bluuebunny/crossref_metadata_2025 huggingface dataset:
Facilitation of Thymic and Bone Marrow Engraftment by Hematopoietic Progenitors Expanded Ex-Vivo Using the Notch Ligand Delta1
Authors: Dallas Mari , Delaney Colleen , Varnum-Finney Barbara , Bernstein Irwin D.
Published: October 2019

Previously, we demonstrated that activation of Notch receptors by culture of murine lin− Sca-1+c-kit+ (LSK) hematopoietic progenitors with the Notch ligand Delta1ext-IgG, consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG, promoted early T cell differentiation and increased the number of progenitors capable of short-term lymphoid and myeloid reconstitution. Here we show accelerated thymic engraftment and T cell recovery after hematopoietic cell transplantation (HCT) in recipients of LSK cells cultured with Delta1ext-IgG compared to recipients of non-cultured LSK cells or control human IgG-cultured LSK cells. Furthermore, data suggest that addition of Delta1ext-IgG cultured LSK cells to non-cultured LSK cells have the potential to accelerated T cell recovery by the non-cultured LSK cells by facilitating thymic engraftment by the non-cultured LSK cells. 103 Ly5a LSK cells cultured with Delta1ext-IgG for 21 days generated a 108-fold increase in total number of cells compared to culture with control IgG. Lethally irradiated C57Bl/6 mice received 105 C57Bl/6 GFP+ LSK cells alone or along with 103 Ly5a LSK cells or 106 Lya5a Delta1ext-IgG-cultured cells or 106 Ly5a IgG cultured cells. We monitored egraftment by difference in CD45 as well as GFP. Mice were sacraficed and blood, bone marrow and thymus were harvested at 1,2,3 and 4 wks after HCT. At 3 wks after HCT, blood samples from recipients of Delta1ext-IgG-cultured cells contained at least 4-fold higher total number of CD3+ cells compared to the other control groups (p<0.05). Furthermore, recipients of Delta1ext-IgG-cultured cells contained at least 3-fold higher numbers of CD3+ cells derived from the GFP+ LSK cells compared to the other control groups (p<0.05). 1 wk after HCT, thymuses and BM from recipients of Delta1ext-IgG-cultured cells showed at lest a 3- and 7-fold increase, respectively, in the number of GFP+ cells compared to the other control groups (p<0.05and p<0.007). Previously, we have shown our system generates committed and non-committed T cell progenitors with enhanced T cell reconstitution potential. Here, we demonstrate that the addition of Delta1ext-IgG-cultured LSK cells to a non-cultured LSK graft has the potential to facilitate BM and thymic engraftment of the non-cultured LSK cells. By using the GFP+ LSK cells, we were able to demonstrate that the facilitation of early BM and thymic recovery was due to the increased engraftment/homing of the GFP+ cells rather than autologous recovery of the host. Thus this report describes a novel and clinically feasible ex-vivo culture system using Delta1 for the generation of T cell progenitors as a means to accelerate initial T-cell recovery as well as facilitating BM and thymic engraftment after HCT.
ChIP-on-Chip Analysis Using Dominant Negative Form of PAX5 Fusion Gene Revealed Genes Associated with Tumorigenesis Were Directly Regulated by PAX5 in a Human B Cell Line, NALM6
Authors: Kawamata Norihiko , Isken Fabienne , Goellner Stefanie , Müller-Tidow C. , Koeffler H. Phillip
Published: October 2019

PAX5 is a transcriptional factor playing an important role in B-cell development. Overexpression of PAX5 induced by translocation to the enhancer region of immunoglobulin heavy chain gene occurs in non-Hodgkin lymphomas (NHL), suggesting that PAX5 can be also associated with development of NHL. To identify genes associated with tumorigenesis in malignancies overexpressing PAX5, we performed ChIP-on-chip analysis using PAX5 specific antibody. Non-specifically immunoprecipitated DNA by antibodies can cause false positive results using ChIP-on-chip analysis (background). To reduce the background in ChIP-on chip analysis, we used a dominant negative form of PAX5 and a wild-type PAX5 specific antibody for our ChIP-on-chip analysis. We have previously found a PAX5 chimeric protein expressed in acute lymphoblastic leukemia in which the C-terminal end of PAX5 was replaced by C20ORF112 protein (Kawamata N et al, Proc Natl Acad Sci U S A. Aug. 12, 2008). We have also found that this chimeric protein behaved in a dominant negative fashion over the wild-type PAX5 and suppressed expression of target genes of wild-type PAX5. PAX5 chimeric protein can compete with wild-type PAX5 for binding on the promoter region of direct down-stream target genes. To identify the genes directly regulated by PAX5 in human B-cells, we transfected the dominant-negative form of PAX5 chmeric protein, PAX5-C20ORF112 (PAX5-C20S) into NALM6 human B-cell leukemia cells which constitutively express abundant PAX5. Transfected cells were collected and chromatin immunoprecipitation (ChIP) assay was performed using PAX5 C-terminal specific antibody which can recognize only wild-type PAX5, but not the chimeric PAX5 protein, PAX5C20S. As a control, we also performed ChIP assay using NALM6 cells transfected with an empty vector. Immunoprecipitated DNA was recovered and amplified using the whole genome amplification technique. The DNAs were hybridized with oligonucleotide probes containing the promoter regions of the human genome. The levels of hybridized DNA were quantified and genes directly bound by PAX5 were identified. Comparison between NALM6 cells transfected with the empty vector and PAX5C20S significantly reduced the background and allowed identification of genes directly regulated by PAX5 in NALM6, including BUB1B, SSSCA1, CEP68, and BAG1. BUB1B, CEP68 and SSSCA1 are proteins involved in mitosis; BAG1 is a protein associated with apoptosis. Dysregulation of these genes by overexpressed PAX5 may be associated with development of B-cell malignancies.
Gray Zone Lymphomas: Clinical and Histological Characteristics and Treatment with Dose-Adjusted-EPOCH-R
Authors: Dunleavy Kieron , Pittaluga Stefania , Grant Nicole , Steinberg Seth , Shovlin Margaret , Tay Kevin , Janik John E. , Jaffe Elaine S. , Wilson Wyndham H.
Published: October 2019

