Accumulation Of Myeloid Precursors Research Articles

Spleen mediates a distinct hematopoietic progenitor response supporting tumor-promoting myelopoiesis.

Cancer progression is associated with alterations of intra- and extramedullary hematopoiesis to support a systemic tumor-promoting myeloid response. However, the functional specialty, mechanism, and clinical relevance of extramedullary hematopoiesis (EMH) remain unclear. Here, we showed that the heightened splenic myelopoiesis in tumor-bearing hosts was not only characterized by the accumulation of myeloid precursors, but also associated with profound functional alterations of splenic early hematopoietic stem/progenitor cells (HSPCs). With the distinct capability to produce and respond to granulocyte-macrophage CSF (GM-CSF), these splenic HSPCs were "primed" and committed to generating immunosuppressive myeloid cells. Mechanistically, the CCL2/CCR2 axis-dependent recruitment and the subsequent local education by the splenic stroma were critical for eliciting this splenic HSPC response. Selective abrogation of this splenic EMH was sufficient to synergistically enhance the therapeutic efficacy of immune checkpoint blockade. Clinically, patients with different types of solid tumors exhibited increased splenic HSPC levels associated with poor survival. These findings reveal a unique and important role of splenic hematopoiesis in tumor-associated myelopoiesis.

Abstract A38: Gene expression profile of the Wnt signaling pathway in mesenchymal stromal cells from acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a heterogeneous hematologic disease characterized by proliferation and accumulation of myeloid precursors in the bone marrow, decrease in apoptosis level, and differentiation arrest of these cells. Although there are several studies in the area, events related to the beginning of disease as well as its progression are still unknown. It is believed that malignant transformation in normal hematopoietic stem cells (HSC) can give rise to a leukemic stem cell (LSC) and this transformation could be related to changes in mesenchymal stromal cells (hMSC) signaling. Previous studies showed that mesenchymal stromal cells from acute myeloid leukemia patients (hMSC-AML) have a common molecular signature, compared to healthy donors' hMSCs (hMSC-HD), and these differentially expressed genes could be related to malignant transformation. Among the 55 differentially expressed genes, BMP4 has its expression decreased in hMSC-AML, and this decrease could be regulated by the Wnt pathway. In this context, the aim of this work was to evaluate the gene expression profile of the Wnt pathway in hMSC-AML. To address this hypothesis, we used a PCR Array assay approach to evaluate the gene expression profile of hMSC-AML compared to hMSC-HD. For this purpose, bone marrow samples from healthy donors (HD) and patients diagnosed with AML previously classified into subtypes were placed in culture. For PCR array we used 4 samples from different subtypes of AML and compared with two pools of HD. After the 3rd passage, total RNA from MSCs cultures was obtained using RNeasy Mini Kit according to the manufacturer's instructions (Qiagen). After that, the RNA was processed according to the Human WNT Signaling Pathway RT2 ProfilerTM PCR Array (Qiagen) according to the manufacturer's protocols. Data were analyzed using GeneGlobal data analysis center (Qiagen) and differentially expressed genes with ≥ 1.5 fold-change was used as a criterion to define overexpression or downregulation. To confirm the results we performed real-time PCR with a large number of patients with different subtypes. The pathway analysis and related processes were obtained using the MetaCoreTM software. To confirm the protein levels of some differentially expressed genes, Western blot analysis was performed. The results obtained in PCR array assay showed changes in the gene expression profile of the Wnt pathway when comparing hMSC-AML to hMSC-HD. Among the 84 genes presented on the PCR Array, 26 genes were found to be differentially expressed. To confirm the results, real-time PCR with some genes was performed (KREMEN1, NKD1, and PRICKLE1- overexpressed and TCF7, LEF, and PORCN downregulated). The results confirmed that the WNT pathway is altered in all hMSC-AML from AML patients instead of subtypes. TCF7 and LEF are transcription factors, and the decrease of their expression could be related to the BMP4 regulation. To verify if these proteins are differentially expressed in hMSC-AML compared to hMSC-HD, we also performed Western blot analysis. The results confirmed a decrease in these transcription factors in hMSC-AML. Moreover, we performed in silico analysis and identified four consensus sites Tcf/Lef in 2kb from BMP4 promoter region, suggesting that they could regulate BMP4. Altogether, these results suggest that the WNT signaling pathway is changed in hMSC-AML from AML patients instead of subtypes and this pathway regulation could be related to the decrease of BMP4 in hMSC-AML. Citation Format: Pedro Azevedo, Nathalia Oliveira, Eliana Abdelhay, Renata Binato. Gene expression profile of the Wnt signaling pathway in mesenchymal stromal cells from acute myeloid leukemia [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A38.

