Chromatin Remodeling Activities Research Articles

Gain-of-Function Chromatin Remodeling Activity of Oncogenic FOXL2-C134W Reprograms Glucocorticoid Receptor Occupancy to Drive Granulosa Cell Tumors.

Adult type ovarian granulosa cell tumors (AGCTs) are rare malignancies with the near universal c.C402G (p.Cys134Trp) somatic mutation in FOXL2, a Forkhead box-family transcription factor important for ovarian function. Relapsed AGCT is incurable, but the mechanism of the unique FOXL2 mutation could confer therapeutic vulnerabilities. To identify FOXL2-C134W-dependent pharmacologic synergies, we created and characterized endogenous FOXL2 isogenic AGCT cells and an AGCT tumoroid biobank. A drug screen identified that glucocorticoids promote FOXL2-C134W-dependent AGCT growth. Epigenetic investigation revealed that the Cys134Trp mutation exposes latent DNA sequence-specific chromatin remodeling activity in FOXL2. FOXL2-C134W-dependent chromatin remodeling activity redirected glucocorticoid receptor chromatin occupancy to drive hyaluronan synthase 2 gene expression and increase extracellular hyaluronan secretion. Treatment of AGCT models with hyaluronidase reduced viability, and dexamethasone rescued this effect. Combinatorial drug-drug interaction experiments demonstrated that dexamethasone antagonizes the potency of paclitaxel, a chemotherapy agent frequently used in the treatment of AGCT. Thus, gain-of-function pioneering activity contributes to the oncogenic mechanism of FOXL2-C134W and creates a potentially targetable synergy with glucocorticoid signaling.

EOMES establishes mesoderm and endoderm differentiation potential through SWI/SNF-mediated global enhancer remodeling.

Mammalian pluripotent cells first segregate into neuroectoderm (NE), or mesoderm and endoderm (ME), characterized by lineage-specific transcriptional programs and chromatin states. To date, the relationship between transcription factor activities and dynamic chromatin changes that guide cell specification remains ill-defined. In this study, we employ mouse embryonic stem cell differentiation toward ME lineages to reveal crucial roles of the Tbx factor Eomes to globally establish ME enhancer accessibility as the prerequisite for ME lineage competence and ME-specific gene expression. EOMES cooperates with the SWItch/sucrose non-fermentable (SWI/SNF) complex to drive chromatin rewiring that is essential to overcome default NE differentiation, which is favored by asymmetries in chromatin accessibility at pluripotent state. Following global ME enhancer remodeling, ME-specific gene transcription is controlled by additional signals such as Wnt and transforming growth factor β (TGF-β)/NODAL, as a second layer of gene expression regulation, which can be mechanistically separated from initial chromatin remodeling activities.

Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy.

The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well-known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae , the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). We have previously reported that in the absence of SIR2 , a derepressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both, activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30 . Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

Identification of a Novel Frameshift variant of the ATRX gene: a Case Report and Review of the genotype–phenotype relationship

BackgroundX-linked intellectual disability-hypotonic facies syndrome-1 (MRXHF1) and Alpha-thalassemia X-linked intellectual disability (ATR-X) syndrome are caused by pathogenic variant in the ATRX gene, a member of the switch/sucrose non-fermentable (SWI-SNF) protein family that exhibits chromatin remodeling activity. These syndromes show a wide spectrum of clinical manifestations, such as distinctive dysmorphic features, mild-to-profound intellectual disability, motor development delay, seizures, urogenital abnormalities, and gastrointestinal disorders.Case presentation and literature reviewA 3-year-old boy from a Chinese non-consanguineous family was diagnosed with MRXHF1 by whole-exome sequencing. Comprehensive family history information was obtained. The Medline database was searched until 1st Aug 2023 for articles related to ATRX pathogenic variant. Data on gene/protein mutations and clinical symptoms were extracted. The proband showed intellectual disability, motor development delay, typical facial abnormalities, urogenital defect, behavior problems, and optical nerve dysplasia. A novel frameshift mutation c.399_400dup, (p.Leu134Cysfs*2) in the ATRX gene was the primary cause, which occurs right before the ATRXDNMT3-DNMT3L (ADD) domain of ATRX protein. Missense mutation is the most common variation type. The ADD and helicase-like domains are the most frequently affected domains. Epilepsy, congenital heart disease, urogenital defect, acoustic defect, and optical defect are more prevalent in patients with frameshift mutations compared to those with missense mutations. There are more urogenital defects with C-terminal frameshift mutations than with N-terminal frameshift mutations.ConclusionWe described a novel frameshift mutation in the ATRX gene in a patient with MRXHF1 syndrome and summarized the genotype–phenotype relationship of ATRX pathogenic variant by variation type and affected protein domain. The regulatory mechanism underlying ATRX variant requires comprehensive analysis in future studies.

