bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Proteins Related to Therapeutic Outcome, Including Drug Resistance, in Acute Myeloid Leukemia Using Mass Spectrometry and Gel Based Proteomic Profiling.
Authors: Stenke Leif , Orre Lukas , Dhar Sumeer , Larsson Rolf , Lewensohn Rolf , Lehtiö Janne
Published: April 2020

To further improve and individualize curative treatment approaches for patients with AML, novel proteins and protein functions need to be discovered. In particular, the mechanisms responsible for chemotherapy resistance appear essential to unravel. Clinical proteomics may help to provide such information. We have used a chip-based technique, surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS), to compare protein expression profiles in AML. With this technique proteins in the molecular weight area of 2–40 kDa can be spotted. For selected samples, a two-dimensional electrophoresis (2DE) method was employed, allowing detection of proteins between 15–120kDa. In the first part of the study, mononuclear peripheral blood cells were obtained from 8 AML patients clinically sensitive to and 7 patients clinically resistant to doxorubicin-containing induction chemotherapy. The clinical outcome correlated to results from parallel in vitro drug testing using the FMCA method. In addition, two variants of the human leukemic cell line CEM, one sensitive and one resistant to teniposide (CEM/VMI), were analysed. In the second part of the study, global proteomic profiles from 24 selected AML patients with good vs. poor clinical outcome (remission duration) were investigated. A total of 812 proteins were detected in the mononuclear cells obtained from the 15 doxorubicin-treated patients. Twenty of these proteins were distinguished as different between the sensitive vs. resistant patient groups. Assessing the CEM cell lines by utilizing four different affinity surfaces on ProteinChip™ arrays, approximately 450 peptides and proteins were detected. Between 29 and up to 65 proteins were detected as differentially expressed between the sensitive and the resistant CEM variants. Using the 2DE technology, approximately 1000 spots were matched on the CEM cell lines, with 47 clear expression level differences between the sub cell lines. In clustering analysis using the protein profile data we could demonstrate clear clustering of sensitive and resistant patient and cell line cells according to sensitivity measurements (IC50). Using these methods we have selected a panel of putative resistance related proteins, which are currently being identified and validated. Data from the second phase of the study, derived from 24 selected AML cell populations, are presently being analyzed. In conclusion, significant differences in protein profile expression were observed between cells obtained from AML patients with clinically divergent treatment responses. Further studies aim at identifying key proteins and protein functions to allow for improved therapeutic efficacy.
The First Fully Experimental In Vivo Model of Human Leukemogenesis: Human Hematopoietic Stem Cells Infected with MLL-ENL Induce Pro-B Acute Lymphoblastic Leukemia in NOD/SCID Mice.
Authors: Barabe Frederic , Kennedy James A. , Dick John E.
Published: April 2020

Identification of genes and translocations involved in human leukemia, as well as classification and clustering by gene arrays, have greatly evolved in the past years. However, the mechanisms of human leukemogenesis remain to be elucidated and the failure to develop an in vivo model where primary human hematopoietic cells are transformed into leukemic cells represents a significant limitation.

Using a retrovirus encoding the oncogene MLL-ENL resulting from the t(11;19)(q23;p13.3) translocation found in acute myeloid leukemias (AML) as well as in acute lymphoblastic leukemias (ALL) of B or T cell origin, we infected lineage-negative cord blood cells and injected those cells into sub-lethally irradiated NOD/SCID mice. 15 to 20 weeks after injection, all the mice developed an aggressive pro-B acute lymphoblastic leukemia characterized by immature B cells (CD10+, CD19+, CD20−, IgD−, IgM−) involving more than 90% of bone marrow. Spleen and thymus were increased in size and infiltrated with >90% leukemic cells. Furthermore, analysis of the lungs and liver showed significant infiltration of these organs. Transplantation of leukemic cells from primary mice to secondary recipients was able to recapitulate the disease with the same phenotype and the same organ involvement in a shorter period of time.

If MLL-ENL transduced cells are grown in suspension culture with IL-3 and SCF, there is massive proliferation of cells blocked in differentiation along the monocytic lineage. In contrast to untransduced cells, colony-forming progenitors were maintained long term in these cultures and could be serially replated, suggestive of an enhanced capacity for self-renewal. After 50 to 70 days in culture, these cells were injected in NOD/SCID mice and mice were analyzed after 12 to 15 weeks. Monoblastic cells were engrafted in the bone marrow and spleen with the same phenotype of the cultured cells (CD33+, CD11b+, CD15+, HLA DR+). These cells were able to engraft secondary and tertiary recipients formally demostrating increased self-renewal capacity of the transformed stem cell. In a limited number of primary mice, transplanted with high cell doses, AML developed at 15 weeks post-transplant.

To our knowledge, these results provide the first in vivo model where human hematopietic stem/progenitor cells are transformed into leukemia. Remarkably, depending on the cellular environment, MLL-ENL can induce ALL or AML in primary cells as a sole genetic event, although we cannot rule out the spontaneous acquistion of additional co-operating genetic or epigenetic abnormalities. This model provides a significant step forward to understand the mechanisms involved in human leukemogenesis.
Estimating Immunoregulatory Gene Networks in Human Herpesvirus Type 6 (HHV-6)-Infected T Cells.
Authors: Takaku Tomoiku , Ohyashiki Junko H. , Zhang Yu , Ohyashiki Kazuma
Published: April 2020

