CFU-MK Formation Research Articles

Whole-genome CRISPR screening identifies N-glycosylation as a genetic and therapeutic vulnerability in CALR-mutant MPNs

AbstractCalreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPNs). Although the biological mechanism by which CALR mutations cause MPNs has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPNs. To identify unique genetic dependencies in CALR-mutant MPNs, we performed a whole-genome clustered regularly interspaced short palindromic repeats (CRISPR) knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (among others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells, through a reduction in MPL cell surface expression. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor 2-deoxy-glucose (2-DG) and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared with wild-type cells and normalization of key MPNs disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow as compared with bone marrow derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPNs.

Abivertinib inhibits megakaryocyte differentiation and platelet biogenesis.

Abivertinib, a third-generation tyrosine kinase inhibitor, is originally designed to target epidermal growth factor receptor (EGFR)-activating mutations. Previous studies have shown that abivertinib has promising antitumor activity and a well-tolerated safety profile in patients with non-small-cell lung cancer. However, abivertinib also exhibited high inhibitory activity against Bruton's tyrosine kinase and Janus kinase 3. Given that these kinases play some roles in the progression of megakaryopoiesis, we speculate that abivertinib can affect megakaryocyte (MK) differentiation and platelet biogenesis. We treated cord blood CD34+ hematopoietic stem cells, Meg-01 cells, and C57BL/6 mice with abivertinib and observed megakaryopoiesis to determine the biological effect of abivertinib on MK differentiation and platelet biogenesis. Our in vitro results showed that abivertinib impaired the CFU-MK formation, proliferation of CD34+ HSC-derived MK progenitor cells, and differentiation and functions of MKs and inhibited Meg-01-derived MK differentiation. These results suggested that megakaryopoiesis was inhibited by abivertinib. We also demonstrated in vivo that abivertinib decreased the number of MKs in bone marrow and platelet counts in mice, which suggested that thrombopoiesis was also inhibited. Thus, these preclinical data collectively suggested that abivertinib could inhibit MK differentiation and platelet biogenesis and might be an agent for thrombocythemia.

Tanshinone Iia Inhibits Megakaryopoiesis in Immune Vasculitis

Introduction Tanshinone IIA, an active component of Danshen (Salvia miltiorrhiza), has been used for centuries to treat hypercoagulation-related diseases, which attributed to its anti-platelet and anti-inflammatory effects. However, the role of Tanshinone IIA in megakaryocytes, the precursor of platelet within the bone marrow, remains unclear. Therefore, the present study established a rabbit model with immune vasculitis to examine the effect of Tanshinone IIA on megakaryopoiesis and to identify the underlying mechanism(s).Methods Immune vasculitis was established in rabbits (3-4 weeks old) by two intravenous injection of 10% bovine serum albumin (2.5 ml/kg) at two-week interval. Those rabbits were randomly treated with Tanshinone IIA (5 mg/kg/d, 7 d, iv) or aspirin (100 mg/kg/d, 7 d, ig). Megakaryocyte count and CFU-MK formation were measured by Wright's and AChE staining, respectively. Human megakaryotic cell lines Meg-01 and CHRF-288-11 were used to examine the effect of Tanshinone IIA on apoptosis by Annexin V-FITC/PI, mitochondrial membrane potential/JC-1 and Caspase-3 activity assays using flow cytometry.ResultsIn rabbits with immune vasculitis, the platelet count, platelet aggregation and the serum levels of inflammatory cytokines IL-1β, TNF-α and IL-6 were significantly increased when compared to their healthy controls. After 7 days of Tanshinone IIA treatment, all these parameters were significantly reduced, with the inhibitions comparable to those caused by aspirin. In addition, the number of megakaryocytes and the formation of CFU-MK were also statistically increased in rabbits with immune vasculitis, which could be significantly reduced by Tanshinone IIA. In vitro, Tanshinone IIA (1, 3, 10 and 30 μg/ml) also significantly inhibited the formation of CFU-MK of bone marrow cells of BALB/c mice (6-10 weeks) in a dose-dependent manner. In human megakaryocytic cell line Meg-01, Tanshinone ⅡA (10 μg/ml, 72 h) induced apoptosis; both early and late apoptotic rates were significantly increased. In another human megakaryocytic cell line CHRF-288-11, Tanshinone ⅡA (10 μg/ml, 72 h) statistically increased the proportion of depolarized cells, from 9.70% to 14.13%, according to mitochondrial membrane potential using JC-1 assay. The expression of active Caspase-3 in CHRF-288-11 was also significantly increased by Tanshinone ⅡA (10 μg/ml, 72 h) from 5.25% to 15.86%.Conclusion The present study shows that Tanshinone IIA ameliorates immune vasculitis by inhibiting megakaryopoiesis and inducing apoptosis of megakaryocytes, which might explain the anti-platelet and anti-inflammatory effects of Tanshinone IIA. DisclosuresNo relevant conflicts of interest to declare.

Anti-Apoptotic Effect of Angelica Polysaccharide (APS) on Cryopreservation of Platelets

