Reversal of the glycolytic phenotype of primary effusion lymphoma cells by combined targeting of cellular metabolism and PI3K/Akt/ mTOR signaling.

PEL is a B-cell non-Hodgkin lymphoma, occurring predominantly as a lymphomatous effusion in body cavities, characterized by aggressive clinical course, with no standard therapy. Based on previous reports that PEL cells display a Warburg phenotype, we hypothesized that the highly hypoxic environment in which they grow in vivo makes them more reliant on glycolysis, and more vulnerable to drugs targeting this pathway. We established here that indeed PEL cells in hypoxia are more sensitive to glycolysis inhibition. Furthermore, since PI3K/Akt/mTOR has been proposed as a drug target in PEL, we ascertained that pathway-specific inhibitors, namely the dual PI3K and mTOR inhibitor, PF-04691502, and the Akt inhibitor, Akti 1/2, display improved cytotoxicity to PEL cells in hypoxic conditions. Unexpectedly, we found that these drugs reduce lactate production/extracellular acidification rate, and, in combination with the glycolysis inhibitor 2-deoxyglucose (2-DG), they shift PEL cells metabolism from aerobic glycolysis towards oxidative respiration. Moreover, the associations possess strong synergistic cytotoxicity towards PEL cells, and thus may reduce adverse reaction in vivo, while displaying very low toxicity to normal lymphocytes. Finally, we showed that the association of 2-DG and PF-04691502 maintains its cytotoxic and proapoptotic effect also in PEL cells co-cultured with human primary mesothelial cells, a condition known to mimic the in vivo environment and to exert a protective and pro-survival action. All together, these results provide a compelling rationale for the clinical development of new therapies for the treatment of PEL, based on combined targeting of glycolytic metabolism and constitutively activated signaling pathways.

CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administration.

This study examined the pharmacologic characterization of CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide], a novel phosphodiesterase (PDE)4 inhibitor designed for treating pulmonary inflammatory diseases via inhaled administration. CHF6001 was 7- and 923-fold more potent than roflumilast and cilomilast, respectively, in inhibiting PDE4 enzymatic activity (IC50 = 0.026 ± 0.006 nM). CHF6001 inhibited PDE4 isoforms A-D with equal potency, showed an elevated ratio of high-affinity rolipram binding site versus low-affinity rolipram binding site (i.e., >40) and displayed >20,000-fold selectivity versus PDE4 compared with a panel of PDEs. CHF6001 effectively inhibited (subnanomolar IC50 values) the release of tumor necrosis factor-α from human peripheral blood mononuclear cells, human acute monocytic leukemia cell line macrophages (THP-1), and rodent macrophages (RAW264.7 and NR8383). Moreover, CHF6001 potently inhibited the activation of oxidative burst in neutrophils and eosinophils, neutrophil chemotaxis, and the release of interferon-γ from CD4(+) T cells. In all these functional assays, CHF6001 was more potent than previously described PDE4 inhibitors, including roflumilast, UK-500,001 [2-(3,4-difluorophenoxy)-5-fluoro-N-((1S,4S)-4-(2-hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide], and cilomilast, and it was comparable to GSK256066 [6-((3-(dimethylcarbamoyl)phenyl)sulfonyl)-4-((3-methoxyphenyl)amino)-8-methylquinoline-3-carboxamide]. When administered intratracheally to rats as a micronized dry powder, CHF6001 inhibited liposaccharide-induced pulmonary neutrophilia (ED50 = 0.205 µmol/kg) and leukocyte infiltration (ED50 = 0.188 µmol/kg) with an efficacy comparable to a high dose of budesonide (1 µmol/kg i.p.). In sum, CHF6001 has the potential to be an effective topical treatment of conditions associated with pulmonary inflammation, including asthma and chronic obstructive pulmonary disease.

Kaposi's sarcoma herpesvirus K15 protein contributes to virus-induced angiogenesis by recruiting PLCγ1 and activating NFAT1-dependent RCAN1 expression.

