bluuebunny/crossref_metadata_2025 huggingface dataset:
Exon-12 Nucleophosmin (NPM) Mutation and Aberrant Cytoplasmic Expression of NPM Protein in Leukemia Cell Line OCI-AML3.
Authors: Quentmeier Hilmar , Martelli Maria P. , Dirks Wilhelm G. , Bolli Niccolo , Liso Arcangelo , MacLeod Roderick A.F. , Nicoletti Ildo , Mannucci Roberta , Pucciarini Alessandra , Bigerna Barbara , Martelli Massimo F. , Mecucci Cristina , Drexler Hans G. , Falini Brunangelo
Published: April 2020

Wild-type nucleophosmin (NPM) is a multifunctional protein shuttling between the nucleus and the cytoplasm. Chromosomal rearrangements leading to NPM fusion proteins occur in leukemias and lymphomas (e.g. with partners RARA, ALK). Recently, Falini et al. reported that 60% of acute myeloid leukemia (AML) patients with normal karyotype carry mutations at exon-12 of the NPM gene. This results in frame shifts that lead to alterations of the C-terminus of NPM resulting in the aberrant cytoplasmic localization of the mutated protein (NPMc+) (1). The effects of a mutationally altered protein on cellular functions like proliferation, differentiation or apoptosis, have often been revealed using immortalized cell lines that carry the mutation in question. Therefore, we screened a panel of 79 myeloid leukemia cell lines for presence of mutations - 4 bp insertions - at the exon-12 of the NPM gene. We performed polymerase chain reaction (PCR) analysis with fluorescent dye-labeled primers. For fragment size determination, the PCR products were mixed with dye-labeled size standards and separated by capillary electrophoresis. OCI-AML3 was the only cell line that expressed a signal in addition to and 4 bp larger than the wild-type NPM signal. Sequencing of the cloned NPM-mutated PCR product showed TCTG duplication at positions 956–959 of exon-12. This mutation was heterozygous and corresponded to the type that occurs in 77% of primary NPMc+ AMLs. OCI-AML3 cells have a hyperdiploid karyotype with 48(45–50)<2n>X/XY, +1, +5, +8, der(1)t(1;18)(p11;q11), i(5p),del(13)(q13q21), dup(17)(q21q25); sideline with r(Y)x1-2 and show the following immunoprofile: CD3−, CD4+, CD7−, CD8−, CD10−, CD13+, CD14−, CD15+, CD19−, CD30−, CD33−, CD34−, CD41+, CD42b−, CD68+, CD235a+, HLA-DR-. Especially the presence of myeloid markers and absence of CD34 is typical for NPMc+ cells (1). Furthermore, immunostaining with anti-NPM antibodies confirmed that the OCI-AML3 cells, like primary NPMc+ AML and in contrast to NPM wild-type cells, show cytoplasmic expression of NPM. Functional studies showed that the altered nucleo-cytoplasmic transport of NPM was nuclear export signalling-dependent and could be blocked by using the specific CRM1/exportin-1 inhibitor leptomycin B. In conclusion, cell line OCI-AML3 promises to be an important tool for studying the biological properties and response to new drugs of NPMc+ AML.
IL-7 Differentially Regulates Cell Cycle Entry and Exit of Naive and Memory CD4+ T Lymphocytes: Effects on HIV-Mediated Infection.
Authors: Verhoeyen E.L.S. , Swainson Louise , Cosset Francois-Loic , Taylor Naomi
Published: April 2020

The IL-7 cytokine plays a major role in the in vivo maintenance of polyclonal naive and memory T-cells, positively regulating the survival, differentiation and proliferation of thymocyte and peripheral T lymphocyte populations. Under conditions of lymphopenia due to HIV infection, it has been shown that increased peripheral IL-7 levels correlate with T cell differentiation. Moreover, a short-term exogenous IL-7 treatment enhances T-cell differentiation as well as expansion of naive and memory T-cells. These properties have lead to the proposal that recombinant IL-7 be used as an adjuvant immune therapy in HIV-infected individuals. Nevertheless, primate studies have shown that after an initial increase in in vivo T cell proliferation, there is a substantial drop in the absolute lymphocyte numbers (Fry et al., 2003; Nugeyre et al., 2003). As the bases for this decrease have not yet been elucidated, it is important to assess the long-term biological effects of IL-7 on quiescent human CD4+ T lymphocyte subsets. Here, we demonstrated that although IL-7-stimulated memory T-cells enter into cycle much more rapidly than their naive counterpart (3 days as compared to 6 days), they also exit the cell cycle much earlier, by day 10 vs. day 18. Importantly, cell cycle exit occurred despite the continuous replenishment of IL-7 and was inversely correlated with IL-7 receptor levels. Specifically, IL-7Rα levels were completely downregulated on both naive and memory T subsets within 12 hours post cytokine treatment but were significantly re-upregulated in memory T cell within 8–10 days. In naive lymphocytes, IL7Rα expression was absent during the first 14 days of continuous IL-7 stimulation after which time it slowly increased, with expression coinciding with cell cycle exit. Importantly, the permissivity of IL-7-stimulated CD4+ lymphocytes to infection with an HIV-1 vector was not related to cell cycle entry per se. After extended IL-7 stimulation of the naive T cell subset, they remained more refractory to HIV-1 vector infection than memory cells even though they demonstrated a higher level of cell cycle progression. Moreover, under conditions mimicking the lymph node environment, cell cycle entry was not required for IL-7 mediated infection. The differential effects of recombinant IL-7 on the cell cycle entry-exit status of naive and memory CD4+ T lymphocytes as well as the relative susceptibilities of these CD4+ subsets to HIV-1 vector infection have important implications for the use of this cytokine as an adjuvant immunotherapy.
Adoptive T Cell Therapy Using Antigen-Specific CD8+ T Cells for the Treatment of Patients with Cancer: A Phase I Clinical Study.
Authors: Mackensen Andreas , Meidenbauer Norbert , Vogl Sandra , Laumer Monika , Andreesen Reinhard
Published: April 2020