Background: Angelica Polysaccharide (APS) is from the root of Radix Angelicae Sinensis (Danggui). Danggui has been used for centuries to treat blood-deficiency related diseases. The hematopoietic effect of Danggui may be related to its constituent, polysaccharide. The effects of angelica polysaccharide on cryopreservation of platelets and megakaryocytes have not been well studied. This study focused on anti-apoptotic effect of APS and TPO on cryopreservation of platelets and megakaryocytes and provided new methods for prolonging the preservation time of platelets in vitro.Methods: The expression of platelet membrane glycoprotein CD41 and CD61, as well as the platelet apoptotic rate, Caspase 3 expression and mitochondrial membrane potential (MMP) were detected by flow cytometry; the anti-apoptotic mechanism of APS by PI3K /AKT signaling pathway was analyzed by Western blot assay. CFU assays were used to determine the effects of APS on megakaryocytic progenitor cells. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were conducted in megakaryocytic cell line M-07e. The effects of APS on cells treated with Ly294002, PI3K inhibitor and the effect of APS on the p-AKT were also studied.Results: The platelets were divided into 4 group: control group (4 ℃ stored platelets), APS group (APS-treated platelets stored at 4 ℃), LY294002 group (LY294002-treated platelets stored at 4 ℃) and LY294002+APS group (LY294002+APS treated platelets stored at 4 ℃). The apoptotic rate of platelets in LY294002 group was obviously increased. Compared with control group, the expression of CD41 and CD61 gradually decreased along with the enhancement of LY294002 concentrations (r=-0.953). The apoptotic rate of platelets in LY294002 group was enhanced significantly (P<0.05). While in LY294002+APS group, the apoptotic rate of platelets was significantly reduced (P<0.05) as compare with LY294002 group, which suggest that APS has an anti-apoptotic effect on the cryopreserved platelets. APS decreased the expression of Caspase-3 and inhibited the reduction of mitochondrial membrane potential induced by LY294002. Moreover, APS increased the activation of PI3K /AKT pathway in Platelets . We further analyzed the in vitro effect of APS on CFU-MK formation. APS (50 ug/ml) enhanced TPO (50 ng/ml) -induced CFU-MK formation (p=0.06, n=4). APS also significantly enhanced PDGF, bFGF and VEGF-induced CFU-MK formation (n=4). Moreover, the anti-apoptotic effect of APS in M-07e cells was also demonstrated by Annexin-V, Caspase-3, and JC-1 assays. Adding LY294002 alone increased the percentage of cells undergoing apoptosis. However, additional of APS to LY294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of APS significantly increased the p-AKT.Conclusion: APS, like TPO, has an anti-apoptotic effect on the cryopreserved platelets and megakaryocytes through activating PI3K/AKT, decreasing the expression of Caspase-3 and inhibiting the reduction of MMP. DisclosuresNo relevant conflicts of interest to declare.

Synthesis of Inflammatory Cytokines and Expression of Adhesion Molecules in Megakaryocytes

Inflammatory cytokines and platelet adhesion molecules may play a major role in hemostasis and immuno-pathological processes. Hematopoietic cells of myeloid and lymphoid lineages are known to be important sources of a variety of inflammatory cytokines. However, the immunological role of megakaryocytes is not well known. This project studied the involvement of megakaryocytes on immuno-inflammatory processes and the possible mechanism via the adhesion molecules and the synthesis of relevant cytokines. The expression of interleukin (IL)-1 to IL-10, TNF-a, TNF-g and IFN-g in primary megakaryocytes and four megakaryocytic cell lines: Meg-01, Dami, CHRF-288-11 and M-07e were determined by RT-PCR. The effect of IL-1b, IL-3, IL-6 and TPO on megakaryocyte colony formation (CFU-MK) was studied by using a plasma clot culture system. The CFU-MK was confirmed by AchE staining. The expression of adhesion protein CD36 on human platelets, megakaryocytes and megakaryocytic cell lines was also analyzed by using flow cytometry, ELISA and immunocytochemical methods. Our results showed that CD36 was expressed on platelets, megakaryocytes and the four megakaryocytic cell lines. The relative expression level is as follows: platelets > megakaryocytes > Meg-01 > Dami > CHRF-288-11 > M-07e, suggesting that the level of CD36 expression correlates with the degree of maturity of megakaryocytic differentiation. Inflammatory cytokines TNF-a, IL-1b, IL-3 and IL-6 were detected in primary megakaryocytes and all the four megakaryocytic cell lines, suggesting that different stages of megakaryocytes can be as a source of inflammatory cytokines. IL-1b, IL-3 and IL-6, as well as TPO, play a stimulating effect on CFU-MK formation, suggesting that there is an “autocrine” effect on megakaryopoiesis. Megakaryocytes may involve in immuno-inflammatory processes via the synthesis of inflammatory cytokines and platelet adhesion molecules. DisclosuresNo relevant conflicts of interest to declare.

Melatonin protects against apoptosis of megakaryocytic cells via its receptors and the AKT/mitochondrial/caspase pathway.

Clinical studies have shown that melatonin lowers the frequency of thrombocytopenia in patients with cancer undergoing radiotherapy or chemotherapy. Here, we investigated the mechanisms by which melatonin promotes platelet formation and survival. Our results show that melatonin exerted protective effects on serum-free induced apoptosis of CHRF megakaryocytes (MKs). Melatonin promoted the formation of MK colony forming units (CFUs) in a dose-dependent manner. Using doxorubicin-treated CHRF cells, we found that melatonin rescued G2/M cell cycle arrest and cell apoptosis induced by doxorubicin. The expression of p-AKT was increased by melatonin treatment, an effect that was abolished by melatonin receptor blocker. In addition, we demonstrated that melatonin enhanced the recovery of platelets in an irradiated mouse model. Megakaryopoiesis was largely preserved in melatonin-treated mice. We obtained the same results in vivo from bone marrow histology and CFU-MK formation assays. Melatonin may exert these protective effects by directly stimulating megakaryopoiesis and inhibiting megakaryocyte apoptosis through activation of its receptors and AKT signaling.

Dysregulated IL-6/GP130/JAK Signaling in Calreticulin Mutated Myeloproliferative Neoplasms (MPN)

