Histone Modifications H3K4me3 Research Articles

Abstract 6146: Characterizing underlying molecular mechanisms of prostate cancer risk variants identified from multi-ethnic GWAS

Abstract Prostate cancer (PCa) is a complex disease with the second leading cause of cancer death in US males. Multi-ethnic genome-wide association studies (GWAS) identified a thousand genetic variants associated with PCa risk in different races. However, the majority of risk variants are located in non-coding regions, making it challenging to understand their contribution to PCa risk. Non-coding regions include functional regulatory elements (REs) such as promoters and enhancers, whose activity can be evaluated by histone modification H3K4me3 and H3K27ac, respectively. In this study, we integrated PCa risk variants with hundreds of H3K4me3 and H3K27ac ChIP-seq data from normal and cancer prostate cell lines and tissues, finding 122 variants in promoters and 301 variants in enhancers. Enhancers are distal to promoters, and they form loops to regulate gene expression, mediated by transcription factors (TFs). Enhancer activities are tightly associated with cell fate. Genetic variants can affect the activity of enhancers by changing the binding preference of TFs and such changes can affect chromatin interactions and dysregulate gene expression. We hypothesize that allele changes of enhancer variants contribute to PCa risk differently among individuals from different ancestries, leading to the development of heterogeneous PCa molecular subgroups, characterized by genetic alterations including TMPRSS2-ERG fusion. By integrating H3K27ac ChIP-seq data from prostate cell lines derived from European (EUR) and African (AFR) ancestries, we found 185 risk variants in enhancers in EUR cell lines and 69 risk variants in enhancers in AFR cell lines. Next, we integrated patient tissue-derived H3K27ac ChIP-seq data and performed chromatin quantitative trait loci (cQTL) and allelic imbalance (AI) analyses to determine risk variants associated with enhancer activity. Among the 185 risk enhancer variants in EUR and 69 in AFR cell lines, we found 130 and 46 with significant association, respectively. To determine TFs that change binding upon allele change of enhancer risk variants, we integrated hundreds of TF ChIP-seq data from prostate cell lines and tissues. Among the 185 risk enhancer variants in EUR and 69 in AFR cell lines, we found 142 and 44 variants bound by TFs, respectively. When we further performed cQTL and AI analyses for FOXA1, HOXB13, and AR, we found 87 and 29 variants significantly associated with TF binding affinity, respectively. Moreover, we found that these associations differ between TMPRSS2-ERG fusion positive and negative tumors. To identify the target genes of enhancer risk variants, we are currently integrating chromatin interaction data including Hi-C and Micro-C. Overall, this study provides valuable insights into the underlying molecular mechanisms of risk variants in various populations and individuals which lead to the development of heterogeneous PCa subgroups. Citation Format: Zexun Wu, Andrew Vu, Ethan S. Nelson-Moore, Huan Cao, Suhn K. Rhie. Characterizing underlying molecular mechanisms of prostate cancer risk variants identified from multi-ethnic GWAS [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 6146.

Abstract 561: Targeting Y box binding protein 1 (YBX1) in MYC-amplified medulloblastoma

Abstract Medulloblastoma is the most common malignant pediatric brain tumor. Patients diagnosed with MYC-amplified group 3 medulloblastoma (G3 MB) have a devastating prognosis with a 5-year survival rate of <50%. Standard of care (SOC) consists of maximal resection surgery followed by chemoradiotherapy, but these extensive treatments leave patients with neurocognitive and developmental deficits. Therefore, the identification of novel targets that can be used alone or in combination with SOC is of utmost importance. Using a batch-normalized bulk RNA-seq dataset (GSE124814) comprised of 1350 MB samples and 291 normal cerebella, we reveal upregulation of Y-box binding protein 1 (YBX1) in MB compared to normal cerebellum. Further analysis of this dataset and others demonstrates G3 tumors that display high MYC levels also have the highest YBX1 expression. Analysis of ChIP-seq data shows strong binding of MYC to the YBX1 promoter in G3 MB, concurrent with H3K27ac and H3K4me3 histone modifications. Using a well-established ex vivo model, we demonstrate increased YBX1 expression as normal cerebellar progenitor cells transition to tumorigenic G3 MB stem-like cells. Recent work in leukemia reveals a reciprocal regulation of MYC by YBX1, however, this mechanism is not well established in G3 MB. We utilized an orthogonal pharmacogenetic approach to inhibit YBX1 function and determine its effects on G3 MB tumor biology. Suppression of YBX1 significantly reduced the proliferation and decreased MYC protein expression of G3 MB cells in vitro. These findings support a reciprocal MYC-YBX1 axis, which could influence tumor growth in vivo. Orthotopic implantation of YBX1 KO G3 MB cells leads to increased animal survival. We next evaluated the therapeutic potential of two recently characterized YBX1 inhibitors which display single agent efficacy in vitro and in vivo. YBX1 inhibitors synergized with SOC in vitro lowering the IC50 for both chemo and radiotherapy, which could reduce adverse effects experienced by patients. In silico molecular docking using PyMOL predicts YBX1 inhibitors interact with amino acids within the cold shock domain of YBX1, likely impairing its ability to bind RNA. We performed formaldehyde RNA immunoprecipitation (fRIP)-sequencing to identify changes in RNA transcript levels following YBX1 pharmacological inhibition. Disruption of YBX1-RNA binding impacts the stability of known oncogenic transcripts which may be required for tumorigenesis. Our findings impart an alluring prospect of targeting YBX1 in combination with standard-of-care, and possibly other compounds to improve patients’ outcomes. Citation Format: Stephen Carney, John Hemenway, Ashmitha Rajendran, Matt Hathaway, Eric Nealy, Christian Bonatto, Dhanir Tailor, Sanjay Malhotra, Myron Evans. Targeting Y box binding protein 1 (YBX1) in MYC-amplified medulloblastoma [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 561.

