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Abstract Phospholipid Hydroperoxide Gluthatione Peroxidase also called Glutathione Peroxidase 4 is one of the 25 described human selenoproteins. It plays an essential role in eliminating toxic lipid hydroxy peroxides, thus inhibiting ferroptosis and favoring cell survival. GPX4 is differentially expressed according to myeloid differentiation stage, exhibiting lower expression in hematopoietic stem cells and polymorphonuclear leucocytes, while harboring higher level of expression in common myeloid progenitors and monocytes. In addition, GPX4 is highly expressed in most of acute myeloid leukemia (AML) subtypes compared to normal hematopoietic stem cells. High GPX4 expression is consistently correlated to poor prognosis in patients suffering AML. However, the role of GPX4 in the development of the myeloid lineage and in the initiation and progression of myeloid leukemia remains poorly explored. Given its essential role in the detoxification of lipid hydroperoxides, and its overexpression in most of myeloid malignancies, GPX4 inhibition has emerged as a promising therapeutic strategy to specifically trigger ferroptosis and eradicate myeloid leukemia cells. In this review, we describe the most recent advances concerning the role of GPX4 and, more generally ferroptosis in the myeloid lineage and in the emergence of AML. We also discuss the therapeutic interest and limitations of GPX4 inhibition alone or in combination with other drugs as innovative therapies to treat AML patients.

Acute myeloid leukemia (AML) is characterized by the abnormal proliferation and differentiation arrest of myeloid progenitor cells. The clinical treatment of AML remains challenging. Promoting AML cell differentiation is a valid strategy, but effective differentiation drugs are lacking for most types of AML. In this study, we generated Tg(drl:hoxa9) zebrafish, in which hoxa9 overexpression was driven in hematopoietic cells and myeloid differentiation arrest was exhibited. Using Tg(drl:hoxa9) embryos, we performed chemical screening and identified four FDA-approved drugs, ethacrynic acid, khellin, oxcarbazepine, and alendronate, that efficiently restored myeloid differentiation. The four drugs also induced AML cell differentiation, with ethacrynic acid being the most effective. By an RNA-seq analysis, we found that during differentiation, ethacrynic acid activated the IL-17 and MAPK signaling pathways, which are known to promote granulopoiesis. Furthermore, we found that ethacrynic acid enhanced all-trans retinoic acid (ATRA)-induced differentiation, and both types of signaling converged on the IL-17/MAPK pathways. Inhibiting the IL-17/MAPK pathways impaired ethacrynic acid and ATRA-induced differentiation. In addition, we showed that ethacrynic acid is less toxic to embryogenesis and less disruptive to normal hematopoiesis than ATRA. Thus, the combination of ethacrynic acid and ATRA may have broader clinical applications. In conclusion, through zebrafish-aided screening, our study identified four drugs that can be repurposed to induce AML differentiation, thus providing new agents for AML therapy. [ABSTRACT FROM AUTHOR]

Phospholipid Hydroperoxide Gluthatione Peroxidase also called Glutathione Peroxidase 4 is one of the 25 described human selenoproteins. It plays an essential role in eliminating toxic lipid hydroxy peroxides, thus inhibiting ferroptosis and favoring cell survival. GPX4 is differentially expressed according to myeloid differentiation stage, exhibiting lower expression in hematopoietic stem cells and polymorphonuclear leucocytes, while harboring higher level of expression in common myeloid progenitors and monocytes. In addition, GPX4 is highly expressed in most of acute myeloid leukemia (AML) subtypes compared to normal hematopoietic stem cells. High GPX4 expression is consistently correlated to poor prognosis in patients suffering AML. However, the role of GPX4 in the development of the myeloid lineage and in the initiation and progression of myeloid leukemia remains poorly explored. Given its essential role in the detoxification of lipid hydroperoxides, and its overexpression in most of myeloid malignancies, GPX4 inhibition has emerged as a promising therapeutic strategy to specifically trigger ferroptosis and eradicate myeloid leukemia cells. In this review, we describe the most recent advances concerning the role of GPX4 and, more generally ferroptosis in the myeloid lineage and in the emergence of AML. We also discuss the therapeutic interest and limitations of GPX4 inhibition alone or in combination with other drugs as innovative therapies to treat AML patients. [ABSTRACT FROM AUTHOR]

Introduction: Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapies are focused toward tumor debulking, all-trans retinoic acid (ATRA) induces neutrophil differentiation in the AML subtype acute promyelocytic leukemia (APL). Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML remains elusive. Methods: We took advantage of human AML primary samples and databases to analyze CMA gene expression and activity. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) APL cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cyclic adenosine monophosphate. Results: Here, we report that CMA-related mRNA transcripts are significantly higher expressed in immature hematopoietic cells as compared to neutrophils, contrasting the macroautophagy gene expression patterns. Accordingly, lysosomal degradation of an mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed ATRA-resistant NB4-R1 cells to differentiate upon ATRA treatment but reduced the association of lysosome-associated membrane protein type 2A (LAMP-2A) and heat shock protein family A (Hsp70) member 8 (HSPA8), necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL cell differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation. Conclusion: Overall, our findings suggest that APL neutrophil differentiation requires CMA inactivation and that this pathway predominantly depends on HSPA8 and is possibly assisted by other co-chaperones. [ABSTRACT FROM AUTHOR]

