Dll4 Blockade Modifies the Bone Marrow Vascular Niche and Improves Donor Bone Marrow Cells for Bone Marrow Transplant

Abstract 1321

The Delta:Notch signaling pathway regulates multiple aspects of hematopoietic cell differentiation and function and more recently Delta-like 4 (Dll4) has been shown also to regulate adult neo-vascularization (angiogenesis), namely in tumors, which led to the development of therapeutic strategies targeting Dll4 for the treatment of different cancers. Nevertheless, the current therapeutic strategies to target Dll4, which were suggested to result in non-functional vessels in the tumors, appear also to affect the vasculature of normal organs. Given the importance of the Bone marrow (BM) vascular niche in regulating hematopoiesis, we hypothesized Dll4 targeting might also affect BM function. In the present study, we describe 2 means of targeting Dll4: one using Dll4 specific neutralizing monoclonal antibodies (systemic treatment), and another using VECad^CreERT2Dll4^lox/lox mice, where Dll4 is specifically ablated on VE-Cadherin expressing cells. Using these 2 models, we investigated if Dll4 targeting affected the BM vascular niche and consequently affected hematopoietic recovery following irradiation and BM recovery in a transplant setting.

First, we show that the genetic ablation of Dll4 on VE-Cadherin⁺ vessels (VECad^CreERT2Dll4^lox/lox mice) and also anti-Dll4 antibody treatment of WT sub-lethally irradiated mice (300rad) does affect the BM vascular niche, resulting in increased megakaryocytes (CD41⁺), CD31⁺ and VE-Cadherin⁺ vessels, without significant changes on CD105⁺ vessels. Interestingly, the systemic anti-Dll4 treatment of non-irradiated and sub-lethally irradiated WT mice increased the BM and peripheral blood myeloid (CD11b⁺) content and promoted hematopoietic progenitor cell (Sca1⁺Flk1⁻) mobilization, as determined by FACS analysis. The genetic model (VECad^CreERT2Dll4^lox/lox mice) showed no changes in hematopoietic cells percentage or mobilization.

However, BM recovery in a transplant setting was improved in both models. Lethally irradiated WT mice transplanted with anti-Dll4 treated BM following sub-lethal irradiation or BM from VECad^CreERT2Dll4^lox/lox mice showed faster hematopoietic recovery than control mice transplanted with WT BM alone.

Taken together, we show that Dll4 blockade perturbs the BM vascular niche, increasing VE-Cadherin⁺ and CD31⁺ vessel number and also megakaryocyte BM content, both in the setting of Dll4 genetic ablation (using VECad^CreERT2Dll4^lox/lox mice) and also using a systemic approach to target Dll4. However, these vascular changes do not, at least following sub-lethal irradiation, significantly affect the BM recovery or affect the normal distribution of the hematopoietic populations. Nevertheless, the significant effects observed in a BM transplant setting (both where donor had been exposed to the systemic anti-Dll4 blockade and where Dll4 had been specifically ablated on VE-Cadherin⁺ vessels) suggest the existence of cell autonomous (and BM vascular niche-autonomous) effects of Dll4 targeting which deserve further detailed analysis. These data also suggest targeting Dll4-expressing vessels in the setting of BM malignancies may have therapeutic benefit, since it appears to have limited effects on normal BM/hematopoietic function. We are currently investigating the therapeutic efficacy of Dll4 targeting using the genetic model and the antibody-based approaches, in the setting of malignant myeloma and also acute leukemias.