Iris Gehrke

Targeting the WNT/β-catenin/TCF/LEF1 axis in solid and haematological cancers: Multiplicity of therapeutic options

Among aberrantly regulated signalling pathways in cancer the WNT/beta-catenin pathway plays an outstanding role, since it was shown to be critically involved in a wide range of neoplasias. While the underlying mechanisms vary, overexpression of WNTs was found to mediate active signalling in some of these diseases. Other cancers show a mutation in pathway members further downstream, such as APC, Axin or beta-catenin, leading to aberrant signalling activation. Another mechanism initiating activation of WNT/beta-catenin signalling is the silencing of expression of negative WNT/beta-catenin regulators, such as DKK and WIF1, by, for example, promoter hypermethylation. All these mechanisms result in a common consequence, the activation of TCF/LEF1 transcription factors and subsequent target gene expression. Several target genes are known to be key players in tumourigenesis, such as c-myc, cyclin D1 or survivin. The variety of possible underlying mechanisms leading to beta-catenin/TCF/LEF1 activation offers multiple options to target the aberrantly activated pathway in order to prevent target gene expression and/or their gene products to exert their tumourigenic function. Here, we summarise the physiological role of WNT/beta-catenin signalling and the consequences of its aberrant activation during tumourigenesis. Furthermore, we discuss the possible strategies to target this pathway and their potential importance in cancer treatment.

Monoclonal and bispecific antibodies as novel therapeutics

Abstract.Gene amplification, over-expression, and mutation of growth factors, or the receptors themselves, causes increased signaling through receptor kinases, which has been implicated in many human cancers and is associated with poor prognosis. Tumor growth has been shown to be decreased by interrupting this process of extensive growth factor-mediated signaling by directly targeting either the surface receptor or the ligand and thereby preventing cell survival and promoting apoptosis. Monoclonal antibodies have long been eyed as a potential new class of therapeutics targeting cancer and other diseases. Antibody-based therapy initially entered clinical practice when trastuzumab/Herceptin became the first clinically approved drug against an oncogene product as a well-established blocking reagent for tumors with hyperactivity of epidermal growth factor signaling pathways. In the first part of this review we explain basic terms related to the development of antibody-based drugs, give a brief historic perspective of the field, and also touch on topics such as the “humanization of antibodie” or creation of hybrid antibodies. The second part of the review gives an overview of the clinical usage of bispecific antibodies and antibodies “armed” with cytotoxic agents or enzymes. Further within this section, cancer-specific, site-specific, or signaling pathway-specific therapies are discussed in detail. Among other antibody-based therapeutic products, we discuss: Avastin (bevacizumab), CG76030, Theragyn (pemtumomab), daclizumab (Zenapax), TriAb, MDX-210, Herceptin (trastuzumab), panitumumab (ABX-EGF), mastuzimab (EMD-72000), Erbitux (certuximab, IMC225), Panorex (edrecolomab), STI571, CeaVac, Campath (alemtuizumab), Mylotarg (gemtuzumab, ozogamicin), and many others. The end of the review deliberates upon potential problems associated with cancer immunotherapy.

Small molecule inhibitors of WNT signaling effectively induce apoptosis in acute myeloid leukemia cells

In a significant proportion of acute myeloid leukemia (AML) cases the canonical WNT pathway is upregulated and targeting the WNT/LEF1 signaling cascade in AML may be a promising approach to develop new treatments for this entity. Recently two compounds (CGP049090 and PFK115‐584) have been identified, which specifically inhibit complexation of β‐catenin (CTNNB1) and lymphoid enhancer‐binding factor 1 (LEF1) leading to transcriptional inactivation of LEF1 in colon carcinoma cell lines. To evaluate the effect of WNT inhibition utilizing theses compounds with regard to their effectivity in AML we treated the AML cell lines Kasumi‐1 and HL‐60, primary AML blasts and healthy peripheral blood mononuclear cells (PBMCs) with varying concentrations of both substances. Treatment with both compounds for 24 h resulted in a significant killing of AML cell lines and primary AML blasts with 50% effective concentration doses (EC50) within the submicromolar range. PBMCs were not significantly affected as indicated by EC50‐values 100‐fold higher than for AML cells. Cell kill was mediated by apoptosis as indicated by induction of caspases 3 and 7 and cleavage of poly(ADP‐ribose) polymerase (PARP) upon treatment. Furthermore, we could show that both compounds substantially decrease expression of CTNNB1/LEF1 target genes c‐myc, cyclin D1 and survivin, proofing the specificity of the substances. This was shown in both, AML cell lines and most of the tested primary samples. Our data demonstrate that targeting this pathway seems to be an innovative approach in the treatment of AML.

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. Clin Cancer Res; 16(13); 3390–8. ©2010 AACR.

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.

Physiological inhibitors of Wnt signaling

Wnt signaling is crucial for cell proliferation and differentiation. It represents a complex network with mechanisms of self‐regulation through positive and negative feedback. Recent increasing interest in this signaling pathway has led to the discovery of many new proteins that down‐regulate Wnt activity. Here, we provide a short description of the most important and best‐studied inhibitors, group them according to the target molecule within the Wnt cascade, and discuss their clinical potential. Although most of the inhibitors discussed here may also interact with proteins from other signaling pathways, we focus only on their ability to modulate Wnt signaling.

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-/β-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/β-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 LC50 (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 β-catenin and downregulation of β-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. Clin Cancer Res; 17(2); 286–93. ©2010 AACR.

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.

Evidence for non-functional Dickkopf-1 (DKK-1) signaling in chronic lymphocytic leukemia (CLL).

Purpose:  Wnt signaling was demonstrated to be activated in chronic lymphocytic leukemia (CLL). It is thought to be responsible for the extended survival of CLL cells in vivo. Dickkopf1 (DKK1) is known to antagonize Wnt signaling by direct high‐affinity binding to the extracellular domain of WNT coreceptor lipoprotein receptor‐related protein 6 (LRP6). The purpose of this study was to investigate the effect of DKK1 in B‐CLL cells in vitro.

A rare case of t(11;22) in a mantle cell lymphoma like B-cell neoplasia resulting in a fusion of IGL and CCND1: case report

The chromosomal translocation (11;14)(q13;q32) rearranging the locus for cyclin D1 (CCND1) to that of the immunoglobulin heavy chain (IGH) can be found in virtually all cases of mantle cell lymphoma (MCL), while other CCND1 translocations are extremely rare. As CCND1 overexpression and activation is a hallmark of MCL it is regarded as a central biological mechanism in the development and maintenance of this disease.Here we present a patient initially diagnosed with chronic lymphocytic leukemia (CLL) where chromosome banding analysis revealed, among other aberrations, a translocation (11;22)(q13;q11.2). We show by fluorescence in situ hybridization (FISH) analysis that on chromosome 22 the immunoglobulin light chain lambda (IGL) is involved in this cytogenetic aberration. Additionally, we demonstrate the resulting overexpression of CCND1 on the RNA and protein level, thereby consolidating the new diagnosis of a MCL-like B-cell neoplasia. Summing up, we described a rare case of t(11;22)(q13;q11.2) in a MCL-like neoplasia and showed that this aberration leads to an overexpression of CCND1 which is regarded as a key biological feature in MCL. This case underlines the importance of cytogenetic analyses especially in atypical cases of B cell lymphomas.