AIM: This study aim to investigate the effects of lipopolysaccharide （LPS）-induced inflammation on the aging phenotype of hematopoietic stem/progenitor cells （HSPCs） in the bone marrow （BM） and spleen of mice. METHODS: （1） Young （2-month old） wild-type （WT） mice were treated with LPS to establish an actue inflammation model. The percentage of HSPCs in the BM and spleen of mice after LPS stimulation, as well as the ratio of mature cells in peripheral blood （PB） and spleen, were analyzed using flow cytometry. The proliferation of HSPCs in the BM and spleen was evaluated by examining the expression of the proliferation marker Ki67. In addition, changes in CD45 expression on HSPCs in the spleen of mice following LPS exposure were investigated by flow cytometry.（ 2） The percentage of HSPCs in BM and mature cells in PB and spleen of both young （2-month old） and old （24-month old） WT mice were analyzed by flow cytometry. （3） The transcriptome changes of hematopoietic stem cells （HSCs） after LPS stimulation was performed by an in silico analysis. RESULTS:（ 1） Mice exposed to LPS exhibited a significant increase in the percentage of HSPCs in BM and a marked elevation in the percentages of myeloid cells in PB and spleen compared to the mice in control group （P<0. 05）. （2） LPS exposure resulted in increased spleen weight and cell counts （P<0. 05）, along with a higher per-centage of HSPCs in the spleen compared to controls（ P<0. 05）.（ 3） LPS stimulation promoted the proliferation of HSPCs in the BM and spleen（ P<0. 05）.（ 4） The expression of CD45 was reduced on HSPCs from spleen of mice after LPS stimulation（ P<0. 01）.（ 5） In comparison to young mice, aged mice showed an increase in spleen weight and a higher percentage of HSPCs in the spleen（ P<0. 05）.（ 6） Aged mice, in comparison to young mice, demonstrated a significantly higher percentage of HSPCs in the BM and myeloid skewing in the PB and spleen（ P<0. 01）.（ 7） The silico analysis revealed upregualtion of reactive oxygen species （ROS） and apoptosis signaling in HSPCs following LPS stimulation. CONCLUSION: Young HSPCs stimulated by LPS exhibited an increase in cell number, a bias towards myeloid differentiation, enhanced extramedullary hematopoiesis, and elevated levels of ROS and apoptosis, all of which collectively manifested the aging phenotype of HSPCs. [ABSTRACT FROM AUTHOR]

Bone marrow‐derived cells (BMDCs) infiltrate hypoxic tumors at a pre‐angiogenic state and differentiate into mature macrophages, thereby inducing pro‐tumorigenic immunity. A critical factor regulating this differentiation is activation of SREBP2—a well‐known transcription factor participating in tumorigenesis progression—through unknown cellular mechanisms. Here, we show that hypoxia‐induced Golgi disassembly and Golgi‐ER fusion in monocytic myeloid cells result in nuclear translocation and activation of SREBP2 in a SCAP‐independent manner. Notably, hypoxia‐induced SREBP2 activation was only observed in an immature lineage of bone marrow‐derived cells. Single‐cell RNA‐seq analysis revealed that SREBP2‐mediated cholesterol biosynthesis was upregulated in HSCs and monocytes but not in macrophages in the hypoxic bone marrow niche. Moreover, inhibition of cholesterol biosynthesis impaired tumor growth through suppression of pro‐tumorigenic immunity and angiogenesis. Thus, our findings indicate that Golgi‐ER fusion regulates SREBP2‐mediated metabolic alteration in lineage‐specific BMDCs under hypoxia for tumor progression. Synopsis: Bone marrow‐derived cells (BMDCs) infiltrate hypoxic tumors enhancing malignant angiogenesis and immunity, but how low‐oxygen conditions alter BMDC activity remains unclear. This study reports hypoxia‐induced Golgi disassembly and cholesterol biosynthesis as a novel organelle‐mediated regulation of monocytic BMDCs for enhanced tumorigenesis. Hypoxia triggers cholesterol biosynthesis in BMDCs and cancer cell line‐induced tumors in mice.Hypoxia‐induced cholesterol biosynthesis requires Golgi disassembly‐mediated activation of SREBP2 in monocytic myeloid cells.Hypoxia‐induced SREBP2 activation is lineage‐specific and reduced during monocyte differentiation.Inhibition of cholesterol biosynthesis suppresses tumor growth through reduced infiltration of pro‐tumorigenic BMDCs. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by abnormal cell proliferation, apoptosis repression and myeloid differentiation blockade of hematopoietic stem/progenitor cells. Developing and identifying novel therapeutic agents to reverse the pathological processes of AML are of great significance. Here in this study, we found that a fungus-derived histone deacetylase inhibitor, Apicidin, presents promising therapeutic effect on AML by inhibiting cell proliferation, facilitating apoptosis and inducing myeloid differentiation of AML cells. Mechanistic investigation revealed that QPCT is identified as a potential downstream target of Apicidin, which exhibits significantly decreased expression in AML samples compared with the normal controls and is remarkably up-regulated in AML cells upon Apicidin management. Functional study and rescue assay demonstrated that QPCT depletion further promotes cell proliferation, inhibits apoptosis and impairs myeloid differentiation of AML cells, alleviating the anti-leukemic effect of Apicidin on AML. Our findings not only provide novel therapeutic target for AML, but also lay theoretical and experimental foundation for the clinical application of Apicidin in AML patients.

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 FROM AUTHOR]

Ormeloxifene (ORM) (3,4‐trans‐2,2‐dimethyl‐3‐phenyl‐4‐p‐(β‐pyrrolidinoethoxy) phenyl‐7‐methoxychroman), world's first nonsteroidal selective estrogen receptor modulator approved for contraception in India has been shown to have potential anticancer activities. Here, we show that ORM can induce megakaryocyte and myeloid (granulocytic) but not erythroid differentiation in multipotent human myeloid leukemia cell line K562. We show that ORM at an IC50 of 7.5 µM can induce morphological changes similar to megakaryocytes in K562 cells. ORM led to increase in levels of megakaryocytic differentiation markers (CD41 and CD61) as well as key transcription factors GATA1 and AML1. We further show that ORM induces megakaryocytic differentiation in K562 cells through ERK activation and induction of autophagy in a fashion similar to other known inducers of megakaryocytic differentiation such as phorbol esters. In addition, as shown earlier, we yet again observed that ORM led to activation of caspases since their inhibition through pan‐caspase inhibitor mitigated megakaryocytic differentiation as they led to significant decrease in CD41 and CD61. Because induction of megakaryocytic differentiation in K562 involves growth arrest and exit from cell cycle, we also observed an increase in levels of p21 and p27 with decrease in c‐Myc protein levels in K562 cells treated with 7.5 µM ORM for 24 and 48 h, respectively. Taken together, these findings indicate that ORM can markedly induce megakaryocytic differentiation in K562 cells. [ABSTRACT FROM AUTHOR]

The study of the mechanisms of resistance of tumor cells to TRAIL-induced death remains an urgent task since this cytokine is an important highly selective molecular effector of antitumor immunity. Our study showed that human leukemia cells THP-1, HL-60, and K562 increased their resistance to TRAIL-induced death in vitro as a result of induction of myeloid differentiation in them by exogenous factors in all directions of myelopoiesis, except for erythroid, by reducing the expression of DR4 and DR5 receptors on the cell surface. It was also found that ONC 201, tunicamycin, and SAHA (hydroxamic acid suberoylanilide), capable of causing an increase in the expression of DR5 in leukemic cells, suppressed their TRAIL resistance induced by differentiation factors. The results obtained are of interest for the development of drugs and strategies to improve the effectiveness of the treatment of myeloid leukemia. [ABSTRACT FROM AUTHOR]

