bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of Anti-Cancer Effects by Targeting Alloreactive Intentionally Mismatched T Cells and NK Cells to Tumor Cells Using Bi-Specific Trifunctional Antibodies.
Authors: Morecki Shoshana , Lindhofer Horst , Yacovlev Elena , Gelfand Yael , Slavin Shimon
Published: October 2019

Engraftment of donor stem cells induces host vs graft unresponsiveness thus allowing durable engraftment of donor lymphocytes which can induce graft vs malignancy (GVM) effects following transplantation of MHC compatible donor stem cells. Unfortunately, graft vs host disease (GVHD) frequently results in major toxicity and mortality and limits the clinical efficacy of cell therapy. Although the intensity of GVM effects can be improved by using mismatched donors GVM effects cannot be induced in recipients of haploidentically mismatched allografts because of anticipated lethal GVHD. We have attempted to induce GVM effects by intentionally mismatched donor lymphocytes by targeting donor derived T cells and NK cells activated by rIL-2 to cancer cells, in order to improve the efficacy of the GVL effects on the one hand and prevent or minimize GVHD by targeting alloreactive cells to the tumor cells that need to be eliminated using bispecific tri-functional antibodies, on the other. A trifunctional bispecific antibody (BiLu) directed against murine CD3 and human epithelial-cell adhesion molecule (EpCAM), was administered concomitantly with naïve or IL-2 activated donor splenocytes to mice inoculated with a murine model of melanoma cells transfected with human EpCAM (B16-EpCAM). A total of 10/20 and 32/38 mice treated with BiLu and naïve or IL-2 activated donor splenocytes, respectively, were tumor-free survivors without GVHD for >250 days following tumor inoculation. Out of 28 of the disease free survivors (DFS) without GVHD, treated with IL-2 activated splenocytes and BiLu, 24 mice were resistant to a second tumor inoculum (104) given >250 days following the first tumor inoculation. In contrast, only 8 of the 18 DFS mice treated with naïve splenocytes and BiLu, and 5 of the 10 DFS controls treated with BiLu only, resisted the second tumor challenge. Induction of anti-tumor immunity by IL-2 activated cells and BiLu was more efficient and long-lasting in mice previously injected with a lethal first tumor cell dose (5×104) than in mice inoculated with a sub-lethal tumor cell dose (5×103). Interestingly, a similar infusion of donor cells labeled with bi-specific anti-EpCAM and anti-CD3 following allogeneic stem cell transplantation for metastatic breast cancer did not result in augmentation of GVHD. In conclusion, concomitant inoculation of alloreactive donor lymphocytes and trifunctional bispecific antibody can be effective for targeting of killer T and NK cells to tumor cells while avoiding GVHD, as well as for induction of long-lasting anti-cancer immunity, most likely due to presentation of tumor antigen to T cells by antigen presenting cells (dendritic cells) expressing Fc receptor binding to the Fc portion of the bi-specific antibody.
Six-Year Follow-Up of Inhibitor Prone Hemophilia B Dogs Treated with Muscle and Liver-Directed AAV2 Mediated Factor IX Gene Therapy.
Authors: Niemeyer Glenn P. , Herzog Roland W. , Mount Jane D. , Arruda Valder , Tillson Michael , Hathcock John , High Katherine A. , Lothrop Clinton D.
Published: October 2019

Hemophilia B, caused by factor IX (FIX) deficiency, is intensively studied as a model for gene therapy. Preclinical studies in mice, dogs and initial clinical trials have documented the feasibility of adeno-associated virus (AAV) mediated gene therapy for hemophilia B. The purpose of this study is to report 6 year follow-up of inhibitor prone hemophilia B dogs treated with muscle or liver directed AAV2 FIX gene therapy. Short term, one year follow-up of these dogs (n=6) has been previously reported. The dogs treated with liver directed AAV2 FIX have not had a single bleed requiring FIX replacement in the 6 years since vector administration. The dogs undergoing muscle directed gene therapy have continued to have a bleed frequency similar to untreated FIX deficient dogs. The whole blood clotting time (WBCT), activated clotting time (ACT), and activated partial thromboplastin time (APTT) have remained at the upper limits of the normal ranges for the six year follow-up in the two liver treated dogs. The FIX activity has remained stable between 4–10% in both liver treated dogs but undetectable in the dogs undergoing muscle directed therapy. The vector/FIX sequences have persisted in liver biopsies but were undetectable in WBC and sperm DNA. Administration of purified canine FIX protein resulted in an immune response to FIX in the muscle treated dogs but not the dogs treated with liver directed gene therapy, suggesting FIX tolerance had been established in the hemophilia B dogs with liver directed gene therapy. A complete clinical evaluation of the dogs undergoing liver directed gene therapy including CBC, serum chemistries, bile acid profile, hepatic MRI and CT scans and liver biopsy was normal with no evidence for tumor formation. These results demonstrate that AAV mediated liver directed gene therapy completely corrects the hemophilia phenotype without toxicity or inhibitor development in the inhibitor prone null mutation dogs for more than six years.
Hematologic Outcomes and Erythropoiesis-Stimulating Therapy Costs in Epoetin Alfa (EPO)- and Darbepoetin Alfa (DARB)-Treated Cancer Patients: Results of the Dosing and Outcomes Study of Erythropoiesis-Stimulating Therapies (D.O.S.E. Registry).
Authors: Chen Er , Peake Cyrus , Buscaino Erminia , Forlenza Jamie , Bookhart Brahim , McKenzie Scott
Published: October 2019

Background: National Comprehensive Cancer Network (NCCN) anemia treatment guidelines recommend maintenance of hemoglobin (Hb) levels between 11–12 g/dL. To investigate hematologic outcomes and costs of erythropoiesis-stimulating therapies (ESTs), data were analyzed from the D.O.S.E. Registry, an ongoing, prospective registry collecting data on real-world practice patterns and outcomes in cancer patients treated with ESTs.

