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Background: ZNF384‐fusion (Z‐fusion) genes were recently identified in B‐cell acute lymphoblastic leukemia (B‐ALL) and are frequent in Japanese adult patients. The frequency is about 20% in those with Philadelphia chromosome‐negative B‐ALL. ZNF384 is a transcription factor and Z‐fusion proteins have increased transcriptional activity; however, the detailed mechanisms of leukemogenesis of Z‐fusion proteins have yet to be clarified. Methods: We established three transfectants of cell lines expressing different types of Z‐fusion proteins, and analyzed their gene expression profile (GEP) by RNA‐seq. We also analyzed the GEP of clinical ALL samples using our previous RNA‐seq data of 323 Japanese ALL patients. We selected upregulated genes in both Z‐fusion gene‐expressing transfectants and Z‐fusion gene‐positive ALL samples, and investigated the binding of Z‐fusion proteins to regulatory regions of the candidate genes by ChIP‐qPCR. Results: We selected six commonly upregulated genes. After the investigation by ChIP‐qPCR, we finally identified CREB5 and RGS1 as direct and common target genes. RGS1 is an inhibitor of CXCL12‐CXCR4 signaling that is required for the homing of hematopoietic progenitor cells to the bone marrow microenvironment and development of B cells. Consistent with this, Z‐fusion gene transfectants showed impaired migration toward CXCL12. Conclusions: We identified CREB5 and RGS1 as direct and common transcriptional targets of Z‐fusion proteins. The present results provide novel insight into the aberrant transcriptional regulation by Z‐fusion proteins. [ABSTRACT FROM AUTHOR]

Introduction: Nucleoporin 98 (NUP98) fusion proteins are recurrently found in leukemia and are associated with unfavorable clinical outcomes. They are distributed to the nucleus and contribute to leukemogenesis via aberrant transcriptional regulation. We previously identified NUP98‐BPTF (NB) fusion in patients with T‐cell acute lymphoblastic leukemia (T‐ALL) using next‐generation sequencing. The FG‐repeat of NUP98 and the PHD finger and bromodomain of bromodomain PHD finger transcription factor (BPTF) are retained in the fusion. Like other NUP98 fusion proteins, NB is considered to regulate genes that are essential for leukemogenesis. However, its target genes or pathways remain unknown. Materials and Methods: To investigate the potential oncogenic properties of the NB fusion protein, we lentivirally transduced a doxycycline‐inducible NB expression vector into mouse NIH3T3 fibroblasts and human Jurkat T‐ALL cells. Results: NB promoted the transformation of mouse NIH3T3 fibroblasts by upregulating the proto‐oncogene Pim1, which encodes a serine/threonine kinase. NB transcriptionally regulated Pim1 expression by binding to its promoter and activated MYC and mTORC1 signaling. PIM1 knockdown or pharmacological inhibition of mTORC1 signaling suppressed NB‐induced NIH3T3 cell transformation. Furthermore, NB enhanced the survival of human Jurkat T‐ALL cells by inactivating the pro‐apoptotic protein BCL2‐associated agonist of cell death (BAD). Conclusion: We demonstrated the pivotal role of NB in cell transformation and survival and identified PIM1as a key downstream target of NB. These findings propose a promising therapeutic strategy for patients with NB fusion‐positive leukemia. [ABSTRACT FROM AUTHOR]

Gene aberrations of B-cell regulators and growth signal components such as the JAK–STAT pathway are frequently found in B-cell acute lymphoblastic leukemia (B-ALL). EBF1 is a B-cell regulator that regulates the expression of PAX5 and co-operates with PAX5 to regulate B-cell differentiation. Here, we analyzed the function of the fusion protein of EBF1 and JAK2, EBF1–JAK2 (E–J). E–J caused constitutive activation of JAK–STAT and MAPK pathways and induced autonomous cell growth in a cytokine-dependent cell line. E–J did not affect the transcriptional activity of EBF1 but inhibited that of PAX5. Both the physical interaction of E–J with PAX5 and kinase activity of E–J were required for E–J to inhibit PAX5 function, although the detailed mechanism of inhibition remains unclear. Importantly, gene set enrichment analysis using the results of our previous RNA-seq data of 323 primary BCR-ABL1-negative ALL samples demonstrated repression of the transcriptional target genes of PAX5 in E–J-positive ALL cells, which suggests that E–J also inhibited PAX5 function in ALL cells. Our results shed new light on the mechanisms of differentiation block by kinase fusion proteins. [ABSTRACT FROM AUTHOR]

