Abelson Murine Leukemia Virus Research Articles

Cytotoxic and Apoptotic Impacts of Ceranib-2 on RAW 264.7 Macrophage Cells.

Many ceramidase inhibitors have been developed and identified as potential treatment agents for various types of tumors in the last several decades. In recent years, their therapeutic potential against tumors has gained great attention. Inhibition of ceramidase is r eportedly related to apoptosis and cytotoxicity in macrophages, which are closely related to tumor development and progression. However, whether and how ceranib-2, a novel ceramidase inhibitor, can exert its cytotoxic and apoptotic effects on RAW 264.7, a macrophage cell line established from a tumor in a male mouse induced with the Abelson murine leukemia virus, remains unknown. In this study, we aimed to investigate whether and how ceranib-2 can exert cytotoxic, antiproliferative, and apoptotic effects on the RAW264.7 macrophages. We performed the MTT assay, Annexin V staining assay, and confocal microscopy to detect the cytotoxicity, apoptosis, and morphological changes, respectively, in the RAW264.7 cells. The viability of RAW264.7 cells treated with ceranib-2 was decreased as the doses of ceranib-2 increased at 24 h and 48 h due to apoptosis resulting from ceranib-2-reduced integrity of the mitochondrial membrane. Moreover, morphological changes were observed in these ceranib-2 exposed cells, further indicating the role of ceranib-2 in inducing apoptosis in these cells. Ceranib-2 is cytotoxic to RAW 264.7 macrophages and can induce apoptosis in these cells.

Analysis of early molecular response at 3 months in predicting overall response in newly diagnosed patients with chronic myeloid leukemia on imatinib

Objectives: This study aimed to study the correlation between Breakpoint Cluster Region- ABelson Leukemia virus 1 transcript levels at 3 months with the treatment responses at 6 and 12 months in patients on imatinib. Around 30% of patients with chronic myeloid leukemia (CML) might have treatment failure with the first-line tyrosine kinase inhibitors (TKI). Patients with a “warning response” at 3 months can continue therapy with the same TKI while monitoring for disease progression. However, newer pieces of evidence suggest that patients who fail treatment with imatinib do have suboptimal responses in the early time points, and hence, 1st 3-month assessment might be a useful indicator for future treatment failure. Material and Methods: It is a single-center prospective observational study involving 60 treatment-naive consecutive patients with CML-chronic phase who attended Hematology Outpatient Department at IHTM, Kolkata. Treatment responses were assessed by cytogenetics and BCR-ABL1 transcript levels by real-time quantitative polymerase chain reaction at 3 monthly time points. Results: About 51% and 70.2% of the study participants achieved complete cytogenetic response at 6 and 12 months, respectively. About 74% of the participants had achieved early molecular response (EMR) at 3 months. The failure rates of cytogenetic responses were 13% and 20% at 6 and 12 months, respectively. Patients who failed to achieve EMR at 3 months had higher failure rates at 6 months. The major, warning and failure of molecular responses at 6 and 12 months were found to be 15%, 25%, and 9%, and 34%, 39%, and 27%, respectively. The analyses showed that patients who failed to achieve EMR at 3 months are also more likely to have the failure of molecular response at 12 months, with a statistical significance of P < 0.01. Failure of EMR at 3 months also correlated with failure of overall responses (both cytogenetic and molecular at 12 months) with a statistical significance of P = 0.006. When followed up, there was a progression of disease in three including a death in the suboptimal response group. Conclusion: Our patients had inferior treatment responses to imatinib than that observed in the previous studies. The majority have baseline fibrosis of the marrow and splenomegaly at presentation which might contribute to adverse outcomes. The molecular response at 3 months was found to be a consistent and powerful indicator of treatment responses at later time points.

Regorafenib: A narrative drug review

The process of angiogenesis is a complex balance of positive and negative regulators, and vascular endothelial growth factor (VEGF) is one of the most important proangiogenic factors involved in tumor angiogenesis. One of the drugs targeting this process is regorafenib, a multi-kinase inhibitor. It inhibits VEGF receptors 1, 2, and 3; rearranged during transfection (RET); receptor tyrosine kinase (KIT); platelet-derived growth factor receptor (PDGFR) alpha and beta; fibroblast growth factor receptor (FGFR) 1 and 2; angiopoietin-1 receptor (Tie2); discoidin domain-containing receptor 2 (DDR2); Ephrin type-A receptor 2 (Eph 2A); tropomyosin receptor kinase A (TrkA); rapidly accelerated fibrosarcoma (RAF-1); v-RAF murine sarcoma viral oncogene homolog B1 (BRAF); stress-activated protein kinase-2 (SAPK2); protein tyrosine kinase 5 (PTK5); and Abelson murine leukemia virus (Abl). Regorafenib has been approved for patients with metastatic colorectal cancer (CRC), locally advanced or metastatic gastrointestinal stromal tumor (GIST), and hepatocellular carcinoma (HCC). We present a review of regorafenib which includes the history, basic chemistry, pharmacology, pharmacokinetics, clinical indications, drug interactions, and safety profile. We performed a search in PubMed and other sources using the search terms “CRC,” “GIST,” “HCC,” “osteosarcoma,” and “regorafenib” and searched for full-text articles published in the past 10 years. Out of the total 65 studies identified, we included 46 studies. We combined all the abstracts and conference proceedings to generate a comprehensive review.

