Study of intracellular signaling pathways in Chronic Myeloproliferative Neoplasms

Mtor Pathway ◽

Janus Kinase ◽

Pi3k Inhibitor ◽

Chronic Myeloproliferative Neoplasms ◽

Intracellular Signaling Pathways

A gain-of-function mutation in Janus kinase 2 (JAK2V617F) is at the basis of the majority of chronic myeloproliferative neoplasms (MPN). Enhanced activation of other downstream pathways including the PI3K/mTOR pathway has been documented as well. In this study we evaluated the effects of JAK1/2 inhibitors, alone and in combination with mTOR, with a dual mTOR/PI3K inhibitor and with a pan PI3K inhibitor in in-vitro and in-vivo MPN models. Our findings of strong synergy between the JAK2 inhibitors and mTOR/PI3K inhibitor suggested that we might be able to administer these drugs at lower concentrations than when the drugs are used individually. This provides a framework for combination trials using compounds in patients with myeloproliferative neoplasms

Caffeine Inhibits EGF-Stimulated Trophoblast Cell Motility through the Inhibition of mTORC2 and Akt

Endocrinology ◽

10.1210/en.2011-1930 ◽

2012 ◽

Vol 153 (9) ◽

pp. 4502-4510 ◽

Cited By ~ 8

Author(s):

Isobelle Grant ◽

Judith E. Cartwright ◽

Brooke Lumicisi ◽

Alison E. Wallace ◽

Guy S. Whitley

Keyword(s):

Cell Motility ◽

Signaling Pathways ◽

Intracellular Signaling ◽

Pregnancy Loss ◽

Mammalian Target Of Rapamycin ◽

First Trimester ◽

Trophoblast Invasion ◽

Intracellular Signaling Pathways

Impaired trophoblast invasion is associated with pregnancy disorders such as early pregnancy loss and preeclampsia. There is evidence to suggest that the consumption of caffeine during pregnancy may increase the risk of pregnancy loss; however, little is known about the direct effect of caffeine on normal trophoblast biology. Our objectives were to examine the effect of caffeine on trophoblast migration and motility after stimulation with epidermal growth factor (EGF) and to investigate the intracellular signaling pathways involved in this process. Primary first-trimester extravillous trophoblasts (EVT) and the EVT-derived cell line SGHPL-4 were used to study the effect of caffeine on EGF-stimulated cellular motility using time-lapse microscopy. SGHPL-4 cells were further used to study the effect of caffeine and cAMP on EGF-stimulated invasion of fibrin gels. The influence of caffeine and cAMP on EGF-stimulated intracellular signaling pathways leading to the activation of Akt were investigated by Western blot analysis. Caffeine inhibits both EGF-stimulated primary EVT and SGHPL-4 cell motility. EGF stimulation activates phosphatidylinositol 3-kinase, and Akt and caffeine inhibit this activation. Although cAMP inhibits both motility and invasion, it does not inhibit the activation of Akt, indicating that the effects of caffeine seen in this study are independent of cAMP. Further investigation indicated a role for mammalian target of rapamycin complex 2 (mTORC2) as a target for the inhibitory effect of caffeine. In conclusion, we demonstrate that caffeine inhibits EGF-stimulated trophoblast invasion and motility in vitro and so could adversely influence trophoblast biology in vivo.

Direct Activation of STAT5 by TEL-Lyn Fusion Protein Promotes Induction of Myeloproliferative Neoplasms with Myelofibrosis

Blood ◽

10.1182/blood.v116.21.4114.4114 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4114-4114

Author(s):

Yusuke Takeda ◽

Chiaki Nakaseko ◽

Hiroaki Tanaka ◽

Masahiro Takeuchi ◽

Makiko Yui ◽

...

