Bortezomib overcomes cell-adhesion-mediated drug resistance through downregulation of VLA-4 expression in multiple myeloma.

Multiple myeloma (MM) is incurable, mainly because of cell adhesion-mediated drug resistance (CAM-DR). In this study, we performed functional screening using short hairpin RNA (shRNA) to define the molecule(s) responsible for CAM-DR of MM. Using four bona fide myeloma cell lines (KHM-1B, KMS12-BM, RPMI8226 and U266) and primary myeloma cells, we identified CD29 (beta1-integrin), CD44, CD49d (alpha4-integrin, a subunit of VLA-4), CD54 (intercellular adhesion molecule-1 (ICAM-1)), CD138 (syndecan-1) and CD184 (CXC chemokine receptor-4 (CXCR4)) as major adhesion molecules expressed on MM. shRNA-mediated knockdown of CD49d but not CD44, CD54, CD138 and CD184 significantly reversed CAM-DR of myeloma cells to bortezomib, vincristine, doxorubicin and dexamethasone. Experiments using blocking antibodies yielded almost identical results. Bortezomib was relatively resistant to CAM-DR because of its ability to specifically downregulate CD49d expression. This property was unique to bortezomib and was not observed in other anti-myeloma drugs. Pretreatment with bortezomib was able to ameliorate CAM-DR of myeloma cells to vincristine and dexamethasone. These results suggest that VLA-4 plays a critical role in CAM-DR of MM cells. The combination of bortezomib with conventional anti-myeloma drugs may be effective in overcoming CAM-DR of MM.

Analysis of origin and optimization of expansion and transduction of circulating peripheral blood endothelial progenitor cells in the rhesus macaque model.

Adult marrow-derived cells have been shown to contribute to various nonhematologic tissues and, conversely, primitive cells isolated from nonhematopoietic tissues have been shown to reconstitute hematopoiesis. Circulating endothelial progenitor cells (EPCs) have been reported to be at least partially donor derived after allogeneic bone marrow transplantation, and shown to contribute to neovascularization in murine ischemia models. However, it is unknown whether these EPCs are actually clonally derived from the same population of stem and progenitor cells that reconstitute hematopoiesis, or from another cell population found in the marrow or mobilized blood that is transferred during transplantation. To approach this question, we characterized circulating EPCs and also endothelial cells from large vessels harvested at autopsy from rhesus macaques previously transplanted with retrovirally transduced autologous CD34-enriched peripheral blood stem cells (PBSCs). Endothelial cells were grown in culture for 21-28 days and were characterized as CD31(+) CD14(-) via flow cytometry, as acLDL(+) UEA-1(+) via immunohistochemistry, and as Flk-1(+) by reverse transcriptase-polymerase chain reaction (RT-PCR). Animals had stable vector marking in hematopoietic lineages of 2-15%. Neither cultured circulating EPCs collected in steady state (n = 3), nor endothelial cells grown from large vessels (n = 2), had detectable retroviral marking. EPCs were CD34(+) and could be mobilized into the circulation with granulocyte colony-stimulating factor. Under ex vivo culture conditions, in which CD34(+) cells were optimized to transduce hematopoietic progenitor and stem cells, there was a marked depletion of EPCs. Transduction of EPCs was much more efficient under conditions supporting endothelial cell growth. Further elucidation of the origin and in vivo behavior of EPCs may be possible, using optimized transduction conditions and a vascular injury model.

In vivo selective expansion of gene-modified hematopoietic cells in a nonhuman primate model.

A major problem limiting hematopoietic stem cell (HSC) gene therapy is the low efficiency of gene transfer into human HSCs using retroviral vectors. Strategies, which would allow in vivo expansion of gene-modified hematopoietic cells, could circumvent the problem. To this end, we developed a selective amplifier gene (SAG) consisting of a chimeric gene composed of the granulocyte colony-stimulating factor (G-CSF) receptor gene and the estrogen receptor gene hormone-binding domain. We have previously demonstrated that primary bone marrow progenitor cells transduced with the SAG could be expanded in response to estrogen in vitro. In the present study, we evaluated the efficacy of the SAG in the setting of a clinically applicable cynomolgus monkey transplantation protocol. Cynomolgus bone marrow CD34(+) cells were transduced with retroviral vectors encoding the SAG and reinfused into each myeloablated monkey. Three of the six monkeys that received SAG transduced HSCs showed an increase in the levels of circulating progeny containing the provirus in vivo following administration of estrogen or tamoxifen without any serious adverse effects. In one monkey examined in detail, transduced hematopoietic progenitor cells were increased by several-fold (from 5% to 30%). Retroviral integration site analysis revealed that this observed increase was polyclonal and no outgrowth of a dominant single clonal population was observed. These results demonstrate that the inclusion of our SAG in the retroviral construct allows selective in vivo expansion of genetically modified cells by a non-toxic hormone treatment.

