AML Regression by Vascular Disruption with OXi4503 and Anti-Angiogenesis with Bevacizumab.
Authors: Madlambayan Gerard J , Meacham Amy , Hosaka Koji , Mir Saad , Jorgensen Marda , Scott Edward W , Siemann Dietmar W. , Cogle Christopher R. 
Published: October 2019

Abstract
Abstract 1031
Poster Board I-53
Acute myelogenous leukemia (AML) cells and endothelial cells depend on each other for survival and proliferation. Strategies that target single pathways in this co-dependent relationship have demonstrated limited efficacy in treating AML. Thus, it was hypothesized that a multi-target anti-vascular strategy is effective in treating leukemia. Recently, a novel tubulin-binding combretastatin, OXi4503, was identified and displays potent vascular disruption. To investigate the effects of vascular disruption with OXi4503 on leukemia proliferation in vivo, we first established KG-1 subcutaneous chloromas in NOG mice. Chloromas in mice treated with OXi4503 (n=5) grew significantly slower after treatment compared to control animals (n=5) (p=0.05). Furthermore, OXi4503-treated chloromas displayed a central core of necrotic cells with a surrounding rim of viable and highly vascularized tissue. When compared to control chloromas, OXi4503-treated leukemias showed significantly decreased blood vessels within leukemic cores (p<0.0001); however, viable rims showed increased microvessel density (p<0.01). Given this reactive angiogenic process and persistence of a viable leukemic rim after OXi4503 monotherapy, bevacizumab was added to inhibit VEGF activity. KG-1 chloromas in mice treated with bevacizumab alone (n=5) grew at the same rate as compared to control animals (p=0.58). In contrast, chloromas in combination-treated mice regressed in overall size and were significantly smaller in comparison to control mice (p=0.01) as well as bevacizumab monotherapy treated mice (p=0.0006). The chloromas in combination-treated mice were softer to palpation and were mainly necrotic without a persistent viable rim as observed with the OXi4503 alone treatments. TUNEL staining revealed widespread apoptosis throughout the entire leukemic mass. Microvessel density after combination treatment was markedly decreased when compared to control cohorts (p<0.0001) as well as bevacizumab-treated (p<0.0001) chloromas. To test the effects of OXi4503 and bevacizumab in a setting that more closely resembles the clinical presentation of leukemic infiltration of bone marrow, a systemic, primary AML model was used. Moreover, the primary human AML specimen harbored a high risk FLT3 ITD mutation. Sublethally irradiated NOG mice were transplanted intravenously with primary AML cells and then examined at 6-8 weeks to confirm leukemia engraftment. Mice were then randomly assigned to one of four treatment groups: bevacizumab alone (n=5), OXi4503 alone (n=5), combination of OXi4503 and bevacizumab at the same dosing regimens (n=5) or control (n=5). AML engraftment in bone marrow of bevacizumab-treated mice was comparable to control mice (2.25% vs. 4.02%; p=0.4472) with all mice showing engraftment. Furthermore, using PCR to detect FLT3 ITD mutations, there were no molecular remissions in any of the control and bevacizumab-treated animals. Mice treated with OXi4503 monotherapy resulted in a significant decrease of AML in bone marrow as compared to controls (0.02% vs. 4.02%; p=0.0082) with only 1 of 5 OXi4503-treated mice showing engraftment. In animals treated with combination OXi4503 and bevacizumab, none of five animals showed AML engraftment by flow cytometry. Molecular remissions were evident in both OXi4503 monotherapy and combination-treated mice (2 of 5 (40%) in each group). Bone marrow cellularity among treatment groups was similar. However, when compared to control bone marrows, microvessel density was decreased by bevacizumab alone (p=0.03), OXi4503 alone (p=0.0001) and combination OXi4503 plus bevacizumab (p<0.0001). Additionally, both Oxi4503 monotherapy and combination treatment resulted in a further decrease in microvessel density than bevacizumab alone (p=0.0008 and p=0.0002, respectively). Bone marrows from AML-bearing control mice revealed no HIF-1αa expression. In contrast, bone marrows from leukemias treated with bevacizumab, OXi4503 and combination showed expression of HIF-1αa in keeping with the decreased microvessel density observed in the BM of treated mice. Together, the data highlight the importance of blood vessels in primary AML proliferation and engraftment. In addition, a multi-target anti-vascular strategy of vascular disruption and anti-angiogenesis may be a promising strategy in the treatment of AML, potentially including high-risk leukemia.

Disclosures:
Siemann: OXiGene: Membership on an entity's Board of Directors or advisory committees.