Chemosensitizing Leukemia By Targeting the Leukemia Microenvironment with Vascular Disrupting Combretastatins

Background: Refractory disease is the greatest challenge in treating leukemia. We recently discovered that blood vessels serve as a protective sanctuary for acute myeloid leukemia (AML) and developed a novel combretastatin, OXi4503, to disrupt this vascular niche. We translated our findings into a phase I clinical study for patients with refractory AML (ClinicalTrials.gov NCT01085656). An interim analysis of this trial suggested that optimal treatment of AML will likely require more than a single microenvironment targeting agent. Other AML trials, such as those with single agent sunitinib, bevacizumab, or pazopanib also support this concept. Therefore, we examined the effects of combining microenvironment targeting agents with cytotoxic chemotherapy.

Methods: Bone marrow endothelial cells (BMECs) were isolated from bone marrow of healthy individuals. In vitro co-cultures of human AML (KG1) and BMECs were used to examine various treatments with cytarabine (Ara-C), azacitidine, and vascular disrupting combretastatins (CA1 and CA4). Cell death was analyzed by PI staining and flow cytometry. Cell cycle distribution was analyzed using 7-AADand BrdU staining. Given that combretastatins bind reversibly to B-tubulin, immunostaining of treated cells was performed using antibodies to B-tubulin and VCAM-1 and analyzed by fluorescence microscopy. Finally, the drug combinations with optimal in vitro effects were then tested in vivo using a xenograft model of human AML.

Results: Co-culturing AML cells with BMECs afforded the leukemia cells a protective survival advantage despite treatments with Ara-C or azacitidine, further supporting the evolving understanding that leukemia cells located in or near the vascular niche may be a source of refractory disease. BMECs treated with combretastatins showed distinct degradation and disruption of microtubule cytoskeleton as well as an internalization of the adhesion molecule VCAM-1 one hour after combretastatin treatment. The combretastatin-treated BMECs were still viable but showed dramatic reduction in migratory function and adhesion molecule expression. When AML cells were co-cultured on combretastatin-treated BMECs, leukemia cell adhesion was significantly reduced. Treatment with chemotherapy agents, Ara-C or azacitidine, in co-cultures of AML + combretastatin-treated BMECs showed significant reductions in AML cell viability (Ara-C: 71.4% (Vehicle) vs 54.4% (CA1) and 59% (CA4), p < .001; azacitidine: 73.5% (Vehicle) vs 56.7% (CA1) and 59.2% (CA4), p < .001), suggesting that combretastatin pre-treatment reverses EC protection of AML cells. Furthermore, treatment of leukemia cells with combretastatins induced a bottleneck at the G2/M checkpoint, resulting in increased proportion of leukemia cells at the S phase (39.1% (CA1) and 38.4% (CA4) vs. 29%, p < .001) and at the G2/M phase (48.2% (CA1) and 45.9% (CA4) vs. 10.4%, p < .0005), suggesting that combretastatins sensitize AML cells against cell-cycle cytotoxic agents. Finally, human AML xenograft experiments showed a complete clearance of AML cells in the bone marrow after combination treatment with low dose CA1P (OXi4503) and Ara-C or azacitidine; whereas Ara-C or azacitidine alone showed limited effect on AML engraftment in the bone marrow.

Conclusion: BMECs protect AML cells from conventional cytotoxic chemotherapy partly through adhesion factors. However, this protection can be removed by pretreating the leukemia microenvironment with vascular disrupting combretastatins. Combination combretastatin + cytotoxic chemotherapy sensitizes AML cells and leads to improved leukemia regression. Results of this study support a phase IB study of OXi4503 and chemotherapy in patients with AML and advanced myelodysplastic syndrome.