Differentiation Of Immature Myeloid Cells Research Articles

Inhibition of Enhancer of Zeste Homolog 2 Induces Blast Differentiation, Impairs Engraftment and Prolongs Survival in Murine Models of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the malignant proliferation of immature myeloid cells characterized by a block in differentiation. As such, novel therapeutic strategies to promote the differentiation of immature myeloid cells have been successful in AML, although these agents are targeted to a specific mutation that is only present in a subset of AML patients. In the current study, we show that targeting the epigenetic modifier enhancer of zeste homolog 2 (EZH2) can induce the differentiation of immature blast cells into a more mature myeloid phenotype and promote survival in AML murine models. The EZH2 inhibitor EPZ011989 (EPZ) was studied in AML cell lines, primary in AML cells and normal CD34+ stem cells. A pharmacodynamic assessment of H3K27me3; studies of differentiation, cell growth, and colony formation; and in vivo therapeutic studies including the influence on primary AML cell engraftment were also conducted. EPZ inhibited H3K27me3 in AML cell lines and primary AML samples in vitro. EZH2 inhibition reduced colony formation in multiple AML cell lines and primary AML samples, while exhibiting no effect on colony formation in normal CD34+ stem cells. In AML cells, EPZ promoted phenotypic evidence of differentiation. Finally, the pretreatment of primary AML cells with EPZ significantly delayed engraftment and prolonged the overall survival when engrafted into immunodeficient mice. Despite evidence that EZH2 silencing in MDS/MPN can promote AML pathogenesis, our data demonstrate that the therapeutic inhibition of EZH2 in established AML has the potential to improve survival.

ADRB3 induces mobilization and inhibits differentiation of both breast cancer cells and myeloid-derived suppressor cells

Metastatic tumors are mainly composed of neoplastic cells escaping from the primary tumor and inflammatory cells egressing from bone marrow. Cancer cell and inflammatory cell are remained in the state of immaturity during migration to distant organs. Here, we show that ADRB3 is crucial in cell mobilization and differentiation. Immunohistochemistry revealed ADRB3 expression is significantly more frequent in breast cancer tissues than in adjacent noncancerous tissues (92.1% vs. 31.5%). Expression of ADRB3 correlated with malignant degree, TNM stage and poor prognosis. Moreover, ADRB3 expression was markedly high in activated disseminated tumor cells, myeloid-derived suppressor cells (MDSCs), lymphocytes and neutrophil extracellular traps of patients. Importantly, ADRB3 promoted the expansion of MDSC through stimulation of bone marrow mobilization and inhibiting of the differentiation of immature myeloid cells. Furthermore, ADRB3 promoted MCF-7 cells proliferation and inhibited transdifferentiation into adipocyte-like cell by activating mTOR pathway. Ultimately, the MDSC-deficient phenotype of ADRB3 -/- PyMT mice was associated with impairment of mammary tumorigenesis and reduction in pulmonary metastasis. Collectively, ADRB3 promotes metastasis by inducing mobilization and inhibiting differentiation of both breast cancer cells and MDSCs.

MicroRNAs: Regulatory Biomarkers in Acute Myeloid Leukemia and Graft Versus Host Disease.

MicroRNAs are a group of small non-coding RNAs with about 19 - 22 nucleotides and have a crucial role in different biologic processes such as cell proliferation, differentiation, and cell death at the post-transcriptional level. Disruption in these molecules can play an important role in tumorigenesis, and they can act as oncogenes or tumor suppressors. Acute myeloid leukemia (AML) is a hematologic malignancy with abnormal proliferation and differentiation of immature myeloid cells. MicroRNAs can be considered as biomarkers for diagnosis, prognosis, and treatment in AML patients. One of the treatments in these patients is hematopoietic stem cell transplantation (HSCT), and acute graft versus host disease (aGVHD) is the most common complication of HSCT in these patients. Patients with aGVHD appear with different clinical symptoms. Some microRNAs can predict the risk of aGVHD in these patients. The resources of this study are from different sites and journals such as ncbi.nlm.nih.gov/pubmed, scopus.com, Blood Journal, British Journal of Haematology, etc. Results: The expression of various microRNAs is different in AML patients. Also, these differences can be observed in patients with aGVHD. Identification of microRNAs can be useful in the diagnosis and prognosis of AML and aGVHD in these patients. In this review, we discuss the role of microRNAs in the pathogenesis of AML and aGVHD in patients who have undergone HSCT.

