Differentiation Block Research Articles

The BCAT1 CXXC Motif Provides Protection against ROS in Acute Myeloid Leukaemia Cells.

The cytosolic branched-chain aminotransferase (BCAT1) has received attention for its role in myeloid leukaemia development, where studies indicate metabolic adaptations due to BCAT1 up-regulation. BCAT1, like the mitochondria isoform (BCAT2), shares a conserved CXXC motif ~10 Å from the active site. This CXXC motif has been shown to act as a ‘redox-switch’ in the enzymatic regulation of the BCAT proteins, however the response to reactive oxygen species (ROS) differs between BCAT isoforms. Studies indicate that the BCAT1 CXXC motif is several orders of magnitude less sensitive to the effects of ROS compared with BCAT2. Moreover, estimation of the reduction mid-point potential of BCAT1, indicates that BCAT1 is more reductive in nature and may possess antioxidant properties. Therefore, the aim of this study was to further characterise the BCAT1 CXXC motif and evaluate its role in acute myeloid leukaemia. Our biochemical analyses show that purified wild-type (WT) BCAT1 protein could metabolise H2O2 in vitro, whereas CXXC motif mutant or WT BCAT2 could not, demonstrating for the first time a novel antioxidant role for the BCAT1 CXXC motif. Transformed U937 AML cells over-expressing WT BCAT1, showed lower levels of intracellular ROS compared with cells over-expressing the CXXC motif mutant (CXXS) or Vector Controls, indicating that the BCAT1 CXXC motif may buffer intracellular ROS, impacting on cell proliferation. U937 AML cells over-expressing WT BCAT1 displayed less cellular differentiation, as observed by a reduction of the myeloid markers; CD11b, CD14, CD68, and CD36. This finding suggests a role for the BCAT1 CXXC motif in cell development, which is an important pathological feature of myeloid leukaemia, a disease characterised by a block in myeloid differentiation. Furthermore, WT BCAT1 cells were more resistant to apoptosis compared with CXXS BCAT1 cells, an important observation given the role of ROS in apoptotic signalling and myeloid leukaemia development. Since CD36 has been shown to be Nrf2 regulated, we investigated the expression of the Nrf2 regulated gene, TrxRD1. Our data show that the expression of TrxRD1 was downregulated in transformed U937 AML cells overexpressing WT BCAT1, which taken with the reduction in CD36 implicates less Nrf2 activation. Therefore, this finding may implicate the BCAT1 CXXC motif in wider cellular redox-mediated processes. Altogether, this study provides the first evidence to suggest that the BCAT1 CXXC motif may contribute to the buffering of ROS levels inside AML cells, which may impact ROS-mediated processes in the development of myeloid leukaemia.

Chromatin-associated orphan snoRNA regulates DNA damage-mediated differentiation via a non-canonical complex.

Small nucleolar RNAs (snoRNAs) are commonly acknowledged as a class of homogeneous non-coding RNAs that guide ribosomal RNA modifications. However, snoRNAs referred to as orphans have largely unknown functions. Here, we systematically profile chromatin-associated snoRNAs (casnoRNAs) in mammalian cells and identify a subgroup of orphan casnoRNAs responding to DNA damage stress, among which SNORA73 shows the most marked reduction in chromatin enrichment. Downregulated SNORA73 maintains cancer genome stability and differentiation block in hematopoietic malignancy. Mechanistically, casnoRNA the 5' end non-canonical structure of SNORA73 is critical for its function and binding to poly (ADP-ribose) polymerase 1 (PARP1). SNORA73 inhibits PARP1 auto-PARylation to affect cancer genome stability by forming a small nucleolar ribonucleoprotein (snoRNP) with PARP1 and canonical H/ACA proteins DKC1/NHP2. Our findings reveal the role of an orphan snoRNA serving as casnoRNA and highlights a link between non-canonical structure of snoRNA and their functional diversity.

Novel Molecular Therapeutics Targeting Signaling Pathway to Control Hepatitis B Viral Infection.