Keyword(s):

Bone Marrow ◽

Jak2 V617f ◽

Signaling Molecules ◽

Janus Kinase ◽

Idiopathic Myelofibrosis ◽

Lyn Kinase

Abstract Abstract 4114 Background Myeloproliferative neoplasms (MPN), a group of hematopoietic stem cell (HSC) disorders, are often accompanied by myelofibrosis. The V617F somatic mutation in the Janus kinase 2 (JAK2) gene has recently been found in the majority of patients with polycythemia vera (PV) and more than half of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). The expression of JAK2 V617F causes a PV-like disease with myelofibrosis in a murine bone marrow (BM) transplant model. In addition, a gain-of-function c-MPL W515 mutation was described in nearly 10% of patients with JAK2 V617F-negative IMF. However, the mechanism responsible for MPD and the formation of myelofibrosis in patients without JAK2 or c-MPL mutations is still unclear. We previously identified the fusion of the TEL gene to the Lyn gene (TEL-Lyn) in idiopathic myelofibrosis with ins(12;8)(p13;q11q21). The introduction of TEL-Lyn into HSCs resulted in fatal MPN with massive myelofibrosis in mice, implicating the rearranged Lyn kinase in the pathogenesis of MPN with myelofibrosis. However, the signaling molecules directly downstream from and activated by TEL-Lyn remain unknown. Design and Methods We examined the signaling pathways activated by TEL-Lyn by Western blotting, immunoprecipitation, and in vitro kinase assay using a TEL-Lyn kinase-dead mutant as a control. We further characterized the functional properties of Stat5-deficient HSCs transduced with TEL-Lyn by colony-forming assay and bone marrow transplantation to evaluate the role of STAT5 in TEL-Lyn-induced MPN. Results TEL-Lyn was demonstrated to be constitutively active as a kinase through autophosphorylation. In TEL-Lyn-expressing cells, STAT5, STAT3, and Akt were constitutively activated. Among these signaling molecules, STAT5 was activated most prominently and this occurred without the activation of Jak2, the major kinase for STAT5. TEL-Lyn was co-immunoprecipitated with STAT5, and STAT5 was phosphorylated when incubated with TEL-Lyn, but not with TEL-Lyn kinase-dead mutant. These results indicate that TEL-Lyn interacts with STAT5 and directly activates STAT5 both in vitro and in vivo. Of note, the capacity of TEL-Lyn to support the formation of hematopoietic colonies under cytokine-free conditions in vitro and to induce MPN with myelofibrosis in vivo was profoundly attenuated in a Stat5-null background. Conclusions In this study, we clearly showed that TEL-Lyn directly activates STAT5 and the capacity of TEL-Lyn to induce MPN with myelofibrosis was profoundly attenuated in the absence of STAT5. Our findings of TEL-Lyn in this study support the role of the Src family kinases in the regulation of STAT pathways and implicate active Lyn in the alternative pathway for STAT activation in pathological cytokine signaling. Our mouse model of MPD with myelofibrosis would be beneficial for the analysis of therapeutic approaches for myelofibrosis. Disclosures: No relevant conflicts of interest to declare.

The PI3K Specific Inhibitor BKM120 Results Effective and Synergizes With Ruxolitinib In Preclinical Models Of Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v122.21.1599.1599 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1599-1599 ◽

Cited By ~ 1

Author(s):

Niccolò Bartalucci ◽

Costanza Bogani ◽

Serena Martinelli ◽

Carmela Mannarelli ◽

Jean-Luc Villeval ◽

...

Keyword(s):

