Expression Of PU Research Articles

PU.1 induces tumor-associated macrophages promoting glioma progression through BTK-mediated Akt/mTOR pathway activation.

Glioma, the most common primary malignant brain tumor, is characterized by infiltrating immune cells that contribute to tumor progression and therapeutic resistance. Tumor-associated macrophages (TAMs) constitute a significant proportion of these infiltrating immune cells and have been implicated in glioma progression. However, the underlying molecular mechanisms by which TAMs promote glioma progression remain elusive. In this study, we investigated the role of PU.1, a crucial transcription factor involved in myeloid cell development, in glioma-associated macrophage polarization and activation. First, bioinformatics and analysis of clinical glioma samples demonstrated a positive correlation between PU.1 expression in TAMs and disease severity. Further experiments using in vitro coculture systems revealed that the expression of PU.1 is increased in glioma cells vs. control cells. Importantly, PU.1-overexpressing macrophages exhibited a protumorigenic phenotype characterized by enhanced migration, invasion, and proliferation. Mechanistically, we found that PU.1-induced activation of the Bruton tyrosine kinase (BTK) signaling pathway led to Akt/mTOR pathway activation in macrophages, which further enhanced their protumorigenic functions. Furthermore, pharmacological inhibition of the BTK or Akt/mTOR pathway reversed the protumorigenic effects of macrophages in vitro and impaired their ability to promote glioma progression in vivo. In conclusion, our study elucidates a novel mechanism by which PU.1 induces the polarization and activation of TAMs in the glioma microenvironment. We highlight the significance of BTK-mediated Akt/mTOR pathway activation in driving the protumorigenic functions of TAMs. Targeting PU.1 and its downstream signaling pathways in TAMs may provide a promising therapeutic strategy to suppress glioma progression and improve patient outcomes.

<i>JMJD3</i> Exerts Oncorepressor Activity in Acute Promyelocytic Leukemia by Promoting PU.1 Expression

All-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL) has been the most famous differentiation induction therapy during which the expression of PU.1, a key transcription factor (TF) for myeloid lineage determination in normal hematopoiesis is restored. In our previous studies, we found a stress-inducible H3K27 demethylase, JMJD3, to directly upregulate PU.1 expression to promote myeloid commitment during normal myelopoiesis. In addition, JMJD3 acts as an oncorepressor and plays a critical regulatory role in the initiation and progression of malignant hematopoiesis. In this study, we further resolved the relationship between JMJD3 and PU.1 in APL therein JMJD3 exerts oncorepressor activity via promoting PU.1 expression.

Abstract 1594: SMARCA2 (BRM) degraders promotes differentiation and inhibit proliferation in AML models

Abstract Dysregulated cellular differentiation is a major pathological feature of myeloid malignancies such as acute myeloid leukemia (AML). Targeting cellular differentiation programs has emerged as a novel therapeutic approach to treat patients with AML. Advantages of such differentiation therapy may include fewer systemic side-effects as well as opportunities to target leukemic stem cells (LSCs) and a broader range of clonal populations, likely resulting in lower frequencies of resistance and relapse in AML patients. The success of ATRA and decitabine in subsets of AML patients has proven that inducing differentiation can play a critical role in long-term durable responses. More agents targeting epigenetic regulators have been increasingly studied as differentiation inducers, including LSD1, DNMT1, Menin, and BET inhibitors. Recently, targeting SWI/SNF chromatin remodeling complexes has also been shown to regulate key leukemic gene expression signatures and induce AML differentiation. Small molecule inhibitors as well as gene knockdown for ATP-dependent SWI/SNF subunits SMARCA2 (BRM) and SMARCA4 (BRG1) are associated with re-direction of oncogenic transcriptional regulation to drive cellular differentiation and apoptosis in AML models. We have previously described the activity of highly potent, bispecific SMARCA2 degraders that efficiently promote SMARCA2 protein degradation in preclinical models. In the present study, we investigated the effects of our SMARCA2 selective degraders in AML models. Treatment with SMARCA2 degraders significantly inhibits AML cell line proliferation in vitro with IC50 ranging from 10 to 50 nM. In the SMARCA2 degrader treated cells, expression of PU.1 (SPI1), a key transcription factor in myeloid leukemias, was downregulated. In an in vivo OCI-AML3 xenograft model, treatment with a SMARCA2 selective degrader showed moderate tumor growth inhibition accompanied by robust increases in monocytic maturation markers CD11b and CD14. Further analyses of SMARCA2 degrader effects on global transcriptome and AML immunophenotypes as well as combination effects with other therapies are currently in progress. These findings highlight the potential of SMARCA2 degraders to target AML differentiation blocks and to improve the effectiveness of other therapeutic agents such as decitabine and venetoclax in AML patients. Citation Format: Anjana Agarwal, Olusola Peace Osinubi, Komali Vykuntam, Norman Fultang, Neha Bhagwat, Diane Heiser, Kris Vaddi, Koichi Ito, Peggy Scherle. SMARCA2 (BRM) degraders promotes differentiation and inhibit proliferation in AML models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1594.

