ILF2 enhances the DNA cytosine deaminase activity of tumor mutator APOBEC3B in multiple myeloma cells.

DNA cytosine deaminase APOBEC3B (A3B) is an endogenous source of mutations in many human cancers, including multiple myeloma. A3B proteins form catalytically inactive high molecular mass (HMM) complexes in nuclei, however, the regulatory mechanisms of A3B deaminase activity in HMM complexes are still unclear. Here, we performed mass spectrometry analysis of A3B-interacting proteins from nuclear extracts of myeloma cell lines and identified 30 putative interacting proteins. These proteins are involved in RNA metabolism, including RNA binding, mRNA splicing, translation, and regulation of gene expression. Except for SAFB, these proteins interact with A3B in an RNA-dependent manner. Most of these interacting proteins are detected in A3B HMM complexes by density gradient sedimentation assays. We focused on two interacting proteins, ILF2 and SAFB. We found that overexpressed ILF2 enhanced the deaminase activity of A3B by 30%, while SAFB did not. Additionally, siRNA-mediated knockdown of ILF2 suppressed A3B deaminase activity by 30% in HEK293T cell lysates. Based on these findings, we conclude that ILF2 can interact with A3B and enhance its deaminase activity in HMM complexes.

FRET-Based Detection and Quantification of HIV-1 Virion Maturation.

HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation in vitro, we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.

APOBEC3B is preferentially expressed at the G2/M phase of cell cycle.

APOBEC3B (A3B) is a cytosine deaminase that converts cytosine to uracil in single-stranded DNA. Cytosine-to-thymine and cytosine-to-guanine base substitution mutations in trinucleotide motifs (APOBEC mutational signatures) were found in various cancers including lymphoid hematological malignancies such as multiple myeloma and A3B has been shown to be an enzymatic source of mutations in those cancers. Although the importance of A3B is being increasingly recognized, it is unclear how A3B expression is regulated in cancer cells as well as normal cells. To answer these fundamental questions, we analyzed 1276 primary myeloma cells using single-cell RNA-sequencing (scRNA-seq) and found that A3B was preferentially expressed at the G2/M phase, in sharp contrast to the expression patterns of other APOBEC3 genes. Consistently, we demonstrated that A3B protein was preferentially expressed at the G2/M phase in myeloma cells by cell sorting. We also demonstrated that normal blood cells expressing A3B were also enriched in G2/M-phase cells by analyzing scRNA-seq data from 86,493 normal bone marrow mononuclear cells. Furthermore, we revealed that A3B was expressed mainly in plasma cells, CD10+ B cells and erythroid cells, but not in granulocyte-macrophage progenitors. A3B expression profiling in normal blood cells may contribute to understanding the defense mechanism of A3B against viruses, and partially explain the bias of APOBEC mutational signatures in lymphoid but not myeloid malignancies. This study identified the cells and cellular phase in which A3B is highly expressed, which may help reveal the mechanisms behind carcinogenesis and cancer heterogeneity, as well as the biological functions of A3B in normal blood cells.

Risk factors and appropriate therapeutic strategies for thrombotic microangiopathy after allogeneic HSCT.

Transplant-associated thrombotic microangiopathy (TA-TMA) is a fatal complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, so far, no large cohort study determined the risk factors and the most effective therapeutic strategies for TA-TMA. Thus, the present study aimed to clarify these clinical aspects based on a large multicenter cohort. This retrospective cohort study was performed by the Kyoto Stem Cell Transplantation Group (KSCTG). A total of 2425 patients were enrolled from 14 institutions. All patients were aged ≥16 years, presented with hematological diseases, and received allo-HSCT after the year 2000. TA-TMA was observed in 121 patients (5.0%) on day 35 (median) and was clearly correlated with inferior overall survival (OS) (hazard ratio [HR], 4.93). Pre- and post-HSCT statistically significant risk factors identified by multivariate analyses included poorer performance status (HR, 1.69), HLA mismatch (HR, 2.17), acute graft-versus-host disease (aGVHD; grades 3-4) (HR, 4.02), Aspergillus infection (HR, 2.29), and veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS; HR, 4.47). The response rate and OS significantly better with the continuation or careful reduction of calcineurin inhibitors (CNI) than the conventional treatment strategy of switching from CNI to corticosteroids (response rate, 64.7% vs 20.0%). In summary, we identified the risk factors and the most appropriate therapeutic strategies for TA-TMA. The described treatment strategy could improve the outcomes of patients with TA-TMA in the future.

APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.

Protein kinase A inhibits tumor mutator APOBEC3B through phosphorylation.

APOBEC3B cytidine deaminase (A3B) catalyzes cytosine into uracil in single-strand DNA and induces C-to-T mutations in genomic DNA of various types of tumors. Accumulation of APOBEC signature mutations is correlated with a worse prognosis for patients with breast cancer or multiple myeloma, suggesting that A3B activity might be a cause of the unfavorable DNA mutations and clonal evolution in these tumors. Phosphorylation of conserved threonine residues of other cytidine deaminases, activation induced deaminase (AID) and APOBEC3G, inhibits their activity. Here we show that protein kinase A (PKA) physically binds to A3B and phosphorylates Thr214. In vitro deaminase assays and foreign DNA editing assays in cells confirm that phosphomimetic A3B mutants, T214D and T214E, completely lose deaminase activity. Molecular dynamics simulation of A3B phosphorylation reveals that Thr214 phosphorylation disrupts binding between the phospho-A3B catalytic core and ssDNA. These mutants still inhibit retroviral infectivity at least partially, and also retain full anti-retrotransposition activity. These results imply that PKA-mediated phosphorylation inhibits A3B mutagenic activity without destructing its innate immune functions. Therefore, PKA activation could reduce further accumulation of mutations in A3B overexpressing tumors.

Endogenous APOBEC3B Overexpression Constitutively Generates DNA Substitutions and Deletions in Myeloma Cells.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminases have emerged as potential genomic mutators in various cancers. Multiple myeloma accumulates APOBEC signature mutations as it progresses; however, the mechanisms underlying APOBEC signature acquisition and its consequences remain elusive. In this study, we examined the significance and clinical impact of APOBEC3B (A3B) activity in multiple myeloma. Among APOBECs, only highly expressed A3B was associated with poor prognosis in myeloma patients, independent of other known poor prognostic factors. Quantitative PCR revealed that CD138-positive primary myeloma cells and myeloma cell lines exhibited remarkably high A3B expression levels. Interestingly, lentiviral A3B knockdown prevented the generation of deletion and loss-of-function mutations in exogenous DNA, whereas in control cells, these mutations accumulated with time. A3B knockdown also decreased the basal levels of γ-H2AX foci, suggesting that A3B promotes constitutive DNA double-strand breaks in myeloma cells. Importantly, among control shRNA-transduced cells, we observed the generation of clones that harboured diverse mutations in exogenous genes and several endogenous genes frequently mutated in myeloma, including TP53. Taken together, the results suggest that A3B constitutively mutates the tumour genome beyond the protection of the DNA repair system, which may lead to clonal evolution and genomic instability in myeloma.

Efficacy of antithymocyte globulin for allogeneic hematopoietic cell transplantation: a systematic review and meta-analysis.

The efficacy of rabbit antithymocyte globulin (ATG) for the prevention of graft-versus-host disease (GVHD) has been evaluated in several randomized control trials, but the results show some discrepancies. Therefore, we performed a systematic review and meta-analysis covering the latest RCTs including six trials (total 845 patients). The incidence of acute and chronic GVHD was significantly lower in the ATG arms (risk ratio, 0.75 and 0.54, respectively). No significant differences were found regarding overall survival, the incidence of relapse, and non-relapse mortality; however, the incidence of cytomegalovirus and Epstein-Barr virus reactivation increased (risk ratio, 1.25 and 1.33), and neutrophil engraftment was significantly delayed (median, 2.66 days). In conclusion, rabbit ATG should be beneficial as a GVHD prophylaxis in addition to conventional regimens, with close monitoring of virus reactivation and enough attention to delayed engraftment. Studies comparing the timing and dosage of ATG are essential to determine the suitable prophylactic regimens.

Classical NF-κB pathway is responsible for APOBEC3B expression in cancer cells.