Chromatin profiling reveals TFAP4 as a critical transcriptional regulator of bovine satellite cell differentiation

BackgroundSatellite cells are myogenic precursor cells in adult skeletal muscle and play a crucial role in skeletal muscle regeneration, maintenance, and growth. Like embryonic myoblasts, satellite cells have the ability to proliferate, differentiate, and fuse to form multinucleated myofibers. In this study, we aimed to identify additional transcription factors that control gene expression during bovine satellite cell proliferation and differentiation.ResultsUsing chromatin immunoprecipitation followed by sequencing, we identified 56,973 and 54,470 genomic regions marked with both the histone modifications H3K4me1 and H3K27ac, which were considered active enhancers, and 50,956 and 59,174 genomic regions marked with H3K27me3, which were considered repressed enhancers, in proliferating and differentiating bovine satellite cells, respectively. In addition, we identified 1,216 and 1,171 super-enhancers in proliferating and differentiating bovine satellite cells, respectively. Analyzing these enhancers showed that in proliferating bovine satellite cells, active enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation whereas repressed enhancers were associated with genes essential for myoblast differentiation, and that in differentiating satellite cells, active enhancers were associated with genes essential for myoblast differentiation or muscle contraction whereas repressed enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation. Active enhancers in proliferating bovine satellite cells were enriched with binding sites for many transcription factors such as MYF5 and the AP-1 family transcription factors; active enhancers in differentiating bovine satellite cells were enriched with binding sites for many transcription factors such as MYOG and TFAP4; and repressed enhancers in both proliferating and differentiating bovine satellite cells were enriched with binding sites for NF-kB, ZEB-1, and several other transcription factors. The role of TFAP4 in satellite cell or myoblast differentiation was previously unknown, and through gene knockdown and overexpression, we experimentally validated a critical role for TFAP4 in the differentiation and fusion of bovine satellite cells into myofibers.ConclusionsSatellite cell proliferation and differentiation are controlled by many transcription factors such as AP-1, TFAP4, NF-kB, and ZEB-1 whose roles in these processes were previously unknown in addition to those transcription factors such as MYF5 and MYOG whose roles in these processes are widely known.

Three-dimensional chromatin analysis reveals Sp1 as a mediator to program and reprogram HPV-host epigenetic architecture in cervical cancer

Human papillomavirus (HPV) is predominantly associated with HPV-related cancers, however, the precise mechanisms underlying the HPV-host epigenetic architectures in HPV carcinogenesis remain elusive. Here, we employed high-throughput chromosome conformation capture (Hi-C) to comprehensively map HPV16/18-host chromatin interactions. Our study identified the transcription factor Sp1 as a pivotal mediator in programming HPV-host interactions. By targeting Sp1, the active histone modifications (H3K27ac, H3K4me1, and H3K4me3) and the HPV-host chromatin interactions are reprogrammed, which leads to the downregulation of oncogenes located near the integration sites in both HPV (E6/E7) and the host genome (KLF5/MYC). Additionally, Sp1 inhibition led to the upregulation of immune checkpoint genes by reprogramming histone modifications in host cells. Notably, humanized patient-derived xenograft (PDX-HuHSC-NSG) models demonstrated that Sp1 inhibition promoted anti-PD-1 immunotherapy via remodeling the tumor immune microenvironment in cervical cancer. Moreover, single-cell transcriptomic analysis validated the enrichment of transcription factor Sp1 in epithelial cells of cervical cancer. In summary, our findings elucidate Sp1 as a key mediator involved in the programming and reprogramming of HPV-host epigenetic architecture. Inhibiting Sp1 with plicamycin may represent a promising therapeutic option for HPV-related carcinoma.