The immune response to viral infection involves complex network of dynamic gene and protein interactions. Comprehensive gene expression analysis of the host immune response against viruses has been extensively studied, however, the mechanism of virus-induced immune response is not completely understood. This might be due in part to the difficulty of finding pathologically relevant genes, despite the fact that DNA microarray technology can simultaneously monitor the expression of thousands of genes. Likewise, it is hard to estimate how each gene interferes during the viral infection. Thus, construction of gene networks from microarray gene expression data is becoming an important challenge in the post-genome era. Human herpesvirus 6 (HHV-6) is a β-herpesvirus that is closely related to human cytomegalovirus. HHV-6 shows a predominant tropism for CD4 T lymphocytes, on which it exerts marked cytopathic effects. Understanding of the clinical spectrum of HHV-6 is still evolving, however, in vitro interactions between HHV-6 and other viruses, such as the human immunodeficiency virus (HIV), and their relevance to the in vivo situation has become increasingly apparent. We present here the dynamic gene network of the host immune response during human herpesvirus type 6 (HHV-6) infection in an adult T cell leukemia (ATL) cell line. Using a pathway-focused oligonucleotide DNA microarray, we found a possible association between chemokine genes regulating Th1/Th2 balance and genes regulating T-cell proliferation during HHV-6B infection. Gene network analysis using an integrated comprehensive workbench, VoyaGene® revealed that a gene encoding a TEC-family kinase, ITK, might be a putative modulator in the host immune response against HHV-6B infection. We conclude that Th2-dominated inflammatory reaction in host cells may play an important role in HHV-6B infected T cells, thereby suggesting the possibility that ITK might be a therapeutic target in diseases related to dysregulation of Th1/Th2 balance. This study describes a novel approach to find genes related with the complex host-virus interaction using microarray data employing the Bayesian statistical framework.

Figure Figure
Robust Expansion of Cytomegalovirus (CMV) Antigen-Specific CD4+ and CD8+ T Cells Using Gene-Modified Activated T Cells as Antigen Presenting Cells.
Authors: Melenhorst J. Joseph , Solomon Scott R. , Mielke Stephan , Hensel Nancy F. , Barrett Austin J.
Published: April 2020

CMV reactivation after stem cell transplantation can be treated with CMV-specific T cells but current in vitro techniques using dendritic cells as antigen-presenting cells are time-consuming and expensive. To simplify the production of clinical grade CMV specific T cells, we evaluated gene-modified activated T cells (T-APC) as a reliable and easily produced source of APC to boost CD4 and CD8 T cell responses against the immunodominant CMV antigen pp65. Peripheral blood mononuclear cells from CMV seropositive donors were activated for 2–3 days in complete medium (IMDM, AB serum, glutamine, and antibiotics) supplemented with 0.8 mg/ml phytohaemagglutinin and 100 IU IL-2/ml. The cells were transduced with Phoenix-A derived recombinant virus encoding pp65 in retronectin-coated 6-well plates, and further expanded in anti-CD3 plus CD28-coated flasks for 3–7 more days. Cultured cells expressed high levels of HLA-DR, and the costimulatory molecules CD80 and CD86. Autologous PBMC (0.5 – 1.0 x 106 cells) were stimulated with 106 irradiated (25 Gy) transduced T-APC in a 24-well plate. After 1–3 days IL-2 and IL-7 were added to a final concentration of 20 IU/ml and 10 ng/ml, respectively. Two weeks later the T cell lines were tested for antigen specificity using the flow cytometric intracellular detection of interferon-gamma following stimulation for 6 hours with a pp65 peptide library of 15-mers, overlapping by 11 amino acids. This technique induced a 135-fold (median; range, 20–120,000) and a 255-fold (median; range, 17–20,000) expansion of pp65-specific CD4 and CD8 responder cells, respectively, in 10/10 seropositive donors (figure). To further improve proliferation, CD25-expressing T regulatory cells were removed from the PBMC at the start of the culture by immunomagnetic depletion (Miltenyi). In 7/10 donors, CD25 depletion resulted in increased CD4 and/or CD8 responder numbers (p>0.05; Mann Whitney paired t-test). Median increase in responder cell numbers was 4.25-fold (range, 1.4–6) for CD4+ T cells, and 4.2-fold (range, 3–7.5) for CD8+ T cells. These data indicate that T-APC efficiently boost pp65-specific CD4 and CD8 T cell numbers to clinically useful levels and that removal of CD25-expressing cells can further augment the total yield of antigen-specific T cells in most donors. The approach has the advantage of using a single leukocyte collection from the donor to generate large numbers of CMV-specific T cells within a total 3 week culture period using only one stimulation of antigen.

Fold-change in the total number of pp-65 specific CD4 and CD8 T cells from PBMC Fold-change in the total number of pp-65 specific CD4 and CD8 T cells from PBMC
The Immunomodulatory Drug Lenalidomide (CC5013; Revlimid), Enhances Antigen-Presenting Cell’s Function Leading to Effective Priming of CD4+ T-Lymphocytes.
Authors: Wang Hongwei , Naing Aung , Cheng Fengdong , Horna Pedro , Suarez Ildelfonso , Brayer Jason , List Alan F. , Sotomayor Eduardo M.
Published: April 2020

Professional antigen-presenting cells (APCs) play an important role in the initiation of antigen-specific T-cell responses. The demonstration that these cells are also required for the induction of T-cell tolerance, placed APCs at the crossroads of immune activation versus immune tolerance. Recent studies have demonstrated that the inflammatory status of the APC at the time of antigen presentation is the central determinant of T-cell priming versus T-cell tolerance. As such, therapeutic induction of inflammatory APCs might override immune tolerance and enhance the efficacy of immunotherapeutic strategies targeting hematologic tumors.