CEBPA‐IGH, a fusion gene of the immunoglobulin heavy‐chain locus (IGH) and the CCAAT enhancer‐binding protein α (C/EBPα) gene, is recurrently found in B‐ALL cases and causes aberrant expression of C/EBPα, a master regulator of granulocyte differentiation, in B cells. Forced expression of C/EBPα in B cells was reported to cause loss of B‐cell identity due to the inhibition of Pax5, a master regulator of B‐cell differentiation; however, it is not known whether the same mechanism is applicable for B‐ALL development by CEBPA‐IGH. It is known that a full‐length isoform of C/EBPα, p42, promotes myeloid differentiation, whereas its N‐terminal truncated isoform, p30, inhibits myeloid differentiation through the inhibition of p42; however, the differential role between p42 and p30 in ALL development has not been clarified. In the present study, we examined the effect of the expression of p42 and p30 in B cells by performing RNA‐seq of mRNA from LCL stably transfected with p42 or p30. Unexpectedly, suppression of PAX5 target genes was barely observed. Instead, both isoforms suppressed the target genes of MEF2 family members (MEF2s), other regulators of B‐cell differentiation. Similarly, MEF2s target genes rather than PAX5 target genes were suppressed in CEBP‐IGH‐positive ALL (n = 8) compared with other B‐ALL (n = 315). Furthermore, binding of both isoforms to MEF2s target genes and the reduction of surrounding histone acetylation were observed in ChIP‐qPCR. Our data suggest that the inhibition of MEF2s by C/EBPα plays a role in the development of CEBPA‐IGH‐positive ALL and that both isoforms work co‐operatively to achieve it. [ABSTRACT FROM AUTHOR]

B‐cell acute lymphoblastic leukemia (B‐ALL), a genetically heterogeneous disease, is classified into different molecular subtypes that are defined by recurrent gene rearrangements, gross chromosomal abnormalities, or specific gene mutations. Cells with these genetic alterations acquire a leukemia‐initiating ability and show unique expression profiles. The distribution of B‐ALL molecular subtypes is greatly dependent on age, which also affects treatment responsiveness and long‐term survival, partly accounting for the inferior outcome in adolescents and young adults (AYA) and (older) adults with B‐ALL. Recent advances in sequencing technology, especially RNA sequencing and the application of these technologies in large B‐ALL cohorts have uncovered B‐ALL molecular subtypes prevalent in AYA and adults. These new insights supply more precise estimations of prognoses and targeted therapies informed by sequencing results, as well as a deeper understanding of the genetic basis of AYA/adult B‐ALL. This article provides an account of these technological advances and an overview of the recent major findings of B‐ALL molecular subtypes in adults. [ABSTRACT FROM AUTHOR]

The optimal treatment for Philadelphia chromosome (Ph)-negative acute lymphoblastic leukemia (ALL) in first complete remission (CR1) has not been established in the high-intensity chemotherapy era. The outcomes of patients with Ph-negative ALL who underwent allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen-matched related or unrelated donor in CR1 (HSCT-MRD group and HSCT-MUD group) were obtained from a Japanese registry database. Patients aged 16–24 years and 25–65 years were analyzed separately, and their outcomes were compared to those of patients who continued high-intensity chemotherapy in CR1 in studies (202U group and 202O group) by the Japan Adult Leukemia Study Group (JALSG). In the HSCT-MRD group, patients younger than 25 years had lower overall survival (OS) than the 202U group, presumably due to the higher non-relapse mortality (NRM) in the HSCT-MRD group. Patients 25 years and older had similar OS to the 202O group. The lower relapse rate was counterbalanced by higher NRM in the HSCT-MRD group. In the HSCT-MUD group, patients in both age groups had similar OS to their corresponding groups in the JALSG studies. In conclusion, high-intensity chemotherapy may change the role of HSCT for Ph-negative ALL. [ABSTRACT FROM AUTHOR]

