P472: THE FLT3-ITD RECEPTOR CAN PROMOTE PROTEIN FOLDING IN THE ENDOPLASMIC RETICULUM THROUGH THE OXIDOREDUCTASE ERO1

Abstract

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy and the most common myeloid tumour in adults. Internal tandem duplications (ITD) in FLT3 receptor appear in 30% of cases and is associated with poor disease outcome. This receptor is synthesized and processed in the endoplasmic reticulum (ER) before being transported to the plasma membrane, where it is activated by ligand binding. Upon activation, it regulates cell differentiation, proliferation, and survival. Mutations in FLT3, such as ITD mutations, not only constitutively activate this receptor, but also prevent its correct processing in the endoplasmic reticulum. Therefore, it is mostly retained in this organelle. Aims: We decided to study if the aberrant accumulation of FLT3-ITD affects the functioning of the ER. 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, reactive oxygen species (ROS) and glutathione (GSH) levels were determined by Thioflavin T, DCFH-DA and Thioltracker Violet fluorescence, respectively. Protein detection was performed by Western Blot. Cell viability and death was analysed by cell count and Trypan Blue exclusion. Results: Protein folding in the ER generates reactive oxygen species (ROS). Antioxidant molecules such as GSH are needed to maintain redox homeostasis. We found that FLT3-ITD cell lines have lower levels of protein aggregates, as well as ROS and GSH. This data could suggest that mutant cell lines have more efficient protein folding mechanisms. In fact, among the ER proteins that are involved in the protein folding process, we found the oxidoreductase ERO1 to be increased in FLT3-ITD cell lines. Supporting our findings, we saw that FLT3-ITD cell lines were less sensitive to protein folding blocking agents, DTT and 2-mercaptoethanol. This was reflected by a slight decrease in cell proliferation compared to wild-type cell lines. We confirmed that the decrease in cell viability in FLT3-ITD cell lines was not accompanied by cell death, as opposed to wild-type cell lines. Under ER stress conditions, such as an accumulation of misfolded proteins in the ER, the unfolded protein response (UPR) is activated. We decided to study if there were differences in the basal levels of UPR by western blot. We found FLT3-ITD cell lines to have lower levels of UPR, reflected by lower protein expression of the active forms of PERK and IRE (p-PERK and p-IRE respectively), together with the protein BiP, the major chaperone found in the ER and binds to misfolded proteins. Altogether, our data suggest that FLT3-ITD cell lines have less ER stress, reflected by lower levels of chaperones and UPR proteins, 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 (EN460), and found it to have a greater impact in FLT3-ITD cell lines. In order to determine a possible relation between the ITD mutation in the FLT3 receptor and ERO1, we used stably transfected Ba/F3 cell lines carrying the normal or mutated FLT3 receptor. We found ERO1 to be overexpressed on FLT3-ITD Ba/F3 cell lines. Summary/Conclusion: Current AML treatments are mainly based on the administration of FLT3 inhibitors, with limited success. Our data suggest that FLT3-ITD cell lines, with greater resistance to treatments, have more efficient protein folding mechanisms. This process should be studied in more detail to determine its potential as a therapeutic target for AML treatment.