菊池 次郎

Not available.

Abstract Background The immunomodulatory drug lenalidomide, which is now widely used for the treatment of multiple myeloma (MM), exerts pharmacological action through the ubiquitin‐dependent degradation of IKZF1 and subsequent down‐regulation of interferon regulatory factor 4 (IRF4), a critical factor for the survival of MM cells. IKZF1 acts principally as a tumour suppressor via transcriptional repression of oncogenes in normal lymphoid lineages. In contrast, IKZF1 activates IRF4 and other oncogenes in MM cells, suggesting the involvement of unknown co‐factors in switching the IKZF1 complex from a transcriptional repressor to an activator. The transactivating components of the IKZF1 complex might promote lenalidomide resistance by residing on regulatory regions of the IRF4 gene to maintain its transcription after IKZF1 degradation. Methods To identify unknown components of the IKZF1 complex, we analyzed the genome‐wide binding of IKZF1 in MM cells using chromatin immunoprecipitation‐sequencing (ChIP‐seq) and screened for the co‐occupancy of IKZF1 with other DNA‐binding factors on the myeloma genome using the ChIP‐Atlas platform. Results We found that c‐FOS, a member of the activator protein‐1 (AP‐1) family, is an integral component of the IKZF1 complex and is primarily responsible for the activator function of the complex in MM cells. The genome‐wide screening revealed the co‐occupancy of c‐FOS with IKZF1 on the regulatory regions of IKZF1‐target genes, including IRF4 and SLAMF7, in MM cells but not normal bone marrow progenitors, pre‐B cells or mature T‐lymphocytes. c‐FOS and IKZF1 bound to the same consensus sequence as the IKZF1 complex through direct protein‐protein interactions. The complex also includes c‐JUN and IKZF3 but not IRF4. Treatment of MM cells with short‐hairpin RNA against FOS or a selective AP‐1 inhibitor significantly enhanced the anti‐MM activity of lenalidomide in vitro and in two murine MM models. Furthermore, an AP‐1 inhibitor mitigated the lenalidomide resistance of MM cells. Conclusions C‐FOS determines lenalidomide sensitivity and mediates drug resistance in MM cells as a co‐factor of IKZF1 and thus, could be a novel therapeutic target for further improvement of the prognosis of MM patients.

Extramedullary disease (EMD) is known to be associated with chemoresistance and poor prognosis in multiple myeloma (MM); however, the mechanisms of its development are not fully understood. Elucidating the mechanism of EMD development and its therapeutic targeting would greatly contribute to further improvement of treatment outcome in MM patients. Here, we show that bone marrow stroma cell-derived hyaluronan elicits homophilic interactions of MM cells by binding to surface CD44, especially long-stretch variants, under physiological shear stress and generates cell clusters that might develop into EMD. We recapitulated the development of EMD via administration of hyaluronan in a syngeneic murine MM model in a CD44-dependent manner. Hyaluronan-induced MM cell clusters exhibited the specific resistance to proteasome inhibitors (PIs) in vitro and in murine models via γ-secretase-mediated cleavage of the intracellular domains of CD44, which in turn transactivated PI resistance-inducible genes. Treatment of hyaluronan-injected mice with anti-CD44 antibody or γ-secretase inhibitors readily suppressed the development of EMD from transplanted MM cells and significantly prolonged the survival of recipients by overcoming PI resistance. The hyaluronan-CD44 axis represents a novel pathway to trigger EMD development and could be a target of the prediction, prevention, and treatment of EMD in MM patients.

We report expression and purification of a FLT3 protein with ITD mutation (FLT3-ITD) with a steady tyrosine kinase activity using a silkworm-baculovirus system, and its application as a fast screening system of tyrosine kinase inhibitors. The FLT3-ITD protein was expressed in Bombyx mori L. pupae infected by gene-modified nucleopolyhedrovirus, and was purified as an active state. We performed an inhibition assay using 17 kinase inhibitors, and succeeded in screening two inhibitors for FLT3-ITD. The result has paved the way for screening FLT3-ITD inhibitors in a fast and easy manner, and also for structural studies.

Mantle cell lymphoma (MCL) is usually resistant to the current standard-of-care regimens and also to novel agents such as the proteasome inhibitor bortezomib. A better prognosis of leukemic variants of MCL suggests that MCL cells acquire drug resistance in nodal and/or bone marrow microenvironments via interaction with supporting cells. Bortezomib exerts cytotoxic action in MCL cells via stabilization of the pro-apoptotic BCL-2 family protein NOXA. Here we show that autophagic degradation of NOXA is a mechanism of bortezomib resistance in MCL cells in a tumor microenvironment. First, we demonstrated that interaction with bone marrow-derived or nodal stromal cells conferred bortezomib resistance to MCL cells in vitro and in a murine model. Co-culture of MCL cells with stromal cells enhanced bortezomib-induced ubiquitination and subsequent binding of NOXA to the p62 adaptor, which escorted NOXA to the lysosome for autophagic degradation. Finally, we found that not only direct contact with stromal cells but also stroma-derived humoral factors, especially interleukin-6, promoted selective autophagy and NOXA degradation in MCL cells. Targeting protective autophagy, for example, using the lysosome inhibitor chloroquine, might increase the efficacy of bortezomib-containing regimens in MCL.