Molecular and structural basis of the chromatin remodeling activity by Arabidopsis DDM1

The histone H2A variant H2A.W occupies transposons and thus prevents access to them in Arabidopsis thaliana. H2A.W is deposited by the chromatin remodeler DDM1, which also promotes the accessibility of chromatin writers to heterochromatin by an unknown mechanism. To shed light on this question, we solve the cryo-EM structures of nucleosomes containing H2A and H2A.W, and the DDM1-H2A.W nucleosome complex. These structures show that the DNA end flexibility of the H2A nucleosome is higher than that of the H2A.W nucleosome. In the DDM1-H2A.W nucleosome complex, DDM1 binds to the N-terminal tail of H4 and the nucleosomal DNA and increases the DNA end flexibility of H2A.W nucleosomes. Based on these biochemical and structural results, we propose that DDM1 counters the low accessibility caused by nucleosomes containing H2A.W to enable the maintenance of repressive epigenetic marks on transposons and prevent their activity.

The importance of DNA sequence for nucleosome positioning in transcriptional regulation.

Nucleosome positioning is a key factor for transcriptional regulation. Nucleosomes regulate the dynamic accessibility of chromatin and interact with the transcription machinery at every stage. Influences to steer nucleosome positioning are diverse, and the according importance of the DNA sequence in contrast to active chromatin remodeling has been the subject of long discussion. In this study, we evaluate the functional role of DNA sequence for all major elements along the process of transcription. We developed a random forest classifier based on local DNA structure that assesses the sequence-intrinsic support for nucleosome positioning. On this basis, we created a simple data resource that we applied genome-wide to the human genome. In our comprehensive analysis, we found a special role of DNA in mediating the competition of nucleosomes with cis-regulatory elements, in enabling steady transcription, for positioning of stable nucleosomes in exons, and for repelling nucleosomes during transcription termination. In contrast, we relate these findings to concurrent processes that generate strongly positioned nucleosomes in vivo that are not mediated by sequence, such as energy-dependent remodeling of chromatin.

Acute depletion of BRG1 reveals its primary function as an activator of transcription

The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization of human gene mutations implicate that the complexes both repress and activate a large number of genes. However, the direct function of the complexes in cells remains largely unclear due to the relatively long-term nature of gene deletion or natural mutation. Here we generate a mouse line by knocking in the auxin-inducible degron tag (AID) to the Smarca4 gene, which encodes BRG1, the essential ATPase subunit of the BAF complexes. We show that the tagged BRG1 can be efficiently depleted by osTIR1 expression and auxin treatment for 6 to 10 h in CD4 + T cells, hepatocytes, and fibroblasts isolated from the knock-in mice. The acute depletion of BRG1 leads to decreases in nascent RNAs and RNA polymerase II binding at a large number of genes, which are positively correlated with the loss of BRG1. Further, these changes are correlated with diminished accessibility at DNase I Hypersensitive Sites (DHSs) and p300 binding. The acute BRG1 depletion results in three major patterns of nucleosome shifts leading to narrower nucleosome spacing surrounding transcription factor motifs and at enhancers and transcription start sites (TSSs), which are correlated with loss of BRG1, decreased chromatin accessibility and decreased nascent RNAs. Acute depletion of BRG1 severely compromises the Trichostatin A (TSA) -induced histone acetylation, suggesting a substantial interplay between the chromatin remodeling activity of BRG1 and histone acetylation. Our data suggest BRG1 mainly plays a direct positive role in chromatin accessibility, RNAPII binding, and nascent RNA production by regulating nucleosome positioning and facilitating transcription factor binding to their target sites.

Non-canonical role for the BAF complex subunit DPF3 in mitosis and ciliogenesis.

DPF3, along with other subunits, is a well-known component of the BAF chromatin remodeling complex, which plays a key role in regulating chromatin remodeling activity and gene expression. Here, we elucidated a non-canonical localization and role for DPF3. We showed that DPF3 dynamically localizes to the centriolar satellites in interphase and to the centrosome, spindle midzone and bridging fiber area, and midbodies during mitosis. Loss of DPF3 causes kinetochore fiber instability, unstable kinetochore-microtubule attachment and defects in chromosome alignment, resulting in altered mitotic progression, cell death and genomic instability. In addition, we also demonstrated that DPF3 localizes to centriolar satellites at the base of primary cilia and is required for ciliogenesis by regulating axoneme extension. Taken together, these findings uncover a moonlighting dual function for DPF3 during mitosis and ciliogenesis.

Small molecules that disrupt RAD54-BLM interaction hamper tumor proliferation in colon cancer chemoresistance models.

