bluuebunny/crossref_metadata_2025 huggingface dataset:
CD26/Dipeptidyl Peptidase IV (DPPIV) Regulates p38 Phosphorylation and Topoisomerase II Alpha Expression in the B-Lymphoma Line Jiyoye, Associated with Enhanced In Vitro and In Vivo Sensitivity to Doxorubicin.
Authors: Yamochi Toshiko , Yamochi Tadanori , Aytac Ugur , Sato Tsutomu , Morimoto Chikao , Dang Nam H.
Published: October 2019

CD26 is a 110 kDa surface glycoprotein with diverse functional properties, including having a key role in normal T-cell biology, being a marker of aggressive disease for selected T-cell malignancies and being involved in the development of certain cancers. Its extracellular domain encodes a membrane-associated dipeptidyl peptidase IV (DPPIV) activity capable of processing biological factors to alter their functional profiles. We have shown previously that expression of CD26 on the T cell line Jurkat is associated with increased topoisomerase II alpha level and in vitro sensitivity to topoisomerase II inhibitors (Aytac U et al. Cancer Res61:7204, 2001; Aytac U et al. Br J Cancer88:455, 2003; Sato K et al. Br J Cancer89:1366, 2003). We now show that CD26 expression, particularly its DPPIV enzyme activity, on the B-lymphoma line Jiyoye results in increased topoisomerase II alpha level and in vitro sensitivity to doxorubicin-induced apoptosis. Examining the molecular mechanisms involved in CD26-associated signaling, our present findings also indicate that CD26/DPPIV expression on Jiyoye cells is associated with increased phosphorylation of p38 and its upstream regulators MKK3/6 and ASK1. Importantly, inhibition of p38 phosphorylation decreases topoisomerase II alpha expression, suggesting a role for p38 in the regulation of topoisomerase II alpha. Finally, studies using a SCID mouse xenograft model with CD26 Jiyoye transfectants show that CD26 expression is associated with enhanced survival following treatment with low doses of doxorubicin. In particular, treatment with low-dose doxorubicin of SCID mice injected with CD26-negative parental Jiyoye cells does not lead to a statistically significant survival advantage over those treated with saline. On the other hand, SCID mice injected with CD26 Jiyoye transfectants show significantly greater survival when treated with low-dose doxorubicin than with saline alone, indicating that CD26 presence renders tumor cells more sensitive to doxorubicin in an in vivo model. Our data thus characterize the biochemical linkage among CD26 and other key intracellular molecules, while demonstrating that CD26 may have a role in tumor sensitivity to antineoplastic agents targeting topoisomerase II alpha. In addition, our work suggests that CD26/DPPIV may be an appropriate target for therapy for selected hematological malignancies of both B- and T-cell lineages.
Molecular Mechanisms Underlying the Development of Drug Resistance in Multiple Myeloma.
Authors: Shammas Masood A. , Koley Hemant , Li Cheng , Anderson Kenneth C. , Reis Robert J. S. , Munshi Nikhil C.
Published: October 2019

A prominent feature of most cancers including multiple myeloma (MM) is a striking genetic instability, leading to ongoing accrual of mutational changes some of which underlie tumor progression, including development of drug resistance and metastasis. The molecular basis for the generation of genetic diversity in cancer cells has thus emerged as an important focus of investigation and a target for successful eradication. We have previously observed that homologous recombination (HR) is upregulated in MM. Utilizing a genomewide LOH assay based on SNP genotyping (Affymetrix) as a tool to estimate the rate of mutation and genomic instability, we have observed that over time elevated HR leads to progressive accumulation of genetic variation in MM cell lines and patient cells; and inhibition of HR activity in MM cells by altering components of the HR pathway concordantly affects the acquisition of new genetic changes. As HR activity is dependent on concerted action of number of genes, instead of over expressing single HR related gene, we utilized nickel chloride, a known recombinogen to evaluate effects of increased HR activity on the development of genomic diversity. We cultured ARP cells in the presence or absence of nickel chloride, over a period of 90 days. Genome-wide LOH was evaluated by comparing genotypes before and after the 90-day interval. In three independent experiments treatment of cells with nickel chloride increased the number of new LOH sites by more than 12-fold. We next evaluated the effect of induction of HR and the consequent increase in genetic aberrations, on development of drug resistance in MM. Myeloma cells were cultured with nickel chloride as a potent inducer of HR and dexamethasone (10−8M); control cells were exposed to dexamethasone alone. The cell viability was measured weekly. No live cells were detected in cultures exposed to dexamethasone alone; while >95% cells exposed to both nickel chloride and dexamethasone were alive following 2 weeks culture. These findings were confirmed by 3 independent experiments. The development of drug resistance was further confirmed by demonstrating no significant effects of dexamethasone on these cells at 10−6M concentrations for >1 week. Dexamethasone at this concentration kills all control cells by day 3. Evaluation of development of resistance to other agents is underway. We propose that continued accumulation of new genetic changes mediated by HR, as demonstrated here, provides the molecular events required to develop drug resistance; and its inhibition may allow us to successfully treat MM cells without the currently observed development of resistance. HR may be a potential therapeutic target to maintain chemo sensitivity of the tumors.
JS-K, a Novel Nitric Oxide (NO) Generator, Induces Cytochrome c Release and Caspase Activation in HL-60 Myeloid Leukemia Cells.
Authors: Shami Paul J. , Udupi Vidya , Yu Margaret , Malaviya Swati , Saavedra Joseph E. , Keefer Larry K.
Published: October 2019

