Human herpesvirus 6 reactivation on the 30th day after allogeneic hematopoietic stem cell transplantation can predict grade 2-4 acute graft-versus-host disease.

BACKGROUND: Viral infections and their occult reactivation occasionally cause not only organ damage, but also exacerbation of acute graft-versus-host disease (aGVHD), which may increase transplantation-related mortality synergistically. To determine correlations between viral reactivation and transplantation-related complications, we performed various viral screening tests on the 30th day after allogeneic hematopoietic stem cell transplantation (HSCT), and assessed the clinical implications. PATIENTS AND METHODS: Between August 2007 and January 2013, 49 patients (37 men, 12 women) underwent HSCT in our hospital. The stem cell sources were bone marrow (n = 21), peripheral blood (n = 13), and cord blood (n = 15). The presence of cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpesvirus (HHV) 6, and HHV7 in plasma samples prospectively collected from HSCT recipients on day 30 after HSCT was assayed by quantitative polymerase chain reaction, and the correlations with transplantation-related complications were evaluated. RESULTS: The positivities of CMV, EBV, HHV6, and HHV7 were 44.9%, 22.4%, 53.1%, and 18.3%, respectively. We analyzed transplantation-related complications, and a significant correlation was found only between HHV6 and grade 2-4 aGVHD from day 30 to day 100 (P < 0.001). Using a receiver operating characteristic curve, the area under the curve was calculated as 0.86 (95% confidence interval [CI], 0.74-0.98) between the viral load (VL) of HHV6 and grade 2-4 aGVHD. The sensitivity and specificity were 79% and 93%, respectively, when a cutoff value of 87 copies/mL was used. In multivariate analysis using the Fine and Gray proportional hazards model, the clinically determined high-risk patients (P = 0.004; hazard ratio [HR], 3.69; 95% CI, 1.52-9.00) and the positivity of HHV6 (P < 0.001; HR, 9.957; 95% CI, 2.68-37.06) were extracted as independent risk factors for the cumulative incidence of grade 2-4 aGVHD on or after post-HSCT day 30. The only risk factor extracted for the elevation of HHV6 VL >87 copies/mL was cord blood transplantation (P = 0.0032; odds ratio, 7.10; 95% CI, 1.98-30.00). CONCLUSION: All of the risk factors previously reported to predict severe aGVHD were obtained only during, but not after, HSCT. Our study suggests that the reactivation of HHV6 (≥ 87 copies/mL) at 30 days after HSCT is a possible predictive marker for grade 2-4 aGVHD on or after post-HSCT day 30.

A novel reduced-intensity umbilical cord blood transplantation using a recombinant G-CSF combined with high-dose Ara-C for active myeloid malignancies.

Non-remitting patients with hematologic myeloid malignancies have poor prognosis. To overcome this problem, we investigated the use of reduced-intensity preconditioning umbilical cord blood transplantation (RICBT) combined with recombinant G-CSF (rG-CSF) with high-dose Ara-C, fludarabine, melphalan, and 4 Gy of TBI in a phase I/II study in patients with non-remitting myeloid hematologic malignancies. Thirteen patients were enrolled, including 12 with non-remitting AML and one patient with blastic crisis CML (CML-BC). The patients' median age was 45 years, with a median comorbidity index of 4. All patients received 4/6 serological HLA-antigen matched unrelated umbilical cord blood. All patients were engrafted within 30 days after RICBT (median, 20 days; range, 14-29) and achieved complete remission without prior hematopoiesis. Common grade III non-hematologic toxicities included eight cases of transient mucositis (62%) and six cases of febrile neutropenia (46%). Transplant-related mortality was 7.7%. The 1-year overall survival was 28.6% in cases without post-RICBT treatment and 83.3% in cases with post-RICBT treatment. These data suggest that in active AML and CML-BC, the combination of rG-CSF with high-dose Ara-C and fludarabine/melphalan/4 Gy TBI with a reduced-intensity preconditioning regimen is well tolerated, secures engraftment and has significant anti-leukemia activity. In addition, performing post-RICBT treatment may provide high-quality long-term survival and remission.

