Derangements of Toll-like Receptors, Inflammatory Cytokines, and Reactive Oxygen Species in Philadelphia Chromosome-Negative Myeloproliferative Neoplasm: Implicate Roles of Inflammation in the Pathogenesis

Abstract Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) including essential thrombocythemia(ET), polycythemia vera(PV), and myelofibrosis (MF) have been regarded as a stem cell diseases with malignant clonal proliferation; but recently, inflammatory processes have been proposed as playing found a major role in the pathogenesis of MPN. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that function as key initiators of innate immunity signaling, then induce inflammatorycytokines, and ROS formation. Therefore we measured TLRs, inflammatory cytokines, and ROS in patients with MPN to assess the role of inflammation in MPN. Methods: TLR assay.TLR-2, 4, 7, 9 quantification was performed by immuno-staining of 1×106 mononuclear cells which were incubated with fluorescence-conjugated anti-TLR-2, 4, 7, 9 antibodies and assayed by flow cytometry. Monocytes culture and dendritic cells differentiation.Human monocytes were isolated from human peripheral blood mononuclear cells (PBMNC) using Pan Monocyte Isolation Kit. Isolated monocytes were incubated with IL-4 and GM-CSF in cultures to differentiate to dendric cells . Immature dendritic cells were either left untreated or stimulated with Pam3CSK4. The maturation of dendritic cells was determined by flow cytometry using CD80, CD83, CD11c, and HLA-DR. Multiplex ELISA. Human plasma and cell culture supernatants were analyzed in duplicates by Meso Scale Discovery Multi-Spot Assay system. In total, ten cytokines were assayed: IFN-g, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-a. Cell ROS measurement. Cellular ROS was determined by a dichlorofluorescein (DCF) assay. PBMNCs were incubated with 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) (Life Technology) for 30 min at 37°C. The oxidation of H2DCFDA was measured and analyzed by flowcytometery. Results. 1)TLR2 was the only TLR found to be elevated in PV or ET patients (Fig 1). 2) PlasmaIL-1b levels were elevated in TLR2 high patients than TLR-2 low patients. 3) No difference between TLR-2 high and low patients in the maturation of monocytes to dendritic cells. 4) Monocyte derived dendritic cells with high TLR-2 patientsreleased more IL-8 and TNF-a after Pam3CSK4 stimulation (Fig 2). 5) ROS were more elevated in MF patients than PV, ET patients, and controls (Fig 3). Conclusion. 1) TLR 2 is significantly elevated in PV and some ET patients and TLR-2 high patients were found to have elevated plasma IL-1b,and IL-8, and TNF-α in monocytes-derived dendric cell cultures afterPam3CSK4 stimulation than TLR-2 low patients. This confirms that TLR-2 is deranged in PV and ET. 2) ROS is elevated in MF patients compared to ET and PV patients, or controls. Thus, this study suggests that inflammatory processes likely play a role in the pathogenesis of Ph- MPN through first TLR-2 derangement in PV and ET , then through years of chronic inflammatory process , with the accumulation of more ROS seen in MF, which caused more damage to the DNA resulting more malignant clonal proliferation. A similar phenomenon was observed that in JAK2V617F mutation , allele-burden was observed gradually increased from ET , PV then to MF . Disclosures No relevant conflicts of interest to declare.

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TLR, RAGE, and HMGB1 in the Inflammatory Response in Ph(-) Myeloproliferative Neoplasm

Blood ◽

10.1182/blood-2020-134988 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-32

Author(s):

Guanfang Shi ◽

Kiron Nair ◽

Preethi Ramachandran ◽

Chi Chen ◽

Ching Wong ◽

...

Keyword(s):

Inflammatory Response ◽

Real Time ◽

Inflammatory Cytokines ◽

Internal Control ◽

Real Time Pcr ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Philadelphia Chromosome ◽

