bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Hypoxia on the Expression of BRIT1 in K562 Cell Line: Implication for Resistance to Imatinib.
Authors: Giallongo Cesarina , Tibullo Daniele , La Cava Piera , Chiarenza Annalisa , Berretta Salvatore , Palumbo Giuseppe A. , Di Raimondo Francesco
Published: October 2019

BRIT1 (Microcephalin, MCPH1) is a chromatin binding protein that forms ionizing irradiation-induced nuclear foci (IRIF); it is a crucial DNA damage regulator in the ATM/ATR pathways. Here we have analyzed BRIT1 expression on K562 cell line after treatment with imatinib 0,1-1 μM, UV irradiation (12 μJ/cm2) and after exposure to an hypoxic condition (3% O2). K562 in standard culture conditions (21% O2) were used to establish BRIT1 basal expression. Total RNA from K562 cultures was prepared at 24 and 48 hs. 1 μg total RNA was reverse transcribed from each sample. TaqMan Gene expression assay (Applied Biosystems) in Real-Time PCR was employed to examine the expression of BRIT1. Cellular count was assayed by trypan blue. Treatment with IM (0,1 and 1 μM) and UV induced a 2–3 fold increased expression of BRIT1 at 24 h and a minor increase at 48 h. Cell mortality at 24 h was 30% for IM and 20% for UV while at 48 h cells started to grow again, possibly as a consequence of the DNA damage repair by BRIT1. Exposure of K562 cells to hypoxia increased BRIT1 expression about 5- and 6-folds at 24 and 48 h respectively. Cell mortality at 24 h was 30% while after 48 h it was 20%. We therefore concluded that BRIT1 expression increases in hypoxia more than after IM and UV exposure. Since we have found that K562 in hypoxic conditions are relatively resistant to imatinib, (K562 cell mortality after 24 h exposure to IM 1μM was 30% ± 0,3 in normoxia vs 9% ± 0,8 in hypoxia, P<0,005) we have hypothesized that BRIT1 could be involved in resistance to IM. Therefore, we exposed K562 to UV and, after 48 h we incubated the cells with imatinib 1 μM. Pre-exposure of K562 cells to UV reduced imatinib-induced mortality (9,7% ± 1,6 vs 21,3% ± 1,5 of control, p< 0,005) thus indicating that upregulation of BRIT1 (as we have observed especially in hypoxia) could contribute to resistance to imatinib in K562 cell line. Understanding of BRIT1 function may well contribute to novel therapeutic approaches for cancer.
CMV Colitis in a Patient Receiving Dasatinib.
Authors: Khan Mudussara A. , Walling Terri , Cumpston Aaron , Craig Michael
Published: October 2019

Introduction: CMV generally produces an asymptomatic or minimally symptomatic acute illness in immunocompetent patients. CMV reactivation commonly occurs in patients with CML after allogeneic transplant but has not been described in those who are treated with tyrosine kinase inhibitors. We report a case of a patient with CML in lymphoid blast phase treated with dasatinib to cytogenetic remission that presented with CMV Colitis.

Method: 73-year-old male diagnosed with CML in lymphoid blast crisis 2/2006 treated with chemotherapy and imatinib with good response. He relapsed in 2/2007 with leukostasis and underwent leukopharesis. His BCR/ABL mutation analysis was positive for F359V mutation conferring resistance to imatinib. He was started on dasatinib 70 mg twice daily and had a cytogenetic remission within four months. He presented five months after starting therapy with four weeks of recurrent fevers, chills, malnutrition and diarrhea.

Results: CBC and differential revealed only mild thrombocytopenia, Absolute CD4 count of 209; absolute CD8 count of 1495. CMV DNA PCR Quantification of 1500 copies/ml. CT chest abdomen and pelvis showed right sided pleural effusion but no pneumonitis. Flex sigmoidoscopy showed inflamed edematous mucosa with submucosal hemorrhages scattered throughout colon and rectum. Pathology showed chronic colitis, focal viral cytopathic changes consistent with CMV presence. Presence of CMV was confirmed by immunoperoxidase stain for CMV. Treatment was started with IV ganciclovir then switched to oral valganciclovir with good response and resolution of CMV infection.

Conclusion: CMV colitis is a well-known complication in immunocompromised hosts after HCT. We are reporting a case of CMV colitis in a patient with CML and had hematologic and cytogenetic remission on a novel tyrosine kinase inhibitor - dasatinib. CMV reactivation may occur in patients who are receiving dasatinib. Furthur evaluation is necessary to determine the incidence of this complication.
Expression of Genes Regulating Iron Metabolism in Hepatocyte Cell-Line HepG2 Induced by Sera from MDS Patients.
Authors: Breda Laura , Ghoti Hussam , Rivella Stefano , Rechavi Gideon , Cabantchik Ioav , Rachmilewitz Eliezer A.
Published: October 2019

Liver expression of hepcidin, a key factor in the regulation of iron absorption and recycling, responds commensurately to plasma iron levels. However, in thalassemic humans and mouse models, despite the overt iron overload found, there is a low expression of liver hepcidin and low levels in urine. We have recently shown that hepcidin expression induced by plasma factors in hemochromatosis is overriden in thalassemic patients (Weiser-Stern et al, Brit. J. Haemat.135:129,2006). As low urinary hepcidin levels were also found in patients with Myelodysplastic Syndrome (MDS), mainly in those with refractory anemia (RA) with or without ring sideroblasts (RARS) and iron overload, we set out to examine whether plasma factor(s) in MDS might also affect the expression of liver genes of iron metabolism.

Patients and methods. Serum samples were collected from 19 MDS patients, average age: 75.7 y, Hb 6.5–12.0 gm/dl; type and #: RA 11, RARS 5 and 3 RA with excess of blasts (RAEB). The international prognostic score (IPSS) was low in 13/19 and intermediate 1 (Int 1) in 6/19. Only 7/19 patients received less that 5 blood units, while 12/19 received 20–140 units and in 11/19 serum ferritin was >1000 ng/ml. The serum samples were incubated with human hepatoma HepG2 cells, which were harvested and their RNA isolated and analyzed by quantitative RT-PCR for: hepcidin (Hamp), the lipocalin Ngal, HFE, transferrin receptors (TfR) 1 and 2, and DMT1-IRE(−). Values were normalized against those obtained from 3 healthy individuals.

