Development of a novel companion diagnostic for IRE1α activity and its application to investigate the role of IRE1α in acute myeloid leukaemia cells

Abstract

The unfolded protein response (UPR) is an evolutionarily conserved mechanism activated in response to accumulation of unfolded protein (termed ER stress). Activation of the UPR is observed during many physiological processes but also in multiple disease states, including various cancers, neurodegenerative diseases and diabetes mellitus. The UPR has both a pro-survival and pro-apoptotic role in cells, depending on the severity and duration of ER stress. The most conserved and well described transducer of the UPR is inositol-requiring enzyme 1 (IRE1) which is capable of signalling through both its kinase and RNase domains. The RNase domain of IRE1’s most studied role is in the unconventional splicing of xbox binding protein (XBP1) mRNA. Spliced XBP1 (XBP1s) mRNA allows for translation of a unique, potent transcription factor termed (XBP1s) normally associated with IRE1’s pro-survival role. Here we outline the development and application of a clinically compatible test for the detection of XBP1s and its unspliced counterpart XBP1u in cell or tissue lysates. This test, the XBP1 biochip, utilises biochip array technology (BAT) to deliver a quantitative, simultaneous result for both analytes from a single sample. We used the XBP1 biochip to investigate the effects of small molecule (MKC-8866) inhibition of the IRE1 RNase domain in cell models of acute myeloid leukaemia (AML). Investigation of extracellular effects under sub-cytotoxic ER stress revealed universal IL8 and ERdj3 release. When experiencing cytotoxic levels of ER stress due to proteasome inhibition, AML cell lines were partially protected by co-culture with immortalised bone marrow stromal cells (BMSCs). MKC-8866 was able to ablate this protection in cell lines when combined with carfilzomib (CFZ). In AML patient sample-BMSC co-cultures XBP1s expression correlated with MKC-8866 mediated enhancement of CFZ treatment. This thesis demonstrates the potential of the XBP1 biochip in research and clinical applications and CFZ with MKC-8866 co-treatment in AML.2024-08-2