Hematopoietic stem and progenitor cells (HSPCs) are a heterogeneous group of cells with expansion, differentiation, and repopulation capacities. How HSPCs orchestrate the stemness state with diverse lineage differentiation at steady condition or acute stress remains largely unknown. Here, we show that zebrafish mutants that are deficient in an epigenetic regulator Atf7ip or Setdb1 methyltransferase undergo excessive myeloid differentiation with impaired HSPC expansion, manifesting a decline in T cells and erythroid lineage. We find that Atf7ip regulates hematopoiesis through Setdb1-mediated H3K9me3 modification and chromatin remodeling. During hematopoiesis, the interaction of Atf7ip and Setdb1 triggers H3K9me3 depositions in hematopoietic regulatory genes including cebpß and cdkn1a, preventing HSPCs from loss of expansion and premature differentiation into myeloid lineage. Concomitantly, loss of Atf7ip or Setdb1 derepresses retrotransposons that instigate the viral sensor Mda5/Rig-I like receptor (RLR) signaling, leading to stress-driven myelopoiesis and inflammation. We find that ATF7IP or SETDB1 depletion represses human leukemic cell growth and induces myeloid differentiation with retrotransposon-triggered inflammation. These findings establish that Atf7ip/Setdb1-mediated H3K9me3 deposition constitutes a genomewide checkpoint that impedes the myeloid potential and maintains HSPC stemness for diverse blood cell production, providing unique insights into potential intervention in hematological malignancy. [ABSTRACT FROM AUTHOR]

Myeloid differentiation blockage at immature and self-renewing stages is a common hallmark across all subtypes of acute myeloid leukemia (AML), despite their genetic heterogeneity. Metabolic state is an important regulator of hematopoietic stem cell (HSC) self-renewal and lineage-specific differentiation as well as several aggressive cancers. However, how O-GlcNAcylation, a nutrient-sensitive posttranslational modification of proteins, contributes to both normal myelopoiesis and AML pathogenesis remains largely unknown. Using small molecule inhibitors and the CRISPR/Cas9 system, we reveal for the first time that inhibition of either OGA or OGT, which subsequently caused an increase or decrease in cellular O-GlcNAcylation, inhibits the self-renewal and maintenance of CD34+ hematopoietic stem/progenitor cells (HSPCs) and leukemic stem/progenitor cells and drives normal and malignant myeloid differentiation. We further unveiled the distinct roles of OGA and OGT inhibition in lineage-specific differentiation. While OGT inhibition induces macrophage differentiation, OGA inhibition promotes the differentiation of both CD34+ HSPCs and AML cells into dendritic cells (DCs), in agreement with an upregulation of a multitude of genes involved in DC development and function and their ability to induce T-cell proliferation, via STAT3/5 signaling. Our novel findings provide significant basic knowledge that could be important in understanding AML pathogenesis and overcoming differentiation blockage—agnostic to the genetic background of AML. Additionally, the parallel findings in normal HSPCs may lay the groundwork for future cellular therapy as a means to improve the ex vivo differentiation of normal DCs and macrophages. Normal and pathogenic stem cell self-renewal and myeloid differentiation can be regulated via O-GlcNAc homeostasis. Inhibition of O-GlcNAcase (OGA) and subsequent increase in O-GlcNAcylation, which also occurs spontaneously during dendritic cell (DC) differentiation, activates STAT3/5 at the posttranslational level through tyrosine phosphorylation and promotes DC differentiation. [ABSTRACT FROM AUTHOR]

The reconstruction of gene regulatory networks underlying cell differentiation from high-throughput gene expression and chromatin data remains a challenge. Here, we derive dynamic gene regulatory networks for human myeloid differentiation using a 5-day time series of RNA-seq and ATAC-seq data. We profile HL-60 promyelocytes differentiating into macrophages, neutrophils, monocytes, and monocyte-derived macrophages. We find a rapid response in the expression of key transcription factors and lineage markers that only regulate a subset of their targets at a given time, which is followed by chromatin accessibility changes that occur later along with further gene expression changes. We observe differences between promyelocyte- and monocyte-derived macrophages at both the transcriptional and chromatin landscape level, despite using the same differentiation stimulus, which suggest that the path taken by cells in the differentiation landscape defines their end cell state. More generally, our approach of combining neighboring time points and replicates to achieve greater sequencing depth can efficiently infer footprint-based regulatory networks from long series data.

Abstract RhoH is an unusual member of the Rho family of small GTP‐binding proteins in that it lacks GTPase activity. Since the RhoH protein is constantly bound by GTP, it is constitutively active and controlled predominantly by changes in quantitative expression. Abnormal levels of RHOH gene transcripts have been linked to a range of malignancies including acute myeloid leukemia (AML). One of the hallmarks of AML is a block in the normal program of myeloid differentiation. Here we investigate how myeloid differentiation is controlled by the quantitative expression of RHOH. Our analysis demonstrates that increasingly mature myeloid cells express progressively lower levels of RHOH. However, as monocytic myeloid cells terminally differentiate into macrophages, RHOH expression is up‐regulated. This up‐regulation is not apparent in AML where myeloid differentiation is blocked at stages of low RHOH expression. Nevertheless, when the up‐regulation of RHOH is forced, then terminal macrophage differentiation is induced and the Cdc42 and Wnt intracellular signalling pathways are repressed. These results indicate that RHOH induction is a driver of terminal differentiation and might represent a means of effecting AML differentiation therapy. The potential of this therapeutic strategy is supported by forced up‐regulation of RHOH reducing the ability of AML cells to produce tumours in vivo.