BCR-ABL regulates death receptor expression for TNF-related apoptosis-inducing ligand (TRAIL) in Philadelphia chromosome-positive leukemia.

Allogeneic stem cell transplantation (allo-SCT) is a potentially curative therapy for chronic myeloid leukemia and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia, and the graft-vs-leukemia (GVL) effect can eradicate residual leukemia after allo-SCT. Ph(+) leukemia cells frequently express death-inducing receptors (DR4 and DR5) for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is one of the cytotoxic ligands expressed on cytotoxic T cells and natural killer cells mediating the GVL effect. Here we demonstrate that imatinib specifically downregulated DR4 and DR5 expression in cell lines and clinical samples of Ph(+) leukemia. Second-generation tyrosine kinase inhibitors (dasatinib and nilotinib) and short hairpin RNA against bcr-abl also downregulated DR4 and DR5 expression in Ph(+) leukemia cells, and transfection of bcr-abl into a Ph(-) leukemia cell line induced DR4 and DR5 expression, which was abrogated by imatinib treatment. Accordingly, Ph(+) leukemia cells that had been pretreated with imatinib showed resistance to the pro-apoptotic activity of recombinant human soluble TRAIL. These observations demonstrate that BCR-ABL is critically involved in the leukemia-specific expression of DR4 and DR5 and in the susceptibility of Ph(+) leukemia to TRAIL-mediated anti-leukemic activity, providing new insight into the mechanisms of the tumor-specific cytotoxic activities of TRAIL.

Combined effects of novel heat shock protein 90 inhibitor NVP-AUY922 and nilotinib in a random mutagenesis screen.

To overcome imatinib resistance, more potent ABL tyrosine kinase inhibitors (TKIs), such as nilotinib and dasatinib have been developed, with demonstrable preclinical activity against most imatinib-resistant BCR-ABL kinase domain mutations, with the exception of T315I. However, imatinib-resistant patients already harboring mutations have a higher likelihood of developing further mutations under the selective pressure of potent ABL TKIs. NVP-AUY922 (Novartis) is a novel 4,5-diaryloxazole adenosine triphosphate-binding site heat shock protein 90 (HSP90) inhibitor, which has been shown to inhibit the chaperone function of HSP90 and deplete the levels of HSP90 client protein including BCR-ABL. In this study, we investigated the combined effects of AUY922 and nilotinib on random mutagenesis for BCR-ABL mutation (Blood, 109; 5011, 2007). Compared with single agents, combination with AUY922 and nilotinib was more effective at reducing the outgrowth of resistant cell clones. No outgrowth was observed in the presence of 2 µM of nilotinib and 20 nM of AUY922. The observed data from the isobologram indicated the synergistic effect of simultaneous exposure to AUY922 and nilotinib even in BaF3 cells expressing BCR-ABL mutants including T315I. In vivo studies also demonstrated that the combination of AUY922 and nilotinib prolonged the survival of mice transplanted with mixture of BaF3 cells expressing wild-type BCR-ABL and mutant forms. Taken together, this study shows that the combination of AUY922 and nilotinib exhibits a desirable therapeutic index that can reduce the in vivo growth of mutant forms of BCR-ABL-expressing cells.

Identification and functional signature of genes regulated by structurally different ABL kinase inhibitors.

Dasatinib is an ATP-competitive, multi-targeted SRC and ABL kinase inhibitor that can bind BCR-ABL in both the active and inactive conformations. From a clinical standpoint, dasatinib is particularly attractive because it has been shown to induce hematologic and cytogenetic responses in imatinib-resistant chronic myeloid leukemia patients. The fact because the combination of imatinib and dasatinib shows the additive/synergistic growth inhibition on wild-type p210 BCR-ABL-expressing cells, we reasoned that these ABL kinase inhibitors might induce the different molecular pathways. To address this question, we used DNA microarrays to identify genes whose transcription was altered by imatinib and dasatinib. K562 cells were cultured with imatinib or dasatinib for 16 h, and gene expression data were obtained from three independent microarray hybridizations. Almost all of the imatinib- and dasatinib-responsive genes appeared to be similarly increased or decreased in K562 cells; however, small subsets of genes were identified as selectively altered expression by either imatinib or dasatinib. The distinct genes that are selectively modulated by dasatinib are cyclin-dependent kinase 2 (CDK2) and CDK8, which had a maximal reduction of <5-fold in microarray screen. To assess the functional importance of dasatinib regulated genes, we used RNA interference to determine whether reduction of CDK2 and CDK8 affected the growth inhibition. K562 and TF-1BCR-ABL cells, pretreated with CDK2 or CDK8 small interfering RNA, showed additive growth inhibition with imatinib, but not with dasatinib. These findings demonstrate that the additive/synergistic growth inhibition by imatinib and dasatinib may be mediated in part by CDK2 and CDK8.

