Yuki Kagoya

Positive feedback between NF-κB and TNF-α promotes leukemia-initiating cell capacity

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy that originates from leukemia-initiating cells (LICs). The identification of common mechanisms underlying LIC development will be important in establishing broadly effective therapeutics for AML. Constitutive NF-κB pathway activation has been reported in different types of AML; however, the mechanism of NF-κB activation and its importance in leukemia progression are poorly understood. Here, we analyzed myeloid leukemia mouse models to assess NF-κB activity in AML LICs. We found that LICs, but not normal hematopoietic stem cells or non-LIC fractions within leukemia cells, exhibited constitutive NF-κB activity. This activity was maintained through autocrine TNF-α secretion, which formed an NF-κB/TNF-α positive feedback loop. LICs had increased levels of active proteasome machinery, which promoted the degradation of IκBα and further supported NF-κB activity. Pharmacological inhibition of the proteasome complex markedly suppressed leukemia progression in vivo. Conversely, enhanced activation of NF-κB signaling expanded LIC frequency within leukemia cell populations. We also demonstrated a strong correlation between NF-κB activity and TNF-α secretion in human AML samples. Our findings indicate that NF-κB/TNF-α signaling in LICs contributes to leukemia progression and provide a widely applicable approach for targeting LICs.

BAALC potentiates oncogenic ERK pathway through interactions with MEKK1 and KLF4

Although high brain and acute leukemia, cytoplasmic (BAALC) expression is a well-characterized poor prognostic factor in acute myeloid leukemia (AML), neither the exact mechanisms by which BAALC drives leukemogenesis and drug resistance nor therapeutic approaches against BAALC-high AML have been properly elucidated. In this study, we found that BAALC induced cell-cycle progression of leukemia cells by sustaining extracellular signal-regulated kinase (ERK) activity through an interaction with a scaffold protein MEK kinase-1 (MEKK1), which inhibits the interaction between ERK and MAP kinase phosphatase 3 (MKP3/DUSP6). BAALC conferred chemoresistance in AML cells by upregulating ATP-binding cassette proteins in an ERK-dependent manner, which can be therapeutically targeted by MEK inhibitor. We also demonstrated that BAALC blocks ERK-mediated monocytic differentiation of AML cells by trapping Krüppel-like factor 4 (KLF4) in the cytoplasm and inhibiting its function in the nucleus. Consequently, MEK inhibition therapy synergizes with KLF4 induction and is highly effective against BAALC-high AML cells both in vitro and in vivo. Our data provide a molecular basis for the role of BAALC in regulating proliferation and differentiation of AML cells and highlight the unique dual function of BAALC as an attractive therapeutic target against BAALC-high AML.

Thalidomide maintenance therapy for patients with multiple myeloma: Meta-analysis

We performed a meta-analysis of randomized controlled trials comparing thalidomide maintenance with other regimens after induction chemotherapy for multiple myeloma. Overall, 6 trials including 2786 patients were identified. Patients treated with thalidomide maintenance had marginally better overall survival (hazard ratio HR 0.83, P=0.07). The improvement was especially prominent in a subgroup of studies using corticosteroids with thalidomide (HR 0.70, P=0.02). Thalidomide improved progression-free survival (HR 0.65, P<0.01), but had more frequent venous thrombosis (risk difference 0.024, P<0.05) and peripheral neuropathy (risk difference 0.072, P<0.01). These results suggest that thalidomide maintenance with corticosteroids is effective in prolonging survival for multiple myeloma.

BET bromodomain inhibition enhances T cell persistence and function in adoptive immunotherapy models

Adoptive immunotherapy is a potentially curative therapeutic approach for patients with advanced cancer. However, the in vitro expansion of antitumor T cells prior to infusion inevitably incurs differentiation towards effector T cells and impairs persistence following adoptive transfer. Epigenetic profiles regulate gene expression of key transcription factors over the course of immune cell differentiation, proliferation, and function. Using comprehensive screening of chemical probes with defined epigenetic targets, we found that JQ1, an inhibitor of bromodomain and extra-terminal motif (BET) proteins, maintained CD8+ T cells with functional properties of stem cell-like and central memory T cells. Mechanistically, the BET protein BRD4 directly regulated expression of the transcription factor BATF in CD8+ T cells, which was associated with differentiation of T cells into an effector memory phenotype. JQ1-treated T cells showed enhanced persistence and antitumor effects in murine T cell receptor and chimeric antigen receptor gene therapy models. Furthermore, we found that histone acetyltransferase p300 supported the recruitment of BRD4 to the BATF promoter region, and p300 inhibition similarly augmented antitumor effects of the adoptively transferred T cells. These results demonstrate that targeting the BRD4-p300 signaling cascade supports the generation of superior antitumor T cell grafts for adoptive immunotherapy.

