bluuebunny/crossref_metadata_2025 huggingface dataset:
Fucoidan down Regulates the Expression of CXCR4 In Vitro on Human Hematopoietic CD34+ Cells.
Authors: Irhimeh Mohammad R. , Nordon Robert E. , Ko Kap-Hyoun E. , Fitton Helen , Lowenthal Raymond M.
Published: October 2019

Sulfated glycans such as fucoidan have been shown to induce rapid hematopoietic progenitor stem cell (HPCs) mobilization in mice and primates. Our previous clinical study has demonstrated up-regulation of CXCR4 on peripheral CD34+ cells in volunteers who had received oral doses of 75% pure fucoidan. The aim of this study was to investigate the in vitro effects of fucoidan on a cytokine mediated CD34+ cell expansion system and to characterize the fucoidan effects on CD34+ proliferation and differentiation using a factorial design experiment and flow cytometry. Isolex selected CD34+ cells harvested from multiple myeloma patients were used in the study. There is institutional approval for the in vitro growth of CD34+ cells from mobilized peripheral blood.

Fractional (25−1 resolution V, 16 runs, 2 blocks, E=ABCD) and full (24, 16 runs, 2 blocks) factorial design experiments were used to investigate the effects of depyrogenated fucoidan (DP-GFS) and cytokines on the growth of CD34+ cells. The cells were grown in 24 well dishes (1 ml/well) at an inoculum density of 20,000 cells/ml in serum free media and cultured for 10 days in a cytokine cocktail including G-CSF, FL, SCF, TPO and SDF1 each at two levels 100 ng/ml or 1000 ng/ml. DP-GFS was used in the cultures at four different levels including 0, 10, 100 and 500 μg/ml. Cells were then harvested and analyzed using three color flow cytometry and a panel of antibodies that includes CD15, CD14, CD41a, CD38, CD34, and CXCR4.

The in vitro effect of DP-GFS on CXCR4 expression was opposite to that found in vivo using 75% fucoidan. At 100 ng/ml and at saturating levels of FL, SCF, TPO and G-CSF (1000 ng/ml), DP-GFS at 10 μg/ml was observed to reduce the number of CD34+ cells produced per input CD34+ cell (1.09±0.07 versus 0.84±0.05, n=16, p=0.002) and have a negative effect on the percentage of CD34+ cells that express CXCR4 (87.2±1.8 versus 77.4±2.3, n=16, p=0.001). There was no significant effect of DP-GFS on the total cellular output nor other cell subsets examined (CD41a+CD14−, CD15+, CD14+). Our in vitro results are more consistent with a recent clinical study that demonstrates down-regulation of CXCR4 receptors on CD34+ cells in G-CSF or GM-CSF mobilized blood (Dlubek et al, 2006). We hypothesize that fucoidan acts in vitro and in vivo via different mechanisms.
Angiogenesis in Multiple Myeloma (MM): Angiogenic Switch or Reflexion of Plasma Cell Number? A Gene Expression Based Survey in Primary Myeloma Cells and the Bone Marrow Microenvironment.
Authors: Hose Dirk , DeVos John , Heiß Christiane , Rossi Jean-Francois , Rösen-Wolff Angela , Benner Axel , Mahtouk Karène , Hundemer Michael , Reme Thierry X. , Hillengass Jens , Herde Katja , Mazitschek Uta , Moreaux Jérôme , Wenisch Sabine , Pantesco Véronique , Jauch Anna , Jourdan Eric , Goldschmidt Hartmut , Klein Bernard , Möhler Thomas
Published: October 2019

BACKGROUND. Angiogenesis is a hallmark of active multiple myeloma. However, two etiologic hypotheses have been proposed:

an angiogenic switch (i.e. differential or de novo expression of pro/antiangiogenic genes in MM), and, alternatively an effect of increased plasma cell number.

AIM of this study was to investigate the angiogenic signature of multiple myeloma cells (MMC), normal bone marrow plasma cells (BMPC), the bone marrow microenvironment (BMME) and cellular subfractions therein.

PATIENTS AND METHODS. 128 newly diagnosed MM-patients (65 training (TG) / 63 independent validation group (VG)) and 14 normal donors (ND) were included. Bone marrow aspirates were CD138-purified by activated magnetic cell sorting. Whole bone marrow (n=49) and FACSAria sorted subfractions thereof (n=5) were investigated. RNA was in-vitro transcribed and hybridised to Affymetrix HG U133 A+B GeneChip (TG) and HG U133 2.0 plus arrays (VG). Expression data were gcrma-normalised and the empirical Bayes algorithm used. p-Values were adjusted using the Benjamini-Hochberg method (Bioconductor). iFISH was performed on purified MM-cells using probesets for chromosomes 1q21, 9q34, 11q23, 11q13, 13q14, 15q22, 17p13, 19q13, 22q11 and the translocations t(4;14) and t(11;14). HGF expression was verified by real time RT-PCR and western blotting. Based on Medline review, we established a list of 89 pro- and 56 antiangiogenic genes and investigated their expression according to the stage of disease: BMPC vs. MGUS, SD stage I (asymptomatic myeloma) vs. SD stage II/III (symptomatic myeloma requiring therapy).

RESULTS.

BMPC express pro- (e.g. VEGFA) and antiangiogenic genes (e.g. TIMP2). Only one pro-angiogenic gene (hepatocyte growth factor, HGF) is significantly overexpressed in MMC compared to BMPC. HGF has previously been linked with myeloma progression and induction of angiogenesis. Six antiangiogenic genes (TIMP2, SERPINF1, COL18A1, PF4, THBS1, CXCL14) are downregulated in MMC compared with BMPC. Compared to healthy donors, the BMME of MM shows a significant downregulation of PLAU (urokinase, antiangiogenic) and upregulation of TNF(proangiogenic).