Numerous canonical cellular signaling pathways modulate hepatitis B virus (HBV) replication. HBV genome products are known to play a significant role in regulating these cellular pathways for the liver’s viral-related pathology and physiology and have been identified as the main factor in hepatocarcinogenesis. Signaling changes during viral replication ultimately affect cellular persistence, multiplication, migration, genome instability, and genome damage, leading to proliferation, evasion of apoptosis, block of differentiation, and immortality. Recent studies have documented that numerous signaling pathway agonists or inhibitors play an important role in reducing HBV replication in vitro and in vivo, and some have been used in phase I or phase II clinical trials. These optional agents as molecular therapeutics target cellular pathways that could limit the replication and transcription of HBV or inhibit the secretion of the small surface antigen of HBV in a signaling-independent manner. As principle-based available information, a combined strategy including antiviral therapy and immunomodulation will be needed to control HBV infection effectively. In this review, we summarize recent findings on interventions of molecular regulators in viral replication and the interactions of HBV proteins with the components of the various targeting cellular pathways, which may assist in designing novel agents to modulate signaling pathways to prevent HBV replication or carcinogenesis.

Specific effects of somatic GATA2 zinc finger mutations on erythroid differentiation

GATA2 zinc-finger (ZF) mutations are associated with distinct entities of myeloid malignancies. The specific distribution of these mutations points toward different mechanisms of leukemogenesis depending on the ZF domain affected. In this study, we compared recurring somatic mutations in ZF1 and ZF2. All tested ZF mutants disrupted DNA binding in vitro. In transcription assays, co-expression of FOG1 counteracted GATA2-dependent transcriptional activation, while a variable response to FOG1-mediated repression was observed for individual GATA2 mutants. In primary murine bone marrow cells, GATA2 wild-type (WT) expression inhibited colony formation, while this effect was reduced for both mutants A318T (ZF1) and L359V (ZF2) with a shift toward granulopoiesis. In primary human CD34+ bone marrow cells and in the myeloid cell line K562, ectopic expression of GATA2 L359V, but not A318T or G320D, caused a block of erythroid differentiation accompanied by downregulation of GATA1, STAT5B, and PLCG1. Our findings may explain the role of GATA2 L359V during the progression of chronic myeloid leukemia and the collaboration of GATA2 ZF1 alterations with CEBPA double mutations in erythroleukemia.

Reduced Plasmacytoid Dendritic Cell Output Is Associated With High Risk in Low-grade Myelodysplastic Syndrome.

Reduced Plasmacytoid Dendritic Cell Output Is Associated With High Risk in Low-grade Myelodysplastic Syndrome.

Inflammasome Activation in Myeloid Malignancies-Friend or Foe?

Myeloid malignancies including myelodysplastic syndromes, myeloproliferative neoplasms and acute myeloid leukemia are heterogeneous disorders originating from mutated hematopoietic stem and progenitor cells (HSPCs). Genetically, they are very heterogeneous and characterized by uncontrolled proliferation and/or blockage of differentiation of abnormal HSPCs. Recent studies suggest the involvement of inflammasome activation in disease initiation and clonal progression. Inflammasomes are cytosolic innate immune sensors that, upon activation, induce caspase-1 mediated processing of interleukin (IL) -1-cytokine members IL-1β and IL-18, as well as initiation of gasdermin D-dependent pyroptosis. Inflammasome activation leads to a pro-inflammatory microenvironment in the bone marrow, which drives proliferation and may induce clonal selection of mutated HSPCs. However, there are also contradictory data showing that inflammasome activation actually counteracts leukemogenesis. Overall, the beneficial or detrimental effect of inflammasome activation seems to be highly dependent on mutational, environmental, and immunological contexts and an improved understanding is fundamental to advance specific therapeutic targeting strategies. This review summarizes current knowledge about this dichotomous effect of inflammasome activation in myeloid malignancies and provides further perspectives on therapeutic targeting.

