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The stem/progenitor landscape is reshaped in a mouse model of essential thrombocythaemia and causes excess megakaryocyte production

ABSTRACT: Frameshift mutations in CALR (calreticulin) are associated with essential thrombocythaemia (ET), but the stages at and mechanisms by which mutant CALR drives transformation remain incompletely defined. Here, we use single-cell approaches to examine the haematopoietic stem/progenitor cell (HSPC) landscape in a mouse model of mutant CALR-driven ET. We identify a trajectory linking HSCs with megakaryocytes, and prospectively identify a novel intermediate population that is overrepresented in the disease state. We also show that mutant CALR drives transformation primarily from the earliest stem cell compartment, with some contribution from megakaryocyte progenitors. Finally, relative to wild-type HSCs, mutant CALR HSCs showed increases in JAK-STAT signalling, the unfolded protein response, cell cycle, and a previously undescribed upregulation of cholesterol biosynthesis. Overall, we have identified a novel megakaryocyte-biased cell population that is increased in a mouse model of ET and described transcriptomic changes linking CALR mutations to increased HSC proliferation and megakaryopoiesis.

ORGANISM(S): Mus musculus

PROVIDER: GSE160466 | GEO | 2020/10/30

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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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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Hematopoietic Stem Cell Heterogeneity in JAK2 V617F+ Essential Thrombocythemia

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Calreticulin mutated hematopoietic cells are vulnerable to ATR-CHK1 pathway inhibition

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2014-06-03 | E-GEOD-54644 | biostudies-arrayexpress

JAK2V617F mutant HSC microarray

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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.

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