Sphingomyelin metabolism underlies Ras excitability for efficient cell migration and chemotaxis.

In eukaryotic motile cells, the active Ras (Ras-GTP)-enriched domain is generated in an asymmetric manner on the cell membrane through the excitable dynamics of an intracellular signaling network. This asymmetric Ras signaling regulates pseudopod formation for both spontaneous random migration and chemoattractant-induced directional migration. While membrane lipids, such as sphingomyelin and phosphatidylserine, contribute to Ras signaling in various cell types, whether they are involved in the Ras excitability for cell motility is unknown. Here we report that functional Ras excitability requires the normal metabolism of sphingomyelin for efficient cell motility and chemotaxis. The pharmacological blockade of sphingomyelin metabolism by an acid-sphingomyelinase inhibitor, fendiline, and other inhibitors suppressed the excitable generation of the stable Ras-GTP-enriched domain. The suppressed excitability failed to invoke enough basal motility to achieve directed migration under shallow chemoattractant gradients. The fendiline-induced defects in Ras excitability, motility and stimulation-elicited directionality were due to an accumulation of sphingomyelin on the membrane, which could be recovered by exogenous sphingomyelinase or phosphatidylserine without changing the expression of Ras. These results indicate a novel regulatory mechanism of the excitable system by membrane lipids, in which sphingomyelin metabolism provides a membrane environment to ensure Ras excitation for efficient cellular motility and chemotaxis.Key words: cell polarity, cell migration, Ras, excitability, sphingomyelin.

Clinical implications of NUP98::NSD1 fusion at diagnosis in adult FLT3-ITD positive AML.

OBJECTIVES: The cryptic fusion oncogene NUP98::NSD1 is known to be associated with FLT3-ITD mutation in acute myeloid leukemia (AML), and an independent poor prognostic factor in pediatric AML. However, there are little data regarding the clinical significance of NUP98::NSD1 in adult cohort. METHODS: We conducted a multicenter retrospective study to investigate the prevalence, clinical characteristics, and prognostic impact of NUP98::NSD1 in adult FLT3-ITD-positive AML patients. RESULTS: In a total of 97 FLT3-ITD-positive AML patients, six cases (6.2%) were found to harbor the NUP98::NSD1 fusion transcript. NUP98::NSD1 positive cases had significantly higher platelet counts and a higher frequency of FAB-M4 morphology than NUP98::NSD1 negative cases. NUP98::NSD1 was found to be mutually exclusive with NPM1 mutation, and was accompanied by the WT1 mutation in three of the six cases. The presence of NUP98::NSD1 fusion at the time of diagnosis predicted poor response to cytarabine-anthracycline-based intensive induction chemotherapy (induction failure rate: 83% vs. 36%, p = .038). Five of the six cases with NUP98::NSD1 underwent allogeneic hematopoietic stem cell transplantation (HSCT). Two of the five cases have successfully maintained remission, with one of them being rescued through a second HSCT. CONCLUSIONS: Detecting NUP98::NSD1 in adult FLT3-ITD-positive AML is crucial to recognizing chemotherapy-resistant group.

[Prompt cytogenetic response by venetoclax plus azacitidine regimen in a patient with AML harboring double-minute chromosomes with MYC gene amplification].

Double minute chromosomes (dmin) are small, acentric, and extrachromosomal fragments that frequently mediate oncogene amplification and induce rapid disease progression with poor prognosis, although they are infrequent in myeloid neoplasms. An 81-year-old woman with anemia and thrombocytopenia was admitted to our hospital. Bone marrow examination showed 54.0% of the blasts. She was diagnosed with acute myeloid leukemia (French-American-British classification, M2; World Health Organization classification, acute myeloid leukemia [AML], not otherwise specified, AML with maturation). Chromosomal analysis revealed the presence of 3-45 dmin in the background of 46 and XX in 14 out of 20 metaphases examined. Spectral karyotyping examination demonstrated that the dmins were derived from chromosome 8. Fluorescence in situ hybridization (FISH) targeting the MYC gene demonstrated that dmins contained full-length MYC genes with multiple signals. Finally, she was diagnosed with AML with dmin via MYC amplification and was administered with venetoclax plus azacitidine chemotherapy. After two cycles of the regimen, FISH found no MYC amplification signals, indicating her state being in cytogenetic remission. At present, she has finished four cycles of the regimen and remained in complete remission. Venetoclax plus azacitidine could be an effective regimen for the poor prognosis of AML with dmin through MYC amplification.

Excitable dynamics of Ras triggers spontaneous symmetry breaking of PIP3 signaling in motile cells.