Gray zone lymphomas (GZL) are diseases with transitional morphology and immunophenotypic features between Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). Their pathological and clinical characteristics are not well studied and the best treatment strategy (using HL or DLBCL regimens) has not been defined. Small previous series of HL-like ALCL, which would include GZL, suggest they have a poor outcome with HL treatments. We present GZL’s treated on studies of DA-EPOCH-R at the National Cancer Institute and describe their clinical and histological features and outcome. Overall, 14 patients with GZL were identified. Characteristics included median (range) age 30 (12–51) years; male sex 10 (71%); stage III/IV 2 (14%) and; elevated LDH 7 (50%). These cases could be divided into three Gray zone groups:

classical HL (cHL) and primary mediastinal B-cell lymphoma (PMBL) in 9 (64%) patients; cHL and DLBCL in 2 (14%) and; lymphocyte predominant HL (LPHL) and T-cell histiocyte-rich large cell lymphoma (TCRBCL) in 2 (14%).

Pathological characteristics are shown below. All but one case was CD 10 negative. Markers of cHL included CD15 in 33–50% and CD30 in 66–100% of cases. Morphologically, Reed-Sternberg like cells were typically seen in GZL with cHL features. Thirteen newly diagnosed patients received DA-EPOCH-R. Of 11 patients evaluable for response (2TE), 10 (91%) achieved CR and 1 PR. At a median follow-up time of 4 years, OS and PFS are is 86% and 57%, respectively. Of 9 patients with GZL between cHL and PMBL, 4 (44%) also required radiation therapy compared to only 3/31 (10%) patients with PMBL to achieve durable remissions. Gray zone lymphomas represent a biological and clinical continuum between HL and B-cell lymphomas. Clinically, they appear to be more resistant to treatment than either HL or DLBCL and may require aggressive treatment strategies including radiation. Accrual continues.

Gray Zone Total 14 CD 20 CD 15 CD 30 cHL- PMBL 9 8 (89%) 7 (50%) 9 (100%) cHL-DLBCL 2 2 (100%) 1 (50%) 2(100%) LPHL-TCRBCL 3 3 (100%) 1 (33%) 2 (66%)
Nocodazole Induces Myeloma Cell Death by Disrupting the Microtubular Network, Resulting in G2/M Arrest
Authors: Feng Rentian , Rios Jorge A , Mapara Markus , Lentzsch Suzanne
Published: October 2019

Patients with relapsed multiple myeloma (MM) previously treated with bortezomib and lenalidomide often fail to respond to further therapies. To identify potential new treatment approaches for MM, we used Luminex technology to screen a library of 1,120 compounds provided by the Multiple Myeloma Research Foundation.

By multiplex cytokine array, we identified benzimidazoles including the anthelmintics mebendazole, fenbendazole, albendazole, nocodazole and pyrvinium pamoate, as inhibiting the production of cytokines essential for MM cell growth and survival, such as IL-6 (inhibition rate 40–70%), MIP-1α (inhibition rate 65–75%), VEGF (inhibition rate 75%), and soluble IL-6R (inhibition rate 40–52%). Consequently, these anthelmintics demonstrated dose-dependent inhibition of myeloma cell (RPMI-8226, H929, U266 and MM1S) proliferation. The lead compound, nocodazole, caused nuclear fragmentation and caspase-8 activation in MM cell lines and primary CD138+ cells in dose- and time-dependent fashion (IC50: 30–60 nM). Importantly, growth and survival signals provided by bone marrow stromal cells in bone marrow co-cultures failed to protect MM cells from nocodazole-induced cell death. In the apoptotic cells, caspase-8 was more activated than caspase-9, suggesting that mitochondrial signaling is not a major apoptotic pathway. Cell cycle analysis indicated that G2/M cell cycle arrest reached a peak at 17 hr. Sub-G1 proportion was strongly increased after treatment for 24 hr in all tested cell lines. Electron microscope (EM) and nuclear staining studies consistently showed the accumulation of metaphase cells, and morphologic elongation at 7 hr, at which time G2/M arrest was obvious. Most of the elongated cells had only one nucleus, suggesting that they failed to progress to mitosis due to overall microtubular network disarray.

We conclude that nocodazole exposure induced microtubular network disarray with cell elongation, and G2/M arrest with a late stage mitotic block resulting in cell death.

Benzimidazoles including nocodazole, traditionally used as antihelmintic drugs, have shown antitumor activity against hepatocellular, lung and adrenocortical carcinoma, and melanoma. In our study, we identified the anthelmintic compound nocodazole as a new anti-myeloma agent. Nocodazole warrants further investigation for its anti-MM effects in vitro and in vivo.
Bak Binding to Bcl-xL and Not to Mcl-1 Is Associated with ABT-737 Sensitivity in Multiple Myeloma Cell Lines
Authors: Morales Alejo A , Siefker David , Matulis Shannon M , Gutman Delia M , Boise Lawrence H
Published: October 2019