Résumé: Les cellules souches hématopoïétiques (CSH) se trouvent au sommet du système hématopoïétique et assurent le renouvellement des cellules sanguines matures tout au long de la vie. Des études menées aussi bien chez l'homme que chez la souris ont démontré que les propriétés fonctionnelles des CSH sont altérées au cours du vieillissement. La capacité des CSH à reconstituer le système hématopoïétique ainsi que leur potentiel de différenciation lymphoïde diminuent avec l'âge, conduisant à une hématopoïèse biaisée, myéloïde, chez les individus âgés. De nombreux facteurs contribuent au déclin fonctionnel des CSH observé avec l'âge, qu'il s'agisse d'altérations intrinsèques aux CSH, ou extrinsèques, survenant dans le microenvironnement médullaire des cellules souches. Les modifications intrinsèques comprennent l'accumulation de dommages à l'ADN, l'érosion des télomères, ainsi que des altérations métaboliques, déclenchant des mécanismes de réponse au stress et d'épuisement cellulaire dans les CSH âgées. Des études récentes indiquent que le vieillissement est également associé à une altération de l'état épigénétique des CSH, qui semble être un moteur important dans le déclin fonctionnel des cellules souches. Enfin, le vieillissement se traduit par des modifications du microenvironnement de la moelle osseuse, qui entraînent une diminution de la formation osseuse et des niches endostéales, favorisant là encore la différenciation myéloïde aux dépens de la lymphopoïèse. Il s'y ajoute l'installation, à bas bruit, d'une inflammation chronique, connue sous le nom d' inflammaging. L'ensemble de ces phénomènes ont notamment pour effet une restriction clonale au sein du système hématopoïétique, favorisant l'apparition de malignités myéloïdes et le déclin de la fonction immunitaire adaptative chez les individus âgés. L'élucidation des mécanismes à l'origine du vieillissement des CSH a permis de proposer de nombreuses stratégies thérapeutiques visant à rajeunir la fonction des CSH, qui montrent des résultats prometteurs dans des modèles animaux. Haematopoietic stem cells (HSCs) occupy the apex of the haematopoietic system, and ensure the renewal of all mature blood cells throughout life. Studies in both mice and humans have demonstrated that the functional properties of HSCs change with age, altering their capacity to reconstitute the haematopoietic system, and diminishing their lymphoid differentiation potential, giving rise to a myeloid-biased haematopoiesis in aged individuals. A range of different events are now known to contribute to the decline of HSC function observed with age, including alterations within the HSC themselves, as well as changes occurring in the bone marrow microenvironment where HSC reside. Cell intrinsic changes include the accumulation of DNA damage and telomere erosion, as well as alterations in HSC metabolism which trigger stress-response mechanisms and cellular exhaustion. More recently, age-associated changes in the epigenetic state have also been implicated as an important driver of HSC functional decline. Within the bone marrow microenvironment, the onset of low-level chronic inflammation and remodelling of HSC niches which take place with age also influence HSC functional capacity and contribute to a myeloid-biased differentiation. HSC aging has important downstream consequences for the health of the elderly, contributing to the chronic inflammatory state and decline in adaptive immune function observed in aged individuals, and predisposing to the onset of myeloid malignancies. Understanding the mechanisms driving HSC aging has suggested numerous therapeutic strategies to rejuvenate HSC function, which have shown promising results in aging mice. [ABSTRACT FROM AUTHOR]

Transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPα) regulates myelopoiesis by coupling growth arrest with differentiation of myeloid progenitors. Mutations in one or both alleles are observed in 10-14% AML cases that render C/EBPα functionally inactive. Besides, antagonistic protein-protein interactions also impair C/EBPα expression and function. In recent independent studies, we showed that CDK2 and SKP2 downregulated C/EBPα expression in an ubiquitin-dependent proteasome degradation manner leading to differentiation block in AML. Here, we demonstrate that CDK2-instigated C/EBPα downregulation is actually mediated by SKP2. Mechanistically, we show that CDK2 stabilizes SKP2 by phosphorylating it at Ser64 and thereby potentiates C/EBPα ubiquitination and subsequent degradation in AML cells. Immunoblot experiments showed that CDK2 inhibition downregulated SKP2 levels and concomitantly enhanced C/EBPα levels in myeloid cells. We further show that while CDK2 promoted C/EBPα ubiquitination and inhibited its protein levels, negatively affected its transactivation potential and DNA binding ability, simultaneous SKP2 depletion abrogated CDK2-promoted ubiquitination and restored C/EBPα expression and function. Taken together, these findings consolidate that CDK2 potentiates SKP2-mediated C/EBPα degradation in AML and targeting CDK2-SKP2 axis can be harnessed for therapeutic benefit in AML. Hypothetical model depicts that SKP2-mediated C/EBPα proteasomal degradation is reinforced by CDK2. CDK2 phopshorylates SKP2 leading to its enhanced stabilization which in turn exaggerates C/EBPα degradation leading to differentiation arrest in AML. [ABSTRACT FROM AUTHOR]

High mobility group box 1 (HMGB1) is a non-histone nuclear protein which has been intensively studied in various physiological and pathological processes including leukemia. Here in this study, we further demonstrated that HMGB1 presents higher expression in the bone marrow mononuclear cells of acute myeloid leukemia (AML) patients compared with the normal controls and contributes to the AML pathogenesis and progression by inhibiting apoptosis, facilitating proliferation, and inducing myeloid differentiation blockade of AML cells. Mechanistic investigation revealed that transforming growth factor beta-induced (TGFBI) acts as a potential downstream target of HMGB1 and lentivirus-mediated knockdown of TGFBI expression impaired phorbol-12-myristate-13-acetate (PMA) and all-trans retinoic acid (ATRA)–induced myeloid differentiation of AML cell lines. On the other hand, chidamide, an orally histone deacetylase inhibitor, decreases HMGB1 expression significantly in AML cells with concomitant upregulation of TGFBI expression, and confers therapeutic effect on AML by inducing cell differentiation, apoptosis and inhibiting cell proliferation. In conclusion, our findings provide additional insights that HMGB1 is a promising therapeutic target of AML, and also present experimental evidence for the clinical application of chidamide as a novel agent in AML therapy by downregulating HMGB1 expression. Key messages: HMGB1 induces cell proliferation and myeloid differentiation blockade and inhibits apoptosis of AML cells. TGFBI acts as a potential target of HMGB1. Chidamide, a selective HDAC inhibitor, confers promising therapeutic effect for AML via downregulating HMGB1 expression. [ABSTRACT FROM AUTHOR]

