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Due to an increased incidence of copper deficiency, we investigated adult patients who had low serum levels of copper with cytopenia at our hospital from March 2014 to March 2021.We retrospectively reviewed the clinical data of patients who had been diagnosed with cytopenia due to copper deficiency at the Aichi Medical University Hospital from March 2014 to March 2021.In the 15 patients with cytopenia secondary to low serum copper level, 11 had cytopenia of two to three lineages; three (27%) had pancytopenia, and eight (73%) had bicytopenia. Of the 15 patients, nine (60%) underwent bone marrow examinations; three (30%) showed typical morphologic features associated with copper deficiency, such as multiple clear cytoplasmic vacuoles in erythroblasts and myeloid cells, and three (30%) showed dysplastic features as observed in myelodysplastic syndrome. Among the 14 (93%) patients who were treated with copper supplements, had cessation of zinc supplements, or both, 11 (73%) and eight (53%) showed normal copper levels and hematological improvement, respectively.Copper deficiency is more common than expected and should be considered in patients with unexplained cytopenia.

Chromosome band 8q24 is the most frequently amplified locus in various types of cancers. MYC has been identified as the primary oncogene at the 8q24 locus, whereas a long noncoding gene, PVT1, which lies adjacent to MYC, has recently emerged as another potential oncogenic regulator at this position. In this study, we established and characterized a novel cell line, AMU-ML2, from a patient with diffuse large B-cell lymphoma (DLBCL), displaying homogeneously staining regions at the 8q24 locus. Fluorescence in situ hybridization clearly detected an elevation in MYC copy numbers corresponding to the homogenously staining region. In addition, a comparative genomic hybridization analysis using high-resolution arrays revealed that the 8q24 amplicon size was 1.4 Mb, containing the entire MYC and PVT1 regions. We also demonstrated a loss of heterozygosity for TP53 at 17p13 in conjunction with a TP53 frameshift mutation. Notably, AMU-ML2 cells exhibited resistance to vincristine, and cell proliferation was markedly inhibited by MYC-shRNA-mediated knockdown. Furthermore, genes involved in cyclin D, mTOR, and Ras signaling were downregulated following MYC knockdown, suggesting that MYC expression was closely associated with tumor cell growth. In conclusion, AMU-ML2 cells are uniquely characterized by homogenously staining regions at the 8q24 locus, thus providing useful insights into the pathogenesis of DLBCL with 8q24 abnormalities.

Amylase-producing myeloma exhibits refractoriness to chemotherapy and a dismal prognosis. In this study, we established a human myeloma cell line, 8226/AMY1, in which a lentivirally transfected AMY1 gene was stably expressed and explored its biological characteristics. 8226/AMY1 showed a survival advantage over mock control when treated with dexamethasone, bortezomib, and lenalidomide in vitro partly through inhibition of apoptosis induced by these reagents. In a xenograft murine model, 8226/AMY1 showed rapid tumor growth and reduced sensitivity to bortezomib compared with mock. A microarray gene expression analysis identified TCL1A, which functions as a coactivator of the cell survival kinase Akt, differentially up-regulated in 8226/AMY1. The expression of phosphorylated Akt was increased in the 8226/AMY1 cells following bortezomib treatment, but not in the mock cells. In addition, treatment with perifosine, an inhibitor of Akt phosphorylation, enhanced the anti-myeloma effect of bortezomib in the 8226/AMY1 cells. Our data suggest that amylase-producing myeloma reduced the sensitivity to bortezomib in vitro and in vivo, and the up-regulation of TCL1A may influence the drug susceptibility of 8226/AMY1 via the phosphorylation of Akt. These findings provide clues for developing treatment approaches for not only amylase-producing myeloma, but also relapsed and refractory myelomas.

Some cancer cells depend on glutamine despite of pronounced glycolysis. We examined the glutamine metabolism in leukemia cells, and found that HL-60 cells most depended on glutamine in the 4 acute myelogenous leukemia (AML) cell lines examined: growth of HL-60 cells was most suppressed by glutamine deprivation and by inhibition of glutaminolysis, which was rescued by tricarboxylic acid (TCA) cycle intermediate, oxaloacetic acid. Glutamine is also involved in antioxidant defense function by increasing glutathione. Glutamine deprivation suppressed the glutathione content and elevated reactive oxygen species most evidently in HL-60 cells. Glutamine metabolism might be a therapeutic target in some leukemia.

