bluuebunny/crossref_metadata_2025 huggingface dataset:
CCR2 Mediates Hematopoietic Stem/Progenitor Cell Trafficking to Peripheral Tissues after Inflammation
Authors: Si Yue , Tsou Chia-Lin , Charo Israel
Published: April 2020

Hematopoietic stem cells (HSCs) are bone-marrow derived, self-renewing pluripotent cells that give rise to terminally differentiated circulating blood cells. HSCs have been implicated in parenchymal tissue repair in the setting of inflammation. In response to the antagonist of the chemokine receptor CXCR4, HSCs and their progenitors migrate from bone marrow to the blood. However, little is known about the signals that mediate their trafficking from the blood into peripheral tissues. Recently, we showed that mice genetically deficient in chemokine receptor CCR2 (CCR2−/− mice) have a marked decrease in the number of circulating “inflammatory” (7/4+, Ly6c+) monocytes, but no decrease in myeloid progenitor cells in the bone marrow (Tsou et al, J Clin Invest, 2007, 902). These data indicated that although CCR2 is not necessary for HSCs to differentiate into mature monocytes, it does play a role in monocyte egress from bone marrow to blood. In the current study, we extend this work and investigate the expression of CCR2 on HSCs, and tested the hypothesis that CCR2 is critical for the recruitment of circulating HSCs to sites of inflammation. We found that CCR2 was expressed on subsets of primitive HSCs and myeloid progenitors and mediated HSC movement in response to inflammation. Using traditional transwell chambers, we found that c-Kit+Lin− cells derived from bone marrow underwent chemotaxis in response to the CCR2 ligands MCP-1 (CCL2) and MCP- 3 (CCL7). To determine whether CCR2 mediates HSC movement in vivo, we treated wildtype mice with thioglycollate to induce aseptic inflammation. HSCs were actively recruited to the peritoneum, as shown by fluorescence-activated cell sorting and functional colony formation assays. In contrast, this response was profoundly impaired in CCR2−/− mice. To determine whether the clonogenic cells recruited to peritoneum were true HSCs, we performed competitive transplantation assays. Thioglycollate was instilled into wildtype CD45.2+ mice, and peritoneal Lin− cells were collected, purified, and infused, together with CD45.1+ bone marrow cells, into lethally irradiated CD45.1+ mice. Four months later, up to 12% of the leukocytes in the peripheral blood of these primary recipient mice were CD45.2+. At the time of sacrifice, bone marrow cells were collected from these mice and injected into lethally irradiated secondary CD45.1+ recipient mice. Two months following the transplantation, up to 9% of the blood leukocytes in these secondary recipient mice were CD45.2+, confirming that long-term repopulating HSCs were recruited to the inflamed peritoneum of the donor mice. These findings suggest a novel role for CCR2 in the recruitment of long-term repopulating HSCs to sites of inflammation and injury. We are currently investigating whether recruited HSCs and their progenitors hasten the resolution of the inflammatory response or promote the repair of injured tissue.
Tashinone IIA Can Trigger the Apoptosis of a Megakaryocytic Cell Line CHRF- 288 through the TNFRSF9 Mediated Caspase-3 Activation Pathway
Authors: Liu Chang Charlie , Wang LiangJie Vicky , Zhou Min , Ye JieYu , Chan Godfrey ChiFung , Yang Mo
Published: April 2020

Tanshinone IIA (TIIA), a purified compound and an active component of Salvia miltiorrhiza (DanShen) has previously been shown to induce apoptosis on several human cancer cell lines. In the current study, we studied the cytotoxic effect of TIIA on a megakaryocytic leukemic cell line CHRF-288. First, we tested the cytotoxic effect of TIIA on CHRF-288 cells by Trypan blue assay and the cell viability was reduced to 80.8% after 72hrs of TIIA treatment (30ug/ml). Using Annexin V/PI staining and flow cytometry, we confirmed that TIIA induced apoptosis on CHRF-288 cells in a dose dependent manner. At the concentrations of 1, 3, 10 and 30ug/ml, the early apoptosis cells proportion showed a stepwise increment of 11.8±1.2%, 13.2±2.8%, 16.3±0.8% and 22.4±1.5% at 72 hrs respectively. This was verified by Caspase 3 assay which showed the activation of Caspase 3 increased from 5.1% to 16.2% after 10ug/ml of TIIA treatment. Activation of Caspase 3 implied the involvement of common apoptotic pathway. Then, we studied the mechanisms of apoptosis. Using JC-1 assay, we found that the depolarized cells increased from 9.06% to 16.6% after 10ug/ml TIIA treatment suggestive the involvement of intrinsic apoptotic pathway. To further determine the molecular mechanisms involved in the pro-apoptosis effect, Microarray studies using Affymetrix 133 plus genechips were conducted to identify the genes that were differentially expressed after TIIA treatment. Several groups of genes involved in apoptosis, calcium regulation and cell cycle checkpoints were found to be differentially expressed after the treatment. The differential expressions of these genes were validated using quantitative PCR. The most significantly upregulated genes (5.9±0.333 folds) was TNF Receptor Super-Family 9 (TNFRSF9). In addition, Receptor Interacting Protein Kinase (RIPK), a protein that likely interacts with TNFRSF9, was upregulated to 1.7±0.167 folds. And lastly, Jun, a transcriptional factor of TNFRSF9 was up-regulated to 1.7±0.03 folds. Since TNF and RIPK1 both involve in caspase-8 activation, this was suggestive of the activation of external apoptotic pathway. Our findings suggested that TIIA could induce significant apoptosis on mekakaryoctic leukemic cells via both intrinsic and extrinsic apoptotic pathways. Its role in treating this poor prognostic leukemia deserves further exploration.
Role for Chemokines in the Vasculopathy Associated with Sickle Cell Dsease and Pulmonary Hypertension
Authors: Tailor Anitaben , Kato Gregory J.
Published: April 2020