Integrative epigenome-transcriptome analysis unravels cancer-specific over-expressed genes potentially regulating immune microenvironment in clear cell renal cell carcinoma

BackgroundClear cell Renal Cell Carcinoma (ccRCC) is the frequently diagnosed histological life-threatening tumor subtype in the urinary system. Integrating multi-omics data is emerging as a tool to provide a comprehensive view of biology and disease for better therapeutic interventions. MethodWe have integrated freely available ccRCC data sets of genome-wide DNA methylome, transcriptome, and active histone modification marks, H3K27ac, H3K4me1, and H3K4me3 specific ChIP-seq data to screen genes with higher expression. Further, these genes were filtered based on their effect on survival upon alteration in expression. ResultsThe six multi-omics-based identified genes, RUNX1, MSC, ADA, TREML1, TGFA, and VWF, showed higher expression with enrichment of active histone marks and hypomethylated CpG in ccRCC. In continuation, the identified genes were validated by an independent dataset and showed a correlation with nodal and metastatic status. Furthermore, gene ontology and pathway analysis revealed that immune-related pathways are activated in ccRCC patients. ConclusionsThe network analysis of six overexpressed genes suggests their potential role in an immunosuppressive environment, leading to tumor progression and poor prognosis. Our study shows that the multi-omics approach helps unravel complex biology for patient subtyping and proposes combination strategies with epi-drugs for more precise immunotherapy in ccRCC.

Histone modification-dependent production of peptide hormones facilitates acquisition of pluripotency during leaf-to-callus transition in Arabidopsis.

Chromatin configuration is critical for establishing tissue identity and changes substantially during tissue identity transitions. The crucial scientific and agricultural technology of in vitro tissue culture exploits callus formation from diverse tissue explants and tissue regeneration via de novo organogenesis. We investigated the dynamic changes in H3ac and H3K4me3 histone modifications during leaf-to-callus transition in Arabidopsis thaliana. We analyzed changes in the global distribution of H3ac and H3K4me3 during the leaf-to-callus transition, focusing on transcriptionally active regions in calli relative to leaf explants, defined by increased accumulation of both H3ac and H3K4me3. Peptide signaling was particularly activated during callus formation; the peptide hormones RGF3, RGF8, PIP1 and PIPL3 were upregulated, promoting callus proliferation and conferring competence for de novo shoot organogenesis. The corresponding peptide receptors were also implicated in peptide-regulated callus proliferation and regeneration capacity. The effect of peptide hormones in plant regeneration is likely at least partly conserved in crop plants. Our results indicate that chromatin-dependent regulation of peptide hormone production not only stimulates callus proliferation but also establishes pluripotency, improving the overall efficiency of two-step regeneration in plant systems.

Regulation of effector gene expression as concerted waves in Leptosphaeria maculans: a two-player game.

Effector genes, encoding molecules involved in disease establishment, are concertedly expressed throughout the lifecycle of plant-pathogenic fungi. However, little is known about how effector gene expression is regulated. Since many effector genes are located in repeat-rich regions, the role of chromatin remodeling in their regulation was recently investigated, notably establishing that the repressive histone modification H3K9me3, deposited by KMT1, was involved in several fungal species including Leptosphaeria maculans. Nevertheless, previous data suggest that a second regulatory layer, probably involving a specific transcription factor (TF), might be required. In L. maculans, a Dothideomycete causing stem canker of oilseed rape, we identified the ortholog of Pf2, a TF belonging to the Zn2Cys6 fungal-specific family, and described as essential for pathogenicity and effector gene expression. We investigated its role together with KMT1, by inactivating and over-expressing LmPf2 in a wild-type strain and a ∆kmt1 mutant. Functional analyses of the corresponding transformants highlighted an essential role of LmPf2 in the establishment of pathogenesis and we found a major effect of LmPf2 on the induction of effector gene expression once KMT1 repression is lifted. Our results show, for the first time, a dual control of effector gene expression.

Leveraging chromatin state transitions for the identification of regulatory networks orchestrating heart regeneration.

The limited regenerative capacity of the human heart contributes to high morbidity and mortality worldwide. In contrast, zebrafish exhibit robust regenerative capacity, providing a powerful model for studying how to overcome intrinsic epigenetic barriers maintaining cardiac homeostasis and initiate regeneration. Here, we present a comprehensive analysis of the histone modifications H3K4me1, H3K4me3, H3K27me3and H3K27ac during various stages of zebrafish heart regeneration. We found a vast gain of repressive chromatin marks one day after myocardial injury, followed by the acquisition of active chromatin characteristics on day four and a transition to a repressive state on day 14, and identified distinct transcription factor ensembles associated with these events. The rapid transcriptional response involves the engagement of super-enhancers at genes implicated in extracellular matrix reorganization and TOR signaling, while H3K4me3 breadth highly correlates with transcriptional activity and dynamic changes at genes involved in proteolysis, cell cycle activity, and cell differentiation. Using loss- and gain-of-function approaches, we identified transcription factors in cardiomyocytes and endothelial cells influencing cardiomyocyte dedifferentiation or proliferation. Finally, we detected significant evolutionary conservation between regulatory regions that drive zebrafish and neonatal mouse heart regeneration, suggesting that reactivating transcriptional and epigenetic networks converging on these regulatory elements might unlock the regenerative potential of adult human hearts.

