bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the Endoplasmic Reticulum (ER) Unfolded Protein Response (UPR) in Aggressive B-Cell Lymphomas.
Authors: Balague Olga , Colomo Luis , Lopez-Guillermo Armando , Campo Elias , Martinez Antonio
Published: October 2019

BACKGROUND The UPR is a prosurvival pathway activated in cells under ER stress induced by the accumulation of unfolded proteins. UPR activation in B cells normally occurs during the differentiation to antibody secreting plasma cells and requires XBP1activation. XBP-1 is a member of the TREB family of transcription factors that exists in the endoplasmic reticulum (ER) as a 33kDa protein, and in the nucleus as an active 50kDa transcription factor. The UPR stimulates two different ER proteins, ATF-6 and Ire-1, to increase XBP-1 transcription and XBP-1 mRNA splicing resulting in the accumulation of the active 50kDa nuclear protein. Moreover XBP1 is a target of proteosome inhibitors and is related to the aggressive behaviour of some carcinomas. The role of the activation of XBP-1 in lymphomas is still unknown.

DESIGN: Reactive lymphoid tissues and 25 neoplastic human B-cell lines representing different stages of B-cell development were studied for XBP-1 expression by western blot and XBP-1, PAX-5, Blimp-1/prdm1, MUM-1/IRF-4 and ICSBP1/IRF-8 by immunohistochemistry. XBP-1 activation was assessed in 225 B-cell lymphomas from the archives of the laboratory of pathology by western blot, RT-PCR and immunohistochemistry . To further evaluate whether XBP-1 activation was related to the plasmacytic program or to ER stress signals we analyzed the cell lines by Western blot for XBP-1 and ATF-6 expression.

RESULTS We characterize XBP-1 expression in reactive lymphoid tissues, 25 human cell lines and 225 B-cell tumors. In nearly all tonsillar lymphoid cells XBP-1 was detected as a cytoplasmic protein with a paranuclear dot pattern. Nuclear positivity was observed only in scattered centrocytes in the light zone of the germinal centers and in plasma cells, always coexpressed with plasma cell related transcription factors as MUM-1/IRF-4 and Blimp1/prdm1. Active p50XBP-1 was found in 24/25 cell lines by western blot regardless ATF-6 expression and confirmed by immunohistochemistry . Moreover p50XBP1 was found in 27/31(87%) plasmacytomas, 36/64(56%) DLBCL-ABC and in 3/10(30%) DLBCL-GCB and 22/43(51%) plasmablastic lymphomas. Intriguingly, p50XBP1 was detected also in 2/11(18%)BL and 4/25(16%)MCL with blastic features.

CONCLUSIONS.XBP-1 is activated in a subset of follicular centre cells committed to plasma cell differentiation and in plasma cells.UPR prosurvival pathways in the neoplastic cell lines are activated independently of the extent of the ATF-6 activation.p50XBP1 is mostly activated in aggressive B-cell lymphomas regardless to the plasmacytic differentiation of the tumours.

Thus, p50XBP-1 may be a new molecular target in the treatment of aggressive B-cell malignancies.
Cell Adhesion Induces p27Kip1-Associated Cell-Cycle Arrest through Down-Regulating SCFSkp2 Ubiquitin Ligase Pathway in Mantle Cell and Other Non-Hodgkin’s B-Cell Lymphomas.
Authors: Lwin Tint , Hazlehurst Lori A. , Moscinski Lynn C. , Dalton William S. , Tao Jianguo
Published: October 2019

Mounting evidence now suggests that dynamic interactions between the tumor cell and its local and systemic microenvironment play a critical role in tumor development and that all of the clinical properties of a tumor, including cell cycle progression and response to therapy, depend heavily on the tumor stroma. However, the molecular mechanisms of the interaction between stroma and tumor cells are not well defined. In this study we used mantle cell lymphoma (MCL) as a model to characterize the mechanisms by which stroma regulate cell-cycle progression. We demonstrated that adhesion of MCL and other non-Hodgkin lymphoma (NHL) cells to bone marrow stroma cell (HS-5) resulted in a reversible G1 arrest associated with elevated p27Kip1 protein level. The adhesion mediated p27Kip1 increase was posttranslational and regulated through down-regulating Skp2, a substrate-recognition subunit of SCFSkp2 ubiquitin ligase. Overexpression of wild-type Skp2 in MCL decreases p27Kip1, whereas inhibition of Skp2 expression by siRNA increases p27Kip1 level. Furthermore, we found that cell adhesion up-regulated Cdh1, an activating subunit of anaphase-promoting complex (APC) ubiquitin ligase, and reducing Cdh1 by siRNA induced Skp2 accumulation and hence p27Kip1 degradation, implicating Cdh1 as an upstream effector of Skp2/p27Kip1 signaling pathway. Overall, this is a first report demonstrating that the bone marrow stroma controls tumor cell cycle via ubiquitin-proteasome proteolytic pathways in MCL and other NHL. The understanding of this novel molecular pathway may prove valuable in designing new therapeutic approaches modifying tumor cell growth and response to therapy.
Clinicopathologic Subtypes of Mantle Cell Lymphoma (MCL) Show Distinct Patterns of Genetic Copy Number Alteration.
Authors: deLeeuw Ronald J. , Malloff Chad A. , Kimm Lindsey R. , Connors Joseph M. , Dyer Martin J.S. , Gascoyne Randy D. , Lam Wan L.
Published: October 2019

Introduction: MCL is an aggressive NHL characterized by a t(11;14)(q13;q32). However this translocation alone cannot initiate lymphomagenesis and multiple secondary genomic alterations are necessary to induce the neoplastic phenotype. In addition, three distinct clinicopathologic subtypes of MCL (classic, blastoid, and leukemic) have been identified with differing prognosis. Secondary genomic alterations associated with each subtype have been poorly defined. Experimental

Design: Genomic profiles from a panel of MCL samples comprised of 20 classic, 5 leukemic (no nodal involvement), and 12 blastoid cases, were created using a tiling-resolution BAC array CGH platform. Alignment of profiles to identify regions of recurrent alteration was accomplished using a custom developed software package (SeeGH). In addition, specific genes were analyzed for copy number status. Statistically significant distributions were determined using Chi square and Fisher’s exact analyses.