Myeloproliferative Neoplasm ◽

Significant Difference

Recent evidence of increased constitutional symptoms and inflammatory cytokines in Philadelphia chromosome negative (Ph (-)) MPN suggests that an inflammatory response is important in the pathogenesis of Ph (-) MPN. Toll-like receptors (TLR), Receptor for Advanced Glycation End products (RAGE) and High mobility group protein B1 (HMGB1) are the important pathways for the inflammatory response. All these three important pathway proteins were studied in MPN diseases in the current studies. Materials and Methods: TLR assay. TLR 2,3, 4, 7, 9 quantification was performed by immuno-staining of 1×106 mononuclear cells (peripheral blood) which were incubated with fluorescence-conjugated anti-TLR-2,3, 4, 7, 9 antibodies and assayed by flow cytometry. HMGB1assay:HMGB1 ELISA kit from Immuno-Biological Laboratories, Inc. (IBL-America) were used. The plasma samples were diluted four times with the provided sample dilution buffer, and assayed in duplicate according to the manufacturer's suggestion. RAGE (RT-PCR) Assay: Total RNA was extracted from normal control or patient mononuclear cells. Predesigned primers for RAGE, and internal control genes were ordered from Qiagen (Germantown, MD). Real-time PCR was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. At least three house-keeping genes (ribosomal protein L4, TATA box binding protein, and tubulin-α 1b) were used as normalization controls. The expression of RAGE were compared with each internal control. Average of three was used to calculate the ratio of final patient to normal Results: Total of 97 patients with MPN were studied 1) TLR: TLR 3,7,9 was not significantly different from controls. But TLR 2 was significantly increased in both PV, as well as in the MPN group when PV, ET and MF were grouped together as MPN (Fig A). TLR 4 was not significantly increased in PV, ET, MF individually but was found to be significantly increased than the controls, when they are grouped together as MPN (Fig B). 2) RAGE: No significant difference was found between ET, PV, MF individually or when they were grouped together as MPN than the controls (Fig C). 3) HMGB1: No significant difference was seen between ET, PV, MF or when they were grouped as MPN (Fig D). Conclusion: Current study suggests that TLR pathway especially TLR2, and to a lesser extent TLR4 are the important pathways for inflammatory response with increased inflammatory cytokines in MPN, while HMGB1 and RAGE pathways were not different from controls. Figure Disclosures No relevant conflicts of interest to declare.

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TLR-2 Signal Pathway Is Enhanced in Myeloproliferative Neoplasm - Related to the Increased Infammatory Cytokine and Pathogenesis?

Blood ◽

10.1182/blood.v126.23.5202.5202 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5202-5202 ◽

Cited By ~ 1

Author(s):

Guanfang Shi ◽

Maryna Yarotska ◽

Hui Chen ◽

Abdelmalek Cherif ◽

Ching Wong ◽

...

Keyword(s):

Dendritic Cells ◽

Inflammatory Cytokines ◽

Inflammatory Cytokine ◽

Magnetic Beads ◽

Mononuclear Cells ◽

Cytokine Production ◽

Myeloproliferative Neoplasms ◽

Clonal Evolution ◽

Myeloproliferative Neoplasm ◽

Stimulation Of

Abstract Introduction Myeloproliferative neoplasms (MPNs) are associated with constitutional symptoms and high levels of circulating inflammatory cytokines. The etiology of this increase in inflammatory cytokines could be secondary to Jak2 mutation resulting in enhanced transduction signaling of cytokines. Toll-like receptors (TLRs) represent another activating mechanism by which their expression on antigen presenting cells, such as dendritic cells (DCs) and macrophages, serve as key pattern recognition receptors (PRR) which play a central role in induction of innate and adaptive immune responses resulting in the signaling of inflammatory cytokines. TNFα is one of the inflammatory cytokine which is significantly raised in MPN patients. Recent evidence suggests that TNFα may facilitate clonal expansion of JAK2 V617F mutation positive cells which leads to hypothesis suggesting that chronic inflammation is involved in the pathogenesis and clonal evolution in Philadelphia-negative chronic MPNs. Therefore, we investigated the role of TLRs and related cytokines in MPNs and we found that only TLR-2 was significantly elevated in MPNs. We further used the TLR-2 ligand, Pam3CSK4, to study its effects on the production inflammatory cytokines. Materials & Methods TLR assay: TLR-2, 4, 7, 9 quantification was performed by TLR staining of 5×105 mononuclear cells which were incubated with 5 μg/mlof conjugated TLR-2, 4, 7, 9 antibody and assayed by flow cytometry. Monocyte-derived dendritic cells (mdDC) and Inflammatory Cytokines. mdDC were generated from isolated monocytes (anti-CD14-coated magnetic beads) then by incubation with IL-4 and GM-CSF for 4 days. Immature mdDC (1×104/well) were either left untreated or stimulated with Pam3CSK4, in 96 well plates. After 48h, cells were incubated with 20% human AB serum in PBS containing 0.1% BSA and 0.1% NaN3 for 20 min at 4¡C. Thereafter, cells were stained with fluorescence-labeled mAb specific to CD80, CD83, (BD Biosciences). Cytokine levels were determined in supernatants harvested after 48h using the MSD (Meso Scale Discovery) System. Results. Levels ofTLR-2 were significantly elevated in patients with MPNs especially in those with polycythemia vera and essential thrombocythemia, but only marginally elevated in myelofibrosis (MF) patients, which likely may be attributable to the relatively lack in number of MF patients (Fig 1). In all patient subsets, TLR 4, 7, 9 levels were not significantly different from controls as shown in Fig 1. Fig 2 shows maturation of mdDC by TLR-2 ligand, Pam3CSK4. Higher numbers of CD83+ cells were generated more from patients with elevated TLR levels, confirming these MPN patients has elevated TLR2 levels to be able to generated more dendric cells by TLR-2 ligand. Fig 3 a and b shows in elevated TLR-2 level MPN patients, Pam3CSK4 stimulated more cytokins of IFNγ, IL12p70, TNFα, IL6, IL8, IL10, IL13. Fig 4 showed in elevated TLR-2 levels MPN patients have more of IL8, TNFα, IL13, IL6, in the plasma, consistent with inflammatory cytokines likely from elevated elevated TLR-2.Therefore TLR-2 likely plays a very important role in the inflammatory cytokine production in MPN diseases. Conclusion. TLR-2 and not TLR-4, 7, 9 were found to be significantly elevated in MPNs. Stimulation of the TLR-2 ligand promoted the generation of more mature DCs in TLR-2 elevated MPN patients, demonstrating TLR-2 were elevated in these patients. Further, the stimulation of mdDC cultures with TLR2 ligand led to greater cytokine production and the level of plasma cytokines correlated with TLR-2 levels. We conclude that TLR-2 is important in the production of inflammatory cytokines in MPNs and it may play a significant role in the pathogenesis of MPNs. Disclosures Wang: Celgene: Research Funding.