The adoptive transfer of in vitro induced and expanded tumor antigen-specific cytotoxic T lymphocytes (CTL) provides a promising approach to the immunotherpy of cancer. We have previously shown that antigen-specific CTL can be generated from HLA-A2.1+ cancer patients by 4 rounds of in vitro stimulation of purified CD8+ T cells with autologous dendritic cells pulsed with HLA-A2 binding tumor-associated peptides. Based on these results we have initiated a pilot study of adoptive T cell therapy in advanced melanoma patients demonstrating that in vitro generated Melan-A (ELAGIGILTV)-specific CTL survive intact in vivo for several weeks and localize preferentially to tumor. Here we report on the clinical results of a phase I study of 11 HLA-A2+ melanoma patients that received at least three i.v. infusions of Melan-A-specific CTL i.v. at 2-week intervals. Each T cell infusion was accompanied by a 6-day course of s.c. IL-2 (3x106 IU daily). A total of 51 T-cell infusions were administered, averaging 2.1 x108 Melan-A specific T cells per infusion, with a range from 0.11 – 13.1 x108 T cells per infusion. Clinical side effects were mild and consisted of chills and low-grade fever (WHO grade I–II) in 7 out of 11 patients that typically occurred within 6 to 8 h post infusion. Hematological effects, observed after T cell transfer, included an increase in eosinophils up to 50% in 7 out of 11 patients, peaking 24h post transfer. Clinical and immunological responses consisted of antitumor responses in 3 out of 11 patients (1 CR, 1 PR, 1 mixed response), an elevated frequency of circulating Melan-A multimer+ T cells up to 2% of total CD8+ T cells for 2 weeks post T cell infusion, suggesting long-term survival and/or proliferation of transferred CTL, and a complete loss of Melan-A expression in lymph node metastases of 2 patients after T cell transfer. Our data indicate that the adoptive transfer of antigen-specific T cells in cancer patients is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects.
Immunotherapy with Cytokine Induced Killer Cells in Solid and Hematopoietic Tumors: Preliminary Data.
Authors: Olioso Paola , Giancola Raffaella , Di Riti Maria , Accorsi Patrizia , Spadano Antonio , Natale Dino , Rossetti Roberto , Di Bartolomeo Paolo , Iacone Antonio
Published: April 2020

CIK cells are a unique population of CD3+CD56+ immune effector cells capable of lysing a broad variety of tumor cells targeted through a perforin based mechanism mediated by NKG2D.

We started a pilot clinical trial in patients with refractory lymphoma and solid tumors according to GMP guidelines that is currently ongoing.

Methods: CIK cells were generated from PBMNC and incubated in LifeCell culture bags in the presence of IFN-γ followed by IL-1β, OKT3 and IL-2. Expansion was assessed between day 21 and 28 and flow cytometric analysis was performed every week. Patients were monitored before and after treatment. Unpaired t-test was used to analyze for statistical significance. A p-value <0.05 was considered statistically significant.

Results: At present 10 patients are enrolled: 6 advanced lymphomas, 3 metastatic kidney carcinoma and 1 hepatocellular carcinoma. The median number of transferred cells per patient was 19 x109 (6–37 x109) and the absolute number of CD3+CD56+ cells infused ranged from 1 to 16 x109 (median value 5 x107/Kg). Patients affected by solid tumors received in association low doses of rhIL-2 or α-interferon. Protocol adherence was excellent and the toxicity profile was favourable. Only 2 patients developed low-grade fever during the first cycle of infusion (5%) but promptly resolved without antibiotic treatment. After CIK cell infusion, in patient’s peripheral blood the absolute median count of PBLs, CD3+, CD8+ and CD3+CD56+ cells significantly increased with a p-value of 0.034, 0.025, 0.034 and 0.038, respectively. Clinical outcome appeared promising: 2 patients had complete response (1 metastatic kidney carcinoma and 1 hepatocellular carcinoma) and 2 patients had stabilization of disease (1 metastatic kidney carcinoma and 1 NHL) with a median follow-up of 12 months (range 4–14).

Conclusions: These preliminary data showed that adoptive immunotherapy with CIK cells is a safe therapy with some suggestion of efficacy that significantly enhances immune functions increasing absolute numbers of effector cells without side effects. If confirmed in larger scale studies, these promising results may have a favourable impact on conventional treatment strategy of malignancies.
A Better Strategy To Extend the Proliferative Lifespan of Human T-Cells In Vitro.
Authors: Roeth Alexander , Schneider Lisa , Baerlocher Gabriela , Duehrsen Ulrich
Published: April 2020