MicroRNAs: pivotal regulators in acute myeloid leukemia.

MicroRNAs are a class of small non-coding RNAs that are 19-22 nucleotides in length and regulate a variety of biological processes at the post-transcriptional level. MicroRNA dysregulation disrupts normal biological processes, resulting in tumorigenesis. Acute myeloid leukemia is an invasive hematological malignancy characterized by the abnormal proliferation and differentiation of immature myeloid cells. Due to the low 5-year survival rate, there is an urgent need to discover novel diagnostic markers and therapeutic targets. In recent years, microRNAs have been shown to play important roles in hematological malignancies by acting as tumor suppressors and oncogenes. MicroRNAs have the potential to be a breakthrough in the diagnosis and treatment of acute myeloid leukemia. In this review, we summarize the biology of microRNAs and discuss the relationships between microRNA dysregulation and acute myeloid leukemia in the following aspects: signaling pathways, the abnormal biological behavior of acute myeloid leukemia cells, the clinical application of microRNAs and competing endogenous RNA regulatory networks.

Abstract 3391: Dissecting intratumoral cell-cell interactions in myeloid reprogramming by single cell RNA-seq

Abstract Tumor-infiltrating myeloid cells are the most abundant leukocyte population within tumors. Molecular cues from the tumor microenvironment promote the differentiation of immature myeloid cells toward an immunosuppressive phenotype. However, the in situ dynamics of the transcriptional reprogramming underlying this process are poorly understood. Therefore, we applied single cell RNA-seq (scRNAseq) to computationally investigate the cellular composition and transcriptional dynamics of tumor and adjacent normal tissues from 4 early-stage non-small cell lung cancer (NSCLC) patients. Our scRNA-seq analyses identified 11,485 cells that varied in identity and gene expression traits between normal and tumor tissues. Among these, myeloid cell populations exhibited the most diverse changes between tumor and normal tissues, consistent with tumor-mediated reprogramming. Through trajectory analysis, we identified a differentiation path from CD14+ monocytes to M2 macrophages (monocyte-to-M2). This differentiation path was reproducible across patients, accompanied by increased expression of genes (e.g. MRC1/CD206, MSR1/CD204, PPARG, TREM2) with significantly enriched functions (Oxidative phosphorylation and P53 pathway) and decreased expression of genes (e.g. CXCL2, IL1B) with significantly enriched functions (TNFa signaling via NF-kB and inflammatory response). Our analysis further identified a co-regulatory network implicating upstream transcription factors (JUN, NFKBIA) in monocyte-to-M2 differentiation, and activated ligand-receptor interactions (e.g. SFTPA1-TLR2, ICAM1-ITGAM) suggesting intratumoral mechanisms whereby epithelial cells stimulate monocyte-to-M2 differentiation. Overall, our analysis identified the prevalent monocyte-to-M2 differentiation in NSCLC, accompanied by an intricate transcriptional reprogramming mediated by specific transcriptional activators and intercellular crosstalk involving ligand-receptor interactions. Citation Format: Qianqian Song, Gregory A. Hawkins, Leonard Wudel, Ping-Chieh Chou, Elizabeth Forbes, Ashok K. Pullikuth, Liang Liu, Guangxu Jin, Lou Craddock, Umit Topaloglu, Gregory Kucera, Stacey O’Neill, Edward A. Levine, Peiqing Sun, Kounosuke Watabe, Yong Lu, Martha A. Alexander-Miller, Boris Pasche, Lance D. Miller, Wei Zhang. Dissecting intratumoral cell-cell interactions in myeloid reprogramming by single cell RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3391.

Dissecting intratumoral myeloid cell plasticity by single cell RNA-seq.