AML Mesenchymal Stem Cells Signature

Background: Acute myeloid leukemia(AML) is a heterogeneous hematological disease characterized by proliferation and accumulation of myeloid precursors in the bone marrow, decrease of apoptosis level and differentiation arrest of these cells. The French, American and British group of leukemia experts (FAB) divided acute myeloid leukemias into eight subtypes based on the characteristics and behavior of leukocytes and the in cell differentiation blockage. Although several studies in the area, events related to the beginning of disease as well as its progression is still unknown. It is believed that malignant transformation in normal Hematopoietic Stem Cells (HSC) can give rise a Leukemic Stem Cell (LSC) and this transformation could be related to changes in Mesenchymal stromal cells (MSC) signaling. In this context, the aim of this work is to analyze the role of MSCs in the leukemic transformation of hematopoietic stem cell. To address this hypothesis we used ChipArray approaches to evaluate the gene expression profile of MSC from AML patients compared to MSC from Healthy donors (HD).Methods: For this purpose, bone marrow(BM) samples from HD and patients diagnosed with AML previously classified into subtypes were placed in culture. We used 7 samples from different subtypes of AML (four from subtype M2, two from M3 and one from M4/M5) and compared with two pools of HD. After 3rd passage, total RNA from MSCs cultures were obtained using RNeasy Mini Kit according to the manufacturer’s instructions (QIAGEN). After that, RNA was processed according to GeneChip whole transcription (WT) sense target-labeling assay (Affymetrix) and hybridized to GeneChip human gene 1.0 ST array (Affymetrix) washed and stained according to the manufacturer’s protocols. Data were analyzed using Partek® software and differentially expressed genes with ³ 2 fold-change was used as criteria to define overexpression or downregulation.To confirm the array assay we performed real-time PCR with a large number of patients with different subtypes. Pathway analysis and related processes was obtained using the Ingenuity Pathways Analysis (IPA) and MetaCoreTM softwares.Results: The results obtained in ChipArray assay showed that there is a common molecular signature to all MSCs AML patients compared to donors. Fifty five genes differentially expressed were found. To confirm the results obtained in ChipArray real-time PCR with some genes was performed with more patients (CCL2, SPON2, MUC12 - overexpressed and MMP16, BMP4, CLDN1, SDC2 and OPN downregulate). The results confirmed the commom molecular signature in all AML patients instead of subtypes.These genes are involved in processes of cellular adhesion, differentiation of osteoclast and Wnt pathway. We further carried out another ChipArray assay with another samples. At this time, an unsupervised analysis with the 55 differentially expressed genes was done to verify if the samples agrouping in clusters that separate AML patients from HD. The results confirmed that this molecular signature is capable to distinguish patients from HD. Changes in MSC signaling could be related to malignant transformation of HSC and some of these genes found in ChipArray are secreted proteins that could affect HSC. To verify if these secreted proteins are reallly differentially expressed in AML patiens we performed Western blot assay with plasma samples from AML patients and HD. The secreted proteins seleted were CCL2 and BMP4 (overexpressed and downregulated in AML patients, respectively). The results confirmed the increase of CCL2 protein and decrease of BMP4 protein in AML patients plasma. In Acute Lymphoblastic Leukemia (ALL), CCL2 was able to increase the adhesion of ALL cells to MSC and to promote survival and proliferation of MSC.BMP4 has been described as an important component produced by the hematopoietic microenvironment that regulates both HSC number and function. Moreover, BMP4 has also implicated BMP4 in homing and engraftment of human hematopoietic stem/progenitor cells.Conclusions: With these results, we could start to elucidate the participation of AML-MSCs in the leukaemogenic process. Altogether, both CCL2 and BMP4 produced and secreted by MSC from BM microenvironment seems to be important for the biology of AML cells.Financial support: CNPq, FAPERJ, INCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Proposed Steroid Mobilization Test to Separate Margination From Marrow Suppression and to Determine Drug Cessation for Safety in Lenalidomide-Induced Neutropenia in Non-Del(5q) MDS Low and INT-1 Patients

