Endothelial cell derived angiocrine support of acute myeloid leukemia targeted by receptor tyrosine kinase inhibition

Abstract

In acute myeloid leukemia (AML), refractory disease is a major challenge and the leukemia microenvironment may harbor refractory disease. Human AML cell lines KG-1 and HL-60 expressed receptors also found on endothelial cells (ECs) such as VEGFRs, PDGFRs, and cKit. When human AML cells were co-cultured with human umbilical vein endothelial cells (HUVECs) and primary bone marrow endothelial cell (BMECs), the AML cells were more resistant to cytarabine chemotherapy, even in transwell co-culture suggesting angiocrine regulation. Primary BMECs secreted significantly increased levels of VEGF-A and PDGF-AB after exposure to cytarabine. Pazopanib, a receptor tyrosine kinase inhibitor (RTKI) of VEGFRs, PDGFRs, and cKit, removed EC protection of AML cells and enhanced AML cell sensitivity to cytarabine. Xenograft modeling showed significant regression of AML cells and abrogation of BM hypervascularity in RTKI treated cohorts. Together, these results show direct cytotoxicity of RTKIs on AML cells and reversal of EC protection. Combining RTKIs with chemotherapy may serve as promising therapeutic strategy for patients with AML.

Fig. 1.

Endothelial cells protect leukemia cells from chemotherapy induced apoptosis in co-culture. (A) Flow cytometry shows HL-60 cells express VEGFR1 while KG1 cells express VEGFR1 and VEGFR2. PCR demonstrates HL-60 cells express c-Kit while KG-1 cells express c-Kit and PDGFR-a. (B) KG1 cells were seeded in direct co-culture with HUVECs and treated with varying concentrations of cytarabine. After 48 h of treatment the cells were stained with Annexin V and PI, and then analyzed by flow cytometry. KG1 AML cells were more resistant to cytarabine treatment when in co-culture with ECs (p < 0.001). (C) KG1 cells were seeded in transwell co-culture with HUVECs and treated with varying concentrations of cytarabine. After 48 h of treatment the cells were stained with Annexin V and PI, and then analyzed by flow cytometry. KG1 AML cells were more resistant to cytarabine treatment when in transwell co-culture with ECs (p < 0.001). (D) Angiogenic cytokine secretion from primary human BMECs. After cytarabine chemotherapy, BMECs secreted significantly increased VEGF-A (40-fold, p < 0.05) and PDGF-A/B (1.6-fold, p < 0.005). RFI = Relative Fluorescence Intensity. (E) BMEC viability after 24 h treatment with cytarabine chemotherapy at clinically relevant concentration, 20 μM. Flow cytometry analysis revealed no reduction in viability.

Fig. 2.

In vitro flow cytometry apoptosis analysis shows an additive effect of treatment with pazopanib and cytarabine. (A) KG-1 cells exposed to pazopanib and cytarabine demonstrate modest reduction in viability by XTT assay. In combination, the effect is additive, significantly decreasing viability of AML cells relative to controls. *p-values <0.05 for all treatment samples, compared to untreated control. (B) In the presence of an EC feeder layer, the addictive effect of pazopanib and cytarabine on AML cells persists. *p-values <0.05 for all treatment samples, compared to untreated control. (C) Western blot of phosphorylated SRC, a downstream target of RTK signaling, shows a dose-dependent decrease in phosphorylation with pazopanib exposure.

Fig. 3.

Pazopanib treatment decreases engraftment equivalent to cytarabine and disrupts microvessel formation. (A) Pazopanib and cytarabine decreased the level of HL-60 engraftment equally. The decrease in engraftment for all treatment groups was determined by flow cytometry analysis. HL-60 engraftment for untreated mice was approximately 34%. HL-60 engraftment for treated mice ranged 12–18%. *p-values <0.05 for all treatment samples compared to untreated control. (B) Pazopanib treatment leads to a reduction in the number of blood microvessels within leukemic cores compared to other treatment groups. Quantification of microvessels based on MECA-32⁺. Values represent mean ± SEM. p < .05. (C) Representative bone marrow micrographs showing microvessel density (MECA-32 endothelial stain, red) and nuclear staining (DAPI, blue) of mouse femurs after transplant with human AML cells (HL-60) and treatment with pazopanib and cytarabine. Untreated animals with robust AML engraftment showed high microvessel density in the bone marrow. Pazopanib alone markedly decreased microvessel density in the bone marrow. Cytarabine also decreased microvessel density. The combination of pazopanib and cytarabine also caused marked reduction in blood vessels within bone marrow. (D) Pazopanib and cytarabine decreased levels of bone marrow KG1 engraftment equally. The decrease in engraftment for all treatment groups was determined by flow cytometry analysis. KG1 engraftment for untreated mice ranged 59–80%. KG1 engraftment for treated mice ranged 33–78%. **p-values <0.005, *p-value <0.05 for all treatment samples compared to untreated control.