Telomerase maintains telomere length by adding TTAGGG repeats to the 3′-ends of chromosomes. In human T-cells telomerase is transiently expressed upon activation and stimulation and, as shown previously, telomerase levels are able to control their lifespan. To understand the effects of culture parameters on telomerase activity and cell lifespan is of critical importance to improve T-cell expansion. Here, we investigated the influence of culture conditions and of stimulation on lifespan, clonogenicity (number of positive wells), cell cycle and telomerase activity in T-cells in vitro. Isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals were cultured in RPMI 1640 medium containing IL-2, different sera (10%) (fetal calf serum (FCS), AB serum and autologous serum) and stimulated with phytohaemagglutinin (PHA) and irradiated allogeneic mononuclear feeder cells or anti-CD3/CD28 beads. T-cells were counted and passaged weekly until senescence. Expanded T-cells were analyzed for cell cycle by DNA staining with propidium iodide (PI) at various time points after stimulation and telomerase activity was measured using the telomerase PCR ELISA. Clonogenicity was determined by limiting dilution using different culture conditions. The proliferative lifespan of T-cells expanded with PHA/feeder cells and autologous serum from two different donors was significantly increased compared to T-cells stimulated with PHA/feeder cells and AB serum (44.7 vs. 14.2 PDs; 42.4 vs. 27.4 PDs). In contrast, no or only little difference was found for T-cells expanded with anti-CD3/CD28 beads and autologous or AB serum (8.7 vs. 8.1 PDs; 15.5 vs. 9.6 PDs). Most likely the latter findings reflect the loss of CD28 expression on T-cells. Furthermore, the use of autologous serum also increased the clonogenicity to about 4-fold compared to the use of AB serum or FCS without any signs for differences in the fractions of cycling cells. Interestingly, T-cells cultured with autologous serum exhibited a higher telomerase activity at day 3 after stimulation and a reduced decline of telomerase activity at day 6 and 9 compared to cultures with AB serum. Our results demonstrate that the use of autologous serum combined with PHA stimulation and feeder cells remarkably extends the proliferative lifespan and clonogenicity of human T-cells in vitro. In addition, T-cells cultured in medium containing autologous serum exhibit higher and prolonged telomerase activity compared to cultures with AB serum. Further studies are ongoing to elucidate the underlying factors involved in those observations. Nevertheless, we suggest that autologous serum together with PHA stimulation and feeder cells is superior to expand human T-cells especially for clinical applications where large numbers of specific T-cells are required for adoptive transfer strategies in cancer patients with for example severe viral infections.
B-Aggressive Lymphoma (BAL) Family Proteins Have Unique Domains Which Modulate Transcription and Exhibit PARP Activity.
Authors: Aguiar Ricardo C.T. , Takeyama Kunihiko , He Chunyan , Kreinbrink Katherine , Shipp Margaret A.
Published: April 2020

BAL1 was originally identified in a genome-wide search for risk-related genes in diffuse large B-cell lymphoma (DLBCL). In pilot and subsequent independent series of primary DLBCLs, BAL1 expression was significantly higher in chemoresistant tumors. BAL1 encodes a nuclear protein with a duplicated N-terminal domain homologous to the non-histone region of histone macroH2A. Several lines of evidence suggest that macro domains may modulate transcription, including: high concentration of histone macroH2A in the inactive X chromosome, direct interference with transcription factor binding in a positioned nucleosome and structural similarity to DNA binding domains. In addition to N-terminal macro domains, the BAL1 protein includes a C-terminal region with homology to the catalytic domain of poly(ADP-ribose)polymerase (PARP) proteins. PARPs catalyze the transfer of ADP-ribose onto acceptor proteins, using NAD+ as a substrate. Poly(ADP-ribosyl)ation is a critical post-translational modification that regulates chromatin configuration, transcription and DNA repair. After characterizing BAL1, we recently identified two additional BAL family members, BAL2 and BAL3, with multiple N-terminal macro domains and putative C-terminal PARP catalytic domains. Given the emerging evidence that PARP family proteins represent possible cancer treatment targets and the reported association between BAL1 expression and chemoresistance in DLBCL, we performed detailed structure/function analyses of BAL family members. We demonstrate that BAL macro domains repress transcription when tethered via a Gal4 DNA binding domain to a TK promoter in classic transcription repression assays. In addition, we show that BAL2 and BAL3, but not BAL1, exhibit PARP activity. In agreement with these data, BAL1 lacks several critical donor and acceptor residues that are conserved in the BAL2 and 3 PARP active sites. Of interest, BAL family members with inactive or functional PARP domains differed in their ability to repress transcription. BAL family members are the only described proteins with both PARP and macro domains, underscoring the potential functional significance of this unique combination.
Active Combination Therapy of Bortezomib (Velcade) and Ibritumomab Tiuxetan (Zevalin) in an In Vivo Diffuse Large B-Cell Lymphoma Model.
Authors: Pervan Milena , Calimlim Jeffrey , Matso Dan , Said Jonathan W. , McBride William H. , de Vos Sven
Published: April 2020

Chemotherapy-resistant diffuse large B-cell lymphomas (DLBCL) pose an unresolved clinical problem and most patients die of their disease within months. The goal of this study was to test a novel, non-chemotherapy containing regimen for Non Hodgkin’s lymphomas, including DLBCL, by combining the proteasome inhibitor bortezomib (PS-341, VELCADE®) with radio-immunotherapy using ibritumomab tiuxetan (Zevalin™). Previously, we have shown that the combination of bortezomib and rituximab is an active and at least additive regimen in an in vitro and in vivo DLBCL model. These data supported an ongoing clinical trial using this combination in patients with low grade NHL. In addition, Pervan et al. identified the proteasome as a novel, sensitive target for ionizing radiation (Mol Cancer Res. 2005 (7):381–90), providing further rationale for this combination study. The combination treatment was evaluated in vivo in a SCID diffuse large B-cell lymphoma model. Bortezomib treatment did not alter the CD20 expression levels of SUDHL-16 cells in vitro. MMT assays showed an additive effect of bortezomib and gamma radiation on SUDHL-16 cell proliferation. In preliminary experiments, we identified the maximal tolerated dose of ibritumomab tiuxetan in our mouse model as 50 μCi/mouse. 2x106 SUDHL-16 cells injected s.q. in the flank resulted in local tumor growth within 4 weeks. When tumors became palpable, animals were stratified in 4 different treatment groups of 12 mice each. Mice received i.v. injections of either diluent, single agent bortezomib, single agent ibritumomab tiuxetan, or the combination of both. Ibritumomab tiuxetan was injected once i.v. at a dose of 20 μCi /mouse. Bortezomib was given i.v. as single dose of 0.01 mg/mouse. The combined treatment group received i.v. bortezomib as a radio-sensitizer 3 hrs prior to the injection of ibritumomab tiuxetan. All treatment schedules delayed tumor growth compared with controls. After 16 days of treatment, bortezomib reduced tumor progression by 52%, ibritumomab tiuxetan by 69%, and the combination treatment by 91%. Selected mice were sacrificed at day two and their tumors studied for potential immediate therapeutic effects. The Y90 content of tumors and normal tissues was measured and demonstrated targeting of Y90 to the xenograft. The tumors of the control and treatment groups did not differ by microscopic morphological appearance. In situ Tunel assays did not reveal increased apoptosis at treatment day two. In summary, bortezomib as a radio-sensitizer in combination with ibritumomab tiuxetan is an active and at least additive regimen in an in vivo DLBCL model.
Inhibition of Protein Kinase C beta by Enzastaurin (LY 317615) Induces Alterations of Key Regulators of Cell Cycle and Apoptosis in Mantle Cell Lymphoma and Synergizes with Chemotherapeutic Agents in a Sequence Dependent Manner.
Authors: Rieken Malte , Weigert Oliver , Pastore Alessandro , Hutter Grit , Zimmermann Yvonne , Weinkauf Marc , Hiddemann Wolfgang , Dreyling Martin
Published: April 2020

