Emily Sims

Rho Kinase Regulates the Survival and Transformation of Cells Bearing Oncogenic Forms of KIT, FLT3, and BCR-ABL

We show constitutive activation of Rho kinase (ROCK) in cells bearing oncogenic forms of KIT, FLT3, and BCR-ABL, which is dependent on PI3K and Rho GTPase. Genetic or pharmacologic inhibition of ROCK in oncogene-bearing cells impaired their growth as well as the growth of acute myeloid leukemia patient-derived blasts and prolonged the life span of mice bearing myeloproliferative disease. Downstream from ROCK, rapid dephosphorylation or loss of expression of myosin light chain resulted in enhanced apoptosis, reduced growth, and loss of actin polymerization in oncogene-bearing cells leading to significantly prolonged life span of leukemic mice. In summary we describe a pathway involving PI3K/Rho/ROCK/MLC that may contribute to myeloproliferative disease and/or acute myeloid leukemia in humans.

The p85α Subunit of Class IA Phosphatidylinositol 3-Kinase Regulates the Expression of Multiple Genes Involved in Osteoclast Maturation and Migration

ABSTRACT Intracellular signals involved in the maturation and function of osteoclasts are poorly understood. Here, we demonstrate that osteoclasts express multiple regulatory subunits of class IA phosphatidylinositol 3-kinase (PI3-K) although the expression of the full-length form of p85α is most abundant. In vivo, deficiency of p85α results in a significantly greater number of trabeculae and significantly lower spacing between trabeculae as well as increased bone mass in both males and females compared to their sex-matched wild-type controls. Consistently, p85α−/− osteoclast progenitors show impaired growth and differentiation, which is associated with reduced activation of Akt and mitogen-activated protein kinase extracellular signal-regulated kinase 1 (Erk1)/Erk2 in vitro. Furthermore, a significant reduction in the ability of p85α−/− osteoclasts to adhere to as well as to migrate via integrin αvβ3 was observed, which was associated with reduced bone resorption. Microarray as well as quantitative real-time PCR analysis of p85α−/− osteoclasts revealed a significant reduction in the expression of several genes associated with the maturation and migration of osteoclasts, including microphathalmia-associated transcription factor, tartrate-resistant acid phosphatase, cathepsin K, and β3 integrin. Restoring the expression of the full-length form of p85α but not the version with a deletion of the Src homology-3 domain restored the maturation of p85α−/− osteoclasts to wild-type levels. These results highlight the importance of the full-length version of the p85α subunit of class IA PI3-K in controlling multiple aspects of osteoclast functions.

Pak and Rac GTPases promote oncogenic KIT–induced neoplasms

An acquired somatic mutation at codon 816 in the KIT receptor tyrosine kinase is associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemic cells bearing this mutation with an allosteric inhibitor of p21-activated kinase (Pak) or its genetic inactivation results in growth repression due to enhanced apoptosis. Inhibition of the upstream effector Rac abrogates the oncogene-induced growth and activity of Pak. Although both Rac1 and Rac2 are constitutively activated via the guanine nucleotide exchange factor (GEF) Vav1, loss of Rac1 or Rac2 alone moderately corrected the growth of KIT-bearing leukemic cells, whereas the combined loss resulted in 75% growth repression. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of myeloproliferative neoplasms (MPNs) and corrected the associated pathology in mice. To assess the role of Rac GEFs in oncogene-induced transformation, we used an inhibitor of Rac, EHop-016, which specifically targets Vav1 and found that EHop-016 was a potent inhibitor of human and murine leukemic cell growth. These studies identify Pak and Rac GTPases, including Vav1, as potential therapeutic targets in MPN and AML involving an oncogenic form of KIT.

The PI3K pathway drives the maturation of mast cells via microphthalmia transcription factor

Mast cell maturation is poorly understood. We show that enhanced PI3K activation results in accelerated maturation of mast cells by inducing the expression of microphthalmia transcription factor (Mitf). Conversely, loss of PI3K activation reduces the maturation of mast cells by inhibiting the activation of AKT, leading to reduced Mitf but enhanced Gata-2 expression and accumulation of Gr1(+)Mac1(+) myeloid cells as opposed to mast cells. Consistently, overexpression of Mitf accelerates the maturation of mast cells, whereas Gata-2 overexpression mimics the loss of the PI3K phenotype. Expressing the full-length or the src homology 3- or BCR homology domain-deleted or shorter splice variant of the p85α regulatory subunit of PI3K or activated AKT or Mitf in p85α-deficient cells restores the maturation but not growth. Although deficiency of both SHIP and p85α rescues the maturation of SHIP(-/-) and p85α(-/-) mast cells and expression of Mitf; in vivo, mast cells are rescued in some, but not all tissues, due in part to defective KIT signaling, which is dependent on an intact src homology 3 and BCR homology domain of p85α. Thus, p85α-induced maturation, and growth and survival signals, in mast cells can be uncoupled.