CONCLUSION. Upregulation of HGF-expression, downregulation of TIMP2, SERPINF1, COLA18A1, PF4, THBS1 and CXCL14 expression in MMC as well as downregulation of PLAU and upregulation of TNFα in the BMME seem to indicate an “angiogenic switch”. However, given the relatively low number of differentially expressed genes (7/145) and the expression of angiogenic genes by BMPC, an effect caused by an increasing number of plasma cells might be evenly important.
Akt Inhibitor Perifosine-Induced Cytotoxicity Is Associated with Significant Downregulation of Survivin in Human Multiple Myeloma (MM) Cells.
Authors: Hideshima Teru , Yasui Hiroshi , Catley Laurence , Raje Noopur , Chauhan Dharminder , Ishitsuka Kenji , Podar Klaus , Mitsiades Constantine , Tai Yu-Tzu , Vallet Sonia , Kizitepe Tanyel , Ocio Enrique , Ikeda Hiroshi , Okawa Yutaka , Hideshima Hiromasa , Munshi Nikhil C. , Richardson Paul G. , Anderson Kenneth C.
Published: October 2019

Perifosine (NSC 639966; Keryx Biopharmaceuticals, New York, NY) is a synthetic novel alkylphospholipid, a new class of anti-tumor agents which potently inhibits Akt (PKB) activity. Our previous studies have shown that Perifosine induces significant cytotoxicity in MM cells triggered by c-Jun NH2-terminal kinase (JNK) activation followed by caspase-8, caspase-9, and PARP cleavage even in the presence of cytokines (ie, IL-6 and IGF-1) or bone marrow stromal cell (BMSCs). Importantly, MEK inhibitor and bortezomib enhance Perifosine-induced cytotoxicity. It has also shown significant anti-tumor activity in a human MM cell xenograft mouse model (Hideshima et al. Blood2006, 107:4053–4062). In this study, we further delineated molecular mechanisms whereby Perifosine triggers cytotoxicity as a single agent and in combination with bortezomib in MM cells. In most MM cell lines, the IC50 for Perifosine-induced cytotoxicity is 5–10 μM range assessed by MTT assay at 24h; however, apoptosis assessed by APO2.7 staining, varied in each cell line. Moreover, neither the degree of JNK phosphorylation nor caspase-8/9/PARP cleavage correlated with Perifosine-induced cytotoxicity. Therefore we further examined expression level of anti-apoptotic proteins in MM cell lines and found that survivin, which has a crucial role in regulation of caspase-3 activity, was markedly downregulated by Perifosine treatment in a time- and dose-dependent fashion, without affecting expression of other anti-apoptotic proteins (ie, cIAP, XIAP, Bcl-2, Bcl-xL). Since survivin is a known downstream protein of β-catenin/TCF-4 cascade, we next hypothesized that Perifosine may inhibit β-catenin activity. As expected, Perifosine significantly downregulated both phosphorylation and protein expression of β-catenin, associated with downregulation of survivin and enhanced caspase-3 cleavage. Real-time PCR confirmed that gene expression of survivin was suppressed 35% and 55% after 3h and 6h Perifosine treatment, respectively. Since β-catenin is a substrate of proteasomes, we further examined whether bortezomib could augment survivin expression by blocking its degradation. Importantly, bortezomib significantly upregulated β-catenin and survivin, which was blocked in the presence of Perifosine. These results suggest that inhibition of bortezomib-induced survivin expression, at least in part, accounts for enhanced bortezomib-induced cytotoxicity by Perifosine. Based upon these preclinical studies, a rational combination trial of bortezomib with Perifosine to treat relapsed refractory MM is currently ongoing.
Inhibition of Kinesin Spindle Protein Induces Apoptosis and Overcomes Drug Resistance in Models of Multiple Myeloma.
Authors: Chen Qing , Pamarthi Vishwan , Kuhn Deborah J. , Voorhees Peter M. , Strader John S. , Orlowski Robert Z.
Published: October 2019

Introduction: Kinesin spindle protein is a mitotic kinesin that is expressed only in proliferating cells and plays a key role in spindle pole separation, formation of a bipolar mitotic spindle, and thus in satisfaction of the mitotic checkpoint. Ispinesib (SB-715992) is a potent and selective inhibitor of kinesin spindle protein with a Ki of 0.6 nM, has cytotoxic activity at less than 10 nM in a spectrum of tumor cell lines, and disrupts the assembly of functional bipolar mitotic spindles.

Methods: This study sought to examine whether spindle disruption by inhibition of kinesin spindle protein with ispinesib may have therapeutic potential in the treatment of multiple myeloma.

Results: Ispinesib reduced cell viability in both interleukin-6-independent (RPMI 8226 and U266) and interleukin-6-dependent (ANBL-6 and KAS-6/1) models of multiple myeloma in a time- and concentration-dependent fashion. The average IC50 for ispinesib against these cell lines was 3.0 nM, 1.7 nM, 1.8 nM, and 1.8 nM, respectively. Cell cycle analysis showed that ispinesib induced growth arrest of myeloma cells with 4N DNA content (in M phase) within 24-hours. Two days after treatment at a 1 nM concentration, cells were able to recover from M phase arrest and resume normal cycling but, after exposure to 10 nM, treated cells could not escape M phase arrest, and instead entered apoptosis as determined by an increased sub-G1 population. Ispinesib was able to overcome resistance to melphalan in that the IC50 in melphalan-resistant RPMI 8226/LR5 cells (1.6 ± 0.2 nM) was comparable to that in parental RPMI 8226 controls (3.0 ± 0.9 nM). Similarly, ispinesib was also able to overcome dexamethasone resistance and bortezomib resistance. In regard to the latter, KAS-6/VR5 bortezomib-resistant cells (IC50 of 12.5 nM for bortezomib) retained sensitivity to ispinesib (IC50 1.6 ± 0.2 nM). Combination therapy with ispinesib and bortezomib in these cells resulted in enhanced levels of specific apoptosis (24%) that were greater than the sum of either agents alone (7% for ispinesib and 1% for bortezomib), suggesting synergy. Importantly, ispinesib was also active against freshly isolated CD138+ patient-derived multiple myeloma cells, while relatively sparing CD138− cells.

