SET-domain containing protein 2 (SETD2) is the primary methyltransferase responsible for generating H3K36me3, an epigenetic mark that is essential for transcriptional regulation and chromatin integrity. SETD2 mutations are frequently observed in various cancers and tend to cluster within its catalytic SET domain. Despite the clinical relevance of SETD2 missense mutations in cancer, their biochemical and structural consequences remain insufficiently characterized. Here, we present the enzymatic and structural characterization of the SETD2 L1609P mutant enzyme identified in leukemia. The L1609 residue is located in the SET domain within a conserved hydrophobic pocket that is involved in substrate H3K36 recognition. Interestingly, site-directed mutagenesis of residues within this hydrophobic pocket leads to SETD2 enzyme variants with either decreased or increased H3K36me3 methyltransferase activity, suggesting that cancer mutations affecting the L1609 residue could result in a loss-or gain-of-function enzyme variant. Using molecular and cellular approaches, we show that the SETD2 L1609P mutant exhibits reduced H3K36 methyltransferase activity, decreased protein stability, and poor cellular expression. Consistently, the crystal structure of the SETD2 L1609P in complex with a H3K36M peptide shows remodeling of the active site. These findings support the pivotal role of SETD2 inactivation and subsequent disruption of H3K36me3 deposition in oncogenesis, particularly in hematologic malignancies. Our study provides the first mechanistic and three-dimensional protein structure information on how SETD2-associated cancer mutations can lead to altered H3K36 methyltransferase activity.

Epitranscriptomics, the study of RNA modifications, together with their functional characterization, is emerging as an important area of investigation in RNA biology. Of the over 170 RNA modifications that have been identified on mRNA and non-coding RNAs, N6-methyladenosine (m6A) modification to mRNA is recognized as a key regulator of gene expression, splicing and protein translation. Functional readout of m6A is mediated by m6A readers mostly in the cytoplasm except for the nuclear-localized YTHDC1. m6A-YTHDC1 function has recently been extended to include short and long-range fine-tuning of genome activity via chromatin-associated mechanisms. This review summarizes YTHDC1-m6A nuclear functions in normal and cancer cells with special focus on its chromatin-associated roles and the ability of YTHDC1 to assemble into higher order nuclear structures called condensates. These processes are disturbed in cancer.

Despite recent therapeutic advances, acute myeloid leukemia (AML) confers poor survival. Inflammatory signaling critically influences AML progression and treatment resistance. We previously showed that protein arginine methyltransferase 2 (PRMT2) modulates inflammatory pathways in AML cells. We sought to delineate the role of PRMT2 in AML by mapping its protein interactome and assessing its functional impact on AML cells.Our proteomic analysis revealed VAV1 and the WASP/WIPF1 complex as principal PRMT2 interactors. VAV1, a hematopoietic-specific guanine nucleotide exchange factor (GEF) pivotal for Rho GTPase-mediated actin remodeling, forms a cytoplasmic complex with PRMT2. PRMT2 knockout (KO) resulted in changes of cytoskeletal architecture in AML cells, reducing cortical F-actin and cell adhesion. Transcriptomic analysis of PRMT2KO cells identified 976 differentially expressed genes related to cell adhesion and inflammatory pathways. Western blotting demonstrated lower JNK phosphorylation upon TNF stimulation in PRMT2KO cells, underscoring the role of PRMT2 in JNK activation. In contrast, nuclear accumulation of phosphorylated NF-κB p65 was enhanced, suggesting PRMT2 constrains NF-κB-driven inflammation.Supporting data in AML cells indicated that VAV1 is dimethylated at R422, located in the pleckstrin homology domain, a critical site for GEF activity. Our findings suggest PRMT2 directly methylates VAV1 at R422. Moreover, PRMT2 and VAV1 expression in 371 AML samples (Leucegene) was correlated with patient survival.Our findings identify PRMT2 as a pivotal regulator of AML progression through its interactions with VAV1 and the WASP/WIPF1 complex. The methylation of VAV1 at R422 by PRMT2 may control its GEF function. As VAV1 dysregulation is linked to aggressive leukemia, targeting PRMT2-VAV1 interactions could offer novel therapeutic strategies to counteract inflammation-driven AML progression.

