Blocking cholesterol efflux mechanism is a potential target for antilymphoma therapy.

Cholesterol is an essential plasma membrane lipid for the maintenance of cellular homeostasis and cancer cell proliferation. Free cholesterol is harmful to cells; therefore, excessive free cholesterol must be quickly esterified by acetyl-coenzyme A:cholesterol acetyltransferase (ACAT) and exported by scavenger receptor class B member I (SR-BI) or ATP-binding cassette protein A1 from specific cells such as macrophage foam cells, which contain cholesteryl ester-derived vacuoles. Many vacuoles are present in the cytoplasm of Burkitt lymphoma cells. In this study, we observed that these vacuoles are often seen in high-grade lymphomas. Cell culture study using lymphoma cell lines found that esterified cholesterol is the main component of these vacuoles and the expression of cholesterol metabolism-related molecules was significantly upregulated in lymphoma cell lines, with SR-BI and ACAT inhibitors (BLT-1 and CI-976, respectively) impeding lymphoma cell proliferation. Cytoplasmic free cholesterol was increased by ACAT and SR-BI inhibitors, and the accumulation of free cholesterol induced lymphoma cell apoptosis by inducing endoplasmic reticulum stress. Furthermore, synergistic effects of SR-BI and ACAT inhibitors were observed in a preclinical study. Treatment with SR-BI inhibitor suppressed lymphoma progression in a tumor-bearing mouse model, whereas ACAT inhibitor did not. Therefore, SR-BI inhibitors are potential new antilymphoma therapeutics that target cholesterol metabolism.

Novel p97/VCP inhibitor induces endoplasmic reticulum stress and apoptosis in both bortezomib-sensitive and -resistant multiple myeloma cells.

p97/VCP is an endoplasmic reticulum (ER)-associated protein that belongs to the AAA (ATPases associated with diverse cellular activities) ATPase family. It has a variety of cellular functions including ER-associated protein degradation, autophagy, and aggresome formation. Recent studies have shown emerging roles of p97/VCP and its potential as a therapeutic target in several cancer subtypes including multiple myeloma (MM). We conducted a cell-based compound screen to exploit novel small compounds that have cytotoxic activity in myeloma cells. Among approximately 2000 compounds, OSSL_325096 showed relatively strong antiproliferative activity in MM cell lines (IC50 , 100-500 nmol/L). OSSL_325096 induced apoptosis in myeloma cell lines, including a bortezomib-resistant cell line and primary myeloma cells purified from patients. Accumulation of poly-ubiquitinated proteins, PERK, CHOP, and IREα, was observed in MM cell lines treated with OSSL_325096, suggesting that it induces ER stress in MM cells. OSSL_325096 has a similar chemical structure to DBeQ, a known p97/VCP inhibitor. Knockdown of the gene encoding p97/VCP induced apoptosis in myeloma cells, accompanied by accumulation of poly-ubiquitinated protein. IC50 of OSSL_325096 to myeloma cell lines were found to be lower (0.1-0.8 µmol/L) than those of DBeQ (2-5 µmol/L). In silico protein-drug-binding simulation suggested possible binding of OSSL_325096 to the ATP binding site in the D2 domain of p97/VCP. In cell-free ATPase assays, OSSL_325096 showed dose-dependent inhibition of p97/VCP ATPase activity. Finally, OSSL_325096 inhibited the growth of subcutaneous myeloma cell tumors in vivo. The present data suggest that OSSL_325096 exerts anti-myeloma activity, at least in part through p97/VCP inhibition.

[Spontaneous remission of acquired factor XIII inhibitor concurrent to development of IgA-λ type multiple myeloma].

Coagulation factor XIII is a fibrin-stabilizing factor that leads to the crosslinking of fibrin when activated by thrombin. Acquired factor XIII inhibitor is caused when antibodies are generated against factor XIII, reducing its activity. Here we report a case of acquired factor XIII inhibitor. Although prednisolone was administered, factor XIII activity was not recovered. Interestingly, the activity normalized following the onset of multiple myeloma. The presence of inhibitors was evaluated in the patient's plasma by absorption tests and enzyme-linked immunosorbent assay. Immunoglobulin G inhibitors of factor XIII were present at admission, but later decreased with the onset of the IgA-λ-type myeloma. Thus, it is possible that the level of factor XIII inhibitors and polyclonal immunoglobulins could have been suppressed by the progression of myeloma, resulting in the normalization of factor XIII activity.

MUC1/KL-6 expression confers an aggressive phenotype upon myeloma cells.

