bluuebunny/crossref_metadata_2025 huggingface dataset:
Phase I Trial of Bortezomib (NSC 681239) and Flavopiridol (NSC 649890) in Patients with Recurrent or Refractory Indolent B-Cell Neoplasms.
Authors: Grant Steven , Sullivan Daniel , Roodman David , Stuart Robert , Perkins Edward B. , Ramakrishnan Viswanathan , Wright John , Colevas A. Dimitrios , Roberts John D.
Published: April 2020

Preclinical studies suggest that neoplastic cells may be particularly sensitive to simultaneous interruption of cell-cycle and survival signaling pathways. In accord with this concept, we have shown that flavopiridol (F), a CDK inhibitor, interacts with Bortezomib (B), a proteasome inhibitor, to induce mitochondrial injury and apoptosis in human leukemia, myeloma, and lymphoma cells (Dai et al, Oncogene22:7108, 2003; Dai et al, Blood104,509,2004). These actions were associated with inhibition of NF-kappaB DNA binding, and diminished expression of phospho-JNK, XIAP, and Mcl-1. Based on these findings, we have initiated a phase I trial to identify appropriate doses of B+F for further investigation. Eligible patients (pts) include those with multiple myeloma, indolent B-cell lymphoma, or a related disorder, and must have recurrent or refractory disease following at least 1 prior systemic therapy (excluding allogeneic stem cell transplantation). Pts receive B (iv bolus) immediately followed by F (1 hour infusion) on days 1, 4, 8, and 11; courses are repeated every 21 days. Targeted dose levels are (B/F; mg/m2): 1.0/15, 1.3/15, 1.3/22, 1.3/30, and 1.3/40. Dose limiting toxicity (DLT) is defined as NCI CTCAE grade 4 ANC/platelet for > 1 week or grade ≥ 3 non-heme toxicity. 14 pts have been treated at 4 dose levels. There have been no DLT to date. There have been 3 episodes of herpes zoster (2 disseminated), 4 cases of exacerbation of pre-existing peripheral neuropathy, and 8 pts have discontinued study treatment after 1 to 5 cycles due to non-DLT toxicities. Among 11 patients evaluable for response and who are Bortezomib-naive, there has been 1 complete response (mantle cell lymphoma; MCL), 3 partial responses (2 myeloma, 1 lymphoma), and 5 patients with stable disease (3 myeloma, 1 lymphoma, 1 Waldenstrom’s). The patient with MCL who achieved a CR had previously received CHOP, Rituximab-CHOP, and fludarabine-Rituximab x 2. Correlative laboratory studies involving bone marrow CD138+ cells from patients with myeloma revealed a reduction in post-treatment NF-kappaB nuclear localization in 4/5 evaulable patients. Variable effects on myeloma cell expression of phospho-JNK, Mcl-1, and XIAP have been observed. Additional patients who previously received Bortezomib have now been entered on the study, but it is too early to evaluate their responses. Collectively, these findings indicate that a regimen combining Bortezomib and flavopiridol is well tolerated in patients with progressive B-cell malignancies, and has clear activity in some patients refractory to standard therapy. Pending identification of the MTD and RPTD (recommended phase II dose), Phase II evaluation of this therapeutic strategy should define its activity more definitively.
Phase I Study of Noscapine for Patients with Non-Hodgkin’s Lymphoma or Chronic Lymphocytic Leukemia Refractory to Chemotherapy.
Authors: Tulpule Anil , Levine Alexandra M. , Berman Nancy E. , Smith Lynne , Auerbach Alan , Douer Dan
Published: April 2020

Noscapine is an alkaloid derived from opium, which lacks sedative, euphoric, analgesic or respiratory depressant properties. Antitumor activity of Noscapine has been demonstrated both in vivo, against human lymphoid tumor cells, and in vitro, in human tumors implanted in athymic mice. Noscapine induces conformational changes in tubulin, which results in altered microtubule assembly. In this Phase I trial, cohorts of subjects with relapsed/refractory non-Hodgkin’s lymphoma (NHL) or chronic lymphocytic leukemia (CLL/SLL) were treated at three different dose levels of total daily doses of 1 g, 2 g, and 3 g. At each dose level the drug was administered orally on a three times a day schedule. The total duration of treatment was 49 days. Results: Twelve subjects with a median age of 65 years (range 38–71) have been accrued. Four subjects had CLL/SLL, 2 had mantle cell lymphoma, one had follicular grade III, 4 had DLC, and one had lymphoplasmacytic low grade lymphoma. 10 subjects are evaluable for response. Partial response was seen in 1 subject with follicular grade III disease. The duration of this PR is 56+ months. Stable disease was seen in a case of mantle cell lymphoma (duration 30 days) and a case of DLC (duration 77 days). The remaining 7 subjects had progression of disease. Noscapine has been well tolerated, with no grade 3 or 4 hematological toxicities. One grade 3 neurotoxicity consisting of depressed level of consciousness was experienced at the 3 g dose level. We conclude that a larger study of Noscapine is warranted to evaluate the efficacy of the compound in patients with lymphoma. The oral formulation, suggestion of efficacy and relative lack of toxicity make Noscapine an attractive candidate for further study.
HuMax-CD4 (Zanolimumab), a Fully Human Monoclonal Antibody: Early Results of an Ongoing Clinical Trial in CD4+ Peripheral T-Cell Lymphoma of Non-Cutaneous Type.
Authors: d’Amore Francesco , Relander T. , Hagberg H. , Osterborg A. , Hansen M. , Moeller L. , Johnson P. , Radford J. , Hancock B. , Gramatzki M. , Krause S. , Dreyling M. , Schmitz N. , Lisby S. , Baadsgaard O.
Published: April 2020

