scholarly journals Targeting M2-Tumor Associated Macrophages By Arginase-1 and PD-L1 in Regulating Juvenile Myelomonocytic Leukemia (JMML) Development and Relapse

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1471-1471
Author(s):  
Santhosh Kumar Pasupuleti ◽  
Baskar Ramdas ◽  
Kai Yang ◽  
Chujing Zhang ◽  
Elliot Stieglitz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Cells ◽  
Juvenile Myelomonocytic Leukemia ◽  
M2 Macrophages ◽  
Arginase 1 ◽  
Donor Cells ◽  
Transplantation Experiment ◽  
Leukemia Relapse ◽  
Myelomonocytic Leukemia

Abstract Tumor-associated macrophages (TAMs) are a key component of tumor-infiltrating immune cells. Macrophages are largely characterized as M1 or M2 types, and TAMs have been shown to express an M2-like phenotype. TAMs endorse tumor progression and contribute to resistance to chemotherapies. However, it is unclear what the composition of M2 macrophages is in patients with Juvenile myelomonocytic leukemia (JMML) and how do these cells mechanistically contribute to JMML and/or relapse after bone marrow transplantation. To study the role of M2- TAMs in JMML development, we first examined the bulk RNA-sequence data in 90 JMML patients. These data demonstrated a significant increase in the expression of arginase-1 (Arg-1) and programmed cell death-1 (PD-1). Furthermore, single cell RNA-sequencing analysis of monocytes/macrophages from 4 JMML patients revealed higher expression of M2- macrophage markers/genes such as IL-10, CD163, MRC1/CD206, TGF-β1 and IL-1R1 compared to M1 macrophage (CD80, CCR7, IL-6, CXCL10, CXCL11 and TNF) expression. We hypothesized that in JMML, inflammatory myeloid cells including neutrophils and M2-macrophages express higher levels of arginase and PD-1, which may contribute to the local suppression of immune responses and damage the bone marrow microenvironment (BME) leading to poor engraftment of normal donor cells, resulting in relapse. To study how alterations in bone marrow (BM) macrophages (M1/M2) contribute to JMML development and relapse, we utilized a mouse model bearing Shp2 E76K mutation (Ptpn11 E76K/+) driven by lysosome-cre (Ptpn11 E76K/+; LysM-Cre+, indicated as Shp2* mice hereafter). This model is frequently used to study JMML as it manifests cardinal features of human JMML. In a competitive transplantation experiment using, Shp2* + Boy/J BM cells (1:1 ratio) transplanted into lethally irradiated Shp2* recipient mice, we show that Shp2* mutant cells out compete WT BoyJ cells and result in rapid growth of CD45.2+ Shp2* mutant mature myeloid cells, hematopoietic stem and progenitors (HSC/Ps) and M2- macrophages (F4/80+/CD206+) in the BM and spleen leading to leukemia relapse. To determine if modulating Arg-1 and PD-1/PD-L1 levels in the background of Shp2* mutant leukemic stem cells in Shp2* recipients would alter the overall engraftment and JMML development and relapse, we again performed a competitive transplantation experiment using, Shp2* + Boy/J (BM cells, 1:1 ratio) into Shp2* and WT recipient mice. After 8 weeks post transplantation, we investigated the role of Arg-1 and PD-L1 in Shp2* recipients using pharmacological inhibitors, CB-1158 (Arg-1 inhibitor; 100 mg/kg, orally) + anti-PD-L1 antibody (10 mg/kg, i.p) for 30 days. The Arg-1 + PD-L1 treatment significantly reduced the number of white blood cells, neutrophils, monocytes and improved RBC and platelet counts. The spleen and liver weights were significantly rescued as well. Interestingly, CD45.1 WT donor cells in the PB, BM, and spleen were significantly increased and a significant reduction of Shp2* mutant CD45.2+ mature myeloid cells in the PB, BM, and spleen was observed. Importantly, the frequency and absolute number of leukemic blasts, LSK (Lin-/Sca1+/c-KIT+) cells, short term hematopoietic cells (ST-HSCs), common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid progenitors (MEP) were significantly reduced. Furthermore, the M2- TAMs were significantly reduced in the BM and spleen of Arg-1 + PD-L1 drug treated group compared to vehicle treated mice. Notably the CD8+ T-cells (IFN-γ+ and TNF-α+) were significantly improved in the drug treated mice. These data suggest that the suppression of arginase-1 allows for the arginine levels to increase, which promotes the proliferation of T-cells. Increasing arginine levels also promotes an anti-tumor immune response resulting in the emergence of CD45.1 WT HSCs as opposed to mutant CD45.2 HSCs, suggesting that Arg-1 + PD-L1 treatment is a novel therapeutic approach to treat patients with JMML and for preventing leukemia relapse after BM transplantation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of lncRNA Morrbid in PTPN11(Shp2)E76K-driven juvenile myelomonocytic leukemia