Inhibition of histone methyltransferase EZH2 depletes leukemia stem cell of mixed lineage leukemia fusion leukemia through upregulation of p16

Leukemia stem cells (LSC) are resistant to conventional chemotherapy and persistent LSC after chemotherapy are supposed to be a major cause of relapse. However, information on genetic or epigenetic regulation of stem cell properties is still limited and LSC‐targeted drugs have scarcely been identified. Epigenetic regulators are associated with many cellular processes including maintenance of stem cells. Of note are polycomb group proteins, because they potentially control stemness, and can be pharmacologically targeted by a selective inhibitor (DZNep). Therefore, we investigated the therapeutic potential of EZH2 inhibition in mixed lineage leukemia (MLL) fusion leukemia. Intriguingly, EZH2 inhibition by DZNep or shRNA not only suppressed MLL fusion leukemia proliferation but also reduced leukemia initiating cells (LIC) frequency. Expression analysis suggested that p16 upregulation was responsible for LICs reduction. Knockdown of p16 canceled the survival advantage of mice treated with DZNep. Chromatin immunoprecipitation assays demonstrated that EZH2 was highly enriched around the transcription‐start‐site of p16, together with H3K27 methylation marks in MLL/ENL and Hoxa9/Meis1 transduced cells but not in E2A/HLF transduced cells. Although high expression of Hoxa9 in MLL fusion leukemia is supposed to be responsible for the recruitment of EZH2, our data also suggest that there may be some other mechanisms independent of Hoxa9 activation to suppress p16 expression, because expression levels of Hoxa9 and p16 were not inversely related between MLL/ENL and Hoxa9/Meis1 transduced cells. In summary, our findings show that EZH2 is a potential therapeutic target of MLL fusion leukemia stem cells.

Targeted gene correction of RUNX1 in induced pluripotent stem cells derived from familial platelet disorder with propensity to myeloid malignancy restores normal megakaryopoiesis

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease associated with a germline mutation in the RUNX1 gene and is characterized by thrombocytopenia and an increased risk of developing myeloid malignancies. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a patient with FPD/AML possessing a nonsense mutation R174X in the RUNX1 gene. Consistent with the clinical characteristics of the disease, FPD iPSC-derived hematopoietic progenitor cells were significantly impaired in undergoing megakaryocytic differentiation and subsequent maturation, as determined by colony-forming cell assay and surface marker analysis. Notably, when we corrected the RUNX1 mutation using transcription activator-like effector nucleases in conjunction with a donor plasmid containing normal RUNX1 cDNA sequences, megakaryopoiesis and subsequent maturation were restored in FPD iPSC-derived hematopoietic cells. These findings clearly indicate that the RUNX1 mutation is robustly associated with thrombocytopenia in patients with FPD/AML, and transcription activator-like effector nuclease-mediated gene correction in iPSCs generated from patient-derived cells could provide a promising clinical application for treatment of the disease.

JAK2V617F+ myeloproliferative neoplasm clones evoke paracrine DNA damage to adjacent normal cells through secretion of lipocalin-2.

Genetic instability is strongly involved in cancer development and progression, and elucidating the mechanism could lead to novel therapeutics for preventing carcinogenesis. Philadelphia-negative myeloproliferative neoplasms (MPNs) are clonal myeloid disorders with a high prevalence of JAK2V617F mutation, and transformation to acute myeloid leukemia through accumulation of additional mutations is a major complication in MPNs. Here, we showed that JAK2V617F(+) cells conferred paracrine DNA damage to neighboring normal cells as well as to themselves through increased reactive oxygen species (ROS). We screened candidate factors responsible for the effect and found that lipocalin-2 (Lcn2) is overexpressed in JAK2V617F(+) cells and that short hairpin RNA-mediated knockdown of Lcn2 significantly alleviated the paracrine DNA damage. Normal hematopoietic cells showed elevated ROS levels through increased intracellular iron levels when treated with lipocalin-2, which led to p53 pathway activation, increased apoptosis, and decreased cellular proliferation. In contrast, JAK2V617F(+) cells did not suffer from lipocalin-2-induced growth suppression resulting from attenuated p53 pathway activation, which conferred a relative growth advantage to JAK2V617F(+) clones. In summary, we demonstrated that JAK2V617F-harboring cells cause paracrine DNA damage accumulation through secretion of lipocalin-2, which gives proliferative advantage to themselves and an increased risk for leukemic transformation to both JAK2V617F(+) and JAK2V617F(-) clones.

Pretransplant Predictors and Posttransplant Sequels of Acute Kidney Injury after Allogeneic Stem Cell Transplantation

Acute kidney injury (AKI) is a common complication after allogeneic stem cell transplantation (SCT). Although various risk factors for AKI have been reported, the influence of pretransplant comorbidity on the incidence of AKI has not been well investigated. We performed a retrospective analysis of 207 consecutive patients undergoing myeloablative or nonmyeloablative SCT between 2001 and 2009, using the hematopoietic cell transplantation-specific comorbidity index (HCT-CI) as a representative of pretransplant comorbidities. According to Risk, Injury, Failure, Loss, and End-stage kidney disease (RIFLE) criteria, 158 patients (76.3%) developed AKI, and 92 patients (44.4%) developed severe AKI (RIFLE class I or class F) within 100 days after SCT. The cumulative incidence of severe AKI within 100 days in patients with an HCT-CI score 0, 1-2, and ≥3 was 21.3%, 48.8%, and 73.9%, respectively. In multivariate analysis, the HCT-CI was independently associated with severe AKI (HCT-CI 1-2: adjusted hazard ratio [HR] 2.42, P < .01; HCT-CI ≥3: adjusted HR 4.69, P < .01). In a landmark analysis, patients with severe AKI had a lower 3-year overall survival (OS) (39.3% versus 61.4%, P < .01), and a higher 3-year nonrelapse mortality (NRM) (40.8% versus 5.6%, P < .01) than those without AKI. Multivariate analysis showed that severe AKI was a significant risk factor for worse OS (HR: 2.10, P = .01) and NRM (HR: 6.15, P < .01). Thus, it is important to assess the HCT-CI to predict the incidence of AKI, which is a strong indicator of worse prognosis after SCT.

