Special AT-Rich Sequence-Binding Protein 1 Supports Survival and Maturation of Naive B Cells Stimulated by B Cell Receptors.

Epigenetic mechanisms underpin the elaborate activities of essential transcription factors in lymphocyte development. Special AT-rich sequence-binding protein 1 (SATB1) is a chromatin remodeler that orchestrates the spatial and temporal actions of transcription factors. Previous studies have revealed the significance of SATB1 in T cell lineage. However, whether and how SATB1 controls B cell lineage development is yet to be clarified. In this study, we show that SATB1 is an important factor during splenic B cell maturation. By analyzing SATB1/Tomato reporter mice, we determined the dynamic fluctuation of SATB1 expression in the B cell lineage. Although SATB1 expression decreased to minimal levels during B cell differentiation in the bone marrow, it resurged markedly in naive B cells in the spleen. The expression was dramatically downregulated upon Ag-induced activation. Splenic naive B cells were subdivided into two categories, namely SATB1high and SATB1-/low, according to their SATB1 expression levels. SATB1high naive B cells were less susceptible to death and greater proliferative than were SATB1-/low cells during incubation with an anti-IgM Ab. Additionally, SATB1high cells tended to induce the expression of MHC class II, CD86, and CD83. Accordingly, naive B cells from B lineage-specific SATB1 conditional knockout mice were more susceptible to apoptosis than that in the control group upon anti-IgM Ab stimulation in vitro. Furthermore, conditional knockout mice were less capable of producing Ag-specific B cells after immunization. Collectively, our findings suggest that SATB1 expression increases in naive B cells and plays an important role in their survival and maturation.

Preparation of monovalent follistatin-like 3-Fc-fusion protein and evaluation of its effects on muscle mass in mice.

Follistatin-like 3 (FSTL3) is an endogenous antagonist against transforming growth factor-ß family ligands. Monovalent FSTL3-Fc fusion protein (mono-FSTL3-Fc) generated with knobs-into-holes technology overcomes limitations of current anti-myostatin therapies. We have developed a facile protocol for affinity purification of the Fc-fused protein from the supernatant of HEK293T cells stably expressing the protein. This protocol is advantageous by only requiring readily accessible equipment. We further outline the steps for validation of mono-FSTL3-Fc increasing systemic muscle mass in mice after intraperitoneal administration. For complete details on the use and execution of this protocol, please refer to Ozawa et al. (2021).

Systemic administration of monovalent follistatin-like 3-Fc-fusion protein increases muscle mass in mice.

Targeting the signaling pathway of growth differentiation factor 8 (GDF8), also known as myostatin, has been regarded as a promising strategy to increase muscle mass in the elderly and in patients. Accumulating evidence in animal models and clinical trials has indicated that a rational approach is to inhibit a limited number of transforming growth factor ß (TGF-ß) family ligands, including GDF8 and activin A, without affecting other members. Here, we focused on one of the endogenous antagonists against TGF-ß family ligands, follistatin-like 3 (FSTL3), which mainly binds and neutralizes activins, GDF8, and GDF11. Although bivalent human FSTL3 Fc-fusion protein was rapidly cleared from mouse circulation similar to follistatin (FST)-Fc, monovalent FSTL3-Fc (mono-FSTL3-Fc) generated with the knobs-into-holes technology exhibited longer serum half-life. Systemic administration of mono-FSTL3-Fc in mice induced muscle fiber hypertrophy and increased muscle mass in vivo. Our results indicate that the monovalent FSTL3-based therapy overcomes the difficulties of current anti-GDF8 therapies.

Autonomous TGFß signaling induces phenotypic variation in human acute myeloid leukemia.