Cell Cycle ◽

Colony Formation ◽

Janus Kinase ◽

Preclinical Models ◽

Mtor Inhibition ◽

Jak2 Inhibitor ◽

Cd34 Cells

Abstract Background and Aims A gain-of-function mutation in Janus kinase 2 (JAK2V617F) is at the basis of the majority of chronic myeloproliferative neoplasms (MPN). The dual JAK1/JAK2 inhibitor ruxolitinib (ruxo) determined rapid and sustained responses in splenomegaly and symptomatic improvement in patients with myelofibrosis (MF), supporting the central role of dysregulated JAK2 signaling. Enhanced activation of other downstream pathways including the PI3K/mTOR pathway has been documented as well. We previously reported (Bogani et al, PlosOne 2013;8:54828) that targeting mTOR by the allosteric inhibitor RAD001 resulted in inhibition of JAK2VF mutated cells and produced clinical benefits in a phase I/II trial (Guglielmelli et al, Blood 2011;118:2069). In this study we evaluated the effects of BKM120, a specific PI3K inhibitor, alone and in combination with ruxolitinib, in in-vitro and in-vivo MPN models. Methods To evaluate cell proliferation, colony formation, apoptosis, cell cycle and protein phosphorylation status we used mouse BaF3 and BaF3-EPOR cells expressing wild type (WT) or VF mutated JAK2, the human VF-mutated HEL and SET2 cell lines, and primary MPN CD34+ cells from patients with MF or polycythemia vera (PV). Effect of drug combination was analyzed according to Chou and Talalay calculating the combination index (CI); a CI <1 indicates synergistic activity. For in vivo studies we used two mouse models: (1) SCID mice receiving iv BaF3-EPOR VF-luciferase (luc) cells (gift of T. Radimerski) were randomized on day 6 to different treatment groups based on baseline luminescence. (2) C57Bl6/J JAK2 VF Knock-in mice were generated by insertion of the reversed JAK2V617F exon 13 sequence; mating with Vav-Cre transgenic mice activates the VF allele producing a MPN phenotype in progenies with VF heterozygous expression (Hasan et al, Blood 2013;Epub). Mice were treated for 15 days, then blood, spleen and bone marrow cells were analyzed. Results We found that BKM120 preferential inhibited BAF3 VF and BaF3-EpoR VF cells (IC50: 364±200nM and 1100±207nM, respectively) compared to their respective WT counterpart (5300±800nM and 3122±1000nM: p<.05). HEL and SET2 cells resulted also sensitive to BKM120 (2000±500nM and 1000±300nM). Interestingly we found that BKM120 significantly increased G2/M phase and decreased S phase of cell cycle (p<.01) and induced apoptosis (IC50, SET2=10µM, BaF3-EPOR VF=1.8 µM). Western blot analysis showed marked reduction of phospho-mTOR and its target phospho-4EBP1 as well as downregulation of phospho-STAT5 at 6 and 24h of treatment. BKM120 impaired colony formation from MF and PV CD34+ cells at doses 2 to 8-fold lower than healthy controls (p<.01). BKM120 strongly inhibited EEC colony growth from PV pts (IC50, 9±4nM). Co-treatment of BKM120+ruxo resulted in synergistic inhibition of proliferation of SET2 (median CI=0.45) and BaF3-EPOR VF (median CI=0.8) cells. Triple combinations including BKM120/ruxo plus either RAD001 (Torc1 inhibitor) or PP242 (Torc1/2 inhibitor) resulted highly synergistic (median CI=0.27 and 0.52) to indicate the importance of complete mTOR inhibition. BKM120 at 45mpk and 60mpk increased mean lifespan of BaF3 VF luc mouse model from 21d in control mice to 27.2d and 28d in BMK120 treated mice. In KI mice, co-treatment with 60mpk BKM120 + 60mpk ruxo resulted in improvement of splenomegaly (median spleen weight: 1.4, 0.82, 0.8 and 0.6 g respectively for controls, 60mpk BKM120, 60mpk ruxo and 60mpk BKM120+60mpk ruxo) and reduction of leukocytosis and reticulocyte count. The level of phosho-STAT5 and -4EBP1 in the spleen was significantly reduced in mice receiving BKM120+ruxo as compared to single drug treatment. We finally analyzed the effects of BKM120+/-ruxo on the in-vitro clonogenic growth of BM cells from VF and WT KI mice mixed in a 1:1 ratio. The proportion of VF-positive colonies resulted reduced in a dose dependent manner by 19%, 33% and 44% (p<.03) compared to controls with 50nM, 100nM and 300nM BKM120 respectively. A 25% and 39% of VF-positive colonies reduction was achieved with 50nM and 100nM ruxolitinib. The combined treatment with 100nM BKM120 + 50nM ruxo resulted in a 50% decrease of the number of mutated colonies (p<.02). Conclusions Inhibition of PI3K by BKM120 alone and combined with JAK2 inhibitor ruxolitinib resulted in enhanced activity in preclinical models of MPN, providing a rationale for the ongoing combination clinical trial. Disclosures: Vannucchi: Novartis: Membership on an entity’s Board of Directors or advisory committees.