2020 ◽  
Vol 4 (14) ◽  
pp. 3246-3251
Author(s):  
Zhigang Cai ◽  
Chi Zhang ◽  
Jonathan J. Kotzin ◽  
Adam Williams ◽  
Jorge Henao-Mejia ◽  
...  
Keyword(s):  
Overall Survival ◽  
Tyrosine Phosphatase ◽  
Myeloid Cells ◽  
Erythroid Cell ◽  
Juvenile Myelomonocytic Leukemia ◽  
Poor Overall Survival ◽  
Significant Correction ◽  
Evolutionarily Conserved ◽  
Myelomonocytic Leukemia

Abstract Mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, contribute to ∼35% of cases of juvenile myelomonocytic leukemia (JMML). A common clinical picture in children with JMML is that it presents as a constitutive hyperinflammatory syndrome, partially reminiscent of chronic myelomonocytic leukemia in adults. Thus, a component of JMML is associated with a hyperinflammatory state and abundant innate immune cells such as neutrophils and monocytes. Recently, we showed that the evolutionarily conserved mouse lncRNA Morrbid is specifically expressed in myeloid cells and uniquely represses the expression of the proapoptotic gene Bim to regulate the lifespan of myeloid cells. However, its role in JMML has not been investigated. In this study, we characterized the role of Morrbid and its target Bim, which are significantly dysregulated in Shp2E76K/+-bearing myeloid cells, in driving JMML. Loss of Morrbid in a mouse model of JMML driven by the Shp2E76K/+ mutation resulted in a significant correction of myeloid and erythroid cell abnormalities associated with JMML, including overall survival. Consistently, patients with JMML who had PTPN11, KRAS, and NRAS mutations and high expression of MORRBID manifested poor overall survival. Our results suggest that Morrbid contributes to JMML pathogenesis.

Evidence for PTEN Deficiency in Juvenile Myelomonocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3428-3428
Author(s):  
Y. Lucy Liu ◽  
Likang Xu ◽  
Robert P. Castleberry ◽  
Peter Dean Emanuel
Keyword(s):  
Bone Marrow ◽  
Negative Regulator ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Mrna Levels ◽  
Pten Protein ◽  
Gm Csf ◽  
The Status ◽  
Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is a myelodysplastic/myeloproliferative disorder (MDS/MPD) of young children. It is characterized by monocytosis, leukocytosis, elevated fetal hemoglobin, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), low percentage of myeloblasts in bone marrow, and absence of the Philadelphia chromosome. The pathogenesis of JMML has been linked to dysregulated signal transduction through the NF1/RAS signaling pathway and PTPN11. This dysregulation results in JMML cells demonstrating selective hypersensitivity to GM-CSF in vitro dose-response assays. PTEN, a major negative regulator of the PI3-kinase pathway by virtue of its PIP3 phosphatase activity, was initially isolated as a tumor suppressor in a variety of malignancies. In order to evaluate the role of PTEN in the pathogenesis of JMML, we examined the status of PTEN in JMML patient samples. Peripheral blood or bone marrow was collected from 40 patients. Mononuclear cells (MNCs) were isolated and lysed in lysis buffer at a concentration of 107/ml. Total RNA was extracted from MNCs of patients and 17 normal individuals. Protein and mRNA levels of PTEN were evaluated by Western-blot and relative-quantitative real-time RT-PCR, respectively. We found that PTEN protein was decreased in 18 of 30 (60%) JMML patients, and the patients had significantly lower RNA expression of PTEN than normal controls (p=0.015). With the available samples we also evaluated AKT activity and MAP kinase (MAPK) levels. We found that MAPK levels were correlated well with the status of the PTEN in 12 of 27(44%), and AKT activity in 13 of 25 patients (52%). Our data indicates that PTEN is significantly deficient in JMML patients, and the low PTEN protein level is related to its low transcription of RNA in JMML patients. The role of PTEN in regulation of MAPK and AKT activities in JMML is under further evaluation by studying the upstream status of the RAS pathway prior to PTEN. This is the first investigation of PTEN deficiency in JMML patients, and additional investigations may help to further understand the pathogenetic mechanisms in JMML, as well as to guide the development of targeted therapeutics for JMML.