Sickle cell disease (SCD) is a hemolytic disorder characterized by transient vaso-occlusive crisis (VOC). Moreover, there is a high prevalence of SCD-associated pulmonary arterial hypertension (PAH), indicated by a tricuspid regurgitant jet velocity (TRV) ≥ 2.5 m/sec, leading to increased risk for early mortality. Monocytes have been proposed as pivotal cells in vascular inflammation in animal models of SCD. Preliminary studies in our group have shown a potential role for monocyte chemokines in the vasculopathy of SCD. The objective of this study is to investigate the role of two chemokines: regulated upon activation, normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein-1β (MIP-1β) in SCD and SCD-associated PAH. Plasma samples were collected from patients with SCD at steady state and from healthy African-American control subjects. Plasma levels of RANTES and MIP-1β were measured using an enzyme immunoassay system. Patients with SCD exhibited significantly higher levels of both MIP-1β (median 465.3 vs. 398.5 pg/ml, p<0.0001) and RANTES (median 47774 vs. 17445pg/ml, p<0.0004) than healthy controls. Further characterization of SCD patients with or without PAH demonstrated that whereas MIP-1β exhibited no correlation with PAH, RANTES demonstrated an inverse correlation with PAH (r= −0.25, p<0.03) with significance achieved in patients that exhibited severe PAH (TRV> 2.9, p<0.05). These findings suggest that

both chemokines, RANTES and MIP-1β, are associated with the vasculopathy of SCD, and whereas MIP-1β does not appear to be associated with SCD-associated PAH, RANTES levels are inversely correlated to the severity of PAH. RANTES merits further investigation as a potential marker in PAH, and for a possible role in its progression.
INK4 Locus Deletion in Childhood Acute Lymphoblastic Leukemia (ChALL)-a Preliminary Clinical Evaluation
Authors: Paisiou Anna , Papadhimitriou Stefanos I , Tsagarakis Nikolaos , Papadakis Vassilios , Kentrou Nektaria , Paterakis Georgios , Parcharidou Agapi , Androutsos Georgios , Tsitsikas Konstantinos , Kostopoulos Ioannis , Pavlidis Dimitris , Georgaki Maria , Polychronopoulou Sophia
Published: April 2020

Introduction: Deletions of the INK4 locus (on chromosomal region 9p21) are common in chALL. p16/14 and p15 genes lost with INK4 deletion are tumor suppressors, with a well-documented role in regulating cell cycle and chemosensitivity and in promoting apoptosis in vitro. However, the clinical significance of INK4 deletions remains unclear.

Patients & Methods: The study included 78 children (49 boys and 29 girls) with ALL (medium age: 3,12, range:0,7 to 16,5 ). The disease was of B- and T-cell origin in 70 and 8 cases, respectively. INK4 deletion was investigated by interphase FISH on the diagnostic bone marrow samples and the result was correlated with the patients’ demographic data, biological and clinical parameters, and disease outcome. All patients were treated according to the ALL-BFM-95 protocol and followed-up for 11–105 (medium 41) months.

Results: INK4 locus deletion was found in 24 cases (30.8%). The loss was hemizygous in 15 (19.2%) and homozygous in 9 cases (11.5%). A positive finding was strongly associated with disease of T-cell origin (p=0.008). Apart from a weak correlation between INK4 deletion and advanced age (p=0.068), there was no association with sex, initial leucocytosis level, cytogenetic category, risk group grade according to the ALL-BFM- 95 criteria or the presence of residual disease at days 15 and 33. Nevertheless, INK4 homozygous deletion was significantly associated with disease-free (p=0.004) and overall survival (p=0.002), while the respective associations for an hemizygous deletions were found insignificant (p=0.137 and p=0.131).

Conclusions: The findings from this cohort of children suggest that INK4 homozygous deletion is an unfavorable prognostic factor in chALL, even for cases in which an aggressive course cannot be predicted by the conventional risk estimating criteria. Unclear results with regard to hemizygous deletion may be attributed to the functional state of remaining p16/p14 and p15 genes on the non-deleted chromosome 9. The study of a greater group of patients and the extension of the follow-up period will hopefully clarify whether INK4 deletion is an independent prognostic factor and whether it could be incorporated in risk evaluation guidelines, especially for cases which would otherwise be treated as chALL of standard or medium risk.
Combination of Bortezomib, Dexamethasone, Thalidomide and Zoledronic Acid (VdtZ) Results in a High Rate of Complete Response in High-Risk Non-Hodgkin Lymphoma
Authors: Teoh Gerrard , Ang Ai Leen
Published: April 2020

We previously reported that the combination of low-dose dexamethasone (d) and thalidomide (t) with higher frequency zoledronic acid (ZOMETA, Z) – the “dtZ” regimen – was associated with an overall response rate (RR) of 75.0% in patients with relapsed/refractory multiple myeloma (MM). However, the rate immunofixation (IF)-negative complete reponses (CR) was only 7.7%. In those patients who did not achieve CR, addition of bortezomib (VELCADE, V) to “dtZ” – the “VdtZ” regimen – greatly improved the overall RR to 92.9% and CR to 42.9%. In addition, 21.4% of “dtZ/VdtZ”-treated patients also achieved near-CR (nCR). Amongst 14 consecutive newly-diagnosed patients with MM who were treated with “dtZ/VdtZ”, the RR was 100% and CR/nCR was >75%. Encouraged by these data in MM, we applied the 3-weekly “VdtZ” regimen – intravenous Vel 1.5 mg/m2 days 1, 4, 8 and 11; oral Dex 20 mg days 1, 8 and 15; oral Thal 100 mg days 1–21; and intravenous Zol 4 mg day 1 – in 3 consecutive patients with newly-diagnosed high-risk, difficult-to-treat non-Hodgkin lymphoma (NHL) to determine the feasibility of using “VdtZ” to treat NHL. The patient characteristics and results are summarized in the table below:

In summary, all 3 (100%) patients rapidly reported CR upon initiation of “VdtZ. Although significant tumor lysis was reported in all patients, treatment was fairly welltolerated. These data provide a basis for further studies on the “VdtZ” regimen for the treatment of high-risk NHL.

