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Angiogenesis begins as endothelial cells migrate, forming a sprouting tip and subsequent growth-rich stalk cells. Here, we present a protocol for transcriptomic and epigenomic analyses of tip-like cells in cultured endothelial cells. We describe steps for stimulating human umbilical vein endothelial cells (HUVECs) with vascular endothelial cell growth factor (VEGF) to generate tip-like cells. We then detail procedures for library preparation for single-cell RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq), and data analysis. This scalable protocol is also applicable to diverse omics studies, including proteomics and metabolomics. For complete details on the use and execution of this protocol, please refer to Miyamura et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lenalidomide (LEN) is commonly used as an effective therapeutic agent for multiple myeloma (MM). However, in some patients, primary resistance to LEN is observed, the mechanisms of which remain poorly understood. In this study, we combined a LEN sensitivity assay with proteomics data from 15 MM cell lines to identify protein expression profiles associated with primary LEN resistance. Our findings revealed that CSN5 expression is lower in LEN-resistant cell lines than in LEN-sensitive lines. Moreover, we established that CSN5 is degraded via the cullin-RING ubiquitin ligase (CRL)-mediated ubiquitin-proteasome pathway through ubiquitination at lysine 194. Our data suggest that reduced CSN5 expression leads to abnormalities in the ubiquitination cycle of CRL4A, resulting in the inhibition of LEN-mediated degradation of IKZF1 and IKZF3. These findings delineate an additional mechanism of LEN resistance in MM cells and may contribute to the development of alternative therapeutic strategies to overcome LEN resistance., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Forkhead box O (FOXO) family proteins are expressed in various cells, and play crucial roles in cellular metabolism, apoptosis, and aging. FOXO1-null mice exhibit embryonic lethality due to impaired endothelial cell (EC) maturation and vascular remodeling. However, FOXO1-mediated genome-wide regulation in ECs remains unclear. Here, we demonstrate that VEGF dynamically regulates FOXO1 cytosol-nucleus translocation. FOXO1 re-localizes to the nucleus via PP2A phosphatase. RNA-seq combined with FOXO1 overexpression/knockdown in ECs demonstrated that FOXO1 governs the VEGF-responsive tip cell-enriched genes, and further inhibits DLL4-NOTCH signaling. Endogenous FOXO1 ChIP-seq revealed that FOXO1 binds to the EC-unique tip-enriched genes with co-enrichment of EC master regulators, and the condensed chromatin region as a pioneer factor. We identified new promoter/enhancer regions of the VEGF-responsive tip cell genes regulated by FOXO1: ESM1 and ANGPT2 . This is the first study to identify cell type-specific FOXO1 functions, including VEGF-mediated tip cell definition in primary cultured ECs., Competing Interests: All authors declare that they have no competing interests., (© 2024 The Authors.)

Multiple myeloma (MM) preferentially expands and acquires drug resistance in the bone marrow (BM). We herein examined the role of histone deacetylase 1 (HDAC1) in the constitutive activation of the master transcription factor IRF4 and the prosurvival mediator PIM2 kinase in MM cells. The knockdown or inhibition of HDAC1 by the class I HDAC inhibitor MS-275 reduced the basal expression of IRF4 and PIM2 in MM cells. Mechanistically, the inhibition of HDAC1 decreased IRF4 transcription through histone hyperacetylation and inhibiting the recruitment of RNA polymerase II at the IRF4 locus, thereby reducing IRF4-targeting genes, including PIM2. In addition to the transcriptional regulation of PIM2 by the HDAC1-IRF4 axis, PIM2 was markedly upregulated by external stimuli from BM stromal cells and interleukin-6 (IL-6). Upregulated PIM2 contributed to the attenuation of the cytotoxic effects of MS-275. Class I HDAC and PIM kinase inhibitors cooperatively suppressed MM cell growth in the presence of IL-6 and in vivo. Therefore, the present results demonstrate the potential of the simultaneous targeting of the intrinsic HDAC1-IRF4 axis plus externally activated PIM2 as an efficient therapeutic option for MM fostered in the BM., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Recent studies have revealed the genetic aberrations involved in the initiation and progression of various cancers, including multiple myeloma (MM), via next-generation sequencing analysis. Notably, DIS3 mutations have been identified in approximately 10% of patients with MM. Moreover, deletions of the long arm of chromosome 13, that includes DIS3 , are present in approximately 40% of patients with MM. Regardless of the high incidence of DIS3 mutations and deletions, their contribution to the pathogenesis of MM has not yet been determined. Herein, we summarize the molecular and physiological functions of DIS3, focusing on hematopoiesis, and discuss the characteristics and potential roles of DIS3 mutations in MM. Recent findings highlight the essential roles of DIS3 in RNA homeostasis and normal hematopoiesis and suggest that the reduced activity of DIS3 may be involved in myelomagenesis by increasing genome instability.

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10,000 cells using the in-solution digestion method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

Epigenetic modifications are crucial for chromatin remodeling and transcriptional regulation. Post-translational modifications of histones are epigenetic processes that are fine-tuned by writer and eraser enzymes, and the disorganization of these enzymes alters the cellular state, resulting in human diseases. The KDM5 family is an enzymatic family that removes di- and tri-methyl groups (me2 and me3) from lysine 4 of histone H3 (H3K4), and its dysregulation has been implicated in cancer. Although H3K4me3 is an active chromatin marker, KDM5 proteins serve as not only transcriptional repressors but also transcriptional activators in a demethylase-dependent or -independent manner in different contexts. Notably, KDM5 proteins regulate the H3K4 methylation cycle required for active transcription. Here, we review the recent findings regarding the mechanisms of transcriptional regulation mediated by KDM5 in various contexts, with a focus on cancer, and further shed light on the potential of targeting KDM5 for cancer therapy.

