The Unfolded Protein Response Mediates Resistance in AML and Its Therapeutic Targeting Enhances TKI Induced Cell Death in FLT3-ITD + AML
Authors: Jaquet Timo , Preisinger Christian , Bütow Marlena , Tillmann Stefan , Chatain Nicolas , Huber Michael , Koschmieder Steffen , Kirschner Martin , Jost Edgar , Brümmendorf Tim H , Kharabi Bezhad , Appelmann Iris , Schemionek Mirle 
Published: November 2021

Abstract
Introduction: The unfolded protein response (UPR) is a stress sensing signaling network that is activated upon endoplasmic reticulum (ER) stress, a condition characterized by an accumulation of mis- and unfolded proteins in the ER. To retain a functional cell metabolism, UPR activation increases protein folding and degradation. Acute myeloid leukemia (AML) stem cells are prone to develop ER stress, due to their oncogene-driven metabolism and the bone marrow niche, where they face stressors like hypoxia or nutrient fluctuations. Our preliminary work showed enhanced UPR gene expression levels, especially of IRE1α and XBP1, in different AML subtypes. Patients with high XBP1 mRNA expression had an inferior overall survival rate compared to patients with low XBP1 mRNA expression.
Aims: We studied the role of elevated UPR signaling in AML therapy resistance and assessed the therapeutic potential of IRE1α-XBP1 inhibitor STF-083010 (STF) as a new strategy in different AML subtypes, including FLT3-ITD + AML.
Methods: Human MV4-11 (FLT3-ITD), RS4-11 (FLT3 wildtype; WT), NB-4 (PML-RARα), THP-1 (MLLr) cells, and murine 32D cells transduced with FLT3-ITD or FLT3 WT were analyzed via western blot and RT-PCR. Metabolic activity was assessed by MTT assay, cell death and apoptosis were measured with propidium iodide (PI) or Annexin V staining using flow cytometry. FLT3 cell surface expression was measured via flow cytometry. The clonogenic potential was determined in CFU assays, using patient-derived mononuclear and CD34 + cells. For hypoxic experiments, MV4-11 cells were cultivated under hypoxia (3 % O 2) and cells were subjected to phosphoproteomic analysis, which was performed by mass spectrometry. Conditional Mx1-Cre/XBP1 fl/fl knockout mice were generated and deletion of XBP1 was induced by IP injection of Polyinosinic-polycytidylic acid (Poly(I:C)). Bone marrow and spleen cells were analyzed via flow cytometry and RT-PCR.
Results: Treatment with FLT3 TKI AC220 specifically enhanced IRE1α mRNA (9.3-fold, p&lt;0.05) and increased IRE1α protein in 32D FLT3-ITD cells. Likewise, the percentage of dead cells was significantly elevated in 32D FLT3-ITD upon IRE1α inhibition by STF compared to 32D FLT3 WT cells. Treatment with STF prevented XBP1 splicing and reduced the metabolic activity of human AML cell lines in a dose-dependent manner. Furthermore, IRE1α inhibition significantly induced apoptosis in human MV4-11 (6-fold, p&lt;0.05), NB-4 (8-fold, p&lt;0.01) and THP-1 (7-fold, p&lt;0.01) cells and reduced their clonogenic potential. The combination of STF and AC220 strongly enhanced the percentage of apoptotic cells in MV4-11 cells compared to single treatments (by 3-fold, p&lt;0.001). This strong induction of cell death was specific for FLT3-ITD + MV4-11 cells and not observed in FLT3 WT + RS4-11 cells. Similarly, the clonogenic potential of MV4-11 cells and FLT3-ITD + AML mononuclear patient cells was significantly decreased by the combinatorial treatment, while healthy donor cells were not affected. Likewise, conditional XBP1 knockout did not significantly alter normal hematopoiesis in mice. Hypoxia further enhanced IRE1α signaling in MV4-11 cells and strongly reduced the efficacy of AC220 (normoxia: 58.4-fold induction of dead cells, p&lt;0.01; hypoxia: 2.2-fold induction, p&gt;0.05). Analysis of phosphoproteomics revealed a less active FLT3 signaling under hypoxia. Intriguingly, the combination of IRE1α and FLT3 inhibition overcame the resistance towards AC220 under hypoxia and significantly induced cell death.
Conclusion: IRE1α-XBP1 signaling is activated in different AML subtypes including FLT3-ITD + and is further enhanced by hypoxia present in the bone marrow niche. Targeting IRE1α in FLT3-ITD + cells effectively decreases clonogenic growth and induces apoptosis. Our data demonstrate that hypoxia-mediated resistance against AC220 can be overcome by simultaneous IRE1α inhibition. Genetic deletion of XBP1 does not harm steady-state murine hematopoiesis, rendering XBP1 an excellent therapeutic target.

Disclosures
Koschmieder: CTI: Membership on an entity's Board of Directors or advisory committees, Other; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Baxalta: Membership on an entity's Board of Directors or advisory committees, Other; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); AOP Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Shire: Honoraria, Other; Image Biosciences: Other: Travel support; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Geron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Karthos: Other: Travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Alexion: Other: Travel support; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Abbvie: Other: Travel support; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Brümmendorf: Bristol Myers: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Repeat Diagnostics: Research Funding; Takepart Media: Honoraria.