Corynebacterium pyruviciproducens-peptidoglycan: A novel bacterial peptidoglycan inhibiting overexpression of MyD88 in macrophages

Objectives: Bacterial peptidoglycan (PGN) is an essential ligand of TLR2 inducing inflammatory damage by boosting MyD88 overexpression in pathogen invasion, such as Methicillin-resistant Staphylococcus aureus (MRSA) infection. CP-PGN is a novel PGN from an adjuvant bacterium, displaying anti-infection immune regulation. This study aimed to clarify the unique moderation of MyD88 expression by CP-PGN. Methods: Compared with other ligands of TLR2, high expression of MyD88 in macrophages was established by MRSA and virus to investigate the immunomodulation of CP-PGN. Results: Compared with PGN derived from MRSA (M-PGN) and chemosynthetic Pam3CSK4 of model agonists of TLR2, CP-PGN could inhibit overexpression of MyD88 in a time- and dose-dependent way in infected macrophages by MRSA or Abelson leukemia virus. CP-PGN also promoted more anti-inflammatory IL-10 and less pro-inflammatory TNF-α in immature primary macrophages. Furthermore, IL-10 secretion induced by CP-PGN was reduced most significantly by blocking the dimer formation of MyD88 with ST2825 and lowering down expression by si-MyD88. Conclusion: CP-PGN could inhibit MyD88 overexpression by infection to moderate inflammatory cytokines. Therefore, CP-PGN is a novel potential ligand of TLR2 to induce inflammatory balance in the process of host defense against invading pathogens.

MiR-125a-3p and MiR-320b Differentially Expressed in Patients with Chronic Myeloid Leukemia Treated with Allogeneic Hematopoietic Stem Cell Transplantation and Imatinib Mesylate.

Chronic myeloid leukemia (CML), a hematopoietic neoplasm arising from the fusion of BCR (breakpoint cluster region) gene on chromosome 22 to the ABL (Abelson leukemia virus) gene on chromosome 9 (BCR-ABL1 oncogene), originates from a small population of leukemic stem cells with extensive capacity for self-renewal and an inflammatory microenvironment. Currently, CML treatment is based on tyrosine kinase inhibitors (TKIs). However, allogeneic hematopoietic stem cell transplantation (HSCT-allo) is currently the only effective treatment of CML. The difficulty of finding a compatible donor and high rates of morbidity and mortality limit transplantation treatment. Despite the safety and efficacy of TKIs, patients can develop resistance. Thus, microRNAs (miRNAs) play a prominent role as biomarkers and post-transcriptional regulators of gene expression. The aim of this study was to analyze the miRNA profile in CML patients who achieved cytogenetic remission after treatment with both HSCT-allo and TKI. Expression analyses of the 758 miRNAs were performed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Bioinformatics tools were used for data analysis. We detected miRNA profiles using their possible target genes and target pathways. MiR-125a-3p stood out among the downregulated miRNAs, showing an interaction network with 52 target genes. MiR-320b was the only upregulated miRNA, with an interaction network of 26 genes. The results are expected to aid future studies of miRNAs, residual leukemic cells, and prognosis in CML.

CXCR2, a novel target to overcome tyrosine kinase inhibitor resistance in chronic myelogenous leukemia cells