Lysine demethylase 5A (KDM5A) is a negative regulator of histone H3K4 trimethylation, a histone mark associated with activate gene transcription. We identify that KDM5A interacts with the P-TEFb complex and cooperates with MYC to control MYC targeted genes in multiple myeloma (MM) cells. We develop a cell-permeable and selective KDM5 inhibitor, JQKD82, that increases histone H3K4me3 but paradoxically inhibits downstream MYC-driven transcriptional output in vitro and in vivo . Using genetic ablation together with our inhibitor, we establish that KDM5A supports MYC target gene transcription independent of MYC itself, by supporting TFIIH (CDK7)- and P-TEFb (CDK9)-mediated phosphorylation of RNAPII. These data identify KDM5A as a unique vulnerability in MM functioning through regulation of MYC-target gene transcription, and establish JQKD82 as a tool compound to block KDM5A function as a potential therapeutic strategy for MM., Competing Interests: DECLARATION OF INTERESTS J.Q. is scientific co-founder and consultant of Epiphanes. Other authors declare no potential conflicts of interest.

In the tumor microenvironment, senescent non-malignant cells, including cancer-associated fibroblasts (CAFs), exhibit a secretory profile under stress conditions; this senescence-associated secretory phenotype (SASP) leads to cancer progression and chemoresistance. However, the role of senescent CAFs in metastatic lesions and the molecular mechanism of inflammation-related SASP induction are not well understood. We show that pro-inflammatory cytokine-driven EZH2 downregulation maintains the SASP by demethylating H3K27me3 marks in CAFs and enhances peritoneal tumor formation of gastric cancer (GC) through JAK/STAT3 signaling in a mouse model. A JAK/STAT3 inhibitor blocks the increase in GC cell viability induced by senescent CAFs and peritoneal tumor formation. Single-cell mass cytometry revealed that fibroblasts exist in the ascites of GC patients with peritoneal dissemination, and the fibroblast population shows p16 expression and SASP factors at high levels. These findings provide insights into the inflammation-related SASP maintenance by histone modification and the role of senescent CAFs in GC peritoneal dissemination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Anti-CD38 monoclonal antibody (MoAb) treatments including daratumumab (DARA) are effective therapies for both newly diagnosed and relapsed multiple myeloma (MM). In this study, we examined the soluble factors that modulate CD38 expression and are associated with sensitivity to DARA-mediated antibody-dependent cellular cytotoxicity (ADCC) in the bone marrow (BM) microenvironment. Importantly, primary BM stromal cell (BMSC) culture supernatant (BMSC-sup) and interleukin-6 (IL-6) downregulated CD38 expression and reduced DARA-mediated ADCC. Both cytokine profiling of the BMSC-sup and genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) knockout screening in MM cell lines identified and validated the JAK-STAT3 signaling pathway mediating CD38 downregulation, whereas the JAK-STAT1 pathway mediated CD38 upregulation. STAT3 knockdown abrogated BMSC-sup- and IL-6-induced CD38 downregulation on MM cell lines. We also confirmed that STAT3 and CD38 is negatively correlated in primary MM cells. To assess potential clinical relevance, pharmacological inhibition of the JAK-STAT pathway on BMSC-sup-induced CD38 downregulation was further examined. JAK inhibitor ruxolitinib inhibited STAT3 phosphorylation in MM cell lines, upregulated CD38 expression in MM cell lines and primary patient MM cells, and augmented DARA-mediated ADCC against MM cell lines. Taken together, our results suggest that CD38 expression on MM cells in the BM microenvironment is regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of the JAK-STAT3 pathway represents a novel therapeutic option to enhance CD38 expression and anti-CD38 MoAb-mediated MM cytotoxicity., (© 2020 by The American Society of Hematology.)

Pemafibrate, a selective peroxisome proliferator-activated receptor (PPAR) α modulator, is a new drug that specifically modulates PPARα conformation and co-activator recruitment, thereby lowers plasma triglycerides with less off-target effects. Classical PPARα ligands such as fenofibrate suppress inflammatory cells including microglia. However, effects of pemafibrate on microglia have never been addressed. Here we show that pemafibrate, like other PPARα ligands, potently suppressed NF-κB phosphorylation and cytokine expression in microglial cells. PPARα knockdown significantly amplified LPS-induced cytokine expression. Pemafibrate-induced suppression of IL-6 expression was reversed by PPARα knockdown. However, suppression by fenofibrate was not reversed by PPARα knockdown but by Sirtuin 1 (SIRT1) knockdown. In conclusion, pemafibrate and fenofibrate similarly suppresses microglial activation but through distinct PPARα and SIRT1-dependet pathways., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Endothelial cell (EC) plasticity in pathological settings has recently been recognized as a driver of disease progression. Endothelial-to-mesenchymal transition (EndMT), in which ECs acquire mesenchymal properties, has been described for a wide range of pathologies, including cancer. However, the mechanism regulating EndMT in the tumor microenvironment and the contribution of EndMT in tumor progression are not fully understood. Here, we found that combined knockdown of two ETS family transcription factors, ERG and FLI1, induces EndMT coupled with dynamic epigenetic changes in ECs. Genome-wide analyses revealed that ERG and FLI1 are critical transcriptional activators for EC-specific genes, among which microRNA-126 partially contributes to blocking the induction of EndMT. Moreover, we demonstrated that ERG and FLI1 expression is downregulated in ECs within tumors by soluble factors enriched in the tumor microenvironment. These data provide new insight into the mechanism of EndMT, functions of ERG and FLI1 in ECs, and EC behavior in pathological conditions., Competing Interests: The authors have declared that no competing interests exist.

We herein describe a case of pulmonary tumor thrombotic microangiopathy (PTTM) with rapidly progressing colon cancer. A 61-year-old man who had been receiving treatment for type 2 diabetes mellitus for 3 years was hospitalized due to critical hypoxemia. Computed tomography, which had not shown any abnormalities 3 months previously, revealed a tumor in the ascending colon, multiple nodules in the liver, and the absence of any lung abnormalities. On day 3 of hospitalization, a sudden onset of severe dyspnea and tachycardia occurred, followed by death. Autopsy revealed microscopic metastatic tumor emboli in multiple pulmonary vessels with fibrin thrombus and intimal proliferation, which led to a diagnosis of PTTM.

