S191: CALRETICULIN-MUTATED HEMATOPOIETIC CELLS ARE VULNERABLE TO THE COMBINED INHIBITION OF THE PROTEASOME AND THE IRE1A-XBP1 AXIS OF THE UNFOLDED PROTEIN RESPONSE

Abstract

Background: Mutations in the endoplasmic reticulum (ER) chaperone calreticulin (CALR) are frequent and disease-initiating in myeloproliferative neoplasms (MPN). These mutant CALR proteins have impaired chaperone function. Concordant with this, transcriptional upregulation of the unfolded protein response (UPR) has been reported in patients with CALR-mutated MPN. However, it is not understood how CALR-mutated cells counter-balance ER stress resulting from impaired chaperone function. Despite the frequency of CALR mutations in MPN, there are currently no treatment strategies to preferentially target CALR-mutant cells over healthy cells. Aims: To determine the mechanisms by which mutant CALR cells alleviate ER stress and to exploit these mechanisms selectively by pharmacological intervention in vivo. Methods: Long-term HSCs (LT-HSCs) from CalrΔ52 knockin mice were isolated for RNA-Seq. CalrΔ52 bone marrow (BM) was differentiated ex vivo, subsequently enriched for megakaryocytes, and subjected to quantitative proteomic analysis. Pathway analyses of CALR-mutant megakaryocyte progenitors (MkPs) were performed on Genotyping of Transcriptome (GoT) data from patients with Essential Thrombocytosis (ET). CalrΔ52 VAVCre mice and chimeric transplanted CalrΔ52 mice were treated with a proteasome inhibitor, an IRE1a inhibitor, or both. Results: To identify dysregulated pathways in CalrΔ52/Δ52 LT-HSCs, we performed RNA-seq and found that Xbp1s downstream of IRE1a and the proteasome pathway was significantly upregulated in CalrΔ52/Δ52 animals. We confirmed elevated XBP1s protein level in heterozygous CalrΔ52 knockin mice by flow cytometry. Since elevated and abnormal megakaryopoiesis are hallmarks of CALR-mutant MPN, we performed quantitative proteomics on megakaryocyte-enriched cells from CalrΔ52 mice. We confirmed upregulation of both proteasome and ER chaperone proteins in Calr-mutant cells. To verify these findings in human MPN, we interrogated a published GoT data set and found that in addition to XBP1s, the proteasome pathway was also differentially upregulated in CALR-mutated patient MkPs compared to the wildtype MkPs (p<2e-16). To investigate the functional consequences of IRE1a or proteasome inhibition, we treated CALR-mutant hematopoietic cell lines with KIRA6 (IRE1a inhibitor) and/or bortezomib (proteasome inhibitor) and found CALR-mutant cells to be differentially sensitive as compared to isogenic controls. We subsequently found that treatment with bortezomib leads to the accumulation of misfolded and ubiquitinylated proteins in mutant Calr BM, resulting in pro-apoptotic priming. In vivo treatment of CalrΔ52/+ mice with bortezomib resulted in a significant reduction in platelets. Finally, we combined IRE1a and proteasome inhibition in a chimeric BM transplantation in vivo model using CD45.2 CalrΔ52/+ MxCre GFP and CD45.1 wild-type competitor cells. Following combination therapy, we found a significant reduction in Calr-mutant donor chimerism, specifically in LT-HSCs and platelets, accompanied by reductions in LT-HSC frequency and platelet count. Summary/Conclusion: In summary, we have found that disrupted proteasis in CALR-mutated MPN cells results in a dependency on the IRE1-XPB1 axis of the UPR and proteasome activity. Using pre-clinical MPN models, we found that combined inhibition of both pathways in vivo normalizes important features of MPN and preferentially targets CalrΔ52 over wild-type cells. These findings highlight combined inhibition of IRE1 and the proteasome as a promising therapeutic strategy in CALR-mutant MPN.