Mantle Cell Lymphoma (MCL) is a subtype of malignant lymphoma, characterized by a poor clinical outcome, a median survival time of three years and almost no long-term survivors. In previous studies it could be shown that constitutive overexpression of NFκB pathway plays an important role in the pathogenesis of MCL. The protein kinase C (PKC) family is essential in cell signal regulation effecting cellular growth, proliferation and apoptosis. Previous studies in diffuse large B-cell lymphoma suggest that PKCβ overexpression enhances B-cell proliferation and is associated with poor clinical prognosis. The pivotal role of PKCβ in activation of NFκB renders it a promising therapeutic target in the therapy of MCL. Five MCL cell lines (GRANTA 519, HBL-2, Jeko-1, NCEB-1, Rec-1) and two haematological control cell lines (Jurkat, Karpas 422) were treated with Enzastaurin in a proliferation inhibiting dose of 10μM, defined in initial pilot experiments. Real-time PT-PCR and protein expression (Western blot) levels of various regulators of cell cycle and apoptosis were determined at various time points of enzastaurin treatment (0.5 to 12 hours). In addition analysis of cell cycle and apoptosis were performed by flow-cytometry. All, cell lines were also exposed to different doses of enzastaurin in combination with cytarabine, fludarabine or mitoxantrone and analysed for efficacy by evaluating the combination index. Inhibition of proliferation was achieved in all cell lines in a dose and time-dependent manner, however susceptibility varied strongly within the cell lines characterized by IC 50 (24 hours) ranging from 8,0 μM in Jeko-1 to > 50μM in GRANTA 519, Rec-1 and Karpas 422. Induction of apoptosis was detected in all cell lines according to susceptibility, and cell cycle analysis exhibits a moderate accumulation of and cells in the G2/M phase. Significant alterations of RNA expression was detected for BCL-2, cyclin D1 and p14 applying the Kruskal-Wallis-Test. Preliminary Western blot data confirm the altered levels of protein expressions. Synergistic effects of Enzastaurin combinations were sequence dependent, and resulted in a super-additive effect when the exposure of cells to cytostatic agents was followed by Enzastaurin treatment. No synergy effect was observed in combination with fludarabine. Our study revealed alteration of gene expression, critical for the regulation of cell cycle and apoptosis, after Enzastaurin treatment. Even more important, combination with specific cytostatic drugs demonstrated a synergistic efficacy in vitro. Thus, our data suggest that inhibition of PKCβ can overcome chemotherapy resistance by cellular survival mechanisms supporting the evaluation of such combinations in clinical trials.
Antileukemic Activity of Shepherdin, a Novel Targeted Inhibitor of the Survivin-Hsp90 Complex.
Authors: Gyurkocza Boglarka , Plescia Janet , Toke Madhavi , Greiner Dale , Colombo Giorgio , Lowry Philip A. , Altieri Dario C.
Published: April 2020

Cancer therapy now aims at disabling critical signaling pathways essential for tumor cell maintenance without affecting normal cellular functions. The molecular interaction between heat shock protein 90 (Hsp90) and survivin functions as a crossroad to preserve cell viability and cell proliferation in malignant cells, and offers tangible therapeutic prospects. We recently used structure-based rational design to identify Shepherdin, a cell permeable peptidomimetic from the survivin sequence Lys79-Leu87, which inhibits the complex between survivin and Hsp90 in tumor cells (Kd~80 nM). Here, we tested the activity of Shepherdin against a panel of acute leukemia cell lines (AML). We identified a shorter cell-permeable Shepherdin variant Lys79-Gly83 that docked in the Hsp90 ATP pocket by molecular dynamics simulations, bound recombinant Hsp90 in vitro, and competitively inhibited the survivin-Hsp90 complex. When administered in cell culture, Shepherdin rapidly (<30 min) accumulated intracellularly in all AML cells tested, as well as in quiescent or phytohemagglutinin (PHA)-stimulated normal mononuclear cells. A brief, 30-min exposure of AML cells to Shepherdin was sufficient to induce complete cell killing by apoptotic and non-apoptotic mechanisms, with associated mitochondrial dysfunction and effector caspase activity. Conversely, Shepherdin did not affect colony formation of isolated CD34+ hematopoietic progenitors or viability of resting or proliferating normal mononuclear cells. When compared with known Hsp90 antagonists currently in the clinic, i.e. 17-AAG, Shepherdin was superior for rapidity and efficacy of antileukemic activity. Shepherdin induced collapse of Hsp90 chaperone functions in solid tumor cell types with destabilization of multiple Hsp90 client proteins, but did not affect the levels of pro-survival Hsp70 in AML cells, which is typically upregulated following Hsp90 inhibition by 17-AAG. In conclusion, Shepherdin provides a novel approach for rational therapy of acute leukemia.
The Novel Triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic Acid (CDDO) Induces Apoptosis of Human Diffuse Large B-Cell Lymphoma Cells (DLCL) through a Peroxisome Proliferator-Activated Receptor γ (PPARγ) Independent Pathway Which Is Enhanced by NFκB Inhibition.
Authors: Ray Denise , Morse Kimberly , Hilchey Shannon , Garcia Tatiana , Felgar Raymond , Maggirwar Sanjay , Phipps Richard , Bernstein Steven H.
Published: April 2020