Repurposing Tranexamic Acid as an Anticancer Agent

10.1101/2021.10.17.464714 ◽

2021 ◽

Author(s):

Mary E Law ◽

Bradley J Davis ◽

Amanda F Ghilardi ◽

Elham Yaaghubi ◽

Zaafir M Dulloo ◽

...

Keyword(s):

Tranexamic Acid ◽

Anticancer Agents ◽

Intracellular Signaling ◽

Anticancer Agent ◽

Lead Compounds ◽

Multiple Processes ◽

Broad Array

Tranexamic Acid (TA) is a clinically used antifibrinolytic that acts as a lysine mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic lysine and arginine, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.

Analysis of Differentially Activated Signaling Pathways in Myeloproliferative Disease Using Kinomics Chip Profiling

Blood ◽

10.1182/blood.v112.11.3723.3723 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3723-3723

Author(s):

Daniel B Lipka ◽

Marie-Christine Blum ◽

Florian H Heidel ◽

Thomas Kindler ◽

Thomas Fischer

Keyword(s):

Signaling Pathways ◽

Cell Line ◽

Intracellular Signaling ◽

Jak2v617f Mutation ◽

Myeloproliferative Disease ◽

Differential Activation ◽

Cell Lysate ◽

Chip Analysis

Abstract In a multitude of cases, oncogenic mutations are gain of function mutations that confer a constitutively activated gene product. Currently, evidence from a large body of experimental studies suggests that oncogenic transformation induced by activating kinase mutations is not sufficiently explained by constitutive kinase activation alone but is a result of aberrantly activated signaling pathways in affected cells. The JAK2V617F-mutation is a highly prevalent molecular marker in Ph-negative myeloproliferative disease (MPD). In vitro, Ba/F3-cells expressing both erythropoietin receptor (EpoR) and the JAK2V617F-mutation show constitutive activation of the JAK-STAT pathway and cytokine independent growth. Multiple in-vitro and in-vivo studies demonstrate that the JAK2V617F-mutation mediates many of the phenotypic characteristics of the MPDs. Nevertheless, until now it is largely unclear, which signaling pathways in particular are involved in the process of malignant transformation in JAK2V617F-positive cells. Therefore, we applied a kinomics chip approach to screen for activated intracellular signaling pathways. We used a commercially available peptide chip containing 960 synthetic kinase substrate peptides spotted in triplicates and covering peptide substrates for approximately 50% of the human kinome. Peptides have been selected for their biologic relevance in physiologic processes such as stress response, growth and cell differentiation. With this approach, a broad spectrum of intracellular signaling pathways and kinases can be investigated simultaneously in a single experiment. As a proof of principle, we performed kinomics chip analysis of Ba/F3-cells stably transfected with EpoR and either the JAK2V617F-mutant (Ba/F3-EpoR-VF) or wildtype JAK2 (Ba/F3-EpoR-WT). In brief, cells were seeded and treated with erythropoietin. One chip per cell lysate was incubated with an activation buffer containing the cell lysate and radioactively labelled ATP for a defined time period and washed several times afterwards. The chip was then analyzed by means of autoradiography using a phospho-storage-screen and a phospho-imager. Chip analysis was performed using standard microarray software and Microsoft Excel software. Chip experiments were performed simultaneously for Ba/F3-EpoR-VF and Ba/F3-EpoR-WT and in duplicate. Analysis revealed differential activation of known pathways such as Ras/Raf/MEK/ERK, JAK/STAT, and PI3-Kinase with pronounced activation seen in Ba/F3-EpoR-VF cells as compared to Ba/F3-EpoR-WT cells. This was not a surprising result but strongly underlines the feasibility and validity of this approach and therefore served as an internal control. Differential regulation of a number of other signaling nodes that have not yet been described in the context of mutant JAK2 signaltransduction have been detected. To select for relevant hits among these potential targets, we first excluded all substrates from further analysis that are known to be involved in lymphocyte-specific pathways. For the remaining hits we performed a literature search to learn more about their known functions and their potential impact in JAK2V617F-positive MPD. Validation of selected signaling molecules by means of Western blotting analysis and functional investigations such as siRNA knock-down experiments are currently under way. In addition to the widely used lymphoid Ba/F3-model, we also established a novel cell culture model with simultaneous expression of EpoR and either mutant or wildtype JAK2 in a myeloid 32D-cell background. This model will be helpful to us to determine false-positive results due to cell-line specific changes. We conclude, that kinomic profiling using the above mentioned chip-technology is a valid method to comprehensively investigate differential activation of signaling pathways in cell lysates. In our cell line model, we were able to detect activation of well known signaling pathways in JAK2V617F-positive cells. Furthermore, we were able to identify candidate proteins that appear to be specifically involved in JAK2V617F-signaling.

