VLX1570 induces apoptosis through the generation of ROS and induction of ER stress on leukemia cell lines.

A novel proteasome deubiquitinase inhibitor, VLX1570, has been highlighted as a promising therapeutic agent mainly for lymphoid neoplasms and solid tumors. We examined in vitro effects of VLX1570 on eight myeloid and three lymphoid leukemia cell lines. From cell culture studies, 10 out of 11 cell lines except K562 were found to be susceptible to VLX1570 treatment and it inhibited cell growth mainly by apoptosis. Next, to identify the signaling pathways associated with apoptosis, we performed gene expression profiling using HL-60 with or without 50 nmol/L of VLX1570 for 3 hours and demonstrated that VLX1570 induced the genetic pathway involved in "heat shock transcription factor 1 (HSF1) activation", "HSF1 dependent transactivation", and "Regulation of HSF1 mediated heat shock response". VLX1570 increased the amount of high molecular weight polyubiquitinated proteins and the expression of HSP70 as the result of the suppression of ubiquitin proteasome system, the expression of heme oxygenase-1, and the amount of phosphorylation in JNK and p38 associated with the generation of reactive oxygen species (ROS) induced apoptosis and the amount of phosphorylation in eIF2α, inducing the expression of ATF4 and endoplasmic reticulum (ER) stress dependent apoptosis protein, CHOP, and the amount of phosphorylation slightly in IRE1α, leading to increased expression of XBP-1s in leukemia cell lines. In the present study, we demonstrate that VLX1570 induces apoptosis and exerts a potential anti-leukemic effect through the generation of ROS and induction of ER stress in leukemia cell lines.

Cytotoxic activity of standardized extracts, a fraction, and individual secondary metabolites from fenugreek seeds against SKOV-3, HeLa and MOLT-4 cell lines.

CONTEXT: Trigonella foenum-graecum L. (Fabaceae) has many therapeutic properties and anticancer potential. OBJECTIVE: The cytotoxic activities of standardized extracts and a fraction from fenugreek seeds and their compounds (sapogenins, flavone C-glycosides, alkaloid trigonelline) against human cancer SKOV-3, HeLa and MOLT-4 cells were evaluated. MATERIALS AND METHODS: Fenugreek seeds were extracted with 70% methanol (A) or water (B). Furthermore, the seeds were purified with petroleum ether and chloroform and next extracted with methanol to obtain fraction (C). The quantitative analysis of saponins and flavonoids in the extracts was done with HPLC methods. The extracts (5-120 µg/mL) and compounds (1-50 µg/mL) were tested on the cells by MTT assay and RTCA system. The effect of a fraction on ROS production, mitochondrial membrane potential and caspase-3/7 activity in HeLa and SKOV-3 cells was also evaluated by flow cytometry. RESULTS: The strongest cytotoxic activity on cancer cells showed the fraction C (IC50 was 3.91 ± 0.03 for HeLa, 3.97 ± 0.07 for SKOV-3, and 7.75 ± 0.37 for MOLT-4) with the highest content of steroidal saponins (163.18 ± 11.03 µg/mg) and flavone C-glycosides (820.18 ± 0.05 µg/mg). The fraction significantly increased ROS production (up to four times higher than in keratinocytes as control) and caspases activity in the cells. The examined flavonoids did not exhibit the cytotoxic activity in contrast to yamogenin, tigogenin, and diosgenin. CONCLUSIONS: The obtained results complement the data on the cytotoxic activity of Foenugraeci Semen and synergistic effect of flavonoids and saponins complex contained in the plant.

Apoptosis induced by synthetic compounds containing a 3,4,5-trimethoxyphenyl fragment against lymphoid immature neoplasms.

T-cell acute lymphoblastic leukemia is an aggressive hematological malignancy originating from the malignant transformation of progenitor T cells at different stages of development. The treatment causes severe adverse effects and is associated with relapses and high morbidity and mortality rates. The present study aimed to evaluate the cytotoxic activity of 28 new compounds containing 3,4,5-trimethoxyphenyl analogues on hematological neoplastic cells lines. Cytotoxicity screening by the MTT method revealed that compound 1d was the most promising. Cell viability of neoplastic cells decreased in a concentration- and time-dependent manner, with compound 1d not causing hemolysis or reducing peripheral blood mononuclear cells viability, suggesting a selective cytotoxicity. We also suggested that compound 1d induced apoptotic-like cell death with mitochondrial involvement in Jurkat cells.

