Generation of transgenic mouse models with expression of constitutively active STAT5A

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Pathologic activation of thrombopoietin receptor and JAK2-STAT5 pathway by frameshift mutants of mouse calreticulin

Mutations in the human calreticulin (CALR) gene represented by -1+2 frameshifting deletions and insertions in exon 9 are associated with myeloproliferative neoplasms (MPNs). Mutant CALR proteins induce thrombocytosis in vivo due to their ability to pathologically and persistently activate the thrombopoietin receptor (TpoR) and the JAK2-STAT5/3/1 pathway. Homologous murine Calr frame-shifting mutations generate a C-terminal sequence that is not absolutely identical to that of human CALR mutants, although we show it bears similar biophysical features. We demonstrate that the murine CALR mutants del52, ins5 and del61 also activate the TpoR-JAK-STAT pathway. Using the CRISPR/Cas9 system, we created the del61 endogenous murine Calr mutation, as well as other -1/+2 frameshift mutations in Ba/F3 cells. These led to autonomous growth in Ba/F3 TpoR, but not parental Ba/F3 cells and induced constitutive TpoR, JAK-STAT and weak MAP-kinase signaling. In this heterozygous system we show that excess wild-type CALR does not inhibit mutant CALR signaling. Pathologic signaling is associated with a decrease of cell surface TpoR levels, which explains weaker response of transformed cells to ligand. Our results support the use of mouse models using murine CALR mutants and demonstrate pathologic signaling induced by heterozygous CALR mutations in hematopoietic cells

Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis.

Frameshift mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and myelofibrosis patients. To address the contribution of the CALR mutants to the pathogenesis of myeloproliferative neoplasms (MPNs) we engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing these mutants. In contrast to wild-type CALR, CALRdel52 (type I) and, to a lesser extent, CALRins5 (type II) induced thrombocytosis due to a megakaryocyte (MK) hyperplasia. Disease was transplantable into secondary recipients. After six months, CALRdel52-, in contrast to rare CALRins5-transduced mice, developed a myelofibrosis associated with a splenomegaly and a marked osteosclerosis. Monitoring of virus-transduced populations indicated that CALRdel52 leads to expansion at earlier stages of hematopoiesis than CALRins5. However, both mutants still specifically amplified the MK lineage and platelet production. Moreover a mutant deleted of the entire exon 9 (CALRdelex9) did not induce a disease suggesting that the oncogenic property of CALR mutants was related to the new C-terminus peptide. To understand how the CALR mutants target the MK lineage, we used a cell line model and demonstrated that the CALR mutants, but not CALRdelex9, specifically activate the thrombopoietin (TPO) receptor (MPL) to induce constitutive activation of JAK2 and STAT5/3/1. We confirmed in c-mpl- and tpo-deficient mice that expression of Mpl, but not of Tpo, was essential for the CALR mutants to induce thrombocytosis in vivo, although Tpo ligand contributes to disease penetrance. Thus, CALR mutants are sufficient to induce thrombocytosis through MPL activation

Hematoxylin binds to mutant calreticulin and disrupts its abnormal interaction with thrombopoietin receptor.

Somatic mutations of calreticulin (CALR)have been identified as one of the main disease drivers of myeloproliferative neoplasms (MPNs), suggesting that developing drugs targeting mutant CALR is of great significance. Site-directed mutagenesis in the N-glycan binding domain (GBD)abolishes the ability of mutant CALRto oncogenically activate the thrombopoietin receptor (MPL).We thus hypothesized that a small molecule targeting the GBD might inhibit the oncogenicity of the mutant CALR. Using an in-silico molecular docking study, we identified candidate binders to the GBD of CALR. Further experimental validation of the hits identified a group of catechols inducing selective growth inhibitory effect on cells that depend on oncogenic CALRs for survival and proliferation. Apoptosis-inducing effects by the compound were significantly higher in the CALR mutated cells than in CALR wild type cells. Additionally, knockout or C-terminal truncation of CALR abolished the drug hypersensitivity in CALR mutated cells. We experimentally confirmed the direct binding of the selected compound to CALR, the disruption of the mutant CALR-MPL interaction, the inhibition of the JAK2-STAT5 pathway, and reduction of intracellular level of mutant CALR upon the drug treatment. Our data conclude that small molecules targeting the GBD of CALR can selectively kill CALR mutated cells by disrupting the CALR-MPL interaction and inhibiting the oncogenic signaling

Increased survival and cell cycle progression pathways are required for EWS/FLI1-induced malignant transformation