Results: Twenty two regions of recurrent alteration were identified that were statistically different between subtypes (partial data in Table 1). As expected chromosome 9 losses were more prevalent in blastoid MCL. 13q34 losses were prevalent within classic and blastoid MCL, as opposed to leukemic MCL, which showed gains of this region. Analysis of biological pathways revealed that apoptotic signaling is not disrupted via copy number in a large portion of MCL cases (21%), while chromosomal instability genes show copy number disruption in 67% of MCL cases. Surprisingly, the glioblastoma pathway was preferentially disrupted in blastoid MCL (p=1.09x10−5), affecting all cases; whereas the p53 pathway was disrupted via copy number more often in leukemic MCL (p=0.0207).

Conclusions: Multiple genomic regions and cancer related genes show a differential pattern of altered copy number distribution between MCL subtypes. These regions and genes likely contribute to the different clinicopathologic subtypes of MCL cases.

Differential regions in MCL subtypes +/− chr. band size (Mb) clas% leuk% blast% enriched in deficient in − 1q25.2-q31.2 15.50 0 20 33 blast (p=0.03) class (p=0.01) − 2q26.3 2.91 5 0 33 blast (p=0.03) + 3cent-p12.1 4.18 0 60 25 leuk (p=0.02) class (p=0.005) + 7q11.23 3.24 0 0 33 blast (p=0.008) class (p=0.04) − 9p21.3 1.02 10 20 83 blast (p=0.00001) class (p=0.002) − 9q21.13 1.10 5 40 75 blast (p=0.0003) class (p=0.0002) − 9q21.2-q31.1 22.87 5 40 67 blast (p=0.001) class (p=0.0007) − 9q31.1 0.83 0 40 67 blast (p=0.0004) class (p=0.00006) − 9q34.12-qter 7.27 0 20 42 blast (p=0.009) class (p=0.005) + 10p12.2-p12.31 0.63 5 0 42 blast (p=0.009) − 13q34 1.69 40 0 75 blast (p=0.03) leuk (p=0.05) − 17p13.3-pter 2.28 5 60 25 leuk (p=0.04) − 17p13.1 1.21 5 60 25 leuk (p=0.04)
Prognostic Factors in Childhood Anaplastic Large Cell Lymphoma (ALCL): Results of the European Intergroup Study.
Authors: Le Deley Marie Cécile , Brugières Laurence , Williams Denise , Reiter Alfred
Published: October 2019

Purpose and methods

In order to study prognostic factors of disease progression and relapse, the data of 235 children enrolled for an ALCL between 1987 and 1997 in the BFM (93 patients, pts), SFOP (82 pts) and UKCCSG (60 pts) studies have been merged in 1998. All these children have been treated according to protocols designed to treat childhood ALCL with a short and intensive chemotherapy treatment, similar to those used for B-cell. Slides had been reviewed by the national pathologist panel for 226 patients (96%). Hodgkin-like ALCL were excluded from this series. Follow-up has been updated in April 2006.

Results

Among the 235 pts included in the study, 206 pts (88%) achieved a CR. With a median follow-up of 7.4 years, 77 events were observed (13 early progressions, 55 relapses, 3 secondary leukaemias, 6 toxic deaths) and 46 patients died. The 5-year overall survival and event-free survival of the whole population is 81% [95%CI, 76–86%] and 69% [63–75%] respectively. Event-free survival differs significantly according to the treatment (p=.01) : the risks of failure of children treated in the SFOP studies and in the UKCCSG studies are multiplied by 1.8 [1.05–3.2] and 2.3 [1.3–4.1] respectively, as compared to pts treated in the BFM studies.

Several factors seem to be prognostic of the risk of early progression or relapse in univariate analysis: B-symptoms, mediastinal involvement, skin lesions, visceral involvement, St Jude stage 3–4 or Ann Arbor stage 3–4 or elevated LDH. Patients with bone lesions seem to have a better prognosis than others. Soft tissue masses do not worsen the prognosis.

In multivariate analysis, three factors are found to be associated to the risk of progression/relapse :

* mediastinal involvement (hazard ratio of progression/relapse, HR=2.2 [1.3–3.6], p=.003), * visceral involvement defined as lung, liver or spleen involvement (HR=2.1 [1.3–3.5], p=.005), * skin lesions (HR = 1.9 [1.2–3.2], p=.01).

Based on the results of this Cox model, it is possible to define:

* a good prognosis group (85 patients without skin, mediastinal or visceral involvement) with 89% pts [82–96%] free of progression/relapse at 5 year * a poor prognosis group (150 patients with skin and/or mediastinal and/or visceral involvement) with 61% pts [53–69%] free of progression/relapse at 5 year

This risk group classification appears significant in each of the three treatment groups.