Results and discussion. An apparently normal expression of all the above genes was found in 13/19 patients (group I), whereas in the remaning 6/19 (group II) there was: A. a significant increase in expression relative to normal controls (1.0±0.20) of Ngal (4.64±2.15) p<0.01, TfR1 (5.24±0.59) p<0.01, HFE (1.76±0.12) p<0.05 and DMT1-IRE(−) (1.64±0.24) p<0.05; B. a significant decrease in TfR2 (0.19±0.11) p <0.01 and C. an increased Hamp expression in only 3/6 (7.2±5.0) p<0.05 patients and normal expression in 2/6 (1.03±0.3). No clear correlation could be established between expression of the above mentioned genes and iron overload parameters (serum iron and/or ferritin) or the number of transfusions. This study shows a heterogeneous behavior of MDS sera in eliciting gene expression and highlights that increased Hamp expression of HepG2 cells in response to plasma factors is found only in a small fraction of this class of iron overloaded patients. Thus as in thalassemia (1), most of the MDS sera with ferritin levels >1000 ng/ml failed to induce Hamp, suggesting that also in MDS patients there might be circulating factors overriding the serum iron effect on Hamp expression. It remains to be established whether the putative factors modulating expression of key genes of liver iron metabolism in MDS are of the same origin or biochemical character as in thalassemia.
Decreased Megakaryocytepoiesis and No Excess of Platelet Destruction in Myelodysplastic Syndrome Patients.
Authors: Murai Kazunori , Oyake Tatsuo , Kowata Shugo , Ishiguro Mamiko , Ito Shigeki , Ishida Yoji
Published: October 2019

Myelodysplastic syndrome (MDS) is a malignant disorder of hematopoietic progenitor cells and characterize by peripheral blood cytopenias with normo- to hyper-cellular bone marrow (BM) and morphologically dysplastic changes. Thrombocytopenia is observed in approximately 50% of MDS. The underlying pathophysiology is not fully understood. We analyzed the megakaryocytopoiesis and thrombocytopoiesis state by several parameters, including % reticulated platelet (%RP), which indicated platelet production state, glycoalicine index (GCI), which indicated platelet destruction state, and serum thromopoietin (TPO) levels in 47 refractory anemia in myelodysplastic syndrome (MDS-RCMD) patients with platelet counts less than 100 x 109/L. Furthermore, apoptosis frequency of megakaryocyte progenitors was analyzed in several patients. In all patients, dysmegakaryocytopoiesis findings such as hypolobulated micromegakaryocyte, non-lobulated nuclei in all sizes, and multiple, widely-separated nuclei were observed. The megakaryocytes in BM was normal to decreased in number in 32 patients (70%). Plasma TPO levels were significantly higher (718.7±746.0 pg/ml, n=50) in MDS-RCMD, while they were less than 205.0 pg/ml in normal volunteers (68.3 ±65.3 pg/ml, n=32)(p< 0.01). The %RPs in MDS-RCMD and normal controls were similar (MDS-RCMD 1.7±0.9% vs control 1.2±0.6%), indicating that increased thrombocytopoiesis was not observed in MDS-RCMD, regardless of high TPO levels. GCI was similar to normal controls (MDS-RCMD, 1.5±1.3% vs controls, 1.6±0.3%), indicating no excess of platelet destruction. There was no correlation between %RP and GCI. These data strongly suggested that platelet life span be not shortend in MDS-RCMD. We have reported that excessive apoptosis of CD34(+) cells was observed in MDS patients in the previous ASH meetings. The three-color flow cytometric analysis of bone marrow mononuclear cells using PE labeled Annexin V, PerCP labeled anti-CD34 antibody and FITC labeled anti-CD41 antibody were carried out in 17 MDS-RCMD patients. Much higher frequency of apoptosis was observed in double positive cells for CD34 and CD41 (48.1%:13.4∼78.4%, median: range) in MDS-RCMD, compared to that in normal controls (7.7%: 4.4∼17.2%, median: range) (P<0.05). The frequency of apoptosis was not increased significantly in CD34(−)CD41(+) cells in MDS-RCMD patients (median: 6.9% ; 1.3∼21.5%), n=12), compared to those in normal controls (3.3%: 1.5∼8.1%, n=10). These data strongly suggested that the main cause of thrombocytopenia in MDS-RCMD should be apoptosis in megakaryocytes progenitors, followed by the decreased megakaryocytopoiesis.
Enhanced HDAC (Histone Deacetylase) Enzyme Activities in Blood CD 34 + Cells in Patients with Myeloid Metaplasia Myelofibrosis (MMM).
Authors: Wang Jen-Chin , Chen Chi , Dumlao Theresa , Chang Thong , Xiao Yng-Li , Sominsky Inna , Burton Jack
Published: October 2019