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Clinical Significance Of Circulating Microparticles In Ph- Myeloproliferative Neoplasms (MPN)

Blood ◽

10.1182/blood.v122.21.2368.2368 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2368-2368 ◽

Cited By ~ 2

Author(s):

Yue Han ◽

Shixiang Zhao ◽

Wenjuan Zhang ◽

Jiannong Cen ◽

Wei Zhang ◽

...

Keyword(s):

Plasma Levels ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Jak2v617f Mutation ◽

Polycythaemia Vera ◽

Healthy Donors ◽

Significant Difference ◽

Thrombotic Complications

Abstract Background Microparticles (MPs) are small membrane vesicles that are classified as red blood cell MPs (RMPs), platelet-derived MPs (PMPs), tissue factor MPs (TF+MPs) and endothelial MPs (EMPs) based on their origins. Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPN) are disorders characterized by abnormal hematopoiesis, thrombosis, JAK2V617F mutation. Although MPs are considered as biomarkers reﬂecting procoagulant state in cancer patients, their involvement in the patients with Ph-MPN remains unclear. Our objective in this study was to measure the alterations of the four MPs types in the patients with MPN and to evaluate their correlations with JAK2V617F mutation and some clinical complications, especially for thrombosis and splenomegaly. Methods Sixty-seven patients with MPN were enrolled in this study, including 12 polycythaemia vera (PV), 49 essential thrombocythemia (ET) and 6 primary myelofibrosis (PMF). 30 healthy donors were selected as normal controls. Venous blood was anticoagulated with sodium citrate (1:9). Using flow cytometry, plasma samples were measured for RMPs, PMPs, TF+MPs and EMPs with phycoerythrin (PE)-conjugated monoclonal antibodies CD235a, CD61, CD142, and CD62E, respectively. Forward scatter was set in scale using fluorescent microspheres of 0.8μm and standard fluorescent microbeads (0-0.8μm) in diameter were used to set the microparticle gate. Data were expressed as median (M) and interquartile range (IQR). Meanwhile, genomic DNA was extracted from mononuclear cells and amplified by allele speciﬁc polymerase chain reaction (PCR). Results (1) Patients with MPN showed significantly higher plasma levels for all four MPs compared with healthy donors (P<0.05), namely 49.0/μl (15.8-109.5/μl) vs 21.0/μl (13.8-32.6/μl) for RMPs, 181.2/μl(75.8-1111.6/μl) vs 74.9/μl (55.5-115.4/μl) for PMPs, 48.1/μl (13.1-72.4/μl) vs 31.0/μl (14.9-47.6/μl) for TF+MPs and 310.2/μl (128.6-1130.5/μl) vs 155.9/μl (100.3-227.6/μl) for EMPs. (2) Among different subtypes of MPN, PMPs were higher in patients with PMF than patients with PV and ET (P<0.05), but there was no significant difference between PV and ET group. No obvious difference was found in RMPs, TF+MPs and EMPs among different subtypes of MPN patients. (3) MPN patients with JAK2V617F mutation (n=34) were found to have higher plasma levels of TF+MPs and RMPs than those without mutation (P<0.05) and this difference was not found for PMPs and EMPs. (4) MPN patients with various thrombotic complications (n=10) showed higher levels of all four types of MPs than those without thrombotic complications (n=31) (P<0.05). Elevated MP levels were also found in patients with splenomegaly (n=19) compared to those without splenomegaly (n=14) (P<0.05). Conclusion Higher levels of MPs were observed in MPN patients compared with healthy controls, especially in patients complicated with thrombosis and splenomegaly, which reflects a prothrombotic state. Moreover, significantly increased TF+MPs and RMPs were found in MPN patients with JAK2V617F mutatioin. Disclosures: No relevant conflicts of interest to declare.

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Revisiting the IGF-1R Signaling Pathway in Myeloproliferative Neoplasms: Quantification of IGF-1 Receptor May be Useful in the Differential Diagnosis of Polycythemia

Blood ◽

10.1182/blood.v124.21.3226.3226 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3226-3226 ◽

Cited By ~ 1

Author(s):

Guanfang Shi ◽

Stacey Baptiste ◽

Maryna Yarotska ◽

Hemant Sindhu ◽

Ching Wong ◽

...

