bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a Homeobox-Like Protein Immune Response in Bone Marrow Transplant Patients
Authors: Arden Nilou , Chen George L. , Miklos David
Published: April 2020

The identification of novel immune targets following hematopoietic cell transplantation (HCT) promises to identify clinically important graft-v-leukemia and graft-v-host disease targets. High-density protein microarrays present thousands of human proteins and facilitate broad-band multiplexed quantitative auto and alloreactive antibody screening. Our studies utilized both male and female patient plasma which were collected pre-transplant and one year post-transplant to identify new immune response targets. The tetra-peptide repeat homeobox-like protein (TPRXL) was initially identified by protein microarray as a potential biomarker in one MDS and four AML transplant patients. We developed an ELISA assay by affinity purifying TPRXL expressed in E.coli, and tested plasma samples collected from 80 HCT patients 1–2 years after HCT. A 42% sero-positive incidence was found across the group of post transplant samples (p-value <0.001). Located at 3p25.1 of the human genome, TPRXL encodes a 257 amino acid and 25 kDa protein containing a significant percentage of serine tetra-peptide repeats. Being a member of the TPRX homeobox gene family, the protein’s secondary structure and function lead to direct interaction with DNA and tight regulation of gene expression. Presently, of the 18 reported TPRXL transcript variants a majority are nearly identical and are found to be highly expressed in early embryonic development, in the placenta, oral tissues and the ascites. Results indicate disease specificity with prevalence of post transplant response in patients with Mantle cell lymphoma (MCL). In addition, patients with chronic GVHD of the oral pharyngeal tissues are predominant in our analysis. In silico studies show that TPRXL is indeed differentially expressed in acute pro-myelocytic leukemia (PML) and acute lymphocytic lymphoma (ALL) cell lines as compared to controls. Since bioinformatics expression data available for MCL cell lines are scarce, perhaps due to fewer reports or studies in this area, it is clear that our novel findings will contribute to currently available information for this disease type. In sum, protein microarray technology represents a powerful tool in facilitating the identification of novel disease associated antigenic targets and that TPRXL is potentially one such prognostic marker.
Study on the Dendritic Cell Subsets and Their Relationship with the Expressions of T-Bet and GATA-3 in Peripheral Lymphocytes of Severe Aplastic Anemia Patients
Authors: Shao Zonghong , Jun Wang , Fu Rong , Li Lijuan , Liu Hui , Qu Wen , Rua Erbao , Liang Yong , Liu Hong , Wu Yuhong , Wang Guojin , Song Jia , Wang Huaquan , Xing Limin , Guan Jing , Wang Xiaoming
Published: April 2020

Objective To investigate the effects of the dendritic cell (DC) subsets and the transcriptive factors, T-bet and GATA-3, on immune imbalance in the pathogenesis of acquired severe aplastic anemia (SAA).

Methods The mDCs(HLA-DR+Lin−CD11c+) and pDCs(HLA-DR+Lin−CD123+) in peripheral blood mononuclear cells (PBMNC) were measured with flow cytometry (FCM), the expressions of T-bet mRNA and GATA-3 mRNA in PBMNC with semiquantitive RT-PCR and the plasma levels of IFNγ and IL-4 with ELISA in 29 SAA patients (21 untreated and 8 recovered) and 16 healthy controls.

Results The percentages of mDCs in PBMNC were (0.44±0.24) % and (0.73±0.30) % in untreated and recovered SAA patients respectively, either of which was higher than that of controls (0.29±0.10%) (P<0.05). The percentage of pDCs in the untreated cases was lower than that of the recovered or controls [(0.18±0.14) % vs. (0.28±0.20) % and (0.29±0.13) %] (P<0.05). mDCs/pDCs ratios were 3.45±2.71 and 2.90±0.95 in untreated and recovered groups respectively, either of which was higher than that of controls (1.15±0.56) (P<0.05). No statistical difference of mDCs/pDCs was found between untreated and recovered patients (P<0.05). The relative mRNA expressions of transcriptive factor T-bet were 0.37±0.07, 0.20±0.07 and 0.17±0.05 in 3 groups, respectively. The expression of T-bet of untreated patients was higher than that of recovered patients or healthy controls (P<0.05). There was no statistical difference of GATA-3 expression among 3 groups(P>0.05). T-bet/GATA-3 ratio was 0.72±0.13 in untreated patients, which was higher than that of recovered patients (0.33±0.08) or controls (0.35±0.11). The plasma level of IFNγ in the untreated cases was (50.9±1.1)ng/L, which was higher than that of recovered [(49.7±0.9) ng/L] or controls[(49.7±0.7) ng/L]. There were significant positive correlation between T-bet and mDCs/pDCs (r=0.445, P<0.01), as well as T-bet and IFNγ (r=0.402, P<0.01).

Conclusion Both mDCs/pDCs and T-bet/GATA-3 ratios in SAA were higher and positively correlated which might contribute to the cascade activation of T lymphocytes. Immunosuppressive therapy for SAA should be kept on until both ratios become normal.
Performance Comparison of Commercial Fixing and Permeabilizing Reagents
Authors: Joe Edward , Frey Tom
Published: April 2020