We previously reported that histone deacetylase (HDAC) activity is elevated, but is not correlated to the JAK-2 mutation status, in patients with myelofibrosis myeloid metaplasia (MMM) (Blood 107:319b 2005). Now we have studied more patients: totally, 17 with MMM, 19 with other myeloproliferative disorders (MPD), and 16 normal volunteers as controls. Significantly elevated HDAC levels again was shown in patients with MMM compared with other MPD patients and normal volunteer controls (p<0.05). Sixteen patients with MMM were also studied for correlation between JAK-2 mutation status and HDAC levels; no significant correlation was found. We then studied which members of HDACs were elevated in patients with MMM. cDNA was prepared from total RNA obtained from blood CD 34+ cells; then QRT-PCR was performed using pre-made mixtures of primer and FAMTAMRA-labeled probes from ABI (www.appliedbiosystems.com). Primers and probe to GAPDH were used as internal controls. Cycle threshold (Ct) values were obtained graphically for target genes and internal control GAPDH gene products. Amplification efficiencies were calculated by plotting Ct s from serial diluted cDNAs for target genes and GAPDH and all with slopes below 0.1. Δ Ct values were obtained by subtracting GAPDH Ct from target gene Ct. Relative mRNA levels were determined by subtraction of normal control Δ Ct values from MMM Δ Ct values to give ΔΔ Ct values, which were converted to 2− ΔΔ Ct (Relative Quantitation of Gene Expression). The results showed elevated HDAC1 (2.80), HDAC2 (18.45), HDAC3 (2.10), HDAC 6 (2.03) HDAC 9 (2.71), SIRT3 (70.20), and SIRT6 (39.40) and depressed HDAC4 (0.01), HDAC5 (0.001), HDAC8 (0.001), SIRT2 (0.23), SIRT5 (0.005), and SIRT7 (0.88). () indicates relative mRNA values of MMM to controls. These results suggest that HDAC activities are elevated in patients with MMM and are elevated in many members of HDAC. This study may lay the basis for using HDAC inhibitors in clinical trials treating patients with MMM.
Genome-Wide DNA Profiling Identifies a Stable Profile Although with Aberrations Targeting the Fibroblast Growth Factor Pathway in Hairy Cell Leukemia.
Authors: Forconi Francesco , Poretti Giulia , Kwee Ivo , Sozzi Elisa , Rossi Davide , Capello Daniela , Rinaldi Andrea , Zucca Emanuele , Lauria Francesco , Gaidano Gianluca , Bertoni Francesco
Published: October 2019

Hairy cell leukemia (HCL) is a rare B-cell neoplasm with largely unravelled features of lymphomagenesis. Molecular mechanisms of disease are largely unknown and genomic aberrations have infrequently been investigated in HCL. In order to elucidate the genetic mechanisms of HCL pathogenesis, we analyzed copy number changes and loss of heterozigosity (LOH) by genome wide DNA profiling with the Affymetrix Human Mapping 250k Nsp arrays in 16 HCL. All investigated cases expressed multiple pre- (IgM+/−D) and post-switch (IgG+/−A) isotypes. Tumor IGHV gene transcript homology to germline ranged from 88.4 to 98.6%, with 4/16 HCL having >98% homology. Four of 16 patients were refractory or had progressive disease after first line purine-analogue-based treatments. CD103+ve hairy cells were >60% in all test samples. Genome wide DNA profiling demonstrated gross non recurrent copy number deletions in only 4/16 cases (25%), affecting 14q24-q32, 3q23, 3q27.3-q29, 8p12-pter 10q21-q23, 10q25-q26, 17p11-pter, 17q11-q21, 17q22–23, Xp21.2-p22.11, Xq13.3, Xq21.1-q21.33, Xq22.2–q28, the whole chromosomes 21 and X. Five of 16 patients (31%) presented LOH. Most interestingly, the 5 patients showed LOH regions, with a region of overlap in two patients at 6q25.1, without any copy number changes, indicative of uniparental disomy. Genes involved in bone marrow fibrosis, multiple isotype expression and response to treatment were specifically investigated. Interestingly, among genes critical for fibrosis, FGF12, a member of the fibroblast growth factors family, was specifically targeted by a complex chromosomal rearrangement at 3q27.3-q29, and reverse transcript-polymerase chain reaction confirmed FGF12 transcript expression. Among genes associated with poor response, p53 deletion was documented in a non-responder HCL. Conversely, no abnormalities at 14q32 locus (duplications or gains) were observed at IgH 14q32 region in these multiple IgH isotype expressing HCL. Overall, our analysis showed a highly stable genome with lack of gross recurrent abnormalities, which is an additional unique feature of HCL among B-cell neoplasms. However, our genomic analyses were able to identify specific chromosomal rearrangements targeting genes involved in fibrosis. Lack of gains of 14q32 locus was consistent with our hypothesis that multiple IgH isotypes are generated at RNA level in multiple IgH expressing HCL. The present analysis provides the basis for further molecular and epigenetic investigations.
Efficacy of Dasatinib in Patients with Accelerated-Phase Chronic Myelogenous Leukemia with Resistance or Intolerance to Imatinib: 2-Year Follow-Up Data from START-A (CA180-005).
Authors: Guilhot Francois , Apperley Jane F. , Kim Dong-Wook , Rosti Gianantonio , Yuan Xiaobin , Van Tornout Jan M.A. , Garzon Felix T. , Cortes Jorge
Published: October 2019

Dasatinib (SPRYCEL®), a potent inhibitor of BCR-ABL and SRC-family kinases, has been shown to be effective and safe for patients with accelerated-phase chronic myelogenous leukemia (AP-CML) who are resistant or intolerant to imatinib. START-A is a 39-center, international study to which patients with AP-CML who failed prior imatinib therapy were enrolled between December 2004 and July 2005. Here we report an update of the efficacy and safety of dasatinib (70 mg BID) from this open-label study of 174 patients with imatinib-resistant (n=161) or -intolerant (n=13) AP-CML after a median follow-up of 14.1 mo (range 0.1–21.7). Dose escalation (100 mg BID) or reduction (50 or 40 mg BID) were allowed for a lack of response or toxicity, respectively. Median time from original diagnosis of CML was 82 mo (range 4–359). Prior therapy included interferon-α in 72% of patients and stem-cell transplantation (SCT) in 13%; 52% had received prior imatinib doses >600 mg/d and 59% treatment with imatinib for >3 years. Best response to prior imatinib therapy was complete hematologic response (CHR) in 79% of patients, major cytogenetic response (MCyR) in 33%, and complete cytogenetic response (CCyR) in 21%. CHRs were attained by 45% of patients, while MCyRs and CCyRs were seen in 39% and 32%, respectively. Responses were achieved irrespective of imatinib status (with MCyR of 39% for both imatinib-resistant and -intolerant subgroups), prior stem-cell transplantation (26% MCyR), or the presence of prior BCR-ABL mutations (with the exception of T315I) (40% MCyR for both the mutation-positive and -negative subgroups). Twelve-month progression-free survival and overall survival were 66% and 82%, respectively. Dose interruptions were required for 85% of patients and dose reduction for 65%; the average daily dose administered was 126 mg (range 32–196). Grade 3–4 neutropenia and thrombocytopenia were reported in 76% and 82% of patients. Non-hematologic toxicity was generally mild to moderate and consisted primarily of diarrhea (52% all grades, 8% grade 3–4), headache (29%, <1%), nausea (28%, <1%), pleural effusion (27%, 5%), fatigue (26%, 4%), and superficial edema (22%, 1%). Dasatinib is effective in patients with AP-CML following imatinib treatment failure and the overall benefit-risk evaluation is favorable in this poor prognosis population. Updated analyses corresponding to a minimum follow-up of 2 years on all patients will be presented.
Soluble IL-6 Receptor Synergistically Stimulates the Growth of Multiple Myeloma with IL-6 and Confers Resistance to Bortezomib.
Authors: Lee Young Y. , Park Joowon , Yoon Sung-Soo , Choi Jung H. , Park Byoung B. , Kim Byoung K.
Published: October 2019

