bluuebunny/crossref_metadata_2025 huggingface dataset:
Searching Polymorphic Markers Associated with the Genetic Predisposition To Develop Therapy-Related Myelodysplastic Syndromes and Acute Myelogenous Leukemia T-(MDS/AML) after Myeloablative Chemotherapy.
Authors: Tormo Mar M. , Guillem Vicent V. , Mata Manuel M. , Gonzalez Marcos M. , Nomdedeu Josep J. , Esteve Jordi J. , Calasanz Maria Jose M.J. , Terol Maria Jose M.J. , Marugan Isabel I. , Solano Carlos C.
Published: April 2020

INTRODUCTION: Therapy-related MDS/AML constitutes the most frequent secondary neoplasia and his incidence is increasing in the last years. This incidence is especially high when myeloablative chemotherapy is used before autologous stem cell transplantation (ASCT). Since for similar treatments only a percentage of the patients ends up by developing a secondary leukemia, it has been proposed the existence of some type of genetic predisposition associated to this syndrome.

OBJETIVE: To search single nucleotide polymorphisms (SNPs) associated with the genetic predisposition to develop a t-MDS/AML by means of SNP genome screening using high density microarrays.

MATERIAL AND METHODS: Two groups of individuals were genotyped: Group A, composed by 16 patients with t-MDS/AML post-ASCT (10 NHL, 4 HD, 2 multiple myeloma) and Group B (control), formed by 16 patients submitted to ASCT and that after a period of more than 10 years have not developed t-MDS/AML(14 NHL, 2 HD). For each individual more than 10.000 SNPs were genotyped by means of the use of microarrays of high density (Mapping 10K, Affymetrix®). Those SNPs are distributed along the whole genome and have a high average heterocigosity (0,37). The alele frequencies for each SNP between two groups were compared.

RESULTS: We found a group of 4 SNPs located in the q23.3 band of the chromosome 5 in which the differences of alele frequency between the groups A and B were superior to 50 %. These SNPs are located in a region that comprises the only gene, CSS3 (chondroitin sulphate synthetase 3), being the difference of alele frequency of 57 % in the intronic SNP of this gene.

CONCLUSIONS: The results suggest that the CSS3 gene is a candidate for being studied by its possible relation with the genetic predisposition to develop LMAt/SMDt, although its putative functional implication with the disease must still be elucidated. There is known that a counterpart of CSS3, CSS1, plays a key role in the proliferation and apoptosis of myeloma cells. Thus, it is possible that CSS3 could be somehow related with the hematopoyesis pathways and implied in the development of t-MDS/AML. Financed by FIS G03/008.
CD24High / FLJ/P1Low Expression Correlates with Bad Prognosis and Shorter Survival in Adult ALL.
Authors: Coignet Lionel J. , Johnson Thimothy , Nganga Alain , Zhang Qing , Meyer Corinna , Drexler Hans G. , Ghosh Debashis , Singh Keshav , Baer Maria R.
Published: April 2020

Leukemia is a neoplastic proliferation of cells of hematopoietic origin that arises following somatic mutation in a single hematopoietic stem cell, the progeny of which forms a clone of leukemia cells. Genetic alterations leading to leukemia transformation of a cell are often associated with major alterations of chromosomes that can be detected by studying cells of the leukemic clone in mitosis. One of these alterations is a chromosomal translocation that is often used to identify genes potentially involved in other type of rearrangements such as deletions. Abnormalities of chromosome band 13q14 occur in hematologic malignancies of all lineages and at all stages of differentiation. Unlike other chromosomal translocations, which are usually specific for a given lineage, the chromosomal translocation t(12;13)(p12;q14) has been observed in both B-cell and T-cell precursor acute lymphoblastic leukemia (ALL), in differentiated and undifferentiated acute myeloid leukemia (AML) and in chronic myeloid leukemia at progression to blast crisis (CML-BC). Recently, we have shown the presence of a myeloid- and lymphoid-specific breakpoint cluster regions within chromosome band 13q14 in acute leukemia (Genes Chromosome Cancer 25:222–229,1999). In addition, a new cell line has been established from one of the lymphoid cases, MUTZ5, that carries a single t(12;13) translocation (Leukemia 15:1471–1474, 2001). The molecular characterization of this translocation led to the identification of a new gene, FLJ13639, that is disrupted and lost in the MUTZ5 cell line. This gene shares homologies with the large family of short-chain dehydrogenase reductase (SDR). Furthermore, three transcripts and proteins were found to be differentially expressed for this gene, where P1 is potentially the active form of dehydrogenase, while P2 and P3 are lacking the co-activator site. We report here that one of the consequences of the loss of FLJ13639 is the over-expression of CD24 that appears to provide leukemia cells with a proliferation and invasiveness advantage, as well as a certain degree of chemoresistance. FLJ13639 is a new mitochondrial protein that seems to be important for the respiration and apoptosis processes. In addition, semi-quantitative RT-PCR for CD24 and FLJ13639/P1 was performed in a series of cell lines as well as 29 adult ALL samples at diagnosis. Fifty percent of the samples showed a CD24High / FLJ/P1Low profile whereas the remaining samples showed either a balanced expression or a CD24Low / FLJ/P1High profile. These preliminary data on patient samples indicated a correlation between survival and CD24High / FLJ/P1Low expression profile (p=0.04). The current median time survival for the CD24High / FLJ/P1Low group is 9 months whereas the CD24Low / FLJ/P1High group is 28 months. Therefore, this appears to be a new potential prognostic marker for adult ALL.

