Autonomous Cell Growth Research Articles

The U2AF homology motif kinase 1 (UHMK1) is upregulated upon hematopoietic cell differentiation

UHMK1 (KIS) is a nuclear serine/threonine kinase that possesses a U2AF homology motif and phosphorylates and regulates the activity of the splicing factors SF1 and SF3b155. Mutations in these components of the spliceosome machinery have been recently implicated in leukemogenesis. The fact that UHMK1 regulates these factors suggests that UHMK1 might be involved in RNA processing and perhaps leukemogenesis. Here we analyzed UHMK1 expression in normal hematopoietic and leukemic cells as well as its function in leukemia cell line.In the normal hematopoietic compartment, markedly higher levels of transcripts were observed in differentiated lymphocytes (CD4+, CD8+ and CD19+) compared to the progenitor enriched subpopulation (CD34+) or leukemia cell lines. UHMK1 expression was upregulated in megakaryocytic-, monocytic- and granulocytic-induced differentiation of established leukemia cell lines and in erythrocytic-induced differentiation of CD34+ cells. No aberrant expression was observed in patient samples of myelodysplastic syndrome (MDS), acute myeloid (AML) or lymphoblastic (ALL) leukemia. Nonetheless, in MDS patients, increased levels of UHMK1 expression positively impacted event free and overall survival.Lentivirus mediated UHMK1 knockdown did not affect proliferation, cell cycle progression, apoptosis or migration of U937 leukemia cells, although UHMK1 silencing strikingly increased clonogenicity of these cells. Thus, our results suggest that UHMK1 plays a role in hematopoietic cell differentiation and suppression of autonomous clonal growth of leukemia cells.

BDNF/trkB Induction of Calcium Transients through Cav2.2 Calcium Channels in Motoneurons Corresponds to F-actin Assembly and Growth Cone Formation on β2-Chain Laminin (221).

Spontaneous Ca2+ transients and actin dynamics in primary motoneurons correspond to cellular differentiation such as axon elongation and growth cone formation. Brain-derived neurotrophic factor (BDNF) and its receptor trkB support both motoneuron survival and synaptic differentiation. However, in motoneurons effects of BDNF/trkB signaling on spontaneous Ca2+ influx and actin dynamics at axonal growth cones are not fully unraveled. In our study we addressed the question how neurotrophic factor signaling corresponds to cell autonomous excitability and growth cone formation. Primary motoneurons from mouse embryos were cultured on the synapse specific, β2-chain containing laminin isoform (221) regulating axon elongation through spontaneous Ca2+ transients that are in turn induced by enhanced clustering of N-type specific voltage-gated Ca2+ channels (Cav2.2) in axonal growth cones. TrkB-deficient (trkBTK−/−) mouse motoneurons which express no full-length trkB receptor and wildtype motoneurons cultured without BDNF exhibited reduced spontaneous Ca2+ transients that corresponded to altered axon elongation and defects in growth cone morphology which was accompanied by changes in the local actin cytoskeleton. Vice versa, the acute application of BDNF resulted in the induction of spontaneous Ca2+ transients and Cav2.2 clustering in motor growth cones, as well as the activation of trkB downstream signaling cascades which promoted the stabilization of β-actin via the LIM kinase pathway and phosphorylation of profilin at Tyr129. Finally, we identified a mutual regulation of neuronal excitability and actin dynamics in axonal growth cones of embryonic motoneurons cultured on laminin-221/211. Impaired excitability resulted in dysregulated axon extension and local actin cytoskeleton, whereas upon β-actin knockdown Cav2.2 clustering was affected. We conclude from our data that in embryonic motoneurons BDNF/trkB signaling contributes to axon elongation and growth cone formation through changes in the local actin cytoskeleton accompanied by increased Cav2.2 clustering and local calcium transients. These findings may help to explore cellular mechanisms which might be dysregulated during maturation of embryonic motoneurons leading to motoneuron disease.

The Essential Genome of Burkholderia cenocepacia H111.

