Person:

Sykes, David

Mesenchymal stem cells (MSCs) are multi-potent cells that can differentiate into osteoblasts, adipocytes, chondrocytes and myocytes. This potential declines with aging. We investigated whether the sirtuin SIRT1 had a function in MSCs by creating MSC specific SIRT1 knock-out (MSCKO) mice. Aged MSCKO mice (2.2 years old) showed defects in tissues derived from MSCs; i.e. a reduction in subcutaneous fat, cortical bone thickness and trabecular volume. Young mice showed related but less pronounced effects. MSCs isolated from MSCKO mice showed reduced differentiation towards osteoblasts and chondrocytes in vitro, but no difference in proliferation or apoptosis. Expression of β-catenin targets important for differentiation was reduced in MSCKO cells. Moreover, while β-catenin itself (T41A mutant resistant to cytosolic turnover) accumulated in the nuclei of wild-type MSCs, it was unable to do so in MSCKO cells. However, mutating K49R or K345R in β-catenin to mimic deacetylation restored nuclear localization and differentiation potential in MSCKO cells. We conclude that SIRT1 deacetylates β-catenin to promote its accumulation in the nucleus leading to transcription of genes for MSC differentiation.

The genes encoding Hoxa9 and Meis1 are transcriptionally coactivated in a subset of acute myeloid leukemia (AML) in mice. In marrow reconstitution experiments, coexpression of both genes produces rapid AML, while neither gene alone generates overt leukemia. Although Hoxa9 and Meis1 can bind DNA as heterodimers, both can also heterodimerize with Pbx proteins. Thus, while their coactivation may result from the necessity to bind promoters as heterodimers, it may also result from the necessity of altering independent biochemical pathways that cooperate to generate AML, either as monomers or as heterodimers with Pbx proteins. Here we demonstrate that constitutive expression of Hoxa9 in primary murine marrow immortalizes a late myelomonocytic progenitor, preventing it from executing terminal differentiation to granulocytes or monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3. This immortalized phenotype is achieved in the absence of endogenous or exogenous Meis gene expression. The Hoxa9-immortalized progenitor exhibited a promyelocytic transcriptional profile, expressing PU.1, AML1, c-Myb, C/EBP alpha, and C/EBP epsilon as well as their target genes, the receptors for GM-CSF, G-CSF, and M-CSF and the primary granule proteins myeloperoxidase and neutrophil elastase. G-CSF obviated the differentiation block of Hoxa9, inducing neutrophilic differentiation with accompanying expression of neutrophil gelatinase B and upregulation of gp91phox. M-CSF also obviated the differentiation block, inducing monocytic differentiation with accompanying expression of the macrophage acetyl-low-density lipoprotein scavenger receptor and F4/80 antigen. Versions of Hoxa9 lacking the ANWL Pbx interaction motif (PIM) also immortalized a promyelocytic progenitor with intrinsic biphenotypic differentiation potential. Therefore, Hoxa9 evokes a cytokine-selective block in differentiation by a mechanism that does not require Meisgene expression or interaction with Pbx through the PIM.

The mTOR pathway is a critical determinant of cell persistence and growth wherein mTOR complex 1 (mTORC1) mediates a balance between growth factor stimuli and nutrient availability. Amino acids or glucose facilitates mTORC1 activation by inducing RagA GTPase recruitment of mTORC1 to the lysosomal outer surface, enabling activation of mTOR by the Ras homolog Rheb. Thereby, RagA alters mTORC1-driven growth in times of nutrient abundance or scarcity. Here, we have evaluated differential nutrient-sensing dependence through RagA and mTORC1 in hematopoietic progenitors, which dynamically drive mature cell production, and hematopoietic stem cells (HSC), which provide a quiescent cellular reserve.In nutrient-abundant conditions, RagA-deficient HSC were functionally unimpaired and upregulated mTORC1 via nutrientinsensitive mechanisms. RagA was also dispensable for HSC function under nutritional stress conditions. Similarly, hyperactivation of RagA did not affect HSC function. In contrast, RagA deficiency markedly altered progenitor population function and mature cell output. Therefore, RagA is a molecular mechanism that distinguishes the functional attributes of reactive progenitors from a reserve stem cell pool. The indifference of HSC to nutrient sensing through RagA contributes to their molecular resilience to nutritional stress, a characteristic that is relevant to organismal viability in evolution and in modern HSC transplantation approaches.