Telomerase inhibition with a novel G-quadruplex-interactive agent, telomestatin: in vitro and in vivo studies in acute leukemia.

The telomerase complex is responsible for telomere maintenance and represents a promising neoplasia therapeutic target. Recently, we have demonstrated that treatment with a G-quadruplex-interactive agent, telomestatin reproducibly inhibited telomerase activity in the BCR-ABL-positive leukemic cell lines. In the present study, we investigated the mechanisms of apoptosis induced by telomerase inhibition in acute leukemia. We have found the activation of caspase-3 and poly-(ADP-ribose) polymerase in telomestatin-treated U937 cells (PD20) and dominant-negative DN-hTERT-expressing U937 cells (PD25). Activation of p38 mitogen-activated protein (MAP) kinase and MKK3/6 was also found in telomestatin-treated U937 cells (PD20) and dominant-negative DN-hTERT-expressing U937 cells (PD25); however, activation of JNK and ASK1 was not detected in these cells. To examine the effect of p38 MAP kinase inhibition on growth properties and apoptosis in telomerase-inhibited cells, we cultured DN-hTERT-expressing U937 cells with or without SB203580. Dominant-negative-hTERT-expressing U937 cells stopped proliferation on PD25; however, a significant increase in growth rate was observed in the presence of SB203580. Treatment of SB203580 also reduced the induction of apoptosis in DN-hTERT-expressing U937 cells (PD25). These results suggest that p38 MAP kinase has a critical role for the induction of apoptosis in telomerase-inhibited leukemia cells. Further, we evaluated the effect of telomestatin on the growth of U937 cells in xenograft mouse model. Systemic intraperitoneal administration of telomestatin in U937 xenografts decreased tumor telomerase levels and reduced tumor volumes. Tumor tissue from telomestatin-treated animals exhibited marked apoptosis. None of the mice treated with telomestatin displayed any signs of toxicity. Taken together, these results lay the foundations for a program of drug development to achieve the dual aims of efficacy and selectivity in vivo.

A G-quadruplex-interactive agent, telomestatin (SOT-095), induces telomere shortening with apoptosis and enhances chemosensitivity in acute myeloid leukemia.

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is up-regulated in the vast majority of human neoplasias but not in normal somatic tissues. Therefore, the telomerase complex represents a promising universal therapeutic target in cancer. Telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We examined G-quadruplex interactive agent, telomestatin (SOT-095), for its ability to inhibit the proliferation of human leukemia cells, including freshly obtained leukemia cells. Telomere length was determined by either the terminal restriction fragment method or flow-FISH, and apoptosis was assessed by flow cytometry. Moreover, chemosensitivity was examined in telomestatin-treated U937 cells before ultimate telomere shortening. Treatment with telomestatin reproducibly inhibited telomerase activity in U937 and NB4 cells followed by telomere shortening. Enhanced chemosensitivity toward daunorubicin and cytosine-arabinoside was observed in telomestatin-treated U937 cells, before ultimate telomere shortening. Telomere shortening associated with apoptosis by telomestatin was evident in some freshly obtained leukemia cells from acute myeloid leukemia patients, regardless of sub-types of AML and post-myelodysplasia AML. These results suggest that disruption of telomere maintenance by telomestatin limits the cellular lifespan of AML cells, as well. However, in a minority of AML patients apoptosis was not evident, thus indicating that resistant mechanism might exist in some freshly obtained AML cells. Therefore, further investigation of telomestatin as a therapeutic agent is warranted.