JMJD3 Exerts Oncorepressor Activity in Acute Promyelocytic Leukemia by Promoting PU.1 Expression

All-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL) has been the most famous differentiation induction therapy during which the expression of PU.1, a key transcription factor (TF) for myeloid lineage determination in normal hematopoiesis is restored. In our previous studies, we found a stress-inducible H3K27 demethylase, JMJD3, to directly upregulate PU.1 expression to promote myeloid commitment during normal myelopoiesis. In addition, JMJD3 acts as an oncorepressor and plays a critical regulatory role in the initiation and progression of malignant hematopoiesis. In this study, we further resolved the relationship between JMJD3 and PU.1 in APL therein JMJD3 exerts oncorepressor activity via promoting PU.1 expression.

PU.1 negatively regulates tumorigenesis in non-small-cell lung cancer.

PU.1 is a key transcription factor that modulates hematopoietic cell differentiation and is involved in various physiological and pathological processes. PU.1 has been described to have multiple roles in a diverse range of cancers, but its contribution in non-small-cell lung cancer (NSCLC) has not been clearly elucidated. Fifty pairs of lung adenocarcinoma (LUAD) tissues and paraneoplastic tissues were collected. RT-qPCR assay was used to test PU.1 expression. Expression of PU.1 in LUAD cell lines and control cell lines was detected by RT-qPCR, and the role of PU.1 in LUAD was investigated by in vitro experiment. Levels of the major proteins in the apoptotic pathway were also detected by Western blot. The expression of PU.1 was remarkably downregulated in LUAD. Overexpression of PU.1 impaired the viability of LUAD cells as well as their metastatic function. In addition, PU.1 promoted apoptosis of LUAD cells by decreasing Bcl2 and increasing Bax/Bak1 expression. PU.1 plays an inhibitory role in LUAD, mainly promoting the apoptosis of LUAD cells.

Neuroinflammation after ischemic stroke involves INPP5D expression mediated by the TMPO-AS1-PU.1 complex

ABSTRACT Objectives This study aims to explore the role of lncRNA TMPO-AS1 in ischemic stroke and corresponding mechanism. Methods Adult male C57BL/6 J mice were subjected to a middle cerebral artery occlusion (MCAO) model of ischemic stroke, then TMPO-AS1 shRNA lentivirus were injected into ipsilateral striatum of mice. The neurological score and cerebral infarction volume were evaluatedHypoxia/glucose deprivation/reoxygenation (OGD/R)-induced BV2 cells were transfected with TMPO-AS1 shRNA (sh-TMPO-AS1) or together with pcDNA-INPP5D, as well as transfected with sh-PU.1 or together with pcDNA-INPP5D, then TMPO-AS1 level, the expression of PU.1 and INPP5D proteins, the secretion of inflammatory factors (TNF-α, IL-6 and IL-1β), the levels of iNOS, CD68,Arg1 and CD206 mRNA were detected. RIP and PNA-pull down assays were used to detect the binding of TMPO-AS1 and PU.1, luciferase reporter gene and chromatin immunoprecipitation (ChIP) assays were used to detect the binding activity of PU.1 and INPP5D. Results TMPO-AS1 level was increased in peripheral blood of ischemic stroke patients , brain tissues of MCAO/R model mice and OGD/R-induced BV2 cells. TMPO-AS1 interference inhibited the inflammation of OGD/R-induced BV2 cells. TMPO-AS1 also enhanced the nuclear accumulation of PU.1 by binding to the transcription factor PU.1, and promoted the transcriptional activation of INPP5D. The anti-inflammatory effects of TMPO-AS1 interference were reversed by INPP5D overexpression. In addition, TMPO-AS1 interference improved the infarct volume of MCAO mice, and improved sensorimotor and cognitive functions. Conclusion INPP5D underexpression mediated by TMPO-AS1-PU.1 complex alleviated neuroinflammation after ischemic stroke.

A comprehensive study of immune function and immunophenotyping of white blood cells from β-thalassaemia/HbE patients on hydroxyurea supports the safety of the drug.

Hydroxyurea (HU) (hydroxycarbamide) is used as a therapeutic option in β-thalassaemia to increase fetal haemoglobin, which results in a reduced requirement for blood transfusion. However, a potential serious adverse effect of HU is neutropenia. Abnormal neutrophil maturation and function in β-thalassaemia/HbE patients are well documented. This raises questions about the effect of the drug with regards to the immune response these patients. This study investigated the effects of HU treatment on both innate and adaptive immunity in a cross-sectional study of 28 β-thalassaemia/HbE patients who had received HU treatment (BE+HU) as compared with 22 β-thalassaemia/HbE patients who had not received HU (BE-HU) and 26 normal subjects. The expression of PU.1 and C/EBPβ, transcription factors, which are associated with neutrophil maturation, was significantly reduced in BE+HU patients as compared with BE-HU patients and normal subjects. Interestingly, C3bR expression on neutrophils and their oxidative burst activity in BE+HU were restored to close to normal levels when compared with BE-HU. There was no observed effect of HU on monocytes, myeloid derived suppressor cells (both granulocytic and monocytic subsets), CD4+ T cells, CD8+ T cells, complement levels and serum immunoglobulin levels in this study. The full immunophenotyping analysis in this study indicates that HU therapy in β-thalassaemia/HbE patients does not significantly compromise the immune response.