Conclusion :

Results obtained with BFM-studies are significantly better than those obtained with the French and British pediatric studies. Pts with mediastinal involvement, visceral involvement and/or skin lesions are at a higher risk of disease failure. These results was the basis of ALCL99 aiming to improve EFS of high risk pts by adding vinblastine to BFM protocol.
Array CGH Analysis for PTCL-U Revealed Two Genetically Distinct Subgroups.
Authors: Nakagawa Masao , Oshiro Aya , Tagawa Hiroyuki , Karnan Sivasundaram , Tsuzuki Shinobu , Ohshima Koichi , Nakamura Shigeo , Seto Masao
Published: October 2019

Peripheral T-cell lymphoma, unspecified (PTCL-U) is the most common group among Peripheral T-cell lymphomas (PTCLs). This category consists of the cases which do not belong to any of the recognizable subtypes of PTCLs in WHO classification. PTCL-U comprises heterogeneous groups in morphology and phenotype. Molecular basis of clinical heterogeneity is needed to identify distinct subgroups with clnical relevance. Several reports of conventional cytogenetic studies including comparative genomic hybridization (CGH) showed some recurrent aberrations, but failed to identify the genetic hallmarks to categorize distinct subgroups. So far, no array-based comparative genomic hybridization (array CGH) study for PTCL-U has been reported. Here we analyzed 29 cases of PTCL-U by means of array CGH consisting of 2265 artificial chromosome clones that cover the whole genome at a 1.3 mega base resolution. The analysis clearly divided these cases into two distinct subgroups on the basis of frequency of genomic alterations. One group consists of 17 cases which showed significant lower copy number changes (average copy number gains: 0.5 regions, average copy number losses: 0.1 regions). The other group had average copy number gains of 15.7 regions and losses of 15.0 regions in 12 cases. We designate the former as “simple type” and the latter as “complex type”. In the complex type, regions of recurrent (>20%) gain are detected on chromosome 1q23.3-24.2, 3q25.31-tel, 4p15.1-16.1, 4q28.3-31.23, 5q34, 6p24.1-25.1, 7p21.3-tel, 7p21.1, 7q, 8q24.23, 11q13.4-tel, 12p11.21-11.22, 16p12.3-13.3, 17q11.2-22. Regions of recurrent (>20%) losses are detected on chromosome 1p13.1-13.3, 2q37.3, 4q21.21-21.23, 4q34.3-35.1, 5q21.2-23.1, 6p12.1-q14.3, 6q23.2-24.1, 6q25.1-26, 7p14.3-22.1, 9p21.3, 10p14-qtel, 12p13.1-13.2, 13, 14q12, 16q, 17p, 18p, 20q13-2, 22q11.21-12.2. Median age is 62 years in the simple type and 73 years in the complex type, respectively. Median survival is 27 months in the simple type and 11 months in the complex type. Log-rank test for overall survival between the simple type and the complex type showed inferior survival for the complex type but significance was marginal (p=0.21). Our findings showed that PTCL-U comprised two genetically distinct subgroups, implying that distinct mechanisms underlay in molecular pathogenesis of PTCL-U. Furthermore cilinicopathological features of each group are also being studied.
Outcome-Related Genome Loci in T-Cell Non-Hodgkin’s Lymphoma Identified by High-Resolution SNP-Typing Arrays.
Authors: Fujiwara Shin-ichiro , Yamashita Yoshihiro , Nakamura Naoya , Abe Masafumi , Ozawa Keiya , Mano Hiroyuki
Published: October 2019

Peripheral T-cell lymphoma, unspecified (PTCL) and angioimmunoblastic T-cell lymphoma (AITL) are non-Hodgkin’s lymphoma of T-cell origin. Despite their relatively poor outcome, little is yet understood for the molecular pathogenesis of these disorders. To screen for transforming/tumor-suppressor genes among PTCL/AITL, we here tried to examine copy number alterations (CNAs) among HTLV-I-negative lymph node specimens of PTCL (n = 32) and AITL (n = 42). Utilization of high-density SNP-typing arrays (GeneChip Mapping 100K Hind III, Affymetrix) has allowed us to measure CNA at a mean resolution of 47 kbp among the samples. Interestingly, both of PTCL and AITL cells have frequent CNAs throughout the genome. Among the 55,700 SNP sites examined for PTCL, for instance, chromosome copy number at 20,860 sites was shown to be ≥ 3 in at least 2 specimens, while loss of copy number (≤ 1) was found at 2,287 sites in ≥ 2 samples. Similarly, among the AITL specimens, copy number gain (≥ 3) in ≥ 2 samples was observed at 9,897 SNP sites, and copy number loss in ≥ 2 samples was identified at 43 sites. We further searched for frequent CNAs among the whole dataset, revealing that copy number gain of ≥ 4 was observed in ≥ 10 cases at 1q, 2p, 2q, 8q, 9p, 13q, 15q, and 19q. On the other hand, copy number of ≤ 1 was found among ≥ 10 cases at 14q. Further, the copy number of a genomic region was shown to be linked to a differential diagnosis of PTCL/AITL. From the same dataset, we could also reveal a vast number of loss-of-heterozygosity (LOH) regions with a divergent length. Frequent LOH (≥ 5 cases) was mapped to 2q, 5q, 8p and 9p. By using a Cox proportional hazard analysis, we then investigated whether chromosome copy number or LOH likelihood is statistically linked to prognosis of the patients enrolled in this study. Interestingly, the copy number of narrow regions at 2q, 5p, 5q and 17q was shown to be statistically associated with the clinical outcome (P <0.01) while the LOH-likelihood at 8q was linked to it (P <0.05). The chromosomal regions identified in our screening are the good candidates where transforming- or/and clinical character-related genes are mapped. Especially, the narrow regions thus identified contain a few genes per region, and provide us with a provocative list of PTCL/AITL-related genes.
Genomic Profiling of Chronic Lymphocytic Leukemia Identifies Novel Genetic Subtypes.
Authors: Malek Sami N. , Kujawski Lisa A. , Erba Harry P. , Kaminski Mark S. , Jakubowiak Andrzej , Dressel Amanda , Saddler Chris M. , Shedden Kerby A. , Ouillette Peter D.
Published: October 2019