Background Like normal hematopoietic stem cells, leukemia cells proliferate in bone marrow, where oxygen supply is limited. However, the growth and energy metabolism of leukemia cells under hypoxia have not been well understood. Although it has been known that reactive oxygen species (ROS) is generated under hypoxic conditions, normal and leukemia stem cells were characterized by relatively low levels of ROS. Roles of ROS on leukemia cells under hypoxia also have not been well understood. Methods Four Leukemia cell lines were cultured under normoxia (21% O2) or hypoxia (1% O2), where NB4 and THP-1 were most extensively studied. To evaluate energy metabolism, we estimated whole cell number or apoptotic cells with or without a glycolysis inhibitor or an oxidative phosphorylation (OXPHOS) inhibitor. Glucose consumption and lactate production were also measured. To evaluate oxidative stress in hypoxic condition, the ROS level and GSH (reduced glutathione) / GSSG (oxidized glutathione) ratio was measured. In addition, pyruvate dehydrogenase kinase 1 (PDK1) and cytochrome c oxidase subunit 4 (COX4) were examined by western blotting or RT-PCR. Results NB4, which grows well under normoxia depending on glycolysis, demonstrated prominent apoptosis and growth suppression after 48 hours culture under hypoxia. NB4 cells cultured under hypoxia showed significantly increased ROS. Culture with a ROS scavenger resulted in decrease of apoptotic cell death of NB4 under hypoxia. NB4 cells cultured for longer period (7 days) under hypoxia did not come to extinction, but grew slowly by upregulating GSH synthesis to protect from ROS generated in hypoxic condition. By contrast, THP-1, which largely depends on OXPHOS in mitochondria under normoxia, demonstrated more growth under hypoxia by changing metabolism from OXPHOS to glycolysis through upregulating PDK1. Moreover, THP-1 avoided ROS generation by substituting COX 4 subunit (from COX 4–1 to COX 4–2) through upregulation of LON, a mitochondrial protease under hypoxia. Conclusions We showed that leukemia cells survive and adapt to the hypoxic condition through various pathways. Our results will help understanding energy metabolism of leukemia cells and creating novel therapeutics.

We examined the effects of diet nutrients on xenotransplanted leukemia cells, THP-1 or NB4. THP-1 tumors showed more growth when fed with high fat diet, while NB4 tumors grew more with high carbohydrate diet. Then, administration of 2-deoxyglucose (a glycolysis inhibitor) showed a significant antitumor effect on both tumors: NB4 tumor showed large necrotic areas, while THP-1 tumor did not, but had augmented expression of enzymes for fatty acid oxidation. 2-Deoxyglucose inhibited the growth of NB4 by cell death because main energy producing pathway (glycolysis) was abolished, while 2-deoxyglucose slowed the growth of THP-1 by shifting energy metabolism to fatty acid β-oxidation.