Spontaneous cell movement is underpinned by an asymmetric distribution of signaling molecules including small G proteins and phosphoinositides on the cell membrane. However, the molecular network necessary for spontaneous symmetry breaking has not been fully elucidated. Here, we report that, in Dictyostelium discoideum, the spatiotemporal dynamics of GTP bound Ras (Ras-GTP) breaks the symmetry due its intrinsic excitability even in the absence of extracellular spatial cues and downstream signaling activities. A stochastic excitation of local and transient Ras activation induced phosphatidylinositol (3,4,5)-trisphosphate (PIP3) accumulation via direct interaction with Phosphoinositide 3-kinase (PI3K), causing tightly coupled traveling waves that propagated along the membrane. Comprehensive phase analysis of the waves of Ras-GTP and PIP3 metabolism-related molecules revealed the network structure of the excitable system including positive-feedback regulation of Ras-GTP by the downstream PIP3. A mathematical model reconstituted a series of the observed symmetry-breaking phenomena, illustrating the essential involvement of Ras excitability in the cellular decision-making process.

A sub-population of Dictyostelium discoideum cells shows extremely high sensitivity to cAMP for directional migration.

The chemotaxis of Dictysotelium discoideum cells in response to a chemical gradient of cyclic adenosine 3',5'-monophosphate (cAMP) was studied using a newly designed microfluidic device. The device consists of 800 cell-sized channels in parallel, each 4 µm wide, 5 µm high, and 100 µm long, allowing us to prepare the same chemical gradient in all channels and observe the motility of 500-1000 individual cells simultaneously. The percentage of cells that exhibited directed migration was determined for various cAMP concentrations ranging from 0.1 pM to 10 µM. The results show that chemotaxis was highest at 100 nM cAMP, consistent with previous observations. At concentrations as low as 10 pM, about 16% of cells still exhibited chemotaxis, suggesting that the receptor occupancy of only 6 cAMP molecules/cell can induce chemotaxis in very sensitive cells. At 100 pM cAMP, chemotaxis was suppressed due to the self-production and secretion of intracellular cAMP induced by extracellular cAMP. Overall, systematic observations of a large number of individual cells under the same chemical gradients revealed the heterogeneity of chemotaxis responses in a genetically homogeneous cell population, especially the existence of a sub-population with extremely high sensitivity for chemotaxis.

Talin B regulates collective cell migration via PI3K signaling in Dictyostelium discoideum mounds.

Collective cell migration is a key process during the development of multicellular organisms, in which the migrations of individual cells are coordinated through chemical guidance and physical contact between cells. Talin has been implicated in mechanical linkage between actin-based motile machinery and adhesion molecules, but how talin contributes to collective cell migration is unclear. Here we show that talin B is involved in chemical coordination between cells for collective cell migration at the multicellular mound stage in the development of Dictyostelium discoideum. From early aggregation to the mound formation, talB-null cells exhibited collective migration normally with cAMP relay. Subsequently, talB-null cells showed developmental arrest at the mound stage, and at the same time, they had impaired collective migration and cAMP relay, while wild-type cells exhibited rotational cell migration continuously in concert with cAMP relay during the mound stage. Genetic suppression of PI3K activity partially restored talB-null phenotypes in collective cell migration and cAMP relay. Overall, our observations suggest that talin B regulates chemical coordination via PI3K-mediated signaling in a stage-specific manner for the multicellular development of Dictyostelium cells.

Ruxolitinib protects skin stem cells and maintains skin homeostasis in murine graft-versus-host disease.

Graft-versus-host disease (GVHD) is the major complication after allogeneic stem cell transplantation (SCT). Emerging evidence indicates that GVHD leads to injury of intestinal stem cells. However, it remains to be investigated whether skin stem cells could be targeted in skin GVHD. Lgr5+ hair follicle stem cells (HFSCs) contribute to folliculogenesis and have a multipotent capacity to regenerate all epithelial cells in repair. We studied the fate of Lgr5+ HFSCs after SCT and explored the novel treatment to protect Lgr5+ HFSCs against GVHD using murine models of SCT. We found that GVHD reduced Lgr5+ HFSCs in association with impaired hair regeneration and wound healing in the skin after SCT. Topical corticosteroids, a standard of care for a wide range of skin disorders including GVHD, damaged HFSCs and failed to improve skin homeostasis, despite of their anti-inflammatory effects. In contrast, JAK1/2 inhibitor ruxolitinib significantly ameliorated skin GVHD, protected Lgr5+ HFSCs, and restored hair regeneration and wound healing after SCT. We, for the first time, found that GVHD targets Lgr5+ HFSCs and that topical ruxolitinib represents a novel strategy to protect skin stem cells and maintain skin homeostasis in GVHD.