Figure Figure
In Vitro Synergistic Anti-Tumor Activity by Targeting TRAIL-R1 and CD20 Antigen by Combining HGS-ETR1 (Agonistic Human Monoclonal Antibody to TRAIL Receptor 1) and Rituximab Monoclonal Antibody Against Non-Hodgkin’s Lymphoma Cells (NHL).
Authors: Czuczman Myron S. , Maddipatla Sreeram , Knight Joy , Hernandez-Ilizaliturri Francisco J.
Published: April 2020

Tumor necrosis factor-related apoptosis ligand (TRAIL) or agonist antibodies targeting specific TRAIL-receptors possess significant in vitro and in vivo anti-tumor activity. HGS-ETR1 (mapatumumab) and HGS-ETR2 are two fully human recombinant, high affinity IgG1λ mAbs targeting TRAIL-R1 (Death-receptor 4, DR-4) and TRAIL-R2 (Death-receptor 5, DR-5), respectively. TRAIL-receptor agonist mAbs trigger the extrinsic (mitochondria-independent) apoptotic pathway and enhance the anti-tumor effects of chemotherapy drugs against NHL. Combining TRAIL-receptor agonist mAbs with biological agents (e.g. rituximab) capable of activating the intrinsic (mitochondria-dependent) apoptotic pathway may result in synergistic effects against NHL.

Objective: To study the biological effects of rituximab when combined with either HGS-ETR1 or HGS-ETR2.

Materials and methods: A panel of NHL cell lines [Raji, SUDHL-4, SU-DHL-10, Ramos, and two rituximab resistant cell lines (RRCL), 2R and 4RH] were utilized. Expression of CD20, TRAIL-R1 and TRAIL-R2 in the NHL cells used were evaluated by flow cytometry. NHL cells were exposed to HGS-ETR1 or HGS-ETR2 followed by either rituximab, istoype control antibody or media alone. DNA synthesis and cell growth arrest were quantified by [H3] Thymidine incorporation assays at 24 and 48 hrs. Induction of apoptosis was detected by multiparameter flow cytometric analysis. For antibody-dependent cellular cytotoxicity (ADCC)/Complement mediated cytotoxicity (CMC) studies, 51Cr-labeled NHL cells were exposed to HGS-ETR1 or HGS-ETR2 (5μg/ml) +/− rituximab or isotype control antibody (10μg/ml) and peripheral blood mononuclear cells (Effector: Target ratio 40:1) or human serum, respectively. 51Cr-release was measured and the percentage of lysis was calculated. Statistical analysis of results was performed using the Chi-square test. Results: In sensitive cells in vitro exposure to HGS-ETR1 resulted in significant apoptosis (30–50%) and decrease in DNA synthesis. The combination of HGS-ETR1 and rituximab resulted in significant inhibition of cell proliferation (90% reduction) when compared to either HGS-ETR1 (60% reduction) or rituximab (5% reduction) alone at 24 and 48 hrs. In sensitive cells HGS-ETR1 induced CMC and ADCC as a single agent. No significant anti-tumor activity was observed with HGS-ETR2. The biological activity of anti-TRAIL receptors mAbs did not correlate to TRAIL-R1 or TRAIL-R2 cell surface antigen expression. Conclusions: Targeting TRAIL-R1 with HGS-ETR1 induces apoptosis, cell growth arrest and in some cell lines ADCC/CMC. The combination of HGS-ETR1 with rituximab results in significant synergistic activity (i.e. anti-proliferation) and warrants further pre-clinical and clinical evaluation in B-cell lymphomas.
BCL6, p53 and Molecular Targeted Therapy in B-Cell Lymphomas.
Authors: Cerchietti Leandro C.A. , Polo Jose M. , Da Silva Gustavo F. , Dowdy Steve M. , Giorgio Catoretti M. , Rita Shaknovich M. , Melnick Ari M.
Published: April 2020

The BCL6 transcriptional repressor is an oncogene often constitutively expressed in diffuse large B-cell lymphomas (DLBCL). The oncogenic mechanism of action of BCL6 presumably involves repression of its direct target genes. We recently developed a targeted therapy agent (called BPI - BCL6 peptide inhibitor) that specifically blocks transcriptional repression by BCL6, and which causes apoptosis in lymphoma cells in vitro and in vivo. We present here potent and stable derivatives of BPI able to specifically eradicate lymphoma cells after a single dose in vitro. Expression array studies of BCL6 target genes reactivated by BPI revealed that one such gene is the p53 tumor suppressor. p53 was also recently shown to be BCL6 target gene by Phan et. al., Nature 2004. We find that BCL6 represses p53 in DLBCL cells through recruitment of the SMRT and N-CoR corepressors, which explains how BPI, which blocks recruitment of these corepressors, reactivates p53. We next wished to determine the contribution of BCL6-mediated repression of p53 to lymphomagenesis, and how p53 modulation might affect BCL6 targeted therapy strategies for DLBCL. We found that BPI could induce p53 target gene expression in DLBCL cells with wild-type p53 and that small molecules or peptides that block p53 rescue apoptosis induced by BPI. In contrast, although BPI also induces p53 in DLCBL cells with mutant p53, there was no activation of p53 target genes and no rescue by p53 blocking molecules. However BPI causes apoptosis of DLBCL cells regardless of p53 status indicating the BCL6 mediates its oncogenic actions through both p53 dependent and independent pathways. p53 is usually wild-type in DLBCL and our analysis of >100 patients show that p53 protein is, surprisingly, still expressed in these tumors. These data suggest that p53 is not fully active in DLBCL cells, consistent with the fact that we found that BCL6 also directly represses upstream activators of p53 such as Chk1 and ATR. BCL6 blockade thus can fully restore activity of p53, both by increasing its expression levels and by enhancing its activation by upstream mediators. Accordingly, sequential administration of p53 activating molecules that enhance p53 activity, potently synergizes with BPI in killing lymphoma cells. BPI also synergizes with chemotherapy drugs that act in part through p53, such as doxorubicin. From these studies we conclude that i) BCL6 mediates lymphomagenesis by direct repression of p53 and upstream target gene pathways; ii) BCL6 positive lymphomas are dependent on BCL6 for their survival regardless of whether p53 is wild type or mutated; iii) Sequential targeting of BCL6 and p53 with BPI and a p53 activating molecule or doxorubicin is likely to be a highly effective therapeutic regimen for patients with DLBCL, especially for the majority who have wild-type p53; iv) The new BPI derivatives are sufficiently potent and stable to be tested in the clinical setting.
Antitumor Activity and Mechanism of Action of a Novel Stat3 Inhibitor, WP1066, Against Human B-Cell Non-Hodgkin’s Lymphoma and Multiple Myeloma.
Authors: Kong Ling-Yuan , Kapuria Vaibhav , Bartholomeusz Geoffrey , Talpaz Moshe , Priebe Waldemar , Donato Nicholas J.
Published: April 2020