Introduction. Frameshift mutations in exon 9 of calreticulin (CALR) gene represent the second most frequent alteration in MPN. More than 30 types of CALR mutations are described; the most frequent are a 52bp deletion (Type1, T1) and a 5bp insertion (T2). All mutations cause a recurrent frameshift that originates a novel C-terminal peptide lacking the KDEL Endoplasmic Reticulum-retention sequence. Recent data described the interaction of mutated CALR (CALRm) with the thrombopoietin receptor MPL and the resulting activation of JAK/STATs as well MAPK and PI3K pathways (Araki M et al &amp; Chachoua I et al, Blood 2016; Elf S et al, Blood 2018). However, full characterization of the molecular mechanisms linking CALRm with MPN remains to be fully accomplished. Methods. By using CRISPR/Cas9 technology we generated CALR knock out (KO) and T1 variants from the GM-CSF cytokine dependent UT7 cell line; primary CD34+ cells from CALRm pts were also analyzed. Results. Introduction of CALR T1 mutation or generation of KO status did not appreciably modified the proliferation rate, cell cycle and apoptosis of UT7 cells under standard conditions. However, UT7 T1 and KO cells were similarly able to grow in the absence of GM-CSF, otherwise necessary for WT cells; in these cytokine-depleted conditions, apoptosis was significantly increased in WT cells compared both T1 and KO (4- and 8-fold higher; p&amp;lt;0.01) suggesting acquisition of cytokine independency. Exploring the mechanisms leading to cytokine independence we found that signal transducer and activation of transcription 3 (STAT3) was the main constitutively phosphorylated STAT in T1 and KO cells. Among a panel of STAT3 activation candidates assessed by RT-PCR, we found that mRNA levels of interleukin 6 (IL6), which induces STAT3 phosphorylation and activation through the glycoprotein 130/Janus kinase (GP130/JAK) pathway, were significantly increased in T1 and KO compared to parental cells (2- and 3.5-fold higher; p&amp;lt;0.01). The levels of IL6 released in cell culture medium were also significantly increased (1.5- and 2-fold higher; p&amp;lt;0.05). Blocking GP130 with SC144 or IL6 receptor (IL6R) with Tocilizumab (TCZ), in cytokine-depleted conditions, led to cell growth arrest in T1 and KO with no effects on WT cells. SC144 treatment induced a marked dephosphorylation of STAT3 in T1 and KO cells. Also, the membrane expression of GP130 and IL6R resulted higher in KO and T1 compared to WT cells, whilst intracellular content was similar. Co-immunoprecipitation showed that wild type calreticulin, unlike the mutated T1, formed complexes with GP130 and IL6R suggesting that CALRm lost the capacity to negatively regulate their membrane exposure resulting in downstream increased IL6 synthesis and release. The role of IL6 pathway in CALRm patients was evaluated by assessing megakaryocyte (Mk) colony generation in plasma clot cultures of CD34+ cells in the absence/presence of IL6, as part of cytokine cocktail optimally supporting CFU-Mk growth. We found that CFU-Mk generation by CALRm CD34+ cells was largely independent of exogenously added IL6, unlike control CFU-MK whose number was halved in the absence of IL6 (p&amp;lt;0.05). Moreover, inhibition of GP130 and IL6R by SC144 and TCZ, respectively, significantly reduced CFU-Mk by CALRm CD34+ cells (2.9- and 3.2-fold; p&amp;lt;0.05), with no effect in control cultures. As anticipated, JAK inhibitors ruxolitinib and momelotinib reduced CFU-Mk formation by CALRm CD34+ cells (2- and 3.2-fold, respectively; p&amp;lt;0.01), particularly evident in exogenous IL6-free conditions (5.8- and 4.8-fold, respectively; p&amp;lt;0.01). The combination of either ruxolitinib or momelotinib with TCZ on CFU-Mk formation was also assessed. Both combinations proved synergistic activity in reducing CFU-Mk from CALRm CD34+ cells (4.3- and 5-fold vs control cultures, respectively; p&amp;lt;0.01; combination compared to single agent, 2.8- fold vs ruxolitinib, 4.5-fold vs momelotinib and 4-fold vs TCZ, p&amp;lt;0.05 for all). Conclusions. Overall, our data suggest a novel model of action of mutant CALR resulting from the loss of its capability to complex GP130 and IL6R and regulate their cell membrane expression. Augmented expression of IL6 receptor complex on CALRm CD34+ cells on turn results in deregulated IL6-dependent signaling and augmented megakaryocyte generation. Blockade of IL-6/GP130/JAK signaling pathway might represent a new therapeutic target in pts with CALR mutation. Disclosures Vannucchi: Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

A novel thrombopoietin mimetic RWJ-800088 increases megakaryopoiesis without causing malignant proliferation in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).

e18527 Background: In patients (pts) with AML and MDS, thrombocytopenia is a source of morbidity/mortality and there is a need for safer treatment options. RWJ-800088 (RWJ) is a novel pegylated thrombopoietin (TPO) mimetic peptide that increases megakaryopoiesis preclinically and in humans. Before using a TPO mimetic agent in AML and MDS pts, it is important to evaluate if the agent has potential stimulatory effects on malignant myeloid cells. To assess whether RWJ is likely to stimulate malignant myeloid cells, we evaluated its effects in vitro on primary human samples from healthy controls and AML/MDS pts. Methods: We studied the effect of various RWJ doses on proliferation of AML/MDS cell lines; clonogenic capacity for leukemic colony forming units (CFU) and megakaryocytic CFU (CFU-Mk) of mononuclear cells (MNC) from controls and several MDS and AML pts; and expression of differentiation markers in MNC cultures. Results: RWJ treatment had no effect on proliferation of THP1, MOLM13, MV411 and MDSL cell lines at doses up to 1000 ng/ml. A slight increase in proliferation of CMK, a megakaryocytic cell line, was seen at doses &gt; 5xEC50 (mean 12%, p = 0.03). Primary clonogenic assays from 18 AML/MDS pts did not show increased growth of leukemic CFU at RWJ concentrations up to 10 ng/ml and RWJ had a nonsignificant inhibitory effect in 4 pts. Notably, RWJ significantly increased normal CFU-Mk formation from MNC in 6 of 8 AML and all MDS pts (mean 2.6-fold, p = 0.02), as well as from healthy CD34+ stem cells and healthy MNC controls (mean 1.7-fold, p = 0.03). FACS analysis of primary cultures treated with RWJ showed no increase in the malignant blast compartment, no change in differentiation markers of granulocyte/monocyte or erythrocyte lineages but a significant increase in the number of CD41a+ megakaryocytic cells (mean 1.8-fold, p &lt; 0.01). RWJ treatment increased phosphorylation of STAT5 but not STAT3, demonstrating on target stimulation of the TPO receptor in hematopoietic cells. Conclusions: Our results show that the novel TPO mimetic RWJ stimulates megakaryopoiesis without causing proliferation of the malignant myeloid clone in MDS and AML.

