Georgios V. Georgakis

Activity of selective fully human agonistic antibodies to the TRAIL death receptors TRAIL-R1 and TRAIL-R2 in primary and cultured lymphoma cells: Induction of apoptosis and enhancement of doxorubicin- and bortezomib-induced cell death

Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL/Apo2L) is a death protein that preferentially kills tumour cells while sparing normal cells. TRAIL has four exclusive receptors, two of which (TRAIL‐R1, TRAIL‐R2) are death receptors. Both TRAIL/Apo2L and agonistic antibodies to the TRAIL death receptors are currently being explored for cancer therapy. Although the activity of TRAIL/Apo2L in a variety of haematological malignancies has been examined, the activity of anti‐TRAIL receptor agonistic antibodies in primary and cultured lymphoma cells has not. Using two fully human selective agonistic monoclonal antibodies to the TRAIL death receptors TRAIL‐R1 (HGS‐ETR1) and TRAIL‐R2 (HGS‐ETR2) this study demonstrated that both monoclonal antibodies activated caspase‐8 and induced cell death in five of nine human lymphoma cell lines, and induced >10% cell death in 67% and 70%, respectively, of 27 primary lymphoma cells, and >20% cell death in at least one‐thirds of the samples. HGS‐ETR1 and HGS‐ETR2 demonstrated comparable activity in the fresh tumour samples, which was independent of TRAIL receptor surface expression, Bax, cFLIP, or procaspase‐8 expression, or exposure to prior therapy. Furthermore, both antibodies enhanced the killing effect of doxorubicin and bortezomib. Our data demonstrate that HGS‐ETR1 and HGS‐ETR2 monoclonal antibodies can induce cell death in a variety of cultured and primary lymphoma cells, and may have therapeutic value in lymphoma.

Vorinostat inhibits STAT6-mediated TH2 cytokine and TARC production and induces cell death in Hodgkin lymphoma cell lines

Epigenetic changes have been implicated in silencing several B-cell genes in Hodgkin and Reed-Sternberg cells (HRS) of Hodgkin lymphoma (HL), and this mechanism has been proposed to promote HRS survival and escape from immunosurveillance. However, the molecular and functional consequences of histone deacetylase (HDAC) inhibition in HL have not been previously described. In this study, we report that the HDAC inhibitor vorinostat induced p21 expression and decreased Bcl-xL levels causing cell-cycle arrest and apoptosis. Furthermore, vorinostat inhibited STAT6 phosphorylation and decreased its mRNA levels in a dose- and time-dependent manner, which was associated with a decrease in the expression and secretion of Thymus and Activation-Regulated Chemokine (TARC/CCL17) and interleukin (IL)-5 and an increase in IP-10 levels. Moreover, vorino-stat inhibited TARC secretion by dendritic cells that were activated by the thymic stromal lymphopoietin (TSLP). Collectively, these data suggest that pharmacologic HDAC inhibition in HL may induce favorable antitumor activity by a direct antiproliferative effect on HRS cells, and possibly by an immune mediated effect by altering cytokine and chemokines secretion in the microenvironment.

Induction of Cell Cycle Arrest and Apoptosis by the Proteasome Inhibitor PS-341 in Hodgkin Disease Cell Lines Is Independent of Inhibitor of Nuclear Factor-κB Mutations or Activation of the CD30, CD40, and RANK Receptors