Parameter Case 1 Case 2 Case 3 PTCL = peripheral T cell lymphoma; DLBCL = diffuse large B cell lymphoma Age, Sex 78 years, Male 61 years, Female 61 years, Male NHL PTCL DLBCL DLBCL Stage IV B E S IV B E S III B E S Tumor burden Very bulky Extremely bulky Bulky Additional features Pleural effusion, ascites Massive ascites, pleural effusion Severe autoimmune hemolytic anemia Zubrod score 4 4 2 IPI risk High High High Response to“VdtZ” CR after 2 cycles CR after 3 cycles CR after 1 cycle Survival to date 12 months 4 months 1 month
Murine Hematopoietic Stem Cells Require Akt1 and Akt2 for Long-Term Self-Renewal
Authors: Juntilla Marisa M. , Patil Vineet , Joshi Rohan , Koretzky Gary A.
Published: April 2020

Murine hematopoietic stem cells (HSCs) rely on components of the Akt signaling pathway, such as FOXO family members and PTEN, for efficient self-renewal and continued survival. However, it is unknown whether Akt is also required for murine HSC function. We hypothesized that Akt would be required for HSC self-renewal, and that the absence of Akt would lead to hematopoietic failure resulting in developmental defects in multiple lineages. To address the effect of Akt loss in HSCs we used competitive and noncompetitive murine fetal liver-bone marrow chimeras. In short-term assays, Akt1−/−Akt2−/− fetal liver cells reconstituted the LSK compartment of an irradiated host as well or better than wildtype cells, although failed to generate wildtype levels of more differentiated cells in multiple lineages. When placed in a competitive environment, Akt1−/−Akt2−/− HSCs were outcompeted by wildtype HSCs in serial bone marrow transplant assays, indicating a requirement for Akt1 and Akt2 in the maintainance of long-term hematopoietic stem cells. Akt1−/−Akt2−/− LSKs tend to remain in the G0 phase of the cell cycle compared to wildtype LSKs, suggesting the failure in serial transplant assays may be due to increased quiesence in the absence of Akt1 and Akt2. Additionally, the intracellular content of reactive oxygen species (ROS) in HSCs is dependent on Akt signaling because Akt1−/−Akt2−/− HSCs have decreased ROS levels. Furthermore, pharmacologic augmentation of ROS in the absence of Akt1 and Akt2 results in an exit from quiescence and rescue of differentiation both in vivo and in vitro. Together, these data implicate Akt1 and Akt2 as critical regulators of long-term HSC function and suggest that defective ROS homeostasis may contribute to failed hematopoiesis.
Disulfiram, An Old Drug with New Potential Therapeutic Uses for Human Haematological Malignancies
Authors: Conticello Concetta , Martinetti Daniela , Lombardo Laura , Buccheri Simona , Adamo Luana , Amato Gabriella , Cavalli Maide , Chiarenza Annalisa , de Maria Ruggero , Giustolisi Rosario , Gulisano Massimo , Di Raimondo Francesco
Published: April 2020

Proteasome is a novel, interesting target in cancer drug therapy, and the proteasome inhibitor Bortezomib has been used for its antitumor activity in multiple myeloma and other lymphoid malignancies. The commonly used thiocarbamate alcohol-abuse deterrent disulfiram (DSF) is an aldehyde dehydrogenase (ALDH) inhibitor with documented proteasome-inhibiting activity. Recent reports indicate that DSF and other dithiocarbamates may have a significant potential in the treatment of human cancer without significant side effects. It was previously shown that this compound is able to block the P-glycoprotein extrusion pump, to inhibit the transcription factor nuclear factor-kappaB (NF-kB), to sensitize tumors to chemotherapy, to reduce angiogenesis, and to inhibit tumor growth in mice. In addition a recent work has described several compounds with proteasome-inhibiting activity, among which in the hit was DSF. Here we show that MM (18 samples), AML (14 samples) and ALL (5 samples) primary cells from newly diagnosed and relapsed/resistant patients were significantly sensitive to DSF at doses < 5 mM (dose achievable in vivo). Median IC50 was DSF 0,5 mM for MM and DSF 0,35 mM for AML and r ALL samples after 48 hours of incubation. These cells are ALDH weakly positive and we did not find any difference in the sensitivity to DFS on the basis of amount of ALDH expression or status of disease (diagnosis vs relapse). Conversely, DSF, at the dose of 0,5 mM had only a weak effect on normal CD34 and peripheral blood mononuclear cells (<30% of cell death). We next exposed the cells to the combination of low dose DCF (0,5 mM) plus CuSO4 (another commonly used herbal medicinal product, that makes complex with DCF and reduces the superoxide dismutase, an enzyme involved in the mitochondrial depolarization) and we found that this combination was able to induce about 80–90% of cell death after 48 hours of treatment. The apoptotic effect of the combination (DSF plus CuSO4 0,5 mM) was comparable to that exert by therapy with Bortezomib plus Desametazone for MM samples, Cytarabine, Etoposide, Daunorubicine alone or in combination for AML samples or Vincristine, Desametazone and Daunorubicine alone or in combination for ALL samples. All these drugs were used in vitro at doses compatible with the levels reached in vivo during cancer treatment. In addition we show that DSF plus CuSO4 induce loss of mithocondrial membrane potential thus suggesting the involvement of mitochondrial apoptotic pathway. These results may suggest a novel strategy for treating multiple myeloma and acute leukemias by employing an old drug, with known and mild side-effect, toward a new therapeutic use, probably in combination with other agents.
NTAL Potentiates Glucocorticosteroid-Induced Apoptosis in T-ALL – in Vivo Observations and Experimental Model
Authors: Svojgr Karel , Burjanivova Tatiana , Vaskova Martina , Kalina Tomas , Brdicka Tomas , Stary Jan , Trka Jan , Zuna Jan
Published: April 2020

PAG, LAT, NTAL and LIME belong to category of transmembrane adaptor proteins (TRAPs). They do not possess an enzymatic or kinase function, but they are involved in mediation of signal transmission from surface receptors to cell nucleus. We propose that some of them are engaged in development or maintenance of leukaemia.