Inhibitors of PI3K/Akt and/or mTOR Inhibit the Growth of Cells of Myeloproliferative Neoplasms and Synergize with JAK2 Inhibitor and Interferon,

Blood ◽

10.1182/blood.v118.21.3835.3835 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3835-3835 ◽

Cited By ~ 5

Author(s):

Alessandro M. Vannucchi ◽

Costanza Bogani ◽

Niccolò Bartalucci ◽

Lorenzo Tozzi ◽

Serena Martinelli ◽

...

Keyword(s):

Cell Proliferation ◽

Mtor Inhibitor ◽

Mtor Inhibitors ◽

Mtor Pathway ◽

Simultaneous Treatment ◽

Jak2 Inhibitor ◽

Stat Pathway

Abstract Abstract 3835 Dysregulated JAK/STAT signaling, occurring mainly but not exclusively in cells harboring mutations in JAK2 or other proteins involved in JAK/STAT pathway such as MPL, CBL, or Lnk, represents a pathogenetic event in chronic myeloproliferative neoplasms (MPN). However, activation of other downstream pathways such as the ERK and PI3K/Akt/mTOR pathway has been also documented in JAK2V617F-mutated cells. In this study we explored in-vitro the potential relevance of targeting PI3K/Akt/mTor pathway with specific inhibitors, alone or in combination with JAK2 inhibitor. Indeed, clinical trials have recently documented the effectiveness of JAK1/2 inhibitors (Verstovsek S, NEJM, 2010;363:117; Pardanani A, JCO 2011; 29:789) and RAD001, an mTOR inhibitor (Guglielmelli 2001; Blood, in press), in patients with MPN, mainly with myelofibrosis. The following drugs were used: an allosteric (RAD001) and an ATP (PP242) mTOR competitor; a dual PI3K/mTOR inhibitor (BEZ235); the JAK1/2 kinase ATP competitor AZD1480 and INCB018424. In the BA/F3/EPOR JAK2V617F-mutated cells, cell proliferation was prevented by lower doses of RAD001 (615±50nM, measured as IC50), PP242 (98±5nM) and BEZ235 (87±50nM) compared to BA/F3/EPOR JAK2wild-type (wt) cells (>10,000nM; 5,931±1,000nM; 676±200nM, respectively). In case of JAK inhibitors, IC50 was 313±23nM for AZD1480 and 51±2nM for INCB018424 as compared to 752±30nM and 457±15nM in wt cells, respectively. mTOR inhibitors induced cell cycle arrest in Go but were very poorly inducers of apoptosis (less than 15–20% at maximum); conversely JAK1/2 inhibitors induced dose-dependent increase of Annexin-V +ve cells up to >60% and BEZ235 induced 30–40% apoptosis at highest concentrations. All above drugs were able to prevent short-term cell proliferation and colony formation also in JAK2V617F-mutated HEL and SET2 cells. Western blot analysis demonstrated that, in addition to the expected inhibition of phosphorylation of specific drug targets (mTOR, 4EBP1), all three PI3K/mTOR inhibitors also reduced the degree of phophoSTAT5. siRNA-induced down-regulation of mTOR in SET2, HEL and BA/F3/EPOR JAK2V617F cells resulted in reduced phosphoSTAT5, indicating a direct mTOR-dependent phopshoSTAT5 regulation. Then, the activity of RAD001, BEZ235 and AZD1480 was analyzed in primary cells from MPN patients. All three drugs reduced clonogenic growth of MPN erythroid, myeloid and megakaryocytic progenitors at doses significantly lower (from 5 to 10-fold) than in normal cells, and prevented erythropoietin-independent colony (EEC) formation in the low nM range. Single colony genotyping in JAK2V617F mutated patients showed a median of 30±20% (range, 5–57%) reduction of V617F mutated colonies in favor of wt colonies Overall, these data indicated that inhibitors of PI3K/mTOR prevent proliferation