The study of the minimum set of genes required to sustain life is a fundamental question in biological research. Recent studies on bacterial essential genes suggested that between 350 and 700 genes are essential to support autonomous bacterial cell growth. Essential genes are of interest as potential new antimicrobial drug targets; hence, our aim was to identify the essential genome of the cystic fibrosis (CF) isolate Burkholderia cenocepacia H111. Using a transposon sequencing (Tn-Seq) approach, we identified essential genes required for growth in rich medium under aerobic and microoxic conditions as well as in a defined minimal medium with citrate as a sole carbon source. Our analysis suggests that 398 genes are required for autonomous growth in rich medium, a number that represents only around 5% of the predicted genes of this bacterium. Five hundred twenty-six genes were required to support growth in minimal medium, and 434 genes were essential under microoxic conditions (0.5% O2). A comparison of these data sets identified 339 genes that represent the minimal set of essential genes required for growth under all conditions tested and can be considered the core essential genome of B. cenocepacia H111. The majority of essential genes were found to be located on chromosome 1, and few such genes were located on chromosome 2, where most of them were clustered in one region. This gene cluster is fully conserved in all Burkholderia species but is present on chromosome 1 in members of the closely related genus Ralstonia, suggesting that the transfer of these essential genes to chromosome 2 in a common ancestor contributed toward the separation of the two genera.IMPORTANCE Transposon sequencing (Tn-Seq) is a powerful method used to identify genes that are essential for autonomous growth under various conditions. In this study, we have identified a set of "core essential genes" that are required for growth under multiple conditions, and these genes represent potential antimicrobial targets. We also identified genes specifically required for growth under low-oxygen and nutrient-limited environments. We generated conditional mutants to verify the results of our Tn-Seq analysis and demonstrate that one of the identified genes was not essential per se but was an artifact of the construction of the mutant library. We also present verified examples of genes that were not truly essential but, when inactivated, showed a growth defect. These examples have identified so-far-underestimated shortcomings of this powerful method.

DNA sensing in senescence.

Cellular senescence, a cell-autonomous growth arrest program, also executes pleiotropic non-cell-autonomous activities through the senescence-associated secretory phenotype (SASP). The innate cGAS-STING DNA-sensing pathway is now shown to regulate senescence by recognizing cytosolic DNA and inducing SASP factors, uncovering an unexpected link between these two previously unrelated pathways.

Mechanical Behavior of Cells within a Cell-Based Model of Wheat Leaf Growth.

Understanding the principles and mechanisms of cell growth coordination in plant tissue remains an outstanding challenge for modern developmental biology. Cell-based modeling is a widely used technique for studying the geometric and topological features of plant tissue morphology during growth. We developed a quasi-one-dimensional model of unidirectional growth of a tissue layer in a linear leaf blade that takes cell autonomous growth mode into account. The model allows for fitting of the visible cell length using the experimental cell length distribution along the longitudinal axis of a wheat leaf epidermis. Additionally, it describes changes in turgor and osmotic pressures for each cell in the growing tissue. Our numerical experiments show that the pressures in the cell change over the cell cycle, and in symplastically growing tissue, they vary from cell to cell and strongly depend on the leaf growing zone to which the cells belong. Therefore, we believe that the mechanical signals generated by pressures are important to consider in simulations of tissue growth as possible targets for molecular genetic regulators of individual cell growth.

IGF1R as a Key Target in High Risk, Metastatic Medulloblastoma

Risk or presence of metastasis in medulloblastoma causes substantial treatment-related morbidity and overall mortality. Through the comparison of cytokines and growth factors in the cerebrospinal fluid (CSF) of metastatic medulloblastoma patients with factors also in conditioned media of metastatic MYC amplified medulloblastoma or leptomeningeal cells, we were led to explore the bioactivity of IGF1 in medulloblastoma by elevated CSF levels of IGF1, IGF-sequestering IGFBP3, IGFBP3-cleaving proteases (MMP and tPA), and protease modulators (TIMP1 and PAI-1). IGF1 led not only to receptor phosphorylation but also accelerated migration/adhesion in MYC amplified medulloblastoma cells in the context of appropriate matrix or meningothelial cells. Clinical correlation suggests a peri-/sub-meningothelial source of IGF-liberating proteases that could facilitate leptomeningeal metastasis. In parallel, studies of key factors responsible for cell autonomous growth in MYC amplified medulloblastoma prioritized IGF1R inhibitors. Together, our studies identify IGF1R as a high value target for clinical trials in high risk medulloblastoma.