We have previously demonstrated that expression status of TRAPs at mRNA level is specific in some subgroups of childhood ALL, particularly in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) with the TEL/AML1 fusion gene. Furthermore, we have described that variable expression of NTAL mRNA is related to the response to initial glucocorticosteroid pre-phase in the treatment of childhood T-ALL; patients with high NTAL levels show better treatment response compared to the low-NTAL cases.

In the current study, we aimed to prove experimentally that different levels of NTAL protein influence response of leukaemic T cells to glucocorticosteroids.

In the wild type Jurkat cells (human T-cell leukaemia cell line) the NTAL protein is undetectable. For the in-vitro experiments we created a derivative Jurkat cell line transfected with the NTAL construct. In the derivative cell line, the NTAL positivity at both mRNA and protein level was verified using RT-PCR and Western blotting. The derivative cell line in a cell culture behaves similar as wild type Jurkat cell line. Transfectants and wild type Jurkat cells were incubated with methylprednisolone, dexamethasone or with solvent alone (H2O) as a negative control. Using flow cytometry we determined a percentage of surviving cells after 24, 48 and 72 hours of treatment. Cells were stained with Annexin V and DAPI and living cells were defined as Annexin V, DAPI negative. At each time point, the number of living cells in the negative control was set to 100%. After 48 hours of dexamethasone treatment the number of surviving cells in the Jurkat wild-type was higher by 12% compared to the Jurkat cells expressing NTAL. After 72 hours this difference was even more prominent reaching 46%. The same effect of methylprednisolone treatment was less pronounced (non-significant difference at 48 hours and 5% difference at 72 hours). Based on our experimental data we propose that NTAL acts in T-cells as a putative tumour suppressor. A plausible mechanism of its function is that NTAL competitively inhibits another adaptor LAT, which is required for signal propagation to the nucleus in T-cells. NTAL and LAT can compete for its localisation in proper “lipid rafts” or for palmitoylation. Our data can be used for subsequent functional analysis of signaling pathways in leukaemic blasts as well as in physiological lymphoid cells.
Comparison of the Effects on Reversal of Multi-Drug Resistance of 5-Bromotetrandrine and Tetrandrine in K562/A02 Cell Line in Vivo and in Vitro
Authors: Chen Bao-An , Wang Jue-qiong , Cheng Jian , Gao Feng , Xu Wen-lin , Shen Hui-lin , Ding Jia-hua , Gao Chong , Sun Yun-Yu , Wang Jun , Zhao Gang , Song Hui-hui , Bao Wen , Sun Xin-chen , Cheng Hong-yan , Deng Yu-xia , Li Guo-hong , Liu Li-jie , Chen Wen-ji , Chen Ning-na , Wang Xue-mei
Published: April 2020

Objective This study was to compare the reversal effect of 5-bromotetrandrine (BrTet) with Tetrandrine (Tet) when combined with ADM on multidrug resistance cell line K562/A02 and to investigate the reversal mechanism of this new derivative.

Methods The protein levels of P-glycoprotein (P-gp) were detected by fluorospectrophotometry and Western blot. The mRNA levels of P-gp were determined by RT-PCR. The in vivo effect of Tet was investigated using nude mice grafted with sensitive human leukemia cell line K562 and MDR cell line K562/A02.

Results Flow cytometry assay showed that 1.0 μMol/L BrTet significantly increased the apoptosis percentage. BrTet also enhanced the intracellular accumulation of ADM in K562/A02 cells and its potency was greater than that of Tet at the same concentrations. BrTet inhibited the overexpression of P-gp and down regulated MDR1 mRNA expression in K562/A02 cells in a dose-dependent manner. In nude mice bearing K562 xenografts on the left flank and K562/A02 xenografts on the right flank, i.p. injection of 10 mg/kg BrTet significantly enhanced the antitumor activity of ADM against K562/A02 xenografts with inhibitory rates of 26.1%, while ADM alone inhibited the growth of KBv200 xenografts by only 5.8%.

Conclusion BrTet showed significant MDR reversal activity in vitro and in vivo. Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs, which lead to more K562/A02 cells apoptosis.
Megakaryocytic and Platelet Dysplasia in MDS Patients as Detected by Multiparameter Flow Cytometry Immunophenotyping
Authors: Sandes Alex Freire , Yamamoto Mihoko , Quijano Sandra , López Antonio , Chauffaille Maria de Lourdes L.F. , Oguro Tsutomu , Matarraz Sergio , Silva Marçal C.A. , Kimura Eliza Y.S. , Orfao Alberto
Published: April 2020

Introduction: Multiparameter flow cytometry immunophenotyping (FC) has shown the presence of phenotypic abnormalities in both CD34 precursors and maturing myeloid and erythorid cells in patients with myelodysplastic syndromes (MDS). However, detection of megakaryocytic dysplasia by FC still remains a challenge.

Aims: To evaluate the potential utility of FC analysis of megakaryopoesis and platelet immunophenotype in MDS patients for the diagnosis and prognostic evaluation of the disease.

