P461: the flt3 receptor is involved in the endoplasmic reticulum homeostasis and glucose metabolism status in acute myeloid leukemia

Background: Acute myeloid leukemia (AML) is the most common myeloid tumour in adults. ITD mutations in the FLT3 receptor appear in 30% of cases and is associated with poor disease outcome. This mutation prevents the correct processing of this receptor in the endoplasmic reticulum (ER) and it is retained in this organelle. In addition to being a cellular compartment for protein synthesis and processing, the ER plays an important role in the regulation of tumor metabolism, specifically mitochondrial metabolism, through the mitochondria-associated membranes (MAM). Interestingly, we and other have previously described differences in tumor glucose metabolism in FLT3 wild-type and ITD AML cell lines. Aims: The main objective of our study was to study if the accumulation of FLT3-ITD affects the functioning of the ER, and if it has any role in the regulation of glucose metabolism in AML. Methods: Experiments were performed on wild-type and FLT3-ITD AML cell lines, as well as on stably transfected FLT3 and FLT3-ITD Ba/F3 cell lines. Protein aggregation, ROS, GSH levels, mitochondrial membrane potential and calcium levels were determined by Thioflavin T, DCFH-DA, Thioltracker Violet, Rhodamine and Rhod-2 fluorescence, respectively. Protein detection was performed by Western Blot. Cell viability and death was analysed by cell count and Trypan Blue exclusion. Results: The ER depends on GSH to neutralize reactive oxygen species (ROS) produced in the process of protein folding. We found FLT3-ITD cell lines to have lower levels of protein aggregates, ROS and GSH, additionally to greater levels of ERO1, an ER protein folding oxidorreductase. We verified that FLT3-ITD cell lines were less sensitive to protein folding blocking agents, DTT and 2-mercaptoethanol, compared to wild-type cell lines, which showed high levels of cell death after the treatment. Under ER stress conditions, such as an accumulation of misfolded proteins in the ER, the unfolded protein response (UPR) is activated. We found FLT3-ITD cell lines to have lower levels of UPR proteins (PERK and IRE), together with the main chaperone in the ER, BiP. Altogether, our data suggest that FLT3-ITD cell lines have less ER stress, possibly because they rely on more efficient protein folding mechanisms. To confirm the importance of protein folding process in acute myeloid leukemia, we tested the effect of ERO1 inhibitor and found it to have a greater impact in FLT3-ITD cell lines. In order to determine a possible relation between the mutated FLT3 receptor and ERO1, we used stably transfected Ba/F3 cell line carrying the normal or mutated FLT3 receptor. In agreement with results in human cell lines, we found ERO1 to be overexpressed on FLT3-ITD Ba/F3 cells. The IP3R and GRP75 are proteins involved in MAM formation and promote oxidative phosphorylation through the transfer of calcium from the ER to the mitochondria. We found these proteins to be overexpressed in AML cell lines, as well as the BaF3 cell model, carrying the wild-type FLT3 receptor together with greater mitochondrial activity, reflected by increased levels of mitochondrial calcium, mitochondria membrane potential and lower cytotoxic response to oxamate (LDH inhibitor). Summary/Conclusion: In this study we describe that AML cell lines carrying the ITD mutation have more efficient protein folding mechanisms in which ERO1 seems to play an important role. Additionally, we show that the ER could be involved in the regulation of mitochondria glucose metabolism by directly interacting with this organelle through MAM. Both processes could be directly regulated by the FLT3 receptor. More studies are needed to fully understand this process in view of describing new therapeutic targets. Keywords: Mitochondria, FLT3, Acute myeloid leukemia, Signaling