Molecular Events of Hepatitis C in Hepatocellular Carcinoma

Cancer is defined by a set of more than 100 diseases that have in common disordered autonomous growth of abnormal cells and genetic alterations, including acquired mutations and genetic instability. The infectious etiology of cancer, on the other hand, is the result of virus activity, which induce tumor cell transformation. This transformation results in activation or deactivation of key regulatory genes, cell proliferation and differentiation. The alterations of these events leads to important modulatory changes in the expression of several proteins and/or their structure, causing profound modifications on cellular metabolism. Carcinomas are cancers that occur in epithelial tissues, as skin or mucous. Among those, the hepatocellular carcinoma (HCC) is one of the most relevant carcinomas and is the sixth most common cancer worldwide. Several factors are associated with progression of HCC, among them diabetes, obesity, metabolic syndrome, alcohol, tobacco, aflatoxins, genetic predisposition and hepatitis B and C virus. The hepatitis C virus (HCV) it is one of the most important etiological agents of HCC, resulting in virus induced liver necroinflammation. The infection of hepatitis C virus (HCV) in hepatocytes is an orchestrated event involving viral oncogenic factors and its development in the host cell, which involve viral envelope glycoprotein structures such as E1 and E2. The most interesting trade is that the HCV genetic material does not invade the nucleus of the infected cell, it acts directly as a mRNA in the cytoplasm, where virus transmission is initiated by the internal ribosome entry site. Numerous events are involved in the molecular pathogenesis of HCC by HVC. In this review, we highlight those involved in p53 signaling pathway; Wnt signaling and E-cadherina/β-catenin activation and TGF-β/activin pathway regulation. As these molecular alterations derived from host-pathogen interactions interfere in hepatocyte normal effectors expression, to understand the interactome behind these well-driven mechanistic events could lead to more realistic molecular targets, as well to early screening for possible biomarkers molecules.

Targeted Inhibition of Rictor/mTORC2 in Cancer Treatment: A New Era after Rapamycin.

The evolutionarily conserved mechanistic target of rapamycin (mTOR) forms two functionally distinct complexes, mTORC1 and mTORC2. mTORC1, consisting of mTOR, raptor, and mLST8 (GβL), is sensitive to rapamycin and thought to control autonomous cell growth in response to nutrient availability and growth factors. mTORC2, containing the core components mTOR, mLST8, Rictor, mSIN1, and Protor1/2 is largely insensitive to rapamycin. mTORC2 specifically senses growth factors and regulates cell proliferation, metabolism, actin rearrangement, and survival. Dysregulation of mTOR signaling often occurs in a variety of human malignant diseases, rendering it a crucial and validated target in cancer treatment. However, the effectiveness of rapamycin as single-agent therapy is suppressed, in part, by the numerous strong mTORC1-dependent negative feedback loops. Although preclinical and clinical studies of ATP-competitive mTOR inhibitors that target both mTORC1 and mTORC2 have shown greater effectiveness than rapalogs for cancer treatment, the mTORC1 inhibition-induced negative feedback activation of PI3- K/PDK1 and Akt (Thr308) may be sufficient to promote cell survival. Recent cancer biology studies indicated that mTORC2 is a promising target, since its activity is essential for the development of a number of cancers. These studies provide a rationale for developing inhibitors specifically targeting mTORC2, which do not perturb the mTORC1- dependent negative feedback loops and have a more acceptable therapeutic window. This review summarizes the present understanding of mTORC2 signaling and functions, especially tumorigenic functions, highlighting the current status and future perspectives for targeting mTORC2 in cancer treatment.

Novel chemical library screen identifies naturally occurring plant products that specifically disrupt glioblastoma-endothelial cell interactions.