CLL is the most common leukemia in the Western world and displays considerable clinical heterogeneity. Genetic approaches to elucidate defects in CLL genomes have resulted in the identification of recurrent chromosomal abnormalities with prognostic importance. A genome-wide high-density unbiased view of chromosomal copy number changes or loss of heterozygosity (LOH) may allow for further refinements of genomics-based risk prognostication. We have conducted a genomic profiling study using 50KSNP-oligonucleotide arrays (Affymetrix) on FACS-sorted CD19+ cells and paired buccal DNA from 120 patients with typical CLL (CD5+/CD23+) at various stages of presentation enrolled in a clinical translational protocol. We have found novel genetic subtypes, including novel regions of recurrent LOH and chromosomal copy loss on chromosomes 3p, 14q and 18p at frequencies of 3–6%. Fine mapping of the clinically important regions del(6q), del(11q), del(13q14) and del(17p) suggests molecular heterogeneity of these lesions. Anatomic criteria suggest existence of multiple distinct subtypes of del13q14. Furthermore, the clinically important del(17p) is associated with almost uniform deletion of most of 17p, occasionally displays copy-neutral LOH (undetectable by FISH) with p53 mutations and occurs without p53 mutations in a subset of patients as previously reported. Finally, using a genome-wide composite genomic instability score of copy gains and losses we found a subset of CLL cases (18%) with high genomic instability values (greater than 2 lesions per genome). These findings may have implications for CLL risk-adapted therapies.
Flavopiridol Decreases Mcl-1 and Initiates Early Mitochondrial Damage in Chronic Lymphocytic Leukemia (CLL) Cells.
Authors: Lucas David M. , Hussain Syed-Rehan A. , Johnson Amy J. , Smith Lisa L. , Wagner Amy J. , Allison Jamie M. , Guster Sara E. , Lin Thomas S. , Byrd John C. , Julian Mark W. , Crouser Elliott D. , Grever Michael R.
Published: October 2019

We have noted impressive activity of the cyclin-dependent kinase inhibitor flavopiridol in advanced-stage CLL patients, including those with a deletion of chromosome 17p13. Using a novel, effective schedule of flavopiridol, substantial and sometimes dramatic evidence of tumor cell death is observed as early as 4–6 hours. This is accompanied by hyperkalemia, hyperphosphatemia, hypocalcemia, and dramatic elevation in LDH consistent with acute tumor lysis syndrome. Studies by several groups including our own have demonstrated that Mcl-1 protein and mRNA is down-regulated with flavopiridol. Mcl-1 is an important protein that contributes to mitochondrial membrane stability. We have therefore sought to determine the role of mitochondria disruption in the mechanism of action of flavopiridol. By flow cytometry using the voltage-sensitive dye JC-1, loss of mitochondrial membrane potential was detected in flavopiridol-treated whole blood as early as five hours, prior to the onset of annexin-V or propidium iodide staining. This is in contrast to in vitro studies using human serum, in which mitochondrial depolarization and annexin-V staining occurred simultaneously. In isolated CLL cells treated with flavopiridol in vitro, loss of mitochondrial membrane potential was not affected by inhibitors of caspases 8 or 9 or by the broad caspase inhibitor Z-VAD-fmk, although apoptosis was effectively blocked by Z-VAD-fmk and caspase-8 inhibitor, and to a lesser extent, caspase-9 inhibitor. Flavopiridol was also able to effectively induce apoptosis and mitochondrial membrane depolarization in Jurkat cell lines deficient in caspase-8 or its adapter protein FADD. Additionally, lymphoid cells overexpressing Bcl-2 are resistant to flavopiridol-mediated apoptosis relative to the vector control. This suggests there is not direct binding of flavopiridol or its metabolites to APAF-1 (cytosolic adapter protein) and apoptosome assembly, as this process is insensitive to Bcl-2 family proteins. Further mechanistic studies were undertaken using isolated liver mitochondria. While the electron transport system was not uncoupled in this system, potential mechanisms of mitochondrial injury in leukemic cells from CLL patients are currently under exploration. Taken together, these observations suggest that mitochondrial perturbation contributes significantly to the death process induced by flavopiridol. Further studies to identify the mechanism of mitochondrial perturbation will be essential to understanding flavopiridol’s mechanism of action and for predicting patients at risk for acute tumor lysis syndrome. (Support for this work was provided by the Samuel Waxman Foundation and the Leukemia & Lymphoma Society.)
The Specificity and Isotype of Monoclonal Anti-D Antibodies Dictates Their Ability To Inhibit Opsonized Platelet Phagocytosis.
Authors: Semple John W. , Aslam Rukhsana , Kim Michael , Speck Edwin R. , Stewart Donald I.H. , Weirsma Erik , Freedman John , Kjaersgaard Mimi
Published: October 2019

The use of polyclonal anti-D is the established treatment for prophylaxis against rhesus immunization in Rh(D) negative pregnant women and is effective in raising the platelet counts in patients with autoimmune thrombocytopenic purpura (AITP). Since the supply of plasma derived anti-D antibodies is limited and plasma products carry the risk of infectious complications, replacement with monoclonal anti-D antibodies is a promising alternative. This study analyzed the efficacy of polyclonal anti-D (WinRho®SDF, Cangene) and six human monoclonal anti-D antibodies with differeing isotypes and specificities (Table 1) for their ability to opsonized erythrocytes and modulate monocyte phagocytosis of opsonized platelets in a flow cytometric assay. Human platelets were labeled with CellTracker CMGreen, opsonized with various HLA or HPA1a specific antibodies, incubated with the monocytic leukemic cell line THP-1 for 2 h and intracellular fluorescence was compared at 4°C and 37°C. Various concentrations of anti-D opsonized erythrocytes were added to the assay and the changes in intracellular fluorescence was compared. The results demonstrate that the THP cells significantly phagocytosed opsonized platelets in an Fc-dependent manner e.g. the fold change in median channel fluorescence between 37°C and 4°C for intact IgG W6/32 opsonized platelets was 51.5 ± 5.2 (mean+SD, n=74) and only 2.1+2.1 if the platelets were labeled with F(ab’)2 fragments. When erythrocytes were opsonized with the monoclonal anti-D antibodies that shared specificity but differed in IgG isotype (RhG1/RhG3 and BRAD3/BRAD5) and added to the assay, the IgG3 isotypes had a significantly greater ability to inhibit opsonized platelet phagocytosis compared with IgG1 isotypes and this inhibition was similar to that observed with polyclonal anti-D opsonized erythrocytes. However, in the one case where the monoclonal anti-D antibodies shared isotypes but differed in specificity (113/178), rRh9B8-113 monoclonal antibody significantly inhibited platelet phagocytosis whereas the rRh429-178 antibody did not. These results suggest that some human monoclonal antibodies can be as efficient as polyclonal anti-D in inhibiting opsonized platelet phagocytosis and the degree inhibition depends on the IgG isotype and specificity of the monoclonal antibodies. Thus, a defined cocktail of monoclonal anti-D antibodies could be prepared to mimic the effects and replace plasma-derived polyclonal anti-D.