A synthetic covalent ligand of the C/EBPβ transactivation domain inhibits acute myeloid leukemia cells

C/EBPβ has recently emerged as a pro-leukemogenic transcription factor that cooperates with oncoprotein MYB to maintain proliferation and differentiation block of AML cells, making C/EBPβ an interesting drug target for AML. Here we have studied the inhibitory potential and biological effects of a synthetic analog of the natural product helenalin, a known inhibitor of C/EBPβ. The synthetic compound inhibits C/EBPβ by covalent binding to cysteine residues in the transactivation domain, thereby causing up-regulation of differentiation-associated genes, cell death and reduced self-renewal potential of AML cells. Suppression of these effects by ectopic expression of C/EBPβ or MYB and gene expression profiling validate C/EBPβ as a relevant target of the helenalin-mimic and highlight its role as a pro-leukemogenic factor. Overall, our work demonstrates that the synthetic helenalin mimic acts as a covalent inhibitor of C/EBPβ and identifies the cysteine residues in the transactivation domain of C/EBPβ as ligandable sites. The helenalin mimic can be considered a potential “lead molecule” but needs further development towards more effective C/EBPβ inhibitors before being used as a therapeutic agent.

MECOM gene overexpression in pediatric patients with acute myeloid leukemia

Background Acute myeloid leukemia (AML) is characterized by blocked or aberrant differentiation of hematopoietic stem cells. The MECOM gene overexpression in hematopoietic progenitors induces myeloid differentiation block, resulting in increased self-renewal and survival of these transformed progenitors. However, its exact role in AML remains unclear. We aimed to estimate the prevalence of MECOM overexpression among pediatric AML patients, and assess its impact on clinical outcome. Patients and Methods Real-time quantitative polymerase chain reaction and Livak method (2ΔΔCt) were used to determine relative MECOM expression level among 243 pediatric patients with AML. MECOM overexpression was considered if the cumulative relative expression was above 1 (2-ΔΔCt) and was designated as MECOMpos . Results Of 243 AML patients tested 57(23.5%) demonstrated MECOMpos . Patients with MECOMpos had significantly lower median age. The frequency of MECOMpos was significantly higher among AML patients with 11q23 abnormalities, complex karyotypes and among high- and intermediate-risk groups compared to low-risk group (p = .014). MECOMpos patients had significantly lower overall survival (OS) (38.7 vs. 78.9%, p < .001), event-free survival (EFS) (37.3% vs. 68.4%, p < .001), and had higher cumulative incidence of relapse (49.5% vs. 23.5%, p = .002) at 36 months compared to MECOMneg patients. Multivariate analysis revealed that MECOMpos was an adverse prognostic factor for OS (hazards ratio (HR) = 2.11, 95% confidence interval (CI) 1.24–3.60, p = .006) and EFS (HR= 1.71, 95% CI 1.07–2.75, p = .025). The logistic regression model showed that MECOMpos was an independent prognostic factor regardless of minimal residual disease status post first induction therapy in the intermediate-risk group (odds ratio 2.89; 95% CI 1.19–6.57, p = .018). Conclusion The aberrant MECOM gene expression is an adverse prognostic factor, especially in patients without previously known cytogenetic risk factors. Our results suggest the potential benefit from pretreatment screening for MECOM gene overexpression in newly diagnosed AML patients for better risk stratification and treatment adjustment.

Lymphocyte cytosolic protein 1 (L-plastin) I232F mutation impairs granulocytic proliferation and causes neutropenia

AbstractNeutrophils migrate into inflamed tissue, engage in phagocytosis, and clear pathogens or apoptotic cells. These processes require well-coordinated events involving the actin cytoskeleton. We describe a child with severe neutropenia and episodes of soft tissue infections and pneumonia. Bone marrow examination showed granulocytic hypoplasia with dysplasia. Whole-exome sequencing revealed a de novo heterozygous missense mutation in LCP1, which encodes the F-actin–binding protein Lymphocyte Cytosolic Protein 1. To determine its pathophysiological significance, we stably transduced cells with doxycycline-inducible wild-type LCP1 and LCP1 I232F lentiviral constructs. We observed dysplastic granulocytic 32D cells expressing LCP1 I232F cells. These cells showed decreased proliferation without a block in differentiation. In addition, expression of LCP1 I232F resulted in a cell cycle arrest at the G2/M phase, but it did not lead to increased levels of genes involved in apoptosis or the unfolded protein response. Both 32D and HeLa cells expressing mutant LCP1 displayed impaired cell motility and invasiveness. Flow cytometry showed increased F-actin. However, mutant LCP1-expressing 32D cells exhibited normal oxidative burst upon stimulation. Confocal imaging and subcellular fractionation revealed diffuse intracellular localization of LCP1, but only the mutant form was found in the nucleus. We conclude that LCP1 is a new gene involved in granulopoiesis, and the missense variant LCP1 I232F leads to neutropenia and granulocytic dysplasia with aberrant actin dynamics. Our work supports a model of neutropenia due to aberrant actin regulation.