Introduction: Primary refractory DLBCL is an extremely difficult condition to treat and represent an unmet medical need. The clarification of the molecular pathogenesis of this disease can contribute the development of new therapeutic possibilities. PVT1 is located adjacent to MYC at 8q24 and a non-protein coding gene. PVT1 produces a variety of long non-coding RNAs (lncRNAs) and has now emerged as a potential regulator in the pathogenesis of a lot of cancers. However, the precise biological and clinical significance of PVT1 remains largely unknown. In this study we established a novel human DLBCL cell line with hsr (homogeneous staining region) of 8q24 inducing high expression of PVT1 lncRNAs, named AMU-ML2. The cell line was established from a patient with primary refractory DLBCL before initiation of chemotherapy. We analyzed the genetic characteristics and investigated the drug sensitivity of the cell line in comparison with other B-cell lymphoma cell lines which had 8q24 abnormalities. Case: A 64-year-old man was diagnosed as DLBCL involved with bone marrow and pleural effusion. He was refractory to initial R-CHOP and subsequent R-hyper-CVAD/MA therapy and died of Trichosporon asahiisepsis 6 months after diagnosis. His lymphoma cells at diagnosis were positive for CD19, CD20, BCL6 and HLA-DR, and negative for CD3, CD5, CD10, cyclinD1, BCL2, MUM1, TP53 and EBER by flow cytometry and/or immunohistochemical staining, indicative of germinal center B-cell-like (GCB) DLBCL. The representative karyotype of cells was 46,XY, including add(6)(p11), add(8)(q24), hsr 8q24, add(9)(p13) and add(17)(p11.2). Establishment of the cell line: After 2 weeks of culture, the cells in pleural effusion collected before chemotherapy started to grow in suspension. The cell line was designated as AMU-ML2 after confirmation that the cells started growing again after the conventional freeze-thaw procedure. Materials and methods: B-cell lymphoma cell lines; AMU-ML2, SU-DHL-10, Raji, P3HR-1 and VAL were used in the present study. These cell lines had t(8;14)(q24;q32) or 8q24 amplification. The genomic aberration of AMU-ML2 was analyzed by cytogenetics including G-banding and FISH (fluorescence in situ hybridization) combined with array-CGH (SurePrint G3, Agilent). The TaqMan real time RT-PCR was used for measurement of expression levels of MYC and PVT1. MTT assay was used for the cell proliferation to analyze the drug sensitivities of cyclophosphamide, doxorubicin, vincristine (VCR) and prednisolone. Results: AMU-ML2 cells showed same immunophenotypic feature and karyotype as the primary sample from the patient. FISH using a MYC/IGH probe set showed no fusion signal for IGH and MYC; however, the MYC copy number was extremely increased, corresponding to hsr on chromosome 8q24. Array-CGH revealed that a 1,462 kb region, containing both the entire MYC and PVT1 genes at 8q24.21, was amplified, with greater than 20 copies present in cells (Figure 1). In addition to amplification of the MYC/PVT1 region at 8q24, 14 additional copy number alterations were detected. These included segment losses on 6p22.1-6p21.31 and 17p13 led to the LOH of human leukocyte antigen (HLA) loci and TP53, respectively (Figure 1). Using the real time RT-PCR, the expression level of the PVT1 lncRNAs were highest in AMU-ML2 among other B-cell lymphoma cell lines that we used, while the expression level of MYC in AMU-ML2 was relatively low. The proliferation rate of AMU-ML2 was significantly higher after 72-h exposure to VCR (100, 500 and 1,000 nM) compared with other cell lines (Figure 2). Discussion: AMU-ML2 was established in 2-week culture from a refractory patient before starting the chemotherapy, therefore this cell line seems to reflect the real nature of primary refractory DLBCL, not related to chemotherapy and/or cell culture. MYC amplification and the LOH of TP53 and HLA may contribute to the development of lymphoma in our case. High expression of PVT1 lncRNAs was likely to be more related with drug resistance to VCR than MYC expression, although AMU-ML2 had co-amplification of MYC and PVT1. Thus, AMU-ML2 can provide insight into the genetic and biological features and the therapeutic approaches in primary refractory DLBCL. Disclosures Ueda: Mundipharma KK: Consultancy; Kyowa Hakko Kirin: Research Funding.

In this study, we established and analyzed a novel human myeloid leukemia cell line, AMU-AML1, from a patient with acute myeloid leukemia with multilineage dysplasia before the initiation of chemotherapy. AMU-AML1 cells were positive for CD13, CD33, CD117, and HLA-DR by flow cytometry analysis and showed a single chromosomal abnormality, 46, XY, t(12;22)(p13;q11.2), by G-banding and spectral karyotyping. Fluorescent in situ hybridization analysis indicated that the chromosomal breakpoint in band 12p13 was in the sequence from the 5' untranslated region to intron 1 of TEL and that the chromosomal breakpoint in band 22q11 was in the 3' untranslated region of MN1. The chimeric transcript and protein of MN1-TEL could not be detected by reverse-transcriptase polymerase chain reaction or Western blot analysis. However, the MN1 gene was amplified to three copies detected by array comparative genomic hybridization analysis, and the expression levels of the MN1 transcript and protein were high in AMU-AML1 cells when compared with other cell lines with t(12;22)(p13;q11-12). Our data showed that AMU-AML1 cells contain t(12;22)(p13;q11.2) without chimeric fusion of MN1 and TEL. The AMU-AML1 cells gained MN1 copies and had high expression levels of MN1. Thus, the AMU-AML1 cell line is useful for studying the biological consequences of t(12;22)(p13;q11.2) lacking chimeric MN1-TEL.