Flow cytometry is commonly used to characterize cellular antigens expressed both on the cell surface and inside the cell. As the technology advances, more researchers are using this tool to study intracellular activity to understand how cells communicate and respond to their environment. Intracellular targets of interest include cytoplasmic antigens and proteins, nuclear antigens and proteins, cytokines, etc. The quality of intracellular staining depends on the ability of the permeabilizing reagent to permeabilize the cell membrane for antibodies to get to the target of interest without destroying the cell. Five different commercial permeabilizing reagents were evaluated in a small study, Caltag Fix and Perm® Kit, BD IntraSure™ Kit*, BD FACS™ Permeabilizing Solution 2 (FACS Perm 2), 1X FACS Lysing Solution, and BD Cytofix/Cytoperm™ Kit. Five categories of performance were evaluated: cell recovery, maintenance of surface staining, preservation of light scattering properties, intracellular staining, and background staining. Cell recovery was assessed using CD45 APC and BD TruCOUNT™ tube; surface staining maintenance was assessed using CD45 APC, CD45 PerCP-Cy5.5, CD19 PE, and CD20 PerCP; intracellular and background staining were assessed using anti-bcl-2 FITC, anti-Zap 70 PE, anti-TdT FITC, anti-Ki-67 FITC, and CD3 FITC/anti-MPO PE/CD79a PerCP-Cy5.5 reagents. Caltag and BD IntraSure Kits performed well in the category of cell recovery. Caltag Kit, BD IntraSure Kit, and FACS Lysing Solution maintained cell surface staining after cell permeabilization. BD IntraSure Kit and FACS Lysing Solution preserved light scattering properties of cells after permeabilization. Using signal-to-noise ratio as the criterion to evaluate background and intracellular positive staining, FACS Lysing Solution and FACS Perm 2 detected anti-bcl-2 FITC; BD IntraSure Kit and FACS Lysing Solution detected anti-TdT FITC; BD IntraSure Kit, FACS Lysing Solution, FACS Perm 2, and BD Cytofix/Cytoperm Kit detected anti-Ki-67 FITC; BD IntraSure Kit, FACS Lysing Solution, and FACS Perm 2 detected anti-Zap 70 PE; all kits detected CD3 FITC/anti-MPO PE/CD79a PerCP-Cy5.5. Results showed that there is no universal permeabilizing reagent for all intracellular staining assays. Each test reagent has its own strength and weakness. Choosing the best permeabilizing reagent for a particular assay depends on the target of interest and the output of the assay.
Does PDG-PET Add Additional Prognostic Information to CT Staging in Lymphoma Patients?
Authors: Sanchez-Gonzalez Blanca , Trampal Carlos , Solano Alberto , Pedro Carmen , Maiques Jm , Alvarez Alberto , Gimeno Eva , Abella Eugenia , Besses Carles , Salar Antonio
Published: April 2020

BACKGROUND: 18-Fluoro-deoxyglucose positron emission tomography (FDG-PET) is a noninvasive imaging tool for initial staging and evaluation of treatment response in Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). PET imaging can provide additional information to conventional imaging as computer tomography (CT).

AIM: The aim of this study was to evaluate the role of PET in improving the staging and if this fact could have an impact in prognosis, in compared to CT.

METHODS: From February 2005 to January 2008, one hundred ten consecutive patients with NHL (49 aggressive, 32 indolent and 3 unclassifficated) and HL (26 patients) were staged according to the Ann Arbor classification with CT and bone marrow biopsy (BMb). As well, all patients underwent to FDG-PET body before treatment. International Prognostic Index (IPI) was calculated following standard staging (IPI-st) and with standard staging plus FDG-PET (IPI-PET) in NHL.

RESULTS: CT and FDG-PET were concordant in 84 patients (66%). In 37 patients (34%) discordance was observed: in 7 patients FDG-PET was negative, in 11 patients FDG-PET detected more bone lesions, in 11 patients FDGPET uptake in other lymph nodes, including spleen infiltration, and in 8 patients FDGPET detected less lymph nodes or organ involvement. However, additional information of FDG-PET changed in staging in 21 out of 37 patients with discordant findings but the IPI risk changed in only 5 patients. Discrepancies CT and FDG-PET were seen in similar proportion in all lymphoma subtypes. Overall survival curves at two years showed no differences between IPI-st and IPI-PET (IPI-st 0–1: 97%, 2–3: 89%, 4–5: 48%; IPI-PET 0–1: 96%, 2–3: 89%, 4–5: 48% ).

CONCLUSIONS: FDG-PET can provide additional information to CT in both aggressive or indolent non-Hodgkin lymphoma and Hodgkin lymphoma. CT and FDG-PET were discordant in 30 % of the patients but only half of them showed change of stage. IPI risk was modified in few patients with NHL. Inclusion of patients is ongoing.
Alternate Splicing Is a Frequent Event and Impacts Clinical Outcome in Myeloma: A High-Density Exon Array Analysis of Uniformly Treated Newly-Diagnosed Myeloma Patients
Authors: Munshi Nikhil C. , Li Cheng , Minvielle Stephane , Amin Samir B , Moreau Philippe , Magrangeas Florence , Anderson Kenneth C , Avet-Loiseau Herve
Published: April 2020