ABT-737 is a Bad-like BH3 mimetic and an effective inhibitor of the anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL and Bcl-w, but not Mcl-1. Recent studies have shown this new drug as a promising anti-cancer agent with activity in multiple myeloma cells. The purpose of this study was to evaluate the role of Bcl-2 family members in both determining the sensitivity and the mechanism of action of ABT-737 in multiple myeloma cell lines. ABT-737, as a single agent, induced apoptosis in six myeloma cell lines, although the sensitivity was quite different among cell lines. Three cell lines 8226/S, MM.1s and KMS18, were highly sensitive to ABT-737 with EC50 values of 0.30, 0.39 and 0.58 μM, respectively. In contrast, three cell lines, KMS11, U266 and OPM2 displayed lower sensitivity to the drug with EC50 values of 1.60, 2.58 and 2.57 μM ABT-737. No correlation between the sensitivity to ABT-737 and the expression pattern of the Bcl-2 family members was found. Interestingly, Mcl-1, a critical anti-apoptotic protein involved in myeloma cell survival that has also been shown to confer resistance to ABT-737, did not correlate with sensitivity to the drug. Bfl-1, an anti-apoptotic Bcl-2 family member with similar functions to Mcl-1, was only expressed in two sensitive cell lines, MM.1s and KMS18. Since the expression pattern did not reveal any strong correlation, we determined the effects of ABT-737 on association of Bcl-2 proteins. Co-immunoprecipitation experiments in MM.1s and KMS11, demonstrated that ABT-737 released Bak and Bim from Bcl-xL and Bim from Bcl-2 while no change was observed for Bak and Bim bound to Mcl-1. A closer look at the interaction of Bcl-2 family members revealed that Bak is equally bound to Mcl-1 and Bcl-xL in the less sensitive cell lines while it is primarily bound to Bcl-xL in the more sensitive cell lines 8226/S and KMS18. Interestingly, Bak in equally bound to Mcl-1 and Bcl-xL in MM.1s, the third sensitive cell line; however, Bim is also highly bound to Bcl-xL, suggesting an easier release of Bak and Bim by ABT-737 from a Bim-primed-Bcl-xL. Consistent with this idea, Bcl-xL overexpression significantly protected 8226/S but not KMS11 from ABT-737-induced death. Additionally, while silencing of Bim significantly protected MM.1s and KMS11 from ABT-737-induced apoptosis, release of Bak from Bcl-xL was not observed after Bim silencing in the MM.1s cells. Together these data suggest that the interaction pattern not the expression pattern of Bcl-2 proteins is a more accurate measure of ABT-737 function in cells. This is important in diseases like multiple myeloma where Mcl-1 in addition to other anti-apoptotic Bcl-2 proteins are typically expressed.
Clinical Efficacy of VEL-CTD (Bortezomib, Cyclophosphamide, Thalidomide, and Dexamethasone) Regimen in Patients with Relapsed or Refractory Multiple Myeloma: A Phase II Study
Authors: Kim Yeo-Kyeoung , Lee Je-Jung , Sohn Sang-Kyun , Shin Ho-Jin , Chung Joo-Seop , Choi Young-Jin , Moon Joon-Ho , Chae Yee-Soo , Kim Jong-Gwang , Ahn Jae-Sook , Yang Deok-Hwan , Kim Hyeoung-Joon , (KMMWP) the Korean Multiple Myeloma Working Party
Published: October 2019

BACKGROUND: Recent studies demonstrates that the combination of bortezomib and several chemotherapeutic agents may have significant activity in myeloma. In this study, we assessed the efficacy and safety of the combination with bortezomib, cyclophosphamide, thalidomide, and dexamethasone (vel-CTD) for the patients with relapsed/refractory myeloma.

METHODS: Fifty-three patients who had received at least four cycles of treatment were enrolled. Bortezomib was given at 1.3 mg/m2 on D1, 4, 8, 11, thalidomide 50 mg/day, daily, cyclophosphamide 150 mg/m2 P.O. on D1–4, and dexamethasone 20 mg/m2 I.V. or P.O. on D1, 4, 8, 11.

RESULTS: There were 26 males (49.1%) and 27 females (50.9%). The median age of patients was 67 years (range, 40–78 years). Median number of the previous treatment regimens was two (range, 1~5). Of total 53 patients, forty-nine patients (92.5%) achieved at least a partial response (PR) including 28 (52.8%) with complete response (CR) and 21 (42.9%) with PR as their best response. Median progression-free survival (PFS) was 14.7 months (range; 12.3–17.1 months) and median overall survival (OS), from the onset of vel-CTD was 31.6 months (range; 23.3–39.9 months). Patients who achieved a good response at least a PR after four cycles of vel-CTD showed longer PFS than those with poor therapeutic responses (2yr PFS, 28±8.7% vs. 0%, p=0.03). Further, patients who achieved a good therapeutic responses also showed significantly longer OS than those with poor responses (2yr OS, 72.3±8.3% vs. 33.3±15.7%, p=0.009). Grade 3–4 toxicities included thrombocytopenia (30.2%), neutropenia (11.3%), peripheral sensory neuropathy (32.1%), and thrombosis (3.8%). There was no treatment-related death.

CONCLUSIONS: Vel-CTD is a highly active salvage therapy in patients with relapsed/refractory myeloma. However, frequent side effects such as peripheral neuropathy should be considered. Furthermore, trials evaluating novel treatment approaches after bortezomib should be offered to reduce the rate of disease progression, particularly in patients who fail to show a good response at least PR after four cycles of therapy.
PI3K/AKT-and JAK3-Dependent Regulation of SKP2 Expression Is Mediated by E2F1 in Anaplastic Large Cell Lymphoma
Authors: Fontaine Jean-Marc , Elenitoba-Johnson Kojo S.J. , Lim Megan S
Published: October 2019

