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Thrombopoietin (Tpo), which binds to its specific receptor, the Mpl protein, is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo binding to Mpl triggers activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor, as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for regulation of major Tpo-stimulated signalling pathways: Mpl-Y565, Mpl-Y599 and Mpl-Y604. Here, we have introduced mutations in the mouse germline and report a consistent physiological requirement for Mpl-Y599, mutation of which resulted in thrombocytopenia, deficient megakaryopoiesis, low hematopoietic stem cell (HSC) number and function, and attenuated responses to myelosuppression. We further show that in models of myeloproliferative neoplasms (MPN), where Mpl is required for pathogenesis, thrombocytosis was dependent on intact Mpl-Y599. In contrast, Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to myelosuppression, and exacerbated thrombocytosis associated with MPN.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.

Genetic alterations disrupting the transcription factor IKZF1 (encoding IKAROS) are associated with poor outcome in B lineage acute lymphoblastic leukemia (B-ALL) and occur in >70% of the high-risk BCR-ABL1+ (Ph+) and Ph-like disease subtypes. To examine IKAROS function in this context, we have developed novel mouse models allowing reversible RNAi-based control of Ikaros expression in established B-ALL in vivo. Notably, leukemias driven by combined BCR-ABL1 expression and Ikaros suppression rapidly regress when endogenous Ikaros is restored, causing sustained disease remission or ablation. Comparison of transcriptional profiles accompanying dynamic Ikaros perturbation in murine B-ALL in vivo with two independent human B-ALL cohorts identified nine evolutionarily conserved IKAROS-repressed genes. Notably, high expression of six of these genes is associated with inferior event–free survival in both patient cohorts. Among them are EMP1, which was recently implicated in B-ALL proliferation and prednisolone resistance, and the novel target CTNND1, encoding P120-catenin. We demonstrate that elevated Ctnnd1 expression contributes to maintenance of murine B-ALL cells with compromised Ikaros function. These results suggest that IKZF1 alterations in B-ALL leads to induction of multiple genes associated with proliferation and treatment resistance, identifying potential new therapeutic targets for high-risk disease.

A pivotal mediator of actin dynamics is the protein cofilin, which promotes filament severing and depolymerization, facilitating the breakdown of existing filaments, and the enhancement of filament growth from newly created barbed ends. It does so in concert with actin interacting protein 1 (Aip1), which serves to accelerate cofilin's activity. While progress has been made in understanding its biochemical functions, the physiologic processes the cofilin/Aip1 complex regulates, particularly in higher organisms, are yet to be determined. We have generated an allelic series for WD40 repeat protein 1 (Wdr1), the mammalian homolog of Aip1, and report that reductions in Wdr1 function produce a dramatic phenotype gradient. While severe loss of function at the Wdr1 locus causes embryonic lethality, macrothrombocytopenia and autoinflammatory disease develop in mice carrying hypomorphic alleles. Macrothrombocytopenia is the result of megakaryocyte maturation defects, which lead to a failure of normal platelet shedding. Autoinflammatory disease, which is bone marrow–derived yet nonlymphoid in origin, is characterized by a massive infiltration of neutrophils into inflammatory lesions. Cytoskeletal responses are impaired in Wdr1 mutant neutrophils. These studies establish an essential requirement for Wdr1 in megakaryocytes and neutrophils, indicating that cofilin-mediated actin dynamics are critically important to the development and function of both cell types.

A pivotal mediator of actin dynamics is the protein cofilin, which promotes filament severing and depolymerization, facilitating the breakdown of existing filaments, and the enhancement of filament growth from newly created barbed ends. It does so in concert with actin interacting protein 1 (Aip1), which serves to accelerate cofilin’s activity. While progress has been made in understanding its biochemical functions, the physiologic processes the cofilin/Aip1 complex regulates, particularly in higher organisms, are yet to be determined. We have generated an allelic series for WD40 repeat protein 1 (Wdr1), the mammalian homolog of Aip1, and report that reductions in Wdr1 function produce a dramatic phenotype gradient. While severe loss of function at the Wdr1locus causes embryonic lethality, macrothrombocytopenia and autoinflammatory disease develop in mice carrying hypomorphic alleles. Macrothrombocytopenia is the result of megakaryocyte maturation defects, which lead to a failure of normal platelet shedding. Autoinflammatory disease, which is bone marrow–derived yet nonlymphoid in origin, is characterized by a massive infiltration of neutrophils into inflammatory lesions. Cytoskeletal responses are impaired in Wdr1 mutant neutrophils. These studies establish an essential requirement for Wdr1 in megakaryocytes and neutrophils, indicating that cofilin-mediated actin dynamics are critically important to the development and function of both cell types.