Alternate splicing is an important post translational change that alters specificity of gene function. Misregulation of alternative splicing has been implicated in number of disease processes including cancer. We have here analyzed alternate splicing in myeloma using high throughput exon array analysis. The GeneChip Human Exon 1.0 ST Arrays used in this investigation not only provides information on expression levels for genes, but as the probe sets are spread evenly over each exon, the array data also provides information on presence of each exon and identify recurrent alternately spliced genes. We conducted a study in series of 170 newly-diagnosed patients with multiple myeloma treated homogeneously in tandem transplantation IFM trials. RNA isolated from CD138 purified MM cells collected at the time of diagnosis were analyzed using the GeneChip Human Exon 1.0 ST Arrays. The dChip software was modified to analyze exon array data. We first normalized gene-level expression values across samples, and then identified differentially expressed exons between two sample groups. These exons are candidates for alternatively splicing events. We observed over 100 genes with candidate alternate splicing events, which have up or down-regulation of an exon relative to the baseline group in more than 20% patients. Dividing the group based on response, 52 alternately spliced exons were identified as influencing response to therapy. The genes and their exons with highest frequency of alternate splicing were CD79A exon 5 and 6, EDNRB exon 2, RASSF1 exon 8, CD1d exon9, TGFBetaRII exon 1, Calmin exon2, CEACAM1 exon 7, MADH1 exon 7, TBX5 exon 2, Amyloid beta exon 14, Nit protein 2 exon 10, Thymidylate synthetase exon 7. Amongst these genes, 32 genes had the most influence on response with over 50% differential frequency in patients achieving CR. Similarly we have identified 85 alternately spliced genes as influencing overall survival between groups divided by less than or more than 48 month survival. The genes with highest frequency of alternate splicing were NOTCH2 exon 18, CXCL3 exon 8, CD9 exon 3 Calmin exon 2, TIMELESS exon 12, SPOUTY homolog 1 exon 4, Amyloid beta exon 14, Nit protein 2 exon 10, Cyclin A2 exon7, lymphocyte antigen 6 exon 7. Forty-nine genes within this group had the most influence on overall survival. Number of spliced variants were shared between both response and survival groups suggesting their biological and functional significance. Further validation of these alternate splicing is under investigations. This study highlights significant frequency of alternate splicing and points to the need for evaluation of not only the expression level of genes but also post translational modifications. The genes identified here are important target for therapy as well as possible immune modulation.
Clinical Efficacy of ATRA for Adult T-Cell Leukemia
Authors: Maeda Yasuhiro , Yamaguchi Terufumi , Hirase Chikara , Kanamaru Akihisa
Published: April 2020

We previously reported that all-trans retinoic acid (ATRA) inhibited growth in HTLV-I- positive T-cell lines and fresh cells from patients with adult T-cell leukemia. We here confirmed the clinical effects of ATRA in 20 patients with ATL. Twenty patients (n=20) with median age of 56 years (range 35–68 years) diagnosed with ATL received ATRA orally. ATRA was administered for a median of 25.7 days (range 14–56 days). Efficacy was described below; no CR case, PR case was 55%, NR case was 45%. In 7 acute cases, PR case was 4 (20%) and NR case was 3 (15%). In 3 lymphoma cases, no NR case and 3 PR cases (15%) was found. In 4 chronic cases, PR case was 1 (4%) and NR case was 3 (15%). In 6 skin type. PR case was 3 (15 %) and NR case was 3 (15%). Major side effects were headache (n=5), transient liver dysfunction (n=2), hyperlipidemia (n=2) and anorexia (n=1). No major toxicity was observed. These results indicated that ATRA might be a useful agent for skin involvement of ATL with safety.
Pilot Study of Peginterferon Alfa-2a (PEGASYS®) Concurrent Administration during Induction Chemoradiotherapy and Maintenance Therapy in Patients with EBV-Associated Extranodal NK/T Cell Lymphoma
Authors: Cho Seok-Goo , Kim Sung-Won , Park Kyung-Sin , Lim Ji-Hyang , Min Chang-Ki , Kim Yong-Goo , Lee Jong-Wook , Min Woo-Sung
Published: April 2020

Extranodal NK/T-cell lymphoma, nasal type is common in Asians rather than in Western populations. This special subtype responds poorly to intensive chemotherapy regimens or has a significant relapse rate in spite of low IPI and loco-regional staging. The presence of the EBV genome within tumor cells raised the possibility of developing therapeutic strategies directed at viral targets. The primary objectives of this prospective, single center, nonrandomized phase II trial are the evaluation of efficacy by determination of progression-free survival after concurrent administration of peginterferon alfa-2a (PEGASYS®, provided by Roche Korea) and chemoradiotherapy in patients with extranodal NK/T cell lymphoma. In addition, we evaluated the efficacy of peginterferon alfa-2a maintenance therapy for 3 years. All seven patients with newly diagnosed CD56+ EBV+ extranodal NK/T-cell lymphoma were enrolled onto a prospective clinical trial. Step 1; PEGASYS® 180 mcg was administered once weekly on D1, D8 of ProMACE-CytaBOM. 1, 2 cycles. Step 2; PEGASYS® 180 mcg will be administered once weekly on D1, D8, D15, D22 of involved-filed irradiation 3600 cGy (180 cGy × 20, for 4 weeks). Step 3; PEGASYS® 180 mcg will be administered once weekly on D1, D8 of ProMACE-CytaBOM. 3, 4 cycles. Step 4; PEGASYS® 180 mcg will be administered every two weeks after hematologic recovery of high-dose therapy and autologous PBSCT for 3 years. In case of advanced stage and non-nasal lesion, Step 2 was omitted and ProMACE-CytaBOM was extended to 6 cycles. EBV DNA copy based on real-time PCR was monitored. Of all 7 patients, 6 patients achieved complete response and 1 patient was dead due to pneumonia during chemotherapy. With a median follow-up of 26 months (3–36 months), the 3-year PFS were 67%. Of 6 assessable patients, five patients extended to peginterferon alfa-2a maintenance therapy and four patients showed PFS. Among two relapsed patients, one patient refused a peginterferon alfa-2a maintenance therapy and eventually relapsed 3 months later. Titer of EBV DNA copy in whole blood was significantly decreased compared with the titer on initial diagnosis. The main toxicities of peginterferon alfa-2a were mild flu-like symptoms. This pilot study suggests that larger prospective randomized studies are needed to define the role of peginterferon alfa-2a concurrent administration during induction therapy and maintenance therapy in EBV-associated extranodal NK/T-cell lymphoma.
Trans-Cinnamaldehyde Induces Granulocytic Differentiation and Impairs Survival and Migration of Acute Myeloid Leukemic Cells
Authors: Liu Liqiong , Wang Ping , Wang Xingbin , Gu Junxia , Li Xiaoqing , Huang Shiang
Published: April 2020

