Novel mechanisms for STAT5 activation in myeloid neoplasms and acute myeloid leukemia

Myeloproliferative neoplasms (MPNs) include polycythemia vera, essential thrombocythemia and myelofibrosis. The common feature of these disorders is persistent activation of the JAK-STAT pathway, which is mainly induced by mutations in JAK2, MPL and CALR. This thesis addresses the role of STAT5 in both development of CALR-related MPNs and the evolution to secondary acute myeloid leukemia (sAML). Firstly, we show that STAT5 is activated by CALR mutants specifically via MPL and JAK2. This activation occurs in the secretory pathway and on the cell surface. We show that the latter is crucial for transformation. CALR mutants act as rogue chaperones and rescue cell surface localization of MPLs defective in traffic. Secondly, we demonstrate that the release of STAT5 activity from the inhibitory effect of p53 is key for the evolution of MPNs to sAML. STAT5 release requires the loss of both p53 alleles, and it does not require the abolishment of p53 transcriptional activity.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

A p53-JAK-STAT connection involved in myeloproliferative neoplasm pathogenesis and progression to secondary acute myeloid leukemia

Since the discovery of JAK2 V617F as a highly prevalent somatic acquired mutation in the majority of myeloproliferative neoplasms (MPNs), it has become clear that these diseases are driven by pathologic activation of JAK2 and eventually of STAT5 and other members of the STAT family. The concept was strengthened by the discovery of the other activating driver mutations in MPL (thrombopoietin receptor, TpoR) and in calreticulin gene, which all lead to persistent activation of wild type JAK2. Although with a rare frequency, MPNs can evolve to secondary acute myeloid leukemia (sAML), a condition that is resistant to treatment. Here we focus on the role of p53 in this transition. In sAML mutations in TP53 or amplification in genes coding for negative regulators of p53 are much more frequent than in de novo AML. We review studies that explore a signaling and biochemical interaction between activated STATs and p53 in MPNs and other cancers. With the development of advanced sequencing efforts, strong evidence has been presented for dominant negative effects of mutated p53 in leukemia. In other studies, gain of function effects have been described that might be cell type specific. A more profound understanding of the potential interaction between p53 and activated STATs is necessary in order to take full advantage of novel p53–targeted therapies

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

Crispr/Cas9 Engineered 61bp Deletion in the Calr Gene of Mice Leads to Development of Thrombocytosis

In a subset of patients suffering from myeloproliferative neoplasms (MPNs), calreticulin (CALR) exon 9 frameshift mutations are known to be responsible for the development of either essential thrombocythemia (ET) or primary myelofibrosis (PMF) (1, 2). The most prevalent mutations are a 52-bp deletion (del52, type-1 mutation) and a 5-bp TTGTC insertion (ins5, type-2 mutation). In these patients, the mutational status is almost always heterozygous. Our group and collaborators have recently shown that the pathogenic mutant CALR proteins require interaction with and activation of the thrombopoietin receptor (TpoR) for activation of the JAK-STAT pathway (3, 4). Until now, no knock-in mouse model of these diseases has been published. In this abstract, we show how we succeeded in creating such a model. We had shown that the murine CALR mutant proteins behave just like their human counterparts (5). Specifically, the del52, ins5 and del61 (61bp deletion, type-1) Calr mutations were able to transform Ba/F3 cells (murine pro-B lymphocytic cells normally dependent on IL-3 for growth) expressing the thrombopoietin receptor (TpoR) and render them cytokine-independent. Importantly, we also mutated the Ba/F3 genome using the widely adopted CRISPR/Cas9 system in order to create a 61-bp deletion of the exon 9 of Calr. This too successfully transformed the Ba/F3 cells, showing that endogenous levels of expression of a mutant CALR protein are sufficient to induce phenotype in vitro. Now, using the same approach, we injected C57BL/6J mouse zygotes with the same CRISPR/Cas9 constructs to create the same 61-bp deletion in the murine Calr gene. Out of 46 pups born from the procedure, one male pup was heterozygous for the 61-bp deletion. By in vitro fertilization, we subsequently obtained heterozygous Calr del61/WT pups. After inter-breeding the mice, we analyzed the blood of 12 Calr del61/WT males and 12 Calr WT/WT males (littermates) at three different timepoints (15, 18 and 22 weeks old) and found that the Calr del61/WT mice showed significantly higher levels of circulating platelets. Conversely, red blood and white blood cell numbers were the same between both groups at all time points. We further show that expression of a mutant CALR protein, in a heterozygous state, is sufficient to induce abnormal proliferation of megakaryocytes and develop an ET phenotype in vivo in mice. Follow-up in dynamics of the phenotype and bone marrow and spleen pathology (examination of myeloproliferation and fibrosis) allow comparison with the retroviral murine models of CALR-mutant MPNs and with the known features of the human disease. The only limitation of our model is the fact that the Calr del61 mutation is parentally acquired and widespread throughout the organism. With this new model, we aim to test the efficiency of various drugs to prevent or cure the MPN phenotype, such as ruxolitinib, a JAK2 type-1 inhibitor that is already used in clinics in patients suffering from CALR-mutated MPNs. We also now have a means to generate a high number of Calr del61/WT bone marrow cells to extensively study the oncogenic properties of the Calr mutations at different stages of the hematopoeisis. It will also be of great interest to study, if generated, a homozygous mutational status of Calr del61 in vivo. Thus, our system will shed light on the importance of the negatively charged tail of CALR and on the effects of the novel positively charged tail on myeloproliferation

