bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct and Enhanced Cytotoxicity of the Bcl-2 Family Inhibitor GX15-070 on Rituximab-Sensitive and Rituximab-Resistant B-NHL Clones.
Authors: Martinez-Paniagua Melisa A. , Vega Mario I. , Huerta-Yepez Sara , Martinez-Miguel Bernardo , Hernandez-Pando Rogelio , Gonzalez-Bonilla Cesar R. , Bonavida Benjamin
Published: October 2019

Rituximab (chimeric anti-CD20 mAb) has been used for the treatment of Non Hodgkin B cell lymphomas (B-NHL), alone or in combination with CHOP. However, a subset of patients does not respond to treatment or develops refractoriness to further treatments. Therefore, there is an urgent need to develop new alternatives to treat these patients. We have reported that treatment of B-NHL cell lines with rituximab inhibits anti-apoptotic survival pathways and down regulates the expression of anti-apoptotic Bcl-2 family proteins (i.e. Bcl-2/Bcl-xl) resulting in sensitization to chemotherapeutic drugs. Further, rituximab-resistant clones showed over-expression of anti-apoptotic gene products (Jazirehi et al., Cancer Research1:1270–81, 2007). Therefore, we hypothesize that inhibitors of anti-apoptotic Bcl-2 family may reverse the resistance to apoptotic stimuli. We examined chemical inhibitors that mimic natural ligands of the anti-apoptotic BH3-only proteins. GX15–070 (Gemin X Biotechnologies, Inc., Canada) inhibits Bcl-2 protein-protein interactions resulting in Bak and Bax oligomerization, release of cytochrome C, and activation of caspases (Shore and Viallet, Hematology, 2005; ASH, 226–230). Treatment of B-NHL cell lines (Raji, Ramos, 2F7, DHL-4) with subtoxic concentrations of GX15–070 (20–50 uM) resulted in inhibition of cell proliferation and subsequently induction of apoptosis as determined by TUNEL. There was a time and concentration-dependent effect of GX15–070 on cytostasis and apoptosis. Analysis of cells treated with GX15–070 by western blotting revealed that Bcl-2, Bcl-xl, and Mcl-1 protein expressions were significantly inhibited as compared to controls. The protein inhibition by GX15–070 was not expected and needs further investigation. We also examined the effect of combination treatment of GX15–070 with the chemotherapeutic drug CDDP and there was an additive or synergistic cytotoxic effect. Treatment of rituximab-resistant clones (generated from 2F7, Raji and Ramos) treated with GX15–070 resulted in significant inhibition of cell growth and apoptosis. The cytotoxicity of GX15–070 in the B-NHL cell lines was tumor specific, because treatment of human peripheral blood leukocytes from different donors did not show any cytotoxic effect. Likewise, treatment of nude mice with different concentrations of GX15–070 did not show any detectable toxicity. These findings demonstrate that GX15–070 is cytotoxic to various drug/rituximab-resistant B-NHL cell lines and is not toxic to normal human leukocytes. This study suggests that combination of GX15–070 with subtoxic concentrations of chemotherapeutic drugs may have additive/synergistic effects. The present findings support the potential therapeutic application of GX15–070 in the treatment of patients with B-NHL that are resistant to current therapies.
Hematopoiesis in Regenerated Bone Marrow on the Hydroxyapatite Scaffold.
Authors: Fujita Atsushi , Migita Makoto , Ueda Takahiro , Fukunaga Yoshitaka , Shimada Takashi
Published: October 2019

Background: In bone marrow (BM), osteoblastic cells lining the endosteal surface are a key component of the niche to promote and regulate hematopoietic stem cells (HSCs). Anatomical architecture of BM could be regenerated by culturing BM derived stromal cells on the hydroxyapatite (HA) scaffold. In the present study, we examined whether the regenerated BM has the ability to support HSCs in vivo.

Methods: Our study was designed as follows;

BM stromal cells from C57/BL6 mice (Ly5.2) were cultured on the HA scaffold with numerous small pores for 3 days in vitro and the scaffold with attached cells was implanted subcutaneously onto the back of C57/BL6 recipient mice. 4.0x105 Lineage negative (Lin−) Ly5.1 BM cells transduced with a lentiviral vector containing the luciferase (Luc) gene were intravenously administered into the recipient mice after lethal irradiation. Eight weeks after transplantation, the scaffolds were removed from the first recipient mice and subcutaneously transplanted into the lethally irradiated second recipient mice. The mice also received fresh Ly5.2 BM cells for survival. Biodistribution and kinetics of Luc+ Ly5.1 cells were monitored by in vivo bioluminescence imaging and FACS.

Results: In the secondary transplanted mice, Luc+ hematopoitic cells were detected in the scaffolds for at least 6 months after transplantation. Subcutaneous injection of G-CSF resulted in wide distribution of bioluminescence signals from the original scaffolds to whole body including the head, extremities, chest, and abdomen. The presence of Ly5.1 B and T lymphocytes in the circulation was confirmed by FACS analysis 5 months after secondary transplantation.