Conclusions: Taken together, these studies demonstrate that kinesin spindle protein inhibition with ispinesib was able to induce growth arrest and apoptosis in myeloma cells, and overcome resistance to both conventional drugs and novel agents such as bortezomib. Moreover, the preferential activity against transformed plasma cells with sparing of normal bone marrow cells provides a strong rationale for translation of this agent into the clinic to combat relapsed/refractory multiple myeloma.
Inhibitors of mToR Mediate Anti-Myeloma Activity by Apoptosis through the Extrinsic Pathway.
Authors: Günther Andreas , Tiemann Markus , Bakker Frank , Baum Wolfgang , Brocke-Heidrich Katja , Horn Friedemann , Burger Renate , Gramatzki Martin
Published: October 2019

The mammalian target of rapamycin (mToR) plays a crucial role in cell growth due to its role as nutrient dependent regulator of important cytokine signalling pathways. In multiple myeloma (MM) mToR is involved in the AKT pathway which can be activated by the loss of the tumor suppressor PTEN or stimulation with growth and survival factors such as interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1). mToR can be blocked by rapamycin or everolimus. When various human plasmacytoma lines were tested, rapamycin and everolimus induced a dose-dependent growth inhibition. The addition of bone marrow stromal cells, IL-6 or IGF-1 was unable to prevent the inhibitory activity. Growth inhibition was mediated not only by G1 cell cycle arrest but also by an induction of apoptosis as measured by propidium iodide staining. Further analysis was performed to elucidate the mechanism of apoptosis in the particular sensitive plasma cell line INA-6. mToR inhibition induced exclusively cleavage of pro-caspase 8 (extrinsic pathway) but not of pro-caspase 9 (intrinsic pathway). To confirm this observation, INA-6 cells were transfected with BCL-XL, an inhibitor of the intrinsic pathway, and treated with rapamycin. The transfectants showed similar sensitivity to rapamycin as the paternal cell line. Rapamycin was also tested in the INA-6 SCID mouse xenograft model.

Rapamycin was given orally for two weeks starting 24 h after tumor inoculation. A significant survival benefit of the rapamycin treated group compared to control animals (p= 0.0004) was observed. Six out of 14 treated mice did not develop plasmacytomas during the observation period of 149 days. In addition, even short term treatment of plasmacytoma bearing mice led to a significant shrinkage of the tumor mass. Histological examination revealed an increase of apoptotic plasma cells according to morphology after 72 h of treatment compared to controls. Induction of apoptosis in tumor cells was confirmed by immunohistological staining using an antibody specific for the human cleaved form of poly (ADP-ribose) polymerase. Since drugs inhibiting mToR are inducing apoptosis in human malignant plasma cells not only in vitro but also in vivo, the inhibition of mToR may represent an attractive new concept for multiple myeloma therapy.
On the Molecular Mechanism of DKK1 Inhibition of Osteoblast Differentiation in Multiple Myeloma.
Authors: Qiang Ya-Wei , Chen Yu , Stephens Owen , Rubin Jeff , Rudikoff Stuart , Barlogie Bart , Shaughnessy John D.
Published: October 2019

Lytic bone destruction is a severe complication of multiple myeloma growth in the bone. We have demonstrated that Dkk1, an antagonist of Wnt signaling which is essential for osteoblast growth and differentiation, is expressed by myeloma plasma cells leading to increased levels of this protein in serum. Myeloma serum can block osteoblast differentiation in-vitro and a neutralizing anti-DKK1 antibody can inhibit this. These data suggest that MM cells disrupt Wnt signaling and that this promotes uncoupled bone turnover. However, the molecular mechanism responsible for this processes remains to be elucidated. Here we investigated Wnt signaling in osteoblast differentiation and identified the molecular mechanism regulating this process. Using qRT-PCR and sequence confirmation we showed that multiple Frizzled (Fz) mRNAs and LRP5/6 co-receptors were expressed in C2C12 cells and three human osteoblast cell lines, hFOB1.19, MG63 and Saos-2. Furthermore, these cells had high levels of total beta-catenin an indicator of active Wnt signaling. Wnt3a conditioned medium (CM) and recombinant Wnt3a protein increased uncomplexed beta-catenin level as assessed by GST-E-cadherin binding assay. Blocking autocrine Wnt signaling with Dkk1 blocked Wnt-3a-induced increase in beta-catenin levels in dose-dependent fashion. Co-culturing of OPM-2 MM cells ectopically expressing Dkk1 inhibited the Wnt-3a induced beta-catenin stabilization in C2C12 cells. Transfection of C2C12 cells with a dominant negative beta-catenin (DNBC) completely blocked endogenous and Wnt-3a-induced TCF/LEF transcriptional activity as evidenced by TOPflash luciferase activity. Furthermore, BMP-2 induced increase in alkaline phosphatase (ALP) activity was completed abrogated in C2C12 cells expressing DNCB. Silencing LRP5/6 mRNA with small inference RNAs blocked BMP-2 induced increase in ALP expression, suggesting that canonical Wnt pathway contributes to BPM-2 induced OB differentiation. BMP-2 induced phosphorylation of Smad 1, 5 and 8 whereas Wnt-3a had no effect on phosphorylation of these Smads. Moreover, Dkk1 did not block BMP-2 induced phosphorylation of Smad-1, −5 and −8 and did not alter transcriptional activity of Cbfa1, an essential transcriptional factor for OB differentiation. Taken together, these data suggest that Wnt induction of OB cell differentiation through canonical Wnt-beta-catenin pathway is independent of the activation of the Smad pathway. Our results suggested that canonical Wnt signaling pathway plays an important role in osteoblastogenesis and that Dkk1 inhibits OB cell differentiation in a way that may contribute to MM bone lesions. New therapies targeting DKK1 in myeloma may represent a novel treatment strategy.
ZIO-101, a Novel Organic Arsenic, Inhibits Human Myeloma Cell Growth in a SCID-hu Model.
Authors: Campbell Richard A. , Sanchez Eric , Chen Haiming , Turker Lauren , Li Mingjie , Trac Olivia , Shalitin Dror , Gordon Melinda S. , Pang Shen , Bonavida Benjamin , Said Jonathan , Wallner Barbara , Gale Robert P. , Berenson James R.
Published: October 2019