Chronic myeloid leukemia (CML) is a reciprocal translocation disorder driven by a breakpoint cluster region (BCR)-Abelson leukemia virus (ABL) fusion gene that stimulates abnormal tyrosine kinase activity. Tyrosine kinase inhibitors (TKIs) are effective in treating Philadelphia chromosome (Ph) + CML patients. However, the appearance of TKI-resistant CML cells is a hurdle in CML treatment. Therefore, it is necessary to identify novel alternative treatments targeting tyrosine kinases. This study was designed to determine whether C-X-C chemokine receptor 2 (CXCR2) could be a novel target for TKI-resistant CML treatment. Interleukin 8 (IL-8), a CXCR2 ligand, was significantly increased in the bone marrow serum of initially diagnosed CML patients and TKI-resistant CML cell conditioned media. CXCR2 antagonists suppressed the proliferation of CML cells via cell cycle arrest in the G2/M phase. CXCR2 inhibition also attenuated mTOR, c-Myc, and BCR-ABL expression, leading to CML cell apoptosis, irrespective of TKI responsiveness. Moreover, SB225002, a CXCR2 antagonist, caused higher cell death in TKI-resistant CML cells than TKIs. Using a mouse xenograft model, we confirmed that SB225002 suppresses tumor growth, with a prominent effect on TKI-resistant CML cells. Our findings demonstrate that IL-8 is a prognostic factor for the progression of CML. Inhibiting the CXCR2-mTOR-c-Myc cascade is a promising therapeutic strategy to overcome TKI-sensitive and TKI-insensitive CML. Thus, CXCR2 blockade is a novel therapeutic strategy to treat CML, and SB225002, a commercially available CXCR2 antagonist, might be a candidate drug that could be used to treat TKI-resistant CML.

Abstract 2600: Development of TCR-stimulating 300.19 and 300.19-hB7-1 cells using modified anti-CD3 scFv constructs

Abstract To facilitate immunological assays which require T cell activation, we made two membrane anchored anti-CD3 single chain fragment variable (scFv) constructs: one named Steinberger (St, which includes the hTIM-3 mucin domain as linker and medium strength human PGK promoter) and the other named Riley (Ri, which includes hCD8 stem as linker and weaker human UBC promoter). Isolating T cells activated using adherent CHO-TCR-Activator cells is labor intensive, results in variable amounts of CHO cells, and makes it difficult to examine early time points after TCR activation. To combat this issue, we established stable expressing cell lines by transfecting St and Ri into 300.19 cells (Abelson leukemia virus transformed murine pre-B cell line that grows as non-adherent single cells) as well as 300.19 cells stably transfected with hB7-1 and selected 300.19-hB7-1-St, 300.19-hB7-1-Ri, 300.19-St, and 300.19-Ri using the following procedures. We electroporated 300.19 and 300.19-hB7-1 cells with three different amounts of St and Ri linearized plasmid: 0.5µg, 5µg, and 50µg. After selection for resistant cells with hygromycin and puromycin, we stained each cell line with goat anti-mouse IgG (Fab2-specific)-Alexa647 and sorted into three levels of scFv expression (high, medium, and low) and cultured single cell clones. We tested the T cell-stimulatory capacities of our anti-CD3 cells in a Jurkat T cell model through assay of increased IL-2 promoter mediated transcription of luciferase compared to control and ranked the stimulatory capacity of each clone. We found that T cell ligation by 300-hB7-1-Ri and 300-hB7-1-St cells induced the highest IL-2 promoter luciferase reporter activity while the 300-Ri and 300-St induced significant but lesser amounts. Surprisingly, Ri construct cells were generally stronger than the St clones though the literature says the PGK promoter (St plasmid) is stronger than the UBC promoter (Ri plasmid). This difference in stimulatory capacity may be due to the Ri construct (scFv-CD8 linker) being more stable than the St construct (scFv-hTIM3 linker) or to different promoter activity in mouse pre-B cells. Our results also suggest that the amount of DNA transfected may be predictive of construct expression level. The TCR-engaging capacity of our cell lines makes them a widely applicable tool in cancer immunotherapy research. These cells improve the TCR-engaging assay technique by avoiding the use of adherent CHO-TCR-Activator cells and/or CD28 antibodies. Collectively, our anti-CD3 scFv construct-expressing cells may enable researchers to better understand the cascading processes involved in T cell proliferation and differentiation into effector T cells, assess the dynamics of co-stimulatory and co-inhibitory receptors under various controlled levels of T cell stimulation, and understand tumor microenvironments and cytokine production with and without CD28 co-stimulation. Citation Format: Melissa T. Bu, Baogong Zhu, Gordon J. Freeman. Development of TCR-stimulating 300.19 and 300.19-hB7-1 cells using modified anti-CD3 scFv constructs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2600.