Methods: Data come from U.S. hospital and community-based outpatient practices assessed from 1/04–6/06. Adult patients with a non-myeloid malignancy and receipt of at least 2 doses of either EPO or DARB were included. Outcomes assessed included mean treatment duration; mean cumulative dose; maintenance of Hb between 11–12g/dL; mean Hb level at Weeks 4, 8, 12, and 16; and proportion of patients receiving blood transfusions. EST costs were based on May 2006 wholesale acquisition costs.

Results: 861 patients (312 EPO, 549 DARB) from 45 sites were identified. Mean baseline characteristics were similar between the groups (entire cohort: age 62.4 years, 64.1% women, weight 75.9 kg, and Hb 10.4 g/dL) with the exception of a significantly higher proportion of patients receiving iron supplementation in the DARB-treated group (EPO 18%, DARB 29%, p<0.01). The most common malignancies were breast, lung and gastrointestinal. Both groups had similar mean treatment duration (approximately 8 weeks), number of Hb assessments (approximately 8) and proportion of patients requiring blood transfusion following the initial four weeks of treatment (EPO 9%, DARB 11%, p=0.32). The mean cumulative doses for EPO (373,827 Units) and DARB (1,185 mcg) were associated with EST drug costs of $4,550 for EPO and $5,267 for DARB, (p<0.001). Mean Hb level was ≥ 11 g/dL at all post-baseline time points in the EPO-treated group, however <11 g/dL in the DARB-treated group at Weeks 12 and 16. Mean Hb level was significantly higher in the EPO-treated group at Week 12 (EPO 11.3 g/dL, DARB 10.8 g/dL, p=0.03).

Conclusions: In this prospective observational study, EPO-treated patients achieved and maintained NCCN target Hb levels at all timepoints. These findings also showed EST cost to be 16% higher in the DARB-treated group than in the EPO-treated group.
Hematology-Related Direct-to-Consumer Advertising: A Content Analysis and Verification of Claims from Print Media.
Authors: Abel Gregory A. , Sorel Marie-Adele , Neufeld Ellis J. , Weeks Jane C.
Published: October 2019

Background: Patient-directed marketing of hematology medications is an increasingly prevalent and controversial practice. Content analysis of hematology-related direct-to-consumer advertising (DTCA), with attention to how benefit and risk/side effect information is presented as well as assessment of the appeals and claims made, is essential to understanding its potential effect on patient outcomes.

Methods: We reviewed all DTCA for hematological medications appearing in 2005 in the largest circulation patient-focused bleeding-disorder and cancer magazines (Hemaware and CURE), as well as a sample of popular magazines. We assessed the Flesch reading ease score (FRES) for the benefit and risk/side effect text presented in each advertisement. Ranging from 0 to 100, a FRES of 65 or higher is considered understandable for the average person. We also assessed the proportion of text dedicated to, and the largest type size of, benefit and risk/side effect information, as well as the nature and content of the advertisements’ appeals. We then attempted to link the advertisements’ main efficacy or safety appeal to the hematological literature based upon references available from a Medline search for the claim and the drug name.

Results: Of 134 total ads reviewed, 20 unique advertisements were analyzed (the remainder were repeats). Overall, the ratio of number of words devoted to benefit text versus risk/side effect text was 1.33. The mean FRES for benefit information was 39.0, while the mean FRES for risks/side effects was 23.7, a difference of 15.3 [95% CI: 6.4, 24.2]. The mean largest benefit type size was 6.1 mm, while the mean largest risk/side effect type size was 2.3 mm, a difference of 3.8 mm [95% CI: 1.7, 5.6]. 65% of ads made appeals to social-psychological enhancements (eg: regaining a normal life). 88% of the ads made appeals to efficacy, and 53% to safety; we were able to verify all of these except one through Medline.

Conclusion: Hematology print DTCA is difficult to read. When compared to risks/side effects, benefits often comprise more text, appear in larger typeface, and are presented in language that is somewhat more readable. Appeals to efficacy and safety are common, and, for the most part, easy to verify with Medline. Although the ultimate effects of hematology DTCA for patients and providers remain to be elucidated, awareness of its characteristics is a vital first step toward better informing its regulation.
Economic Burden of Hematologic Adverse Events in Cancer Patients Undergoing Chemotherapy: A Systematic Review.
Authors: Stephens Jennifer M. , Liou Sarah Y. , Tran Kimbach T. , Botteman Marc F.
Published: October 2019

OBJECTIVES: Hematologic adverse events such as neutropenia, thrombocytopenia, and anemia are commonly experienced by cancer patients receiving chemotherapy. These cytopenias are associated with increased morbidity and mortality, high healthcare costs, and decreases in quality of life. The objective of this study was to review the economic burden of these hematologic adverse events associated with chemotherapy in cancer patients.

METHODS: A systematic search of the English-language medical literature published between 1990 and 2006 was conducted. Online conference proceedings and a review of article bibliographies were included in the review. Articles selected included prospective or retrospective studies specifically designed to examine burden of illness, direct medical costs, indirect costs, or cost drivers associated with neutropenia, thrombocytopenia, and anemia in adult cancer patients treated with chemotherapy. All original costs were reported, with adjusted figures (to 2006 US dollars) presented in parentheses using the medical care component of the consumer price index from the US Bureau of Labor Statistics.