Not available.

Chronic myeloid leukemia is driven by the BCR-ABL oncoprotein, a constitutively active protein tyrosine kinase. Although tyrosine kinase inhibitors (TKIs) have greatly improved the prognosis of CML patients, the emergence of TKI resistance is an important clinical problem, which deserves additional treatment options based on unique biological properties to CML cells. In this study, we show that metabolic homeostasis is critical for survival of CML cells, especially when the disease is in advanced stages. The BCR-ABL protein activates AMP-activated protein kinase (AMPK) for ATP production and the mTOR pathway to suppress autophagy. BCR-ABL is detected in the nuclei of advanced-stage CML cells, in which ATP is sufficiently supplied by enhanced glucose metabolism. AMP-activated protein kinase is further activated under energy-deprived conditions and triggers autophagy through ULK1 phosphorylation and mTOR inhibition. In addition, AMPK phosphorylates 14-3-3 and Beclin 1 to facilitate cytoplasmic translocation of nuclear BCR-ABL in a BCR-ABL/14-3-3τ/Beclin1/XPO1 complex. Cytoplasmic BCR-ABL protein undergoes autophagic degradation when intracellular ATP is exhausted by disruption of the energy balance or forced autophagy flux with AMP mimetics, mTOR inhibitors, or arsenic trioxide, leading to apoptotic cell death. This pathway represents a novel therapeutic vulnerability that could be useful for treating TKI-resistant CML.

The treatment outcome of multiple myeloma (MM) is worse than expected from the average numbers of non-synonymous mutations, which are roughly correlated with the prognosis of cancer patients. The refractoriness of MM may be ascribed to the complex genomic architecture and clonal behavior of the disease. In MM, disease progression is accomplished by branching patterns of subclonal evolution from reservoir clones with a propagating potential and/or the emergence of minor clones, which already exist at the MGUS stage and outcompete other clones through selective pressure mainly by therapeutic agents. Each subclone harbors novel mutations and distinct phenotypes including drug sensitivities. In general, mature clones are highly sensitive to proteasome inhibitors (PIs), whereas immature clones are resistant to PIs but could be eradicated by immunomodulatory drugs (IMiDs). The branching evolution is a result of the fitness of different clones to microenvironment and their evasion of immune surveillance; therefore, IMiDs are effective for MM with this pattern of evolution. In contrast, ~ 20% of MM evolve neutrally in the context of strong oncogenic drivers, such as high-risk IgH translocations, and are relatively resistant to IMiDs. Further understanding of the genomic landscape and the pattern of clonal evolution may contribute to the development of more effective treatment strategies for MM.

SLAMF7 is expressed mainly on multiple myeloma (MM) cells and considered an ideal target for immunotherapeutic approaches. Indeed, elotuzumab, an anti-SLAMF7 antibody, is used for the treatment of MM in combination with immunomodulatory drugs. SLAMF7 is cleaved via unknown mechanisms and detected as a soluble form (sSLAMF7) exclusively in the serum of MM patients; however, little is known about the role of sSLAMF7 in MM biology. In this study, we found that sSLAMF7 enhanced the growth of MM cells via homophilic interaction with surface SLAMF7 and subsequent activation of the SHP-2 and ERK signaling pathways. Elotuzumab suppressed sSLAMF7-induced MM cell growth both in vitro and in vivo. Promoter analyses identified IKZF1 (Ikaros) as a pivotal transcriptional activator of the SLAMF7 gene. Pharmacological targeting of Ikaros by lenalidomide and its analog pomalidomide downregulated SLAMF7 expression and ameliorated the response of MM cells to sSLAMF7. Elotuzumab blocked the growth-promoting function of sSLAMF7 when combined with lenalidomide in a murine xenograft model. Neutralization of sSLAMF7 is a novel antimyeloma mechanism of elotuzumab, which is enhanced by immunomodulatory drugs via downregulation of surface SLAMF7 expression on MM cells. These findings may provide important information for the optimal use of elotuzumab in MM treatment.