Lenalidomide (CC5013) is a thalidomide analogue with immunomodulatory properties. Phase I and Phase II clinical trials in patients with myelodysplastic syndrome (MDS) have shown high frequency of erythropoietic responses, particularly in patients with 5q31 deletion associated with emergence of polyclonal lymphoid infiltrate in responding patient bone marrows. This observation raised the question as to whether immunological mechanism(s) may mediate, at least in part, the beneficial effect of CC5013 in patients with MDS. To gain further insight into the effects of Lenalidomide on APC’s function and regulation of antigen-specific CD4+ T-cell responses, we treated peritoneal elicited macrophages (PEM) and bone marrow-derived dendritic cells (DCs) with escalating concentration of Lenalidomide in vitro. Enhanced expression of both B7.1 and B7.2 co-stimulatory molecules was observed in Lenalidomide-treated APCs relative to untreated APCs. No difference in the expression of MHC class II molecules or CD40 was detected. Assessment of cytokine production by ELISA showed that Lenalidomide-treated APCs produce higher levels of TNF-a, IL-6 and IL-10 in response to LPS stimulation as compared to untreated APCs. Next, we evaluated the ability of Lenalidomide-treated APCs to present cognate antigen to naïve and tolerant CD4+ T-cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA). We found that treatment of either PEM or DC with low doses of Lenalidomide (range: 1.5–12.5 uM) significantly enhanced their antigen-presenting capabilities leading to effective priming of naïve CD4+ T-cells confirmed by their increased production of IL-2 and IFN-gamma in response to cognate antigen. Taken together, our results shows that by inducing inflammatory APCs, Lenalidomide directs the outcome of antigen-specific T-cell responses. Furthermore, they have broadened the scope of this drug as a promising adjuvant in cancer immunotherapy.
Cell Based Assays Completed with the Mantle Cell Lymphoma Cell Lines Z138 and NCEB-1 Indicate That Combinations of Bortezomid and Flavopiridol Interact To Achieve Synergistic Activity.
Authors: Kapanen Anita , Tucker Catherine , Chikh Ghania , Bally Marcel , Klasa Richard
Published: April 2020

Mantle cell lymphoma (MCL) has the poorest long-term survival of all the lymphoma subtypes. CHOP-like regimens currently represent the standard of care for individuals with MCL, but traditional chemotherapeutic regimens do not elicit disease free survival times that are comparable to other sub classes of B-cell lymphomas. Median survival rate of a MCL patient still is approximately 3 years. Therefore new, targeted therapeutics are needed to improve the clinical outcome of MCL. The aim of the study was to identify and characterize combination effects achieved when flavopiridol, a Cyclin D1 inhibitor, and bortezomib, a proteosome inhibitor, are used in a combination setting together or with other agents contained in the CHOP regimen. Two MCL cell lines, Z138 and NCEB-1, were tested for cytotoxicity of drugs alone and in combinations, where alamar blue was used to assess cell viability. The resulting data were analyzed by using the median effect principle introduced by Chou and Talalay. In addition, mechanisms governing measured cytotoxic effects and combination effects were studied by DNA staining (propidium iodine), Caspase 3/7 activation and by western analysis of cyclinD1, NfkB, Bcl-xl, Bax and Bcl-2. The combination of bortezomib and flavopiridol was found to be synergistic in both cell lines studied. Synergistic interactions were dependent on drug-to-drug ratio. In NCEB-1, a ratio 1:12000 (bortezomib:flavopiridol) determined based on the IC90 of each of the individual agents showed synergy over a broad range of fa values (representing the fraction of affected cells). At ratios based on the IC10 and IC50 of the agents when used alone produced effects that were estimated to be antagonistic by the median effect principle. In the Z138 cell line, drug to drug ratios based on the individual agent IC50 and IC90 resulted in synergistic interactions. Consistent with the objective of identifying synergistic combinations, a clear dose reduction was observed to achieve a given therapeutic goal (e.g. 90% cell kill). 10-fold less bortezomib and 55-fold less flavopiridol would be required to achieve 90% cell death/cytostasis based on addition of the agents alone. Mechanistic studies showed that bortezomib in the combination and alone activated the caspase-dependent apoptotic pathway. The combination of bortezomib and flavopiridol for treatment of MCL would appear to an appropriate choice for clinical evaluation, however novel strategies must be developed in order to insure that combination effects observed in cell based screening assays are capture in patients. In this regard it is important to consider drug-drug ratio effects as well as combination engendered toxicities, both parameters that can be evaluated through careful pharmacodynamic assessments in well defined animal models of MCL.
Heat Shock Protein 90 (HSP90) Is Overexpressed in Primary and Cultured Hodgkin Lymphoma (HL) Cells: Role of Therapeutic Targeting Using the Small Molecule Inhibitor 17-AAG.
Authors: Georgakis Georgios V. , Li Yang , Rassidakis George Z. , Medeiros L. Jeffrey , Younes Anas
Published: April 2020

Conventional chemotherapy is the golden standard for therapy of Hodgkin Lymphoma (HL). Nevertheless, considerable toxicity and secondary malignancies indicate the need for targeted therapy that preferentially kills the malignant cells. The molecular chaperone heat shock protein 90 (HSP90) is expressed in all mammalian cells, but it is overexpressed in malignancy. 17-AAG, a small molecule inhibitor of HSP90, has been shown to induce apoptosis and cell cycle arrest in a variety of tumor types. In the present study we show that HSP90 is overexpressed in the primary Hodgkin and Reed-Sternberg (HRS) cells, as well as in HL derived cells lines. Inhibition of HSP90 17-AAG showed antiproliferative effect in HL derived cell lines in a dose dependent manner. Cell death was due to apoptosis, as determined by Annexin-V staining and FACS analysis. Apoptosis was mediated by the activation of the caspase pathway, especially by caspase 8, 9, and 3. Inhibition of caspase activity by the pancaspase inhibitor Z-VAD-FMK partially reversed the 17-AAG lethal effect. 17-AAG had no significant on the level of the antiapoptotic Bcl-2 family members or the cellular or X-Linked inhibitors of apoptosis. In contrast, there was considerable degradation of cFLIP. Moreover, 17-AAG treatment reduced the intracellular levels of molecules that have been shown to be of key importance in HRS cell survival and proliferation, including AKT and the phosphorylated ERK1/2, but with minimal change in total ERK1/2. Cell cycle arrest was observed at G0/G1 or at G2/M phase, and was mediated by reduction in the levels of MDM2, cyclin D1 with cdk4 and cdk6, and cyclin B1. The potential synergy of 17-AAG with conventional chemotherapy and anti-TRAIL death receptor monoclonal antibody, was explored by the simultaneous incubation of HL derived cells with both doxorubicin or antibodies against TRAIL receptors R1 and R2, respectively. The combination of 17-AAG with doxorubicin or anti-TRAIL antibodies was significantly more effective than either agent alone. Based on these data we are conducting a phase II study of 17-AAG in patients with relapsed classical HL.
Preclinical Anti-Tumor Efficacy of ABT-510 (Thrombospondin-1 Mimetic Peptide), a Novel Anti-Angiogenic Agent, in Hematological Malignancies.
Authors: Cikojevic Anica , Wang Eunice S.
Published: April 2020

