VE-cadherin Regulates Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Sensitivity to Apoptosis

Abstract

The mechanisms by which the bone marrow microenvironment regulates tumor cell survival are diverse. This study describes the novel observation that in addition to Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) cell lines, primary patient cells also express Hypoxia Inducible Factor-2α (HIF-2α) and Vascular Endothelial Cadherin (VE-cadherin), which are regulated by Abl kinase. Tumor expression of the classical endothelial protein, VE-cadherin, has been associated with aggressive phenotype and poor prognosis in other models, but has not been investigated in hematopoietic malignancies. Targeted knockdown of VE-cadherin rendered Ph+ ALL cells more susceptible to chemotherapy, even in the presence of bone marrow stromal cell (BMSC) derived survival cues. Pre-treatment of Ph+ ALL cells with ADH100191, a VE-cadherin antagonist, resulted in increased apoptosis during in vitro chemotherapy exposure. Consistent with a role for VE-cadherin in modulation of leukemia cell viability, lentiviral-mediated expression of VE-cadherin in Ph- ALL cells resulted in increased resistance to treatment-induced apoptosis. These observations suggest a novel role for VE-cadherin in modulation of chemoresistance in Ph+ ALL.

Keywords: Bcr/Abl; Bone marrow stromal cells; HIF-2α; Philadelphia chromosome (Ph+); VE-cadherin.

Fig. 1

Patient derived ALL cells express VE-cadherin and HIF-2α. a RNA was isolated from 5 × 10⁶ patient derived cells and RT-PCR was performed for HIF-2α, VE-cadherin or actin. b 1 × 10⁶ patient derived Ph+ (black line) and Ph− (gray line) cells were obtained from leukapheresis and stained to detect HIF-2α (intracellular), VE-cadherin (surface) , or matched isotype control and analyzed by FACS. c Ph+ and Ph− primary patient bone marrow core biopsies were stained for VE-cadherin or matched isotype control. 11/12 samples were positive for VE-cadherin and four representative samples are shown. All samples were positive for CD19, CD10, CD22, HLADR and TDT

Fig. 2

Bone marrow stromal cells maintain Ph+ ALL expression of VE-cadherin during Imatinib treatment. a SUP-B15 cells were cultured in media alone or co-cultured with BMSC and subsequently treated with 10 µM Imatinib Mesylate (IM). RT-PCR was performed for VE-cadherin, HIF-2α and actin. b SUP-B15 cells were exposed to DMSO as the control solvent, or to 10 uM IM, for 24 h. HIF-2α and VE-cadherin proteins were evaluated by FACS. c SUP-B15 cells were cultured in either media alone, in contact with BMSC, or in contact with BMSC and Imatinib for 24 h. Tumor cells were subsequently fixed with 4% PFA, permeabilized with 0.5% Triton-X 100 and stained with anti-VE-cadherin antibody or a matched isotype control for evaluation by confocal microscopy (images shown are 63× with no zoom) or following the standard protocol described in materials and methods for flow cytometric analysis. d SUP-B15 cells were cultured in media alone, or in the presence of BMSC for 24 h, and subsequently treated with 0.1 mg/ml DNR overnight. Cells were collected, lysed and western blots were completed to analyze Bcr/abl activity as well as VE-cadherin and β-catenin expression. GAPDH was used as a loading control. Loading of gels is as follows: Lane 1 is media alone, Lane 2 is DNR treatment, Lane 3 is co-culture with BMSC and Lane 4 is co-culture with BMSC and DNR treatment

Fig. 3

Bone marrow stromal cells modulate HIF-2α. a & b LTMC and LTCC SUP-B15 cells were cultured in media alone or in the presence of BMSC. HIF-2α mRNA and protein were evaluated by RT-PCR and FACS, respectively. c LTMC and LTCC cells were placed in media for 24 h and their baseline expression of HIF-2α was evaluated by FACS. d SUP-B15 cells were cultured in media alone or in the presence of BMSC and evaluated for expression of Oct-4

Fig. 4

Modulation of VE-cadherin results in altered chemosensitivity. a SUP-B15 cells were transiently transfected with scrambled, HIF-2α, or VE-cadherin siRNA and conformation of down-regulation was determined using FACS. b & c siRNA transfected cells were subsequently cultured in media alone or in the presence of BMSC and exposed to 10 uM IM or DMSO and 0.01 mg/ml DNR. Cell viability was determined by trypan blue exclusion and Annexin-V-FITC

Fig. 5

Expression of VE-cadherin decreases leukemic cell sensitivity to chemotherapy. a Ph−/surface VE-cadherin- REH cells were transduced with virus containing empty vector control (REH^Vect) or wild type CDH5 (REH^CDH5). Western blot was completed to demonstrate expression of VE-cadherin. b REH^Vect and REH^CDH5 cells were challenged with 0.6 mg/ml DNR for 48 h and viability was evaluated by trypan blue exclusion

Fig. 6

Disruption of VE-cadherin signaling using the VE-cadherin antagonist ADH increases Ph+ ALL sensitivity to apoptosis. a & b SUP-B15 were cultured in media alone or co-cultured with BMSC for 24 h. The cells were then pre-treated with 1 mg/ml ADH, or media control, for 6 h and subsequently challenged with chemotherapy (0.1 mg/ml Daunorubicin (DNR)/24 h). Viability was determined by trypan blue exclusion and Annexin-V-FITC. c SUP-B15 cells we cultured in media alone or in the presence of BMSC for 24 h, pre-treated with either media/DMSO, IM, ADH or a combination of IM & ADH for 6 h prior to treatment with DNR. Cell viability was then determined by trypan blue analysis. Statistical significance is shown comparing both the SUP-B15 on BMSC treated with DNR to the IM/DNR, ADH/DNR or IM/ADH/DNR groups as well as the comparison between the IM/DNR or ADH/DNR to IM/ADH/DNR. d Ph− REH cells were pre-treated with 1 mg/ml or 2 mg/ml ADH for 6 h and subsequently treated with 0.6 mg/ml DNR. Viability was determined by Annexin-V-FITC. e SUP-B15 cells were treated with media alone or with 1 mg/ml ADH for 24 h and surface stained for VE-cadherin

Fig. 7

Inhibition of VE-cadherin signaling leads to targeted degradation of β-catenin. a SUP-B15 cells were treated with 1 mg/ml ADH or media control for 4 h and subsequently stained for phospho-β-catenin (Ser33/37 and Thr41) or matched isotype control. Cells were analyzed by confocal microscopy (images shown are 40× with zoom). b SUP- B15 cells were treated with 1 mg/ml ADH or media control for 24 h and analyzed by western blot to observe changes in total β-catenin. Samples shown were run on the same gel, in outer lanes, and therefore the image was cut to show only bands relevant to the current study