This phase 1/2 study aimed to identify the maximum tolerated dose, the recommended phase 2 dose (RP2D), and efficacy of the clofarabine, etoposide, and cyclophosphamide combination regimen in adult patients with relapsed/refractory acute lymphoblastic leukemia (ALL). Patients aged ≥ 15 years with relapsed/refractory ALL were enrolled. Escalating doses of clofarabine (20–30 mg/m2/day × 5 days), etoposide (50–100 mg/m2/day × 5 days), and cyclophosphamide (200–440 mg/m2/day × 5 days) were administered. Dose-limiting toxicity was defined as Grade 3 or more non-hematological toxicities and others. A total of 18 patients (B-ALL; n = 13, T-ALL; n = 5) were recruited in phase 1; however, the protocol was amended to close study without proceeding to phase 2. Three patients were enrolled in cohort 1, three in cohort 2, six in cohort 3, and six in cohort 4. The RP2D of clofarabine, etoposide, and cyclophosphamide was 30, 100, and 440 mg/m2 daily, respectively. Complete remission (CR) was achieved in four patients (22%) and CR without platelet recovery in four patients (22%), with an overall response rate of 44%. The RP2D of the combination therapy was successfully determined in this study. [ABSTRACT FROM AUTHOR]

PAX5-KIDINS220 (PAX5-K220) is a novel chimeric fusion gene identified in a pediatric Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) patient, but the function of the encoded fusion protein has not yet been analyzed. Here, we report the functional analysis of PAX5-K220 in vitro. We successfully generated PAX5-K220 expressing cells and demonstrate that PAX5-K220 is a nuclear protein. Luciferase reporter assay reveals that PAX5-K220 inhibits wild-type PAX5 transcriptional activity in a dominant-negative fashion like other PAX5-related fusion proteins, and may contribute to lymphocyte differentiation block. However, although identified in Ph-like ALL, PAX5-K220 does not induce IL-3-independent proliferation when transduced in the IL-3-dependent Ba/F3 murine leukemia cells, but rather attenuates growth. These results reveal that PAX5-K220 certainly shares the character with other PAX5-related fusion proteins rather than other fusion proteins with tyrosine kinase activity identified in Ph-like ALL, and did not contribute to proliferation activity. Precise functional analysis of each differently partnered PAX5 fusion protein is warranted in the future for better understanding of PAX5-related translocations and their effects. [ABSTRACT FROM AUTHOR]

Background: Hemophilia A (HA) is an X‐linked recessive bleeding disorder caused by pathogenic variants of the coagulation factor VIII gene (F8). Half of the patients with severe HA have a recurrent inversion in the X chromosome, that is, F8 intron 22 or intron 1 inversion. Here, we characterized an abnormal F8 due to atypical complex X chromosome rearrangements in a Japanese patient with severe HA. Methods: Recurrent F8 inversions were tested with inverse shifting‐PCR. The genomic structure was investigated using PCR‐based direct sequencing or quantitative PCR. Results: The proband's X chromosome had a 119.5 kb insertion, a reverse duplex of an extragenic sequence on the F8 telomere region into the F8 intron 1 with two breakpoints. The telomeric breakpoint was a joining from the F8 intron 1 to the inverted FUNDC2 via a two‐base microhomology, and the centromeric breakpoint was a recombination between F8 intron 1 homologous sequences. The rearrangement mechanism was suggested as a multi‐step rearrangement with template switching such as fork stalling and template switching (FoSTeS)/microhomology‐mediated break‐induced replication (MMBIR) and/or homologous sequence‐associated recombination during a sister chromatid formation. Conclusion: We identified the aberrant X chromosome with a split F8 due to a multi‐step rearrangement in a patient with severe HA. [ABSTRACT FROM AUTHOR]

Zinc‐finger protein 384 (ZNF384) fusion (Z‐fusion) genes have recently been identified as recurrent fusion genes in B‐cell precursor acute lymphoblastic leukaemia (BCP‐ALL) and have been detected in 7–17% of Philadelphia chromosome‐negative BCP‐ALL cases. We selected SALL4 and ID2 as potential Z‐fusion‐specific transcriptional targets that might lead to the differentiation disorder of Z‐fusion‐positive ALL. The introduction of EP300‐ZNF384 and SYNRG‐ZNF384 induced the expression of these genes. Z‐fusion proteins exhibited stronger transcriptional activities on the promoter or enhancer region of these genes than Wild‐Z. Furthermore, GST pull‐down assay revealed that Z‐fusion proteins associated more strongly with EP300 than Wild‐Z. Coexpression of EP300 specifically enhanced the transcriptional activities of Z‐fusion proteins. We propose the increased EP300 binding of Z‐fusion proteins as a mechanism for their increased transcriptional activities. [ABSTRACT FROM AUTHOR]