Kaposi's Sarcoma (KS), caused by Kaposi's Sarcoma Herpesvirus (KSHV), is a highly vascularised angiogenic tumor of endothelial cells, characterized by latently KSHV-infected spindle cells and a pronounced inflammatory infiltrate. Several KSHV proteins, including LANA-1 (ORF73), vCyclin (ORF72), vGPCR (ORF74), vIL6 (ORF-K2), vCCL-1 (ORF-K6), vCCL-2 (ORF-K4) and K1 have been shown to exert effects that can lead to the proliferation and atypical differentiation of endothelial cells and/or the secretion of cytokines with angiogenic and inflammatory properties (VEGF, bFGF, IL6, IL8, GROα, and TNFß). To investigate a role of the KSHV K15 protein in KSHV-mediated angiogenesis, we carried out a genome wide gene expression analysis on primary endothelial cells infected with KSHV wildtype (KSHVwt) and a KSHV K15 deletion mutant (KSHVΔK15). We found RCAN1/DSCR1 (Regulator of Calcineurin 1/Down Syndrome critical region 1), a cellular gene involved in angiogenesis, to be differentially expressed in KSHVwt- vs KSHVΔK15-infected cells. During physiological angiogenesis, expression of RCAN1 in endothelial cells is regulated by VEGF (vascular endothelial growth factor) through a pathway involving the activation of PLCγ1, Calcineurin and NFAT1. We found that K15 directly recruits PLCγ1, and thereby activates Calcineurin/NFAT1-dependent RCAN1 expression which results in the formation of angiogenic tubes. Primary endothelial cells infected with KSHVwt form angiogenic tubes upon activation of the lytic replication cycle. This effect is abrogated when K15 is deleted (KSHVΔK15) or silenced by an siRNA targeting the K15 expression. Our study establishes K15 as one of the KSHV proteins that contribute to KSHV-induced angiogenesis.

Anti-leukemic activity of dasatinib in both p53(wild-type) and p53(mutated) B malignant cells.

The multi-kinase inhibitor dasatinib induced a variable but significant decrease of viability in both p53(wild-type) (EHEB, JVM-2, JVM-3) and p53(mutated) (MEC-1, MEC-2, BJAB) prolymphocytic B leukemic cells, due to a combination of cell cycle block in G1 and apoptosis. Antibody phospho-kinase array analysis revealed that dasatinib inhibited the phosphorylation of various kinases, including ERK1/2 and p38/MAPK as well as of STAT3 transcription factors, in both p53(wild-type) and p53(mutated) cells. Therefore, dasatinib might offer a novel therapeutic strategy not only for p53(wild-type), but also for p53(mutated) B malignancies that have the worst prognosis and urgently need innovative therapeutic approaches.

Deletion of Kaposi's sarcoma-associated herpesvirus FLICE inhibitory protein, vFLIP, from the viral genome compromises the activation of STAT1-responsive cellular genes and spindle cell formation in endothelial cells.

Kaposi's sarcoma herpesvirus (KSHV) Fas-associated death domain (FADD)-like interleukin-1 beta-converting enzyme (FLICE)-inhibitory protein, vFLIP, has antiapoptotic properties, is a potent activator of the NF-κB pathway, and induces the formation of endothelial spindle cells, the hallmark of Kaposi's sarcoma, when overexpressed in primary endothelial cells. We used a reverse genetics approach to study several functions of KSHV vFLIP in the context of the whole viral genome. Deletion of the gene encoding vFLIP from a KSHV genome cloned in a bacterial artificial chromosome (BAC) reduced the ability of the virus to persist and induce spindle cell formation in primary human umbilical vein endothelial cells (HUVECs). Only a few, mainly interferon (IFN)-responsive, genes were expressed in wild-type KSHV (KSHV-wt)-infected endothelial cells at levels higher than those in KSHV-ΔFLIP-infected endothelial cells, in contrast to the plethora of cellular genes induced by overexpressed vFLIP. In keeping with this observation, vFLIP induces the phosphorylation of STAT1 and STAT2 in an NF-κB-dependent manner in endothelial cells. vFLIP-dependent phosphorylation of STAT1 and STAT2 could be demonstrated after endothelial cells were infected with KSHV-wt, KSHV-ΔFLIP, and a KSHV-vFLIP revertant virus. These findings document the impact of KSHV vFLIP on the transcriptome of primary endothelial cells during viral persistence and highlight the role of vFLIP in the activation of STAT1/STAT2 and STAT-responsive cellular genes by KSHV.