B-cell non-Hodgkin’s lymphomas (B-NHL) have the highest incidence rates among all lymphomas, comprising more than 85% of malignant lymphomas worldwide. Multiple myeloma (MM) is a plasma cell malignancy that affects 14,000 patients per year. Although new therapies have recently been introduced to treat these malignancies, patient survival has not significantly improved, illustrating the need for additional agents that target and suppress this disease. B-cell malignancies over-express c-myc and frequently retain a constitutively activated Stat3, resulting in increased survival gene expression and apoptotic protection. Targeting these transcription factors may reduce apoptotic thresholds and suppress the growth and survival of these malignancies. In this study, we refined and tested the anti-tumor activity of a novel small MW compound discovered by screening a synthetic library for agents that bock Stat3 activation. WP1066, blocked IL-6 mediated Stat3 activation in numerous human B-NHL and MM tumor cells including the Mino, LP, MM-1 and OCI-My5 at low micromolar concentrations. This compound blocks Jak2-depedent cytokine signaling (IL-3, GM-CSF, IL-6) through the rapid (15 min) down-regulation of the upstream tyrosine kinase, Jak2. WP1066 also causes rapid reduction in c-myc in B-cell malignancies but other transcription factors and signaling kinases are not affected by WP1066. The apoptotic effects of WP1066 on B-NHL and MM cells parallel its Stat inhibitory, Jak2 down-regulatory activity. Jak2 down-regulation is not dependent on proteosomal processing but requires activation of a tyrosine phosphatase, suggesting a unique mechanism of WP1066 action. Based on activities described in vitro, we further examined the antitumor activity of WP1066 in a B-NHL xenograft tumor model. Mice were inoculated (i.p.) with 25 × 106 Mino cells (CB17 SCID mice, 4–5 weeks of age) and 13 days later were treated with 10, 20, or 40 mg/kg of WP1066 (i.p.) qd x 5/week for 2 weeks. WP1066 significantly decreased tumor burden and increased the survival of Mino tumor bearing mice treated with 20 or 40 mg/kg of WP1066. These results suggest that WP1066 reduces Stat activation through a unique mechanism and may have therapeutic potential for the treatment of human B-cell non-Hodgkin’s lymphomas and multiple myeloma.
Development of Small Molecule Inhibitor of the AML1-ETO Oncoprotein.
Authors: Grembecka Jolanta , Graf Kristin , Newman Miki , Zhou Yunpeng , Cheney Mathew , Speck Nancy A. , Brown Milton L. , Bushweller John H.
Published: April 2020

Core binding factor (CBF) is a heterodimeric transcription factor composed of RUNX1 (CBFa) and CBFb subunits which are essential for normal blood cell development. RUNX1 binds DNA and the affinity of this interaction is regulated by the binding of CBFb to the RUNX1 subunit. The RUNX1(AML1) gene is disrupted by a variety of chromosomal translocations, including t(8;21)(q22;q22), which produces the chimeric protein AML1-ETO associated with AML (M2 subtype). The AML1-ETO fusion protein is a dominant inhibitor of wildtype RUNX1-CBFb activity in vivo and functions as a transcriptional repressor of RUNX1-CBFb target genes, causing a blockage in normal hematopoietic development and predisposing for the development of leukemia. We have validated the importance of the RUNX1 Runt domain - CBFb interface as a potential therapeutic target by assessing the effect of mutations introduced into the Runt domain of AML1-ETO on AML1-ETO activity. Therefore, treatments targeting the AML1-ETO protein and blocking its interactions with CBFb are likely to be therapeutically beneficial. We initiated efforts to develop small molecule inhibitors of the RUNX1-CBFb interaction as possible therapeutics for the treatment of the associated leukemia. Based on the X-ray structures of the RUNX1 Runt domain in complex with CBFb and on mutagenesis data, the interaction areas for the virtual screening searches were defined. The computer program LUDI was utilized for virtual screening using CAP (Chemicals Available for Purchase) database of 70,000 commercially available compounds with drug-like properties. The resultant hits were subjected to further scoring and visual inspection of the potential interactions with the RUNX1 Runt domain to select compounds for experimental testing by FRET (Fluorescence Resonance Energy Transfer). Experimental screening resulted in identification of six initial lead compounds that inhibit the interaction of RUNX1 with CBFb. These compounds were further optimized using standard medicinal chemistry approaches to increase their affinity and determine the structure-activity relationship (SAR). This resulted in a number of compounds with low micromolar affinity which effectively block the heterodimerization of CBF. These compounds represent the first small molecule inhibitors targeting RUNX1 and inhibiting its interaction with CBFb. They represent a good starting point for the development of a therapeutically useful inhibitor. Several approaches are being explored to modify these compounds to achieve selectivity towards AML1-ETO versus wild type RUNX1.
Simultaneous Targeting of Lyn and Bcr-Abl Kinases by NS-187 Cures Mice Bearing Imatinib-Resistant Leukemic Cells.
Authors: Naito Haruna , Kimura Shinya , Nakaya Yohei , Naruoka Haruna , Wakayama Tatsushi , Kimura Sachie , Ito Shinsaku , Ashihara Eishi , Maekawa Taira , Hirabayashi Kazuko
Published: April 2020

