Keita Kirito

FOXO3A as a key molecule for all-trans retinoic acid–induced granulocytic differentiation and apoptosis in acute promyelocytic leukemia

All-trans retinoic acid (ATRA) induces granulocytic differentiation and apoptosis in acute promyelocytic leukemia (APL) cells, although the detailed mechanisms are not fully understood. We investigated ATRA-induced cellular responses mediated by the transcription factor FOXO3A in APL cells. FOXO3A was constitutively phosphorylated and localized in the cytoplasm in both APL-derived NB4 cells and primary APL cells. Upon treating the cells with ATRA, FOXO3A phosphorylation was reduced and FOXO3A translocated into the nucleus. In addition, the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a target molecule for FOXO3A, was increased at the transcriptional and protein levels. As expected, transfection of a short hairpin RNA (shRNA) oligonucleotide specific for FOXO3A significantly inhibited ATRA-induced granulocytic differentiation and apoptosis in NB4 cells. In NB4-derived ATRA-resistant NB4/RA cells, neither FOXO3A nuclear localization nor subsequent TRAIL induction was observed after ATRA treatment. Furthermore, forced expression of active FOXO3A in the nucleus induced TRAIL production and apoptosis in NB4/RA cells. We conclude that activation of FOXO3A is an essential event for ATRA-induced cellular responses in NB4 cells. FOXO3A is a promising target for therapeutic approaches to overcome ATRA resistance in APL.

Romiplostim for the treatment of chronic immune thrombocytopenia in adult Japanese patients: a double-blind, randomized Phase III clinical trial

The efficacy and safety of romiplostim, a thrombopoietin-mimetic peptibody, were evaluated in a double-blind, placebo-controlled, randomized trial of Japanese patients with chronic immune thrombocytopenia (ITP). Thirty-four ITP patients received romiplostim (nxa0=xa022) or placebo (nxa0=xa012) for 12xa0weeks, with a starting romiplostim dose of 3xa0μg/kg weekly. The primary end point was the number of weeks with platelet response, defined as a platelet count ≥50xa0×xa0109/L (not including the 4xa0weeks after rescue medication administration). Patients received a median of 4 (range 1–19) prior ITP therapies including splenectomy in 44%. On study, 68% also received concomitant ITP therapy. Weekly responses occurred for a median of 11xa0weeks with romiplostim as compared to 0xa0weeks with placebo (pxa0<xa00.0001). Most romiplostim-treated patients (95%) achieved platelet responses; two showed extended responses after the treatment period. The use of rescue medication was required in 9% of romiplostim-treated patients as compared with 17% of placebo-treated patients. Both treatment groups had similar incidences of adverse events (91% romiplostim, 92% placebo). Adverse events that occurred more frequently (>10%) in romiplostim-treated patients included nasopharyngitis, headache, peripheral edema, back pain, and extremity pain. In conclusion, romiplostim significantly increased and maintained platelet counts and was well tolerated in Japanese patients with ITP.

Ruxolitinib versus best available therapy in patients with Polycythemia Vera: 80 Week follow up from the RESPONSE trial

RESPONSE is an open-label phase 3 study evaluating the Janus kinase 1/Janus kinase 2 inhibitor ruxolitinib versus best available therapy for efficacy/safety in hydroxyurea-resistant or intolerant patients with polycythemia vera. This preplanned analysis occurred when all patients completed the Week 80 visit or discontinued. Objectives included evaluating the durability of the primary response (Week 32 phlebotomy-independent hematocrit control plus ≥35% spleen volume reduction), its components, and that of complete hematologic remission; and long-term safety. Median exposure was 111 weeks; 91/110 (82.7%) patients randomized to ruxolitinib remained on treatment. No patients continued best available therapy (98/112 [87.5%] crossed over to ruxolitinib, most at/soon after Week 32). At Week 32, primary response was achieved by 22.7% vs. 0.9% of patients randomized to ruxolitinib and best available therapy, respectively (hematocrit control, 60.0% vs. 18.8%; spleen response, 40.0% vs. 0.9%). The probability of maintaining primary and hematocrit responses for ≥80 weeks was 92% and 89%, respectively; 43/44 spleen responses were maintained until Week 80. Complete hematologic remission at Week 32 was achieved in 23.6% of ruxolitinib-randomized patients; the probability of maintaining complete hematologic remission for ≥80 weeks was 69%. Among ruxolitinib crossover patients, 79.2% were not phlebotomized, and 18.8% achieved a ≥35% reduction from baseline in spleen volume after 32 weeks of treatment. New or worsening hematologic laboratory abnormalities in ruxolitinib-treated patients were primarily grade 1/2 decreases in hemoglobin, lymphocytes, and platelets. The thromboembolic event rate per 100 patient-years was 1.8 with randomized ruxolitinib treatment vs. 8.2 with best available therapy. These data support ruxolitinib as an effective long-term treatment option for hydroxyurea-resistant or intolerant patients with polycythemia vera. This trial was registered at clinicaltrials.gov identifier: 01243944.