APOBEC3B (A3B) is a DNA cytosine deaminase and catalyzes cytosine deamination, resulting in mutations in genomic DNA. A3B is aberrantly expressed in a variety of cancers and considered to be a source of genomic mutations that contribute to cancer progression and metastasis. However, the mechanisms through which A3B expression is dysregulated in cancer cells are not fully elucidated. Here we report that the classical NF-κB pathway plays a crucial role in the transcriptional regulation of A3B in various cancer cells, including lymphoid malignancies. PMA, a strong activator of PKC, induces A3B at both mRNA and protein levels in cancer cell lines, and specific inhibitors of both PKC and IKK downregulate A3B expression. Using luciferase reporter and EMSA assays, we identify 3 NF-κΒ binding sites in the A3B promoter and reveal that NF-κB p65/p50 and p65/c-Rel heterodimers are important for A3B transcription. These results suggest that the classical NF-κB pathway is responsible for activation of A3B mRNA expression and further imply that inhibition of PKC and IKK might augment cancer treatment by reducing cancer progression and metastasis through downregulation of A3B expression.

A-Raf and C-Raf differentially regulate mechanobiological response of osteoblasts to guide mechanical stress-induced differentiation.

Regulation of osteoblast activity by mechanical stress is important for bone remodeling. However, the precise mechanotransduction mechanism that triggers the anabolic reaction of osteoblasts is largely unknown. In this study, we performed RNA interference (RNAi) screening to identify the signaling molecules upstream of ERK, which was responsible for osteogenesis. Of twenty-two mitogen-activated protein kinase (MAPK) kinase kinases (MAP3Ks), we identified A-Raf and C-Raf as upstream MAP3Ks of the mechanical stretch-activated ERK pathway. Subsequently we screened the mechanosensitive cation channel, and identified P2X7 as an upstream molecule of the ERK pathway. Intriguingly, P2X7 functioned as an upstream activator of A-Raf but not of C-Raf. Furthermore, A-Raf contributed to mechanical stretch-induced osteoblast differentiation. In contrast, C-Raf but not A-Raf protected osteoblasts from mechanical stretch-induced apoptosis. These results suggested that A-Raf and C-Raf were involved in mechanobiological osteogenesis in a distinct way: A-Raf was responsible for osteogenesis while C-Raf for anti-apoptotic protection and promotion.

Hairy Cell Leukemia with Systemic Lymphadenopathy: Detection of BRAF Mutations in Both Lymph Node and Peripheral Blood Specimens.

A 47-year-old woman with pancytopenia, excessive systemic lymphadenopathy and splenomegaly was referred to our hospital. The peripheral blood (PB) smear findings indicated neutropenia with lymphoid cells exhibiting hairy projections, while the histological findings of the cervical lymph node (LN) suggested hairy cell leukemia (HCL). In addition, the BRAF V600E mutation was detected, and the immunoglobulin gene rearrangement patterns were identical in both the cervical LN and PB specimens. Based on these findings, we diagnosed the patient with systemic lymphadenopathy due to HCL. This is the first report of a BRAF mutation detected in both the PB and LN at the onset of HCL.

The expression of Fn14 via mechanical stress-activated JNK contributes to apoptosis induction in osteoblasts.

Bone mass is maintained by the balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. It is well known that adequate mechanical stress is essential for the maintenance of bone mass, whereas excess mechanical stress induces bone resorption. However, it has not been clarified how osteoblasts respond to different magnitudes of mechanical stress. Here we report that large-magnitude (12%) cyclic stretch induced Ca(2+) influx, which activated reactive oxygen species generation in MC3T3-E1 osteoblasts. Reactive oxygen species then activated the ASK1-JNK/p38 pathways. The activated JNK led to transiently enhanced expression of FGF-inducible 14 (Fn14, a member of the TNF receptor superfamily) gene. Cells with enhanced expression of Fn14 subsequently acquired sensitivity to the ligand of Fn14, TNF-related weak inducer of apoptosis, and underwent apoptosis. On the other hand, the ASK1-p38 pathway induced expression of the monocyte chemoattractant protein 3 (MCP-3) gene, which promoted chemotaxis of preosteoclasts. In contrast, the ERK pathway was activated by small-magnitude stretching (1%) and induced expression of two osteogenic genes, collagen Ia (Col1a) and osteopontin (OPN). Moreover, activated JNK suppressed Col1a and OPN induction in large-magnitude mechanical stretch-loaded cells. The enhanced expression of Fn14 and MCP-3 by 12% stretch and the enhanced expression of Col1a and OPN by 1% stretch were also observed in mouse primary osteoblasts. These results suggest that differences in the response of osteoblasts to varying magnitudes of mechanical stress play a key role in switching the mode of bone metabolism between formation and resorption.