Ewing sarcoma (ES) is the second most frequent childhood bone cancer driven by the EWS/FLI1 (EF) fusion protein. Genetically defined ES models are needed to understand how EF expression changes bone precursor cell differentiation, how ES arises and through which mechanisms of inhibition it can be targeted. We used mesenchymal Prx1-directed conditional EF expression in mice to study bone development and to establish a reliable sarcoma model. EF expression arrested early chondrocyte and osteoblast differentiation due to changed signaling pathways such as hedgehog, WNT or growth factor signaling. Mesenchymal stem cells (MSCs) expressing EF showed high self-renewal capacity and maintained an undifferentiated state despite high apoptosis. Blocking apoptosis through enforced BCL2 family member expression in MSCs promoted efficient and rapid sarcoma formation when transplanted to immunocompromised mice. Mechanistically, high BCL2 family member and CDK4, but low P53 and INK4A protein expression synergized in Ewing-like sarcoma development. Functionally, knockdown of Mcl1 or Cdk4 or their combined pharmacologic inhibition resulted in growth arrest and apoptosis in both established human ES cell lines and EF-transformed mouse MSCs. Combinatorial targeting of survival and cell cycle progression pathways could counteract this aggressive childhood cancer

Activated STAT5 Confers Resistance to Intestinal Injury by Increasing Intestinal Stem Cell Proliferation and Regeneration

Intestinal epithelial stem cells (IESCs) control the intestinal homeostatic response to inflammation and regeneration. The underlying mechanisms are unclear. Cytokine-STAT5 signaling regulates intestinal epithelial homeostasis and responses to injury. We link STAT5 signaling to IESC replenishment upon injury by depletion or activation of Stat5 transcription factor. We found that depletion of Stat5 led to deregulation of IESC marker expression and decreased LGR5+ IESC proliferation. STAT5-deficient mice exhibited worse intestinal histology and impaired crypt regeneration after γ-irradiation. We generated a transgenic mouse model with inducible expression of constitutively active Stat5. In contrast to Stat5 depletion, activation of STAT5 increased IESC proliferation, accelerated crypt regeneration, and conferred resistance to intestinal injury. Furthermore, ectopic activation of STAT5 in mouse or human stem cells promoted LGR5+ IESC self-renewal. Accordingly, STAT5 promotes IESC proliferation and regeneration to mitigate intestinal inflammation. STAT5 is a functional therapeutic target to improve the IESC regenerative response to gut injury

Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants.

Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a -1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except G-CSF receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3'-kinase (PI-3K) and Mitogen Activated Protein (MAP) Kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells were such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation CALR mutants exhibit different intracellular localization from that of wild type CALR. Finally, knocking-down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling

Whole exome sequencing identifies novel MPL and JAK2 mutations in triple negative myeloproliferative neoplasms.

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR, exon 10 of MPL or JAK2-V617F in >90% of the cases, while the remaining cases are termed "triple negative". We aimed to identify the disease causing mutations in the triple negative cases of ET and PMF by applying whole exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5/8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3/8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis, analyzed by WES, we identified a somatic MPL-S204P and a germline MPL-V285E mutation, as well as a germline JAK2-G571S variant. Sequencing of entire coding region of MPL and JAK2 was performed in additional 62 and 49 triple negative cases of ET or PMF, respectively. We detected new somatic (T119I, S204F, E230G, Y591D) and one germline (R321W) MPL mutation in 5/62 cases. All the mutations were gain-of-function mutations when analyzed in functional assays. JAK2 variants were identified in 5/57 triple negative cases and 3 of them were germline. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders

Calreticulin mutants as oncogenic rogue chaperones for TpoR and traffic-defective pathogenic TpoR mutants

Calreticulin (CALR) +1 frameshift mutations in exon 9 are prevalent in myeloproliferative neoplasms. Mutant CALRs possess a new C-terminal sequence rich in positively charged amino acids, leading to activation of the thrombopoietin receptor (TpoR/MPL). We show that the new sequence endows the mutant CALR with rogue chaperone activity, stabilizing a dimeric state and transporting TpoR and mutants thereof to the cell-surface in states that would not pass quality control, and this function is absolutely required for oncogenic transformation. Mutant CALRs determine traffic via the secretory pathway of partially immature TpoR, as they protect N117-linked glycans from further processing in the Golgi. A number of engineered or disease-associated TpoRs, such as TpoR/MPL R102P, which causes congenital thrombocytopenia are rescued for traffic and function by mutant CALRs, which can also overcome endoplasmic reticulum retention signals on TpoR. Besides requiring N-glycosylation of TpoR, mutant CALRs require a hydrophobic patch located in the extracellular domain of TpoR to induce TpoR thermal stability and initial intracellular activation, while full activation requires cell surface localization of TpoR. Thus, mutant CALRs are rogue chaperones for TpoR and traffic-defective TpoR mutants, a function required for the oncogenic effect