Heterogeneity of leukemia stem cells (LSCs) is involved in their collective chemoresistance. To eradicate LSCs, it is necessary to understand the mechanisms underlying their heterogeneity. Here, we aimed to identify signals responsible for heterogeneity and variation of LSCs in human acute myeloid leukemia (AML). Monitoring expression levels of endothelial cell-selective adhesion molecule (ESAM), a hematopoietic stem cell-related marker, was useful to detect the plasticity of AML cells. While healthy human hematopoietic stem/progenitor cells robustly expressed ESAM, AML cells exhibited heterogeneous ESAM expression. Interestingly, ESAM- and ESAM+ leukemia cells obtained from AML patients were mutually interconvertible in culture. KG1a and CMK, human AML clones, also represented the heterogeneity in terms of ESAM expression. Single cell culture with ESAM- or ESAM+ AML clones recapitulated the phenotypic interconversion. The phenotypic alteration was regulated at the gene expression level, and RNA sequencing revealed activation of TGFß signaling in these cells. AML cells secreted TGFß1, which autonomously activated TGFß pathway and induced their phenotypic variation. Surprisingly, TGFß signaling blockade inhibited not only the variation but also the proliferation of AML cells. Therefore, autonomous activation of TGFß signaling underlies the LSC heterogeneity, which may be a promising therapeutic target for AML.

A multi-center retrospective analysis of patients with relapsed/refractory follicular lymphoma after third-line chemotherapy.

The overall outcome of patients with advanced-stage follicular lymphoma (FL) has improved significantly. However, some patients still develop multiple relapsed/refractory FL (RRFL). To address the still-limited data on this population, we performed this multi-center retrospective study. We analyzed 41 patients who received third-line treatment for RRFL at 8 institutes. The median age at diagnosis was 59 years (range, 38-70). The median progression-free survival (PFS) and probability of PFS at 2 years were 1.61 years and 39.4%, respectively, after third-line chemotherapy, and 0.45 years and 19.0%, respectively, after fourth-line chemotherapy. Objective response (OR) after third-line chemotherapy was achieved in 24 patients (53.7%). Bendamustine (Ben)-based regimens were associated with a significantly higher OR rate than other regimens (77.8% vs. 40.0%, respectively, P = 0.025). The median overall survival (OS) and probability of OS at 2 years were 4.71 years and 65.9%, respectively, after third-line chemotherapy, and 1.01 year and 45.1%, respectively, after fourth-line chemotherapy. In conclusion, this study had a small sample size and retrospective design, but it was able to demonstrate poor response rate and duration in patients with multiple RRFL, particularly after fourth-line chemotherapy. The optimal treatment strategy in this population should be clarified, including possibly hematopoietic stem cell transplantation.

Long-term Effects of the Janus Kinase 1/2 Inhibitor Ruxolitinib on Pulmonary Hypertension and the Cardiac Function in a Patient with Myelofibrosis.

Constitutive activation of the Janus kinase/signal transduction and activator of transcription (JAK-STAT) signaling pathway plays a central role in the pathogenesis of myelofibrosis (MF) and pulmonary hypertension (PH) is a known complication of MF. On the other hand, it has been proposed that the JAK-STAT pathway, especially signal transducer and activation of transcription (STAT) 3 activation, protects cardiomyocytes from various stresses. We describe the case of a patient with MF-associated PH who developed left ventricular dysfunction after five years of treatment with the JAK 1/2 inhibitor, ruxolitinib. This is the first report with histopathological findings that demonstrate possible contradictory effects of a JAK 1/2 inhibitor: improvement of MF-associated PH and cardiotoxicity.

Endothelial Cell-Selective Adhesion Molecule Contributes to the Development of Definitive Hematopoiesis in the Fetal Liver.

Endothelial cell-selective adhesion molecule (ESAM) is a lifelong marker of hematopoietic stem cells (HSCs). Although we previously elucidated the functional importance of ESAM in HSCs in stress-induced hematopoiesis in adults, it is unclear how ESAM affects hematopoietic development during fetal life. To address this issue, we analyzed fetuses from conventional or conditional ESAM-knockout mice. Approximately half of ESAM-null fetuses died after mid-gestation due to anemia. RNA sequencing analyses revealed downregulation of adult-type globins and Alas2, a heme biosynthesis enzyme, in ESAM-null fetal livers. These abnormalities were attributed to malfunction of ESAM-null HSCs, which was demonstrated in culture and transplantation experiments. Although crosslinking ESAM directly influenced gene transcription in HSCs, observations in conditional ESAM-knockout fetuses revealed the critical involvement of ESAM expressed in endothelial cells in fetal lethality. Thus, we showed that ESAM had important roles in developing definitive hematopoiesis. Furthermore, we unveiled the importance of endothelial ESAM in this process.