PU.1 inhibition attenuates atrial fibrosis and atrial fibrillation vulnerability induced by angiotensin-II by reducing TGF-β1/Smads pathway activation.

Fibrosis serves a critical role in driving atrial remodelling‐mediated atrial fibrillation (AF). Abnormal levels of the transcription factor PU.1, a key regulator of fibrosis, are associated with cardiac injury and dysfunction following acute viral myocarditis. However, the role of PU.1 in atrial fibrosis and vulnerability to AF remain unclear. Here, an in vivo atrial fibrosis model was developed by the continuous infusion of C57 mice with subcutaneous Ang‐II, while the in vitro model comprised atrial fibroblasts that were isolated and cultured. The expression of PU.1 was significantly up‐regulated in the Ang‐II‐induced group compared with the sham/control group in vivo and in vitro. Moreover, protein expression along the TGF‐β1/Smads pathway and the proliferation and differentiation of atrial fibroblasts induced by Ang‐II were significantly higher in the Ang‐II‐induced group than in the sham/control group. These effects were attenuated by exposure to DB1976, a PU.1 inhibitor, both in vivo and in vitro. Importantly, in vitro treatment with small interfering RNA against Smad3 (key protein of TGF‐β1/Smads signalling pathway) diminished these Ang‐II‐mediated effects, and the si‐Smad3‐mediated effects were, in turn, antagonized by the addition of a PU.1‐overexpression adenoviral vector. Finally, PU.1 inhibition reduced the atrial fibrosis induced by Ang‐II and attenuated vulnerability to AF, at least in part through the TGF‐β1/Smads pathway. Overall, the study implicates PU.1 as a potential therapeutic target to inhibit Ang‐II‐induced atrial fibrosis and vulnerability to AF.

Expression profile of PU.1 in CD4+T cells from patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with complex genetic predisposing factors involved. PU.1 is an important member of the ETS transcription factors family which has diverse functions such as regulating the proliferation, differentiation of immune cells and multiple inflammatory cytokines. Previous studies preliminary explored the relation between PU.1 and SLE. To further explain the potential role of PU.1 in the pathogenesis of SLE, 40 SLE patients and 20 age-sex matched healthy controls (HC) were recruited in this study. Flow cytometry was used to test the percentages of CD4+PU.1+T cells in peripheral blood mononuclear cells (PBMCs) from patients with SLE and HC. Expression levels of PU.1 mRNA in CD4+T cells from SLE patients and HC were analyzed by real-time transcription-polymerase chain reaction. Expression levels of plasma IL-1β, IL-9, IL-18, IL-6, IFN-α, TNF-α, IL-10 and TGF-β1 were measured by enzyme-linked immunosorbent assay. The percentage of CD4+PU.1+T cells in PBMCs from patients with SLE was significantly higher than that from HC (P < 0.001). In addition, the PU.1 mRNA expression in CD4+T cells from SLE patients was increased than that from HC (P = 0.002). In SLE patients, no significant correlation was found between the percentage of CD4+PU.1+T cells and the expression of PU.1 mRNA in CD4+T cells (P > 0.05). Associations of PU.1 mRNA expression in CD4+T cells with major clinical and laboratory parameters of SLE patients were also analyzed, but no significant correlations were found. Consistent with previous studies, SLE patients had increased IL-1β, IL-18, IL-6, IFN-α, TNF-α and IL-10 plasma concentrations than HC (P < 0.01). The expression level of plasma TGF-β1 was significantly decreased in SLE patients than in HC (P < 0.001). In SLE patients, the expression level of IL-1β was positive correlated with PU.1 mRNA expression in CD4+T cells (P = 0.001). Our study first time evaluated the expression profile of PU.1 in CD4+T cells from SLE patients confirming that PU.1 may participate in the pathogenesis of SLE.