Conclusions: The regenerated BM on the HA scaffold is capable of supporting HSCs in vivo suggesting that the functional niche is reconstituted. Hematopoiesis in the regenerated BM may have a significant impact for development of new therapeutic strategies for various hematopoietic diseases.
Three Restricted Forms of Epstein-Barr Virus Latency Counteracting Apoptosis in c-Myc Expressing Burkitt Lymphoma Cells.
Authors: Kelly Gemma L. , Stylianou Julianna , Bell Andrew I. , Wei Wenbin , Rowe Martin , Rickinson Alan B.
Published: October 2019

Epstein-Barr virus (EBV) is aetiologically linked with Burkitt Lymphoma (BL) but its contribution to lymphomagenesis, versus that of the chromosomal translocation activating c-myc expression, remains unclear. This is in part because the full virus growth transforming programme that is expressed when EBV infects normal resting B cells, is not expressed in BL. Instead EBV in BL normally exhibits a restricted Latency I form of infection characterised by expression of only one latent antigen EBNA1 from the BamHI Q promoter. Here we describe an endemic BL, Awia, which uniquely is heterogeneous at the single cell level for EBV gene expression. Analysis of single cell clones of Awia-BL revealed cells displaying three forms of restricted EBV latency:

classical Latency I, ’Wp-restricted latency’ expressing EBNAs 1, 3A, 3B, 3C and -LP, and a novel ’EBNA2+/LMP1- latency’ in which all EBNAs including EBNA2 are expressed without the latent membrane proteins LMPs 1 and 2.

Comparison with rare EBV-negative clones from the same tumour showed that each form of infection provides the c-myc-expressing BL cells with a specific degree of protection from apoptosis. Microarray analysis was carried out on the isogenic Awia-BL clones to determine the influence of EBV gene expression on cellular transcription. Interestingly it was found that expression of the pro-apoptotic Bcl2 family protein BIM, which has previously been implicated in c-myc induced apoptosis, was downregulated in the apoptosis resistant BL cells. Our work suggests that EBV acts as an anti-apoptotic rather than a growth-promoting agent in BL by downregulating expression of the cellular BIM protein. In addition the microarray analysis on these isogenic Awia-BL clones showed that the EBNA profile influences the differentiation status of the BL cell on the germinal centre to plasmacytoid differentiation pathway. These findings may reflect viral functions that are important not just for BL pathogenesis but also for EBV’s normal strategy of persistence in the B cell system.
SB1317, a Potent and Orally Active FLT3-CDK Inhibitor with High Anti-Tumor Efficacy in Models of Hematological Malignancies.
Authors: Goh Kee C. , Ong Wai C. , Hu Changyong , Liang Ai L. , Stunkel Walter , Tan Yong C. , Sangthongpitag Kanda , Goh Siok K. , Bonday Zahid Q. , William Anthony D. , Dymock Brian W. , Kantharaj Ethirajulu , Wood Jeanette M.
Published: October 2019

FLT3 is the most common mutated gene in acute myeloid leukemia (AML), and FLT3 mutations are strongly correlated with poor prognosis. Small-molecule inhibitors of FLT3 kinase have been evaluated in clinical trials but with limited success due to the emergence of activating mutants other than FLT3. CDK1, 2, 4 and 6 are well-established anti-cancer targets due to their direct role in cell cycle control. CDK7 and 9 are transcriptional CDKs that are emerging anti-cancer targets as a result of recent advances in the understanding of their effects on apoptotic regulators. Recent evidence suggests that the combined targeting of CDK1, 2 and 9 enhances apoptotic killing of tumor cells. Most CDK active compounds currently in clinical development do not target all of these CDKs and have poor pharmacokinetic/pharmacodynamic properties. Compounds targeting these CDKs, in addition to FLT3, may be more effective in AML and also be effective against other hematological as well as solid tumors. SB1317 is a novel potent inhibitor of FLT3 kinase (IC50 = 45 nM) and CDK2 (IC50 = 11 nM). The CDK spectrum also includes potent inhibition of CDK1 and 9 (IC50 = 19 and 10 nM respectively). SB1317 inhibits proliferation of a broad panel of tumor cell lines, and is particularly potent against the mutant FLT3-dependent AML cell line, MV4-11 (proliferation IC50 = 18 nM). SB1317 reduced phospho-Rb and phospho-FLT3 levels in MV4-11 cells in a dose-dependent manner. It induced apoptosis in MV4-11 cells as well as in primary AML cells from patients. The pharmaceutical, metabolic and pharmacokinetic properties of SB1317 render it amenable to oral dosing. In a nude mouse subcutaneous model of AML (MV4-11), SB1317 induced complete tumor regression after oral daily dosing (40 mg/kg for 21 days). In an orthotopic model of AML (HL60), SB1317 significantly prolonged median survival time (59 days versus 40 days) after treatment at 100 mg/kg p.o. 2d on/5d off. It also showed significant anti-tumor activity in a nude mouse model of B-cell lymphoma (Ramos) with a tumor growth inhibition of 63% (15 mg/kg i.p. 5d on/5d off). These results demonstrate the therapeutic potential of this novel kinase inhibitor for the treatment of hematological malignancies.
Use of Enoxaparin Versus Unfractionated Heparin in the Prevention of Venous Thromboembolic Events in Acutely Ill Medical Patients: A Meta-Analysis of Individual Patient Data.
Authors: Mismetti Patrick , Liotier Jérôme , Kleber Franz-Xaver , Sherman David G. , Moulin Nathalie , Laporte Silvy
Published: October 2019

Background The ACCP guidelines recommend prophylaxis with low-molecular-weight-heparin (LMWH) or unfractionated heparin (UFH) to prevent venous thromboembolism (VTE) in acutely ill medical patients. However, there is a paucity of data on the relative benefits of using LMWH versus UFH. A meta-analysis was performed using individual patient data to assess the efficacy of the LMWH enoxaparin versus UFH for VTE prophylaxis in hospitalized medical patients.

Methods A systematic literature review was conducted to identify randomized clinical trials comparing enoxaparin 4000 IU S.C. once-daily versus UFH administered S.C. twice (2 × 5000 IU) or three-times daily (3 × 5000 IU) for VTE prophylaxis. Data from electronic database searches were supplemented by information from trial registries, abstract books, meeting proceedings and the trial sponsors. All investigators or sponsors were contacted to provide individual patient data. Recorded outcomes were VTE, major bleeding, and deaths at day 14 as adjudicated by the adjudication committee of each trial. All trial analyses were performed on an intention-to-treat basis. The pooled analysis was based on the logarithm of the relative risk (RR) weighted by the inverse of the variance.

