Severe Bone Marrow Aplasia Following Macrophage Activation Syndrome in Systemic Lupus Erythematosus.

Macrophage activation syndrome (MAS) is a potentially fatal complication of rheumatic diseases, characterized by activated macrophages with hemophagocytosis and multiple organ damage. We report a case of MAS associated with systemic lupus erythematosus that initially presented with severe liver dysfunction. Although it was improved with steroids and plasmapheresis, severe pancytopenia was subsequently experienced, and the bone marrow showed severe aplasia similar to aplastic anemia. Nevertheless, the administration of immunosuppressants resulted in the recovery of blood counts within two weeks. When severe MAS results in cytokine overproduction, bone marrow aplasia may occur, for which immunosuppressive therapy may be highly effective.

Elucidation of the Role of FAM210B in Mitochondrial Metabolism and Erythropoiesis.

Mitochondria play essential and specific roles during erythroid differentiation. Recently, FAM210B, encoding a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1, as well as an erythropoietin-inducible gene. While FAM210B protein is involved in regulate mitochondrial metabolism and heme biosynthesis, its detailed function remains unknown. Here, we generated both knockout and knockdown of endogenous FAM210B in human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells using CRISPR/Cas9 methodology. Intriguingly, erythroid differentiation was more pronounced in the FAM210B-depleted cells, and this resulted in increased frequency of orthochromatic erythroblasts and decreased frequencies of basophilic/polychromatic erythroblasts. Comprehensive metabolite analysis and functional analysis indicated that oxygen consumption rates and the NAD (NAD+)/NADH ratio were significantly decreased, while lactate production was significantly increased in FAM210B deletion HiDEP cells, indicating involvement of FAM210B in mitochondrial energy metabolism in erythroblasts. Finally, we purified FAM210B-interacting protein from K562 cells that stably expressed His/biotin-tagged FAM210B. Mass spectrometry analysis of the His/biotin-purified material indicated interactions with multiple subunits of mitochondrial ATP synthases, such as subunit alpha (ATP5A) and beta (ATP5B). Our results suggested that FAM210B contributes prominently to erythroid differentiation by regulating mitochondrial energy metabolism. Our results provide insights into the pathophysiology of dysregulated hematopoiesis.

Exploring the mechanistic link between SF3B1 mutation and ring sideroblast formation in myelodysplastic syndrome.

Acquired sideroblastic anemia, characterized by bone marrow ring sideroblasts (RS), is predominantly associated with myelodysplastic syndrome (MDS). Although somatic mutations in splicing factor 3b subunit 1 (SF3B1), which is involved in the RNA splicing machinery, are frequently found in MDS-RS, the detailed mechanism contributing to RS formation is unknown. To explore the mechanism, we established human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells stably expressing SF3B1K700E. SF3B1K700E expressing cells showed higher proportion of RS than the control cells along with erythroid differentiation, indicating the direct contribution of mutant SF3B1 expression in erythroblasts to RS formation. In SF3B1K700E expressing cells, ABCB7 and ALAS2, known causative genes for congenital sideroblastic anemia, were downregulated. Additionally, mis-splicing of ABCB7 was observed in SF3B1K700E expressing cells. ABCB7-knockdown HUDEP-2 cells revealed an increased frequency of RS formation along with erythroid differentiation, demonstrating the direct molecular link between ABCB7 defects and RS formation. ALAS2 protein levels were obviously decreased in ABCB7-knockdown cells, indicating decreased ALAS2 translation owing to impaired Fe-S cluster export by ABCB7 defects. Finally, RNA-seq analysis of MDS clinical samples demonstrated decreased expression of ABCB7 by the SF3B1 mutation. Our findings contribute to the elucidation of the complex mechanisms of RS formation in MDS-RS.

Congenital sideroblastic anemia model due to ALAS2 mutation is susceptible to ferroptosis.