HuMax-CD4 (zanolimumab) is a fully human monoclonal IgG1k antibody, targeting the CD4 molecule on T-cells. It exhibits cytotoxic and anti-proliferative effects and has previously shown efficacy in cutaneous T-cell lymphoma. We report initial clinical efficacy and safety of treatment with 980 mg of HuMax-CD4 in 8 patients diagnosed with biopsy-proven, treatment-refractory or relapsed CD4+ peripheral T-cell lymphoma (PTCL) of non-cutaneous type. Patients were enrolled with the following diagnoses; enteropathy-type T-cell lymphoma (n=1), anaplastic large T-cell lymphoma (n=3, 2 ALK− subtype, 1 unknown), angioimmunoblastic T-cell lymphoma (n=1) and PTCL, unspecified (n=3). Primary endpoint was objective tumor response. Responses were based on CT scan and clinical examination and classified according to the Cheson criteria. Out of the 8 patients enrolled, 2 responses have been seen; 1 PR and 1 CRu (verified by CT scan). Further, clinical improvements (marked decrease in the size of peripheral lymph nodes) were recorded by the physicians in 3 other patients. In total, 9 SAEs have been reported. Of these, 1 case of febrile neutropenia was judged related. 8 unrelated SAEs were recorded; knee pain, viremia (n=2), condition aggravated, and disease progression (n=4). Three unrelated deaths have been reported, all due to disease progression. In conclusion, HuMax-CD4 was well tolerated in heavily pre-treated patients and the present results indicate efficacy in the treatment of PTCL.
Primary Gastric T-Cell Lymphoma: Clinicopathologic Features and Treatment Outcome.
Authors: Park Yeon Hee , Kim Won Seog , Bang Soo-Mee , Lee Soon Il , Uhm Ji Eun , Kang Hye Jin , Na Im Il , Ryoo Baek-Yeol , Yang Sung Hyun , Kim Kihyun , Lee Seung Sook , Ko Young H. , Park Keunchil
Published: April 2020

Primary gastric lymphoma is the most frequent type of extranodal malignant lymphoma. The large majority of cases are of B-cell lineage, while primary gastric T-cell lymphomas are exceedingly rare and least well characterized malignancy and the clinical features have been described only in a few case reports. In this study, we conducted a retrospective analysis to investigate the natural history and the clinical outcome after treatment. Between 1990 and 2004, 444 patients with primary gastric lymphoma had been diagnosed at four institutes. Among these 444 patients, 17 patients (3.8%) were identified as primary gastric lymphoma with T-cell immunophenotype. The pathologic review was performed by two experienced hematopathologist to confirm T-cell lymphoma with typical immunohistochemical features including CD3. All of the patients were staged according to the Mushoff system. Among these 17 gastirc T-cell lymphoma patients, 14 patients were treated with combination chemotherapy including CHOP with or without radiotherapy. The two patients were treated with high-dose chemotherapy with autologous peripheral hematopoietic stem cell transplantation. The median age of the 13 male and 4 female patients was 49 years (range 22–76 years). The pathologic subtypes of these patients with T-cell immunophenotype were as follows; precursor T-lymphoblastic lymphoma in 2 patients, peripheral T-cell lymphoma, unspecified in 6 patients, NK/T cell lymphoma in 3 patients, and T-cell others in 5 patients. The many of the patients showed poor performance status (PS), and high LDH level (65%). 12 patients (71%) showed stage III or IV diseases, and 9 patients (53%) showed high or high-risk according to the International Prognostic Index (IPI). A response to treatment was observed in 8 patients (47%), with 6 Complete Response (CR) and 2 Partial Response (PR). The median Progression-Free Survival (PFS) and Overall Survival (OS) were only 5 months (95% C.I.; 4–6 months), and 14 months (95% C.I.; 0–189 months). 5 patients of these 17 patients (29%) presented only in stomach and regional lymph nodes. The patients with localized gastric T-cell lymphoma showed good therapeutic outcome (median PFS; 53 months, median OS; 123 months, p =0.024 by log-rank test). Other clinical factors including PS, age, and LDH did not show clinical significance in this series. Although our series had limitation to interpret due to small population, this study showed that the incidence of this subtype of T-cell gastric lymphoma was very rare, and poor prognosis, like as other T-cell lymphoma. However, about 30% of the patients had localized diseases limited to gastric area and regional lymph nodes with relative good prognosis. New therapeutic strategies need to improve the outcome of patients with gastric T-cell lymphoma.
SGN-30 (Anti-CD30 Monoclonal Antibody) Is Active and Well Tolerated in Patients with Refractory or Recurrent Systemic Anaplastic Large Cell Lymphoma (ALCL).
Authors: Forero-Torres Andres , Bernstein Steven H. , Gopal Ajay K. , Foss Francine , Leonard John P. , Rosenblatt Joseph D. , Bartlett Nancy L. , Cheson Bruce D. , Epner Elliott , Hernandez-Illizaliturri Francisco J. , Proctor Stephen J. , Brice Pauline , Tilly Harve , Haioun Corinne , Jerkeman Mats , Bosly Andre , Lorenz Jennie M. , Barton Jeremy
Published: April 2020