and clonogenic capacity of MPN cells mainly through a cytostatic rather apoptotic effect (as JAK1/2 inhibitors do). To exploit whether simultaneous treatment with PI3K/AKt and JAK1/2 inhibitor displayed synergism we treated SET2 cells with different drug doses and measured their proliferation and apoptotic rate. Synergism was calculated as the combination index (CI) according to Chou and Talalay. Evidence of synergism was obtained for AZD1480 and INCB018424 with RAD001 (CI: 0.13 and 0.20, respectively), PP242 (CI: 0.13 and 0.20, respectively) and BEZ235 (CI: 0.77 and 0.37, respectively). Synergism was similarly demonstrated in BA/F3/EPOR JAK2V617F-mutated cells. Activity of RAD001 with AZD1480 and INCB018242 was also assessed in a EEC assay. We found that addition of RAD001 (5nM) or BEZ235 (50nM) to very low doses of JAK1/2 inhibitors (in the range of 5 to 50 nM) resulted in significant synergism and almost completely prevented EEC formation. In summary, these in vitro data indicate that PI3K/mTOR inhibitors are active against MPN cells and their combination with JAK1/2 inhibitors produced synergism, allowing to use lower doses of each drug; studies in murine models are ongoing to confirm these results in vivo. Thus, concurrent targeting of PI3K/mTOR and JAK/STAT pathway might represent a new therapeutic strategy to optimize efficacy and reduce toxicity in patients with MPN. Disclosures: Vannucchi: Italfarmaco: Consultancy; Novartis: Honoraria.

Protosappanin B Exerts Anti-tumor Effects on Colon Cancer Cells via Inhibiting GOLPH3 Expression

Integrative Cancer Therapies ◽

10.1177/1534735420972477 ◽

2020 ◽

Vol 19 ◽

pp. 153473542097247

Author(s):

Xue-Cong Zheng ◽

Ze-Sheng Shi ◽

Cheng-Zhi Qiu ◽

Zhong-Shi Hong ◽

Chun-Xiao Wang ◽

...

Keyword(s):

Colon Cancer ◽

Cancer Cells ◽

Signaling Pathways ◽

Intracellular Signaling ◽

Dependent Manner ◽

Colon Cancer Cells ◽

Human Colon Cancer ◽

Sw620 Cells

Protosappanin B (PSB) is a key active component of Lignum Sappan extract. Although the antiproliferative effects of Lignum Sappan extract have been demonstrated in various cancer cells, relatively little is known about the effects of PSB on tumor progression. The aim of this study was to explore the anti-tumor effects of PSB on human colon cancer cells by regulation of intracellular signaling pathways and Golgi phosphoprotein 3 (GOLPH3) expression in vitro and in vivo. Our results showed that PSB effectively inhibited the viability and migration of SW620 cells and induced apoptosis, but had poor effect on HCT116 cells. Furthermore, PSB significantly reduced the expression of p-AKT, p-p70S6K, β-catenin, and p-ERK1/2 proteins in SW620 cells, and this effect was reversed by the corresponding signaling pathway agonists. Interestingly, PSB could also suppress GOLPH3 expression of SW620 cells in a concentration-dependent manner, but SW620 cells transfected with lentiviral vectors overexpressing GOLPH3 can effectively resist the cytotoxic activity of PSB in vitro. The xenograft experiment of SW620 cells with LV-GOLPH3 confirmed that PSB distinctly inhibited the tumor growth via suppressing GOLPH3 expression. Collectively, these findings clarified a new anti-cancer mechanism of PSB through inhibition of GOLPH3 expression and intracellular signaling pathways in colon cancer cells. PSB may be a potential new drug for colon cancer.