RESULTS: Of 160 studies initially identified, 64 met selection criteria and were reviewed in detail. The cost of neutropenia ranged from $1,893 (2006 US $2,632) per episode in the outpatient setting to $38,583 ($54,807) for a febrile neutropenia hospitalization. The cost of treating thrombocytopenia ranged from $1,037 ($1,395) to $7,550 ($9,336) per cycle or episode. Costs attributable to treating anemia ranged from $18,418 ($22,775) to $69,478 ($93,454) per year. Key cost drivers include hospitalization, drugs (e.g., granulocyte colony-stimulating factors and antibiotics), and diagnostic tests for neutropenia; hospitalization, major bleeding episodes, and platelet transfusions for thrombocytopenia; and inpatient and outpatient services, erythropoietic agents, and red blood cell transfusions for anemia. Another finding was that the costs of hematologic adverse events for patients with hematologic malignancies were up to twice that of patients with solid tumors.

CONCLUSIONS: Chemotherapy-related cytopenias result in a substantial economic burden on patients, payers, caregivers, and society in general. This burden is particularly high for patients with hematologic malignancies due to the underlying malignancy. Furthermore, AEs affect the ability to deliver planned treatments, resulting in potentially suboptimal clinical outcomes. An evaluation of both clinical outcomes of chemotherapy and economic consequences as a result of chemotherapy-induced toxicities is recommended in determining optimal treatments for patients with cancer.
Incidence, Risk Factors and Outcomes of Bronchiolitis Obliterans after Allogeneic Stem Cell Transplantation.
Authors: Nakaseko Chiaki , Ozawa Shinnichi , Nishimura Miki , Sakai Miwa , Ohshima Kumi , Kanda Yoshinobu , Takahashi Satoshi , Shimizu Takayuki , Okamoto Shinichiro , Yokota Akira , Yoshiba Fumiaki , Fujimaki Katsumichi , Kanamori Heiwa , Sakai Rika , Matsushima Takafumi , Sakura Tohru , Sakamaki Hisashi
Published: October 2019

Background: Bronchiolitis obliterans (BO) after allogeneic stem cell transplantation (allo-SCT) is a late-onset, life-threatening respiratory complication which significantly reduces patients’ quality of life. Despite different therapeutic protocols, BO mortality remains high and most patients die of respiratory failure or infections. In recent practice, the source of stem cells and the conditioning regimen for allo-SCT have become more varied, but their influence on the incidence of BO is not established. Here, we retrospectively analyzed incidence of and risk factors for BO in allo-SCT protocols.

Patients and methods: Between Jan 1994 and June 2005, 2692 patients underwent allo-SCT in 14 facilities of the Kanto Study Group for Cell Therapy (KSGCT) in Japan, and 2154 surviving at least 100 days after transplantation were evaluated in this study. Clinical diagnosis of BO was made by pulmonary function tests (PFT) revealing a forced expiratory volume for 1 second (FEV1) less than 70% and FEV1/forced vital capacity less than 80% of the predicted value, along with typical changes on high-resolution computed tomography.

Results: BO developed in 57 patients with a cumulative incidence at 5 years post transplant of 2.6%. The Kaplan Meier estimate of median time interval from transplant to diagnosis of BO was 335 days (83–907). The cumulative incidence of BO at 5 years was 1.62% (12/691) in bone marrow transplants from related donors (R-BMT), 3.83% (16/424) in peripheral blood stem cell transplantation from related donors (R-PBSCT), 2.91% (24/808) in BMT from unrelated donors (UR-BMT), and 2.65% (5/199) in unrelated cord blood transplantation (CBT). The incidence of BO after R-PBSCT was significantly higher than after any other type of allo-SCT (p=0.02). At BO diagnosis, the mean value of FEV1% decreased to 52.1% from 82.2% pre-transplant. 94% of patients had already developed chronic GVHD before the onset of BO. Risk factors for BO by univariate analysis were R-PBSCT (p=0.019) and preceding chronic GVHD (p=0.000). Twenty eight patients died after developing BO, 21 of respiratory failure. Only one patient died of relapse of primary disease. Overall 5 yr-survival of patients with BO from the time of diagnosis was 46.5%, significantly less than for those without (76.2% from day 335, p=0.000) by semi-landmark analysis.

Conclusions: The incidence of BO in CBT recipients was higher than R-BMT recipients and not significantly different with UR-BMT recipients. R-PBSCT recipients who have already developed chronic GVHD have a higher risk for developing BO and need extensive care and repeated PFT examinations.
Gene Expression Profiling of the Clinical Significant CD57+CD8+ Cytotoxic T Cell Expansions in Patients with Waldenstrom’s Macroglobulinemia.
Authors: Sze Daniel , Yamagishi Tetsuo , Kaplan Warren , Brown Ross D. , Ho Phoebe Joy , Gibson John , Joshua Douglas E.
Published: October 2019

Previous studies have suggested that expanded T-cell clones are found in the blood of 59% of patients with multiple myeloma. These expanded T-cell clones are associated with prolonged overall survival and thus it has been suggested that they may have anti-tumor activity. We have previously reported similar T-cell clones exist in the peripheral blood of patients with Waldenstrom’s Macroglobulinemia (WM) by using flow cytometry to determine the T cell receptor (TCR) Vβ repertoire. Expanded T-cell clones were detected in 9 of 15 (60%) patient samples. Of the nine patients with TCR Vβ clones, four patients had multiple clones. The TCR Vβ clones were not identical, representing a variety of families across the TCR Vβ repertoire.

We have previously found that while the TCRVβ+CD8+CD57 negative subset represents polyclonal populations, the CD57 positive subset represents either monoclonal or biclonal populations. By comparing the genetic profiling of these two subsets from a statistically significant gene list, two genes have been found to be highly upregulated in the CD57 negative polyclonal subset. These two genes are i.) SESN3, a member in the Sorting Nexin (SNX) protein family which is implicated in regulating membrane traffic capable of interaction with phosphatidylinositol-3-phosphate (10.4 fold, p=0.0241); ii.) Epstein-Barr virus induced gene 2 (lymphocyte-specific G protein-coupled receptor) EBI2 (7.4 fold, p=0.0207): This finding is in contrast to previous report that EBI2 is expressed in B-lymphocyte cell lines and in lymphoid tissues but not in T-lymphocyte cell lines or peripheral blood T lymphocytes.