PDGF-BB Promotes CFU-GM and CFU-MK Formation and Its Possible Mechnism

To investigate the effect of platelet-derived growth factor (PDGF-BB) on the formation of granulocyte-monocyte colony forming unit (CFU-GM) and megakaryocyte colony forming unit (CFU-MK) and its anti-apoptotic effect on CHRF cells. The CFU-GM and CFU-MK of murine and human bone marrow cells were cultured in vitro by using plasma clot culture system. The anti-apoptotic effect of PDGF-BB on CHRF cells and its mechanism were clarified by flow cytometry. PDGF-BB 0-100 ng/ml stimulated the proliferation of murine and human CFU-GM and CFU-MK in a dose-dependent manner. The maximal stimulation effect was obtained at 50 ng/ml of PDGF-BB (P<0.01). Furthermore, PDGF-BB had an anti-apoptotic effect on CHRF cells as shown by the flow cytometry with AnnexinV/PI double staining, Caspase-3 expression and JC-1 detection (P<0.05). PDGF-BB significantly stimulates the proliferation of CFU-GM and CFU-MK in vitro, and has an anti-apoptotic effect on CHRF cells.

Notoginsenoside R1 Has Thrombopoietic Effect in Irradiated Mice

Objective: Chinese herbs have been reported to be effective and safe as a treatment for thrombocytopenia. Sanqi, Radix Notoginseng, is the dried roots of Panax notoginseng (Burk.) F. H. Chen (Araliaceae). It has been prescribed in several Chinese formulas including "Yunnan Bai Yao", which is used for treatment of trauma and bleeding due to internal and external injury. Its main constituents are ginsenosides (a kind of saponins), as well as notoginsenosides (only rich in Notoginseng species). Although Sanqi is a well-known haemostatic drug, its effects and mechanisms on megakaryocyte/platelet production have not been well studied. The objective of this study was to compare the effect of a purified notoginsenoside R1 (NR1) and thrombopoietin (TPO) on thrombopoiesis in irradiated mice.Methods: NR1 (2.5 mg) and TPO (0.25 ug) were dissolved in distilled water and given by intra-peritoneal injection daily for 14 days starting from the day after radiotherapy. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from NR1, TPO, and vehicle control groups on day 0, 7 and 14. On day 14, the mice were sacrificed and bone marrow cells were harvested for CFU-MK, CFU-GM, BFU-E and CFU-F (fibroblastoid) assays.Results: Our results showed that NR1 enhanced the recovery of platelets, WBC, and RBC count. Moreover, NR1 also promoted the CFU-F (12 + 0.8 vs 20 + 0.4 colonies/2 x 106 cells, p=0.003), CFU-MK (22 + 1.8 vs 26 + 3.6 colonies/2 x 105 cells, p=0.025), CFU-GM (26 + 5.0 vs 38 + 4.2 colonies/2 x 105 cells, p=0.002), and BFU-E (12 + 2.6 vs 18 + 1.8 colonies/2 x 105 cells, p=0.003) formation. Similar results were obtained in TPO-treated group. In in-vitro study, we further analyzed the effect of NR1 (0-50mM) on mouse CFU-MK formation using a plasma clot colony assay. The results showed that NR1 (20 mM) enhanced TPO (50 ng/ml)-induced CFU-MK formation (18 + 2.2 vs 30 + 6.0 colonies/2 x 105 cells, p=0.022, n=6). Furthermore, the effect of NR1 (5-50 mM) on the growth of bone marrow stromal cells was also investigated using CFU-F assay. NR1 (50mM) had a promoting effect on CFU-F growth (18 + 3.6 vs 24 + 1.8 colonies/2 x 106 cells, p=0.04, n=6). Our studies showed that NR1 enhances thrombopoiesis in vivo and the growth of bone marrow stromal cells as well as megakaryocytes in vitro. Therefore, we speculate that the thrombopoietic activity of NR1 may be mediated via promoting the progenitor of platelet, megakaryocytes, and bone marrow stromal cells. Consequently, we tested the anti-apoptotic effect of NR1 using megakaryocytic cell line M-07e. Data demonstrated that nutrient deprivation significantly increased cell death (n = 6, control vs. normal), and NR1 significantly reduced apoptosis and total cell death (n = 6, NR1 vs. control). TPO treatment also significantly reduced M-07e cell death (n = 6, TPO vs. control). Data also demonstrated that nutrient deprivation increased caspase-3 expression (n = 6, normal vs. control), and NR1 (NR1 vs. control) or TPO (TPO vs. control) treatment significantly reduced caspase-3 expression. M-07e cells were stained with JC-1 reagent. Control cultures had increased proportion of cells containing JC-1 monomers, the percentage of R2 populations are significant higher in the normal, NR1 and TPO treated groups comparing to those of the control group (n = 6). R1, a transitional cell subset containing both monomers and aggregates, was not significantly affected by these reagents. R2: a cell subset showing mitochondria membrane damage.Conclusions: Here we reported that the effect of NR1 is comparable with TPO on the production of platelets in irradiated mice. Therefore, the effect of NR1 on thrombopoiesis may be mediated via anti-apoptosis on megakaryocytic cells. DisclosuresYang:National Natural Science Foundation of China: Other: National Natural Science Foundation of China(81270580).

Micro-concentration Lipopolysaccharide as a Novel Stimulator of Megakaryocytopoiesis that Synergizes with IL-6 for Platelet Production.

Lipopolysaccharide (LPS) induces platelet activation and enhances platelet sensitivity to aggregation, which might alter platelet counts. We found that serial doses of micro-concentration LPS significantly increased the platelet count in mice treated with kanamycin, along with increased expression of IL-6 compared with IL-3 and TPO in megakaryocytes obtained from the mouse bone morrow following LPS administration. Furthermore, LPS at lower levels ranging plus IL-6 effectively stimulated CFU-MK formation and increased CD41 expression and megakaryocyte polyploidization. Meanwhile, there was a sustained rise in the percentage of reticulated platelets in the whole blood in response to low-dosage LPS combined with IL-6. In vivo experiments also demonstrated that the administration of LPS combined with IL-6 substantially enhanced the number of circulating platelets in normal and thrombocytopenic mice. Notably, the optimal LPS concentration in combination with IL-6 might be a novel stimulator of TLR4 and IL-6R expression in Dami cell lines, which initially occurs through TLR4-IL-6R crosstalk and then involves the activation of NF-κB and phosphorylation of p38 MAPK. These data suggest a new paradigm for the regulation of megakaryocytopoiesis and platelet production via a synergistic effect of LPS and IL-6, which has the potential to be used for the design of new therapies.