AbstractAbstract 4942 BackgroundThe mechanism of lenalidomide-induced neutropenia, a common reason for cessation of therapy, has recently been shown to involve down-regulation of transcription factor PU. 1, resulting in granulocyte maturation arrest and accumulation of myeloid precursors (Pal S, et al. Blood 2010; 115(3):605–614). Data have also exhibited a correlation between cytopenias and response to therapy in del(5q) patients alone, revealing lenalidomide-induced neutropenia or thrombocytopenia as a predictor of transfusion independence (Sekeres MA, et al. J Clin Oncol 2008. 26(36):5943–5949). Because preliminary data from our phase I/II optimal dose study in non-5q- low and int-1 risk patients has shown a lower degree of both neutropenia and thrombocytopenia than that observed in patients treated with the same 10mg/d dose for del(5q) disease, we hypothesized that the comparably poor response of non-del(5q) patients may be related to the lesser degree of associated cytopenias in the setting of a suboptimal dose. Anticipating the limitation of optimal dosing by neutropenias, we set out to determine whether we could avoid withholding the drug for safety by demonstrating the patients’ ability to mobilize neutrophils with corticosteroid administration, in contrast to limitation by true marrow suppression. MethodsIn the course of our phase I/II optimal dose study, we examined whether cytopenias observed with high dose lenalidomide may be attributed to margination rather than entirely to true marrow suppression. Eight patients with low or int-1 risk non-del(5q) MDS received lenalidomide at an increased dose of 15mg to 20mg, administered for 21d of each 28d cycle. A baseline absolute neutrophil count along with a pre-therapy response of peripheral granulocytes to prednisone was established. Patients whose absolute neutrophil count decreased during therapy to less than 50 percent of the pre-study baseline underwent a granulocyte mobilization test, receiving a 1mg/kg dose of prednisone with a complete blood count drawn both before and twenty four hours after administration to assess change in absolute neutrophil count. In patients with a positive steroid test, we proceeded with therapy. In those with a negative steroid test, lenalidomide was held until recovery of ANC. This study is listed on clinicaltrials. gov under the identifier NCT00699842. ResultsOf the 8 patients enrolled in the study, 4 (50%) of these patients developed neutropenia with an absolute neutrophil count (ANC) falling to less than 50% of their pre-treatment baseline. Of these patients, 2 had a marked response to the prednisone granulocyte mobilization test, with 24-hour post-steroid ANC levels approaching and in most cases exceeding the patients' baseline values (Fig. 1). A third patient had a less notable and delayed response, but did exhibit an increase in ANC from less than 50% to within 5% of pre-study baseline. The fourth patient responded minimally and was taken off of the study to receive G-CSF (Fig. 3). [Display omitted] [Display omitted] [Display omitted] There was no increase in neutropenic fever during the course of the study, although the observation was limited to only 8 patients. ConclusionsDetermining increments of ANC (31000) following steroid administration in 3 (75%) of the studied patients who developed marked neutropenia in the setting of an elevated dose of lenalidomide demonstrated a return of the absolute neutrophil count to pre-treatment levels. The drug was continued on these patients with no neutropenic fever nor other adverse event. The results of the study provide evidence that margination of neutrophils is a contributing mechanism of lenalidomide-induced neutropenia, providing an alternative explanation to true marrow suppression as the primary etiology of observed cytopenias. This may allow for increased tolerance and determination of the optimal dose of lenalidomide in the treatment of patients with non-5q myelodysplastic syndrome. Disclosures:Besa:Celgene: Research Funding. Quan:Celgene: Employment.

