No abstract

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.

We determined lymphoid enhancer-binding factor-1 (LEF1) mRNA expression in 112 chronic lymphocytic leukemia (CLL) samples and assessed correlations with the prognostic markers ZAP70 and CD38, Binet stages, the percentage of lymphocytes in the peripheral blood, and fibromodulin (FMOD) transcripts. The mean LEF1 relative expression ratios (RER) were 53.72 and 37.10 in ZAP70-positive and ZAP70-negative patients, respectively (P=0.004). However, we did not observe a significant difference in LEF1 expression between CD38-positive and CD38-negative patients. Moreover, patients requiring treatment showed a mean LEF1 RER of 85.61 whereas patients in recently diagnosed Binet A stage had a mean of only 22.01 (P<0.001). We also found significant correlations of LEF1 with the percentage of lymphocytes and FMOD expression. Our results suggest that high LEF1 expression is associated with poor prognosis and disease progression. Thus, LEF1 might be involved in the process of disease progression and possibly can serve as a molecular parameter for risk assessment and/or monitoring of CLL.

3451 Poster Board III-339 We and others have shown that vascular endothelial growth factor (VEGF) plays a pivotal role in growth, survival and migration of chronic lymphocytic leukemia (CLL). VEGF dependent survival benefits in CLL are thereby mediated via autocrine and paracrine loops. However, so far no approach was studied focusing on the selective inhibition of VEGF in CLL. Vatalanib (PTK787/ZK 222584) and pazopanib (GW786034) are potent orally available, selective VEGF tyrosine kinase inhibitors. The aim of the present investigation was i) to study the efficacy and selectivity of both inhibitors in CLL cells, ii) to simulate potential combination with conventional cytostatics in vitro and iii) to test the effect on CLL like tumor xenografts in a mouse model. Primary CLL as well as healthy B cells were incubated with varying concentrations of both inhibitors for different time periods. Cells were then treated with combinations of each inhibitor and fludarabine, vincristin and doxorubicin, respectively. Apoptosis induction was analysed by flow cytometry (annexin V-FITC/PI staining) and cell survival was additionally investigated by an ATP dependent fluorescence assay. Percentage of surviving cells was defined as double annexin/PI negative cells in flow cytometry analysis. For in vivo experiments, four-week old BALB/c nu/nu mice were grafted with cells of a human chronic B cell leukemia cell line (JVM3). After tumors reached a mean volume of 100 mm3 per group (10 mice), drugs were administered once daily by oral gavage at 100 mg/kg bodyweight. Tumor volume was measured every second day by calliper. Vatalanib and pazopanib effectively induced apoptosis in CLL cells in vitro in a dose and time dependent fashion. During 24h incubation of CLL cells vatalanib showed a 50% lethal concentration (LC50) of 46.7 μM (n=26) and pazopanib of 32.7μM (n=19). In contrast, survival of B cells derived from healthy donors was only slightly affected at high concentrations of both drugs, thereby suggesting a large therapeutic range. Healthy B cells survived 80.5 ± 4.5% after 24h incubation with 100 μM vatalanib. In contrast, CLL cells showed a survival of 27.8 ± 6.0% (n=5; p=0.0001). Survival of healthy B cells treated with 100 μM of pazopanib was 89.1 ± 2.2% whereas survival of CLL cells was only 40.8 ± 2.3% (n=5; p<0.0001). Combination of the low dosed drugs with conventional cytostatics like fludarabin, vincristin and doxorubicin showed synergistic effects and significantly increased apoptosis rates in vitro. Single drug treatment showed survival rates of 80.1 ± 5.7% for 10 μM vatalanib, 86.2 ± 10.1 for 10 μM fludarabin, 69.8 ± 6.1 % for 0.10 μM vincristin and 51.6 ± 5.2% for 10 μM of doxorubicin. The combination of 10 μM vatalanib with 10 μM fludarabin showed survival rates of 59.4 ± 11.6% (p=0.0167), with 0.1 μM vincristin 32.5 ± 7.2% (p=0.0095) and with doxorubicin 26.0 ± 11.8% (p=0.0381). Survival rates for single treatment with 10 μM pazopanib were 84.3 ± 4.3%. The combination of 10 μM pazopanib with 10 μM fludarabin showed survival rates of 55.0 ± 6.1% (p=0.0384), with 0.1 μM vincristin 42.8 ± 5.3 (p=0.0473) and with doxorubicin 41.5 ± 11.8% (n.s.). After three weeks of treatment of the xenograft mice with each inhibitor the mean tumor volume was 645.0 ± 241.2 mm3 with pazopanib (p=0.002), 671.8 ± 198.0 mm3 with vatalanib (p=0.002) and 2,458.3 ± 742.39 mm3 in the vehicle treated group. This translated into a mean tumor inhibition rate of 77.3% for pazopanib and 71.3% for vatalanib after 21 days of treatment. Summing up, specific inhibition of VEGF by vatalanib or pazopanib might be a promising new therapeutic approach in CLL. Both compounds are orally available and showed acceptable in vivo toxicities in clinical trials, promising good compliance for the treatment. Therefore, they are attractive candidates for further testing in CLL. Disclosures Hallek: BayerScheringAG: Honoraria, Research Funding.