The sialic glycoprotein, MUC1, is known to be involved in the pathogenesis of various types of cancers. KL-6 is one of the surface antigens of MUC1 and also a marker of interstitial pneumonitis. A fraction of patients with myeloma (3.9%) have elevated serum KL-6 levels without any evidence of interstitial pneumonitis and their myeloma cells have high MUC1 expression. We established a myeloma cell line designated EMM1 from a patient with multiple myeloma accompanied with elevated serum KL-6. EMM1 cells expressed high levels of MUC1 compared with other myeloma cell lines. Knockdown of MUC1 in EMM1 cells induced cell cycle arrest during S phase and apoptosis, suggesting that the MUC1 expression is involved in accelerated growth of EMM1 cells. RNA-seq analysis suggests that MUC1 expression activates k-ras and TNFα-induced NFκB pathways in EMM1 cells. We injected EMM1 cells subcutaneously into Rag2-/-Jak3-/- Balb/c mice to establish a mouse xenograft model. These mice had aggressive tumor growth that was accompanied by high serum KL-6 levels. In addition, MUC1 knockdown in EMM1 cells led to inhibited tumor growth. These findings demonstrate that MUC1 serves as a potential target for developing drugs for treatment of patients with KL-6+ myeloma, and EMM1 cells and EMM1-engrafted mice are useful tools for the development of such novel agents.

A xenograft model reveals that PU.1 functions as a tumor suppressor for multiple myeloma in vivo.

We previously demonstrated that PU.1 expression is down-regulated in the majority of myeloma cell lines and primary myeloma cells from patients. We introduced the tet-off system into the human myeloma cell lines U266 and KMS12PE that conditionally express PU.1 and demonstrated that PU.1 induces cell cycle arrest and apoptosis in myeloma cells in vitro. Here, we established a mouse xenograft model of myeloma using these cell lines to analyze the effects of PU.1 on the phenotype of myeloma cells in vivo. When doxycycline was added to the drinking water of mice engrafted with these myeloma cells, all mice had continuous growth of subcutaneous tumors and could not survived more than 65 days. In contrast, mice that were not exposed to doxycycline did not develop subcutaneous tumors and survived for at least 100 days. We next generated mice engrafted with subcutaneous tumors 5-10 mm in diameter that were induced by exposure to doxycycline. Half of the mice stopped taking doxycycline-containing water, whereas the other half kept taking the water. Although the tumors in the mice taking doxycycline continued to grow, tumor growth in the mice not taking doxycycline was significantly suppressed. The myeloma cells in the tumors of the mice not taking doxycycline expressed PU.1 and TRAIL and many of such cells were apoptotic. Moreover, the expression of a cell proliferation marker Ki67 was significantly decreased in tumors from the mice not taking doxycycline, compared with that of tumors from the mice continuously taking doxycycline. The present data strongly suggest that PU.1 functions as a tumor suppressor of myeloma cells in vivo.

Analysis of deposited peptides in amyloid lesions of AL amyloidosis by mass spectrometry.

Although immunoglobulin light chain (LC) plays a critical role in AL amyloidosis, detailed characteristics of deposited LC are not well known. In this study, LC peptides were analyzed by a combination of laser microdissection, liquid chromatography, and mass spectrometry (LC-MS/MS) in 65 patients with AL amyloidosis. Constant regions of LC were detected in all patients. Kappa- or lambda-types were detected in 20 and 45 patients, respectively. Various types of constant regions of LC were found; however, IGLC3 and IGKC4 were the most frequently detected. Besides LC, apolipoproteins and vitronectin were repeatedly found in amyloid lesions. These results suggest marked heterogeneity in terms of subtype of constant regions of LC in amyloid lesions. Immunohistochemistry identified LC in approximately half of patients in whom LC was detected by LC-MS/MS. This finding indicates superiority of LC-MS/MS over IHC for the detection of LC.

An IL-27/Stat3 axis induces expression of programmed cell death 1 ligands (PD-L1/2) on infiltrating macrophages in lymphoma.