SGN-30 is a chimeric monoclonal antibody (mAb) directed against the CD30 antigen expressed on hematologic malignancies, notably Hodgkin’s disease and ALCL. SGN-30 was well tolerated in phase I studies and objective responses were seen. In this multicenter phase II study, refractory or recurrent CD30-positive ALCL patients, ECOG performance status 0–2, are treated with SGN-30 at 6 mg/kg administered by IV over a 90 minute infusion weekly for 6 consecutive doses. A two stage statistical design provides a 90% power to detect an objective tumor response rate of at least 30%. Twenty patients (11M, 9F) with ALCL have been enrolled, with a median age of 53 (range 33–78) and 3 median prior therapies (range 2–5). Eighteen of 20 patients were Anaplastic Lymphoma Kinase (ALK) negative. Response evaluations demonstrate that one patient achieved a complete response and remains in remission more than one year after therapy. Three patients achieved a partial response, of which two had a lasting response of 27 and 53 days respectively. The third patient is currently still on treatment. Of the other 16 patients, 3 had stable disease, 10 progressed and 3 are not yet evaluable. SGN-30 has been well tolerated. Drug-related adverse events have been typically mild and consistent with the phase I experience. There were 2 grade 3/4 toxicities possibly related to SGN-30. No significant hematological or biochemical toxicities have been observed. One patient had a catheter related infection and another experienced urticaria. There was 1 definitely related grade 3/4 event in a patient who experienced an exacerbation of his cutaneous lesions after 2 doses of SGN-30 and followed by a partial response. The requisite number of responses have now been obtained to allow the study to continue.
The JAK Inhibitor INCB20 Induces Antiproliferative and Apoptotic Effects in Human Myeloma Cells In Vitro and In Vivo.
Authors: Burger Renate , Legouill Steven , Tai Yu-Tzu , Shringarpure Reshma , Podar Klaus , Catley Laurence , Tassone Pierfrancesco , Neri Paola , Hideshima Teru , Chauhan Dharminder , Caulder Eian , Vaddi Kris , Neilan Claire L. , Gramatzki Martin , Fridman Jordan S. , Anderson Kenneth C.
Published: April 2020

In multiple myeloma (MM), IL-6 plays an important role for tumor cell growth, survival, and drug resistance. Janus kinases (JAKs) are protein tyrosine kinases and constitutively associated with the gp130 chain of the IL-6 receptor complex. Their activation is one of the first steps in cytokine receptor-mediated signaling and critical for virtually all subsequent downstream signaling cascades. INCB20 is a small-molecule synthetic compound which, in biochemical assays, potently inhibited all four JAKs with IC50 values between 0.3 nM and 1.2 nM (for comparison, IC50 of AG490, another JAK inhibitor, was >50 μM). Consistent with the central role of JAKs in gp130-mediated signaling, INCB20 inhibited IL-6 induced phosphorylation of SHP-2, STAT1, STAT3, ERK1/2, and AKT in MM1.S cells. In contrast, AKT phosphorylation induced by IGF-1 remained unchanged. Evaluation of the cellular efficacy of INCB20 was performed using the IL-6 dependent INA -6 cell line. Growth of INA-6 cells was inhibited in a dose-dependent manner with an IC50 of approx. 0.5 μM, as measured by [3H]-thymidine uptake and an MTS-based assay (for comparison, the cellular IC50 of AG490 was 15–20 μM). This correlated with an increase in the percentage of apoptotic cells, as evaluated by Apo2.7 staining after 48 hours. Importantly, INA-6 growth was inhibited in the presence of bone marrow stromal cells accompanied by a decrease in phospho-STAT3 levels. Furthermore, in a subcutaneous INA-6-SCID model, INCB20 inhibited tumor growth (and phosphorylated STAT3) in a dose-dependent manner. Our studies provide the conceptual basis for the use of JAK inhibitors as a therapeutic approach in MM.
Nucleoside Analogues of Cladribine Produce Enhanced Responses in Cell Lines.
Authors: Walsby Elisabeth , Congiatu Constantino , Schwappach Anna , Walsh Val , Burnett Alan K. , McGuigan Christopher , Mills Kenneth I.
Published: April 2020

Nucleoside analogues are antimetabolites that can be used to treat AML. These compounds mimic the effects of physiological nucleotides in terms of uptake and metabolism and become incorporated into nascent DNA resulting in synthesis inhibition and chain termination. Nucleoside drugs often suffer from poor metabolism to the active triphosphate forms, deactivation and clinical toxicities. Phosphate prodrugs (protides) have modified monophosphate groups on the drug which potentially enables them to enter cells and subsequently become metabolised into the monophosphate form. Production of the monophosphate form of the drug is the rate limiting step in the production of the active triphosphate form.

Cladribine (2-chlorodeoxyadenosine) has been used as a parent drug to produce a series of protides with altered monophosphate groups. The parent drug and the protides were used at concentrations between 10μM to 0.002μM on a panel of six myeloid cell lines (HL-60, NB4, NB4R2, U937, KG1 and K562) for 72 hours. The effects of the protides were measured by proliferative assays (MTS) and IC50 values calculated. Two protides, CC122 and CC124, were assessed in combination with cytarabine (Ara-C) at ratios of 1:1 and 1:10 in the same manner as previously described.

Fourteen protides of cladribine were analysed in addition to cladribine itself and Ara-C. The average IC50 value for each protide, across all the cell lines, tested were reduced by 2 to 11 fold compared to cladribine and Ara-C. One protide (CPF193) had no effect on any of the cell lines, whilst CPF194 was found only to be active in the KG1 cell line with a 2.7 fold reduction in the concentration required kill 50% of the cells treated compared to cladribine. In general, K562 cells did not respond to treatment with Ara-C, cladribine or 6 of the protides although they did respond to the remaining protides with IC50 values slightly higher than the other cell lines.

The IC50 values for each protide in each cell line individually was also calculated and some cell lines, particularly NB4, showed dramatic reductions in IC50. In particular protide CC124 showed over 40-fold reduction from 9.4μM for cladribine to 0.23μM for CC124. This was also lower than the IC50 for Ara-C in this cell line (0.53μM).

Ara-C and cladribine combinations, at a ratio of 1:1, gave an average combination index (CI) at the IC50 of 0.9857 across all the cell lines, excluding K562, indicating an additive effect only. However, protide CC124 and Ara-C in combination, at the same ratio, produced an average CI of 0.6334 indicating synergistic activity.

