Anthracycline Treatment Induces Extracellular Calreticulin in Acute Myeloid Leukemia

Abstract 1376

The myeloid key transcription factor CEBPA (CCAAT-enhancer binding protein alpha) is blocked by the mRNA binding protein calreticulin (CRT) thereby affecting myeloid differentiation. We previously reported that the unfolded protein response (UPR) is activated in 24.5% of AML patients, involving the up-regulation of endoplasmatic reticulum (ER) stressors, such as CRT. In addition, anthracycline treatment was shown to trigger the UPR and to specifically induce CRT cell membrane surface expression in some solid tumor types. This ecto-CRT is specifically targeting dendritic cells and initiates immunogenic cell death of cancer cells by acting as an “eat-me” signal. However, UPR induced translocation of CRT in leukemogenesis has hardly been investigated so far.

This study aimed at analyzing anthracycline induced CRT exposure and extracellular release of CRT in leukemic cells as well as at elucidating the candidate pathways involved such as the UPR transducers PERK, ATF6 and IRE1.

In leukemic cell lines, we observed that anthracycline treatment was inducing CRT expression on the cell membrane surface within 2 hours. This effect was caused by intracellular translocation, but not de novo synthesis. Exposure of CRT was decreased by Brefeldin A, which blocks the vesicle transport from the ER to the Golgi apparatus within 2 hours of treatment, suggesting that exocytosis is critical for CRT delocalization. By specific inhibition of the PERK pathway of the UPR using Salubrinal, we observed reduced CRT expression on the cell membrane. In contrast, blocking the ATF6 or the IRE1 pathway did not affect CRT exposure.

Mean CRT levels on the cell membrane of blasts from 53 AML patients at diagnosis were significantly (21-fold; p<0.0005) increased compared to CRT levels of myeloid cells from healthy volunteers (n=75). Using an ELISA assay investigating serum from 104 AML patients at diagnosis and 86 healthy volunteers, we found that extracellular CRT was significantly (2.2-fold; p<0.0001) increased in AML patients compared to healthy controls. We observed a more favorable outcome in AML patients with increased CRT expression on the cell membrane, as well as in patients with higher levels of extracellular CRT in the serum. Finally, we identified a strong correlation between CRT serum levels and CRT expression on the cell surface of leukemic cells. Ongoing studies aim at elucidating the mechanisms of CRT secretion from the cell membrane to the serum.

In conclusion, CRT translocation to the cell membrane and increasing levels of secreted CRT in the serum represent early events of anthracycline treatment in leukemic cells, and both are associated with a more favorable outcome in AML patients.