Immune escape and tolerance in the tumor microenvironment are closely involved in tumor progression, and are caused by T-cell exhaustion and mediated by the inhibitory signaling of immune checkpoint molecules including programmed death-1 (PD-1), cytotoxic T-lymphocyte associated protein 4, and T-cell immunoglobulin and mucin domaincontaining molecule-3. In the present study, we investigated the expression of the PD-1 ligand 1 (PD-L1) in a lymphoma microenvironment using paraffin-embedded tissue samples, and subsequently studied the detailed mechanism of upregulation of PD-L1 on macrophages using cultured human macrophages and lymphoma cell lines. We found that macrophages in lymphoma tissues of almost all cases of adult T-cell leukemia/lymphoma (ATLL), follicular lymphoma and diffuse large B-cell lymphoma expressed PD-L1. Cell culture studies showed that the conditioned medium of ATL-T and SLVL cell lines induced increased expression of PD-L1/2 on macrophages, and that this PD-L1/2 overexpression was dependent on activation of signal transducer and activator of transcription 3 (Stat3). In vitro studies including cytokine array analysis showed that IL-27 (heterodimer of p28 and EBI3) induced overexpression of PD-L1/2 on macrophages via Stat3 activation. Because lymphoma cell lines produced IL-27B (EBI3) but not IL-27p28, it was proposed that the IL-27p28 derived from macrophages and the IL-27B (EBI3) derived from lymphoma cells formed an IL-27 (heterodimer) that induced PD-L1/2 overexpression. Although the significance of PD-L1/2 expressions on macrophages in lymphoma progression has never been clarified, an IL-27-Stat3 axis might be a target for immunotherapy for lymphoma patients.

Immunomodulatory drugs act as inhibitors of DNA methyltransferases and induce PU.1 up-regulation in myeloma cells.

Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide are efficacious in the treatment of multiple myeloma and significantly prolong their survival. However, the mechanisms of such effects of IMiDs have not been fully elucidated. Recently, cereblon has been identified as a target binding protein of thalidomide. Lenalidomide-resistant myeloma cell lines often lose the expression of cereblon, suggesting that IMiDs act as an anti-myeloma agent through interacting with cereblon. Cereblon binds to damaged DNA-binding protein and functions as a ubiquitin ligase, inducing degradation of IKZF1 and IKZF3 that are essential transcription factors for B and T cell development. Degradation of both IKZF1 and IKZF3 reportedly suppresses myeloma cell growth. Here, we found that IMiDs act as inhibitors of DNA methyltransferases (DMNTs). We previously reported that PU.1, which is an ETS family transcription factor and essential for myeloid and lymphoid development, functions as a tumor suppressor in myeloma cells. PU.1 induces growth arrest and apoptosis of myeloma cell lines. In this study, we found that low-dose lenalidomide and pomalidomide up-regulate PU.1 expression through inducing demethylation of the PU.1 promoter. In addition, IMiDs inhibited DNMT1, DNMT3a, and DNMT3b activities in vitro. Furthermore, lenalidomide and pomalidomide decreased the methylation status of the whole genome in myeloma cells. Collectively, IMiDs exert demethylation activity through inhibiting DNMT1, 3a, and 3b, and up-regulating PU.1 expression, which may be one of the mechanisms of the anti-myeloma activity of IMiDs.

Transcriptional dysregulation of the deleted in colorectal carcinoma gene in multiple myeloma and monoclonal gammopathy of undetermined significance.

The deleted in colorectal carcinoma (DCC) gene at 18q21 encodes a netrin-1 receptor, a tumor suppressor that prevents cell growth. While allele loss or decreased expression of DCC has been associated with the progression of solid tumors and hematologic malignancies, including leukemias and malignant lymphomas, its involvement has not been evaluated in multiple myeloma (MM), a plasma cell malignancy characterized by complex and heterogenous molecular abnormalities. We here show that 10 of 11 human myeloma-derived cell lines (HMCLs) expressed non-translated aberrant DCC transcriptional variants, in which exon 2 fuses with intron 1 instead of exon 1 (mt.DCC). Among them, two co-expressed wild type transcripts (wt.DCC), while eight co-expressed the splicing variant (sv.DCC) lacking exon 1. The remaining HMCL expressed only sv.DCC. In addition, analyses revealed that there were two types of mt.DCC that differed in their fusion of intron 1 with exon 2. In patient-derived samples from 30 MM and 8 monoclonal gammopathy of undetermined significance (MGUS) patients, wt.DCC was expressed in 53% of MM, but not in MGUS, while 23% of MM and 75% of MGUS expressed only sv.DCC. Considering that 25% of MGUS, 57% of MM, and 91% HMCLs expressed mt.DCC, our results suggest that the acquisition of mt.DCC might be a secondary genetic change in plasma cell dyscrasia.

RUNX1 regulates the CD34 gene in haematopoietic stem cells by mediating interactions with a distal regulatory element.