This study has shown that altered monophosphate groups on the nucleoside inhibitor cladribine reduces the IC50, particularly when used in combination with Ara-C. The reduced doses would be beneficial in a clinical environment.
Inhibition of Bcl-2 Signaling by Small Molecule BH3 Inhibitor GX15-070 as a Novel Therapeutic Strategy in AML.
Authors: Contractor Rooha , Konopleva Marina , Samudio Ismael , Kantarjian Hagop , Andreeff Michael
Published: April 2020

GX15-070 is a novel cycloprodigiosin derived small molecule BH3 inhibitor that binds with moderate affinity to all antiapoptotic Bcl-2 family members, including Mcl-1, and is currently undergoing Phase I clinical trials in CLL. In this study, we investigated anti-leukemic activity of GX15-070 in acute myeloid leukemia (AML) cell lines and primary AML samples. GX15-070 inhibited cell growth of HL-60, U937, OCI-AML3 and KG-1 at IC50’s of 0.1, 0.5, 0.5 and 2.5μM, respectively as determined by trypan blue exclusion method at 72 hours. Overexpression of Bcl-2 or Bcl-XL did not confer resistance to GX15-070 (IC50’s of HL-60/neo, HL-60/Bcl-2 and HL-60/Bcl-XL cells 0.25μM). MDR-overexpressing HL-60/Dox cells were similarly sensitive compared to their non-MDR counterpart, and specific MDR inhibitor (PSC 833) did not enhance apoptosis caused by GX15-070. GX15-070 (250nM) inhibited Mcl-1/Bak heterodimerization in HL-60 cells starting at 3 hours as evidenced by IP/Western blot analysis, however it did not affect Bax/Bcl-2 heterodimerization. This was associated with cytosolic release of cytochrome c followed by an increase in annexin positivity (21±2.5% annexin + cells with 250nM) and a decrease in mitochondrial inner membrane potential with 64±4.8% of cells losing membrane potential at 72 hours. GX15-070 did not affect cell cycle distribution of HL-60 cells. In 6/7 primary AML samples, GX15-070 induced apoptosis in CD34+ progenitor cells at an average IC50 of 3.6±1.2μM at 24 hours. In conclusion, the small molecule BH3 inhibitor GX15-070 potently induces apoptosis in myeloid leukemia cells via disruption of Mcl-1/Bak dimerization and activation of the intrinsic apoptotic cascade. Our findings provide rationale for the further development of this BH3 inhibitor as a novel therapeutic strategy for AML.
Fully Human CD38 Antibodies Efficiently Trigger ADCC of Multiple Myeloma Cell Lines and Primary Tumor Cells.
Authors: Peipp Matthias , de Weers Michel , Beyer Thomas , Repp Roland , Parren Paul , Valerius Thomas , van de Winkel Jan G.J.
Published: April 2020

Although new treatment modalities have recently been added to the standard regimens for multiple myeloma, the clinical outcome for patients with advanced disease is often limited. Monoclonal antibodies are increasingly used for tumor therapy, and may also represent interesting options for multiple myeloma patients. CD38 is one of the most promising target antigens on malignant plasma cells, which are evaluated in preclinical and early clinical studies as targets for antibody therapy. CD38 is a type II transmembrane protein with ectoenzymatic activity, which is involved in calcium mobilization. Human CD38 is predominantly expressed by bone marrow precursor cells and by terminally differentiated plasma cells. Multiple myeloma cells show moderate to high expression levels - making CD38 a potential candidate as target for immunotherapy.

A panel of 42 fully human CD38 antibodies was generated by immunizing human Ig transgenic mice. Immunofluorescence studies with CD38 transfected cells demonstrated antigen-specific, high affinity binding, and cross-blocking experiments revealed four distinct epitope groups. Seven antibodies, representing each of the four groups, were selected for further analyses. ADCC and CDC activity against CD38-positive myeloma cell lines (AMO-1 and JK6), and against freshly-isolated primary multiple myeloma cells was investigated. Human whole blood served as effector source, which was then fractionated into plasma (containing human complement), mononuclear (MNC) or granulocytic (PMN) effector cells. All antibodies mediated concentration-dependent killing of both multiple myeloma cell lines - using human mononuclear cells as effector source. Also complement-dependent killing of freshly isolated myeloma cells was observed. However, none of the antibodies recruited PMN for tumor cell lysis. Importantly, CD38 antibodies also killed freshly isolated tumor cells from a rare patient with a CD38/138- positive plasma cell leukemia, which was chemotherapy- refractory at the time of analysis. Furthermore, CD38 antibodies effectively prevented outgrowth of CD38-positive tumor cells in SCID mouse xenograft models. Antibody 005 was significantly more effective in these assays compared to the remaining panel of CD38 antibodies.

In conclusion, CD38 antibodies efficiently mediated killing of multiple myeloma cell lines as well as freshly isolated tumor cells and prevented tumor outgrowth in xenografted SCID mice. Antibody 005 was superior in mediating CDC and ADCC via MNC - particularly at low antibody concentrations.
The Y-box Binding Protein YB-1 Is Associated with Progressive Disease and Mediates Survival and Drug Resistance in Multiple Myeloma.
Authors: Chatterjee Manik , Ransco Christoph , Stühmer Thorsten , Eckstein Niels , Royer Hans-Dieter , Bargou Ralf
Published: April 2020

Introduction: The Y-box binding protein YB-1 is a member of the cold shock domain protein superfamily and represents one of the most evolutionary conserved nucleic-acid binding proteins. Yb-1 is involved in a wide variety of cellular functions, such as regulation of transcription and translation, but also in DNA-repair and stress response to extracellular signals. Furthermore, recent reports from our group could show that overexpressed YB-1 plays a role in drug resistance of breast cancer cells and might act as an oncogene. The goal of this study was to investigate a potential pathogenetic role of YB-1 in MM.