Methods: A total of 54 peripheral blood (PB) samples corresponding to 39 patients with MDS and 15 controls (6 healthy subjects, 3 reactive cytopenias, 3 patients with May-Heglin syndrome and 3 myeloproliferative disorders) were collected in sodium citrate. In all cases, the median amount of expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b, CD61, CD62P, CD63, PAC-1 and Fibrinogen receptor was evaluated on circulating platelets as refleted by the median fluorescence intensity (MFI). Morphologic megakaryocytic dysplasia was evaluated in parallel in bone marrow samples from the same MDS individuals.

Results: Morphological megakaryocytic dysplasia was detected in 49% of MDS patients who showed a shorter median overall survival (50 ± 8 vs 107 ± 12 months; p = 0.04). The following FC abnormalities were detected in circulating PB platelets of MDS patients: increased FSC and SSC (36%), overexpression of CD36 (18%), CD31 (36%), CD41b (20%), CD42b (2.5%) and CD61 (20%) and underexpression of CD36 (10%) and CD61 (5%). One case of aberrant CD34 expression was detected. A total of 27 (69.2%) patients presented at least one of these abnormalities by FC. Eighty percent of patients with morphological dysplasia and 64% without showed immunophenotypic abnormalities. According to IPSS, 6 patients constituted a high risk group (HRG) (Int-2 and High) and 28 a low risk group (LRG) (Int-1 and Low). Platelet phenotypic aberrations were detected in 83% of the HRG and in 75% of the LRG. Immunophenotypic abnormalities were not detected in myeloproliferative disorders. All May-Heglin cases and 2 reactive cytopenias presented overexpression of at least one of the following: FSC, SSC, CD31, CD36, CD41a, CD41b, CD42b and CD61. Deficient expression of CD36, CD61 and aberrant expression of CD34 were unique to MDS.

Conclusion: Our results show the presence of immunophenotypic abnormalities in PB platelets from MDS patients, the abnormally decreased reactivity for CD36 and CD61 together with aberrant CD34 expression being of potential diagnostic utility in MDS.
The Sequence-Selective DNA Cross-Linking Agent SJG-136 (SG2000, BN2629) Is Highly Potent in Multiple Myeloma Cells and Is Synergistic with Bortezimib
Authors: Walsby Elisabeth J , Burnett Alan , Knapper Steven , Fegan Chris , Pepper Chris
Published: April 2020

Despite recent advances in the treatment of multiple myeloma (MM), including the introduction of bortezimib, it remains an incurable disease with a short median survival. The development of new agents with efficacy in MM therefore remains both important and urgent. SJG-136 (SG2000, BN2629) is a novel DNA cross-linking agent that binds in a sequence-selective manner in the minor groove of the DNA helix. It is structurally novel compared with other clinically used DNA cross-linking agents and has exhibited a unique pattern of activity in the NCI 60-cell line screen. We have previously shown that this agent is highly effective against primary chronic lymphocytic leukaemia cells. In this study we evaluated its potential as a therapy for MM in cell lines and plasma cells derived from patients. The MM cell lines H929, U266 and JJN3 were shown to have a mean LD50 of 5.1nM (±3.4) following in vitro culture with SJG-136 for 48 hours. This was accompanied by a dose-dependent increase in the percentage of apoptotic cells as evidenced by Annexin V positivity and caspase-3 activation. In comparison to the standard therapies for MM, SJG-136 demonstrated significantly lower LD50 values than doxorubicin (P=0.004), Melphalan (P=0.004) and bortezimib (P=0.03). We also assessed the ability of SJG-136 to kill plasma cells from primary myeloma samples (identified by CD38 and CD138 positivity). These cells were sensitive to SJG-136 with a mean LD50 of 2.3nM (± 0.96) following 48 hour exposures to SJG-136. In contrast, normal bone marrow was significantly less affected by SJG-136 than the myeloma samples (P<0.0001). Finally, we assessed the potential for synergy between SJG-136 and these standard treatments using the H929 cell line as a model. SJG-136 was combined with doxorubicin (1:125), melphalan (1:4000) and bortezimib (1:8) and the combination index (CI) was calculated to assess synergy. A CI of less than 1 was considered synergistic. We found no evidence for synergy between SJG-136 and doxorubicin or melphalan (CI=1.29 and 1.75 respectively). However, SJG-136 was synergistic with bortezimib (CI=0.47) possibly reflecting their different mechanisms of action. In conclusion, SJG-136 is a potentially valuable addition to the battery of drugs available for treatment of MM. Not only was SJG-136 highly potent as a single agent in MM cell lines and primary plasma cells but it also showed a high level of synergy with the proteasome inhibitor bortezimib.
Pentameric IgM Monoclonal Antibodies Specific for Human β2-Microglobulin Are More Potent at Inducing Apoptosis in Multiple Myeloma Cells
Authors: Yang Jing , Cao Yabing , Hong Sungyongl , Li Haiyan , Kwak Larry W. , Yi Qing
Published: April 2020