Results Individual patient data were obtained from 3600 patients enrolled in 4 randomized clinical trials, 3 in stroke patients and 2 in patients with various acute illnesses. The data from 1 additional trial is currently unavailable (100 patients). The median age of evaluated patients was 71 years; 51% are male; 32% presented with a creatinine clearance <50 mL/min. Meta-analysis of the overall incidence of VTE (including asymptomatic DVT, symptomatic VTE and PE-related death) showed a 38% significant reduction in favor of enoxaparin (RR=0.62 [95% CI 0.50–0.77], p<0.001, heterogeneity test pHet=NS). A non-significant 13% increase in major bleeding was also observed with enoxaparin (RR=1.13 [95% CI 0.53–2.44], p=0.75, pHet=NS). Finally, there was a non-significant 16% reduction in death with enoxaparin (RR=0.84 [95% CI 0.63–1.10], p=0.20, pHet=NS).

Conclusion The results of this meta-analysis support superior efficacy of enoxaparin, with a non-significant increase in major bleeding, compared with UFH for VTE prophylaxis in acutely ill medical patients.
Bleeding and Thromboembolic Events after Termination of Therapy with Idraparinux for Prevention of Recurrent Venous Thromboembolism-Observation after the van Gogh Trials.
Authors: Harenberg Job , Joerg Ingrid , Hagedorn Antje , Giese Christina
Published: October 2019

The long acting polymethylated antithrombotic compound Idraparinux has been investigated in the vanGogh trials for prevention of recurrent venous thromboembolism (VTE) using once weekly subcutaneous injections for 3–6 months (vanGogh PE and DVT trial) and for additional 6 months (vanGogh Extension trial). After termination of the studies Idraparinux was eliminated with a half live of 60 days or longer (Harenberg et al, submitted). We followed-up the patients (n=23) at our center for bleeding events, recurrent VTE and other severe events over 15 months. During the study period 2 major bleeding complications occurred in patients randomized to warfarin (days 70 and 186). During follow-up, 2 patients initially randomized to Idraparinux suffered from major bleeding events (days 65 and 140 during follow-up). Bleeding was related to an additional therapy of warfarin or low-molecular-weight heparin. At that time, the long elimination half life time of Idraparinux was unknown. One patient required continuation of anticoagulation due to homocygous factor V Leiden mutation and received warfarin starting one week after termination of idraparinux. At day 65 he was hospitalized because of a major intramuscular bleeding. One patient underwent total hip replacement at day 140 of follow-up receiving nadroparin for prophylaxis of VTE and developed a major postoperative bleeding with reoperation and transfusion. The concentrations of Idraparinux were 0,16μg/ml in each patient. Thereafter the long elimination time of Idraparinux was taken into consideration. In 1 of the patients warfarin was started at a 0.1μg/ml Idrparinux and titrated slowly into an INR range of 1.6 to 2.0 until Idrapaninux decreased to 0.05μg/ml. In the other patient postoperative prophylaxis of VTE was performed with LMWH at 0,01μg/ml Idraparinux (day 571 of follow-up). No adverse events occurred in both patients. During follow-up VTE occurred in 2 patients each initially randomized to warfarin (days 58 and 406) and Idraparinux (days 266 and 381), respectively. The concentrations of idraparinux were <0.01μg/ml in both patients. The data show that the very long half life of Idraparinux may lead to serious bleeding complications, if anticoagulation is given on top of the circulating compound within 3 or 4 months after termination of therapy. Cautious anticoagulation during this period may avoid bleeding. Recurrent VTE occurs late after termination of Idraparinux but in probably to the same frequence as after termination of warfarin.
Development and Performance of the GTI Diagnostics ADAMTS-13 Activity Assay (ATS-13™) and ADAMTS-13 Inhibitor Assay (ATS-13™ INH).
Authors: Pressman Melissa A. , Wuitschick Elizabeth , Chance Suzette C.
Published: October 2019

Thrombotic Thrombocytopenic Purpura (TTP) is a disease characterized by the sudden onset of a classical pentad of symptoms: thrombocytopenia, fever, renal insufficiency, neurologic deficit, and microangiopathic hemolytic anemia. ADAMTS-13 is the protease responsible for cleaving von Willebrand Factor (VWF); decreased levels or deficiency of ADAMTS-13 activity has been demonstrated in the plasma of TTP patients. The lack of ADAMTS-13 activity results in the accumulation of multimers of VWF in the plasma and ultimately intravascular platelet aggregation resulting in the clinical symptoms associated with TTP. The presence or absence of inhibitory antibodies directed against ADAMTS-13 influences the treatment protocol for these patients. GTI has developed a rapid, quantitative assay for ADAMTS-13 activity that is based on the cleavage of a von Willebrand Factor substrate and subsequent fluorescent detection. A method comparison was done of GTI’s ADAMTS-13 Activity Assay (n=397) using normal plasma, plasma from suspected TTP patients, and plasma from patients suspected to have other coagulation diseases (ITP, HUS, HIT, APS). All samples were also run on the collagen-binding (n=197) or the Furlan method (n=200) for comparison. This data demonstrated good correlation. Assay precision (with-in run, n=20 and assay-to-assay, n=40) was evaluated and there was less than 20% cv total imprecision for samples with levels of activity across the assay range. In addition, GTI has developed an assay for the semi-quantitative detection of inhibitory antibodies directed against the ADAMTS-13 protease. This assay is similar in format and principle to our ATS-13™ kit; however inhibition of the ADAMTS-13 protease activity is determined in the patient’s heat-inactivated sample following mixing with a provided control at a single dilution. Assay precision and preliminary method comparison data will be presented.
Localization of Stereo-Specific Phosphatidylserine Binding Sites within the Factor V C2 Domain.
Authors: Liang Xiaoe , Quinn-Allen Mary Ann , Kane William H.
Published: October 2019