Material and Methods: For the detection of YB-1 expression in vivo, bone marrow biopsies of MM patients were analyzed by immunohistochemistry. The expression of YB-1 protein in a number of MM cell lines was analyzed by Western Blotting. The regulation of YB-1 expression through major signaling pathways, e.g. IL-6R/STAT3, Ras/MAPK and PI3K/AKT, was analyzed using specific inhibitors of these pathways (Sant7, PD98059 and Ly294002). To determine the role of YB-1 for survival and proliferation, siRNA technology was exploited to transiently knockdown the expression of YB-1 in the MM cell lines INA-6 and MM.1s. In addition, these YB-1 knockdown cells were exposed to doxorubicin and melphalan to evaluate the influence of YB-1 on drug resistance.

Results: Immunohistochemical analyses of bone marrow biopsies revealed that YB-1 is strongly expressed only in MM cells of samples that show a highly proliferative phenotype. This subgroup of MM patients was characterized by an aggressive clinical course. In contrast, MM cells of samples with a slow proliferative status did not show YB-1 expression. Interestingly, tumor cells of patients that responded to chemotherapy did not express YB-1. Furthermore, neither normal bone marrow plasma cells nor premalignant plasma cells of MGUS patients showed YB-1 expression. YB-1 was detected in all of the evaluated MM cell lines. YB-1 expression was not regulated by the IL-6R/STAT3, Ras/MAPK and PI3K/AKT pathways. Knockdown of YB-1 in INA-6 and MM1.s cells by siRNA resulted in a slow proliferation phenotype with a higher apoptotic cell fraction. In addition, we observed that YB-1 knockdown remarkably sensitized cells towards drug-induced apoptosis.

Conclusions: YB-1 expression in MM appears to be correlated with a highly proliferative phenotype and with disease progression. YB-1 contributes to the malignant growth and drug resistance and might be therefore an attractive therapeutical target to circumvent aquired drug resistance in MM.
Extracellular Endosulfatases (Sulfs) Inhibit Myeloma Tumor Growth In Vivo.
Authors: Dai Yuemeng , Yang Yang , MacLeod Veronica , Yue Xinping , Rapraeger Alan C. , Shriver Zachary , Venkataraman Ganesh , Sasisekharan Ram , Sanderson Ralph D.
Published: April 2020

Human sulfatases Hsulf-1 and Hsulf-2 are extracellular endosulfatases that specifically remove 6-O sulfates from heparan sulfate chains. Published studies have shown that expression of Hsulf-1 in some tumor cell lines diminishes their response to heparin-binding growth factors (e.g., FGF-2, HGF) leading to speculation that these enzymes may regulate tumor growth. To examine this possibility in vivo, the cDNA for either Hsulf-1 or Hsulf-2 was expressed in the human myeloma cell line CAG. Disaccharide composition analysis of heparan sulfate chains isolated from the surface of the transfected cells reveals that 6-O sulfation of trisulfated disaccharides is diminished by 70% and 50% on cells expressing Hsulf-1 and Hsulf-2, respectively, as compared to control cells transfected with empty vector only. When stimulated with exogenous FGF-2, downstream signaling as measured by the level of phosphorylated ERK1/2, is reduced in HSulf-1 or Hsulf-2 transfectants as compared to controls. To determine if Hsulf-1 and Hsulf-2 regulate myeloma tumor cell growth in vivo, cells were injected subcutaneously into the left flank of SCID mice. Monitoring of whole animal tumor burden by analysis of human kappa light chain present in the serum revealed that tumors formed from cells expressing either Hsulf-1 or Hsulf-2 exhibited a dramatic inhibition of tumor growth as compared to tumors formed from control cells (p<0.0004). Upon sacrifice, mean weights of primary tumors were 0.46 g (n=12), 0.18 g (n=17) and 2.44 g (n=16) for Hsulf-1, Hsulf-2 and control tumors, respectively (p<0.0001). To confirm that the endosulfatase enzymes were active in vivo, assembly of the FGF-2 ternary signaling complex (FGF-2/heparan sulfate/FGF Receptor 1) was analyzed on frozen tissue sections of the tumors. Incorporation of the FGFR1 into the complex requires the presence of 6-O sulfate groups in the endogenous heparan sulfate chains. Experiments show that FGF-2 binds equally well to the heparan sulfate present in all tumors, but the FGFR1 is not incorporated into the complex on tumors expressing either Hsulf-1 or Hsulf-2. This indicates that the Sulfs are active in vivo and that this activity alters heparin-binding growth factor signaling of the tumor cells. Additionally, we find that the elevation of Sulf expression causes a marked increase in extracellular matrix (ECM) deposition within tumors which may, along with attenuated growth factor signaling, contribute to the reduction in tumor growth. These studies indicate that dynamic regulation of heparan sulfate structure by Sulfs present within the tumor microenvironment can have a dramatic impact on the growth and progression of myeloma tumors in vivo.
Critical Role of flt3 in Fetal and Adult T Lymphopoiesis.
Authors: Sitnicka Ewa , Buza-Vidas Natalija , Ahlenius Henrik , Cilio Corrado M. , Gekas Christos , Nygren Jens M. , Jensen Christina T. , Svensson Marcus , Agace William W. , Jacobsen Sten Eirik W.
Published: April 2020