Multiple myeloma is still an incurable plasma malignancy because patients are prone to quickly relapse after conventional treatment or even high-dose chemotherapy. At the present time, monoclonal antibodies (mAbs) are being successfully used to treat cancers. We recently discovered that anti-β2-microglobulin (β2M) mAbs (IgG1 isotype) induced myeloma cell apoptosis (Yang et al., Cancer Cell2006; 10:295–307). The mAbs bind, cross-link, and recruit surface β2M/MHC class I molecules on myeloma cells to lipid rafts where downstream signaling pathways are activated. Therefore, it is possible that enhancing the capacity of the mAbs to cross-link surface MHC class I could further improve the efficacy of mAb-induced myeloma cell apoptosis. To examine this hypothesis, we generated anti-β2M mAbs of IgM isotype with a pentameric structure. By using Annexin V and TUNEL assays, we showed that, compared with monomeric IgG mAbs, IgM anti-β2M mAbs exhibit stronger tumoricidal activity on all six myeloma cell lines and primary myeloma cells isolated from five myeloma patients in dose- and time-dependent manner. About 80% to 90% of myeloma cells were apoptotic when treated with IgM mAb at a concentration as low as 20 mg/mL in a 12-hour culture. Furthermore, IgM anti-β2M mAbs, which at the same dose had stronger therapeutic efficacy than IgG mAbs in vivo, had greatly reduced tumor burdens and retarded tumor growth in SCID mice. To examine whether the pentameric structure plays an important role in IgM mAb-mediated tumoricidal activity, IgM mAbs were treated with β-mercaptoethanol (2ME), a specific agent that breaks the J-chain of IgM antibody leading to irreversible disruption of the IgM pentameric structure. By using native gel electrophoresis, we confirmed that without 2ME treatment, IgM mAbs were pentamers with one band of 950 kDa. After treatment with 2ME, IgM mAbs displayed a strong band in 175 kDa (monomer), indicating that 2ME completely broke pentamers to monomers. Furthermore, we found that the addition of 2ME-pretreated IgM mAbs to cell culture induced much weaker myeloma cell apoptosis than pentameric IgM mAbs. By using Western blot analysis, we further showed that 2ME-pretreated IgM mAbs induce weaker JNK activation and caspase-9, -8, -3, and PARP cleavage. By using confocal microscopy, we showed that 2ME-pretreated IgM mAbs are much less efficient at recruiting β2M/MHC class I molecules into lipid rafts on myeloma cells, although 2ME-pretreated IgM mAbs bound well to myeloma cell surface. Thus, our findings indicate that enhancing cross-linking of surface β2M/MHC class I molecules may be a novel approach to improve the antimyeloma efficacy of the mAbs. This study also suggests that our IgM anti-β2M mAbs may be a better therapeutic agent for future clinical application.
A Staged Approach to the Use of Bortezomib/Thalidomide/Dexamethasone (VTD) in Multiple Myeloma Patients with Suboptimal Initial Cytoreduction Led to a High Complete Remission Rate
Authors: Chim Chor Sang James , Liang Raymond
Published: April 2020

Autologous hematopoietic stem cell transplantation (AHSCT) and bortezomib are the two most important advances in the treatment of multiple myeloma (MM) in the last decade. Autologous HSC harvest is preferably performed at complete remission (CR) or after maximal reduction of myeloma tumor load. Owing to the high cost of bortezomib, we devised a total therapy incorporating VAD (vincristine, adriamycin, dexamethasone), followed by VTD in those with inadequate cytoreduction (i.e. <75% reduction in paraprotein) to maximize eradication of myeloma cells in the marrow prior to HSC harvest. Those achieving >75% paraprotein reduction proceeded to AHSCT directly. Those with measurable disease after the first AHSCT proceeded to second AHSCT. Response was based on the EBMT criteria (Blade et al, 1998). There were 16 patients with a median age of 50 years (33–64). Majority had advanced stage disease by International staging system. After VAD, 6 patients had >75% response (>90% response, n=3; 75%–90% response, n=3) and hence proceeded to first AHSCT directly. The other 10 patients had VTD salvage therapy, which upgraded response in 9 cases. After VAD and VTD treatments, there were 2, 6,7 and 1 patients achieving CR, >90% response, 75%–90% response and no response respectively. Thirteen patients proceeded to the first AHSCT, which rendered upgrading of response in 9 patients (69%) with CR/near CR in 7 cases (54%) and >90% response in 4 cases (31%). The latter 4 patients proceeded to second AHSCT, resulting in CR/near CR in 9 cases (70%) and >90% in 2 cases (15%). Of the 11 patients with >90% response after first and/or second AHSCT, 5 patients had maintained response with continual CR in 4 cases, whereas 6 patients relapsed (with asymptomatic disease in 4 cases and fatality in 2 cases). Therefore, this strategy of employing bortezomib only in those with insufficient cytoreduction achieved a high CR/near CR rate comparable to that with upfront bortezomib use. However, the frequent relapse implied that the quality of CR is equally important. Moreover, the aggressive fatal relapse in CR patients demonstrated a biologically aggressive subgroup in which CR might be inadequate.
Validation of a Predictive Model for Indentifying An Increased Risk for Thromboembolism in Children with Acute Lymphoblastic Leukemia: Results of a Multicenter Cohort Study
Authors: Flege Silke , Mitchell Lesley , Kenet Gili , Heller Christine , Fruhwald Michael , Nowak-Gottl Ulrike
Published: April 2020

Children with acute lymphoblastic leukemia (ALL) are at increased risk for venous thromboembolism (VTE), however, not all children experience a VTE. Developing a predictive model for determining children at increased risk would be beneficial in targeting interventional studies to only high risk groups. A recent meta-analysis of studies in VTE in children with ALL identified four potential risk factors:

treatment with Escherichia coli asparaginase (CASP), concomitant use of steroids, presence of central venous lines and thrombophilic genetic abnormalities.

As VTE in childhood ALL is well recognized as serious clinical problem and due to the lack of studies on prevention, the standard of practice varies and some centres use enoxaparin prophylaxis for these children. However, the risks and benefits of the intervention are unknown. The aim of the study was

to develop a simple model for predicting ALL-chemotherapy-associated VTE using baseline clinical and laboratory variables, and to evaluate, on an explorative basis, the increasing off-label use of enoxaparin for VTE prophylaxis in ALL children.