PU.1 interaction with p50 promotes microglial-mediated inflammation in secondary spinal cord injury in SCI rats

Purpose/aim of the study Secondary spinal cord injury is the inflammatory damage to surrounding tissues caused by activated microglial-mediated neuroinflammatory responses. The nuclear factor-κB (p65/p50) pathway and PU.1 are closely correlated with inflammatory responses; thus, we examined the relationship and function between PU.1 and p50 in secondary spinal cord injury.Materials and methods In this study, we established an adult rat acute spinal cord injury model to simulate the pathological process of spinal cord injury.Results: We found that the expression of PU.1 was significantly increased at three days after spinal cord injury and mainly expressed in activated microglia. Moreover, p-p50 expression was increased in SCI rats and the protein interacted with PU.1. Lipopolysaccharide was used to induce microglia activation in vitro.Conclusions: The results showed that PU.1 and p-p50 expression was significantly increased and PU.1 interacted with p50 in the nucleus. The levels of tumor necrosis factor-α and interleukin-1β secreted by microglia were detected by enzyme-linked immunosorbent assay. The results showed that when both PU.1 and p50 were overexpressed, tumor necrosis factor-α and interleukin-1β secretion was significantly increased to levels higher than in cells overexpressing PU.1 or p50 alone. These results suggest that PU.1 and p50 interact to promote p65 transcription and the expression of inflammatory factors, which is an important mechanism of the microglial-mediated inflammatory response to secondary injury after spinal cord injury.

Expression of Transcription Factor PU.1 in Stromal Cells as a Prognostic Marker in Non-Small Cell Lung Cancer.

Transcription factor PU.1 is involved in hematopoiesis and is expressed in various cells of the inflammatory infiltrate of the tumor stroma, mainly in macrophages. The expression of PU.1 in 100 samples of non-small cell lung cancer was analyzed by the immunohistochemical method. The number of PU.1+ cells did not correlate with clinical and morphological parameters of the tumors. However, increased number of PU.1+ cells significantly correlated with favorable prognosis in adenocarcinoma and poor prognosis in squamous cell carcinoma. Thus, transcription factor PU.1 can serve as a stromal prognostic marker of non-small cell lung cancer depending on the histological type of the tumor.

Protective low expression of PU.1 reduces microglial inflammatory and phagocytic response

AbstractBackgroundThe genetic risk for sporadic Alzheimer’s disease (AD) is polygenic, driven by more than 40 loci. Pathway analysis implicates immune response in the etiology of AD, and given that genetic risk alleles are enriched in myeloid cell types, it has been proposed that impaired microglial function is a major contributing factor to AD risk. Microglial homeostasis is affected by the myeloid lineage‐determining transcription factor SPI1/PU.1, with lower SPI1 expression associated with protection and higher expression with risk for AD based on the rs1057233 genotype.MethodsTo understand how modulation of PU.1 expression affects microglial function we generated stable Spi1/PU.1 overexpression and knock‐down BV2 mouse microglial cell lines and assessed their response in functional assays under baseline and challenged conditions using staurosporine, rotenone and LPS.ResultAnalysis of RNA sequencing data from BV2 lines showed that knock‐down of PU.1 repressed the microglial homeostatic gene signature in a manner similar to that described in disease‐associated microglia in AD mouse models. Pathways of LXR/RXR signaling, antioxidant action and protein translation were upregulated, while inflammatory gene expression was decreased, which may underlie the protective effect of microglia with reduced PU.1 expression. BV2 microglial cells with PU.1 overexpression showed increased, while PU.1 knock‐down led to decreased phagocytosis of bioparticles independent of their type, e.g. zymosan, myelin, early or late apoptotic cells. PU.1 overexpression led to resistance to staurosporine‐induced apoptosis because of decreased caspase cascade activity, while PU.1 knock‐down cells were more susceptible to apoptosis‐induced cell death independent of caspase activation. Increased viability of PU.1 overexpressing cells resulted in potentiation of production of reactive oxygen species, nitric oxide and inflammatory cytokines under stimulated conditions. Furthermore, conditioned media from these cells led to propagation of the reactive phenotype to mouse primary astrocytes in culture. In contrast, PU.1 knock‐down repressed the inflammatory response of BV2 cells after LPS stimulation, and repressed the reactive phenotype in astrocytes.ConclusionReduced expression of PU.1 represses the homeostatic signature of microglia and decreases stimulus‐mediated cell viability, phagocytic and inflammatory responses, which may lead to increased turnover of microglia in the brain underlying the protective effect of SPI1 genotype against AD risk.

Folate Receptor β (FRβ) Expression in Tissue-Resident and Tumor-Associated Macrophages Associates with and Depends on the Expression of PU.1.