Melatonin Protects Against Apoptosis of Megakaryocytes Via Its Receptors and Activation of PI3k/Akt Pathway

Melatonin has been shown to be able to lower the frequency of thrombocytopenia in patients with cancer undergoing radiotherapy or chemotherepy in clinical studies (Lissoni et al, J Pineal Res, 1999). Melatonin is an acetylated product of serotonin. Our previous study demonstrated that serotonin stimulates megakaryopoiesis and thrombopoiesis (Yang et al, Stem Cells, 2007; 2014). However, the underlying mechanism of melatonin on platelet formation/survival has not been well investigated. We hypothesized that melatonin has therapeutic effects on thrombocytopenia by reducing apoptosis of megakaryocytes (MKs)/platelets via melatonin receptors and activation of Akt signaling. We investigated the in-vitro effect of melatonin on CFU-MK formation at different doses (0-500 nM). The results showed that melatonin significantly promoted CFU-MK formation at a dose dependant manner. The maximal concentration was at 200 nM (P<0.01, n=6). The size of CFU-MK with melatonin treatment was much larger and each MK cell was more mature looking. Using megakaryocytic cell CHRF-288-11, we identified the expression of melatonin receptors MT2 on these MK cells by RT-PCR and western blotting. We further found that melatonin exerted protective effect on serum-free induced apoptosis of CHRF cells. The cell viability was significantly increased with the treatment of melatonin. Flow cytometric dot-plot analysis demonstrated that the population of apoptotic cells (Annexin V/PI staining) was significantly decreased with melatonin treatment (200nmol/L) for 72 hrs (n=4). It also showed trends of amelioration of caspase-3 expression and the proportion of cells containing JC-1 monomers (indicating a trend in the drop of mitochondrial membrane potential). The population of phospho-Akt were significantly increased after treatment by melatonin and this effect was abolished by melatonin receptor MT2 blocker. The effect of melatonin on in-vivo thrombopoiesis was also investigated in an irradiated mouse model. Eighteen mice were randomly divided into three groups (6 each). Group 1 (normal control) received no irradiation or melatonin; Group 2 (model control) and Group 3 (melatonin treated) both received 4 Gy total body irradiation. After irradiation, melatonin (10mg/kg.d) was injected intraperitoneally into Group 3 consecutively for 21 days. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from the three groups on day 0, 7, 14, and 21.We found that melatonin enhanced the recovery of platelets and WBC production in Group 3. Our results showed that melatonin enhanced platelet formation and survival in radiation induced thrombocytopenia. The underlying mechanisms might involve in direct stimulation of megakaryopoiesis and anti-apoptotic effect on megakaryocytes via it receptors and activation of Akt signaling. DisclosuresNo relevant conflicts of interest to declare.

Imetelstat (GRN163L), a Telomerase Inhibitor Selectively Affects Malignant Megakaryopoiesis in Myeloproliferative Neoplasms (MPN)

Imetelstat is a telomerase inhibitor which has been shown to have therapeutic activity in patients with myelofibrosis (MF) (Tefferi A. et al., 2013 ASH Annual Meeting, Abstract 662) and essential thrombocythemia (ET) (Baerlocher G. et al., 2012 ASH Annual Meeting, Abstract 179). In clinical trials involving patients with a variety of other malignancies, the primary dose-limiting toxicity of GRN163L has been thrombocytopenia. We utilized GRN163L in order to explore the possible role of telomerase on human megakaryocytes (MK) and the mechanisms underlying the drug’s inhibitory effects on platelet production. MK were generated from normal primary CD34+ cells using an ex vivo culture system previously described by our laboratory (Iancu-Rubin C. et al, Blood 2011, 117:17; 4580-4589). We first showed that both telomerase activity and the expression of its catalytic unit hTERT were elevated in proliferating normal CD34+ cells, declined transiently during the initial stages of MK differentiation but were then partially restored during the final stages of terminal maturation. Treatment of normal CD34+ cells with GRN163L did not affect the numbers of CFU-MK assayed. Furthermore, exposure to GRN163L during the first 7 days in a liquid culture system did not interfere with the ability of CD34+ cells to commit to MK. When the same cultures were allowed to mature for 7 additional days, the proportion of CD34+/CD41+ MK precursors in drug-treated cultures was twice that observed in control and the drug-treated cultures contained 70% fewer mature CD41+/CD42b+MK than control cultures. The inhibitory effects of GRN163L on MK maturation were supported by observations showing that the cultures treated with the drug contained 60% fewer polyploid MK than control cultures. These results suggest that that GRN163L-mediated inhibition of telomerase does not affect normal MK progenitors but blocks the maturation of MK precursor cells.Previous studies have shown that GRN163L inhibits neoplastic CFU-MK colony formation by CD34+ cells from patients with ET (Brunold C. et al., 2011 ASH Annual Meeting, Abstract 3843). We, therefore, examined the effects of GRN163L on malignant MK from patients with MF and ET. Unlike normal CD34+ cells, treatment of MPN PB-MNCs with GRN163L decreased the numbers of assayable CFU-MK from 6 out of 11 patients. CFU-MK colony formation by PB-MNCs from these 6 patients was reduced by 33% (ranging from 13% to 50% reduction in CFU-MK formation) as compared to PB-MNCs treated with an inactive form of the drug (p value= 0.00473). Of note, in two out of 5 patients in which the total number of CFU-MK was not affected by GRN163L treatment, the drug decreased the size of the CFU-MK colonies formed. The ability of MPN PB-MNCs to differentiate into MK was next assessed. Although the total number of cells in PB-MNC liquid cultures exposed to GRN163L was decreased as compared to those treated with the inactive drug (n=6; p value=0.03204), the proportion of CD34+/CD41+MK precursors generated was not significantly affected. We then evaluated the effects of GRN163L on the genotype of MPN MK generated in the presence and absence of GRN163L by assessing the JAK2V617F allele burden. Treatment with GRN163L but not the inactive form of the drug reduced the JAK2V617F allele burden in MK derived from two patients: in one patient, JAK2V617F allele burden in MK generated in the presence of the inactive drug was 91.86% while MK generated in the presence of GRN163L was 19.75%; MK generated from a second patient had a JAK2V617F allele burden of 10.84% in the presence of the inactive drug which was reduced to 2.14% in the presence of GRN163L.We conclude that GRN163L-mediated inhibition of telomerase affects normal megakaryopoiesis by blocking the terminal maturation of CD34+/CD41+ MK precursors, providing a possible explanation for GRN163L’s propensity to induce thrombocytopenia in patients with normal bone marrow. By contrast, GRN163L treatment inhibited the ability of MPN CD34+ cells but not normal CD34+ cells to form CFU-MK colonies and drug treatment reduced the numbers of malignant MK generated. We propose that the amelioration of fibrosis observed in a clinical trial of MF patients treated with GRN163L might be due to, at least in part, two potential modes of action: 1) inhibiting malignant MK progenitors cells and 2) preventing terminal MK maturation thus depleting the pool of mature MKs which are the major source of fibrogenic cytokines in MF. DisclosuresIancu-Rubin:Geron Corporation: Research Funding. Parker:Geron Corporation: Employment. Eng:Geron Corporation: Employment. Hoffman:Geron Corporation: Consultancy.