We have identified a specific dual Bcr-Abl/Lyn inhibitor, NS-187 (elsewhere described as CNS-9), which is 25–55 times more potent than imatinib against wild type Bcr-Abl in vitro. To evaluate the potential of NS-187 as a therapeutic agent, we assessed its in vivo activity. When Balb/c mice were given NS-187 orally at a dose of 30 mg/kg, the pharmacokinetic parameters were as follows: Tmax, 2 h; Cmax, 586 ng/ml; AUC0-∝, 2999 ng•h/ml; T1/2, 1.0 h; and bioavailability value (BA), 33%. The maximal tolerated dose (MTD) of NS-187 in Balb/c or Balb/c-nu/nu mice was 200 mg/kg/day (100 mg/kg, twice daily). To test the effect of NS-187 on in vivo tumor growth, Balb/c-nu/nu mice were injected subcutaneously with Bcr-Abl-positive KU812 cells on Day 0 and given NS-187 or imatinib orally twice a day from Day 7 to Day 17. At 20 mg/kg/day, imatinib inhibited tumor growth slightly, while at 200 mg/kg/day, it inhibited tumor growth almost completely. In contrast, at only 0.2 mg/kg/day NS-187 significantly inhibited tumor growth, while at 20 mg/kg/day it completely inhibited tumor growth without any adverse effects. The body weights of the treated tumor-bearing mice were not significantly different from those of untreated mice, even at a dosage of 200 mg/kg/day NS-187. Thus, NS-187 was at least 10-fold more potent than imatinib in vivo with complete inhibition of tumor growth as the end-point. We also tested the ability of NS-187 to suppress tumor growth in another murine tumor model, namely, Balb/c-nu/nu mice intravenously transplanted with BaF3 cells harboring wild type Bcr-Abl. The mice were treated orally with NS-187 or imatinib for 11 days starting on Day 1. All eight untreated mice and all eight mice treated with 400 mg/kg/day imatinib had died by Day 25 due to leukemic cell expansion, and NS-187 significantly prolonged the survival of the mice in a dose-dependent manner. We next examined the ability of NS-187 to block the in vivo growth of BaF3 cells harboring one of the Abl point-mutants M244V, G250E, Q252H, Y253F, T315I, M351T and H396P in Balb/c-nu/nu mice. These mice were treated with NS-187 or imatinib for 11 days starting on Day 1. NS-187 at 200 mg/kg/day significantly prolonged the survival of mice inoculated with BaF3 cells harboring any of these mutants except T315I compared with untreated or imatinib-treated mice (see Figure for an example). Thus, NS-187 was more potent than imatinib and could override the point-mutation-based imatinib-resistance mechanism in vivo. The efficacy and safety of NS-187 for Ph+ leukemias is expected to be verified by early-phase clinical trials.

Figure Figure
Cloning and Characterization of a Tipifarnib-Resistant BCR/ABL-Positive Cell Line, K562/RR.
Authors: Miyoshi Takuji , Nagai Tadashi , Ohmine Ken , Kikuchi Satoru , Nakamura Makiko , Ozawa Keiya
Published: April 2020

The results of preclinical studies and clinical trials have suggested that Tipifarnib, a farnesyltransferase inhibitor, might be effective for treating various hematological disorders, including chronic myeloid leukemia. However, period for which Tipifarnib is effective may be short term, indicating that drug resistance is an important problem for patients being treated with Tipifarnib. To analyze the mechanisms of resistance to Tipifarnib, we have generated a new Tipifarnib-resistant BCR/ABL-positive cell line, K562/RR. The IC50 of Tipifarnib was 7.4-fold higher in K562/RR than in the parental cell line K562. The level of unprocessed HDJ-2, which is a substrate of farnesyltransferase, was increased by Tipifarnib treatment in K562/RR as well as in K562 cells. This result suggests that Tipifarnib inhibits protein farnesylation in K562/RR cells in a same manner and that mechanisms independent of farnesyltransferase activity are involved in the acquisition of resistance to Tipifarnib in K562/RR cells. While the levels of cleaved caspase 3, cleaved caspase 7, cleaved caspase 9 and cleaved PARP were significantly increased in K562 cells, those were not changed in K562/RR cells with Tipifarnib treatment. Furthermore, Tipifarnib-induced decrease in the level of phospho-Bcl-2 was not observed in K562/RR cells, indicating that induction of apoptosis by Tipifarnib is much less in K562/RR than in K562 cells. Tipifarnib significantly increased the percentage of S phase cells and decreased that of G1 phase cells in K562 cells, which was probably caused by activation of G2/M check point. In contrast, addition of Tipifarnib had no remarkable effect on cell cycle in K562/RR cells, suggesting that Tipifarnib-mediated induction of cell cycle blockage is also abrogated in K562/RR cells. From DNA microarray analyses using a cDNA microarray comprising 25,000 genes, we identified 5 genes whose expression levels were higher in K562/RR than in K562 cells. These genes include _-globin, calcium channel Caveolin 2 and FEN1, which is involved in DNA replication and repair, and CUGBP2, which may affect expression of cyclogenase 1. These results suggest that various mechanisms are involved in Tipifarnib resistance in K562/RR cells and that this cell line is a useful model for investigating the farnesyltransferase activity-independent mechanisms of resistance to Tipifarnib.
Glucocorticoid Receptor in Lymphocytic Malignancies.
Authors: Tang JingYan , Miao Yan , Jiang LiMin
Published: April 2020

Objective To determine the level of glucocorticoid(GC) receptor (GR) at the membrane and cytoplasma of lymphocytic malignancy cell. To study the relationship among clinical characters, membrane glucocorticoid receptor (mGR) and cytoplasma glucocorticoid receptor (cGR).

