Inhibition of FAO in AML Co-Cultured with BM Adipocytes: Mechanisms of Survival and Chemosensitization to Cytarabine

Adipocytes are the prevalent type of stromal cells in adult bone marrow (BM), and fatty acids produced by adipocytes modulate the activity of signaling molecules. Leukemia cells are forced to continuously adapt to deficiency of nutrients and acquire chemoresistant profiles in the BM environment. We have previously shown that fatty acid metabolism is a key energy pathway for survival of acute myeloid leukemia (AML) cells in the adipocyte-abundant BM microenvironment (Tabe, Cancer Res. 2017) .

The novel fatty acid oxidation (FAO) inhibitor avocatin B is an odd-numbered carbon lipid derived from the avocado fruit, and avocatin B-induced apoptosis and growth inhibition in mono-cultured AML cells has been demonstrated previously (Lee, Cancer Res. 2015, Tabe, ASH. 2016) . However, in AML cells co-cultured with mesenchymal stem cells (MSC)-derived BM-adipocyte (BM adipocyte) mimicking BM microenvironment, the cytotoxic effects of avocatin B were significantly diminished compared to mono-cultured condition (decrease of specific cell death; THP1 14.4%, U937 25.0%, OCI-AML3 36.2%, 48 h, trypan blue cell count). We therefore investigated the adaptive molecular mechanisms against avocatin B cytotoxicity in AML cells co-cultured with BM adipocytes. Under BM adipocyte co-culture conditions, avocatin B induced increase in reactive oxygen species (ROS) (cellROX, flow cytometry) and activation of stress-induced ATF4 and AMPK signaling; increase in phospho- (p-) AMPK and repression of p-S6, p-4E-BP1 (immunoblotting) in AML cells (THP1, U937, OCI-AML3). On the other hand, free fatty acid (FFA) and glucose uptake was stimulated after avocatin B treatment (fluorometric assay), which was accompanied by upregulation of FABP4 and CPT1 mRNA (Q-RT-PCR) and lactate and alanine (CE-MS). These changes might reflect the compensatory response to a shortage of FFA supply to the mitochondria.

AMPK is known to regulate energy metabolism including FAO and glycolysis dependent / independent gene transcription through mTOR inhibition. To access the role of AMPK in anti-leukemic effects of avocatin B, we utilized AMPK knockdown (sh AMPK ) OCI-AML3 cells. In mono-culture condition, sh AMPK OCI-AML3 cells were less sensitive to avocatin B, showing higher baseline level of glucose uptake than parental shC OCI-AML3 cells, which was further increased by avocatin B. Of note, co-culture with BM adipocytes induced significant increase of glucose uptake in sh AMPK cells, which was not affected by avocatin B. In parental OCI-AML3 cells, FFA uptake was repressed by avocatin B in mono-culture but increased in BM adipocyte co-culture conditions, concordant with other tested AML cells. However, avocatin B-stimulated FFA uptake was not observed in sh AMPK OCI-AML3 cells regardless of the absence or presence of BM adipocytes. These results indicate that AMPK plays a role in adaptive uptake regulation of glucose and FFA in response to avocatin B-induced FAO suppression. We next performed ATF4 knockdown experiments to determine the role of ATF4. Whereas no significant difference in apoptotic responses to avocatin B was observed between mono-cultured sh ATF4 - and shC-OCI-AML3 cells, sh ATF4 OCI-AML3 cells demonstrated lower sensitivity to avocatin B compared to shC OCI-AML3 cells under the BM adipocyte co-culture condition (p<0.05). These findings suggest that avocatin B induced ATF4 activation facilitates apoptosis of AML cells co-cultured with BM adipocytes

We further investigated the effects of combinational treatment of avocatin B and conventional anti-AML therapeutic agent cytarabine (AraC). AraC itself increased ROS, and avocatin B / AraC combination synergistically induced cell growth inhibition of THP1 cells under BM adipocyte co-culture condition (Combination Index; mono-culture 0.7, co-culture 0.4). Immunoblotting analysis revealed that avocatin B induced p-S6 repression was accelerated by co-treatment with AraC irrespective of BM adipocyte presence, suggesting the efficient inhibition of mTOR by avocatin B / AraC combination under BM adipocyte co-culture condition.

Taken together, these findings highlight the potential of AraC and FAO inhibitor combination which abrogates the adaptive pro-survival mechanisms and chemoresistance of BM residual AML cells.

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