Abstract A direct link between chronic inflammation and development of Acute Myeloid Leukemia (AML) has been highlighted in the past few years, demonstrating an interconnection between marked inflammatory phenotype and aberrant myeloproliferation in AML patients. Treating AML patients exhibiting a higher inflammatory signature with anti-inflammatory molecules resulted in significant increase of overall survival. Protein Arginine Methyltransferases (PRMTs) are epigenetic factors known to regulate gene expression through methylation of histone tails. It has been previously reported that PRMT1, 4, and 5 inhibition exhibit anti-proliferative effects on AML models. In this study, we investigated the role of another PRMT, called PRMT2, in the development of AML through its regulatory roles in inflammatory pathways. We first determined from an AML cohort (The Leucegene project, IRIC, Montréal, QC, Canada) that patients with a low PRMT2 expression display an enrichment of proinflammatory pathways compared to patients with a high PRMT2 expression. Therefore, we hypothesized that PRMT2 could be a key regulator of inflammatory processes in AML. We thus used a PRMT2 knockout mouse model (Prmt2 KO) and a PRMT2 knockout human AML cell line to validate our hypothesis. Although we demonstrated no difference in the bone marrow progenitors or mature cell populations of Prmt2 KO mice compared to control, we observed that Prmt2 KO Bone-Marrow Derived Macrophages (BMDMs) are more sensitive to LPS stimulation and express higher levels of pro-inflammatory cytokines, supporting our previous findings for a role of PRMT2 in the negative regulation of inflammatory processes. PRMT2 depleted human AML cells displayed an increased pro-inflammatory signature due to overactivation of STAT3, which is caused by an enhanced activation of the NFkB signaling pathway, leading to an overproduction of IL6. Together, these findings demonstrate that PRMT2 is a key regulator of the control of inflammation in AML. Recognition of PRMT2 as a biomarker of inflammation in AML would help to adapt treatment possibly through the synergistic use of anti-inflammatory molecules with other cytotoxic drugs. Citation Format: Camille Sauter, Thomas Morin, Fabien Guidez, John Simonet, Cyril Fournier, Céline Row, Denis Masnikov, Baptiste Pernon, Anne Largeot, Aziza Aznague, Yann Herault, Guy Sauvageau, Marc Maynadie, Mary Callanan, Jean-Noël Bastie, Romain Aucagne, Laurent Levadny Delva. Protein Arginine Methyltransferase 2 is involved in the control of inflammatory processes in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5588.