For the CD57 positive clonal T cell expansions, consistent with our previous reports, CD28 expression was found to be down regulated by 2.6 fold. There are two genes found to be highly upregulated. They are i.) Granzyme B (4.3 fold, p=0.0337) also called Cytotoxic T-lymphocyte proteinase 2. This enzyme is necessary for target cell lysis in cell-mediated immune responses through caspase-dependent apoptosis; ii.) Granzyme H, also called Cytotoxic T-lymphocyte proteinase and probably necessary for target cell lysis in cell-mediated immune responses.

In summary, we have shown that CD57 positive clonal T cell populations exist in some patients with WM. Importantly, microarray results have indicated some genes and proteins that may related to better patients survival as previously demonstrated in patients with Multiple Myeloma.
Hematopoietic Stroma Confers Resistance to Immune Control by TRAIL in Multiple Myeloma through Induction of FLIP.
Authors: Perez Lia , Parquet Nancy , Shain Ken , Nimmanapalli Ramadevi , Alsina Melissa , Anasetti Claudio , Dalton William S.
Published: October 2019

TNF-related apoptosis inducing ligand/Apo-2L (TRAIL), a member of the TNF superfamily of death ligands, is preferentially cytotoxic against neoplastic cells, while sparing normal tissue. TRAIL has been implicated in tumor immune-surveillance and mediates murine allogeneic graft-versus-tumor responses. The bone marrow microenvironment provides tumor protection from chemotherapy and Fas-death receptor mediated apoptosis through a process known as environmental mediated death resistance (EM-DR). We tested whether the tumor microenvironment inhibits TRAIL-mediated killing of myeloma cells, as this process may contribute to multiple myeloma escape from immune-surveillance. Three drug-sensitive myeloma cell lines (RPMI-8226, U266 and MM1s) exhibit apoptosis resistance to recombinant human (rh) TRAIL while adhered to HS5 stromal cells that expresses green-fluorescence-protein (HS5-GFP). Apoptosis resistance to TRAIL was time- and dose- dependent. Evaluation of TRAIL apoptosis in a transwell (TW) assay, with HS5-GFP cells on the bottom and RPMI-8226 cells in the upper well (TW+HS5) revealed that HS5-GFP stromal cells blocked TRAIL-induced apotosis through soluble factors. We then assessed the modifications in the TRAIL signaling pathway induced by TW+HS5 and responsible for the observed phenotype. RPMI-8226 treated in TW+HS5 exhibited attenuated pro-caspase-8, pro-caspase-3, PARP, and BID cleavage, with diminished mitochondrial membrane potential changes, but without alterations on TRAIL receptors and other Bcl-2 family members. We found increased levels of Fas-associated death domain like IL-1 converting enzyme-like inhibitory protein (FLIP), a regulatory factor that competes with caspase-8 inhibiting apoptosis. Subcellular fractionation of RPMI-8226 cells showed that FLIPL is maintained in or associated with organelle membranes and is released to the cytosol when exposed to soluble factor (TW+HS5) suggesting that that soluble factor signaling may influence FLIPL localization and availability. Furthermore, FLIP reduction by FLIP-RNA interference increases TRAIL sensitivity of RPMI-8226 treated in TW+HS5. Bortezomib is a proteosome inhibitor that inhibits NF-KB and reduces FLIP levels. To this end, pretreatment of RPMI-8226 cells with bortezomib for 20 hours followed by TRAIL for 4 hours overcame TRAIL EM-DR through inhibition of FLIP, thus providing rationale for testing this drug combination in a clinical trial. In addition, rh-IL-6 induced FLIP levels in RPMI-8226 cells conferring resistance to TRAIL mediated apoptosis. Our results suggest that IL-6 and other soluble factors produced by marrow stromal cells promote myeloma cell survival by upregulating FLIP, thereby mitigating the influence of the microenvironment on TRAIL-induced apoptosis. We conclude that the cytotoxic effect of TRAIL may be enhanced by FLIP inhibitors.
IL-6, IGF-1 and IL-21 Act in Synergy with HGF in Myeloma Cells by Inducing the HGF-Receptor c-Met.
Authors: Hov Håkon , Fagerli Unn-Merethe , Tian Erming , Waage Anders , Borset Magne , Sundan Anders
Published: October 2019