Cancer‐associated fibroblasts (CAF) are a key component in the tumor microenvironment and play functional roles in tumor metastasis and resistance to chemotherapies. We have previously reported that CAF isolated from lymphoma samples increase anaerobic glycolysis and decrease intracellular production of reactive oxygen species, promoting the survival of tumor cells. Herein, we analyzed the mechanisms underlying this support of tumor‐cell survival by CAF. As direct contact between lymphoma cells and CAF was not indispensable to survival support, we identified that the humoral factor pyruvate was significantly secreted by CAF. Moreover, survival of lymphoma cells was promoted by the presence of pyruvate, and this promotion was canceled by inhibition of monocarboxylate transporters. Metabolome analysis of lymphoma cells in coculture with CAF demonstrated that intermediates in the citric acid cycle were significantly increased, indicating that tumor cells produced energy by aerobic metabolism. These findings indicate that energy production in lymphoma cells is regulated in coordination not only with anaerobic glycolysis, but also with aerobic metabolism termed the reverse‐Warburg effect, involving the secretion of pyruvate from CAF resulting in increased use of the citric acid cycle in lymphoma cells. Figure 2 shows that CAF and conditioned media support survival of primary lymphoma cells. We show CAF support survival of various disease types and lineages of primary lymphoma cells in Figure 2. [ABSTRACT FROM AUTHOR]

The Japan Adult Leukemia Study Group (JALSG) Ph+ALL202 study reported a high complete remission (CR) rate for Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) patients treated with imatinib-combined chemotherapy. However, the long-term treatment efficacy remains uncertain. Here, we report a final analysis of the JALSG Ph+ALL202 study. The outcomes were compared with those of the JALSG ALL93 and ALL97 studies, which were conducted in the pre-imatinib era. Ninety-nine newly diagnosed Ph+ALL patients were enrolled in Ph+ALL202 (median age, 45 years; median follow-up, 4.5 years). CR was achieved in 96/99 (97%) patients. Fifty-nine of these 96 patients (61%) underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) in their first CR (CR1). The 5-year overall and disease-free survival (DFS) rates were 50 and 43%, respectively, which were significantly higher compared to those in the pre-imatinib era (15 and 19%, respectively). Multivariate analysis revealed that imatinib administration, allo-HSCT in CR1, and a white blood cell count < 30 × 109/L were favorable independent prognostic factors for long-term DFS. Improved odds of receiving allo-HSCT and a lower relapse rate leaded to good long-term outcomes. The 3-year DFS tended to be higher in PCR-negative than that in PCR-positive patients (29 vs. 14%) in the non-HSCT patients, and this tendency was also seen in the allo-HSCT patients (59 vs. 50%). The higher rate of CR upon imatinib use may have contributed to these improvements. [ABSTRACT FROM AUTHOR]

Regenerating islet-derived protein 3 alpha (REG3A) is a biomarker of lower gastrointestinal graft-versus-host disease (GVHD); however, the biological role of REG3A in the pathophysiology of GVHD is not understood. Here, we examined the association between a single nucleotide polymorphism in the REG3A gene, rs7588571, which is located upstream and within 2 kb of the REG3A gene, and transplant outcomes including the incidence of GVHD. The study population consisted of 126 adult Japanese patients who had undergone bone marrow transplantation from a HLA-matched sibling. There was no association between rs7588571 polymorphism and the incidence of acute GVHD. However, a significantly higher incidence of extensive chronic GVHD was observed in patients with the rs7588571 non-GG genotype than in those with the GG genotype (Odds ratio 2.6; 95% confidence interval, 1.1–6.0; P = 0.029). Semi-quantitative reverse transcription PCR demonstrated that the rs7588571 non-GG genotype exhibited a significantly lower REG3A mRNA expression level than the GG genotype (P = 0.032), and Western blot analysis demonstrated that the rs7588571 non-GG genotype exhibited a trend toward lower REG3A protein expression level than the GG genotype (P = 0.053). Since REG proteins have several activities that function to control intestinal microbiota, and since intestinal dysbiosis is in part responsible for the development of GVHD, our findings lead to the novel concept that REG3A could have some protective effect in the pathogenesis of GVHD through the regulation of gut microbiota. [ABSTRACT FROM AUTHOR]

