Myeloproliferative disorders are clonal haematopoietic stem cell malignancies characterized by independency or hypersensitivity of haematopoietic progenitors to numerous cytokines. The molecular basis of most myeloproliferative disorders is unknown. On the basis of the model of chronic myeloid leukaemia, it is expected that a constitutive tyrosine kinase activity could be at the origin of these diseases. Polycythaemia vera is an acquired myeloproliferative disorder, characterized by the presence of polycythaemia diversely associated with thrombocytosis, leukocytosis and splenomegaly. Polycythaemia vera progenitors are hypersensitive to erythropoietin and other cytokines. Here, we describe a clonal and recurrent mutation in the JH2 pseudo-kinase domain of the Janus kinase 2 (JAK2) gene in most (> 80%) polycythaemia vera patients. The mutation, a valine-to-phenylalanine substitution at amino acid position 617, leads to constitutive tyrosine phosphorylation activity that promotes cytokine hypersensitivity and induces erythrocytosis in a mouse model. As this mutation is also found in other myeloproliferative disorders, this unique mutation will permit a new molecular classification of these disorders and novel therapeutical approaches.

We recently reported (April 18 issue) 1 the sustained correction of X-linked severe combined immunodeficiency disease by ex vivo, retrovirally mediated transfer of the g c gene into CD34+ cells in four of five patients with the disease. These results have since been confirmed in four additional patients with typical X-linked severe combined immunodeficiency. Of the first four successfully treated patients, three continue to do well up to 3.6 years after gene therapy, whereas a serious adverse event occurred in the fourth patient. At a routine checkup 30 months after gene therapy, lymphocytosis consisting of a monoclonal population of V g 9/V d 1, g / d T cells of mature phenotype was detected. One proviral integration site was found, located on the short arm of chromosome 11 within the LMO-2 locus, as determined with the use of linearamplification mediated polymerase-chain-reaction analysis. 2 This proviral integration within the LMO-2 locus was associated with aberrant expression of the LMO-2 transcript in the monoclonal T-cell population. Aberrant expression of LMO-2 has been reported in acute lymphoblastic leukemia arising from T cells with a / b receptors, usually with the chromosomal translocation t(11;14). 3 Tests for replicationcompetent retrovirus were repeatedly negative in our patient's lymphocytes. Between 30 and 34 months after gene therapy, the patient's lymphocyte count rose to 300,000 per cubic millimeter, and hepatosplenomegaly developed. Further investigations showed the presence of a t(6;13) translocation, which had not been detected 30 months after the therapy. Treatment with a chemotherapy regimen based on a high-risk protocol for acute lymphocytic leukemia (a protocol of the Dutch Childhood Leukemia Study Group) was initiated and has resulted, to date, in a dramatic reduction in the abnormal cells.

A JAK2 V617F mutation is frequently found in several BCR/ABL-negative myeloproliferative disorders. To address the contribution of this mutant to the pathogenesis of these different myeloproliferative disorders, we used an adoptive transfer of marrow cells transduced with a retrovirus expressing JAK2 V617F in recipient irradiated mice. Hosts were analyzed during the 6 months after transplantation. For a period of 3 months, mice developed polycythemia, macrocytosis and usually peripheral blood granulocytosis. Transient thrombocytosis was only observed in a low-expresser group. All mice displayed trilineage hyperplasia in marrow and spleen along with an amplification of myeloid and erythroid progenitor cells and a formation of endogenous erythroid colonies. After 3 to 4 months, polycythemia regressed, abnormally shaped red blood cells and platelets were seen in circulation, and a deposition of reticulin fibers was observed in marrow and spleen. Development of fibrosis was associated with anemia, thrombocytopenia, high neutrophilia, and massive splenomegaly. These features mimic human polycythemia vera and its evolution toward myelofibrosis. This work demonstrates that JAK2 V617F is sufficient for polycythemia and fibrosis development and offers an in vivo model to assess novel therapeutic approaches for JAK2 V617F

Key Points Calreticulin type I and type II mutants are drivers of the disease as they induce thrombocytosis in a retroviral mouse model. Thrombopoietin receptor MPL is required for calreticulin mutants to induce an essential thrombocythemia phenotype in transplanted mice.

Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34 + progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.

Although other mutations may predate the acquisition of the JAK2(V617F) mutation, the latter is sufficient to drive the disease phenotype observed in BCR-ABL-negative myeloproliferative neoplasms (MPNs). One of the consequences of JAK2(V617F) is genetic instability that could explain JAK2(V617F)-mediated MPN progression and heterogeneity. Here, we show that JAK2(V617F) induces the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment of a knock-in (KI) mouse model and in patients with JAK2(V617F) MPNs. JAK2(V617F)-dependent ROS elevation was partly mediated by an AKT-induced decrease in catalase expression and was accompanied by an increased number of 8-oxo-guanines and DNA double-strand breaks (DSBs). Moreover, there was evidence for a mitotic recombination event in mice resulting in loss of heterozygosity of Jak2(V617F). Mice engrafted with 30% of Jak2(V617F) KI bone marrow (BM) cells developed a polycythemia vera-like disorder. Treatment with the anti-oxidant N-acetylcysteine (NAC) substantially restored blood parameters and reduced damages to DNA. Furthermore, NAC induced a marked decrease in splenomegaly with reduction in the frequency of the Jak2(V617F)-positive hematopoietic progenitors in BM and spleen. Altogether, overproduction of ROS is a mediator of JAK2(V617F)-induced DNA damages that promote disease progression. Targeting ROS accumulation might prevent the development of JAK2(V617F) MPNs.

Key Points JAK2V617F amplifies in mouse early hematopoietic cells, giving them a proliferative advantage through high cell cycling and low apoptosis. IFNα prevented myeloproliferative neoplasm development by specifically inhibiting JAK2V617F cells at an early differentiation stage.

Oncogenic activation of tyrosine kinase signaling pathway is recurrent in human leukemia. To gain insight into the oncogenic process leading to acute megakaryoblastic leukemia (AMKL), we performed sequence analyses of a subset of oncogenes known to be activated in human myeloid and myeloproliferative disorders. In a series of human AMKL samples from both Down syndrome and non-Down syndrome patients, mutations were identified within KIT, FLT3, JAK2, JAK3, and MPL genes, with a higher frequency in DS than in non-DS patients. The novel mutations were analyzed using