Enrichment of histone tail methylated lysine residues via cavitand-decorated magnetic nanoparticles for ultra-sensitive proteomics.

Nearly every protein in the human body is modified with post-translational modifications (PTMs). PTMs affect proteins on many levels, including their function, interaction, half-life, and localization. Specifically, for histone proteins, PTMs such as lysine methylation and acetylation play essential roles in chromatin dynamic regulations. For this reason, methods to accurately detect and quantify PTMs are of paramount importance in cell biology, biochemistry, and disease biology. Most protein modifications are sub-stoichiometric, so, to be analyzed, they need methods of enrichment, which are mostly based on antibodies. Antibodies are produced using animals, resulting in high costs, ecological concerns, significant batch variations, and ethical implications. We propose using ferromagnetic nanoparticles functionalized with synthetic receptors, namely tetraphosphonate cavitands, as a tool for selective enrichment of methylated lysines present on histone tails. Before the enrichment step, histone proteins from calf thymus were digested to facilitate the recognition process and to obtain small peptides suitable for mass analyses. Cavitands were anchored on ferromagnetic nanoparticles to easily separate the PTM-peptides of interest from the rest of the proteolytic peptides. Our approach detects more modified peptides with higher signal intensity, rivaling commercial antibodies. This chemical strategy offers a cost-effective and efficient alternative for PTM detection, potentially advancing proteomic research.

Productive mRNA Chromatin Escape is Promoted by PRMT5 Methylation of SNRPB.

Protein Arginine Methyltransferase 5 (PRMT5) regulates RNA splicing and transcription by symmetric dimethylation of arginine residues (Rme2s/SDMA) in many RNA binding proteins. However, the mechanism by which PRMT5 couples splicing to transcriptional output is unknown. Here, we demonstrate that a major function of PRMT5 activity is to promote chromatin escape of a novel, large class of mRNAs that we term Genomically Retained Incompletely Processed Polyadenylated Transcripts (GRIPPs). Using nascent and total transcriptomics, spike-in controlled fractionated cell transcriptomics, and total and fractionated cell proteomics, we show that PRMT5 inhibition and knockdown of the PRMT5 SNRP (Sm protein) adapter protein pICln (CLNS1A) -but not type I PRMT inhibition-leads to gross detention of mRNA, SNRPB, and SNRPD3 proteins on chromatin. Compared to most transcripts, these chromatin-trapped polyadenylated RNA transcripts have more introns, are spliced slower, and are enriched in detained introns. Using a combination of PRMT5 inhibition and inducible isogenic wildtype and arginine-mutant SNRPB, we show that arginine methylation of these snRNPs is critical for mediating their homeostatic chromatin and RNA interactions. Overall, we conclude that a major role for PRMT5 is in controlling transcript processing and splicing completion to promote chromatin escape and subsequent nuclear export.

KAT2A and KAT2B prevent double-stranded RNA accumulation and interferon signaling to maintain intestinal stem cell renewal.

Histone acetyltransferases KAT2A and KAT2B are paralogs highly expressed in the intestinal epithelium, but their functions are not well understood. In this study, double knockout of murine Kat2 genes in the intestinal epithelium was lethal, resulting in robust activation of interferon signaling and interferon-associated phenotypes including the loss of intestinal stem cells. Use of pharmacological agents and sterile organoid cultures indicated a cell-intrinsic double-stranded RNA trigger for interferon signaling. Acetyl-proteomics and sequencing of immunoprecipitated double-stranded RNA were used to interrogate the mechanism behind this response, which identified mitochondria-encoded double-stranded RNA as the source of intrinsic interferon signaling. Kat2a and Kat2b therefore play an essential role in regulating mitochondrial functions and maintaining intestinal health.

Mono-methylation of lysine 27 at histone 3 confers lifelong susceptibility to stress.

