Molecular Mechanisms Of Adipocyte-Leukemia Interactions Revealed By The Quantitative Proteomics Technology: Pro-Survival Function Of Adipocyte-Derived Free Fatty Acids On Acute Monoblastic Leukemia Mitochondrial Biogenesis
Authors: Tabe Yoko , Jin Linhua , Kazuno Saiko , Fujimura Tsutomu , Matsushita Hiromichi , Ueno Takashi , Miida Takashi , Andreeff Michael , Konopleva Marina 
Published: October 2019

Abstract
Acute monocytic leukemia (AMoL) is a distinct subtype of AML with an average 3-year overall survival of 31% (Tallman, J Clin Oncol 2004), and the majority of patients die from disease progression after relapse. Adipocytes represent an essential component of the adults bone marrow (BM) microenvironment (Battula, Blood 2013, Tabe, Blood 2004, Konopleva, Blood 1999), and promote survival of monoblastic leukemia cells (Tabe, ASH, 2012).
In this study, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ, Applied Biosystems) to examine the molecular mechanism of pro-survival properties of BM adipocytes in co-culture with AMoL. The specific pathway alterations were identified by Metacore (GeneGo, St. Joseph, MI). We first confirmed the more prominent protective role of BM-derived adipocytes as compared to BM stromal cells (MSC) on AMoL cells. Co-culture with adipocytes derived from differentiated MSCs significantly protected U937 and primary AMoL cells (n = 5) from serum starvation-induced apoptosis compared to MSCs (U937 p = 0.029, primary samples p = 0.034). Gene expression analysis demonstrated striking upregulation of mRNA expression levels of CD36 (4.1+0.4-fold),FABP4 (569.3+40.1-fold), and PPARG (2.1+0.2-fold) in U937 cells co-cultured with adipocytes compared to the MSC co-cultured cells. Notably, Bcl-2 mRNA expression in U937 cells was significantly upregulated after adipocyte co-culture compared to MSCs (5.4+0.3-fold). We previously reported that fatty acids (FAs) promote leukemic cell survival via leukemia cells metabolic shifting from pyruvate oxidation to fatty acid oxidation for glycolysis, which links to the Bcl-2 anti-apoptotic machinery (Samudio, J Clin Invest. 2010). Mature adipocytes are capable of releasing abundant free fatty acids (Fas), the essential ligands of a nuclear receptor PPARgamma (PPARG), which are internalized by leukemic cells via scavenger receptor CD36 and ligate PPARG through fatty acid binding protein 4 (FABP4). Enhanced transcriptional activity of FA-ligated PPARG may in turn stimulate glucose metabolism, regulate fatty acids storage, maintain mitochondrial membrane potential, and prevents apoptosis by upregulating anti-apoptotic Bcl-2 resulting in further increase of its downstream target genes including CD36 and FABP4. To gain further insights into metabolic alterations induced in adipocyte-leukemia co-cultures, we performed iTRAQ proteomic analysis of U937 cells cultured in the presence of MSCs or adipocytes. A total of 1,610 proteins were detected by this technology, with 106 proteins differentially expressed between co-culture conditions; 50 genes were up-regulated and 56 genes were down-regulated by adipocytes co-culture compared to MSCs. Among the upregulated proteins, FA synthase (p = 0.007) ATP-citrate synthase responsible for cytosolic acetyl-CoA synthesis (p = 0.019); and glyceraldehyde-3-phosphate dehydrogenase (p = 0.028) involved in the activation of glycolysis and gluconeogenesis pathways (p< 0.0001) were identified. Of note, 20% of the upregulated proteins (10/50) were ribosomal proteins, accompanied by an increase in six DNA elongation or replication related proteins. Anti-apoptotic chaperone proteins known to assist ribosome biogenesis, HSP70, HSP90alpha and HSP90beta were also increased by adipocyte co-culture (p<0.01). The mass spectrometry further revealed more prominent repression of Cytochrome c (p=0.01) as well as the proline metabolism pathway (p=0.0011) under adipocyte co-culture conditions, consistent with inhibition of mitochondrial apoptosis of U937 cells.

Summary
our results indicate that abundant FAs produced by BM adipocytes support AMoL cell survival via stimulation of PPARG transcription which directly increases the FA metabolism through uptake and translocation of FAs by CD36 and FABP4 in AMoL cells. FA metabolism in turn facilitates supply of Acetyl-CoA via oxidative phosphorylation, which drives the TCA cycle; and enhances ribosomal biogenesis of AMoL cells, resulting in pro-survival signaling in AMoL cells. It is conceivable that increased adipocyte content of the BM in adult AML patients may promote leukemogenesis and negatively affect the responsiveness to chemotherapy.

Disclosures:
No relevant conflicts of interest to declare.