The Unfolded Protein Response (UPR) Is Activated in Human Acute Myeloid Leukemia (AML) and Suppresses Translation of the CCAAT/Enhancer Binding Protein-Alpha (CEBPA) by Induction of the RNA-Binding Protein Calreticulin

Deregulation of the myeloid key transcription factor CCAAT/enhancer binding protein alpha (CEBPA) is a common event in AML patients. We previously reported that the RNA-binding protein calreticulin efficiently blocks CEBPA translation and is specifically induced in core binding factor (CBF) leukemias. In addition, calreticulin is a crucial component of the unfolded protein response (UPR) which is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). In vitro studies suggested that the UPR is activated in some solid cancers and thereby involved in tumor development. The role of the UPR during leukemogenesis has not been addressed so far. Here, we investigated the induction of the spliced variant of the X-box binding protein 1 (XBP1s) as a marker for activated ER stress and determined the expression of key mediators of the UPR such as calreticulin and the 78-kDa glucose-regulated protein (GRP78) in leukemic cells from 92 consecutive AML patients. Increased expression of the XBP1 spliced variant was detected in 16 of 92 AML patients. Consistently, this group also had increased mRNA and protein levels of calreticulin and GRP78. In patients expressing the XBP1 spliced variant, CEBPA protein was hardly detectable in contrast to AML patients not expressing the XBP1 spliced variant. Moreover, treatment of myeloid leukemic cells with compounds activating the UPR – such as thapsigargin - confirmed rapid induction of XBP1s, GRP78 and calreticulin in myeloid cells whereas CEBPA protein levels decreased. In addition, conditional expression of calreticulin in U937 cells suppressed CEBPA protein. At the molecular level, we identified two functional ER stress response elements (ERSE) in the calreticulin promoter, and both elements were found to be necessary for full induction of calreticulin following ER stress. The presence of the tripartite nuclear transcription factor Y (NFY) and activating transcription factor 6 (ATF6), as well as an intact binding site for the YY1 transcription factor (YY1) within these ERSE motifs appeared to be crucial for mediating sensitivity to ER stress. Finally, chromatin-immunoprecipitation assays indicated that binding of NFY and YY1 to the ERSE motifs is induced after induction of ER stress in vivo. Therefore, we conclude that the UPR is activated in a subgroup of AML patients. Activation of the UPR involves induction of calreticulin expression by the ATF6 pathway and ultimately leads to suppressed CEBPA translation, thus contributing to the block in myeloid differentiation in these leukemias.