Multiple myeloma (MM) is a clonal plasma cell disorder that is characterized by a variety of genetic alterations. Recent studies have highlighted not only the importance of these genetic events but also epigenetic aberrations including DNA methylation, histone modifications, and non-coding RNAs in the biology of MM. Post-translational modifications of histone, such as methylation and acetylation, contribute to chromatin dynamics, and are modulated by histone modifying enzymes, and dysregulation of these enzymes is implicated in the pathogenesis of cancers, including MM. Histone modifiers also have non-histone substrates and enzymatically independent roles, which are also involved in tumorigenesis. Here we review and provide comprehensive insight into the biologic significance of histone methyl- and acetyl-modifiers in MM, and further provide an overview of the clinical applications of histone modifier inhibitors, especially histone deacetylase inhibitors. These findings underline the emerging roles of histone modifiers in the pathogenesis of MM, and further highlight the possibility of novel epigenetic therapies in MM.

We report of a case of Graves' ophthalmopathy presented solely with symptoms of the eyes with normal thyroid function tests and negative immunoreactive TSH receptor autoantibody. 40-year-old male was referred to our hospital due to 2-month history of ocular focusing deficit without any signs or symptoms of hyper- or hypothyroidism. Serum thyroid function tests and 99m Tc uptake were both within the normal range. Anti-thyroid autoantibodies were all negative except for the cell-based assay for serum TSH receptor stimulating activity. Since orbital CT scan and MRI gave typical results compatible with Graves' ophthalmopathy, we treated the patients with corticosteroid pulse therapy and orbital radiation therapy, leading to a partial improvement of the symptoms. This case gives insights into the potential pathophysiologic mechanism underlying Graves' ophthalmopathy and casts light upon the difficulties of establishing the diagnosis in a euthyroid case with minimal positive results for anti-thyroid autoantibodies.

Background: Parathyroid hormone-related protein (PTHrP) that causes hypercalcemia of malignancy appears to function as an endogenous smooth muscle relaxant. For example, PTHrP released upon bladder wall distension relaxes detrusor smooth muscle to accommodate urine. Here, we explored mechanisms underlying PTHrP-induced suppression of the smooth muscle contractility in the gastric antrum that also undergoes a passive distension., Methods: Effects of PTHrP on phasic contractions and electrical slow waves in the antral smooth muscle of the guinea pig stomach were studied using isometric tension and intracellular microelectrode recordings, respectively. Fluorescent immunohistochemistry was also carried out to identify the distribution of PTH/PTHrP receptors., Key Results: Parathyroid hormone-related protein (1-100 nM) reduced the amplitude of phasic contractions and the basal tension. N ω -nitro-l-arginine (L-NA, 100 μM), a nitric oxide (NO) synthase inhibitor, or 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ, 10 µM), a guanylate cyclase inhibitor, diminished the PTHrP (10 nM)-induced reduction in the amplitude of phasic contractions. SQ22536 (300 μM), an adenylate cyclase inhibitor, attenuated the PTHrP-induced reduction in basal tension. The combination of ODQ (10 μM) and SQ22536 (300 μM) inhibited the PTHrP-induced reductions in both phasic contractions and basal tension. PTHrP (100 nM) had no inhibitory effect on the electrical slow waves in the antral smooth muscle. PTH/PTHrP receptors were expressed in cell bodies of PGP9.5-positive neurons in the myenteric plexus., Conclusions & Inferences: Parathyroid hormone-related protein exerts its inhibitory actions on the antral smooth muscle via both nitric oxide-cyclic guanosine monophosphate (NO-cGMP) and cyclic adenosine monophosphate (AMP) pathways. Thus, PTHrP may act as an endogenous relaxant of the gastric antrum employing the two complementary signaling pathways to ensure the adaptive relaxation of stomach., (© 2017 John Wiley & Sons Ltd.)

Epigenetic signaling pathways are implicated in tumorigenesis and therefore histone deacetylases (HDACs) represent novel therapeutic targets for cancers, including multiple myeloma (MM). Although non-selective HDAC inhibitors show anti-MM activities, unfavorable side effects limit their clinical efficacy. Isoform- and/or class-selective HDAC inhibition offers the possibility to maintain clinical activity while avoiding adverse events attendant to broad non-selective HDAC inhibition. We have previously reported that HDAC3 inhibition, either by genetic knockdown or selective inhibitor BG45, abrogates MM cell proliferation. Here we show that knockdown of HDAC3, but not HDAC1 or HDAC2, as well as BG45, downregulate expression of DNA methyltransferase 1 (DNMT1) mediating MM cell proliferation. DNMT1 expression is regulated by c-Myc, and HDAC3 inhibition triggers degradation of c-Myc protein. Moreover, HDAC3 inhibition results in hyperacetylation of DNMT1, thereby reducing the stability of DNMT1 protein. Combined inhibition of HDAC3 and DNMT1 with BG45 and DNMT1 inhibitor 5-azacytidine (AZA), respectively, triggers synergistic downregulation of DNMT1, growth inhibition and apoptosis in both MM cell lines and patient MM cells. Efficacy of this combination treatment is confirmed in a murine xenograft MM model. Our results therefore provide the rationale for combination treatment using HDAC3 inhibitor with DNMT1 inhibitor to improve patient outcome in MM.