HGF is known to be a prognostic factor in multiple myeloma, however the proliferative effect of HGF alone has been moderate. We investigated the effects of the myeloma growth factors IL-6, IGF-1 and IL-21 in combination with HGF in the myeloma cell lines IH-1, INA-6 and OH-2. In proliferation experiments the cells were grown in serum free media with 3 different HGF-concentrations together with various concentrations of IL-6, IGF-1 or IL-21. HGF alone had low effect. However, 200 ng/ml HGF in combination with IL-6 doubled the effect of IL-6 and tripled the effect of IGF-1 in INA-6. Similar results were obtained in OH-2 combining IL-6, IGF-1 and IL-21 with HGF and in IH-1 combining IL-6 with HGF. A combination of IL-6 and IGF-1 in INA-6 gave no synergistic effects, so the synergism seemed to be a specific effect for HGF in combination with the other cytokines.The myeloma cell line ANBL-6 harbours an autocrine growth promoting HGF-loop. We inhibited this autocrine HGF loop with a specific c-Met receptor tyrosine kinase inhibitor PHA-665752 in the presence of IL-6 and IGF-1. IL-6 and IGF-1 potentiated the decrease in proliferation caused by PHA-665752. These results demonstrate synergistic effects between the three cytokines (IL6, IGF-1, IL-21) and HGF. We also combined PHA-665752 and IL-6 in INA-6 cells, and 200 nM of the c-Met inhibitor halved the effect of IL-6. We have not seen any unspecific effects of PHA-665752 at 200 nM. This result raises the very interesting possibility that some of the effects of IL-6 can be due to c-Met signalling. At the protein level the cell lines responding to IL-6, IGF-1 or IL-21 also increased the level of c-Met expression in response to the individual cytokines. In OH-2 there was also an increase in the HGF transcript after IL-6, IGF-1 and IL-21 stimulation, as investigated by RT-PCR, indicating another mechanism for synergy between the HGF-c-Met pathway and the other cytokines in this cell line. In conclusion our results demonstrates the induction of c-Met by IL-6, IGF-1 and IL-21, and proliferative synergy between these cytokines and HGF in myeloma cell lines. Inhibition of c-Met with a specific tyrosine kinase inhibitor decreased the proliferative effect of IL-6. Together these results indicate that inhibition of c-Met signalling would target several pathways and could therefore be a promising treatment strategy in multiple myeloma.
BIRB796 Inhibits p38 MAPK/Hsp27 Pathway and Enhances Cytotoxicity Triggered by Bortezomib, Hsp90 Inhibitor, and Dexamethasone in Multiple Myeloma.
Authors: Yasui Hiroshi , Hideshima Teru , Ikeda Hiroshi , Jin Janice , Ocio Enrique M. , Kiziltepe Tanyel , Okawa Yutaka , Vallet Sonia , Chhetri Shweta , Ishitsuka Kenji , Tai Yu-Tzu , Podar Klaus , Chauhan Dharminder , Pargellis Chris , Moss Neil , Raje Noopur , Anderson Kenneth C.
Published: October 2019

We have previously shown that heat shock protein (Hsp) 27 or its upstream molecule p38 mitogen-activated protein kinase (MAPK) confers resistance to bortezomib and dexamethasone (Dex) in multiple myeloma (MM). In this study, we evaluate the anti-tumor activity of combination treatment with novel p38 MAPK inhibitor BIRB796 and other therapeutics agents in MM. Although BIRB796 alone triggers a marginal growth inhibitory effect in MM cells, it blocked baseline and bortezomib-triggered upregulated phosphorylation of p38 MAPK and Hsp27, associated with enhanced cytotoxicity in combination with bortezomib. BIRB796 augmented bortezomib- triggered cleavage of caspase-8, caspase-9, and poly(ADP)-ribose polymerase (PARP). We next examined the combination of BIRB796 with Hsp90 inhibitor 17-AAG. Surprisingly, 17-AAG up-regulates protein expression and phosphorylation of Hsp27; conversely, BIRB796 inhibits this phosphorylation and enhances 17-AAG-induced cytotoxicity. Importantly, BIRB796 enhances cytotoxicity induced by 17-AAG plus bortezomib. BIRB796 also augments cytotoxicity of Dex in MM cells, associated with inhibition of Hsp27 phosphorylation. In bone marrow stromal cells (BMSCs), BIRB796 inhibited phosphorylation of p38 MAPK and secretion of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) triggered by either tumor necrosis factor-α or tumor growth factor-β 1. BIRB796 also inhibits IL-6 secretion in BMSCs triggered by adherence to MM cells, thereby inhibiting MM cell proliferation. These studies therefore suggest that BIRB796 overcomes drug-resistance in the BM microenvironment, providing the framework for clinical trials of a p38 MAPK inhibitor alone, and in combination with bortezomib, Hep90 inhibitor, or Dex, to improve patient outcome in MM.
Multiple Myeloma: Interleukin-15 Antagonizes Bone Morphogenetic Protein-Induced Apoptosis and Growth Inhibition.
Authors: Rekvig Magne , Brenne Anne-Tove , Ro Torstein Baade , Waage Anders , Borset Magne , Sundan Anders
Published: October 2019

Multiple myeloma has two distinct features: Expansion of malignant plasma cells within the bone marrow accompanied by skeletal destruction. Bone morphogenetic proteins (BMPs) have been shown to induce apoptosis and inhibit growth in myeloma cells. BMPs are members of the TGF-β superfamily of proteins capable of inducing bone formation, and regulate proliferation, differentiation and apoptosis. We have investigated myeloma cell apoptosis and proliferation with BMP-4 and −6 in concert with the myeloma cell growth factors interleukin (IL)-2, IL-6, IL-10, IL-15, IL-21, tumor necrosis factor (TNF)-α and insulin-like growth factor (IGF)-1. Eight samples of highly purified myeloma cells from patients and a human myeloma cell line, IH-1 (Brenne AT et al. Blood. 2002 May 15;99(10):3756–62.), were used in this study. Cytokine concentrations used in the referred experiments were for BMP-4 20ng/ml, BMP-6 250ng/ml, IL-15 20ng/ml and IL-6 0,1ng/ml, respectively. Growth inhibition was measured in a proliferation assay by methyl-[3H]-thymidine incorporation and apoptosis by annexin V- FITC-binding/PI-uptake on flow cytometry. IL-15 antagonized growth inhibition (Figure A) and prevented apoptosis induced by BMP-4 (Figure B) and BMP-6 in the myeloma cell line IH-1. IL-15 also antagonized the growth inhibition induced by BMP-4 and/or BMP-6 in three out of eight patient samples. Neither IL-6, nor any of the other investigated cytokines were able to rescue the myeloma cells from growth inhibition and apoptosis induced by BMP-4 and -6. Among the investigated cytokines, we found that IL-15 has a unique capability to antagonize BMP- induced apoptosis and growth inhibition in myeloma cells. We examined cleavage of the proapoptotic protein caspase-3 and found that BMP-4 activated caspase-3 in the IH-1 cell line. This activation of caspase-3 was blocked by IL-15 but not by IL-6. We have demonstrated a possible mechanism for myeloma cells to escape apoptosis and growth-inhibition within the bone marrow. Intramedullar levels of IL-15 and BMPs may play a role in the pathogenesis of multiple myeloma.