Eradication of leukemia stem cells by inhibitors of DNA methyltransferase, EZH2 and G9a histone methyltransferases

Complete eradication of leukemic stem cells (LSCs) in patients with acute myeloid leukemia (AML) is required for curative therapy. Epigenetic alterations that involve gene-silencing by DNA methylation by DNMT1, methylation of H3K27 by EZH2 histone methyltransferase (HMT) and methylation of H3K9 by G9a HMT may play a major role in the development of AML. The major action of these epigenetic alterations is the silencing of the genes that program differentiation of AML cells. Inhibitors of DNA and histone methylation have the potential to reverse this block in differentiation. If tumor suppressor genes (TSGs) contain two gene-silencing markers, such as DNA methylation and H3K27me3, they may not be fully reactivated with only an inhibitor of DNA methylation, such as 5-aza-2’-deoxycytidine (5-AZA-CdR), but may also require an inhibitor of EZH2. In support of this model is the synergistic antileukemic action as shown by a colony assay on AML cells using 5-AZA-CdR in combination with 3-deazaneplanocin A (DZNep), a potent inhibitor of EZH2. A similar type of interaction can occur when TSGs are silenced by DNA methylation and the G9a methylation of H3K9me2, a second gene-silencing marker. Treatment of these AML cells with 5-AZA-CdR and BIX01294, an inhibitor of G9a, also results in a synergistic antileukemic action. Leukemic cells that contain 3 different gene-silencing markers: DNA methylation, H3K27me3 and H3K9me2 may require 3 different inhibitors for maximal antineoplastic activity. This result was observed when the AML cells were treated in with 5-AZA-CdR, DZNep and BIX01294. The aim of this study was to demonstrate that epigenetic agents that target DNA and histone methylation have remarkable antineoplastic activity against myeloid leukemia cells. The second aim was to propose a dose-schedule for these epigenetic agents that can be evaluated in a clinical trial in patients with advanced AML for its potential to eradicate LSCs. One of the most sensitive targets for chemotherapeutic intervention in LSCs is the block in differentiation due to gene-silencing by DNA and histone methylation. Epigenetic agents that have the potential to reverse this block merit clinical investigation with high priority.

3065 – CRISPR MODELING OF ACUTE MYELOID LEUKEMIA MUTATIONS IN PRIMARY HEMATOPOIETIC STEM CELLS TO STUDY (PRE-)LEUKEMIA DEVELOPMENT