Nutlin-3 downregulates the expression of the oncogene TCL1 in primary B chronic lymphocytic leukemic cells.

PURPOSE: The oncogene TCL1 plays a key role in the development of B chronic lymphocytic leukemia (B-CLL), but it is not known whether TCL1 could be modulated by therapeutic approaches. EXPERIMENTAL DESIGN: B-CLL patient samples (n = 35) and B leukemic cell lines (EHEB, JVM2, JVM3, MEC1, MEC2, and BJAB) with different p53 status were exposed to Nutlin-3, a small-molecule inhibitor of the p53-MDM2 interaction. Modulations of the steady-state mRNA levels of TCL1 were analyzed by quantitative real-time PCR and Western blotting in both primary B-CLL samples and leukemic cell lines. In addition, transfection experiments with either p53 siRNA or with a TCL1 expression plasmid were carried out in the EHEB B-CLL cell line. RESULTS: Upon ex vivo treatment with Nutlin-3, TCL1 was significantly (P < 0.05) decreased in 23 of 28 B-CLL p53(wild-type). The functionality of the p53 pathway in the same leukemic cell samples was underscored by the concomitant ability of Nutlin-3 to significantly (P < 0.05) upregulate the p53 target gene MDM2 in the p53(wild-type) leukemic cells. The dependence of TCL1 downregulation by a functional p53 pathway was confirmed in a panel of B lymphoblastoid cell lines and by p53 knockdown experiments with p53 siRNA. The importance of TCL1 in promoting leukemic cell survival was underscored in transfection experiments, in which TCL1 overexpression significantly counteracted the Nutlin-3-mediated induction of apoptosis in EHEB. CONCLUSIONS: Our data indicate that the Nutlin-3 downregulates TCL1 mRNA and protein, which likely represents an important molecular determinant in the proapoptotic activity of Nutlin-3.

Molecular targets for selective killing of TRAIL-resistant leukemic cells.

INTRODUCTION: TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines, and shows promising therapeutic activity against solid tumors and lymphomas, in a variety of Phase I and II clinical trials. In contrast, primary leukemias have shown poor susceptibility to TRAIL-mediated cytotoxicity, suggesting the need for sensitizing TRAIL-resistant leukemic cells, by combining soluble recombinant TRAIL either with chemotherapeutic drugs, or with targeted small molecules. AREAS COVERED: This review discusses potential therapeutic applications of combinations able to restore the sensitivity of leukemic cells to either recombinant TRAIL or anti-TRAIL-receptor agonistic antibodies for the treatment of hematological malignancies. EXPERT OPINION: Up-to-date knowledge of the most innovative anti-leukemic therapies including functional screening of specific-sensitizers, enhancing TRAIL-mediated cytotoxicity. Strategies aimed to enhance TRAIL-mediated apoptosis, include the combination of novel sensitizers, functionally identified from libraries of pharmaceutically active, synthetic or naturally derived compounds. Other approaches aim to employ the administration of stem cells engineered to express TRAIL, in the leukemic stem cell niche, and promise to be a successful treatment with reduced specific toxicity.

Recent advances in the therapeutic perspectives of Nutlin-3.