Hematopoietic stem cell transplantation (HSCT) offers curative therapy for patients with hemoglobinopathies, congenital immunodeficiencies, and other conditions, possibly including AIDS. Autologous HSCT using genetically corrected cells would avoid the risk of graft-versus-host disease (GVHD), but the genotoxicity of conditioning remains a substantial barrier to the development of this approach. Here we report an internalizing immunotoxin targeting the hematopoietic-cell-restricted CD45 receptor that effectively conditions immunocompetent mice. A single dose of the immunotoxin, CD45–saporin (SAP), enabled efficient (>90%) engraftment of donor cells and full correction of a sickle-cell anemia model. In contrast to irradiation, CD45–SAP completely avoided neutropenia and anemia, spared bone marrow and thymic niches, enabling rapid recovery of T and B cells, preserved anti-fungal immunity, and had minimal overall toxicity. This non-genotoxic conditioning method may provide an attractive alternative to current conditioning regimens for HSCT in the treatment of non-malignant blood diseases.

Regulation of STAT3 activation is critical for normal and malignant hematopoietic cell proliferation. Here, we have reported that the endogenous transmembrane protein upstream-of-mTORC2 (UT2) negatively regulates activation of STAT3. Specifically, we determined that UT2 interacts directly with GP130 and inhibits phosphorylation of STAT3 on tyrosine 705 (STAT3Y705). This reduces cytokine signaling including IL6 that is implicated in multiple myeloma and other hematopoietic malignancies. Modulation of UT2 resulted in inverse effects on animal survival in myeloma models. Samples from multiple myeloma patients also revealed a decreased copy number of UT2 and decreased expression of UT2 in genomic and transcriptomic analyses, respectively. Together, these studies identify a transmembrane protein that functions to negatively regulate cytokine signaling through GP130 and pSTAT3Y705 and is molecularly and mechanistically distinct from the suppressors of cytokine signaling (SOCS) family of genes. Moreover, this work provides evidence that perturbations of this activation-dampening molecule participate in hematologic malignancies and may serve as a key determinant of multiple myeloma pathophysiology. UT2 is a negative regulator shared across STAT3 and mTORC2 signaling cascades, functioning as a tumor suppressor in hematologic malignancies driven by those pathways.

JAK2V617F is the most common oncogenic lesion in patients with myeloproliferative neoplasms (MPNs). Despite the ability of JAK2V617F to instigate DNA damage in vitro, MPNs are nevertheless characterized by genomic stability. In this study, we address this paradox by identifying the DNA helicase RECQL5 as a suppressor of genomic instability in MPNs. We report increased RECQL5 expression in JAK2V617F-expressing cells and demonstrate that RECQL5 is required to counteract JAK2V617F-induced replication stress. Moreover, RECQL5 depletion sensitizes JAK2V617F mutant cells to hydroxyurea (HU), a pharmacological inducer of replication stress and the most common treatment for MPNs. Using single-fiber chromosome combing, we show that RECQL5 depletion in JAK2V617F mutant cells impairs replication dynamics following HU treatment, resulting in increased double-stranded breaks and apoptosis. Cumulatively, these findings identify RECQL5 as a critical regulator of genome stability in MPNs and demonstrate that replication stress-associated cytotoxicity can be amplified specifically in JAK2V617F mutant cells through RECQL5-targeted synthetic lethality.

The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses.

Neuroinflammation contributes to the pathophysiology of diverse diseases including stroke, traumatic brain injury, Alzheimer's Disease, Parkinson's Disease, and multiple sclerosis, resulting in neurodegeneration and loss of neurological function. The response of the microvascular endothelium often contributes to neuroinflammation. One such response is the up-regulation of endothelial adhesion molecules which facilitate neutrophil adhesion to the endothelium and their migration from blood to tissue. Neuregulin-1 (NRG1) is an endogenous growth factor which has been reported to have anti-inflammatory effects in experimental stroke models. We hypothesized that NRG1 would decrease the endothelial response to inflammation, and result in a decrease in neutrophil adhesion to endothelial cells. We tested this hypothesis in an in-vitro model of cytokine-induced endothelial injury, in which human brain microvascular endothelial cells (BMECs) were treated with IL-1β, along with co-incubation with vehicle or NRG1-β. Outcome measures included protein levels of endothelial ICAM-1, VCAM-1, and E-selectin; as well as the number of neutrophils that adhere to the endothelial monolayer. Our data show that NRG1-β decreased the levels of VCAM-1, E-selectin, and neutrophil adhesion to brain microvascular endothelial cells activated by IL1-β. These findings open new possibilities for investigating NRG1 in neuroprotective strategies in brain injury.

Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876’s mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets.

The TEMPI syndrome was recently described in 2011, and is characterized by the constellation of five hallmarks: Telangiectasias, Erythrocytosis and elevated Erythropoietin, Monoclonal gammopathy, Perinephric fluids collections, and Intrapulmonary shunting. The underlying pathophysiology is unknown, though it has been postulated that the monoclonal gammopathy may play a causal role.