Recurrent CDC25C mutations drive malignant transformation in FPD/AML

Familial platelet disorder (FPD) with predisposition to acute myelogenous leukaemia (AML) is characterized by platelet defects with a propensity for the development of haematological malignancies. Its molecular pathogenesis is poorly understood, except for the role of germline RUNX1 mutations. Here we show that CDC25C mutations are frequently found in FPD/AML patients (53%). Mutated CDC25C disrupts the G2/M checkpoint and promotes cell cycle progression even in the presence of DNA damage, suggesting a critical role for CDC25C in malignant transformation in FPD/AML. The predicted hierarchical architecture shows that CDC25C mutations define a founding pre-leukaemic clone, followed by stepwise acquisition of subclonal mutations that contribute to leukaemia progression. In three of seven individuals with CDC25C mutations, GATA2 is the target of subsequent mutation. Thus, CDC25C is a novel gene target identified in haematological malignancies. CDC25C is also useful as a clinical biomarker that predicts progression of FPD/AML in the early stage.

Inhibition of hypoxia-inducible factor-1 function enhances the sensitivity of multiple myeloma cells to melphalan

Abnormal activation of hypoxia-inducible factor-1 (HIF-1), one of the most important transcription factors for the adaptation of cells to hypoxia, is frequently observed in numerous types of solid tumors. Dysregulation of HIF-1 induces tumor angiogenesis and enhances the expression of anti-apoptotic proteins and glycolysis-associated enzymes in cancer cells, which in turn leads to the promotion of tumor growth. In the present study, we examined the pathophysiologic role of HIF-1 in multiple myeloma. Furthermore, we explored the possibility that HIF-1 may be a molecular target for myeloma therapy. We identified constitutive expression of the hypoxia-inducible factor-1 α (HIF-1α)-subunit in established myeloma cell lines and in primary myeloma cells. Treatment with insulin-like growth factor-1 (IGF-1) significantly increased HIF-1α expression through activation of the AKT and mitogen-activated protein kinase signaling pathways. Inhibition of HIF-1 function either by echinomycin, a specific HIF-1 inhibitor, or a siRNA against HIF-1α resulted in enhanced sensitivity to melphalan in myeloma cells. This inhibition of HIF-1 also reversed the protective effect of IGF-1 on melphalan-induced apoptosis. Inhibition of HIF-1 drastically reduced both basal and IGF-1–induced expression of survivin, one of the most important anti-apoptotic proteins in myeloma cells. We conclude that HIF-1 inhibition may be an attractive therapeutic strategy for multiple myeloma. [Mol Cancer Ther 2009;8(8):2329–38]

HIF-1 prevents the overproduction of mitochondrial ROS after cytokine stimulation through induction of PDK-1

Excessive reactive oxygen species (ROS) are toxic to hematopoietic cells. The majority of cellular ROS are derived from mitochondria and glucose metabolism, and cytokines stimulate this process. During hypoxia, hypoxia inducible factor-1 (HIF-1) attenuates hypoxia-induced mitochondrial ROS production through the induction of pyruvate dehydrogenase kinase-1 (PDK-1). Previously, we found that thrombopoietin (TPO) induces the generation of mitochondrial ROS. Interestingly, the TPO-induced production of mitochondrial ROS promotes the activation of HIF-1. Based on these findings, we speculated that TPO-activated HIF-1 functions as a feedback mechanism to block the overproduction of ROS following TPO stimulation. We found that TPO induces the expression of PDK-1 in a TPO-dependent cell line, UT-7/TPO, in a HIF-1-dependent manner. Inhibition of either HIF-1 or PDK-1 resulted in the increased production of ROS following TPO stimulation. Our observations suggest that HIF-1 functions as a ROS sensor to prevent the overproduction of mitochondrial ROS following cytokine stimulation.

Thrombopoietin (TPO) regulates HIF-1α levels through generation of mitochondrial reactive oxygen species

Hypoxia inducible factor (HIF)-1 is a master transcriptional regulator mediating the cellular adaptation to hypoxia. In addition, HIF-1 is also vital for the development of hematopoietic stem cells (HSCs). In a previous study we found that thrombopoietin (TPO), an important and non-redundant cytokine for HSC maintenance and expansion, induces HIF-1α expression in HSCs by enhancing the stability of HIF-1α under normoxic conditions. However, the molecular mechanisms of these effects are not yet fully understood. In this study, we explored the mechanisms and found that TPO-induced mitochondrial reactive oxygen species (ROS) played a crucial role in stabilization of HIF-1. Both ROS scavengers and inhibitors of mitochondrial electron transport completely blocked HIF-1α induction by TPO in UT-7/TPO cells and in primary immature mouse bone marrow cells. We also found that TPO-induced HIF-1α induction was tightly coupled with glucose metabolism. Inhibition of glucose transporter or glycolytic enzyme blocked HIF-1α elevation of TPO. These results indicate that TPO induces HIF-1α expression in a manner very similar to that of hypoxia.