Intratracheal and oral administration of SM-276001: a selective TLR7 agonist, leads to antitumor efficacy in primary and metastatic models of cancer.

Topical TLR7 agonists such as imiquimod are highly effective for the treatment of dermatological malignancies; however, their efficacy in the treatment of nondermatological tumors has been less successful. We report that oral administration of the novel TLR7-selective small molecule agonist; SM-276001, leads to the induction of an inflammatory cytokine and chemokine milieu and to the activation of a diverse population of immune effector cells including T and B lymphocytes, NK and NKT cells. Oral administration of SM-276001 leads to the induction of IFNα, TNFα and IL-12p40 and a reduction in tumor burden in the Balb/c syngeneic Renca and CT26 models. Using the OV2944-HM-1 model of ovarian cancer which spontaneously metastasizes to the lungs following subcutaneous implantation, we evaluated the efficacy of intratracheal and oral administration of SM-276001 in an adjuvant setting following surgical resection of the primary tumor. We show that both oral and intratracheal TLR7 therapy can reduce the frequency of pulmonary metastasis, and metastasis to the axillary lymph nodes. These results demonstrate that SM-276001 is a potent selective TLR7 agonist that can induce antitumor immune responses when dosed either intratracheally or orally.

Src, p130Cas, and Mechanotransduction in Cancer Cells.

Anchorage-independent growth is the most significant hallmark of cell transformation, which has an intimate relevance to cancer. Anchorage or adhesion physically links cells to the extracellular matrix and allows the transmission of external mechanical cues to intracellular signaling machineries. Transformation involves acquiring the ability to proliferate without requiring mechanically initiated signal transduction, known as mechanotransduction. A number of signaling and cytoskeletal molecules are located at focal adhesions. Src and its related proteins, including p130Cas, localize to adhesion sites, where their functions can be mechanically regulated. In addition, the aberrant activation and expression of Src and p130Cas are linked to transformation and malignancy both in vitro and in vivo. These findings shed light on the importance of mechanotransduction in tumorigenesis and the regulation of cancer progression and also provide insights into the mechanical aspects of cancer signaling.

Risk factors for and prognosis of hemorrhagic cystitis after allogeneic stem cell transplantation: retrospective analysis in a single institution.

Hemorrhagic cystitis (HC) is a major complication after allogeneic stem cell transplantation (allo-SCT) and can be life threatening. To analyze risk factors and prognosis, we retrospectively reviewed 249 cases receiving allo-SCT in our institution. Median age was 47 years (13-72 years). Disease status at SCT was progressive in 73 cases. Conditioning was myeloablative (MAC) in 146 cases. Acute graft-versus-host disease (aGVHD) grade II-IV treated with prednisolone occurred in 82 cases, and cytomegalovirus (CMV) was reactivated in 91 cases. HC was reported in 47 cases at a median of 35 days (7-469 days) after SCT, and 34 (72.3%) cases recovered after a median of 19.5 days (2-252 days). In univariate analysis, the identified risk factors for HC included age over 45 years, progressive disease status, MAC, aGVHD treated with prednisolone, and CMV reactivation. In multivariate analysis, older age, MAC, and CMV remained independent predictors (hazard ratios: 2.35, 3.50, and 2.87). In patients with severe HC, percentage recovery was lower (3 in 13 cases; 23.1%) and the median duration was longer (54 days) than in those with moderate HC (31 in 36 cases; 86.1%, 17 days, P < 0.01). Treatment-related mortality was also higher (59.1%, P = 0.03) and overall survival was poorer (16.7%, P < 0.01) at 1 year after SCT. Prospective studies should be started considering prophylactic antiviral administration in high-risk patients such as those identified in this study.

TGF-ß-activated kinase 1 mediates mechanical stress-induced IL-6 expression in osteoblasts.