Myeloid differentiation factor 88 signaling in donor T cells accelerates graft-versus-host disease.

Myeloid differentiation factor 88 (MyD88) signaling has a crucial role in activation of both innate and adoptive immunity. MyD88 transduces signals via Toll-like receptor and interleukin-1 receptor superfamily to the NFκB pathway and inflammasome by forming a molecular complex with interleukin-1 receptor-associated kinase 4. The MyD88/interleukin-1 receptor-associated kinase 4 pathway plays an important role, not only in innate immunity, but also T-cell immunity; however, its role in donor T cells on the pathophysiology of graft-versus-host disease (GvHD) remains to be elucidated. We addressed this issue by using MyD88-deficient T cells in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-SCT). While MyD88-deficient and wild-type T cells proliferated equivalently after transplantation, MyD88-deficient T cells demonstrated impaired survival and differentiation toward Th1, Tc1, and Th17, and induced less severe GvHD compared to wild-type T cells. Administration of interleukin-1 receptor-associated kinase 4 inhibitor PF-06650833 significantly ameliorated GvHD after allo-SCT. These results thus demonstrate that donor T-cell MyD88/interleukin-1 receptor-associated kinase 4 pathway is a novel therapeutic target against GvHD after allo-SCT.

[Thrombotic microangiopathy developing subsequent to tocilizumab therapy in a patient with TAFRO syndrome].

We report a case of a 60-year-old male who presented with fever and anasarca as well as hepatosplenomegaly, general lymphadenopathy, and disseminated intravascular coagulation (DIC), and was, therefore, admitted to our hospital. In addition, the patient suffered from respiratory failure and renal dysfunction and had pleural effusion and ascites. The pathological diagnosis from lymph node biopsy suggested multicentric Castleman's disease of the plasma cell type; however, the presence of high IL-6 levels, myelofibrosis, thrombocytopenia, anasarca, renal dysfunction, and hepatosplenomegaly led to a definitive diagnosis of TAFRO syndrome. Tocilizumab was administered on day 15 of disease diagnosis, resulting in the improvement in DIC but not other symptoms. As schizocytes were detected in the peripheral blood, he also experienced disturbance of consciousness and thrombotic microangiopathy (TMA) was considered. Following plasma exchange (PE) and continuous hemodiafiltration (CHDF), his symptoms temporarily improved. However, his condition worsened again, and he eventually died on day 33. Pathological autopsy revealed that although the lymph nodes were not enlarged, he had organomegaly, gastrointestinal and omental hemorrhage, and acute necrotizing pancreatitis. Since TMA developed after the administration of tocilizumab, the possibility of drug-induced secondary TMA cannot be ruled out.

α-Mannan induces Th17-mediated pulmonary graft-versus-host disease in mice.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy for various hematopoietic disorders. Graft-versus-host disease (GVHD) and infections are the major obstacles of HSCT, and their close relationship has been suggested. Although roles of bacterial and viral infections in the pathophysiology of GVHD are well described, impacts of fungal infection on GVHD remain to be elucidated. In mouse models of GVHD, injection of α-mannan (Mn), a major component of fungal cell wall, or heat-killed Candida albicans exacerbated GVHD, particularly in the lung. Mn-induced donor T-cell polarization toward Th17 and lung-specific chemokine environment in GVHD led to accumulation of Th17 cells in the lung. The detrimental effects of Mn on GVHD depended on donor IL-17A production and host C-type lectin receptor Dectin-2. These results suggest a previously unrecognized link between pulmonary GVHD and fungal infection after allogeneic HSCT.

Efficacy and prognosis of antiviral therapy on hepatitis C following treatment of lymphoma in HCV-positive diffuse large-cell lymphoma.

The purpose of this study is to study the usefulness of post-remission antiviral therapy in cases of HCV-RNA-positive diffuse large-cell lymphoma. Antiviral therapy against HCV was performed after remission using CHOP or CHOP-like chemotherapy in combination with rituximab in five successive cases of HCV-RNA-positive diffuse large-cell lymphoma. The control groups consisted of a group of HCV-RNA-positive diffuse large-cell lymphoma cases prior to this trial (control 1), and a group of cases that tested negative for HIV, HCV, and HBV (control 2). All the cases were in remission at the time of initial treatment. There were no significant differences between the three groups in terms of age, sex, treatment, stage, or International Prognosis Index (IPI). When HCV antiviral therapy was performed after treatment for diffuse large-cell lymphoma, we observed no recurrence or deaths, and the 2-year overall survival and progression-free survival rates were significantly greater than those in the control 1 group (P = 0.0246). It is possible that a better prognosis can be achieved by performing HCV antiviral therapy after achieving remission in cases of HCV-RNA-positive diffuse large-cell lymphoma through the use of R-CHOP or similar treatments.