For development of the risk model the predictive variables were scored as follows: treatment with CASP (5000–10000/m2) in combination with prednisone or dexamethasone, presence of central venous lines, thrombophilic genetic abnormalities, e.g. positive family history for VTE or identification of a single thrombophilic trait (1 point each), or carrier status of combined thrombophilic traits (2 points). A definition of VTE risk by score was low (1–2) and high (□ 3). The risk score was than prospectively validated in an independent cohort of 136 newly recruited patients enrolled into the German database. Seven patients were excluded (lost to follow-up n=2; death n=2, secondary malignancy, VTE before ALL-onset, infant < 12 months of age: each n=1). The cumulative VTE rates at 3.5 months in the validation cohorts were 3.6% (95% CI 1%–9%) in the low-risk group (4 of 112), and 47% (95%CI 23%–72%) in the high-risk category (8 of 17). In multivariate analysis [Cox regression] the high risk group was significantly associated with VTE when compared to the low risk group even after adjusting for age at ALL-onset, duration of CASP administration, steroid administered (prednisone/dexamethasone), and presence or absence of enoxaparin prophylaxis [hazard/95%CI: 4.16/1.13–15.34]. The negative predictive value for VTE was 96.3% [95%CI: 92.9–99.8]. Early enoxaparin prophylaxis reduced the absolute VTE-risk about 60% [95%CI: 23–96]. Therefore, the model can identify ALL-children with an increased risk for symptomatic VTE.
Elevated Leukocyte Alkaline Phosphatase Scores Induced by Jak2 V617F Mutation
Authors: Oku Seido , Takenaka Katsuto , Kumano Takashi , Kuriyama Takuro , Urata Shingo , Shide Kotaro , Shimoda Haruko , Iwasaki Hiromi , Miyamoto Toshihiro , Teshima Takanori , Nagafuji Koji , Shimoda Kazuya , Akashi Koichi
Published: April 2020

Leukocyte alkaline phosphatase (LAP) enzymatic activity is a marker of the last stages of myeloid differentiation. The level of LAP is quantitated as the LAP score. Estimation of the LAP score has been useful for distinguishing chronic myelogenous leukemia (CML) from BCR-ABL–negative chronic myeloproliferative disorders (MPDs) and neutrophilic reactions in severe infections. CML patients usually have a low LAP score, whereas elevated LAP scores are seen in patients with polycythemia vera (PV), primary myelofibrosis (PMF), and leukocytosis caused by infections. An acquired Jak2 V617F mutation is seen in approximately 95% of patients with PV and in about 50% of patients with essential thrombocythemia or PMF. It has been shown that Jak2 V617F mutation induced constitutive activation of the JAK-STAT signaling pathway. We speculated that an elevated LAP score might be caused due to activation of JAK-STAT signaling through a Jak2 V617F mutation, and conducted this study to address this question. We analyzed the LAP scores in Jak2 V617F-positive and -negative MPD patients. Jak2 V617F-positive MPD patients had higher LAP scores than Jak2 V617F-negative patients. Moreover, patients carrying homozygous mutations had higher LAP scores than patients with heterozygous mutations. AG490, the Jak2 inhibitor, was shown to significantly decrease the LAP expression in neutrophils of Jak2 V617F-positive patients. We lentivirally transfected the acute promyelocytic leukemia cell line NB4 with the Jak2 V617F mutation and wild-type Jak2 V617F. The expression level of Jak2 was not significantly different between the Jak2 V617F mutation and wild-type Jak2 V617F. We then examined the LAP scores of transfected NB4 cells after these cells were differentiated by all-trans retinoic acid and granulocyte colony stimulating factor. It was observed that the Jak2 V617F mutation and not the wild-type Jak2 induced elevated LAP scores. Furthermore, we showed that Jak2 followed the MAP kinase pathway and not the PI3 kinase pathway, as a downstream signaling pathway to elevate the LAP scores using MEK 1/2 (U0126) and PI3 kinase (LY294002) inhibitors. In conclusion, we obtained direct evidence that Jak2 V617F mutation induces elevated LAP scores via the MAP kinase pathway.
Clinical Significance of the Presence of Occult CNS Involvement Assessed by Cytofluorometry in Patients with Non Hodgkin’s Lymphoma and High Risk of CNS Relapse
Authors: Sancho Juan-Manuel , Orfao Alberto , García Olga , Panizo Carlos , Pérez Elena , Morgades Mireia , Salar Antonio , García-Vela José-Antonio , Alonso Natalia , González-Barca Eva , Plaza Josefa , Caballero Dolores , Provencio Mariano , Marin Miguel , Molero Teresa , Ribera Josep-Maria
Published: April 2020

Background and objective. Several studies have demonstrated that flow cytometry (FCM) improves the sensitivity of conventional cytology (CC) for the identification of leptomeningeal disease in aggressive lymphomas with high risk of central nervous system (CNS) involvement. A previous report from our group showed infiltration by FCM study in 27 out of 123 (22%) newly diagnosed aggressive B-cell lymphomas, while CC was positive in only 7 (6%). However, the clinical significance of the occult disease in CNS (FCM positive and CC negative) remains unknown.

Patients and Methods. At the time of the analysis, 68 out of 123 patients of the previous study had adequate follow-up (median of follow-up for the alive patients [n=42] of 18 months [range 12–30]). In this cohort of patients the CNS involvement during follow-up was assessed according to CC and FCM results in cerebrospinal fluid at the time of diagnosis.

Results. Three groups were defined according to the results of CC and CFM: Group 1, patients without CNS involvement (CC and FCM negatives, n=49); Group 2, patients with occult CNS disease (FCM positive and CC negative, n=11); Group 3, patients with CNS disease (CC and FCM positive, n=8). The groups were comparable for the main clinical and biological characteristics and histological subtypes of NHL. In the Group 1, 40 patients received intrathecal (IT) prophylaxis, 2 received CNS therapy due to the presence neurological symptoms and 7 did not receive any prophylaxis. In the Group 2, CNS prophylaxis was administered to 7 patients, whereas the 4 remaining received active therapy. The 8 patients of the Group 3 received active CNS therapy. Overall, 6 patients showed CNS relapse/progression, 2 in the Group 1 (4.1%) (both with diffuse large B cell lymphoma [DLBCL]) and 4 in the Group 3 (50%) (3 with Burkitt’s lymphoma and the remaining with DLBCL). None of the patients of the Group 2 showed CNS relapse or progression during follow-up. As expected, patients of Group 3 showed a significant increase in CNS relapse compared to those from Groups 1 (p<0.001) and 2 (p=0.038). However, the incidence of CNS relapse in patients of Groups 1 and 2 was not statistically different (p=0.496).