NO induces differentiation and apoptosis in Acute Myelogenous Leukemia (AML) cells. Glutathione S-Transferases (GST) play an important role in multidrug resistance and are upregulated in 90% of AML cells. We have designed a novel prodrug class that releases NO on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antileukemic activity. We have previously shown that JS-K induces apoptosis in HL-60 cells by a caspase dependent mechanism (Molecular Cancer Therapeutics2:409-417,2003). The purpose of this study was to determine the pathway through which JS-K induces apoptosis. Western blot analysis showed that treatment of HL-60 cells with JS-K (0 – 1 μM) for 6 hours results in release of Cytochrome c from mitochondria in a dose dependent fashion. Treatment with JS-K resulted in a dose dependent activation of Caspase 9. Sixteen and 24 hours after exposure to 1 μM JS-K, Caspase 9 activity was induced by 393 ± 93% and 237 ± 13% of control, respectively (p = 0.03 at the 24 hours time point). Treatment with JS-K resulted in a dose dependent activation of Caspase 3. Twenty four hours after exposure to 1 μM JS-K, Caspase 3 activity was 208 ± 3.4 % of control (p = 0.02). Treatment with JS-K also resulted in a dose dependent activation of Caspase 8, but to a lesser extent than Caspase 9 and 3. Twenty four hours after exposure to 1 μM JS-K, Caspase 8 activity was 144 ± 5.3 % of control (p = 0.04). We conclude that JS-K activates the intrinsic pathway of apoptosis in leukemia cells by inducing the release of Cytochrome c from mitochondria. (NO1-CO-12400).
CD40-Activation of Chronic Lymphocytic Leukemia Cells Induces Latent Sensitivity to Fas/TRAIL-Mediated Apoptosis Via a P53-Independent, C-Abl-Dependent Pathway.
Authors: Kater Arnon P. , Dicker Frank , Fukuda Tetsuya , Kipps Thomas J.
Published: October 2019

Patients with chronic lymphocytic leukemia (CLL) experienced rapid reductions in leukemia cell count and lymph node size following an intravenous infusion of autologous CLL cells that were transduced to express a recombinant ligand for CD40 (CD154), including patients with leukemia cells that had inactivating mutations in P53 . Subsequent studies demonstrated that ligation of CD40 on CLL cells induced latent sensitivity to Fas (CD95)-dependent apoptosis and that this might account, in part, for the early reductions in leukemia cell counts observed in patients treated with CD154 gene therapy (Proc Natl Acad Sci U S A 100:3854–9, 2002). We found that ligation of CD40 induces CLL cells to express P53, as well as P53-dependent genes encoding CD95, DR5, and p21. Similar effects were observed following treatment of CLL cells with γ-radiation. In contrast to the effects of γ-radiation, however, CD40-ligation also induced CLL cells to express high-levels of Bid, a pro-apoptotic protein that mediates cross talk between extrinsic death receptors and mitochondrial-dependent apoptotic pathways. CLL cells that had inactivating mutations in P53 also were induced to express CD95, DR5, p21, and Bid following CD40-ligation, but not following γ-radiation, demonstrating that the gene expression response to CD40-ligation is dependent on a factor(s) other than p53. A good candidate for this is P73, a member of the P53 gene family that has been implicated in lymphocyte activation-induced cell death. Consistent with this notion, we found that ligation of CD40 on CLL cells induced expression of the α isoform of P73. The induction of P73 and latent sensitivity to CD95 (and TRAIL)-mediated apoptosis could be inhibited with the c-abl kinase inhibitor, imatinib mesylate, when added at the time of CD40-ligation. However, transduction of leukemia cells with a mutant gene encoding a c-abl kinase resistant to imatinib mesylate resulted in CLL cells that could be induced by CD40-ligation to express P73 and Bid even in the presence of imatinib mesylate. We conclude that sensitization to CD95-mediated apoptosis following CD40-activation is via a p53-independent, c-abl-kinase dependent process that is associated with activation of p73. Unlike p53, p73 mutations are extremely rare in human cancer. As such, CD154-based immune gene therapy may have activity even in patients who have CLL cells with dysfunctional P53 that are resistant to many of the anti-leukemia drugs currently used in the treatment of this disease.
Bmi-1 Expression Predicts the Progression of Malignant Hematopoietic Stem Cells in Myelodysplastic Syndrome.
Authors: Mihara Keichiro , Nakaju Nanae , Hidani Sachiko , Chowdhury Moniruddin , Ihara Akihiro , Kimura Akiro
Published: October 2019