The Effect of Tanshinone-IIa on IL-1β Induced-Thrombocytosis in an Immune Vasculitis Model

Inflammation cytokines may be involved in the pathogenesis of thrombocytosis with vasculitis. Our previous study showed that inflammation cytokine IL-1β plays an important role on in-vitro megakaryopoiesis (Yang M et al, Br J Haematol 2000). The role of IL-1β and Tanshinone IIA (TIIA) (Isolated from Danshen, Radix Salviae Miltiorrhiza Bge) on platelets and megakaryocytes (MKs) in immune vasculitis model was investigated in this study. Rabbit model with immune vasculitis was established by injection (iv) of BSA. After treatment with BSA for 7 days, the platelet count, platelet aggregation and the expression of AnnexinⅤ were significantly increased in this vasculitis model group compared with normal control group (n=7). IL-1β levels was also significantly higher in vasculitis model. There were positive correlations between platelet count and IL-1β levels (R=0.65), platelet aggregation and IL-1β levels (R=0.60). Treatment with TIIA (5 mg/kg/day, iv) and aspirin significantly decreased all these parameters. MKs and CFU-MK number were also significantly increased in vasculitis group as compared to normal group. Treatment with TIIA and aspirin significantly reduced the number of MKs and CFU-MK in this model. Study further demonstrated that IL-1β alone or in combination with TPO induced in-vitro CFU-MK formation. The mRNA of of IL-1 type I and type II receptors (IL-1 RI and RII) were detected in cultured MK (CD61+ CD41+) cells. The expression of IL-1 RI and RII was also confirmed by immunofluorescence staining in bone marrow MKs. Moreover, the IL-1R bloker can reduced IL-1β induced megakaryopoiesis. TIIA on in-vitro megakaryopoiesis was also investigated. TIIA at 10-30 ug/ml significantly inhibited CFU-MK formation. TIIA also induced the apoptosis of MKs in a dose dependent manner by Anexin V assay. Caspase 3 assay showed the activation of Caspase 3 increased from 5% to 16%. Using JC-1 assay found that the depolarized cells increased from 9% to 17% suggesting the involvement of intrinsic apoptotic pathway. IL-1β may play an important role in the thrombocytosis of immune vasculitis by inducing megakaryopoiesis. TIIA has anti-platelet effect in this model which may be mediated via inhibiting IL-1β induced-megakaryopoiesis. DisclosuresNo relevant conflicts of interest to declare.

The Expression of Functional Dopamine and Serotonin Receptors on Megakaryocytes

Monoamine dopamine (DA) and serotonin (5-HT) can affect the functions of many cells including platelets. Our previous studies have shown that 5-HT has potent effects on promoting megakaryopoiesis and platelet formation (Yang et al, Stem Cells, 2007; 2014). In this study, our hypothesis is that both DA and 5-HT receptors may have the development expression on platelets, megakaryocytes (MK) and hematopoietic stem cells (HSCs). The expression of DA receptors: DA1, DA2 and 5-HT receptors: 5-HT2A, 2B and 2C on these hematopoietic cells, and the effect of DA and 5-HT on the cell proliferation and apoptosis were investigated using RT-PCR, flow cytometry, immunofluorescence staining, annexin V, JC-1 and caspase-3 assay. Our results showed that dopamine DA1, DA2 receptors, 5-HT2A, 2B and 2C receptors were identified on human platelets, MK and HSCs using RT-PCR method. Using flow cytometry with monoclonal antibodies, these receptors were also identified on human platelets and MK. But these receptors expressed on human HSCs at a low levels. The expression of the five receptors were further confirmed on human bone marrow MK and HSC by using immunofluorescence staining. The functions of receptors were further studied in human MK and HSCs. DA at a low dose induced cell differention, but at a high dose induced cell apoptosis. This effect could be reduced by DA2 receptor blocker L-Sulpiride (L-SULP). In the other hand, 5-HT had an anti-apoptotic effect in MK cells. 5-HT also stimulated CFU-MK formation, this effect was reduced by 5-HT2R antagonist ketanserin. 5-HT also enhanced Akt phosphorylation, while addition of ketanserin compromised the 5-HT effect. 5-HT-driven MK effect was likely mediated via 5-HT2R and p-Akt. A higher number of proplatelet bearing MK was also detected in 5-HT-treated. Polymerization F-actin level was higher in 5-HT group. Adding ketanserin/PD98059, the intensity was reduced. Treated with 5-HT for 30 minutes ERK1/2 was activated. The adding of ketanserin/PD98059 to 5-HT cultures nullified the 5-HT effect. This effect was likely mediated via 5HT2R, p-ERK1/2, which leads to F-actin reorganization and proplatelet formation. This study demonstrated that the expression of DA and 5-HT functional receptors on human platelets, MKs and HSCs. DA and 5-HT may be involved in the regulation of thrombopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Activation of p53 by the MDM2 inhibitor RG7112 impairs thrombopoiesis