Cysteine and hydrophobic residues in CDR3 serve as distinct T-cell self-reactivity indices

Cysteine and hydrophobic residues in CDR3 serve as distinct T-cell self-reactivity indices

Research progress of Ph-like acute lymphoblastic leukemia

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a new high-risk subtype of acute lymphoblastic leukemia (ALL) proposed in recent years. It is characterized by a gene-expression profile similar to Ph-positive ALL, involving abnormal activation of cytokine receptor and tyrosine kinase, and commonly harbor genetic alterations targeting lymphoid transcription factors, and associated with a poor prognosis. Clinical studies showed that targeted inhibitors of related signaling pathways can significantly improve the prognosis in specific patients. The research progress of Ph-like ALL is summarized in this review. Key words: Philadelphia chromosome; Precursor cell lymphoblastic leukemia-lymphoma; Abelson murine leukemia virus; Prognosis; CRLF2 gene

Mechanism underlying tumorigenesis induced by Bcr-Abl oncogene and A-MuLV virus

The Bcr-Abl oncogene is produced by the reciprocal translocation between c-Abl gene on chromosome 9 and the Bcr gene on chromosome 22 in human genome. The encoded Bcr-Abl fusion protein is responsible for the pathogenesis of certain human leukemias. Abelson murine leukemia virus (A-MuLV) is a retrovirus that could lead to transformation of B lymphocyte in mice, and v-Abl is the oncogene of A-MuLV. Abl oncoproteins (such as Bcr-Abl and v-Abl) play critical roles in tumorigenesis of certain cell types. Several signal transduction pathways, including JAK/STAT/Pim, PI3K/AKT/mTOR and RAS/RAF/MEK signaling pathway, are involved in Abl-mediated tumorigenesis. In addition, Abl-mediated tumorigenesis is associated with mutation or abnormal modification of key signal molecules as well as dysregulation of some critical long noncoding RNAs (lncRNAs). Here, we review the molecular mechanisms by which Abl oncogenes activate three major signaling pathways, and provide a scientific basis for therapy of Abl oncoprotein-induced tumors.

Evaluation of Antioxidant, Anti-Inflammatory and Cytoprotective Properties of Ethanolic Mint Extracts from Algeria on 7-Ketocholesterol-Treated Murine RAW 264.7 Macrophages.

The present study consisted in evaluating the antioxidant, anti-inflammatory and cytoprotective properties of ethanolic extracts from three mint species (Mentha spicata L. (MS), Mentha pulegium L. (MP) and Mentha rotundifolia (L.) Huds (MR)) with biochemical methods on murine RAW 264.7 macrophages (a transformed macrophage cell line isolated from ascites of BALB/c mice infected by the Abelson leukemia virus). The total phenolic, flavonoid and carotenoid contents were determined with spectrophotometric methods. The antioxidant activities were quantified with the Kit Radicaux Libres (KRLTM), the ferric reducing antioxidant power (FRAP) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. The MS extract showed the highest total phenolic content, and the highest antioxidant capacity, while the MR extract showed the lowest total phenolic content and the lowest antioxidant capacity. The cytoprotective and anti-inflammatory activities of the extracts were quantified on murine RAW 264.7 macrophages treated with 7-ketocholesterol (7KC; 20 µg/mL: 50 µM) associated or not for 24 h and 48 h with ethanolic mint extracts used at different concentrations (25, 50, 100, 200 and 400 µg/mL). Under treatment with 7KC, an important inhibition of cell growth was revealed with the crystal violet test. This side effect was strongly attenuated in a dose dependent manner with the different ethanolic mint extracts, mainly at 48 h. The most important cytoprotective effect was observed with the MS extract. In addition, the effects of ethanolic mint extracts on cytokine secretion (Interleukin (IL)-6, IL-10, Monocyte Chemoattractant Protein (MCP)-1, Interferon (IFN)-ϒ, Tumor necrosis factor (TNF)-α) were determined at 24 h on lipopolysaccharide (LPS, 0.2 µg/mL)-, 7KC (20 µg/mL)- and (7KC + LPS)-treated RAW 264.7 cells. Complex effects of mint extracts were observed on cytokine secretion. However, comparatively to LPS-treated cells, all the extracts strongly reduce IL-6 secretion and two of them (MP and MR) also decrease MCP-1 and TNF-α secretion. However, no anti-inflammatory effects were observed on 7KC- and (7KC + LPS)-treated cells. Altogether, these data bring new evidences on the potential benefits (especially antioxidant and cytoprotective properties) of Algerian mint on human health.

Efficacy of Early Convert to Dasatinib in Chronic Myeloid Leukemia Patients Who Inresponse or Intolerance to Imatinib