Bmi-1 is a member of the Polycomb group (PcG) of transcriptional repressor genes, which could have an essential role in embryogenesis and be expressed restrictedly in the stem cells and proliferating cells. Some studies have recently reported that Bmi-1 is required to regulate the adult self-renewing hematopoietic stem cells. Others have shown that overexpressed Bmi-1 gene could cause the neoplastic proliferation of cells. A series of evidence on Bmi-1 suggests that it might be closely associated with the progression of the hematopoietic malignancies. Myelodysplastic syndrome (MDS) is a clonal bone marrow disorder characterized by aberrant hematopoiesis susceptible to acute leukemia. This category is quite heterogeneous with a variable clinical course and prognosis. Therefore, it is difficult to decide when to initiate therapies because some patients survive for years without treatment whereas others come to die in rapid progress in overt leukemia. Thus, we investigated whether the expression of Bmi-1 protein in CD34+ cells could be correlated with disease progression of MDS. Primary cells from patients with RA, RAEB, and MDS-AML, were stained with anti-CD34 antibody-PE and then fixed in PFA followed by staining with anti-Bmi-1-antibody-FITC. These cells were subjected to flow cytometric analysis. Bmi-1 expression in CD34+ cells was preferentially seen in RAEB and MDS-AML compared with RA (13.86%±6.40% in RA(n=4), 63.05%±8.19% in RAEB(n=6), 73.99%±33.40% in MDS-AML(n=6)). Patients were dead with >70% at Bmi-1 expression level in CD34+. Moreover, two patients with RA at >10% of Bmi-1 positivity made disease progression to RAEB. One patient with RAEB at <10% at the Bmi-1 expression in CD34+ is still alive. Furthermore, we are dissecting gene profiling of cells from different types of MDS. These results show that the expression of Bmi-1 can distinguish the proliferating malignant stem cells from resting cells by using flow cytometry. Alternatively, IPSS in MDS patients is closely correlated with Bmi-1 expression of CD34+ cells. It may lead to the prediction of prognosis in patients with MDS in quality. Taken together, Bmi-1 expression could predict the disease progression and prognosis of MDS. Finally, Bmi-1 gene might be a target to control leukemogenesis by immunotherapy or gene therapy.
Identification and Hematopoietic Potential of CD45-Negative Clonal Cells with Very Immature Phenotype (CD45−CD34−CD38−Lin−) in Patients with Myelodysplastic Syndromes.
Authors: Ogata Kiyoyuki , Satoh Chikako , Tachibana Mikiko , Hyodo Hideya , Tamura Hideto , Dan Kazuo , Kimura Takafumi , Sonoda Yoshiaki , Tsuji Takashi
Published: October 2019

CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. Cells expressing CD45 and CD34 but lacking CD38 and lineage antigens (CD45+CD34+CD38−Lin− cells) are well-documented hematopoietic stem cells (HSCs), and CD45+CD34−CD38−Lin− cells are probably less mature HSCs. In myelodysplastic syndromes (MDS), the malignant transformation site was reported to be committed myeloid progenitors and, more recently, the CD45+CD34+CD38−Lin− HSCs. In this study, we detected CD45−CD34−CD38−Lin− cells in the peripheral blood and bone marrow of MDS patients. Fluorescence in situ hybridization showed that CD45−CD34−CD38−Lin− cells had the same chromosomal aberration as the myeloblasts. In addition to CD45- and CD34-negativity, they lacked CD117 and CD133 expressions. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal hematopoietic cells, but we documented the following in some cases. Freshly-isolated CD45−CD34−CD38−Lin− cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When these cells were co-cultured with stroma cells, CD45−CD34−CD38−Lin− cells showed only weak potential for proliferation/differentiation, yet differentiated to CD34+ cells and then mature myeloid cells. This newly-identified cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.
Evidence for PTEN Deficiency in Juvenile Myelomonocytic Leukemia.
Authors: Liu Y. Lucy , Xu Likang , Castleberry Robert P. , Emanuel Peter Dean
Published: October 2019

Juvenile myelomonocytic leukemia (JMML) is a myelodysplastic/myeloproliferative disorder (MDS/MPD) of young children. It is characterized by monocytosis, leukocytosis, elevated fetal hemoglobin, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), low percentage of myeloblasts in bone marrow, and absence of the Philadelphia chromosome. The pathogenesis of JMML has been linked to dysregulated signal transduction through the NF1/RAS signaling pathway and PTPN11. This dysregulation results in JMML cells demonstrating selective hypersensitivity to GM-CSF in vitro dose-response assays. PTEN, a major negative regulator of the PI3-kinase pathway by virtue of its PIP3 phosphatase activity, was initially isolated as a tumor suppressor in a variety of malignancies. In order to evaluate the role of PTEN in the pathogenesis of JMML, we examined the status of PTEN in JMML patient samples. Peripheral blood or bone marrow was collected from 40 patients. Mononuclear cells (MNCs) were isolated and lysed in lysis buffer at a concentration of 107/ml. Total RNA was extracted from MNCs of patients and 17 normal individuals. Protein and mRNA levels of PTEN were evaluated by Western-blot and relative-quantitative real-time RT-PCR, respectively. We found that PTEN protein was decreased in 18 of 30 (60%) JMML patients, and the patients had significantly lower RNA expression of PTEN than normal controls (p=0.015). With the available samples we also evaluated AKT activity and MAP kinase (MAPK) levels. We found that MAPK levels were correlated well with the status of the PTEN in 12 of 27(44%), and AKT activity in 13 of 25 patients (52%). Our data indicates that PTEN is significantly deficient in JMML patients, and the low PTEN protein level is related to its low transcription of RNA in JMML patients. The role of PTEN in regulation of MAPK and AKT activities in JMML is under further evaluation by studying the upstream status of the RAS pathway prior to PTEN. This is the first investigation of PTEN deficiency in JMML patients, and additional investigations may help to further understand the pathogenetic mechanisms in JMML, as well as to guide the development of targeted therapeutics for JMML.
The STK Receptor Tyrosine Kinase Regulates the Response of Primary Macrophages to LPS in a Ligand-Independent Manner.
Authors: Correll Pamela , Liu Qingping Liu
Published: October 2019