CAM-cytarabine, aclarubicin plus macrophage colony-stimulating factor in the treatment of acute myelogenous leukemia with trilineage dysplasia: usefulness of in vitro apoptosis in leukemic cells.

A 67-year-old woman was treated for acute myelogenous leukemia with trilineage dysplasia (AML-TLD) by combination chemotherapy with cytarabine, aclarubicin plus macrophage colony-stimulating factor (M-CSF) (referred to as CAM therapy). Complete remission was achieved after two courses of CAM therapy. After coculture of her bone marrow mononuclear cells with M-CSF in vitro, differentiation of leukemic cells into macrophages with apoptotis was observed. This case confirms an earlier report that an effect of M-CSF inducible by differentiation with apoptotic phenomena, against human leukemic cells was shown both in vitro and in vivo when achieving complete remission.

Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates.

Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investigate this finding, which has direct implications for clinical transplantation and gene therapy applications. Transfer of rhesus CD34(+) cells to culture in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but decreased cycling. Using retroviral marking with two different gene transfer vectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) before reinfusion. In vivo competitive repopulation studies showed that the level of marking originating from the cells continued in culture for 2 days with SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days and reinfused without the 2-day culture under nonstimulatory conditions. We observed stable in vivo overall gene marking levels of up to 29%. This approach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.

The impact of ex vivo cytokine stimulation on engraftment of primitive hematopoietic cells in a non-human primate model.

The impairment of engraftment ability after ex vivo or in vivo stimulation of hematopoietic stem cells, potentially related to induction of active cell cycling, has recently been a topic of intense interest. Our group has used the non-human primate autologous transplantation model and genetic marking to investigate a number of questions in hematopoiesis with direct relevance to human clinical applications. The issue of a potential reversible engraftment defect would have many implications for gene therapy and allogeneic or autologous transplantation. Initial in vitro studies with rhesus CD34+ cells indicated that after 4 days of stimulatory culture in stem cell factor (SCF), megakaryocyte growth and development factor (MDGF), and flt3 ligand (FLT), transfer of the cells to SCF alone on retronectin (FN) support resulted in decreased active cycling and a halt to proliferation, without a loss of viability or induction of apoptosis. We then directly compared the engraftment potential of cytokine-stimulated cells versus those transferred to SCF on FN alone before reinfusion, SCF/G-CSF mobilized CD34+ cells from three animals were split into two parts and transduced with either of two retroviral marking vectors for 4 days in the presence of SCF/FLT/MGDF on FN. One aliquot was cryopreserved, and the other was continued in culture without transduction for 2 days in the presence of SCF alone on FN. After total body irradiation, both aliquots were thawed and reinfused into each animal. In all animals, the level of marking from the fraction continued in culture for 2 days with SCF on FN was significantly higher than the level of marking from the aliquot transduced for 4 days without the 2-day period in SCF alone. This approach may allow more efficient engraftment of successfully transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.

A de novo philadelphia chromosome-positive acute mixed-lineage leukemia with both major and minor BCR/ABL mRNA transcripts.

A patient with a Philadelphia chromosome (Ph)-positive acute mixed-lineage leukemia (AMLL) expressing both major and minor BCR/ABL mRNA transcripts is described. Phenotypic analysis of the leukemic blasts revealed positivity for both myeloid and B-cell lineages. Southern blot analysis showed a rearrangement of the immunoglobulin heavy chain (IgH) gene. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed the expression of both major and minor BCR/ABL mRNA transcripts. To our knowledge, this is the first report of AMLL expressing both major and minor BCR/ABL mRNA transcripts and rearrangement of the IgH gene.

Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates.