The majority of anaplastic large cell lymphomas (ALCL) are characterized by the chromosomal translocation t(2;5)(p23;q35) leading to the expression of NPM/ALK. The constitutive activation of the NPM/ALK tyrosine kinase induces downstream mediators such as phosphoinositide 3-kinase (PI3-kinase)/AKT, JAK3 and STAT3 that result in increased cell proliferation and enhanced survival. Although the molecular mechanism by which these pathways deregulate the cell cycle machinery is not fully understood, previous studies have shown that NPM/ALK-mediated PI3K/AKT activation is required for cell cycle progression and that inhibition of PI3K/AKT results in decreased p27Kip1 degradation and cell cycle arrest. The expression of S-phase kinase protein 2 (SKP2), an F-box motif-containing protein which targets cell cycle regulators including cyclin-dependent kinase inhibitor p27Kip1 via ubiquitin-mediated degradation, was evaluated in a panel of ALCL cell lines. Western blot analysis of five t(2;5)-positive ALCL-derived cell lines demonstrated an inverse pattern of expression between F-box protein SKP2 and p27Kip1. We hypothesized that SKP2 deregulation contributes to the oncogenic activity of NPM/ALK by regulating the degradation of p27Kip1. In this study we investigated regulation of SKP2 and p27Kip1 expression as a consequence of inhibition of two well-known pathways downstream of NPM/ALK. Inhibition of PI3K/AKT with Ly294002 (20 mM) or JAK3 with WHI-P154 (10 mM) resulted in a dose and time-dependent decrease in cell viability (50% or 20% respectively at 24h). To determine the mechanism of SKP2 transcriptional regulation by PI3K, we performed quantitative RT-PCR and western blot analysis which demonstrated a decrease in both SKP2 transcript and protein levels after PI3K/AKT and JAK2 inhibition (33% or 47% at 24h respectively), with increase in the levels of p27 transcript and protein (47% or 71% at 24h respectively). Furthermore, the levels of E2F1 (a transcription factor associated with cell cycle progression) also decreased upon PI3K/AKT and JAK3 inhibition. Chromatin immunoprecipitation (ChIP) assays revealed that E2F1 binding to the SKP2 gene promoter was reduced as early as 4 hours after inhibition of PI3K/AKT or JAK3 (80% and 59% respectively) while no binding was detected with the GAPDH gene promoter (control). In conclusion, these results indicate that the expression of the F-box protein SKP2 is regulated by NPM/ALK mediators, PI3K/AKT and JAK3, and that E2F1 mediates the transcriptional control of SKP2 expression. Our data supports the role of SKP2–mediated regulation of p27Kip1 in ALCLs and implicates SKP2 and E2F1 as a potential therapeutic target in ALCLs.
IgA and IgG Hypogammaglobulinemia Is a Constitutive Feature in Most Waldenstrom’s Macroglobulinemia Patients and May Be Related to Mutations Associated with Common Variable Immunodeficiency Disorder (CVID)
Authors: Treon Steven , Hunter Zachary , Ciccarelli Bryan , Zhou Yangsheng , Hanzis Christina , Manning Robert , Ioakimidis Leukothea , Xu Lian , Patterson Christopher , Morra Massimo
Published: October 2019

Background: Hypogammaglobulinemia of the “uninvolved” immunoglobulins is commonly observed in Waldenstrom’s macroglobulinemia (WM), and has often been attributed to disease-related suppression. However, there is a paucity of information related to the pathogenesis of hypogammaglobulinemia in these disorders.

Methods: We evaluated the incidence of IgA and IgG hypogammaglobulinemia in 207 patients with WM, and addressed the impact of therapy and response on IgA and IgG levels for 93 of these patients who required subsequent treatment. We also performed extensive sequence analysis of the promoter, all exonic, and flanking intronic regions from peripheral blood of 19 untreated WM patients who demonstrated IgA and/or IgG hypogammaglobulinemia for 8 genes often observed in common variable immunodeficiency disorders (CVID) and B cell deficiency i.e. AICDA, BTK, CD40, CD154, NEMO(IkBkG), TACI, SH2D1A, UNG.

Results: At baseline, 120/207 (58.0%), 131/207 (63.3%), and 102/196 (49.3%) patients had abnormally low levels of serum IgG (<700 mg/dL), IgA (<70 mg/dL), or both, respectively. No correlation between baseline bone marrow disease involvement and immunoglobulin levels was observed. With a median follow-up of 12 months following completion of therapy, IgA and/or IgG levels remained abnormally low for 92.1% and 87.3% of responding patients, respectively, including those who achieved a complete remission. Sequence analysis of the NEMO and CVID gene panel demonstrated intronic variation at position c.1056-6T>C (n=2) and a hemizygous missense mutation at codon 113 for NEMO (n=1), and a heterozygous missense mutation at codon 142 in UNG (n=1).

Conclusions: The results of these studies demonstrate that IgA and IgG hypogammaglobulinemia is a constitutive feature of patients with WM, which neither correlates with, nor is impacted by disease burden, despite therapeutic intervention and response. The results also suggest that patients with WM may harbor sequence mutations, which may be a contributor to the pathogenesis and/or morbidity of WM.
Early Response Using Serum Immunoglobulin Free Light Chain (sFLC) Measurement Predicts Response to Therapy in Waldenstrom Macroglobulinemia (WM)
Authors: Leleu Xavier , Roccaro Aldo M. , Leduc Renee , Poulain Stephanie , Moreau Anne-Sophie , Gay Julie , Dulery Remy , Richardson Paul G. , Ghobrial Irene M.
Published: October 2019

Background. Measurement of serum M-spike is used to determine response to therapy and treatment free survival in Waldenstrom Macroglobulinemia (WM). However, there are many limitations to the use of IgM M-spike, and new sensitive markers are needed to determine early response or progression in these patients. We have previously shown that involved serum free light chain (sFLC) accurately diagnosed patients with WM, and correlated with markers of poor prognosis, specifically beta- 2 microglobulin (B2M). In this study, we sought to determine the role of levels of involved in the response to therapy in patients with WM treated on clinical trials, and compare it to the response observed using the traditional M-spike measurement.

Methods. We prospectively studied involved sFLC in 61 WM patients enrolled on 2 clinical trials, at diagnosis (N=8) and with relapse/refractory disease (N=53). Patients were treated on one of two clinical trials: either perifosine (N=30; given 150mg oral daily) or combination of bortezomib and rituximab (N=30; given IV bortezomib 1.6mg/m2 at days 1, 8, 15 q 28 days × 6 cycles and rituxan 375 mg/m2 at days 1, 8, 15, 22 on cycles 1 and 4). Response was assessed after cycle 2, confirmed on 2 consecutive measurements, and included minor response (MR), partial response (PR) or complete remission (CR).