We have shown previously that activation of the STK/RON receptor tyrosine kinase expressed on tissue resident macrophages, by it’s ligand macrophage stimulating protein (MSP), results in the inhibition of NFkB activation, inducible nitric oxide synthase (iNOS) expression and TNFa production, as well as the induction of arginase expression, suggesting a role for this receptor in the regulation of classical vs. alternative macrophage activation. Furthermore, mice with a targeted deletion in this receptor exhibit increased sensitivity to endotoxic shock and DTH responses. More recently, we have demonstrated that MSP stimulation of primary peritoneal macrophages inhibits the production of IL-12. In order to map the domains of STK responsible for the inhibition of classical macrophage activation by MSP, we generated mutant forms of the receptor and expressed wild-type and mutant receptors in primary bone marrow derived macrophages by retroviral transduction. Expression of wild-type STK in these primary cells resulted in the ligand-independent reduction in IL-12p40 production in response to LPS stimulation, which was further inhibited by MSP treatment. This is consistent with the lack of a requirement for MSP in regulating responses to endotoxin in vivo. Surprisingly, a kinase dead receptor, which fails to signal in 293T cells, was fully functional in this assay, suggesting that the kinase activity of the receptor is not required for the inhibition of IL-12p40 under these conditions. However, the docking site tyrosines in the c-terminal tail of the receptor are essential for the inhibition of IL-12p40 by STK, suggesting that STK may be phosphorylated by an another kinase in this system. STK/RON has been shown to associate both physically and functionally with a number of other cell-surface receptors including EpoR, IL-3R bc, EGFR, MET as well as a number of integrins and cadherins. We have shown previously that STK regulates the activity of the aMb2 integrin (CR3) in peritoneal macrophages in a PI3K, PKCz-dependent manner. Here we show that STK also physically associates with CR3, as well as CD14, in RAW264.7 cells in the absence of ligand. Both CR3 and CD14 are capable of directly binding to LPS. Thus, we speculate that STK may exist as part of a receptor complex in macrophages and that signalling through STK might be induced directly by LPS. This would provide a means by which STK could temper the response of tissue-resident macrophages to LPS thereby preventing damage to host tissues.
Deletion of a Key PU.1 Gene Regulatory Element Induces T-Cell Lymphoma.
Authors: Rosenbauer Frank , Steidl Ulrich , Owens Bronwyn , Clayton Linda , Wagner Katharina , Kutok Jeffery L. , Le Beau Michelle M. , Tenen Daniel G.
Published: October 2019

The transcription factor PU.1 is indispensable for the generation of myeloid and B-lymphoid cells. PU.1 expression is highly regulated, and enforced expression of PU.1 in early erythroid or T-cell progenitors leads to erythroleukemia or an early block in T-cell differentiation, respectively. While PU.1 function in hematopoietic differentiation is well characterized, the regulation of the PU.1 gene is still poorly understood. We previously identified a -14 kb upstream regulatory element (URE) in the PU.1 gene. Replacement of this element with a neomycin cassette led to mice with hypomorphic PU.1 alleles, and uncovered a dosage effect of this lineage specific transcription factor in acute myeloid leukemia. However, since insertion of a neomycin gene might interfere with PU.1 expression, it was excised from the genome to eliminate any effects other than deletion of the URE. We found that UREΔ/Δ animals exhibited markedly decreased PU.1 expression in multiple lineages, resulting in a profound reduction in macrophage and B-cell numbers in bone marrow and spleen. Thus, normal PU.1 levels are required for development of both lineages in vivo. Furthermore, UREΔ/Δ animals exhibited decreased thymocyte numbers due to a partial block in double negative T-cell differentiation at the transition from DN1/2 to DN3/4 cells. Within a period of 3 to 8 months, many animals developed aggressive, clonal, lymphoblastic T-cell lymphomas, which were dominant over the myeloid leukemia in this model. Gene profiling analysis of early thymocyte populations sorted from preleukemic mice revealed several key genes known to be involved in normal T-cell development and transformation. The role of these novel PU.1 downstream targets is currently being analyzed and the data will be presented. Together, this murine model demonstrates (1) a crucial function of the URE for endogenous PU.1 expression, and (2) a PU.1 dosage effect in the induction of fatal T-cell malignancies. These results show that deletion of a key regulatory element can be a sufficient event for malignant transformation, thus highlighting the need to increase the search for such elements in tumor suppressor genes and proto-oncogenes and to validate their contribution to human disease.
PPARγ Regulates the Anti-Inflammatory Effects of Carbon Monoxide on Macrophages: A Gene Profiling Study.
Authors: Bilban Martin , Otterbein Sherrie L. , Ifedigbo Emeka , Enjyoji Keiji , Usheva Anny , Robson Simon C. , Bach Fritz H. , Otterbein Leo E. , Wagner Oswald
Published: October 2019