Inorganic arsenics like arsenic trioxide (ATO) are novel anti-cancer drugs active in acute promyelocytic leukemia (APL) and multiple myeloma (MM). ATO induces apoptosis of plasma cells by several mechanisms including down-regulation of BCL-2 expression and inhibition of DNA-binding by NF-κB. The amount of ATO that can be safely given is low because of QTc-prolongation. ZIO-101, a new organic arsenic, is in phase-1/-2 trials. ZIO-101 can be safely given at much higher doses than ATO. We evaluated the in vivo anti-myeloma activity of ZIO-101 in a SCID-hu mouse model of human myeloma. LAGλ-1 was developed from a person with melphalan-resistant and LAGλ-1B from a person with bortezomib-resistant myeloma. Each severe combined immunodeficient (SCID) mouse was implanted with a 2–4 mm3 fragment of LAGλ-1B or LAGλ-1 into the left superficial gluteal muscle. Fragments were allowed to grow for 14 d when human IgG was first detectable. ZIO-101 was given IV 1 or 2 times/d using three different schedules: one day/w, 3 days/w or 5 days/w at doses of 50– 200 mg/kg/d. Tumor volume and human IgG were assessed weekly. Doses up to 200 mg/kg were well-tolerated. Anti-myeloma effects were observed in both models at doses of 100 – 200 mg/kg on all 3 schedules. Mice receiving 100 mg/kg twice daily thrice weekly and those receiving 200 mg/kg once weekly showed marked anti-myeloma activity (100 mg/kg, P = 0.03; 200 mg/kg, P = 0.001) and reduced human IgG levels (100 mg/kg, P < 0.001; 200 mg/kg, P = 0.01) compared to controls. We are evaluating ZIO-101 in other SCID-hu models of human myeloma and exploring different doses and schedules of ZIO-101 alone or combined with other anti-myeloma agents. In summary, these data show activity of ZIO-101 in human myeloma in vivo. These studies provide the bases for future clinical trials.
Inhibition of Multiple Myeloma Cells Growth by AS602868, a Pharmacological Inhibitor of IKK2.
Authors: Jourdan Michel , Moreaux Jerome , Hose Dirk , Rossi Jean-Francois , Mahtouk Karène , Aboulaze Mathieu , Robert Nicolas , Baudard Marion , Reme Thierry , Romanelli Angela , Goldschmidt Hartmut , Dreano Michel , Klein Bernard
Published: October 2019

The NF-κB pathway is involved in the physiological regulation of cell proliferation in many cell types as well as in the resistance of several malignancies to cell death. The pathophysiologic basis for multiple myeloma (MM) has been attributed to the dysregulation of various paracrine or autocrine growth factor loops and to perturbations in several signal transduction pathways including IKK/NF-κB. The aim of the present study was to investigate the effect of a pharmaceutical IKK2 inhibitor (IKK2-I), the anilinopyrimidine derivative AS602868 (Serono International SA), on the in vitro growth of human MM cell lines (HMCL) and primary MM cells. We evaluated the effect of AS602868 on the proliferation and the survival of 12 IL-6-dependent HCML and 2 autonomously growing HCML as well as on the survival of total bone marrow mononuclear cells from patients with newly diagnosed MM (n = 6) or with relapsing MM (n = 7). Results show that using HMCL or primary MM cells, AS602868 induces a clear dose-dependent inhibition of MM cell growth (the 50% inhibitory concentration (IC50) ranging from 0.28 to 8.3 μM, mean IC50 = 2.6 μM on HCML). It was shown using HMCL that the growth inhibition induced by AS602868 is the result of a simultaneous induction of apoptosis and inhibition of the cell cycle progression. Importantly, AS602868 does not alter the survival of other bone marrow mononuclear cells (CD138−) co-cultured with primary MM (CD138+) cells except on CD34+ hematopoietic stem cells. Interestingly, using gene expression profiling with Affymetrix microarrays on 13 HMCL, we show that the resistance (high IC50) to AS602868 inhibitor is strongly correlated to APRIL gene expression (r =.7603, p <.01). Thus, an autocrine production of APRIL confers a resistance to the AS602868 IKK2-I. This can be explained since APRIL has been shown to activate the alternative NF-κB pathway which implicates an IKK complex composed of IKK1 homodimers instead of the IKK1/IKK2/NEMO complex involved in the canonical NF-κB pathway. The results demonstrate the important role of NF-κB in maintaining survival of MM cells and suggest that a pharmacological inhibition of the NF-κB pathway by an IKK2-I, AS602868, can efficiently kill myeloma cell lines or primary myeloma cells and might represent an innovative approach for treating MM patients.
BSc2118, a Novel Proteasome Inhibitor, Shows Anti-Tumor Activity in Multiple Myeloma and Mantle Cell Lymphoma.
Authors: Sterz Jan , Jakob Christian , Kuckelkorn Ulrike , Braun Hannes A. , Kaiser Martin , Fleissner Claudia , Kleeberg Lorenz , Heider Ulrike , Possinger Kurt , Kloetzel Peter M. , Sezer Orhan , Zavrski Ivana
Published: October 2019