Multiple myeloma (MM) is among the most intractable of malignancies and is characterized by uncontrolled growth of malignant plasma cells in the bone marrow (BM). Elucidation of the mechanisms underlying cell adhesion-mediated drug resistance (CAM-DR) may prolong remission and ultimately improve the survival of MM patients. Toward this goal, we identified trimethylation of histone H3 at lysine-27 (H3K27me3) as a critical histone modification associated with CAM-DR. Cell adhesion counteracted drug-induced hypermethylation of H3K27 via inhibiting phosphorylation of enhancer of zeste homolog 2 (EZH2), and promoted sustained expression of anti-apoptotic genes. In addition, we found that CD180, a non-canonical lipopolysaccharide (LPS) receptor, was markedly up-regulated in response to adherence and/or hypoxic conditions. Bacterial LPS enhanced the growth of MM cells both in vitro and in vivo, correlating with expression of CD180. Promoter analyses identified Ikaros (IKZF1) as a pivotal transcriptional activator of the CD180 gene; expression of CD180 was activated via cell adhesion- and/or hypoxia-mediated increases in IKZF1 expression. Administration of lenalidomide prevented the LPS-triggered activation of MM cells by targeting CD180. Taken together, our results suggest that lenalidomide-mediated prevention of LPS-triggered disease progression may be an effective means for prolonging survival in patients with MM.

Multiple myeloma (MM) cells acquire dormancy and drug resistance via interaction with bone marrow stroma cells (BMSC) in a hypoxic microenvironment. Elucidating the mechanisms underlying the regrowth of dormant clones may contribute to further improvement of the prognosis of MM patients. In this study, we find that the CD180/MD-1 complex, a noncanonical lipopolysaccharide (LPS) receptor, is expressed on MM cells but not on normal counterparts, and its abundance is markedly upregulated under adherent and hypoxic conditions. Bacterial LPS and anti-CD180 antibody, but not other Toll-like receptor ligands, enhanced the growth of MM cells via activation of MAP kinases ERK and JNK in positive correlation with expression levels of CD180. Administration of LPS significantly increased the number of CD180/CD138 double-positive cells in a murine xenograft model when MM cells were inoculated with direct attachment to BMSC. Knockdown of CD180 canceled the LPS response in vitro and in vivo. Promoter analyses identified IKZF1 (Ikaros) as a pivotal transcriptional activator of the CD180 gene. Both cell adhesion and hypoxia activated transcription of the CD180 gene by increasing Ikaros expression and its binding to the promoter region. Pharmacological targeting of Ikaros by the immunomodulatory drug lenalidomide ameliorated the response of MM cells to LPS in a CD180-dependent manner in vitro and in vivo. Thus, the CD180/MD-1 pathway may represent a novel mechanism of growth regulation of MM cells in a BM milieu and may be a therapeutic target of preventing the regrowth of dormant MM cells. Significance: This study describes a novel mechanism by which myeloma cells are regulated in the bone marrow, where drug resistance and dormancy can evolve after treatment, with potential therapeutic implications for treating this often untreatable blood cancer.

Human induced pluripotent stem cells (hiPSCs) are creating great expectations for regenerative medicine. However, safety strategies must be put in place to guard against teratoma formation after transplantation of hiPSC-derived cells into patients. Recent studies indicate that epigenetic regulators act at the initial step of tumorigenesis. Using gain-of-function and loss-of-function approaches, we show here that the expression and function of lysine-specific demethylase 1 (LSD1) are tightly regulated in hiPSCs, and their deregulation underlies the development of teratomas. Consistent with these results, we demonstrate that an LSD1 inhibitor, S2157, prevented teratoma formation from hiPSCs transplanted into immunodeficient mice. This novel action of LSD1 and the effects of its inhibition potentially allow for the development of new clinical applications and therapeutic strategies using hiPSCs.

Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin) in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17; 19) ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17; 19) ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17; 19) ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.

Multiple myeloma cells acquire the resistance to anti-cancer drugs through physical and functional interactions with the bone marrow microenvironment via two overlapping mechanisms. First, bone marrow stromal cells (BMSCs) produce soluble factors, such as interleukin-6 and insulin-like growth factor-1, to activate signal transduction pathways leading to drug resistance (soluble factor-mediated drug resistance). Second, BMSCs up-regulate the expression of cell cycle inhibitors, anti-apoptotic members of the Bcl-2 family and ABC drug transporters in myeloma cells upon direct adhesion [cell adhesion-mediated drug resistance (CAM-DR)]. Elucidation of the mechanisms underlying drug resistance may greatly contribute to the advancement of cancer therapies. Recent investigations, including ours, have revealed the involvement of epigenetic alterations in drug resistance especially CAM-DR. For example, we found that class I histone deacetylases (HDACs) determine the sensitivity of proteasome inhibitors and the histone methyltransferase EZH2 regulates the transcription of anti-apoptotic genes during the acquisition of CAM-DR by myeloma cells. In addition, another histone methyltransferase MMSET was shown to confer drug resistance to myeloma cells by facilitating DNA repair. These findings provide a rationale for the inclusion of epigenetic drugs, such as HDAC inhibitors and histone methylation modifiers, in combination chemotherapy for MM patients to increase the therapeutic index.