Aberrant activation of the Hedgehog signaling pathway has been implicated in the maintenance of leukemia stem cell populations in several model systems. PF-04449913 ( PF-913) is a selective, small-molecule inhibitor of Smoothened, a membrane protein that regulates the Hedgehog pathway. However, details of the proof-of-concept and mechanism of action of PF-913 following administration to patients with acute myeloid leukemia ( AML) are unclear. This study examined the role of the Hedgehog signaling pathway in AML cells, and evaluated the in vitro and in vivo effects of the Smoothened inhibitor PF-913. In primary AML cells, activation of the Hedgehog signaling pathway was more pronounced in CD34+ cells than CD34− cells. In vitro treatment with PF-913 induced a decrease in the quiescent cell population accompanied by minimal cell death. In vivo treatment with PF-913 attenuated the leukemia-initiation potential of AML cells in a serial transplantation mouse model, while limiting reduction of tumor burden in a primary xenotransplant system. Comprehensive gene set enrichment analysis revealed that PF-913 modulated self-renewal signatures and cell cycle progression. Furthermore, PF-913 sensitized AML cells to cytosine arabinoside, and abrogated resistance to cytosine arabinoside in AML cells cocultured with HS-5 stromal cells. These findings imply that pharmacologic inhibition of Hedgehog signaling attenuates the leukemia-initiation potential, and also enhanced AML therapy by sensitizing dormant leukemia stem cells to chemotherapy and overcoming resistance in the bone marrow microenvironment. [ABSTRACT FROM AUTHOR]

Although the clinical outcomes of diffuse large B-cell lymphoma ( DLBCL) have improved in the immunochemotherapy era, approximately one-third of patients develop intractable disease. To improve clinical outcomes for these patients, it is important to identify those with poor prognosis prior to initial treatment in order to select optimal therapies. Here, we investigated the clinical and biological significance of SPIB, an Ets family transcription factor linked to lymphomagenesis, in DLBCL. We classified 134 DLBCL patients into SPIB negative ( n = 108) or SPIB positive ( n = 26) groups by immunohistochemical staining. SPIB positive patients had a significantly worse treatment response and poor prognosis compared with SPIB negative patients. Multivariate analysis for patient survival indicated that SPIB expression was an independent poor prognostic factor for both progression free survival ( PFS) and overall survival ( OS) ( PFS, hazard ratio [ HR] 2.65, 95% confidence interval [ CI] 1.31-5.33, P = 0.006; OS, HR 3.56, 95% CI 1.43-8.91, P = 0.007). Subsequent analyses of the roles of SPIB expression in DLBCL pathogenesis revealed that SPIB expression in lymphoma cells resulted in resistance to the BH3-mimetic ABT-263 and contributed to apoptosis resistance via the PI3K- AKT pathway. The inhibition of AKT phosphorylation re-sensitized SPIB expressing lymphoma cells to ABT-263-induced cell death. Together, our data indicate that SPIB expression is a clinically novel poor prognostic factor in DLBCL that contributes to treatment resistance, at least in part, through an anti-apoptotic mechanism. [ABSTRACT FROM AUTHOR]

We carried out a multicenter dose-escalation phase I study of oral OPB-51602, a signal transducer and activator of transcription 3 phosphorylation inhibitor, in patients with relapsed or refractory hematological malignancies to evaluate the safety, maximum tolerated dose ( MTD), pharmacokinetics, and preliminary antitumor activity. Twenty patients were treated with OPB-51602 at doses of 1, 2, 3, 4, and 6 mg in the '3 + 3' dose escalation design. The most common treatment-related adverse events included nausea (55%), peripheral sensory neuropathy (45%), and diarrhea (40%). The most frequently observed grade 3 or 4 drug-related adverse events were neutropenia (20%), leukopenia (15%), lymphopenia (10%), and thrombocytopenia (10%). The MTD was 6 mg, with dose-limiting toxicities of grade 3 lactic acidosis and increased blood lactic acid levels observed in one of three patients and grade 1-2 peripheral neuropathy in three of three patients. The recommended dose was determined to be 4 mg. OPB-51602 was rapidly absorbed, and exposure tended to increase in a dose-dependent manner. Accumulation of OPB-51602 was seen with 4 weeks of multiple treatments. No clear therapeutic response was observed. Durable stable disease was observed in two patients with acute myeloid leukemia and one with myeloma. In conclusion, the MTD of OPB-51602 was 6 mg. OPB-51602 was safe and well tolerated in a dose range of 1-4 mg. However, long-term administration at higher doses was difficult with the daily dosing schedule, and no response was seen. Therefore, further clinical development of OPB-51602 for hematological malignancies with a daily dosing schedule was terminated. [ABSTRACT FROM AUTHOR]