Results. The baseline characteristics of the patients were as follows: the median age was 65 years (44–83), male/female ratio 2.38, serum B2M 3.0mg/L (1.00–7.30), hemoglobin 11.0g/dL (7.00–14.90), platelet count 216 ×109/mm3 (46–563), serum M-spike 2.24g/L (0.41–4.62), and involved sFLC 95.2mg/L (4.92–868). The WM International staging system (WM-ISS) showed that 28% of patients had low ISS, 31% intermediate, and 41% high ISS scores. The overall response rate for the entire cohort was 62.3%, assessed by M-spike measurement (MR=23, PR=13, nCR=2). The overall response rate using involved sFLC level was 72.1% (MR=14, PR=29, and CR=1), (p<0.001). We then investigated the role of sFLC in predicting early response to therapy in patients treated with bortezomib and rituximab. We studied the level of sFLC weekly for the first month of therapy. Interestingly, early measurement of sFLC in the first month of therapy significantly predicted response; 93% of the patients who obtained at least an MR within the first 3 weeks of therapy by sFLC demonstrated a response on further follow up using serum M-spike (p=0.024).

Conclusion. In this study, we identified that involved sFLC is a new and reliable marker for monitoring response to therapy in WM disease, especially to determine early response in these patients.
A Prognostic Role for Absolute Lymphocyte Counts in Pediatric Non- Hodgkin’s Lymphoma
Authors: Levy Alejandro G. , Sabella-River Romina , Ghisoli Maurizio L. , Nunez Cesar A. , Anderson Pete M. , Palla Shana L , De Angulo Guillermo , Zweidler-McKay Patrick A.
Published: October 2019

A role for the immune system during treatment for cancer therapy has not been widely established. However, we have previously reported that lymphocyte counts during chemotherapy independently predict outcome in pediatric patients with acute lymphoblastic leukemia (ALL), acute myelogenous leukemia, and Ewing’s sarcoma. Others have reported similar observations in adults with AML, follicular lymphoma and diffuse large B cell lymphoma. This led us to evaluate the prognostic ability of lymphocyte counts in pediatric patients with non-Hodgkin’s lymphoma (NHL). Seventy-six consecutive patients with de novo NHL, age 1–21 years, who were treated at M. D. Anderson Cancer Center were reviewed. We analyzed absolute lymphocyte counts (ALC) at diagnosis and days 7, 15, 21 and 28 of induction chemotherapy. We found that similar to children with ALL, lymphocyte counts in young patients with NHL during induction chemotherapy are independently prognostic for both overall survival and relapse-free survival. For example, children with low ALC on day 7 (ALC-7) of induction had poor RFS and OS. Specifically, patients with ALC-7 <600 cells/μL had a 2-year RFS of 56% versus 95% for those with ALC-7 >600 cells/mcL (HR=4.4, p=0.003). Strikingly, the 19% (12/63) of children with low ALC on both day 7 and 15 (<600 cells/mcL) had a 2-year RFS of 23% compared with 90% for those with either an ALC-7 or -15 >600/mcL (HR=8, p=0.0004). This effect was so dramatic that when ALC-7 of treatment was evaluated as a continuous variable, each cell/mcL increase in ALC was protective for OS decreasing the hazard ratio by 0.002 (p=0.04–0.05). This predicts that an increased ALC of only 250 cells/mcL would yield an HR of 0.5. Importantly, in multivariate analysis with disease stage (I+II versus III+IV), ALC-7<600 remained significantly prognostic for OS and RFS (HRs 6.2–12.2, p=0.006–0.018). Interestingly, lymphocyte counts prior to starting treatment also were prognostic with ALC<500–1000/mcL predicting poor OS (HRs 3.4–4, p=0.04–0.05).

These data reveal that low lymphocyte counts prior to and during induction chemotherapy in children with NHL are significant independent predictors of poor prognosis. These findings support a potential role for the immune system in the treatment of pediatric NHL which should be explored. Outcome differences of 39% are highly clinically relevant and suggest that ALC during induction should be evaluated prospectively and may contribute to future risk stratification of pediatric NHL.

Figure Figure
From Signalosome to Enhanceosome: The Translocation of CD40 from Membrane to Nucleus Involves Multiple Mechanisms
Authors: Zhou Hai-Jun , Tamayo Archie , Pham Lan , Lin-Lee Yen-Chiu , Ford Richard J.
Published: October 2019

CD40 plays important roles in the proliferation, survival and differentiation of lymphocytes. Constitutively active CD40 recruits TRAFs and IKKs within the lipid rafts to form a signalosome that mediates pivotal downstream proliferation and survival mechanisms involving NF-kB. Recently, we have reported that nuclear localization of CD40, through its interaction with c-Rel, promotes growth, cell cycle progression and survival in large B cell lymphoma. Our studies have opened a new paradigm in the functional role of CD40 in non-Hodgkin lymphomas of B cell origin (NHL-B). However, the mechanism about how CD40 enters nuclear still remains elusive. In this study, we show that CD40 ligation enhances its nuclear accumulation with activation of c-Rel in both normal B-lymphocytes and B cell lymphoma cells with cell fractionation assay and con-focal microscopy. Over-expression of c-Rel in B cell lymphoma cells drives CD40 into cell nucleus. We hypothesize that the route CD40 enters nucleus may involve endosome-endoplasmic reticulum-nuclear pore complex. Indeed, further studies show CD40 co-localizes with endosome marker-EEA1 and endoplasmic reticulum marker-Sec61. Furthermore, our co-immunoprecipitation assay has demonstrated CD40 interacts with Sec61. CD40 also co-localizes and immuno-precipitates with nuclear pore complex (NPC) proteins-NUP62 in normal B-lymphocytes and B lymphoma cells, which suggests NPC proteins may facilitate the nuclear translocation of CD40 protein. Overall, our study suggests that translocation of CD40 into cell nucleus involves multiple pathways. Blocking nuclear localization may modulate the function of CD40 in lymphoma cells; which could provide a new-targeted therapeutic approach for lymphoma therapy.
Dissecting Proto-Oncogenic PIM Serine/Threonine Kinases in FLT3-ITDInduced Leukemogenesis: PIM1 Regulates CXCL12/CXCR4-Mediated Homing and Migration
Authors: Gasser Christelle , Grundler Rebekka , Brault Laurent , Bullock Alec , Dechow Tobias , Nawijn Martjin , Woetzel Sabine , Ehret Sabine , Berridge Georgina , Spoo Anke , Dierks Christine , Biondi Andrea , Knapp Stefan , Duyster Justus , Schwaller Juerg
Published: October 2019