Proinflammatory cytokines including Interleukin-6 (IL-6), TNFa, and IL-8, involved in a variety of cellular responses including proliferation, differentiation, and inflammatory processes. They play a pivotal role in the pathogenesis of multiple myeloma (MM). Elevated IL-6 and souble IL-6 receptor (sIL-6R) levels have been found in MM patients indicating that its production is not only coordinated as part of a disease progression but also strongly associated with outcome of anti-cancer therapy. Even though Bortezomib, one of proteosome inhibitors, is known to effectively induce the apoptosis of U266 myeloma cells via caspase-dependent pathway and inactivation of NF-kB, these apoptotic effects by Bortezomib could be decreased in U266 myeloma cells exposed to various proinflammatory cytokines. However, roles of IL-6 and sIL-6R in the progression of MM and response to anti-cancer treatment still remain to be explored. In this study, ELISA assay showed higher levels of IL-6, sIL-6R, and IL-8 in bone marrow plasma of MM patients compared to control group, which is the similar results of other investigators. Bortezomib-induced apoptotic effect in U266 myeloma cells was reduced by treatment of IL-6. This Bortezomib-induced apoptotic effect was more reduced in U266 myeloma cells following combined treatment with IL-6 and sIL-6R, although sIL-6R only did not exert on apoptotic effect. Caspase-3 activity by Bortezomib treatment decreased when U266 cells exposed to combined IL-6 and sIL-6R. In addition, U266 cells treated with combined IL-6 and sIL-6R synergically activated the phosphorylation of STAT-3 and ERK resulting in prolong survival of U266 cells. Phosphorylation of STAT-3 and ERK in U266 myeloma cells following combined treatment of IL-6 and sIL-6R did not affect by Bortexomib treatment. These results suggest that the levels of IL-6 and sIL-6R in bone marrow plasma may strongly be associated with clinical outcome of Bortezomib treatment in MM patients.
Transplant Centre Experience and Patient Selection Are the Key Elements Associated with Outcome of RIC and MAC HSCT for CML.
Authors: Crawley Charles R. , Brand Ronald , Olavarria Eduardo , de Witte Theo M. , Apperley Jane F. , Niederwieser Dietger W. , Guglielmi Cesare , Ljungman Per T. , Gratwohl Alois
Published: October 2019

Allogeneic transplantation continues to have a role in the management of high risk CML. However the place of reduced intensity (RIC) versus myeloablative conditioning (MAC) is uncertain in younger patients. A total of 2331 first HSCT’s performed from 1998–2005 were reported to the European Group for Blood and Marrow Transplantation Registry, RIC was used in 276 and MAC in 2055. Overall survivals were similar for the first 9 months. After 9 months the survival curves separated (HR for RIC 1.8, 95% CI 1.2–2.8 p=0.01). All estimates were based on time dependent Cox models and landmark analyses at 9 months. EBMT score was higher in the RIC group (score 0–2, 27% vs 52% p<0.001) and was associated with 5-year survival estimates: EBMT score 0–2, 68% vs 68%, 3–4, 48% vs 56% and 5–7, 27% vs 33% for RIC and MAC respectively. Comparison of RIC and MAC is complicated by differences in patient characteristics. A propensity score was developed to adjust for these differences from a logistic regression model based on the following factors: age, use of PBSC, T cell depletion, disease phase, year, CMV status, sex mismatch, time to transplant and donor type. The propensity score predicts the likelihood of receiving a RIC (high score, RIC more likely). Adjusting for the propensity score compensates for the differences in the underlying covariates. Forty nine percent of RIC allografts were performed in centres reporting ≥5 RIC transplants. There was a significant interaction between propensity score, a centre’s RIC-activity and conditioning intensity on survival. Excluding centres not performing RIC to avoid bias and adjusting for propensity score, survival >9 months was worse in RIC patients with a low propensity score when treated in centres with <5 RICS (HR 2.4 CI 1.3–4.4) whereas survival >9 months was equal in centres treating ≥5 patients with RIC who had a high propensity score (HR 0.8 CI 0.4–1.6). The figures below illustrate this showing the cumulative risk of non-relapse mortality, relapse and death after relapse for these groups estimated in a competing risk setting.

Conclusion: Transplant centre experience and patient selection impact on outcome. Survival with RIC HSCT is inferior to MAC except for the subgroup of patients with a high propensity score transplanted at centres with ≥5 RIC HSCT.