S-phase progression of the cell cycle is accelerated in tumors through various genetic abnormalities, and, thus, pharmacologic inhibition of altered cell-cycle progression would be an effective strategy to control tumors. In the current study, we analyzed the antileukemic activity of three available small molecules targeting CDK4/CDK6 against lymphoid crisis of chronic myeloid leukemia (CML-LC) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), and found that all three molecules showed specific activities against leukemic cell lines derived from CML-LC and Ph+ ALL. In particular, PD0332991 exhibited extremely high antileukemic activity against CML-LC and Ph+ ALL cell lines in the nanomolar range by the induction of G(0)-G(1) arrest and partially cell death through dephosphorylation of pRb and downregulation of the genes that are involved in S-phase transition. As an underlying mechanism for favorable sensitivity to the small molecules targeting CDK4/CDK6, cell-cycle progression of Ph+ lymphoid leukemia cells was regulated by transcriptional and posttranscriptional modulation of CDK4 as well as Cyclin D2 gene expression under the control of BCR-ABL probably through the PI3K pathway. Consistently, the gene expression level of Cyclin D2 in Ph+ lymphoid leukemia cells was significantly higher than that in Ph+ lymphoid leukemia cells. Of note, three Ph+ ALL cell lines having the T315I mutation also showed sensitivity to PD0332991. In a xenograft model, PD0332991, but not imatinib, suppressed dissemination of Ph+ ALL having the T315I mutation and prolonged survival, demonstrating that this reagent would be a new therapeutic modality for relapsed CML-LC and Ph+ ALL patients after treatment with tyrosine kinase inhibitors. (C) 2015 AACR.

Alterations in chromatin modifications, such as histone methylation, have been suggested as mediating chemotherapy resistance in several cancer types; therefore, elucidation of the epigenetic mechanisms that underlie drug resistance may greatly contribute to the advancement of cancer therapies. In the present study, we identified histone H3-lysine 27 (H3K27) as a critical residue for epigenetic modification in multiple myeloma. We determined that abrogation of drug-induced H3K27 hypermethylation is associated with cell adhesion-mediated drug resistance (CAM-DR), which is the most important form of drug resistance, using a coculture system to evaluate stroma cell adhesion-dependent alterations in multiple myeloma cells. Cell adhesion counteracted anticancer drug-induced hypermethylation of H3K27 via inactivating phosphorylation of the transcription regulator EZH2 at serine 21, leading to the sustained expression of antiapoptotic genes, including IGF1, B cell CLL/Iymphoma 2 (BCL2), and hypoxia inducible factor 1, alpha subunit (H/F1A). Pharmacological and genetic inhibition of the IGF-1R/P13K/AKT pathway reversed CAM-DR by promoting EZH2 dephosphorylation and H3K27 hypermethylation both in vitro and in refractory murine myeloma models. Together, our findings identify and characterize an epigenetic mechanism that underlies CAM-DR and suggest that kinase inhibitors to counteract EZH2 phosphorylation should be included in combination chemotherapy to increase therapeutic index.

Recent investigations indicate that epigenetic regulators act at the initial step of myeloid leukemogenesis by forming preleukemic hematopoietic stem cells (HSCs), which possess the increased self-renewal potential but retain multidifferentiation ability, and synergize with genetic abnormalities in later stages to develop full-blown acute myeloid leukemias. However, it is still unknown whether this theory is applicable to other malignancies. In this study, we demonstrate that lysine-specific demethylase 1 (LSD1) overexpression is a founder abnormality for the development of T-cell lymphoblastic leukemia/lymphoma (T-LBL) using LSD1 transgenic mice. LSD1 expression is tightly regulated via alternative splicing and transcriptional repression in HSCs and is altered in most leukemias, especially T-LBL. Overexpression of the shortest isoform of LSD1, which is specifically repressed in quiescent HSCs and demethylates histone H3K9 more efficiently than other isoforms, increases self-renewal potential via upregulation of the HoxAfamily but retains multidifferentiation ability with a skewed differentiation to T-cell lineages at transcriptome levels in HSCs. Transgenic mice overexpressing LSD1 in HSCs did not show obvious abnormalities but developed T-LBL at very high frequency after g-irradiation. LSD1 overexpression appears to be the first hit in T-cell leukemogenesis and provides an insight into novel strategies for early diagnosis and effective treatment of the disease.

Multiple myeloma (MM), one of the most intractable malignancies, is characterized by the infiltration and growth of plasma cells, the most differentiated cells in the B-cell lineage, in the bone marrow. Despite the introduction of novel therapeutic agents, including proteasome inhibitors and immunomodulatory drugs, the prognosis of patients with MM is still worse than that of most hematological malignancies. A better understanding of the molecular pathogenesis of the disease is essential to achieve any improvement of treatment outcome of MM patients. All MM cases pass through the phase of asymptomatic expansion of clonal plasma cells, referred to as monoclonal gammopathy of undetermined significance (MGUS). It has long been believed that MM evolves linearly from MGUS to terminal phases, such as extramedullary tumors and plasma cell leukemia via the accumulation of novel mutations. However, recent studies using next-generation sequencing have disclosed the complex genomic architecture of the disease. At each step of progression, the acquisition of novel mutations is accompanied by subclonal evolution from reservoir clones with branching patterns. Each subclone may carry novel mutations and distinct phenotypes, including drug sensitivity. In addition, minor clones already exist at the MGUS stage, which could expand later in the clinical course, resulting in relapse and/or leukemic conversion. The ultimate goal of treatment is to eradicate all clones, including subclonal populations with distinct biological characteristics. This goal could be achieved by further improving treatment strategies that reflect the genomic landscape of the disease.