Retinoic acid-inducible gene I (RIG-I) is up-regulated during granulocytic differentiation of acute promyelocytic leukemia (APL) cells induced by all-trans retinoic acid (ATRA). It has been reported that RIG-I recognizes virus-specific 5'-ppp-double-stranded RNA (dsRNA) and activates the type I interferons signaling pathways in innate immunity. However, the functions of RIG-I in hematopoiesis remain unclear, especially regarding its possible interaction with endogenous RNAs and the associated pathways that could contribute to the cellular differentiation and maturation. Herein, we identified a number of RIG-I-binding endogenous RNAs in APL cells following ATRA treatment, including the tripartite motif-containing protein 25 (TRIM2S) messenger RNA (mRNA). TRIM25 encodes the protein known as an E3 ligase for ubiquitin/interferon (IFN)-induced 15-kDa protein (ISG15) that is involved in RIG-I-mediated antiviral signaling. We show that RIG-I could bind TRIM2S mRNA via its helicase domain and C-terminal regulatory domain, enhancing the stability of TRIM2S transcripts. RIG-I could increase the transcriptional expression of TRIM2S by caspase recruitment domain (CARD) domain through an IFN-stimulated response element. In addition, RIG-I activated other key genes in the ISGylation pathway by activating signal transducer and activator of transcription 1 (STAT1), including the modifier ISG15 and several enzymes responsible for the conjugation of ISG15 to protein substrates. RIG-I cooperated with STAT1/2 and interferon regulatory factor 1 (IRF1) to promote the activation of the ISGylation pathway. The integrity of ISGylation in ATRA or RIG-I-induced cell differentiation was essential given that knockdown of TRIM25 or ISG15 resulted in significant inhibition of this process. Our results provide insight into the role of the RIG-I-TRIM25-ISGylation axis in myeloid differentiation. [ABSTRACT FROM AUTHOR]

For acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) is well established. However, the narrow application and tolerance development of ATRA remain to be improved. In this study, we investigated the effects of combinations of glycosylation inhibitors with ATRA to achieve better efficiency than ATRA alone. We found that the combination of fucosylation inhibitor 6-alkynylfucose (6AF) and ATRA had an additional effect on cell differentiation, as revealed by expression changes in two differentiation markers, CD11b and CD11c, and significant morphological changes in NB4 APL and HL-60 acute myeloid leukemia (AML) cells. In AAL lectin blot analyses, ATRA or 6AF alone could decrease fucosylation, while their combination decreased fucosylation more efficiently. To clarify the molecular mechanism for the 6AF effect on ATRA-induced differentiation, we performed microarray analyses using NB4 cells. In a pathway analysis using DAVID software, we found that the C-type lectin receptor (CLR) signaling pathway was enriched with high significance. In real-time PCR analyses using NB4 and HL-60 cells, FcεRIγ , CLEC6A , CLEC7A , CASP1 , IL-1β , and EGR3 , as components of the CLR pathway, as well as CD45 and AKT3 were upregulated by 6AF in ATRA-induced differentiation. Taken together, the present findings suggest that the CLR signaling pathway is involved in the 6AF effect on ATRA-induced differentiation. • ・6AF with ATRA has a profound effect on differentiation in NB4 and HL-60 cells. • ・The combination of 6AF with ATRA efficiently decreases the expression of fucosylation. • ・Microarray shows the enrichment of CLR signaling pathway by 6AF. • ・ FcεRIγ , CLEC6A , CLEC7A , CASP1 , IL-1β , EGR3 , CD45 and AKT3 genes are induced by 6AF. [ABSTRACT FROM AUTHOR]

Myeloid-derived suppressor cells (MDSCs) and dendritic cells (DCs) arise from common progenitors. Tumor-derived factors redirect differentiation from immune-promoting DCs to tolerogenic MDSCs, an immunological hallmark of cancer. Indeed, in vitro differentiation of DCs from human primary monocytes results in the generation of MDSCs under tumor-associated conditions (PGE2 or tumor cell-conditioned media). Comparison of MDSC and DC DNA methylomes now reveals extensive demethylation with specific gains of DNA methylation and repression of immunogenic-associated genes occurring in MDSCs specifically, concomitant with increased DNA methyltransferase 3A (DNMT3A) levels. DNMT3A downregulation erases MDSC-specific hypermethylation, and it abolishes their immunosuppressive capacity. Primary MDSCs isolated from ovarian cancer patients display a similar hypermethylation signature in connection with PGE2-dependent DNMT3A overexpression. Our study links PGE2- and DNMT3A-dependent hypermethylation with immunosuppressive MDSC functions, providing a promising target for therapeutic intervention.

The tumor suppressor transcription factor CCAAT enhancer-binding protein α (C/EBPα) expression is downregulated in myeloid leukemias and enhancement of C/EBPα expression induces granulocytic differentiation in leukemic cells. Previously we reported that Styryl quinazolinones induce myeloid differentiation in HL-60 cells by upregulating C/EBPα expression. To identify more potent molecule that can induce leukemic cell differentiation we synthesized and evaluated new series of styryl quinazolinones, ethynyl styryl quinazolinones, styryl quinolinones and thienopyrimidinones. Thienopyrimidinones were found toxic and styryl quinolinones were found inactive. Ethynyl styryl quinazolinone 39 and styryl quinazolinone 5 were found active on par with the earlier reported analogues 1 and 2 suggesting that the 5-nitro furan-2-yl styryl quinazolinones find a real promise in leukemic cell differentiation. The improved potency of 5 suggested that further modifications in the 5-nitro furan-2-yl styryl quinazolinones can be at the phenyl substitution at the 3-position of the quinazolinone ring apart from the 5-position of the heteroaryl ring. [ABSTRACT FROM AUTHOR]

Understanding of the mechanism for myeloid differentiation provides important insights into the hematopoietic developmental processes. By using an ESC-derived myeloid progenitor cell model, we found that CSF2/GM-CSF triggered macrophage differentiation and activation of the MTOR signaling pathway. Activation or inhibition of the MTOR signaling enhanced or attenuated macrophage differentiation, respectively, suggesting a critical function. We further showed that macroautophagy/autophagy was inhibited with the addition of CSF2. Furthermore, pharmacological inhibition and genetic modification of autophagy enhanced macrophage differentiation and rescued the inhibitory effect on differentiation caused by MTOR inhibition. Thus, the MTOR signaling pathway regulates macrophage differentiation of myeloid progenitors by inhibiting autophagy. Our results provide new insights into the mechanisms for myeloid differentiation and may prove useful for therapeutic applications of hematopoietic and myeloid progenitor cells. Abbreviations: 2-DG: 2-deoxy-D-glucose; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; BM: bone marrow; CQ: chloroquine; ECAR: extracellular acidification rate; ESC: embryonic stem cell; CSF2/GM-CSF: colony stimulating factor 2; CSF3/G-CSF: colony stimulating factor 3; HPC: hematopoietic progenitor cell; ITGAM/CD11b: integrin alpha M; LPS: lipopolysaccharide; MFI: median fluorescence intensity; MTOR: mechanistic target of rapamycin kinase; RPS6KB1/p70S6K1: ribosomal protein S6 kinase, polypeptide 1; shRNA: short hairpin RNA; SQSTM1/p62: sequestosome 1. [ABSTRACT FROM AUTHOR]