Low Propensity Score and <5 RIC HSCT Low Propensity Score and <5 RIC HSCT High propensity Score and ≥5 RIC HSCT High propensity Score and ≥5 RIC HSCT
Combination of a Spicamycin Analogue KRN5500 and Chemotherapeutic Agents Synergistically Enhances Their Cytotoxicity in Human Myeloma Cells.
Authors: Miki Hirokazu , Ozaki Shuji , Tanaka Osamu , Fujii Shiro , Nakamura Shingen , Mihara Ai , Kagawa Kumiko , Asano Jin , Takeuchi Kyoko , Yata Kenichiro , Hashimoto Toshihiro , Abe Masahiro , Matsumoto Toshio
Published: October 2019

Multiple myeloma (MM) is a plasma cell malignancy characterized by the accumulation of neoplastic plasma cells in the bone marrow. Although new classes of agents such as thalidomide, lenalidomide, and bortezomib have shown marked anti-MM activity in clinical settings, MM remains an incurable disease due to increased resistance to these agents. Therefore, alternative approaches are necessary to overcome drug resistance in MM. KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus (Kirin Pharma, Tokyo, Japan). This drug potently decreases protein synthesis and inhibits cell growth in human tumor cell lines both in vitro and in vivo. Several phase I studies of KRN5500 were conducted in patients with solid tumors, which showed Cmax values of 1000–3000 nM at the maximum tolerated doses. However, no objective anti-tumor response to KRN5500 alone was observed in these patients. In this study, we examined the anti-tumor activity of KRN5500 against MM cells and evaluated its therapeutic potential in combination with other anti-MM agents. MM cell lines and freshly-isolated MM cells were incubated with various concentrations of KRN5500 for 24 hours. Cell proliferation assay showed marked inhibition of cell growth in MM cells such as RPMI 8226, KMS12-BM, and UTMC-2 (IC50 = 10–40 nM), and U266, MM.1S, and primary MM cells (IC50 = 500–1000 nM). Importantly, a chemotherapy-resistant subclone of RPMI 8226 had a similar sensitivity to KRN5500. Annexin V/propidium iodide staining confirmed that KRN5500 induced apoptosis of MM cells in a dose- and time-dependent manner. Moreover, cleavage of poly (ADP-ribose) polymerase (PARP) was detected after 24 hours with only modest activation of caspase-8, -9, and -3 by immunoblotting. Flow cytometric analysis of anti-apoptotic proteins revealed that apoptosis induced by KRN5500 was associated with down-regulation of Mcl-1 and Bcl-2 expression. To determine the effect of KRN5500 on the unfolded protein response (UPR), splicing of XBP-1 mRNA was analyzed by reverse transcription-polymerase chain reaction. In response to stimulation with KRN5500, splicing of XBP-1 mRNA occurred after 24 hours in RPMI 8226 cells, suggesting that KRN5500-induced apoptosis is mediated in part by the inhibition of UPR. Furthermore, synergistic effects on MM cells were observed when KRN5500 was combined with anti-MM agents including melphalan, dexamethasone, and bortezomib. These results suggest that KRN5500 induces apoptosis in MM cells mainly by the caspase-independent pathway and that its unique mechanism of action provides a valuable therapeutic option to overcome drug resistance in patients with MM.
A New Generation XIAP Inhibitor Induces Apoptosis of Human Myeloma Cells and Synergises with Conventional and Novel Anti-Myeloma Therapeutics.
Authors: Khong Tiffany , Spencer Andrew
Published: October 2019

We have demonstrated that a new generation of IAP inhibitor (Novartis) induces apoptosis of a range (n = 8) of genetically heterogeneous human myeloma cell lines (HMCLs) in a dose and time dependent fashion. Dose responsiveness to the IAP inhibitor (IAPI) was determined via MTS assays using IAPI at doses 5 to 50μM for 24 and 72 hours with IC50s of 25 to 50μM evident in 7 out of 8 HMCLs at 72 hours. Mechanistic studies were undertaken utilising 3 HMCLs with high (NCI H929), intermediate (LP-1) or low (OPM2) sensitivity to IAPI with QRT-PCR demonstrating the highest level of XIAP expression in the most IAPI sensitive HMCL NCI H929. Following IAPI exposure immunoblot analyses of all 3 HMCLs exhibited rapid falls in ICAD and CAD as well as cleavage of ROCK-1 and PARP, confirming apoptosis induction. IAPI - induced apoptosis was mediated via both caspase 7 and caspase 3 as demonstrated by rapid cleavage of both caspases following IAPI exposure. Studies combining IAPI with conventional therapies (bortezomib plus doxorubicin) and (bortezomib plus etoposide) against all 3 HMCLs demonstrated synergistic killing with the optimum sequence (IAPI administered 24 hours before or after drug partners) varying between the 3 HMCLs. Similarly, TNF-related-apoptosis-inducing-ligand (TRAIL) and IAPI also demonstrated synergistic killing irrespective of drug sequence. Finally, IAPI combined with LBY135 (Novartis), a chimeric agonistic TRAIL-R2 monoclonal antibody, demonstrated synergistic killing maximally with LBY135 for 24 hours followed by IAPI rather than the reverse sequence. We conclude that IAPI induces down-regulation of both ICAD/CAD signalling as well as ROCK-1 and PARP cleavage via a caspase 3 and 7 dependent process. Furthermore, our data suggest that IAPI is synergistic with a range of conventional and a novel therapeutic agent. While the mechanism of XIAP inhibition in MM warrants further clarification IAPI clearly represents a potentially novel therapeutic approach to MM.
Deficiency of Mannose-Binding Lectin Is a Risk Factor for Invasive Pulmonary Aspergillosis in Patients with Multiple Myeloma: An Analysis of 482 Patients
Authors: Anaissie Elias J. , Zhao Wei-zhi , Wen Yue-Jin , Grazziutti Monica , Ling Wen , Lee Jeanette , van Rhee Frits , Barlogie Bart
Published: October 2019

Invasive pulmonary aspergillosis (IPA) is a major cause of morbidity and mortality in neutropenic cancer patients. Mannose-binding lectin (MBL) is a circulating pattern-recognition molecule that recognizes microbial carbohydrate motifs, leading to complement activation, opsonization, phagocytosis, and cell lysis and is considered an important component of innate immunity. Deficiency of MBL is common due to genetic polymorphisms and has been identified as a risk factor for severe infections among myeloma patients undergoing autologous stem cell transplantation (ASCT). MBL deficiency has also been reported as a predisposing factor for chronic necrotizing pulmonary aspergillosis, a locally invasive infection typically seen in patients with chronic lung disease. The association between MBL deficiency in cancer patients and susceptibility to IPA has never been evaluated.