Tumor suppressor protein p19(ARF) (Arf; p14(ARF) in humans) functions in both p53-dependent and -independent modes to counteract hyper-proliferative signals caused by proto-oncogene activation, but its p53-independent activities remain poorly understood. Using the tandem affinity purification-tag technique, we purified Arf-containing protein complexes and identified p68 DEAD-box protein (DDX5) as a novel interacting protein of Arf. In this study, we found that DDX5 interacts with c-Myc, and harbors essential roles for c-Myc-mediated transcription and its transforming activity. Furthermore, when c-Myc was forcibly expressed, the expression level of DDX5 protein was drastically increased through the acceleration of protein synthesis of DDX5, suggesting the presence of an oncogenic positive feedback loop including c-Myc and DDX5. Strikingly, Arf blocked the physical interaction between DDX5 and c-Myc, and drove away DDX5 from the promoter of c-Myc target genes. These observations most likely indicate the mechanism by which Arf causes p53-independent tumor-suppressive activity.

The Notch signaling pathway has been recognized as a key factor for the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), because of the high incidence of activating mutations of Notch1. Notch inhibition could serve as a new treatment strategy for T-ALL; however, the attempts to perturb Notch signaling pathways have been unsuccessful so far. In this study, we found that proteasome inhibitors exert cytotoxic effects on T-ALL cells with constitutive activation of Notch1 to a similar extent as myeloma cells. The proteasome inhibitor bortezomib repressed the transcription of Notch1 and downstream effectors including Hes1, GATA3, RUNX3 and nuclear factor-kappa B (NF-kappa B) (p65 and p50), coincided with downregulation of the major transactivator Sp1 and its dissociation from Notch1 promoter. Overexpression of the Notch1 intracellular domain (NICD) significantly ameliorated bortezomib-induced cytotoxicity against T-ALL cells. Drug combination studies revealed that bortezomib showed synergistic or additive effects with key drugs for the treatment of T-ALL such as dexamethasone (DEX), doxorubicin and cyclophosphamide, which were readily abolished by NICD overexpression. The synergy of bortezomib and DEX was confirmed in vivo using a murine xenograft model. Our findings provide a molecular basis and rationale for the inclusion of proteasome inhibitors in treatment strategies for T-ALL.

Several clinical trials have demonstrated the effectiveness of bortezomib in combination with various anti-myeloma agents; however, no definitive information is available regarding drugs best suited for use in combination with bortezomib. Using isobologram analysis, we investigated the combined effects of bortezomib with four key anti-myeloma drugs (melphalan, cyclophosphamide, doxorubicin and lenalidomide), which represent components of major bortezomib-based regimens with corticosteroids, in three myeloma cell lines (U266, RPMI8226 and KMS-12BM) under various conditions. Melphalan showed the best performance with bortezomib under all culture conditions tested (liquid culture, on fibronectin-coated plates, and co-culture with bone marrow stromal cells), whereas cyclophosphamide was antagonistic with bortezomib especially in the presence of stromal cells. Doxorubicin showed additive effects under stroma-free conditions and in contact with fibronectin, but was rather antagonistic in the presence of stromal cells. In contrast, lenalidomide exerted the most favorable effect with bortezomib in contact with stromal cells. Consistent with these results, caspase-3 was activated more strongly by melphalan than by other agents in combination with bortezomib. Moreover, bortezomib-induced up-regulation of CHOP was readily enhanced by lenalidomide in contact with stromal cells. The present findings may provide fundamental information for the selection of bortezomib-based regimens for myeloma patients.