Tumor‐infiltrating myeloid cells are the most abundant leukocyte population within tumors. Molecular cues from the tumor microenvironment promote the differentiation of immature myeloid cells toward an immunosuppressive phenotype. However, the in situ dynamics of the transcriptional reprogramming underlying this process are poorly understood. Therefore, we applied single cell RNA‐seq (scRNA‐seq) to computationally investigate the cellular composition and transcriptional dynamics of tumor and adjacent normal tissues from 4 early‐stage non‐small cell lung cancer (NSCLC) patients. Our scRNA‐seq analyses identified 11 485 cells that varied in identity and gene expression traits between normal and tumor tissues. Among these, myeloid cell populations exhibited the most diverse changes between tumor and normal tissues, consistent with tumor‐mediated reprogramming. Through trajectory analysis, we identified a differentiation path from CD14+ monocytes to M2 macrophages (monocyte‐to‐M2). This differentiation path was reproducible across patients, accompanied by increased expression of genes (eg, MRC1/CD206, MSR1/CD204, PPARG, TREM2) with significantly enriched functions (Oxidative phosphorylation and P53 pathway) and decreased expression of genes (eg, CXCL2, IL1B) with significantly enriched functions (TNF‐α signaling via NF‐κB and inflammatory response). Our analysis further identified a co‐regulatory network implicating upstream transcription factors (JUN, NFKBIA) in monocyte‐to‐M2 differentiation, and activated ligand‐receptor interactions (eg, SFTPA1‐TLR2, ICAM1‐ITGAM) suggesting intratumoral mechanisms whereby epithelial cells stimulate monocyte‐to‐M2 differentiation. Overall, our study identified the prevalent monocyte‐to‐M2 differentiation in NSCLC, accompanied by an intricate transcriptional reprogramming mediated by specific transcriptional activators and intercellular crosstalk involving ligand‐receptor interactions.

9: Progesterone promotes the expansion of proangiogenic immature myeloid cells and prevents their differentiation into inflammatory cells

Immature myeloid cells (IMCs) are bone marrow-derived cells that normally differentiate into granulocytes, macrophages, and dendritic cells (DCs) but expand in pathological conditions such as malignancy. DCs are antigen-presenting cells that regulate the immune response. We have shown that IMCs accumulate in the placenta and actively promote angiogenesis. IMCs peak in concentration during mid-pregnancy and their presence correlates with neonatal birthweight. Furthermore, labor and delivery are preceded by a decrease in IMCs and an increase in DCs populating the placenta. We hypothesized that progesterone may promote IMC population expansion while preventing their differentiation into inflammatory cells. We derived bone marrow cells from C57Bl6 pregnant mice. We isolated the CD11b+ myeloid subset by magnetic based immuno-separation, and cultured them in the presence of GMCSF and progesterone (10-7, 10-8, and 0 mM). After 5 days we analyzed cultures for the presence of Ly6CintLy6Ghi granulocytic IMCs, Ly6ChiLy6Gint monocytic IMCs and CD11c+MHCII+ DCs by flow cytometry. We also compared their presence in Lewis Lung Carcinoma tumors that were implanted subcutaneously in mice to that in placentas derived from pregnant mice. Progesterone treatment caused a dose dependent increase in granulocytic IMCs (n=3, p<0.05), accompanied by a concomitant decline in the monocytic IMCs (n=3, p<0.001). Importantly, these changes were paralleled by a decrease in DCs (n=3, p<0.05). When analyzing CD45+ hematopoietic cells in tumors and placentas, we detected a significant enrichment (P<0.01) of monocytic IMCs subpopulation in tumors compared to that in placentas. This was paralleled by a concomitant, more than 2-fold decrease (P<0.01) in granulocytic IMCs. Progesterone enhances proliferation and/or survival of placenta specific- granulocytic IMCs but not that of tumor specific- monocytic IMCs, in a dose dependent manner. Importantly, the differentiation of IMCs into DCs was abrogated by progesterone. We thus speculate that progesterone may play role in the maintenance of proangiogenic IMCs in the placenta. Inhibition of their differentiation into inflammatory cells such as DCs and neutrophils might explain, at least in part, the protective effect of progesterone in preventing preterm labor.