Histone post-translational modifications are critical for mediating persistent alterations in gene expression. By combining unbiased proteomics profiling and genome-wide approaches, we uncovered a role for mono-methylation of lysine 27 at histone H3 (H3K27me1) in the enduring effects of stress. Specifically, mice susceptible to early life stress (ELS) or chronic social defeat stress (CSDS) displayed increased H3K27me1 enrichment in the nucleus accumbens (NAc), a key brain-reward region. Stress-induced H3K27me1 accumulation occurred at genes that control neuronal excitability and was mediated by the VEFS domain of SUZ12, a core subunit of the polycomb repressive complex-2, which controls H3K27 methylation patterns. Viral VEFS expression changed the transcriptional profile of the NAc, led to social, emotional, and cognitive abnormalities, and altered excitability and synaptic transmission of NAc D1-medium spiny neurons. Together, we describe a novel function of H3K27me1 in the brain and demonstrate its role as a "chromatin scar" that mediates lifelong stress susceptibility.

Effect of dynamic exclusion and the use of FAIMS, DIA and MALDI-mass spectrometry imaging with ion mobility on amyloid protein identification.

Amyloidosis is a disease characterized by local and systemic extracellular deposition of amyloid protein fibrils where its excessive accumulation in tissues and resistance to degradation can lead to organ failure. Diagnosis is challenging because of approximately 36 different amyloid protein subtypes. Imaging methods like immunohistochemistry and the use of Congo red staining of amyloid proteins for laser capture microdissection combined with liquid chromatography tandem mass spectrometry (LMD/LC-MS/MS) are two diagnostic methods currently used depending on the expertise of the pathology laboratory. Here, we demonstrate a streamlined in situ amyloid peptide spatial mapping by Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging (MALDI-MSI) combined with Trapped Ion Mobility Spectrometry for potential transthyretin (ATTR) amyloidosis subtyping. While we utilized the standard LMD/LC-MS/MS workflow for amyloid subtyping of 31 specimens from different organs, we also evaluated the potential introduction in the MS workflow variations in data acquisition parameters like dynamic exclusion, or testing Data Dependent Acquisition combined with High-Field Asymmetric Waveform Ion Mobility Spectrometry (DDA FAIMS) versus Data Independent Acquisition (DIA) for enhanced amyloid protein identification at shorter acquisition times. We also demonstrate the use of Mascot's Error Tolerant Search and PEAKS de novo sequencing for the sequence variant analysis of amyloidosis specimens.

Patient-specific mutation of Dync1h1 in mice causes brain and behavioral deficits.

AIMS: Cytoplasmic dynein heavy chain (DYNC1H1) is a multi-subunit protein complex that provides motor force for movement of cargo on microtubules and traffics them back to the soma. In humans, mutations along the DYNC1H1 gene result in intellectual disabilities, cognitive delays, and neurologic and motor deficits. The aim of the study was to generate a mouse model to a newly identified de novo heterozygous DYNC1H1 mutation, within a functional ATPase domain (c9052C > T(P3018S)), identified in a child with motor deficits, and intellectual disabilities. RESULTS: P3018S heterozygous (HET) knockin mice are viable; homozygotes are lethal. Metabolic and EchoMRI™ testing show that HET mice have a higher metabolic rate, are more active, and have less body fat compared to wildtype mice. Neurobehavioral studies show that HET mice perform worse when traversing elevated balance beams, and on the negative geotaxis test. Immunofluorescent staining shows neuronal migration abnormalities in the dorsal and lateral neocortex with heterotopia in layer I. Neuron-subtype specific transcription factors CUX1 and CTGF identified neurons from layers II/III and VI respectively in cortical layer I, and abnormal pyramidal neurons with MAP2+ dendrites projecting downward from the pial surface. CONCLUSION: The HET mice are a good model for the motor deficits seen in the child, and highlights the importance of cytoplasmic dynein in the maintenance of cortical function and dendritic orientation relative to the pial surface. Our results are discussed in the context of other dynein mutant mice and in relation to clinical presentation in humans with DYNC1H1 mutations.