Trans-cinnamaldehyde (TCA), the major active component of oil isolated from the stem bark of Cinnamomum cassia traditionally used to treat dyspepsia, gastritis and inflammatory disease world widely, has been shown to inhibit proliferation and promote apoptosis in a number of cancer cells. However, the functional roles TCA plays in hematopoietic system have not been fully investigated. In this study, we show the effects of TCA on acute promyelocytic leukemia (APL) cell line HL60 and primary bone marrow mononuclear cells (BMMNC) as well as bone marrow stromal cells (BMSC) from acute myeloid leukemia (AML) patients. We found that TCA affected HL60 cells in a time-and dose-dependent fashion. Low concentration of TCA (20 μM) arrested HL60 cells at G0/G1 phase at 72 h without significant apoptosis. Middle concentration of TCA (60 μM) accumulated HL60 cells at G2/M at 24 h with increased apoptosis when the treated time was prolonged. Both low and middle concentrations of TCA induced HL60 cells to differentiate toward mature granule cells characterized with up-regulation of CD11b on cells accompanied by decreased c-Myc protein and increased p27 protein. Consistently, the expression and cellular distribution of p16 and Cdc6 were also significantly changed in differentiated HL60 cells treated with TCA. On the other hand, high concentration of TCA (100 μM) rapidly inhibited NF-kappaB activity and induced apoptosis in HL60 cells. Importantly, TCA induced apoptosis of AML CD34+ cells and suppressed colony formation of AML BMMNC, while its cytotoxicity on normal BMMNC was minor. In addition, TCA synergized with AraC to kill AML BMMNC and AML CD34+ cells. Finally, TCA also decreased CXCR4 expression on HL60 cells, consistent with it ability to depress migration and invasion of HL60 cells induced by rhSDF-1α as well as the adhesion of HL60 cells to AML BMSC. Of note, TCA also impaired survival and SDF-1α secretion of AML BMSC, which may further suppress the interaction of HL60 with AML BMSC. Taken together, our data show that TCA is an effective agent for the treatment of hematopoietic malignancies, not only being the direct inducer of terminal differentiation and apoptosis of acute myeloid leukemic cells, but attenuating the protective effect of AML BMSC on leukemia cells via inhibiting SDF-CXCR4 axis, which highlights the potential of TCA to be a promising therapeutic agent for hematopoietic malignancies treatment.
Interferon-γ Enhances PML Protein Expression in Acute Promyelocytic Leukemia Cells and Cooperates with All-Trans Retinoic Acid to Induce Maturation of NB4 and MR2 Cells
Authors: He Pengcheng , Zhang Mei , Wang Xiaoning , Qi Jun , Xi Jieying , Li Jing , Wu Di , Cao Yunxin
Published: April 2020

More than 90% patients with untreated acute promyelocytic leukemia(APL) obtain complete remission clinically by using All-trans retinoic acid(ATRA), a strong differentiation inducer. However, the rapid development of ATRA-resistance brings a new problem to the treatment of APL. Interferon(IFN), as an important cytokine, has broad biological activities. It can not only inhibit the growth of tumor cells, but also reverse the drug-resistance of chemotherapy. As proved by some research, the mechanisms of ATRA-resistance are probably related to lacking some important proteins which synthesized by Interferon-γ (IFN-γ). In order to explore a new way to solve the problem of ATRA-resistance in APL, we investigate the effect and mechanisms of IFN-γ in combination with ATRA on the proliferation/differentiation of NB4 cells(APL cell line with ATRA-sensitiveness) and MR2 cells(APL cell line with ATRA-resistance) respectively. ATRA, IFN-γ and IFN-γ in combination with ATRA were incubated with NB4 and MR2 cells respectively. The cell proliferation was tested by MTT assay, the cell differentiation was tested through light microscope, by NBT reduction test and flow cytometry(FCM). The results showed that ATRA could inhibit the growth of NB4 cells significantly (P<0.05), but it had no effect on the growth of MR2 cells (P>0.05). IFN-γ could inhibit the growth of both NB4 cells and MR2 cells slightly (P<0.05). Moreover, the growth inhibition effect of IFN-γ in combination with ATRA on NB4 and MR2 cells was obviously stronger than that of any single drug group (P<0.05). The results of cell morphology observation, NBT reduction test and CD11b antigen detection showed that ATRA could induce differentiation of NB4 cells significantly (P<0.05), but it had no effect on MR2 cells (P>0.05). Although IFN-γ alone had no effect on the differentiation of either NB4 or MR2 cells (P>0.05), it could augment the differentiation of NB4 cells induced by ATRA (P<0.05) and induce the differentiation of MR2 cells slightly (P<0.05) when it combined with ATRA. Furthermore, we have observed the expression of promyelocytic leukemia(PML) protein by indirect immune fluorescent test. The results showed that the number of fluorescent particles in both NB4 and MR2 cells’ nucleus was increased significantly (P<0.05) when they were incubated with IFN-γ respectively, which indicated IFN-γ could induce the expression of PML protein, a tumor growth inhibitor. It can be seen that IFN-γ could augment the proliferation inhibition effect of ATRA on NB4 and MR2 cells through enhancing the expression of PML protein. Moreover, IFN-γ in combination with ATRA not only can strengthen the induction differentiation effect of ATRA on NB4 cells, but also can partially induce the maturation of MR2 cells with ATRA-resistance.
Upregulation of XAF1 by 5-Aza-2′-Deoxycytidine Sensitizes Busulfan-Resistant Human CML Cells; Implications for Pretransplant Conditioning Therapy in Myeloid Leukemia
Authors: Valdez Benigno C. , Li Yang , Murray David , Champlin Richard E. , Andersson Borje S.
Published: April 2020