Binding of activated factor V (Fva) to phosphatidylserine (PS) is required for formation of the prothrombinase complex on the surface of activated platelets. The interaction between Fva and PS has been shown to be stereo-specific with bovine Fva favoring L-PS over D-PS. Our previous low resolution alanine scan data localized PS binding sites to the FV-C2 domain and identified two tryptophan residues, W2063 and W2064, which are required for high affinity binding. Two putative PS binding sites were also predicted based on modeling studies using the FV-C2 structure. In this study, the stereo-specific interaction of Fva with PS was evaluated with plasma derived human Fva (pd-HFVa), recombinant human Fva (rHFVa), rHFVa-C2 domain and rHFVa mutants. Quantitative fluorescence binding assays and functional prothrombinase assays were conducted using phospholipid vesicles containing 20 mol% dioleoyl phosphatidyl-L-serine (L-PS) or dioleoyl phosphatidyl-D-serine (D-PS). Direct binding experiments demonstrated that pd-HFVa and rHFVa bound to membranes containing L-PS with 6 to 9 fold higher affinity than to membranes containing D-PS. Analysis of prothrombinase activity on these membranes demonstrated the difference in Vmax/K1/2Va between L-PS and D-PS was 15-fold and 23-fold higher for pd-HFVa and rHFVa, respectively. These data confirm that HFVa, similar to bovine FVa, prefers L-PS over D-PS. The rHFV-C2 domain bound to L-PS membranes with an affinity of 5.3 ± 1.7 nM. In contrast, the binding of C2 domain to D-PS membranes was undetectable. This indicates that the rHFV-C2 domain contains a stereo-specific binding site for PS. We next analyzed the membrane binding of four rHFVa mutants containing alanine substitutions within the FV-C2 domain. rHFVa mutants W2063A and W2064A both bound to membranes containing 20% L-PS with high affinity similar to native rHFVa with Kd’s of 0.97 ± 0.30 nM and 0.38 ± 0.18 nM, respectively. In contrast no detectable binding of W2063A or W2064A could be detected on membranes containing 20% D-PS. Mutants were also prepared containing alanine substitutions for amino acid side chains predicted to participate in two stereospecific PS binding sites based on modeling studies using the rHFV-C2 structure. The binding of (K2060, Q2085, S2115)A and (Q2078, N2082, R2187)A to L-PS and D-PS were characterized as described above. These mutants bound to both L-PS and D-PS membranes with relatively high affinity. The Kd values for binding of (K2060, Q2085, S2115)A and (Q2078, N2082, R2187)A to 20% D-PS membranes were 1.19 ± 0.31 nM and 1.11 ± 0.32 nM, respectively. These results suggest that these two sites play little or no role in the binding of FVa to PS membranes. In conclusion, the HFV-C2 domain contains stereo-specific binding sites for L-PS which require the indole side chains of W2063 and W2064. These findings provide new insights into the regulation of prothrombinase activity by PS.
Molecular Background of “Smeary” von Willebrand Factor Multimers.
Authors: Schneppenheim Reinhard , Marggraf Olivier , Eckert Heike , Obser Tobias , Oyen Florian , Pieconka Antje , Schneppenheim Sonja , Will Kerstin , Budde Ulrich
Published: October 2019

Background: Multimer analysis of von Willebrand factor (VWF) is a most important technique to classify patients with von Willebrand disease (VWD) type 2. Besides “classical” multimer patterns a “smeary” appearance of individual VWF oligomers is increasingly observed and has previously been regarded as a pre-analytical artifact.

Objective: To phenotypically and genotypically assess the molecular background of “smeary” VWF multimers.

Patients and methods: Samples of 8 VWD patients were analyzed in our reference lab (UB) for further classification and molecular testing. Multimer profiles were assessed by intermediate resolution gels. VWF:CB and VWF:GpIb binding were used as functional assays. VWF gene mutation analysis was performed in all index cases (IC). The causal relationship between genotype and phenotype was studied by analyzing recombinant mutants in comparison to wildtype VWF.

Results: In all IC the phenotype correlated with particular mutations in the VWF D3 domain (G1172D), the A1 domain (R1315C, R1374S, R1374C, R1399C), the D4 domain (C2257R), the C1 domain (R2464C) and in the region close to the CK domain (C2671Y), respectively. The multimer patterns of recombinant mutant VWF was of a “smeary” appearance and closely resembled those of mutant plasma VWF. Mutations in the A1 domain additionally correlated with severe GpIb binding deficiency.