The transcription factor RUNX1 is essential to establish the haematopoietic gene expression programme; however, the mechanism of how it activates transcription of haematopoietic stem cell (HSC) genes is still elusive. Here, we obtained novel insights into RUNX1 function by studying regulation of the human CD34 gene, which is expressed in HSCs. Using transgenic mice carrying human CD34 PAC constructs, we identified a novel downstream regulatory element (DRE), which is bound by RUNX1 and is necessary for human CD34 expression in long-term (LT)-HSCs. Conditional deletion of Runx1 in mice harbouring human CD34 promoter-DRE constructs abrogates human CD34 expression. We demonstrate by chromosome conformation capture assays in LT-HSCs that the DRE physically interacts with the human CD34 promoter. Targeted mutagenesis of RUNX binding sites leads to perturbation of this interaction and decreased human CD34 expression in LT-HSCs. Overall, our in vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34.

PU.1 is a potent tumor suppressor in classical Hodgkin lymphoma cells.

PU.1 has previously been shown to be down-regulated in classical Hodgkin lymphoma (cHL) cells via promoter methylation. We performed bisulfite sequencing and proved that the promoter region and the -17 kb upstream regulatory element of the PU.1 gene were highly methylated. To evaluate whether down-regulation of PU.1 is essential for the growth of cHL cells, we conditionally expressed PU.1 in 2 cHL cell lines, L428 and KM-H2. Overexpression of PU.1 induced complete growth arrest and apoptosis in both cell lines. Furthermore, in a Hodgkin lymphoma tumor xenograft model using L428 and KM-H2 cell lines, overexpression of PU.1 led to tumor regression or stable disease. Lentiviral transduction of PU.1 into primary cHL cells also induced apoptosis. DNA microarray analysis revealed that among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428 cells after PU.1 induction. Stable knockdown of p21 rescued PU.1-induced growth arrest in L428 cells, suggesting that the growth arrest and apoptosis observed are at least partially dependent on p21 up-regulation. These data strongly suggest that PU.1 is a potent tumor suppressor in cHL and that induction of PU.1 with demethylation agents and/or histone deacetylase inhibitors is worth exploring as a possible therapeutic option for patients with cHL.

Complete remission achieved in a multiple myeloma patient with elevated serum KL-6 level by a combination regimen with bortezomib, cyclophosphamide, and dexamethasone.

A 79-year-old woman suffering from double vision after a 4-year history of MGUS was referred to our hospital. MRI revealed that she had three intracranial plasmacytoma masses and one spinal plasmacytoma mass. Bone marrow aspirates showed 52.4% plasma cell infiltration and immunoelectrophoresis identified serum IgG-M protein, leading to a diagnosis of IgG-type multiple myeloma. IgG was elevated to 3,355 mg/dl and urine type Bence-Jones protein was positive. KL-6, a membrane-bound glycoprotein encoded by Mucin 1 and a marker of interstitial pneumonia, was also elevated to 1,409 mg/dl, but computed tomography of the lungs revealed no obvious pulmonary lesions. Previously reported studies showing that myeloma patients with elevated KL-6 might have a poor prognosis prompted us to treat this patient with a three-drug (bortezomib, cyclophosphamide, and dexamethasone: VCD) combination regimen. When 6 cycles of the regimen had been completed, no M-protein was detectable in her serum. Furthermore, κ free light chain had significantly decreased from 12,700 to 24.8 mg/l. In addition, (18)F-FDG PET/CT revealed reduced mass sizes and no (18)F-FDG uptakes by plasmacytomas. Thus, she was defined as having achieved a stringent complete remission (sCR). We therefore concluded that the VCD combination regimen was highly effective in this multiple myeloma patient with KL-6 elevation.

[Regression of primary rectal MALT lymphoma after Helicobacter pylori eradication].

A 74-year-old-man was referred to our hospital to undergo examination and therapy for a rectal polypoid lesion. MALT lymphoma of the rectum (stage IE) was diagnosed by performing a biopsy of the tumor during colonoscopy. The patient received eradication therapy against Helicobacter pylori (H. pylori) because of chronic gastritis due to H. pylori, and a complete remission was documented after this therapy. However, MALT lymphoma recurred 18 months later. Our observations in this case suggest that eradication therapy against H. pylori may be useful not only for gastric MALT lymphoma but also for colorectal MALT lymphoma.

Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1.

