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Type 1 calreticulin mutations activate the IRE1α-XBP1 pathway of the unfolded protein response to drive MPN

ABSTRACT: Experimental data indicates unique activation of the unfolded response in calreticulin (CALR) mutant myeloproliferative neoplasms (MPN). To dissect UPR activation in an MPN, calreticulin-mutant context at the transcriptional level, RNA-seq was performed. Cell lines were generated with the addition of a plasmid containing the thrombopoietin receptor (MPL) in addition to a CALR variant: either wild-type calreticulin, calreticulin with a deletion of fifty-two base pairs/type I mutant, or calreticulin with an insertion of five base pairs/type II mutant. Cells were cultured in growth media containing 10% FBS RPMI with penicillin/streptomycin addition in a 5% CO2 incubator. Cells were washed three times with PBS followed by media replacement using growth media with or without IL3 addition. In unstarved conditions, cells were supplied with the cytokine IL3 at a 1:10,000 dilution for a final concentration of 2 ng/mL; in starved conditions, cells were depleted of the IL3 cytokine addition for twenty-four hours. RNA-seq was performed on both unstarved and starved cells in triplicates for each genotype. Additionally, for preliminary data purposes, a cell line was generated with MPL plasmid transduction and with the transduction of a plasmid containing the JAK2 kinase with the V617F mutation to examine MPN activation in a JAK2 mutant, MPN context; these samples (unstarved and starved) were not sequenced in triplicates but with one sample each.

ORGANISM(S): Mus musculus

PROVIDER: GSE173805 | GEO | 2022/03/31

REPOSITORIES: GEO

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Physical interaction between mutant calreticulin and the thrombopoietin receptor is required for transformation of hematopoietic cells

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Targeting the CALR Interactome in Myeloproliferative Neoplasms

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Project description:To understand transcriptional aberrations induced by oncogenic Calreticulin (Calr) in myeloproliferative neoplasms we used a cell line – 32D, generated from the murine myeloid precursor cells, to overexpress human thrombopoietin receptor (MPL) together with human Calr. Cells expressing oncogenic Calr show increased JAK/STAT signaling independent of extrinsic stimuli like IL3.

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Project description:Cancer is driven by somatic mutations that provide a fitness advantage. While targeted therapies often focus on the mutated gene or its direct downstream effectors, imbalances brought on by cell-state alterations may also confer unique vulnerabilities. In myeloproliferative neoplasms (MPN), somatic mutations in the calreticulin (CALR) gene are disease-initiating through aberrant binding of mutant CALR to the thrombopoietin receptor MPL and ligand-independent activation of JAK-STAT signaling. Despite these mechanistic insights into the pathogenesis of CALR-mutant MPN, there are currently no mutant CALR-selective therapies available. Here, we identified differential upregulation of unfolded and ubiquitinylated proteins, the proteasome and the ER stress response in CALR-mutant hematopoietic stem cells and megakaryocyte progenitors. We further found that combined pharmacological inhibition of the proteasome and IRE1-XBP1 axis of the ER stress response preferentially targets Calr-mutated stem and progenitors over wild-type cells in vivo, resulting in an amelioration in the MPN phenotype. Together, these findings leverage loss of proteostasis in Calr-mutant MPN to identify combinatorial dependencies that may be targeted for therapeutic benefit.

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Expression data from C. elegans harboring type 1-like and type 2-like calreticulin mutations of MPN patients

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Whole-genome CRISPR screening identifies N-glycosylation as an essential pathway and a potential novel therapeutic target in CALR-mutant MPN (Whole Genome CRISPR Screen).

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2022-09-21 | GSE203456 | GEO

Whole-genome CRISPR screening identifies N-glycosylation as an essential pathway and a potential novel therapeutic target in CALR-mutant MPN (Pooled CRISPR Screen).

2022-09-21 | GSE203455 | GEO

Calreticulin Ins5 and Del52 mutations impair unfolded protein and oxidative stress responses in K562 cells expressing CALR mutants.

Project description:Somatic mutations of calreticulin (CALR) have been described in approximately 30-40% of JAK2 and MPL unmutated Essential Thrombocythemia and Primary Myelofibrosis patients. CALR is an endoplasmic reticulum (ER) chaperone responsible for proper protein folding and calcium retention. Recent data demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven Myeloproliferative Neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven't been fully unraveled. In this study, we showed that CALR mutants impair the ability to respond to the ER stress and reduce the activation of the pro-apoptotic pathway of the unfolded protein response (UPR). Moreover, our data demonstrated that CALR mutations induce increased sensitivity to oxidative stress, leading to increase oxidative DNA damage. We finally demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by mutant CALR. Altogether, our data identify novel mechanisms collaborating with MPL activation in CALR-mediated cellular transformation. CALR mutants negatively impact on the capability of cells to respond to oxidative stress leading to genomic instability and on the ability to react to ER stress, causing resistance to UPR-induced apoptosis.

2019-08-04 | GSE127250 | GEO

Calreticulin Ins5 and Del52 mutations impair unfolded protein and oxidative stress responses in hematopoietic cells

Project description:Somatic mutations of calreticulin (CALR) have been described in approximately 30-40% of JAK2 and MPL unmutated essential trombocytemia and primary myelofibrosis patients. CALR is a chaperone that localizes primarily in the endoplasmic reticulum (ER) where it is responsible for the control of proper protein folding and for the calcium retention. Recent data have demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven myeloproliferative neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven't been fully unraveled. In this study, we attempted to clarify whether CALR mutations may affect the functions of CALR in the ER under homeostatic conditions. Our results showed that CALR mutants impair the ability to respond to the ER stress and reduce the activation of the pro-apoptotic pathway of the unfolded protein response, therefore allowing the accumulation of unfolded proteins and conferring resistance to ER-stress induced apoptosis. Moreover, our data demonstrated that CALR mutations induce increased sensitivity to oxidative stress, reducing the ability to counteract ROS intracellular accumulation and thus leading to increase oxidative DNA damage. We finally demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by CALR mutations. Altogether, our data identify a novel MPL-independent mechanism involved in the development of MPNs mediated by CALR mutants: CALR mutations negatively impact on the capability of cells to respond to oxidative stress, and this in turn leads to increase DNA damage and genomic instability. We used microarrays to detail the change of gene expression caused by calreticulin mutations in K562 cells

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Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis [HEL cell lines]

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