Table 1: Characteristic of the monoclonal anti-D antibodies. Antibody Isotype Specificity rRh9B8-113 IgG1 Loop 3 rRh429-178 IgG1 Loop 4 RhG1 IgG1 Loop 6 RhG3 IgG3 Loop 6 BRAD3 IgG3 Loop 6 BRAD5 IgG1 Loop 6
The Geldanamycin Derivative DMAG Demonstrates Improved Cytotoxicity and Down-Modulation of Hsp90 Client Proteins Relative to 17-AAG in Chronic Lymphocytic Leukemia (CLL) Cells: Justification for Clinical Trials in CLL.
Authors: Johnson Amy J. , Wagner Amy J. , Smith Lisa L. , Lucas David M. , De Lay Michael D. , Allison Jamie M. , Ivy S. Percy , Lin Thomas S. , Byrd John C.
Published: October 2019

The heat shock protein Hsp90 functions to stabilize important cell survival- and proliferation-related kinases such as AKT, IKK, c-Src, Raf-1, and cdk9. Cancer cells have activated Hsp90 as compared to normal cells, making this a relevant therapeutic target to eliminate these kinases. The semi-synthetic geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) binds to and inhibits the activity of Hsp90, and previous work demonstrated the ability of 17-AAG to deplete AKT in several cancer types in vitro. A newer geldanamycin derivative, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) has improved pharmacological properties including solubility and oral bioavailability, and was shown to be more effective than 17-AAG in melanoma and pancreatic carcinoma mouse xenograft mouse models. We therefore examined the effects of 17-AAG and DMAG against CD19-positive tumor cells from CLL patients. Cell viability was examined by the MTT assay, and AKT and IKK protein expression was examined by immunoblot analysis. In samples from seven CLL patients, 1.0uM DMAG resulted in 31.5% viability (95% CI: 13.1–50.0%), compared to 61.5% viability (95% CI: 45.0–78.0%) using the same concentration of 17-AAG. In four CLL patient samples treated with 1.0uM DMAG for 24 hours, AKT was decreased an average of 72.5% (95% CI: 57.7–87.3%) relative to the untreated controls. This is in comparison to 1.0uM 17-AAG, which caused a 52.7% decrease in AKT (95%CI: 39.7–65.6%). IKK protein was also decreased at similar levels in all patient samples examined. This data indicates that in CLL cells, DMAG has superior activity both in cytotoxicity and in reduction of relevant Hsp90 client proteins. 17-AAG is currently undergoing Phase I clinical testing in CLL, and DMAG is completing phase I clinical development in solid tumor malignancies. Overall, our data and that of others support clinical development of DMAG in CLL, based on the improved pharmacologic properties, enhanced efficacy relative to 17-AAG, and expected down-regulation of target proteins. In addition, our in vitro observations support using measurement of protein down-regulation of AKT and IKK as pharmacodynamic biomarkers of activity in patients undergoing therapy with these agents.
Attacking Oncogene Addiction in B-CLL: Seliciclib First Engages the Mcl-1/Noxa Axis, after Which Gradual Exhaustion of Bcl-2 Protection Leads to Bax Activation.
Authors: Hallaert Delfine Y.H. , Spijker Rene , Jak Margot , Jaspers Annelieke , Derks Ingrid A.M. , Alves Nuno L. , de Boer Jan Paul , de Jong Daphne , Green Simon R. , van Lier Rene A.W. , van Oers Marinus H.J. , Eldering Eric
Published: October 2019

Background : Seliciclib (R-roscovitine) is a cyclin-dependent kinase inhibitor in clinical development. It triggers apoptosis by inhibiting de novo transcription of the short-lived anti-apoptotic Mcl-1 protein, but it is unclear if and how this leads to Bax or Bak activation that is required for most forms of cell death.

Aim: To study the effects of seliciclib on apoptosis gene expression and Mcl-1 and Bcl-2 protein interactions in B cell chronic lymphocytic leukemia (B-CLL), a malignancy with known aberrant apoptosis regulation.

Methods: Purified B-CLL cells (PBMC consisting of >90% B-CLL cells; n=20) and Ramos cell lines overexpressing different apoptosis regulators were used in this study. The effect of seliciclib on viability, apoptosis gene expression pattern, and protein associations was investigated via RT-Multiplex-Ligation-dependent Probe Amplification (RT-MLPA), Western blotting and co-immunoprecipitation assays. Ramos cells were transduced with retroviral vectors expressing either Noxa siRNA, Bim siRNA or control-GFP virus, and tested for different apoptosis stimuli.