Background: While acute promyelocytic leukemia (APL) can generally be cured by current treatment (combining all-trans retinoic acid (ATRA) and arsenic (ATO)), it can serve as a model to develop immunotherapeutic approaches for other hematological malignancies. We have previously used an APL mouse model bearing the PML-RARA fusion gene to show that a non-specific immunotherapy comprising of a plasmid, pVAX14, coding for immunogenic open reading frames, which effectively code for neoantigens, in combination with ATRA, has a similar efficacy as a specific vaccine with PML-RARA fusion sequences with decreased bone marrow (BM) blasts, reduced minimal residual disease (PML-RARA transcripts) and measured immune responses to include an increase in anti-RARA antibody levels (Le Pogam et al Oncotarget 2015) - with long term survivors surviving up to 2 years (the mouse lifespan). The protective effect was shown to be T-cell mediated. In order to visualize cytotoxic killing we have imaged the effectors from immunized APL mice co-cultured with their APL targets in real time. Gene expression profiles revealed the activation of a gene list regulated in immune pathways. Methods: APL mice were treated with pVAX14 (3x100 micrograms every 20 days intramuscularly) + ATRA (5mg 21-day release). Effectors (total WBC or CD3+ T cells) were isolated from spleens of immunized mice and labelled (red) and co-cultured with APL BM cells labelled (green) (effector:target, E:T was 2:1) and visualized via timelapse imaging. Normal FVB/N BM were assayed as controls. Additional analyses were carried out by the incucyte Live-Cell Analysis Systems, which is like a flow cytometer that measures fluorescence in real time. Furthermore, APL mice were treated with ATRA + ATO (5 micrograms/g/mouse intraperitoneally for 28 consecutive days) without or with pVAX14 as previously described (Patel et al BCJ 2015). Bone marrows were harvested after the end of treatment and RNA-sequencing was performed on mice treated with ATRA+ATO (n=5) or ATRA + ATO + pVAX14 (n=7). Functional analysis using David identified immune related pathways. The genes regulated were ranked according to the frequency upregulated in the pathways, with confirmation of some by RQ-PCR. Results: Effectors originating from immunized mice were shown to kill APL BM target, sparing wild type BM cells. Two types of killing were observed, lytic or programmed cell death. Incucyte data showed an increase in apoptosis when the effectors were incubated with their APL targets compared with FVB/N BM marrow cells. Gene expression profiles showed distinct treatment signatures. Principle component analysis showed that the DNA treated mice had a more homogeneous expression pattern than mice without DNA treatment. The functional David pathway analysis identified 29 genes regulated in 16 immune pathways. RQ-PCR confirmed NLRP3 active in the inflammasome pathway as the most frequently regulated (13 pathways with >6-fold increased expression in the DNA treated mice relative to no DNA samples). Increased expression of Caspase 1 protein, also in the inflammasome pathway was additionally detected (3-fold increased expression in extracts from AML effectors co-cultured with AML targets compared with AML effectors with FVB/N targets). RQ-PCR also identified significant activation of innate immune pathway genes in immunized diseased mice compared to placebo such as the nucleic acid sensor, Hmgb1 (p=0.0002), the adapter molecule Myd88 (p=0.0017) and ATRA inducible Rig-I (p<0.0001). Conclusions: Visualization of cytotoxic T-cell kill in real time confirms one of the mechanisms of the protective effect of the immunotherapy. The gene list provides potential biomarkers of response to DNA treatment. Activation of the innate immune pathway suggests that this strategy can turn “cold” tumors “hot” and responsive to treatment. The findings using this APL model may be useful to treat other malignancies where the results of achieving remissions and cures are more challenging.

Background The outcome of Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remain dismal despite the development of treatment. Targeted therapy is gaining more and more attention in improving prognosis. Methods Expression of BRAF was analyzed by RT-qPCR in AML and MDS patients. Cells viability treated by drugs was measured by CCK-8 assay. Network pharmacology and RNA-sequence were used to analyze the mechanism of drugs and verified in vitro and xenograft tumor model. Results Here we showed that BRAF was overexpressed in AML and MDS patients, and correlated with poor prognosis. The BRAF inhibitor-Vemurafenib (VEM) could significantly induce senescence, proliferation inhibition and apoptosis in AML cells, which can be enhanced by Bortezomib (BOR). This inhibitory effect was also verified in CD34 + cells derived from AML patients. Mechanistically, we showed that VEM combined with BOR could turn on HIPPO signaling pathway, thereby inducing cellular senescence in AML cells and xenograft mouse. Conclusions Taken together, our findings demonstrate a significant upregulation of BRAF expression in AML and MDS patients, which is associated with unfavorable clinical outcomes. We also discovered that the BRAF inhibitor Vemurafenib induces cellular senescence through activation of the HIPPO signaling pathway. Analysis of BRAF expression holds promise as a prognostic indicator and potential therapeutic target for individuals with AML and MDS.

Arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and is involved in various cellular processes, including cancer development. PRMT2 expression is increased in several cancer types although its role in acute myeloid leukemia (AML) remains unknown. Here, we investigate the role of PRMT2 in a cohort of patients with AML, PRMT2 knockout AML cell lines as well as a Prmt2 knockout mouse model. In patients, low PRMT2 expressors are enriched for inflammatory signatures, including the NF-κB pathway, and show inferior survival. In keeping with a role for PRMT2 in control of inflammatory signaling, bone marrow-derived macrophages from Prmt2 KO mice display increased pro-inflammatory cytokine signaling upon LPS treatment. In PRMT2-depleted AML cell lines, aberrant inflammatory signaling has been linked to overproduction of IL6, resulting from a deregulation of the NF-κB signaling pathway, therefore leading to hyperactivation of STAT3. Together, these findings identify PRMT2 as a key regulator of inflammation in AML.