The DNA-alkylating drug busulfan (Bu) is commonly used in myeloablative pretransplant conditioning therapy in patients with chronic myelogenous leukemia (CML). A major obstacle to successful treatment is inherent or acquired cellular Bu-resistance. We hypothesized that cellular Bu-resistance can be reversed by epigenetic upregulation of pro-apoptotic genes. We established a Bu-resistant CML cell line B5/Bu2506 which is 4-fold more resistant to Bu than the parental B5 cells but 4-fold collaterally more sensitive to DNA demethylating agent 5-aza-2′-deoxycytidine (DAC). Exposure to DAC synergistically increased Bu-mediated cytotoxicity in B5/Bu2506 cells as evaluated by the MTT assay and analyzed by the median-effect method (combination index CI < 0.5). The DAC-induced sensitivity to Bu of B5/Bu2506 cells was associated with PARP1 cleavage, phosphorylation of histone 2AX and activation of caspase 8. Real-time PCR and immunostaining analyses of the expressions of various pro-apoptotic genes, which are known to be epigenetically regulated, showed a highly significant increase (approximately 40-fold) in the expression of XAF1 (X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1) in B5/Bu2506 cells that were exposed to 0.5 microM DAC for 48 hrs. Analysis of the XAF1 promoter by methylation-specific PCR showed a significant decrease in its methylation status which correlates with the alteration in its gene expression in the presence of DAC. These findings will be discussed relative to our analysis of XAF1 gene methylation status in leukemia cell samples obtained from patients who underwent allogeneic stem cell transplantation after high dose Bu-based conditioning therapy and either remained in remission for more than one year or who experienced rapidly progressive leukemia. Our results suggest that DNA methylation status (eg. XAF1 gene) may be used to identify leukemia patients who could benefit from Bu-DAC combinations to achieve improved leukemic cytoreduction with the conditioning program.
A Newly Developed IAP Inhibitor (IAPi) Induces Apoptosis of Human Myeloma Cells and Synergises with Conventional and Novel Anti-Myeloma Therapeutics
Authors: Khong Tiffany T. , Spencer Andrew
Published: April 2020

The IAPs (inhibitor of apoptosis proteins) are a family of 8 structurally related caspase inhibitors that efficiently block the execution phase of apoptosis induced by various stimuli including death receptor activation. Based on reports of elevated expression of IAPs in multiple myeloma (MM) we evaluated the therapeutic potential of a novel IAP (IAPi) in MM. A panel of 9 genetically heterogeneous human myeloma cell lines (HMCL) were screened and found to be sensitive to IAPi with an IC50 of 25 to 50μM in a dose and time dependent manner. Induction of cell death was confirmed by Annexin V and propidium iodide staining of treated HMCL. Mechanistic studies were then undertaken utilising 3 HMCL with high (NCI H929), intermediate (LP-1) or low (OPM2) sensitivity to IAPi with QRT-PCR demonstrating the highest expression of XIAP in the most IAPi sensitive HMCL NCI H929. In all 3 HMCL IAPi exposure was associated with rapid cleavage of caspase 3, caspase 7, ROCK-1, cytosolic degradation of ICAD, nuclear translocation of CAD and PARP cleavage confirming cell death via apoptosis. Replicate experiments utilising caspase specific inhibitors and siRNA knock-down of caspases 3 and 7 confirmed this to be both caspase 3 and caspase 7 dependent. Co-culture of HMCL with conditioned media from the human stromal cell line HS-5 or primary MM bone marrow mononuclear cells reproducibly demonstrated enhanced IAPi-induced HMCL apoptosis with a median increase in HMCL killing of 1.2 fold compared to HMCL treated in standard culture media. Similarly, IAPi activity was not abrogated but enhanced in the presence of either IL-6 or IGF-1 (1.1 and 1.3 fold, respectively) when compared to HMCL treated without the addition of exogenous growth factors. Primary MM cells from 9 multiply relapsed MM patients were treated with IAPi in an autologous bone marrow co-culture assay with concentrations of IAPi ranging from 10–100μM inducing a median of 23% (range, 8–56% as determined by Apo2.7 staining) MM cell killing at 48 hours. Finally the anti-MM activity of IAPi in combination with other conventional (etoposide, adriamycin, velcade) and novel (HSP90 inhibitor, agonistic TRAIL DR5 monoclonal antibody) therapeutics was investigated against both HMCL and primary MM tumour samples. All combinations showed synergistic cell killing with Combination Indices of <1 (Calcusyn) when compared to either agent alone. We conclude that IAPi induces apoptosis of MM cells via a caspase 3 and caspase 7 dependent process. Furthermore, IAPi retains anti-MM activity in the context of pro-survival cytokine exposure and induces synergistic killing of MM cells when combined with both conventional and novel anti-MM therapeutics. Based on these preliminary observations further investigation of the role of IAPi as a potential tumour sensitising agent for the therapy of MM is justified.
Circulating MUC1 Levels (CA15.3) in Myeloproliferative Disorders (MPD)
Authors: Rughetti Aurelia , Fama Angelo , von Mensdorff-Pouilly Silvia , Taurino Federica , Rahimi Hassan , Ribersani Michela , Natalino F. , D’Elia G.M. , Bizzoni L. , Latagliata Roberto , Breccia Massimo , Foà Robin , Alimena Giuliana , Girelli Gabriella , Frati Luigi , Nuti Marianna , Tafuri Agostino
Published: April 2020