Gene body DNA hydroxymethylation restricts the magnitude of transcriptional changes during aging.

DNA hydroxymethylation (5hmC), the most abundant oxidative derivative of DNA methylation, is typically enriched at enhancers and gene bodies of transcriptionally active and tissue-specific genes. Although aberrant genomic 5hmC has been implicated in age-related diseases, its functional role in aging remains unknown. Here, using mouse liver and cerebellum as model organs, we show that 5hmC accumulates in gene bodies associated with tissue-specific function and restricts the magnitude of gene expression changes with age. Mechanistically, 5hmC decreases the binding of splicing associated factors and correlates with age-related alternative splicing events. We found that various age-related contexts, such as prolonged quiescence and senescence, drive the accumulation of 5hmC with age. We provide evidence that this age-related transcriptionally restrictive function is conserved in mouse and human tissues. Our findings reveal that 5hmC regulates tissue-specific function and may play a role in longevity.

Fibroblast Smad7 Induction Protects the Remodeling Pressure-Overloaded Heart.

BACKGROUND: Cardiac fibroblast activation contributes to adverse remodeling, fibrosis, and dysfunction in the pressure-overloaded heart. Although early fibroblast TGF-ß (transforming growth factor-ß)/Smad (small mother against decapentaplegic)-3 activation protects the pressure-overloaded heart by preserving the matrix, sustained TGF-ß activation is deleterious, accentuating fibrosis and dysfunction. Thus, endogenous mechanisms that negatively regulate the TGF-ß response in fibroblasts may be required to protect from progressive fibrosis and adverse remodeling. We hypothesized that Smad7, an inhibitory Smad that restrains TGF-ß signaling, may be induced in the pressure-overloaded myocardium and may regulate fibrosis, remodeling, and dysfunction. METHODS: The effects of myofibroblast-specific Smad7 loss were studied in a mouse model of transverse aortic constriction, using echocardiography, histological analysis, and molecular analysis. Proteomic studies in S7KO (Smad7 knockout) and overexpressing cells were used to identify fibroblast-derived mediators modulated by Smad7. In vitro experiments using cultured cardiac fibroblasts, fibroblasts populating collagen lattices, and isolated macrophages were used to dissect the molecular signals responsible for the effects of Smad7. RESULTS: Following pressure overload, Smad7 was upregulated in cardiac myofibroblasts. TGF-ß and angiotensin II stimulated fibroblast Smad7 upregulation via Smad3, whereas GDF15 (growth differentiation factor 15) induced Smad7 through GFRAL (glial cell line-derived neurotrophic factor family receptor α-like). MFS7KO (myofibroblast-specific S7KO) mice had increased mortality, accentuated systolic dysfunction and dilative remodeling, and accelerated diastolic dysfunction in response to transverse aortic constriction. Increased dysfunction in MFS7KO hearts was associated with accentuated fibrosis and increased MMP (matrix metalloproteinase)-2 activity and collagen denaturation. Secretomic analysis showed that Smad7 loss accentuates secretion of structural collagens and matricellular proteins and markedly increases MMP2 secretion. In contrast, Smad7 overexpression reduced MMP2 levels. In fibroblasts populating collagen lattices, the effects of Smad7 on fibroblast-induced collagen denaturation and pad contraction were partly mediated via MMP2 downregulation. Surprisingly, MFS7KO mice also exhibited significant macrophage expansion caused by paracrine actions of Smad7 null fibroblasts that stimulate macrophage proliferation and fibrogenic activation. Macrophage activation involved the combined effects of the fibroblast-derived matricellular proteins CD5L (CD5 antigen-like), SPARC (secreted protein acidic and rich in cysteine), CTGF (connective tissue growth factor), ECM1 (extracellular matrix protein 1), and TGFBI (TGFB induced). CONCLUSIONS: The antifibrotic effects of Smad7 in the pressure-overloaded heart protect from dysfunction and involve not only reduction in collagen deposition but also suppression of MMP2-mediated matrix denaturation and paracrine effects that suppress macrophage activation through inhibition of matricellular proteins.