Despite the advances in the understanding and treatment of myeloproliferative neoplasm (MPN), the disease remains incurable with the risk of evolution to acute myeloid leukemia or myelofibrosis (MF). Unfortunately, the evolution of the disease to MF remains poorly understood, impeding preventive and therapeutic options. Recent studies in solid tumor microenvironment and organ fibrosis have shed instrumental insights on their respective pathogenesis and drug resistance, yet such precise data are lacking in MPN. In this study, through a patient sample-driven transcriptomic and epigenetic description of the MF microenvironment landscape and cell-based analyses, we identify homeobox B7 (HOXB7) overexpression and more precisely a potentially novel TGF-β/WNT/HOXB7 pathway as associated to a pro-fibrotic and pro-osteoblastic biased differentiation of mesenchymal stromal cells (MSCs). Using gene-based and chemical inhibition of this pathway, we reversed the abnormal phenotype of MSCs from patients with MF, providing the MPN field a potentially novel target to prevent and manage evolution to MF.

Despite significant advancements in the research of the pathogenesis mechanisms of Myelodysplastic Neoplasm (MDS) in recent years, there are still many gaps to fill. The advancement of metabolomics studies has led to a research booming in clarifying the impact of metabolic abnormalities during the pathogenesis of MDS. The present review primarily focuses on the dysregulated metabolic pathways, exploring the influences on the pathogenesis of MDS and their roles during the course of the disease. Furthermore, we discuss the potential of relevant metabolic pathways as therapeutic targets, along with the latest metabolic-related treatment drugs and approaches.

Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders, representing high risk of progression to acute myeloid leukaemia, and frequently associated to somatic mutations, notably in the epigenetic regulator TET2. Natural Killer (NK) cells play a role in the anti-leukemic immune response via their cytolytic activity. Here we show that patients with MDS clones harbouring mutations in the TET2 gene are characterised by phenotypic defects in their circulating NK cells. Remarkably, NK cells and MDS clones from the same patient share the TET2 genotype, and the NK cells are characterised by increased methylation of genomic DNA and reduced expression of Killer Immunoglobulin-like receptors (KIR), perforin, and TNF-α. In vitro inhibition of TET2 in NK cells of healthy donors reduces their cytotoxicity, supporting its critical role in NK cell function. Conversely, NK cells from patients treated with azacytidine (#NCT02985190; https://clinicaltrials.gov/ ) show increased KIR and cytolytic protein expression, and IFN-γ production. Altogether, our findings show that, in addition to their oncogenic consequences in the myeloid cell subsets, TET2 mutations contribute to repressing NK-cell function in MDS patients.

WHIM Syndrome is a rare immunodeficiency caused by gain-of-function CXCR4 mutations. Here we report a decrease in bone mineral density in 25% of WHIM patients and bone defects leading to osteoporosis in a WHIM mouse model. Imbalanced bone tissue is observed in mutant mice combining reduced osteoprogenitor cells and increased osteoclast numbers. Mechanistically, impaired CXCR4 desensitization disrupts cell cycle progression and osteogenic commitment of skeletal stromal/stem cells, while increasing their pro-osteoclastogenic capacities. Impaired osteogenic differentiation is evidenced in primary bone marrow stromal cells from WHIM patients. In mice, chronic treatment with the CXCR4 antagonist AMD3100 normalizes in vitro osteogenic fate of mutant skeletal stromal/stem cells and reverses in vivo the loss of skeletal cells, demonstrating that proper CXCR4 desensitization is required for the osteogenic specification of skeletal stromal/stem cells. Our study provides mechanistic insights into how CXCR4 signaling regulates the osteogenic fate of skeletal cells and the balance between bone formation and resorption.