Bendamustine has shown considerable clinical activity against indolent lymphoid malignancies as a single agent or in combination with rituximab, but combination with additional anti-cancer drugs may be required for refractory and/or relapsed cases as well as other intractable tumors. In this study, we attempted to determine suitable anti-cancer drugs to be combined with bendamustine for the treatment of mantle cell lymphoma, diffuse large B-cell lymphoma, aggressive lymphomas and multiple myeloma, all of which are relatively resistant to this drug, and investigated the mechanisms underlying synergism. Isobologram analysis revealed that bendamustine had synergistic effects with alkylating agents (4-hydroperoxy-cyclophosphamide, chlorambucil and melphalan) and pyrimidine analogues (cytosine arabinoside, gemcitabine and decitabine) in HBL-2, B104, Namalwa and U266 cell lines, which represent the above entities respectively. In cell cycle analysis, bendamustine induced late S-phase arrest, which was enhanced by 4-hydroperoxy-cyclophosphamide, and potentiated early S-phase arrest by cytosine arabinoside (Ara-C), followed by a robust increase in the size of sub-G1 fractions. Bendamustine was able to elicit DNA damage response and subsequent apoptosis faster and with shorter exposure than other alkylating agents due to rapid intracellular incorporation via equilibrative nucleoside transporters (ENTs). Furthermore, bendamustine increased the expression of ENT1 at both mRNA and protein levels and enhanced the uptake of Ara-C and subsequent increase in Ara-C triphosphate (Ara-CTP) in HBL-2 cells to an extent comparable with the purine analog fludarabine. These purine analog-like properties of bendamustine may underlie favorable combinations with other alkylators and pyrimidine analogues. Our findings may provide a theoretical basis for the development of more effective bendamustine-based combination therapies.

The Lim Domain Only 2 (LMO2) leukaemia oncogene encodes an LIM domain transcriptional cofactor required for early haematopoiesis. During embryogenesis, LMO2 is also expressed in developing tail and limb buds, an expression pattern we now show to be recapitulated in transgenic mice by an enhancer in LMO2 intron 4. Limb bud expression depended on a cluster of HOX binding sites, while posterior tail expression required the HOX sites and two E-boxes. Given the importance of both LMO2 and HOX genes in acute leukaemias, we further demonstrated that the regulatory hierarchy of HOX control of LMO2 is activated in leukaemia mouse models as well as in patient samples. Moreover, Lmo2 knock-down impaired the growth of leukaemic cells, and high LMO2 expression at diagnosis correlated with poor survival in cytogenetically normal AML patients. Taken together, these results establish a regulatory hierarchy of HOX control of LMO2 in normal development, which can be resurrected during leukaemia development. Redeployment of embryonic regulatory hierarchies in an aberrant context is likely to be relevant in human pathologies beyond the specific example of ectopic activation of LMO2.

Bortezomib therapy is now indispensable for multiple myeloma, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. The development of orally active proteasome inhibitors with distinct mechanisms of action is therefore eagerly awaited. Previously, we identified homopiperazine derivatives as a novel class of proteasome inhibitors with a different mode of proteasome binding from bortezomib. In this study, we show that K-7174, one of proteasome inhibitory homopiperazine derivatives, exhibits a therapeutic effect, which is stronger when administered orally than intravenously, without obvious side effects in a murine myeloma model. Moreover, K-7174 kills bortezomib-resistant myeloma cells carrying a beta 5-subunit mutation in vivo and primary cells from a patient resistant to bortezomib. K-7174 induces transcriptional repression of class I histone deacetylases (HDAC1, -2, and -3) via caspase-8-dependent degradation of Sp1, the most potent transactivator of class I HDAC genes. HDAC1 overexpression ameliorates the cytotoxic effect of K-7174 and abrogates histone hyperacetylation without affecting the accumulation of ubiquitinated proteins in K-7174-treated myeloma cells. Conversely, HDAC inhibitors enhance the activity of K-7174 with an increase in histone acetylation. These results suggest that class I HDACs are critical targets of K-7174-induced cytotoxicity. It is highly anticipated that K-7174 increases the tolerability and convenience of patients by oral administration and has the clinical utility in overcoming bortezomib resistance as a single agent or in combination with HDAC inhibitors.

The proteasome is a proteolytic machinery that executes the degradation of polyubiquitinated proteins to maintain cellular homeostasis. Proteasome inhibition is a unique and effective way to kill cancer cells because they are sensitive to proteotoxic stress. Indeed, the proteasome inhibitor bortezomib is now indispensable for the treatment of multiple myeloma and other intractable malignancies, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. To resolve these problems, we attempted to develop orally bioavailable proteasome inhibitors with distinct mechanisms of action and identified homopiperazine derivatives (HPDs) as promising candidates. Biochemical and crystallographic studies revealed that some HPDs inhibit all three catalytic subunits (beta 1, beta 2 and beta 5) of the proteasome by direct binding, whereas bortezomib and other proteasome inhibitors mainly act on the beta 5 subunit. Proteasome-inhibitory HPDs exhibited cytotoxic effects on cell lines from various hematological malignancies including myeloma. Furthermore, K-7174, one of the HPDs, was able to inhibit the growth of bortezomib-resistant myeloma cells carrying a beta 5-subunit mutation. Finally, K-7174 had additive effects with bortezomib on proteasome inhibition and apoptosis induction in myeloma cells. Taken together, HPDs could be a new class of proteasome inhibitors, which compensate for the weak points of conventional ones and overcome the resistance to bortezomib.