As macrophages exhibit a huge functional plasticity under homeostasis and pathological conditions, they have become a therapeutic target for chronic inflammatory diseases. Hence, the identification of macrophage subset-specific markers is a requisite for the development of macrophage-directed therapeutic interventions. In this regard, the macrophage-specific Folate Receptor β (FRβ, encoded by the FOLR2 gene) has been already validated as a target for molecular delivery in cancer as well as in macrophage-targeting therapeutic strategies for chronic inflammatory pathologies. We now show that the transcriptome of human macrophages from healthy and inflamed tissues (tumor; rheumatoid arthritis, RA) share a significant over-representation of the “anti-inflammatory gene set”, which defines the gene profile of M-CSF-dependent IL-10-producing human macrophages (M-MØ). More specifically, FOLR2 expression has been found to strongly correlate with the expression of M-MØ-specific genes in tissue-resident macrophages, tumor-associated macrophages (TAM) and macrophages from inflamed synovium, and also correlates with the presence of the PU.1 transcription factor. In fact, PU.1-binding elements are found upstream of the first exon of FOLR2 and most M-MØ-specific- and TAM-specific genes. The functional relevance of PU.1 binding was demonstrated through analysis of the proximal regulatory region of the FOLR2 gene, whose activity was dependent on a cluster of PU.1-binding sequences. Further, siRNA-mediated knockdown established the importance of PU.1 for FOLR2 gene expression in myeloid cells. Therefore, we provide evidence that FRβ marks tissue-resident macrophages as well as macrophages within inflamed tissues, and its expression is dependent on PU.1.

Stag2 Regulates Hematopoietic Differentiation and Self-Renewal through Alterations in Gene Expression and Topological Control

Cell-type specific transcriptional programs are regulated by the activity of tissue-specific transcription factors and enhancer elements within structurally defined topologically associating domains (TADs). The coordinated and dynamic changes in chromatin architecture are highly regulated as transcriptional output is influenced by chromatin accessibility, histone modification, promoter enhance interactions, and recruitment of transcriptional co-activator complexes. The genes which contribute to transcriptional regulation, including members of the cohesin complex, are frequently mutated in human cancers, including leukemias, Ewing sarcoma, and glioblastoma multiforme. Despite this, the mechanistic role of STAG2 in gene regulation, hematopoietic function, and tumor suppression has not been delineated. We show that somatic Stag2 deletion in hematopoietic stem/progenitor cells (HSPC) results in altered hematopoietic function, increased self-renewal, and impaired differentiation across all three lineages consistent with myelodysplasia. Chromatin immunoprecipitation sequencing of Stag2-deficient HSPCs revealed that Stag2 and Stag1 have both shared and non-redundant cistromes with Stag1/2 common binding sites enriching at TAD boundaries with CTCF occupancy. This maintains TAD integrity in the setting of Stag2 loss of function which we confirmed with Hi-C chromosome capture. High resolution of the Hi-C data at 10kB resolution identified a specific role for Stag2, but not Stag1, in maintaining short-range chromatin interactions, specifically at genes with PU.1 and IRF8 motifs. While co-deletion of Stag2 and Stag1 resulted in synthetic lethality, Stag2 loss alone resulted in decreased chromatin accessibility and reduced transcriptional output at key PU.1 target loci involved in lineage-specification, including reduced Ebf1 and Pax5 expression resulting in impaired B-lineage differentiation. We investigated whether expression of PU.1 could overcome the non-permissive chromatin state at these key downstream targets and rescue hematopoietic differentiation; however, PU.1 expression could not restore Ebf1 expression or B cell differentiation and did not attenuate the serial replating capacity of Stag2 deficient hematopoietic stem/progenitor cells (HSPCs). Given this transcriptional "choke-point" we investigated whether expression of Ebf1 would have a more significant impact on Stag2 deficient cells. Indeed Ebf1 rescue restored B cell colony formation/differentiation in vitro and in vivo and abrogated serial replating of Stag2 deficient HSPCs. These data highlight the non-hierarchical and non-redundant relationship between transcription factors and chromatin architecture and demonstrate a key role for Stag2-regulated local interactions in transcription factor output and hematopoietic differentiation. Nonetheless, the mechanistic underpinnings of the structural basis for transcriptional regulation remain associative. We have recently been able to reduce the cell input for Hi-C assays such that we can now analyze the chromatin architecture of purified populations and model the structural transition from Lin- Sca-1+ Kit+ hematopoietic stem cells to committed granulocyte macrophage precursor cells both in normal hematopoiesis and in the Stag2 deficient setting. Previous studies using in vitro systems have shown that complete cohesin depletion results in the loss of structural loop components; however, cohesin levels are reduced, but not absent, in cancer cells. As such, our studies highlight a key role for locus-specific alterations in gene regulation and DNA interactions in Stag2 deficient cells, which results in altered gene expression and contributes to transformation. Taken together, these data identify a key role for Stag2 loss in transcriptional dysregulation distinct from its shared role with Stag1 in chromosomal segregation. Moreover, we illustrate a critical link between cohesin, chromosomal contacts, and gene regulation that contributes to hematopoietic transformation. Disclosures Viny: Mission Bio: Other: Sponsored travel; Hematology News: Membership on an entity's Board of Directors or advisory committees. Dekker:Arima Genomics: Membership on an entity's Board of Directors or advisory committees. Levine:Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees.

