Activation of the Unfolded Protein Response (UPR) Induces CEBPA mRNA Decay in AML Cells.

The myeloid key transcription factor CEBPA (CCAAT-enhancer binding protein alpha) is crucial for normal neutrophil differentiation, and cebpa-deficient mice lack mature granulocytes. In addition, deregulation of CEBPA function is a frequent event in AML patients. The endoplasmatic reticulum (ER) is involved in proper protein folding. Alterations in calcium levels or insufficient protein folding capacity leads to ER stress, thereby inducing rescue pathways that are commonly summarized as the unfolded protein response (UPR). The UPR aims either to re-establish ER homeostasis or to induce cell death. So far, induction of ER stress has been associated with a number of non-hematopoietic diseases. Here, we analyzed whether activated ER stress in leukemic cell lines and in AML patients affects the myeloid key regulator CEBPA. We induced ER stress in the leukemic HL60 cell line by treatment with 0.4mg/ml of Calcimycin (deregulation of calcium homeostasis) or with 3mg/ml of Tunicamycin (block of protein glycosylation). Consistently, we observed a rapid decrease of CEBPA mRNA to 10% after 6 hours of treatment. In accordance, CEBPA protein was no more detectable after 8 hours. Experiments in HL60 cells with Actinomycin D and Cycloheximide as well as competition experiments with various constructs of wild-type CEBPA sequences suggest a decay mechanism involving one or several elements in the 3’UTR of the CEBPA mRNA. We excluded microRNA-124a, a previously identified CEBPA regulator acting through the 3’UTR, to be involved. The precise elements mediating ER stress induced CEBPA mRNA decay remain to be elucidated. We also determined the expression of CEBPA mRNA and mediators of UPR - such as CHOP and GRP78 as well as alternative splicing of the XBP1 mRNA - in leukemic cells from 76 AML patients of all FAB subtypes. We found that AML patients with induced ER stress (13%) tended to have two-fold lower CEBPA mRNA expression, thus underlining the in vitro results reported above. In conclusion, our experiments indicate activated ER stress to be involved in a significant subgroup of AML patients. Moreover, it suggests that activation of the UPR in myeloid leukemic cells efficiently induces 3’UTR mediated decay of CEBPA mRNA expression, thereby contributing to the block in myeloid differentiation in these leukemias.