Retroviral insertion site analysis was used to track the contribution of retrovirally transduced primitive progenitors to hematopoiesis after autologous transplantation in the rhesus macaque model. CD34-enriched mobilized peripheral blood cells were transduced with retroviral marking vectors containing the neo gene and were reinfused after total body irradiation. High-level gene transfer efficiency allowed insertion site analysis of individual myeloid and erythroid colony-forming units (CFU) and of highly purified B- and T-lymphoid populations in 2 animals. At multiple time points up to 1 year after transplantation, retroviral insertion sites were identified by performing inverse polymerase chain reaction and sequencing vector-containing CFU or more than 99% pure T- and B-cell populations. Forty-eight unique insertion sequences were detected in the first animal and also in the second animal, and multiple clones contributed to hematopoiesis at 2 or more time points. Multipotential clones contributing to myeloid and lymphoid lineages were identified. These results support the concept that hematopoiesis in large animals is polyclonal and that individual multipotential stem or progenitor cells can contribute to hematopoiesis for prolonged periods. Gene transfer to long-lived, multipotent clones is shown and is encouraging for human gene therapy applications.

A case of bilateral heel ulcers associated with hydroxyurea therapy for chronic myelogenous leukemia.

Bilateral heel skin ulcers developed in a 50-year-old male in the chronic phase of chronic myelogenous leukemia who had been receiving hydroxyurea (HU) therapy for 3 years. Histological examination showed perivascular lymphocytic inflammation without vasculitis. After interruption of HU administration, the heel ulcers were completely resolved within 2 months. The clinical course strongly suggested that the heel ulcers were induced by long-term HU therapy.

All-trans-retinoic acid treatment for chemotherapy-resistant acute adult T-cell leukemia.

We report a case in which treatment with all-trans-retinoic acid (ATRA) improved the clinical features of a 47-year-old female patient with acute adult T-cell leukemia (ATL). The patient was first treated several times with combination chemotherapy. but the number of ATL cells increased and other clinical manifestations progressed. ATRA 60 mg was then administered daily. ATRA treatment dramatically improved the patient's clinical features. In vitro examination revealed that ATRA inhibited the growth of ATL cells from the patient. These findings suggest that ATRA may be a useful treatment for patients with chemotherapy-resistant acute ATL.

[Systemic lupus erythematosus presenting as fulminant thrombotic thrombocytopenic purpura].

A 20-year-old woman visited a nearby hospital because of sudden, severe, and unusual genital bleeding. She also exhibited severe anemia and thrombocytopenia. In transit to our hospital, the patient suddenly suffered cardiac arrest and died soon thereafter despite immediate blood transfusion and therapeutic intubation. Thrombotic thrombocytopenic purpura (TTP) was initially diagnosed at autopsy due to the observation of numerous fragmented erythrocytes in peripheral blood, evidence of hemolysis, and thrombotic microangiopathy in multiple organs. In addition, histopathologic and serologic findings disclosed an association with systemic lupus erythematosus (SLE). Test for anticardiolipin antibody was positive, and hemophagocytic findings were detected in lymph node specimens. Reports of TTP in association with SLE have been increasing in recent years. However, the mechanisms correlating these two illnesses have not been identified. We speculated that the rapid clinical course in this case was attributable to TTP that had been provoked by endothelial microangiopathy due to SLE, and moreover, the fact that the patient's general condition had been seriously complicated by excessive menstrual bleeding.

Oculomotor nerve invasion of acute myelogenous leukemia demonstrated by magnetic resonance imaging.

A 53-year-old male was diagnosed as having acute myelogenous leukemia (M2, FAB). He complained of double vision and right blepharoptosis after receiving remission induction chemotherapy. Magnetic resonance imaging (MRI) showed enlargement of the bilateral oculomotor nerves. Intrathecal injections of methotrexate and cytosine arabinoside were partially effective and repeated MRI showed shrinkage of the enlarged oculomotor nerves, after therapy. This case shows the importance of MRI in the early diagnosis of CNS leukemia.

A novel function of Stat1 and Stat3 proteins in erythropoietin-induced erythroid differentiation of a human leukemia cell line.