Ligands for the transcription factor PPARγ are emerging as a new class of anti-tumor agents. Herein we report that the synthetic triterpenoid CDDO, a PPARγ ligand that induces PPARγ transcriptional activity in human DLCL OCI-Ly-19 cells, also induces cell death in human DLCL of both germinal center (OCI-Ly19) and activated B-cell phenotype (OCI-Ly10), cells which express the PPARγ protein. This effect of CDDO appears to be independent of PPARγ stimulated pathways since the functional antagonist of PPARγ, GW9662, which completely inhibits CDDO induced PPARγ transcriptional activity was unable to prevent CDDO induced cell death. Similar findings were seen using the additional PPARγ antagonists T0070907 and BADGE. CDDO induces cell death by inhibiting cell proliferation and inducing apoptosis as shown by Annexin-V and propidium iodide staining. As we have previously shown that PPARγ ligands inhibit NF-κB activity in B lymphocytes (J. Immunol2005; 174(7): 4060–9), we next examined the effect of CDDO on NF-κB in DLCL cells. Surprisingly, exposure of Ly19 cells to CDDO resulted in a dose-, and time-dependent increase in the activity of both the p50 and p65 subunits of NF-κB as determined by ELISA, by direct visualization of the nuclear translocation of p65 using indirect immunofluorescence assays, and by EMSA. The nuclear translocation of both the p50 and p65 NF-κB subunits was also confirmed by performing immunoblot analyses using nuclear fractions of CDDO-treated Ly19 cells. NF-κB activation was also observed in Ly10 cells exposed to CDDO. Follow-up experiments revealed that the activation of NF-κB in Ly19 cells by CDDO was due to proteolysis of inhibitory IκBα molecules. To determine whether the CDDO-induced NF-κB activation was a pro- survival mechanism, Ly19 and Ly10 cells were pre-treated with the NF-κB inhibitors SN50, helenalin or BAY 11-7082 and then exposed to CDDO for 24 hrs. In all cases, the NF-κB inhibitors significantly enhanced CDDO induced cell death suggesting that NF-κB activation is an anti-apoptotic mechanism elicited to protect the cell against CDDO cytotoxicity. Collectively, these studies suggest that; (a) CDDO (which will shortly be entering clinical trials for patients with acute myeloid leukemia) as a single agent may have significant clinical activity in patients with DLCL and; (b) the combination of CDDO with pharmacological inhibitors of NF-κB would be a rationale combination of novel agents to test in the context of clinical trials for patients with DLCL.
Updated Results from a Phase II Study of Galiximab (Anti-CD80) in Combination with Rituximab for Relapsed or Refractory, Follicular NHL.
Authors: Friedberg Jonathan W. , Leonard John P. , Younes Anas , Fisher David C. , Gordon Leo I. , Moore Joseph O. , Czuczman Myron S. , Miller Thomas P. , Stiff Patrick J. , Cheson Bruce D. , Forero-Torres Andres , Finucane Deborah M. , Leigh Bryan R. , Molina Arturo
Published: April 2020

Galiximab is a PRIMATIZED®, anti-CD80, monoclonal antibody with human IgG1 constant regions and macaque variable regions. CD80 is an immune costimulatory molecule that is constitutively expressed on the surface of follicular lymphomas. Modest single-agent clinical activity was demonstrated in a Phase I study in relapsed or refractory, follicular NHL (ORR=11%). Preclinical data show that galiximab + rituximab may be more effective than either antibody alone. Here we report updated results from the Phase II part of a multicenter study (Study 114–21) evaluating galiximab + rituximab for relapsed or refractory, follicular NHL. Patients (pts) were administered galiximab (500 mg/m2 qwk x 4) concurrently with a standard course of rituximab (375 mg/m2 qwk x 4). Rituximab-refractory pts (no response or a response with TTP<6 months) were excluded. Study objectives were to evaluate safety, PK, and efficacy. Sixty-four pts received treatment. The median follow up is 14.5 months. Mean age at study entry was 59 yrs. The majority of pts (88%) were Stage III/IV, and FLIPI risk groups were distributed as good (27%), intermediate (39%), or poor (34%). All pts had received at least 1 prior lymphoma therapy; 42% were rituximab naïve. Galiximab infusions were delivered over 1 hr and were well tolerated. No DLTs were reported. Sixty-one (95%) pts experienced study-related AEs; the most common were lymphopenia (44%), leucopenia (38%), fatigue (38%), neutropenia (23%), and chills (23%). IWRC was used to evaluate response. An ORR of 64% was demonstrated: 17% CR, 14% CRu, and 33% PR. The median PFS was 12.1 months. Univariate analysis showed no correlation between response and baseline characteristics, although there was a trend for fewer responses in pts with elevated LDH, poor FLIPI score, and Grade III follicular histology. Cmax and AUC values were dose-proportional, with a mean serum half-life of 13 to 24 days. These results were retrospectively compared with 3 historical Biogen Idec studies of follicular NHL pts treated with a standard course of rituximab monotherapy. Baseline characteristics were similar; however, there was a higher incidence of rituximab-naïve pts in the rituximab monotherapy group (77%) compared with galiximab + rituximab (42%). The toxicity profiles for the 2 groups were similar. The median PFS was longer in the galiximab + rituximab group (12.1 mo.) than in the historical rituximab monotherapy group (9.4 mo.). These results suggest that galiximab can be safely combined with a standard course of rituximab and produce promising response rates and PFS. The toxicity profile and PFS with this combination compared favorably with historical rituximab monotherapy studies. A Phase III, randomized study is planned.
Rituximab-Mediated Antibody-Dependent Cellular Cytotoxicity Is Enhanced by Interleukin-15 or CpG Oligodeoxynucleotides.
Authors: Moga Esther , Briones Javier , Alvarez Eva , Cantó Elisabet , Vidal Silvia , Rodríguez-Sánchez José Luís , Sierra Jordi
Published: April 2020