Cutting Edge: Hypoxia Sensing by the Histone Demethylase UTX (KDM6A) Limits Colitogenic CD4+ T Cells in Mucosal Inflammation.

Hypoxia is a hallmark of inflammatory conditions (e.g., inflammatory bowel disease [IBD]), and adaptive responses have consequently evolved to protect against hypoxia-associated tissue injury. Because augmenting hypoxia-induced protective responses is a promising therapeutic approach for IBD, a more complete understanding of these pathways is needed. Recent work has demonstrated that the histone demethylase UTX is oxygen-sensitive, but its role in IBD is unclear. In this study, we show that hypoxia-induced deactivation of UTX downregulates T cell responses in mucosal inflammation. Hypoxia results in decreased T cell proinflammatory cytokine production and increased immunosuppressive regulatory T cells, and these findings are recapitulated by UTX deficiency. Hypoxia leads to T cell accumulation of H3K27me3 histone modifications, suggesting that hypoxia impairs UTX's histone demethylase activity to dampen T cell colitogenic activity. Finally, T cell-specific UTX deletion ameliorates colonic inflammation in an IBD mouse model, implicating UTX's oxygen-sensitive demethylase activity in counteracting hypoxic inflammation.

A low-input high resolution sequential chromatin immunoprecipitation method captures genome-wide dynamics of bivalent chromatin

BackgroundBivalent chromatin is an exemplar of epigenetic plasticity. This co-occurrence of active-associated H3K4me3 and inactive-associated H3K27me3 histone modifications on opposite tails of the same nucleosome occurs predominantly at promoters that are poised for future transcriptional upregulation or terminal silencing. We know little of the dynamics, resolution, and regulation of this chromatin state outside of embryonic stem cells where it was first described. This is partly due to the technical challenges distinguishing bone-fide bivalent chromatin, where both marks are on the same nucleosome, from allelic or sample heterogeneity where there is a mix of H3K4me3-only and H3K27me3-only mononucleosomes.ResultsHere, we present a robust and sensitive method to accurately map bivalent chromatin genome-wide, along with controls, from as little as 2 million cells. We optimized and refined the sequential ChIP protocol which uses two sequential overnight immunoprecipitation reactions to robustly purify nucleosomes that are truly bivalent and contain both H3K4me3 and H3K27me3 modifications. Our method generates high quality genome-wide maps with strong peak enrichment and low background, which can be analyzed using standard bioinformatic packages. Using this method, we detect 8,789 bivalent regions in mouse embryonic stem cells corresponding to 3,918 predominantly CpG rich and developmentally regulated gene promoters. Furthermore, profiling Dppa2/4 knockout mouse embryonic stem cells, which lose both H3K4me3 and H3K27me3 at approximately 10% of bivalent promoters, demonstrated the ability of our method to capture bivalent chromatin dynamics.ConclusionsOur optimized sequential reChIP method enables high-resolution genome-wide assessment of bivalent chromatin together with all required controls in as little as 2 million cells. We share a detailed protocol and guidelines that will enable bivalent chromatin landscapes to be generated in a range of cellular contexts, greatly enhancing our understanding of bivalent chromatin and epigenetic plasticity beyond embryonic stem cells.

Evaluating histone modification analysis of individual preimplantation embryos

BackgroundWe previously reported a modification of the CUT&Tag method (NTU-CAT) that allows genome-wide histone modification analysis in individual preimplantation embryos. In the present study, NTU-CAT was further simplified by taking advantage of the Well-of-the-Well (WOW) system, which enables the processing of multiple embryos in a shorter time with less reagent and cell loss during the procedure (WOW-CUT&Tag, WOW-CAT).ResultsWOW-CAT allowed histone modification profiling from not only a single blastocyst but also from a portion of it. WOW-CAT generated similar H3K4me3 profiles as NTU-CAT, but they were closer to the profiles produced by chromatin immunoprecipitation-sequencing, such as a valley-like trend and relatively lower false positive rates, indicating that WOW-CAT may attenuate the bias of Tn5 transposase to cut open chromatin regions. Simultaneous WOW-CAT of two halves of single blastocysts was conducted to analyze two different histone modifications (H3K4me3 and H3K27ac) within the same embryo. Furthermore, trophectoderm cells were biopsied and subjected to WOW-CAT in anticipation of preimplantation diagnosis of histone modifications. WOW-CAT allowed the monitoring of epigenetic modifications in the main body of the embryo. For example, analysis of H3K4me3 modifications of XIST and DDX3Y in trophectoderm biopsies could be used to sex embryos in combination with quantitative PCR, but without the need for deep sequencing.ConclusionsThese results suggest the applicability of WOW-CAT for flexible epigenetic analysis of individual embryos in preimplantation epigenetic diagnosis.

The m6A reader YTHDC2 regulates UVB-induced DNA damage repair and histone modification.