Keyword(s):

Flow Cytometry ◽

Differential Diagnosis ◽

Colony Formation ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Signal Pathway ◽

Interferon Α ◽

Cd34 Cells ◽

Igfbp 3

Abstract Endogenous erythroid colony formation in vitro is one of the important criteria for the diagnosis of polycythemia vera (PV). Extensive studies performed in the last decade suggest that IGF-1 plays a significant role in the erythropoiesis and endogenous colony formation in PV. Therefore, IGF-1R signal pathway is important in the pathogenesis of PV. We elected to revisit IGF-1R pathway by flow cytometry technique in PV and other myeolproliferative neoplasms (MPNs). 32 MPN patients (including 11 PV, 19 myelofibrosis (MF), 12 essential thrombocytopenia (ET), 8 secondary PV , and 17 age-matched normal controls were studied. Methods: 1) plasma IGF-1, IGFBP-1, IGFBP-3 were assayed by ELISA(R&D Systems, Minneapolis, MN) ; 2) quantification of IGF-1R . Peripheral blood mononuclear cells (MNC) harvested from Ficoll-Paque separation were stained with phycoerythrin conjugated antibody against IGF-1 receptor (R&D Systems), and assayed by flow cytometry using FACS Calibur system (BD Biosciences, San Jose, CA; 3) quantification of IGF-1R phosphoration (pIGF-1R) after IGF-1 stimulation. MNC cells were suspended in serum-free α-MEM medium for 2 hours, then starved cells were treated with or without 10 nM IGF-1 for 3 min and assayed for pIGF-1R by flow cytometry using Alexa Fluor 647 conjugated antibody against phosphorylated IGF-1 receptor (BD Biosciences); 4) Interferon alpha (IFNα) Treatment. IFN-α (Schering Corporation, Kenilworth, NJ) was added to the cell culture at 100 unit per ml. Cells were incubated with IFN-α for either 2 hours or 3 days and then collected for further pIGF-1R analysis. The results showed 1) No difference in plasma IGF-1 levels in MPN and controls; IGFBP-1 and IGFBP-3 were significantly elevated in MPN than controls but the change of IGFBP-3 in MF patients was not significant. 2) the PV group had significantly elevated IGF-1R expression than secondary PV (Fig 1). Mean ± SE (MFI expression) were 529 ± 84 and 90 ± 30 in PV versus secondary PV, respectively (p=0.004). 3) The untreated PV group had significantly higher IGF-1R than MF, ET, and controls. MF and ET were not different from controls, but 8/19 (43%) in MF and 6/12 (50%) in ET have elevated IGF-1R than controls. 4) No difference in IGF-1R expression in Jak2 (+) versus Jak2 (-) in MPN patients. Mean ± SE (MFI) were 293.2 ± 62.1 and 164.8 ± 47.7 in JAK2(+) versus JAK2 (-) respectively (p=NS). 5) Preliminary studies showed PV has a significant elevated pIGF-1R than controls in unstimulated PB CD34+ cells, but not in MNC cells. 6) IFN-α decreased IGF-1R expression and pIGF-IR about 10% in the PV group. Conclusions: 1) IGF1R signal pathway is more active in PV than MF, ET and controls. 2) Quantification of IGF-1R expression by flow cytometry may be helpful in differential diagnosis in primary versus secondary PV. 3) IGF-1R expression is not correlated with JAK2 status. 4) IGF-1R phosphorylation is more elevated in unstimulated CD34+ cells of PV patients than in controls suggesting autonomous erythroid colony formation in PV may be associated with IGF-1R activation. 5) interferon-α has some inhibitory effects in IGF-1R expression and phosphorylation but is unlikely to be the main mechanism of its induced remission of JAK2 –mutant allele-burden effects. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Superior Cytotoxicity of Clonal Versus Polyclonal Gamma Delta T Cells against Philadelphia Chromosome Positive and B-CLL Derived Leukemic Cells.

Blood ◽

10.1182/blood.v114.22.3032.3032 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3032-3032

Author(s):