Introduction

Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism in the clinical activity of rituximab for the treatment of B-cell malignancies. The NK cells, through the activating receptor FcγRIIIa, play a major role in the rituximab-mediated ADCC. We have studied the effect of NK stimulators such IL-15 and CpG oligodeoxynucleotides (ODN) in the enhancement of rituximab-mediated ADCC against B-cell lymphoma.

Methods

ADCC was performed by standard 51Cr release assays using peripheral blood mononuclear cells (PBMC) purified from 4 normal volunteers by Ficoll-Hypaque gradient centrifugation. Cells were cultured for 18 hours in the presence of human recombinant interleukin 15 (rhIL-15; 10 ng/ml) or CpG ODN (2,5 μg/ml). The B-lymphoma cell line Raji was used as target in the presence of rituximab (10 μg/ml) or human IgG1 as control (10 μg/ml) for 30 min at room temperature. Target cells and effector cells were incubated at different ratios ranging from 1:1 to 30:1, for 4 hours at 37°C. The polymorphism at amino acid 158 of the activating FcγRIIIa receptor (FcγRIIIa V→F) was analyzed in effector cells from normal donors with a polymerase chain reaction (PCR)-based allele-specific restriction analysis assay.

Results

Lysis of B lymphoma cells treated with rituximab alone was 40% ± 5% (mean ± SD) at the highest ratio, as compared to 12% ± 7% (p<0.001) in the presence of human IgG1 control. When the effector cells were treated with IL-15, a dramatic increase in the lysis of lymphoma cells was observed (70% ± 8% vs. 40% ± 5%, IL-15 plus rituximab vs. rituximab alone) (p<0.001). Effector cells treated with CpG ODN showed a significant increase in rituximab-mediated cytotoxicity (84% ± 2% vs. 53% ± 16%) (CpG ODN vs. control, respectively; p=0.03). The enhancement of rituximab-mediated ADCC when the effector cells were treated with IL-15 or CpG ODN was similar for both FcγRIIIa-V/V and FcγRIIIa-V/F genotypes on effector cells.

Conclusion

PBMC cells activated in the presence of IL-15 or CpG ODN become strong effectors in rituximab-mediated ADCC against B cell lymphoma. These results may be useful to improve the therapeutic activity of rituximab.
BiovaxID™ Vaccine Therapy of Follicular Lymphoma in First Remission: Long-Term Follow-Up of a Phase II Trial and Status of a Controlled, Randomized Phase III Trial.
Authors: Santos Carlos , Stern Lee , Katz Laura , Watson Thelma , Barry Gause
Published: April 2020

Malignant B-cells in Follicular Non-Hodgkin’s Lymphoma expresses a clonal idiotype immunoglobulin which can serve as the basis for a patient-specific anti-idiotype vaccine. In a previous single-arm Phase II study by Bendandi, et al (Nature Med5:1171–1177, 1999), we evaluated the ability of tumor-specific idiotype (Id) conjugated to keyhole limpet hemocyanin (KLH) administered concurrently with granulocyte-monocyte colony-stimulating factor (GM-CSF) adjuvant to induce complete remissions and molecular remissions in treated patients. The vaccine formulation induced a tumor-specific cytotoxic CD8+ and CD4+ T-cell response in patients in first complete remission after standard chemotherapy, as well as achieved molecular remissions in 8 of 11 of these patients. Data available at the time of this abstract for the 20-patient cohort, indicates a median follow-up of 9.167 years. 9 patients (45 %) remain in continuous first CR at their most recent follow-up (either in 2004 or 2005), and overall survival is 95%. The data further indicates the median disease free survival for the cohort is 96.5 months (8.04 years). To date there have been no additional reported mortalities in this cohort. As of August 2005, we report the progress of the Phase III clinical trial for this vaccine, opened in January 2000 by the NCI to evaluate the impact of this hybridoma-based Id vaccine on disease-free survival in a group of up to 375 previously untreated patients who have attained a CR or CRu from PACE [Prednisone, Doxorubicin, Cyclophosphamide, and Etoposide (ProMACE without methotrexate)] chemotherapy, and who are randomized to receive either vaccine or control. To date, 187 patients have been accrued onto the study. Of those patients, 145 (77.5%) achieved a CR or Cru and are being followed in this ongoing clinical trial.
Rituximab Combination with Anthracyclin Based Chemothrapy Significantly Improved the Outcome of Young Patients with Diffuse Large B-Cell Lymphoma in Low as Well in High Risk Subgroups.
Authors: Trneny Marek , Belada David , Vasova Ingrid , Pytlik Robert , Kozak Tomas , Sykorova Alice , Kubackova Katerina , Pirnos Jan , Bolomska Ingrid , Petrova Marketa
Published: April 2020

Background: It has been demonstrated that rituximab with chemotherapy improves the outcome of older pts with DLBCL (Coiffier 2000, 2002, Habermann 2003) regardless risk category as well as the outcome of young patients with low IPI risk (Pfreundschuh, 2004). There is a lack of data on the impact of rituximab in high risk young patients. We have analyzed the outcome of young patients with DLBCL registered in Czech Lymphoma Study Group registry.

