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Targeting the CALR Interactome in Myeloproliferative Neoplasms

ABSTRACT: Mutations in the endoplasmic reticulum (ER) chaperone calreticulin (CALR) are common in myeloproliferative neoplasm (MPN) patients, activate the thrombopoietin receptor (MPL), and mediate constitutive JAK/STAT signaling. The mechanisms by which CALR mutations cause myeloid transformation are incompletely defined. We employed mass spectrometry proteomics to identify novel CALR-mutant interacting proteins. Mutant CALR caused mislocalization of binding partners and increased recruitment of FLI1, ERP57 and CALR to the MPL promoter to enhance transcription. CALR 52 mutant was also found to increase genome-wide recruitment of Fli1 to the chromatin. Overall, these results show that type 1 CALR mutant modulates Fli1 cellular localization and recruitment.

ORGANISM(S): Mus musculus

PROVIDER: GSE120134 | GEO | 2018/09/19

REPOSITORIES: GEO

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CALR-mutated cells are vulnerable to combined inhibition of the proteasome and the endoplasmic reticulum stress response

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.

2023-10-12 | GSE207684 | GEO

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

Project description:Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN). However, the mechanism by which mutant CALR is oncogenic is unknown. Here, we demonstrate that a megakaryocytic-specific MPN phenotype is induced when mutant CALR is over-expressed in mice and that the thrombopoietin receptor, MPL is required for mutant CALR driven transformation. Whole transcriptome analysis reveals enrichment of STAT signatures in mutant CALR transformed cells and JAK2 inhibitor treatment abrogates STAT activation. Employing extensive mutagenesis-based structure-function analysis we demonstrate that the positively charged amino acids within the mutant CALR C-terminus are required for cellular transformation through facilitating physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel mechanism of cancer pathogenesis.

2016-04-14 | GSE74890 | GEO

Oncogenic Calreticulin Induces Immune Escape by Stimulating TGF-β Expression and Regulatory T Cell Expansion in the Bone Marrow Microenvironment - microarray

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.

2024-05-30 | GSE241162 | 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

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

Project description: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.

2022-03-31 | GSE173805 | GEO

Oncogenic CALR Mutant C-terminus Mediates Dual Binding to the Thrombopoietin Receptor Triggering Complex Dimerization and Activation

Project description:Calreticulin (CALR) is a master lectin chaperone that guides the proper folding of integral membrane proteins in the endoplasmic reticulum. In healthy cells, CALR transiently and non-specifically interacts with thousands of immature N-glycosylated proteins through its N-terminal glycan-binding domain. Conversely, frameshift mutants of CALR turn into rogue cytokine by acquiring the ability to stably and specifically interact with the Thrombopoietin Receptor (TpoR). Strikingly, this interaction induces activation of TpoR leading to myeloproliferative neoplasms, blood cancers resulting from the overproduction of blood cells. Using a multidisciplinary approach, we unveil how the CALR frameshift mutations result in structural overhaul of the entire protein and identify the structural basis of its acquired specificty for TpoR. We further describe the mechanisms by zhich complex formation triggers TpoR dimerization and activation. In addition, we provide the first complete dynamic conformational footprints of both wild-type and mutant CALR and identify novel potentially targetable sites.

2023-04-11 | PXD034131 | Pride

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

Project description:There is growing evidence that Ph-negative myeloproliferative neoplasms (MPNs) are blood cancers in which multiple molecular mechanisms are significantly disturbed. Since their discovery in 2016, CALR (calreticulin) type 1 and type 2 driver mutations have been demonstrated to trigger pathogenic mechanisms apart from the well-documented activation of JAK2/MPL-related pathways affecting these diseases. C. elegans seems to be a suitable model for the study of these mechanisms since they have a CALR ortholog (crt-1) but have no JAK or MPL ortholog. We used microarrays in C. elegans strains harboring patient-like calreticulin mutations to identify JAK/MPL-independent molecular mechanisms derived from mutant calreticulin and identified a significant disruption of some of the typically altered biological processes in cancer and Ph-negative MPN patients. Finally, we compared the expression of the genes participating in these processes with a calreticulin knockout strain in order to examine if the alteration of these processes is a consequence of a gain or a loss of function of the mutant protein.

2023-01-09 | GSE201599 | GEO

CALR frameshift mutations accelerate maturation of megakaryocytes in MPN patient-derived iPS cells

Project description:In this dataset, we compare the gene expression data of induced pluripotent stem (iPS) cell-derived CD61+ megakaryocytes carrying heterozygous or homozygous Calreticulin (CALR) ins5 mutations or the CALR wildtype gene.

2021-10-21 | GSE182479 | 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

2020-08-20 | GSE102900 | 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).

Project description:Calreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPN). Although the biological mechanism by which CALR mutations cause MPN has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPN. To identify unique genetic dependencies in CALR-mutant MPN, we performed a whole-genome CRISPR knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (amongst others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells in vitro. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor, 2-deoxy-glucose (2-DG), and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared to wild-type cells, and a normalization of key MPN disease features. These findings advance the development of clonally selective treatments for CALR-mutant MPN.

2022-09-21 | GSE203455 | GEO

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