This study investigated whether MBL deficiency, as measured by serum MBL levels, is associated with the development of IPA in patients with multiple myeloma undergoing multiple cycles of antineoplastic chemotherapy including tandem ASCT and post-transplant consolidation chemotherapy. Baseline serum MBL level was measured using ELISA. The frequency of IPA was assessed retrospectively in 482 adult myeloma patients (2007–2008), 50 of whom developed IPA.

Association between MBL levels and IPA was analyzed using logistic regression analysis (table 1). Patients with normal serum MBL levels (≥ 1000 ng/mL) had significantly fewer episodes of IPA [(21 in 303 patients; 6.9%); Odds ratio, 0.385; 95% CI (0.212–0.699); P = 0.0017] than patients with low (100–999 ng/mL; 18 episodes in 125 patients (14.4%)) and very low (<100 ng/mL) MBL levels (11 episodes in 54 patients; 20.4%) (Table 1). We conclude that MBL deficiency is associated with more frequent episodes of IPA among patients with myeloma undergoing intensive antineoplastic therapy including ASCT and should be added to the list of risk factors for chemotherapy-related IPA. Baseline MBL level may serve as a predictor for the risk of IPA among such patients. This novel finding may have important therapeutic implications because recombinant Human MBL replacement therapy is now available and could therefore be applied prophylactically to decrease the risk of IPA in MBL-deficient patients receiving chemotherapy.

Table 1. Association between serum MBL level and risk of invasive pulmonary aspergillosis (482 pts)

Aspergillosis MBL (ng/mL) No (n=432) Yes (n=50) Rate Odds ratio (95%C.I.) P-value <100 43 (10%) 11 (22%) 11/54 (20% ) Reference Reference 100–1000 107 (25%) 18 (36%) 18/125 (14%) 0.658 (0.287–1.507) 0.3218 1000 282 (65%) 21 (42%) 21/303 (7%) 0.291 (0.131–0.646) 0.0024 MBL <1000 150 (35%) 29 (58%) 29/179 (16%) Reference
Choosing First-Line Therapy for Follicular Lymphoma (FL): A Decision Analysis
Authors: Olin Rebecca L. , Kanetsky Peter A. , Have Thomas Ten , Nasta Sunita Dwivedy , Schuster Stephen J. , Andreadis Charalambos
Published: October 2019

Introduction: There is no standard of care for first-line therapy of low-grade FL. In US practice, the most common strategy is rituximab with combination chemotherapy. However, the optimal choice of regimen remains controversial; options include RCVP, RCHOP and R-Fludarabine-based chemotherapy (RFlu). Because data from randomized clinical trials are not available and unlikely to be generated in the future, we performed a decision analysis comparing RCVP, RCHOP, and RFlu as first-line therapy for FL.

Methods: We constructed a Markov model of sequential first- and second-line therapy based on prescribing patterns in the US. The endpoint of the model was quality-adjusted time to tertiary referral for therapy such as RIT or autologous transplant (≥3rd line). A literature review was performed of the Medline database and international meeting abstracts. Clinical trials of both untreated and previously treated patients were systematically evaluated using explicit eligibility criteria. Data were extracted regarding response rates, treatment-related mortality, and progression-free survival (PFS). Weighted estimates were obtained using a fixed effects meta-analysis. The model also incorporated published data on heath state utilities, risk of anthracycline cardiotoxicity and fludarabine-related delayed cytopenias. Primary and sensitivity analyses were performed using TreeAge software.

Results: The optimal treatment strategy consisted of RCHOP in first-line followed by RFlu in second-line (9.0 quality-adjusted life years; QALYs). Strategies containing RCVP in either first- or second-line were inferior (6.2–7.7 QALYs). The model was sensitive to first-line PFS of RCHOP and RFlu when these were varied over the range of estimates obtained from individual published trials. The model was robust in sensitivity analysis of most other parameters, including rate of delayed cytopenias after RFlu, anthracycline cardiotoxicity, and quality of life adjustments.

Conclusions: Using decision analysis, the optimal first-line therapy for low-grade FL is RCHOP, followed by RFlu in second-line. This strategy maximizes quality-adjusted time to tertiary therapy. Use of RCVP does not improve overall quality-adjusted time relative to more intensive therapies.
TOR Complex 1 and TOR Complex 2 Differentially Regulate Effector and Regulatory T Cell Differentiation
Authors: Delgoffe Greg M , Kole Thomas P. , Zheng Yan , Xiao Bo , Worley Paul F. , Kozma Sara C , Powell Jonathan
Published: October 2019

Effector T cell lineage commitment is determined by the integration of multiple and sometimes opposing signals. Our lab has identified mTOR, an evolutionarily conserved serine/threonine kinase, as a crucial protein dictating the outcome of T cell fate in response to antigen. To do this we utilized a Cre-lox system to conditionally delete mTOR in T cells. In such mice, although mTOR is deleted in the double-positive stage, lymphocyte populations in the spleen and the periphery are comparable to wild-type mice. T cells lacking mTOR proliferate more slowly but secrete appropriate levels of IL-2 upon initial stimulation. However, such cells fail to differentiate into Th1, Th2 or Th17 effector T cells under the appropriate skewing conditions. This failure to differentiate is the result of decreases in appropriate STAT activation and the concomitant lack of upregulation in lineage specific transcription factors. Notably, under normally activating conditions, T cells lacking mTOR preferentially differentiate into Foxp3+ regulatory cells. Supporting this observation, mTOR deficient T cells display hyperactive Smad3 activation, even in the absence of exogenous TGF-β.