MUC1 is a glycoprotein expressed on the luminal surface of simple epithelia. In carcinoma, MUC1 overexpression is associated with malignancy. In breast cancer patients, increased levels of circulating MUC1 (CA 15.3 tumor marker) are associated with poor prognosis (Tumour Biol.2005; 26:217–20). In myeloma, MUC1 overexpression correlates with apoptosis resistance. We have previously shown (Br J Haematol.2003; 120:344–52) that MUC1 is selectively found in differentiating erythroid cells suggesting a possible role as cross-talk molecule between erythroblasts and bone marrow microenvironment during erythropoiesis. In CD34+ cells cultured in the presence of EPO and SCF, MUC1 is expressed before Glycophorin A (GlyA) during the erythroid differentiation process, and disappears following GlyA up-regulation. Aiming to evaluate the role of MUC1 in the erythroid counterpart present in neoplastic haemopoiesis, we studied MUC1 serum circulating levels in patients affected by Ph-negative Myeloproliferative Chronic Disorders (MPD). We analysed serum samples from 42 MPD patients affected by Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) (N 25, 14, and 3, respectively). CA15.3 (soluble MUC1) levels were determined using the commercially available ADVIA Centaur CA 15.3 assay (Bayer Corporation, NY) and cut off level was set at 25 U/mL. As control group serum samples from healthy donors, matched for sex and age, were also analyzed. Evaluation of the humoral immune response to MUC1 protein core was performed in ELISA. Results indicated that CA15.3 levels were statistically higher in MPD patients than in the control group (p<0.005), this difference was more evident in PV patients (p<0.001) and in female cases (p<0.001). The JAK2V617F mutation status was not associated with CA15.3 values; similarly, neither the clinical or hematological features nor the clinical complications (thrombosis) were influenced byCA15.3 levels. Analysis of the humoral immune response to MUC1 core protein showed no substantial changes in serum anti-MUC1 IgG titers in PV patients, suggesting that in PV increase of soluble MUC1 does not induce immune responses against this self-antigen. Further studies are ongoing to investigate the significance of MUC1 in the development and onset of myeloproliferative disorders. In conclusion, MUC1 levels seem to be increased in PV patients, suggesting that further evaluations on large series of cases should include this test in the diagnostic work-up to PV patients.
Peripheral Blood Disease Monitoring in Marginal Zone Lymphoma Using Flow Cytometry
Authors: de Tute Ruth , Rawstron Andy C , Owen Roger G , Jack Andrew
Published: April 2020

The use of sensitive flow cytometric techniques for the primary diagnosis and monitoring of patients with B-CLL is now well established. The aim of this study is to examine the feasibility of extending this approach to other indolent B-cell lymphoproliferative disorders. Such a technique, particularly if applicable to peripheral blood samples, would have significant benefits in allowing minimally invasive sequential monitoring of treatment responsiveness and early detection of relapse. Using a similar statistical approach to that used in the design of the CLL minimal disease flow cytometry panels we identified LAIR1, CCR6, CXCR5 and CD10 as markers that allowed discrimination between normal and neoplastic B-cells. These markers were incorporated into a 2 tube 8 colour assay together with CD19, CD5, CD20, CD22, CD45, kappa and lambda. Leucocytes were prepared using ammonium chloride lysis, incubated with these antibodies and washed before acquisition of 500,000 events on a FacsCanto flow cytometer (BD BioSciences, Oxford). As part of the evaluation of the technique we investigated peripheral blood samples from a total of 28 patients (14 male, median age 69.1 years and 14 female, median age 73.4 years), 22 with Marginal Zone Lymphoma/Waldenstrom’s Macroglobulinaemia (MZL) and a further 6 with Extranodal Marginal Zone Lymphoma (EMZL). Of the 22 MZL patients, 8 had active disease, 5 had stable disease that was untreated, 1 was on treatment and 8 were in remission. Of the extranodal MZL patients 1 had stable untreated disease, with 2 on treatment and 3 in remission. 77% (10/13) of MZL patients with active or stable untreated disease had detectable circulating tumour cells. Abnormal B-cells represented a median 2% of leucocytes (range 0.08 – 39%). The median absolute abnormal B-cell count was 0.34 ×109/l (range 0.003–9.6 ×109/l). The phenotypes of the cells detected showed a high level of heterogeneity. Abnormal cells were detectable in 22% (2/9) of cases on treatment or in remission, with a median % abnormal B-cells of 0.88% (range 0.35–5.37%) and a median absolute B-cell count of 0.09×109/l (range 0.02–0.386×109/l). None of the EMZL patients had abnormal cells detectable. Circulating tumour cells were detectable down to a level of 0.003×109/l and were found in a proportion of patients on treatment or considered to be in clinical remission. Methods used to monitor patients with indolent lymphoproliferative disorder need to take into account the phenotypic diversity found in marginal zone lymphoma compared to CLL. However the results demonstrate the feasibility of extending the use of flow cytometric residual disease assays to marginal zone lymphoma.
IL-6 and Tumor Susceptibility Alleles of Strain BALB/C Cause Phenotypic Shift of MYC-Driven Lymphomas in Mice from Diffuse Large B-Cell Lymphoma (DLBCL) to Plasmacytoma (PCT)
Authors: Neppalli Vishala T. , Janz Siegfried , Rutsch Sebastian , Goldschmidt Hartmut
Published: April 2020

Aims: Deregulation of cellular oncoprotein MYC (c-Myc) and the plasma cell growth, differentiation and survival cytokine, interleukin-6 (IL-6), are key pathogenetic factors in human high grade B-cell lymphomas (Burkitt lymphoma and DLBCL) and PCT respectively. Genomic instability, as a consequence of this deregulation, is poorly understood. Transgenic expression of these factors in the B-cell lineage, in mice, generates a pre-clinical model system of great relevance for human B-cell lymphoma counterparts. In this study we evaluated for the incidence of lymphomas in transgenic mouse models, with deregulated CMYC and IL-6. We performed a morphologic classification and correlation of the malignant lymphoid proliferations with the underlying genetic lesions.