Despite recent progress in treatment for multiple myeloma (MM), a complete cure remains elusive. To further improve the therapeutic outcome of patients with MM, elucidation of the pathology of refractory cases is important. Hyperamylasemia, which is associated with ectopic amylase (AMY) production by MM cells, is a rare condition, and it has been reported to present with poor prognosis showing rapid tumor growth, extramedullary tumor mass formation, and refractoriness of the condition. However, to date, there have been no biological analyses of MM cells ectopically producing AMY. In this study we generated transfectants that stably expressed AMY with human MM cells, and investigated the impact that ectopic AMY production has on tumor proliferation and changes in drug susceptibility in vitro and in vivo. Two human MM cell lines (RPMI8226 and KMS11) and the cDNA encoding AMY1 were used to establish transfectants with ViraPower™ Lentiviral Gateway Expression Kit (Invitrogen), because the increased AMY isotype was salivary type, which is coded in AMY1, in all MM patients previously reported. The constitutive expression and production of AMY1 were confirmed in the AMY-transfectants (8226/AMY and KMS11/AMY), while they were not in the mock controls. These transfectants were assayed for proliferation and apoptosis after exposure to dexamethasone (Dex), bortezomib (Bz) and lenalidomide (Len) in vitro. The anti-myeloma activity of Bz was also tested in vivo in a xenograft model generated by injecting 8226/AMY or the mock cells into NOD-SCID mice. 8226/AMY had no growth advantage in vitro but grew rapidly when subcutaneously transplanted in mice compared with the mock control (2,177±878 vs 970±131 mm3, p = 0.044). 8226/AMY showed a higher cell proliferation rate than the mock control in vitro when treated with Dex (40uM), Bz (2nM), and Len (1mM). The number of apoptotic 8226/AMY cells decreased after exposure to Bz and Len, but the number after exposure to Dex was equivalent compared with the mock control by the Annexin / Propidium Iodide assay. Therefore, 8226/AMY became less sensitive to Bz and Len partly through the inhibition of apoptosis induced by these drugs. 8226/AMY grew rapidly subcutaneously in mice compared with the mock control when treated with Bz (0.3mg/kg, twice weekly) (p = 0.017). As for KMS11/AMY, the AMY-transfectant showed a higher proliferation rate than the mock control in vitro. KMS11/AMY showed reduced susceptibility to Dex, no change in the susceptibility to Bz, and an enhanced susceptibility to Len unexpectedly in comparison with the mock control. The reason for a difference in the effect of ectopic AMY expression on the susceptibility to anti-MM drugs between 8226/AMY and KMS11/AMY is unclear; however, it might be due to the nature of their parental cells. No significant difference was observed in the gene expression profiling between both AMY-transfectants and each of the respective mock controls, except for AMY1, suggesting that ectopic AMY expression did not affect the expression level of the specific gene in MM. In conclusion, we found that 8226/AMY had reduced susceptibility to Dex, Bz, and Len in vitro and also rapid tumor growth with a weakened anti-tumor effect of Bz in vivo. All of these were consistent with the clinical course of previously reported patients with ectopic AMY-producing MM. On the other hand, KMS11/AMY showed an enhanced susceptibility to Len compared with the mock control, indicating that Len might be effective for some patients with AMY-producing MM. Our data provided beneficial clues for elucidating the molecular pathology and developing a treatment strategy for this clinical setting. Disclosures No relevant conflicts of interest to declare.