Interleukin-6 (IL-6) is an important trigger for the expansion and recruitment of myeloid-derived suppressor cells (MDSCs), which are regarded to be major coordinators of the immunosuppressive tumor microenvironment. In this study, we constructed IL-6-knockdown breast cancer mice models to explore the molecular events involved in the IL-6-mediated effects on MDSC development. We defined a subset of early-stage MDSCs (e-MDSCs) with the phenotype of CD11b+Gr-1−F4/80−MHCII− in IL-6 high-expressing 4T1 mice mammary carcinoma models, which were the precursors of CD11b+Gr-1+ conventional MDSCs. Furthermore, sustained suppression of SOCS3 and aberrant hyperactivation of the JAK/STAT signaling pathway was exclusively detected in wide-type 4T1 tumor-bearing mice, which promoted the accumulation of e-MDSCs in situ and their immunosuppressive capability in vitro. After blocking the IL-6/STAT3 signaling pathway with the IL-6 receptor antibody or STAT3 antagonist JSI-124 in tumor-bearing mice, significant shrinkage of primary tumors and decrease in lung metastatic nodules were observed in vivo, accompanied by the dramatic decrease of e-MDSC recruitment and recovery of anti-tumor T cell immunity. Thus, SOCS3 suppression accelerated the IL-6-mediated growth and metastasis of mammary carcinoma via affecting myeloid differentiation in breast cancer. Moreover, the IL-6/STAT3 signaling pathway might be a promising candidate target in developing novel therapeutic strategies to eliminate e-MDSCs and improve breast cancer prognosis.

The α-Dystrobrevin gene encodes at least five different protein isoforms, expressed in diverse tissues. The α-Dystrobrevin-1 isoform (α-Db-1) is a member of the cytoplasmic dystrophin-associated protein complex, which has a C-terminal extension comprising at least three tyrosine residues susceptible to phosphorylation in vivo . We previously described α-Db in stem-progenitor cells and blood neutrophils as playing a scaffolding role and, in association with kinesin and microtubules, α-Db promotes platelet-granule trafficking. Additionally, the microtubules must establish a balanced interaction with the lamina A/C network for appropriate nuclear morphology. Considering that the most outstanding feature during neutrophil differentiation is nuclei lobulation, we hypothesized that α-Db might possess a pivotal function during the neutrophil differentiation process. Western Blot (WB) and confocal microscope assays evidenced a differential pattern expression and a subcellular redistribution of α-Db in neutrophils derived from HL-60 cells. At the end of the differentiation process, we detected an important diminution in the expression of tubulin, kinesin, and α-Db-1. Knockdown of α-Db prevented nuclei lobulation, increased Lamin A/C and syne1 expression and augmented the roughness of derived neutrophil membrane and disturbed filopodia assembly. Our results suggest that HL-60 cells undergo extensive cytoskeletal reorganization including α-Db in order to possess lobulated nuclei when they further differentiate into neutrophils. [ABSTRACT FROM AUTHOR]

Summary Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils. Graphical Abstract Highlights • Dynamic acetylation enforces human neutrophil progenitor differentiation • Neutrophils’ cytotoxic capability is established early at the (pro)myelocyte stage • Coordinated signaling component expression prevents toxic effects to the bone marrow Grassi et al. report that the establishment of transcriptional enhancers drives neutrophil differentiation. Coordinated waves of gene expression establish the cytotoxic capability of these cells at early stages of maturation. A set of super-enhancers is specifically opened at the end of the differentiation process to control neutrophil activation. [ABSTRACT FROM AUTHOR]

Interleukin-6 (IL-6) is an important trigger for the expansion and recruitment of myeloid-derived suppressor cells (MDSCs), which are regarded to be major coordinators of the immunosuppressive tumor microenvironment. In this study, we constructed IL-6-knockdown breast cancer mice models to explore the molecular events involved in the IL-6-mediated effects on MDSC development. We defined a subset of early-stage MDSCs (e-MDSCs) with the phenotype of CD11b+Gr-1−F4/80−MHCII− in IL-6 high-expressing 4T1 mice mammary carcinoma models, which were the precursors of CD11b+Gr-1+ conventional MDSCs. Furthermore, sustained suppression of SOCS3 and aberrant hyperactivation of the JAK/STAT signaling pathway was exclusively detected in wide-type 4T1 tumor-bearing mice, which promoted the accumulation of e-MDSCs in situ and their immunosuppressive capability in vitro. After blocking the IL-6/STAT3 signaling pathway with the IL-6 receptor antibody or STAT3 antagonist JSI-124 in tumor-bearing mice, significant shrinkage of primary tumors and decrease in lung metastatic nodules were observed in vivo, accompanied by the dramatic decrease of e-MDSC recruitment and recovery of anti-tumor T cell immunity. Thus, SOCS3 suppression accelerated the IL-6-mediated growth and metastasis of mammary carcinoma via affecting myeloid differentiation in breast cancer. Moreover, the IL-6/STAT3 signaling pathway might be a promising candidate target in developing novel therapeutic strategies to eliminate e-MDSCs and improve breast cancer prognosis. [ABSTRACT FROM AUTHOR]

Granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is a type I cytokine receptor which is involved in hematopoietic cell maturation. G-CSFR has three putative C -mannosylation sites at W253, W318, and W446; however, it is not elucidated whether G-CSFR is C -mannosylated or not. In this study, we first demonstrated that G-CSFR was C -mannosylated at only W318. We also revealed that C -mannosylation of G-CSFR affects G-CSF-dependent downstream signaling through changing ligand binding capability but not cell surface localization. Moreover, C -mannosylation of G-CSFR was functional and regulated granulocytic differentiation in myeloid 32D cells. In conclusion, we found that G-CSFR is C -mannosylated at W318 and that this C -mannosylation has role(s) for myeloid cell differentiation through regulating downstream signaling. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRs) have been demonstrated to perform important roles in normal hematopoiesis and leukemogenesis. Accumulating evidence suggests that miR-10a and miR-10b may behave as novel oncogenes or tumor suppressors in human cancer. The present study reported the function of the miR-10 family in myeloid differentiation and acute myeloid leukemia (AML). The levels of miR-10a/b expression were increased in AML cases compared with normal controls, particularly in M1, M2 and M3 subtypes. The levels of miR-10a/b expression were also upregulated in patients with nucleophosmin-mutated AML and AML patients with t(8;21) and t(9;11), compared with the normal control. In addition, the role of miR-10a/b in regulating myeloid differentiation and leukemogenesis was investigated. The results indicated that miR-10a/b expression was able to promote the proliferation of human promyelocytic leukemia cells, while suppressing the granulocytic and monocytic differentiation of the leukemia cells. These findings suggested that abnormal high expression of miR-10a/b may result in unlimited proliferation of immature blood progenitors and repression of mature blood cell differentiation and maturation, thus leading to the occurrence of AML. miR-10a/b may be developed as novel therapeutic targets for the treatment of AML. [ABSTRACT FROM AUTHOR]