X-linked sideroblastic anemia (XLSA), the most common form of congenital sideroblastic anemia, is caused by a germline mutation in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. In XLSA, defective heme biosynthesis leads to ring sideroblast formation because of excess mitochondrial iron accumulation. In this study, we introduced ALAS2 missense mutations on human umbilical cord blood-derived erythroblasts; hereafter, we refer to them as XLSA clones. XLSA clones that differentiated into mature erythroblasts showed an increased frequency of ring sideroblast formation with impaired hemoglobin biosynthesis. The expression profiling revealed significant enrichment of genes involved in ferroptosis, which is a form of regulated cell death induced by iron accumulation and lipid peroxidation. Notably, treatment with erastin, a ferroptosis inducer, caused a higher proportion of cell death in XLSA clones. XLSA clones exhibited significantly higher levels of intracellular lipid peroxides and enhanced expression of BACH1, a regulator of iron metabolism and potential accelerator of ferroptosis. In XLSA clones, BACH1 repressed genes involved in iron metabolism and glutathione synthesis. Collectively, defective heme biosynthesis in XLSA clones could confer enhanced BACH1 expression, leading to increased susceptibility to ferroptosis. The results of our study provide important information for the development of novel therapeutic targets for XLSA.

TM5614, an Inhibitor of Plasminogen Activator Inhibitor-1, Exerts an Antitumor Effect on Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is triggered by t(9;22)(q34;q11.2) translocation, leading to the formation of the BCR-ABL1 fusion gene. Although the development of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has dramatically improved the prognosis of CML, the disease could often relapse, presumably because leukemic stem cell fraction of CML (CML-LSC) may reside in specific niches, and also acquire an ability to resist the cytotoxic agents. Recently a study indicated that pharmacological inhibition of plasminogen activator inhibitor-1 (PAI-1, also known as SERPINE1) would cause detachment of CML-LSCs from their niche by inducing maturation of membrane-type matrix metalloprotease-1 (MT1-MMP), leading to increased susceptibility of CML-LSCs against TKIs. However, the direct antitumor effect of PAI-1 inhibition in CML remains unclear. Because PAI-1 mRNA expression was lower in CML cell line (K562) than bone marrow mononuclear cells derived from CML patients, we established K562 cell clones stably expressing exogenous PAI-1 (K562/PAI-1). We found that TM5614 treatment significantly suppressed cell proliferation and induced apoptosis in K562/PAI-1 cells, accompanied by increased activity of Furin protease, which is a known target of PAI-1. Besides processing mature MT1-MMP, Furin is in charge of cleaving the NOTCH receptor to form a heterodimer before exporting it to the cell surface membrane. In K562/PAI-1 cells, TM5614 treatment increased NOTCH1 intracellular domain (NICD) protein expression as well as NOTCH1 target of HEY1 mRNA levels. Finally, forced expression of either Furin or NICD in K562/PAI-1 cells significantly inhibited cell proliferation and induced apoptosis. Collectively, PAI-1 inhibition may have an antitumor effect by modulating the Furin/NICD pathway.

Flow Cytometry-Based Photodynamic Diagnosis with 5-Aminolevulinic Acid for the Detection of Minimal Residual Disease in Multiple Myeloma.

Multiple myeloma is the cancer of plasma cells. Along with the development of new and effective therapies, improved outcomes in patients with multiple myeloma have increased the interest in minimal residual disease (MRD) monitoring. However, the considerable heterogeneity of immunophenotypic and molecular markers of myeloma cells has limited its clinical application. 5-Aminolevulinic acid (ALA) is a natural compound in the heme biosynthesis pathway. Following ALA treatment, tumor cells preferentially accumulate porphyrins because of the differential activities of aerobic glycolysis, known as Warburg effect. Among various porphyrins, protoporphyrine IX is a strong photosensitizer; thus, ALA-based photodynamic diagnosis has been widely used in various solid cancers. Here, the feasibility of flow cytometry-based photodynamic detection of MRD was tested in multiple myeloma. Among various human cell lines of hematological malignancies, including K562 erythroleukemia, Jurkat T-cell leukemia, Nalm6 pre-B cell leukemia, KG1a myeloid leukemia, and U937 monocytic leukemia, human myeloma cell line, KMS18, and OPM2 abundantly expressed ALA transporters, such as SLC36A1 and SLC15A2, and 1 mM ALA treatment for 24 h resulted in nearly 100% porphyrin fluorescence expression, which could be competitively inhibited by ALA transport with gamma-aminobutyric acid. Titration studies revealed that the lowest ALA concentration required to achieve nearly 100% porphyrin fluorescence in KMS18 cells was 0.25 mM, with an incubation period of 2 h. Under these conditions, incubation of primary peripheral blood mononuclear cells resulted in only 1.8 % of the cells exhibiting porphyrin fluorescence. Therefore, flow cytometry-based photodynamic diagnosis is a promising approach for detecting MRD in multiple myeloma.