Protein ubiquitination in lymphoid malignancies.

Human lymphoid malignancies inherit gene expression networks from their normal B-cell counterpart and co-opt them for their own oncogenic purpose, which is usually governed by transcription factors and signaling pathways. These transcription factors and signaling pathways are precisely regulated at multiple steps, including ubiquitin modification. Protein ubiqutination plays a role in almost all cellular events and in many human diseases. In the past few years, multiple studies have expanded the role of ubiquitination in the genesis of diverse lymphoid malignancies. Here, we discuss our current understanding of both proteolytic and non-proteolytic functions of the protein ubiquitination system and describe how it is involved in the pathogenesis of human lymphoid cancers. Lymphoid-restricted ubiquitination mechanisms, including ubiquitin E3 ligases and deubiquitinating enzymes, provide great opportunities for the development of targeted therapies for lymphoid cancers.

PRES in the course of hemato-oncological treatment in children.

INTRODUCTION: Posterior reversible leukoencephalopathy syndrome (PRES) is a clinical syndrome of varying aetiologies, characterised by acute neurological symptoms of brain dysfunction with MRI abnormalities in posterior cerebral white and grey matter. In most cases, symptoms resolve without neurological consequences. AIM: The aim of this paper is the analysis of predisposing factors, clinical outcomes and radiological features of PRES in eight children with hemato-oncological disease. MATERIAL AND METHODS: We analysed the medical records of eight hemato-oncological patients aged from 3.0 to 16.1 years. The mean of age at primary diagnosis was 8.5 years. RESULTS: All patients had both clinical and radiological PRES features. Seven out of eight underwent intensive chemotherapy regimens. Time elapsed from start of treatment to the occurrence of PRES ranged from 6 to 556 days. In one case, PRES occurred before chemotherapy and was the first symptom of cancer. Most (six of eight) patients had history of hypertension (> 95pc) and some (two of eight) occurred electrolyte imbalance-mainly hypomagnesaemia. Patients presented headache (seven of eight), disturbances of consciousness (six of eight), seizures (six of eight), visual changes (four of eight) and vomiting (three of eight). MRI demonstrated abnormalities in seven children: typical cerebral oedema in the white matter of the occipital to the parietal lobes. Most patient lesions in the MRI shrunk after 4 weeks, and clinical symptoms of PRES disappeared completely within a few hours to few days. CONCLUSION: PRES may complicate oncological treatment in children. Hypertension is the most important risk factor of PRES. PRES should be included in differential diagnosis in all patients with acute neurological symptoms.

Incidence of Myelofibrosis in Chronic Myeloid Leukemia, Multiple Myeloma, and Chronic Lymphoid Leukemia during Various Phases of Diseases.

Pathomorphological study of trephinobiopsy specimens from 129 patients with lymphoproliferative and myeloproliferative diseases was carried out over the course of chemotherapy. Combinations of initial and manifest myelofibrosis (loose network of reticulin fibers and extensive network of reticulin and collagen fibers, respectively) predominated at the debut of chronic myeloid leukemia, chronic lymphoid leukemia, and multiple myeloma. Manifest myelofibrosis was detected in patients with chronic myeloid leukemia without hematological response (failure of normalization of hematological values) and in patients with progressing and relapsing multiple myeloma. Combinations of foci of initial and manifest myelofibrosis were most incident in patients with progressing and relapsing chronic lymphoid leukemia. The incidence of myelofibrosis was higher in patients with multiple myeloma and chronic lymphoid leukemia progression and relapses and in patients with chronic myeloid leukemia without hematological response than at the disease debut and in case of response to chemotherapy. The response to chemotherapy in patients with chronic myeloid leukemia and chronic lymphoid leukemia was associated with a decrease in the incidence of myelofibrosis. In patients with multiple myeloma responding to chemotherapy, the incidence of myelofibrosis did not change in comparison with the disease debut.

