Integrin αvβ3 Transduces Survival and Angiogenic Signals to T Cell Lymphomas and Is a Therapeutic Target

CAYROL F; DÍAZ FLAQUÉ MC; FERNANDO T; YANG SN ; BOLONTRADE M; AMORÓS M; FARÍAS RN; INGHIRAMI G ; CREMASCHI GA; CERCHIETTI L

San Diego

Congreso; 56th ASH Meeting; 2014

American Society of Hematology

Malignant T cell proliferation, survival and drug resistant are dependent on a combination of external stimuli delivered by the microenvironment. Studies from us and others have shown that the transmembrane receptor integrin Vβ3 plays a critical role mediating the interaction of T cell lymphoma (TCL) cells with external signals. Integrin Vβ3 ligands include extracellular matrix-associated signaling proteins and soluble factors such as thyroid hormones (TH). We have also shown that THs stimulate the proliferation of TCLs through complimentary intracellular pathways involving the Vβ3 integrin. We therefore hypothesized that targeting integrin Vβ3 could represent a novel strategy to treat TCL pts. To determine survival pathways induced by TH on Vβ3 integrins, we evaluated the transcriptional changes (RNA-seq) induced by physiological concentrations of cell impermeable T3/T4 (TH-AG) vs. control in the TCL cell line CUTLL1. We identified 123 up- and 5 down-regulated transcripts (p< 0.01), belonging to ?angiogenesis? (e.g. VEFGB), ?lymphocyte proliferation/differentiation? and ?DNA replication/transcription? (e.g. DBP, IL4, EDF1, DOK2) pathways. Target-gene promoter analysis suggested that TH acting on Vβ3 integrin activated NFkB (that was later confirmed by EMSA-like assays). We then focused on the angiogenesis pathway since (i) VEGF expression and angiogenesis correlate with survival and prognosis in TCL pts, and (ii) we found a positive correlation between integrin Vβ3 and VEGFA and VEGFB expression in 169 cases of TCLs. We analyzed T3/T4-Vβ3-dependent increase of VEGFB and VEGFA in an extended panel of cell lines (n=5) representing the spectrum of immature and peripheral TCL. Similarly to CUTLL1, treatment with TH-AG increased VEGFB and VEGFA mRNA levels in Jurkat (TCL/L), HuT-78 (CTCL), OCI-Ly12 (PTCL-NOS) and Karpas299 (ALCL-ALK+) cells. Increase in VEGF production was completely abrogated by knocking-down either V or β3 components with specific si-RNAs (vs. siRNA control) in CUTLL1, HuT-78 and OCI-Ly12 cells. Moreover, exposing HMEC1 endothelial cells to conditioned medium from CUTLL1 cells treated with TH-AG vs control increased they proliferation and migration (to 481 ± 118 cells from 206 ± 82 cells, respectively, p= 0.01). Importantly, the migration increase was completely abrogated when conditioned medium was obtained from CUTLL1 cells knocked-down for either V or β3 and treated with TH-AG. To determine whether targeting Vβ3 integrin can be of therapeutic benefit for TCL, we developed TCL xenografts in SCID mice using CUTLL1 cells transfected with si-control, si-V and si-β3, and monitored tumor growth and angiogenesis. We found that CUTLL1 transfected with si-V and si-β3 developed significant smaller tumors than si-control. Also, tumors from integrin knocked-down cells, showed decreased tumor vascularization (by CD31) and VEGF expression. To determine the translational impact of this strategy, we assessed the effect of cilengitide, a selective Vβ3 integrin inhibitor in phase 3 for glioma, in pre-clinical models of PTCL-NOS and ALCL-ALK+. For PTCL-NOS we xenografted OCI-Ly12 cells in NOD/SCID mice (n=12) and for ALCL-ALK+ we developed a patient-derived tumorgraft (PDT) in NSG mice (n=8). Cilentide treatment for 10 days (vs. vehicle), at human equivalent dose, induced tumor remission in both models (p