Tumor growth is not solely a consequence of autonomous tumor cell properties. Rather, tumor cells act upon and are acted upon by their microenvironment. It is tumor tissue biology that ultimately determines tumor growth. Thus, we developed a compound library screen for agents that could block essential tumor-promoting effects of the glioblastoma (GBM) perivascular stem cell niche (PVN). We modeled the PVN with three-dimensional primary cultures of human brain microvascular endothelial cells in Matrigel. We previously demonstrated stimulated growth of GBM cells in this PVN model and used this to assay PVN function. We screened the Microsource Spectrum Collection library for drugs that specifically blocked PVN function, without any direct effect on GBM cells themselves. Three candidate PVN-disrupting agents, Iridin, Tigogenin and Triacetylresveratrol (TAR), were identified and evaluated in secondary in vitro screens against a panel of primary GBM isolates as well as in two different in vivo intracranial models. Iridin and TAR significantly inhibited intracranial tumor growth and prolonged survival in these mouse models. Together these data identify Iridin and TAR as drugs with novel GBM tissue disrupting effects and validate the importance of preclinical screens designed to address tumor tissue function rather than the mechanisms of autonomous tumor cell growth.

SMALL GTPASE ARF6 ACTIVATES mTORCl-DEPENDENT SIGNALING PATHWAY AND STIMULATES GLIOBLASTOMA CELL GROWTH

In this study we first identified role of small GTPase Arf6 in stimulation of proliferation and autonomous growth of human glioblastoma cells. We revealed that Arf6 activates the mTOR complex 1 (mTORCl) and this process is appeared to be an alternative mechanism of mTORC1-signaling activation, independent of PI3K/Akt-signaling pathway. We also showed that Arf6 is a negative regulator of protein kinase ERK1/2 in glioblastoma cells.

Clinical utility and patient consideration in the use of lenalidomide for multiple myeloma in Chinese patients.

Multiple myeloma (MM) is an incurable hematologic malignancy caused by the autonomous growth of malignant plasma cells. In the last decade, the introduction of novel targeted agents such as thalidomide, bortezomib, and lenalidomide has dramatically improved the clinical outcome of MM patients in both the frontline and recurrent settings. Lenalidomide is a synthetic derivative of thalidomide, which has been shown to significantly improve overall survival, time to progression, and overall response rates in patients with MM. The China Food and Drug Administration approved the use of lenalidomide in patients with MM in 2013. In a Phase II trial, lenalidomide plus low-dose dexamethasone was associated with a high response rate and acceptable safety profile in heavily pretreated Chinese patients with relapsed/refractory MM, including those with renal impairment and IgD subtype. However, lenalidomide will remain as a second-line antimyeloma drug in the near future because of its high price and the policy of health insurance reimbursement in People’s Republic of China. In this review, we summarize the clinical utility and patient considerations in the use of lenalidomide for MM in Chinese patients. Further studies with larger sample sizes are required to investigate the better quality, longer duration, and more clinically meaningful outcomes of lenalidomide in the treatment of MM in Chinese patients.

Lymphatic dysfunction attenuates tumor immunity through impaired antigen presentation.

Tumor growth and metastasis of cancer involve autonomous tumor cell growth and host-tumor interactions. While tumor-specific immunity has been intensively studied in vitro, dynamic roles of lymphatic transport on tumor immunity in vivo have not been fully elucidated. In this study, we examined tumor growth and anti-tumor immune responses using kCYC mice, which demonstrate severe lymphatic dysfunction. Primary tumor growth was augmented in kCYC mice (compared to wild-type mice) when B16 melanoma or EL-4 lymphoma cells were subcutaneously injected. Expression of inflammatory cytokines such as IFN-γ, TNF-α, and IL-2 as well as IL-10 expression in draining lymph nodes (LNs) was significantly reduced in kCYC mice after tumor inoculation. Moreover, decreased levels of tumor-associated antigens were detected in draining LNs in kCYC mice, together with impaired antigen presentation. CD8+ T cells in draining LNs derived from kCYC mice bearing B16 melanoma also showed significantly decreased cytotoxic activity in vitro. Finally, tumor suppression activity of CD8+ T cells derived from kCYC mice bearing B16 melanoma was reduced when adoptively transferred to naive wild-type mice. In summary, these findings suggest that lymphatic transport is essential in generating optimal tumor-specific immune responses mediated by CD8+ T cells.

Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis.

Glutaminase (GLS), which converts glutamine to glutamate, plays a key role in cancer cell metabolism, growth, and proliferation. GLS is being explored as a cancer therapeutic target, but whether GLS inhibitors affect cancer cell-autonomous growth or the host microenvironment or have off-target effects is unknown. Here, we report that loss of one copy of Gls blunted tumor progression in an immune-competent MYC-mediated mouse model of hepatocellular carcinoma. Compared with results in untreated animals with MYC-induced hepatocellular carcinoma, administration of the GLS-specific inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) prolonged survival without any apparent toxicities. BPTES also inhibited growth of a MYC-dependent human B cell lymphoma cell line (P493) by blocking DNA replication, leading to cell death and fragmentation. In mice harboring P493 tumor xenografts, BPTES treatment inhibited tumor cell growth; however, P493 xenografts expressing a BPTES-resistant GLS mutant (GLS-K325A) or overexpressing GLS were not affected by BPTES treatment. Moreover, a customized Vivo-Morpholino that targets human GLS mRNA markedly inhibited P493 xenograft growth without affecting mouse Gls expression. Conversely, a Vivo-Morpholino directed at mouse Gls had no antitumor activity in vivo. Collectively, our studies demonstrate that GLS is required for tumorigenesis and support small molecule and genetic inhibition of GLS as potential approaches for targeting the tumor cell-autonomous dependence on GLS for cancer therapy.

Schwann cell Miz without POZ: degeneration meets regeneration.

Peripheral motor and sensory neuropathies are diseases with different etiologies emerging from genetic disorders, diabetes, infection or inflammation, paraneoplastic damage or intoxications including alcohol abuse (Martyn and Hughes, 1997). Hereditary neuropathies, most of which are subsumed under the hypernym Charcot-Marie Tooth (CMT) disease, are clinically heterogeneous: about 50 affected gene loci encoding about 30 genes have been so far identified to be involved in the pathogenesis of different CMTs (Niemann et al., 2006). Depending on structural and electrophysiological features CMTs are divided into axonal and demyelinating forms. Peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ) and connexin 32 (GJB1) are the most frequently affected genes in human demyelinating neuropathies and a variety of animal models have been designed to mimic these human disorders (Bouhy and Timmerman, 2013). These models rely on the deletion, overexpression or mutation of the gene relevant for a human neuropathy and usually clinical symptoms occur early in life and the course of disease is progressive. Recently, Sanz-Moreno et al. (2014) published the partial deletion of the transcription factor Myc interacting zinc finger protein 1 (Miz1) in Schwann cells of mice and observed a late onset demyelinating neuropathy with a spontaneous clinical remission, introducing a unique new model to study peripheral nerve regeneration.

ETV6/ARG Oncoprotein Induces Autonomous Cell Growth By Enhancing c-Myc Expression through STAT5 Activation in the Acute Promyelocytic Leukemia Cell Line HT93A