Helena Dhamko ◽

Gabrielle Melanie Siegers ◽

Julia Schüler ◽

Armand Keating

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Conflicts Of Interest ◽

Philadelphia Chromosome ◽

Leukemic Cells ◽

Small Subset ◽

Target Cells ◽

Gamma Delta T Cells ◽

Gamma Delta ◽

Phenotypic Stability

Abstract Abstract 3032 Poster Board II-1008 Gamma delta T cells (GDTCs), a small subset of T-lymphocytes (<10%) involved in tumor immune surveillance, are promising candidates for adoptive immunotherapy demonstrated by their ability to elicit cytolytic responses against many tumors. We have isolated and expanded GDTCs as a first step in developing a clinical protocol (Siegers, GM et al., ASH 2008). GDTCs exist in subsets whose specificity and function are determined by receptor rearrangement and tissue localization. The Vdelta2 (Vd2) subset in blood recognizes small phosphate containing non-peptide antigens and has been shown to kill myeloma and Burkitt lymphoma cells, whereas Vdelta1 (Vd1) GDTCs are typically found in tissue mucosae and provide defense against epithelial cancers. Although circulating GDTCs are predominantly of the Vdelta2 (Vd2) subset, we found that in 59% of GDTC cultures derived from the peripheral blood of healthy donors (n=17), the Vdelta1 (Vd1) subset was preferentially expanded, comprising 70.5% ± 14.7% (mean ± standard deviation) as determined by flow cytometry. In the remaining cultures, Vd2 GDTCs comprised 75.9 ± 14.2%. Preferential expansion of Vd1 did not correlate with a higher percentage of this subset in donor blood prior to GDTC isolation. In one expanded culture, Vd1 and Vd2 were equally present (40.3% and 41.3% respectively, on day 17). To determine activation status of Vd1 and Vd2 subsets simultaneously when co-incubated for 3 hours at a 1:5 effector:target ratio (E:T) with EM2eGFPluc, Ph(+) leukemic target cells, exposure of the degranulation-induced marker CD107 was determined by flow cytometry. Assays performed on culture days 10 to 17 (n=8) revealed that only 3.4 ± 2.7% Vd1 cells were activated, whereas Vd2 cells exhibited ten-fold activation with 34.1 ± 4.7% expressing CD107. To further investigate the different cytotoxic potential of these GDTC subsets, we generated 3 Vd2 clones from Donor 1 and 7 clones (3 Vd1 and 4 Vd2) from Donor 2. 3 clones were obtained from 200 Vd1-sorted cells, and 4 clones from 600 Vd2-sorted cells, suggesting superior clonogenicity of Vd1. Indeed, Vd1 clones grew faster than Vd2 from this donor. After 40 days in culture, we obtained 57 ± 37 × 106 Vd1 and 37 ± 23 × 106 Vd2 cells from a single cell on day 0. The enhanced growth of Vd1 explains how this subset predominates in most polyclonal GDTC cultures, despite donors having more Vd2 than Vd1 in their blood (Vd2:Vd1 = 5.7±3.2, n=7). Polyclonal expansion of GDTCs from Donor 2 yielded 11.2 × 106 cells on day 20, from 1.7 × 106 on day 0, a 6.7-fold expansion compared to 107-fold achieved with clones from the same donor. Vd2 clones were screened for their ability to lyse EM2eGFPluc in vitro. In a flow-cytometric assay based on propidium iodide staining, Vd2 clones exhibited cytotoxicities ranging 4.5%-10.6% for a 4-hour co-incubation at 2.6:1 E:T. Clones from Donor 1 were tested again and ranking confirmed in a 4-hour cytotoxicity assay at 10:1 E:T, with a range of 23.5%-35.4% for clones A1, B3 and C6, respectively. When C6 was compared to polyclonal GDTCs from the same donor, it was found to be more cytotoxic (9.0% versus 2.0% at 10:1 for 4 hours). Vd2 clones and polyclonal GDTC from Donor 2 were compared; clone E5 exhibited 10-fold (49.2%) and E3 1.4-fold (7.6%) cytotoxicity of polyclonal GDTCs (5.3%). Published reports describe an increase in Vd1 in B-CLL patients, hence we used MEC1, an EBV-positive B-cell line derived from B-CLL, as a target. At a 1.9:1 ratio over 4 hours, % cytotoxicity ranged 7.0% - 13.8% (D3 most cytotoxic). Vd1 clones were compared with polyclonal GDTC cultures derived from Donors 2 and 3, which exhibited 57% and 52% Vd1, respectively. Clone D3 again proved most cytotoxic at 10:1 E:T over 4 hours, with 40.8% compared to 18.6% (Donor 3) and 6.8% (Donor 2). Immunophenotyping indicates phenotypic stability in clones over time that is not evident in polyclonal populations. We conclude that the increased cytotoxicity, superior expansion potential and extended culture duration as well as phenotypic stability of GDTC clones make them a more attractive therapeutic agent than polyclonal cultures for the treatment of hematological malignancies. Our study reveals the potential importance of selecting specific and potent GDT effector cells for treating Ph(+) and B-CLL leukemias with GDTCs. We next plan to test this approach in our established pre-clinical xenogeneic leukemia mouse model. (Dhamko H was the recipient of an ASH Summer Trainee Research Award). Disclosures No relevant conflicts of interest to declare.

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Mechanisms of Co-Operation of DNMT3A Mutations with JAK2 V617F Through Histone H4 Arginine 3 Provides New Insights in MPN Disease Pathogenesis

Blood ◽

10.1182/blood.v118.21.2823.2823 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2823-2823

Author(s):

Nisha Rao ◽

Carolyn M Butcher ◽

Petra J Neufing ◽

Sarah Bray ◽

Chris N Hahn ◽

...