Nutlin-3 is a small molecule inhibitor of the MDM2/p53 interaction, which leads to the non-genotoxic p53 stabilization, activation of cell cycle arrest and apoptosis pathways. A series of recent studies have strengthened the concept that selective, non-genotoxic p53 activation by Nutlin-3 might represent an alternative to the current cytotoxic chemotherapy, in particular for pediatric tumors and for hematological malignancies, which retain a high percentage of p53(wild-type) status at diagnosis. Like most other drugs employed in cancer therapy, it will be unlikely that Nutlin-3 will be used as a monotherapy. In this respect, Nutlin-3 shows a synergistic cytotoxic effect when used in combination with innovative drugs, such as TRAIL or bortozemib. Although Nutlin-3 is currently in phase I clinical trial for the treatment of retinoblastoma, its effects on normal tissues and cell types remain largely to be determined and will require further investigation in the future years.

miR-34a induces the downregulation of both E2F1 and B-Myb oncogenes in leukemic cells.

PURPOSE: To elucidate new molecular mechanisms able to downregulate the mRNA levels of key oncogenes, such as B-Myb and E2F1, in a therapeutic perspective. EXPERIMENTAL DESIGN: B-Myb and E2F1 mRNA levels were evaluated in primary B chronic lymphocytic leukemia (B-CLL, n = 10) and acute myeloid leukemia (AML, n = 5) patient cells, in a variety of p53(wild-type) and p53(mutated/deleted) leukemic cell lines, as well as in primary endothelial cells and fibroblasts. Knockdown experiments with siRNA for p53 and E2F1 and overexpression experiments with miR34a were conducted to elucidate the role of these pathways in promoting B-Myb downregulation. RESULTS: In vitro exposure to Nutlin-3, a nongenotoxic activator of p53, variably downregulated the expression of B-Myb in primary leukemic cells and in p53(wild-type) myeloid (OCI, MOLM) and lymphoblastoid (SKW6.4, EHEB) but not in p53(mutated) (NB4, BJAB, MAVER) or p53(deleted) (HL-60) leukemic cell lines. The transcriptional repression of B-Myb was also observed in primary normal endothelial cells and fibroblasts. B-Myb downregulation played a critical role in the cell-cycle block in G(1) phase induced by Nutlin-3, as shown by transfection experiments with specific siRNA. Moreover, we have provided experimental evidence suggesting that miR-34a is a central mediator in the repression of B-Myb both directly and through E2F1. CONCLUSIONS: Owing to the role of B-Myb and E2F1 transcription factors in controlling cell-cycle progression of leukemic cells, the downregulation of these oncogenes by miR-34a suggests the usefulness of therapeutic approaches aimed to modulate the levels of miR-34a.

Dasatinib plus Nutlin-3 shows synergistic antileukemic activity in both p53 wild-type and p53 mutated B chronic lymphocytic leukemias by inhibiting the Akt pathway.

PURPOSE: To analyze the effect of the combination of Dasatinib, a multikinase inhibitor, plus Nutlin-3, a nongenotoxic activator of the p53 pathway, in primary B chronic lymphocytic leukemia (B-CLL) patient samples and B leukemic cell line models. EXPERIMENTAL DESIGN: The induction of cytotoxicity was evaluated in both primary B-CLL cell samples (n = 20) and in p53(wild-type) (EHEB, JVM-2) and p53(deleted/mutated) (MEC-2, BJAB) B leukemic cell lines. The role of Akt in modulating leukemic cell survival/apoptosis in response to Dasatinib or Dasatinib + Nutlin-3 was documented by functional experiments carried out using specific pharmacological inhibitors and by overexpression of membrane-targeted constitutively active form of Akt. RESULTS: The combination of Dasatinib + Nutlin-3 exhibited a synergistic cytotoxicity in the majority (19 out of 20) of B-CLL samples, including patients carrying 17p- (n = 4), and in both p53(wild-type) and p53(deleted/mutated) B leukemic cell lines. At the molecular level, Dasatinib significantly counteracted the Nutlin-3-mediated induction of the p53 transcriptional targets MDM2 and p21 observed in p53(wild-type) leukemic cells. Conversely, Nutlin-3 did not interfere with the ability of Dasatinib to decrease the phosphorylation levels of ERK1/2, p38/MAPK, and Akt in both p53(wild-type) and p53(deleted/mutated) B leukemic cell lines. A critical role of Akt downregulation in mediating the antileukemic activity of Dasatinib and Dasatinib + Nutlin-3 was demonstrated in experiments carried out by specifically modulating the Akt pathway. CONCLUSIONS: These findings suggest that Dasatinib + Nutlin-3 might represent an innovative therapeutic combination for both p53(wild-type) and p53(deleted/mutated) B-CLL.