Ultraviolet B (UVB) radiation represents a major carcinogen for the development of all skin cancer types. Mechanistically, UVB induces damage to DNA in the form of lesions, including cyclobutane pyrimidine dimers (CPDs). Disruption of the functional repair processes, such as nucleotide excision repair (NER), allows persistence of DNA damage and contributes to skin carcinogenesis. Recent work has implicated m6A RNA methylation and its regulatory proteins as having critical roles in facilitating UVB-induced DNA damage repair. However, the biological functions of the m6A reader YTHDC2 are unknown in this context. Here, we show that YTHDC2 inhibition enhances the repair of UVB-induced DNA damage. We discovered that YTHDC2 inhibition increased the expression of PTEN while it decreased the expression of the PRC2 component SUZ12 and the levels of the histone modification H3K27me3. However, none of these functions were causally linked to the improvements in DNA repair, suggesting that the mechanism utilized by YTHDC2 may be unconventional. Moreover, inhibition of the m6A writer METTL14 reversed the effect of YTHDC2 inhibition on DNA repair while inhibition of the m6A eraser FTO mimicked the effect of YTHDC2 inhibition, indicating that YTHDC2 may regulate DNA repair through the m6A pathway. Finally, compared to normal human skin, YTHDC2 expression was upregulated in human cutaneous squamous cell carcinomas (cSCC), suggesting that it may function as a tumor-promoting factor in skin cancer. Taken together, our findings demonstrate that the m6A reader YTHDC2 plays a role in regulating UVB-induced DNA damage repair and may serve as a potential biomarker in cSCC.

Single-cell joint profiling of multiple epigenetic proteins and gene transcription.

Sculpting the epigenome with a combination of histone modifications and transcription factor occupancy determines gene transcription and cell fate specification. Here, we first develop uCoTarget, utilizing a split-pool barcoding strategy for realizing ultrahigh-throughput single-cell joint profiling of multiple epigenetic proteins. Through extensive optimization for sensitivity and multimodality resolution, we demonstrate that uCoTarget enables simultaneous detection of five histone modifications (H3K27ac, H3K4me3, H3K4me1, H3K36me3, and H3K27me3) in 19,860 single cells. We applied uCoTarget to the in vitro generation of hematopoietic stem/progenitor cells (HSPCs) from human embryonic stem cells, presenting multimodal epigenomic profiles in 26,418 single cells. uCoTarget reveals establishment of pairing of HSPC enhancers (H3K27ac) and promoters (H3K4me3) and RUNX1 engagement priming for H3K27ac activation along the HSPC path. We then develop uCoTargetX, an expansion of uCoTarget to simultaneously measure transcriptome and multiple epigenome targets. Together, our methods enable generalizable, versatile multimodal profiles for reconstructing comprehensive epigenome and transcriptome landscapes and analyzing the regulatory interplay at single-cell level.

Characterization of the distribution and dynamics of chromatin states in the C. elegans germline reveals substantial H3K4me3 remodeling during oogenesis.

Chromatin organization in the C. elegans germline is tightly regulated and critical for germ cell differentiation. Although certain germline epigenetic regulatory mechanisms have been identified, how they influence chromatin structure and ultimately gene expression remains unclear, in part because most genomic studies have focused on data collected from intact worms comprising both somatic and germline tissues. We therefore analyzed histone modification and chromatin accessibility data from isolated germ nuclei representing undifferentiated proliferating and meiosis I populations to define chromatin states. We correlated these states with overall transcript abundance, spatiotemporal expression patterns, and the function of small RNA pathways. Because the essential role of the germline is to transmit genetic information and establish gene expression in the early embryo, we compared epigenetic and transcriptomic profiles from undifferentiated germ cells to those of embryos to define the epigenetic changes during this developmental transition. The active histone modification H3K4me3 shows particularly dynamic remodeling as germ cells differentiate into oocytes, which suggests a mechanism for establishing early transcription of essential genes during zygotic genome activation. This analysis highlights the dynamism of the chromatin landscape across developmental transitions and provides a resource for future investigation into epigenetic regulatory mechanisms in germ cells.

Histone binding of ASF1 is required for fruiting body development but not for genome stability in the filamentous fungus Sordaria macrospora.

Histone chaperones are proteins that are involved in nucleosome assembly and disassembly and can therefore influence all DNA-dependent processes including transcription, DNA replication, and repair. ASF1 is a histone chaperone that is conserved throughout eukaryotes. In contrast to most other multicellular organisms, a deletion mutant of asf1 in the fungus Sordaria macrospora is viable; however, the mutant is sterile. In this study, we could show that the histone-binding ability of ASF1 is required for fertility in S. macrospora, whereas the function of ASF1 in maintenance of genome stability does not require histone binding. We also showed that the histone modifications H3K27me3 and H3K56ac are misregulated in the Δasf1 mutant. Furthermore, we identified a large duplication on chromosome 2 of the mutant strain that is genetically linked to the Δasf1 allele present on chromosome 6, suggesting that viability of the mutant might depend on the presence of the duplicated region.