The development of acute myeloid leukemia (AML) is a stepwise process with early founder mutations that give rise to hematopoietic stem cells (HSCs) with a clonal advantage, followed by subsequent driver mutations that result in transformation and full-blown leukemia. To study the development and progression of AML through time requires consecutive samples of (pre-)leukemic cells, however, this also comes with many practical challenges. Therefore, we implemented a recently published CRISPR methodology that allows modeling of AML mutations including DNMT3A, TET2, ASXL1, RUNX1, GATA2, and CEBPA p30 in primary HSCs. In short, bone marrow (BM) derived CD34+ cells are expanded, electroporated with ribonucleoprotein complexes, and in case of homologous directed repair, directly followed by HDR-template delivery using adeno-associated viruses. The HDR template consists of a SFFV-GFP, which is integrated in an early exon resulting in a loss of function (LOF) that can be traced through GFP expression. In general, we reach editing efficiencies ranging from 60-90% and HDR efficiencies of around 30-50%. Modeling CEBPA p30 in BM CD34+ cells resulted in a block in differentiation and increased colony forming potential in multiple replates, and uncontrolled cell growth. Proteome analysis revealed both known and unknown CEBPA p30 targets being differentially expressed. Injecting CEBPA p30 cells in NSGS mice resulted in a (pre-)leukemic phenotype with increased myeloid output, aberrant plasma membrane marker expression, and an immature phenotype. Currently, we are further evaluating the additional effect of recurrently co-mutated genes TET2 and GATA2. In addition, we also study the consequences of LOF mutations in DNMT3A, ASXL1, TET2, and RUNX1 in NSG and NSGS mice potentially followed by secondary hits, stresses and treatments in order to study the leukemic evolution through time. The development of acute myeloid leukemia (AML) is a stepwise process with early founder mutations that give rise to hematopoietic stem cells (HSCs) with a clonal advantage, followed by subsequent driver mutations that result in transformation and full-blown leukemia. To study the development and progression of AML through time requires consecutive samples of (pre-)leukemic cells, however, this also comes with many practical challenges. Therefore, we implemented a recently published CRISPR methodology that allows modeling of AML mutations including DNMT3A, TET2, ASXL1, RUNX1, GATA2, and CEBPA p30 in primary HSCs. In short, bone marrow (BM) derived CD34+ cells are expanded, electroporated with ribonucleoprotein complexes, and in case of homologous directed repair, directly followed by HDR-template delivery using adeno-associated viruses. The HDR template consists of a SFFV-GFP, which is integrated in an early exon resulting in a loss of function (LOF) that can be traced through GFP expression. In general, we reach editing efficiencies ranging from 60-90% and HDR efficiencies of around 30-50%. Modeling CEBPA p30 in BM CD34+ cells resulted in a block in differentiation and increased colony forming potential in multiple replates, and uncontrolled cell growth. Proteome analysis revealed both known and unknown CEBPA p30 targets being differentially expressed. Injecting CEBPA p30 cells in NSGS mice resulted in a (pre-)leukemic phenotype with increased myeloid output, aberrant plasma membrane marker expression, and an immature phenotype. Currently, we are further evaluating the additional effect of recurrently co-mutated genes TET2 and GATA2. In addition, we also study the consequences of LOF mutations in DNMT3A, ASXL1, TET2, and RUNX1 in NSG and NSGS mice potentially followed by secondary hits, stresses and treatments in order to study the leukemic evolution through time.

Mathematical model for simulation of morphological changes associated to crypt fission in the colon

&lt;p style='text-indent:20px;'&gt;Morphological changes due to colorectal cancer propagation by an abnormal crypt fission is an interesting application arising in medicine and biology, that we try to analyse by a differential equations model coupled with a discrete crypt fission model. A colonic crypt can slowly change its shape in three steps: growth, bifurcation and fission. Fission is a rare event in a normal tissue, however if transit cells are unable to differentiate, due to an activation of Wnt signaling, then it may become fast and uncontrolled and cause a formation of aberrant crypt foci (ACF), defined as clusters of aberrant crypts. The differential equation system is composed by a convective diffusive equation for transit cell density and an elliptic equation for cell pressure, both defined on a manifold. The discrete crypt fission model acts in a set of adjacent crypts in order to investigate the ACF dynamics, due to a differentiation block. By using a Galerkin finite element method we solve numerically the differential equation model and show some interesting results about an ACF formation caused by a deformation and fission of abnormal crypts.&lt;/p&gt;

Inductive Spatiotemporal Graph Convolutional Networks for Short-Term Quantitative Precipitation Forecasting

Short-term quantitative precipitation forecasting (SQPF) using weather radar is an important but challenging problem as one must cope with inherent nonlinearity and spatiotemporal correlation in the data. In this article, we propose a novel deep learning model, named Inductive spatiotemporal Graph Convolutional Networks (InstGCNs), to overcome these issues in SQPF. The proposed InstGCN can learn a nonlinear mapping from historical radar reflectivity to future rainfall amounts and extract informative spatiotemporal representations simultaneously. Specifically, we first provide a formal definition for formulating the SQPF problem from a graph perspective. Then, based on radar reflectivity and rain gauge observation, we propose a novel graph construction approach that utilizes a special elliptic structure to model the spatial dependence of precipitation areas. In addition, a new Node level Differential Block (Node-DB) is introduced to tackle the nonstationary temporal dependence. To execute inductive graph learning for unseen nodes, we design to decompose a whole graph into subgraphs. We conduct extensive experiments on three real-world datasets in East China and a public weather radar dataset in the southeastern parts of France. The experimental results confirm the advantages of InstGCN compared with several state of the arts.

