Trichostatin A effectively induces apoptosis in chronic lymphocytic leukemia cells via inhibition of Wnt signaling and histone deacetylation.

BACKGROUND: The ontogenetic Wnt pathway shows almost no activity in adult tissues. In contrast, chronic lymphocytic leukemia (CLL) cells show constitutionally active Wnt signaling, which is associated with upregulated levels of pathway members such as Wnt3 and lymphoid enhancer-binding factor-1. Functionally, this results in increased resistance to apoptosis. We therefore assumed that targeting members of the pathway could reveal new therapeutic options for the treatment of CLL. METHODS: Screening a Wnt compound library with 75 Wnt modulators via ATP assay revealed Trichostatin A as an outstanding substance with strong viability decreasing effects on CLL cells and little effect on healthy peripheral blood mononuclear cells (PBMCs). Further survival analysis was performed via fluorescence-activated cell sorting analysis. RESULTS: A maximum effect was achieved after 48 h with a wide therapeutic window in contrast to PBMCs (CLL cells: 0.253 µM, PBMCs: 145.22 µM). Trichostatin A induced caspases and acted via a dual mechanism to reveal histone and non-histone targets. Histone targets were displayed in deacetylation inhibition at DNA level, and non-histone targeting was demonstrated by elevated levels of Dickkopf-related protein 1 mRNA. Primary cells of patients with critical mutations such as TP53 or those who had already undergone extensive previous treatment responded well to the treatment. Moreover, the approved histone deacetylase (HDAC) inhibitor suberoylanilidehydroxamic acid (SAHA) was not as effective as Trichostatin A (Trichostatin A: 0.253 µM, SAHA: 7.88 µM). Combining Trichostatin A with established CLL drugs fludarabine or bendamustine showed an additive effect in vitro. CONCLUSION: Taken together, Trichostatin A appears to act via a dual anti-HDAC/Wnt mechanism with a high selectivity and efficacy in CLL and therefore warrants further investigation.

Targeting the vascular endothelial growth factor in hematologic malignancies.

There exists increasing evidence that apart from solid tumors, angiogenic growth factors also play important roles in the development and/or maintenance of hematolymphoid malignancies. Thus, in these cancers, angiogenesis and bone marrow microvessel density often correlate with prognosis and disease burden. Several reports speculated on the role of angiogenesis and the resulting possible therapeutic options in hematologic malignancies. The most prominent angiogenic factor, vascular endothelial growth factor (VEGF), is expressed in a number of established leukemic cell lines as well as in freshly isolated human leukemias and lymphomas, and several human leukemias express VEGF receptor 1 and/or VEGF receptor 2. VEGF/VEGF-receptor interactions are also involved in proliferation, migration, and survival of leukemic cells by autocrine and paracrine mechanisms. As a consequence, a possible drugable effect by inhibiting VEGF signaling in different hematologic malignancies has been discussed. This review focuses on angiogenesis-independent effects of VEGF on survival and proliferation of leukemic or lymphoma cells and on possible therapeutic approaches using anti-VEGF/VEGF-receptor therapies to inhibit proliferation or induce apoptosis of malignant cells in hematologic diseases.

Use of the receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a diagnostic tool in chronic lymphocytic leukemia (CLL).

Flow cytometry is commonly used to establish the diagnosis of chronic lymphocytic leukemia (CLL). A defined combination of antibodies discriminates between normal B cells and CLL cells (coexpression of CD5, CD19, and CD23). The receptor tyrosine-like orphan receptor one (ROR1) is an embryonic glycoprotein involved in several developmental processes. It was shown to be highly and specifically expressed on circulating B lymphoma cells, but not on normal B cells. Here, we examined the potential of ROR1 as a diagnostic marker in initial and follow-up diagnostics of patients with CLL. 105 untreated and 72 treated patients, as well as healthy volunteers were examined using flow cytometry assays. Furthermore, we examined 10 patients with various B cell non-Hodgkin lymphomas (B-NHL). ROR1 was detected using a directly labeled antibody. We detected uniformly high ROR1 expression levels in all CLL samples. In marked contrast, only low or absent ROR1 expression levels were found on B cells from healthy donors. ROR1 expression in CLL patients was not influenced by various treatments. Taken together, ROR1 may be used as a diagnostic marker for CLL. As it is the only antigen which can exclusively be detected on neoplastic B cells it may greatly increase both, specificity as well as sensitivity, in lymphoma diagnostics.