Thrombospondin-1 (TSP-1) is a multi-functional 420 kDa matrix protein implicated in cancer cell adhesion, migration, and invasion and is a potent anti-angiogenic agent. TSP-1 expression is inversely correlated with vascularization and malignant progression of breast and lung cancers but has not been extensively investigated in hematological malignancies. We found low total mRNA levels of TSP-1 expression by RT-PCR in half (7 out of 14) of human cell lines representing lymphoma, leukemia, and myeloma. We also found lower total mRNA TSP-1 levels in two thirds (10 out of 15) of primary patient leukemia samples as compared to human umbilical vein endothelial cells (HUVEC). Treatment of human leukemia and lymphoma cell lines with recombinant TSP-1 (100 mg/ml) or ABT-510 (a novel synthetic peptide derivative of TSP-1) decreased tumor cell proliferation as measured by tritiated thymidine incorporation. Single agent ABT-510 treatment (total daily doses 50–120 mg/kg i.p. daily) resulted in statistically significant reduction in tumor volumes of subcutaneous SKI-DLBCL (diffuse large B cell lymphoma) xenografts. ABT-510 treatment (60 mg/kg IP bid) in disseminated SKI-DLBCL lymphoma mouse models resulted in increased long-term survival consistent with delayed disease progression. We then examined the effects of ABT-510 treatment in combination with anthracycline chemotherapy used in standard lymphoma regimens (doxorubicin) or another anti-angiogenic agent (anti-hVEGF antibody Avastin) in subcutaneous lymphoma xenografts. Our results suggest an additive effect with concomitant ABT-510 (60 mg/kg bid) and doxorubicin (3 mg/kg TIW) treatment, but no additive effect with concomitant ABT-510 (60 mg/kg bid) and Avastin (3 mg/kg TIW) treatment. Conclusion: Our data suggest that decreased levels of thrombospondin-1, a matrix protein with endogenous anti-angiogenic activity, play a role in the growth of human hematological malignancies. Based on these results, the TSP-1 synthetic peptide, ABT-510, represents a novel anti-angiogenic therapeutic strategy for patients with these diseases and warrants additional preclinical investigation in combination regimens. (Of note, a phase II trial of single agent ABT-510 in refractory/relapsed lymphoma patients has recently completed enrollment).
Nordic Mantle Cell Lymphoma (MCL) Project: Preemptive Rituximab Treatment of Molecular Relapse Following Autotransplant Can Reinduce Molecular Remission and Prolonged Disease-Free Survival.
Authors: Geisler Christian H. , Elonen Erkki , Kolstad Arne , Laurell Anna , Pedersen Lone B. , Andersen Niels S. , Jerkeman Mats , Nordstrom Marie , Lauritzen Grethe F. , Ralfkiaer Elisabeth , Akerman Maans , Sundstrom Christer , Langholm Ruth , Karjalainen-Lindsberg Marja-Liisa , Kuittinen Outti , Eriksson Mikael ,   Nordic Lymphoma Group
Published: April 2020

The 2nd. Nordic Lymphoma Group mantle cell lymphoma (MCL2) protocol has demonstrated the importance of Ara-C and Rituximab in the induction chemotherapy and stem-cell mobilisation before high-dose therapy and autologous stem-cell transplant (1). By July 2005, 128 patients (83% stage IV) had completed protocol treatment consisting of 3 series of R-CHOP and 3 series of R-Ara-C, stem-cell harvest and high-dose therapy with BEAM/BEAC with ASCT. The 5-year failure-free and overall survival is 50% and 83% respectively, significantly higher than the historic control group of the Nordic MCL1 protocol with the same treatment without HD-Ara-C and Rituximab (P<0.0001). Patients with a molecular marker (t(11;14) or clonal IgH rearrangement) identified at the time of diagnosis in bone marrow and blood, undergo regular molecular follow-up posttransplant,. Patients who turn PCR-positive or increase their qPCR signal, without clinical disease, are offered preemptive treatment with Rituximab 375 mg/m2 Wx4.

Of 75 patients with molecular markers who had completed treatment, 55 remain PCR-negative and 20 have become/remained PCR-pos. posttransplant. Clinical relapse ocurred significantly more often in the latter group (11 of 20) than in the PCR-neg. patients (4 of 55) (P<0.0001) (Fig.1).

Ten of the 20 PCR-positive patient did not receive preemptive rituximab: five due to immediate clinical relapse, 2 due to stable qPCR signals, one due to protocol error and two await treatment. Of 10 patients who did receive preemptive rituximab 8 again became PCR-negative and 2 remain PCR-positive. Six of the 10 Rituximab treated patients remain in clinical and molecular remission 200–600 days after the Rituximab treatment (Fig. 2).

Conclusions:

In MCL, molecular relapse is a harbinger of imminent clinical relapse, whereas continuous molecular remission is associated with prolonged disease-free survival (89% at 4 years)

Rituximab preemptive treatment can reinduce molecular remission and may delay clinical relapse. Following molecular relapse, only Rituximab treated patients (6 of 8 evaluable) remain disease-free.