Carbon monoxide (CO) at low concentrations has generated recent interest due to its ability to modulate the inflammatory response associated with chronic graft rejection, vascular injury and septic shock. Both in vivo and in vitro CO can inhibit the expression of pro-inflammatory genes such as TNFα in macrophages while simultaneously increasing the expression of the anti-inflammatory cytokine IL-10. The mechanisms by which this occurs are still unclear. To better understand the mechanisms underlying the effects of CO, we employed the Affymetrix GeneChip technology to evaluate the time-dependent expression patterns of >12,000 genes in macrophages stimulated with bacterial endotoxin (LPS) in the presence or absence of a low concentration of CO previously demonstrated to evoke an anti-inflammatory response. We were particularly interested whether CO would, by itself, modulate in a specific manner the expression of proteins that might explain the anti-inflammatory effects observed following subsequent administration of endotoxin. RAW 264.7 murine macrophages were grown to 75% confluency and then exposed to CO (250 ppm) for 3 hr prior to administration of LPS (10 ng/ml). At 0, 15, 30, 60, 120 and 240 min thereafter, total RNA was isolated by standard methods and the RNA was then labeled and hybridized to U74Av2 GeneChips. Of >12,000 genes assessed, 116 of 270 that were LPS-responsive were affected by CO treatment. CO inhibited the majority of LPS-induced pro-inflammatory cytokines and acute phase proteins including expression of early growth response-1 (Egr-1), a transcription factor that serves as a central intermediary regulating many genes. Egr-1 was nearly completely inhibited by CO as was Egr-1-dependent expression of tissue factor (TF) and PAI-1. Treatment of cells with CO alone led to a rapid early increase in PPARγ, the expression of which was essential for the anti-inflammatory effects of CO. Inhibition of PPARγ using the selective chemical inhibitor GW9662 reversed the CO inhibitory effects on LPS-induced Egr-1 and TF expression. Correlative in vivo experiments in mice showed that CO pre-treatment blocked endotoxin-induced Egr-1 expression and decreased markers of lung inflammmation the effects of which were also lost with inhibition of PPARγ. Our analyses of gene expression patterns has led to the first molecular understanding of how treatment with CO, in this case by inducing PPARγ, blocks the pro-inflammatory response. These experiments provide novel insights into the mechanisms and pathophysiology of endotoxic shock and identify cellular targets by which CO mediates these cytoprotective effects.
A Phase I Trial of Dose Escalating Simvastatin Combined with Chemotherapy in End-Stage Myeloma and Lymphoma.
Authors: Lokhorst Henk M. , van de Donk Niels , van de Spek Ellen , de Weerdt Okke , van de Griend Rene , Wittebol Shulamiet , Verdonck Leo F. , Bloem Andries C.
Published: October 2019

We recently described that simvastatin effectively induced apoptosis in myeloma and lymphoma tumor cells by inhibition of proteingeranylgeranylation resulting in the reduction of the anti-apoptosis protein Mcl-1. In addition low concentrations of simvastatin had a chemosensitizing effect in combination with dexamethasone or doxorubicin. Based on these observations we initiated a Phase I study of dose escalating simvastatin combined with chemotherapy in patients with end-stage Myeloma and Lymphoma. Starting dose level of simvastatin was 5 mg/kg/day for 7 days, divided in 2 doses orally, followed by VAD chemotherapy in patients with myeloma and CHOP in patients with lymphoma. Three patients were included per dose level. In the absence of side effects WHO grade III/IV in 3 patients the dose of simvastatin was escalated with 2.5 mg/kg. Twenty-one heavily pretreated patients all refractory to at least 3 lines of chemotherapy (14 myeloma patients and 7 lymphoma patients) were included. No toxicity beyond WHO 2 was recorded with dose level 1–4 (5 mg–12.5mg/kg/day/7days). One patient treated at dose level 5 (15 mg simva/kg/day/7 days) became severely depressed and performed an unsuccessful suicide attempt. Two patients treated at dose level 6 (17.5 mg/kg/day/7 days had severe gastro-intestinal side effects (WHO 3; vomiting, diarrhoea, dehydration), necessitating interrupting simvastatin after 3 and 4 day respectively. The third patient treated at dose level 6 had moderate gastro-intestinal complaints but died on day 13 (2 days after VAD, deeply neutropenic) from overwhelming Gram-Septicaemia although prophylactic antibiotics had been prescribed. Three additional patients were then treated at dose level 5 again without side effects beyond WHO 2. None of the patients complained about muscle pain. No signs of rhabdomyolysis were registered.

Although response was not the primary endpoint of the study, it could be evaluated in 16 patients who completed at least 2 cycles of simvastatin with chemotherapy. Six patients (4 myeloma and 1 transformed low grade lymphoma) responded (32 %) including 5 patients with a Partial Response (> 50 tumor reduction) and 1 patient with a minor response. Four of 8 evaluable (myeloma) patients treated at dose level 4 and 5 responded. Due to toxicity all 3 patients at dose level 6 were not evaluable for response. In patients treated at dose level 4 and 5, in vivo down regulation of Mcl-1 (> 50 %) was observed in PBMC collected after 7 days of simvastatin treatment. These data show that in vivo downregulation of Mcl-1 by high dose (simva) statin in combination with chemotherpy may be a promising new modality for patients with drug resistant myeloma and lymphoma.
VAD-t (Vincristine, Adriamycin, Dexamethasone and Low-Dose Thalidomide) Is an Effective Initial Therapy with High Response Rates for Patients with Treatment Naïve Multiple Myeloma (MM).
Authors: Chanan-Khan Asher Alban , Miller Kena C. , McCarthy Philip , Koryzna Alexandra , Kouides Peter , Donohue Kathleen , Mohr Alice , Bernstein Zale P. , Alam Arif , Czuczman Myron S.
Published: October 2019