Blackground:Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm hallmarked by the presence of fusion protein kinase derived from reciprocal translocation between chromosome 9 and 22, breakpoint cluster region (BCR)-Abelsonleukemia virus (ABL). Since the discovery of a tyrosine kinase inhibitor (TKI), CML has become a chronic illness if managed appropriately. The use of the first generation of TKI such as imatinib, benefit for most CML patients, but there are still a significant number of patients resistance to imatinib , need switched to second generation TKIs, such as dasatinib or nilotinib. however the efficiency and safety of CML patients who inresponse or intolerance to imatinib early convert to dasatinib should clinical studies.Objective: To evaluate the efficiency and safety of CML patients who inresponse or intolerance to imatinib early convert to dasatinib.Methods:We retrospectively analyzed 70 CML patients who inresponse or intolerance to imatinib in Southern Hospital from March 2014 to April 2016;real-time quantitative PCR method was used to detect BCR-ABL1 fusion gene Sanger sequencer.Results: 37 CML patients BCR/ABL (IS)>10% treated by imatinib for 3 to 6 months,had early conversion to dasatinib. Among them, 31 cases had low response to imatinib (sokal score was middle and high risk) and 6 cases intolerance to imatinib. 24 cases (64.9%) reached BCR/ABL (IS) < 10% after convert to dasatinib for 3 months, and 6 cases (40.5%) reached MMR after convert to dasatinib for 6 months.15 cases (40.5%) reached MMR in 12 months after conversion to dasatinib.33 patients (treated with imatinib for 3 to 6 months) did not undergo early transformation. Among them, 31 patients had poor response to imatinib and 2 patients intolerance to imatinib ,these patients continued imatinib or add imatinib dose to 600mg or 800mg qd. 13 patients (51%) did not reach the BCR/ABL (IS) < 10% for next 3 months, only 12 cases (36.3%) achieved MMR after 12 months follow-up, and 4 patients changed to dasatinib or nilatinib becase detected mutation during follow-up.The most common hematological adverse effects during dasatinib treatment were thrombocytopenia, neutropenia, anemia, and non-hematological adverse effects such as edema, pleural effusion, alopecia, gastrointestinal reactions and rare pulmonary hypertension. AEs can be alleviated by reducing or suspending dasatinib use.Conclusion: The cml patients who inresponse or intolerance to imatinib early convert to dasatinib will gain an earlier and greater benefit, and adverse effects of dasatinib are controllable.Key words:Chronic myeloid leukemia, Imatinib, Dasatinib, Early conversion DisclosuresNo relevant conflicts of interest to declare.

Resistance in chronic myeloid leukemia: definitions and novel therapeutic agents.

Chronic myeloid leukemia (CML) is hallmarked by the presence of fusion protein kinase derived from a reciprocal translocation between chromosome 9 and 22, breakpoint cluster region (BCR)-Abelson leukemia virus (ABL) 1, causing aberrant regulation of the downstream pathways leading to unchecked CML leukemia stem cells (LSCs) proliferation. Since the discovery of tyrosine kinase inhibitors (TKI), CML, once a fatal disease, has become a chronic illness if managed appropriately. Changing treatment landscape has unsurfaced the challenge of TKI resistance that is clinically difficult to overcome. In this review, we discuss the concept of TKI resistance and pathways leading to the resistance which allows for a survival advantage to CML LSCs. Aside from BCR-ABL-dependent mechanisms of resistance which involves aberrant expression in the regulatory pumps involving efflux and influx of the TKI affecting drug bioavailability, activation of alternate survival pathways may be accountable for primary or secondary resistance. Activation of these pathways, intrinsically and extrinsically to LSCs, may be mediated through various upstream and downstream signaling as well as conditions affecting the microenvironment. Several therapeutic approaches that combine TKI with an additional agent that inhibits the activation of an alternate pathway have been studied as part of clinical trials which we will discuss here. We categorize the resistance into BCR-ABL-dependent and BCR-ABL-independent subgroups to further describe the complex molecular pathways which can potentially serve as a therapeutic target. We further discuss novel combination strategies currently in early or advanced phase clinical trials aimed to overcome the TKI resistance. We further highlight the need for further research despite the tremendous strides already made in the management of CML.

Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) and IL-23 Induced by Polysaccharide of the Black Hoof Medicinal Mushroom, Phellinus linteus (Agaricomycetes).