Myeloid leukemias are highly diverse diseases and have been shown to be associated with microRNA (miRNA) expression aberrations. The present study involved an in-depth miRNome analysis of two human acute myeloid leukemia (AML) cell lines, HL-60 and THP-1, and one human chronic myeloid leukemia (CML) cell line, K562, via massively parallel signature sequencing. mRNA expression profiles of these cell lines that were established previously in our lab facilitated an integrative analysis of miRNA and mRNA expression patterns. miRNA expression profiling followed by differential expression analysis and target prediction suggested numerous miRNA signatures in AML and CML cell lines. Some miRNAs may act as either tumor suppressors or oncomiRs in AML and CML by targeting key genes in AML and CML pathways. Expression patterns of cell type-specific miRNAs could partially reflect the characteristics of K562, HL-60 and THP-1 cell lines, such as actin filament-based processes, responsiveness to stimulus and phagocytic activity. miRNAs may also regulate myeloid differentiation, since they usually suppress differentiation regulators. Our study provides a resource to further investigate the employment of miRNAs in human leukemia subtyping, leukemogenesis and myeloid development. In addition, the distinctive miRNA signatures may be potential candidates for the clinical diagnosis, prognosis and treatment of myeloid leukemias.

The NM23 gene is overexpressed in many hematological malignancies and its overexpression predicts poor treatment outcomes. NM23 overexpression is thought to suppress myeloid differentiation of leukemia cells, but the molecular mechanism is unknown. In breast cancer cells, the lysophosphatidic acid (LPA) receptor EDG2/ lpa1 was downregulated by NM23-H1 overexpression, and this reciprocal expression pattern was associated with suppressed or induced cell motility/metastasis. Here, we examined the relationship between EDG2 and NM23 expression during myeloid differentiation of leukemia cells. NM23 expression decreased and EDG2 expression increased during all-trans retinoic acid (ATRA)-induced myeloid differentiation of HL-60, NB4, and THP-1 leukemia cells. Moreover, this inverse correlation was more evident when myeloid differentiation was enhanced by ellagic acid, an inhibitor of NM23 activity. In contrast, there was no inverse correlation between EDG2 and NM23 expression during erythroid differentiation of HEL and K562 cells. ATRA plus LPA enhanced the motility of leukemia cells as well as breast cancer cells in an EDG2-dependent manner. These results suggest a common molecular mechanism between myeloid differentiation of leukemia cells and migration of breast cancer cells depending on NM23 and EDG2 expression levels. [ABSTRACT FROM AUTHOR]

Ikaros is a broad transcription factor pointed as a critical regulator of lymphocyte development. Recent reports have emphasized that distinct isoforms of Ikaros control the dichotomy of the hematopoietic system into lymphoid and myeloid lineages. In addition, expression of dominant-negative isoforms of Ikaros is linked to abnormal hematopoiesis, which could culminate in hematological disorders due to loss of function of the protein. B-1 cells are an intriguing subtype of B-lymphocytes that preserves some myeloid characteristics. These cells are able to differentiate into phagocytes (B-1CDP – B-1 cell derived phagocytes) in vitro and in vivo . During such process, reprogramming of gene expression occurs: lymphoid genes are turned off, while expression of myeloid genes is increased. This study aims to investigate whether Ikaros could be related to the control of B-1 cell plasticity. Interestingly, Ikaros expression by B-1CDP cells was found to be relatively low, and the protein is abnormally localized in the cytoplasm. Moreover, the isoforms expressed by B-1 cells are different from those expressed by other lymphocytes, with expression of active isoforms being almost absent in B-1CDP. Based on these findings, Ikaros could be an important factor driving the differentiation and proliferation of B-1 cells. [ABSTRACT FROM AUTHOR]

Summary Myeloid-derived suppressor cells (MDSCs) and dendritic cells (DCs) arise from common progenitors. Tumor-derived factors redirect differentiation from immune-promoting DCs to tolerogenic MDSCs, an immunological hallmark of cancer. Indeed, in vitro differentiation of DCs from human primary monocytes results in the generation of MDSCs under tumor-associated conditions (PGE2 or tumor cell-conditioned media). Comparison of MDSC and DC DNA methylomes now reveals extensive demethylation with specific gains of DNA methylation and repression of immunogenic-associated genes occurring in MDSCs specifically, concomitant with increased DNA methyltransferase 3A (DNMT3A) levels. DNMT3A downregulation erases MDSC-specific hypermethylation, and it abolishes their immunosuppressive capacity. Primary MDSCs isolated from ovarian cancer patients display a similar hypermethylation signature in connection with PGE2-dependent DNMT3A overexpression. Our study links PGE2- and DNMT3A-dependent hypermethylation with immunosuppressive MDSC functions, providing a promising target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Double plant homeodomain finger 2 (DPF2) is a highly evolutionary conserved member of the d4 protein family that is ubiquitously expressed in human tissues and was recently shown to inhibit the myeloid differentiation of hematopoietic stem/progenitor and acute myelogenous leukemia cells. Here, we present the crystal structure of the tandem plant homeodomain finger domain of human DPF2 at 1.6-Å resolution. We show that DPF2 interacts with the acetylated tails of both histones 3 and 4 via bipartite binding pockets on the DPF2 surface. Blocking these interactions through targeted mutagenesis of DPF2 abolishes its recruitment to target chromatin regions as well as its ability to prevent myeloid differentiation in vivo. Our findings suggest that the histone binding of DPF2 plays an important regulatory role in the transcriptional program that drives myeloid differentiation. [ABSTRACT FROM AUTHOR]

