Selective Targeting of Vascular Endothelial Growth Factor (VEGF) Receptor Signaling with Pazopanib and Vatalanib Induces Apoptosis in Chronic Lymphoctic Leukemia (CLL) Cells in Vitro Inhibits Growth of Human CLL Like Tumor Xenografts in Mice.
Authors: Paesler Julian , Gehrke Iris , Razavi Regina , Gandhirajan Rajesh Kumar , Filipovich Alexandra , Hertweck Magdalena , Erdfelder Felix , Uhrmacher Sabrina , Poll-Wolbeck Simon Jonas , Hallek Michael , Kreuzer Karl Anton 
Published: October 2019

Abstract
Abstract 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.