Previous work has shown that FLT3-ITD mediated leukemogenesis is associated with increased expression of PIM1 and PIM2 serine/threonine kinases. Here we show that retroviral expression of FLT3-ITD could not compensate impaired clonogenic in vitro growth of PIM1−/− bone marrow cells. Induction of a lethal myelo- and lymphoproliferative disorder by FLT3-ITD in vivo was independent of PIM2, but rather unexpectedly, lethally irradiated recipients could not be reconstituted with FLT3-ITD expressing bone marrow cells lacking PIM1. Transplants of CSFE-labeled PIM1−/− cells revealed an impaired homing capacity to bone marrow and spleen. Expression of lower surface CXCR4 levels (while maintaining normal total CXCR4 levels) in PIM1−/− bone marrow cells was associated with significantly reduced migration towards a CXCL12 gradient and impaired CXCL12-mediated intracellular Ca2+ release. Using siRNA-mediated knockdown, a small molecule PIM inhibitor, expression of a dominant-negative acting PIM1 mutant or re-expression of PIM1 in knockout cells, we observed that PIM1 activity was critical for CXCR4 surface expression. In vitro kinase assays and masspectrometric analysis further revealed that PIM1 directly phosphorylated serine 339 located in the CXCR4 intracellular domain known to be essential for proper receptor recycling. Interestingly, in leukemic blasts from acute myeloid leukemia (AML) patients, we found an association of increased PIM1 expression and high-level of surface CXCR4. In addition, treatment of the cells with a small molecule PIM inhibitor resulted in decreased surface CXCR4 expression in some patients. Our work suggests that PIM1 exerts its oncogenic activity not only by supporting proliferation and survival but also by regulation of cell homing and migration through direct modification of the CXCL12/CXCR4 axis. As CXCR4 is a key mediator of cancer stem cell homing and metastasis, targeting of PIM1 may offer new therapeutic avenues against tumor progression and relapse.
The C-Terminus of the CBFβ-SMMHC Fusion Protein Is Required for the Myeloblastic Transformation of inv16 Leukemia
Authors: Kamikubo Yasuhiko , Garrett-Beal Lisa , Kirby Martha , Liu Pu Paul
Published: October 2019

Inv(16)(p13q22) is found in almost all human acute myeloid leukemia (AML) subtype M4Eo cases and forms a fusion gene CBFB-MYH11, which encodes a fusion protein CBFβ–SMMHC. CBFβ forms a heterodimeric transcription factor with RUNX1 and both are required for embryonic hematopoiesis, while SMMHC is the smooth muscle myosin heavy chain. Using knock-in mouse strategy, we previously demonstrated that Cbfb+/MYH11 F1 embryos have severe blockage of definitive hematopoiesis and die from CNS hemorrhage. The phenotype was similar to that of embryos with homozygous deletion of Cbfb or Runx1, suggesting that Cbfb-MYH11 dominantly represses Runx1/CBFb function. We further demonstrated that Cbfb-MYH11 is necessary but not sufficient for leukemia to develop, as the Cbfb+/MYH11 mice required additional mutations for leukemogenesis. Several hypotheses have been proposed, based on in vitro studies, to explain how CBFβ-SMMHC dominantly inhibits RUNX1/CBFβ. The C-terminal region of CBFβ-SMMHC is responsible for multimerization and also interacts with corepressors; thus this region might be critical for RUNX1 repression. To determine the importance of this multimerization domain in vivo, we generated knock-in mice expressing CBFβ-SMMHC with a 95 aa C-terminal deletion (CBFβ-SMMHCdC95), which truncates this multimerization/repression domain. CBFβ-SMMHCdC95 expressing F1 heterozygous embryos (Cbfb+/MYH11dC95) developed normally with no hematopoietic defects and no hemorrhage. Hematopoiesis was normal in the adult Cbfb+/MYH11dC95 mice except for mild increase of mature neutrophils and minor T cell developmental defects in the first year. However, the mice proceeded to a lethal myeloproliferative disorder (MPD) during their second year of life. There was a significant increase of Mac1/Gr1 double positive cells in the peripheral blood and the spleen, which were negative for the stem cell/progenitor cell marker, c-kit. Morphologically the erythrocytes and neutrophils were dysplastic, and the mice developed severe splenomegaly. ENU treatment of the Cbfb+/MYH11dC95 mice accelerated the development of the MPD phenotype but could not induce overt, transplantable, myeloid leukemia. These data suggest that the multimerizatin domain of SMMHC is important for both hematopoiesis blockage and leukemogenesis, especially the blastic transformation of inv16 leukemia.
The Role of Hepcidin in Anemia of Cancer
Authors: Rivera Seth , Kim Airie , Ganz Tomas
Published: October 2019