Background: A fusion gene comprising ETS-variant gene 6 (ETV6) and ABL- related gene (ARG) has been identified in some patients with acute leukemia. Although the ETV6/ARG fusion gene is considered to contribute to the leukemogenesis, the detailed underlying mechanism is still unknown. Here, we investigated the role of ETV6/ARG fusion genes by exposing the HT93A cell line, which was established from a patient with relapsed acute promyelocytic leukemia carrying ETV6/ARG fusion gene, to the tyrosine kinase inhibitor, nilotinib.Material and Methods: HT93A cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum with or without nilotinib ± 50 ng/ml of granulocyte colony-stimulating factor (G-CSF). The cells were intracellularly stained anti-phosphorylated ARG (Y429), anti-phosphorylated STAT3 (Y705), anti-phosphorylated STAT5 (Y694), anti-p21/Waf1, anti-p27/Kip1, and anti-c-Myc polyclonal antibodies. Thereafter, protein expression and the degree of phosphorylation were evaluated with a flow cytometer. Cell growth was evaluated by trypan blue exclusion test, cell cycle analysis, and by intracellular staining with propidium iodide followed by flow cytometric analysis. Experiments were independently repeated at least thrice. Results shown are the mean ± standard deviation (SD) values. Data were analyzed using Student’s t-test, and p < 0.05 was considered statistically significant.Results: ARG was predominantly activated in HT93A cells and nilotinib treatment inhibited ARG phosphorylation. HT93A cells were sensitive to nilotinib, which inhibited cell growth in a dose-dependent manner for 96 h of incubation. Nilotinib treatment for 48 h significantly increased the probability of cells in the G0/G1 phase and decreased that in the S+G2/M phase. Further, nilotinib alone inhibited the expression of both phosphorylated STAT3 and STAT5. Moreover, nilotinib downregulated the expression levels of p21/Waf1 and c-Myc. However, p27/Kip expression level did not alter. Because the activation of STAT3 and/or STAT5 was assumed as the major pathway induced by ETV6/ARG oncoprotein, HT93A cells were coincubated with 50 ng/mL G-CSF in the presence or absence of nilotinib. After stimulation with G-CSF for 60 min, STAT5 was significantly phosphorylated in both nilotinib-treated and untreated cells; the expression level of phosphorylated STAT5 returned to the control level when treated with nilotinib and G-CSF together. In contrast, STAT3 phosphorylation was unaltered by G-CSF stimulation in nilotinib-treated and untreated cells. G-CSF alone had an insignificant effect on cell growth. The downregulation of the expression levels of p21/Waf1 and c-Myc by nilotinib was abolished by the combination of G-CSF. G-CSF had slight effect on HT93A cell cycle distribution; results of the combination therapy with nilotinib and G-CSF returned the cell cycle distribution to the control level.Conclusions: We demonstrated that ETV6/ARG inhibition by nilotinib abrogates autonomous cell growth via the downregulation of c-Myc through the inactivation of STAT5 but not of STAT3. Our data suggest that ETV6/ARG oncoprotein has a contributory effect for the autonomous cell growth and is a potential target in the treatment of leukemia. DisclosuresIriyama:Novartis: Honoraria; Kyowa Hakko Kirin: Honoraria. Hatta:Novartis: Honoraria; Kyowa Hakko Kirin: Honoraria. Takei:Novartis: Research Funding; Kyowa Hakko Kirin: Research Funding.