Results: We found that although seliciclib resulted in proteasome-dependent Mcl-1 degradation within 4 hrs in B-CLL cells, Bax and Bak activation and apoptosis occurred with a considerable delay, i.e. at 16 hrs. During this period, there was no evidence of transcriptional changes in p53-responsive or apoptosis-related genes. In freshly isolated, viable B-CLL cells, pro-survival Mcl-1 was engaged by the pro-apoptotic proteins Noxa and Bim but not by Bak. The contribution of Noxa [Figure1] and Bim (liberated from McL-1 within 4 hours) as specific mediators of seliciclib-induced apoptosis was demonstrated via RNAi in two model systems. Interestingly, 16 hrs after seliciclib treatment, there was a clear accumulation of Bcl-2, Bim, and Bax in the detergent insoluble (mitochondria containing) fraction of B-CLL cells. This suggests that after Mcl-1 degradation, the remaining apoptosis neutralizing capacity of Bcl-2 is gradually overwhelmed, probably resulting in Bax multimerisation and pore formation in the mitochondria.

Conclusions: These data support the ’oncogene-addiction’ model in which malignant cells depend on increased Bcl-2 levels, and extend it to include Mcl-1. Furthermore, since Noxa is elevated in B-CLL, its involvement in p53-independent apoptosis suggests this BH3-only protein may be a therapeutic target.

Figure Figure
Deletions of the Derivative Chromosome 9 Do Not Influence Response to Imatinib of Early Chronic Phase Chronic Myeloid Leukemia Patients (A GIMEMA Working Party Analysis).
Authors: Castagnetti Fausto , Marzocchi Giulia , Luatti Simona , Buontempo Francesca , Baldazzi Carmen , Palandri Francesca , Giannoulia Panagiota , Amabile Marilina , Iacobucci Ilaria , Mancini Marco , Giussani Ursula , Specchia Giorgina , Cuneo Antonio , Gugliano Emilia , Kerim Simonetta , Modaferri Bruno , Abruzzese Elisabetta , Martinelli Giovanni , Testoni Nicoletta , Rosti Gianantonio , Baccarani Michele
Published: October 2019

Extensive submicroscopic deletions adjacent to the breakpoint on derivative chromosome 9 [der(9)] have been reported in a subset of Chronic Myeloyd Leukemia (CML) patients and have been associated with an adverse outcome with conventional drugs and α-interferon (α-IFN). Huntly et al (Blood.2003; 102.2205–12) reported 275 CML pts who were treated with imatinib in CP, suggesting that der(9) deletions were associated with lower response rates and a shorter time to progression. Different data were reported by Quintas-Cardama et al (Blood.2005; 105:2281–6), who did not find any difference related with der(9) deletions in other 320 patients treated with imatinib. In these 2 studies, some patients began imatinib in early CP (51 and 152, respectively) while many patients (224 and 168, respectively) were treated in late CP. To establish the relationship of der(9) deletions with the response to imatinib in early CP patients, we planned a prospective study involving 3 consecutive multicentric national studies of the GIMEMA (Gruppo Italiano Malattie Ematologiche dell’Adulto) CML Working Party. 421 CML patients in early chronic phase were enrolled between January, 2004 and January, 2006; Fluorescence in situ hybridization (FISH) analysis of bone marrow cells was performed using BCR/ABL extra-signal, D-FISH or dual-color dual-fusion probes. At diagnosis, 52 (12%) of them had der(9) deletion and 369 (88%) had not. The 2 groups, with/without deletions, were comparable (no difference in age, Sokal risk, imatinib dose). Median observation time is 12 months. At 3 months, the CHR rates in with/without deletions patients were 87%/92%. At 6 months, the complete cytogenetic response (0% Ph-pos; CCgR) rates were 80%/80%, with major molecular response (MMolR, defined as a Bcr-Abl/Abl x 100 ratio < 0.1%) rates of 52%/51%, respectively. At 12 months, CCgR rates were 89%/86% and MMolR 52%/61%. No difference is significant. We conclude that the presence of der(9) deletions at diagnosis do not constitute a negative factor for response to imatinib: the haematological, cytogenetic and molecular response rates resulted equal between the 2 groups of patients with and without der(9) deletions. This finding is relevant to the long term effect of imatinib treatment, since both the CCgR and the MMolR are important and established indicator of long term survival.
Extracellular Signal-Regulated Protein Kinase 5 Mediates Resistance of Human Chronic Myeloid Leukemia K562 Cells to Imatinib.
Authors: Sovershaev Mikhail A. , Egorina Elena M. , Gruber Franz X. , Bjørkøy Geir , Johansen Terje
Published: October 2019

Mutations in the Abl kinase domain and Bcr-Abl gene amplification have been recognised as the most common mechanisms of resistance to Imatinib (Gleevec) in chronic myeloid leukemia (CML). However, Imatinib resistance by Bcr-Abl independent mechanisms have also been reported. The extracellular signal-regulated protein kinase 5 (Erk5) signalling pathway is involved in cell survival and cell cycle control functions. Interestingly, it was recently reported that ERK5 is important in CML (Buschbeck M., 2005). Bcr/Abl was found to increase the level of Erk5 by stabilizing the protein thus contributing to increased cell survival. In this study we asked whether small interfering RNA (siRNA)-mediated depletion of endogenous Erk5 or overexpression of wild type Erk5 would affect the survival of Imatinib-treated human K562 CML cells. These cells express Bcr-Abl with a wild type kinase domain. Fluorescence activated cell sorting analyses of propidium iodide-stained K562 cells revealed that treatment with 1 μM Imatinib for 24 hrs led to an increase of the apoptotic cell population from 9 to 27 %. The number of cells, arrested in G0/1 phase was also elevated following Imatinib treatment from 66 to 85 %. Overexpression of wild type Erk5 in K562 cells reduced the frequency of Imatinib-induced apoptosis from 27 to 18 % without affecting cell cycle distribution of the living cells. After having established that overexpression of Erk5 renders cells partially resistant to Imatinib treatment we tested the effect of siRNA-mediated depletion of endogenous Erk5 on the viability of K562 cells challenged with Imatinib. Indeed, 42% of K562 cells transfected with siRNA against Erk5 underwent apoptosis after Imatinib treatment. Imatinib induced apoptosis in only 20% of cells transfected with a control siRNA. Furthermore, Erk5 depletion strongly inhibited cell cycle progression in Imatinib-treated K562 cells. Thus, the cell populations in S phase and mitosis were reduced from 22 to 16 % and from 24 to 9 %, respectively. Taken together, our data and those of Buschbeck et al. suggest that ERK5 levels may strongly affect cell survival during Imatinib treatment of CML cells.
First Overall Molecular Response of the Randomized French SPIRIT Study on Behalf the Fi-LMC Group.
Authors: Roche-Lestienne Catherine , Mahon François-Xavier , Crinquette Antoine , Guilhot Joëlle , Guilhot Francois , Preudhomme Claude
Published: October 2019