Human bone marrow mesenchymal stem cells display anti-cancer activity in SCID mice bearing disseminated non-Hodgkin's lymphoma xenografts.

BACKGROUND: Although multimodality treatment can induce high rate of remission in many subtypes of non-Hodgkin's lymphoma (NHL), significant proportions of patients relapse with incurable disease. The effect of human bone marrow (BM) mesenchymal stem cells (MSC) on tumor cell growth is controversial, and no specific information is available on the effect of BM-MSC on NHL. METHODOLOGY/PRINCIPAL FINDINGS: The effect of BM-MSC was analyzed in two in vivo models of disseminated non-Hodgkin's lymphomas with an indolent (EBV(-) Burkitt-type BJAB, median survival = 46 days) and an aggressive (EBV(+) B lymphoblastoid SKW6.4, median survival = 27 days) behavior in nude-SCID mice. Intra-peritoneal (i.p.) injection of MSC (4 days after i.p. injection of lymphoma cells) significantly increased the overall survival at an optimal MSC:lymphoma ratio of 1:10 in both xenograft models (BJAB+MSC, median survival = 58.5 days; SKW6.4+MSC, median survival = 40 days). Upon MSC injection, i.p. tumor masses developed more slowly and, at the histopathological observation, exhibited a massive stromal infiltration coupled to extensive intra-tumor necrosis. In in vitro experiments, we found that: i) MSC/lymphoma co-cultures modestly affected lymphoma cell survival and were characterized by increased release of pro-angiogenic cytokines with respect to the MSC, or lymphoma, cultures; ii) MSC induce the migration of endothelial cells in transwell assays, but promoted endothelial cell apoptosis in direct MSC/endothelial cell co-cultures. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that BM-MSC exhibit anti-lymphoma activity in two distinct xenograft SCID mouse models of disseminated NHL.

Baseline serum concentrations of TRAIL in early rheumatoid arthritis: relationship with response to disease-modifying antirheumatic drugs.

OBJECTIVE: To assess the relationship between serum concentrations of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and osteoprotegerin (OPG) and the therapeutic response to disease-modifying antirheumatic drugs (DMARD) in patients with early rheumatoid arthritis (RA). METHODS: Circulating levels of TRAIL and its soluble receptor OPG were measured by ELISA in paired serum samples obtained from 66 patients with early RA at their first visit (baseline) and after 1 year of therapy. Levels of TRAIL and OPG were analyzed in relation to the clinical response, defined by the 28-joint count Disease Activity Score (DAS28). RESULTS: Both serum TRAIL and OPG increased after DMARD therapy. Baseline levels of TRAIL, but not OPG, were significantly higher (p < 0.05) in the patients that achieved a clinical response by DAS28 after 1 year of therapy, versus patients without clinical response to DMARD. Baseline serum levels of TRAIL were higher (p < 0.01) in rheumatoid factor-negative patients. CONCLUSION: Our data suggest that the basal level of circulating TRAIL is an important determinant in the therapeutic response to DMARD in patients with early RA.

Changes in T-cell responses against human herpesvirus-8 correlate with the disease course of iatrogenic Kaposi's sarcoma in a patient with undifferentiated arthritis.