Transcription factors are believed to have a dominant role in acute myeloid leukemia (AML). This idea is supported by analysis of gene-knockout mice, which uncovered crucial roles of several transcription factors in normal hematopoiesis, and of individuals with leukemia, in whom transcription factors are frequently downregulated or mutated. However, analysis of knockout animals has not shown a direct link between abrogated transcription factors and the pathogenesis of AML. Sfpi1, encoding the lineage-specific transcription factor PU.1, is indispensable for normal myeloid and lymphoid development. We found that mice carrying hypomorphic Sfpi1 alleles that reduce PU.1 expression to 20% of normal levels, unlike mice carrying homo- or heterozygous deletions of Sfpi1, developed AML. Unlike complete or 50% loss, 80% loss of PU.1 induced a precancerous state characterized by accumulation of an abnormal precursor pool retaining responsiveness to G-CSF with disruption of M- and GM-CSF pathways. Malignant transformation was associated with a high frequency of clonal chromosomal changes. Retroviral restoration of PU.1 expression rescued myeloid differentiation of mutant progenitors and AML blasts. These results suggest that tightly graded reduction, rather than complete loss, of a lineage-indispensable transcription factor can induce AML.

Relationship between Serum Bortezomib Concentration and Emergence of Diarrhea in Patients with Multiple Myeloma and/or AL Amyloidosis.

(1) Background: multiple myeloma patients have benefited from bortezomib therapy, though it has often been discontinued owing to diarrhea. The objective of this study was to verify serum bortezomib concentration in the emergence of diarrhea. (2) Methods: this prospective, observational case-control, and monocentric study was performed with an approval by the Ethics Committee of Kumamoto University Hospital in 2015 (No. 1121) from February 2015 to April 2017. (3) Results: twenty-four patients with bortezomib therapy were recruited; eight patients (33.3%) developed diarrhea at day 3 as median. Median measured trough bortezomib concentration at 24 h after first or second dose for patients with or without diarrhea was 0.87 or 0.48 ng/mL, respectively (p = 0.04, Wilcoxon signed rank test). Receiver operation characteristic (ROC) analysis produced the cut-off concentration of 0.857 ng/mL (area under the ROC curve of 0.797, sensitivity of 0.625, specificity of 0.875). The survival curves between patients with and without diarrhea were similar (p = 0.667); those between patients with higher and lower concentration than median value (0.61 ng/mL) were also similar (p = 0.940). (4) Conclusions: this study indicated the possible involvement of serum bortezomib concentration in the emergence of diarrhea in bortezomib therapy in patients with multiple myeloma.

Down-regulation of PU.1 by methylation of distal regulatory elements and the promoter is required for myeloma cell growth.

The transcription factor PU.1 is essential for myeloid and B-cell development. Down-regulation of PU.1 by disruption of its 14-kb 5' upstream regulatory element induced acute myeloid leukemia, T-cell lymphoma, and chronic lymphocytic leukemia-like disease in murine models. In the present study, we found that PU.1 was down-regulated in the majority of human myeloma cell lines and a subset of freshly isolated myeloma cells, in contrast to relatively high expression of PU.1 in normal plasma cells. Patients in this low PU.1 expression subset may have a poor prognosis. In human myeloma cell lines, the 17-kb 5' upstream enhancer and the promoter region of the PU.1 gene were highly methylated, and this is consistent with disappearance of DNase I-hypersensitive sites in these regions. To elucidate the significance of down-regulation of PU.1, we generated stable myeloma cell lines with an inducible PU.1 expression system. Exogenous expression of PU.1 in PU.1 null myeloma cell lines, U266 and KMS12PE, induced complete growth arrest and cell death. Up-regulation of PU.1 by 5-aza-2'-deoxycytidine also induced growth arrest of KMS12PE and KHM11 myeloma cells. These data suggest that down-regulation of PU.1 is an essential step for the survival of a subset of myeloma cells and that up-regulation of PU.1 by demethylation agents or other types of agents may represent a new therapeutic strategy for treatment of multiple myeloma patients.

Base-to-apex gradient pattern of cardiac impairment identified on myocardial T1 mapping in cardiac amyloidosis.

Late gadolinium enhancement imaging by cardiac magnetic resonance imaging (CMR) is the most reliable method for identifying cardiac involvement in patients with amyloidosis, and myocardial T1 mapping is a novel CMR technique that enables the noninvasive detection and quantification of myocardial amyloid burden. Although, base-to-apex gradient patterns of impairment in patients with cardiac amyloidosis have been reported on myocardial strain analysis using echocardiography, we could not find any other reports to demonstrate that myocardial T1 mapping on CMR can clearly identify a base-to-apex gradient pattern of cardiac impairment in a patient with cardiac amyloidosis.