Keyword(s):

Dna Methyltransferase ◽

Somatic Mutations ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Chronic Phase ◽

Site Directed Mutagenesis ◽

Hek293 Cells ◽

Histone H4 ◽

Critical Residue

Abstract Abstract 2823 Myeloproliferative neoplasms (MPN), including Polycythemia vera (PV), Essential Thrombocythemia (ET) and Primary myelofibrosis (PMF), are clonal stem cell disorders characterised by an excess of mature cells of one or more myeloid lineages in the chronic phase, and an associated risk of progression to myelofibrosis and leukemic transformation. Highly specific somatic mutations of JAK2 and MPL are found in the majority of MPN patients, while numerous other somatic mutations have now been identified to be common between MPN and de novo acute myeloid leukemia (AML), including somatic mutations of TET2, ASXL1, CBL, IKZF1, EZH2, IDH1, IDH2 (reviewed in Tefferi, 2010 (Tefferi)), and most recently the DNA methyltransferase, DNMT3A (Abdel-Wahab, et al, Ley, et al, Stegelmann, et al, Walter, et al, Yan et al). We identified somatic heterozygous mutations in DNMT3A affecting amino acids R882 (R882C) and M880 (M880V), detected in peripheral blood mononuclear cells from two JAKV617F-positive PV patients (Rao et al, 2011, accepted for publication). At the level of chromatin and gene regulation, cooperation of DNMT3A mutations with JAK2V617F can occur through direct interaction of DNMT3A with the chromatin modifying protein PRMT5 which is a direct phosphorylation target of JAK2V617F (Liu F et al, 2011). PRMT5 is an arginine methyltransferase which associates with chromatin and methylates the key arginine (R) residue at the N-terminus of histone H4 (R3), which in turn recruits a DNMT3A complex resulting in methylation of adjacent CpG residues and gene-silencing (Zhao Q et al., 2009). To test the functional affects of DNMT3A mutations M880V and R882C, we have performed site- directed mutagenesis to generate GFP-tagged DNMT3A wild-type and mutant retroviral constructs. These DNMT3A clones will be transduced into cord blood CD34+ve cells and GFP-positive cells selected with flow cytometry. For functional analysis, we will examine DNA methyltransferase activity, changes in histone H4 modifications and CpG methylation. In a complementary analysis we have identified a change in a JAK2V617F+ve PV patient in the highly conserved HIST1H4C gene resulting in a substitution of Arg3 for Cys in Histone H4. Although there are 15 histone H4 genes encoding identical proteins, gene expression varies significantly in tissues and in cancer cells (W.F Holmes et. al, J.Biol Chem, 2005). We have used Q-PCR, using long primers to differentiate H4 transcripts and measured relative expression levels. We have confirmed that HIST1H4C is the major contributor of the H4 pool making ≥50% of the total H4 RNA in normal CD34+ve cells, Monocytes, lymphocytes and a variety of haemopoietic cell lines, indicating that the change to this critical residue in this gene may be functionally important. An analysis of a matched buccal sample showed that the HIST1H4C R3C variant in the PV patient was germline. Subsequent sequencing of the entire open reading frame of HIST1H4C in 232 normal individuals did not identify any other amino acid substitutions affecting this gene. Given the interaction of this residue with DNMT3A, we are currently investigating functional effects associated with this histone H4 variant. We have generated HEK293 cells with 4-Hydroxy tamoxifen (4-HT) inducible expression of the H4 R3C variant, and following the addition of 4-HT, we are initially assessing changes in histone H4 post-translational modifications, and markers of DNA damage such as gamma H2AX serine 139 phosphorylation. Disclosures: No relevant conflicts of interest to declare.

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Loss of Wild-Type Jak2 Allele Enhances Myeloid Cell Expansion and Accelerates Myelofibrosis in Jak2V617F Knock-in Mice

Blood ◽

10.1182/blood.v120.21.809.809 ◽

2012 ◽

Vol 120 (21) ◽

pp. 809-809

Author(s):

Hajime Akada ◽

Saeko Akada ◽

Dongqing Yan ◽

Robert Hutchison ◽

Golam Mohi

Keyword(s):

Polycythemia Vera ◽

Cell Expansion ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Myeloid Cell ◽