Selinexor Disrupts Epigenetic Programing and Modulates Immunogenicity in Multiple Myeloma

Introduction: Multiple myeloma (MM), remains an incurable, but treatable, malignancy that has benefitted from the integration of novel immunologic agents in clinical use, such as monoclonal antibody, bispecific T cell engager (TCE) and chimeric antigen receptor T (CAR-T) cell and antibody-drug conjugate based therapies. While these agents have shown great promise in treating relapsed/refractory MM patients, there is heterogeneity in response to therapy due to differences in patients' immune tumor microenvironments (iTME) and immunogenicity of the tumor cells. Previously (Sudalagunta et. al., ASH 2021), we used functional genomic screening of 53 MM samples to characterize the transcriptomic profile associated with sensitivity to the selective nuclear export inhibitor, selinexor (SELI), which was shown to be inversely correlated with the transcriptomic profile associated with sensitivity to an anti-CD38 monoclonal antibody in 106 MM samples. In this study, we investigate the cellular mechanisms of SELI resistance leading to an immunogenic cell state. Methods: Primary cells from an additional set of MM patients (n=65) were used for paired bulk RNAseq and an organotypic ex vivo drug sensitivity assay. Cellular viability was assessed using digital analysis of brightfield longitudinal images, and drug sensitivity was assessed as area under the dose-time-response curve (AUC) for each sample. We conducted gene set enrichment analysis (GSEA) on paired RNAseq data and ex vivo AUC of SELI. Individual genes and their respective correlations with ex vivo AUC of SELI were ranked from highest to lowest. We used RNAseq to examine the role of acute exposure to SELI on CD138-selected cells from paired pre/post treatment bone marrow specimens of five MM patients enrolled in the NCT02186834 clinical trial, taken prior to treatment with SELI and dexamethasone (DEX) and one/two weeks post-induction. Paired t-tests were carried out to quantify the changes. Results: In our previous analysis of 844 MM samples, a subset of the genes whose expression is positively correlated with SELI AUC are controlled by H3K27me3, which is catalyzed by nuclear accumulation of EZH2. Tripathi et. al. (AACR Annual Meeting 2022 & 2023) have demonstrated that XPO1 and EZH2 form a complex, and inhibition of XPO1 activity blocks export of EZH2. GSEA of a new cohort of 65 MM patient samples demonstrated increased ex vivo SELI sensitivity (lower AUC, hence negative correlation) in samples with higher expression of known epigenetic regulators of the polycomb repressive complex 2 (PRC2), such as EZH2, SUZ12, and EED; as shown in Figure a, which led to lowering of expression of genes bound by these proteins due to gene silencing via H3K27me3, shown in red in Figure a, according to GSEA of ChIPseq databases. Furthermore, gene expression profiles of paired pre/post SELI/DEX treatment samples showed reduced expression of genes associated with H3K27me3 histone modification (paired t-test p=2.06e-83, 1.1061e-81, 2.56e-80, 5.15e-12, 6.22e-4) in all five patients. These observations suggest that XPO1 inhibition leads to nuclear accumulation of epigenetic modulators like EZH2, SUZ12, and EED, which catalyze H3K27me3 activity leading to silencing of H3K27me3-regulated genes. GSEA of paired ex vivo AUC and RNAseq data for an EZH2 inhibitor (EPZ-6438) resulted in negative correlation between expression of genes in PRC2 and ex vivo sensitivity, similar to SELI. Interestingly, we observed that this epigenetic mechanism of resistance to SELI is inversely associated with key immunological targets in MM such as CD74, SLAMF7, CD38, CD46, TNFRSF13B, and LY9 as shown in Figure a. In Figure b, we present a graphical visualization of intracellular changes due to exposure to SELI in a naïve and a SELI-resistant MM cell. Conclusions: Here we propose that SELI disrupts EZH2 subcellular localization, dysregulating H3K27me3-gene suppression. Sequential BM biopsies from SELI treated patients demonstrate decreased expression of H3K27me3-regulated genes, suggesting increased EZH2 activity by nuclear accumulation. Finally, SELI-resistant primary cells have increased expression of EZH2 regulated immunotherapy target genes (e.g. CD38, SLAMF7), suggesting a possible therapeutic strategy of sequential treatment of SELI followed by immunotherapy. Using multiparameter flow cytometry of samples pre/post SELI/DEX induction, we will study SELI's effect on iTME.