5-Azacytidine Reduces Leukemic Burden in a Xenograft Model of Juvenile Myelomonocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1655-1655
Author(s):  
Christopher Felix Krombholz ◽  
Angelina Meier ◽  
Konrad Aumann ◽  
Silvia Fluhr ◽  
Matthias Kollek ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Dna Methyltransferase ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Patient Specific ◽  
Juvenile Myelomonocytic Leukemia ◽  
Methylation Patterns ◽  
Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood with often fatal outcome. Despite many attempts to develop alternative treatment options allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative modality. In the past our group has linked the prognosis of JMML to differential DNA methylation patterns (Olk-Batz, Blood 2011;117:4871-80 and Poetsch, Epigenetics 2014;9:1252-60), suggesting a key role of epigenetic modifications in JMML pathophysiology. To overcome the lack of suitable preclinical JMML research models we have developed an ex vivo JMML xenotransplantation system using neonatal Rag2-/- gamma-c-/- mice. Transplantation of 1x106 primary JMML cells resulted in stable xenologous engraftment and reproduced a characteristic JMML phenotype including myelomonocytic expansion; infiltration of spleen, liver and, notably, lung; splenomegaly; and reduced survival (median 26 weeks). Persistent human engraftment and leukemic organ infiltration was confirmed by both flow cytometry and immunohistology. Ras pathway mutations present in xenotransplanted patient samples were invariably confirmed in engrafted tissues. In addition, the model sustained serial transplantations and can therefore be used to amplify scarce patient material. We first tested if DNA methylation patterns in JMML cells were stable even after xenologous engraftment because such stability would be a prerequisite if the model were to be used for preclinical investigation of DNA methyltransferase inhibitors. JMML cells before xenotransplantation and those retrieved from the bone marrow of engrafted mice were profiled for global CpG methylation using Illumina 450K arrays. DNA methylation patterns in JMML were patient-specific and surprisingly robust in functional regions over several months of engraftment time (on average, 0.29% of 30877 promoters and 0.25 % of 30725 intragenic regions were called as "differentially methylated" between source and xenograft; 0.2 β-value change cutoff). These findings confirm the suitability of the xenograft model to investigate JMML epigenetics and, more importantly, indicate that patient-specific epigenetic profiles originate in leukemia-initiating stem cells, reinforcing a fundamental role of these alterations in JMML biology. Our group recently published a retrospective case series demonstrating unprecedented clinical efficacy of the DNA methyltransferase inhibitor 5-azacytidine (5AC) to induce partial or complete remissions in JMML before allogeneic HSCT (Cseh, Blood 2015;125:2311-3). To further investigate the drug on the preclinical level we administered 5AC to Rag2-/- gamma-c-/- mice xenografted with primary JMML cells. After a leukemia establishment phase the mice were divided into treatment or mock groups and treated with 5AC (3mg/kg body weight i.p., N=6) or saline (N=6) for 2 cycles (1 dose daily for 5 days; 9 days of recovery). This regimen was tolerated well by the animals. We found that 5AC reduced JMML infiltration in all organs analyzed, with most pronounced effects in spleen (human CD45+ fraction of all CD45+ cells, 0.24% +/- 0.04% vs 39.78% +/- 10.72%; p<0.01) and lung (0.41% +/-0.18% vs 42.88% +/-8.42%; p<0.01). The proportion of early progenitor cells (CD34+) within the human leukemia population in murine bone marrow was dramatically reduced after 5AC treatment (7.89% +/-0.74% vs 32.65% +/-3.76%; p<0.01) while the amount of granulocytes increased simultaneously (44.90% +/-1.74% vs 9.35% +/-1.95%; p<0.01). These findings suggest a loss of JMML cells induced by forced differentiation of more immature cells into mature myelomonocytic cells with reduced proliferation potential. Bisulfite pyrosequencing of the human BMP4 promoter CpG island, a locus frequently hypermethylated in JMML, showed significantly reduced DNA methylation in JMML cells retrieved from 5AC-treated mice (31.32% +/-2.66% vs 52.46% +/-1.39%; p<0.001). In summary we created an ex vivo JMML xenograft model in immunodeficient mice that reflects many important aspects of this disorder and proved its usefulness for preclinical research of DNA methyltransferase inhibition because of extraordinary stability of leukemic DNA methylation patterns. 5AC showed clear preclinical efficacy in this model, supporting its further development in clinical treatment strategies for JMML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Korszakváltás a gyermekkori szerzett csontvelő-elégtelenséggel járó kórképek kezelésében Magyarországon