Purpose: The malignant Hodgkin and Reed-Sternberg cells of Hodgkin disease (HD) are known to constitutively express high levels of activated nuclear factor κB (NF-κB), which plays an important role in their survival. The proteasome inhibitor PS-341 has been recently shown to modulate tumor cell proliferation and survival by inhibiting NF-κB and modulating critical cellular regulatory proteins, but its activity in cells carrying IκBα gene mutations has not been reported previously. Experimental Design: The activity of PS-341 in four well-characterized, HD-derived cell lines. Cell proliferation and apoptosis were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) and Annexin-V binding methods, respectively. Cell cycle analysis was determined by flow cytometry. Intracellular protein levels were determined by Western blot. Results: PS-341 demonstrated a strong antiproliferative activity, which was irrespective of the status of mutations in IκBα and even the presence of CD30, CD40, or RANK receptor activation. This effect was attributable to the induction of apoptosis and cell cycle arrest at the G2-M phase. PS-341 not only inhibited nuclear localization of NF-κB but also activated the caspase cascade, increased p21 and Bax levels, and decreased Bcl-2 levels. Furthermore, PS-341 enhanced the effect of gemcitabine chemotherapy and potentiated the effect of tumor necrosis factor-related apoptosis-inducing ligand/APO2L and two agonistic antibodies to tumor necrosis factor-related apoptosis-inducing ligand death receptors R1 and R2. Conclusions: The in vitro activity of PS-341 against HD-derived cell lines suggests that PS-341 may have a therapeutic value for the treatment of HD.

Temsirolimus downregulates p21 without altering cyclin D1 expression and induces autophagy and synergizes with vorinostat in mantle cell lymphoma

OBJECTIVE To investigate the mechanisms of antiproliferative effect induced by the mammalian target of rapamycin (mTOR) inhibitor temsirolimus in mantle cell lymphoma (MCL). MATERIALS AND METHODS The antiproliferative effect of temsirolimus on three well-defined MCL cell lines was examined by the MTS assay. Induction of cell-cycle arrest, autophagy, and apoptosis were determined by fluorescence-activated cell sorting analysis. The molecular mechanisms underlining these effects were determined by Western blot. Synergy between temsirolimus and vorinostat were examined by MTS assay and the combination index was calculated. RESULTS Temsirolimus has antiproliferative activity in three MCL cell lines in a dose- and time-dependent manner. Mechanistically, temsirolimus inhibited mTOR, as evidenced by inhibition of ribosomal S6 phosphorylation, and induced cell-cycle arrest in the G(0)/G(1) phase and a decrease in p21 expression without altering p27 or cyclin D1 levels. Furthermore, temsirolimus increased the number of acidic vesicular organelles and the amount of microtubule-associated protein 1 light-chain 3 processing, which are characteristic of autophagy, without induction of apoptosis. These changes were not associated with alteration in phosphorylated extracellular signal-regulated kinase (ERK), beclin-1, Bax, or Bak levels. In contrast, treatment of these cell lines with the histone deacetylase inhibitor vorinostat decreased ERK phosphorylation, activated caspase 3, and induced apoptosis. Moreover, temsirolimus synergized with submaximal concentrations of vorinostat in all MCL cell lines. CONCLUSION This is the first report of temsirolimus-induced autophagy in MCL, and of vorinostat inhibition of ERK phosphorylation in MCL. Collectively, these data suggest that the combination of temsirolimus and vorinostat have synergistic antiproliferative activity in MCL cells by distinctively targeting apoptosis and autophagy.

Inhibition of heat shock protein 90 function by 17-allylamino-17-demethoxy-geldanamycin in Hodgkin's lymphoma cells down-regulates Akt kinase, dephosphorylates extracellular signal-regulated kinase, and induces cell cycle arrest and cell death.

