C-Jun Regulates ER Stress Signaling to Promote Chemotherapy Resistance in Acute Myeloid Leukemia
Authors: Zhou Chun , Di Marcantonio Daniela , Martinez Esteban , Solanki-Patel Nehal , Peri Suraj , Scholl Claudia , Fröhling Stefan , Wiest David L. , Sykes Stephen Matthew 
Published: October 2019

Abstract
The first-line treatment for acute myeloid leukemia (AML) patients is a combination of cytarabine (Ara-C) and an antracycline, such as daunorubicin (Dnr). However, one third of AML patients are refractory to first-line therapy and 35-60% of those who do initially respond to chemotherapy relapse with resistant disease. Therefore, identifying and deciphering the molecular abnormalities that contribute to chemotherapy resistance in AML is the critical first step for developing new therapies that overcome chemotherapy resistance. In our study, we found that expression of the proto-oncogene c-JUN is elevated in patient-derived AML samples compared with normal hematopoietic progenitor cells. Additionally, expression and phosphorylation of c-JUN further increased in human AML cells exposed to Ara-C or Dnr. Confirming functional functional importance of c-JUN in human AML cells, we found that short hairpin RNAs (shRNAs) that specifically target c-JUN suppress cell growth and induce apoptosis in several genetically distinct human AML cell lines (Kasumi-1, MOLM-14, MV4-11, NOMO-1, OCI-AML3, SKM-1, THP-1 and U937). Furthermore, we have observed that shRNA-mediated inhibition of c-JUN also significantly potentiated the cytotoxic effects of Ara-C (p<0.05), suggesting that c-JUN is required for AML cell survival and may play a critical role in chemotherapy resistance.
To elucidate the molecular mechanism by which c-JUN promotes AML cell survival and chemotherapy resistance, we performed global gene expression analyses (RNA-seq) of THP-1 cells expressing either non-targeting control or c-JUN shRNAs. From these analyses, we found that c-JUN inhibition results in decreased expression of numerous genes that are activated in response to endoplasmic reticulum (ER) stress, including a significant reduction in the expression of genes regulated by the ER stress-activated transcription factors ATF4 and DDIT3 (p=2.44x10-20).
We therefore hypothesized that c-JUN may control ER stress signaling pathways to promote cell survival and chemotherapy resistance in AML. In support of this hypothesis, we have found that c-JUN inhibition blunts the activation of ER-stress related pathways stimulated by chemical inducers of ER stress, thapsigargin and tunicamycin. Additionally, we found that Ara-C treatment activates ER stress-related signaling pathways and that this activation can be reversed by c-JUN inhibition, which correlates with the increased death of c-JUN shRNA-expressing AML cells exposed to Ara-C.
To further elucidate the role of ER-stress related pathways in AML, we focused on the serine/threonine kinase, PERK (encoded by EIF2AK3), which activates signaling pathways that aim to ameliorate ER stress and promote cell survival. Similar to c-JUN inhibition, shRNA-mediated inhibition of PERK not only increased cell death but also enhanced the cytotoxicity of Ara-C in AML cells, indicating that this ER stress pathway is a potential therapeutic target to increase the effectiveness of standard chemotherapy.
To investigate the therapeutic potential of targeting the PERK pathway, we treated human AML cells with a chemical inhibitor of PERK, GSK2656157, in combination with Ara-C and found that certain concentrations of the two drugs synergistically increased AML cell killing versus each drug alone. Using leukemia cells derived from a mouse model of AML driven by the leukemogenic fusion allele MLL-AF9 alone or in combination with a FLT3 internal tandem duplication, we observed that the combination of GSK2656157 and Ara-C significantly reduced colony growth in methylcellulose by 80% compared with each drug alone (p<0.05).
These findings suggest that c-JUN plays a vital role in mitigating ER stress in AML cells and that targeting c-JUN and/or ER stress signaling pathways is a novel potential therapeutic approach for improving the effectiveness of current first-line AML therapies.

Disclosures
No relevant conflicts of interest to declare.