Sunitinib effectively reduces clonogenic acute myeloid leukemia cells in vitro

Background Acute myeloid leukemia (AML) is a clonal hematopoietic disorder characterized by enhanced proliferation and block in differentiation of immature myeloid cells. AML pathogenesis is attributed to several recurrent chromosomal abnormalities in nearly half of the AML cases while the rest of the AML cases show at least one or more molecular abnormalities. Activating mutations and/or high expression of receptor tyrosine kinase (RTK) family members (FLT3, cKIT) have been well documented in AML. Specific RTK inhibitors (TKI) have been developed for targeted therapy of AML and many are currently under study [1]. Sunitinib is a potent multi-TKI that has been FDA approved for renal carcinoma and imatinib resistant gastro intestinal tumors. Although some studies show the effect of Sunitinib on AML cell line growth, there has been no investigation of its effect on AML clonogenic cells [2]. In the current study, we investigated the effect of Sunitinib on clonogenic AML cells and normal hematopoietic progenitors in vitro.

Abstract 3649: Contribution of fatty acid accumulation to myeloid-derived suppressor cell function in cancer

Abstract MDSCs are a heterogeneous population of cells that consists of myeloid progenitor cells and immature myeloid cells (IMCs). In healthy individuals, IMCs that are generated in the bone marrow quickly differentiate into mature granulocytes, macrophages or dendritic cells (DCs). By contrast, under pathological conditions, such as cancer, a partial block in the differentiation of IMCs and activation of these cells results in the expansion of the population with potent immune suppressive activity – myeloid-derived suppressor cells. We investigated the mechanism of MDSC effect on immune system and focused our attention on possible role of fatty acids (FA) in this process. Recently, we found FA accumulation in dendritic cells (DCs) from tumor bearing mice or cancer patients. It was caused by increased uptake of extracellular lipids due to up-regulation of scavenger receptor A. FA accumulation in DCs resulted in the dysfunction of DCs. Pharmacological normalization of lipid abundance in DCs with an inhibitor of acetyl-CoA carboxylase restored the functional activity of DCs and substantially enhanced the effects of cancer vaccines. In parallel, we also observed dramatic lipid accumulation in MDSC from tumor bearing mice. Interestingly, MDSCs don't have up-regulation of scavenger receptor A, but instead have increased expression of fatty acid transporter FATP4. Over-expression of FATP4 in MDSCs induced lipid accumulation. This suggested that FATP4 may be involved in the accumulation of lipid in these cells. MDSCs with lipid overload exerted stronger inhibitory effect on immune cells than MDSC with normal lipid level. MDSCs treated with unsaturated FA linoleic acid displayed stronger inhibitory effect than MDSCs treated with saturated FA palmitic acid. Since MDSCs has strong oxidizing environment, the data suggested that oxidized FA may be involved in the inhibitory effect of MDSCs. Elucidation of this mechanism in detail will be helpful for the development of potential therapeutic targets regulating immune function in tumor-bearing hosts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3649. doi:10.1158/1538-7445.AM2011-3649

Identification and Characterization of 6-Benzylthioinosine as a Novel Myeloid Differentiation-Inducing Compound.