Conclusions: Our data suggest a molecular cause of the “smeary” multimer structure rather than pre-analytical artifacts. Most of the mutations identified involved cysteine residues suggesting an influence on the VWF secondary structure which is determined by intra- and intermolecular disulfide bonds. This could explain the peculiar multimer appearance. The functional deficits, however, seem to depend on the location of the mutations with a significant impact on GpIb binding of mutants in the A1 domain.
Bortezomib Retreatment in Relapsed Multiple Mycelia - A Retrospective Multicenter Survey.
Authors: Hrusovsky Irena , Emmerich Berthold , Engelhardt Monika , Kornacker Martin , Gann Claudia-Nanette , Pliskat Harold , Hess Georg
Published: October 2019

Bortezomib (Velcade) has demonstrated highest single agent response rates in anti-myeloma therapy. There is no data from preclinical studies to suggest that resistance develops from repeated treatment with bortezomib. It was therefore of interest to investigate, whether repeated bortezomib treatment is safe, feasible and efficient. Here, we have retrospectively collected data from multiple myeloma patients who had previously responded to bortezomib (previous bortezomib treatment), presented with relapsed disease and who received bortezomib for a second time (retreatment). Treatment and retreatment had been on discretion of the physician according to prescribing information, and no diagnostic or therapeutic instructions were given during the course of this multicenter non-interventional survey (26866138MMY4014). Data from from a total of 15 centers and 65 patients were obtained: these patients had all received bortezomib and were eligible for safety analyses. Sixteen patients had to be excluded due to major protocol violations, leaving 49 patients for the modified intention to treat population. Patients had a median age of 66 years and had been treated with a median of 4 prior therapies before receiving bortezomib for the first time. Median cycle number for previous bortezomib therapy and retreatment was 5 and 4, respectively. The majority of patients (85.7%) received doses of 1.3 mg/m2 body surface area. Concomitant dexamethasone was given in 38.8% of patients with previous bortezomib treatment, and in 62.2% with retreatment. Six patients (12.2%) received various anti-myeloma therapies between bortezomib treatment and retreatment. Efficacy data is summarized below, revealing very encouraging responses and similar response durations for previous bortezomib therapy and retreatment.

Thirty-three (50.8%) patients experienced a total of 109 adverse drug reactions (ADRs). Three (4.6%) patients experienced a total of 12 life-threatening or disabling ADRs and six (9.2%) experienced 13 severe ADRs (related to bortezomib = 6 SADRs). By the end of the documentation period, 21 patients had died (32.8%). This retrospective survey suggests that the safety profile of bortezomib retreatment is in line with the current SmPC of Velcade and that high remission rates can be achieved. It remains to be shown if these bortezomib retreatment results can be repeated in prospective trials and larger patient cohorts.

Previous bortezomib treatment Retreatment Overall response rate 100% (per protocol) 63.3% Complete remission rate 12.2% 10.2% Time to response (median) 3.2 months 3.0 months Duration of response (median) 6.3 months 4.5 months Treatment free interval (median) 6.6 months 4.1 months Time to progression (median) 10.9 months 6.7 months
Longer Duration of Thalidomide Monotherapy Results in Improved Outcome in Relapsed/Refractory Multiple Myeloma.
Authors: von Lilienfeld-Toal Marie , Hahn-Ast Corinna , Bertolini Francesco , Bila Jelena , Boulin Mathieu , Oakervee Heather , Cibeira Teresa , Cook Gordon , Dmoszynska Anna , Fenk Roland , Guglielmelli Tommasina , Goldschmidt Hartmut , Hattori Yukata , Myers Bethan , Offidani Massimo , Patriarca Francesca , Petrucci Maria Teresa , Pini Massimo , Prince Miles , Schey Steve , Sonneveld Pieter , Yakoub-Agha Ibrahim , Waage Anders , Glasmacher Axel
Published: October 2019

Although efficacy of thalidomide (thal) monotherapy in relapsed/refractory Multiple Myeloma (MM) is proven, the optimal duration of treatment is unknown. Here, we analysed individual patient data of 852 patients (pts.) with relapsed/refractory MM from 21 centres who received thal monotherapy in a trial or in a compassionate use program. To assess the influence of treatment duration, pts. who discontinued thal because of lack of efficacy, progression or death were excluded, so we analysed only pts. who continued on thal as long as they tolerated it or until the study they were enrolled in was complete. 162 pts. were evaluable; 32 completed the trial, 57 had severe neurotoxicity, 2 severe somnolence, 72 other adverse events leading to discontinuation of thal. The median age was 65 years (interquartile range 58–72), 92 (56%) were male, 96 (59%) had relapsed MM. Sixteen (10%) achieved complete remission (CR), 64 (39%) partial remission (PR), 29 (18%) minimal response, 34 (21%) no change, 9 (6%) had progressive disease, 9 (6%) were unevaluable for response. The median daily dose at 3 months was 200 mg/d (100–400), the cumulative dose after 3 months 23200 mg (9025–34150). Median time to response was 3.6 months (3–7), median treatment duration 5 months (2.3–12). Median event-free survival (EFS) was 16 months (95%CI 12–21), median overall survival (OS) 33 months (95%CI 22–44). In univariate analysis, prognostic factors for EFS/OS were leukocyte/platelet count, haemoglobin, cumulative dose at 3 months, duration of treatment and CR/PR. In multivariate analysis, duration of treatment, CR/PR and platelet count remained independent prognostic factors for EFS/OS. If pts. took thal for ≥ 10 months (n=51), median EFS was 31 (95%CI 14–48) vs 12 (9–14, p<0.001) months and median OS 83 (95%CI 49–116) vs 24 (95%CI 17–31, figure 1, p<0.001) months. We analysed responding pts. (n=80) separately to assess the role of treatment duration in maintenance. Patients in CR/PR who took thal ≥ 10 months (n=41) had a longer EFS (39 (95%CI 19–59) vs 18 (95%CI 9–28, p<0.001) and OS (83 (95%CI 49–116) vs 32 (95%CI 20–44, p<0.001) months). This analysis of patients whose duration of treatment was determined by factors independent of disease demonstrates a clear survival benefit of longer duration of thal treatment for relapsed/refractory MM and supports its value as maintenance therapy.