Mechanical stress plays a key role in bone remodeling. Previous studies showed that loading of mechanical stretch induces a rapid Ca(2+) influx and subsequent activation of stress-activated protein kinase pathways in osteoblasts. However, the activation mechanism and its significance in bone remodeling have not been fully elucidated. Here we show that TAK1 MAPKKK was activated by cyclic stretch loading of MC3T3-E1 cells. Knockdown of TAK1 attenuated the stretch-induced activation of JNK, p38, and NF-κB. Extracellular (EGTA) or intracellular (BAPTA/AM) Ca(2+) chelator prevented the stretch-induced activation of TAK1. Activation of TAK1 and its associated downstream signaling pathways were also suppressed by CaMKII inhibitors (KN-93 and KN-62). Furthermore, TAK1-mediated downstream pathways cooperatively induced the expression of IL-6 mRNA in the stretched MC3T3-E1 cells. We also confirmed that TAK1 mediates cyclic stretch-induced IL-6 protein synthesis in the cells using immunoblotting and ELISA. Finally, stretch loading of murine primary osteoblasts induced the expression of IL-6 mRNA via TAK1. Collectively, these data suggest that stretch-dependent Ca(2+) influx activates TAK1 via CaMKII, leading to the enhanced expression of IL-6 through JNK, p38, and NF-κB pathways in osteoblasts.

Spontaneous rupture of liver plasmacytoma mimicking hepatocellular carcinoma.

Extramedullary plasmacytoma of the liver is rare. Here, we report a case presenting with rupture of extramedullary plasmacytoma of the liver. She had a past history of multiple myeloma with IgA lambda type. Her serum was positive for hepatitis C virus infection and exhibited elevated levels of serum protein induced by vitamin K absence or antagonist-II. She was initially diagnosed as rupture of hepatocellular carcinoma (HCC) and then treated with transarterial chemoembolization (TACE) since bloody ascites and formation of hematoma were seen around hyper-vascular liver tumors on computed tomography. However, the clinical course of this case after TACE was atypical for HCC rupture, as shown by the development of a huge intra-abdominal abscess extending from the liver tumor. Immuno-histochemical analysis of the tumor biopsy specimen revealed massive infiltration of plasma cells expressing IgA and lambda chain. To our knowledge, this is the first case of rupture of extramedullary liver plasmacytoma.

Intracoronary transplantation of non-expanded peripheral blood-derived mononuclear cells promotes improvement of cardiac function in patients with acute myocardial infarction.

BACKGROUND: Transplantation of non-expanded peripheral blood mononuclear cells (PBMNCs) enhances neovessel formation in ischemic myocardium and limbs by releasing angiogenic factors. This study was designed to examine whether intracoronary transplantation of PBMNCs improves cardiac function after acute myocardial infarction (AMI). METHODS AND RESULTS: After successful percutaneous coronary intervention (PCI) for a ST-elevation AMI with occlusion of proximal left anterior descending coronary artery within 24 h, patients received an intracoronary infusion of PBMNCs within 5 days after PCI (PBMNC group). PBMNCs were obtained from patients by COBE spectra-apheresis and concentrated to 10 ml, 3.3 ml of which was infused via over-the-wire catheter. The global left ventricular ejection fraction (LVEF) change from baseline to 6 months followup in th ePBMNC group that underwent standard PCI for similar AMI [corrected]. The primary endpoint was the global left ventricular ejection fraction (LVEF) change from baseline to 6 months' follow-up. The data showed that the absolute increase in LVEF was 7.4% in the control group and 13.4% (p=0.037 vs control) in the PBMNC group. Cell therapy resulted in a greater tendency of DeltaRegional ejection fraction (EF) or significant improvement in the wall motion score index and Tc-99m-tetrofosmin perfusion defect score associated with the infarct area, compared with controls. Moreover, intracoronary administration of PBMNCs did not exacerbate either left ventricular (LV) end-diastolic and end-systolic volume expansion or high-risk arrhythmia, without any adverse clinical events. CONCLUSION: Intracoronary infusion of non-expanded PBMNCs promotes improvement of LV systolic function. This less invasive and more feasible approach to collecting endothelial progenitor cells may provide a novel therapeutic option for improving cardiac function after AMI.