In September 2003, the French CML study group (Fi-LMC) activated the four armed randomized SPIRIT trial comparing imatinib 400mg with imatinib 600mg, imatinib 400mg + ara-C and imatinib 400mg + pegylated interferon in newly diagnosed CML in chronic phase. The molecular monitoring was centralized and performed, according to the EAC protocol, in duplicate on the same BCR-ABL cDNA among two French laboratories (Bordeaux and Lille). Data were expressed as BCR-ABL/ABLx100 on the new internationally agreed scale (IS) using a conversion factor of 1.33 and 0.80 for Bordeaux and Lille respectively. A good correlation between the two laboratories was observed. During the first year of follow-up the quantification was performed each three months (M0 to M12). In May 2006, 297 of the 492 enrolled pts had a follow up of more than 12 months, and 263 pts were analysed at M12. Using the IS, the median values of the BCR-ABL/ABL normalized ratios were 90%, 6,4%, 0.68%, 0.32% and 0.16% at M0, M3, M6, M9, and M12 respectively. At M12, 15%, 29%, 27%, 17% and 12% of the pts presented a BCR-ABL/ABL ratio lower than 0.01%, 0.1%, 1%, 10%, and 100% respectively. Overall, 44% of the pts reached the Major Molecular Response (MMR). Results were also expressed using individual M12/M0 ratio. At M12 13%, 29%, 28%, 17% and 13% of the pts presented a 4 log, 3 log, 2 log, 1 log and 0 log reduction respectively. Overall, 42% of the pts presented an individual 3 log reduction.

Despite very similar results between the two means of expression, the pts distribution was different due to unlike individual BCR-ABL transcript level at MO. Furthermore, the analysis of 75 paired samples before (at diagnosis) and after (at inclusion) hydroxyurea treatment (5 days to 1.2 months) revealed BCR-ABL/ABL ratios of 129% and 108% respectively, confirming the 0.2 log difference previously reported by A. Hochhaus et al.

In conclusion, expression of the data using the new IS or the individual M12/M0 ratio was very similar in our experience regarding the percentage of each subgroup but the pts distribution was different and a longer follow up would be useful to define the best method. However, the good correlation observed in the IRIS trial between MMR and progression, the influence of hydroxyurea treatment on BCR-ABL level, the good correlation observed among laboratories and the need of harmonisation of expressed results influenced our choice for the new IS. The application of this recommendation is in progress in the great majority of the molecular French laboratories.
Sokal and Hasford Scores Are Able To Predict Cytogenetic Response and Survival in CML Patients Who Failed Prior Therapy and Are Receiving Salvage Therapy with Imatinib or Other Tyrosine Kinase Inhibitors.
Authors: Tam Constantine S. , Kantarjian Hagop M. , Shan Jenny , Wierda William G. , Ferrajoli Alessandra , Koller Charles Asa , Cortes Jorge
Published: October 2019

Sokal and Hasford scores have been used to estimate the survival of CML patients following alkylator-based or interferon-based therapy, and may also predict the outcome of patients receiving first-line imatinib. These scores were designed for patient characteristics at the time of diagnosis and they have been assumed to have no clinical value at other times during the course of the disease (eg. second-line or subsequent therapy). We seek to determine the applicability of the Sokal and Hasford scores in predicting outcomes in patients receiving second or subsequent-line therapy with imatinib and other novel tyrosine kinase inhibitors (TKI). This analysis is based on 379 CML patients in chronic phase (90%) or accelerated phase as defined by cytogenetic clonal evolution (10%), who were enrolled on clinical trials of imatinib following interferon failure (n=294), or nilotinib or dasatinib following imatinib failure (n=85). Significance of relationship between Sokal and Hasford risk categories and response were tested using the Chi-Square test, and differences in survival assessed using the log-rank method. P-values are summarised below:

Imatinib following Interferon Failure Nilotinib/Dasatinib Following Imatinib Failure Sokal Hasford Sokal Hasford *comparison between low and intermediate risk groups only Low Risk 219 (74%) 176 (60%) 39 (46%) 45 (53%) Intermediate Risk 65 (22%) 111 (38%) 34 (40%) 38 (45%) High Risk 10 (3%) 6 (2%) 12 (14%) 2 (2%) Any Cytogenetic Response p<0.001 p<0.001 p=0.01 p=NS Major Cytogenetic Response p<0.001 p<0.001 p=0.002 p=0.04* Complete Cytogenetic Response p<0.001 p<0.001 p=0.005 p=0.07* Survival p<0.0001 p<0.0001 Insuff Events Insuff Events