Recent studies have delineated cancer-type-specific roles of histone 3 lysine 27 (H3K27) demethylase KDM6B/JMJD3 depending on its H3K27 demethylase activity. Here we show that KDM6B is expressed in multiple myeloma (MM) cells; and that shRNA-mediated knockdown and CRISPR-mediated knockout of KDM6B abrogate MM cell growth and survival. Tumor necrosis factor-α or bone marrow stromal cell culture supernatants induce KDM6B, which is blocked by IKKβ inhibitor MLN120B, suggesting that KDM6B is regulated by NF-κB signaling in MM cells. RNA-seq and subsequent ChIP-qPCR analyses reveal that KDM6B is recruited to the loci of genes encoding components of MAPK signaling pathway including ELK1 and FOS, and upregulates expression of these genes without affecting H3K27 methylation level. Overexpression of catalytically inactive KDM6B activates expression of MAPK pathway-related genes, confirming its function independent of demethylase activity. We further demonstrate that downstream targets of KDM6B, ELK1 and FOS, confer MM cell growth. Our study therefore delineates KDM6B function that links NF-κB and MAPK signaling pathway mediating MM cell growth and survival, and validates KDM6B as a novel therapeutic target in MM.

Clinicians sometimes encounter difficulty in diagnosing hypoglycaemia. Here, we present a case report of a 53-year-old woman with recurrent nocturnal hypoglycaemia. A continuous glucose monitoring system (CGMS) revealed postprandial hyperglycaemia and subsequent hypoglycaemia, and an oral glucose tolerance test showed an impaired glycaemic and delayed hyperinsulinaemic pattern. On the basis of these clinical findings, we diagnosed her unexplained hypoglycaemia as reactive hypoglycaemia. CGMS showed a sharp contrast of diurnal variation in blood glucose levels including hypoglycaemia between before and after treatment with an alpha-glucosidase inhibitor, voglibose. Her hypoglycaemic attacks disappeared., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Since the International Commission on Radiological Protection recommended reducing the occupational equivalent dose limit for the lens of the eye in 2011, there have been extensive discussions in various countries. This paper reviews the current situation in radiation protection of the ocular lens and the discussions on the potential impact of the new lens dose limit in Japan. Topics include historical changes to the lens dose limit, the current situation with occupational lens exposures (e.g., in medical workers, nuclear workers, and Fukushima nuclear power plant workers) and measurements, and the current status of biological studies and epidemiological studies on radiation cataracts. Our focus is on the situation in Japan, but we believe such information sharing will be useful in many other countries.

Purpose: To investigate the biological and clinical significance of ribonucleotide reductase (RR) in multiple myeloma. Experimental Design: We assessed the impact of RR expression on patient outcome in multiple myeloma. We then characterized the effect of genetic and pharmacologic inhibition of ribonucleotide reductase catalytic subunit M1 (RRM1) on multiple myeloma growth and survival using siRNA and clofarabine, respectively, in both in vitro and in vivo mouse xenograft models. Results: Newly diagnosed multiple myeloma patients with higher RRM1 expression have shortened survival. Knockdown of RRM1 triggered significant growth inhibition and apoptosis in multiple myeloma cells, even in the context of the bone marrow microenvironment. Gene expression profiling showed upregulation of DNA damage response genes and p53-regulated genes after RRM1 knockdown. Immunoblot and qRT-PCR analysis confirmed that γ-H2A.X, ATM, ATR, Chk1, Chk2, RAD51, 53BP1, BRCA1, and BRCA2 were upregulated/activated. Moreover, immunoblots showed that p53, p21, Noxa, and Puma were activated in p53 wild-type multiple myeloma cells. Clofarabine, a purine nucleoside analogue that inhibits RRM1, induced growth arrest and apoptosis in p53 wild-type cell lines. Although clofarabine did not induce cell death in p53-mutant cells, it did trigger synergistic toxicity in combination with DNA-damaging agent melphalan. Finally, we demonstrated that tumor growth of RRM1-knockdown multiple myeloma cells was significantly reduced in a murine human multiple myeloma cell xenograft model. Conclusions: Our results therefore demonstrate that RRM1 is a novel therapeutic target in multiple myeloma in the preclinical setting and provide the basis for clinical evaluation of RRM1 inhibitor, alone or in combination with DNA-damaging agents, to improve patient outcome in multiple myeloma. Clin Cancer Res; 23(17); 5225-37. ©2017 AACR ., (©2017 American Association for Cancer Research.)

p53-related protein kinase (TP53RK, also known as PRPK) is an upstream kinase that phosphorylates (serine residue Ser15) and mediates p53 activity. Here we show that TP53RK confers poor prognosis in multiple myeloma (MM) patients, and, conversely, that TP53RK knockdown inhibits p53 phosphorylation and triggers MM cell apoptosis, associated with downregulation of c-Myc and E2F-1-mediated upregulation of pro-apoptotic Bim. We further demonstrate that TP53RK downregulation also triggers growth inhibition in p53-deficient and p53-mutant MM cell lines and identify novel downstream targets of TP53RK including ribonucleotide reductase-1, telomerase reverse transcriptase, and cyclin-dependent kinase inhibitor 2C. Our previous studies showed that immunomodulatory drugs (IMiDs) downregulate p21 and trigger apoptosis in wild-type-p53 MM.1S cells, Importantly, we demonstrate by pull-down, nuclear magnetic resonance spectroscopy, differential scanning fluorimetry, and isothermal titration calorimetry that IMiDs bind and inhibit TP53RK, with biologic sequelae similar to TP53RK knockdown. Our studies therefore demonstrate that either genetic or pharmacological inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and axis-independent pathways, validating TP53RK as a novel therapeutic target in patients with poor-prognosis MM.