Telomerase inhibition enhances apoptosis in human acute leukemia cells: possibility of antitelomerase therapy.

Telomerase is a ribonucleoprotein enzyme that maintains protective structures at the ends of eukaryotic chromosomes. We examined the impact of telomerase inhibition by the dominant-negative human catalytic subunit of telomerase (DN-hTERT) on the biological features of acute leukemia. We introduced vectors encoding dominant- negative (DN)-hTERT, wild-type (WT)-hTERT, or a control vector expressing only a drug-resistant marker into a telomerase-positive human acute lymphoblastic leukemia cell line, HAL-01. Expression of DN-hTERT dramatically inhibited telomerase activity, leading to apoptotic cell death. Mutant telomerase expression also enhanced daunorubicin-induced apoptosis. Nude mice (n=5 per group) received subcutanous implants of HAL-01 cells expressing the control vector or DN-hTERT or WT-hTERT. Implantation of HAL-01 cells expressing control vector (n=5) rapidly produced tumors, whereas implantation of those expressing DN-hTERT (n=5) did not. Thus, telomerase inhibition both growth of HAL-01 cells in vitro and tumorigenic capacity in vivo. Furthermore, the G-quadruplex-interactive telomerase-specific inhibitor, telomestatin, shortened the telomere length and induced apoptosis in freshly isolated primary acute leukemia cells. These results suggest that antitelomerase therapy may be useful in some acute leukemias in combination with antileukemic agents such as daunorubicin.

Telomere dynamics in myelodysplastic syndromes and acute leukemic transformation.

Myelodysplastic syndromes (MDS) are characterized by cytopenias in the blood and dysplastic features in the hematopoietic cells. Although the impact of cytogenetic abnormalities is considerable for prognosis, the exact genetic mechanism of MDS remains undetermined. In this study we assessed cytogenetic changes, microsatellite alterations, and telomere dynamics in order to obtain further insight into the pathogenesis of MDS. Thirty-three percentage of MDS patients and 60% of post-MDS acute leukemia (post-MDS AML) had de novo microsatellite changes. In the MDS phase, however, > 60% of patients showed reduction of telomere lengths without microsatellite changes, indicating that telomere reduction in most MDS patients does not seem to be directly linked to genome instability, or that reduction of telomere length does not induce microsatellite changes in the MDS phase. Some MDS patients had microsatellite changes without telomerase elevation, indicating that genome instability might accumulate during the disease progression in some MDS patients, and this condition (cellular senescence) may be related to ineffective hemopoiesis in MDS patients. In contrast, 40% of post-MDS AML patients had elevated telomerase activity with microsatellite changes, indicating that approximately 40% of patients with post-MDS AML patients had accumulation of genome instability resulting in elevated telomerase activity in an attempt to obtain genetic stability. However, the remaining MDS patients had microsatellite changes without telomerase up-regulation, suggesting that some MDS had genome instability even after leukemic transformation. Most MDS patients with elevated telomerase activity in the AML phase had elevated telomerase activity even in the MDS phase without apparent change in telomere length before and after leukemic transformation. These findings indicate that telomerase activity in the MDS phase may be independent of telomere length, although telomere shortening seems to be related to genomic instability, and this process may be linked to apoptosis of MDS cells.

Successful treatment with cyclosporin A for myelodysplastic syndrome with erythroid hypoplasia associated with T-cell receptor gene rearrangements.

Myelodysplastic syndrome (MDS) with erythroid hypoplasia, a rare form of MDS, has not yet been clearly defined. We report four patients with MDS with erythroid hypoplasia who received immunosuppressive therapy. All were elderly, had severe transfusion-dependent anaemia, morphological evidence of myelodysplasia and a low percentage (3.2-13.6%) of erythroid precursors. Administration of cyclosporin A (CsA) improved their anaemia; all transfusion-dependent patients achieved transfusion-independence. An inverted CD4/8 ratio was seen in three patients who also demonstrated T-cell receptor (TCR)-beta and -gamma gene rearrangements by Southern blotting and clonality by polymerase chain reaction. Treatment with CsA can be an attractive alternative treatment for patients with MDS with erythroid hypoplasia, which may be associated with a clonal abnormality in T cells.