Arginine methylation is a common post-translational modification affecting protein activity and the transcription of target genes when methylation occurs on histone tails. There are nine protein arginine methyltransferases (PRMTs) in mammals, divided into subgroups depending on the methylation they form on a molecule of arginine. During the formation and maturation of the different types of blood cells, PRMTs play a central role by controlling cell differentiation at the transcriptional level. PRMT enzymatic activity is necessary for many cellular processes in hematological malignancies, such as the activation of cell cycle and proliferation, inhibition of apoptosis, DNA repair processes, RNA splicing, and transcription by methylating histone tails' arginine. Chemical tools have been developed to inhibit the activity of PRMTs and have been tested in several models of hematological malignancies, including primary samples from patients, xenografts into immunodeficient mice, mouse models, and human cell lines. They show a significant effect by reducing cell viability and increasing the overall survival of mice. PRMT5 inhibitors have a strong therapeutic potential, as phase I clinical trials in hematological malignancies that use these molecules show promising results, thus, underlining PRMT inhibitors as useful therapeutic tools for cancer treatment in the future.

Introduction: Despite the advances in the treatment of MPN, the disease remains incurable with little remission rates and evolution to AML or Myelofibrosis (MF). MPN pathogenesis has been extensively studied but the evolution to MF remains poorly understood. In this study, using multiomic and functional approaches we comprehensively characterized mesenchymal stromal cells (MSCs) from fibrotic MPN patients (F-MPN) and identified a novel potentially druggable axis involved in the fibrosis phenotype. Methods: We characterized MSCs expanded from 13 MF patients (WHO criteria) (F-MSC) and from 17 age-matched healthy controls. The expanded F-MSCs were analyzed by immunophenotyping, Luminex cytokine array, RNA-seq and ATAC-seq techniques. The findings were validated using RT-qPCR, ChIP and differentiation assays while functional in vitro assays leaned on the HS-5 cell line and knock-down assays. Results: Immunophenotyping of F-MSCs, showed a significant differential expression of MSC markers compared to controls (decrease of CD90, CD73, CD106, CD140a, increase of CD105, CD295). As expected in a fibrotic tissue, F-MSCs secreted higher levels of inflammatory cytokines such as IL-1a, IL-1b, IL-8, Lipocalin, Leptin, VEGF, PDGF, , IGFBP2 and TGF-B (p<0.05, (fig a). The transcriptomic analysis of F-MPN samples further confirmed their fibrotic phenotype with enrichment of genes associated with fibrosis in signaling pathways such as actin filament organization, TGF-B receptor and Wnt (GSEA) (fig b). RNA-Seq highlighted two novel correlated findings of F-MPN stroma. First: an enrichment for genes of the osteoblast differentiation (fig b&c), underscored by ATAC-seq pointing to an enrichment for osteoblast differentiation transcription factor motifs (RUNX2, SMAD, SP5), an increased accessibility for ACTA2 (fibrotic/osteoblast gene) and decreased accessibility for adipocyte (PPARG) and chondrocyte (SOX9) transcription factors (P<0.002) (fig d). In vitro differentiation confirmed the F-MPN stroma biased differentiation towards osteoblast differentiation with reduced adipocyte and chondrocyte differentiation (P=0.0001, fig e). Second, a gene not previously linked to MPN fibrosis, HOXB7, was identified among the top 20 highly deregulated genes (fig f); RT-qPCR confirmed the significant upregulation of HOXB7 in F-MPN samples (P=0.002, fig f). HOXB7 is known as the most relevant HOXB gene associated with osteoblast differentiation. To functionally link these two novel gene profiles (ie the increased osteoblast differentiation pathway and HOXB7 gene expression) functional analyses of TGF-B treated F-MSCs showed, along with the reported activation of the Wnt pathway (increase, stabilization and translocation to the nucleus of B-Catenin), an increased binding of B-catenin to the promoter of HOXB7 (ChIP assay) followed by increased expression of HOXB7 (P=0.05) (fig g). Finally, shRNA-mediated knockdown or incubation with inhibitors (Cardamonin or