Multiple myeloma (MM) has proven clinically susceptible to modulation of pathways of protein homeostasis. Blockade of proteasomal degradation of polyubiquitinated misfolded proteins by the proteasome inhibitor bortezomib (BTZ) achieves responses and prolongs survival in MM, but long-term treatment with BTZ leads to drug-resistant relapse in most patients. In a proof-of-concept study, we previously demonstrated that blocking aggresomal breakdown of polyubiquitinated misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro. However, these foundational studies were limited by the pharmacologic liabilities of tubacin as a chemical probe with only in vitro utility. Emerging from a focused library synthesis, a potent, selective, and bioavailable HDAC6 inhibitor, WT161, was created to study the mechanism of action of HDAC6 inhibition in MM alone and in combination with BTZ. WT161 in combination with BTZ triggers significant accumulation of polyubiquitinated proteins and cell stress, followed by caspase activation and apoptosis. More importantly, this combination treatment was effective in BTZ-resistant cells and in the presence of bone marrow stromal cells, which have been shown to mediate MM cell drug resistance. The activity of WT161 was confirmed in our human MM cell xenograft mouse model and established the framework for clinical trials of the combination treatment to improve patient outcomes in MM., Competing Interests: T.H. is a consultant for Acetylon Pharmaceuticals. R.M. has financial interests in SHAPE Pharmaceuticals and Acetylon Pharmaceuticals and is the inventor on intellectual property licensed to these two entities. K.C.A. is an advisor for Celgene, Millennium Pharmaceuticals, and Gilead Sciences and is a Scientific Founder of OncoPep, Acetylon Pharmaceuticals, and C4 Therapeutics. J.E.B. is a Scientific Founder of SHAPE Pharmaceuticals, Acetylon Pharmaceuticals, Tensha Therapeutics, and C4 Therapeutics and is the inventor on intellectual property licensed to these entities. As of January 1, 2016, J.E.B. is an employee of the Novartis Institutes of Biomedical Research.

Multiple myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage response. Here, we show that the NAD(+)-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response to genomic stability, and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with the ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of mitogen-activated protein kinase (MAPK) pathway genes, MAPK signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1 and confers resistance to DNA damage. Using genetic and biochemical studies in vitro and in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA-damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications for both tumorigenesis and the treatment of MM., (© 2016 by The American Society of Hematology.)

Twelve high schools in Japan (of which six are in Fukushima Prefecture), four in France, eight in Poland and two in Belarus cooperated in the measurement and comparison of individual external doses in 2014. In total 216 high-school students and teachers participated in the study. Each participant wore an electronic personal dosimeter 'D-shuttle' for two weeks, and kept a journal of his/her whereabouts and activities. The distributions of annual external doses estimated for each region overlap with each other, demonstrating that the personal external individual doses in locations where residence is currently allowed in Fukushima Prefecture and in Belarus are well within the range of estimated annual doses due to the terrestrial background radiation level of other regions/countries.

KDM3A is implicated in tumorigenesis; however, its biological role in multiple myeloma (MM) has not been elucidated. Here we identify KDM3A-KLF2-IRF4 axis dependence in MM. Knockdown of KDM3A is toxic to MM cells in vitro and in vivo. KDM3A maintains expression of KLF2 and IRF4 through H3K9 demethylation, and knockdown of KLF2 triggers apoptosis. Moreover, KLF2 directly activates IRF4 and IRF4 reciprocally upregulates KLF2, forming a positive autoregulatory circuit. The interaction of MM cells with bone marrow milieu mediates survival of MM cells. Importantly, silencing of KDM3A, KLF2 or IRF4 both decreases MM cell adhesion to bone marrow stromal cells and reduces MM cell homing to the bone marrow, in association with decreased ITGB7 expression in MAF-translocated MM cell lines. Our results indicate that the KDM3A-KLF2-IRF4 pathway plays an essential role in MM cell survival and homing to the bone marrow, and therefore represents a therapeutic target.

Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM.

Purpose: PD-1/PD-L1 signaling promotes tumor growth while inhibiting effector cell-mediated antitumor immune responses. Here, we assessed the impact of single and dual blockade of PD-1/PD-L1, alone or in combination with lenalidomide, on accessory and immune cell function as well as multiple myeloma cell growth in the bone marrow (BM) milieu., Experimental Design: Surface expression of PD-1 on immune effector cells, and PD-L1 expression on CD138(+) multiple myeloma cells and myeloid-derived suppressor cells (MDSC) were determined in BM from newly diagnosed (ND) multiple myeloma and relapsed/refractory (RR) multiple myeloma versus healthy donor (HD). We defined the impact of single and dual blockade of PD-1/PD-L1, alone and with lenalidomide, on autologous anti-multiple myeloma immune response and tumor cell growth., Results: Both ND and RR patient multiple myeloma cells have increased PD-L1 mRNA and surface expression compared with HD. There is also a significant increase in PD-1 expression on effector cells in multiple myeloma. Importantly, PD-1/PD-L1 blockade abrogates BM stromal cell (BMSC)-induced multiple myeloma growth, and combined blockade of PD-1/PD-L1 with lenalidomide further inhibits BMSC-induced tumor growth. These effects are associated with induction of intracellular expression of IFNγ and granzyme B in effector cells. Importantly, PD-L1 expression in multiple myeloma is higher on MDSC than on antigen-presenting cells, and PD-1/PD-L1 blockade inhibits MDSC-mediated multiple myeloma growth. Finally, lenalidomide with PD-1/PD-L1 blockade inhibits MDSC-mediated immune suppression., Conclusions: Our data therefore demonstrate that checkpoint signaling plays an important role in providing the tumor-promoting, immune-suppressive microenvironment in multiple myeloma, and that PD-1/PD-L1 blockade induces anti-multiple myeloma immune response that can be enhanced by lenalidomide, providing the framework for clinical evaluation of combination therapy., (©2015 American Association for Cancer Research.)

Histone deacetylase (HDAC) inhibitors have been extensively investigated as therapeutic agents in cancer. However, the biological role of class IIa HDACs (HDAC4, 5, 7 and 9) in cancer cells, including multiple myeloma (MM), remains unclear. Recent studies show HDAC4 interacts with activating transcription factor 4 (ATF4) and inhibits activation of endoplasmic reticulum (ER) stress-associated proapoptotic transcription factor C/EBP homologous protein (CHOP). In this study, we hypothesized that HDAC4 knockdown and/or inhibition could enhance apoptosis in MM cells under ER stress condition by upregulating ATF4, followed by CHOP. HDAC4 knockdown showed modest cell growth inhibition; however, it markedly enhanced cytotoxicity induced by either tunicamycin or carfilzomib (CFZ), associated with upregulating ATF4 and CHOP. For pharmacological inhibition of HDAC4, we employed a novel and selective class IIa HDAC inhibitor TMP269, alone and in combination with CFZ. As with HDAC4 knockdown, TMP269 significantly enhanced cytotoxicity induced by CFZ in MM cell lines, upregulating ATF4 and CHOP and inducing apoptosis. Conversely, enhanced cytotoxicity was abrogated by ATF4 knockdown, confirming that ATF4 has a pivotal role mediating cytotoxicity in this setting. These results provide the rationale for novel treatment strategies combining class IIa HDAC inhibitors with ER stressors, including proteasome inhibitors, to improve patient outcome in MM.