Objective: We investigated the mechanism responsible for imatinib (IM) resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) cell lines. Methods: We established cell lines from a patient with Ph+ ALL at the time of first diagnosis and relapsed phase and designated as NPhA1 and NPhA2, respectively. We also derived IM-resistant cells, NPhA2/STIR, from NPhA2 under gradually increasing IM concentrations. Results: NPhA1 was sensitive to IM (IC50 0.05 μm) and NPhA2 showed mild IM resistance (IC50 0.3 μm). NPhA2/STIR could be maintained in the presence of 10 μm IM. Phosphorylation of MEK and ERK was slightly elevated in NPhA2 and significantly elevated in NPhA2/STIR compared to NPhA1 cells. After treatment with IM, phosphorylation of MEK and ERK was not suppressed but rather increased in NPhA2 and NPhA2/STIR. Active RAS was also increased markedly in NPhA2/STIR after IM treatment. The expression of BCL-2 was increased in NPhA2 compared to NPhA1, but no further increase in NPhA2/STIR. Proliferation of NPhA2/STIR was significantly inhibited by a combination of MEK inhibitor and IM. Analysis of tyrosine phosphorylation status with a protein tyrosine kinase array showed increased phosphorylation of EphB4 in NPhA2/STIR after IM treatment. Although transcription of EphB4 was suppressed in NPhA1 and NPhA2 after IM treatment, it was not suppressed and its ligand, ephrinB2, was increased in NPhA2/STIR. Suppression of EphB4 transcripts by introducing short hairpin RNA into NPhA2/STIR partially restored their sensitivity to IM. Conclusions: These results suggest a new mechanism of IM resistance mediated by the activation of RAS/MAPK pathway and EphB4. [ABSTRACT FROM AUTHOR]

The article discusses the association of Wnt signaling with cell survival in the interaction between acute myeloid leukemia cells and stromal cells. It demonstrates the protection of TRL-01 cells from cell death, which is mediated by Wnts secreted from HTS cells. It also investigates the regulation of the antiapoptotic downstream pathway molecules in TRL-01 cells, and the effects of other inhibitors targeted for Wnt signaling.

Chronic myelogenous leukemia (CML) is effectively treated with imatinib mesylate (IM), a small molecule inhibitor of the BCR-ABL tyrosine kinase that is expressed in the entire hematopoietic compartment including stem cells (HSC) and progenitors in CML patients. While IM induces disease remission, it does not appear to eradicate BCR-ABL-positive stem cells. We investigated the residual CML cells in HSC and myeloid progenitors isolated using fluorescence-activated cell sorting after IM-therapy. Quantitative real-time polymerase chain reaction detecting BCR-ABL transcripts showed that CML progenitors were eradicated within 12 months while the BCR-ABL-positive HSC remained. However, IM-therapy continuation could significantly decrease the ratio of BCR-ABL to BCR also in the HSC population. Our results implicate that the sorted and purified stem cells are useful for more sensitive quantification of BCR-ABL-positive minimal residual disease. [ABSTRACT FROM AUTHOR]

The RUNX1 (also known as AML1) gene is observed frequently as the target of chromosomal rearrangements in human acute leukemia. We describe here a previously unreported rearrangement, t(11;21)(q13;q22), that disrupts the RUNX1 gene in a patient with acute leukemia and the molecular analysis of the fusion gene. Methods: We have established a monocytic leukemia cell line, ELAM-1, from a patient with acute leukemia evolving from myelodysplastic syndrome (MDS). Translocation (11;21) (q13;q22) was observed in both patient leukemia cells and ELAM-1. Results: The split signal of RUNX1 was detected by fluorescence in situ hybridization and indicated the involvement of RUNX1 in ELAM-1. Using 3′-Rapid amplification of cDNA ends and reverse transcription-Polymerase chain reaction analysis, we detected both RUNX1 (exon 5)-LRP16 and RUNX1 (exon 6)-LRP16 transcripts, suggesting that the RUNX1 breakpoint lies in intron 6 and that alternative fusion splice variants are generated. Reciprocal LRP16-RUNX1 fusion was also detected. Conclusions: We identified a novel RUNX1 fusion partner, LRP16 on 11q13 involving t(11;21)(q13;q22). Although it was reported that overexpression of LRP16 promotes human breast cancer cell proliferation, the function of LRP16 in leukemia remains to be studied. This fusion gene and cell line may provide a new research tool to investigate the mechanism of leukemogenesis generated by the RUNX1 fusion gene. [ABSTRACT FROM AUTHOR]