2018 ◽  
Vol 159 (42) ◽  
pp. 1710-1719
Author(s):  
Krisztián Kállay ◽  
Judit Csomor ◽  
Emma Ádám ◽  
Csaba Bödör ◽  
Csaba Kassa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Survival Rate ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Severe Aplastic Anemia ◽  
Juvenile Myelomonocytic Leukemia ◽  
Reduced Intensity Conditioning ◽  
Myelomonocytic Leukemia ◽  
Reduced Intensity

Abstract: Introduction: Acquired bone marrow failures are rare but fatal diseases in childhood. Since 2013, Hungary has been participating as a full member in the work of the European Working Group on uniform diagnostics and therapy in patients with acquired bone marrow failure syndromes. Hypocellular refractory cytopenia of childhood has been emphasized as a frequent entity, transplanted by reduced intensity conditioning with excellent outcomes. Aim: To analyse and compare the results of treatment before and after our joining. Method: A total of 55 patients have been treated in the 8 centres of the Hungarian Pediatric Oncology Network during 5 years between 2013 and 2017 (severe aplastic anemia: 9, myelodysplastic syndrome: 41, juvenile myelomonocytic leukemia: 5 patients). Allogeneic hematopoietic stem cell transplantation was performed in severe aplastic anemia in 7 cases, while antithymocyte globulin was administered in one case and one patient died before diagnosis. In patients with myelodysplastic syndromes, watch and wait strategy was applied in 4, while transplantation in 37 cases. Reduced intensity conditioning was used in 54 percent of these cases. Transplantation was the treatment of choice in all 5 patients with juvenile myelomonocytic leukemia. Results: In the whole patient cohort, the time from diagnosis to treatment was median 92 (3–393) days, while in severe aplastic anemia median 28 (3–327) days only. Grade II–IV acute graft versus host disease occurred in 22.6%, grade III–IV in 6.8% and chronic in 11.2%. All the patients treated with severe aplastic anemia are alive and in complete remission (100%). The overall estimated survival rate is 85.1% in myelodysplastic syndrome, while 75% in juvenile myelomonocytic leukemia. The median follow-up was 30.4 (1.1–62.5) months. There was a remarkable increase in overall survival comparing the data before (1992–2012) and after (2013) joining the international group, 70% vs. 100% (p = 0.133) in severe aplastic anemia and 31.3% vs. 85.1% (p = 0.000026) in myelodysplastic syndrome. Conclusion: Due to a change in the paradigm of the conditioning regimen in hypocellular refractory cytopenia of childhood, the overall survival rate has significantly increased. Orv Hetil. 2018; 159(42): 1710–1719.

scholarly journals Crosstalk between adipocytes and M2 macrophages compensates for osteopenic phenotype in the Lrp5-deficient mice

2020 ◽  
pp. 153537022097232
Author(s):  
Lisha Li ◽  
Xuemin Qiu ◽  
Na Zhang ◽  
Yan Sun ◽  
Yan Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Wnt Signaling ◽  
Macrophage Polarization ◽  
Myeloid Cells ◽  
Osteogenic Potential ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tartrate Resistant Acid Phosphatase ◽  
M2 Macrophage ◽  
M2 Macrophages ◽  
Alizarin Red