FIG. 1. NORDIC NCL-2 PROTOCOL: RELAPSE-FREE SURVIVAL ACC. TO MOLECULAR STATUS POSTTRANSPALNT FIG. 1. NORDIC NCL-2 PROTOCOL: RELAPSE-FREE SURVIVAL ACC. TO MOLECULAR STATUS POSTTRANSPALNT FIG. 2. NORDIC NCL-2 PROTOCOL: RELAPSE-FREE SURVIVAL FROM TIME OF MOLECULAR RELAPSE. FIG. 2. NORDIC NCL-2 PROTOCOL: RELAPSE-FREE SURVIVAL FROM TIME OF MOLECULAR RELAPSE.
Is First Line Single Agent Rituximab the Best Treatment for Indolent Non-Hodgkin’s Lymphoma? Update of a Multicentric Study Comparing Rituximab vs CNOP vs Rituximab Plus CNOP.
Authors: Rivas-Vera Silvia , Baez Enrique , Sobrevilla-Calvo Pedro , Baltazar Severiano , Tripp Francisco , Vela Jorge , Garces Oscar , Aguilar Luis , Ignacio Gregorio , Duque Jorge , Rodriguez Petty , Reyes Gustavo
Published: April 2020

Purpose: To evaluate efficacy, safety, Disease-Free Survival (DFS) and Overall Survival (OS) in patients with indolent non-Hodgkin’s lymphoma (NHL) treated with chemotherapy vs. immunotherapy vs immunochemotherapy as first-line therapy, an up-date report.

Methods: Patients with indolent NHL were randomized to receive: (A) Rituximab x 6/w, (B) CNOP (cyclophosphamide, mitoxantrone, vincristine and prednisone) x 6 or (C) R-CNOP x 6, at standard doses.

Results: 195 patients were included, 183 are evaluable for OS and toxicity (A:62, B:55 and C:66), 144 are evaluable for overall response rate (ORR) and DFS (A:53, B:41 and C:50).

Clinical characteristics: 89 male (45.6%), mean age 59±14 (±SD), 148 (75.9%) in stage (III/IV), without significant differences between groups. Overall Response Rate (CR+PR) was: A: 84.9%, B:83.4% and C:90% (P=0.545). Neutropenia grade 3/4 was more frequent in the chemotherapy groups: A: 4.8%, B: 23.6% and C:18.2% (P=0.001) as it was the infectious toxicity (grade 2/4): A:4.8%, B:5.5% and C:15.2% (P=0.07). DFS at 24 months was: A 68%, B:65% and C:70%, (P=0.93) and the OS was A:87%, B:84% and C:78%. P=0.89.

Conclusions: We did not find any important differences, between groups, regarding the Overall Response Rate, Disease Free Survival and Overall Survival at 24 months. However, single agent rituximab was better tolerated, with less toxicity in comparison with the chemotherapy containing groups. Based on these findings, it maybe reasonable to use immunotherapy only, as first-line therapy for patients with indolent NHL.
γδ T Cell-Mediated Immunotherapy of Malignancies with Zoledronate and IL-2.
Authors: Kunzmann Volker , Smetak Manfred , Kimmel Brigitte , Weissinger Florian , Weigang-Koehler Karin , Birkmann Josef , Einsele Hermann , Wilhelm Martin
Published: April 2020

Despite major advances in our understanding of the adaptive immunity towards tumors and the introduction of vaccine-based strategies, durable responses are rare and adaptive immunotherapeutic approaches are still not an established treatment modality. Several lines of evidence indicate that MHC-independent effector cells of the innate immune system such as natural killer (NK) cells or γδ T cells significantly contribute to the immune surveillance of tumors. As we have shown previously, aminobisphosphonates (ABP) such as pamidronate or zoledronate are potent γδ T cell stimulatory compounds by inducing secretion of proinflammatory cytokines (i.e. IFN-γ) and cell-mediated cytotoxicity against lymphoma and myeloma cells in vitro. The detection of ABP as γδ T cell stimulating drugs at pharmacologically achievable concentrations in humans opened the possibility to evaluate the consequences of selective γδ T cell stimulation in vivo. The concept of γδ T cell-mediated immunotherapy is currently validated in a Phase II clinical trial with zoledronate (4mg i.v., d 1) and low dose IL-2 (2 x 106 IU/m2 s.c., d 1–6) for patients with hematological (NHL, myeloma, AML) and non-hematological malignancies (renal cell carcinoma and malignant melanoma). The results of our first clinical pilot study with pamidronate/IL-2 in patients with lymphoid malignancies showed that selective activation and expansion of γδ T cells can be induced in vivo. However, 50% of patients with hematological malignancies failed to respond to pamidronate/IL-2 in vitro. Therefore, positive in vitro sensitivity testing was an essential inclusion criterion in this trial. Immunomonitoring of the first 12 patients included in the study showed that zoledronate/IL-2 is highly effective in activating and expanding γδ T cells in vivo (104 TCRδ2+/HLA-DR+ cells/μl (range 11-323) at day 8 of cycle 1 compared to 3 TCRδ2+/HLA-DR+ cells (range 0-15) before treatment). In addition, IFN-γ serum levels increased from 7 to 110 pg/ml (mean 44, n=6) at day 2 at cycle 1 (day 0: 2, range 0–5). So far, objective tumor responses were observed only in hematological malignancies (updated data will be presented). The application of zoledronate/IL-2 is generally well tolerated. In conclusion, effective γδ T cell activation/expansion can be achieved in vivo by the combination of zoledronate and low dose IL-2. Because of the potent anti-tumor effects of γδ T cells this strategy might be a new attractive immunotherapy approach for malignancies with preserved γδ T cell function.
Mechanisms and Activity of PPARγ-Active Triterpenoids CDDO and CDDO-Me in Leukemias.
Authors: Konopleva Marina , Samudio Ismael , Tsao Twee , Kornblau Steven M. , Shi Yue-Xi , McQueen Teresa , Contractor Rooha , Sporn Michael B. , Meyer Colin J. , Huff Warren , Safe Stephen H. , Kantarjian Hagop , Andreeff Michael
Published: April 2020