Balanced Interactions between Lyn, the p85α Regulatory Subunit of Class IA Phosphatidylinositol-3-Kinase, and SHIP Are Essential for Mast Cell Growth and Maturation

ABSTRACT The growth and maturation of bone marrow-derived mast cells (BMMCs) from precursors are regulated by coordinated signals from multiple cytokine receptors, including KIT. While studies conducted using mutant forms of these receptors lacking the binding sites for Src family kinases (SFKs) and phosphatidylinositol-3-kinase (PI3K) suggest a role for these signaling molecules in regulating growth and survival, how complete loss of these molecules in early BMMC progenitors (MCps) impacts maturation and growth during all phases of mast cell development is not fully understood. We show that the Lyn SFK and the p85α subunit of class IA PI3K play opposing roles in regulating the growth and maturation of BMMCs in part by regulating the level of PI3K. Loss of Lyn in BMMCs results in elevated PI3K activity and hyperactivation of AKT, which accelerates the rate of BMMC maturation due in part to impaired binding and phosphorylation of SHIP via Lyns unique domain. In the absence of Lyns unique domain, BMMCs behave in a manner similar to that of Lyn- or SHIP-deficient BMMCs. Importantly, loss of p85α in Lyn-deficient BMMCs not only represses the hyperproliferation associated with the loss of Lyn but also represses their accelerated maturation. The accelerated maturation of BMMCs due to loss of Lyn is associated with increased expression of microphthalmia-associated transcription factor (Mitf), which is repressed in MCps deficient in the expression of both Lyn and p85α relative to controls. Our results demonstrate a crucial interplay of Lyn, SHIP, and p85α in regulating the normal growth and maturation of BMMCs, in part by regulating the activation of AKT and the expression of Mitf.

Requirement for p85α regulatory subunit of class IA PI3K in myeloproliferative disease driven by an activation loop mutant of KIT

OBJECTIVE Oncogenic activation loop mutations of KIT are observed in acute myeloid leukemia (AML) and in myeloproliferative disorders (MPD); however, the signaling pathways that contribute to transformation via these mutations in vivo are not known. Previous studies have demonstrated hyperactivation of p85alpha regulatory subunit of class IA phosphatidylinositol-3-kinase (PI3K) in cell lines expressing the activation loop mutant of KIT (KITD816V [human] and KITD814V [murine]). Although p85alpha is hyperphosphorylated and constitutively bound to KITD814V in cell-line models; the physiologic significance of this biochemical phenomenon in KITD814V-induced transformation is not known. MATERIALS AND METHODS Here, we describe the generation of a new mouse model to study KITD814V-induced transformation in myeloid cells as opposed to previously described models that primarily result in the generation of disease resembling acute lymphocytic leukemia. RESULTS Our results show that transplantation of KITD814V expressing bone marrow cells from C57/BL6 strain of mice into syngeneic recipients results in a fatal MPD. Importantly, in this model, transplantation of KITD814V expressing p85alpha-deficient bone marrow cells rescues the MPD phenotype. CONCLUSIONS Our results describe the generation of a new murine transplant model to study KITD814V-induced transformation and identify p85alpha as potential therapeutic target for the treatment of KITD814V-bearing diseases.

Role of intracellular tyrosines in activating KIT-induced myeloproliferative disease

Gain-of-function mutations in KIT receptor in humans are associated with gastrointestinal stromal tumors, systemic mastocytosis and acute myelogenous leukemia. The intracellular signals that contribute to oncogenic KIT-induced myeloproliferative disease (MPD) are poorly understood. Here, we show that oncogenic KITD814V-induced MPD occurs in the absence of ligand stimulation. The intracellular tyrosine residues are important for KITD814V-induced MPD, albeit to varying degrees. Among the seven intracellular tyrosines examined, tyrosine 719 alone has a unique role in regulating KITD814V-induced proliferation and survival in vitro, and MPD in vivo. Importantly, the extent to which AKT, extracellular signal-regulated kinase and Stat5 signaling pathways are activated via the seven intracellular tyrosines in KITD814V impacts the latency of MPD and severity of the disease. Our results identify critical signaling molecules involved in regulating KITD814V-induced MPD, which might be useful for developing novel therapeutic targets for hematologic malignancies involving this mutation.