Figure A. Proliferation in response to BMP-4 stimulus Figure A. Proliferation in response to BMP-4 stimulus Figure B. Apoptosis in response to BMP-4 stimulus Figure B. Apoptosis in response to BMP-4 stimulus
The BAFF Inhibitor AMG523 Blocks Adhesion and Survival of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment: Clinical Implication.
Authors: Tai Yu-Tzu , Xu Jiangchun , Li Xian-Feng , Breitkreutz Iris , Podar Klaus , Hideshima Teru , Schlossman Robert , Richardson Paul , Munshi Nikhil C. , Anderson Kenneth C.
Published: October 2019

We previously identified a role of B-cell activating factor (BAFF), a member of the tumor necrosis factor superfamily, in localization and survival of MM cells in the BM microenvironment (Cancer Res2006, 66:6675–82). In the present study, we examined the potential therapeutic utility of the BAFF inhibitor, AMG523, for treating human MM using MM lines, either sensitive or resistant to conventional chemotherapy, as well as freshly isolated patient MM cells, in the presence or absence of bone marrow stromal cells (BMSCs). AMG523 induces modest cytotoxicity in MM cell lines and patient MM cells, suggesting a minor role of autocrine mechanism of BAFF for MM growth and survival. In the presence of BMSCs, AMG523 significantly decreased growth and survival in dexamethasone (Dex)-sensitive MM1S, Dex-resistant MM1R, INA6 MM cells and in patient MM cells (n=7), in a dose-dependent manner (0.1–10 μg/ml). BAFF-augmented MM adhesion to BMSCs is also blocked by AMG523 at 0.1 mg/ml in MM lines (MM1S, 28PE, INA6), as well as in freshly isolated patient MM cells (n=4). BAFF protects MM cells against dex- and lenalidomide-induced cytotoxicity; conversely, AMG523 blocks BAFF-induced protection against drug-induced apoptosis. Importantly, pretreatment of AMG523 blocks BAFF-induced activation of AKT, nuclear factor kB, and ERK in MM cells, confirming its inhibitory effect on BAFF-mediated adhesion and survival. We next asked whether AMG523 enhances Dex-, bortezomib-, Lenalidomide-induced MM cell cytotoxicity. AMG523 augments the inhibitory effect of Dex and lenalidomide in patient MM cells in the presence of BMSCs. Since osteoclasts (OCLs) secrete BAFF in the bone marrow microenvironment, we further asked whether AMG523 inhibits protection by MM-OCL interaction. OCLs were derived from peripheral blood mononuclear cells from MM patients after 2-week culture with M-CSF and RANKL, and MM cells were added in the presence or absence of AMG523. OCLs significantly increased MM cell survival, evidenced by annexin V and PI staining followed by flow cytometric analysis; conversely, AMG523 blocked MM cell survival by coculture with OCLs. Taken together, our data demonstrate that the novel therapeutic AMG523 blocks the interaction between BAFF and its receptors in human MM, thereby providing the rationale for clinical trials of AMG523 to improve patient outcome in MM.
Mcl-1, Bcl-xL and Cell Cycle Inhibition by GCS-100: A Novel Therapy for Myeloma.
Authors: Streetly Matthew , Su Bo , Carver Bradley , Schey Stephen A. , Cotter Finbarr E.
Published: October 2019

The marrow stromal cells in multiple myeloma activate multiple signalling pathways via stimulation by the cytokines they secrete. This leads to upregulation of anti-apoptosis, drug resistance, cell cycle and metabolic pathways. Disruption of these signalling networks can lead to myeloma cell death and make them attractive targets for novel therapies. In addition, Bcl-2 family proteins, mcl-1 and bcl-xL are important for the survival of myeloma cells. The modified citrus pectin, GCS-100 has been used safely in Phase I studies for solid tumors, can induce cell death in lymphoma via blocking galectin-3 / Bcl-2 dimerisation and in myeloma cells by mechanisms not fully understood. GCS-100 as well as pro-apoptotic effects, also demonstrates anti-proliferative and anti-angiogenic properties, all of which may be useful in the therapy of myeloma. This study was aimed at elucidating the mechanism of action of this novel agent so as to apply it most effectively to myeloma therapy. Myeloma cell lines RPMI8226 and U266 treated with GCS-100 show induction of apoptosis in a dose and time dependent manner with a loss of cells in the G1 and S phase of cell cycle and a significant increase in G0 / sub-G1 cells. Treatment of RPMI8226 led to a marked reduction of mcl-1 and bcl-xL after 24 hours of treatment. There was no change observed in protein levels of bcl-2 or galectin-3. Activation of NFκB in myeloma cells is associated with proliferation and adhesion molecule upregulation. GCS-100 treatment led to a time dependent reduction in activated IκBα and p65NFκB. Furthermore pre-treatment of myeloma cells with GCS-100 prior to stimulation with TNFα, a known activator of IκBα, inhibited this activation. Similarly, treatment with GCS-100 led to a reduction in the amount of activated Akt (a regulator of cell cycle) and inhibited IGF-1 associated activation of Akt. The effect of GCS-100 on cell cycle regulatory proteins revealed downregulation of cyclin D1, p16INK4A and CDK6 at 24hrs, however, there was no change in expression of CDK4 and p15INK4B. The p21CIP1 was upregulated with a corresponding decrease in protein levels of cyclin E2 and CDK2 but there was no change in p27KIP1. GCS-100 clearly inhibits progression through G1 / S phase of cell cycle. Studies are currently ongoing for primary cells. In conclusion GCS-100 is a novel complex carbohydrate that is effective for the induction of myeloma cell death. It has multi faceted effects in reducing proliferation and induces apoptosis by down regulating crucial anti-apoptotic proteins, cell cycle regulators and signalling proteins and may also act by interfering with the myeloma cell microenvironment. Phase I studies in myeloma have been commenced.
The Pan-Bcl-2 Inhibitor GX15-O70 Induces Apoptosis in Human Myeloma Cells by Noxa Induction and Strongly Enhances Melphalan, Bortezomib or TRAIL-R1 Antibody Apoptotic Effect.
Authors: Gomez-Bougie Patricia , Maïga Sophie , Pellat-Deceunynck Catherine , Jouan Sandrine , Bataille Regis , Amiot Martine
Published: October 2019