3022 – METABOLIC AND EPIGENETIC IMPACT OF CD44 ANTIBODY-INDUCED DIFFERENTIATION IMPLICATES THE USE OF EPIGENETIC THERAPIES IN AML TREATMENT

Acute myeloid leukemia (AML) is a subset of leukemia characterized by the clonal expansion of abnormal myeloid blasts. The poor prognosis of AML patients caused by low tolerance to intensive chemotherapy and high relapse rates triggered by leukemic stem cells has encouraged the pursuit of alternative therapies. Differentiation therapy, which involves the use of agents that can release the differentiation block in these leukemic stem/blasts has emerged as a promising therapy following the success of All-trans retinoic acid (ATRA) on one AML subtype. Efforts focused on finding differentiation agents that are effective on other AML subtypes brought into light the possibility of using anti-CD44 antibodies to target AML cells. Anti-CD44 has been shown to release the differentiation block in varying AML subtypes and work in our lab is focused on understanding the mechanism by which it induces its effects. Recent studies highlight the role that epigenetic and metabolic regulation plays during hematopoiesis and leukemogenesis, therefore, in this work we investigated the epigenetic and metabolic mechanisms associated with anti-CD44 induced differentiation. Using AML cell lines from different subtypes, we demonstrated that anti-CD44 treatment results in an extensive change of histone modifications. We found that inhibiting CBP/p300 (responsible for H3K9ac) and MLL (responsible for H3K4me1/2/3) attenuated the differentiation promoting effects of anti-CD44. Conversely, inhibition of EZH1/2 (responsible for H3K27me3) enhanced anti-CD44’s effects. The observed increase in H3K4me can be caused by an accumulation of succinate, a metabolite that is part of the tricarboxylic acid cycle. Taken together, these data highlight the promising potential of using anti-CD44 either alone or in combination with epigenetic inhibitor as a therapeutic agent across multiple subtypes in AML. Acute myeloid leukemia (AML) is a subset of leukemia characterized by the clonal expansion of abnormal myeloid blasts. The poor prognosis of AML patients caused by low tolerance to intensive chemotherapy and high relapse rates triggered by leukemic stem cells has encouraged the pursuit of alternative therapies. Differentiation therapy, which involves the use of agents that can release the differentiation block in these leukemic stem/blasts has emerged as a promising therapy following the success of All-trans retinoic acid (ATRA) on one AML subtype. Efforts focused on finding differentiation agents that are effective on other AML subtypes brought into light the possibility of using anti-CD44 antibodies to target AML cells. Anti-CD44 has been shown to release the differentiation block in varying AML subtypes and work in our lab is focused on understanding the mechanism by which it induces its effects. Recent studies highlight the role that epigenetic and metabolic regulation plays during hematopoiesis and leukemogenesis, therefore, in this work we investigated the epigenetic and metabolic mechanisms associated with anti-CD44 induced differentiation. Using AML cell lines from different subtypes, we demonstrated that anti-CD44 treatment results in an extensive change of histone modifications. We found that inhibiting CBP/p300 (responsible for H3K9ac) and MLL (responsible for H3K4me1/2/3) attenuated the differentiation promoting effects of anti-CD44. Conversely, inhibition of EZH1/2 (responsible for H3K27me3) enhanced anti-CD44’s effects. The observed increase in H3K4me can be caused by an accumulation of succinate, a metabolite that is part of the tricarboxylic acid cycle. Taken together, these data highlight the promising potential of using anti-CD44 either alone or in combination with epigenetic inhibitor as a therapeutic agent across multiple subtypes in AML.

IDH1/2 Mutations in Cancer Stem Cells and Their Implications for Differentiation Therapy.