Allogeneic stem cell transplantation (allo-SCT) is a potentially curative therapy for chronic myeloid leukemia and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia, and the graft-vs-leukemia (GVL) effect can eradicate residual leukemia after allo-SCT. Ph(+) leukemia cells frequently express death-inducing receptors (DR4 and DR5) for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is one of the cytotoxic ligands expressed on cytotoxic T cells and natural killer cells mediating the GVL effect. Here we demonstrate that imatinib specifically downregulated DR4 and DR5 expression in cell lines and clinical samples of Ph(+) leukemia. Second-generation tyrosine kinase inhibitors (dasatinib and nilotinib) and short hairpin RNA against bcr-abl also downregulated DR4 and DR5 expression in Ph(+) leukemia cells, and transfection of bcr-abl into a Ph(-) leukemia cell line induced DR4 and DR5 expression, which was abrogated by imatinib treatment. Accordingly, Ph(+) leukemia cells that had been pretreated with imatinib showed resistance to the pro-apoptotic activity of recombinant human soluble TRAIL. These observations demonstrate that BCR-ABL is critically involved in the leukemia-specific expression of DR4 and DR5 and in the susceptibility of Ph(+) leukemia to TRAIL-mediated anti-leukemic activity, providing new insight into the mechanisms of the tumor-specific cytotoxic activities of TRAIL. © 2013 Macmillan Publishers Limited All rights reserved.

The aging suppressor geneKlotho is predominantly expressed in the kidney irrespective of species. Because Klotho protein is an essential component of an endocrine axis that regulates renal phosphate handling, the kidney-specific expression is biologically relevant; however, little is known about its underlying mechanisms. Here we provide in vitro and in vivo evidence indicating that promoter methylation restricts the expression of the Klotho gene in the kidney. Based on evolutionary conservation and histone methylation patterns, the region up to -1200 bp was defined as a major promoter element of the human Klotho gene. This region displayed promoter activity equally in Klotho-expressing and -nonexpressing cells in transient reporter assays, but the activity was reduced to ∼20% when the constructs were integrated into the chromatin in the latter. Both endogenous and transfected Klotho promoters were 30-40% methylated in Klotho-nonexpressing cells, but unmethylated in Klotho-expressing renal tubular cells. DNA demethylating agents increased Klotho expression 1.5- to 3.0-fold in nonexpressing cells and restored the activity of silenced reporter constructs. Finally, we demonstrated that a severe hypomorphic allele of Klotho had aberrant CpG methylation in kl/kl mice. These findings might be useful in therapeutic intervention for accelerated aging and several complications caused by Klotho down-regulation.

The aging suppressor geneKlotho is predominantly expressed in the kidney irrespective of species. Because Klotho protein is an essential component of an endocrine axis that regulates renal phosphate handling, the kidney-specific expression is biologically relevant however, little is known about its underlying mechanisms. Here we provide in vitro and in vivo evidence indicating that promoter methylation restricts the expression of the Klotho gene in the kidney. Based on evolutionary conservation and histone methylation patterns, the region up to - 1200 bp was defined as a major promoter element of the human Klotho gene. This region displayed promoter activity equally in Klotho-expressing and -nonexpressing cells in transient reporter assays, but the activity was reduced to ∼20% when the constructs were integrated into the chromatin in the latter. Both endogenous and transfected Klotho promoters were 30-40% methylated in Klotho-nonexpressing cells, but unmethylated in Klotho-expressing renal tubular cells. DNA demethylating agents increased Klotho expression 1.5- to 3.0-fold in nonexpressing cells and restored the activity of silenced reporter constructs. Finally, we demonstrated that a severe hypomorphic allele of Klotho had aberrant CpG methylation in kl/kl mice. These findings might be useful in therapeutic intervention for accelerated aging and several complications caused by Klotho downregulation. © FASEB.

Latexin is the only known carboxypeptidase A inhibitor in mammals and shares structural similarity with cystatin C, suggesting that latexin regulates the abundance of as yet unidentified target proteins. A forward genetic approach revealed that latexin is involved in homeostasis of hematopoietic stem cells (HSCs) in mice; however, little is known about the mechanisms by which latexin negatively affects the numbers of HSCs. In this study, we found that latexin is preferentially expressed in hematopoietic stem/progenitor cells, and is co-localized with the molecules responsible for the interaction of HSCs with a bone marrow niche, such as N-cadherin, Tie2, and Roundabout 4. Latexin-knockout young female mice showed an increase in the numbers of KSL (c-Kit+/Sca-1+/linegae marker-negative) cells, which may be attributable to enhanced self-renewal because latexin-deficient KSL cells formed more colonies than their wild-type counterparts in methylcellulose culture. Proteomic analysis of Sca-1+ bone marrow cells demonstrated that latexin ablation reduced the abundance of multiple cellular proteins, including N-cadherin, Tie2, and Roundabout 4. Finally, we found that latexin expression was lost or greatly reduced in approximately 50% of human leukemia/lymphoma cell lines. These results imply that latexin inhibits the self-renewal of HSCs by facilitating the lodgment of HSCs within a bone marrow niche to maintain HSC homeostasis. J. Cell. Physiol. 227: 11381147, 2012. (C) 2011 Wiley Periodicals, Inc.