Purpose: Heat shock protein 90 (HSP90) is a chaperone for several client proteins involved in transcriptional regulation, signal transduction, and cell cycle control. HSP90 is abundantly expressed by a variety of tumor types and has been recently targeted for cancer therapy. The objective of this study was to determine the role of HSP90 in promoting growth and survival of Hodgkins lymphoma and to determine the molecular consequences of inhibiting HSP90 function by the small-molecule 17-allylamino-17-demethoxy-geldanamycin (17-AAG) in Hodgkins lymphoma. Experimental Design: HSP90 expression in Hodgkins lymphoma cell lines was determined by Western blot and in primary lymph node sections from patients with Hodgkins lymphoma by immunohistochemistry. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Apoptosis and cell cycle fractions were determined by flow cytometry. Expression of intracellular proteins was determined by Western blot. Results: HSP90 is overexpressed in primary and cultured Hodgkins lymphoma cells. Inhibition of HSP90 function by 17-AAG showed a time- and dose-dependent growth inhibition of Hodgkins lymphoma cell lines. 17-AAG induced cell cycle arrest and apoptosis, which were associated with a decrease in cyclin-dependent kinase (CDK) 4, CDK 6, and polo-like kinase 1 (PLK1), and induced apoptosis by caspase-dependent and caspase-independent mechanisms. Furthermore, 17-AAG depleted cellular contents of Akt, decreased extracellular signal–regulated kinase (ERK) phosphorylation, and reduced cellular FLICE-like inhibitory protein levels (FLIP), and thus enhanced the cytotoxic effect of doxorubicin and agonistic anti–tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) death receptor antibodies. Conclusion: Inhibition of HSP90 function induces cell death and enhances the activity of chemotherapy and anti–tumor necrosis factor–related apoptosis-inducing ligand death receptor antibodies, suggesting that targeting HSP90 function might be of therapeutic value in Hodgkins lymphoma.

The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells

B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder characterized by the surface expression of CD20, CD5 antigens, as well as the receptor CD40. Activation of CD40 by its ligand (CD40L) induces proliferation and rescues the cells from spontaneous and chemotherapy-induced apoptosis. CD40 activation also induces secretion of cytokines, such as IL-6, IL-10, TNF-alpha, IL-8, and GM-CSF, which are involved in tumor cell survival, migration, and interaction with cells in the tumor microenvironment. Here we demonstrate that in primary B-CLL tumor cells, the novel antagonist anti-CD40 monoclonal antibody, HCD122, inhibits CD40L-induced activation of signaling pathways, proliferation and survival, and secretion of cytokines. Furthermore, HCD122 is also a potent mediator of antibody-dependent cellular cytotoxicity (ADCC), lysing B-CLL cells more efficiently than rituximab in vitro, despite a significantly higher number of cell surface CD20 binding sites compared with CD40. Unlike rituximab, however, HCD122 (formerly CHIR-12.12) does not internalize upon binding to the cells. Our data suggest that HCD122 may inhibit B-CLL growth by blocking CD40 signaling and by ADCC-mediated cell lysis.

Inhibition of the phosphatidylinositol-3 kinase/Akt promotes G1 cell cycle arrest and apoptosis in Hodgkin lymphoma.

Activation of the phosphatidylinositol 3‐kinase (PI3K) pathway has been linked with tumour cell growth, survival and resistance to therapy in several cancer types. The active, phosphorylated form of Akt (pAkt) was found to be aberrantly expressed in Hodgkin lymphoma (HL)‐derived cell lines and in Hodgkin–Reed–Sternberg (HRS) cells in 27 of 42 (64·3%) of primary lymph node sections of HL, indicative of PI3K activity. Akt phosphorylation was not associated with loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression, but with its phosphorylation in HL‐cell lines, suggesting that its biological function is impaired. Akt phosphorylation was further induced by CD30 ligand (CD30L), CD40L and receptor activator of nuclear factor kappa B (RANK) ligand. The PI3K inhibitor LY294002 demonstrated antiproliferative effects in a dose‐ and time‐dependent manner, which was associated with Akt dephosphorylation on Thr308 and Ser473 sites and dephosphorylation of the downstream ribosomal protein S6. LY209002 induced cell cycle arrest in the G0/G1 phase and apoptosis, which were associated with upregulation of MDM2, downregulation of cyclin D1, activation of caspase 9 and poly‐ADP‐ribose polymerase cleavage. The Akt inhibitor QLT394 also demonstrated antiproliferative effects in a dose‐ and time‐dependent manner, dephosphorylated ribosomal S6 and cleaved caspase 9. Collectively, these data suggest that the aberrant activation of the PI3K/Akt survival pathway in HRS cells is not because of loss of PTEN expression. Our data suggest that PTEN phosphorylation and activation of CD30, CD40 and RANK may play a role in activating Akt in HRS cells.