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide (Len) and pomalidomide trigger anti-tumor activities in multiple myeloma (MM) by targetting cereblon and thereby impacting IZF1/3, c-Myc and IRF4. Histone deacetylase inhibitors (HDACi) also downregulate c-Myc. We therefore determined whether IMiDs with HDACi trigger significant MM cell growth inhibition by inhibiting or downregulating c-Myc. Combination treatment of Len with non-selective HDACi suberoylanilide hydroxamic acid or class-I HDAC-selective inhibitor MS275 induces synergic cytotoxicity, associated with downregulation of c-Myc. Unexpectedly, we observed that decreased levels of cereblon (CRBN), a primary target protein of IMiDs, was triggered by these agents. Indeed, sequential treatment of MM cells with MS275 followed by Len shows less efficacy than simultaneous treatment with this combination. Importantly ACY1215, an HDAC6 inhibitor with minimal effects on class-I HDACs, together with Len induces synergistic MM cytotoxicity without alteration of CRBN expression. Our results showed that only modest class-I HDAC inhibition is able to induce synergistic MM cytotoxicity in combination with Len. These studies may provide the framework for utilizing HDACi in combination with Len to both avoid CRBN downregulation and enhance anti-MM activities.

Recently, the bone marrow (BM) microenvironment has been reported to support the survival of multiple myeloma (MM) cells. In this study, we examined changes in gene expression profile in human mesenchymal stem cells (MSCs) by co-culture with MM cells. cDNA array analysis indicated that co-culture induced multiple factors which have positive effects on the survival of MM cells, such as interleukin-6 (IL-6) and insulin-like growth factor-1. Other than these factors, thymic stromal lymphopoietin (TSLP), which is a cytokine involved in the progression of pancreatic and breast cancer through induction of T helper 2 cell responses, was up-regulated in MSCs. TSLP exhibited no effect on proliferation or drug resistance of MM cells, but TSLP secreted from MSCs by co-culture expanded IL-13+ CD4+ T cells through stimulation of dendritic cells. These results suggested that MM cells positively act to induce various molecules in MSCs, leading to the formation of a more advantageous BM microenvironment for their survival.

Despite promising preclinical results with mTOR kinase inhibitors in multiple myeloma, resistance to these drugs may arise via feedback activation loops. This concern is especially true for insulin-like growth factor 1 receptor (IGF1R), because IGF1R signaling is downregulated by multiple AKT and mTOR feedback mechanisms. We have tested this hypothesis in multiple myeloma using the novel selective mTOR kinase inhibitor AZD8055. We evaluated p-mTOR S(2481) as the readout for mTORC2/Akt activity in multiple myeloma cells in the context of mTOR inhibition via AZD8055 or rapamycin. We next validated AZD8055 inhibition of mTORC1 and mTORC2 functions in multiple myeloma cells alone or in culture with bone marrow stroma cells and growth factors. Unlike rapamycin, AZD8055 resulted in apoptosis of multiple myeloma cells. AZD8055 treatment, however, induced upregulation of IGF1R phosphorylation in p-Akt S(473)-expressing multiple myeloma cell lines. Furthermore, exposure of AZD8055-treated cells to IGF1 induced p-Akt S(473) and rescued multiple myeloma cells from apoptosis despite mTOR kinase inhibition and TORC2/Akt blockage. The addition of blocking IGF1R antibody resulted in reversing this effect and increased AZD8055-induced apoptosis. Our study suggests that combination treatment with AZD8055 and IGF1R-blocking agents is a promising strategy in multiple myeloma with potential IGF1R/Akt signaling-mediated survival., (©2014 American Association for Cancer Research.)

The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM., (©2014 American Association for Cancer Research.)

Histone deacetylases (HDACs) represent novel molecular targets for the treatment of various types of cancers, including multiple myeloma (MM). Many HDAC inhibitors have already shown remarkable antitumor activities in the preclinical setting; however, their clinical utility is limited because of unfavorable toxicities associated with their broad range HDAC inhibitory effects. Isoform-selective HDAC inhibition may allow for MM cytotoxicity without attendant side effects. In this study, we demonstrated that HDAC3 knockdown and a small-molecule HDAC3 inhibitor BG45 trigger significant MM cell growth inhibition via apoptosis, evidenced by caspase and poly (ADP-ribose) polymerase cleavage. Importantly, HDAC3 inhibition downregulates phosphorylation (tyrosine 705 and serine 727) of signal transducers and activators of transcription 3 (STAT3). Neither interleukin-6 nor bone marrow stromal cells overcome this inhibitory effect of HDAC3 inhibition on phospho-STAT3 and MM cell growth. Moreover, HDAC3 inhibition also triggers hyperacetylation of STAT3, suggesting crosstalk signaling between phosphorylation and acetylation of STAT3. Importantly, inhibition of HDAC3, but not HDAC1 or 2, significantly enhances bortezomib-induced cytotoxicity. Finally, we confirm that BG45 alone and in combination with bortezomib trigger significant tumor growth inhibition in vivo in a murine xenograft model of human MM. Our results indicate that HDAC3 represents a promising therapeutic target, and validate a prototype novel HDAC3 inhibitor BG45 in MM.

