CBL linker region and RING finger mutations lead to enhanced granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling via elevated levels of JAK2 and LYN.

Juvenile myelomonocytic leukemia (JMML) is characterized by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). SHP2, NF-1, KRAS, and NRAS are mutated in JMML patients, leading to aberrant regulation of RAS signaling. A subset of JMML patients harbor CBL mutations associated with 11q acquired uniparental disomy. Many of these mutations are in the linker region and the RING finger of CBL, leading to a loss of E3 ligase activity. We investigated the mechanism by which CBL-Y371H, a linker region mutant, and CBL-C384R, a RING finger mutant, lead to enhanced GM-CSF signaling. Expression of CBL mutants in the TF-1 cell line resulted in enhanced survival in the absence of GM-CSF. Cells expressing CBL mutations displayed increased phosphorylation of GM-CSF receptor ßc subunit in response to stimulation, although expression of total GM-CSFR ßc was lower. This suggested enhanced kinase activity downstream of GM-CSFR. JAK2 and LYN kinase expression is elevated in CBL-Y371H and CBL-C384R mutant cells, resulting in enhanced phosphorylation of CBL and S6 in response to GM-CSF stimulation. Incubation with the JAK2 inhibitor, TG101348, abolished the increased phosphorylation of GM-CSFR ßc in cells expressing CBL mutants, whereas treatment with the SRC kinase inhibitor dasatinib resulted in equalization of GM-CSFR ßc phosphorylation signal between wild type CBL and CBL mutant samples. Dasatinib treatment inhibited the elevated phosphorylation of CBL-Y371H and CBL-C384R mutants. Our study indicates that CBL linker and RING finger mutants lead to enhanced GM-CSF signaling due to elevated kinase expression, which can be blocked using small molecule inhibitors targeting specific downstream pathways.

Loss of JAK2 regulation via a heterodimeric VHL-SOCS1 E3 ubiquitin ligase underlies Chuvash polycythemia.

Chuvash polycythemia is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the VHL (von Hippel-Lindau) gene, whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark abnormalities of Chuvash polycythemia, such as hypersensitivity to erythropoietin, are unclear. Here we show that VHL directly binds suppressor of cytokine signaling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated JAK2 (pJAK2) for ubiquitin-mediated destruction. In contrast, Chuvash polycythemia-associated VHL mutants have altered affinity for SOCS1 and do not engage with and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reversed the disease phenotype in Vhl(R200W/R200W) knock-in mice, an experimental model that recapitulates human Chuvash polycythemia. These results show that VHL is a SOCS1-cooperative negative regulator of JAK2 and provide biochemical and preclinical support for JAK2-targeted therapy in individuals with Chuvash polycythemia.

The current status and future potential of personalized diagnostics: Streamlining a customized process.

Recent genetic discoveries and related developments in genomic techniques have led to the commercialization of novel diagnostic platforms for studying disease or gauging therapeutic outcomes in individual patients. This newly emerging field is called "personalized medicine," and uses the patient's genetic composition to tailor strategies for patient-specific disease detection, treatment, or prevention. Personalized diagnostic tests are used to detect patient-to-patient variations in gene or protein expression levels, which act as indicators for drug treatments or disease prognosis. In turn, medical professionals can better answer questions such as: "Who should be treated with which drug?" and "How should the treatment be administered?" The regulations governing personalized medicine can be complicated because they encompass in vitro diagnostic systems and laboratory tests as well as methods of disease treatment and patient care. Industry, academia, medicine, and the Food and Drug Administration (FDA) are all involved in the cultivation of the field: substantial collaborations between drug developers and regulatory authorities are required to consider and shape emerging regulations as personalized drug strategies mature. Some of the regulatory issues identified by industry and the FDA about personalized medicine and personalized diagnostics will be addressed. In addition, relevant collaborations, advances, and current and draft regulatory guidances will be discussed with respect to the future of personalized medicine.

STAT1 promotes megakaryopoiesis downstream of GATA-1 in mice.

Thrombocytosis is associated with inflammation, and certain inflammatory cytokines, including IFN-gamma, stimulate megakaryocyte and platelet production. However, the roles of IFN-gamma and its downstream effector STAT1 in megakaryocyte development are poorly understood. We previously reported that STAT1 expression was significantly downregulated in Gata1-knockdown murine megakaryocytes, which also have impaired terminal maturation. Here, we show that ectopic expression of STAT1, or its target effector IRF-1, rescued multiple defects in Gata1-deficient megakaryopoiesis in mice, inducing polyploidization and expression of a subset of platelet-expressing genes. Enforced expression of STAT1, IRF-1, or GATA-1 enhanced phosphorylation of STAT1, STAT3, and STAT5 in cultured Gata1-deficient murine megakaryocytes, with concomitant megakaryocyte maturation. In contrast, enhanced thrombopoietin signaling, conferred by enforced expression of constitutively active JAK2 or c-MPL, induced phosphorylation of STAT3 and STAT5, but not STAT1, and failed to rescue megakaryocyte maturation. Finally, megakaryocytes from Stat1(-/-) mice were defective in polyploidization. Together, these findings reveal a unique role for STAT1 in megakaryopoiesis and provide new insights into how GATA-1 regulates this process. Our studies elucidate potential mechanisms by which various inflammatory disorders can cause elevated platelet counts.

Turning cells red: signal transduction mediated by erythropoietin.

Erythropoietin (EPO) is the crucial cytokine regulator of red blood-cell production. Since the discovery of EPO in 1985 and the isolation of its cognate receptor four years later, there has been significant interest in understanding the unique ability of this ligand-receptor pair to promote erythroid mitogenesis, survival and differentiation. The development of knockout mice has elucidated the precise role of the ligand, receptor and downstream players in murine erythroid development. In this review, we summarize EPO-mediated signaling pathways and examine their significance in vivo.