Bone marrow stromal cell-derived vascular endothelial growth factor (VEGF) rather than chronic lymphocytic leukemia (CLL) cell-derived VEGF is essential for the apoptotic resistance of cultured CLL cells.

Chronic lymphocytic leukemia (CLL) cells feature a pronounced apoptotic resistance. The vascular endothelial growth factor (VEGF) possesses a role in this apoptotic block, although underlying functional mechanisms and the involvement of the microenvironment are unclear. In this study, the VEGF status in CLL was assessed by enzyme-linked immunosorbent assay and immunofluorescence. VEGF receptor 2 (VEGFR2) phosphorylation was determined flow cytometrically and by immunofluorescence. For co-culture, CLL cells were cultivated on a monolayer of the bone marrow-derived stromal cell (BMSC) line HS5. Secreted VEGF was neutralized using the monoclonal antibody mAb293 (R&D Systems, Minneapolis, MN, USA). To block protein secretion, we used Brefeldin A. VEGF was downregulated in BMSCs by small interfering RNA (siRNA), and we assessed survival by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. CLL cells express and secrete VEGF and possess phosphorylated VEGFR2. This positive VEGF status is not sufficient to prevent spontaneous apoptosis in vitro. Coculture with BMSCs, which secrete vast amounts of VEGF, maintains in vitro CLL cell survival. Blockage of secreted VEGF using the monoclonal antibody mAb293 significantly reduced the survival support for cocultured CLL cells. Both general blockage of protein secretion by Brefeldin A in BMSCs, but not in CLL cells, and siRNA-mediated downregulation of VEGF in BMSCs, significantly reduced the coculture-mediated survival support for CLL cells. It can be concluded that BMSC-derived proteins and VEGF, in particular, but not CLL cell-derived VEGF, is essentially involved in the coculture-mediated survival support for CLL cells. Hence, therapeutic targeting of VEGF signaling might be a promising approach to overcome the apoptotic resistance CLL cells feature within their natural microenvironment.

The Antineoplastic Effect of Nitric Oxide-Donating Acetylsalicylic Acid (NO-ASA) in Chronic Lymphocytic Leukemia (CLL) Cells is Highly Dependent on its Positional Isomerism.

BACKGROUND: Chronic Lymphocytic Leukemia (CLL) is not curable in patients that are not eligible for allogeneic stem cell transplantation. Therefore, new treatment options are highly desirable. Chemically modified nonsteroidal anti-inflammatory drugs (NSAIDs), such as nitric-oxide-donating acetylsalicylic acid (NO-ASA), have been described to possess antineoplastic capacity. Recently, we could demonstrate a potent apoptosis induction in primary CLL cells in vitro and tumor growth inhibition by para-NO-ASA in a xenograft mouse model. However, little is known about the impact of positional isomerism of NO-ASA on its antineoplastic capacity in CLL. METHODS: Primary CLL cells were treated with the meta-or para-isomer of NO-ASA at varying concentrations and durations. Viability was assessed flow cytometrically by annexin V-FITC/PI staining and by CellTiter-Glo luminescence cell viability assay. Caspase and PARP cleavage as well as involvement of ß-catenin/Lef-1 signaling was determined by immunoblotting. For caspase inhibition, BD™ ApoBlock was used. Nude mice were xenografted with JVM3 cells and treated with meta-NO-ASA, para-NO-ASA or vehicle control. RESULTS: The meta-isomer was entirely ineffective in inducing CLL cell apoptosis in concentrations up to 100 µM, while para-NO-ASA acted in the low micromolar range. meta-NO-ASA, in contrast to para-NO-ASA, did not alter caspase activity. While para-NO-ASA action involved inhibition of ß-catenin/Lef-1 signaling, meta-NO-ASA did not show any impact on this signaling pathway. Further, meta-NO-ASA did not significantly reduce tumor growth in a CLL xenograft mouse model, while para-NO-ASA was highly potent. CONCLUSION: We conclude that positional isomerism is crucial for the antineoplastic effect of NO-ASA in CLL. It can be suggested that the para-isomer, but not the meta-isomer, generates a chemical structure which is essential for the neoplastic effect of NO-ASA.