The ALK-1/SMAD/ATOH8 axis attenuates hypoxic responses and protects against the development of pulmonary arterial hypertension.

Dysregulated bone morphogenetic protein (BMP) signaling in endothelial cells (ECs) is implicated in vascular diseases such as pulmonary arterial hypertension (PAH). Here, we showed that the transcription factor ATOH8 was a direct target of SMAD1/5 and was induced in a manner dependent on BMP but independent of Notch, another critical signaling pathway in ECs. In zebrafish and mice, inactivation of Atoh8 did not cause an arteriovenous malformation-like phenotype, which may arise because of dysregulated Notch signaling. In contrast, Atoh8-deficient mice exhibited a phenotype mimicking PAH, which included increased pulmonary arterial pressure and right ventricular hypertrophy. Moreover, ATOH8 expression was decreased in PAH patient lungs. We showed that in cells, ATOH8 interacted with hypoxia-inducible factor 2α (HIF-2α) and decreased its abundance, leading to reduced induction of HIF-2α target genes in response to hypoxia. Together, these findings suggest that the BMP receptor type II/ALK-1/SMAD/ATOH8 axis may attenuate hypoxic responses in ECs in the pulmonary circulation and may help prevent the development of PAH.

Palbociclib enhances activin-SMAD-induced cytostasis in estrogen receptor-positive breast cancer.

Cyclin-dependent kinase (CDK) 4 and CDK6 inhibitors are effective therapeutic options for hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer. Although CDK4/6 inhibitors mainly target the cyclin D-CDK4/6-retinoblastoma tumor suppressor protein (RB) axis, little is known about the clinical impact of inhibiting phosphorylation of other CDK4/6 target proteins. Here, we focused on other CDK4/6 targets, SMAD proteins. We showed that a CDK4/6 inhibitor palbociclib and activin-SMAD2 signaling cooperatively inhibited cell cycle progression of a luminal-type breast cancer cell line T47D. Palbociclib enhanced SMAD2 binding to the genome by inhibiting CDK4/6-mediated linker phosphorylation of the SMAD2 protein. We also showed that cyclin G2 plays essential roles in SMAD2-dependent cytostatic response. Moreover, comparison of the SMAD2 ChIP-seq data of T47D cells with those of Hs578T (triple-negative breast cancer cells) indicated that palbociclib augmented different SMAD2-mediated functions based on cell type, and enhanced SMAD2 binding to the target regions on the genome without affecting its binding pattern. In summary, palbociclib enhances the cytostatic effects of the activin-SMAD2 signaling pathway, whereas it possibly strengthens the tumor-promoting aspect in aggressive breast cancer.

Gut Microbiota Promotes Obesity-Associated Liver Cancer through PGE2-Mediated Suppression of Antitumor Immunity.

Obesity increases the risk of cancers, including hepatocellular carcinomas (HCC). However, the precise molecular mechanisms through which obesity promotes HCC development are still unclear. Recent studies have shown that gut microbiota may influence liver diseases by transferring its metabolites and components. Here, we show that the hepatic translocation of obesity-induced lipoteichoic acid (LTA), a Gram-positive gut microbial component, promotes HCC development by creating a tumor-promoting microenvironment. LTA enhances the senescence-associated secretory phenotype (SASP) of hepatic stellate cells (HSC) collaboratively with an obesity-induced gut microbial metabolite, deoxycholic acid, to upregulate the expression of SASP factors and COX2 through Toll-like receptor 2. Interestingly, COX2-mediated prostaglandin E2 (PGE2) production suppresses the antitumor immunity through a PTGER4 receptor, thereby contributing to HCC progression. Moreover, COX2 overexpression and excess PGE2 production were detected in HSCs in human HCCs with noncirrhotic, nonalcoholic steatohepatitis (NASH), indicating that a similar mechanism could function in humans.Significance: We showed the importance of the gut-liver axis in obesity-associated HCC. The gut microbiota-driven COX2 pathway produced the lipid mediator PGE2 in senescent HSCs in the tumor microenvironment, which plays a pivotal role in suppressing antitumor immunity, suggesting that PGE2 and its receptor may be novel therapeutic targets for noncirrhotic NASH-associated HCC. Cancer Discov; 7(5); 522-38. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 443.