Isocitrate dehydrogenase 1 and 2 (IDH1/2) are enzymes recurrently mutated in various types of cancer, including glioma, cholangiocarcinoma, chondrosarcoma, and acute myeloid leukemia. Mutant IDH1/2 induce a block in differentiation and thereby contribute to the stemness and oncogenesis of their cells of origin. Recently, small-molecule inhibitors of mutant IDH1/2 have been Food and Drug Administration–approved for the treatment of IDH1/2-mutated acute myeloid leukemia. These inhibitors decrease the stemness of the targeted IDH1/2-mutated cancer cells and induce their differentiation to more mature cells. In this review, we elucidate the mechanisms by which mutant IDH1/2 induce a block in differentiation and the biological and clinical effects of the release into differentiation by mutant-IDH1/2 inhibitors. (J Histochem Cytochem 70:83–97, 2022)

The Hochschild complex of a finite tensor category

Modular functors, i.e. consistent systems of projective representations of mapping class groups of surfaces, have been constructed for non-semisimple modular categories already decades ago. Concepts from homological algebra have not been used in this construction although it is an obvious question how they should enter in the non-semisimple case. In the present paper, we elucidate the interplay between the structures from topological field theory and from homological algebra by constructing a homotopy coherent projective action of the mapping class group $\text{SL}(2,\mathbb{Z})$ of the torus on the Hochschild complex of a modular category. This is a further step towards understanding the Hochschild complex of a modular category as a differential graded conformal block for the torus. Moreover, we describe a differential graded version of the Verlinde algebra.

The distinct effects of P18 overexpression on different stages of hematopoiesis involve TGF-\u03b2 and NF-\u03baB signaling

Deficiency of P18 can significantly improve the self-renewal potential of hematopoietic stem cells (HSC) and the success of long-term engraftment. However, the effects of P18 overexpression, which is involved in the inhibitory effects of RUNX1b at the early stage of hematopoiesis, have not been examined in detail. In this study, we established inducible P18/hESC lines and monitored the effects of P18 overexpression on hematopoietic differentiation. Induction of P18 from day 0 (D0) dramatically decreased production of CD34highCD43− cells and derivative populations, but not that of CD34lowCD43− cells, changed the cell cycle status and apoptosis of KDR+ cells and downregulated the key hematopoietic genes at D4, which might cause the severe blockage of hematopoietic differentiation at the early stage. By contrast, induction of P18 from D10 dramatically increased production of classic hematopoietic populations and changed the cell cycle status and apoptosis of CD45+ cells at D14. These effects can be counteracted by inhibition of TGF-β or NF-κB signaling respectively. This is the first evidence that P18 promotes hematopoiesis, a rare property among cyclin-dependent kinase inhibitors (CKIs).

MS4A3 promotes differentiation in chronic myeloid leukemia by enhancing common β-chain cytokine receptor endocytosis

AbstractThe chronic phase of chronic myeloid leukemia (CP-CML) is characterized by the excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase (BP). Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor but promotes endocytosis of common β-chain (βc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade βc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown (KD) or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhances leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and BP-CML. Promoting MS4A3 reexpression or delivery of ectopic MS4A3 may help eliminate LSPCs in vivo.

IMMU-04. UNVEILING THE TUMOR-METABOLOME-IMMUNITY AXIS OF GLIOMA

Abstract The glioma microenvironment orchestrates tumor evolution, progression, and resistance to therapy. In high-grade gliomas, microglia and monocyte-derived macrophages constitute up to 70% of the tumor mass. However, the dynamics and phenotypes of intratumoral myeloid cells during tumor progression are poorly understood. Here we define myeloid cellular states in gliomas by longitudinal single-cell profiling and demonstrate their strict control by the tumor genotype. We report the unexpected and clinically highly relevant finding that human as well as murine gliomas with Isocitrate Dehydrogenase (IDH)1-R132H, a key oncogenic driver mutation of glioma, subdue their innate immune microenvironment by prompting a multifaceted reprogramming of myeloid and T cell metabolism. We employed integrated single-cell transcriptomic, time-of-flight mass cytometry and proteomic analyses of human healthy cortex control and glioma samples to identify myeloid cell subsets with distinct fates in IDH-mutated glioma that diverge from canonical trajectories of antigen-presenting cells as a result of a monocyte-to-macrophage differentiation block. Moving beyond single time point assessments, we now longitudinally describe differential immune cell infiltration and phenotype dynamics during glioma progression that are orchestrated by a fluctuating network of resident microglial cells and educated recruited immune cells. IDH mutations in glioma induce a tolerogenic alignment of their immune microenvironment through increased tryptophan uptake via large neutral amino acid transporter (LAT1)-CD98 and subsequent activation of the aryl hydrocarbon receptor (AHR) in educated blood-borne macrophages. In experimental tumor models, this immunosuppressive phenotype was reverted by LAT1-CD98 and AHR inhibitors. Taken together with direct effects on T cell activation, our findings not only link this oncogenic metabolic pathway to distinct immunosuppressive pathways but also provide the rationale and novel molecular targets for the development of immunotherapeutic concepts addressing the disease-defining microenvironmental effects of IDH mutations.