<div>Abstract<p><b>Purpose:</b> 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.</p><p><b>Experimental Design:</b> 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 <i>in vitro</i>. For <i>in vivo</i> testing, mice grafted with the CLL-like cell line JVM-3 were treated orally with each inhibitor.</p><p><b>Results:</b> 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.</p><p><b>Conclusion:</b> 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.</p></div>

<p>Supplementary Figure S1 and Supplementary Table S1.</p>

B-cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, but incompetent B-cells due to a decrease of apoptosis rather than an increase in proliferation. Vascular endothelial growth factor (VEGF) has been suggested to play an important role in this so called apoptotic block. However, so far little is understood whether VEGF is acting mainly as a microenvironmental stimulus and/or whether CLL cells themselves contribute to the enhanced apoptotic resistance by maintaining an autocrine VEGF loop. Moreover, it is unknown by which mechanisms VEGF prevents apoptosis and whether this can be circumvented by inhibition of VEGF signaling. By quantitative real time PCR we found no significant difference in mRNA VEGF levels in B-cells from CLL patients and healthy donors after isolation from blood. In contrast, ELISA revealed clearly increased levels of secreted VEGF in plasma of CLL patients and in the supernatant under culture conditions compared to healthy individuals. In addition, we found the VEGF receptor 2 (VEGFR2), which is existent in CLL and healthy B-cells, in a phosphorylated, hence activated state, to a significantly higher extent in CLL cells as assessed by intracellular phospho flow cytometry. In conclusion, despite its expression in healthy B-cells VEGF does not seem to be secreted and therefore, no VEGF receptor phosphorylation takes place. Whereas CLL cells exhibit a long life span in vivo, they die rapidly in vitro, suggesting major survival factors being existent in the CLL cells microenvironment. We found levels of secreted VEGF in supernatant decreasing with time in culture, going along with decreasing levels of phosphorylated VEGFR2 and increasing cell death as assessed by Annexin V-FITC/PI staining. This further supports the role of VEGF in CLL cell survival. Coculturing primary CLL cells with the bone marrow stromal derived cell line HS5 dramatically increased VEGF transcription and secretion and improved cell survival. Hence, VEGF expression in CLL cells is not only mediated by autocrine, but also paracrine stimuli involving bone marrow stromal. Knocking down VEGF in HS5 cells and subsequent coculture with CLL cells might prove the major role of VEGF in this survival supporting coculture setting. Besides coculturing also supplement of culture medium with recombinant human VEGF (rhVEGF) increased survival, but to a lesser extent than coculture, indicating a direct cell-cell interaction as advantageous. Furthermore, we found a downregulation of anti apoptotic proteins, such as X-linked inhibitor of apoptosis protein (XIAP), myeloid cell leukemia 1 (MCL1) and BclXL upon VEGF stimulation. Also cyclinD1 was upregulated as seen by immunoblotting. We further tried to discover the underlying mechanism of how VEGF mediates its pro survival effect and found STAT3 to become phosphorylated on tyrosine 705 upon VEGF stimulation. In CLL STAT3 is known to be constitutively phosphorylated on serine 727. This phosphorylation is not sufficient to induce target gene expression though. We could show that Y705 phosphorylation of STAT3 is responsible for upregulation of anti apoptotic BCLXL and cyclinD1. A PCR array detecting mRNA levels of 84 transcription factors in untreated and VEGF stimulated CLL cells shall provide more information about mechanistical details how VEGF mediates it pro survival effect. Since VEGF seems to be a major player in CLL cell survival it might be a suitable target to overcome the apoptotic block. In first experiments we found an induction of apoptosis after neutralization of VEGF or inhibition of the VEGF receptor. This additionally highlights the severe importance of VEGF in the apoptotic block in CLL cells. Therefore, VEGF might serve as an excellent therapeutic target in CLL.

4589 Chronic lymphocytic leukemia (CLL) is characterized by accumulation of monoclonal CD5+ B lymphocytes. Fibromodulin is a secreted extracellular matrix protein usually found in articular cartilage, bones, connective tissue and collagen rich tissues but has been shown to be excessively expressed in CLL. Moreover, we and others could show that WNT signaling is highly activated in CLL. The aim of our study was to investigate a possible connection between fibromodulin overexpression and the WNT pathway. Moreover, we wanted to explore possible relations between these parameters and the prognosis of CLL. Fibromodulin mRNA transcripts were correlated with the mRNA expression of the WNT pathway transcription factors lymphoid enhancer binding factor-1 (LEF-1) and T cell factor-4 (TCF-4) in primary CLL cells. Furthermore, we assessed correlations between the mRNA expression levels with ZAP-70 and CD38 protein expression. These parameters have been shown to be associated with a poor prognosis. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used and calculated PCR-efficiency corrected relative expression values of Fibromodulin, LEF-1 and TCF-4 mRNA in primary CLL-cells determined. ZAP-70 and CD38 expression was determined by flow cytometry analysis. Based on 20 primary CLL samples we found a significant positive correlation between Fibromodulin and LEF-1 mRNA levels (Spearman's rho = 0.65, p = 0.009). Also, LEF-1 and TCF-4 were found to be strongly correlated (Spearman's rho = 0.61, p = 0.027). In contrast, fibromodulin and TCF-4 mRNA levels were only weakly correlated (Spearman's rho = 0.33, p = 0.271). CD38 and ZAP-70 did not correlate significantly to any of the other values. Both parameters did also not exhibit significant correlations with fibromodulin, LEF-1, and TCF-4 mRNA. The positive correlation of TCF-4 and LEF-1 was expected as LEF-1 has been shown to be a target gene of TCF transcription factors. The strong positive correlation of fibromodulin and LEF-1 indicates that fibromodulin might be a target gene of LEF-1 or part of the same (TCF-4 independent) transcription regulation pathway. Studies investigating theses functional relationships are underway. Disclosures: Hallek: BayerScheringAG: Honoraria, Research Funding.