Signal transducers and activators of transcription (STAT) proteins play a crucial role in mediating signals from a diverse spectrum of cytokine receptors. STAT is thought to be activated by JAK family kinases (JFK) in many cytokine receptor signal pathways; however, recent studies have demonstrated an alternative pathway to activate STAT by Src family kinases (SFK) in growth factor receptor signal. We also observed STAT5 phosphorylation by Lyn, a member of SFK, in our two recent studies. We introduce these studies and review the literature of STAT activation by SFK and aberrant activation of STAT by oncogenic signals. [ABSTRACT FROM AUTHOR]

We have established a stroma-dependent myelomonocytic cell line, NAMO-2, with FLT3 internal tandem duplication (FLT3/ITD). Leukemia cells from a patient with acute myelomonocytic leukemia were administered to form subcutaneous tumors in nude mice, which were maintained successively, although we failed to establish continuously growing cells from the original leukemia cell culture. In the cultures of cells from subcutaneous tumors, there were stroma cells that had originated from the nude mice and showed continuous growth. The leukemia cells showed continuous growth dependent on this stroma, and this cell line was named NAMO-2. Detection of FLT3/ITD by the reverse transcriptase polymerase chain reaction (PCR) and genomic PCR showed that NAMO-2 was homozygous for FLT3/ITD. Constitutive activation of FLT3 was detected by Western blotting, and the phosphorylation of Akt, MEK, and STAT5 was also observed. FLT3 kinase inhibitor AG1296 specifically inhibited cell growth. NAMO-2 provides a useful tool to analyze adherence-dependent survival signaling of leukemia with FLT3/ITD and a model for the screening of FLT3 kinase inhibitors. [ABSTRACT FROM AUTHOR]

We have recently identified an internal tandem duplication of the human Flt3 gene in approximately 20% of acute myeloid leukemia (AML) cases. In the present study, the wild-type and the mutant Flt3 genes were transfected into two IL-3-dependent cell lines, 32D and BA/F3 cells. Mutant Flt3-transfected cells exhibited autonomous growth while wild-type Flt3-transfected cells with the continuous stimulation of Flt3 ligand exhibited a minimal proliferation. Cells expressing mutant Flt3 showed constitutive activation of STAT5 and MAP kinase. In contrast, Flt3 ligand stimulation caused rapid activation of MAP kinase but not STAT5 in cells expressing wild-type Flt3. Finally, we found constitutive activation of MAP kinase and STAT5 in all clinical samples of AML patients with mutant Flt3. Our study shows the significance of internal tandem duplication of Flt3 receptors for leukemia cell expansion. Oncogene (2000) 19, 624–631. [ABSTRACT FROM AUTHOR]

Transcription factor c-Myb plays important roles in cell survival and differentiation in immature hematopoietic cells. Here we demonstrate that c-Myb is acetylated at the carboxyl-terminal conserved domain by histone acetyltransferase p300 both in vitro and in vivo. The acetylation sites in vivo have been located at the lysine residues of the conserved domain (K471, K480, K485) by the use of the mutant Myb (Myb-KAmut), in which all three lysine residues are substituted into alanine. Electrophoretic mobility shift assay reveals that Myb-KAmut shows higher DNA binding activity than wild type c-Myb and that acetylation of c-Myb in vitro by p300 causes dramatic increase in DNA binding activity. Accordingly, transactivation activity of both mim-1 and CD34 promoters by Myb-KAmut is higher than that driven by wild type c-Myb. Furthermore, the bromodomain of p300, in addition to the histone acetyltransferase (HAT) domain, is required for effective acetylation of c-Myb, and hGCN5 is revealed to be a factor acetyltransferase for c-Myb in vitro. We present a new manner of post-translational modification of the c-Myb protein and the potential significance of the acetylation in c-Myb. Oncogene (2000) 19, 444–451. [ABSTRACT FROM AUTHOR]