EDA Fibronectin-TLR4 Axis Sustains Megakaryocyte Expansion and Inflammation during Bone Marrow Fibrosis Progression

Blood ◽

10.1182/blood-2018-99-116454 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1781-1781

Author(s):

Alessandro Malara ◽

Cristian Gruppi ◽

Vittorio Abbonante ◽

Daniele Cattaneo ◽

Luigi De Marco ◽

...

Keyword(s):

Bone Marrow ◽

Primary Myelofibrosis ◽

Janus Kinase ◽

Soluble Form ◽

Signalling Pathways ◽

Hematopoietic Stem ◽

Fibrosis Progression

Abstract Fibronectin (FN) is a glycoprotein of approximately 220 kDa, whose mRNA has three alternative splicing sites (termed EDA, EDB and IIICS) that allow 20 different isoforms of FN mRNA. Circulating plasma FN lacks both EDA and EDB segments and is a soluble form secreted by the hepatocytes, while cellular FN contains variable proportions of EDA and EDB segments and is organized as fibrils in the tissue matrix. FN containing EDA segment (EDA FN) presents unique biochemical properties as compared to the isoform lacking this domain, specifically: 1) pro-fibrotic, as the EDA-containing isoform becomes predominantly expressed during wound healing to sustain myofibroblast differentiation; 2) pro-inflammatory, as inclusion of EDA domain activates Toll Like Receptor 4 (TLR4), involved in the defense of the innate immune response following recognition of pathogens associated molecular patterns resulting in NF-κβ-dependent cytokine release; 3) pro-thrombotic, as EDA FN was shown to promote agonist-induced platelet aggregation and thrombus formation in vivo through TLR4 dependent mechanisms. EDA FN is instrumental in fibrogenesis but, to date, its expression and function in bone marrow (BM) fibrosis have not been explored. We identified the up-regulation, at both molecular and protein levels, of EDA FN in an experimental mouse model of BM fibrosis treated with supra-pharmacological doses of the thrombopoietin (TPO) mimetic Romiplostim (TPOhigh). Higher expression levels of EDA FN were detected in endothelial and stromal cells after inducing experimental fibrosis in wild type (WT) mice as compared to untreated controls. To unravel the role of EDA segment on the progression of BM fibrosis in vivo, we exploited two unique transgenic mouse models that present an aberrant splicing of the EDA exon. Mice with constitutive inclusion of EDA exon (EIIIA+/+), in tissues, were more prone to BM fibrosis development as compared to knockout mice (EIIIA-/-). Upon fibrosis induction with TPO mimetic treatment, EIIIA+/+ mice presented extensive reticulin deposition, thrombocytosis, splenomegaly and increased extra-medullary hematopoiesis. Applying numerous in vitro methods, we demonstrated that EDA FN as opposed to plasma FN lacking the EDA domain, induces megakaryocyte (Mk) proliferation in a TPO-independent fashion through engagement of TLR4 and activation of STAT5 and ERK 1/2 signalling pathways. Additionally, in vitro activation of EDA FN/TLR4 axis on Mks resulted in lipopolysaccharide-like responses, such as NF-kb activation, the release of pro-fibrotic Interleukin-6 (IL-6), and no anti-fibrotic TNF-a. Pharmacological inhibition of TLR4 in TPOhigh and EIIIA+/+/TPOhigh mice or TLR4 deletion in TPOhigh mice abrogated Mk hyperplasia, BM fibrosis, IL-6 release, extramedullary hematopoiesis and splenomegaly. Finally, developing a novel ELISA assay, we analysed samples from patients affected by primary myelofibrosis (PMF). PMF is one of the Philadelphia-negative Myeloproliferative Neoplasms (MPNs), a group of heterogeneous clonal disorders affecting the hematopoietic stem cell. MPNs are characterised by neoplastic proliferation of the myeloid lineage leading to an abnormal increase of platelet count in essential thrombocythemia (ET), red blood cells in polycythemia vera (PV), or Mks with BM fibrosis in primary myelofibrosis (PMF). To date, three major driver mutations have been identified affecting, respectively, the TPO receptor gene (MPL), the intracellular Janus Kinase 2 gene (JAK2) and the latest discovered mutations affecting the endoplasmic reticulum (ER) molecular chaperone calreticulin gene (CALR). All of these mutations induce a permanent activation of the JAK/STAT signalling pathways. We found that the EDA FN is increased in plasma and BM biopsies of PMF patients, as compared to healthy controls and ET patients, correlating with the fibrotic phase. In conclusion, we identified EDA FN/TLR4 as a new pathological axis that sustains Mk expansion and inflammation during BM fibrosis progression. EDA FN and TLR4 targeting has very promising potential for new therapeutic approaches in PMF patients with high phase BM fibrosis. Disclosures No relevant conflicts of interest to declare.