Matrix metalloproteinase-9 (MMP-9) has diverse roles associated with cell growth, migration, invasion, and angiogenesis. Tissue inhibitor of metalloproteinase-1 (TIMP-1) is known to inhibit MMP-9 by complexing with it at a 1:1 ratio. Suppressing MMP-9 activity through the overexpression of TIMP-1 allows for regulation of tumor growth and metastasis by blocking invasion and angiogenesis in the tumor microenvironment. We found that TIMP-1 and interleukin (IL)-23 are induced in RAW264.7 macrophage cells, a cell line established by Abelson leukemia virus transformation from the BALB/c mouse strain, in a dose-dependent pattern, at the transcriptional level by treatment with a crude polysaccharide fraction of Phellinus linteus (CPP) at a range of 10 to 1000 μg/mL. We purified CPP into 2 polysaccharide fractions, Fr-I and Fr-II, and one protein fraction, Fr-III. Among the 3 fractions, Fr-II increased TIMP-1 expression 6.8-fold compared with the control, according to quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis in the RAW264.7 culture system. On the other hand, all 3 fractions increased IL-23 expression, with the highest increase brought about by Fr-II. qRT-PCR analysis showed that Fr-I and Fr-II increased IL-17 expression in RAW264.7 cells by 13.3-fold and 19.6-fold, respectively. IL-17 expression in lung tissue was increased 2.1-fold compared with the control group, whereas that in liver tissue was unaltered by oral administration of CPP for 7 days. In a mouse model, qRT-PCR analysis showed that CPP induced liver TIMP-1 and lung IL-17 expressions 8.9-fold and 2.1-fold, respectively, without affecting MMP-9 expression. Our in vitro and in vivo data suggest that inducing TIMP-1 without altering MMP-9 expression by administering the polysaccharide fraction of Ph. linteus could be a novel antitumor or antimetastasis mechanism of polysaccharide from the medicinal mushroom Ph. linteus.

Dasatinib Inhibits Actin Fiber Reorganization and Promotes Endothelial Cell Permeability through RhoA-Rock Pathway

BackgroundThe balanced translocation of 5′ segment of the breakpoint cluster region (BCR) gene on chromosome 22 to 3′ segment of the Abelson leukemia virus (ABL) gene on chromosome 9 in chronic myeloid leukemia (CML), results in the formation of the BCR-ABL hybrid fusion gene, which has constitutively upregulated tyrosine kinase activity. Imatinib mesylate, a small molecule developed specifically to inhibit BCR-ABL kinase activity, has revolutionized the treatment of CML. Despite the overwhelming success, about 20% of patients develop resistance to imatinib due to point mutations in the BCR-ABL kinase domain. Dasatinib is one of the second generation tyrosine kinase inhibitors (TKI) that was developed to overcome the resistance to imatinib. However, dasatinib inhibits, in addition to BCR-ABL kinase, other tyrosine kinases such as Src family kinases (SFKs), leading to its unique side effects. Clinically, one of the most common non-hematologic side effects of dasatinib is peripheral edema, pleural effusion and pulmonary hypertension and it is seen 20% of patients treated with dasatinib. We investigated the effect of dasatinib on the barrier function of human microvascular endothelial cells in vitro and in vivo.MethodsIn vitro endothelial permeability assays were performed with endothelial cells grown on transwell polycarbonate membranes inserts. FITC-Dextran was added to the upper chamber followed by dasatinib (0-100 ng/ml) or vehicle (DMSO) and at various time intervals, the medium in the lower chamber was collected and the fluorescence intensity was measured. In some experiments, the monolayers were incubated with ROCK1 inhibitor y27632 for 10 minutes before the addition of dasatinib. RhoA activity assay was performed and quantified using the RhoA activation assay kit. Spreading of control and dasatinib treated endothelial cells were quantified by Image J software. Actin cytoskeleton was visualized by Immunocytochemistry. Phosphorylation of cytoskeletal proteins were assessed by Western blot with phospho specific antibodies. In vivo endothelial permeability was measured by quantifying the extravasation of intravenously administered Evans Blue to lung parenchyma in mice.ResultsThe permeability of human microvascular endothelial cells (HMEC1) to FITC-dextran increases in Transwell chambers within 5 minutes following the addition of therapeutic concentrations of dasatinib. These changes in permeability are associated with increased activation of RhoA GTPase and its effector Rho associated coiled-coil kinase I (ROCKI). RhoA inhibitor C3 almost completely inhibits dasatinib-induced increase in permeability. Under similar conditions, imatinib had no effect on permeability and it does not activate RhoA. Since integrin-induced cell spreading suppresses RhoA activation, we examined the effect of dasatinib on cell spreading on fibronectin substrate. Dasatinib impairs endothelial cell spreading in a concentration-dependent manner and induces disorganization of actin fibers. Tyrosine kinases play an essential role in transmitting signals from integrins to RhoA and we examined tyrosine phosphorylation of several cytoskeletal proteins. Dasatinib markedly inhibits tyrosine phosphorylation of p130 Crk associated substrate (p130cas), paxillin and vinculin. These results suggest inhibition of tyrosine phosphorylation of the focal adhesion plaque components by dasatinib may alter the assembly of actin fibers resulting in the activation of RhoA/ROCK pathway. In vitro, consistent with these findings, dasatinib-induced increase in the permeability is blocked by ROCK inhibitor y-23632. In vivo administration of y-27632 significantly inhibits the dasatinib-induced extravasation of Evans Blue to lung parenchyma in mice. Furthermore, dasatinib-induced microvascular permeability is attenuated in ROCK1-deficient mice.ConclusionsOur findings suggest that dasatinib inhibits actin fiber reorganization and promotes endothelial permeability through RhoA-ROCK pathway and ROCK inhibitors could serve as a therapeutic modality to ameliorate the dasatinib-induced pulmonary changes. DisclosuresNo relevant conflicts of interest to declare.