The ubiquitin-proteasome pathway was recently identified as a promising new therapeutic target in cancer treatment. An increased proteasome activity was described in certain cancer types, especially in multiple myeloma (MM) and also in mantle cell lymphoma (MCL). The proteasome inhibitor bortezomib has been approved for the treatment of refractory MM and has also shown reproducible activity in MCL. Recently we described a novel tripeptide compound, BSc2118, with inhibitory activity against all three proteolytic activities (post-glutamyl peptide hydrolase-like, trypsin-like and chymotrypsin-like activities) of the 20S proteasome (Cancer Res2006;66:7598–605). We investigated the in vitro effects of BSc2118 in MM and MCL cell lines by MTT cell viability and AnnexinV apoptosis-assays. Furthermore, the intracellular chymotrypsin-like proteasome activity and NF-κB activity were detected in MM and MCL cells by detection of proteasome-degraded peptides or NF-κB p65 subunit. In MM cell lines OPM-2 and U266 we could show a significant time and dose-dependent reduction of cell viability by BSc2118 with an IC50 at 48hrs of 52nM and 65nM, respectively, whereas the MM cell line RPMI-S was less sensitive with an IC50 of 287nM. Using AnnexinV assay, a dose-dependent induction of apoptosis by BSc2118 was shown after 48hrs incubation. Comparably, in MCL we also found a time and dose-dependent reduction of cell viability in the cell lines HBL-2, JeKo-1 and Granta-519 with an IC50 of 82nM, 130nM and 262nM, respectively. Furthermore, BSc2118 induced apoptosis in all three MCL cell lines. Additionally, we detected a significant dose-dependent inhibition of intracellular chymotrypsin-like proteasome activity in MM and MCL cells, and a dose-dependent inhibition of TNFα-induced NF-κB activation in the MM cell lines OPM-2 and RPMI-S. This is the first report of anti-tumor effects of the novel proteasome inhibitor BSc2118 in MM and MCL cells. The compound effectively reduces the cell survival and shows a high pro-apoptotic activity in the MM cell lines OPM-2 and U266 and a significant activity in MCL cell lines HBL-2 and JeKo-1. Mechanisms of action are the inhibition of proteasome and NF-κB activity. Since previous clinical trials have shown an activity of the proteasome inhibitor bortezomib in MM and MCL, and adverse effects of other proteasome inhibitors may differ, our preclinical data support the idea to consider BSc2118 as a promising new agent in anti-tumor drug development.
Thymoglobulin (Polyclonal Rabbit Anti Thymocyte Globulin) Has In Vivo Activity in a Mouse Model of Myeloma.
Authors: Timm Michael , Wellik Linda , Kimlinger Teresa , Haug Jessica , Kline Michael , Rajkumar S. Vincent , Greipp Philip , Kumar Shaji
Published: October 2019

Background: Multiple myeloma remains incurable with current approaches and newer therapies are needed to improve the outcome of these patients. While monoclonal antibody based therapies have been successful in some of the hematological malignancies, such approaches have had limited efficacy in the setting of myeloma. Thymoglobulin (polyclonal rabbit antithymocyte globulin, Genzyme) (Thymo) has been extensively evaluated in the setting of allogeneic blood and marrow transplantation and solid organ transplants. Given the polyclonal nature of this product, with antibodies against different B cell antigens, we evaluated the in vitro and in vivo activity of Thymo in myeloma.

Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity following drug treatment was measured using the MTT viability assay. Apoptosis was measured by flow cytometry using Annexin V/PI in cell lines and Apo 2.7 in primary patient plasma cells. Shifts in expression of a variety of different B cell and plasma cell antigens were examined on several different myeloma cell lines following Thymo treatment in order to identify the potential antigenic targets. In vivo activity of thymo was evaluated in a SCID plasmacytoma model injected with RPMI myeloma cell lines.

Results: rATG was cytotoxic in vitro to several MM cell lines (RPMI 8226, U266, OPM1, OPM2) including the IL-6 dependent cell line Kas6/1 with LC50 of around 1 mg/mL. Additionally, thymo was cytotoxic MM cell lines resistant to conventional agents such as doxorubicin (Dox40), melphalan (LR5) and dexamethasone (MM1R). Thymo induced apoptosis in MM cell lines and in patient derived primary myeloma cells. When tested in combination with other anti-myeloma agents an additive effect was seen with doxorubicin, PS341 and melphalan. Using competitive flow cytometry, we identified CD138, CD38, Cd45, CD126, CD49d (VLA4), as well as CD20 as antigens likely to be targeted by Thymo. Tumor bearing mice injected with Thymo at two different doses (5 mg/kg and 10 mg/kg for five days) had significantly delayed tumor growth compared to non-injected mice, and this translated into a better survival for these mice. Mice receiving 10 mg/kg dose had a slower tumor growth compared to 5 mg/kg dose (Figure).

Conclusions: Thymoglobulin has promising in vitro and in vivo activity in the setting of myeloma. These studies will provide the rational for future clinical development of this agent in myeloma alone or in combination with other agents. Based on these results, we are in the process of initiating a clinical trial combining Thymo with Melphalan.

Figure Figure
Changes in Bone Marrow Plasma Cell Surface Markers Following CD138-Magnetic Bead Based Positive Selection.
Authors: Kimlinger Teresa K. , Rajkumar S. Vincent , Kline Michael P. , Haug Jessica L. , Timm Michael M. , Witzig Thomas E. , Kumar Shaji
Published: October 2019

Background: Isolation of malignant plasma cells from bone marrow of patients with monoclonal gammopathies is critical for studies into the disease biology. The isolation of plasma cells have generally been performed by positive selection using plasma cell markers such as CD138 or by negative selection by removing other marrow cells using a cocktail of antibodies. We have previously demonstrated differences in apoptotic rates in plasma cells with and without this enrichment step. Here we have examined the effect of CD138 magnetic bead selection on the surface phenotype of plasma cells by flow cytometry.

Methods: Bone marrow aspirates from patients with myeloma (n=12) were first washed, and then lysed with ACK to eliminate red cells. The samples were split with a portion of the cells further processed for CD138 selection. ACK lysed only whole bone marrow (WBM) and sorted cells were stained with CD38, CD45, CD56, activation markers CD71 and CD69, adhesion markers CD49d, CD11b, and CD66, B cell markers CD19 and CD20, and clonality (kappa and lambda). Gates were drawn around the plasma cells and plasma cell subsets on the basis of CD38/45 expression for both sorted and unsorted samples. In addition, an aliquot of the sorted preparation was examined by immunohistochemistry to calculate the purity of the sorted sample.