mTOR signals through two known signaling complexes, TORC1 and TORC2. TORC1 contains Rheb, mTOR, GβL, and raptor, while TORC2 contains mSin1, mTOR, GβL, and rictor. In order to determine the specific role of TORC1 in T cell lineage commitment we conditionally deleted Rheb in T cells. Upon activation such cells fail to phosphorylate the TORC1 substrate S6K-1 while demonstrating normal TORC2 activity. As was the case for the mTOR−/− T cells, Rheb−/− T cells fail to differentiate into Th1 and Th17 cells when skewed in vitro. However, unlike mTOR−/− T cells, the Rheb deficient T cells are capable of becoming Th2 cells. In spite of lacking TORC1 activity, T cells lacking Rheb do not spontaneously develop into Foxp3+ cells. Such observations implicate a specific and novel role for Rheb in regulating T cell lineage commitment. Overall, our data identify mTOR as a regulator of T cell lineage commitment through which TORC1 and TORC2 signaling differentially regulate T cell fate. These findings support a novel paradigm whereby T cell activation induces a default pathway of differentiation to regulatory T cells and that TORC2 signaling is required to divert differentiation to appropriately programmed effector lineages.
Bone Marrow (BM) Minimal Residual Disease (MRD) at End of Induction and Interim Maintenance Is Highly Predictive of Outcome in Children with Standard Risk (SR) Acute Lymphoblastic Leukemia (ALL) Treated on the Children’s Oncology Group Study 1991
Authors: Matloub Yousif , Gaynon Paul S. , Jayabose Somasundaram , Bostrom Bruce C. , Hunger Stephen P. , Angiolillo Anne L , Devidas Meenakshi , Linda Stephen , Carroll William L. , Winick Naomi , Sather Harland , Pine Sharon R
Published: October 2019

Three thousand fifty four children with NCI SR ALL were enrolled on CCG-1991; 2075 eligible patients were randomized and began treatment with intrathecal cytarabine, vincristine (V), dexamethasone (DX), and pegylated asparaginase (ASP). Bone marrow status was assessed at Day 7 and 14, and 28 of Induction. Slow early responders (SER’s) (Day 7/14 M3-M3, or M3-M2; and M2 at Day 28 Induction) received rescue daunorubicin and were assigned to augmented BFM therapy (N Engl J Med1998; 338:1663). Other patients were designated rapid early responders (RER’s) and randomly allocated to V/ intravenous methotrexate versus oral 6MP/oral methotrexate, and DX pulses in months 3–4 and 7–8 of therapy and single or double delayed intensification. The 5-year EFS for RER’s and SER’s was 90.5% (SE ± 1.0%) and 84.7% (SE = ±3.7%). Eight hundred three patients elected to participate in a companion study of minimal residual disease (MRD), which was successfully performed on BM samples of 750 patients (93.4%). Out of 1362 BM submitted samples, 1360 were successfully tested. MRD was assessed by real-time quantitative PCR of clone-specific immunoglobulin heavy chain, immuno-globulin kappa deleting element, and T-cell receptor gene rearrangements on Day 14 Time Point (TP #1) for patients not achieving M1 status at Day 7, end Induction (TP #2) and Day 84 (RER’s) or Day 119 (SER’s) (TP #3), i.e., Day 28 of Interim Maintenance). Various levels of MRD positivity were explored for prognostic significance (see Table). At TP’s 1, 2, and 3, 14%, 57%, and 78% had undetectable MRD with sensitivity of 0.01% or better. At the three time points patients with detectable MRD were 2.7 to 4.3 times more likely to fail than patients with undetectable MRD. TP #1, however, unlike TP #2 and TP #3 was not predictive of EFS in our study. Patients who had MRD > 0.01% at TP#1 had a much lower EFS at 4 years if at TP #3 MRD persisted at > 0.01% vs ≤ 0.01% (78 ± 0.1% vs 94 ± 0.05%, p = 0.01). We assessed MRD by PCR at three TP’s in a homogeneous population of children with SR ALL receiving V/ DX/ASP. At end of induction, 57% of patients were MRD negative but still accrued 1/3 of adverse events.

Time Point 1 Time Point 2 Time Point 3 MRD Day 14 (Induction Day 14) Day 28 (RER) or 35 (SER) (End Induction) Day 84 (RER) or 119 (SER) (Interim Maintenance Day 28) *Sensitivity < 0.01% “absolute” negative* 1/44 13/340 15/350 low positive < 0.01% 1/27 8/89 5/50 positive 0.01 %–0.1% 6/87 6/104 3/37 positive 0.1%–1% 6/91 4/43 4/9 positive > 1% 6/67 7/21 1/1 total 20/316 45/597 30/448
The POZ/BTB Domain Transcription Factor Miz-1 (Zbtb17) Is Required during Early B Cell Development for the Survival of B-Cell Progenitors and Is Essential for the Formation of Mature Follicular B Cells
Authors: Kosan Christian , Moroy Tarik
Published: October 2019