Hyperlipidemia after allogeneic stem cell transplantation: prevalence, risk factors, and impact on prognosis

Hyperlipidemia is one of the late complications after allogeneic stem cell transplantation (SCT). Although intrahepatic cholestasis caused by chronic graft‐versus‐host disease (GVHD) or calcineurin inhibitors has been considered as possible etiologies, its prevalence, risk factors, and impact on prognosis have not been investigated well. We performed a retrospective analysis of 194 patients who underwent allogeneic SCT between 1995 and 2008 in our institute and survived more than 100 d after SCT. Overall, 83 (42.8%) and 99 (50.8%) patients developed hypercholesterolemia (≥240 mg/dL) and hypertriglyceridemia (≥200 mg/dL), respectively. In multivariate analysis, the development of chronic GVHD (hazard ratio [HR] 2.04, p < 0.05) and steroid use (HR 2.24, p < 0.01) were independently associated with hypercholesterolemia, while administration of calcineurin inhibitors was not. As for the prognostic impact, multivariate analysis showed that the patients with hypercholesterolemia had a tendency of lower rate of relapse (HR: 0.44, p = 0.07). There was no difference in non‐relapse mortality or overall survival between the groups. In conclusion, the development of hypercholesterolemia is regarded as one of the symptoms accompanied with chronic GVHD and might indicate a better control of the primary disease.

Recurrent pericardial effusion after treatment for primary effusion lymphoma-like lymphoma: an autopsied case

Dear Editor, Primary effusion lymphoma (PEL) is a rare type of nonHodgkins lymphoma characterized by lymphomatous effusion confined to the serous body cavities such as pleura, pericardium, or peritoneum [1]. Although it is usually associated with human herpes virus 8 (HHV-8) and human immunodeficiency virus (HIV) infection, there are several reports of HHV-8-unrelated, HIV-negative PELlike lymphoma [2]. The prognosis of PEL is generally poor, and re-accumulation of the fluid is an ominous sign of relapse [3]. Here, we present an autopsied case of PEL-like lymphoma in the pericardium, which was unique in that the effusion accumulated repeatedly in spite of the disappearance of lymphoma cells. A 74-year-old male was admitted to our hospital with complaints of dyspnea on exertion and lower leg edema. Echocardiography revealed a marked pericardial effusion. Pericardiocentesis was performed for relief of his symptoms. Cytology of the fluid demonstrated numerous middleto large-sized atypical lymphoid cells with prominent nucleoli (Fig. 1a). Immunohistochemical analysis for the cell block section of the effusion showed that the lymphoid cells were positive for CD20 with high MIB-1 index, and negative for CD3, CD5, CD10, or HHV-8 latent nuclear antigen (stained using the monoclonal antibody 13B10; Novocastra; Fig. 1b, c). Epstein–Barr virus-encoded RNA in situ hybridization was negative. No other lesion was detected in computed tomography, fluorodeoxyglucose positron emission tomography, and bone marrow biopsy. Serum antibody for HIV or hepatitis C virus was negative. He was diagnosed as having HHV-8-unrelated, HIVnegative PEL-like lymphoma and treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). During the treatment, pericardial effusion did not decrease significantly. Because he had a repeated episode of heart failure due to the marked pericardial effusion, pericardiocentesis was performed after five and seven courses of R-CHOP, but cytology showed no lymphoma cells involvement each time. After seven courses of R-CHOP, he suffered from pneumonia and died. At autopsy, histological examination of the pericardium showed fibrinous pericarditis. No residual lymphoma cells were detected in the pericardial effusion, pericardium, or any other organ. In this case, repeated pericardial effusion was initially considered to be caused by the involvement of residual lymphoma cells. Upon autopsy, however, no lymphoma cells were found in the pericardium, indicating successful eradication of lymphoma by chemotherapy. In contrast to PEL, which is known to have poor prognosis, HHV-8unrelated PEL-like lymphoma can be cured by systemic chemotherapy [4, 5]. On the other hand, pericardial involvement of lymphoma causes pericarditis, which sometimes continues even after the treatment and leads to posttreatment morbidity [6]. In this case, persistent inflammation of the pericardium caused decreased absorption rate of the membrane, leading to chronic accumulation of the fluid. Y. Kagoya : T. Takahashi :M. Ichikawa :A. Hangaishi : M. Kurokawa (*) Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan e-mail: kurokawa-tky@umin.ac.jp