Figure Figure
Recruitment of Rad50 and MCM Complexes on the Human Beta Globin Locus Control Region (LCR): A Novel Role for LCR Hypersensitive Site 4 (HS4).
Authors: Karmakar Subhradip , Mahajan Milind C. , Weissman Sherman M.
Published: October 2019

The human beta globin locus control region (LCR) aids in the establishment of an open chromatin configuration thereby facilitating the expression of linked downstream globin genes in a temporal and spatial manner along the developmental ontogeny. The LCR also functions as a classical enhancer of the beta like globin genes. The four DNAase-I hypersensitive regions (HS 1–4) on the LCR are believed to act as nucleation centers for the assembly of multi-protein transcriptional complexes that interact with the distant gene promoters. Present work is focused on characterizing the protein complex assemblies on the HS4 region and delineating their function in the context of goblin gene regulation. Our finding shows an assembly of a 2–5 MDa complex on the HS4 locus that is both erythroid restricted and sequence specific. Electrophoretic mobility shift assays (EMSA) were performed using K562 and HeLa nuclear extracts and ∼35mer double stranded oligonucleotides with 7 bp overlaps tiling the entire core HS4 sequence. We detected prominent K562 specific bands formed with an AT rich 39 bp HS4 sequence. Sequence specificity of DNA-protein complex formation on this AT rich sequence was determined by competitive EMSA assays. Gel supershift assays with a wide range of antibodies and subsequent partial purification of this multiprotein complex followed by the LC-MS/MS analysis reveal the presence of a mini chromosome maintenance (MCM) complex that is associated with the DNA replication, DNA repair associated Rad50-Mre-11-NBS-1 complex, histone acetyl transferase p300 and DNA binding transcription factors ILF2 (NF45) and ILF3 (NF90). The association of these proteins was determined by co-immunoprecipitation experiments. Chromatin immunoprecipitation (ChIP) experiments demonstrate the in vivo recruitment of these proteins on the HS4 region of the LCR. We are currently working on further characterization and functional studies of this complex and its significance on the role of the LCR in DNA replication and repair.
Bortezomib, Doxorubicin and Dexamethasone (PAD) Combination Therapy Followed by Thalidomide and Dexamethasone (TD) as a Salvage Treatment for Relapsed Multiple Myeloma (MM): Preliminary Analysis of Efficacy and Safety.
Authors: Lee Sung Sook , Suh Cheolwon , Kim Bong-Seog , Chung Jooseop , Joo Young-Don , Ryoo Hun-Mo , Do Young Rok , Jin Jong-Youl , Kang Hye Jin , Lee Gyeong-Won , Lee Moon Hee , Shim Hyeok , Kim Kihyun , Yoon Sung-Soo , Kim Ho Young , Lee Dong Soon , Lee Jae-Hoon
Published: October 2019

Introduction: PAD was reported to be highly effective regimen as an induction therapy before high dose therapy. TD is an another effective regimen with no cross resistance. We conducted a phase II study with PAD followed by TD in relapsed MM to test effectiveness of this combination.

Method: Patients were planned to receive 6 cycles of PAD, (bortezomib 1.3 mg/m2 days 1, 4, 8 and 11, doxorubicin 4.5 mg/m2 days 1–4, dexamethasone 40 mg days 1–4, every 21 days). Responders following 6 cylces of PAD received 12 cycles of TD (thalidomide 100 mg days 1–28 and dexamethasone 40 mg days 1–4, every 28 days). In patients with progression during PAD therapy, regimen was changed to 12 cycles of thalidomide 200 mg days 1–28 and dexamethasone 40 mg days 1–4, every 28 days.

Result: This study aimed to enroll 40 patients till Oct 2007 and we are reporting preliminary result with 35 patients. Efficacy could be assessed in 29 patients. After two cycles of PAD, 29 patients showed response with 5 CR. Overall response rate to 6 cylces of PAD was 96.4% with 39.3% CR. Six of total 13 patients with TD showed further improvement of response status with 5 additional CR. Overall response to PAD followed by TD was 92.8%: CR 57.1%, nCR 10.7%, VGPR 7.1%, PR 17.9%, SD 3.6%, PD 3.6%. There was no prognostic factor for CR+nCR achieving in the univariate analysis. The median follow-up was 8.4 months with 1 year PFS 66.8% and 1 year OS 59.4%. One hundred fourty-six PAD cycles (median 5, range 1–6) in 35 patients were assessable for safety. The most common hematologic toxicity was thrombocytopenia, with grade 3/4 in 37.1%. Grade 3/4 neutropenia was observed in 5.8%. Sensory neuropathy occurred with grade 2 in 31.4% and grade 3 in 8.6%. The median dose intensity was 1.44 mg/m2/week for bortezomib and 5.25 mg/m2/week for doxorubicin, which correspond 83.2% and 87.5% of the planned dose intensities, respectively. A total of 112 TD treatment cycles (median 0, range 0–12 cycles) was administered. Two patients developed grade 3 neutropenia. One patient die by the pneumonia.

Conclusion: PAD followed by TD in patients with relapsed multiple myeloma is very active and tolerable. Updated results will be presented at the meeting.