B-cell development takes place in the bone marrow and is defined by a number of sequential steps that include the up-regulation of CD19, the rearrangement of immunoglobulin heavy and light chain genes (V(D)J recombination) and the expression of surface immunoglobulin. The early steps are regulated by cytokine signaling and the hierarchical expression of transcription factors, among them EBF, Pax5 and E2A and any interference with these critical elements leads to partial or total abrogation of B cell development. Here we present evidence that the POZ/BTB domain transcription factor Miz-1 (Zbtb17) represents an important novel regulator of the early development of follicular B cells. We have used gene targeting in mice to generate a non-functional allele of Miz-1 in all hematopoietic cells. In these mice, the development of adult follicular B cells is almost entirely abrogated, whereas the formation of marginal zone B-cells remain unaffected. Miz-1 deficiency correlated with the absence of CD19+ pro B-cells from the bone marrow and a block at the transition of the pre-pro-B cell to the pro-B cell stage. Although common lymphoid progenitors (CLPs) that are at the origin of B-cell development were present in Miz-1 deficient mice, they showed decreased expression of E2A, EBF and Pax5 compared to their wild type counterparts. Moreover, they were unable to differentiate in culture into more mature B cells even on stroma cells (OP9) and the presence or absence of IL-7. Interestingly, a forced expression of EBF or PAX5 in Miz-1 deficient progenitor cells did not rescue this phenotype. Furthermore, fetal B cell development, which has been shown to depend on EBF and Pax5, is not altered in Miz-1 deficient mice, suggesting that Miz-1 acts in a pathway that is independent of these critical B-cell regulators. In contrast, however, to EBF and Paxc5, the co-expression of a Bcl-2 transgene almost completely restored the development of more mature CD19+ or IgM+ B-cells in Miz-1 deficient mice. This indicated that Miz-1 is implicated in the regulation of cell survival at early stages of B cell development. Since it has been shown before that Bcl-2 is a downstream effector of Miz-1, it is conceivable that Miz-1 regulates Bcl-2 in the early B cell precursors, possibly as an element of the IL-7 signaling pathway, and thereby ensures their survival and proper development. We conclude that Miz-1 represents a novel regulator of early B cell development that exerts its function at a precise step in adult mice independently of other well-established regulators of B-cell development such as EBF or Pax5.
The Rho GTPase Cdc42 is essential for B lyphocyte development and Activation
Authors: Guo Fukun , Zheng Yi
Published: October 2019

Cdc42 of the Rho GTPase family has been implicated in a wide range of fundamental cell functions including actin cytoskeleton dynamics, proliferation, and migration, but its cell-type specific functions have just begun to be appreciated. Previous studies of Cdc42 in B cell regulation by using dominant mutant forms of Cdc42 suggest that Cdc42 is involved in B cell cytoskeleton organization and antigen-receptor signaling, however, its contribution to the B-lineage development and B cell physiology remains unknown. In this study we have achieved B cell- and hematopoietic stem cell-deletion of cdc42 by conditional gene targeting with the CD19 and Mx promoter-driven Cre expression, respectively, in the cdc42loxP/loxP mice. Deletion of cdc42 after the pre-proB cell stage significantly inhibited late B cell development, resulting in reduced mature B cell populations in spleen, bone marrow, peripheral blood, lymph node, and peritoneal cavity. Accordingly, antigen-specific IgM, IgG1 and IgG3, was reduced in the Cdc42-deificient mice. The reduction of Cdc42 knockout B cells are associated with impaired proliferation and survival. Cdc42 deficiency caused a BCR signaling defect with increased Erk and decreased Akt activation, and a defect in BCR-mediated BAFF receptor upregulation and subsequent BAFF receptor signaling in the mature resting B cells. Interestingly, Cdc42 is dispensable for SDF-1α- or BLC-induced B cell migration. Further, deletion of Cdc42 from hematopoietic stem cells resulted in an unaltered common lymphoid progenitor production but severely reduced pre-proB/proB/preB and immature B cell populations, indicating that Cdc42 is also critically involved in the B cell precursor differentiation. Together our results reveal multi-facet roles of Cdc42 in B cell development and activation.
Early Consolidation with Myeloablative Radiochemotherapy Followed by Autologous Stem Cell Transplantation in First Remission in Mantle Cell Lymphoma: Long Term Follow up of a Randomized Trial of the
Authors: Dreyling Martin H. , Hoster Eva , Van Hoof Achiel , Metzner Bernd , Gisselbrecht Christian , Pfreundschuh Michael , Reiser Marcel , Truemper Lorenz H. , Steinhauer Hjalmar , Boiron Jean-Michel , Boogaerts Marc , Silingardi Vittorio , Nelemans J.C. Kluin- , Unterhalt Michael , Hiddemann Wolfgang
Published: October 2019

Background: Mantle cell lymphoma (MCL) is a distinct subtype of malignant lymphoma with an especially poor prognosis and a median survival of only 3–4 years.

Methods: In 1996, the European MCL Network initiated a randomized trial to improve the dismal outcome of MCL, comparing early consolidation with myeloablative radiochemotherapy (TBI 12 gray, cyclophosphamide 120 mg/kg bw) followed by autologous stem cell transplantation (ASCT) to a conventional α-interferon maintenance (6×106 IE IFN- α3x weekly) in first remission after a CHOP-like induction regimen (Dreyling et al, Blood 2005). Here we report the long term results of this prospective trial.

Results: Until March 2004, a total of 232 previously untreated patients up to 65 years with advanced stage MCL were randomized upfront. 173 (76%) of 228 patients evaluable according to the intention to treat (ITT) responded to initial induction chemotherapy and 151 of these (87%) proceeded to the assigned consolidation therapy. 144 patients were evaluable per protocol, 75 pts in the ASCT arm and 69 patients in the IFN maintenance arm. Baseline characteristics were comparable in the per protocol as well as in the ITT cohorts. Patients in the ASCT study arm experienced a significantly longer response duration (RD, median 3.7 years) as compared to patients in the IFNa arm (median 1.6 years, p = 0.0004). These differences were even more pronounced in the CR patients (median response duration 4.5 years vs. 1.6 years) and the intent to treat analysis (median time to treatment failure 2.6 vs. 1.4 years, p = 0.0001). Most importantly, this advantage resulted in a pronounced trend towards an improved overall survival (OS) in the ASCT arm. After a prolonged follow-up of up to 10 years (median 6.1 years), median survival after ASCT was 7.5 years in comparison to 5.4 years under IFN-α maintenance (p = 0.075). In the intent to treat analysis median OS was 7.5 vs. 5.3 years (p = 0.031). As expected, acute toxicity was higher in the ASCT group, whereas long-term effects were more frequently encountered under IFN-α maintenance.

Conclusion: In first line treatment of advanced stage MCL, dose-intensified consolidation with myeloablative radiochemotherapy followed by ASCT after CHOP-like induction results in a significantly prolonged response duration and may also result in an improved overall survival. Accordingly, dose-intensified regimens represent the current therapeutic standard in younger MCL patients. Future study concepts will evaluate the addition of molecular targeted approaches to further improve long term outcome.