RAD54 and BLM helicase play pivotal roles during homologous recombination repair (HRR) to ensure genome maintenance. BLM amino acids (aa 181-212) interact with RAD54 and enhance its chromatin remodeling activity. Functionally, this interaction heightens HRR, leading to a decrease in residual DNA damage in colon cancer cells. This contributes to chemoresistance in colon cancer cells against cisplatin, camptothecin, and oxaliplatin, eventually promoting tumorigenesis in preclinical colon cancer mouse models. ChIP-Seq analysis and validation revealed increased BLM and RAD54 corecruitment on the MRP2 promoter in camptothecin-resistant colon cancer cells, leading to BLM-dependent enhancement of RAD54-mediated chromatin remodeling. We screened the Prestwick small-molecule library, with the intent to revert camptothecin- and oxaliplatin-induced chemoresistance by disrupting the RAD54-BLM interaction. Three FDA/European Medicines Agency-approved candidates were identified that could disrupt this interaction. These drugs bound to RAD54, altered its conformation, and abrogated RAD54-BLM-dependent chromatin remodeling on G5E4 and MRP2 arrays. Notably, the small molecules also reduced HRR efficiency in resistant lines, diminished anchorage-independent growth, and hampered the proliferation of tumors generated using camptothecin- and oxaliplatin-resistant colon cancer cells in both xenograft and syngeneic mouse models in BLM-dependent manner. Therefore, the 3 identified small molecules can serve as possible viable candidates for adjunct therapy in colon cancer treatment.

Differential requirements for Smarca5 expression during hematopoietic stem cell commitment

The formation of hematopoietic cells relies on the chromatin remodeling activities of ISWI ATPase SMARCA5 (SNF2H) and its complexes. The Smarca5 null and conditional alleles have been used to study its functions in embryonic and organ development in mice. These mouse model phenotypes vary from embryonic lethality of constitutive knockout to less severe phenotypes observed in tissue-specific Smarca5 deletions, e.g., in the hematopoietic system. Here we show that, in a gene dosage-dependent manner, the hypomorphic allele of SMARCA5 (S5tg) can rescue not only the developmental arrest in hematopoiesis in the hCD2iCre model but also the lethal phenotypes associated with constitutive Smarca5 deletion or Vav1iCre-driven conditional knockout in hematopoietic progenitor cells. Interestingly, the latter model also provided evidence for the role of SMARCA5 expression level in hematopoietic stem cells, as the Vav1iCre S5tg animals accumulate stem and progenitor cells. Furthermore, their hematopoietic stem cells exhibited impaired lymphoid lineage entry and differentiation. This observation contrasts with the myeloid lineage which is developing without significant disturbances. Our findings indicate that animals with low expression of SMARCA5 exhibit normal embryonic development with altered lymphoid entry within the hematopoietic stem cell compartment.

BCL7A and BCL7B potentiate SWI/SNF-complex-mediated chromatin accessibility to regulate gene expression and vegetative phase transition in plants

Switch defective/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes are multi-subunit machineries that establish and maintain chromatin accessibility and gene expression by regulating chromatin structure. However, how the remodeling activities of SWI/SNF complexes are regulated in eukaryotes remains elusive. B-cell lymphoma/leukemia protein 7 A/B/C (BCL7A/B/C) have been reported as subunits of SWI/SNF complexes for decades in animals and recently in plants; however, the role of BCL7 subunits in SWI/SNF function remains undefined. Here, we identify a unique role for plant BCL7A and BCL7B homologous subunits in potentiating the genome-wide chromatin remodeling activities of SWI/SNF complexes in plants. BCL7A/B require the catalytic ATPase BRAHMA (BRM) to assemble with the signature subunits of the BRM-Associated SWI/SNF complexes (BAS) and for genomic binding at a subset of target genes. Loss of BCL7A and BCL7B diminishes BAS-mediated genome-wide chromatin accessibility without changing the stability and genomic targeting of the BAS complex, highlighting the specialized role of BCL7A/B in regulating remodeling activity. We further show that BCL7A/B fine-tune the remodeling activity of BAS complexes to generate accessible chromatin at the juvenility resetting region (JRR) of the microRNAs MIR156A/C for plant juvenile identity maintenance. In summary, our work uncovers the function of previously elusive SWI/SNF subunits in multicellular eukaryotes and provides insights into the mechanisms whereby plants memorize the juvenile identity through SWI/SNF-mediated control of chromatin accessibility.

Development of Complementary Photo-arginine/lysine to Promote Discovery of Arg/Lys hPTMs Interactomes.