Methods The expression level of membrane glucocorticoid receptor (mGR) and cytoplasma glucocorticoid receptor (cGR) at lymphocyte from 51 patients with lymphocytic malignancies, 19 patients with acute myelogenous leukemia (AML) and 22 normal children were measured by flow cytometry. The levels and relationship of mGR and cGR in different diseases, in 3 ALL risk groups, in both poor and good early clinical response groups to prednision in children with ALL, are analyzed.

Results There were no significant differences among the expression of mGR in lymphocytic malignancies, AML and normal group. However, significant increase of cGR expression was observed in lymphocytic malignancies when compared with AML and normal group(P<0.001). Of the 51 lymphocytic malignancies cases, 17 were diagnosed as high risk (HR), 30 as medium risk (MR), and 4 as low risk (LR). There were no significant differences among the expression of mGR in HR, MR and LR group[75.06 (15.51~377.14)vs. 69.89(8.31~527.60)vs.29.64 (28.53~ 571.19); P>0.05]. Significant decrease of cGR expression was observed in HR when compared with MR and LR group[63.57(28.39~473.29)vs.107.78(53.60~545.46)vs.348.67(53.79~582.33);P<0.01]. Of the 47 ALL cases, 42 had good response to predinisone(day 8, pGR), 5 poor response(ppR). There were no significant differences between the expression of mGR in pGR and ppR group[71.60(10.83~529.47)vs. 38.34(15.51~69.81);P>0.05]. But the expression of cGR was significantly increased in pGR group when compared with ppR group[100.76(39.77~580.47)vs. 45.09(28.39~64.05);P<0.01]. Of the 51 lymphocytic malignancies cases, 40 had complete response to early combined chemotherapy(day 19) and 11 had partial or no response. There were no significant differences between the expression of mGR in complete response and resistance group [74.19(10.07~501.94)vs. 66.21(15.51~590.01);P>0.05]. However, the expression of cGR was significantly increased in complete response group when compared with resistance group[107.78(53.83~583.44)vs. 54.10(28.39~430.26);P<0.01].

Conclusion In acute lymphoblastic leukenia, the expression of cGR was positively related to the risk group, the early response to therapy and may work as a predictor for evaluation of prognosis.
An In Vivo Model To Investigate the Identity of the Cell-of-Origin of Multiple Myeloma.
Authors: Asimakopoulos Fotios A. , Varmus Harold E.
Published: April 2020

Multiple myeloma (MM) is characterized by monoclonal expansion of bone marrow plasma cells. However, long-lived plasma cells resident in the marrow are terminally differentiated and possess a limited replicative lifespan; it is puzzling how they could be the source of aggressive and relapsing neoplasms. We postulate that the myeloma clonogenic progenitor may reside in a more immature compartment with greater self-renewal capacity, most probably a cell participating in, or having shortly exited the germinal center reaction. However, it is unclear whether critical mutations occur in the target cell prior to, or following commitment to the plasma cell fate. To investigate the nature of the MM cell-of-origin, we have created a novel flexible mouse model system that enables the delivery of stochastic, sequential, somatic mutations to precisely defined compartments of the germinal center in secondary lymphoid tissues. To this end, we have used BAC transgenic technology to express distinct types of avian leukosis virus (ALV) receptors, TVA and TVB, in the expanding centroblast of the dark zone and the committed plasmablast of the light zone, respectively. Mammalian tissues are refractory to transduction by retroviruses of the ALV family unless they ectopically express the cognate avian-derived receptors. Thus, the coding sequences for the TVA receptor, fused to a fluorescent protein tag were placed under the control of transcription factor A-myb, expressed in centroblasts of the dark zone. Similarly, sequences encoding a fluorescent-tagged TVB receptor were placed under the control of transcription factor Blimp1, expressed in the earliest committed plasmablasts as well as mature plasma cells. Analysis of the Blimp1: TVB mice showed that expression of the avian retroviral receptor in the hematopoietic system is limited to the light zone of germinal centers, extrafollicular collections of CD138+ cells in the spleen and lymph nodes as well as long-lived bone marrow plasma cells. Analysis of A-myb: TVA transgenic mice is currently underway. The system permits the introduction of a variety of molecular lesions to specific plasma cell precursors via retroviral transduction of oncogenes, shRNAs against tumor suppressor genes or inducible regulators of gene expression in an attempt to re-create the sequence of molecular lesions leading to MM in the relevant cellular context.
One Half of Patients with Waldenström Macroglobulinemia Have Large Deletions of Chromosome 6q.
Authors: Van Wier Scott , Chng Wee-Joo , Ahmann Greg , Rempel Rachel , Al Masri Azzah , Gertz Morie , Dispenzieri Angela , Greipp Philip , Schop Roelandt F.J. , Fonseca Rafael
Published: April 2020

Background: Waldenström macroglobulinemia (WM) is a plasma cell dyscrasia characterized by a monoclonal IgM paraproteinemia. The most common genomic abnormality in the clonal cells of WM is loss of portions of the long arm of chromosome 6 (6q-), however a minimal area of deletion has yet to be identified. We decided to further clarify the area of deletion on the 6q-arm in WM along with its prevalence.