HIV-Induced Abnormalities in Myelopoiesis and their Recovery Following Antiretroviral Therapy

HIV-1 infection is associated with hematologic abnormalities including defective myelopoiesis. Most studies of myelopoiesis during HIV-1 infection were performed using unfractionated bone marrow-derived mononuclear cells, thus resulting in significant inter-individual variability in the numbers of cultured precursors. Here we evaluated the myelopoietic potential of circulating CD34+ progenitors by conducting a longitudinal analysis of antiretroviral therapy (ART)-induced changes of colony forming units-granulocyte and monocyte (CFU-GM) growth. Twelve HIV-infected individuals were studied longitudinally before and after initiation of ART (i.e. at a time when plasma HIV-RNA levels had become undetectable); thirty-one HIV-uninfected healthy individuals were enrolled as controls. Peripheral blood-derived CD34+ progenitors were purified by immunomagnetic sorting, and cultured in methylcellulose-based medium containing stem cell factor, granulocyte-monocyte colony-stimulating factor and interleukin-3. ART-induced changes in the proportion of CD8+ T cells expressing surface HLA-DR were also evaluated. We found that CFU-GM levels were increased in untreated HIV-infected individuals when compared to uninfected controls but declined significantly following ART, in parallel with the decline of HIV-RNA levels in plasma and with the down-regulation of HLA-DR expression on CD8+ T cells. These findings suggest that, in untreated HIV-infected individuals, chronic inflammation and/or immune activation is associated with defective myelopoiesis and accumulation of myeloid precursors. ART-induced suppression of HIV-1 replication is associated with normalization of CFU-GM levels.

Tumor Exosomes Inhibit Differentiation of Bone Marrow Dendritic Cells

The production of exosomes by tumor cells has been implicated in tumor-associated immune suppression. In this study, we show that, in mice, exosomes produced by TS/A murine mammary tumor cells target CD11b(+) myeloid precursors in the bone marrow (BM) in vivo, and that this is associated with an accumulation of myeloid precursors in the spleen. Moreover, we demonstrate that TS/A exosomes block the differentiation of murine myeloid precursor cells into dendritic cells (DC) in vitro. Addition of tumor exosomes at day 0 led to a significant block of differentiation into DC, whereas addition at later time points was less effective. Similarly, exosomes produced by human breast tumor cells inhibited the differentiation of human monocytes in vitro. The levels of IL-6 and phosphorylated Stat3 were elevated 12 h after the tumor exosome stimulation of murine myeloid precursors, and tumor exosomes were less effective in inhibiting differentiation of BM cells isolated from IL-6 knockout mice. Addition of a rIL-6 to the IL-6 knockout BM cell culture restored the tumor exosome-mediated inhibition of DC differentiation. These data suggest that tumor exosome-mediated induction of IL-6 plays a role in blocking BM DC differentiation.