Acute myeloid leukemia (AML) is the most common form of leukemia in adults and despite advances in treatment the 5 year survival is less than 20–50% in adults and significantly lower in the elderly. The remarkable success in treating one relatively uncommon subset of AML, APL, with all trans-retinoic acid (ATRA) illustrates the great promise for agents with greater efficacy and less toxicity. Utilizing ATRA, the presumed cure of 75–85% of patients is possible. ATRA's remarkable success stems from the fact that AML is a disease characterized by the arrest of differentiation of immature myeloid cells. ATRA overcomes this block in differentiation by forcing leukemic cells to mature. Unfortunately, ATRA is not clinically useful for patients with AML that do not have the APL subtype. Through a compound library screen, we have recently identified several novel leukemia differentiation-inducing compounds structurally unrelated to previously described differentiation-inducers including a particularly promising compound, 6-benzylthioinosine (6BT). 6BT is a nucleoside analogue that induces monocytic differentiation of multiple AML cell lines as well as primary leukemic patient samples as measured by immunophenotyping, morphology, and NBT reduction. 6BT can induce terminal differentiation of leukemic cells as evidenced by complete prevention of colony formation in soft agar assays after pretreatment with low doses of 6BT (5μM) for 72 hours. In addition 6BT has potent in vivo activity. 6BT completely prevents subcutaneous tumor formation in myeloid leukemia mouse xenograft models in nude mice using HL-60 or MV4-11 leukemic cell lines (n=5) and significantly inhibits myeloid leukemia tumor growth in an HL-60 established tumor xenograft model (n=5). In the established tumor model at the end of the 4 week study period, tumors were significantly smaller after 6BT treatment as compared to vehicle treated mice (0.16g +/− 0.05g vs 0.73g +/− 0.28g). Evidence of 6BT-mediated in vivo differentiation exists as 92% of the tumor cells expressed the mature myeloid cell surface marker CD11b as compared to only 16% in the vehicle control after 4 weeks. Early studies suggest 6BT partially depletes purine nucleotide stores leading to growth inhibition and subsequent myeloid differentiation. Treatment of HL-60 cells with 6BT (5μM) leads to rapid depletion of approximately 75% of ATP stores within 24 hours. The depletion of ATP is not due to the differentiation process itself as after 6BT treatment HL-60 cells contain 24.6% +/− 2.8 of the ATP levels of vehicle treated cells compared to 91.2% +/− 7.2 in ATRA (1μM) treated cells. Preliminary studies also indicate that 6BT exhibits relatively low toxicity with an LD50 at 5 days greater than 100μM in human umbilical vein endothelial cells, mouse embryonic fibroblasts, and primary human lymphocytes. The low toxicity is likely related to its unique chemical structure that prevents its uptake into most mammalian cells as it acts as an inhibitor of one of the main nucleoside transporters, ent1. It is known that leukemic cells can express nucleoside transporters that are not widely expressed by other cell types. As differentiation therapy has already been demonstrated in the case of ATRA to significantly improve the prognosis of patients with AML, further characterization of 6BT could lead to a novel AML therapy that is more efficacious, less toxic, and better tolerated especially for elderly patients.

Leukomogenic factors downregulate heparanase expression in acute myeloid leukemia cells

Heparanase is a heparan sulfate-degrading endoglycosidase expressed by mature monocytes and myeloid cells, but not by immature hematopoietic progenitors. Heparanase gene expression is upregulated during differentiation of immature myeloid cells. PML-RARα and PLZF-RARα fusion gene products associated with acute promyelocytic leukemia abrogate myeloid differentiation and heparanase expression. AML-Eto, a translocation product associated with AML FAB M2, also downregulates heparanase gene expression. The common mechanism that underlines the activity of these three fusion gene products involves the recruitment of histone deacetylase complexes to specific locations within the DNA. We found that retinoic acid that dissociates PML-RARα from the DNA, and which is used to treat acute promyelocytic leukemia patients, restores heparanase expression to normal levels in an acute promyelocytic leukemia cell line. The retinoic acid effects were also observed in primary acute promyelocytic leukemia cells and in a retinoic acid-treated acute promyelocytic leukemia patient. Histone deacetylase inhibitor reverses the downregulation of heparanase expression induced by the AML-Eto fusion gene product in M2 type AML. In summary, we have characterized a link between leukomogenic factors and the downregulation of heparanase in myeloid leukemic cells.

Role of immature myeloid Gr-1+ cells in the development of antitumor immunity.