Methods: Patients (n=41) were required to have a serum IgM paraproteinemia of 1.5 g/dl or greater and a monoclonal lymphoplasmacytic infiltrate. We used interphase fluorescence in situ hybridization (FISH) combined with cytoplasmic immunoglobulin (cIg) staining with an AMCA conjugated anti-IgM antibody along with cytomorphology for the detection of the clonal cells on cytospin slides. Ten probes were used including two commercial (CEP 6 and Telomere 6q) and eight Bacteria Artificial Chromosome (BAC). BAC clones were selected based on a specific gene encompassed in the insert and/or localization on the q arm. Genes of interest included BLIMP1, c-myb, BTF, SHPRH, LATS1 and FOP. BAC’s were grown in selective media; DNA was extracted and directly labeled with a SpectrumGreen or SpectrumOrange fluorophore by standard nick translation. Primers were designed to confirm the insert contained the gene of interest and probes were tested on normal metaphases to reaffirm localization to the correct area of the chromosome and rule out any cross hybridization.

Results: Deletions of 6q were noted in 21 (51%) patients. Of the 19 abnormal patients with all probes tested 1 (5%) case had ten probes deleted, 7 (37%) cases had nine probes, 1 (5%) had eight, 4 (21%) had seven, 3 (16%) had six and 3 (16%) had four probes deleted. Only 3 cases retained the end of the q arm indicating an interstitial deletion while the remaining 16 cases indicate terminal deletions. Deletions of the BAC containing the gene SHPRH at 6q24 were seen in each of the 19 abnormal cases. The majority of patients exhibited little heterogeneity for any given deletion having greater than 80% involvement of the clone.

Conclusion: This study demonstrates that large deletions of the long arm of chromosome 6 in WM are frequent and are usually terminal deletions rather than interstitial and have a common area of deletion of the q24 portion of the q arm. Using the given number of probes localizing along the q arm makes it highly unlikely of any areas of noncontiguous deletion. The presence of deletions in the majority of the clonal cells may suggest that clones with 6q- emerge early in the course of the disease and is selected because of growth advantage or apoptosis resistance. Whether 6q deletions are important for disease pathogenesis has yet to be determined. Clinical prognosis studies and microarray data analysis are pending.
Azacytidine Suppressors Autocrine IL-6 Secretion and Demonstrates In Vitro and In Vivo Activity Against Multiple Myeloma.
Authors: Khong Tiffany , Sharkey Janelle , Spencer Andrew
Published: April 2020

Azacytidine (AZA), a DNA methyltransferase inhibitor, has been shown to inhibit cell growth and induce apoptosis in some cancer cells. We determined the impact of AZA on a panel of human myeloma cell lines (HMCL); KMS 12PE, KMS 18, LP-1, NCI-H929, OPM-2, RPMI-8226 and U266 and in an in vivo murine model of multiple myeloma (5T33 model). Dose responsiveness to AZA was determined via MTS assays with a range of AZA doses (1–10mM) for 72 hours. FACS and cell cycle analysis were used to evaluate the profile of the cells after exposure to AZA for 72 hours. MTS assays demonstrated a dose and time dependent AZA-induced inhibition of HMCL viability with effective concentrations of AZA ranging from 1–10 mM. This was associated with accumulation of cells in the Go/G1 phase with decreasing number of cells in the S and G2/M phases. Western Blot analysis using antibodies against caspases 3,8,10, PARP, phospho-ERK, ERK, Stat3 and phospho -Stat3 were performed to help characterize the mechanism(s) of cell killing. Cleavage of caspases 3,8,10 and PARP within 24 hours of AZA treatment confirmed early AZA-induced HMCL apoptosis. phospho-ERK which was absent in untreated U266 appeared after 48 hours exposure to 5mM AZA. Similarly inhibitors of caspases 3,8 and 9 were used to determine which apoptotic pathway was being preferentially activated by AZA. Inhibitors of both caspase 3 and 9 effectively abrogated AZA-induced apoptosis in U266 and NCI-H929. In contrast caspase 8 inhibitor was less effective which is consistent with AZA acting via the mitochondrial apoptotic pathway. Reactivation of p16 gene by AZA-induced hypomethylation was assessed with methylation specific PCR. MSP-PCR of the p16 gene indicated a loss of methylation and up-regulated transcription after 48 hours treatment with 5 mM AZA. The level of IL-6 in conditioned media from U266 cells treated with AZA was determined by ELISA assay and demonstrated a rapid fall in autocrine IL-6 production. RT-PCR demonstrated rapid AZA-induced cessation of IL-6 transcription temporarily associated with the disappearance of upstream phospho -Stat3. Addition of exogenous IL-6 did not rescue U266 from AZA-induced apoptosis. AZA was also administered to a 5T33 murine model of multiple myeloma at increasing concentrations (1, 3, 10 mg/kg). At 10 mg/kg the median survival of vehicle versus AZA treated mice was 28 days versus 30+ days (p=0.003). These findings justify further evaluation of AZA as a potential therapeutic agent for multiple myeloma.
Combination of Akt/PKB Inhibition (Perifosine) and Farnesyl Transferase Inhibition (Tipifarnib) Results in Increased Cell Death in Myeloma Cells Lines.
Authors: Sinha Rajni , David Ebenezer , Zeilter Emily , Torre Claire , Kaufman Jonathan L. , Lonial Sagar
Published: April 2020

Introduction

Multiple myeloma is a clonal plasma cell malignancy characterized by proliferation and accumulation of plasma cells in the bone marrow. Most patients are incurable with the current treatment modalities. Clearly novel agents are needed to improve the outcome for patients with myeloma. We have previously shown that the combination of bortezomib and tipifarnib results in synergistic myeloma cell death. This increase in apoptosis is associated with down regulation of phosphorylated AKT, a potent anti-apoptotic signaling molecule. Therefore, agents that target AKT represent ideal compounds for further study in myeloma. Perifosine is a novel, oral bioavailable alkylphospholipid. Perifosine has displayed apoptotic and antipropliferative activity in vitro and in vivo in several human cancer models including leukemia. Perifosine exerts its actions by interfering with key intracellular pathways including AKT, MAPK, JNK, p21waf1. Our hypothesis is that targeting AKT via multiple upstream pathways will result in increased myeloma cell apoptosis. Therefore, we assessed the effects of single agent perifosine with and without tipifarnib on multiple myeloma cell lines.