Patients: 422 pts with newly diagnosed DLBCL younger than 60y have been registered between Jan 1999- Aug 2004. Pts with primary CNS lymphoma, pts with missing data, without anthracyclin based therapy were excluded from the analysis. Total 376 pts were analyzed. All patients received anthracyclin based therapy, 120 received rituximab (R-CT) and 256 chemotherapy only (CT). There were no significant differences between both group, R-CT and CT resp. Age median was 47.5y and 49y. Advanced CS was found in 73 pts (60.8%) and in 137 (53.5%), higher LDH was in 72 pts (60%) and 133 pts (51.9%), extranodal involvement was found in 76 pts (63.3%) and in 66 (25.8%) pts. The aaIPI risk distribution was as follows (R-CT vs CT): low risk 31 (25.8%) and 96 (31.5%), low-intermed. 34 (28.3%) and 70 (27.3%), intermed.-high 38 (31.7%) and 55 (21.5%), high 17 (14.2%) and 35 (13.7%). CHOP reg was used in 79 (65.8%) and 186 (72.7%) resp., intensified CHOP or CHOP with other combination was used in 36 (30.0%) and 52 (20.3%), other CT was used in 1 (0.8%) and 18 (7.0%). HDT with ASCT was used in 38 (32.2%) and 55 (22.0%) (ns). The median follow up was 22 m for R-CT and 44 m for CT group.

Results: The estimates for 2y PFS was 85.5 % for R-CT and 66.4% for CT group resp. (p 0.0001). The estimates for 2y OS was 90.7% and 77.6% resp. (0.0007). The differences were significant in low risk (L and LI) as well as high risk (IH and H) group. PFS at 2y was 93.4% and 80.2% resp (p 0.005) and OS was 96.9% and 88.8% resp. (0.02) for low risk group. PFS at 2y was 75.8% and 41.8% resp. (0.0003) and OS was 83.4% and 57.0% resp. (0.0007) for high risk group.

Conclusion: We have demonstrated the significance of rituximab combination with CT in young pts with DLBCL in this comparison. Rituximab has significant impact both in low risk pts (consistent with MINT data) as well as in high risk young pts, which has not been described in any prospective trial yet.

Figure Figure
Activity of Yttrium 90 (90Y) Ibritumomab Tiuxetan (Zevalin®) in 22 Patients with Relapsed and Refractory Mantle Cell Lymphoma (MCL).
Authors: Younes Anas , Pro Barbara , Rodriguez Maria A. , Romaguera Jorge E. , McLaughlin Peter , Wang Michael , Hagemeister Fredrick B. , Dang Nam H. , Neelapu Sattva , Podoloff Donald
Published: April 2020

Background: The management of MCL is a significant therapeutic challenge, especially in patients with relapsed or refractory disease, who are generally refractory to salvage chemotherapy. Although recent studies have shown the clinical utility of radioimmunotherapy (RIT) in relapsed and transformed indolent B-cell lymphoma, the clinical efficacy of this treatment modality in patients with MCL is unknown. We report the results of an ongoing phase II clinical trial of yttrium 90 ibritumomab tiuxetan (Zevalin®) in patients with relapsed and refractory MCL.

Patients and Methods: Patients with relapsed or refractory MCL with measurable disease, age ≥18 years, and performance status <3 were eligible. Patients were required to have adequate function of the bone marrow (ANC ≥1,500/mm3, platelets ≥100,000/mm3), liver, and kidneys. Patients were excluded if they had prior stem cell transplantation, RIT, CNS lymphoma, HIV infection, pleural effusion, HAMA reactivity, or circulating lymphoma cell count >5000/mm3. Patients with pretreatment platelet counts ≥150,000/mm3 received a dose of Zevalin at 0.4 mCi 90Y/kg (maximum dose 32 mCi), whereas those with platelet counts <150,000/mm3 received 0.3 mCi 90Y/kg.

Results: Twenty-two patients were enrolled and all qualified for evaluation of treatment response and toxicity. The median age was 67 years (range 51–77), and 18 patients were male. All patients had an ECOG performance status of 0 or 1 and had been previously treated with rituximab with or without other chemotherapy. The median number of prior regimens was 3 (range 1–6). Fourteen patients were previously treated with Hyper-CVAD alternating with MTX/Ara-C, 21 previously received rituximab, and 5 previously received bortezomib. Zevalin treatment was generally well tolerated, with the most common toxicities being hematologic. Objective responses were observed in 8 of 22 patients (36%), including 3 CR and 2 CRu. Seven of the eight responding patients were previously treated with 3 or less treatment regimens, and all responding patients did not have bulky disease, with the largest lesion measuring 3 cm or less. Median time to progression for CR/CRu patients was 6 months.

Conclusion: The observed responses to Zevalin in heavily pretreated patients with MCL are promising, but the duration of responses has been short. Furtherinvestigation is warranted after first or second relapse, and in conjunction with front-line therapy.
Rituximab-Mediated ADCC Is Augmented by Concomitant Interference with Inhibitory Self-Recognition by Human NK Cells.
Authors: Binyamin Liat , Alpaugh R. Katherine , Campbell Kerry S. , Borghaei Hossein , Weiner Louis M.
Published: April 2020