Genetic Ablation of Nuclear Receptor Interacting Protein 1 (NRIP1) Sensitizes Acute Myeloid Leukemia Cells to Retinoic Acids

The success of differentiation therapy is limited to acute promyelocytic leukemia (APL), and approaches to overcome the differentiation block in non-M3 AML have been unsuccessful. Nuclear hormone receptors (NHR) belong to ligand-inducible transcription factors that govern many cellular functions like differentiation, metabolism, and development. Retinoic Acid Receptor Alpha (RXRA) is a class of NHR that, when activated by all-trans retinoic acid (ATRA), successfully alleviates differentiation block in APL.To identify the NHRs/cofactors that could mediate or prevent differentiation in AML, we examined the differentially expressed NHRs and cofactors between ATRA sensitive (ATs) (NB4 and HL60) vs. ATRA resistant (ATr) AML cell lines (KG1a, Hel, K562, MV4-11, and OCI-AML3). Nuclear Receptor Interacting Protein 1 (NRIP1), a corepressor known to prevent transactivation of ligand-activated NHRs preferentially, was one of the top upregulated targets in the ATr cell lines (3.5 fold increase in RNA expression, figure 1a ). Immunoblot analysis also showed a significant increase in NRIP1 protein expression in the ATr than ATs cell lines (Figure 1b). Further, probing for NRIP1 expression in the publicly available TCGA and MILE AML study cohorts showed decreased NRIP1 expression in the APL cohort compared to other AML subtypes. Methylation profile from CCLE database of the NRIP1 promoter in AML cell lines showed ATs cell lines to be highly methylated compared to the ATr cell lines, suggesting the involvement of NRIP1 in mediating differentiation block in non-M3 AML (Figure 1c). To further dissect the role of NRIP1 in mediating this differentiation block, we carried out experiments in the AML cell line KG1a (having primitive blast features, high expression of NRIP1, and unresponsive to ATRA). Using CRISPR-cas9, we developed an NRIP1 knock-out (KO) cell line (Figure 1d). NRIP1 KO cell line showed a significant reduction in proliferation rate (Doubling time 26.2 vs. 36.5Hrs p<0.05). Further, cell cycle analysis revealed that NRIP1 KO leads to increased accumulation of cells in the G0 phase than in the S-phase (Figure 1e & f). We next assessed the sensitivity of the NRIP1 WT/KO cells to retinoic acids ATRA and bexarotene. Cells were treated with 1µM ATRA / bexarotene or in combination for 72 hours and evaluated for differentiation using CD11b marker by flow cytometry. NRIP1 KO alone leads to a marginal increase in basal CD11b expression compared to the WT cells (Mean CD11b expression 2.03% Vs 0.91%). ATRA treatment further increased the CD11b expression to 3.8% in KO cells compared to 1.6% in the WT cells. A similar increase in CD11b expression was observed in bexarotene-treated cells (3.7% Vs 1.24%). Combination of ATRA with bexarotene showed a 3-fold increase in CD11b expression in the KO cells compared to the WT (23.9% Vs 7.2%, Figure 1g). NRIP1 KO diminishes its repressive action on ligand-activated RARA (ATRA activated) and RXRA (Bexarotene-activated), thereby allowing synergistic differentiation induction by retinoic acids in AML cells.This study suggests a potential mechanism of differentiation inhibition mediated by corepressor NRIP1 in AML cells unresponsive to retinoic acids. Further in-depth analyses of molecular pathways governed by NRIP1 during ligand activation of NHRs are warranted to design differentiation therapies for AML. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.