*Protocol Number: KMM55-NCT00319865
Effect of Disease Stage and Time Since Diagnosis on Time to Progression for Pegylated Liposomal Doxorubicin + Bortezomib vs Bortezomib Alone in Relapsed or Refractory Multiple Myeloma.
Authors: Sutherland Heather J. , Blade Joan , Miguel Jesus San , Nagler Arnon , Sonneveld Pieter , Spencer Andrew , Robak Tadeusz , O’Neal Wane , Mundle Suneel D. , Zhuang Sen H. , Harousseau Jean-Luc , Orlowski Robert Z.
Published: October 2019

A recent report of a phase III randomized trial with pegylated liposomal doxorubicin (PLD)+bortezomib vs. bortezomib alone in relapsed or refractory multiple myeloma (RRMM) demonstrated improved time to progression (TTP) with PLD+bortezomib (Orlowski, JCO 2007). The present post-hoc analyses evaluated the efficacy of PLD+bortezomib according to International Staging System (ISS) disease stage, and time since initial myeloma diagnosis (TSD). Patients with ≥1 prior therapy were randomized to receive PLD at 30 mg/m2 on day 4 and bortezomib at 1.3 mg/m2 on days 1, 4, 8, and 11, or bortezomib alone for up to eight 21-day cycles, or at least 2 cycles beyond CR, or optimal response unless disease progression or unacceptable toxicities occurred. The improved TTP reported previously with PLD+bortezomib over bortezomib alone in the total study population was also observed in the higher risk groups based on disease stage (ISS 2 & 3; Table). TTP was also significantly longer with PLD+bortezomib vs. bortezomib for TSD >2 yrs despite more protracted disease. The therapeutic advantage of prolonged TTP with the PLD+bortezomib combination was maintained consistently across subgroups (heterogeneity tests: ISS 1 vs. 2 vs. 3, p=0.407; TSD ≤2 yrs. vs. >2 yrs., p=0.946). Incidence of grade 3/4 neuropathies was low (<10%) in the two treatment arms in all subgroups. PLD-related hand-foot syndrome was also <10% in all PLD+bortezomib subgroups. Despite high-grade disease or protracted disease history, the PLD+bortezomib combination shows significantly improved TTP as compared to bortezomib alone. Also, the combination therapy was well-tolerated.
Use of Gene-Modified T-Cells as Antigen Presenting Cells (T-APC) for Vaccination Against Myeloma Antigens.
Authors: Anderson Larry D. , Cook Danielle R. , Berger Carolina , Maloney David G. , Riddell Stanley R.
Published: October 2019

Multiple myeloma is incurable with currently available standard treatments, and novel therapies with lower toxicity, including immunological therapies, are needed. Myeloma cells express class I MHC molecules and several potentially immunogenic proteins. Candidate target antigens, other than the patient-specific immunoglobulin idiotype, include cancer-testis antigens, such as MAGE-C1 (CT-7), and tissue differentiation antigens, such as B-Cell Maturation Antigen (BCMA). We have isolated, from normal donors, CD8+ T-cells that are CT-7 and BCMA peptide-specific, suggesting that immunologic tolerance to these proteins is incomplete. A subset of these CD8+ T-cells with the highest avidity are capable of recognizing myeloma cells. However, the frequency of T-cells recognizing these peptides is very low. Vaccination offers a potential approach for augmenting the frequency of tumor-reactive T-cells, provided that a vaccine strategy can be developed that elicits high-avidity T-cells. We have previously shown that adoptive transfer of autologous gene-modified T-cells leads to the rapid development of potent CD8+ T-cell immunity against transgene products expressed in the transferred T-cells. The aims of this study were to determine if vaccination with T-cells modified to express the CT-7 or BCMA genes (T-APC) could induce anti-myeloma immunity in an HLA-A2 transgenic mouse model, and to map additional immunogenic epitopes of these proteins presented by HLA-A2. To address this we genetically modified HLA-A2 transgenic mouse T-cells with retroviral vectors encoding the candidate myeloma antigens CT-7 and BCMA, or with a control vector encoding green fluorescent protein (GFP). The gene-modified T-APC were injected intravenously into HLA-A2 transgenic mice, and ELISPOT and cytotoxicity assays were performed on splenocytes from immunized mice to evaluate T-cell recognition of target cells expressing CT-7 or BCMA (including human myeloma cell lines). T-cell responses specific for CT-7, BCMA, and control antigens were elicited following gene-modified T-APC immunization, and in some cases these T-cells were capable of recognizing human myeloma cells. Furthermore, responses to CT-7 have been mapped to two different dominant CT-7 peptides. CD8+ T-cells from some normal A2+ donors also recognized these two epitopes after in vitro peptide stimulation, suggesting that these epitopes may be applicable to human myeloma therapy. Characterization of the BCMA target peptide(s) is ongoing. The ease with which T-cells can be obtained, genetically-modified, and expanded in vitro, together with data showing potent immunogenicity, suggests that T-APC vaccination should be evaluated as an alternative cell based vaccine strategy for human cancer therapy.
Inhibition of KSP by ARRY-520 Induces Cell Cycle Block and Cell Death Via the Mitochondrial Pathway in AML Cells.
Authors: Carter Bing Z. , Mak Duncan H. , Schober Wendy D. , Woessner Richard , Gross Stefan , Harris David , Estrov Zeev , Andreeff Michael
Published: October 2019