Among patients receiving imatinib following failure of interferon therapy, both Sokal and Hasford scores were highly predictive of cytogenetic response and overall survival. Due to the low number of deaths (n=5) on the nilotinib and dasatinib trials, the effect of Sokal and Hasford scores on predicting survival could not be assessed but they remain predictive of cytogenetic response. We conclude that the Sokal and Hasford scores calculated at the time of therapy failure are useful predictors of subsequent response to salvage therapy with imatinib and other TKIs.
A Phase II Study of Nilotinib, a Novel Tyrosine Kinase Inhibitor Administered to Patients with Imatinib Resistant or Intolerant Chronic Myelogenous Leukemia (CML) in Chronic Phase (CP), Accelerated Phase (AP) or Blast Crisis (BC) Who Have Also Failed Dasatinib Therapy.
Authors: Giles Francis , le Coutre Philipp , Bhalla Kapil , Rosti Gianantonio , Ossenkopplele G.J. , Alimena Giuliana , Weitzman Aaron , Rafferty Teresa , Zheng Ming , Kantarjian Hagop
Published: October 2019

Nilotinib is a potent, highly selective, aminopyrimidine inhibitor which in vitro is 30-fold more potent than imatinib and active against 32/33 imatinib resistant cell lines with Bcr-Abl mutations. This open-label study was designed to evaluate the efficacy and safety as defined by hematologic and cytogenetic response rates (HR/CyR) of nilotinib administered at a daily dose of 400 mg bid to patients who previously received and either failed or were intolerant to imatinib and dasatinib. A total of 50 patients were enrolled and included CP (n=26), AP (n=10), and BC (n=14) patients. Of the 14 BC patients 10 were myeloid and 4 were lymphoid. Overall 8 (16%) patients had extramedullary disease at baseline. The median age was 57 (range 19–78) years and the median time from first diagnosis of CP, AP, or BC to treatment was 65 (range 8–213) months. The median duration of nilotinib exposure was 226 (range 3–379) days. A total of 28 (56%) patients remain on treatment, 22 (44%) discontinued (6 for adverse events, 11 for disease progression, 3 withdrew consent, and there were 2 deaths related to cerebral hemorrhages). Complete (HR) was reported in 9 of the 20 (45%) CP patients who did not have a CHR at baseline. Of the 26 CP patients, 8 (31%) had a major CyR (2 complete and 6 partial), 2 (8%) patients had minimal responses, and 9 (35%) patients had no response. Disease progression occurred in one CP patient and 6 patients were not evaluable. Confirmed hematologic responses were observed in 2 of 10 (20%) AP patients who had a return to chronic phase (RTC), 7 patients were not evaluable and there was one patient death. Of the 10 AP patients 1 (10%) each had CyR (partial, minor, minimal and no response). Of the 14 BC patients 1 had a CHR (7%), 2 (14%) had a return to chronic phase (RTC), 6 (43%) were not assessable for response, and 5 (36%) had progressive disease. Overall the most frequent Grade 3 or 4 adverse events reported were thrombocytopenia in 12 (24%), neutropenia in 11 (22%), and anemia in 5 (10%) patient. In summary, nilotinib has significant clinical activity and an acceptable safety and tolerability in CML-CP, AP, and BC patients who were resistant or intolerant to imatinib and have also failed dasatinib therapy.
The ATP Competitor INNO-406 (NS-187) Inhibits Proliferation and Survival of Mouse Cells Expressing Several Immatinib Mesylate Resistant Bcr/Abl Mutants and Cells from CML Patients.
Authors: Tao Wenjing , Sun Tong , Craig Adam R. , Cortes Jorge E. , Ottmann Oliver G. , Hochhaus Andreas , Reddy Premkumar E. , Kantarjian Hagop M. , Arlinghaus Ralph B.
Published: October 2019

Imatinib mesylate (IM) resistance during the therapy of chronic myeloid leukemia (CML) patients is associated with point mutations in the Bcr/Abl kinase, which strongly interferes with IM binding to Abl kinase domain. INNO-406 (NS-187) is reported to be a new specific inhibitor of the Bcr/Abl and Lyn kinase, and is a candidate for treatment of IM resistant CML patients (Kimura et al, Blood2005, 106:3948). We tested INNO-406 on both IM resistant mouse cell lines and CML patient samples. The MTT assay was used to detect the anti-proliferation effects of INNO-406 on Bcr/Abl wild type cells (32Dp210 wt) and Bcr/Abl mutants (L387M, H396R, Q252R, F311L, T315I and Y253H) after 72 hours treatment. IM or dasatinib treatment was included as controls.

The MTT results showed 32Dp210 wt and four of the six Bcr/Abl mutants were sensitive to INNO-406, with the IC50 values of 0.6 nM (wild type), 15 nM (L387M ), 35 nM (H396R), 0.8 nM (Q252R) and 8.2 nM (F311L). The IC50 values of those four cell lines treated with IM were much higher than with INNO-406 treatment. The IC50 values for IM were: 100 nM (wild type), 500 nM (L387M), 1220 nM (H396R), 100 nM (Q252R) and 100 nM (F311L). But neither INNO-406 nor IM exhibited inhibitory effects on the proliferation of T315I and Y253H mutants (IC50 > 10000 nM). These results indicated that INNO-406 suppressed the growth of Bcr/Abl mouse leukemia cell lines expressing wt Bcr/Abl and Bcr/Abl mutants except T315I and Y253H. We note that the Y253F mutant was sensitive to INNO-406 (IC50 64 nM, Nippon Shinyaku Co. Ltd.). Annexin V/PI apoptosis assays were conducted on cells from CML patient samples after 72 hours treatment with various concentrations of INNO-406. Flow cytometry results showed that INNO-406 (10–20 nM) induced apoptosis in blood/marrow cells from two blast crisis patients, one of which had the G250E mutation. INNO-406 also induced apoptosis in marrow cells from two patients in IM induced remission. One of these samples with a Bcr/Abl mutation at M244V showed a weak response to IM but a strong apoptosis with INNO-406 (20 nM), indicating that NS-187 worked more efficiently in the M244V mutant. Based on these results, INNO-406 (NS-187) appears to be a candidate for the treatment of some forms of imatinib resistant CML. A phase I study of INNO-406 has been initiated in patients with CML who have failed prior imatinib therapy and the first cohort has been safely treated.