Management of Chronic Myeloid Leukemia Patients Resistant to Tyrosine Kinase Inhibitors Treatment.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with a characteristic chromosomal translocation called the Philadelphia chromosome. This oncogene is generated by the fusion of breakpoint cluster region (BCR) and Abelson leukemia virus (ABL) genes and encodes a novel fusion gene translating into a protein with constitutive tyrosine kinase activity. The discovery and introduction of tyrosine kinase inhibitors (TKIs) irreversibly changed the landscape of CML treatment, leading to dramatic improvement in long-term survival rates. The majority of patients with CML in the chronic phase have a life expectancy comparable with that of healthy age-matched individuals. Although an enormous therapeutic improvement has been accomplished, there are still some unresolved issues in the treatment of patients with CML. One of the most important problems is based on the fact that TKIs can efficiently target proliferating mature cells but do not eradicate leukemic stem cells, allowing persistence of the malignant clone. Owing to the resistance mechanisms arising during the course of the disease, treatment with most of the approved BCR-ABL1 TKIs may become ineffective in a proportion of patients. This article highlights the different molecular mechanisms of acquired resistance being developed during treatment with TKIs as well as the pharmacological strategies to overcome it. Moreover, it gives an overview of novel drugs and therapies that are aiming in overcoming drug resistance, loss of response, and kinase domain mutations.

Erratum to: CIP2A is overexpressed and involved in the pathogenesis of chronic myelocytic leukemia by interacting with breakpoint cluster region-Abelson leukemia virus

To detect the expression of cancerous inhibitor of phosphatase 2A (CIP2A) in chronic myelocytic leukemia (CML) and investigate the mechanism underlying CIP2A knockdown-mediated cell proliferation and apoptosis as well as the interaction of CIP2A with breakpoint cluster region- Abelson leukemia virus (BCR-ABL). CIP2A mRNA and protein expression in chronic myelocytic leukemia-chronic (CML-CP) patients and healthy controls were determined by RT-PCR and Western blot. In vivo, c-Myc expression, PP2A activity, cell proliferation, and apoptosis of CML cells were detected with CIP2A depletion. In addition, the relationship among CIP2A, BCR-ABL, and tyrosine phosphatase SHP-1 was explored by depleting/overexpressing CIP2A or inhib- iting BCR-ABL. The level of CIP2A mRNA was higher in CML-CP patients than healthy controls (56/74, 75.7 % vs. 1/35, 2.9 %, P 0.001), and CIP2A protein was overex- pressed in corresponding specimens. CIP2A knockdown by siRNA reduced the level of c-Myc protein and clonogenic formation, inhibited the activity of PP2A, K562 cell prolif- eration, and promoted cell apoptosis. Suppressing BCR- ABL by imatinib mesylate (IM) significantly decreased CIP2A expression. CIP2A knockdown decreased BCR-ABL but increased SHP-1 expression, and CIP2A overexpression had the reverse effect. CIP2A is overexpressed in CML-CP patients, and its expression may promote CML pathogenesis. CIP2A and BCR-ABL can regulate each other in a positive feedback loop. CIP2A may be a useful therapeutic target in CML-CP, particularly in patients with IM resistance. How- ever, further studies are needed to validate the interaction between CIP2A and BCR-ABL using other tyrosine kinase inhibitors than IM.

The Capable ABL: What Is Its Biological Function?

The mammalian ABL1 gene encodes the ubiquitously expressed nonreceptor tyrosine kinase ABL. In response to growth factors, cytokines, cell adhesion, DNA damage, oxidative stress, and other signals, ABL is activated to stimulate cell proliferation or differentiation, survival or death, retraction, or migration. ABL also regulates specialized functions such as antigen receptor signaling in lymphocytes, synapse formation in neurons, and bacterial adhesion to intestinal epithelial cells. Although discovered as the proto-oncogene from which the Abelson leukemia virus derived its Gag-v-Abl oncogene, recent results have linked ABL kinase activation to neuronal degeneration. This body of knowledge on ABL seems confusing because it does not fit the one-gene-one-function paradigm. Without question, ABL capabilities are encoded by its gene sequence and that molecular blueprint designs this kinase to be regulated by subcellular location-dependent interactions with inhibitors and substrate activators. Furthermore, ABL shuttles between the nucleus and the cytoplasm where it binds DNA and actin--two biopolymers with fundamental roles in almost all biological processes. Taken together, the cumulated results from analyses of ABL structure-function, ABL mutant mouse phenotypes, and ABL substrates suggest that this tyrosine kinase does not have its own agenda but that, instead, it has evolved to serve a variety of tissue-specific and context-dependent biological functions.