OBJECTIVES: To describe the first in-depth analysis of both the T-cell responses against human herpesvirus-8 (HHV-8) and the HHV-8 viral load in 1 patient who developed iatrogenic HHV-8-associated-Kaposi's sarcoma (KS) following immunosuppressive treatment for undifferentiated arthritis and to review the literature on iatrogenic KS (IKS). METHODS: T-cell responses against HHV-8 lytic and latent antigens were analyzed by ex vivo enzyme-linked immunospot (Elispot) and HHV-8 viral load was assessed by quantitative polymerase chain reaction, in sequential peripheral blood samples from a 55-year-old woman who developed skin/mucosal and visceral KS, while receiving treatment with cyclosporine, methotrexate, and methylprednisolone for undifferentiated arthritis. RESULTS: KS may result from HHV-8 infection in patients undergoing immunosuppressive treatment for rheumatic diseases and this is the first case of IKS occurring in undifferentiated arthritis. A role for immune surveillance in the pathogenesis of IKS is supported by the observation of disease regression following discontinuation of immunosuppressive therapy. In a 4-year follow-up, we showed that variations of the virus-specific immune responses but not of the viral load correlated well with the disease course, characterized by 2 remission and subsequent relapse phases, following changes of immunosuppressive therapy. CONCLUSIONS: We have provided evidence of a clear-cut correlation between changes in immunologic markers of HHV-8 infection and the disease course of this viral associated tumor, concomitant with variations of immunosuppressive treatment. Thus, ex vivo enzyme-linked immunospot for HHV-8-specific T-cell responses represents a new tool for the clinical management of rheumatic patients with IKS.

Changes in the immune responses against human herpesvirus-8 in the disease course of posttransplant Kaposi sarcoma.

In nine patients with posttransplant Kaposi sarcoma (KS) T-cell responses to human herpesvirus (HHV)-8 latent and lytic antigens, as detected by enzyme-linked-immunospot (Elispot) assay, were absent at disease onset. Virus-specific T-cell responses were detected in six renal recipients at remission after a reduction of calcineurin inhibitors (CIs), and in two HHV-8 seropositive renal recipients without KS. In two liver recipients undergoing switch from CIs to sirolimus (SRL), normalization of the T-cell repertoire and recovery of both HHV-8-specific effector and memory T lymphocytes were associated with complete KS remission. In a renal recipient undergoing SRL conversion, the early recovery of HHV-8-specific effector but not of memory T lymphocytes, was associated only with partial remission. Neither rejection nor changes in graft function were observed after SRL conversion. HHV-8-specific T-cell responses are required to achieve posttransplant KS remission, and may be restored under SRL, while maintaining effective immunosuppression.

B cells and herpesviruses: a model of lymphoproliferation.

Unlike alpha- and beta-herpesviruses, human gamma-herpesviruses, including the Epstein-Barr virus (EBV) and the human herpesvirus-8 (HHV-8), are B lymphotropic viruses. Primary infection with EBV, in otherwise healthy subjects, causes a benign lymphoproliferative syndrome, the mononucleosis syndrome. However, several epidemiologic and biologic studies have shown a pathogenetic role of EBV in the development of human B cell lymphomas, both in immunocompetent and in immunosuppressed individuals. HHV-8 is the necessary etiologic agent of a lymph vascular tumor, the Kaposi sarcoma, but it is also implicated in the pathogenesis of rare B cell lymphoproliferative disorders, mainly occurring in the setting of immunosuppression. The aim of this review is to provide an updated description of the different strategies used by these two herpesviruses to influence B cell fate decisions. Both EBV and HHV-8 have evolved specific mechanisms in order to: (1) interact with the B cell developmental machinery; (2) allow infected B cells to escape from the control of the immune system; (3) affect the B cell cycle checkpoints; (4) mimic and influence B cellular proliferation and differentiation pathways. Understanding the mechanisms of herpesvirus induced B cell lymphoproliferation will provide the basis for novel treatment approaches in patients with EBV and HHV-8 related lymphomas.