Anemia is a common occurrence in a wide variety of malignancies. When the anemia is not due to bleeding, bone marrow infiltration or chemotherapy, it is termed anemia of cancer (AC). AC is presumed to be a form of anemia of inflammation (AI, aka anemia of chronic disease). By limiting the availability of iron absorption from diet and release from stores, hepcidin causes AI. To test whether hepcidin contributes to AC, we created two syngeneic mouse models of lung cancer (TC-1 and LLC) and a syngeneic mouse model of melanoma (B16-F10). In all three models, significant anemia developed (Hgb 10.9±2.0, 9.1±2.0, 7.8±2.0 for TC-1, LLC, and B16-F10 vs. 13.7±1.2 for controls; p<0.001 for all compared to controls). There was no evidence of bleeding in the tumors or intraperitoneally. The anemia was microcytic in TC-1 mice but not LLC or B16-F10. Hepcidin mRNA (hepatic production measured by qRT-PCR) was increased in TC-1 mice but not LLC or B16-F10. To test whether hepcidin was necessary for the development of anemia in the TC-1 model, we generated tumors in hepcidin knockout mice. Hgb did not fall significantly in hepcidin knockout mice with TC-1 induced tumors (Hgb 14.5±2.4 vs. 15.5±2.2, p=0.3).

CONCLUSIONS: Hepcidin is necessary for the development of AC when hepcidin production is induced by the tumor and microcytosis is present. However, AC is likely a heterogenous disease; experiments are underway to determine whether hepcidin plays a role in cancers that do not upregulate hepcidin production or cause microcytosis.
Inhibition of Hamp1 Expression by TMPRSS6, GDF15, PIAS4 and SMAD6 Utilize Several Target Sites in the Hamp1 Promoter
Authors: Truksa Jaroslav , Lee Pauline , Beutler Ernest
Published: October 2019

Hepcidin, the key regulator of iron homeostasis, is up-regulated by iron excess, bone morphogenetic proteins (BMPs) and inflammatory cytokines and down-regulated by hypoxia and anemia. Known positive regulators at the level of transcription factors include SMAD1/4, STAT3 and CEBPα. In this study, we focused on negative regulators of hepcidin regulation:

Matriptase II/TMPRSS6 (Transmembrane serine protease 6, a recently identified negative regulator in which disruption leads to anemia in mice as well in humans); Protein inhibitor of activated STATs no. 4 (PIAS4); Growth and differentiation factor 15 (GDF15, a potential erythroid negative regulator); and SMAD6 (Mothers against decapentaplegic homolog 6, an inhibitory SMAD blocking the SMAD/BMP pathway).

All tested inhibitors significantly decreased expression of the luciferase reporter under the control of 2.5 Kb murine Hamp1 promoter with GDF15 and PIAS4 Hamp1 specific since none of the inhibitors were able to reduce expression of the luciferase reporter under the control of the murine Hamp2 promoter. Inhibition of the luciferase reporter under the control of the 2.5 Kb murine Hamp1 promoter by SMAD6, unlike TMPRSS6, PIAS4 and GDF15, did not require liver specific transcription factors since the inhibition could also be observed in transfected HEK293T cells. GDF15, PIAS4, TMPRSS6 and SMAD6 all reduced basal level expression of the luciferase reporter under the control of the 2.5 Kb murine Hamp1 promoter as well as the total level of reporter expression induced by IL-6 and BMP-4. Nevertheless, GDF15 did not affect responsiveness (fold induction) to IL-6 and BMP-4. PIAS4 and TMPRSS6 inhibited responsiveness to IL-6 but had little effect on responsiveness to BMP-4. In contrast, SMAD6 did not affect responsiveness to IL-6 but reduced responsiveness to BMP-4. Deletion of the −140 bp −260 bp region of the murine Hamp1 or double deletion of the BMP-RE1 and BMP-RE2 motifs severely reduced the ability of all inhibitors to reduce reporter expression. Deletion of the STAT site abrogated PIAS4 inhibition while deletion of either BMP-RE1 or BMP-RE2 motifs alone partially reduced inhibition by TMPRSS6 and SMAD6. We conclude that there are several independent pathways that inhibit hepcidin expression.
cIAP1-Dependent TRAF2 Degradation Is Required for Macrophage Differentiation and Function
Authors: Dupoux Alban , Cartier Jessy , Marivin Arthur , Cathelin Séverine , Filomenko Rodolphe , Solary Eric , Dubrez-Daloz Laurence
Published: October 2019

TRAF2 (Tumor necrosis factor (TNF) Receptor associated factor) is an adaptor molecule involved in TNF family membrane receptor signalling pathway. We demonstrate that TRAF2 is required for macrophage differentiation. Its silencing prevents Iκ-Bα degradation, NF-κB p65 nuclear translocation and the differentiation process. Then, TRAF2 is degraded by the proteasome system in the cytoplasm. Inhibition or silencing of the E3 ubiquitine ligase cIAP1 from the IAP (Inhibitor of apoptosis protein) family of proteins, prevents the differentiation-associated TRAF2-degradation. In the presence of proteasome inhibitor, TRAF2 colocalizes with cIAP1 in a golgi-associated compartment, suggesting that cIAP-1-mediated TRAF2 degradation occurred in this subcellular compartment. An overexpression of TRAF2 or silencing of cIAP1 prevents the outcome of differentiation as revealed by the expression of differentiation markers at the cell surface, and inhibits the secretion of pro-inflammatory cytokines such as interleukin-8 and MCP-1 by fully differentiated-macrophages in response to CD40 stimulation. Our data suggest that TRAF2 expression and its subsequent cIAP1-dependent degradation is required for the differentiation of monocytes into macrophages and allows the macrophages to be fully functional.
Conditional T-Cell Activation for Tumor Under Hypoxia
Authors: Ang Sonny O , Olivares Simon , Shpall Elizabeth J. , Lee Dean A. , Champlin Richard , Yotnda Patricia , Gottschalk Stephen , Cooper Laurence
Published: October 2019