First-line therapy of acute myeloid leukemia (AML) consists of combinations of cytarabine and an anthracycline. While initial complete remissions are frequent, most patients succumb to resistant disease underlining the need for novel, more effective agents. The most striking progress in AML therapy was achieved by targeting the nuclear receptor RARα with ATRA. Research in our laboratory has demonstrated that the novel synthetic triterpenoid CDDO (2-cyano-3,12- dioxooleana-1,9-dien-28-oic acid) and its more active C28 methyl ester derivative CDDO-Me inhibit growth and induce apoptosis in a variety of cancers including AML, CLL and blast crisis CML. CDDO and to a much higher degree CDDO-Me are potent activators of the nuclear transcription factor Peroxisome Proliferator-Activated Receptor gamma (PPARγ). In a mammalian two-hybrid assay, the CDDO and CDDO-Me induced activation of PPARγ was associated with a marked increase in multiple coactivator recruitment (SRC-1, SRC-2, SRC-3, TRAP220/DRIP205, CARM-1 and PGC-1) that is qualitatively different from that induced by other PPARγ ligands. CDDO induced a higher degree of myelo-monocytic differentiation in DRIP205-overexpressing HL-60 cells suggesting that high cellular levels of DRIP205 co-activator modulate differentiation response to PPARγ ligation. CDDO induced p21 mRNA and protein in leukemic cells and transactivation of the p21 promoter in a p53-independent fashion. We have recently identified the PPARγ-independent depletion of mitochondrial glutathione (GSHm) as a novel mechanism of action resulting in redox disbalance and mitochondrial damage as mechanisms of pro-apoptotic effects of CDDO and CDDO-Me. Gene expression studies using cDNA arrays demonstrate that CDDO induces genes involved in the antioxidant response (AR) including phase II detoxifying enzymes (glutamate cysteine ligase, GSH transferase, etc.) and antioxidant enzymes (heme oxygenase 1, thioredoxin reductase). Cotreatment with the GSH precursor, n-acetyl cystein prevented apoptosis and loss of viability induced by CDDOs, whereas alkylation of intracellular thiols by diethylmaleate decreased the accumulation of a biotinylated derivative of CDDO, TP-301, in U937 leukemic cells suggesting that intracellular reduced thiols are functional targets of CDDO and its derivatives. The in vivo studies using liposomal CDDO-Me in a conditional leukemia model demonstrated significant reduction of leukemia burden as measured by bioluminescence imaging and prolongation of survival. Based on the ample pre-clinical evidence of anti-leukemia effects and on the favorable PK/toxicity profile of the parental compound, CDDO will enter Phase I clinical trials in hematologic malignancies in 3Q 2005 and CDDO-Me in 1Q 2006.
The HIV-Protease Inhibitor Ritonavir(Norvir®) Is Active Against Human AML Blasts In Vitro and In Vivo and Has Synergistic Cytotoxic Activity with the Proteaseome Inhibitor Bortezomib (Velcade®).
Authors: Driessen Christoph , Hendrik Müller-Ide , Jeanette Gogel , Marianne Kraus , Lothar Kanz , Thomas Rückrich
Published: April 2020

Upregulation of alternative proteolytic pathways characterizes malignant cells that overcome proteasome inhibition in vitro. The proteasome inhibitor Bortezomib (Velcade®), which selectively targets only one of the three active subunits of the proteasome, has shown limited activity in AML. Ritonavir (Norvir®) is an aspartate protease inhibitor used in intensive HIV-therapy, where therapeutic levels of 5–20μM are reached with an oral dose of 1200 mg/d. A cytotoxic effect of Ritonavir against malignant cells due to proteasome inhibition has been suggested (Gaedicke et al., Cancer Research 62, December 1, 2002). We have here tested the effect of Ritonavir on AML cells, both as single agent and in combination with Bortezomib. Ritonavir induced cytotoxic death in AML cell lines and primary AML blasts with an IC50 of 30–40 μM in vitro. The combination of Ritonavir and Bortezomib was synergistic in vitro, i.e. subtoxic concentrations of Ritonavir at 10 μM combined with subtoxic Bortezomib 5–10 nM induced robust cytotoxicity in AML cell lines and freshly isolated primary AML blasts. Using a novel chemical probe that for the first time allows to visualize the individual activity of proteasomal subunits in intact AML blasts, we show that Velcade selectively abrogates β5 proteasomal activity at 20 nM in AML cells, as expected. Ritonavir, by contrast, had no effect on active proteasomal subunits up to 50 μM. Thus, the synergistic effect of Ritonavir with Bortezomib on AML cells is not due to inhibition of the same proteasomal target by both drugs, but more likely mediated by blocking alternative proteolytic pathways. One individual patient aged 72 years with an early relaps of AML was treated with Ritonavir 400–600 mg/d p.o.. During treatment, the absolute leukocyte count dropped from 24000/μl to 8000/μl while the ANC raised from 185/μl to 1530/μl. Ritonavir was withdrawn due to diarrhoea and abdominal cramps, leading to a sharp increase in peripheral blood blasts and leukocytes. Retreatment with Ritonavir at a reduced dose of 200 mg/d combined with Velcade 1mg/sqm was tolerated and stabilized leukocyte counts for a short period of time. We conclude that Ritonavir has activity against chemotherapy-refractory AML in vitro and in vivo. The combination of Velcade and Ritonavir might allow to synergistically target the proteolytic machinery of AML blasts with tolerable toxicity.
NF-κB Blockade Induces Apoptosis and Decreases Costimulatory Molecules and IL-12 Secretion of Dendritic Cells, Altering DC Maturation: Role in Induction of Tolerance after Transplantation.
Authors: Abarca Luis I. Sanchez , Perez-Simon Jose A. , Blanco Belen , Gutierrez Norma , Mateos Juan , Vidriales Belen , Lopez-Holgado Natalia , Alberca Mercedes , Villaron Eva , Schenkein D. , Canizo Consuelo , San Miguel Jesus F.
Published: April 2020