Recent understanding of MM biology suggests that MM cells rely on their microenvironment for disease progression and dissemination. MM cell interaction with stromal cells in the bone marrow microenvironment fosters chemotherapy resistance. Targeting the microenvironment with novel biologic agents such as thalidomide (T) has shown encouraging clinical results in patients with previously untreated as well as relapsed/refractory MM. We have previously shown here that VAD plus low dose T (t) is an effective salvage regimen for VAD refractory pts. We report the preliminary results of an ongoing phase II trial studying a combined chemoimmunotherapeutic approach - standard VAD plus t (200 mg) in treatment-naïve MM pts. This regimen was designed to target concurrently the malignant cell and its microenvironment. MM pts, ≥ Stage I with no prior therapy were eligible for study. Monthly infusional VAD (standard dose) was given for 4 cycles every 28 days concurrently with t for 4 months. t (100mg) was started 1 wk prior to VAD and was escalated in 1–2 wks to 200mg. Pts were evaluated for response after each cycle and after cycle 4 using SWOG criteria. Pts characteristics include: enrolled n=16, 8M, 8F; median age 58 yrs range 46–77; Stage III disease n=11, Stage II, n=3 and Stage 1, n=1; and Median B2M, 1.9 mg/L (range 1.2–11.5). Low dose coumadin (1–2 mg) was used for venous thromboembolism (VTE) prophylaxis, t was also held during the 4 days of each infusion of VAD. All pts are evaluable for toxicity and 11 are evaluable for response after completing 4 cyc. 10/11 (91%) responded clinically; CR, n=3, PR, n=7. One pt had progressive disease. Three pts with responding disease were removed from study for Grade III toxicity (1 adriamycin associated cardiomyopathy, 1 pulmonary hypertension and 1 recurrent infections) but are not included in response evaluation. Toxicities: fatigue (Gr. II) was the most common side effect (25%) and DVT was noted in 2 (12%). All pts eligible for stem cell (SC) transplant (n=7), were able to collect adequate amount of SC post VAD-t. Preliminary results of this study suggest that VAD-t is an effective and well-tolerated regimen with improved ORR compared to standard VAD regimen in pts with treatment naïve MM. No increase in incidence of VTE was noted with the combination. Adequate SC collection was achieved in transplant eligible pts. Accrual is ongoing on this study and updated results will be presented at the annual meeting.
Use of Patient-Reported Outcomes (PROs) to Assess Prognosis, Toxicity, and Response: The Bortezomib Experience in Relapsed and Refractory Multiple Myeloma (MM).
Authors: Dubois Dominique , Ravinder Dhawan , van de Velde Helgi , Esseltine Dixie , Gupta Sanjay , Viala Muriel , de la Loge Christine
Published: October 2019

PROs provide patients’ own appraisal of their health status and can be used to measure aspects of health that are generally not captured by ‘traditional’ clinical measures such as physician assessments and test results. This study analyzed PRO data from a multicenter, pivotal phase II trial of bortezomib in 202 patients (pts) with relapsed, refractory MM (NEJM2003;348:2609–17). PRO questionnaires (EORTC-QLQ C30 and MY24, FACT/GOG-Neurotoxicity (Ntx) and FACIT-Fatigue subscales) were administered at the screening visit (baseline) and day 1 of cycles 3, 5, 7 and at the end of the study.

Results: In this population with relapsed, refractory MM, poorer baseline pre-treatment multi-dimensional quality of life (QoL) scores were significantly correlated (range of r= 0.22 to r= 0.77) with fatigue, pain, dyspnea, appetite loss, neurotoxicity, MM disease symptoms, and treatment side effects. Clinical response to bortezomib (Complete Response or Partial Response) was associated with statistically significant (p<0.05) improvements in global QoL, physical, emotional and social functioning, fatigue, dyspnea, constipation, disease symptoms, social support and future perspective, whereas pts with a minimal response, no change or disease progression usually reported stable or worsening scores. Despite concern about peripheral neuropathy (12% for grade 3), FACT/GOG-Ntx change scores were stable in all responder groups. The prognostic value of baseline PRO data relative to clinical information was assessed using Partial Least Square (PLS) regression. First a model predicting death during the study (17 patients died during the study out of 202; 8.42%) identified that at baseline high total serum protein, bone marrow plasma cells and ß2-microglobulin and low platelet count, serum albumin, hemoglobin, Karnofsky score, body surface area and weight were predictive of death. In a separate model, the following baseline PRO scores predicted death during the study: high fatigue, appetite loss, treatment side effects, sleep disturbance, dyspnea and pain scores and low FACIT-Fatigue, physical, role, social, global QoL, cognitive, emotional and FACT/GOG-Ntx scores. The combination of the clinical and PRO PLS components into a hierarchical PLS regression resulted in an increase in the predictive value of the model, suggesting complementary information.

Conclusion: PRO data can serve as independent measures of clinical benefit, toxicity, and even mortality risk, complementing traditional physician assessments and test results. In this trial of relapsed and refractory MM pts, PRO results suggested improvements in QoL for clinical responders, tolerable side effects, and independent prognostic value for mortality.
MALT1 Is an Essential Regulator of Antigen Receptor Mediated NF-kappaB Activation.
Authors: Ruland Jürgen , Mak Tak
Published: October 2019