New mutations and pathogenesis of myeloproliferative neoplasms

Blood ◽

10.1182/blood-2011-02-292102 ◽

2011 ◽

Vol 118 (7) ◽

pp. 1723-1735 ◽

Cited By ~ 269

Author(s):

William Vainchenker ◽

François Delhommeau ◽

Stefan N. Constantinescu ◽

Olivier A. Bernard

Keyword(s):

Signaling Pathways ◽

Intracellular Signaling ◽

De Novo ◽

Clonal Selection ◽

Blast Crisis ◽

Chronic Phase ◽

Janus Kinase ◽

Prognostic Impact ◽

Loss Of Function

Abstract Myeloproliferative neoplasms (MPNs) are clonal disorders characterized by excessive production of mature blood cells. In the majority of classic MPN—polycythemia vera, essential thrombocythemia, and primitive myelofibrosis—driver oncogenic mutations affecting Janus kinase 2 (JAK2) or MPL lead to constitutive activation of cytokine-regulated intracellular signaling pathways. LNK, c-CBL, or SOCSs (all negative regulators of signaling pathways), although infrequently targeted, may either drive the disease or synergize with JAK2 and MPL mutations. IZF1 deletions or TP53 mutations are mainly found at transformation phases and are present at greater frequency than in de novo acute myeloid leukemias. Loss-of-function mutations in 3 genes involved in epigenetic regulation, TET2, ASXL1, and EZH2, may be early events preceding JAK2V617F but may also occur late during disease progression. They are more frequently observed in PMF than PV and ET and are also present in other types of malignant myeloid diseases. A likely hypothesis is that they facilitate clonal selection, allowing the dominance of the JAK2V617F subclone during the chronic phase and, together with cooperating mutations, promote blast crisis. Their precise roles in hematopoiesis and in the pathogenesis of MPN, as well as their prognostic impact and potential as a therapeutic target, are currently under investigation.

Repurposing Tranexamic Acid as an Anticancer Agent

Frontiers in Pharmacology ◽

10.3389/fphar.2021.792600 ◽

2022 ◽

Vol 12 ◽

Author(s):

Mary E. Law ◽

Bradley J. Davis ◽

Amanda F. Ghilardi ◽

Elham Yaaghubi ◽

Zaafir M. Dulloo ◽

...

Keyword(s):

Tranexamic Acid ◽

Anticancer Agents ◽

Intracellular Signaling ◽

Anticancer Agent ◽

Lead Compounds ◽

Multiple Processes ◽

Broad Array

Tranexamic Acid (TA) is a clinically used antifibrinolytic agent that acts as a Lys mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic Lys and Arg, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. Further, TA inhibited uptake of Lys and Arg by cancer cells. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.