Treatment of acute myeloid leukemia (AML) largely depends on chemotherapy, but current regimens have been unsatisfactory for long-term remission. Although differentiation induction therapy utilizing 1,25(OH)2D3 (VD3) has shown great promise for the improvement of AML treatment efficacy, severe side effects caused by its supraphysiological dose limit its clinical application. Here we investigated the combinatorial effect of l-asparaginase (ASNase)-mediated amino acid depletion and the latent alternation of VD3 activity on the induction of myeloid differentiation. ASNase treatment enhanced VD3-driven phenotypic and functional differentiation of three-different AML cell lines into monocyte/macrophages, along with c-Myc downregulation. Using gene silencing with shRNA and a chemical blocker, we found that reduced c-Myc is a critical factor for improving VD3 efficacy. c-Myc-dependent inhibition of mTORC1 signaling and induction of autophagy were involved in the enhanced AML cell differentiation. In addition, in a postculture of AML cells after each treatment, ASNase supports the antileukemic effect of VD3 by inhibiting cell growth and inducing apoptosis. Finally, we confirmed that the administration of ASNase significantly improved VD3 efficacy in the prolongation of survival time in mice bearing tumor xenograft. Our results are the first to demonstrate the extended application of ASNase, which is currently used for acute lymphoid leukemia, in VD3-mediated differentiation induction therapy for AML, and suggest that this drug combination may be a promising novel strategy for curing AML. [ABSTRACT FROM AUTHOR]

Although acute myeloid leukaemia (AML) is assumed to be driven by transformed haematopoietic stem and progenitor cells, we have described recently a new pathway leading to AML from T-cell progenitors. Furthermore, we could identify a subgroup of human AML with gene expression profile suggesting T-lymphoid origin and potentially novel treatment.

The Natural resistance-associated macrophage protein 1 (Nramp1 or Solute carrier 11 member 1, Slc11a1) transports divalent metals across the membrane of late endosomes and lysosomes in professional phagocytes. Nramp1 represents an ancient eukaryotic cell-autonomous defense whereas the gene duplication that yielded Nramp1 and Nramp2 predated the origin of Sarcopterygians (lobe-finned fishes and tetrapods). SLC11A1 genetic polymorphisms associated with human resistance to tuberculosis consist of potential regulatory variants. Herein, current knowledge of the regulation of SLC11A1 gene expression is reviewed and comprehensive analysis of ENCODE data available for hematopoietic cell-types suggests a hypothesis for the regulation of SLC11A1 expression during myeloid development and phagocyte functional polarization. SLC11A1 is part of a 34.6 kb CTCF-insulated locus scattered with predicted regulatory elements: a 3' enhancer, a large 5' enhancer domain and four elements spread around the transcription start site (TSS), including several C/EBP and PU.1 sites. SLC11A1 locus ends appear mobilized by ETS-related factors early during myelopoiesis; activation of both 5' and 3' enhancers in myelo-monocytic cells correlate with transcription factor binding at the TSS. Characterizing the corresponding cis/trans determinants functionally will establish the mechanisms involved and possibly reveal genetic variation that impacts susceptibility to infectious or immune diseases.

The CCAAT enhancer-binding protein α (C/EBPα) plays an important role in myeloid cell differentiation and in the enhancement of C/EBPα expression/activity, which can lead to granulocytic differentiation in acute myeloid leukemia (AML) cells. We found that styryl quinazolinones induce upregulation of C/EBPα expression, and thereby induce myeloid differentiation in human myeloid leukemia cell lines. We screened a series of active styryl quinazolinones and evaluated the structure–activity relationship (SAR) of these small molecules in inducing C/EBPα expression—thereby prompting the leukemic cells to differentiate. We observed that compound 78 causes differentiation at 3 μM concentration, while 1 induces differentiation at 10 μM concentration. We also observed an increase in the expression of neutrophil differentiation marker CD11b upon treatment with 78. Both the C/EBPα and C/EBPε levels were found to be upregulated by treatment with 78. These SAR findings are inspiration to develop further modified styryl quinazolinones, in the path of this novel differentiation therapy, which can contribute to the care of patients with AML.

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.

Unfavorable patient survival coincides with lineage plasticity observed in human acute leukemias. These cases are assumed to arise from hematopoietic stem cells, which have stable multipotent differentiation potential. However, here we report that plasticity in leukemia can result from instable lineage identity states inherited from differentiating progenitor cells. Using mice with enhanced c-Myc expression, we show, at the single-cell level, that T-lymphoid progenitors retain broad malignant lineage potential with a high capacity to differentiate into myeloid leukemia. These T-cell-derived myeloid blasts retain expression of a defined set of T-cell transcription factors, creating a lymphoid epigenetic memory that confers growth and propagates myeloid/T-lymphoid plasticity. Based on these characteristics, we identified a correlating human leukemia cohort and revealed targeting of Jak2/Stat3 signaling as a therapeutic possibility. Collectively, our study suggests the thymus as a source for myeloid leukemia and proposes leukemic plasticity as a driving mechanism. Moreover, our results reveal a pathway-directed therapy option against thymus-derived myeloid leukemogenesis and propose a model in which dynamic progenitor differentiation states shape unique neoplastic identities and therapy responses. [ABSTRACT FROM AUTHOR]

The aorta-gonad-mesonephros (AGM) region contains intra-aortic clusters (IACs) thought to have acquired hematopoietic stem cell (HSC) potential in vertebrate embryos. To assess extrinsic regulation of IACs in the AGM region, we employed mouse embryos harboring a Sall1-GFP reporter gene, which allows identification of mesonephros cells based on GFP expression. Analysis of AGM region tissue sections confirmed mesonephros GFP expression. Mesonephric cells sorted at E10.5 expressed mRNA encoding Csf1, a hematopoietic cytokine, and corresponding protein, based on real-time PCR and immunocytochemistry, respectively. Further analysis indicated that some IACs express the CSF1 receptor, CSF1R. Expression of Cebpa and Irf8 mRNAs was higher in CSF1R-positive IACs, whereas that of Cebpε and Gfi1 mRNAs was lower relative to CSF1R-negative IACs, suggesting that CSF1/CSF1R signaling functions in IAC myeloid differentiation by modulating expression of these transcription factors. Colony formation assays using CSF1R-positive IACs revealed increased numbers of myeloid colonies in the presence of CSF1. Analysis using an intra-cellular signaling array indicated the greatest fold increase of Cleaved Caspase-3 in AGM cells in the presence of CSF1. Immunohistochemistry revealed that Cleaved Caspase-3 is primarily expressed in IACs in the AGM region, and incubation of IACs with CSF1 up-regulated Cleaved Caspase-3. Overall, our findings suggest that CSF1 secreted from mesonephros accelerates IAC myeloid differentiation in the AGM region, possibly via Caspase-3 cleavage. [ABSTRACT FROM AUTHOR]