Kinesin spindle protein (KSP or Eg5), a microtubule-associated motor protein, plays an important role in establishing a bipolar spindle during mitosis and is essential for cell cycle progression. It has been demonstrated that inhibition of KSP prevents bipolar spindle formation leading to mitotic arrest and cell death. As such, a KSP inhibitor may have anti-tumor potential without toxicities associated with anti-microtubule agents such as taxanes. We found that KSP is highly expressed in various acute leukemic cells. Inhibition of KSP by a specific inhibitor, ARRY-520, at low nanomolar concentrations, blocked cell cycle progression and led to subsequent cell death in OCI-AML3, Molm13, HL-60, U937, and Jurkat cells. Knocking down p53 by p53shRNA in OCI-AML3 cells did not alter the effectiveness of ARRY-520. U937 cells overexpressing XIAP (U937XIAP) and Jurkat cells lacking caspase 8 (JurkatI2.1) showed unchanged sensitivities to ARRY-520 compared with their respective control cells, suggesting that cell cycle block and cell death induced by KSP inhibition is independent of p53 status, XIAP levels, and the activation of the extrinsic apoptotic pathway. However, although ARRY-520 blocked cell growth and induced mitotic arrest in both HL-60 cells and HL-60 cells overexpressing Bcl-2 (HL-60Bcl-2), cell death (determined by annexin V staining and changes in mitochondrial potential) was significantly abolished in HL-60Bcl-2 cells. These results suggest that cell death following cell cycle blockade by KSP inhibition is mediated through the intrinsic mitochondrial pathway. Furthermore, ARRY-520 induced the protein level of Bim, a proapoptotic BH3-only Bcl-2 family protein, prior to the activation of caspases in HL-60 cells. Although KSP inhibition by ARRY-520 had no effect on the survival of non-dividing AML blasts in vitro, ARRY-520 significantly inhibited the colony formation capacities of AML blasts, further supporting the critical role of KSP in cell proliferation. Our studies demonstrated that inhibition of KSP by ARRY-520 potently induces blockage of cell cycle progression which leads to cell death of various leukemic cells via the mitochondrial pathway and has the potential to eradicate AML progenitor cells.
RNAi-Mediated Silencing of p190Bcr-Abl Inactivates Stat5 and Cooperates with Imatinib Mesylate and 17-Allylamino-17-Demetoxygeldanamycin in Selective Killing of p190Bcr-Abl-Expressing Leukemia Cells.
Authors: Futami Muneyoshi , Hatano Toshiyuki , Soda Yasushi , Kobayashi Seiichiro , Miyagishi Makoto , Tojo Arinobu
Published: October 2019

The 190kD (p190) and 210kD (p210) Bcr-Abl proteins are responsible for the development and progression of Philadelphia chromosome (Ph)-positive leukemia. We applied RNA interference (RNAi) technology to specific killing of p190+ cells. After screening a series of small hairpin RNA (shRNA) targeting p190, we determined the optimal sequences for gene silencing in the BCR (shBCR), junctional (shE1A2) and ABL (shABL) region of p190, respectively. Then, p190+ (KOPN30), p210+ (K562) and both negative (HL60) cell lines were infected with lentiviral vectors encoding these shRNAs, resulting in an efficient killing of KOPN30 cells. K562 cells were also sensitive to the two other than junction-specific shRNA and none of the anti-p190 shRNAs could affect survival of HL-60 cells, although shABL appeared to have some inhibitory effects on their growth, suggesting that Abl may be positively involved in proliferation of HL60 cells. Furthermore, colony formation derived from CFU-GEMM, BFU-E and CFU-GM was not influenced by shBCR. On the contrary, CFU-GEMM and BFU-E-derived colony formation was significantly suppressed by shABL, suggesting that Abl may play a certain role especially in normal erythropoiesis. Nonetheless, anti-p190 shRNAs may become therapeutic options in Ph-ALL by improvement of their delivery system such as CD19-targeted liposome formulation which we previously developed. In p190-transformed Ba/F3 cells, RNAi-mediated silencing of p190 specifically inhibited tyrosine phospohorylation of stat5 prior to their death, but did not affect phosphorylation of either Jak2, Akt, MEK1/2 or ERK1/2. These results offer another evidence for the critical role of Stat5 in BCR-ABL-induced transformation of hematopoietic cells. It is likely that p190 directly phosphorylates Stat5 and that Jak2 is remote from activation of Stat5 in this cell context, although the possible involvement of Src family kinases such as Hck and Lyn has not been excluded. In contrast, down-regulation of p190 by their treatment with 17-allyl-amino-geldanamycin (17-AAG) was associated with reduced phosphorylation of not only Stat5 but also Jak2 and Akt. shRNA targeting p190 also collaborated additively with imatinib and 17-AAG in growth inhibition of Ba/F3-p190 and BaF/3-p190Y253H cells. Collectively, RNAi-mediated silencing of p190 is a promising option both for therapeutic application and for delineating signal transduction in p190-expressing leukemia.

Figure Figure Figure Figure
The Novel Histone Deacetylase Inhibitor, LBH589, Induces Expression of DNA Damage Response Genes and Apoptosis in Ph-Acute Lymphoblastic Leukemia Cells.
Authors: Anna Scuto , Kirschbaum Mark H. , Atadja Peter , Juhasz Agnes , Pullarkat Vinod , Bhatia Ravi , Forman Stephen J. , Yen Yun , Jove Richard
Published: October 2019

We investigated the mechanism of action of LBH589, a novel broad-spectrum HDAC inhibitor belonging to the hydroxamate class, in Philadelphia chromosome-negative (Ph−) acute lymphoblastic leukemia (ALL). Two model human Ph− ALL cell lines (T-cell MOLT-4, and non-T non-B cell Reh) were treated with LBH589 and evaluated for biological and gene expression responses. Low nM concentrations (IC50 5–20 nM) of LBH589 induced cell cycle arrest, apoptosis and histone (H3 and H4) hyperacetylation. PCR array analysis revealed that LBH589 treatment increased mRNA levels of pro-apoptosis, growth arrest and DNA damage repair genes. Quantitative real-time PCR confirmed that LBH589 induces expression of FANCG, FOXO3A, GADD45A, GADD45B and GADD45G. The most dramatically expressed gene (up to 45-fold induction) observed after treatment with LBH589 is GADD45G. Chromatin immunoprecipitation (ChIP) assays demonstrated increased histone acetylation at the GADD45G promoter following LBH589 treatment. Finally, treatment with LBH589 was active against cultured primary Ph− ALL cells, including those from a relapsed patient, inducing loss of cell viability (up to 70%). Thus, LBH589 possesses potent growth inhibitory activity against Ph− ALL cells associated with upregulation of genes critical for DNA repair. These findings provide a rationale for exploring the clinical activity of LBH589 in the treatment of patients with Ph− ALL.