The signalling cascade including Raf, mitogen-activated protein kinase (MAPK) kinase and extracellular-signal-regulated kinase (ERK) is important in many facets of cellular regulation. Raf is activated through both Ras-dependent and Ras-independent mechanisms, but the regulatory mechanisms of Raf activation remain unclear. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified and characterized as negative regulators of growth-factor-induced ERK activation. But the molecular functions of mammalian Sproutys have not been clarified. Here we show that mammalian Sprouty4 suppresses vascular epithelial growth factor (VEGF)-induced, Ras-independent activation of Raf1 but does not affect epidermal growth factor (EGF)-induced, Ras-dependent activation of Raf1. Sprouty4 binds to Raf1 through its carboxy-terminal cysteine-rich domain, and this binding is necessary for the inhibitory activity of Sprouty4. In addition, Sprouty4 mutants of the amino-terminal region containing the conserved tyrosine residue, which is necessary for suppressing fibroblast growth factor signalling, still inhibit the VEGF-induced ERK pathway. Our results show that receptor tyrosine kinases use distinct pathways for Raf and ERK activation and that Sprouty4 differentially regulates these pathways.

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Key Points• CDK6 is a critical effector of MLL fusions in myeloid leukemogenesis.• Genetic and pharmacologic inhibition of CDK6 overcome the differentiation block associated with MLLrearranged AML.Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusionmediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9-driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts. (Blood. 2014;124(1):13-23) Introduction A substantial proportion of acute myeloid leukemia (AML) cases harbor balanced translocations of chromosome 11q23, and AML with t(9;11)(p22;q23) is recognized as a distinct entity by the World Health Organization Classification of Tumors of Hematopoietic and Lymphoid Tissues.1,2 On the molecular level, t(11q23) results in fusion of the MLL gene, which encodes an H3K4 methyltransferase, to a broad spectrum of partner genes, such as MLLT3 (also called AF9), MLLT4 (AF6), MLLT1 (ENL), and MLLT10 (AF10) on chromosomes 9p22, 6q27, 19p13.3, and 10p12, respectively. 3,4 A key functional feature of mixed-lineage leukemia (MLL) rearrangements is their ability to confer leukemia-initiating activity to hematopoietic stem and progenitor cells (HSPC). 5,6 MLL fusions are characterized by loss of the C-terminal H3K4 methyltransferase domain, and their leukemogenic activity is dependent on both features of the remaining N-terminal portion, such as a binding motif for the menin tumor suppressor that mediates the contact between MLL and chromatin as well as aberrant transactivation of target genes through heterologous domains contributed by the various partner proteins.7 For example, MLL fusions involving AF9, ENL, and AF10, which account for the majority of MLLrearranged AML, recruit multiprotein complexes essential for transcriptional activation/elongation...

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Sprouty/Spred family proteins have been identified as negative regulators of growth factor-induced ERK/mitogen-activated protein (MAP) kinase activation. However, it has not been clarified whether these proteins regulate cytokine-induced ERK activity. We found that Spred-1 is highly expressed in interleukin-3 (IL-3)-dependent hematopoietic cell lines and bone marrow-derived mast cells. To investigate the roles of Spred-1 in hematopoiesis, we expressed wild-type Spred-1 and a dominant negative form of Spred-1, ⌬C-Spred, in IL-3-and stem cell factor (SCF)-dependent cell lines as well as hematopoietic progenitor cells from mouse bone marrow by retrovirus gene transfer. In IL-3-dependent Ba/F3 cells expressing c-kit, forced expression of Spred-1 resulted in a reduced proliferation rate and ERK activation in response to not only SCF but also IL-3. In contrast, ⌬C-Spred augmented IL-3-induced cell proliferation and ERK activation. Wild-type Spred-1 inhibited colony formation of bone marrow cells in the presence of cytokines, whereas ⌬C-Spred-1 expression enhanced colony formation. Augmentation of ERK activation and proliferation in response to IL-3 was also observed in Spred-1-deficient bone marrow-derived mast cells. These data suggest that Spred-1 negatively regulates hematopoiesis by suppressing not only SCFinduced but also IL-3-induced ERK activation.Receptor tyrosine kinases, such as stem cell factor (SCF) 1 receptor (c-kit), as well as cytokine receptors including interleukin (IL)-3 or erythropoietin (EPO) receptor activate the extracellular signal-regulated kinase (ERK) cascade. ERK activation is initiated by binding of Grb2 to the phosphorylated tyrosine residues of the receptor or phosphorylated adaptor molecules such as Shc, FRS-2, IRS-1/2, SHP-2, and Gab-1. The complex of Grb2 and SOS (son of sevenless) activates Ras by GTP loading. Ras-GTP recruits Raf1 to the plasma membrane (1, 2), which is then phosphorylated and activated by several, not well defined, kinases with complex regulatory mechanisms (3-5). Activated Raf then phosphorylates and activates the dual-specific kinase MEK, which phosphorylates and activates ERKs. In addition, the Ras-independent Raf1-ERK activation mechanism has been recently demonstrated, and members of the protein kinase C family of serine/threonine kinases have been implicated as potential activators of Raf (6).Mitogen-activated protein (MAP) kinases including ERKs play important roles in hematopoiesis. Most hematopoietic cytokines (hematopoietins) activate the JAK-STAT and Ras-ERK pathways, both being required for a satisfactory level of proliferation and differentiation of hematopoietic cells. For example, STAT5 activation is not sufficient for EPO-dependent growth of CTLL2 cells expressing EPO receptor, but additional activation of MAP kinases can support their cellular proliferation in response to EPO (7). MAP kinases have also been shown to play a critical role in megakaryopoiesis by c-mpl (8). However, little is known about how MAP kinase is regulated in hematopoietic cell...