Expression of histone deacetylases in lymphoma: Implication for the development of selective inhibitors

Unselective histone deacetylase (HDAC) inhibitors are a promising novel therapy for lymphoid malignancies. However, these treatments remain empiric as the pattern of HDAC enzymes in different types of cancer, including lymphoid malignancies, remains unknown. We examined the expression of class I and class II HDACs in a panel of cell lines and tissue sections from primary lymphoid tumours. Class I enzymes were highly expressed in all cell lines and primary tumours studied, including the non‐malignant reactive cells in the Hodgkin lymphoma (HL) microenvironment. The most frequently altered HDAC expression was HDAC6, as it was either weakly expressed or undetected in 9/14 (64%) of lymphoid cell lines and in 83/89 (93%) of primary lymphoma tissue specimens, including 50/52 (96%) cases of diffuse large B‐cell lymphoma, and 18/22 (82%) cases of classical HL. Cell lines that had low expression level of HDAC6 demonstrated aberrant expression of hyper‐acetylated tubulin, and were found to be more sensitive to the growth inhibitory effects of the class I HDAC inhibitor MGCD0103. Collectively, our data demonstrate that HDAC6 is rarely expressed in primary lymphoma cases, suggesting that it may not be an important therapeutic target in these lymphoid malignancies.

Successful Nonoperative Management of the Most Severe Blunt Liver Injuries: A Multicenter Study of the Research Consortium of New England Centers for Trauma

HYPOTHESIS Grade 4 and grade 5 blunt liver injuries can be safely treated by nonoperative management (NOM). DESIGN Retrospective case series. SETTING Eleven level I and level II trauma centers in New England. PATIENTS Three hundred ninety-three adult patients with grade 4 or grade 5 blunt liver injury who were admitted between January 1, 2000, and January 31, 2010. MAIN OUTCOME MEASURE Failure of NOM (f-NOM), defined as the need for a delayed operation. RESULTS One hundred thirty-one patients (33.3%) were operated on immediately, typically because of hemodynamic instability. Among 262 patients (66.7%) who were offered a trial of NOM, treatment failed in 23 patients (8.8%) (attributed to the liver in 17, with recurrent liver bleeding in 7 patients and biliary peritonitis in 10 patients). Multivariate analysis identified the following 2 independent predictors of f-NOM: systolic blood pressure on admission of 100 mm Hg or less and the presence of other abdominal organ injury. Failure of NOM was observed in 23% of patients with both independent predictors and in 4% of those with neither of the 2 independent predictors. No patients in the f-NOM group experienced life-threatening events because of f-NOM, and mortality was similar between patients with successful NOM (5.4%) and patients with f-NOM (8.7%) (P = .52). Among patients with successful NOM, liver-specific complications developed in 10.0% and were managed definitively without major sequelae. CONCLUSIONS Nonoperative management was offered safely in two-thirds of grade 4 and grade 5 blunt liver injuries, with a 91.3% success rate. Only 6.5% of patients with NOM required a delayed operation because of liver-specific issues, and none experienced life-threatening complications because of the delay.

The heat shock protein 90 inhibitor 17-AAG induces cell cycle arrest and apoptosis in mantle cell lymphoma cell lines by depleting cyclin D1, Akt, Bid and activating caspase 9

Mantle cell lymphoma (MCL), a distinct type of non‐Hodgkin lymphoma, is characterised by the overexpression of cyclin D1. Heat shock protein 90 (HSP90) is a molecular chaperon to proteins that regulate cell cycle and survival. 17‐allylamino‐17‐demethoxy‐geldanamycin (17‐AAG), a HSP90 small molecule inhibitor, induced G0/1 cell cycle arrest and cell death in a dose‐ and time‐dependent manner in MCL cell lines. This effect was associated with the downregulation of cyclin D1, cdk4 and Akt, depletion of Bid, and activation of the intrinsic/mitochondrial caspase pathway. These data suggest that 17‐AAG may have a potential therapeutic value in patients with MCL.