Results: Significant differences were observed in terms of the percentage of plasma cells expressing the different antigens when the cells were selected using CD138. This difference included a greater than 10% difference in expression between the two preparations as well as a change from positive to negative in several cases. There was a substantial loss in the expression of CD20, CD71 and CD11b on plasma cells following CD138 based sorting (Table). Among the other markers, CD49d remains unchanged and changes are variable for the other markers. In addition, in 8 of the 12 cases, there was a nearly complete loss of the CD45 positive subset with a loss of discrimination between CD45 negative and CD45 positive plasma cell subsets in the remaining CD138 sorted preparations (figure: unsorted left, sorted right).

Conclusion: In conclusion, the process of CD138 sorting of plasma cells appears to change important markers on the plasma cells and may even eliminate a key subset from further analysis. This should be kept in mind when isolating plasma cells using CD138 positive selection for analysis such as gene expression profiling. Consideration should be given to negative selection using antibodies against other cell types to deplete them.

Marker Unsorted % Sorted % CD56 74 67 CD69 11 2 CD71 23 8 CD49d 95 92 CD11b 33 4 CD66 5 1 CD20 16 6 Figure Figure
New Criteria To Identify Risk of Progression in Monoclonal Gammopathy of Uncertain Significance: Multiparametric Flow Cytometry Analysis of Bone Marrow Plasma Cells.
Authors: Perez-Persona Ernesto , Vidriales María-Belén , Mateo Gema , Sanz Ramón Garcia , Mateos Marivi , de Coca Alfonso Garcia , Galende Josefina , Martin-Nunez Guillermo , Alonso Jose M. , Heras Natalia , Hernandez Jose M. , Martin Alejandro , Lopez-Berges Consuelo , Orfao Alberto , Miguel J.F. San
Published: October 2019

Monoclonal Gammopathy of Uncertain Significance (MGUS) is a monoclonal disorder defined by the presence of a serum monoclonal protein <3g/dL, bone marrow plasma cells < 10% and absence of end-organ damage. The risk of progression to multiple myeloma (MM) is about 1% per year, and therefore these patients require long follow-up. Accordingly, the definition of new parameters that could be used for the identification of patients at risk of progression could be of great value. The aim of the present study is to evaluate the utility of multiparameter flow cytometry analysis of bone marrow (BM) plasma cells (PC) for predicting the risk of progression of MGUS patients. From January 1996 to September 2004, bone marrow aspirate samples from 350 patients, who fulfil the criteria of MGUS according to the International Myeloma Working Group criteria, were analysed by multiparametric flow cytometry. A specific gate on PC was performed based on CD138/CD38 expression and FSC/SSC characteristics and PC were immunophenotypically classified as normal (polyclonal) or aberrant (clonal) according to the expression of CD138, CD38, CD45, CD19 and CD56 antigens. Twenty seven patients (8 %) progressed to MM, with a median time to progression (TTP) of 46 months (range 9 to 109 months). Interestingly, the percentage of aberrant PC within the total BM PC compartment (aPC/BMPCc) clearly identify patients at different risk of progression. Thus, TTP in patients with ≥ 95% aPC/BMPCc was 85 months vs not reached cases with <95% aPC/BMPCc (p=0.0000). Other parameters with a significant influence on progression in the univariate analysis were: paraprotein level (higher vs lower of 2 mg/dl; p= 0.0004), the presence of immunoparesis (no paresis vs. decreased levels in one or two Ig. p= 0.0005), Bence-Jones proteinuria (p= 0.0003), PC BM infiltration assessed both by morphology and flow cytometry (p=0.0074; and p= 0.001, respectively), and DNA index assessed by flow cytometry (diploid vs aneuploid; p=0.0064). Moreover, the cut off level of 95% aPC/BMPCc, also allows the discrimination of two risk categories upon considering only patients at low risk of progression, based on a low paraprotein level or absence of inmunoparesis (p= 0.0000 and p= 0.0000, respectively). On multivariate analysis only the percentage of aPC/BMPCc (≥95%) (p=0.000), the DNA index (p=0.007), and the Bence-Jones proteinuria (p=0.000) showed independent prognostic value. In summary, our results show that multiparameter FC evaluation of BMPC at diagnosis is a simple, cost-effective and valuable tool for predicting the risk of progression of MGUS patients.
Low Efficacy of Thalidomide in Improving Response after Induction in Myeloma Patients Candidate to High-Dose Therapy.
Authors: Corso Alessandro , Mangiacavalli Silvia , Barbarano Luciana , Zappasodi Patrizia , Banfi Luciano , Montalbetti Luigi , Mazzone Antonio , Fava Sergio , Frigerio Guido , Isa Luciano , Montanara Sergio , Perego Daniele , Savarè Maria , Uziel Lilj , Vismara Alessandro , Campiotti Leonardo , Fiumanò Mario , Lazzarino Mario , Morra Enrica
Published: October 2019

Background: In multiple myeloma (MM) patients (pts) undergone high dose therapy, the outcome of the transplant is better if a good response is achieved before the procedure. Therefore, different attempts have been made in intensifying pre-transplant chemotherapy to improve the response. Aim of the study was to evaluate the safety and efficacy of Thal-Dex in improving the response rate after initial VAD therapy.

Methods: 61 untreated MM pts aged ≤65 years were addressed to high dose program with a debulking therapy with 3 pulse-VAD cycles followed by Thal-Dex for 3 months at the following schedule: Thal 100 mg/d orally at bed time, continuously for 3 months, Dex 20 mg/d orally on days 1–4 and 14–17 every 28 days. Response at the end of each phase was defined as follows: complete remission (CR), disappearance of serum and urine monoclonal component (MC) by immunofixation and bone marrow plasmocytosis (BMPC) <5%; very good partial remission (VGPR)/partial remission (PR)/minimal response (MR)/stable disease (SD), reduction of at least 90%/50%/25%/<25% of serum and urine MC, and of BMPC; progression, increase of serum and urine MC or BMPC. Toxicity was registered according to WHO classification.