The tumor suppressor p53 is thought to play a role in megakaryocyte (MK) development. To assess the influence of the p53 regulatory pathway further, we studied the effect of RG7112, a small molecule MDM2 antagonist that activates p53 by preventing its interaction with MDM2, on normal megakaryocytopoiesis and platelet production. This drug has been previously been evaluated in clinical trials of cancer patients where thrombocytopenia was one of the major dose-limiting toxicities. In this study, we demonstrated that administration of RG7112 in vivo in rats and monkeys results in thrombocytopenia. In addition, we identified two distinct mechanisms by which RG7112-mediated activation of p53 affected human megakaryocytopoiesis and platelet production in vitro. RG7112 promoted apoptosis of MK progenitor cells, resulting in a reduction of their numbers and RG7112 affected mature MK by blocking DNA synthesis during endomitosis and impairing platelet production. Together, the disruption of these events provides an explanation for RG7112-induced thrombocytopenia and insight into the role of the p53-MDM2 auto-regulatory loop in normal megakaryocytopoiesis.

Astragalus Polysaccharide has hematopoietic and thrombopoietic activities in an irradiation mouse model

A herbal decoction (Danggui Buxue Tong, DBT) is a traditional Chinese medicine formula comprising Danggui (Radix Angelicae) and Huang Qi (Radix Astragali), which has been used for promotion of “blood production” for centuries and have shown favorable effects on thrombocytopenia. Previously, we have showed that it has significant effects on promoting hematopoiesis and thrombopoiesis (Yang M et al. J Ethnopharmacol, 2009). Many chemical components have been identified in DBT. Among them is the polysaccharides fraction of Angelica sinensis and Radix Astragali. Our study has demonstrated that Angelica Polysaccharide (APS) promotes hematopoiesis and platelet recovery in a mouse model (Liu et al, BMC Complement Altern Med, 2010). In this present study, we further assess the effect of Astragalus Polysaccharide (ASPS) on hematopoiesis and thrombopoiesis in-vivo and in vitro. A myelosuppression mouse model (4-Gy-irradiated) was treated with ASPS (10 mg/kg/day) and TPO (1 mg/kg/day). Peripheral blood cells from ASPS, TPO and vehicle-treated samples were counted on days 0, 7, 14 and 21. Using the colony-forming unit (CFU) assays, we determine the effects of ASPS on the hematopoietic stem/progenitor cells and megakaryocytic lineages. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were also conducted in HL-60 cells and megakaryocytic cell line M-07e. The effects of ASPS on cells treated with Ly294002, a PI3K inhibitor and the effect of ASPS on the phosphorylation of AKT were further studied. Results showed that ASPS, like TPO, significantly enhanced the recovery of WBC and platelet count, and bone marrow CFU-GM and CFU-MK formation (n=8) in this model. Morphological examination of bone marrows also showed that ASPS treatment significantly increased the recovery of hematopoietic stem/progenitor cells and megakaryocytic series. We further analyzed the in vitro effect of ASPS on CFU-MK and CFU-GM formation. ASPS (50-100 ug/ml) enhanced TPO (50 ng/ml) -induced CFU-MK (p=0.05, n=4), and CFU-GM formation (p=0.04, n=4). However, ASPS alone (1-500 ug/ml) did not show significant effect on CFU-MK and CFU-GM proliferation (n=4). Moreover, the anti-apoptotic effect of ASPS in HL-60 and M-07e cells were also demonstrated by using Annexin-V, Caspase-3, and JC-1 assays. Addition of Ly294002 alone increased the percentage of cells undergoing apoptosis. However, additional of ASPS to Ly294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of ASPS significantly increased the phosphorylation of AKT. Here, we showed that ASPS, the polysaccharide from the root of Radix Astragali, has hematopoietic and thrombopoietic activities in a mouse model. This effect is likely to result from the stimulation of cell proliferation through the activation of PI3K/AKT pathway, the activation of which prevents cells from undergoing apoptosis. Disclosures:No relevant conflicts of interest to declare.

IL-1β Plays An Important Role On Thrombocytosis In An Immune Vasculitis Model Via Promoting Megakaryopoiesis

Thrombocytosis and inflammation cytokines may be involved in the pathogenesis of vasculitis. Our previous study have showed that major inflammation cytokine IL-1β play an important role on in-vitro megakaryopoiesis (Yang M et al, Br J Haematol 2000). In this study, we investigated the changes of IL-1β and megakaryopoiesis and the effect of aspirin in an immune vasculitis model. Rabbit immune vasculitis model was established by intravenous injection of bovine serum albumin. In this model, platelet number and function of periphery blood, megakaryocyte number and the CFU-MK formation of the bone marrow, and serum levels of inflammatory cytokines were investigated. After treatment with BSA for 7 days, the platelet count, platelet aggregation and the expression of AnnexinⅤ were significantly increased in this vasculitis model group compared with normal control group (n=6). The serum levels of inflammatory cytokine IL-1β was also significantly higher in vasculitis model. There were positive correlations between platelet count and IL-1β levels (R=0.55), platelet aggregation and IL-1β levels (R=0.603). Treatment with aspirin (100 mg/kg/d) significantly decreased all these parameters, showing aspirin had anti-platelets and anti-inflammation effects. Our results also demonstrated that megakaryocyte number and the formation of CFU-MK were significantly increased in vasculitis group as compared to those in normal group. Treatment with aspirin significantly reduced the number of megakaryocytes and the formations of CFU-MK in bone marrow in this immune vasculitis model. Our study further demonstrated that IL-1β alone or in combination with TPO induced in-vitro CFU-MK formation. Using RT-PCR techniques, the mRNA of of IL-1 type I and type II receptors (IL-1 RI and RII) were detected in cultured CD61+ CD41+ cells and four megakaryocytic cell lines. The expression of IL-1 RI and RII was also confirmed by flow cytometry and immunofluorescence staining in bone marrow megakaryocytes. Moreover, the IL-1R bloker can reduced IL-1β induced megakaryopoiesis. This sudy showed that IL-1β may play an important role in the pathogenesis of immune vasculitis. Aspirin has anti-inflammation effects in this model which may be mediated via inhibiting megakaryopoiesis and platelet formation. Disclosures:No relevant conflicts of interest to declare.