The translocation t(11;18)(q21;q21) is the most common chromosomal abnormality in lymphomas of mucosa associated lymphoid tissue (MALT lymphoma). Molecular cloning of the breakpoint demonstrated that t(11;18) leads to the fusion of the IAP2 gene to the novel gene MALT1. MALT1 encodes a paracaspase that can bind to BCL10 but its cellular function was unknown. We have previously demonstrated that BCL10, which is involved in independent MALT lymphoma translocations, functions as a positive regulator of lymphocyte proliferation. BCL10 connects antigen receptor signaling in B and T cells to NF-kappa B activation. To identify the physiological role of MALT1 we have now disrupted the MALT1 gene in mice. Here we are using both MALT1 and BCL10 deficient animals and show that MALT1 is essential for functional T cell activation, proliferation and cytokine production in response to T cell receptor (TCR) ligation. Similar to BCL10 we found MALT1 to be strictly required for signal-specific NF-kappaB activation in response to TCR ligation but not for TNF-alpha or IL-1 signaling. MALT1 operates downstream of BCL10, controls the catalytic activity of IKK and additionally regulates signaling of p38 and JNK MAP kinases. Moreover we found MALT1 to be critical for B1 and marginal zone (MZ) B cell development. In contrast to BCL10 disruption however, inactivation of MALT1 has only mild effects on conventional B2 B cell activation, BCR induced NF-kappaB signaling in splenic B lymphocytes and does not cause defects during embryonic development. Thus, MALT1 is an essential regulator of BCL10 mediated NF-kB induction that is differentially required depending on the cellular context. Together these data support the view that normal expression of BCL10 and MALT1 are necessary for controlled NF-kB signaling in lymphocytes whereas deregulation of either BCL10 or MALT1 through chromosomal translocation results in increased lymphocyte growth and survival
Global Assessment of Promoter Methylation in a Murine Model of Cancer Identifies ID4 as a Putative Novel Tumor Suppressor Gene in Human Leukemia.
Authors: Yu Li , Liu Chunhui , Vandeusen Jeff , Becknell Brian , Dai Zunyan , Wu Yue-Zhong , Raval Aparna , Liu Te-Hui , Ding Wei , Mao Charlene , Liu Shujun , Smith Laura T. , Lee Stephen , Rassenti Laura , Marcucci Guido , Byrd John , Caligiuri Michael A. , Plass Christoph
Published: October 2019

We developed a mouse model of acute T/natural killer (NK) acute lymphoblastic leukemia (ALL) that is always preceded by polyclonal lymphocyte expansion to determine how aberrant promoter DNA methylation and consequent gene silencing might be contributing to leukemic transformation. We demonstrated the non-random nature of methylation in the transformation from benign polyclonal expansion to leukemia, and identified a novel tumor suppressor gene silenced in the majority of human leukemia but not solid tumors. To this, we utilized restriction landmark genomic scanning (RLGS) on eight IL-15 transgenic (tg) mice with T/NK ALL, and performed RLGS on an additional five spleens from IL-15tg mice without ALL, but with polyclonal T/NK expansion, and on four spleens from wild type (wt) mice. A total of 2447 RLGS fragments with good resolution on each RLGS profile were analyzed. Only 1–2 variable changes were detected in either wt or polyclonal T/NK expansion controls. In contrast, the eight spleens with clonal T/NK leukemia had 45 to 209 changes (1.8 to 8.5% of total fragments) consistent with aberrant DNA methylation. The association of RLGS fragment losses or reduced intensity with leukemic transformation versus polyclonal expansion was highly significant (P<0.001). The promoter of Inhibitor of DNA binding 4 (Id4) was found methylated in 87.5% of the mouse T/NK ALLs. ID4 was found methylated in 85.7% (72/84) of primary human acute myeloid leukemias (AML) and in 100% (61/61) of chronic lymphocytic leukemias (CLL). Id4 was silenced secondary to promoter methylation in 100% mouse T/NK ALLs (n=5) and human acute leukemias (n=6). Overexpression of Id4 induced apoptosis in mouse cell line YAC-1 and inhibited tumor growth both in vitro and in vivo. These data, and an analysis of 219 RLGS profiles from different types of human tumors demonstrate that the ID4 promoter is preferentially methylated and functions as a putative novel tumor suppressor gene in mouse and human leukemia. This study highlights the use of a novel animal model, novel mouse NotI-EcoRV arrayed library, and RLGS to examine the role of methylation in leukemic transformation and to identify new targets for prevention and therapeutic strategies.
Aberrantly Hypermethylated Genes in Adult T-Cell Leukemia Cells: The Implications in the Leukemogenesis.
Authors: Yasunaga Jun-ichirou , Taniguchi Yuko , Nosaka Kisato , Satou Yorifumi , Yoshida Mlka , Takai Ken , Sakai Tatsunori , Mitsuya Hiroaki , Matsuoka Masao
Published: October 2019

Adult T-cell leukemia (ATL) is a highly aggressive neoplasm of helper T lymphocytes and is etiologically associated with human T-cell leukemia virus type I (HTLV-I). Although HTLV-I encoded Tax protein is thought to play a central role in the leukemogenesis of ATL, ATL cells frequently could not produce Tax protein by the somatic mutations, deletion and loss of 5′-LTR. Moreover, a long-term latent period (almost 60 years in Japan) precedes the onset of ATL, suggesting that multistep tumorigenesis is involved in development of ATL in addition to role of viral protein. Such transformation process is thought to include alterations of host genome: genetic and epigenetic changes. The epigenetic alteration, such as DNA methylation and histone modification, is commonly observed in various cancer cells. Recently, we reported that the aberrant expression of MEL1S gene, which is hypomethylated in ATL cells, confers resistance against transforming growth factor-beta.

In this study, we identified 53 aberrantly hypermethylated DNA sequences in ATL cells using methylated CpG island amplification/representational difference analysis (MCA/RDA) method. In addition, we found that the DNA methylation of these regions tends to accumulate with disease progression. Seven genes, which were expressed in normal T-cells, but suppressed in ATL cells, were identified near the hypermethylated regions. Among these silenced genes, Kruppel-like factor 4 (KLF4) gene is a cell cycle regulator and early growth response 3 (EGR3) gene is a critical transcriptional factor for induction of Fas ligand (FasL) expression. Treatment with 5-aza-2′-deoxycytidine resulted in the recovery of their transcription, indicating that their silencing is associated with DNA hypermethylation. To study their role in oncogenesis of ATL, we transfected recombinant adenovirus vectors expressing KLF4 and EGR3 genes into ATL cell lines. Expression of KLF4 induced apoptosis of ATL cells whereas enforced expression of EGR3 induced the expression of FasL gene, resulting in apoptosis. This EGR3-induced apoptosis has been inhibited by stable transfection of cellular and viral FLIP, which suppress the activation of caspase-8 by preventing pro-caspase-8 in Fas-mediated apoptosis pathway. Therefore, suppressed expression of EGR3 enabled ATL cells to escape from activation-induced cell death mediated by Fas-FasL signaling. Our results showed that the DNA hypermethylation plays an important role in the leukemogenesis of ATL by silencing transcriptions of genes that inhibit the abnormal growth of ATL cells.
Loss of the INK4A Locus Accelerates tal1/scl-Induced Leukemia in Mice.
Authors: Calvo Jennifer A. , Kelliher Michelle
Published: October 2019