Materials & Methods: Deregulation of CMYC was observed in gene-targeted C57BL/6 mice that harbor a His6-tagged mouse Myc cDNA gene in themouse immunoglobulin heavy-chain locus (B6.iMyc mice). A double transgenic mouse model, using BALB/c mice, carry the same gene insertion, in addition to a widely expressed human IL-6 transgene (C.iMyc/IL-6 mice). Tumor tissue from the mice was processed by formalin fixation and embedded in paraffin. Sections from the paraffin blocks were stained with hematoxylin and eosin and evaluated for morphology. The morphologic evaluation of the lymphomas was performed blind to the genetic make up of the host, followed by genetic correlation with morphology.

Results: B6.iMyc mice developed nodal and extranodal lymphomas. Tumor incidence was 50% (70/140) at 18 months of age. The lymphomas demonstrated sheets of large cells with centroblastic morphology, consistent with diffuse large B-cell lymphoma. The mitotic index was high, with the presence of frequent atypical mitotic figures and apoptotic debris laden macrophages, giving a starry sky pattern. C.iMyc/IL-6 mice developed nodal and extra nodal plasma cell tumors (PCT). Tumor incidence was 100% (20/20) at 4–5 months of age. PCT demonstrated sheets of monotonous and atypical plasma cells with prominent nucleoli. In lymphoid tissue containing germinal centers, the sheets of plasma cells were located in the interfollicular areas. There was no bone marrow involvement.

Conclusions: Our findings demonstrate that

B6.iMyc mice with CMYC deregulation develop high grade B-cell lymphomas similar to human DLBCL. The activity of deregulated IL-6 appears to enhance and accelerate the oncogenic potential of deregulated CMYC, in the pathogenesis of lymphoid malignancy. IL-6 and/or tumor susceptibility alleles of strain BALB/c cause a remarkable shift in the phenotype of MYC-driven lymphomas from DLBCL to PCT.
Rsp1 Is Indispensable for mRNA Stability during Erythroid Terminal Differentiation
Authors: Tian Meng , Campagna Dean R. , Langer Nathaniel , Choe Seong-Kyu , Paw Barry H. , Justice Monica J. , Fleming Mark D.
Published: April 2020

Mouse models have proven invaluable for understanding erythropoiesis. Here, we describe an autosomal recessive inherited anemia in the mouse mutant hem6. Hematologic and transplantation analyses revealed a mild, congenital, hypochromic, microcytic anemia intrinsic to the hematopoietic system that is associated with a decreased red blood cell zinc protoporphyrin to heme ratio, indicative of porphyrin insufficiency. Iron uptake experiments showed that hem6 reticulocytes are defective in heme production, but not cellular iron uptake defects. Male hem6 mice are infertile due to defects in sperm structure and motility. Through positional cloning and BAC complementation, we identified the gene responsible for the hem6 anemia to be Rsp1 (RNAStabilityProtein1) on Chr.3. The anemia phenotype was reproduced by injecting zebrafish embryos with morpholinos specifically targeting the Rsp1 transcript. Multi-tissue, real-time PCR shows that Rsp1 is abundantly expressed in bone marrow and testis, which correlates with the anemia and infertility defects in the hem6 mutant. To further study the function of Rsp1, we conducted yeast two hybrid analysis, employing a human bone marrow library. We found that Rsp1 interacts with the poly (A)-binding protein cytoplasmic 1(PABPC1) and, using microarray analysis, showed that many abundant erythroid specific transcripts, including Hbb-b1, Hba-a1, Alas2, and Mitoferrin1, were significantly down regulated in hem6 reticulocytes. Since PABPC1 is responsible for stabilizing the Hba-a1 transcript [Wang et al. (1999) Mol Cell Biol19:4552–60 and Wang and Kiledjian (2000) Mol Cell Biol20:6334–41], we hypothesized that the relative deficiency in erythroid-specific mRNAs in hem6 reticulocytes might be due to decreased mRNA stability. Indeed, serial microarray analysis of reticulocytes aged in vitro showed that numerous, abundantly expressed erythroid-specific transcripts decayed at faster rates in hem6 reticulocytes compared to control reticulocytes. Furthermore, these mRNAs also have progressively shorter poly (A) tails, suggesting a mechanism for the increased rate of decay. In sum, the evidence supports the conclusion that Rsp1 is a regulator of mRNA stability and gene expression during the terminal differentiation of erythroid cells.
MicroRNA Profiling of EVI1 Deregulated Myeloid Leukemia
Authors: De Weer An , Poppe Bruce , Mestdagh Pieter , De Preter Katleen , Van Vlierberghe Pieter , Cauwelier Barbara , Van Roy Nadine , Verhasselt Bruno , Vandesompele Jo , Dastugue Nicole , Speleman Frank
Published: April 2020

Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression of, or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) patients, and is a prognostic marker predictive of a poor outcome. Recently, microRNA deregulation was identified as a major contributor to cancer initiation and progression. Furthermore, miRNA genes were shown to be directly regulated by activated proto-oncogenes. In this study, we investigated which miRNAs are implicated in the transcriptional pathways governed by the EVI1 oncogene as well as possible microRNAs regulating the expression of the EVI1 gene itself. A total of 384 miRNAs were profiled through automated qRT-PCR using high-throughput quantitative stem-loop RT-PCR. Our patient series consisted of 18 EVI1 rearranged and overexpressing samples confirmed by FISH, karyotyping and qRT-PCR, 11 normal bone marrow samples and 2 CD34+ cord blood fractions. Through integrated statistical analysis we were able to identify 36 significantly upregulated and 44 significantly downregulated miRNAs (p<0.05) in EVI1 rearranged samples compared to normal bone marrow samples. Among these up- and downregulated miRNAs several have already been associated with leukemia or other types of cancer acting as oncogenes or tumor suppressor genes, respectively. Interestingly, in the panel of downregulated miRNAs, we found 3 miRNAs that have predicted binding sites on the 3′UTR of the EVI1 gene. Currently, no mechanism has been described to explain EVI1 overexpression, therefore loss of the inhibitory effect of miRNAs targeting the 3′UTR of EVI1 might be an important mechanism contributing to EVI1 ectopic expression. Further analyses will include vector based luminescence experiments to confirm the association between EVI1 and the 3 downregulated miRNAs. Functional studies will also be performed in order to assess the contribution of the differentially expressed miRNAs to the leukemic phenotype. Since patients with EVI1 involvement have a poor prognosis, elucidating the downstream EVI1 controlled miRNA targets and pathways could not only yield new insights into EVI1 etiogenesis, but could also add additional prognostic and diagnostic information. We anticipate that these findings could provide new targets for therapeutic intervention.
Bcl-2 Family Dysregulation in AML1-ETO-Expressing Human Hematopoietic Cells
Authors: Chou Fu-Sheng , Wunderlich Mark , Mulloy James C.
Published: April 2020