Expression of IL-34 correlates with macrophage infiltration and prognosis of diffuse large B-cell lymphoma.

OBJECTIVES: Infiltration of macrophages through the tyrosine kinase receptor CSF1R is a poor prognosis factor in various solid tumors. Indeed, these tumors produce CSF1R ligand, macrophage colony-stimulating factor (M-CSF) or interleukin-34 (IL-34). However, the significance of these cytokines, particularly, the newly discovered IL-34 in haematological malignancies, is not fully understood. We therefore analysed the role of IL-34 in diffuse large B-cell lymphoma (DLBCL), the most common subtype of malignant lymphoma. METHODS: We analysed formalin-fixed paraffin-embedded lymphoma tissues of 135 DLBCL patients for the expression of IL-34 and the number of macrophages, and the survival of these patients. The expression of IL-34 in DLBCL cell lines and the activity of IL-34 to induce the migration of monocytic cells were also characterised. RESULTS: Several lymphoma tissues showed a clear IL-34 signal, and such signal was detectable in 36% of patients. DLBCL cell lines also expressed IL-34. Interestingly, the percentage of IL-34+ patients in the activated B-cell subtype was significantly higher than that in the germinal centre B-cell subtype. More interestingly, IL-34+ patients showed shorter survival periods and higher number of macrophages in lymphoma tissues. The recruitment of monocytes is likely the first step for the higher macrophage density in the IL-34+ lymphoma tissues. Indeed, IL-34 induced the migration of monocytic cells. CONCLUSION: Our results raise the possibility that IL-34 in lymphoma tissues of DLBCL patients recruits monocytes, leading to the higher number of macrophages in the tissues and poor prognosis of patients. IL-34 may be an additional therapeutic target of DLBCL.

Induction of cell cycle arrest and apoptosis in myeloma cells by cepharanthine, a biscoclaurine alkaloid.

Cepharanthine (CEP), a biscoclaurine alkaloid extracted from Stephania Cepharantha Hayata, has been used in Japan for treating patients with radiation-induced leucopenia or thrombocytopenia. We treated a patient with multiple myeloma (MM), who was not responding to preceding chemotherapy, who coincidently received therapy with CEP due to thrombocytopenia. Since the case showed a marked reduction of tumor load, direct anti-tumor effects of CEP to myeloma cells were investigated in vitro. Anti-tumor effects were observed in all myeloma cell lines tested, including a line resistant to melphalan. Exposure to CEP of a myeloma cell line induced the production of reactive oxygen species, activated the caspase-3 pathway and eventually induced apoptosis. Pre-exposure of cells to a pan-caspase inhibitor, Z-VAD-FMK, or a free radical scavenger, Tiron, effectively blocked CEP-induced apoptosis. Interestingly, CEP also inhibited cell growth of myeloma cells by inducing CDK inhibitors. These data show, for the first time, that CEP has anti-myeloma effects by the activation of apoptotic pathways and blocking cell cycle progression via CDK inhibitors. Although analysis of these two pathways should be clarified further, the use of CEP may be considered as a potential therapeutic agent for a subset of MM.

Potential autoregulation of transcription factor PU.1 by an upstream regulatory element.

Regulation of the hematopoietic transcription factor PU.1 (Spi-1) plays a critical role in the development of white cells, and abnormal expression of PU.1 can lead to leukemia. We previously reported that the PU.1 promoter cannot induce expression of a reporter gene in vivo, and cell-type-specific expression of PU.1 in stable lines was conferred by a 3.4-kb DNA fragment including a DNase I hypersensitive site located 14 kb upstream of the transcription start site. Here we demonstrate that this kb -14 site confers lineage-specific reporter gene expression in vivo. This kb -14 upstream regulatory element contains two 300-bp regions which are highly conserved in five mammalian species. In Friend virus-induced erythroleukemia, the spleen focus-forming virus integrates into the PU.1 locus between these two conserved regions. DNA binding experiments demonstrated that PU.1 itself and Elf-1 bind to a highly conserved site within the proximal homologous region in vivo. A mutation of this site abolishing binding of PU.1 and Elf-1 led to a marked decrease in the ability of this upstream element to direct activity of reporter gene in myelomonocytic cell lines. These data suggest that a potential positive autoregulatory loop mediated through an upstream regulatory element is essential for proper PU.1 gene expression.