Disseminated toxoplasmosis after hematopoietic stem cell transplantation showing unusual magnetic resonance images.

We herein report a patient who had disseminated toxoplasmosis after hematopoietic stem cell transplantation showing atypical clinical presentation and neuroimaging. Parkinsonism symptoms such as muscle rigidity, bradykinesia, tremor, and postural instability were initial manifestations. Magnetic resonance imaging showed diffuse symmetrical lesions of bilateral basal ganglia lacking ringed enhancement. Post-mortem analysis revealed multiple tachyzoites of Toxoplasma gondii in the basal ganglia, mid brain, cerebellum, and cardiac muscle.

Integrin LFA-1 regulates cell adhesion via transient clutch formation.

Integrin LFA-1 regulates immune cell adhesion and trafficking by binding to ICAM-1 upon chemokine stimulation. Integrin-mediated clutch formation between extracellular ICAM-1 and the intracellular actin cytoskeleton is important for cell adhesion. We applied single-molecule tracking analysis to LFA-1 and ICAM-1 in living cells to examine the ligand-binding kinetics and mobility of the molecular clutch under chemokine-induced physiological adhesion and Mn(2+)-induced tight adhesion. Our results show a transient LFA-1-mediated clutch formation that lasts a few seconds and leads to a transient lower-mobility is sufficient to promote cell adhesion. Stable clutch formation was observed for Mn(2+)-induced high affinity LFA-1, but was not required for physiological adhesion. We propose that fast cycling of the clutch formation by intermediate-affinity integrin enables dynamic cell adhesion and migration.

PTEN hopping on the cell membrane is regulated via a positively-charged C2 domain.

PTEN, a tumor suppressor that is frequently mutated in a wide spectrum of cancers, exerts PI(3,4,5)P3 phosphatase activities that are regulated by its dynamic shuttling between the membrane and cytoplasm. Direct observation of PTEN in the interfacial environment can offer quantitative information about the shuttling dynamics, but remains elusive. Here we show that positively charged residues located in the cα2 helix of the C2 domain are necessary for the membrane localization of PTEN via stable electrostatic interactions in Dictyostelium discoideum. Single-molecule imaging analyses revealed that PTEN molecules moved distances much larger than expected had they been caused by lateral diffusion, a phenomenon we call "hopping." Our novel single-particle tracking analysis method found that the cα2 helix aids in regulating the hopping and stable-binding states. The dynamically established membrane localization of PTEN was revealed to be essential for developmental processes and clarified a fundamental regulation mechanism of the protein quantity and activity on the plasma membrane.

Modeling the self-organized phosphatidylinositol lipid signaling system in chemotactic cells using quantitative image analysis.

A key signaling event that is responsible for gradient sensing in eukaryotic cell chemotaxis is a phosphatidylinositol (PtdIns) lipid reaction system. The self-organization activity of this PtdIns lipid system induces an inherent polarity, even in the absence of an external chemoattractant gradient, by producing a localized PtdIns (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)]-enriched domain on the membrane. Experimentally, we found that such a domain could exhibit two types of behavior: (1) it could be persistent and travel on the membrane, or (2) be stochastic and transient. Taking advantage of the simultaneous visualization of PtdIns(3,4,5)P(3) and the enzyme phosphatase and tensin homolog (PTEN), for which PtdIns(3,4,5)P(3) is a substrate, we statistically demonstrated the inter-dependence of their spatiotemporal dynamics. On the basis of this statistical analysis, we developed a theoretical model for the self-organization of PtdIns lipid signaling that can accurately reproduce both persistent and transient domain formation; these types of formations can be explained by the oscillatory and excitability properties of the system, respectively.

Asymmetric PTEN distribution regulated by spatial heterogeneity in membrane-binding state transitions.