Spatial hepatocyte plasticity of gluconeogenesis during the metabolic transitions between fed, fasted and starvation states.

The liver acts as a master regulator of metabolic homeostasis in part by performing gluconeogenesis. This process is dysregulated in type 2 diabetes, leading to elevated hepatic glucose output. The parenchymal cells of the liver (hepatocytes) are heterogeneous, existing on an axis between the portal triad and the central vein, and perform distinct functions depending on location in the lobule. Here, using single cell analysis of hepatocytes across the liver lobule, we demonstrate that gluconeogenic gene expression ( Pck1 and G6pc ) is relatively low in the fed state and gradually increases first in the periportal hepatocytes during the initial fasting period. As the time of fasting progresses, pericentral hepatocyte gluconeogenic gene expression increases, and following entry into the starvation state, the pericentral hepatocytes show similar gluconeogenic gene expression to the periportal hepatocytes. Similarly, pyruvate-dependent gluconeogenic activity is approximately 10-fold higher in the periportal hepatocytes during the initial fasting state but only 1.5-fold higher in the starvation state. In parallel, starvation suppresses canonical beta-catenin signaling and modulates expression of pericentral and periportal glutamine synthetase and glutaminase, resulting in an enhanced pericentral glutamine-dependent gluconeogenesis. These findings demonstrate that hepatocyte gluconeogenic gene expression and gluconeogenic activity are highly spatially and temporally plastic across the liver lobule, underscoring the critical importance of using well-defined feeding and fasting conditions to define the basis of hepatic insulin resistance and glucose production.

Linking Aging to Cancer: The Role of Chromatin Biology.

Epigenetic changes have been established to be a hallmark of aging, which implies that aging science requires collaborating with the field of chromatin biology. DNA methylation patterns, changes in relative abundance of histone post-translational modifications, and chromatin remodeling are the central players in modifying chromatin structure. Aging is commonly associated with an overall increase in chromatin instability, loss of homeostasis, and decondensation. However, numerous publications have highlighted that the link between aging and chromatin changes is not nearly as linear as previously expected. This complex interplay of these epigenetic elements during the lifetime of an organism likely contributes to cellular senescence, genomic instability, and disease susceptibility. Yet, the causal links between these phenomena still need to be fully unraveled. In this perspective article, we discuss potential future directions of aging chromatin biology.

Decoding cocaine-induced proteomic adaptations in the mouse nucleus accumbens.

Cocaine use disorder (CUD) is a chronic neuropsychiatric condition that results from enduring cellular and molecular adaptations. Among substance use disorders, CUD is notable for its rising prevalence and the lack of approved pharmacotherapies. The nucleus accumbens (NAc), a region that is integral to the brain's reward circuitry, plays a crucial role in the initiation and continuation of maladaptive behaviors that are intrinsic to CUD. Leveraging advancements in neuroproteomics, we undertook a proteomic analysis that spanned membrane, cytosolic, nuclear, and chromatin compartments of the NAc in a mouse model. The results unveiled immediate and sustained proteomic modifications after cocaine exposure and during prolonged withdrawal. We identified congruent protein regulatory patterns during initial cocaine exposure and reexposure after withdrawal, which contrasted with distinct patterns during withdrawal. Pronounced proteomic shifts within the membrane compartment indicated adaptive and long-lasting molecular responses prompted by cocaine withdrawal. In addition, we identified potential protein translocation events between soluble-nuclear and chromatin-bound compartments, thus providing insight into intracellular protein dynamics after cocaine exposure. Together, our findings illuminate the intricate proteomic landscape that is altered in the NAc by cocaine use and provide a dataset for future research toward potential therapeutics.

KDM5-mediated transcriptional activation of ribosomal protein genes alters translation efficiency to regulate mitochondrial metabolism in neurons.