One of the mechanisms by which tumor cells evade the immune system is the lack of proper antigen-presenting cells. Improvement in host immunity against tumor cells can be achieved by promoting the differentiation of dendritic cells (DCs) from immature myeloid cells (Gr-1(+)Ly-6C(+)F4/80(+)) that accumulate in the bone marrow and lymphoid organs of mice with large tumor burdens. The enriched immature myeloid cells inhibit T-cell proliferation and tumor-specific T-cell response, which can be reversed by the differentiation of immature myeloid cells or depletion of F4/80(+) cells. Sorted Gr-1(+)/F4/80(+) immature myeloid cells differentiated into CD11c(+) cells that express CD80 and I-A/I-E (MHC class II) in the presence of recombinant murine granulocyte macrophage colony-stimulating factor (GM-CSF). Furthermore, intratumoral gene delivery of GM-CSF not only promoted the differentiation of carboxyfluoroscein succinimidyl ester-labeled immature myeloid cells into CD11c(+) cells with the characteristics of mature DCs (CD80(+), I-A/I-E(+)) but also enhanced innate natural killer and adaptive cytolytic T-cell activities in mice treated with interleukin (IL)-12 and anti-4-1BB combination therapy. More importantly, intratumoral delivery of GM-CSF and IL-12 genes in combination with 4-1BB costimulation greatly improved the long-term survival rate of mice bearing large tumors and eradicated the untreated existing hepatic tumor. The results suggest that inducing the maturation of immature myeloid cells, thus preventing their inhibitory activity and enhancing their antigen-presenting capability, by GM-CSF gene therapy is a critically important step in the development of effective antitumor responses in hosts with advanced tumors.

Hyperactivation of STAT3 is involved in abnormal differentiation of dendritic cells in cancer.

Abnormal differentiation of myeloid cells is one of the hallmarks of cancer. However, the molecular mechanisms of this process remain elusive. In this study, we investigated the effect of tumor-derived factors on Janus kinase (Jak)/STAT signaling in myeloid cells during their differentiation into dendritic cells. Tumor cell conditioned medium induced activation of Jak2 and STAT3, which was associated with an accumulation of immature myeloid cells. Jak2/STAT3 activity was localized primarily in these myeloid cells, which prevented the differentiation of immature myeloid cells into mature dendritic cells. This differentiation was restored after removal of tumor-derived factors. Inhibition of STAT3 abrogated the negative effects of these factors on myeloid cell differentiation, and overexpression of STAT3 reproduced the effects of tumor-derived factors. Thus, this is a first demonstration that tumor-derived factors may affect myeloid cell differentiation in cancer via constitutive activation of Jak2/STAT3.

Function of RARalpha during the maturation of neutrophils.

The retinoic acid receptor alpha gene is the target of chromosomal rearrangements in all cases of acute promyelocytic leukemia (APL). This recurrent involvement of RARalpha in the pathogenesis of APL is likely to reflect an important role played by this receptor during the differentiation of immature myeloid cells to neutrophils. RARalpha is a negative regulator of promyelocyte differentiation when not complexed with RA, and stimulates this differentiation when bound to RA. Since RARs are dispensable for the generation of mature neutrophils, their role thus appears to be to modulatory, rather than obligatory, for the control of neutrophil differentiation. In vitro, retinoic acid is also a potent inducer of neutrophil cell fate, suggesting that it might play a role in the commitment of pluripotent hematopoietic progenitors to the neutrophil lineage. Thus, the APL translocations target an important regulator of myeloid cell differentiation.

Studies of the proliferation and differentiation of immature myeloid cells in vitro. II. Acute myelogenous leukemia.

Immature myeloid cells prepared from patients with AML were placed into suspension culture and studied over a 2-week period. Cell numbers usually fell and viability was not well maintained. Some degree of differentiation was observed in most cultures. Considering these observations, together with those derived from parallel studies of immature CML cells, the data suggest that AML cells are more dependent than CML cells on environmental conditions for the maintenance of cell viability and proliferation. This is especially the case for AML cells obtained at the time of initial diagnosis. On the other hand, AML cells and myeloid blastic crisis CML cells are similar with respect to their apparent greater ability to differentiate in vitro than in vivo. The addition of recombinant hemopoietins to the suspension cultures of AML cells is associated with either increased proliferation or differentiation but not both. The cells of different patients respond differently to the different hemopoietins and the different hemopoietins produce different affects in cultures of cells obtained from the same patient.

Studies of the proliferation and differentiation of immature myeloid cells in vitro. I. Chronic myelogenous leukemia.

Immature cells prepared from chronic myelogenous leukemic patients in the chronic or blastic phase of their disease were placed into suspension culture in vitro and studied over a 2-week period. Regardless of the status of the disease, the immature cells proliferated and maintained good viability. In contrast, evolution of the disease toward blastic crisis was associated with a progressive loss in the ability of the immature cells to differentiate. Cell proliferation and differentiation were independent of added growth factors.