Pathogenetic importance and therapeutic implications of NF-κB in lymphoid malignancies.

Derangement of the nuclear factor κB (NF-κB) pathway initiates and/or sustains many types of human cancer. B-cell malignancies are particularly affected by oncogenic mutations, translocations, and copy number alterations affecting key components the NF-κB pathway, most likely owing to the pervasive role of this pathway in normal B cells. These genetic aberrations cause tumors to be 'addicted' to NF-κB, which can be exploited therapeutically. Since each subtype of lymphoid cancer utilizes different mechanisms to activate NF-κB, several different therapeutic strategies are needed to address this pathogenetic heterogeneity. Fortunately, a number of drugs that block signaling cascades leading to NF-κB are in early phase clinical trials, several of which are already showing activity in lymphoid malignancies.

The long journey of TCL1 transgenic mice: lessons learned in the last 15 years.

The first transgenic mouse of the TCL1 oncogene was described more than 15 years ago, and since then, the overexpression of the gene in T- and B-cells in vivo has been extensively studied to reveal the molecular details in the pathogenesis of some lymphocytic leukemias. This review discusses the main features of the original TCL1 models and the different lines of research successively developed with particular attention to genetically compound mice and the therapeutic applications in drug development.

Targeting CXCR4 with cell-penetrating pepducins in lymphoma and lymphocytic leukemia.

The chemokine receptor CXCR4, which normally regulates stromal stem cell interactions in the bone marrow, is highly expressed on a variety of malignant hematologic cells, including lymphoma and lymphocytic leukemias. A new treatment concept has arisen wherein CXCR4 may be an effective therapeutic target as an adjunct to treatment of hematologic neoplasms with chemo- and immunotherapy. In the present study, we developed pepducins, cell-penetrating lipopeptide antagonists of CXCR4, to interdict CXCL12-CXCR4 transmembrane signaling to intracellular G-proteins. We demonstrate that pepducins targeting the first (i1) or third (i3) intracellular loops of CXCR4 completely abrogate CXCL12-mediated cell migration of lymphocytic leukemias and lymphomas. Stromal-cell coculture protects lymphoma cells from apoptosis in response to treatment with the CD20-targeted Ab rituximab. However, combination treatment with CXCR4 pepducins and rituximab significantly increases the apoptotic effect of rituximab. Furthermore, treatment of mice bearing disseminated lymphoma xenografts with pepducins alone or in combination with rituximab significantly increased their survival. These data demonstrate that CXCL12-CXCR4 signaling can be effectively inhibited by cell-penetrating pepducins, which represents a potential new treatment strategy for lymphoid malignancies.

Modulating the structure of phenylalanine-incorporated ascidiacyclamide through fluorination.

We designed four fluorinated Phe-incorporated ascidiacyclamide ([Phe]ASC) analogs, (cyclo(-Xxx1-oxazoline2-D-Val3-thiazole4-Ile5-oxazoline6-D-Val7-thiazole8-)), [(4-F)Phe]ASC (Xxx1: 4-fluorophenylalanine), [(3,5-F2)Phe]ASC (Xxx1: 3,5-difluorophenylalanine), [(3,4,5-F3)Phe]ASC (Xxx1: 3,4,5-trifluorophenylalanine) and [(F5)Phe]ASC (Xxx1: pentafluorophenylalanine), to modulate the π-electron density of the aromatic ring of the Phe residue. X-ray diffraction analysis, ¹H NMR and CD spectra all suggested that the interactions between the benzene ring of the Xxx1 residue and the alkyl groups of oxazoline2 contribute to the stability of the folded structure of these analogs. Substituting fluorines for the hydrogens progressively weakened those interactions through reducing the π-electron density, thereby mediating transformation from the folded to square structure. As a result, [(F5)Phe]ASC preferred the square form more than the other analogs did. Also contributing to the preference for the square form may be the hindrance of the rotation around the Cα-Cß bond by the two ortho-fluoro substituents of [(F5)Phe]ASC. These findings demonstrate that the structure of ASC can be modulated by using fluorine as an electron-withdrawing group.

Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy.

Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase that participates in numerous signalling pathways involved in diverse physiological processes. Several of these pathways are implicated in disease pathogenesis, which has prompted efforts to develop GSK3-specific inhibitors for therapeutic applications. However, before now, there has been no strong rationale for targeting GSK3 in malignancies. Here we report pharmacological, physiological and genetic studies that demonstrate an oncogenic requirement for GSK3 in the maintenance of a specific subtype of poor prognosis human leukaemia, genetically defined by mutations of the MLL proto-oncogene. In contrast to its previously characterized roles in suppression of neoplasia-associated signalling pathways, GSK3 paradoxically supports MLL leukaemia cell proliferation and transformation by a mechanism that ultimately involves destabilization of the cyclin-dependent kinase inhibitor p27(Kip1). Inhibition of GSK3 in a preclinical murine model of MLL leukaemia provides promising evidence of efficacy and earmarks GSK3 as a candidate cancer drug target.

Prescriptions for a toxic combination: use of methotrexate plus trimethoprim-sulfamethoxazole in the United States.

OBJECTIVES: To determine the frequency of using the potentially toxic combination of methotrexate and trimethoprim-sulfamethoxazole (TMP-SMX) in outpatient practice in the United States. METHODS: Data from the National Ambulatory Medical Care Survey for 1993-2010 were used to assess the frequency of using methotrexate with TMP-SMX and associated physician specialties and diagnoses. RESULTS: TMP-SMX was coprescribed in 22,000 methotrexate visits per year (1.0% of methotrexate visits). Pediatricians prescribed the combination most frequently, and the most common diagnosis was acute lymphoblastic leukemia. There was no significant change over time in coprescription of TMP-SMX with methotrexate (P = 0.4). CONCLUSIONS: Low-dose TMP-SMX with methotrexate chemotherapy appears to be standard for patients with acute lymphoblastic leukemia; however, other uses appear questionable, and clinicians should be cognizant of the risk for fatal interactions, especially when medications are prescribed by multiple providers.

Evaluation of selected honey and one of its phenolic constituent eugenol against L1210 lymphoid leukemia.

People affected with leukemia are on the rise and several strategies were employed to thwart this deadly disease. Recent decade of research focuses on phenolic constituents as a tool for combating various inflammatory, cancer, and cardiac diseases. Our research showed honey and its phenolic constituents as crusaders against cancer. In this work, we explored the antileukemic activity of selected honey and one of its phenolic constituent eugenol against L1210 leukemia animal model. Results of this experiment showed that the selected honey samples as well as eugenol after intraperitoneal injection could not increase the median survival time (MST) of animals. Further, there was only slight marginal increase in the %T/C values of honey and eugenol treated groups. The number of phenolics present in the honey may not be a prime factor to promote antileukemic effect since there was no difference in the MST of two different honeys tested. This study limits the use of selected honey and eugenol against leukemia animal model.

[Molecular targeted therapy in lymphoid leukemias].

Recent advances in the treatment of lymphoid leukemias have incorporated molecular targeted drugs (CD20-targeting rituximab and BCR-ABL tyrosine kinase inhibitors) into the traditional chemotherapeutic agents. This article reviews novel molecular targeted therapies for patients with lymphoid leukemias including acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairly cell leukemia and HTLV-I-related adult T-cell leukemia. Investigational agents that will be discussed in this review include inotuzumab, blinatumomab, alemtuzumab, ofatumumab, ibrutinib, idelalisib, bafetinib, lenalidomide, ABT-199 and mogamulizumab. Novel approaches warrant continued research to improve outcomes for patients with lymphoid leukemias.

Structural basis for WDR5 interaction (Win) motif recognition in human SET1 family histone methyltransferases.