Modulation of FLT3 through decitabine-activated C/EBPa-PU.1 signal pathway in FLT3-ITD positive cells

FMS-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia (AML) which occurs in approximately 30% of all AML patients still has a poor prognosis. This study aimed to examine the effect of decitabine (DAC) on FLT3-ITD positive AML. In our study, we found that expression of FLT3 and its downstream targets was decreased in FLT3-ITD mutant cell lines treated with DAC. DAC treatment could increase the percentage of apoptotic cells and CD11b positive cells tested by flow cytometry and upregulate the expression of cleaved caspase3, cleaved PARP, C/EBPa and PU.1 detected by western blot. To explore the effect of increased expression of PU.1 on FLT3 protein, we transiently transfected MOLM13 and MV4-11 cells with siRNA against PU.1 and a siRNA control. In both FLT3-ITD positive cells, the effect of DAC on downregulation of FLT3 was diminished in PU.1-konckdown MOLM13 and MV4-11 cells and there was a decrease of CD11b expression after PU.1 knockdown. Furthermore, the percentage of apoptotic cells was also decreased in PU.1-konckdown cells compared with siRNA control-expressing cells with the same dose of DAC. These findings indicated that DAC upregulated PU.1 to induce downregulation of FLT3 to trigger apoptosis. DAC was also found efficacious in mouse xenograft models of FLT3-ITD AML in our study. These findings may provide a novel theoretical basis for treatment of FLT3-ITD positive AML patients.

Protective Effect and Mechanisms of New Gelatin on Chemotherapy-Induced Hematopoietic Injury Zebrafish Model.

The aim of the study is to explore the protective effect of new gelatin (NG, Xin'ejiao in China) on hematopoietic injury caused by chemotherapy. Zebrafish, at 48 hours post fertilization (hpf), was treated with different chemotherapeutic drugs to establish the zebrafish hematopoietic damage model with reduced thrombocytes and erythrocytes. The protecting effects of NG on the thrombocytes and erythrocytes were observed, respectively, on zebrafish models. Then, the RT-PCR method was used to detect the change of mRNA level of the hematopoiesis-related cytokines scl1, c-myb, pu.1, GATA1, and runx1 genes. The results showed that 50 μg·mL−1 and 100 μg·mL−1 NG rescued and increased the thrombocytes numbers induced by vinorelbine (NVB) and chloramphenicol (CHL) and the erythrocytes numbers induced by methotrexate (MTX), doxorubicin (ADM), and mechlorethamine hydrochloride (MH) in zebrafish models. Meanwhile, the mRNA expression of scl1, c-myb, and GATA1 genes in the NG treatment group was raised compared with the MTX treatment group. Also, the mRNA expression of pu.1 and Runx1 in the NG treatment group was reduced compared with the MTX treatment group. In consequence, traditional Chinese medicine NG showed a certain degree protective effect on hematopoiesis injury induced by chemotherapy in this study, which may depend on the promotion of erythrocytes proliferation and the regulation of the hematopoietic genes level.

Comprehensive Genomic Analysis of IDH Inhibitor-Treated AML Samples Delineates Molecular Mechanisms of Differentiation and Heterogeneous Patterns of Acquired Resistance