MPL Mutations in Essential Thrombocythemia Uncover A Common Path of Activation with Eltrombopag Dependent on W491.

Mutations in the gene (MPL) encoding the human thrombopoietin receptor (TpoR) drive sporadic and familial essential thrombocythemia (ET). We identified two ET patients that harbor double mutations in cis in MPL, namely L498W-H499C and H499Y-S505N. Using biochemical and signaling assays along with partial saturation mutagenesis we show that L498W is an activating mutation potentiated by H499C, and that H499C/Y enhance the activity of the canonical S505N mutation. L498W and H499C can activate a truncated TpoR mutant, which lacks the extracellular domain, indicating these mutations act on the transmembrane (TM)-cytosolic domain. Using a protein complementation assay we show that L498W and H499C strongly drive dimerization of TpoR. Activation by tryptophan substitution is exquisitely specific for position 498. Using structure-guided mutagenesis we identify upstream amino acid W491 as a key residue required for activation by L498W or canonical activating mutations such as S505N and W515K, as well as by eltrombopag. Structural data point to a common dimerization and activation path for TpoR via its TM domain that is shared between the small molecule agonist eltrombopag and canonical and novel activating TpoR mutations that all depend on W491, a potentially accessible extracellular residue that could become a target for therapeutic intervention

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

Oncogenic CALR mutant C-terminus mediates dual binding to the thrombopoietin receptor triggering complex dimerization and activation

In myeloproliferative neoplasms, frameshift mutants of calreticulin turn into rogue cytokines by inducing constitutive activation of the Thrombopoietin Receptor (TpoR). Here, the authors define how mutant calreticulin acquires specificity for TpoR binding and triggers its constitutive activation

Persistent STAT5 activation in myeloid neoplasms recruits p53 into gene regulation.

STAT (Signal Transducer and Activator of Transcription) transcription factors are constitutively activated in most hematopoietic cancers. We previously identified a target gene, LPP/miR-28 (LIM domain containing preferred translocation partner in lipoma), induced by constitutive activation of STAT5, but not by transient cytokine-activated STAT5. miR-28 exerts negative effects on thrombopoietin receptor signaling and platelet formation. Here, we demonstrate that, in transformed hematopoietic cells, STAT5 and p53 must be synergistically bound to chromatin for induction of LPP/miR-28 transcription. Genome-wide association studies show that both STAT5 and p53 are co-localized on the chromatin at 463 genomic positions in proximal promoters. Chromatin binding of p53 is dependent on persistent STAT5 activation at these proximal promoters. The transcriptional activity of selected promoters bound by STAT5 and p53 was significantly changed upon STAT5 or p53 inhibition. Abnormal expression of several STAT5-p53 target genes (LEP, ATP5J, GTF2A2, VEGFC, NPY1R and NPY5R) is frequently detected in platelets of myeloproliferative neoplasm (MPN) patients, but not in platelets from healthy controls. In conclusion, persistently active STAT5 can recruit normal p53, like in the case of MPN cells, but also p53 mutants, such as p53 M133K in human erythroleukemia cells, leading to pathologic gene expression that differs from canonical STAT5 or p53 transcriptional programs.Oncogene advance online publication, 31 March 2014; doi:10.1038/onc.2014.60

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