Method

The myeloma cell line RPMI8226 was used. Cell viability and proliferation were assessed using MTT assays. Cells were incubated with increasing concentrations of both agents alone and in combination. Cell proliferation was assayed at 24, 48 and 72 hours. Western blots were then carried out to evaluate the effects of the intracellular protein PDK1, one of the critical signaling molecules that phosphorylates and activates AKT.

Results

As we and others have previously shown, tipifarnib at concentrations that can be achieved clinically is associated with minimal cytotoxicity. At 5 μM, tipifarnib decrease proliferation by only 20%. In contrast, there is a potent dose response effect of single agent perifosine (Fig. 1). These results were apparent as early as 24 hours. When tipifarnib at 5 μM is used in combination with a subtherapeutic dose of perifosine (2 μM), there is a marked decrease in cell proliferation (Fig. 2). In addition, combination therapy resulted in a reduction in the phosphorylated form of PDK1, a critical finding that was not seen with either drug alone.

Conclusion

Combination therapy with tipifarnib and perifosine results in less cell proliferation compared to either agent used alone in the RPMI8226 myeloma cell line. The dosages employed in these in-vitro studies are lower than those used in previously published data and are clinically achievable. Studies targeting other cell lines including MM.1R, MM.1S, and U266 are in progress. Analysis of AKT, Caspase 3, 8 and 9 are being explored to help delineate the mechanism of this novel combination. The goal is to develop further effective treatment options for patients with myeloma.

Figure 1 Figure 1. Figure 2 Figure 2.
The PKC- Inhibitor Enzastaurin Inhibits MM Cell Growth, Survival and Migration in the Bone Marrow Microenvironment.
Authors: Podar Klaus , Raab Marc S. , Abtahi Dean , Tai Yu-Tzu , Lin Boris , Munshi Nikhil C. , Hideshima Teru , Chauhan Dharminder , Anderson Kenneth C.
Published: April 2020

Members of the protein kinase C (PKC) family of serine- threonine protein kinases mediate multiple physiological functions including differentiation, growth and survival, invasiveness, angiogenesis and drug efflux. Dysregulation of PKC signaling has been implicated in tumor progression and prompted the development of novel anticancer therapeutics. In multiple myeloma (MM) PKC isoforms are: (1) involved in MM cell apoptosis; (2) associated with VEGF- and Wnt- induced MM cell migration; and (3) controlling shedding of IL-6 receptor alpha. However, to date the potential of targeting PKC signaling sequelae in MM has not been evaluated. Here we investigated the novel orally available protein- kinase C (PKC) inhibitor Enzastaurin (Eli Lilly and Company) for its therapeutic efficacy in MM. We first tested the ability of Enzastaurin to suppress MM cell proliferation in a wide array of MM cell lines. Our data show that Enzastaurin inhibits 3H[dT] uptake in all cell lines tested in a low micromolar range equivalent to the concentration range achieved in the patient plasma during clinical trials. Importantly, Enzastaurin also abrogates MM cell proliferation in a BMSC-MM coculture system. We next sought to determine whether Enzastaurin can inhibit cell survival and found dose- dependent induction of MM cell apoptosis in MM cell lines MM.1S, MM.1R, OPM-1, OPM-2, RPMI-8226, and RPMI-dox40. Moreover, Enzastaurin significantly inhibited VEGF- induced MM cell migration on fibronectin. Importantly, IGF-1- induced MM cell migration was abrogated by Enzastaurin, demonstrating the requirement of PKC. Signaling pathways mediating these effects were next examined: Our data show that Enzastaurin abrogates phosphorylation of Akt and GSK3beta, which is required for MM cell growth and migration. Furthermore, ongoing studies are evaluating the efficacy of Enzastaurin in a murine model of human MM. Taken together, these studies show for the first time the preclinical efficacy of the orally available PKC inhibitor Enzastaurin providing the basis for its clinical evaluation to improve patient outcome in MM.
Intensified CHOP with Rituximab for Intermediate or High-Risk Non-Hodgkin’s Lymphoma: Interim Analysis of a Randomized Phase III Trial in Elderly Patients by the Dutch HOVON and Nordic Lymphoma Groups.
Authors: Sonneveld Pieter , van Putten Wim , Holte Harald , Biesma Douwe , van Marwijk-Kooy Marinus , Kramer Mark , Wijermans Pierre , Steijaert Monique , Huijgens Peter , Eriksson Mikael
Published: April 2020

High-risk Non-Hodgkin lymphoma (NHL) in elderly patients (> 60 yrs) is associated with a relatively low complete response (CR) rate and a poor overall survival (OS). Previous studies have shown that addition of Rituximab to standard CHOP chemotherapy may improve CR and/or OS. A similar improvement has been shown for intensification of CHOP from 21 day intervals to 14 day intervals. The Dutch HOVON group and the Nordic lymphoma group have performed a multi-center, randomized phase III trial to compare 8 cycles of intensified CHOP (CHOP14) with the same regimen plus 6 administrations of Rituximab in previously untreated elderly patients with intermediate or high-risk NHL. Inclusion criteria were diffuse large B-cell lymphoma, mantle cell lymphoma or follicular lymphoma grade III; intermediate or high-risk NHL according to age adjusted IPI score; CD20-positive NHL; age 65 yrs or higher. The target number was 400 patients to be accrued in 5 years based on an expected increase in event-free survival with hazard ratio HR=0.70.