Ebf1 and c-Myb Repress Rag Transcription Downstream of Stat5 during Early B Cell Development

The temporal control of RAG (Rag) expression in developing lymphocytes prevents DNA breaks during periods of proliferation that could threaten genomic integrity. In developing B cells, the IL-7R and precursor B cell Ag receptor (pre-BCR) synergize to induce proliferation and the repression of Rag at the protein and mRNA levels for a brief period following successful Ig H chain gene rearrangement. Whereas the mechanism of RAG2 protein downregulation is well defined, little is known about the pathways and transcription factors that mediate transcriptional repression of Rag. Using Abelson murine leukemia virus-transformed B cells to model this stage of development, we identified early B cell factor 1 (Ebf1) as a strong repressor of Rag transcription. Short hairpin RNA-mediated knockdown of either Ebf1 or its downstream target c-Myb was sufficient to induce Rag transcription in these highly proliferative cells. Ebf1 and c-Myb antagonize Rag transcription by negatively regulating the binding of Foxo1 to the Rag locus. Ebf1 accomplishes this through both direct negative regulation of Foxo1 expression and direct positive regulation of Gfi1b expression. Ebf1 expression is driven by the IL-7R downstream effector Stat5, providing a link between the negative regulation of Rag transcription by IL-7 and a novel repressive pathway involving Ebf1 and c-Myb.

Cesarean delivery in a parturient with chronic myeloid leukemia

To the Editor, Leukemia rarely occurs during the course of pregnancy, with a reported incidence of 1:75,000-1,000,000 pregnancies. Chronic leukemias account for only 10% of these cases, with the majority being chronic myeloid leukemia (CML). This disease is characterized by a reciprocal translocation between chromosomes 9 and 22 with the resultant fusion of the breakpoint cluster region (BCR) gene with the Abelson leukemia virus (ABL) gene – ‘‘Philadelphia chromosome’’; t(9;22)(q34;q11). We report a case of newly diagnosed CML in a patient’s third trimester. She had been treated for two weeks with hydroxyurea prior to lower uterine segment Cesarean delivery. At that time, the patient’s white blood cell count was 194.4 9 10 L with 5% myeloblasts in the peripheral blood. General endotracheal anesthesia was administered uneventfully, and a normal healthy baby was delivered uneventfully. Two weeks later, the patient was started on molecular targeted therapy. Both mother and child were doing well after two years of follow-up. One important consideration for choosing general anesthesia in this case was the potential for blast crisis (BC), a major challenge in the management of leukemia. Blast crisis is defined as the presence of a high blast count, 30% blasts cells, in the blood/marrow or, in a small proportion of patients, presence of extramedullary blastic infiltrates in the lymph nodes, skin, soft tissues, serosal surfaces, bone, and gastrointestinal and genitourinary tracts. Although rare, leukemic infiltration of meninges and brain, including nerves, can occur even while on therapy. Development of neurological involvement predicts a poorer prognosis with poorer response to treatment and a three-month median survival after the onset. Chronic myeloid leukemia is routinely diagnosed by an elevated white blood cell count with left shift, frequently with basophilia, and splenomegaly. The presence of BCRABL is confirmed with fluorescence in situ hybridization or with polymerase chain reaction (PCR). Bone marrow aspiration and cytogenetic analysis is mandatory for staging the disease as chronic phase (CP), accelerated, or blastic phase (BP). In comparison, all newly diagnosed patients with acute leukemia are routinely subjected to a spinal tap for prognostication. When circulating blast cells are present in the blood in acute lymphocytic leukemias, it is well documented that a traumatic lumbar puncture (TLP) may introduce leukemic blasts from the systemic circulation into the cerebrospinal fluid (CSF) with a resultant two to threefold higher risk of treatment failure. Accordingly, instituting intrathecal therapy early in the course is part of the treatment protocol in patients who have undergone more than one TLP in the presence of peripheral circulating blast cells. A diagnostic spinal tap is not part of the routine investigations in CML unless there is a clinical suspicion of involvement of the central nervous system. Reports of initiating a BC through a TLP for CML are lacking, and the incidence of diagnostic spinal punctures being performed in CML-CP is also not known. There is a lack of consensus on what constitutes TLP based on CSF rheology because there is no specific threshold for the number of red blood R. Datta, MD (&) Department of Anesthesiology and Critical Care, Army Hospital (Research & Referral) Delhi, Delhi, India e-mail: drrashmidatta@gmail.com