We recently demonstrated that signalling through the cytokine tyrosine kinase receptor flt3 and interleukin-7 receptor α (IL-7Rα ) is indispensable for fetal and adult B cell commitment and development (Sitnicka et al., J. Exp. Med. 198: 1495, 2003). We have also shown that flt3 ligand (FL)-deficient mice have reduced levels of the common lymphoid progenitor (CLP) but normal levels of mature T cells (Sitnicka et al., Immunity, 17:463, 2002). However, previous studies failed to demonstrate a role of flt3 and its ligand in T lymphopoiesis. Herein, although having normal numbers of thymocytes, FL-deficient mice were shown to have distinct reductions in the earliest double negative (DN) progenitors in the fetal, postnatal and adult thymus. A critical role of flt3 in thymocyte development was most evident in the absence of Interleukin-7 receptor a (IL-7Rα ) signalling. Strikingly, in adult FL−/−IL-7Rα −/− (double deficient) mice thymic cellularity was reduced 400 fold as compared to FL−/− and wild type (WT) controls and 30 fold as compared to IL-7Rα −/− mice. In agreement with previous studies, IL-7Rα −/− thymocytes revealed a partial block at the progression from the DN2 (CD4−CD8−CD44+CD25+) to DN3 (CD4−CD8−CD44−CD25+) stage, while in FL−/−IL-7Rα −/− mice DN1 (CD4−CD8−CD44+CD25−), DN2 and DN3 thymic progenitors were undetectable. The DP CD4+CD8+ thymocytes were reduced more than 1000 fold as compared to IL-7Rα −/− mice and low levels of peripheral T cells remaining in FL−/−IL-7Rα −/− mice had exclusively an activated/memory phenotype. Furthermore, FL−/−IL-7Rα −/− bone marrow cells lacked T cell reconstituting potential after transplantation into lethally irradiated recipients. Taken together these data demonstrate indispensable and complementary roles of flt3 and IL-7Rα in T lymphopoiesis.
TRAILR Triggering by Human Antibodies Induces Apoptosis through an Early Cleavage of Mcl-1 and Caspase 3 in Myeloma Cells.
Authors: Pellat-Deceunynck Catherine , Gomez-Bougie Patricia , Daniels Sylvanne , Geffroy-Luseau Alexandrine , Bataille Regis , Amiot Martine
Published: April 2020

We investigated TRAILR expression and sensitivity of myeloma cells in vitro. This study was done using a panel of 20 myeloma cell lines that are representative of primary myeloma cells (14q chromosomal translocation, IL-6 dependency, phenotype, oncogenes mutation). TRAILR were stimulated with agonistic human antibodies directed against either TRAIL-R1/DR4 (HGS-ETR1, mapatumumab) or TRAIL-R2/DR5 (HGS-ETR2), provided by Human Genome Sciences, Rockville, MD. This approach allowed us to analyze the contribution of each receptor separately. We show that a wide majority of cell lines, 16 of 20 were killed upon either TRAIL-R1 or R2 stimulation in the presence or absence of IL-6. However, 4 cell lines were resistant to HGS-ETR1 and 6 to HGS-ETR2 and 3 to both. Activation of both caspase 8 and Bid has been extensively described as being associated with TRAIL response. Indeed, we observed an activation of both caspase 8 and Bid. Cleaved molecules were detected 6 to 18h after antibody addition but after detection of cellular apoptosis. However, we show that Mcl-1L, a key molecule for myeloma survival, was downregulated and cleaved as soon as 3h after Ab addition. The cleaved form of Mcl-1 has been shown to behave like a proapoptotic molecule. Since caspase 3 has been reported to cleave Mcl-1, we looked at caspase 3 activation. Indeed, we observed that caspase 3 cleavage occured early and concomitantly to the one of Mcl-1. Our data show that in a wide majority of myeloma cell lines (80%) TRAILR triggering induces massive apoptosis that was not prevented by IL-6, the major myeloma cell growth and survival factor. Moreover, apoptosis induced upon TRAILR triggering was fully correlated to an early cleavage of both caspase 3 and Mcl-1 and to a delayed one of both caspase 8 and Bid.
TACI and BCMA, Unlike BAFF-R, Are Expressed by Human Myeloma Cell Lines and TACI+ Cell Lines Have a Gene Signature Indicative of Mature Bone Marrow Plasma Cells and Bone Marrow Environment Dependence.
Authors: Moreaux Jerome , Cremer Friedrich W. , Reme Thierry , Mahtouk Karene , Pantesco Veronique , de Vos John , Moos Marion , Robert Nicolas , Goldschmidt Hartmut , Rossi Jean-François , Hose Dirk , Klein Bernard
Published: April 2020