We recently determined that erythropoietin (EPO) activates 3 members of the signal transducer and activator of transcription (STAT) family, Stat1alpha, Stat3, and Stat5, in the human EPO-dependent cell lines, UT-7 and UT-7/EPO (Kirito et al, J Biol Chem 272:16507, 1997). In addition, we have shown that Stat1alpha, but not Stat3, is involved in EPO-induced cellular proliferation. In this study, we examined the roles of Stat1alpha and Stat3 in EPO-induced erythroid differentiation. UT-7/GM was used as a model system, because this cell line can differentiate into erythroid-lineage cells with EPO treatment (Komatsu et al, Blood 89:4021, 1997). We found that EPO did not activate Stat1alpha or Stat3 in UT-7/GM cells. Transfection experiments showed that both Stat1alpha and Stat3 inhibited the induction by EPO of gamma-globin and erythroid-specific 5-aminolevulinate synthetase transcripts, resulting in a reduction of the percentage of hemoglobin-positive cells. Dominant negative forms of Stat1alpha or Stat3 promoted the EPO-induced erythroid differentiation of UT-7/GM cells, even in the presence of granulocyte-macrophage colony-stimulating factor, although this cytokine never induced erythroid differentiation of the parent UT-7/GM cells with or without EPO. A cell cycle analysis showed that the constitutive activation of Stat1alpha, but not Stat3, shortened the period of G0/G1 prolongation caused by EPO stimulation. Taken together, our data suggest that Stat1alpha and Stat3 act as negative regulators in EPO-induced erythroid differentiation. Specifically, Stat1alpha may activate a cell cycle-associated gene(s), leading to the entry of cells into the cell cycle.

[Retrospective analysis of elderly patients > or = 60 years of age with acute leukemia].

A retrospective analysis was performed on 76 consecutive elderly patients with acute leukemia aged 60 years or more (48 men, 28 women). Forty patients were 60-69 years old, 28 were 70-79 years old and 8 were > or = 80 years old. There were 55 patients with acute myelogenous leukemia (AML), 13 acute lymphoblastic leukemia (ALL) and 8 AML from myelodysplastic syndrome (MDS/AML). Patients were treated with the JALSG protocol, CAG regimen, or low-dose Ara-C regimen for AML, and DVP/M-CHOP protocol for ALL. The complete remission (CR) rates were 52.7% (29 of 55) in AML, 61.5% (8 of 13) in ALL, and 0% in MDS/AML. The median CR durations were 226, 85, 0 days, and the median survivals were 204, 177, 99 days, respectively. CR rates were 65.3% for the JALSG protocol, 62.5% for the CAG regimen and 25.0% for low-dose Ara-C regimen. According to age, CR was obtained 62.5% in patients aged 60-69 years and 33.3% in patients over 70 years old. Our results indicated that patients aged 60-69 years should be treated with intensive chemotherapy.

Establishment, characterization, and chromosomal analysis of new leukemic cell lines derived from MT/p210bcr/abl transgenic mice.

The p210bcr/abl chimeric protein is implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. Previously, we generated transgenic mice expressing p210bcr/abl by the metallothionein enhancer/promoter (MT/p210bcr/abl) and observed that these mice reproducibly developed T cell leukemia. In this report, we describe the establishment, characterization, and chromosomal analysis of two novel leukemic cell lines derived from MT/p210bcr/abl leukemic mice. Both cell lines carried the transgene and showed the same gene rearrangement patterns as observed in the parental leukemic cells. Expression, tyrosine-phosphorylation, and enhanced kinase activity of the p210bcr/abl were also detected. RT-PCR/SSCP for p53 transcript revealed that one of the cell lines carried a mutation, in contrast to the normal pattern shown by the parental leukemic cells. In addition, the other cell line showed a karyotype of trisomy 15. These results suggest that the p53 mutation and chromosomal abnormality may confer a proliferative ability on leukemic cells in vitro. These new cell lines are considered to be a valuable model not only for examining the biologic properties of p210bcr/abl but also for investigating the malignant process that promotes the proliferation of the leukemic cells expressing p210bcr/abl. Furthermore, these cell lines could be used in therapeutic studies, including adoptive immunotherapy.

In vitro development of erythroid and megakaryocytic cells from a UT-7 subline, UT-7/GM.