A loss-of-function mutation in the Lrp5 gene in mice leads to a low bone mass disorder due to the inhibition of the canonical Wnt signaling pathway; however, the role of bone marrow microenvironment in mice with this mutation remains unclear. In this study, we evaluated proliferation and osteogenic potential of mouse osteoblasts using the MTT assay and Alizarin red staining. The levels of alkaline phosphatase, tartrate-resistant acid phosphatase, and adiponectin in culture supernatants were measured using the enzyme-linked immunosorbent assay. Osteoclast bone resorbing activity was evaluated by toluidine staining and the number and area of bone resorption pits were determined. We observed increased osteogenesis in osteoblasts co-cultured with the BM-derived myeloid cells compared to the osteoblasts cultured alone. Mice with global Lrp5 deletion had a relatively higher bone density compared to the mice carrying osteoblast/osteocyte-specific Lrp5 deletion. An increased frequency of M2 macrophages and reduced expression of inflammatory cytokines were detected in the myeloid cells derived from the bone marrow of mice with global Lrp5 deletion. Higher adipogenic potential and elevated levels of adiponectin in the global Lrp5 deletion mice contributed to the preferential M2 macrophage polarization. Here, we identified a novel systemic regulatory mechanism of bone formation and degradation in mice with global Lrp5 deletion. This mechanism depends on a crosstalk between the adipocytes and M2 macrophages in the bone marrow and is responsible for partly rescuing osteopenia developed as a result of decreased Wnt signaling.

scholarly journals Development of an allele-specific minimal residual disease assay for patients with juvenile myelomonocytic leukemia

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1124-1127 ◽  
Author(s):  
Sophie Archambeault ◽  
Nikki J. Flores ◽  
Ayami Yoshimi ◽  
Christian P. Kratz ◽  
Miriam Reising ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Juvenile Myelomonocytic Leukemia ◽  
Hematopoietic Stem ◽  
Allele Specific ◽  
Myelomonocytic Leukemia

AbstractJuvenile myelomonocytic leukemia is an aggressive and frequently lethal myeloproliferative disorder of childhood. Somatic mutations in NRAS, KRAS, or PTPN11 occur in 60% of cases. Monitoring disease status is difficult because of the lack of characteristic leukemic blasts at diagnosis. We designed a fluorescently based, allele-specific polymerase chain reaction assay called TaqMAMA to detect the most common RAS or PTPN11 mutations. We analyzed peripheral blood and/or bone marrow of 25 patients for levels of mutant alleles over time. Analysis of pre–hematopoietic stem-cell transplantation, samples revealed a broad distribution of the quantity of the mutant alleles. After hematopoietic stem-cell transplantation, the level of the mutant allele rose rapidly in patients who relapsed and correlated well with falling donor chimerism. Simultaneously analyzed peripheral blood and bone marrow samples demonstrate that blood can be monitored for residual disease. Importantly, these assays provide a sensitive strategy to evaluate molecular responses to new therapeutic strategies.

scholarly journals Neonatal bone marrow transplantation of ADA-deficient SCID mice results in immunologic reconstitution despite low levels of engraftment and an absence of selective donor T lymphoid expansion

Blood ◽  
2008 ◽  
Vol 111 (12) ◽  
pp. 5745-5754 ◽  
Author(s):  
Denise A. Carbonaro ◽  
Xiangyang Jin ◽  
Daniel Cotoi ◽  
Tiejuan Mi ◽  
Xiao-Jin Yu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Immune Reconstitution ◽  
Myeloid Cells ◽  
Normal Donor ◽  
Dependent Manner ◽  
Term Survival ◽  
Hematopoietic Stem ◽  
Donor Cells

Abstract Adenosine deaminase (ADA)–deficient severe combined immune deficiency (SCID) may be treated by allogeneic hematopoietic stem cell transplantation without prior cytoreductive conditioning, although the mechanism of immune reconstitution is unclear. We studied this process in a murine gene knockout model of ADA-deficient SCID. Newborn ADA-deficient pups received transplants of intravenous infusion of normal congenic bone marrow, without prior cytoreductive conditioning, which resulted in long-term survival, multisystem correction, and nearly normal lymphocyte numbers and mitogenic proliferative responses. Only 1% to 3% of lymphocytes and myeloid cells were of donor origin without a selective expansion of donor-derived lymphocytes; immune reconstitution was by endogenous, host-derived ADA-deficient lymphocytes. Preconditioning of neonates with 100 to 400 cGy of total body irradiation before normal donor marrow transplant increased the levels of engrafted donor cells in a radiation dose–dependent manner, but the chimerism levels were similar for lymphoid and myeloid cells. The absence of selective reconstitution by donor T lymphocytes in the ADA-deficient mice indicates that restoration of immune function occurred by rescue of endogenous ADA-deficient lymphocytes through cross-correction from the engrafted ADA-replete donor cells. Thus, ADA-deficient SCID is unique in its responses to nonmyeloablative bone marrow transplantation, which has implications for clinical bone marrow transplantation or gene therapy.

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