FABP4 Regulates Fatty Acid Transfer from Bone Marrow Adipocytes to Acute Myeloid Leukemia Blasts

Introduction

The incidence of acute myeloid leukemia (AML) is strongly related to age with poor survival rates in the elderly population. AML blasts isolated from the bone marrow undergo spontaneous apoptosis in culture suggesting its microenvironment in the bone marrow (BM) contains cells and factors favorable to the progression of the disease. The BM microenvironment consists of many cell types not directly of the hematopoietic lineage, including bone marrow adipose tissue (MAT), which accounts for circa 50% of the BM volume in the axial skeleton of adults. We hypothesize that MAT may contribute to the cancerous hallmarks exhibited by the AML blasts in the bone marrow. Here we examine the relationship between cancer associated MAT and AML blast survival and proliferation. We describe how this association favors the survival of AML blasts by the transfer of free fatty acids (FFA) released from MAT to AML blasts via the chaperone protein fatty acid binding protein 4 (FABP4). We also show FABP4 to be a critical player in fatty acid transport and therefore the survival of AML blasts in the bone marrow.

Methods

To investigate the role of MAT in regulating AML survival we used primary AML blasts and marrow derived adipose tissue obtained from patient's bone marrow following an informed consent. MAT was co-cultured with primary AML blasts in vitro and the proliferation and survival of the blasts was determined by BrdU incorporation and Annexin V/PI staining respectively. Immunocytochemistry using lipid specific dye, CD34 antibody and nuclear staining was performed to determine lipid storage in primary AML blasts. Western blot analysis was used to determine the level of phosphorylated hormone sensitive lipase (HSL) and fatty acid binding protein 4 (FABP4) in adipocytes. ß oxidation in AML blasts was determined by transcript levels of CPT-1a and ACOX-1. FABP4 concentration was also assayed in MAT and MAT/AML co-cultures by ELISA. Adipocyte FABP4 was knocked down by shRNA and a small molecule inhibitor (BMS309403) was used for pharmacological inhibition.

Results

Results show that in vitro primary AML blasts demonstrate increased survival and proliferation when co-cultured on MAT. Immunocytochemistry using lipid specific dye on CD34+ primary AML blasts revealed presence of neutral lipids within the blasts, which following a 48 hour incubation in monoculture was significantly depleted. Primary AML blasts/MAT co-cultivation caused lipid accumulation in AML blasts and lipolysis of MAT indicated by increased levels of glycerol and FFA in the co-culture media compared to control. Immunoblotting of co-cultured MAT showed increased levels of lipolysis associated factors, phosphorylated HSL and perilipin with a decreased expression of FABP4 compared to MAT monocultures. An apparent increase in ß oxidation was also revealed in AML blasts indicated by the transcriptional activation of ß oxidation related genes. FABP4 MAT transcript levels were shown to increase significantly suggesting an increased production and subsequent trafficking of FABP4 from MAT. Pharmacological inhibition and shRNA mediated knock-down of FABP4 showed a significant decrease in survival and proliferation of AML blasts cultured with adipocytes. Finally investigations also revealed that AML blasts cultured with adipocytes had depleted lipid accumulation in response to FABP4 inhibition.

Conclusion

Here we report that lipid trafficking between MAT and AML supports survival and proliferation of the leukemic blasts in-vitro. We show that FABP4 is transcriptionally up-regulated in both AML and MAT which mediates the transport of FFA from adipocytes to the leukemic blast. Furthermore, the inhibition of FABP4 significantly reduces AML survival. Together, our data shows evidence for the relationship between MAT and AML blasts by fatty acid transfer identifying FA metabolism as a potential therapeutic target for this aggressive disease.