Genes encoding the KDM5 family of transcriptional regulators are disrupted in individuals with intellectual disability (ID). To understand the link between KDM5 and ID, we characterized five Drosophila strains harboring missense alleles analogous to those observed in patients. These alleles disrupted neuroanatomical development, cognition and other behaviors, and displayed a transcriptional signature characterized by the downregulation of many ribosomal protein genes. A similar transcriptional profile was observed in KDM5C knockout iPSC-induced human glutamatergic neurons, suggesting an evolutionarily conserved role for KDM5 proteins in regulating this class of gene. In Drosophila, reducing KDM5 changed neuronal ribosome composition, lowered the translation efficiency of mRNAs required for mitochondrial function, and altered mitochondrial metabolism. These data highlight the cellular consequences of altered KDM5-regulated transcriptional programs that could contribute to cognitive and behavioral phenotypes. Moreover, they suggest that KDM5 may be part of a broader network of proteins that influence cognition by regulating protein synthesis.

Evidence for negative selection in human primary fibroblasts to tolerate high somatic mutation loads upon treatment with multiple low doses of N-ethyl-N-nitrosourea.

High-throughput sequencing at the single-cell and single-molecule level has shown that mutation rate is much higher in somatic cells than in the germline, with thousands of mutations accumulating with age in most human tissues. While there is now ample evidence that some of these mutations can clonally amplify and lead to disease, most notably cancer, the total burden of mutations a cell can tolerate without functional decline remains unknown. Here we addressed this question by exposing human primary fibroblasts multiple times to low doses of N-ethyl-N-nitrosourea (ENU) and quantitatively analyzing somatic mutation burden using single-cell whole genome sequencing. The results indicate that individual cells can sustain ∼60,000 single-nucleotide variants (SNVs) with only a slight adverse effect on growth rate. We found evidence for selection against potentially deleterious variants in gene coding regions as well as depletion of mutations in sequences associated with genetic pathways expressed in these human fibroblasts, most notably those relevant for maintaining basic cellular function and growth. However, no evidence of negative selection was found for variants in non-coding regions. We conclude that actively proliferating fibroblasts can tolerate very high levels of somatic mutations without major adverse effects on growth rate via negative selection against damaging coding mutations. Since most tissues in adult organisms have very limited capacity to select against mutations based on a growth disadvantage, these results suggest that a causal effect of somatic mutations in aging and disease cannot be ruled out.

Involution of brown adipose tissue through a Syntaxin 4 dependent pyroptosis pathway.

Aging, chronic high-fat diet feeding, or housing at thermoneutrality induces brown adipose tissue (BAT) involution, a process characterized by reduction of BAT mass and function with increased lipid droplet size. Single nuclei RNA sequencing of aged mice identifies a specific brown adipocyte population of Ucp1-low cells that are pyroptotic and display a reduction in the longevity gene syntaxin 4 (Stx4a). Similar to aged brown adipocytes, Ucp1-STX4KO mice display loss of brown adipose tissue mass and thermogenic dysfunction concomitant with increased pyroptosis. Restoration of STX4 expression or suppression of pyroptosis activation protects against the decline in both mass and thermogenic activity in the aged and Ucp1-STX4KO mice. Mechanistically, STX4 deficiency reduces oxidative phosphorylation, glucose uptake, and glycolysis leading to reduced ATP levels, a known triggering signal for pyroptosis. Together, these data demonstrate an understanding of rapid brown adipocyte involution and that physiologic aging and thermogenic dysfunction result from pyroptotic signaling activation.

RNA polymerase II promotes the organization of chromatin following DNA replication.

Understanding how chromatin organisation is duplicated on the two daughter strands is a central question in epigenetics. In mammals, following the passage of the replisome, nucleosomes lose their defined positioning and transcription contributes to their re-organisation. However, whether transcription plays a greater role in the organization of chromatin following DNA replication remains unclear. Here we analysed protein re-association with newly replicated DNA upon inhibition of transcription using iPOND coupled to quantitative mass spectrometry. We show that nucleosome assembly and the re-establishment of most histone modifications are uncoupled from transcription. However, RNAPII acts to promote the re-association of hundreds of proteins with newly replicated chromatin via pathways that are not observed in steady-state chromatin. These include ATP-dependent remodellers, transcription factors and histone methyltransferases. We also identify a set of DNA repair factors that may handle transcription-replication conflicts during normal transcription in human non-transformed cells. Our study reveals that transcription plays a greater role in the organization of chromatin post-replication than previously anticipated.