Negative Regulator ◽

Jak2v617f Mutation ◽

Common Mutation ◽

Wild Type

Abstract Abstract 809 The activating JAK2V617F mutation is the most common mutation found in Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs), which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Although a majority of MPN patients carry heterozygous JAK2V617F mutation, loss of heterozygosity (LOH) on chromosome 9p involving JAK2 has been observed in ∼30% of patients with MPNs particularly in PV and PMF. JAK2V617F homozygosity through 9pLOH has been linked to more severe MPN phenotype. However, the contribution of 9pLOH in the pathogenesis of MPNs remains unclear. To investigate the role of wild-type JAK2 in MPNs induced by JAK2V617F, we have utilized conditional Jak2 knock-out and Jak2V617F knock-in alleles and generated heterozygous, hemizygous and homozygous Jak2V617F mice. Whereas heterozygous Jak2V617F expression results in a polycythemia vera-like disease in mice, loss of wild-type Jak2 allele in hemizygous or homozygous Jak2V617F mice results in a significantly greater increase in reticulocytes, white blood cells, neutrophils and platelets in the peripheral blood and larger spleen size. We also have found that hemizygous or homozygous Jak2V617F expression significantly increased megakaryocyte-erythroid progenitors in the bone marrow and spleens and marked infiltration of neutrophils in the liver compared with heterozygous Jak2V617F. More importantly, hemizygous or homozygous Jak2V617F mice show accelerated myelofibrosis compared with heterozygous Jak2V617F-expressing mice. Thus, loss of wild type Jak2 allele increases myeloid cell expansion and enhances the severity of the MPN. Together, these results suggest that wild-type Jak2 serves as a negative regulator of MPN induced by Jak2V617F. Disclosures: No relevant conflicts of interest to declare.

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5-Aza-2-Deoxycytidine Regulates Wnt Beta-Catenin Pathway in B Cell Lymphoma

Blood ◽

10.1182/blood.v126.23.4810.4810 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4810-4810

Author(s):

Xinyu Li ◽

Lingyu Geng ◽

Xiangxiang Zhou ◽

Kang Lu ◽

Peipei Li ◽

...

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Cell Lines ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Beta Catenin ◽

Cell Functions ◽

Protein Expressions

Abstract Introduction: The Wnt/beta-catenin pathway is aberrantly activated in B cell lymphomas, unphosphorylated beta-catenin accumulates and translocates into the nucleus, regulates the expression of c-myc, cyclinD1 and many other target genes which govern fundamental cell functions, such as proliferation, cell cycle regulation and apoptosis. Methylation is a highlight of epigenetic regulation research which also occurred in lymphoma, but the concrete mechanism of how the demethylation drug 5-aza-2-deoxycytidine affect Wnt/beta-catenin pathway is still unknown. This study was designed to illuminate the implications on Wnt/beta-catenin pathway via demethylation 5-aza in B cell lymphoma. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from samples of 30 primary CLL patients. The PBMCs contained more than 90% of CD19+ B lymphocytes, which were detected by flow cytometry and were referred to as primary CLL cells. The activation of Wnt/beta-catenin pathway and DNMT-1 of B cell lymphoma cells lines (MEC-1, LY8, Jeko-1, Grant519, mino and sp53) and the 30 patients were detected by qPCR and western blot. The expressions of beta-catenin in 20 cases of B cell lymphoma tissues were measured by IHC. The B cell lines and PBMCs from 10 primary CLL patients were given 5-aza-2-deoxycytidine in different concentrations, the effects in the pathway and apoptosis were observed by WB and flow cytometry. Results: The expressions of beta-catenin, c-myc, cyclinD1 and DNMT-1 were aberrantly higher in all cell lines we used ( MEC-1,LY8, Jeko-1, Grant519, mino and sp53 Fig.1-A,B), most primary CLL patients (Fig.1-C), and B cell lymphoma tissues (Fig.1-D). The protein expressions of beta-catenin in MEC-1 were higer than primary CLL patients. 0, 0.5, 1.0, 2.0¦ÌM 5-aza-2-deoxycytidine were given to the B cells lines and PBMCs from primary CLL patients for 48h, beta-catenin were found accumulated, but c-myc and cyclinD1 in the downstream were reduced (Fig.2-A,B,C,D). For further understanding of aberrant accumulation ofbeta-catenin, we extracted the nuclear protein of MEC-1, nuclear beta-catenin protein expressions were found decreased and cytoplasmic were increased (Fig.2-E). After 5-aza treatment, the apoptosis rate increased and caspase pathway were activated (Fig.2-A,F). Conclusions: The enhanced expressions of beta-catenin, c-myc, cyclinD1 in the B cell lines and the B cell lymphoma samples indicated the Wnt/beta-catenin was aberrantly activated. After 5-aza treatment with the cell lines (MEC-1, Jeko-1, LY8) and primary CLL cells, the abnormal accumulation of beta-catenin protein was observed which was discrepancy with previous reports, but the decrease of c-myc and cyclinD1 suggested the pathway was inhibited, apoptosis also occurred. The increase of totalbeta-catenin protein was supposed to be an stress reaction of the 5-aza treatment, however, the redundant beta-catenin protein in B cell lymphoma was speculated to be combined with demethylated genes and resulted in dormancy of this pathway. Our results indicated that 5-aza played a demethylation role through Wnt/beta-catenin pathway in B cell lymphoma. The data are of interest in the context of epigenetic-based therapy in B cell lymphoma. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

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Expression of CD47 and Calr in Myeloproliferative Neoplasms: Potential New Therapeutical Targets

Blood ◽

10.1182/blood-2021-144482 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4598-4598

Author(s):

Kristian Boasman ◽

Matthew J Simmonds ◽

Ciro R Rinaldi

Keyword(s):

Cell Membrane ◽

Protein Expression ◽

Cell Membranes ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Membrane Expression ◽