We report here that loss of the Sprouty2 gene (also known as Spry2) in mice resulted in enteric nerve hyperplasia, which led to esophageal achalasia and intestinal pseudo-obstruction. Glial cell line-derived neurotrophic factor (GDNF) induced hyperactivation of ERK and Akt in enteric nerve cells. Anti-GDNF antibody administration corrected nerve hyperplasia in Sprouty2-deficient mice. We show Sprouty2 to be a negative regulator of GDNF for the neonatal development or survival of enteric nerve cells.

Large chromosomal deletions are among the most common molecular abnormalities in cancer, yet the identification of relevant genes has proven difficult. The 5q؊ syndrome, a subtype of myelodysplastic syndrome (MDS), is a chromosomal deletion syndrome characterized by anemia and thrombocytosis. Although we have previously shown that hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q؊ syndrome, it does not affect thrombocytosis. Here we show that a microRNA located in the common deletion region of 5q؊ syndrome, miR-145, affects megakaryocyte and erythroid differentiation. We find that miR-145 functions through repression of Fli-1, a megakaryocyte and erythroid regulatory transcription factor. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 decreases the production of megakaryocytic cells relative to erythroid cells, whereas inhibition of miR-145 or overexpression of Fli-1 has a reciprocal effect. Moreover, combined loss of miR-145 and RPS14 cooperates to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q؊ syndrome. Taken together, these findings demonstrate that coordinate deletion of a miRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q؊ syndrome. IntroductionThe 5qϪ syndrome is a subtype of myelodysplastic syndrome (MDS) characterized by a macrocytic anemia, a normal or elevated platelet count, and hypolobated micromegakaryocytes. 1 Patients have heterozygous deletions of chromosome 5q, and no genetic lesions have been identified on the intact allele that would cause homozygous inactivation of a gene, raising the hypothesis that haploinsufficiency for one or more genes is sufficient to cause the clinical phenotype of the 5qϪ syndrome. A common deleted region (CDR) for the 5qϪ syndrome has been mapped that spans approximately 1.5 megabases, encompassing 40 protein-coding genes. 2 In a screen of each of the genes within this CDR, we found that decreased expression of RPS14, encoding a member of the 40S ribosomal subunit, causes the erythroid phenotype of the 5qϪ syndrome. 3 Similarly, inherited mutations that inactivate one allele of other ribosomal proteins cause Diamond-Blackfan anemia (DBA), another disease in which patients have a severe macrocytic anemia. 4 Whereas macrocytic anemia is a defining feature of the 5qϪ syndrome and DBA, both of which are associated with haploinsufficiency for ribosomal genes, thrombocytosis is characteristic of the 5qϪ syndrome but not DBA. 5 We hypothesized that additional genes must contribute to the molecular basis of the 5qϪ syndrome given that RPS14 deficiency probably does not explain the full clinical phenotype of the 5qϪ syndrome, that deletions are universally large, and that no mutations have been found that inactivate RPS14 or any other single gene within the 5qϪ syndrome CDR.In our genetic screen of the 5qϪ syndrome CDR, we evaluated all protein coding genes in the CDR fo...

T helper 2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, play a critical role in allergic asthma. These cytokines transmit signals through the Janus kinase/signal transducer and activator of transcription (STAT) and the Ras–extracellular signal-regulated kinase (ERK) signaling pathways. Although the suppressor of cytokine signaling (SOCS) family proteins have been shown to regulate the STAT pathway, the mechanism regulating the ERK pathway has not been clarified. The Sprouty-related Ena/VASP homology 1–domain-containing protein (Spred)-1 has recently been identified as a negative regulator of growth factor–mediated, Ras-dependent ERK activation. Here, using Spred-1–deficient mice, we demonstrated that Spred-1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness, without affecting helper T cell differentiation. Biochemical assays indicate that Spred-1 suppresses IL-5–dependent cell proliferation and ERK activation. These data indicate that Spred-1 negatively controls eosinophil numbers and functions by modulating IL-5 signaling in allergic asthma.