Bone Marrow Failure in Fanconi Anemia from Hyperactive TGF-β Signaling

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life due to attrition of hematopoietic stem and progenitor cells (HSPCs). FA patients also develop other hematologic manifestations, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) due to clonal evolution. FA is caused by biallelic mutants in one of sixteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to DNA cross-linking agents. Bone marrow failure in FA may result, directly or indirectly, from hyperactivation of cell autonomous or microenvironmental growth suppressive pathways induced due to genotoxic stress. Recent studies suggest that one suppressive pathway may be the hyperactive p53 response observed in HSPCs from FA patients.In order to further identify suppressive mechanisms accounting for bone marrow failure in FA, we performed a whole genome-wide shRNA screen in FA cells. Specifically, we screened for candidate genes whose knockdown would rescue cellular growth inhibition and genotoxic stress induced by a DNA cross-linking agent mitomycin C (MMC). We transduced a FA-deficient human fibroblast line with pools of shRNAs and screened for rescue of MMC-inhibited growth. Selected shRNA inserts were identified by next generation sequencing.The top hits in the screen were shRNAs directed against multiple components of the TGF-β signaling pathway. Consistent with this, disruption of the TGF-β signaling pathway by shRNA/sgRNA-mediated knockdown of SMAD3 or TGFR1 (downstream components of the TGF- β pathway) rescued growth of multiple cell lines from several FA complementation groups in presence of genotoxic agents (e.g. MMC or acetaldehyde). Pharmacologic inhibition of the TGF- β pathway using small molecule inhibitors resulted in improved survival of FA-deficient lymphoblast cells in presence of MMC or acetaldehyde, suggesting that a hyperactive, TGF-β-mediated, suppression pathway may account, at least in part, for reduced FA cell growth. Interestingly, genes encoding TGF-β pathway signaling components were highly expressed in the bone marrow from FA patients and FA mice. Moreover, disruption of the TGF- β pathway by shRNA-mediated knockdown of SMAD3 rescued the growth defects of primary HSPCs from FA-deficient murine bone marrow. To further implicate the TGF-β pathway, we established primary stromal cell lines from the bone marrow of FA-deficient mice as well as human FA patients. We confirmed that TGF-β signaling was hyperactive in these stroma cells resulting in growth suppression and elevated phospho-ERK levels due to non-canonical signaling of the pathway. Inhibitors of TGF-β signaling partially rescued the growth defects and reduced phospho-ERK levels in these FA stroma cells.The deficiency of FA DNA repair pathway leads to cellular defects in homologous recombination (HR) repair and hyperactivation of toxic non-homologous end joining (NHEJ)-mediated repair. We therefore tested whether inhibition of the TGF-β pathway in FA cells could rescue HR defects and account for the improvement of FA cellular growth. Interestingly, disruption of the TGF-β signaling pathway caused a decrease in NHEJ activity. Disruption of the TGF-β pathway also resulted in reduced MMC-mediated DNA damage and increased HR.Taken together, our results demonstrate that primary FA hematopoietic and bone marrow stromal cells exhibit hyperactive TGF-β signaling accounting at least in part for the bone marrow failure in FA. Inhibitors of the TGF-β signaling pathway may therefore be useful in the clinical treatment of patients with bone marrow failure and Fanconi anemia. DisclosuresNo relevant conflicts of interest to declare.

ETV6/ARG oncoprotein confers autonomous cell growth by enhancing c-Myc expression via signal transducer and activator of transcription 5 activation in the acute promyelocytic leukemia cell line HT93A

We investigated the role of ETV6/ARG fusion gene by exposing the HT93A cell line to nilotinib. HT93A cells were cultured with or without nilotinib ± 50 ng/mL of granulocyte colony-stimulating factor (G-CSF). Nilotinib treatment inhibited cell growth by increasing the percentage of cells in G0/G1 phase through the decrease of phosphorylated signal transducer and activator of transcription 3 (STAT3) (Y705), STAT5 (Y694) and c-Myc expression. After stimulation with G-CSF, STAT5 but not STAT3 was significantly phosphorylated in both nilotinib-treated and untreated cells. Moreover, combination therapy with nilotinib and G-CSF returned the expression level of c-Myc, cell growth and cell cycle distribution to the control level. These findings suggest that the ETV6/ARG oncoprotein contributes to autonomous cell growth by compensating for the requirement of growth factor through activating STAT5 signaling, which leads to the up-regulation of c-Myc. Our data suggest that ETV6/ARG oncoprotein is a potential target in the treatment of leukemia.

Abstract 1169: TPL2 kinase regulates the inflammatory milieu of the myeloma niche