GX15-070 belongs to small molecule pan-Bcl-2 inhibitors that mimic BH3-only proteins, therefore it may bind the groove of anti-apoptotic Bcl-2 family members and induce cell death. Mcl-1, an anti-apoptotic protein from the Bcl-2 family, is an essential survival protein for Multiple Myeloma (MM) cells. Mcl-1 displays specific affinity for some BH3-only proteins. In viable MM cells, Mcl-1 strongly interacts with BH3-only Bim protein, keeping in check its pro-apoptotic properties. Consequently, we tested a panel of human MM cell lines (HMCL) (n=9) for GX15-070 apoptosis induction. Apoptosis of HMCL was induced at doses ranging from 0,15 μM to 5 μM. HMCL were differentially sensitive to GX15-07. For most HMCL the maximal apoptotic effect was observed at 2,5 μM of GX15-070. Thus we found 2 very sensitive HMCL (> 60% of death cells), 3 intermediate sensitive HMCL (>30% <60%) and 4 resistant HMCL (<30%). Sensitivity to GX15-070 was not correlated with IL-6 dependence. For further studies 4 HMCL were selected according to GX15-070 apoptosis induction, namely RPMI-8826 (71% ± 4), MDN (64% ± 4), LP1 (43% ± 3) and U266 (22%± 2). After 24 hours treatment, no substantial modifications of the anti-apoptotic proteins (Bcl-2, Bcl-xL and Mcl-1) were induced. Among the BH3-only pro-apopototic proteins examined, only Noxa was consistently induced by GX15-070 in all sensitive HMCL. In contrast, Noxa was not induced in the GX15-070 resistant U266 cell line. Pro-apoptotic multidomain molecules Bax and Bak were not modified by GX15-070.

We also evaluated GX15-070 in combination with other drugs currently used in MM treatment. We wondered if GX15-070 could sensitize HMCL LP1 and/or L363 cells to either Melphalan or PS-341 since both HMCL are weakly sensitive to both agents. HMCL were pre-incubated with GX15-070 for 7h at doses ranging from 0,15 to 5 μM followed by either melphalan or Bortezomib during 24h. GX15-070 displayed an additive apoptotic effect either in combination with melphalan or with Bortezomib for both HMCL. Interestingly, GX15-070 had a synergistic apoptotic effect in combination with a fully human antibody directed against TRAIL-R1, Mapatumumab, Human Genome Sciences, Rockville, MD. Of note, GX15-070/Mapatumumab combination exhibited a synergistic apoptotic effect on the GX15-070 resistant U266 cell line. Ongoing studies are conducted to unravel the contribution of GX15-070 either on the intrinsic or the extrinsic apoptotic pathways and to explore the feasibility of GX15-070 as a MM therapeutic agent.
Bortezomib Down-Regulates HLA Class I and Enhances Natural Killer Cell Mediated Lysis of Myeloma.
Authors: Shi Jumei , Garg Tarun K. , Kellum Rachel E. , Szmania Susann M. , Barlogie Bart , Tricot Guido , van Rhee Frits
Published: October 2019

Introduction: Immunotherapy with Natural Killer (NK) cells may eradicate residual chemotherapy-resistant myeloma (MM) after a state of minimal residual disease has been achieved by autologous stem cell supported high-dose chemotherapy. NK cell activity is regulated by Killer Immunoglobulin-Like Receptors (KIRs). The principal inhibitory ligands of KIRs are HLA Class I molecules (-C and -Bw4) expressed by target cells. NK cells lyse tumor cells that do not display such inhibitory KIR-ligands (KIR-L), such as the K562 cell line. The proteasome is responsible for the generation of peptides that bind to and stabilize Class I molecules at the cell surface. We hypothesized that treatment of MM cells with the proteasome inhibitor bortezomib results in the down-regulation of Class I and thereby sensitizes MM to NK cell mediated lysis.

Methods: MM cell lines and primary MM cells were treated with concentrations of bortezomib easily achievable in vivo. The objectives were to determine a) the optimal concentration of bortezomib to down-regulate Class I, b) the time to maximum down-regulation of Class I, and c) a bortezomib concentration that increases NK-mediated MM killing without affecting NK cell function.

Results: We found that down-regulation of Class I post-bortezomib treatment of the MM cell line, JJN3, and primary MM cells was dose and time dependent. Bortezomib at 10–20nM down-regulated HLA-class I by 63% compared to untreated control cells with maximal down-regulation at 24hrs (Fig.1a). Bortezomib treatment of primary MM cells greatly increased sensitivity to NK cell-mediated lysis, compared to controls (6% vs. 61%) at an E:T ratio of 20:1 (Fig.1b). These bortezomib concentrations did not affect NK cell function.

Conclusions: We observed that bortezomib down-regulates Class I and enhances the sensitivity of MM to NK cell-mediated lysis. Our findings have clear therapeutic implications for MM and other NK cell-sensitive malignancies.