In steady state, dendritic cells (DC) are in an immature status and maintain tolerance to self antigens. By contrast, if DC maturation is stimulated, as it occurs in the proinflamatory milieu induced by allogeneic transplantation, they trigger effector and memory T cells and, for this reason, they play an essential role in the development of graft versus host disease (GVHD). DC maturation and survival is dependent on NF-kB. Thus, we explored the ability of the proteosome inhibitor bortezomib (B), which prevents Nuclear Factor kB (NF-kB) activation, to induce DC apoptosis and block maturation as a new strategy to prevent GVHD. DC were obtained from PBMN after culture with GM-CSF and IL-4 and were stimulated to maturate and express costimulatory molecules (CD80, CD83, CD86, CD40L) adding TNF and LPS. Even after this stimulation, B at doses ranging from 10 to 50 nM was able to decrease costimulatory molecules as well as HLA-DR expression, as assessed by flow cytometry. In addition, at 50 nM, B increased DC apoptosis (2650 vs 1404 anex/7AAD positive events among DC cultured with or w/o B, respectively; p=0.05). Moreover, B significantly decreased the production of IL-12, while IL10 secretion was unaffected (1,4% vs 6,2% DC cultured with or w/o B secreting IL-12, respectively; p=0.01). Cytokines and proinflamatory molecules genes expression profiling was also analysed by microarrays in DC cultured with or without B. Finally, after culture with bortezomib, DC were cocultured with T lymphocytes and, interestingly, allogeneic mixed lymphocyte reaction was markedly inhibited also inducing a decrease in Th1 cytokine secretion among T cells.

In conclusion, bortezomib induces apoptosis and decreases DC maturation and cytokine secretion and favours tolerance to alloantigens. This new strategy based on DC therapy supports the use of proteosome inhibitors in the management of GVHD.
Intra-Pathway Inhibition of Upstream (PI3K) and Downstream (mTOR) Kinases Synergistically Induces Apoptosis in AML.
Authors: Zeng Zhihong , Estrov Zeev , Harris David , Giles Frank , Andreeff Michael , Konopleva Marina
Published: April 2020

Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway regulates the growth and survival of acute myeloid leukemia (AML). We hypothesized that targeting this pathway with both PI3K and mTOR inhibitors may greatly enhance the effectiveness of these two inhibitors in the treatment of AML. PI3KI1 is a novel PI3K inhibitor that induced apoptosis in AML cell lines and primary AML cells at an IC50 of 5μM. It directly inhibited AKT at Ser473, however had limited effects on pGSK3b and on the mTOR downstream target p70S6K at Thr389. Colony-forming assays demonstrated that PI3KI1 decreased the viability of primary AML samples but spared normal bone marrow progenitor cells. mTOR inhibitor CCI779 inhibited phosphorylation of downstream mTOR targets p70S6K and 4EBP, however showed only minor cytotoxicity to AML cell lines and primary samples, suggesting that inhibition of mTOR signaling is not sufficient to cause growth inhibition in the majority of AML. Combined use of PI3KI1 and CCI779 synergistically induced apoptosis in U937 cells, with a combination index of 0.061±0.02. Western blot analysis demonstrated enhanced suppression of pP70S6K, pAKT and p4EBP1(Thr70) when PI3KI1 and CCI779 were used in combination. In primary AML samples, combined inhibition of PI3K and mTOR pathways enhanced apoptosis induction in 8/12 samples, with true synergistic responses in 3 samples. Importantly, the combination, but not PI3KI1 or CCI779 alone, was able to overcome the growth advantage conferred to AML cell lines or primary AML samples by adherence to bone marrow stromal cells. Taken together, our results indicate that PI3K and mTOR are relevant molecular targets in AML and that intra-pathway inhibition of both, upstream and downstream proteins may be required for maximal inhibition of leukemia cell growth.
A Phase 1 Trial of STA-5312, a Microtubule Inhibitor with a Novel Binding Site, in Hematologic and Solid Malignancies.
Authors: Burris Howard , Abramson Neil , Martel Cynthia L. , Schwartzberg Lee V. , Dooley William , McLeod Matthew W. , Dahl Thomas , Sherman Matthew , Limentani Steven
Published: April 2020

BACKGROUND: STA-5312, an inhibitor of tubulin polymerization, with a binding site distinct from other agents such as vincristine, colchicine, or paclitaxel, is not a P-gp substrate. STA-5312 shows in vitro and in vivo activity against leukemia and a range of solid tumors and prolonged survival in models of chemotherapy-resistant cancer.

METHODS: A phase 1 trial was initiated in patients with advanced hematologic cancers or solid tumors to determine the safety, toxicity, pharmacokinetics (PK) and maximum tolerated dose (MTD) of STA-5312. Assessment of activity is a secondary objective. STA-5312 is administered iv on days 1, 3 and 5 of a two week cycle. Dose escalation is based on evaluation of toxicity through the first 3 weeks on the study. In the absence of grade (gr) 2 or greater toxicity, 100% escalations are made. If gr 2 or greater toxicity occurs, all future cohorts are expanded to 3 patients, and further escalations limited to 50%. In the event of any dose limiting toxicity (DLT), the cohort is expanded to 6 patients and escalations limited to 33%. The dose below the lowest dose at which 1/3 of 6 or more patients show DLT will be declared the MTD and expanded to at least 12 patients. PK samples were obtained throughout day 1, and trough and peak samples were taken on day 5. Response is assessed by NCI Working Group Response Criteria, International Workshop Response Criteria Recommendations, or RECIST as appropriate.