αKG-mediated carnitine synthesis promotes homologous recombination via histone acetylation.

Homologous recombination (HR) deficiency enhances sensitivity to DNA damaging agents commonly used to treat cancer. In HR-proficient cancers, metabolic mechanisms driving response or resistance to DNA damaging agents remain unclear. Here we identified that depletion of alpha-ketoglutarate (αKG) sensitizes HR-proficient cells to DNA damaging agents by metabolic regulation of histone acetylation. αKG is required for the activity of αKG-dependent dioxygenases (αKGDDs), and prior work has shown that changes in αKGDD affect demethylases. Using a targeted CRISPR knockout library consisting of 64 αKGDDs, we discovered that Trimethyllysine Hydroxylase Epsilon (TMLHE), the first and rate-limiting enzyme in de novo carnitine synthesis, is necessary for proliferation of HR-proficient cells in the presence of DNA damaging agents. Unexpectedly, αKG-mediated TMLHE-dependent carnitine synthesis was required for histone acetylation, while histone methylation was affected but dispensable. The increase in histone acetylation via αKG-dependent carnitine synthesis promoted HR-mediated DNA repair through site- and substrate-specific histone acetylation. These data demonstrate for the first time that HR-proficiency is mediated through αKG directly influencing histone acetylation via carnitine synthesis and provide a metabolic avenue to induce HR-deficiency and sensitivity to DNA damaging agents.

An iron rheostat controls hematopoietic stem cell fate.

Mechanisms governing the maintenance of blood-producing hematopoietic stem and multipotent progenitor cells (HSPCs) are incompletely understood, particularly those regulating fate, ensuring long-term maintenance, and preventing aging-associated stem cell dysfunction. We uncovered a role for transitory free cytoplasmic iron as a rheostat for adult stem cell fate control. We found that HSPCs harbor comparatively small amounts of free iron and show the activation of a conserved molecular response to limited iron-particularly during mitosis. To study the functional and molecular consequences of iron restriction, we developed models allowing for transient iron bioavailability limitation and combined single-molecule RNA quantification, metabolomics, and single-cell transcriptomic analyses with functional studies. Our data reveal that the activation of the limited iron response triggers coordinated metabolic and epigenetic events, establishing stemness-conferring gene regulation. Notably, we find that aging-associated cytoplasmic iron loading reversibly attenuates iron-dependent cell fate control, explicating intervention strategies for dysfunctional aged stem cells.

Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCCs) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The former group exhibits reduced proliferation, genome instability, and heightened sensitivity to genotoxic agents like PARP1/2 inhibitors. Conversely, H3K36M HNSCC models with constant H3K27me3 levels lack these characteristics unless H3K27me3 is elevated by DNA hypomethylating agents or inhibiting H3K27me3 demethylases KDM6A/B. Mechanistically, H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, aberrant H3K27me3 levels induced by H3K36M expression are not a bona fide epigenetic mark because they require continuous expression of H3K36M to be inherited. Moreover, increased sensitivity to PARP1/2 inhibitors in H3K36M HNSCC models depends solely on elevated H3K27me3 levels and diminishing BRCA1- and FANCD2-dependent DNA repair. Finally, a PARP1/2 inhibitor alone reduces tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a model with consistent H3K27me3, a combination of PARP1/2 inhibitors and agents that up-regulate H3K27me3 proves to be successful. These findings underscore the crucial balance between H3K36 and H3K27 methylation in maintaining genome instability, offering new therapeutic options for patients with H3K36me-deficient tumors.

FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.