Small-molecule multi-targeted cyclin-dependent kinase (CDK) inhibitors (CDKIs) are of particular interest due to their potent antitumor activity independent of p53 gene alterations. P53 deletion is associated with a very poor prognosis in multiple myeloma (MM). In this regard, we tested the anti-MM activity of RGB-286638, an indenopyrazole-derived CDKI with Ki-nanomolar activity against transcriptional CDKs. We examined RGB-286638's mode-of-action in MM cell lines with wild-type (wt)-p53 and those expressing mutant p53. RGB-286638 treatment resulted in MM cytotoxicity in vitro associated with inhibition of MM tumor growth and prolonged survival in vivo. RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription. RGB-286638 triggered p53 accumulation via nucleolar stress and loss of Mdm2, accompanied by induction of p53 DNA-binding activity. In addition, RGB-286638 mediated p53-independent activity, which was confirmed by cytotoxicity in p53-knockdown and p53-mutant cells. We also demonstrated downregulation of oncogenic miR-19, miR-92a-1 and miR-21. Our data provide the rationale for the development of transcriptional CDKIs as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells.

Purpose: NF-κB transcription factor plays a key role in the pathogenesis of multiple myeloma in the context of the bone marrow microenvironment. Both canonical and noncanonical pathways contribute to total NF-κB activity. Recent studies have shown a critical role for the noncanonical pathway: selective inhibitors of the canonical pathway present a limited activity, mutations of the noncanonical pathway are frequent, and bortezomib-induced cytotoxicity cannot be fully attributed to inhibition of canonical NF-κB activity., Experimental Design: Multiple myeloma cell lines, primary patient cells, and the human multiple myeloma xenograft murine model were used to examine the biologic impact of dual inhibition of both canonical and noncanonical NF-κB pathways., Results: We show that PBS-1086 induces potent cytotoxicity in multiple myeloma cells but not in peripheral blood mononuclear cells. PBS-1086 overcomes the proliferative and antiapoptotic effects of the bone marrow milieu, associated with inhibition of NF-κB activity. Moreover, PBS-1086 strongly enhances the cytotoxicity of bortezomib in bortezomib-resistant multiple myeloma cell lines and patient multiple myeloma cells. PBS-1086 also inhibits osteoclastogenesis through an inhibition of RANK ligand (RANKL)-induced NF-κB activation. Finally, in a xenograft model of human multiple myeloma in the bone marrow milieu, PBS-1086 shows significant in vivo anti-multiple myeloma activity and prolongs host survival, associated with apoptosis and inhibition of both NF-κB pathways in tumor cells., Conclusions: Our data show that PBS-1086 is a promising dual inhibitor of the canonical and noncanonical NF-κB pathways. Our preclinical study therefore provides the framework for clinical evaluation of PBS-1086 in combination with bortezomib for the treatment of multiple myeloma and related bone lesions., (©2012 AACR.)

Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1α (IRE1α) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1α endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Importantly, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG, even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress, evidenced by induction of XBP1s, which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946, associated with increased CHOP. Finally, MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential therapeutic option in MM.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL) is one of the highest-risk ALL groups. Whenever possible, patients with Ph(+)ALL should undergo allogeneic hematopoietic stem cell transplantation (HSCT) after induction of remission. Although unrelated cord blood transplantation (CBT) has become a common treatment in adult patients who lack a sibling donor, data on the efficacy of CBT for Ph(+)ALL are limited. We analyzed the clinical outcomes of 20 Ph(+)ALL patients who underwent CBT (n = 8) or unrelated bone marrow transplantation (BMT) (n = 12). The median age was 41 years (range, 17-55 years). All but one of the patients were treated with an imatinib-based regimen before HSCT, and 19 patients were in first complete remission (CR) and 1 patient was in second CR at the time of HSCT. Seventeen patients received a myeloablative conditioning regimen containing 12 Gy of total-body irradiation, and 3 received a reduced-intensity conditioning regimen. After a median of 26 months of follow-up, estimated 3-year overall and leukemia-free survival rates were 100% and 85%, respectively, after CBT, and 49% and 38%, respectively, after unrelated BMT. The CBT group had significantly better overall survival than the BMT group (P = .02). Although BCR-ABL transcript was detected in 4 of 8 CBT patients at transplantation, 7 patients remained in molecular CR. Our findings suggest that CBT may be a viable option as postinduction therapy for Ph(+)ALL in patients lacking a sibling donor., (2011 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

The prognosis of advanced extranodal NK/T cell lymphoma (ENKTL) is poor. Allogeneic hematopoietic stem cell transplant (allo-HSCT) has been suggested to be a promising treatment for this disease, but its utility has yet to be established. Here we retrospectively analyzed five cases of ENKTL treated with allo-HSCT in our institute. After induction chemotherapy, disease status at allo-HSCT was second CR in three patients and refractory in two patients. All patients received a myeloablative conditioning regimen, and GVHD prophylaxis consisted of tacrolimus or cyclosporine with short-term methotrexate. Only one patient who received conventional induction chemotherapy developed severe complications, which needed long-term treatment, while others who received chemotherapy containing l-asparaginase did not have severe complications. There were no cases of treatment-related mortality, and all patients survived without disease for a median follow-up period of 1911 days. These results suggested that allo-HSCT following l-asparaginase-containing induction chemotherapy might improve the outcome of advanced ENKTL.

Thrombotic thrombocytopenic purpura (TTP), scleroderma renal crisis (SRC), and hemolysis, elevated liver enzyme levels, and a low platelet count (HELLP) syndrome display common symptoms that include microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Therefore, it is important to distinguish between them because their treatments vary: however, the differential diagnosis is sometimes difficult. We report a 32-year-old woman who was referred to our department for further examination of microangiopathic hemolytic anemia, thrombocytopenia, and a slightly elevated serum creatinine level with anti-centromere antibody-positive Raynaud's syndrome in the early puerperal period. TTP, SRC, and HELLP syndrome were considered in the differential diagnosis, but the measurement of a disintegrin-like metalloprotease with thrombospondin type 1 motifs 13 (ADAMTS 13) activity and its inhibitor level led to the diagnosis of TTP. She was successfully treated by plasma exchange and high-dose prednisolone and angiotensin-converting enzyme inhibitor. If microangiopathic hemolytic anemia and thrombocytopenia are observed in perinatal women or patients with signs of systemic sclerosis, the measurement of ADAMTS13 activity and its inhibitor level are essential for diagnosis and therapeutic choice.