Results: Responses after VAD were evaluable in 60 pts: RC 7%, VGPR 38%, PR 27%, MR 10%, SD 10%, progression 8%. Four pts dropped out after pulse-VAD for progression and in 1 for toxicity. Responses after Thal-Dex in 52 evaluable pts were: RC 12%, VGPR 40%, PR 17%, MR 4%, SD 4%, Progression 23%. Three pts dropped-out during Thal-Dex, 1 for progression and 2 for toxicity. Of note, pts who obtained a scarce response to VAD, namely a MR or SD, showed a higher probability (85% of cases) to have a progression during or after the administration of Thal-Dex. Statistical analysis (Kendall Concordance Coefficient (p<0.0001); and non parametric Wilcoxon matched pairs test (p=0.12)) did not show any improvement of the response rate after Thal-Dex with respect to the evaluation after pulse-VAD cycles.The pulse-VAD related toxicity was as follows: 1 died for sepsis after the first pulse-VAD, 5 developed grade 2 WHO infections, 4 developed mild constipation. Side effects during Thal-Dex therapy were: grade 1–2 peripheral neuropathy in 7 pts, grade 1 constipation in 6 pts; 4 pts developed thrombotic events (TVP in 3, and a bowel infarction with subsequent colectomy in one).

Conclusions: The combination thalidomide-dexamethasone after pulse-VAD is not effective in improving response rate, and shows an additional toxicity. Thus it does not seem useful for the intensification before transplant.
Essential Thrombocythemia in Young Individuals: Frequency and Risk Factors for Vascular Events and Evolution to Myelofibrosis in 126 Patients.
Authors: Alvarez-Larran Alberto , Cervantes Francisco , Bellosillo Beatriz , Giralt Manuel , Julia Antoni , Hernandez-Boluda Juan Carlos , Hernandez-Nieto Luis , Clapes Victoria , Burgaleta Carmen , Bosch Alba , Salvador Carlos , Arellano-Rodrigo Eduardo , Colomer Dolors , Besses Carlos
Published: October 2019

Vascular events and evolution to either myelofibrosis (MF) and acute leukemia (AL) are the main causes of morbidity and mortality in individuals with essential thrombocythemia (ET). However, the frequency of these complications in young ET patients is not well known. The objective of the present study was to assess the frequency of vascular events and the incidence of MF and AL in young patients with ET and to identify the factors associated with the development of such complications. In 126 subjects diagnosed with ET at a median age of 31 years (range: 5–40), overall survival and probability of survival free of either thrombosis, bleeding, MF, AL, and polycythemia vera (PV) were analyzed by the Kaplan-Meier method, followed by the log-rank test. With a median follow-up of eleven years (range: 4–25) three patients have died, being the probability of survival 98% at ten years. A total of 31 thrombotic events were registered in 25 patients; thrombosis-free survival (TFS) was 84% at ten years. Tobacco use was the only factor associated with an increased thrombotic risk, since TFS at 10 years was 72% in smokers versus 90% in non-smokers (p=0.03). Severe hemorrhagic complications were observed in 11 patients, and the estimated probability of bleeding-free survival was 92% at ten years. Evolution to MF was seen in 6 patients, four of whom had never received treatment for ET. MF-free survival was 97% at 10 years, with the risk being higher in patients showing an increased reticulin network in the bone marrow biopsy performed at diagnosis of ET (p=0.005). Transformation to AL was registered in one patient. JAK2 was mutated in 33 out of the 87 assessable patients (38%) and the mutation was associated with higher Hb values at diagnosis (p = 0.001). ET evolved into PV in five patients, being the probability of evolution into PV of 15% in JAK2 V617F positive patients versus 0% in JAK2 V617F negative patients (p=0.01). In conclusion, severe vascular complications are not infrequent in young subjects with ET, whereas transformation to MF or AL is a rare event.
Risk Stratification for Survival and Leukemic Transformation in Essential Thrombocythemia: A Single Institutional Study of 605 Patients.
Authors: Gangat Naseema , Wolanskyj Alexandra , McClure Rebecca F. , Li Chin Y. , Schwager Susan M. , Wu Wenting , Tefferi Ayalew
Published: October 2019

Background

It is widely recognized that advanced age and prior thrombosis predict recurrent thrombosis in essential thrombocythemia (ET) and are used to risk-stratify patients. However, the paucity of large sample size and long-term follow-up has limited the development of similar prognostic models for survival and leukemic transformation (LT).

Methods

Data was abstracted from the medical records of a consecutive cohort of patients with WHO-defined ET seen at the Mayo Clinic. Cox proportional hazards was used to determine the impact of clinical and laboratory variables on survival and LT. Overall survival and leukemia-free survival was estimated by Kaplan-Meier plots.

Results

i. Patient characteristics and outcome

The study cohort included 605 patients of which 399 (66%) were females (median age, 57 years; range 5–91). Median follow-up was 84 months (range; 0–424). During this period, 155 patients (26%) have died and LT was documented in 20 patients (3.3%) occurring at a median of 138 months (range; 23–422) from ET diagnosis.

ii. Prognostic variables for overall survival

Univariate analysis of parameters at diagnosis identified age ≥ 60 years, hemoglobin less than normal (defined as < 12 g/dL in females and < 13.5 g/dL in males), leukocyte count ≥ 15 x 109/L, tobacco use, diabetes mellitus, thrombosis, male sex, and the absence of microvascular symptoms as independent predictors of inferior survival. All of the above except the last two (i.e. male sex and the absence of microvascular symptoms) sustained their prognostic significance on multivariate analysis. Based on the first three prognostic variables: age, hemoglobin level, and leukocyte count, we constructed a prognostic model for survival: low-risk (none of the risk factors), intermediate-risk (1of 3 risk factors), and high-risk (≥ 2 risk factors). The respective median survivals were 278, 200, and 111 months (p<0.0001; Figure 1)

iii. Prognostic variables for leukemic transformation

On univariate analysis of parameters at ET diagnosis, LT was significantly associated with platelet count ≥ 1000 x 109/L, hemoglobin less than normal, and exposure to P-32. However, on multivariate analysis, only hemoglobin less than normal and platelet count ≥ 1000 x 109/L maintained independent prognostic value. Accordingly, we utilized these two variables, to construct a prognostic model for LT: low-risk (none of the risk factors), intermediate-risk (1 risk factor), and high-risk (both risk factors). Only 1 of the 239 patients (0.4%) in the low-risk group vs. 14 of the 289 (4.8%) in the intermediate-risk and 5 of the 77 (6.5%) in the high-risk group underwent LT (p=0.0009; Figure 2).