Myeloid Malignancies ◽

Response To Chemotherapy ◽

Significant Difference

Abstract Myeloproliferative neoplasms (MPN) are myeloid malignancies characterized by overproduction of mature blood cells, hyperplastic bone marrow and tendency to evolve into acute myeloid leukaemia. In solid tumours, calreticulin (CALR) overexpression produces a pro-phagocytic signal and is counteracted by concomitant expression of anti-phagocytic CD47, reflecting an apoptosis vs survival mechanism. Increases of both CALR and CD47 on the cell membrane have been observed in response to chemotherapy, however their role in myeloid malignancies is poorly understood. Aims: Investigate the expression and cellular localisation of CALR and CD47 in untreated and treated patients with essential thrombocythemia (ET), polycythemia vera (PV) myelofibrosis (MF), in comparison with healthy controls. Methods: Mononuclear cells were collected by Ficoll separation, from peripheral blood of 30 MPN (8 PV, 16 ET, 6 MF); 18 MPN patients received cyto-reductive therapies (Hydroxyurea, Anagrelide or Ruxolitinib); and 4 controls. Cells were fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus. Proteins were extracted using TRIzol, with CALR and CD47 protein expression analysed by western blotting. Results: Total CALR and CD47 protein expression increased in MPN samples compared with controls (CALR- 7.9 vs 5.1; CD47- 2.7 vs 2.2 fold, respectively). CD47 showed higher expression of its overall protein on MPN cell membranes when compared with CALR (22% vs 13.9%). We observed a significant reduction of CALR expression in all MPN subtypes when patients were treated with cyto-reductive agents (ET- untreated 43.3% vs treated 2%, PV- 3.6% vs 2.2%, ET- 21% vs 11%). Interestingly we have observed a significant increase in CD47 cell membrane expression after treatment in MF and PV (CD47 in MF- untreated 11.8% vs treated 34.3%, PV-11.4% vs 35.9%), suggesting an anti-phagocytic effect induced by cytotoxic drugs. In ET cell membranes however, CD47 expression is reduced after cyto-reductive treatment (22% vs 16.6%), suggesting instead a prophagocytic effect. Summary/Conclusion: CD47, but not CALR, is overexpressed on the membrane of patients with MPN, suggesting a role for CD47 as a strong antiphagocytic signal responsible for immune survival in MPN. We observed a significant difference in CD47 expression across different MPN subtypes with a significant increase in CD47 expression in PV and MF but not ET when patients were exposed to cytoreduction. The use of anti-CD47 antibodies could represent a new strategy to enhance the treatment response in particular in PV and MF. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Expression and functionality study of 9 Toll-like receptors in drug-naïve non-affective first episode psychosis individuals: a 3-month study

10.21203/rs.3.rs-17979/v1 ◽

2020 ◽

Author(s):

Maria Juncal-Ruiz ◽

Laura Riesco-Davila ◽

Mariluz Ramirez-Bonilla ◽

Victor Ortiz-Garcia de la Foz ◽

Javier Vazquez-Bourgon ◽

...

Keyword(s):

Healthy Volunteers ◽

Inflammatory Cytokines ◽

Mononuclear Cells ◽

Immune Cell ◽

First Episode ◽

Toll Like Receptors ◽

Tnf Α ◽

Drug Naïve ◽

Lower Expression ◽

Pro Inflammatory Cytokines

Abstract Background: Toll-like receptors (TLRs) are a pivotal component of the innate immune system, which are expressed by various subsets of immune cell types, included central nervous system. There are few publications that have studied TLR expression and/or functionality in psychosis, of which most of them have been based on chronic schizophrenia individuals.Objectives: To compare the expression and functionality of 9TLRs in three peripheral blood mononuclear cells (PBMCs) (monocytes, B cells and T cells) within a sample of 33 drug-naïve FEP individuals and 26 healthy volunteers, at baseline and after 3-month of antipsychotic treatment.Methods: The expression of TLR1-9 was assessed by ﬂow cytometry. For the assessment of the TLR functionality (measured as intracellular production of IL-1β, IL-6 and TNF-α following TLR stimulation), cells collected in sodium heparin tubes were polyclonally stimulated for 18h with different agonists for human TLR1–9.Results: Patients showed a lower expression of TLR5 and TLR8 on the three PBMCs at baseline and after 3-month of treatment regarding healthy volunteers (all ps <0.01). We also found less production of some intracellular pro-inflammatory cytokines (especially TNF-α) after TLR stimulation in patients at both baseline and following the medication (all ps <0.01). We have not found differences in the intra-subject analyses after 3-month of treatment.Conclusions: Drug-naive patients with schizophrenia spectrum disorders show lower expression of specific TLR receptors as well as lower intracellular concentrations of some pro-inflammatory cytokines after TLR stimulation. These findings may be a consequence of an excessive cell stimulation via exogenous ligands (such as pathogens) and/or endogenous ligands (such as autoimmunity) in such a way that PBMCs could be exhausted to be activated in the in vitro analyses.

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