CIS1, a cytokine-inducible SH2 protein, suppresses BCR/ABL-mediated transformation. Involvement of the ubiquitin proteasome pathway.

OBJECTIVE: BCR/ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive leukemia. A general understanding of BCR/ABL signaling events is emerging, but little is known about the endogenous inhibitors of p210 BCR/ABL. The present study focused attention on CIS1, a cytokine-inducible SH2 protein, as a potential physiologic antagonist for BCR/ABL. MATERIALS AND METHODS: The murine hematopoietic cell line NSF/N1.H7 stably transfected with BCR/ABL was compared to the parental counterparts for induction of CIS1 by immunoblotting and immunoprecipitation. Cells were treated with a proteasome inhibitor to examine the effect of a proteasome inhibitor on CIS1 protein expression. To determine the effect of CIS1 on BCR/ABL-mediated transformation, we generated Rat-1 fibroblasts transfected with either a control vector, CIS1, BCR/ABL p210, or CIS1 plus BCR/ABL p210. RESULTS: Three forms of CIS1 with molecular masses of 32, 37, and 47 kDa were detected in BCR/ABL-transformed cells. The 47-kDa protein was a ubiquitinated protein. The proteasome inhibitor increased the formation of complexes between CIS1 and BCR/ABL. Transformation of p210 BCR/ABL was significantly suppressed in cells overexpressing CIS1. CONCLUSION: The results suggest that CIS1 is an endogenous inhibitor of p210 BCR/ABL and is likely to be important in the pathogenesis of Ph-positive leukemia.

Impaired telomere regulation mechanism by TRF1 (telomere-binding protein), but not TRF2 expression, in acute leukemia cells.

Telomere regulation is suggested to be an important mechanism in cellular proliferation and cellular senescence not only in normal diploid cells but also in neoplastic cells, including human leukemia cells. We studied the possible correlation among telomere length, telomerase (a ribonuclear protein that synthesizes the telemeres de novo) activity, hTERT (a catalytic subunit of telomerase) expression, and TRF1 and TRF2 (telomere DNA binding proteins) expression in human acute leukemia cells. The hTERT expression level was strongly associated with telomerase activity (P=0.0001), indicating that the expression level of the catalytic subunit (hTERT) regulates telomerase activity in human acute leukemia cells. TRF1 expression, which is believed to control telomere length, was significantly elevated in patients with acute lymphoblastic leukemia (ALL) (P=0.0232) compared to those in acute myeloid leukemia (AML); TRF1 expression tended to be higher in patients without telomere shortening (P=0.077) and in those with hTERT expression (P=0.055). This indicates that TRF1 may act to monitor telomere length under the condition of up-regulated telomerase activity in some neoplastic cells. In contrast, TRF2 expression in acute leukemia did not show any correlation with telomere parameters in this study. Although the precise regulation mechanism of telomere length is still uncertain, these results may suggest that regulation of telomere length is partially associated with TRF1 expression, whereas dysfunction of TRF1 expression may be speculated in a subset of acute leukemia.

Chronic lymphocytic leukemia complicated by plasmacytoma originating from different clones.

In a woman with chronic lymphocytic leukemia (CLL), a plasmacytoma developed on the back region after four years. CLL cases complicated with plasmacytoma are rare. In the present case, the plasmacytoma showed kappa cytoplasmic immunoglobulin (Ig), and the CLL showed gamma lambda surface Ig. To reveal the clonal origin of CLL and plasmacytoma, we analyzed Ig gene rearrangements in the patient's peripheral blood and plasmacytoma. Ig gene DNA analysis confirmed the presence of different rearrangements in the heavy and light chain genes of CLL and plasmacytoma. These findings suggest that in this patient, the two B cell malignancies arose from expansion of two phenotypically and genotypically distinct clones.