Dietary fructose-induced hepatocellular carcinoma development manifested in mice lacking apoptosis inhibitor of macrophage (AIM).

The consumption of fructose, including the use of high-fructose corn syrup as a sweetener, has increased continuously in recent decades. Although the involvement of fructose in the development of metabolic diseases has been emphasized recently, whether fructose intake increases susceptibility to steatosis-associated hepatocellular carcinoma (HCC) is unclear. Here, we investigated this issue using mice lacking a circulating protein, apoptosis inhibitor of macrophage (AIM, encoded by cd5l). AIM does not induce carcinogenesis of hepatocytes, but provokes necrotic death specifically in AIM-bound cancer cells through complement cascade activation, thereby preventing HCC tumor development in wild-type mice. When subjected to a high-fructose diet (HFrD), AIM-deficient (AIM-/- ) mice showed liver steatosis and subsequent liver inflammation as well as fibrosis, but at much milder levels compared with mice fed a high-fat diet. However, AIM-/- mice were markedly susceptible to HCC tumor development, whereas no wild-type mice developed the disease. Systemic metabolic states, including obesity and insulin resistance, were similar in both types of mice after HFrD challenge, indicating no influence of AIM on HFrD-induced metabolic changes. Our results suggest that dietary fructose increases the risk for liver carcinogenesis and that individuals with low blood AIM levels may be susceptible to HCC under chronic fructose intake.

[Efficacy of rituximab for TAFRO syndrome, a variant type of multicentric Castleman's disease].

A 48-year-old woman was hospitalized because of severe thrombocytopenia, leg edema, and fever. Intravenous immunoglobulin therapy was administered, but no efficacy was obtained. Her bone marrow was dry-tap, and fibrosis was found in the biopsy specimens. A positron emission tomographic study showed FDG-avid lymphadenopathy and hepatomegaly. Biopsy specimens of axillary lymph nodes showed Castleman's disease-like findings. Since she then developed severe proteinuria and massive pleural effusion, steroid therapy was started, providing temporary relief of symptoms other than the thrombocytopenia. However, rapid worsening of her general condition prompted us to attempt rituximab as salvage therapy. The pleural effusion, edema, and proteinuria disappeared soon after starting rituximab administration. Platelet counts also normalized and fibrosis of the bone marrow showed amelioration. Recently, a variant of multicentric Castleman's disease, termed the TAFRO syndrome, has been proposed, and our patient's features fit the diagnosis of this syndrome. Rituximab might be considered as a therapeutic option in such cases.

Hypoxia and nitric oxide treatment confer tolerance to glucose starvation in a 5'-AMP-activated protein kinase-dependent manner.

Hypoxia is a critical event for higher organisms, and cells and tissues react by increasing the oxygen supply by vasodilatation, angiogenesis, and erythropoiesis and maintaining cellular energy by increasing glycolysis and inhibiting anabolic pathways. Stimulation of glycolysis has been regarded as the main response that increases energy production during hypoxia; however, there is an obvious conflict during ischemia, because both the oxygen and glucose supply are insufficient. In this study, we found that exposure of HepG2 cells and normal fibroblasts to hypoxia induces cellular tolerance to glucose starvation. The tolerance induced by hypoxia is dependent on several amino acids, indicating a switch from glucose to amino acids as the energy source. When antisense RNA expression vector for 5'-AMP-activated protein kinase or protein kinase B/Akt was transfected into HepG2 cells, the induction of tolerance to glucose was greatly inhibited, indicating that the tolerance was dependent on 5'-AMP-activated protein kinase and protein kinase B/Akt. Similar tolerance was induced by nitric oxide exposure. The tolerance induced was observed in various cells and may represent a previously unknown physiological response related to hypoxia-preconditioning and tumor progression:austerity.