Figure 1a. Bortezomib down-regulates HLA-class I expression on patient myeloma cells. Figure 1a. Bortezomib down-regulates HLA-class I expression on patient myeloma cells. Figure 1b. Increased killing of bortezomib-treated patient myeloma by NK in vitro. Figure 1b. Increased killing of bortezomib-treated patient myeloma by NK in vitro.
An Observational, Retrospective Analysis of Retreatment with Bortezomib of Mulitple Myeloma (MM) Patients.
Authors: Conner Therese M. , Doan QuynhChau D. , LeBlanc Annette L. , Walters Ian B. , Beveridge Roy A.
Published: October 2019

BACKGROUND: The efficacy and safety of single agent bortezomib (btz) in relapsed MM is well established. In that setting, typically 8 cycles of therapy (6 months) are given and treatment stops. However, no therapy is curative and upon relapse, patients are more refractory to subsequent therapies. Until recently, there has been little information on the feasibility and benefits of retreatment with btz. This study was designed to assess the safety and efficacy of btz used as retreatment either as a single agent or in combination with other agents in community oncology practices.

METHODS: MM pts who received at least two btz-based therapies between May 2003 and November 2005 and who had a ≥60-day gap between each treatment were identified from 22 sites using the US Oncology electronic claims warehouse. Data were transcribed from the patient records to a prespecified case report form for analysis. Best response was determined using M-protein values when available: partial response (PR), 50–89% decrease; very good PR (VGPR) or better, ≥90% decrease. In some cases, physician assessments were available without laboratory confirmation in the chart.

RESULTS: Ninety-four patients with relapsed MM met the eligibility requirements. Mean age was 65.9 years, 43% were female, median time from diagnosis to initial btz treatment was 3 years, and median number of therapies prior to first btz treatment was three. Patients received a median of 16 btz doses during the first treatment and a median of 12 doses in the second treatment; the median time between first and second btz treatment was 5 months (range 2–17). Notably, two patients in each treatment period received over 60 btz doses. The majority (55%) of patients received single agent btz without intervening therapies between first and second btz treatment. Of patients evaluable for response, a PR or better was achieved in 44 of 78 (56%) patients in response to initial therapy (26% achieved a VGPR or better) and in 17 of 85 (20%) in response to retreatment. Of patients with a ≥PR, 12 of 39 (31%) responded upon retreatment. Nine percent of patients who did not respond initially achieved a PR when retreated. Of those who achieved a VGPR or better on initial treatment, 18% achieved at least a VGPR on retreatment. The number of patients who stopped treatment due to toxicity was lower with retreatment (27%) compared with initial treatment (39%). Neuropathy and thrombocytopenia were the most common toxicities leading to discontinuation; treatment discontinuation due to neuropathy decreased from 17% on initial treatment to 5% on retreatment. Nine patients received ≥2 btz retreatments.

CONCLUSIONS: Data from the US Oncology claims database for 94 patients retreated with btz (more than two retreatments in some) suggest that retreatment is safe and effective and not associated with new or cumulative toxicities. Btz is an option for repeated therapy in relapsed patients.
The Combination of Cyclophosphamide, Velcade and Dexamethasone (CVD) Induces High Response Rates with Minimal Toxicity Compared to Velcade Alone (V) and Velcade Plus Dexamethasone (VD).
Authors: Davies Faith E. , Wu Ping , Srikanth M. , Jenner Matthew W. , Dines Sharon , Saso Radovan , Morgan Gareth J.
Published: October 2019

Bortezomib (Velcade®), as the first-in-class proteasome inhibitor, has shown to be effective for the treatment of relapsed refractory myeloma. Preclinical and/or clinical studies showed improved activity by combining this agent with a number of conventional chemotherapeutic agents, suggesting synergistic effects. We conducted a retrospective study to assess the efficacy and toxicity profile with Velcade alone and in combination with dexamethasone or cyclophosphamide, and dexamethasone for patients with multiply relapsed myeloma. 11 patients were treated with Velcade alone, 20 with the combination of Velcade and dexamethasone (VD), and 11 with the regimen comprising Velcade, dexamethasone and cyclophosphamide (CVD). Velcade 1.3mg/m2 was given as a single bolus IV on days 1, 4, 8 and 11, dexamethasone 40mg po on the day of Velcade injection and the day thereafter, and cyclophosphamide 500 mg po days 1, 8, 15 of a 21 day cycle for a maximum of 9 cycles of treatment. No patient received prophylactic anticoagulation. Toxicity profiles and response were assessed every 3 weeks. There was no statistical difference of baseline characteristics (age, ISS, number of previous lines of treatment) among the three treatment groups (P>0.05). The overall response rates (CR+PR) within the three groups are 30% (V), 47% (VD), and 64% (CVD) respectively. The CR rate of CVD group is impressive at 27% compared to 5% with VD and 0% with V. The median duration of treatment of three groups (V vs VD vs CVD) are 115 days, 98 days and 116 days respectively (P>0.05). Thrombocytopenia and new/worsening peripheral neuropathy are the most common side effects in each group. Although thrombocytopenia and neutropenia occurred in the CVD group (36% and 27% respectively), it was at a similar frequency as in the groups of VD (50% and 15%) and V (64% and 45%). Grade 3 infection rates were also similar at 18%, 30% and 18% (V vs VD vs CVD). Peripheral neuropathy is the most troublesome side effect, and is the main reason for Velcade dose reduction and/or discontinuation in each group (27% V, 45% VD, 54% CVD). 27% of patients within CVD group required dose reduction of cyclophosphamide due to neutropenia. Median follow up is currently too limited to comment on whether the improved CR rate with CVD translates into an improved progression free survival. In conclusion CVD is a well tolerated regimen producing high overall and complete response rates, with no increase in toxicity compared to VD or V alone, and lacks the toxicity associated with Velcade- melphalan combinations.