20% blasts could benefit most from upfront intensive chemotherapy for AML rather than from moderate intensity MDS-directed therapies, although no firm conclusion can currently be drawn on best therapeutic approaches, due to the limited available data, obtained from small and mainly retrospective series. Caution is also suggested in definitely diagnosing NPM1-mutated MNs with blast count <20%, since NPM1-mutated AML cases frequently present dysplastic features and multilineage bone marrow cells showing abnormal cytoplasmic NPM1 protein delocalization by immunohistochemical staining, therefore belonging to NPM1-mutated clone regardless of blast morphology. Further prospective studies are warranted to definitely assess whether NPM1 mutations may become sufficient to diagnose AML, irrespective of blast percentage.     

Hypoxia-Inducible Factor-2 Alpha Regulates the Migration of Fibroblast-like Synoviocytes via Oxidative Stress-Induced CD70 Expression in Patients with Rheumatoid Arthritis

This study aimed to examine the role of CD70, which is highly expressed on fibroblast-like synoviocytes (FLS), in rheumatoid arthritis (RA) patients. FLS isolated from RA (n = 14) and osteoarthritis (OA, n = 4) patients were stimulated with recombinant interleukin-17 (IL-17; 5 ng/mL) and tumor necrosis factor alpha (TNF-α; 5 ng/mL) for 24 h. Expression of CD70, CD27/soluble CD27 (sCD27), and hypoxia-inducible factor-2 alpha (HIF-2α) was analyzed by RT-qPCR, flow cytometry, and ELISA assays, respectively. Reactive oxygen species (ROS) expression and cell migration were also examined. The HIF-2α inhibitor PT-2385 and CD70 inhibitor BU69 were used to specifically suppress these pathways. Stimulation with IL-17 and TNF-α significantly induced CD70 expression in RA FLS. Although the synovial fluids from patients with RA contained high levels of sCD27, surface expression of CD27, a ligand of CD70, was rarely detected in RA FLS. Cytokine-induced CD70 expression was significantly decreased following antioxidant treatment. Following HIF-2α inhibition, RA FLS had decreased expression of CD70 and ROS levels. Migration of RA FLS was also inhibited by inhibition of CD70 or HIF-2α. The surface expression of CD70 is regulated by HIF-2α and ROS levels and is a key contributor to cytokine-enhanced migration in RA FLS.     

Myeloid Lineage Ablation of Phlpp1 Regulates M-CSF Signaling and Tempers Bone Resorption in Female Mice

Prior work demonstrated that Phlpp1 deficiency alters trabecular bone mass and enhances M-CSF responsiveness, but the cell types and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within myeloid lineage cells, we crossed Phlpp1 floxed mice with mice harboring LysM-Cre. Micro-computed tomography of the distal femur of 12-week-old mice revealed a 30% increase in bone volume per total volume of Phlpp1 female conditional knockouts, but we did not observe significant changes within male Phlpp1 cKO_LysM mice. Bone histomorphmetry of the proximal tibia further revealed that Phlpp1 cKO_LysM females exhibited elevated osteoclast numbers, but conversely had reduced levels of serum markers of bone resorption as compared to littermate controls. Osteoblast number and serum markers of bone formation were unchanged. In vitro assays confirmed that Phlpp1 ablation enhanced osteoclast number and area, but limited bone resorption. Additionally, reconstitution with exogenous Phlpp1 suppressed osteoclast numbers. Dose response assays demonstrated that Phlpp1^−/− cells are more responsive to M-CSF, but reconstitution with Phlpp1 abrogated this effect. Furthermore, small molecule-mediated Phlpp inhibition enhanced osteoclast numbers and size. Enhanced phosphorylation of Phlpp substrates—including Akt, ERK1/2, and PKCζ—accompanied these observations. In contrast, actin cytoskeleton disruption occurred within Phlpp inhibitor treated osteoclasts. Moreover, Phlpp inhibition reduced resorption of cells cultured on bovine bone slices in vitro. Our results demonstrate that Phlpp1 deficiency within myeloid lineage cells enhances bone mass by limiting bone resorption while leaving osteoclast numbers intact; moreover, we show that Phlpp1 represses osteoclastogenesis and controls responses to M-CSF.