Conclusions. Up to now, and different from previous studies, the presence of occult CNS disease in our cohort of patients with NHL and high risk of CNS disease was not associated with a higher risk of CNS relapse. This feature could be due to the fact that most of the patients received adequate CNS prophylaxis, that could be sufficient to eliminate the occult disease.
Epstein-Barr Virus Status as Prognostic Factor in Nodal Diffuse Large B-Cell Lymphoma in Elderly Patients
Authors: Beltran Brady , Carrasco Antonio , Salas Renzo , Morales Domingo , Quinones Pilar , Castillo Jorge
Published: April 2020

BACKGROUND: Epstein-Barr vitus (EBV)-positive status is related to a poor survival in patients with Hodgkin lymphoma, T-cell non-Hodgkin lymphoma (NHL) and, in a recent publication from Asia, in patients with diffuse large B-cell lymphoma (DLBCL).

AIM: In order to define the impact of EBV infection on survival in patients with nodal DLBCL, we investigated the EBV status in elderly Peruvian patients with a diagnosis of DLBCL. PATIENTS AND

METHODS: Between January 2002 and December 2004, thirty-eight patients older than 70 years who were diagnosed with nodal DLBCL were included in the analysis. Clinical data were reviewed retrospectively from the patients’ records. Nodal tissue from all cases was analyzed for the presence of EBV encoded RNA (EBER) using the in situ hybridization (ISH) method. Survival analysis was performed using the Kaplan-Meier method.

RESULTS: Eight cases (21%) were identified as EBV-positive; the remaining 30 cases (79%) were EBV-negative. Within clinical characteristics, EBV-positivity was associated with poorer performance status, more advanced stage and higher IPI scores. From the 8 EBV-positive cases, two cases (25%) had a germinal center (GC)-like phenotype, whereas the other six (75%) had a non-germinal center phenotype. Overall survival for EBV-positive DLBCL patients was 0.5 months and for EBV-negative patients was 14 months (p = 0.002). In the multivariate analysis, EBV status emerged as an independent variable for survival when evaluated against the IPI score (p = 0.001). There were no differences in survival between DLBCL phenotype (GC versus non-GC).

CONCLUSION: EBV status in elderly patients with nodal DLBCL is a powerful survival prognostic factor and its role in the survival of other lymphoproliferative disorders and age groups should be investigated. New therapeutic strategies should be defined in patients with EBV-positive DLBCL.
Gastric Primary Non-Hodgkin Lymphoma: A Retrospective Study in a Brazilian Population
Authors: Costa Renata Oliveira , Chamone Dalton , Pracchia Luis Fernando , Pereira Juliana
Published: April 2020

About 40% of the non-Hodgkin’s Lymphoma (NHL) arises from extranodal tissues. Gastrointestinal tract (GIT) is the most common site and is represented more frequently by MALT lymphoma and diffuse large B-cell lymphoma (DLBCL). In Brazil, despite its importance and frequency there are very few epidemiological data concerning this lymphoma. In order to study the primary gastric lymphoma features in a Brazilian population, 60 patients were retrospectively evaluated. Thirty-one patients (51.7%) were female; median age was 61 years (range 20–86) and median follow-up was seven years. Thirty eight (63.3%) cases were classified as DLBCL and 22 (36.6%) as MALT lymphoma and were submitted to different treatment. There were no significant differences between these two groups about gender, age, GIT symptoms, constitutional symptoms, bulky disease, bone marrow infiltration, Lugano stage, Helicobacter pylori infection and endoscopic findings. The complete remission rate was 73.1% for DLBCL and 95% for MALT lymphoma. The disease free survival in seven years was 84.8% for DLBCL and 94.1% for MALT lymphoma and overall survival rate for DLBCL was 65.7% and 92.9% for MALT lymphoma. We conclude that both DLBCL and MALT lymphoma have similar clinical presentation and seems to have a good prognosis.
MicroRNA-17-92 Downregulates Expression of Distinct Targets in Different B-Cell Lymphoma Subtypes
Authors: Tagawa Hiroyuki , Inomata Mika , Guo Yong-Mei , Kameoka Yoshihiro , Takahashi Naoto , Sawada Kenichi
Published: April 2020

Aberrant overexpression of the miR-17-92 polycistron is strongly associated with B-cell lymphomagenesis. Recent studies have shown that miR-17-92 downregulates the pro-apoptotic protein Bim, leading to overexpression of Bcl2, which likely plays a key role in lymphomagenesis. However, the fact that Jeko-1 cells derived from mantle cell lymphoma exhibit both homozygous deletion of BIM and overexpression of miR-17-92 suggests other targets are also involved in B-cell lymphomagenesis. To identify essential target(s) of miR-17-92 in lymphomagenesis, we first transfected miR-17-92 into genetically distinct B-cell lymphoma cell lines, including Raji cells, which overexpress c-Myc, and SUDHL4 cells, which overexpress Bcl2. Raji cells transfected with miR- 17-19b exhibited downregulation of Bim and reversible upregulation of Bcl2. On the other hand, SUDHL4 cells transfected with miR-17-19b showed aggressive cell growth reflecting facilitated cell cycle progression at the G1-S transition, and decreased expression of CDKN1A mRNA and p21 protein (CDKN1A/p21) that was independent of p53 expression. Conversely, transfection of antisense oligonucleotides against miR-17 and miR-20a into Jeko-1 cells led to upregulation of CDKN1A/p21, resulting in decreased cell growth with G1-S arrest. Thus, CDKN1A/p21 appears to be an essential target of miR-17-92 during B-cell lymphomagenesis, which suggests the polycistron has distinct targets in different B-cell lymphoma subtypes.