B-cell activating factor (BAFF) and A Proliferation-Inducing Ligand (APRIL) are produced by the BM microenvironment of patients with multiple myeloma (MM) and are growth factors of multiple myeloma cells (MMC). BAFF has three receptors: BAFF-R, TACI and BCMA, and APRIL has 2 receptors: TACI and BCMA. Using real time RT-PCR and FACS analysis, we found that TACI and BCMA are expressed by MMC, unlike BAFF-R. Gene expression profiling (GEP) of purified MMC from newly-diagnosed patients has indicated that primary MMC with high TACI expression (TACI[suphigh] MMC) have a mature plasma cell gene signature linked to a dependence on the bone marrow environment. In contrast, primary MMC with a low TACI expression (TACI[suplow] MMC) have a gene signature of proliferating plasmablasts. TACI expression was either undetectable (11 HMCLs) or present (7 HMCLs) on a panel of 18 HMCLs, whereas BCMA expression was detected in all HMCLs with sensitive real-time RT-PCR and FACS analysis. GEP of the 18 HMCLs were determined with Affymetrix U133 DNA microarrays that interrogate 33000 genes or ESTs. 109 out of the 33000 genes/ESTs were differentially expressed between TACI[sup+] and TACI[sup-] HMCLs (ratio ≤ 0.5 and ≥ 2, P ≤ 0.05). TACI[sup+] HMCLs have a gene signature indicative of a BM environment dependence close to that of normal bone marrow plasma cells, including genes involved in intercellular communication and signal transduction genes. TACI[sup+] HMCLs with a BM environment dependence signature include the RPMI8226 and LP1 HMCLs that produce BAFF or APRIL as autocrine growth factors, a property of the bone marrow environment in patients with intramedullary MM. The microenvironment dependence signature was also found in 5 HMCLs (XG-2, XG-12, XG-13, XG-19 and XG-20) whose growth requires addition of exogenous IL-6 or BAFF/APRIL in vitro. These data indicate that a high TACI expression and the associated microenvironment dependence gene signature found in primary MMC is kept in HMCLs that have been maintained in culture for 10–30 years, and whose growth does not need anymore interaction with BM environment. One hypothesis is that this gene signature may be a characteristic of the MMC clone at the occurrence of the disease. The MM stem cell could be immortalized at different stages of normal plasma cell differentiation. Transformation at the plasmablastic stage would give rise to a MMC clone with a plasmablastic gene signature. Transformation at a later plasma cell differentiation stage would give rise to a MMC clone with a gene signature of mature BM plasma cells.
Anti-Angiongenic Effects of mTOR-Inhibitors Is Regulated by AKT Activity in Multiple Myeloma Cells.
Authors: Frost Patrick , Hoang Bao , Shi Yijiang , Yan Huajun , Lichtenstein Alan
Published: April 2020

Inhibitors of the mammalian target of rapamycin (mTOR), such as rapamycin (RAPA) and CCI-779 (CCI), have potential as anti-tumor agents against multiple myeloma (MM). Since other tumor models have demonstrated that heightened AKT activity induces hypersensitivity to mTOR inhibitors, we stably transfected U266 human MM cells with a constitutively activated AKT allele (U266-AKT) or empty vector control (U266-EV) in order to further explore the underlying mechanisms of this phenomena. Analysis of cell death demonstrated that U266-AKT were significantly more sensitive to RAPA in vitro, with an ED50 of 0.01 nM versus an ED50 of >100 nM for U266-EV control cells. A similar alteration of sensitivity to CCI was demonstrated in U266 isogenic tumors grown in NOD/SCID mice and treated with CCI in vivo. Analysis of the excised tumor nodules demonstrated a >5 fold inclease in apoptotic nuclei in U266-AKT tumors treated with CCI compared to isogenic control tumors, despite previous reports that mTOR inhibitors do not induce apoptosis in MM cells in vitro. One potential explanation for this is that AKT regulates the ability of CCI to inhibit angiogenesis, which is only relevent in vivo, and thereby indirectly induces apoptotic cell death. In support of this hypothesis, we demonstrated that CCI significantly decreased angiogenesis (measued by in situ staining of excised tumor nodules with CD34, a marker for endothelial cells) by 80% in U266-AKT, and only by 67% in isogenic controls. Since previous studies demonstrated that AKT/mTOR regulates the expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1a (HIF1a), we hypothesized that MM cells with heightened AKT activity may be more sensitive to the CCI-mediated inhibition of these critical angiogenic factors. In vitro, RAPA was markedly more effective at inhibiting HIF-1a and VEGF expression from U266-AKT compared to U266-EV control cells. One possible explanation for the regulatory role of AKT in the RAPA/CCI response is that MM cells with hyperactive AKT function depend upon mTOR-mediated (i.e. cap-dependent) translational pathway to express critical proteins like VEGF and HIF-1a, while “low-AKT” MM cells may be able to utilize non-mTOR dependent (i.e. cap-independent) salvage pathways to express these critical proteins and are thereby resistant to mTOR inhibitors.
Induction of Cyclin D1 Gene in Myeloma Cells Down-Regulates the Expression of Cyclin D2.
Authors: Kuroda Yoshiaki , Sakai Akira , Tsuyama Naohiro , Mizuno Mami , Nakaju Nanae , Munemasa Shoso , Okikawa Yoshiko , Katayama Yuta , Kimura Akiro
Published: April 2020

Cyclin D is dysregulated in at least two-thirds of multiple myeloma (MM) tumors. In addition, recent reports showed that the dysregulation of cyclin D1 is frequent in the absence of a t(11;14) translocation in MM. However, as we also reported (Int J Oncol, 2004), there appears to be no obvious correlation between the expression of cyclin D1 and the proliferation index (PI) or Ki67 expression. Therefore, we thought that the down-regulation of cyclin D2 might offset the expression of cyclin D1 in myeloma cells with cyclin D1 overexpression in cDNA microarray, since primary myeloma cells or myeloma cell lines express cyclin D3 ubiquitously. Here we transfected cyclin D1 gene into a myeloma cell line (RPMI8226), originally not expressing cyclin D1, using a retrovirus-mediated gene transfer system. In this method we inserted a 1.1 kb fragment containing the open reading frame of cyclin D1 removing from a tet-cyclin D1 plasmid (kindly provided by Dr. Reed SI, MCB, 1994) into a retrovirus vector (pQCXIP). First, we analyzed the expression of cyclin D1 in the bulk culture of cyclin D1 transfectant. We detected the expression of cyclin D1 by western blot, and found that the limited numbers of transfectant expressed cyclin D1 protein by immunohistocytochemical staining. Subsequently, we separated the two types of cyclin D1 transfectant by limiting dilution. Both transfectants showed the expression of cyclin D1 mRNA in RT-PCR, however, one of the two did not show the expression of cyclin D1 protein in western blot and immunohistocytochemical staining. Interestingly, we clearly detected the down-regulation of cyclin D2 mRNA in the transfectant with cyclin D1 protein expression by RQ-PCR. Furthermore, we detected an increase of cells in S phase in the transfectant with cyclin D1 protein by flow cytometry. Unlike in the study of Lamb J et al. (Cell, 2003), we could not observe the induction of IL-6 by the transfection of cyclin D1 gene. Although the mechanism of the impairment of cyclin D1 translation is unclear, here we suggest that the lack of correlation between the expression of cyclin D1 and PI might be due to the impairment of cyclin D1 translation or the offset of the expression of cyclin D1 by the down-regulation of cyclin D2. We are now analyzing the effects of velcade and IMiDs on these transfectants, since we suspect that these differences would affect the response to chemotherapy for MM. Furthermore, we are going to analyze the difference of gene expression between these transfectants using cDNA microaray. Therefore, these transfectants could be useful materials to analyze the cyclin D1 dysregulation in myeloma cells.
Prognostic Value of Serum β2-Microglobulin in Patients with Waldenstrom’s Macroglobulinemia Requiring Treatment.
Authors: Anagnostopoulos Athanasios , Zervas Konstantinos , Terpos Evangelos , Kyrtsonis Marie Christine , Symeonidis Argyris , Gika Dimitra , Bourantas Konstantinos , Zomas Athanasios , Angnostopoulos Nikolaos , Pangalis Gerasimos , Dimopoulos Meletios A.
Published: April 2020