Solid tumors are characterized by cell clusters experiencing chronic and intermittent hypoxia due to haphazard growth. Importantly, highly migratory tumorigenic stem cells are localized within hypoxic niches, shielded by hypoxia-induced metabolites that inhibit tumor-infiltrating T cells, restricting their survival, cytotoxicity, and beneficial immunomodulatory cytokine responses. To eliminate bulky solid tumors including stem cells, T cells must overcome physiological changes distinctive for the tumor microenvironment, such as hypoxia, depleted nutrient levels, and low extracellular pH. To exploit the hypoxic tumor microenivornment, we introduce a new approach that exploits hypoxia as a condition for T-cell activation. We demonstrate a chimeric antigen receptor (CAR), which is specific for CD19 and capable of activating T cells through chimeric CD28 and/or CD3-zeta signaling endodomain, that can be conditionally expresed in a strictly oxygen-sensitive manner. Using the Sleeping Beauty transposon/transposase system, we can achieve stable, persistent CAR transgene expression without the expense and complexity of retroviral production. Cell surface expression of our CAR is high at 1% O and not detectable at 20% O (Figure). Anticipating the circulatory nature of T cells in vivo, our oxygen-sensitive CAR is designed to deactivate upon exiting from hypoxic tumor microenvironment to minimize deleterious off-target effects. When supplemented with one or more survival factors and/or homing receptors, this approach to CAR expression has the advantage of

transforming hypoxia from an adverse factor to a triggering mechanism, efficiency, and high levels of transgene expression.

Figure: Flow cytometry expression of CAR under normoxia and hypoxia. Figure:. Flow cytometry expression of CAR under normoxia and hypoxia.
Identification of a Role for Apolipoprotein E Receptor 2 as a Platelet Receptor for Factor XI
Authors: White Tara C , Berny Michelle A , Tucker Erik I , Pang Jiaqing , Gailani David , Urbanus Rolf T , de Groot Philip G , Gruber András , McCarty Owen JT
Published: October 2019

Factor XI (FXI) promotes hemostasis and thrombosis through the enhancement of thrombin generation, and has been shown to play a critical role in the formation of occlusive thrombi in arterial injury models. Although the binding of FXI to platelets has been demonstrated, the role that FXI binding might play in platelet activation has not been studied. It is also unknown if FXI-platelet binding is solely mediated by GPIbα or if other platelet receptor(s) exist that can support interactions with FXI. This study has identified apolipoprotein E receptor 2 (ApoER2, LRP8), a member of the low-density lipoprotein (LDL) receptor family, as a platelet receptor for FXI. Surface-bound FXI supported platelet spreading and elevation of intracellular Ca2+, while platelet spreading on FXI was eliminated in the presence of either Src-kinase or PI3-kinase inhibitors. Platelet adhesion to immobilized FXI was abrogated in the presence of the universal LDL receptor antagonist, receptor-associated protein (RAP), soluble recombinant ApoER2′ or the LDL-binding domain 1 or 2 of ApoER2. FXI supported wild-type murine platelet binding; in contrast, ApoER2-deficient murine platelets did not adhere to FXI. Soluble FXI binding to immobilized platelets or purified recombinant ApoER2′ was abrogated in the presence of binding domain 1 of ApoER2 or RAP, respectively. These results identify FXI as a novel ligand for the platelet receptor ApoER2. While many of the details regarding the role of FXI in vivo are still undetermined, the ability of FXI to bind and activate platelets via ApoER2 suggests that FXI may contribute to hemostasis and thrombosis in a manner unrelated to contact activation or thrombin generation. Furthermore, these findings suggest that FXI may play a role in promoting cell signaling in other cells that express ApoER2, such as neurons, immune system cells and smooth muscle cells.
Investigation of Parent-Related Etiologic Factors in Children with Acute Lymphoblastic Leukemia
Authors: Yilmaz Oznur , Timur Cetin , Yoruk Asim , Erguven Muferet , Senturk Gunes , Kavakli Kaan
Published: October 2019

In this study, we investigated parental history and habits in children with acute lymphoblastic leukemia and we wanted to point at possible factors that can affect the risk of leukemia. The parents of 105 children diagnosed and treated as ALL between the years 1997–2007 in our Clinic of Pediatric Hematology-Oncology were questioned and results were compared with control group that consisted of 102 healthy children with similar age and gender. There was no significant difference between the groups in terms of maternal and paternal age (p>0.05). Even though the rates of family history of leukemia and other cancer types were higher in the leukemia group, there was no statistically significant difference (p>0.05). Rate of having genetic disease in family history was significantly higher in leukemia group and was associated with an increased leukemia risk (OR: 8.33; %95 CI= 1.02–67.85, p:0.035). Although the rate of miscarriages of mother was higher in leukemia group, there was no statistically significant difference (p>0.05). There was no significant difference between the groups in terms of maternal food consumption. The rate of vitamin and iron supplement use was significantly lower in leukemia group (p:0.017; p:0.002). The rate of folic acid and fish oil use was higher in control group; however there was no significant difference (p>0.05). There was no difference between the groups in terms of oral contraceptive use, X-ray exposure of mothers before conception, infection during pregnancy, hypertension and other diseases in pregnancy. The rate of medication use among mothers was significantly higher in leukemia group (p:0.001). Cigarette smoking was not common among mothers in both groups. When fathers were compared, the rates of cigarette smoking ≥1 package/day (OR: 2.00; %95 CI= 1.02–3.91, p:0.041) an ≥20 package-years (OR: 1.95; %95 CI= 1.00–3.66, p:0.048) were significantly higher in leukemia group. Besides, the rate of routine alcohol consumption was significantly higher among fathers in leukemia group (OR: 6.45; %95 CI= 1.42–29.59, p: 0.029). No significant difference was found between the groups in terms of prenatal x-ray exposure and drug intake when fathers were compared (p>0.05). In conclusion, leukemia is a disease with multi-factorial etiology like most of the other cancer types that arise as a result of interactions between genes and environment. The risk of leukemia may increase as a result of cigarette and alcohol consumption that can lead to chromosomal abnormalities especially in genetically susceptible children.