Translocations and amplifications of the mixed lineage leukemia-1 (MLL1) gene are associated with aggressive myeloid and lymphocytic leukemias in humans. MLL1 is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for transcription of genes involved in hematopoiesis and development. MLL1 associates with a subcomplex containing WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which together form the MLL1 core complex that is required for sequential mono- and dimethylation of H3K4. We previously demonstrated that WDR5 binds the conserved WDR5 interaction (Win) motif of MLL1 in vitro, an interaction that is required for the H3K4 dimethylation activity of the MLL1 core complex. In this investigation, we demonstrate that arginine 3765 of the MLL1 Win motif is required to co-immunoprecipitate WRAD from mammalian cells, suggesting that the WDR5-Win motif interaction is important for the assembly of the MLL1 core complex in vivo. We also demonstrate that peptides that mimic SET1 family Win motif sequences inhibit H3K4 dimethylation by the MLL1 core complex with varying degrees of efficiency. To understand the structural basis for these differences, we determined structures of WDR5 bound to six different naturally occurring Win motif sequences at resolutions ranging from 1.9 to 1.2 Å. Our results reveal that binding energy differences result from interactions between non-conserved residues C-terminal to the Win motif and to a lesser extent from subtle variation of residues within the Win motif. These results highlight a new class of methylation inhibitors that may be useful for the treatment of MLL1-related malignancies.

Antileukemic effects of AMPK activators on BCR-ABL-expressing cells.

The mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in growth and survival of BCR-ABL transformed cells. AMPK kinase is a metabolic sensor that exhibits suppressive effects on the mTOR pathway and negatively regulates mTOR activity. We report that AMPK activators, such as metformin and 5-aminoimidazole-4-carboxamide ribonucleotide, suppress activation of the mTOR pathway in BCR-ABL-expressing cells. Treatment with these inhibitors results in potent suppression of chronic myeloid leukemia leukemic precursors and Ph(+) acute lymphoblastic leukemia cells, including cells expressing the T315I-BCR-ABL mutation. Altogether, our data suggest that AMPK is an attractive target for the treatment of BCR-ABL-expressing malignancies and raise the potential for use of AMPK activators in the treatment of refractory chronic myeloid leukemia and Ph(+) acute lymphoblastic leukemia.

Late-onset Pneumocystis jirovecii pneumonia post-fludarabine, cyclophosphamide and rituximab: implications for prophylaxis.

OBJECTIVE: Fludarabine, cyclophosphamide and rituximab (FCR) therapy for lymphoid malignancies has historically been associated with a low reported incidence of Pneumocystis jirovecii pneumonia (PJP). However, prophylaxis was routinely used in early studies, and molecular diagnostic tools were not employed. The objective of this study was to review the incidence of PJP during and post-FCR in the era of highly sensitive molecular diagnostics and (18) F-fluorodeoxyglucose (FDG) positron emission tomography (PET)-computerised tomography (CT). METHODS: All patients treated with standard FCR at the Peter MacCallum Cancer Centre (March 2009 to June 2012) were identified from a medications management database. Laboratory-confirmed PJP cases during this time were identified from an electronic database. RESULTS: Overall, 66 patients were treated with a median of 5.5 FCR cycles. Eight PJP cases were identified, 6 of whom had received chemotherapy prior to FCR. In 5 cases, (18) F-FDG PET demonstrated bilateral ground-glass infiltrates. Median CD4(+) lymphocyte counts at time of PJP diagnosis and 9-12 months following FCR were 123 and 400 cells/µL, respectively. In patients receiving no prophylaxis, 9.1% developed PJP during FCR. The rate following FCR was 18.4%, with median onset at 6 months (2.4-24 months). CONCLUSION: Given the high rate of late-onset PJP, consideration should be given for extended PJP prophylaxis for up to 12 months post-FCR, particularly in pretreated patients. Further evaluation of the role of CD4(+) monitoring is warranted to quantify risk of disease development and to guide duration of prophylaxis.

Clinical experience of granulocyte transfusion in the management of neutropenic patients with haematological malignancies and severe infection.