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of granulocyte-colony stimulating factor (G-CSF) signaling in vivo. SOCS proteins regulate cytokine signaling by binding, via their SH2 domains, to activated cytokine receptors or their associated Janus kinases. In addition, they bind to the elongin B/C ubiquitin ligase complex via the SOCS box. To ascertain the contribution of the SOCS box of SOCS3 to in vivo regulation of G-CSF signaling, we generated mice expressing a truncated SOCS3 protein lacking the C-terminal SOCS box (SOCS3ΔSB/ΔSB). SOCS3ΔSB/ΔSB mice were viable, had normal steady-state hematopoiesis, and did not develop inflammatory disease. Despite the mild phenotype, STAT3 activation in response to G-CSF signaling was prolonged in SOCS3ΔSB/ΔSB bone marrow. SOCS3ΔSB/ΔSB bone marrow contained increased numbers of colony-forming cells responsive to G-CSF and IL-6. Treatment of the mice with pharmacologic doses of G-CSF, which mimics emergency granulopoiesis and therapeutic use of G-CSF, revealed that SOCS3ΔSB/ΔSB mice were hyperresponsive to G-CSF. Compared with wild-type mice, SOCS3ΔSB/ΔSB mice developed a more florid arthritis when tested using an acute disease model. Overall, the results establish a role for the SOCS box of SOCS3 in the in vivo regulation of G-CSF signaling and the response to inflammatory stimuli.

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of granulocyte-colony stimulating factor (G-CSF) signaling in vivo. SOCS proteins regulate cytokine signaling by binding, via their SH2 domains, to activated cytokine receptors or their associated Janus kinases. In addition, they bind to the elongin B/C ubiquitin ligase complex via the SOCS box. To ascertain the contribution of the SOCS box of SOCS3 to in vivo regulation of G-CSF signaling, we generated mice expressing a truncated SOCS3 protein lacking the C-terminal SOCS box (SOCS3ΔSB/ΔSB). SOCS3ΔSB/ΔSBmice were viable, had normal steady-state hematopoiesis, and did not develop inflammatory disease. Despite the mild phenotype, STAT3 activation in response to G-CSF signaling was prolonged in SOCS3ΔSB/ΔSBbone marrow. SOCS3ΔSB/ΔSBbone marrow contained increased numbers of colony-forming cells responsive to G-CSF and IL-6. Treatment of the mice with pharmacologic doses of G-CSF, which mimics emergency granulopoiesis and therapeutic use of G-CSF, revealed that SOCS3ΔSB/ΔSBmice were hyperresponsive to G-CSF. Compared with wild-type mice, SOCS3ΔSB/ΔSBmice developed a more florid arthritis when tested using an acute disease model. Overall, the results establish a role for the SOCS box of SOCS3 in the in vivo regulation of G-CSF signaling and the response to inflammatory stimuli.

Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2-deficient mice, which grow to one and a half times the size of their wild-type littermates, suggests that SOCS-2 may attenuate growth hormone (GH) signaling. In vitro studies indicate that, while SOCS-2 can inhibit GH action at low concentrations, at higher concentrations it may potentiate signaling. To determine whether a similar enhancement of signaling is observed in vivo or alternatively whether increased SOCS-2 levels repress growth in vivo, we generated and analyzed transgenic mice that overexpress SOCS-2 from a human ubiquitin C promoter. These mice are not growth-deficient and are, in fact, significantly larger than wild-type mice. The overexpressed SOCS-2 was found to bind to endogenous GH receptors in a number of mouse organs, while phosphopeptide binding studies with recombinant SOCS-2 defined phosphorylated tyrosine 595 on the GH receptor as the site of interaction. Together, the data implicate SOCS-2 as having dual effects on GH signaling in vivo.