Many cytokines and growth factors stimulate multiple signal transduction pathways essential for proliferation in human acute leukaemia cells, including a mitogen-activated protein (MAP) kinase pathway and a Janus kinase (JAK)-STAT (signal transducers and activators of transcription) pathway. We have previously shown constitutive activation of MAP kinase in approximately 50% of acute myelogenous leukaemia (AML) samples. Recently, STAT proteins have been reported to be constitutively activated in 10–20% of AML cases. STAT3 and STAT5 are the main STAT proteins activated in haemopoietic progenitors in response to cytokines such as IL-3, GM-CSF, erythropoietin and thrombopoietin. Although the possibility of STAT1 protein as a substrate for MAP kinase at a serine residue has been suggested, the cross-talk between STATs and MAP kinase pathways in vivo, especially in leukaemia cells, remains unknown. We examined the phosphorylation of STAT3 and STAT5 at the tyrosine residues in AML samples in which MAP kinase activity had already been found. 40/50 primary AML cases (80%) exhibited constitutive tyrosine phosphorylation of STAT5. Electrophoretic mobility shift assay showed DNA binding activity of STAT5 correlated with tyrosine phosphorylation of STAT5. Similarly, with respect to STAT3, 17/23 cases examined (74%) showed constitutive tyrosine phosphorylation of STAT3. In addition, we examined the tyrosyl-phosphorylation of STAT5 isoforms, STAT5A and STAT5B, in 20 AML cases, and found selective STAT5B phosphorylation in the absence of STAT5A phosphorylation in three cases. Furthermore, in certain AML cases, constitutive activation of MAP kinase and STAT proteins occurred independently. No significant correlation of MAP kinase activation was observed with either tyrosine phosphorylation of STAT3/STAT5 or positive DNA binding of STAT proteins. These results suggest that constitutive activation of STAT proteins occurs commonly and that the causes of constitutive... [ABSTRACT FROM AUTHOR]

Studies the life-threatening human parvovirus B 19 infection transmitted by intravenous immune globulin. Usage of a double polymerase chain reaction method to detect B 19 DNA; Genotype identity of parvovirus B 19; Information on pathway followed by B 19 infection.

Summary. Infection of human parvovirus B19 (B19) is usually a self-limiting febrile illness, but can sometimes be life-threatening under certain circumstances, such as aplastic crisis in patients with haemolytic anaemia, hydrops fetalis in pregnant women and fulminant hepatitis. B19 canbe transmitted through respiratory secretions, transplacentally and by transfusion of blood or blood products. In the present case, administration of intravenous immune globulin (i.v.Ig) transmitted B19 infection and consequently caused pure red cell aplasia and aggravation of hepatitis to fulminant hepatitis. Our case may raise important questions as to the safety of i.v.Ig and possible contamination by B19. [ABSTRACT FROM AUTHOR]

Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, especially in the points of rapid growth rate and microenvironment independency. Consequently, the majority of conventional anti-cancer drugs are less sensitive to slow growing cells and do not target microenvironmental support, although most primary cancer cells grow slower than cell lines and depend on microenvironmental support. Here, we developed a novel high throughput drug screening system using patient-derived xenograft (PDX) cells of lymphoma that maintained primary cancer cell phenotype more than cell lines. The library containing 2613 known pharmacologically active substance and off-patent drugs were screened by this system. We could find many compounds showing higher cytotoxicity than conventional anti-tumor drugs. Especially, pyruvinium pamoate showed the highest activity and its strong anti-tumor effect was confirmed also in vivo. We extensively investigated its mechanism of action and found that it inhibited glutathione supply from stromal cells to lymphoma cells, implying the importance of the stromal protection from oxidative stress for lymphoma cell survival and a new therapeutic strategy for lymphoma. Our system introduces a primary cancer cell phenotype into cell-based phenotype screening and sheds new light on anti-cancer drug development. [ABSTRACT FROM AUTHOR]

Discusses whether life-threatening human parvovirus b19 infection is transmitted by intravenous immune globulin. Genotype identity of parvovirus b 19; Sequential analysis of DNA infections.