Lack of WNT Pathway Co-Receptors as a Possible Reason for Chronic Lymphoctic Leukemia (CLL) Cells Resistance to Dickkopf-1 (DKK1).
Authors: Filipovich Alexandra , Gandhirajan Rajesh Kumar , Gehrke Iris , Paesler Julian , Poll-Wolbeck Simon Jonas , Uhrmacher Sabrina , Hertweck Magdalena , Erdfelder Felix , Razavi Regina , Hallek Michael , Kreuzer Karl Anton 
Published: October 2019

Abstract
Abstract 1598
Poster Board I-624
WNT signaling is known to regulate cell survival. Components of the WNT signaling cascade were previously shown to be upregulated in CLL cells in comparison to healthy B cells. Accordingly, this pathway is thought to be responsible for the enhanced survival of CLL cells. The canonical WNT pathway is initiated by proteins of the WNT ligand family, which bind to a receptor complex composed of the Frizzled proteins (Fz), and low-density lipoprotein (LDL) receptor-related proteins (LRP5/6). Dickkopf (DKK1) is known to antagonize WNT/beta-catenin signaling by direct high-affinity binding to the WNT coreceptor LRP5/6, thereby inhibiting interaction of LRP5/6 with the WNT-Frizzled complex. DKK1 coreceptor Kremen2 (KRM2) is speculated to play an important role in modulating DKK1 dependent antagonisation of WNT/beta-catenin signaling. Inactivation of WNT pathway by DKK1 could lead to an increased apoptosis in CLL cells. The purpose of this study was to investigate the effect of DKK1 in CLL cells in vitro. B-cells from CLL patients and JVM-3 cells were incubated in presence or absence of DKK1 (0.3 μg/ml) for 24 and 48 hours. Survival was measured by flow cytometry and caspase-3, -7 activation. To prove dose dependency, CLL cells were incubated with different doses of DKK1. Furthermore, the expression status of intracellular WNT signaling components was analysed by immunoblotting. The expression of receptors LRP6 and KRM2 was determined using real time PCR. Furthermore, we measured the expression of DKK1 in healthy and CLL B-cells. The flow cytometry analysis showed that incubating primary B-cells with DKK1 increased overall mean survival after 24 hours (vehicle control 81.3 ± 11.7 % versus DKK11 91.1 ± 5.3%) and after 48 hours (vehicle control 65.8 ± 13.5% versus DKK1 76.2 ± 8.2%). Accordingly, the activity of caspases-3 and -7 in these cultures was significantly reduced after DKK1 stimulation. The level of phosphorylated LRP6 decreased after incubating cells with DKK1 for 24 h in a dose-dependent fashion. β-catenin and c-myc expression was increased compared to untreated samples. Expression of LRP6 and KRM2, acquired by means of real-time PCR was lower in CLL cells, than in healthy B-cells. Similar, the basal amount of DKK1 was higher in CLL cells. Summing up, unlike other WNT active cancers such as multiple myeloma, addition of DKK1 to culture of CLL cells does not lead to inactivation of the WNT pathway. In contrast, in CLL the addition of DKK1 even increases CLL cell survival in vitro. This could be explained by a lack of WNT pathway-coreceptors LRP6 and KRM2 on the membrane of CLL cells. The unexpected increase of WNT pathway proteins might be caused by unspecific binding to other receptors of the WNT pathway than to LRP6. High basal expression of DKK1 in primary CLL cells may be referred to the fact, that DKK1 is itself a target gene of Lef-1, which is known to be highly upregulated in CLL cells. Further investigations on this controversy in WNT-signalling in CLL cells are needed.

Disclosures
Hallek: BayerScheringAG: Honoraria, Research Funding.