FZM1) against HOXB7 or Wnt signalling resulted in decreased expression of osteoblast/fibrosis target genes and reduced osteoblast differentiation (fig h & i). Conclusion: We identify a novel molecular and functional TGFB-WNT-HOXB7 activated axis in the development of a fibrotic phenotype. Invalidation studies demonstrated that this novel axis can be targeted which may counteract fibrotic evolution in MPN. Figure: a) Luminex analysis showing upregulation of inflammatory cytokines secreted from fibrotic MSC in comparison to control MSCs. b-c) GSEA plot of RNA-seq showing enrichment of fibrosis associated pathway and of osteoblast differentiation pathway. d) ATAC-seq motif analysis showing an enrichment of osteoblast associated transcription factors and RT-qPCR showing an upregulation of genes involved in osteoblast differentiation and fibrosis. e) Osteo and Adipo differentiation of expanded stroma: increased osteogenesis and decreased adipogenesis. f) Volcano plot showing differential gene expression in F-MSC: HOXB7 was validated using RT-qPCR. g) increased translocation of B-Catenin subunit in the nucleus of HS-5 cells upon TGF-B treatment and ChIP showing B-Catenin bound to the promoter of HOXB7 h) B-catenin or HOXB7 knock down resulted in reduction of fibrotic/osteoblast genes (ACTA2/a-SMA). i) HOXB7 knockdown in HS-5- cells led to reduced osteoblast differentiation compared to scrambled (SCR) shRNA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The human genome is composed of unique DNA sequences that encode proteins and unique sequence noncoding RNAs that are essential for normal development and cellular differentiation. The human genome also contains over 50% of genome sequences that are repeat in nature (tandem and interspersed repeats) that are now known to contribute dynamically to genetic diversity in populations, to be transcriptionally active under certain physiological conditions, and to be aberrantly active in disease states including cancer, where consequences are pleiotropic with impact on cancer cell phenotypes and on the tumor immune microenvironment. Repeat element-derived RNAs play unique roles in exogenous and endogenous cell signaling under normal and disease conditions. A key component of repeat element-derived transcript-dependent signaling occurs via triggering of innate immune receptor signaling that then feeds forward to inflammatory responses through interferon and NFκB signaling. It has recently been shown that cancer cells display abnormal transcriptional activity of repeat elements and that this is linked to either aggressive disease and treatment failure or to improved prognosis/treatment response, depending on cell context and the amplitude of the so-called 'viral mimicry' response that is engaged. 'Viral mimicry' refers to a cellular state of active antiviral response triggered by endogenous nucleic acids often derived from aberrantly transcribed endogenous retrotransposons and other repeat elements. In this paper, the literature regarding transcriptional activation of repeat elements and engagement of inflammatory signaling in normal (focusing on hematopoiesis) and cancer is reviewed with an emphasis on the role of innate immune receptor signaling, in particular by dsRNA receptors of the RIG-1 like receptor family and interferons/NFκB. How repeat element-derived RNA reprograms cell identity through RNA-guided chromatin state modulation is also discussed.

WHIM Syndrome (WS) is a rare immunodeficiency caused by gain-of-function CXCR4 mutations. Here we report for the first time a substantial decrease in bone mineral density in 25% of WS patients and bone defects leading to osteoporosis in a WS mouse model. Reduction in bone content involved impaired CXCR4 desensitization that disrupts cell cycle progression and osteogenic specification of mouse bone marrow (BM)-residing skeletal stromal/stem cells (SSCs). This was also evidenced in BM stromal cells from WS patients. Consistent with this, chronic treatment with the CXCR4 antagonist AMD3100 normalized in vitro osteogenic fate of mutant SSCs and reversed in vivo loss in skeletal cells, thus demonstrating that proper CXCR4 desensitization is required for the osteogenic specification of BM SSCs. Our study provides novel mechanistic insights into how CXCR4 signaling regulates the osteogenic fate of BM SSCs.