Genetic and Functional Characterization of NUP98-Rearranged Leukemia

NUP98 rearrangements ( NUP98r) are recurrent alterations in pediatric acute myeloid leukemia (AML) and are associated with dismal outcomes (PMID:21813447). These structural variantsresult in fusion oncoproteins comprised of the N-terminus of NUP98 and the C-terminus of fusion partners. Among more than 30 fusion partners discovered so far, the associations between NUP98::NSD1 with myeloblastic AML and NUP98::KDM5A with acute megakaryocytic/erythroid leukemia (AMKL/AEL) have been appreciated, which often harbor distinct cooperating mutations (e.g., NUP98:: NSD1 with FLT3-ITD and WT1 mutations). In contrast, other fusions, such as NUP98:: RAP1GDS1 or NUP98:: HOXA9 can be found also in myelodysplastic syndrome (MDS) or T-cell acute lymphoblastic leukemia (T-ALL) (PMID:32766874). However, how these fusions and cooperating mutations contribute to the specific phenotypes of NUP98r leukemias is poorly understood. To investigate the genetic background of NUP98r leukemia, we collected RNA-seq, WGS, and WES data for 91 pediatric cases from St. Jude (n=71),AEIOP (n=3), and the TARGET study (PMID:29227476, n=17; Panel.A), which included AML (n=82), MDS (n=4), and T-ALL (n=5). NUP98::NSD1 (n=47, 51.6%) and NUP98::KDM5A (n=26, 28.6%) were the most common fusions, in line with previous observations. NUP98::RAP1GDS1 (n=6, 6.6%), an understudied NUP98 fusion, was the third most common fusion in this study. We also confirmed frequent mutations of WT1 and FLT3-ITD in NUP98::NSD1 AML and entire or partial chromosome 13 loss involving RB1 locus in NUP98::KDM5A AMKL, while we observed frequent mutations in RAS or epigenetics-related genes, CCND2/3, and RUNX1 preferentially in cases without alterations of WT1, FLT3 or RB1 loss. NUP98:: RAP1GDS1 and NOTCH1 mutations were strongly enriched in T-ALL. These findings suggest that fusions and cooperating alterations both contribute to the disease phenotypes. To study how NUP98 fusion oncoproteins (FOs) drive leukemogenesis and specific phenotypes, we applied Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to determine NUP98 FO binding for a primary sample (myeloblastic with NUP98::NSD1), CHRF-288-11 cell line (megakaryocytic with NUP98::KDM5A), and HA-tagged CD34 NUP98::KDM5A model (Panel.B-top). CUT&RUN using antibodies against the N-terminus NUP98 and histone modifications (H3K4me3, H3K27ac) revealed that these models shared NUP98 FO binding sites at the HOXA/B and RUNX1 loci, together with active histone marks. In contrast, binding patterns at differentiation-related gene loci were heterogenous. NUP98::NSD1 uniquely binds to CEBPA, which encodes a master regulator of myeloid differentiation, contrary to NUP98::KDM5A binding to GFI1B encoding regulator of erythromegakaryocytic differentiation . These data indicate that NUP98 FOs directly and uniquely regulate differentiation-related genes. We also performed CUT&RUN in AML cells with RUNX1::RUNX1T1 or UBTF tandem duplications, in which the N-terminus NUP98 antibody did not show any detectable peaks, confirming the specificity of the N-terminus NUP98 signals in NUP98r AML models. Experiments using a HA antibody in HA-tagged NUP98::KDM5A CD34 models confirmed that fusion oncoproteins are binding to these target regions. However, the C-terminus NUP98 antibody also detected similar signals as the N-NUP98 antibody, suggesting that wild-type NUP98 is also recruited to NUP98 FO target genes, possibly by a heteromer formation with NUP98 FOs and other NPC components. To understand the possible association between somatic mutations and the genomic binding of NUP98 FOs, we next investigated chromatin immunoprecipitation sequencing (ChIP-seq) data from the ENCODE study (PMID:22955991) for RB1, WT1, and STAT5 (downstream FLT3-ITD). Binding peaks of these proteins partly coincided with NUP98 peaks on representative target genes (Panel.B-bottom), suggesting that frequent mutations in NUP98r leukemia converge on the genomic interaction of NUP98 FOs. In conclusion, genomic characterization of NUP98r leukemia showed the association among fusion partners, cooperating mutations, and disease phenotypes. Epigenetic profiling revealed that NUP98 FOs directly regulate target genes in a fusion and differentiation-specific manner and provide a basis for further study to investigate how NUP98 FOs and cooperating partners drive leukemogenesis and disease phenotypes.

Epigenetic regulation of N-hydroxypipecolic acid biosynthesis by the AIPP3-PHD2-CPL2 complex.

N-Hydroxypipecolic acid (NHP) is a signaling molecule crucial for systemic acquired resistance (SAR), a systemic immune response in plants that provides long-lasting and broad-spectrum protection against secondary pathogen infections. To identify negative regulators of NHP biosynthesis, we performed a forward genetic screen to search for mutants with elevated expression of the NHP biosynthesis gene FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1). Analysis of two constitutive expression of FMO1 (cef) and one induced expression of FMO1 (ief) mutants revealed that the AIPP3-PHD2-CPL2 protein complex, which is involved in the recognition of the histone modification H3K27me3 and transcriptional repression, contributes to the negative regulation of FMO1 expression and NHP biosynthesis. Our study suggests that epigenetic regulation plays a crucial role in controlling FMO1 expression and NHP levels in plants.