Tal1/scl, a basic helix-loop-helix (bHLH) transcription factor essential for hematopoiesis and vasculogenesis, is also found misexpressed in patients with T cell acute lymphoblastic leukemia (T-ALL). In our mouse model of tal1/scl-induced disease, mice develop T cell leukemia after a long latency, suggesting that additional mutations are required for leukemogenesis. In T-ALL patients, the INK4a locus, which encodes both p14ARF and p16INK4a, is often found mutated or methylated, suggesting that loss of p16INK4A/p14ARF expression contributes to the development of T-ALL. Similarly, we fail to detect p16INK4A/p19ARF expression in our mouse tal1/scl leukemic cells. To determine if the loss of INK4a cooperates with tal1/scl to induce leukemia, we mated our tal1/scl transgenic mice to INK4a −/− mice to generate a cohort of tal1/scl/INK4a+/− mice. Mice heterozygous for INK4a are healthy and are not predisposed to the development of leukemia. In contrast, tal1/scl/INK4a+/− mice develop T cell leukemia at an accelerated rate, within a median survival period of 110 days, compared to a median survival of 352 days for the tal1/scl transgenic mice, which are wild type with respect to INK4a. Taken together, these data suggest that mutation of the INK4a locus contributes to tal1/scl-mediated leukemogenesis in mice, similar to the widespread deletion of the INK4a/ARF locus observed in TAL1/SCL-expressing human leukemia (Ferrando, 2002). BrdU labeling of preleukemic tal1/scl thymocytes reveals a 41% increase in the percentage of thymocytes in S phase of the cell cycle, suggesting that tal1/scl may stimulate cell cycle progression. In contrast to wild type thymocytes which exhibit 14.5% (±1.5) of cells in S phase, 24.5% (± 2.9) of preleukemic tal1/scl thymocytes are found in S phase (p<0.05, n=4). Moreover, an increase in the sub-G1 population is also observed in the tal1/scl preleukemic thymus. Preliminary cell cycle analysis of tal1/scl/INK4a−/− thymocytes reveals a decrease in the sub-G1 population, suggesting that the absence of the INK4a locus may contribute to tal1/scl leukemogenesis, in part, by inhibiting the tal1/scl-induced apoptosis.
Association of Protein Z Deficiency with Retinal Artery and Vein Occlusion.
Authors: Koren-Michowitz Maya , Eting Eva , Voltchek Yulia , Rahimi-Levene Naomi , Garach-Jehoshua Osnat , Kornberg Abraham
Published: October 2019

Central retinal vein occlusion (CRVO) and central retinal artery occlusion (CRAO) are common disorders mainly associated with traditional risk factors for atherosclerosis. There is no clear correlation between thrombophilia and CRVO. However it was suggested that hyperhomocysteinemia and the presence of antiphospholipid antibodies are risk factors for the development of CRAO. Protein Z is a vitamin K dependent cofactor for the inactivation of activated factor X (Xa) by the protein Z dependent protease inhibitor (ZPI). Protein Z deficiency presumably promotes thrombosis, in particular arterial thrombosis, as suggested by an association between low plasma protein Z levels and ischemic stroke. The similarity between risk factors for ischemic stroke and CRVO / CRAO and the fact that low protein Z levels are associated with an increased risk for ischemic stroke has led us to assess protein Z levels in patients with CRVO and CRAO. Plasma samples from 29 patients with CRVO and 7 patients with CRAO (total study group= 36, mean age 60±11) were screened for protein Z levels by ELISA. Study group was also screened for the presence of lupus anticoagulant (LAC) by the kaolin clotting time (KCT) index and the dilute Russells’ viper venum test (DRVVT), for activated protein C resistance (APCR) and anticardiolipin antibodies (ACA). Levels of protein Z were compared with a control group of 42 healthy individuals (mean age 43±11). Patients with CRAO and history of ischemic stroke or an embolic source were excluded. Thirty patients in the study group had traditional risk factors for atheroscalerosis including hypertension, diabetes mellitus, hyperlipidemia and smoking and 6 patients had none. There was no significant difference in protein Z levels between the whole study group patients and controls (1995±810 vs. 2010±603 ng/ml p=0.922). However, patients with no risk factors for CRVO/CRAO had significantly lower protein Z levels than controls (1379±682 vs. 2010±603 ng/ml p=0.022) and patients with risk factors (1379±682 vs. 2118±785 ng/ml p=0.04). In the study group three patients had abnormal APCR and 6 had positive LAC. There was no correlation between low protein Z levels and the presence of LAC, APCR or ACA, or a correlation between low protein Z levels and the specific diagnosis (CRVO or CRAO) or age. The data show that low protein Z levels may be an additional risk factor for CRVO and CRAO in patients without traditional risk factors for these disorders. The association should be explored in a larger group of patients.