CSF3R^T618I Is Stabilized By Calnexin and Induces Unfolded Protein Response (UPR) and ER-Phagy: A Pro-Survival Pathway for Oncogenic Misfolded CSF3R Proteins in Chronic Neutrophilic Leukemia (CNL)

Introduction

More than 80 percent of chronic neutrophilic leukemia (CNL) patients harbor the membrane proximal mutation T618I in the colony-stimulating factor 3 receptor (CSF3R), which leads to constitutive activation of the JAK1/STAT3-pathway. However, the molecular mechanisms of oncogenic transformation by CSF3R mutations in CNL are not well understood. We previously established that CSF3R^T618I is trapped in the endoplasmic reticulum (ER) and induces unfolded protein response (UPR) signaling. In this study, we analyzed the function of Calnexin (CNX) for CSF3R^T618I stability, signaling capabilities and UPR-induction, and assessed the impact of ER-phagy on CSF3R^T618I-mediated cell proliferation.

Material and methods

Calnexin knockout (CNX^-/-) mouse embryonic fibroblasts (MEFs), as well as NIH-3T3, Ba/F3 and 32D cells were used to analyze CSF3R mutant stability and oncogenic signaling pathways. Cycloheximide (CHX), Bafilomycin A1 and MG132 treatments were used to determine CSF3R protein turnover. Vitexin and PDI inhibitor 16F16 were used to target UPR and ER-phagy. Western Blots, Co-immunoprecipitation and RNA-seq were performed. Confocal microscopy was used to examine ER-structure and subcellular localization of CSF3R.

Results

While CSF3R^WT is located mostly on the cell surface, confocal microscopy confirmed ER-localization of CSF3R^T618I and showed colocalization of CSF3R^T618I and CNX. CNX^-/- MEFs expressing CSF3R^T618I displayed low levels of CSF3R protein in western blots, and reconstitution of Calnexin (CNX) led to an increase in CSF3R protein levels, suggesting that CNX is involved in the stabilization of CSF3R. Bafilomycin A1, blocking autophagosome-lysosome fusion, significantly increased CSF3R^T618I protein levels, which suggests ER-to-lysosome-associated-degradation (ERLAD) of CSF3R^T618I, while MG132, targeting proteasomal degradation, had no effect on CSF3R^T618I protein stability. CNX overexpression in Ba/F3 cells led to decreased sensitivity towards ruxolitinib and geldanamycin, and led to enhanced JAK1 and GRP94 interaction with CSF3R^T618I, respectively. In contrast to CSF3R^WT, CSF3R^T618I induced ER-stress proteins ATF6 and ERO1-Lα on protein and RNA-level, and CNX expression further enhanced induction of ER-stress markers, such as ATF6, PDI and GRP94. Targeting ATF6 activation and PDI activity using the PDI inhibitor 16F16 led to a decrease in cell proliferation and reduced STAT3 activation in CSF3R^T618I but not CSF3R^WT expressing 32D cells. Confocal microscopy showed alterations regarding ER-architecture in CSF3R^T618I compared to CSF3R^WT expressing NIH-3T3 cells, displaying an increased amount of lysosome-like structures. Additionally, we found significant induction of ER-phagy receptor FAM134B specifically in CSF3R^T618I but not CSF3R^WT expressing Ba/F3 and 32D cells. Preventing the interaction between FAM134B and BiP with vitexin led to decreased cell proliferation and increased activation of ER-stress sensor ATF6 specifically in CSF3R^T618I expressing Ba/F3 and 32D cells.

Conclusions

Taken together, our results reveal that CSF3R^T618I is unstable and undergoes lysosomal degradation. CNX stabilizes CSF3R^T618I and leads to enhanced CSF3R^T618I dependent JAK1/STAT3-pathway activation. CNX overexpression leads to decreased Ruxolitinib sensitivity of CSF3R^T618I expressing cells. In addition, CSF3R^T618I specifically and strongly induces UPR through ATF6. Blocking ATF6 mediated UPR decreases STAT3-mediated signaling, implicating a crucial role of ATF6 in the transformaing activity driven by CSF3R^T618I. CSF3R^T618I expressing cells display striking changes in ER-architecture and upregulation of ER-phagy receptor FAM134B, which points at ERLAD being the key degradational pathway for CSF3R^T618I. Targeting the ERLAD pathway attenuated cell proliferation in CSF3R^T618I expressing cells. These results suggest ER-phagy as a pro survival, oncogene-specific pathway driving CNL, mediated by CSF3R mutations, that might be therapeutically exploited to improve treatment for CNL patients.

Koschmieder:Novartis, BMS, Pfizer, Incyte, Ariad, Shire, Roche, AOP Pharma, Janssen, Geron, Celgene, Kartos, Abbvie, iOMEDICO, MSD: Honoraria; Pfizer, Incyte / Ariad, Novartis, AOP Pharma, BMS, Celgene, Geron, Janssen, CTI, Roche, Baxalta, Sanofi, MPN Hub, Protagonist, Sierra Oncology, Glaxo-Smith Kline, AbbVie, PharmaEssentia, MSD: Consultancy; Novartis Foundation, BMS, Novartis, AOP Pharma, Janssen/Geron: Research Funding; Alexion, Novartis, BMS, Incyte / Ariad, AOP Pharma, Baxalta, CTI, Pfizer, Sanofi, Celgene, Shire, Janssen, Geron, Kartos, Protagonist, Sierra Oncology, Glaxo-Smith Kline, Imago Biosciences, AbbVie, iOMEDICO, MSD: Speakers Bureau; RWTH Aachen University: Patents & Royalties: Patent filed on BET inhibitors; Member of EHA Guidelines Committee, Chairman Hemostasis Working Party of DGHO, Co-Speaker of German Study Group for MPN (GSG-MPN) and GSG-MPN bioregistry: Membership on an entity's Board of Directors or advisory committees. Brummendorf:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Combination of Imatinib with hypusination inhibitors, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria; Merck: Honoraria; Roche: Honoraria; Ariad: Consultancy, Honoraria; Repeat Dx: Consultancy, Research Funding. Heidel:BMS/Celgene, Novartis, CTI: Research Funding; BMS/Celgene, AOP, Novartis, CTI, Janssen, Abbvie, GSK, Merck, Kartos, Telios: Consultancy. Döhner:AbbVie, AOP Health, Janssen, Jazz, Novartis, Bristol Myers Squibb, Celegne: Consultancy, Honoraria; Novartis, AbbVie, Astellas, Bristol Myers Squibb, Celegne, Jazz Pharmaceuticals, Kronos Bio, Servier: Research Funding. Griesshammer:AOP Orphan, Novartis, BMS, AbbVie, Pfizer, Roche, Janssen, Gilead, AstraZeneca, Sierra, Lilly, GSK: Consultancy, Honoraria. Reiter:Novartis: Consultancy, Honoraria, Other: Grants (institution) , Research Funding; Blueprint Medicines Corporation: Consultancy, Other: Grants (institution) , Research Funding; Cogent Therapeutics LLC: Research Funding; Abbvie: Research Funding; AOP: Consultancy, Honoraria, Other: travel grants, Research Funding; Astra Zeneca: Research Funding; BMS: Research Funding; GSK: Consultancy, Honoraria, Other: travel expense, Research Funding; Incyte: Research Funding.