Potent Effects Of The Vascular Disrupting Agent, ASA404 (DMXAA) On The Marrow Microenvironment In Preclinical Human Leukemia and Lymphoma Models
Authors: Portwood Scott , Lal Deepika , Schaefer Christopher W , Uwazurike Odochi , Spernyak Joseph A , Seshadri Mukund , Wang Eunice S. 
Published: October 2019

Abstract

Background
Tumor vascular disrupting agents (VDAs) represent a novel class of anti-cancer therapeutics with the unique ability to rapidly destroy established tumor vasculature and induce ischemic necrosis. In contrast to most VDAs which are derivatives of anti-tubulin agents, ASA404 (5,6-dimethylxanthenone-4-acetic acid, DMXAA) is an unique non-steroidal anti-inflammatory VDA which induces potent host immune responses and disrupts tumor vasculature in solid tumor mouse models.

Methods
The overall aim of these studies was to examine the effects of ASA404 treatment on tumor growth in preclinical human acute myeloid leukemia (HEL, HL60) and lymphoma (Raji) xenograft models. We specifically investigated the effects of drug treatment on host immune and inflammatory responses, marrow vasculature, and initial tumor engraftment. Disease progression in vivo was measured using small animal magnetic resonance imaging and bioluminescent imaging of luciferase-transfected human tumor cells. Host cytokine and chemokine levels in the plasma and other tissues (marrow, spleen, liver) were determined by multiplex flow cytometric assays.

Results
In local acute myeloid leukemia (HEL-luciferase) xenografts, a single dose of ASA404 (25 mg/kg) markedly enhanced tumor vascular permeability, as determined by magnetic resonance imaging at 4 hours. This was followed by vascular disruption and central necrosis at 24 hours, as determined by immunohistochemistry. In systemic leukemia xenografts, a single dose of ASA404 enhanced marrow vascular permeability and induced several-fold elevations in host chemokine levels (G-CSF, IL-6, MCP-1, KC) in circulating plasma as well as bone marrow and spleen cells (MIG, G-CSF, MIP-1alpha, IP-10). Administration of ASA404 4-24 hours prior to inoculation of human acute myeloid leukemia (HEL) or lymphoma (Raji) cells in immunodeficient mice delayed disease progression and prolonged overall survival. Continuous ASA404 treatment (20 mg/kg twice weekly) for up to three weeks in established systemic leukemia and lymphoma xenografts resulted in marrow vascular disruption and significant inhibition of tumor growth. Prolonged ASA404 treatment was associated with elevations in circulating host TNF-a levels and increased numbers of macrophages within the leukemic marrow microenvironment. Combination ASA404 and doxorubicin therapy further improved the overall survival of HEL and Raji-engrafted mice as compared with AS404 or doxorubicin alone. Quantification of doxorubicin levels by HPLC in local tumor xenografts and leukemic marrow samples revealed several-fold higher doxorubicin levels following ASA404 vs. vehicle (PBS) treatment, consistent with ASA404-mediated improvements in leukemia drug delivery. Preliminary data suggest a therapeutic benefit of combination ASA404 and cytarabine therapy in the same xenograft models.

Conclusions
Our results demonstrate that ASA404 exerts anti-tumor activity in human acute myeloid leukemia and lymphoma xenografts via myriad unique effects on the marrow microenvironment. Single dose administration of ASA404 results in rapid and marked alterations in multiple host cytokine/chemokine levels in association with enhanced tumor vascular permeability and impaired in vivo leukemia cell engraftment. Prolonged drug administration leads to pronounced vascular disrupting effects and inhibition of tumor growth. Combination regimens of ASA404 with anthracycline or cytarabine lead to further improvements in anti-tumor activity and overall survival in leukemia-bearing mice. These data suggest that ASA404/DMXAA may be worthy of further clinical investigation for the treatment of aggressive hematological malignancies.

Disclosures:
No relevant conflicts of interest to declare.