A subset of acute myeloid leukemia is caused by the chromosomal translocation (8;21), resulting in the expression of the AML1-ETO (AE) fusion protein. AE expression in human CD34+ umbilical cord blood (UCB) cells causes an expansion of the hematopoietic stem and progenitor cell (HSPC) compartment, but alone does not cause leukemia. We have recently shown that AE represses DNA damage repair genes, resulting in higher mutation frequency as well as p53 activation and increased basal apoptosis. Interestingly, we found that AE expression in CD34+ UCB cells was associated with high Bcl-xL levels, while control cells showed a gradual loss of Bcl-xL protein. Furthermore, expression of AE in aged HSPC that had already downregulated Bcl-xL also led to Bcl-xL protein upregulation. In addition, Bcl-xL protein, but not mRNA, was decreased by pharmacological and RNAi-mediated inhibition of p53 in AE cells and increased by low-intensity γ-irradiation, implying that Bcl-xL turnover may be post-translationally regulated by p53 levels. Bcl-xL knock-down by short-hairpin RNA in AE cells resulted in even higher p53 activity and growth disadvantage, suggesting that AE cells depend largely upon Bcl-xL for survival in the context of p53 activation. AE cells are also more sensitive than long-term cultured UCB cells to GX15-070, a BH3-mimetic drug that binds and inhibits all anti-apoptotic Bcl-2 family proteins. The sensitivity of AE cells to Bcl-xL inhibition and GX15-070 treatment may be due to upregulation of various pro-apoptotic Bcl-2 family proteins. Surprisingly, p53-upregulated modulator of apoptosis (PUMA) mRNA levels were decreased or remained unchanged in association with AE expression, even in the context of an upregulated p53 signaling pathway. Chromatin immunoprecipitation assay revealed that AE binds to and AML1 binding site in the putative promoter of PUMA, in a region distinct from the p53 consensus binding site. Indeed, PUMA expression is increased in response to γ-irradiation in cells expressing AE. This suggests that AE may directly repress PUMA gene expression, but does not prevent p53 binding. Taken together, our data indicate that AE expression may lead to re-establishment of a new balance between the pro-apoptotic signals conveyed by the activated p53 signaling pathway and the pro-survival signals, presumably Bcl-xL upregulation and PUMA repression, within the Bcl-2 family proteins to ensure cell survival and propagation.
Endocytosis and Erythroblast Enucleation Are Linked by the Action of a Survivin-EPS15-Clathrin Complex
Authors: Keerthivasan Ganesan , Small Sara , Liu Hui , Wickrema Amittha , Crispino John
Published: April 2020

The mechanisms that drive the final step in the development of reticulocytes from nucleated erythroblasts remain largely undefined. We recently reported that the chromosome passenger complex (CPC) protein survivin is required for the maintenance of hematopoietic stem cells and for the proliferation and survival of erythroid progenitors. Several lines of evidence suggest that survivin also plays an additional role late in erythropoiesis. First, 20% of survivin heterozygous mice showed a decrease in the proportion of enucleated cells relative to nucleated erythroblasts. Second, survivin is highly expressed in orthochromatic erythroblasts undergoing enucleation, where it partially co-localizes with actin at the junction between the extruding nucleus and cytoplasm. Third, overexpression of survivin promoted enucleation during DMSO-induced murine erythroleukemia (MEL) cell differentiation. Since survivin plays an essential role during normal mitosis and because enucleation has been postulated to occur by asymmetric cytokinesis, we investigated the link between cytokinesis and enucleation. Using primary erythroblast cultures, we found that chemical inhibitors of cytokinesis failed to prevent enucleation. Furthermore, we could not detect co-localization of survivin with aurora-B or INCENP, two other members of the CPC, in enucleating primary human erythroblasts. To determine the mechanism by which survivin contributes to enucleation, we utilized an in vivo biotinylation strategy to isolate survivin protein complexes from extracts of MEL cells. Using mass spectrometry, we discovered that survivin complexes include clathrin and its partner EPS15 (EGFR pathway substrate clone 15). We confirmed the endogenous interaction between these three proteins by co-immunoprecipitation. Treatment of primary erythroblast cultures with an inhibitor of endocytosis led to a decrease in enucleation efficiency. In addition, siRNAs targeting survivin, EPS15, or clathrin each reduced the degree of enucleation of primary human erythroblasts, confirming a requirement for this protein complex in enucleation. Together, our data demonstrate that survivin promotes enucleation by a novel clathrin-mediated process. Furthermore, our data help explain electron microscopy (EM) observations that enucleation is accompanied by an accumulation of vesicles that coalesce to form U-shaped channels at the junction between the nucleus and plasmalemma of the erythroblast (Simpson and Kling, JCB35:237, 1967). These EM studies along with our new data suggest that vesicular trafficking may be one of the major driving forces in enucleating erythroblasts.