Tumor exosomes inhibit differentiation of bone marrow dendritic cells (49.14)

Abstract The production of exosomes by tumor cells has been implicated in tumor-associated immune suppression. Here we show that, in mice, exosomes produced by TS/A murine mammary tumor cells target CD11b+Gr-1+ myeloid precursors in the bone marrow in vivo and that this is associated with an accumulation of myeloid precursors in the spleen. Moreover, we demonstrate that TS/A exosomes block differentiation of murine myeloid precursor cells into dendritic cells in vitro. Addition of tumor exosomes at day 0 led to a complete block of differentiation into dendritic cells, whereas addition at later time points was less effective. Similarly, exosomes produced by human breast tumor cells inhibited differentiation of human monocytes in vitro. The levels of IL-6 and phosphorylated Stat3 were elevated 12 h after tumor exosome stimulation of murine myeloid precursors, and tumor exosomes were less effective in inhibiting differentiation of bone marrow cells isolated from IL-6 knockout mice. These data suggest that tumor exosome-mediated induction of IL-6 plays a role in blocking bone marrow dendritic cell differentiation.

Mice expressing a neutrophil elastase mutation derived from patients with severe congenital neutropenia have normal granulopoiesis

AbstractSevere congenital neutropenia (SCN) is a syndrome characterized by an isolated block in granulocytic differentiation and an increased risk of developing acute myeloid leukemia (AML). Recent studies have demonstrated that the majority of patients with SCN and cyclic neutropenia, a related disorder characterized by periodic oscillations in the number of circulating neutrophils, have heterozygous germline mutations in the ELA2 gene encoding neutrophil elastase (NE). To test the hypothesis that these mutations are causative for SCN, we generated transgenic mice carrying a targeted mutation of theirEla2 gene (“V72M”) reproducing a mutation found in 2 unrelated patients with SCN, one of whom developed AML. Expression of mutant NE mRNA and enzymatically active protein was confirmed. Mice heterozygous and homozygous for the V72M allele have normal numbers of circulating neutrophils, and no accumulation of myeloid precursors in the bone marrow was observed. Serial blood analysis found no evidence of cycling in any of the major hematopoietic lineages. Rates of apoptosis following cytokine deprivation were similar in wild-type and mutant neutrophils, as were the frequency and cytokine responsiveness of myeloid progenitors. The stress granulopoiesis response, as measured by neutrophil recovery after cyclophosphamide-induced myelosuppression, was normal. To define the leukemogenic potential of V72M NE, a tumor watch was established. To date, no cases of leukemia have been detected. Collectively, these data suggest that expression of V72M NE is not sufficient to induce an SCN phenotype or leukemia in mice.

Altered Myeloid Development and Acute Leukemia in Transgenic Mice Expressing PML-RARα Under Control of Cathepsin G Regulatory Sequences

AbstractAcute promyelocytic leukemia (APML) is characterized by abnormal myeloid development, resulting an accumulation of leukemic promyelocytes that are often highly sensitive to retinoic acid. A balanced t(15; 17) (q22; q21) reciprocal chromosomal translocation is found in approximately 90% of APML patients; this translocation fuses the PML gene on chromosome 15 to the retinoic acid receptor α (RARα) gene on chromosome 17, creating two novel fusion genes, PML-RARα and RARα-PML. The PML-RARα fusion gene product, which is expressed in virtually all patients with t(15; 17), is thought to play a direct role in the pathogenesis of APML. To determine whether PML-RARα is sufficient to cause APML in an animal model, we used the promyelocyte-specific targeting sequences of the human cathepsin G (hCG) gene to direct the expression of a PML-RARα cDNA to the early myeloid cells of transgenic mice. Mice expressing the hCG–PML-RARα transgene were found to have altered myeloid development that was characterized by increased percentages of immature and mature myeloid cells in the peripheral blood, bone marrow, and spleen. In addition, approximately 30% of transgene-expressing mice eventually developed acute myeloid leukemia after a long latent period. The splenic promyelocytes of mice with both the nonleukemic and leukemic phenotypes responded to all-trans retinoic acid (ATRA) treatment, which caused apoptosis of myeloid precursors. Although low-level expression of the hCG–PML-RARα transgene is not sufficient to directly cause acute myeloid leukemia in mice, its expression alters myeloid development, resulting in an accumulation of myeloid precursors that may be susceptible to cooperative transforming events.