Nitric oxide-donating acetylsalicylic acid induces apoptosis in chronic lymphocytic leukemia cells and shows strong antitumor efficacy in vivo.

PURPOSE: Nitric oxide-donating acetylsalicylic acid (NO-ASA) has been shown to possess an antineoplastic effect in Wnt-/ß-catenin-active cancers. As chronic lymphocytic leukemia (CLL) cells exhibit aberrantly active Wnt signaling, we investigated the effect of the para-isomer of NO-ASA on CLL cell survival in vitro and in a CLL-like xenograft mouse model. EXPERIMENTAL DESIGN: Apoptosis in primary CLL cells was determined by flow cytometric annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) staining and immunoblotting of caspases, poly(ADP-ribose) polymerase (PARP), and antiapoptotic proteins. Interference of NO-ASA with Wnt/ß-catenin signaling was analyzed through immunoblots of different pathway members. Influence of caspase activation was investigated by pretreatment with a pan-caspase inhibitor. CLL-like JVM3 cells were subcutaneously inoculated into irradiated nude mice that were treated with 100 mg of para-NO-ASA/kg of body weight p.o. (by mouth) for 21 days. RESULTS: para-NO-ASA induced apoptosis in CLL cells with an LC(50) (lethal concentration) of 8.72 + 0.04 µmol/L, whereas healthy blood cells were not affected. Furthermore, the compound induced caspase 9, caspase 3, and PARP cleavage. In addition, cleavage of ß-catenin and downregulation of ß-catenin/lymphoid enhancer factor (Lef)-1 targets was observed. para-NO-ASA demonstrated strong antitumor efficacy in the xenograft mouse model with a tumor inhibtion rate of 83.4%. During therapy, no gross toxicity could be observed. CONCLUSIONS: para-NO-ASA selectively induces apoptosis in primary CLL cells and efficiently reduces tumor growth in a CLL-like xenograft model. As NO-ASA is orally available and is generally well tolerated, para-NO-ASA might be a promising new compound for CLL therapy.

The vascular endothelial growth factor receptor tyrosine kinase inhibitors vatalanib and pazopanib potently induce apoptosis in chronic lymphocytic leukemia cells in vitro and in vivo.

PURPOSE: There is evidence that vascular endothelial growth factor (VEGF) is a critical microenvironmental factor that exerts angiogenesis-independent effects on the survival of chronic lymphocytic leukemia (CLL) cells. Vatalanib and pazopanib are potent orally available VEGF receptor tyrosine kinase inhibitors. We investigated the efficacy and selectivity of both compounds in CLL cells, simulated potential combination with conventional cytostatics, and tested the effect of both substances on CLL-like tumor xenografts. EXPERIMENTAL DESIGN: Primary CLL and normal peripheral blood cells were tested for viability after incubation with varying concentrations of both inhibitors. Further, phosphorylation status of VEGF receptor on treatment, caspase activation, and poly(ADP-ribose) polymerase cleavage were assessed. Combinations of each inhibitor with fludarabine, vincristine, and doxorubicin were analyzed for possible synergistic effects in vitro. For in vivo testing, mice grafted with the CLL-like cell line JVM-3 were treated orally with each inhibitor. RESULTS: Vatalanib and pazopanib decreased phosphorylation of the VEGF receptor, along with induction of apoptosis in CLL cells in clinically achievable concentrations. Healthy B cells were only mildly affected. Immunoblots showed downregulation of the antiapoptotic proteins XIAP and MCL1, whereas poly(ADP-ribose) polymerase cleavage was increased. Combinations with conventional cytostatic agents resulted in synergistic effects. Treatment of xenografted mice with 100 mg/kg of body weight for 21 days resulted in tumor inhibition rates of 76% (vatalanib) and 77% (pazopanib). In two mice, a total tumor eradication could be observed. No gross systemic toxicity occurred. CONCLUSION: We conclude that VEGF inhibition is a promising new therapeutic approach in CLL. Vatalanib and pazopanib seem to be effective and safe candidates to be further evaluated for this purpose.