Conclusion

The current study provides clinician-friendly prognostic models for both survival and LT in ET.

Figure 1 Figure 1. Figure 2 Figure 2.
Recruitment of RXRα by Homo-Oligomeric RARα Is a Key for RARα-Mediated Transformation.
Authors: Zeisig Bernd B. , Kwok Colin , Wilson Amanda J. , Zelent Arthur , Gronemeyer Hinrich , Dong Shuo , So Chi Wai Eric
Published: October 2019

Chimeric fusion proteins involving Retinoic Acid Receptor Alpha (RARα) invariantly associate with almost 100% of Acute Promyelocytic Leukemia (APL). While we and others have recently demonstrated an essential role of aberrant self-association in RARα fusions mediated transformation1,2, forced oligomerization of truncated transcription factors including MLL3 and AML14 has emerged as an important oncogenic property shared by these most common chimeric fusion proteins involved in AML. To further characterize the underlying mechanisms and its modes of action, the present study dissects the essential molecular properties and transformation mechanisms by oligomeric RARα-oncoproteins. Firstly, we define the oncogenic threshold of self-association by showing that homo-oligomerization rather than homo-dimerization is required for Stat5b-RARα mediated transformation of primary hematopoietic cells. Although constitutive transcriptional repression is believed to be the underlying mechanism for RARα-mediated transformation, oligomeric RARα fusion proteins can bind retinoic acid response elements (RAREs) either as homo-oligomers or higher order hetero-oligomers with RXRα. To gain further insights into the transcriptional control mediated by RARα fusions, we successfully constructed various Stat5b-RARα point mutants that maintained the homo-oligomerization properties but lost their abilities to bind DNA as homo-oligomers. These resulting mutants could still efficiently transform primary hematopoietic cells in spite of their lack of homo-oligomeric DNA binding ability. To investigate if this is unique to Stat5b-RARα or is a general phenomena that can also apply to other RARα fusions, we further demonstrated that the synthetic FKBP-RARα fusion, which closely mimick both biochemical and transformation properties of bona fide RARα fusion proteins, was incapable of binding DNA as homo-oligomers, suggesting that homo-oligomeric DNA-binding is dispensable for RARα-mediated transformation. Conversely we reveal that the higher-order RARα-fusion/RXRα hetero-oligomeric complex, which aberrantly recruits transcriptional co-repressor SMRT, represents the major functional DNA-binding module. Specific knockdown of RXRα expression using shRNA approach can suppress transformation mediated by both bona fide and synthetic RARα fusion proteins, thus indicating a critical functional role of RXRα in RARα-mediated transformation of primary hematopoietic cells. Finally to assess the possible therapeutic values of targeting RXRα-mediated pathways in RARα-mediated transformation, we also demonstrated specific inhibition of myeloid transformation mediated by various RARα oncoproteins using the panRXR-selective agonist, SR11237. Taken together, these results not only highlight the functional significance of higher-order RARα-fusion/RXRα hetero-oligomeric complex, but also attest RXRα as a potential therapeutic target for APL.
Identification and Characterization of a Novel Jak2 Tyrosine Kinase Inhibitor.
Authors: Sayyah Jacqueline , Ostrov David , Sayeski Peter
Published: October 2019

Jak2 is a cytoplasmic tyrosine kinase that has been linked to hematological malignancies. Recently, a somatic Jak2 mutation (Jak2-V617F) has been identified in several myeloproliferative disorders such as polycythemia vera, essential thrombocythemia and myeloid metaplasia with myelofibrosis. These myeloproliferative disorders are characterized by unregulated expansion of one or more cells in the blood. The presence of the Jak2-V617F mutation in cells results in uncontrolled cell division and resistance to the negative feedback mechanisms that govern normal cell growth. The availability of a Jak2 tyrosine kinase specific inhibitor would facilitate our understanding of these Jak2-related disorders and perhaps serve a clinical benefit for patients. However, the most widely used Jak2 inhibitor, AG490, suffers from a general lack of specificity. Here, we used a novel approach to identify Jak2-specific inhibitors. We used in silico homology modeling of the Jak2 kinase domain to identify solvent accessible exposed pockets on the surface of the Jak2 protein. We then used a high-throughput program called DOCK to predict the ability of 20,000 small molecules to interact with a structural pocket adjacent to the ATP binding site of murine Jak2. The predicted binding energies of interaction between each compound and the Jak2 kinase domain were calculated and the top six scoring compounds were tested for their ability to inhibit Jak2 tyrosine kinase function in vitro. One of these compounds, 2-methyl-1-phenyl-4-pyridin-2-yl-2-(2-pyridin-2-ylethyl)butan-1-one (Z3) effectively inhibited Jak2-V617F and Jak2-WT autophosphorylation. We were able to show that Z3 inhibits total Jak2 tyrosine phosphorylation as well as Jak2 phosphorylation at the critical tyrosine 1007 residue in both a dose-and time-dependent manner. Z3 is able to reduce growth hormone-dependent Jak2 activation and is a direct inhibitor of Jak2-WT and Jak2-V617F tyrosine kinase autophosphorylation as measured by an in vitro kinase assay. Moreover, we found that Z3 inhibits proliferation of human erythroleukemia (HEL) cells, which express the Jak2-V617F mutation. In summary, this work demonstrates proof-of-principle concept that in silico molecular modeling can be used as a means to identify specific tyrosine kinase inhibitors.

Z3 Inhibits Jak2 Tyrosine Autophosphorylation in a Dose-Dependent Manner Z3 Inhibits Jak2 Tyrosine Autophosphorylation in a Dose-Dependent Manner Z3 Inhibits Growth Hormone Mediated Jak2 Phosphorylation at Tyrosine 1007 Z3 Inhibits Growth Hormone Mediated Jak2 Phosphorylation at Tyrosine 1007