RESULTS: After an initial cohort of 3 patients at the dose level of 3 mg/m2 the dose was successively doubled up to 48 mg/m2 with one patient cohorts. At escalation from 48 mg/m2, only a 50% dose increase was made, as exposures were approaching those where significant toxicity was seen in animals. A DLT (hospitalization with dehydration and thrush, liver enzyme elevations and prolonged neutropenia) was reported for the first patient at 72 mg/m2. Two additional patients were added at a dose of 48 mg/m2 and to date no gr 2 toxicities have been reported during the 21 day evaluation. A cohort has been opened at an intermediate dose (60 mg/m2) and enrollment is ongoing to expand it after DLT (dyspnea) was observed in 1 of 3 patients. Other adverse events attributable to STA-5312 include: fatigue, weakness, decreased sensation and hematuria. STA-5312 exhibited relatively linear PK with regard to AUC. Three patients (2 NHL and 1 multiple myeloma) have experienced durable stable disease, with two patients treated for over 8 months.

CONCLUSIONS: STA-5312 was well tolerated through the first five dose levels, with the few adverse events consistent with microtubule disrupting agents. The patients with durable stable disease are encouraging and planning for Phase 2 clinical trials is underway.
Combination of Proteasome Inhibitor PS 341 (Velcade®) with Histone Acetylase Inhibitor (HDAC) PXD 101 Shows Superior Anti-Myeloma Activity and Inhibits Osteoclastogenesis.
Authors: Lentzsch Suzanne , Anderson Gulsum , Li Cuiling , Belani Chandra P. , Mapara Markus Y. , Roodman David
Published: April 2020

Background: PS 341 has demonstrated activity in multiple myeloma (MM), and PXD 101 is a new HDAC inhibitor, currently being evaluated in Phase II clinical trials for MM. In addition to the anti-myeloma activity of PS 341 and PXD 101, HDAC inhibitors are strong inhibitors of osteoclast formation. Therefore, the combination of the two agents, targeting both the tumor and the development of osteolytic lesions should lead to an optimal treatment strategy for MM.

Methods: MM cell lines (MM.1S, OPM 2, RPMI 8226) were exposed to serial dilutions of PS 341 (1–1000 nM) and PXD 101 (10–1000 nM) for 48 hours. Thymidine uptake was measured to assess inhibition of proliferation. To evaluate the effects of PS 341 and PXD 101 on the formation of osteoclasts from mononuclear hematopoietic precursors, we used a human bone marrow culture system from healthy donors and MM patients to generate osteoclasts using in vitro RANKL/M-CSF stimulation.

Results: Dose-dependent inhibition of proliferation was achieved in all MM cell lines after a 48-hour treatment with PS 341 and PXD 101. For further experiments we used PS 341 at a fixed low concentration (10 nM), which showed no significant inhibition of proliferation of MM cell lines. PXD 101 was used at 100 nM and demonstrated median inhibition of 46% (range 0–48%). The combination of PS 341 with PXD 101 induced synergism with inhibition of proliferation of median 73% (range 44–73%). Although 100 nM PXD 101 and 50 nM PS 341 used as single agents were required to inhibit the proliferation of MM cells, combination of 25 nM PXD 101 and 1 nM PS 341 was sufficient to inhibit OCL formation completely, indicating that the drug combination results also in a highly synergistic inhibition of osteoclast formation.

Conclusions: These studies provide the framework for clinical evaluation of PS 341 combined with PXD 101 in patients with MM and supports the hypothesis that the drug combination targets both the tumor and osteoclastic activity. (Drugs provided by NIH/NCI; Support: UO1 CA099168)
Factor V Level Affects the Host Susceptibility to Group A Streptococcal Infection.
Authors: Sun Hongmin , Yang Angela , Wang Xixi , Ginsburg David
Published: April 2020

Group A streptococci (GAS), a common human pathogen, secrete streptokinase (SK), which activates the host’s plasminogen (PLG). SK is highly specific for human PLG, exhibiting little or no activity against other mammalian species. We demonstrated the major role of the PLG/SK interaction in GAS pathogenicity using a transgenic murine model expressing human plasminogen with increased susceptibility to human pathogenic streptococci. We hypothesize that GAS hijack the host fibrinolytic system in order to circumvent local thrombosis for systemic spread. Markedly increased mortality was also observed following GAS injection in C57BL/6J mice treated with the snake venom Ancrod, which proteolytically degrades plasma fibrinogen, supporting the critical roles of coagulation in host/pathogen interaction.

However, fibrinogen also plays important roles in inflammation and immune response, it is necessary to use independent genetic models to further test the impact of coagulation in host defense against bacterial infection. The effect of variations in FV on mouse susceptibility to streptococcal infection was tested. We established a mouse model with low plasma FV level. These mice have a slightly increased bleeding time, though otherwise phenotypically normal. Subjected to streptococcal infection, these mice exhibited significantly increased mortality than wildtype controls, suggesting that the decreased thrombotic tendency in the low FV mice increases host susceptibility to infection. FV Leiden is a common prothrombotic mutation among Caucasian population with an incidence between 4% and 6%. Previous studies from Kerlin et al demonstrated the FV Leiden conferred survival advantage in patients with severe sepsis and in mice challenged with endotoxin. This may be an example of balanced gene polymorphism that maintains the FV Leiden mutant in the general gene pool by selection of bacterial infections. In order to identify the selective agents responsible for the prevalence of FV Leiden mutation, we took advantage of our plasminogen transgenic murine model for streptococcal infection to test whether streptococci is one of the selective agents. Human plasminogen transgene was introduced into FV Leiden background and the susceptibility to streptococcal infection was measured. No significant improvement of survival was observed in the FV Leiden mouse comparing with the wildtype control. Thus, streptococcal infection is not the selective agent for the prevalence of FV Leiden mutation.

These observations highlight the potential role of variations in blood coagulation factors in host susceptibility to bacterial infection.