Allosteric inhibitors of mutant IDH1 or IDH2 induce differentiation of IDH-mutant AML myeloblasts, which in some patients (pts) can lead to a life-threatening differentiation syndrome (DS). The in vivo mechanism of how IDH inhibitors induce differentiation and occasionally DS is not fully understood. Furthermore, responders to the inhibitors often lose the differentiation effect, after median of 7 months. Although a recent study identified secondary mutations in the dimer-interface of IDH1/2 as a mechanism for acquired IDH-inhibitor resistance, given the rarity of this event, it is unlikely to represent the primary mechanism in the majority of pts.We conducted a comprehensive genomic analysis (DNA sequencing, single-cell DNA sequencing, RNA sequencing and cytosine methylation array) on longitudinally collected bone marrow specimens from IDH1/2-mutant AML pts treated with one of the IDH1/2 inhibitors (ivosidenib, enasidenib, IDH305 or AG881). In total, 146 samples from 39 pts collected at different time points (median 4 [IQR 3-5] samples per patient) were analyzed. 24 pts were IDH1-mutated receiving IDH1 inhibitor and 15 were IDH2-mutated receiving IDH2 inhibitor. 29 pts responded (CR, CRi, CRp or MLFS) and 10 had no response to the inhibitors. 8 of 29 responders developed DS. 22 of 29 responders relapsed after the median of 5 months.Cytosine methylation analysis revealed significant decrease in global methylation at response than baseline (mean beta value 0.54 at baseline vs. 0.45 at response, P < 0.001). This corroborated with a decrease in serum 2HG level, supporting the role of IDH inhibitors in reversing CpG island methylator phenotype (CIMP) likely through TET restoration. Motif enrichment analysis of differentially methylated promoter regions showed significant de-methylation of PU.1(SPI1) and C/EBP binding motifs at response. Consistent with this, RNA sequencing revealed that PU.1 and C/EBP target genes such as CSF2RA, CSF3R, IL34, TREM2, IL6R, CEBPE, GFI1, and KLF5 had significant upregulation at response. Expression of PU.1 itself was also significantly up-regulated upon response, along with other key hematopoietic differentiation genes and pathways such as GM-CSF, IL8-CXCR1 and IL3 pathways. Pts who developed DS had significantly higher upregulation of PU.1 and GM-CSF pathway compared to other DS-free responders, implicating the potential activation of these pathways in the mechanism of DS. At relapse, these pathways that mediated differentiation were all down-regulated when compared to that at response. Interestingly, methylation level remained suppressed at relapse (mean beta value 0.45 at response vs. 0.47 at relapse, P = 0.566), suggesting that the down-regulation of pro-differentiation signals at relapse is independent of methylation.Combined whole exome and targeted deep sequencing revealed the heterogeneous patterns of acquired mutations at relapse. Of 22 cases with baseline and relapse pairs, 18 acquired new driver mutations at relapse (Fig 1). The most frequently acquired mutations at relapse were in BCOR/BCORL1 (N = 6), hematopoietic differentiation genes (CEBPA [N=2] and RUNX1[N=4]), and RTK pathways (NRAS/KRAS [N=5] and PTPN11 [N=1]). We did not identify acquisition of dimer-interface IDH1/2 mutations at relapse. However, in 1 case, relapse was associated with acquisition of mutation in reciprocal IDH gene (IDH2-mutated case acquired IDH1 mutation). Singe-cell DNA sequencing of the double-mutant revealed that the reciprocal acquisition occurred in a different clone.Analysis of differentially expressed genes between baseline and relapse showed significant upregulation of genes in DNA damage response (DDR) pathways at relapse. Consistent with this, relapse samples showed significantly higher mutation burden than baseline, suggesting that accumulation of mutations are upregulating DDR pathway at relapse.In summary, IDH inhibitors induce differentiation and DS through upregulation of PU.1 and C/EBP target genes, which is associated with de-methylation of their binding motifs. The mechanisms of acquired resistance to IDH inhibitors are heterogeneous and involves various genes and pathways including BCOR/BCORL1, hematopoietic differentiation transcription factors (CEBPA, RUNX1), and RTK pathways (RAS, PTPN11). In a rare case, acquired mutation in reciprocal IDH gene was detected, occurring in a different clone. [Display omitted] DisclosuresDiNardo:Agios: Consultancy; Abbvie: Honoraria; Medimmune: Honoraria; Bayer: Honoraria; Celgene: Honoraria; Karyopharm: Honoraria. MacBeth:Celgene Corporation: Employment, Equity Ownership. Tosolini:Celgene Corporation: Employment. Frattini:Celgene Corporation: Employment, Equity Ownership. Kadia:Abbvie: Consultancy; Celgene: Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Novartis: Consultancy; Amgen: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; BMS: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Celgene: Research Funding; Takeda: Consultancy; Abbvie: Consultancy. Ravandi:Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Sunesis: Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Sunesis: Honoraria; Bristol-Myers Squibb: Research Funding; Xencor: Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Orsenix: Honoraria; Macrogenix: Honoraria, Research Funding; Seattle Genetics: Research Funding; Orsenix: Honoraria; Jazz: Honoraria; Jazz: Honoraria; Seattle Genetics: Research Funding; Abbvie: Research Funding; Macrogenix: Honoraria, Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria. Konopleva:Stemline Therapeutics: Research Funding.

Correlation of patient prognosis with PU.1 and JDP2 to provide potential novel prognostic/diagnostic markers in MDS.

e18558 Background: PU.1 is a key transcription factor in haematopoiesis that plays important roles in various haematological malignancies. Previously, significant down-regulation of PU.1 has been reported in high risk myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML) patients. Methods: To clarify PU.1 molecular function we investigated the gene expression of PU.1 and JDP2 (c-Jun dimerization protein-2), a member of the activating protein-1 family located downstream of PU.1, in bone marrow from 12 MDS patients stratified according to IPSS-R score (6-low, 3-intermediate, 3-high risk), 1 AML patient and 10 normal controls. Samples were enriched for the mononuclear fraction by Ficoll separation. Total RNA was extracted and analysed by Real Time PCR for PU.1 and JDP2 expression relative to the housekeeping gene GAPDHusing the 2-ΔΔCT method. Results: We revealed both PU.1 and JDP2 are down regulated in MDS. In addition, our data suggests that PU.1 and JDP2 expression inversely correlates with disease, with expression of these genes consistently reducing according to IPSS-R groups. Furthermore, a positive correlation of PU.1 and JDP2expression &lt; R = O.9333, s = 0.0004 &gt; , provides additional evidence that suppression of JDP2 by PU.1 could contribute to the pathogenesis of AML. Notably, PU.1 and JDP2 do not correlate to the same extent in normal HSCs, indicating that cofactors are required for PU.1 to exert its JDP2-regulating function and that such cofactors are not present under normal conditions. To confirm that JDP2 suppression is a direct result of reduced PU.1 we performed PU.1-knockdown in K562 cells stably expressing PU.1 short interfering RNAs versus control cells. These analyses reveal only a partial reduction in JDP2 expression when analysed by RT-PCR and Western blot, suggesting a more complex regulatory mechanism. Additionally, both PU.1 and JDP2 expression was recovered by treatment with azacitidine, which is routinely used to treat MDS, suggesting an involvement in treatment response. Conclusions: PU.1 and JDP2 expression correlates with patients prognosis highlighting a potential role as new diagnostic and prognostic markers in MDS.