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (EPO) for growth and survival. We isolated a novel subline, UT-7/GM after long-term culture of UT-7 with GM-CSF. The hemoglobin concentration and gamma-globin and EPO-receptor mRNA levels were significantly higher in EPO-treated UT-7/GM cells than in untreated cells. In contrast, the platelet factor 4 and glycoprotein IIb mRNA levels were much higher in thrombopoietin (TPO)-treated UT-7/GM cells than in untreated cells. Some TPO-treated cells had morphologically mature megakaryocytic characteristics such as a developed demarcation membrane in the cytoplasm and multilobular nuclei. These findings indicate that UT-7/GM is a bipotential cell line that can be induced to differentiate into erythroid and megakaryocytic lineages by EPO and TPO, respectively. Moreover, a minority of UT-7/GM cells acquired a high hemoglobin concentration by treatment with TPO, suggesting that TPO in part induced the erythroid differentiation of the UT-7/GM cells. Interestingly, GM-CSF inhibited the EPO- or TPO-induced erythroid differentiation and the TPO-induced megakaryocytic differentiation of UT-7/GM cells. These results support the hypothesis that cytokines influence the programming of gene expression required for lineage commitment or differentiation.

Identification of functional domains of the human thrombopoietin receptor required for growth and differentiation of megakaryocytic cells.

To identify the functional domains of the human thrombopoietin (TPO) receptor essential for proliferation and megakaryocytic differentiation, we introduced human wild type c-mpl cDNA and deletion mutants of c-mpl cDNA into the human erythropoietin (EPO)-dependent cell line UT-7/EPO that does not express endogenous c-Mpl. TPO induced the proliferation and megakaryocytic differentiation of UT-7/EPO expressing wild type c-Mpl, as evidenced by increased levels of the CD41 antigen specific for cells of the megakaryocytic lineage and by changes in morphology. Mutational analysis of the cytoplasmic domain of c-Mpl identified four functional regions: (a) two C-terminal regions (amino acids 575-586 and 615-630) containing a domain essential for cell proliferation and megakaryocytic differentiation but not for DNA synthesis; (b) a region (amino acids 587-614) containing a negative domain for TPO-induced cell proliferation and megakaryocytic differentiation; and (c) a region (amino acids 565-574) including a box2 motif that is required for DNA synthesis. These deletion mutants will provide useful materials for analyzing the signals specific for TPO-induced proliferation and megakaryocytic differentiation.

[Acute myeloid leukemia with monosomy 7 accompanied by central diabetes insipidus].

A 27-year-old female was diagnosed as having atypical aplastic anemia in 1979 because of hypercellular bone marrow with abnormal erythroblasts and megakaryocytes. Afterward the diagnosis was corrected to myelodysplastic syndrome (RA) due to the reevaluation of the bone marrow smears. In March, 1995, thirst and polyurea occurred. In April, 1995, bone marrow aspiration biopsy showed the proliferation of atypical blasts (28%), and two months later, the number of the blasts increased (30%) and leukemic progression was noticed. Only 0.5 percent of the blasts showed weak peroxidase activity, and most of the blasts had CD13, CD33 and several adhesion molecules as CD11a, CD11b, CD44, CD54 and CD56. Karyotype of the bone marrow cells was 45, XX, -7. Her polyurea was caused by central diabetes insipidus. She was also complicated by pleuritis, colon ulcer, sinusitis and hypothalamic dysfunction. The etiology of these signs was due to the leukemic cell infiltration. She died despite of receiving multi-drug chemotherapy.

Expression of p210bcr/abl by metallothionein promoter induced T-cell leukemia in transgenic mice.

The p210bcr/abl chimeric protein is considered to be implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. To investigate its biologic function in vivo, we generated transgenic mice expressing p210bcr/abl driven by the metallothionein enhancer/promoter. Two of six founder mice and the transgenic progeny developed leukemias several months after birth. In the leukemic tissues, the expression of the p210bcr/abl transgene product was detected and the increased tyrosine-phosphorylation of cellular proteins was observed. The expressed p210bcr/abl transgene product was shown to possess an enhanced kinase activity. The leukemic cells showed rearrangements in the T-cell receptor loci, indicating that the leukemic cells were monoclonal and committed to the T-cell lineage. Polymerase chain reaction analysis for tissue distribution of p210bcr/abl expression showed that, in the transgenic line that reproducibly developed leukemias, p210bcr/abl was expressed in the hematopoietic tissues such as thymus and spleen; on the other hand, in the transgenic lines that have not developed leukemias, p210bcr/abl expression was detected only in the nonhematopoietic tissues such as the brain and kidney. These results suggest that the tumorigenicity of the p210bcr/abl chimeric protein is restricted to the hematopoietic tissues in vivo and that an event enhancing p210bcr/abl expression contributed a proliferative advantage to hematopoietic precursor cells and eventually developed T-cell leukemia in transgenic mice.