Bortezomib is approved for the treatment of patients with relapsed or refractory multiple myeloma (MM), but only a few clinical studies for Japanese patients who were treated with bortezomib have been reported. We retrospectively analyzed 40 patients with relapsed or refractory MM who have received bortezomib at three collaborating centers in Miyagi prefecture in Japan. All the patients have been received bortezomib in combination with dexamethasone. Responses were determined using International Myeloma Working Group uniform response criteria. The overall response was observed in 30 patients (75%), including very good partial response in 8 patients (20%), and partial response in 22 patients (55%). The median time to disease progression was 8.7 months, and the median overall survival has not been reached. The factors affecting time to disease progression were International Staging System stage, serum beta2-microglobulin level, and number of treatment cycles. The most common grade 3 and 4 adverse events were thrombocytopenia (50%), peripheral neuropathy (25%), leukopenia (25%), and herpes zoster infection (25%). Thus, bortezomib is well tolerated and effective for Japanese patients with relapsed or refractory MM. Our results suggest that serum beta2-microglobulin level may be a marker of prognosis on bortezomib therapy for patients with relapsed or refractory MM although further studies are needed.

An 80-year-old woman was referred to our hospital because of eosinophilia and thrombocytopenia. She presented with persistent pruritus and cough. Laboratory examinations showed persistent eosinophilia, and there was no underlying cause, consistent with hypereosinophilic syndrome (HES). After admission, she developed a neurological deficit, and microangiopathic hemolytic anemia. She was diagnosed with thrombotic thrombocytopenic purpura (TTP) and successfully treated with corticosteroids and plasmapheresis. Although TTP has been described in association with pregnancy, cancer, collagen diseases, infection, and drug intake, hypereosinophilia is not a well-documented cause of this disorder. To our knowledge, this is only the second case of TTP with HES, proved to be caused by ADAMTS13 inhibitor.

Plasma cell leukemia (PCL) is a rare variant of multiple myeloma, which is very aggressive and resistant to chemotherapy. We report a case of PCL successfully treated with syngeneic peripheral blood stem cell transplantation followed by low-dose thalidomide. As of March 2009, the patient has maintained CR for 39 months posttransplant. The clinical course of the present case suggests that autologous stem cell transplantation using a graft with reduced contamination of malignant cells followed by low-dose thalidomide maintenance therapy may improve the PCL treatment outcome.

We studied clinical outcomes of 25 adult patients with hematological malignancies who underwent cord blood transplantation (CBT) after a myeloablative conditioning regimen, including high-dose cytosine arabinoside (CA) (8 g/m(2)), cyclophosphamide (CY) (120 mg/kg), and total-body irradiation (TBI) (12 Gy). For graft-versus-host disease (GVHD) prophylaxis, all patients received a combination of tacrolimus and short-term methotrexate (sMTX). Neutrophil engraftment was achieved in 20 of 25 patients. Of the 22 evaluable patients, 2 and 7 had grades I and II acute GVHD, respectively, and only 1 developed grade III acute GVHD after discontinuation of tacrolimus due to encephalopathy. Chronic GVHD developed in 13 of 19 evaluable patients, including 4 with the extensive type. However, the Karnofsky scores of survivors at 1 year after CBT were 90% or 100%. Eight of 25 patients died of nonrelapse causes (n = 4) and relapse/progressive disease (n = 4); 17 patients are currently alive with 15 free of disease at the present time (median follow-up, 24 months). The probability of disease-free survival at 2 years among patients with standard risk was 89% and that of high-risk patients was 30%. Transplantation-related mortality within 100 days was 12%. These results suggested that the CA/CY/TBI combination is a promising conditioning regimen for myeloablative CBT. Furthermore, tacrolimus and sMTX seemed to have suppressed severe acute GVHD and chronic GVHD, which may also contribute to the favorable results.

Type 1 iodothyronine deiodinase (Dio1), a selenoenzyme catalyzing the bioactivation of thyroid hormone, is highly expressed in the liver. Dio1 mRNA and enzyme activity levels are markedly reduced in the livers of hepatocyte nuclear factor 4alpha (HNF4alpha)-null mice, thus accounting for its liver-specific expression. Consistent with this deficiency, serum T4 and rT3 concentrations are elevated in these mice compared with those in HNF4alpha-floxed control littermates; however, serum T3 levels are unchanged. Promoter analysis of the mouse Dio1 gene demonstrated that HNF4alpha plays a key role in the transactivation of the mouse Dio1 gene. Deletion and substitution mutation analyses demonstrated that a proximal HNF4alpha site (direct repeat 1 [TGGACAAAGGTGC]; HNF4alpha-RE) is crucial for transactivation of the mouse Dio1 gene by HNF4alpha. Mouse Dio1 is also stimulated by thyroid hormone signaling, but a direct role for thyroid hormone receptor action has not been reported. We also showed that thyroid hormone-inducible Krüppel-like factor 9 (KLF9) stimulates the mouse Dio1 promoter very efficiently through two CACCC sequences that are located on either side of HNF4alpha-RE. Furthermore, KLF9 functions together with HNF4alpha and GATA4 to synergistically activate the mouse Dio1 promoter, suggesting that Dio1 is regulated by thyroid hormone in the mouse through an indirect mechanism requiring prior KLF9 induction. In addition, we showed that physical interactions between the C-terminal zinc finger domain (Cf) of GATA4 and activation function 2 of HNF4alpha and between the basic domain adjacent to Cf of GATA4 and a C-terminal domain of KLF9 are both required for this synergistic response. Taken together, these results suggest that HNF4alpha regulates thyroid hormone homeostasis through transcriptional regulation of the mouse Dio1 gene with GATA4 and KLF9.