Identification of ZFP91 As the Master Transcription Factor for EVI1 Gene Expression

Ecotropic viral integration site 1 (EVI1) is essential for hematopoiesis, particularly for the maintenance of hematopoietic stem cells. Aberrant expression of EVI1 is widely known to be an independent poor prognostic factor in acute myeloid leukemia (AML). Previous studies have shown that enhancer hijacking or gene fusion caused by chromosomal translocation or inversion involving 3q26 leads to EVI1 overexpression. Intriguingly, however, aberrant EVI1 expression is seen in many cases of AML without 3q26 rearrangement. The prognosis of these cases is similarly dismal, but the mechanism that causes aberrant EVI1 expression in the absence of 3q26 chromosomal abnormalities is largely unknown. To identify key regulatory factors essential for EVI1 expression in AML, we performed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout screen. We knocked in the green fluorescent protein (GFP) coding sequence at the EVI1 locus in the human myeloid leukemia cell line K562, which expresses high levels of EVI1 without a 3q26 chromosomal aberration. After transduction of a genome-wide CRISPR/Cas9 knockout library, transduced cells were separated into GFP High and GFP Low subpopulations by flow cytometry. By sequencing the uniquely attached barcodes, we estimated the effect of gene knockout for each gene. Genes counted more frequently in the GFP Low fraction were listed as candidates for activators of EVI1. The top 13 genes, including three apparent positive controls such as EVI1 and two ribosome-related genes, had a false discovery rate (FDR) of less than 1%. We validated these candidate genes in K562 and two other EVI1-overexpressing AML cell lines by transducing individual single guide RNAs targeting these genes. These analyses identified zinc finger protein 91 (ZFP91) as a potential regulatory factor that significantly upregulates EVI1 expression common to the three independent myeloid leukemia cell lines. ZFP91 is an atypical E3 ubiquitin ligase, a DNA-binding protein that functions as a transcription factor. ZFP91 promotes cell growth and inhibits apoptosis in AML and has been shown in previous studies to activate multiple oncogenic pathways, including nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and hypoxia-inducible factor-1α (HIF-1α). We demonstrated that knockout of ZFP91 in K562 substantially reduced EVI1 protein expression to undetectable levels by Western blotting (Figure 1). EVI1 mRNA expression also decreased dramatically after ZFP91 knockout. These results suggest that ZFP91 may play an indispensable role in EVI1 transcription. We performed chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) targeting the EVI1 promoter in K562 and demonstrated that ZFP91 knockout clones showed approximately 10-fold weaker signals for active histone modifications (H3K4me3 and H3K27ac) at the EVI1 promoter (Figure 2). In addition, chromatin immunoprecipitation sequencing (ChIP-seq) for ZFP91 in K562 revealed the presence of a distinct peak as well as the ZFP91-specific motif sequence within the EVI1 promoter region. These data strongly suggest that ZFP91 functions as an essential transcription factor for EVI1 gene overexpression in some EVI1-associated myeloid leukemias. We confirmed that ZFP91 knockout in K562 inhibited cell proliferation, which is consistent with previous studies in other AML cell lines. Based on these results, we believe that ZFP91 has the potential to serve as a therapeutic target in EVI1-associated AML. To conclude, we have identified the transcription factor ZFP91 as a novel prominent activator of EVI1 expression in some EVI1-associated AML. Our study suggests that ZFP91 may be the key to unraveling the pathogenesis of EVI1-associated AML and an attractive therapeutic target in EVI1-associated AML.

Competition for H2A.Z underlies the developmental impacts of repetitive element de-repression.

The histone variant H2A.Z is central to early embryonic development, determining transcriptional competency through chromatin regulation of gene promoters and enhancers. In addition to genic loci, we find that H2A.Z resides at a subset of evolutionarily young repetitive elements, including DNA transposons, long interspersed nuclear elements and long terminal repeats, during early zebrafish development. Moreover, increases in H2A.Z occur when repetitive elements become transcriptionally active. Acquisition of H2A.Z corresponds with a reduction in the levels of the repressive histone modification H3K9me3 and a moderate increase in chromatin accessibility. Notably, however, de-repression of repetitive elements also leads to a significant reduction in H2A.Z over non-repetitive genic loci. Genic loss of H2A.Z is accompanied by transcriptional silencing at adjacent coding sequences, but remarkably, these impacts are mitigated by augmentation of total H2A.Z protein via transgenic overexpression. Our study reveals that levels of H2A.Z protein determine embryonic sensitivity to de-repression of repetitive elements, that repetitive elements can function as a nuclear sink for epigenetic factors and that competition for H2A.Z greatly influences overall transcriptional output during development. These findings uncover general mechanisms in which counteractive biological processes underlie phenotypic outcomes.