BACKGROUND: Prolonged chemotherapy-induced neutropenia is a major risk factor for the development of severe bacterial and fungal infections. Infectious manifestations may progress despite adequate anti-infectious treatment and lead to a very high short-term mortality. Granulocyte transfusion (GT) therapy is often considered. However, its efficacy is not well documented. METHODS: We retrospectively analyzed the clinical characteristics and outcome of a cohort of patients with haematological malignancies receiving GT during neutropenia and severe infection. RESULTS: A total of 30 patients with a median age of 46 y (range 3-82 y) who had received 1 or more GT were included. Acute leukaemia (80%) and non-Hodgkin lymphoma (17%) predominated as the underlying malignancy. All patients had severe and prolonged (median 16 days) neutropenia. The major indications for GT were persistent fever and clinical deterioration despite broad anti-infectious therapy, in combination with progressive pneumonia (n = 16), neutropenic enterocolitis (n = 6), and soft tissue infections (n = 3). GTs were given for a median of 3 transfusions (range 1-14). The median time to fever defervescence after GT was 14 days (range 6-33 days). For 11 patients, the resolution of fever and all signs of infection could directly be related to GT, and 3 of these patients became long-term survivors. Mortality at 30 days post-GT was 40% and at 6 months post-GT was 72%. GT was well tolerated. CONCLUSIONS: A substantial proportion of severely ill neutropenic patients appeared to benefit from GT. The results further underline the need for well- designed, randomized, prospective trials to determine the efficacy of this intervention in patients with life-threatening infectious complications.

Spontaneous and mitogen-induced cytokine production in lymphoproliferative diseases.

The levels of spontaneous and mitogen-induced production of pro- and anti-inflammatory cytokines were studied in patients with chronic lymphoid leukemia, non-Hodgkin's lymphocytic lymphomas, and multiple myeloma during the course of chemotherapy. Cytokine concentrations varied within a great range and did not conform to the normal distribution law. The levels of granulocyte and granulocyte-macrophage CSF were high during the debut, progress, and remission of the lymphoproliferative diseases. Imbalance of a wide spectrum of pro- and anti-inflammatory cytokines was observed during the debut and progress of the lymphoproliferative diseases, more often in chronic lymphoid leukemia and non-Hodgkin's lymphocytic lymphomas than in multiple myeloma.

Efficacy and Safety of Blinatumomab for the Treatment of Relapsed/Refractory Acute Lymphoblastic Leukemia: A Systemic Review and Meta-Analysis.

OBJECTIVE: The aim was to evaluate the efficacy and safety of blinatumomab monotherapy for the treatment of relapsed/refractory acute lymphoblastic leukemia (R/R B-ALL). METHODS: PubMed, Embase, Web of Science, and Cochrane Library were searched to collect clinical studies related to blinatumomab. The primary outcome measures were complete remission (CR), and minimal residual disease (MRD) response. Prognostic indicators included overall survival (OS) and relapse-free survival time (RFS). Grade ≥3 adverse reactions were mainly analyzed for safety, including cytokine release syndrome (CRS), neurological events and hematological toxicity. The heterogeneity was quantified by I2 statistic, which reflected the proportion of the true heterogeneity to the variance of the total effect size. Studies were considered heterogeneous if the I2 statistic was greater than 50%, and conversely, studies were homogeneous. RESULTS: A total of 18 studies involving 1,373 patients were included. The analysis results showed a CR rate of 54% (95%CI:44%-64%) and an MRD response rate of 43% (95%CI:34%-51%). The CR rate was higher in patients with bone marrow (BM) blast <50% than in patients with BM blast ≥50% (71% vs. 34%). The median OS and RFS were 8.16 months (95%CI:6.64-9.69) and 6.02 months (95%CI:4.63-7.41), respectively. For safety analysis, the incidence of grade ≥3 adverse events (AEs) was 80% (95%CI:72%-88%), the incidence of grade ≥3 neurological toxicity was 7% (95%CI:4%-11%), and the incidence of grade ≥3 CRS was 3% (95%CI:2%-5%). However, the mixture of retrospective and prospective studies led to heterogeneity to some extent in this meta-analysis. CONCLUSION: Blinatumomab is effective in the treatment of R/R B-ALL with a controlled occurrence of AEs and a reliable safety profile.