Generation and Molecular Characterization of Human Ring Sideroblasts: a Key Role of Ferrous Iron in Terminal Erythroid Differentiation and Ring Sideroblast Formation.

Ring sideroblasts are a hallmark of sideroblastic anemia, although little is known about their characteristics. Here, we first generated mutant mice by disrupting the GATA-1 binding motif at the intron 1 enhancer of the ALAS2 gene, a gene responsible for X-linked sideroblastic anemia (XLSA). Although heterozygous female mice showed an anemic phenotype, ring sideroblasts were not observed in their bone marrow. We next established human induced pluripotent stem cell-derived proerythroblast clones harboring the same ALAS2 gene mutation. Through coculture with sodium ferrous citrate, mutant clones differentiated into mature erythroblasts and became ring sideroblasts with upregulation of metal transporters (MFRN1, ZIP8, and DMT1), suggesting a key role for ferrous iron in erythroid differentiation. Interestingly, holo-transferrin (holo-Tf) did not induce erythroid differentiation as well as ring sideroblast formation, and mutant cells underwent apoptosis. Despite massive iron granule content, ring sideroblasts were less apoptotic than holo-Tf-treated undifferentiated cells. Microarray analysis revealed upregulation of antiapoptotic genes in ring sideroblasts, a profile partly shared with erythroblasts from a patient with XLSA. These results suggest that ring sideroblasts exert a reaction to avoid cell death by activating antiapoptotic programs. Our model may become an important tool to clarify the pathophysiology of sideroblastic anemia.

Successful Treatment of Aggressive Mature B-cell Lymphoma Mimicking Immune Thrombocytopenic Purpura.

A 55-year-old woman suffered from hemorrhagic tendency. She had severe thrombocytopenia without any hematological or coagulatory abnormalities, and a bone marrow examination revealed an increased number of megakaryocytes without any abnormal cells or blasts. No lymphadenopathy or hepatosplenomegaly was observed on computed tomography. She was initially diagnosed with immune thrombocytopenic purpura (ITP). None of the treatments administered for ITP produced a response. However, abnormal cells were eventually found during the third bone marrow examination. The pathological diagnosis was mature B-cell lymphoma. Rituximab-containing chemotherapy produced a marked increase in the patient's platelet count, and her lymphoma went into complete remission.

Successful Cord Blood Stem Cell Transplantation for Primary Cutaneous CD8-positive Aggressive Epidermotropic Cytotoxic T-cell Lymphoma Complicated with Cerebral Infiltration.

A 16-year-old boy, who had been initially examined for bilateral blepharedema and slight eruption, presented with rapidly deteriorating symptoms in associating with headache and consciousness disturbance. He was diagnosed to have primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma (PCAE-CTL) by a biopsy of the skin and brain. After whole-brain radiation and some courses of chemotherapy, cord blood transplantation was performed with myeloablative conditioning. After transplantation, the cerebral dysfunction gradually improved. Disease remission was confirmed by the disappearance of any abnormal findings on electroencephalogram and magnetic resonance imaging. PCAE-CTL is reported to be an extremely aggressive disease with a poor prognosis, but the timely performance of cord blood transplantation is considered to be a promising treatment strategy.

The Imaging Diagnosis of Less Advanced Cases of Cardiac Amyloidosis: The Relative Apical Sparing Pattern.

An early diagnosis is important for improving the prognosis of cardiac amyloidosis (CA). We herein describe the utility of two-dimensional speckle tracking echocardiography (2-D STE) in diagnosing CA at a less advanced stage. A 63-year-old woman with exertional dyspnea was suspected of having CA based on her echocardiographic and electrocardiographic findings. A myocardial biopsy was negative for amyloid deposits, while the relative apical sparing pattern was detected on 2-D STE, which was highly suggestive of CA. Chemotherapy was initiated as a treatment for CA, and the patient's symptoms were immediately relieved. Thereafter, amyloid deposits were detected in a skin biopsy specimen.