Introduction: Waldenstrom’s macroglobulinemia is a lymphoplasmacytoid lymphoma characterized by the production of monoclonal IgM. This disorder has a relatively indolent course with a median survival ranging from 5 to 10 years in different series. Several clinical and laboratory variables have been associated with an inferior survival such as advanced age, hyperviscosity, presence of cytopenias and hypoalbuminemia. More recently, serum beta2-microglobulin (beta2m) is being investigated for its potential role in stratifying patients with WM into different subsets.

Patients and methods: In order to further evaluate the role of serum beta2m we analyzed 124 patients with available pretreatment value of this variable. These patients received primary treatment consisting of alkylating agents, nucleoside analogues or rituximab. Their median age was 70 years (range 28 to 89 years) and their median survival was 105 months. Multiple clinical and laboratory parameters were evaluated for their possible correlation with overall survival for the whole patient population and for those 70 years of age.

Results: Age more than 70 years, the presence of B-symptoms, serum albumin <4g/dL and beta2m more than 4mg/dL were the main adverse predictive variables for survival. The median survival of patients with beta2m 4 and >4 mg/dL was 115 and 79 months respectively (p=0.01). In patients 70 years of age (N=63), beta2m >4mg/dL at the time of initial treatment was the only statistically significant factor in multivariate analysis associated with shorter survival (median 93 vs 143 months, for beta2m >4 vs ≤4 respsctively p=0,005).

Conclusions: We conclude that elevated serum 2m (>4 mg/dl) is an important parameter associated with inferior survival of patients with WM requiring treatment. Our observation that beta2m is the most significant variable associated with survival in younger patients with WM, indicates that this variable may be used for the selection of poor-risk patients candidates for high-dose therapy.
Enhanced Survival of MDS Progenitor Cells under Hypoxic Conditions.
Authors: Thompson James E. , Carroll Martin
Published: April 2020

The myelodysplastic syndromes (MDS) are a diverse group of primary bone marrow disorders characterized by persistent cytopenias and a variable rate of progression to acute myelogenous leukemia (AML). A consistent finding in the bone marrow of patients with MDS is increased apoptosis and a defect in terminal differentiation of maturing cells. We hypothesized that MDS cells are unable to tolerate the increasing oxygen tension experienced during hematopoiesis as cells move out of the hypoxic stem cell niche and this intolerance leads to increased apoptosis. In order to test this hypothesis, we have studied the growth in methylcellulose colonies of bone marrow progenitors in eight samples from patients with MDS at normoxia (21% O2) and at hypoxia (3% O2). In 5/8 samples there is a greater than 8-fold (range 8.5–15.9) increase in colony number under hypoxic conditions. The remaining three MDS samples, while only demonstrating an average increase in colony number of 2.4-fold, demonstrated significantly larger colonies of various lineages. Comparatively, in six normal patient samples the augmentation averaged 1.5-fold and never exceeded 2-fold, with a statistically significant (p=0.01) difference in the mean fold-change between normal and MDS marrows. These results suggest that MDS cells have an impaired ability to tolerate increasing levels of oxygen and also suggests a methodology for expansion of MDS cells for further biochemical analysis of this phenotype. The cellular response to hypoxia is modulated in part through the mitochondrion and significant genetic data suggests that a mitochondrial abnormality is associated with MDS. In order to examine mitochondrial potential in MDS progenitor cells, we have adapted a multiparameter flow cytometric strategy for the simultaneous analysis of mitochondrial potential (ψm) in defined hematopoietic progenitors using the potentiometric dye tetramethyl rhodamine ethyl ester (TMRE). These studies confirm that in normal bone marrow samples, ψm increases with development from the hematopoietic stem cell (HSC) to differentiated progenitor cells. Analysis of marrow from a patient with early MDS (refractory cytopenia with multilineage dysplasia, IPSS Low) and a 10-fold increase in colony number under hypoxic conditions demonstrates the absence of this progressive increase in ψm through myeloid development when compared to normal controls. Additionally, two samples from patients with progression of their MDS to frank AML demonstrated a markedly elevated ψm in the blast cells, consistent with the elimination of the mitochondrial pathology during the genesis of the leukemic clone. Analysis of mitochondrial potential in additional MDS samples is ongoing and will be presented. These data suggest that a defect in the mitochondrial adaptation to increasing oxygen tension experienced during hematopoietic differentiation may explain the impaired growth of MDS progenitor cells.