SOCS‐1 was originally identified as an inhibitor of interleukin‐6 signal transduction and is a member of a family of proteins (SOCS‐1 to SOCS‐7 and CIS) that contain an SH2 domain and a conserved carboxyl‐terminal SOCS box motif. Mutation studies have established that critical contributions from both the amino‐terminal and SH2 domains are essential for SOCS‐1 and SOCS‐3 to inhibit cytokine signaling. Inhibition of cytokine‐dependent activation of STAT3 occurred in cells expressing either SOCS‐1 or SOCS‐3, but unlike SOCS‐1, SOCS‐3 did not directly interact with or inhibit the activity of JAK kinases. Although the conserved SOCS box motif appeared to be dispensable for SOCS‐1 and SOCS‐3 action when over‐expressed, this domain interacts with elongin proteins and may be important in regulating protein turnover. In gene knockout studies, SOCS‐1−/−mice were born but failed to thrive and died within 3 weeks of age with fatty degeneration of the liver and hemopoietic infiltration of several organs. The thymus in SOCS‐1−/−mice was small, the animals were lymphopenic, and deficiencies in B lymphocytes were evident within hemopoietic organs. We propose that the absence of SOCS‐1 in these mice prevents lymphocytes and liver cells from appropriately controlling signals from cytokines with cytotoxic side effects. J. Leukoc. Biol.66: 588–592; 1999.

Leukemia inhibitory factor [LIF] is a glycoprotein that is able to induce the differentiation of M1 myeloid leukemic cells and prevent the differentiation of murine embryonic stem [ES] cells. This report presents a simple method for the production and purification of large amounts of recombinant murine and human LIF in Escherichia coli. LIF is expressed initially as a fusion product with glutathione Stransferase, separated by a thrombin cleavage site. The fusion product can be rapidly purified on a glutathione-agarose affinity matrix and fully biologically active LIF released from the matrix by cleavage with thrombin. In this form LIF is suitable for many in vitro cell culture uses and in particular for ES cell culture. Further purification to homogeneity of thrombin-released LIF can be achieved by a single fractionation by reversed-phase high performance liquid chromatography; the purified product contains no detectable bacterial endotoxin. Evidence is presented that the intact fusion product has little or no biological activity but biologically active LIF is released by endogenous proteolytic cleavage.

In this study the gene for the murine interleukin-11 receptor α chain (IL-11Rα) has been characterized. The gene spans 9 kilobase pairs of DNA, and the organization of its 14 exons conforms to the pattern observed for other members of the hematopoietin receptor family. Analysis of the 5′ end of the cDNA using 5′ RACE showed that the first two exons, designated exons 1a and 1b, are spliced to form alternate transcripts. Transcripts initiating from exon 1b were not found in adult tissues but were present in embryonic stem cells. S1 nuclease and 5′ rapid amplification of cDNA ends assays demonstrated multiple major and minor sites of transcription initiation for each exon. The putative promoter regions of both exons lacked TATA boxes, although potential recognition sites for several transcription factors including Sp1, AP1, and AP2 were present. A comparison of the murine and human IL-11Rα revealed that the 5′ sequence upstream of the major site of transcription initiation site for exon 1b is highly conserved. Northern analysis showed that IL-11Rα is expressed in many adult murine tissues. A second IL-11Rα-like locus containing a sequence homologous to exons 2-13 was also identified.

Embryonic stem (ES) cells, the totipotent outgrowths of bias-tocysts1,2, can be cultured and manipulated in vitro and then returned to the embryonic environment where they develop normally and can contribute to all cell lineages3–9. Maintenance of the stem-cell phenotype in vitro requires the presence of a feeder layer of fibroblasts1,2,10or of a soluble factor, differentiation inhibitory activity (DIA) produced by a number of sources5,11,12; in the absence of DIA the ES cells differentiate into a wide variety of cell types. We recently noted several similarities between partially purified DIA and a haemopoietic regulator, myeloid leukaemia inhibitory factor (LIF), a molecule which induces differentiation in Ml myeloid leukaemic cells and which we have recently purified, cloned and characterized13–18. We demonstrate here that purified, recombinant LIF can substitute for DIA in the maintenance of totipotent ES cell lines that retain the potential to form chimaeric mice.