Relatively little is known about the regulatory mechanisms of the Drosha/DGCR8 complex, which processes miRNAs at the initial step of biogenesis. We found that histone deacetylase 1 (HDAC1) increases the expression levels of mature miRNAs despite repressing the transcription of host genes. HDAC1 is an integral component of the Drosha/DGCR8 complex and enhances miRNA processing by increasing the affinity of DGCR8 to primary miRNA transcripts via deacetylation of critical lysine residues in the RNA-binding domains of DGCR8. This finding suggests that HDACs have two arms for gene silencing: transcriptional repression by promoter histone deacetylation and post-transcriptional inhibition by increasing miRNA abundance.

LMO2, a critical transcription regulator of hematopoiesis, is involved in human T-cell leukemia. The binding site of proline and acidic amino acid-rich protein (PAR) transcription factors in the promoter of the LMO2 gene plays a central role in hematopoietic-specific expression. E2A-HLF fusion derived from t(17;19) in B-precursor acute lymphoblastic leukemia (ALL) has the transactivation domain of E2A and the basic region/leucine zipper domain of HLF, which is a PAR transcription factor, raising the possibility that E2A-HLF aberrantly induces LMO2 expression. We here demonstrate that cell lines and a primary sample of t(17;19)-ALL expressed LMO2 at significantly higher levels than other B-precursor ALLs did. Transfection of E2A-HLF into a non-t(17;19) B-precursor ALL cell line induced LMO2 gene expression that was dependent on the DNA-binding and transactivation activities of E2A-HLF. The PAR site in the LMO2 gene promoter was critical for E2A-HLF-induced LMO2 expression. Gene silencing of LMO2 in a t(17;19)-ALL cell line by short hairpin RNA-induced apoptotic cell death. These observations indicated that E2A-HLF promotes cell survival of t(17;19)-ALL cells by aberrantly up-regulating LMO2 expression. LMO2 could be a target for a new therapeutic modality for extremely chemo-resistant t(17;19)-ALL. (Blood. 2010;116(6):962-970)

Bortezomib is now widely used for the treatment of multiple myeloma (MM); however, its action mechanisms are not fully understood. Despite the initial results, recent investigations have indicated that bortezomib does not inactivate nuclear factor-kappa B activity in MM cells, suggesting the presence of other critical pathways leading to cytotoxicity. In this study, we show that histone deacetylases (HDACs) are critical targets of bortezomib, which specifically down-regulated the expression of class I HDACs (HDAC1, HDAC2, and HDAC3) in MM cell lines and primary MM cells at the transcriptional level, accompanied by reciprocal histone hyperacetylation. Transcriptional repression of HDACs was mediated by caspase-8-dependent degradation of Sp1 protein, the most potent transactivator of class I HDAC genes. Short-interfering RNA-mediated knockdown of HDAC1 enhanced bortezomib-induced apoptosis and histone hyperacetylation, whereas HDAC1 overexpression inhibited them. HDAC1 overexpression conferred resistance to bortezomib in MM cells, and administration of the HDAC inhibitor romidepsin restored sensitivity to bortezomib in HDAC1-overexpressing cells both in vitro and in vivo. These results suggest that bortezomib targets HDACs via distinct mechanisms from conventional HDAC inhibitors. Our findings provide a novel molecular basis and rationale for the use of bortezomib in MM treatment. (Blood. 2010; 116(3): 406-417)

The E2A-HLF fusion transcription factor generated by t(17;19)(q22;p13) translocation is found in a small subset of pro-B cell acute lymphoblastic leukemias (ALLs) and promotes leukemogenesis by substituting for the antiapoptotic function of cytokines. Here we show that t(17;19)(+) ALL cells express Survivin at high levels and that a dominant negative mutant of E2A-HLF suppresses Survivin expression. Forced expression of E2A-HLF in t(17;19)(-) leukemia cells up-regulated Survivin expression, suggesting that Survivin is a downstream target of E2A-HLF. Analysis using a counterflow centrifugal elutriator revealed that t(17;19)(+) ALL cells express Survivin throughout the cell cycle. Reporter assays revealed that E2A-HLF induces survivin expression at the transcriptional level likely through indirect down-regulation of a cell cycle-dependent cis element in the promoter region. Down-regulation of Survivin function by a dominant negative mutant of Survivin or reduction of Survivin expression induced massive apoptosis throughout the cell cycle in t(17;19)(+) cells mainly through caspase-independent pathways involving translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus. AIF knockdown conferred resistance to apoptosis caused by down-regulation of Survivin function. These data indicated that reversal of AIF translocation by Survivin, which is induced by E2A-HLF throughout the cell cycle, is one of the key mechanisms in the protection of t(17;19)(+) leukemia cells from apoptosis.