The molecular mechanisms that underlie asymmetric PTEN distribution at the posterior of polarized motile cells and regulate anterior pseudopod formation were addressed by novel single-molecule tracking analysis. Heterogeneity in the lateral mobility of PTEN on a membrane indicated the existence of three membrane-binding states with different diffusion coefficients and membrane-binding lifetimes. The stochastic state transition kinetics of PTEN among these three states were suggested to be regulated spatially along the cell polarity such that only the stable binding state is selectively suppressed at the anterior membrane to cause local PTEN depletion. By incorporating experimentally observed kinetic parameters into a simple mathematical model, the asymmetric PTEN distribution can be explained quantitatively to illustrate the regulatory mechanisms for cellular asymmetry based on an essential causal link between individual stochastic reactions and stable localizations of the ensemble.

[Sustained remission over 10 years after rituximab-monotherapy in a patient with primary duodenal follicular lymphoma].

A 79-year-old man received gastrointestinal endoscopy for reexamination of a gastric submucosal tumor in May 2002 and whitish granular mucosa was found near the ampulla of Vater of the duodenum, though biopsy specimens showed only lymphocyte infiltrations. In December 2002, a second gastrointestinal endoscopy revealed an irregular granular elevated lesion around the ampulla of Vater and biopsy specimens showed pathological findings of follicular lymphoma. No other abnormal findings raising suspicion of tumor formation were observed on systemic examinations and the diagnosis of duodenal follicular lymphoma was confirmed. Systemic chemotherapy using rituximab at 375 mg/m(2) weekly for 4 consecutive weeks was started in January 2003. Six months later, endoscopic findings of the lesions revealed nearly normal mucosa around the ampulla of Vater, though histologically the biopsy specimens showed residual lymphoma cells. The same rituximab therapy as before was started in November. There has been no evidence of recurrence and a prolonged, more than 10 years, complete remission has been achieved.

Spontaneous signal generation by an excitable system for cell migration.

Eukaryotic cells exhibit random migration in the absence of extracellular directional cues. This random migration acts as basal motility for various migratory responses such as chemotaxis. The self-organization of random motility requires the internal signals that determine the anterior side of the migrating cell be generated spontaneously from the intrinsic activities of intracellular signaling networks. Recent studies have identified an excitable system as the mechanism of the spontaneous signal generation. Here, we discuss how the excitable system of Ras, a small G protein, regulates signaling networks in Dictyostelium discoideum as a model organism. The excitability produces a domain where an active form of Ras is enriched on the cell membrane without extracellular directional cues, such that Ras serves as the anterior signal. The typical spatiotemporal characteristics are mathematically explained by reaction-diffusion models. These models further enable a quantitative analysis of the dynamics that depends on the internal cellular states and surrounding environments. Downstream of the Ras excitable system, a phosphoinositide metabolic network composed of PI3K, PTEN, PI(3,4,5)P3 and PI(4,5)P2 exhibits bistability to discretize the anterior and posterior regions of the cell membrane. Upstream, a local excitation and global inhibition local excitation global inhibition network, which works for gradient sensing in the presence of chemoattractant gradients, spatiotemporally biases the excitability of Ras for chemotaxis. In parallel with the Ras excitable system, the cGMP signaling pathway constitutes another excitable system of its own periodicity to ensure flexible migratory dynamics. In addition to these intracellular signaling networks, an intercellular signaling network activated by secreted cAMP is coupled with the Ras excitable system for collective cell migration. Finally, we discuss how the excitable system of Ras operates as a platform of information integration by receiving multiple intrinsic and extrinsic signals to ensure spontaneous cellular activity and robust responses in eukaryotic cell migration under natural complex environments.

Intracellular encoding of spatiotemporal guidance cues in a self-organizing signaling system for chemotaxis in Dictyostelium cells.

Even in the absence of guidance cues, chemotactic cells are often spontaneously motile, which should accompany a spontaneous symmetry breaking inside the cells. A shallow chemoattractant gradient can induce these cells to move directionally without much change in cell morphology. As the gradient becomes steeper, the accuracy of chemotaxis increases. It is not clear how the steepness is expressed or encoded internally in the signaling network, which in turn coordinately activates the motile apparatus for chemotaxis. In Dictyostelium cells, self-organizing polarization activities in the signaling network have been reported. In this paper, we conducted a theoretical study of the response of this self-organizing system to guidance cues. Our analyses indicate that self-organizing systems respond sharply to a shallow external gradient by increasing the precision of polarity direction and modulating the frequency of self-polarization. We also show how the precision increase and frequency modulation are achieved. Our results indicate that self-organizing activity, independent of external cues, is the basis for the sensitive and robust response to shallow gradients. Finally, we show that the system can sense the direction of space-time waves of a stimulus, for which Dictyostelium cells exhibit chemotaxis in the developmental process.