Tanshinone IIA has anti-platelet effect and induces apoptosis in megakaryocytes via TNFR and Caspases

Tanshinone IIA (TIIA) isolated from Danshen (Radix Salviae Miltiorrhiza Bge) is a derivative of phenanthrenequinone, which has been used for centuries to treat hypercoagulation related diseases. Our previous study showed that TIIA has neural protective effect (Xia et al, Pediatric Research, 2005). In this study, we investigated the effect of TIIA on platelets and megakaryocytes in a rabbit model, and its molecular mechanisms. Rabbit immune vasculitis model was established by intravenous injection of bovine serum albumin with TIIA treatment (5 mg/kg/day, iv). The platelet count, platelet aggregation, and inflammatory cytokine IL-1β level were significantly increased in vasculitis model compared with normal group at day 7. There were positive correlations between platelet count and IL-1β level. The number of megakaryocytes and CFU-MK formation in bone marrow were significantly increased in this model. Treatment with TIIA significantly decreased all these parameters, and also significantly reduced the injury vessel in immune vasculitis. Furthermore, the effect of IIA on in-vitro megakaryopoiesis was investigated. TIIA at 10-30 ug/ml significantly inhibited CFU-MK formation (p<0.05, n=6). TIIA also induced the apoptosis of megakaryocytic cell line CHRF. The cell viability was reduced to 81% after 72hrs of TIIA treatment. Using Annexin V/PI staining, TIIA induced apoptosis on CHRF cells in a dose dependent manner. This was verified by Caspase 3 assay which showed the activation of Caspase 3 increased from 5% to 16% after TIIA treatment. Using JC-1 assay, we found that the depolarized cells increased from 9% to 17% after TIIA treatment suggesting the involvement of intrinsic apoptotic pathway. To further determine the molecular mechanisms involved in the pro-apoptosis effect, Microarray studies using Affymetrix 133 plus genechips were conducted to identify the genes that were differentially expressed after TIIA treatment. Several groups of genes involved in apoptosis, calcium regulation and cell cycle checkpoints were found to be differentially expressed after the treatment. The differential expressions of these genes were validated using quantitative PCR. The most significantly upregulated gene (6.0±0.333 folds) was TNF Receptor Super-Family 9 (TNFRSF9). In addition, Receptor Interacting Protein Kinase (RIPK), a protein that likely interacts with TNFRSF9, was up-regulated to 2.0±0.167 folds. These results suggest that TIIA has an anti-platelet effect in this model. Its mechanism may be related to its inhibiting megakaryopoiesis and inducing apoptosis of megakaryocytes via TNFR and caspases. Disclosures:No relevant conflicts of interest to declare.

Angelica Polysaccharide and TPO Have a Protective Effect On Hcmv-Induced Apoptosis In Megakaryocytes

Human cytomegalovirus (hCMV) infection is often associated with thrombocytopenia. Megakaryocytes may be one of the major sites of hCMV infection, then inducing this cell apoptosis. Angelica Sinensis (Danggui) is an important ingredient of many commonly used herbal Medicine for promoting blood production. Our previous study has showed that the hematopoietic effect of Angelica Sinensis is related to its constituent, angelica polysaccharide (APS) (Yang M et al, J Ethnopharma, 2009). This present study investigated the anti-apoptotic effect of APS and TPO on hCMV-induced apoptosis in megakaryocytes. Human bone marrow mononuclear cells (MNC) or megakaryocytic cell line CHRF-288-11 and hCMV AD169 strain were co-cultured in this study. hCMV significantly inhibited the formation of CFU-MK as shown in three different concentrations of viral infection groups (103, 104 and 105 pfu/ml), compared with blank control and mock control (n=10, P<0.05). hCMV also significantly inhibited the growth of CHRF cells in these three different concentrations after incubation for 3 days, which compared with control group (n=10, P<0.01). hCMV DNA and mRNA were also positively detected in CHRF cells and the cells of CFU-MK with IS-PCR and RT-PCR respectively, while it was negative in blank and mock control groups. We further studied the effect of APS and TPO on CFU-MK formation. Results showed that APS (50 ug/ml) like TPO (50 ng/ml) enhanced hCMV-reduced CFU-MK (P=0.05, n=6). CHRF cells were also analyzed by Annexin V/PI with flow cytometry at day 3 after infection with hCMV AD169. The percentage of apoptotic cells in group of 103 pfu/ml was 19.0 ± 2.0%; The group of 104 pfu/ml was 23.0 ± 1.5%; The group of 105 pfu/ml was 28.0 ± 3.0%. The control group was 2.0 ± 0.5%. The apoptotic cells were confirmed by morphologic observation. In addition, apoptotic signals from megakaryocytic surface, cytoplasma and mitochondria were detected in hCMV infected cells by flow cytometry with Caspase-3 and JC-1 assay. Compared to mock infection control at day 5, Annexin-V positive cells population increased by 58%; active caspase-3 signal increased by 120% in viable cell population; and cell population with damaged mitochondial membrane showed a 5-times increase. Moreover, the anti-apoptotic effect of APS and TPO on CHRF cells was also demonstrated by using Annexin-V assay. Our studies showed that hCMV induces the apoptosis in megakaryocytes via mitochondrial and caspase-3 signaling, and angelica polysaccharide (APS) like TPO has a protective effect on hCMV-induced apoptosis in these cells. Disclosures:No relevant conflicts of interest to declare.