Deficiency of Src family kinases compromises the repopulating ability of hematopoietic stem cells

OBJECTIVE Src family kinases (SFK) have been implicated in regulating growth factor and integrin-induced proliferation, migration, and gene expression in multiple cell types. However, little is known about the role of these kinases in the growth, homing, and engraftment potential of hematopoietic stem and progenitor cells. RESULTS Here we show that loss of hematopoietic-specific SFKs Hck, Fgr, and Lyn results in increased number of Sca-1(+)Lin(-) cells in the bone marrow, which respond differentially to cytokine-induced growth in vitro and manifest a significant defect in the long-term repopulating potential in vivo. Interestingly, a significant increase in expression of adhesion molecules, known to coincide with the homing potential of wild-type bone marrow cells is also observed on the surface of SFK(-/-) cells, although, this increase did not affect the homing potential of more primitive Lin(-)Sca-1(+) SFK(-/-) cells. The stem cell-repopulating defect observed in mice transplanted with SFK(-/-) bone marrow cells is due to the loss of Lyn Src kinase, because deficiency of Lyn, but not Hck or Fgr, recapitulated the long-term stem cell defect observed in mice transplanted with SFK(-/-) bone marrow cells. CONCLUSIONS Taken together, our results demonstrate an essential role for Lyn kinase in positively regulating the long-term and multilineage engraftment of stem cells, which is distinct from its role in mature B cells and myeloid cells.

p85β regulatory subunit of class IA PI3 kinase negatively regulates mast cell growth, maturation, and leukemogenesis.

We show that loss of p85α inhibits the growth and maturation of mast cells, whereas loss of p85β enhances this process. Whereas restoring the expression of p85α in P85α(-/-) cells restores these functions, overexpression of p85β has the opposite effect. Consistently, overexpression of p85β in WT mast cells represses KIT-induced proliferation and IL-3-mediated maturation by inhibiting the expression of Microphthalmia transcription factor. Because p85α and p85β differ in their N-terminal sequences, chimeric proteins consisting of amino or carboxy-terminal of p85α and/or p85β do not rescue the growth defects of p85α(-/-) cells, suggesting cooperation between these domains for normal mast cell function. Loss of p85β impaired ligand induced KIT receptor internalization and its overexpression enhanced this process, partly because of increased binding of c-Cbl to p85β relative to p85α. In vivo, loss of p85β resulted in increased mast cells, and bone marrow transplantation of cells overexpressing p85β resulted in significant reduction in some tissue mast cells. Overexpression of p85β suppressed the growth of oncogenic KIT-expressing cells in vitro and prolonged the survival of leukemic mice in vivo. Thus, p85α and p85β differentially regulate SCF and oncogenic KIT-induced signals in myeloid lineage-derived mast cells.

Electroacupuncture Promotes Central Nervous System-Dependent Release of Mesenchymal Stem Cells

Electroacupuncture (EA) performed in rats and humans using limb acupuncture sites, LI‐4 and LI‐11, and GV‐14 and GV‐20 (humans) and Bai‐hui (rats) increased functional connectivity between the anterior hypothalamus and the amygdala and mobilized mesenchymal stem cells (MSCs) into the systemic circulation. In human subjects, the source of the MSC was found to be primarily adipose tissue, whereas in rodents the tissue sources were considered more heterogeneous. Pharmacological disinhibition of rat hypothalamus enhanced sympathetic nervous system (SNS) activation and similarly resulted in a release of MSC into the circulation. EA‐mediated SNS activation was further supported by browning of white adipose tissue in rats. EA treatment of rats undergoing partial rupture of the Achilles tendon resulted in reduced mechanical hyperalgesia, increased serum interleukin‐10 levels and tendon remodeling, effects blocked in propranolol‐treated rodents. To distinguish the afferent role of the peripheral nervous system, phosphoinositide‐interacting regulator of transient receptor potential channels (Pirt)‐GCaMP3 (genetically encoded calcium sensor) mice were treated with EA acupuncture points, ST‐36 and LIV‐3, and GV‐14 and Bai‐hui and resulted in a rapid activation of primary sensory neurons. EA activated sensory ganglia and SNS centers to mediate the release of MSC that can enhance tissue repair, increase anti‐inflammatory cytokine production and provide pronounced analgesic relief. Stem Cells 2017;35:1303–1315