Aplanned interim analysis was performed after inclusion of 250 patients, restricted to 211 patients included from 1st September 2001 to 1st October 2004. Forty patients had to be excluded because of lack of treatment and evaluation data, leaving 171 patients for the analysis. The median time off protocol treatment was 119 days (range 4–468 days). The median age was 73 years (range 62–88 years). There was no difference between the two treatment arms regarding histology, age, WHO classification of NHL, age-adjusted IPI score, Ann Arbor stage, WHO performance status and serum LDH. There was no significant difference of toxicity or CR between the two treatment arms. However, a highly significant difference was observed for event-free survival and overall survival in favor of the treatment arm with CHOP plus Rituximab. Based on this interim analysis, the randomization was halted and patients in the control arm were further treated with CHOP plus Rituximab. Since the outcome of the interim analysis could be biased due to incomplete data, a final decision to stop the trial awaits a new analysis which is planned for 26th August 2005 after collection of missing or incomplete data. If the new analysis confirms the results of the interim analysis, i.e. that Rituximab significantly improves the outcome of treatment in elderly patients with intermediate or high-risk NHL, even when an intensified CHOP regimen is used, the study will be closed and outcomes will be presented.
The Role of Oxidative Stress on Telomere Shortening, Telomere Dysfunction and Damage to Telomere G- Strand Overhang - A Putative Model of Carcinogenesis in Fanconi Anemia Cells.
Authors: Lahav Meir , Uziel Orit , Reshef Hagai , Ravid Anat , Fabian Ina
Published: April 2020

Fanconi Anemia is an autosomal recessive disease characterized by various inborn abnormalities and severe genetic instability, which may lead to bone marrow failure and increased incidence of cancer. One possible explanation for this genomic instability may be the cellular hypersensitivity to oxidative stress, leading to chromosomal breaks. The goal of our study was to explore the possible oxidative damage caused by H2O2 to telomeres of Fanconi Anemia (FA) lymphocyte cell line, HSC536/N and its possible relation to carcinogenesis. We surmised that combination of oxygenative damage to telomere function with Overexpression of telomerase may provide a model for malignant transformation in those cells.

The cells were grown in the presence of hTR anti-sense (GRN163) and exposed for one month to hydrogen peroxide combined with a variety of antioxidants. One month of exposure caused marked shortening of telomere (TRF) length (measured by Southern blot), and damage to the single stranded overhang of the telomere [using the newly developed telomeres oligonucleotide ligation assay (T-OLA)]. The telomere and single stranded overhang shortening was associated with significant telomere dysfunction (assessed by karyotype analysis). Control cells did not exhibit this sensitivity to H2O2. Telomere dysfunction did not evoke damage response in FA cells, in contrast to normal p53 up regulation in control cells. Reconstitution of telomerase activity protected FA telomeres from further oxygenative damage. Several antioxidants prevented the H2O2 exerted damage. Based on our data we suggest a model of malignant transformation in FA in which oxidative stress led to a significant shortening of the overall length of the telomere and the single stranded overhang, thus causing telomere dysfunction. P53 mediated damage control was absent in these cells, preventing apoptosis or cycle arrest. Reconstitution of telomerase activity further stabilizes the damaged telomeres preventing crisis and forming the basis for future carcinogenesis. The differential effects of the antioxidants and the significance of our findings to malignancies will be discussed.
Novel Risk Factors for Venous Thromboembolism (VTE): A Population-Based Case-Control Study.
Authors: Heit John A. , Farmer Sara A. , Petterson Tanya M. , Bailey Kent R. , Melton L. Joseph
Published: April 2020

Background: While several uncommon disorders (e.g., myeloproliferative diseases, connective tissue disease, etc.) have been suggested as potential risk factors for VTE, the independence and magnitude of any risk is uncertain.

Objective: To test uncommon characteristics as potential independent risk factors for VTE, and estimate the magnitude of risk for each.

Methods: Using the resources of the Rochester Epidemiology Project, we identified all incident cases of deep vein thrombosis and pulmonary embolism over the 6-year period, 1992–1997. For each objectively confirmed case (n=562), we identified up to two Olmsted County residents without VTE who most closely matched a case on age, gender, and medical record number (n=755). Because medical record numbers are assigned sequentially, such matching assures a similar duration of medical follow-up. For all cases and controls, we reviewed the complete medical records in the community for over 125 baseline characteristics which were tested for an association with VTE using conditional logistic regression.

Results: The mean ± SD (range) ages for cases and controls were 65.71 ± 18.85 (0–102) and 64.90 ± 18.66 (0–102) years, respectively, and 55.5% were female. Novel univariate risk factors for incident VTE included former tobacco smoker, ischemic heart disease, valvular heart disease, chronic lung disease, all-cause pulmonary hypertension, chronic renal disease, myeloproliferative disorders, any infection, femoral artery catheterization (angiography or percutaneous intervention), ICU admission and angiotensin converting enzyme (ACE) inhibitor therapy. Among infections, septicemia, pneumonia, and ENT, cardiovascular, GI, urinary tract and skin/soft tissue infections were univariately associated with VTE; reproductive tract infection was not. Diabetes mellitus, hyperlipidemia, lipid-lowering drugs (including statins), inflammatory bowel disease, asthma, connective tissue disease, and influenza were not univariately associated with VTE, and beta-blocker therapy was not protective. A multivariate analysis will test these characteristics as potential independent VTE risk factors.

Conclusions: Several novel characteristics are univariately associated with VTE. Such novel characteristics may provide new insights into VTE mechanisms if, upon further analysis, these characteristics are independent VTE risk factors.