The anti-CD20 monoclonal antibody, rituximab is widely used in the treatment of non-Hodgkin lymphomas. However, clinical responses to rituximab are variable. It has been demonstrated that rituximab can lead to tumor cell death by engaging the cellular immune system through antibody dependent cellular cytotoxicity (ADCC). NK cells have been shown to play a critical rule in eliminating rituximab coated B-cells, and the efficiency of killing depends on the interaction between the Fc portion of rituximab and the FcγRIII (CD16) activating receptor on NK cells. NK cell function is regulated by a complex balance of inhibitory and activating signals that enable the cells to survey their surrounding and selectively target and kill targets that do not display a “self” ligand (the “missing self hypothesis”). We hypothesized that interference with inhibitory self-recognition would augment rituximab-induced NK cell-mediated ADCC. Initial studies with the 721.221 B51 (HLA Bw4+) CD20+ cell line and NK92.26.5 cells transduced with human CD16 suggested that interference with KIR3DL1 recognition of Bw4 augmented tumor lysis in the presence of rituximab. To further test this hypothesis we employed human NK cells and autologous EBV transformed B cells from normal volunteers, and blocked the KIR3DL1 inhibitory receptor on NK cells using (Fab′)2 fragments of the DX9 antibody, in conjunction with rituximab exposure. Inhibitory blockade promoted rituximab-mediated cytotoxicity by peripheral blood mononuclear cells in three separate HLABw4+, KIR3DL1+ volunteers. These results suggest that manipulating the balance between inhibitory and activating receptors on NK cells might be applied to improve ADCC and ultimately lead to an improvement in response rates to rituximab and related lymphoma-directed antibodies that mediate ADCC. Supported by R01CA50633.
Targeting the Protein Degradation Pathway in Multiple Myeloma with Synergistic, Selective Small Molecules.
Authors: Bradner James , Shen John Paul , Greenberg Edward , Hideshima Teru , Anderson Kenneth C. , Schreiber Stuart L.
Published: April 2020

The response of refractory multiple myeloma to the proteasome inhibitor bortezomib reveals an intriguing sensitivity of this incurable malignancy to perturbations of protein catabolism. However, an overall clinical response rate of approximately 30% as a single agent suggests the importance of chemoresistance mediated by compensatory mechanisms of protein degradation. With proteasome inhibition, juxtanuclear inclusion bodies accumulate. These “aggresomes” are specific cellular structures comprised of chaperones, misfolded proteins, and proteasome components. The cytoplasmic histone deacetylase inhibitor (HDAC6) is essential for aggresome formation. Recently, we have demonstrated robust cytotoxic synergy in multiple myeloma cells between bortezomib and the carboxy-terminal domain-selective inhibitor of HDAC6, tubacin (1). Tubacin is a hydroxamic acid member of a diversity-oriented synthetic chemical library realized and validated previously by members of our laboratory (2). In our study of bortezomib and tubacin in multiple myeloma, we noted the dose-dependent, significant association between cytotoxicity and the marked accumulation of polyubiquitinated proteins in sensitized cells. With an interest in further interruption of the misfolded protein response, we have explored the cytosolic chaperone protein, hsp90, as an additional target using a chemical biologic approach. 17-AAG is an analog of the benzoquinone ansamycin antibiotic geldanamycin, known to bind to the ADP/ATP pocket of hsp90. 17-AAG stabilizes a conformation of the chaperone favoring targeted degradation of its client proteins via recruitment of the hsp70 co-chaperone complex (3). Further supporting this strategy, collaborators have recently identified that HDAC6 binds to and principally mediates the deacetylation of hsp90 (4). Inhibition with non-selective HDAC inhibitors was shown to augment hsp90 acetylation and inhibit ATP binding, resulting in the promotion of protein degradation by polyubiquitination. With the support of these data, we investigated whether the addition of 17-AAG to bortezomib and tubacin results in increased cytotoxicity in multiple myeloma cells. Indeed, we demonstrate potent cytotoxicity in cultured myeloma cells at low concentrations of each small molecule. Focused study of the MM.1S cell line demonstrates that the addition of 17-AAG to tubacin and bortezomib markedly increases the burden of ubiquitinated, cytosolic cellular protein by 24 hours, correlating with enhanced cell killing. These data further credential the protein degradation pathway in multiple myeloma, demonstrate the contribution of targeted, combined approaches with active small molecules, and provide a blueprint for a rational therapeutic strategy.
Redox Regulation of Retinoic Acid Receptor Function in Myeloid Leukemia Cell Differentiation and Apoptosis.
Authors: Robertson Kent A. , Luo Meihua , Chastain Ashley , Colvin Scott , Reed April , Kelley Mark R.
Published: April 2020

Ape1/ref-1 is a multifunctional base excision DNA repair protein that is involved in the repair of abasic sites in DNA. However, it also has a distinct role in the redox regulation of a variety of cellular proteins, such as Fos, Jun, p53, NFkB, PAX, HIF-1a, HLF, and others. Ape-1/ref-1 maintains these proteins in a reduced state thereby facilitating their DNA binding and transcriptional activation capability. HL-60 cells are known to respond to retinoic acid (RA) with terminal granulocytic differentiation and apoptosis, which is mediated through the RA receptors. Previous experiments suggested that Ape1/ref-1 expression is related to apoptosis. To further define this relationship, we used retroviral gene transduction to over-express HA-tagged Ape1/ref-1 in HL-60 myeloid leukemia cells. We observed that the RA-induced growth inhibition of HL-60 cells over-expressing Ape1/ref-1 was significantly enhanced compared to wild type HL-60 cells. To determine if the growth inhibition was related to enhanced programmed cell death and differentiation, we treated Ape1/ref-1 transduced and vector-only (LXSN) transduced HL-60 cells with RA and evaluated the expression of Ape1/ref-1 and the development of apoptosis and markers of differentiation.

Results: 1) RA induced expression of the retroviral Ape1/ref-1 construct as determined by Western blot resulting in a higher (ie retroviral + endogenous Ape1/ref-1) overall expression of Ape1/ref-1 compared to control cells; 2) analysis of RA-treated cells for apoptosis by propidium iodide, TUNEL, and Annexin V staining as well as morphology, unexpectedly demonstrated enhanced programmed cell death in cells expressing the transduced Ape1/ref-1; 3) Ape-1 over-expression enhanced the retinoid differentiation response by morphology and expression of CD11b. Additional mobility shift experiments demonstrated the redox dependence of retinoic acid receptor binding to retinoid response elements mediated by Ape-1/ref-1. In conclusion, our data supports the contention that Ape1/ref-1 expression may be important for mediating RA-induced myeloid differentiation and programmed cell death.