A search of the nucleic acid database of expressed sequence tags (ESTs) revealed several partial cDNA sequences that could encode proteins homologous to the ligands for Eph-related kinases (LERKs). Oligonucleotides designed from the ESTs were used to probe a human brain cDNA library and obtain overlapping clones that encoded two different novel LERKS (NLERK-1 and NLERK-2). NLERK-1 and NLERK-2 are most closely related to human LERK-2/Elk-ligand and they form a subclass of LERKs that contain a transmembrane domain and a conserved cytoplasmic domain. Full-length NLERK-1 was expressed as a glycosylated membrane protein in COS cells and was not secreted into the medium. Full-length NLERK-2 was similarly expressed in COS cells but both membranebound and a truncated, proteolytically-released form were detected. Engineered forms of both NLERK-1 and NLERK-2 lacking transmembrane and cytoplasmic domains were also expressed in COS cells and each was detected in the extracellular medium.

Leukemia inhibitory factor (LIF) induces a variety of disparate biological responses in different cell types. These responses are thought to be mediated through the functional LIF receptor (LIFR), consisting of a heterodimeric complex of LIFR α-chain (LIFRα) and gp130. The present study investigated the relative capacity of the cytoplasmic domains of each receptor subunit to signal particular responses in several cell types. To monitor the signaling potential of LIFRα and gp130 individually, we constructed chimeric receptors by linking the extracellular domain of granulocyte colony-stimulating factor receptor (GCSFR) to the transmembrane and cytoplasmic regions of either LIFRα or gp130. Both chimeric receptors and the full-length GCSFR in expressed in M1 myeloid leukemic cells to measure differentiation induction, in embryonic stem cells to measure differentiation inhibition, and in Ba/F3 cells to measure cell proliferation. Our results demonstrated that whereas GCSFR-gp130 receptor homodimer mediated a GCSF-induced signal in all three cell types, the GCSFR-LIFRα receptor homodimer was only functional in embryonic stem cells. These findings suggest that the signaling potential of gp130 and LIFRα cytoplasmic domains may differ depending upon the tissue and cellular response initiated.

cDNAs encoding the receptors for murine G-CSF, M-CSF or human GM-CSF were inserted into the murine hemopoietic continuous cell lines Ba/F3 or FDC-P1 and sublines selected that were then responsive to proliferative stimulation by these growth factors. When used in microwell assays the Ba/F3 G-CSF receptor-expressing cell line was able to detect 100 pg G-CSF per ml, the Ba/F3 M-CSF receptor-expressing cell line 100-400 pg M-CSF per ml and the FDC-P1 line expressing the α- and β-chains of the human GM-CSF receptor detected 5-10 pg/ml of GM-CSF in test material. These cell lines appear satisfactory for use as selective bioassays for these colony stimulating factors in material potentially containing a mixture of growth factors.

Solving the structure of the hinge domain of an epigenetic regulator highlights residues involved in dimerization and function.

We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hoxgenes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist-HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xistor other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation.

A series of yeast mitochondrial, mit− mutants with defects in the oli2 gene, coding for subunit 6 of the mitochondrial ATPase complex, has been analyzed at the DNA sequence level. Fifteen of sixteen primary mit− mutants were shown to contain frameshift or nonsense mutations predicting truncated subunit 6 polypeptides, in various strains ranging from about 20% to 95% of the wild-type length of 259 amino acids. In only one strain could the defect in subunit 6 function be assigned to amino acid substitution in an otherwise full-length subunit 6. Many mutants carried multiple base substitutions or insertions/deletions, presumably arising from the manganese chloride mutagenesis treatment. Revertants fron three of the mit− mutants were analyzed: all contained full-length subunit 6 proteins with one or more amino acid substitutions. The preponderance of truncated proteins as opposed to substituted full-length proteins in oli8 mit− mutants is suggested to reflect the ability of subunit 6 to accommodate amino acid substitutions at many locations, with little or no change in its functional properties in the membrane Fo-sector of the ATPase complex.