Myeloid and erythroid hematopoietic transcription factor expression decline after knockdown of lmna genes in zebrafish embryos

Objective: To investigate the effect of lmna gene down-regulation on early hematopoietic development of zebrafish. Methods: Phosphorodiamidate morpholino oligomer (PMO) technology was used to downregulate lmna gene expression in Zebrafish. Zebrafish embryos injected phosphorodiamidate morpholino antisense oligonucleotide of lmna gene mRNA by microinjection at unicellular stage were taken as the experimental group, and those injected meaningless phosphorodiamidate morpholino antisense oligonucleotide were taken as the control. The embryos were collected at 18, 24, 30 and 36 hpf after the fertilization. The real-time fluorescent quantitative PCR (RT-PCR) and whole embryo in situ hybridization methods were used to detect the expression of myeloid hematopoietic transcription factor pu. 1 and erythroid hematopoietic transcription factor gata1 in zebrafish. Results: RT-PCR showed that the expressions of pu. 1 and gata1 decreased in the experimental group compared with the control group (all P<0.05). Whole embryo in situ hybridization showed that the blue-black positive hybridization signals of pu. 1 and gata1 in experimental group were shallow than those in the control group. Conclusion: Myeloid hematopoietic and erythroid hematopoietic of zebrafish are blocked with the downregulation of lmna gene.

Azacitidine Blocks GATA-1-Mediated Repression of the PU.1 Gene in Human Leukemic Cells

Introduction: GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. Expression of PU.1 is controlled by several transcription factors including PU.1 itself by binding to the distal URE enhancer (upstream regulatory element) whose deletion leads to acute myeloid leukemia (AML) (Rosenbauer F et al. 2004). Co-expression of PU.1 and GATA-1 in AML-erythroleukemia (EL) blasts prevents efficient differentiation regulated by these transcription factors. Inhibition of transcriptional activity of PU.1 protein by GATA-1 has been reported (Nerlov C et al. 2000), however it is not known whether GATA-1 can inhibit PU.1 gene in human early erythroblasts directly. We have recently found that MDS/AML erythroblasts display repressive histone modifications and DNA methylation status of PU.1 gene that respond to 5-azacitidine (AZA) leading to inhibited blast cell proliferation and stimulated myeloid differentiation (Curik N et al. 2012). We hypothesize that l eukemia blockade during early erythroid differentiation includes direct GATA-1-mediated inhibition of the PU.1 gene.Results: We herein document the GATA-1 mediated repression of the PU.1 gene in human EL cell lines (OCI-M2 and K562) together with the recruitment of DNA methyl transferase I (DNMT1) to the URE known to guide most of the PU.1 gene transcription. Repression of the PU.1 gene involves both DNA methylation at the URE and methylation/deacetylation of the histone H3 lysine-K9 residue and methylation of H3K27 at additional DNA elements and the PU.1 promoter. Inhibition of GATA-1 by siRNA as well as the AZA treatment in AML-EL led to the significant DNA-demethylation of the URE thorough the mechanism of DNMT1 depletion leading to upregulation of the PU.1 expression.Conclusions: Our data indicate that GATA-1 binds to the PU.1 gene at the URE and initiate events leading to the PU.1 gene repression in human ELs. The mechanism includes repressive epigenetic remodeling of the URE that is important for the PU.1 downregulation and leukemogenesis and that is also simultaneously sensitive to the DNA demethylation treatment with AZA. The GATA-1-mediated inhibition likely contributes to the PU.1 downregulation during progenitor cell differentiation that could be employed during leukemogenesis. Importantly, we also observed important differences between murine and human ELs and found that repression of the PU.1 gene in human ELs can become reverted by the epigenetic therapy with AZA. Our work also suggests that hypomethylating therapy using DNA methylation inhibitors in MDS/AML may become potentially effective in MDS/EL patients. We think that during early erythroid differentiation the GATA-1 binds and represses the PU.1 gene, however this is not fully completed in EL and therefore the erythroid as well as myeloid differentiation are blocked. Grants: GACR P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/1. DisclosuresOff Label Use: Azacitidine, DNA demethylation agens tested in vitro in AML/MDS treatment. Stopka:Celgene: Research Funding.