NF-κB mediates aberrant activation of HIF-1 in malignant lymphoma

OBJECTIVEnThe goal of this study was to explore the molecular mechanisms of aberrant hypoxia inducible factor-1 (HIF-1) activation in lymphoma cells.nnnMATERIALS AND METHODSnWe analyzed the expression of the α subunit of HIF-1 in three lymphoma cell lines and in normal CD19-positive B cells by Western blotting. To investigate the role of nuclear factor (NF)-κB in abnormal HIF-1α expression in lymphoma cells, we performed a reporter assay using HIF-1α promoter constructs that contained or lacked an NF-κB binding site. We also used a chromatin immunoprecipitation assay to assess whether NF-κB binds the HIF-1α promoter. In addition, we took advantage of NF-κB inhibitors. To analyze the function of HIF-1 in lymphoma cells, we established stable HIF-1α knockdown cells using short-hairpin RNA.nnnRESULTSnMalignant lymphoma cells, but not normal B cells, demonstrated constitutive expression of HIF-1α. Inhibitors of NF-κB, however, drastically suppressed this HIF-1α expression at both the messenger RNA and protein levels. Furthermore, we found that exposure of lymphoma cells to ionizing radiation clearly induced NF-κB activation and increased HIF-1α expression. Suppressing HIF-1α expression by short-hairpin RNA increased the sensitivity of lymphoma cells to ionizing radiation-induced cell death. In searching for downstream targets of the NF-κB/HIF-1 axis, we identified survivin, a member of the IAP family of anti-apoptotic proteins.nnnCONCLUSIONSnWe found that aberrant activation of HIF-1 in malignant lymphoma cells was mediated, at least in part, by NF-κB activity. Our observations suggest that HIF-1 inhibition may be an effective strategy to improve the outcomes of lymphoma patients treated with radiation.

An open-label extension study evaluating the safety and efficacy of romiplostim for up to 3.5 years in thrombocytopenic Japanese patients with immune thrombocytopenic purpura (ITP)

Long-term use of the thrombopoietin mimetic romiplostim was examined in Japanese patients with chronic immune thrombocytopenic purpura (ITP) in this open-label extension. The starting dose of romiplostim was the previous trial dose or 3xa0μg/kg/week, which was titrated up to 10xa0μg/kg/week to maintain platelet counts between 50 and 200xa0×xa0109/L. As of April 2010, 44 patients had enrolled; 71xa0% women, median age 55.5xa0years, with five patients discontinuing romiplostim due to patient request (2), administrative decision (2), or not achieving study-defined platelet response (1). Median treatment duration was 100xa0weeks; median average weekly dose was 3.8xa0μg/kg. Twenty-eight patients (64xa0%) self-injected romiplostim. The most frequent adverse events were nasopharyngitis and headache. Nine patients (20xa0%) had a total of 14 serious adverse events (0.31/100 patient-weeks); of these, only oral hemorrhage was considered treatment related. Fifty hemorrhagic adverse events were reported in 20 patients (46xa0%) (1.12/100 patient-weeks). Ninety-six percent of patients had a platelet response (doubling of baseline platelet count and platelet count ≥50xa0×xa0109/L). Of the 25 patients receiving concurrent ITP therapy at baseline, all reduced or discontinued the therapy. Eight patients (18xa0%) received rescue medications. Administration of up to 3.5xa0years of romiplostim increased platelet counts and was well tolerated in Japanese patients with chronic ITP.

High prevalence of the MYD88 mutation in testicular lymphoma: Immunohistochemical and genetic analyses

The activating mutation of MYD88 has been identified in diffuse large B‐cell lymphoma (DLBCL). We investigated the mutational status and both the gene amplification and protein expression of MYD88 in 23 cases of testicular DLBCL. To detect the MYD88 mutations, we employed the allele‐specific PCR and Sanger sequencing. MYD88 gene amplification and protein expression were analyzed by quantitative PCR and by immunohistochemistry, respectively. There were 17 cases of primary testicular DLBCL: 94% (16/17) exhibited a non‐Germinal center B‐cell (non‐GCB) subtype, 82% (14/17) showed the MYD88u2005L265P, and 65% (11/17) had intense expression of MYD88. When compared with normal lymph nodes, the MYD88 is significantly amplified in primary testicular DLBCL. However, the amplification status showed no correlation with its mutational status or protein expression. Moreover, neither the MYD88 mutational status nor the expression pattern affected overall survival. Six cases were secondary testicular DLBCL with an 83% (5/6) and an 80% (4/5) incidence of the non‐GCB subtype and of the MYD88u2005L265P, respectively. In conclusion, we demonstrated a high prevalence of the non‐GCB subtype and the common MYD88u2005L265P in both primary and secondary testicular DLBCL. Our data suggest that the MYD88 mutation is a fairly consistent genetic feature in testicular DLBCL.