Abstract Early-stage myeloma tumor cells are critically dependent on paracrine cytokine support originating from their bone marrow microenvironment whereas advanced-stage myeloma tumor cells may elaborate autocrine cytokine production mechanisms and/or cell-autonomous mutations in critical downstream signaling pathways (e.g., NFκB pathway). However, the molecular mechanisms underpinning the paracrine network in myeloma are unclear. The pro-inflammatory cytokine IL-1β has emerged as a major link between inflammation and cancer and has been validated as a therapeutic target in high-risk monoclonal gammopathy, the precursor to myeloma, as well as early-stage myeloma. To determine the source of IL-1β production in early-stage myeloma we analyzed paired purified cell fractions obtained from each of 5 patients at diagnosis: CD138+ tumor cells, CD14+ bone marrow-resident monocyte/macrophages as well as bone marrow-derived stromal/mesenchymal cells (BM-MSC). We found that in all cases, monocytes/macrophages were the predominant IL-1β synthesizer cell type in the myeloma microenvironment. Cytokine production by activated macrophages is controlled by the MAP3 kinase, TPL2 (Cot, MAP3K8). We have previously detected constitutive activation of TPL2-regulated signaling pathways in human myeloma-associated macrophages but its precise functional consequences have been unclear. To dissect the relevant mechanisms, we ablated Tpl2 in the genetically-engineered myeloma in vivo model, Vκ*MYC. Vκ*MYC animals activate MYC sporadically in B lymphocytes participating in germinal center reactions and develop a disease analogous to human multiple myeloma with production of paraprotein (monoclonal immunoglobulin), plasma cell infiltration of the bone marrow as well as end-organ damage (“myeloma kidney”, osteolytic lesions). Vκ*MYC+/Tpl2-null animals developed myeloma with a significantly prolonged latency and the disease burden was lower at all timepoints tested compared to controls. Analysis of monocytic cells from myeloma lesions showed that loss of Tpl2 did not result in macrophage repolarization to an unopposed M1 (tumoricidal/cytotoxic) phenotype. Instead, Tpl2-null myeloma-associated monocytes/macrophages exhibited severe defects in production of inflammatory cytokines, predominantly IL-1β, but also IL-6. Tpl2 loss did not have discernible impacts on tumor cell-autonomous growth and survival. Our results suggest that monocytes/macrophages and TPL2 kinase activity play a central role in orchestrating the inflammatory milieu of the myeloma niche. Disruption of the myeloma cytokine network through pharmacologic TPL2 kinase inhibition could provide novel therapeutic opportunity by interfering with the co-ordinate regulation of key pro-myeloma inflammatory cytokines through a targeted approach. Citation Format: Chelsea Hope, Samuel J. Ollar, Erika Heninger, Jeffrey L. Jensen, Ellen Hebron, Jaehyup Kim, Ioanna Maroulakou, Shigeki Miyamoto, Natalie Callander, Peiman Hematti, Marta Chesi, P. Leif Bergsagel, Fotis Asimakopoulos. TPL2 kinase regulates the inflammatory milieu of the myeloma niche. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1169. doi:10.1158/1538-7445.AM2014-1169

The role of protein kinase PAK1 in the regulation of estrogen-independent growth of breast cancer

The main goal of this work was to study the intracellular signaling pathways responsible for the development of hormone resistance and maintaining the autonomous growth of breast cancer cells. In particular, the role of PAK1 (p21-activated kinase 1), the key mitogenic signaling protein, in the development of cell resistance to estrogens was analyzed. In vitro studies were performed on cultured breast cancer cell lines: estrogen-dependent estrogen receptor (ER)-positive MCF-7 cells and estrogen-resistant ER-negative HBL-100 cells. We found that the resistant HBL-100 cells were characterized by a higher level of PAK1 and demonstrated PAK1 involvement in the maintaining of estrogen-independent cell growth. We have also shown PAK1 ability to up-regulate Snail1, one of the epithelial-mesenchymal transition proteins, and obtained experimental evidence for Snail1 importance in the regulation of cell proliferation. In general, the results obtained in this study demonstrate involvement of PAK1 and Snail1 in the formation of estrogen-independent phenotype of breast cancer cells showing the potential role of both proteins as markers of hormone resistance of breast tumors.

Regulation of Ribosome Biogenesis by Nucleostemin 3 Promotes Local and Systemic Growth in Drosophila

Nucleostemin 3 (NS3) is an evolutionarily conserved protein with profound roles in cell growth and viability. Here we analyze cell-autonomous and non-cell-autonomous growth control roles of NS3 in Drosophila and demonstrate its GTPase activity using genetic and biochemical assays. Two null alleles of ns3, and RNAi, demonstrate the necessity of NS3 for cell autonomous growth. A hypomorphic allele highlights the hypersensitivity of neurons to lowered NS3 function. We propose that NS3 is the functional ortholog of yeast and human Lsg1, which promotes release of the nuclear export adapter from the large ribosomal subunit. Release of the adapter and its recycling to the nucleus are essential for sustained production of ribosomes. The ribosome biogenesis role of NS3 is essential for proper rates of translation in all tissues and is necessary for functions of growth-promoting neurons.