Arginine and lysine, frequently appearing as a pair on histones, have been proven to carry diverse modifications and execute various epigenetic regulatory functions. However, the most context-specific and transient effectors of these marks, while significant, have evaded study as detection methods have thus far not reached a standard to capture these ephemeral events. Herein, a pair of complementary photo-arginine/δ-photo-lysine (R-dz/K-dz) probes is developed and involve these into histone peptide, nucleosome, and chromatin substrates to capture and explore the interactomes of Arg and Lys hPTMs. By means of these developed tools, this study identifies that H3R2me2a can recruit MutS protein homolog 6 (MSH6), otherwise repelDouble PHD fingers 2 (DPF2), Retinoblastoma binding protein 4/7 (RBBP4/7). And it is disclosed that H3R2me2a inhibits the chromatin remodeling activity of the cBAF complex by blocking the interaction between DPF2 (one component of cBAF) and the nucleosome. In addition, the novel pairs of H4K5 PTMs and respective readers are highlighted, namely H4K5me-Lethal(3)malignant brain tumor-like protein 2 (L3MBTL2), H4K5me2-L3MBTL2, and H4K5acK8ac-YEATS domain-containing protein 4 (YEATS4). These powerful tools pave the way for future investigation of related epigenetic mechanisms including but not limited to hPTMs.

Gatad2b, associated with the neurodevelopmental syndrome GAND, plays a critical role in neurodevelopment and cortical patterning

GATAD2B (GATA zinc finger domain containing 2B) variants are associated with the neurodevelopmental syndrome GAND, characterized by intellectual disability (ID), infantile hypotonia, apraxia of speech, epilepsy, macrocephaly and distinct facial features. GATAD2B encodes for a subunit of the Nucleosome Remodeling and Histone Deacetylase (NuRD) complex. NuRD controls transcriptional programs critical for proper neurodevelopment by coupling histone deacetylase with ATP-dependent chromatin remodeling activity. To study mechanisms of pathogenesis for GAND, we characterized a mouse model harboring an inactivating mutation in Gatad2b. Homozygous Gatad2b mutants die perinatally, while haploinsufficient Gatad2b mice exhibit behavioral abnormalities resembling the clinical features of GAND patients. We also observed abnormal cortical patterning, and cellular proportions and cell-specific alterations in the developmental transcriptome in these mice. scRNAseq of embryonic cortex indicated misexpression of genes key for corticogenesis and associated with neurodevelopmental syndromes such as Bcl11b, Nfia and H3f3b and Sox5. These data suggest a crucial role for Gatad2b in brain development.

Abstract A064: Establishing the cellular and molecular impacts of the dual BRM/BRG1 inhibitor FHD-286 on pre-clinical models of non-small cell lung cancer (NSCLC)

Abstract The BRG/BRM-associated factor (BAF) complex plays critical roles in chromatin regulation. Within this complex, the two mutually exclusive ATPases BRM and BRG1 (SMARCA2 and SMARCA4, respectively) drive chromatin remodeling activity. Genetic alterations in the SMARCA4 gene, encoding BRG1, are found in approximately 10% of non-small cell lung cancer (NSCLC) patients, leaving this subset of BRG1-deficient tumors highly susceptible to BRM inhibition. In addition, BRG1 has oncogenic activity in NSCLC, rationalizing the use of a dual BRM/BRG1 ATPase inhibitor, such as the highly potent clinical stage compound FHD-286, in NSCLC patients, regardless of SMARCA4 status. Here, we demonstrate that a panel of NSCLC cell lines respond to dual BAF ATPase inhibition in both in vitro and in vivo settings, regardless of SMARCA4 mutation status. Interestingly, dual BRM/BRG1 inhibition in cell lines reduces chromatin accessibility at motifs normally bound by AP-1 transcription factors, which play tumorigenic roles in NSCLC. We also find that expression of the stemness-associated glycoprotein CD44 is decreased by FHD-286, suggesting that dual BRM/BRG1 inhibition impacts the differentiation state of NSCLC. Moreover, transcriptional analyses of NSCLC xenograft models reveal that a basal cell gene expression signature, which is associated with a less differentiated cell state, is downregulated by FHD-286 treatment. Finally, genome-wide CRISPR knockout screening with FHD-286 in 2 NSCLC lines further elucidates mechanisms of action and molecular vulnerabilities induced by dual BAF ATPase inhibition. Collectively, these results indicate a path forward for the development of FHD-286 for the treatment of NSCLC patients. Citation Format: Molly M. Wilson, Ammar Adam, Victoria Amaral, Oliver Mikse, GiNell Elliott, David L. Lahr, Ashley K. Gartin, Jessica Wan, Liv Johannessen, Kana Ichikawa, Katharine Feldman, Jessica Piel, Gabriel J. Sandoval, Richard C. Centore, Steven F. Bellon, Martin Hentemann. Establishing the cellular and molecular impacts of the dual BRM/BRG1 inhibitor FHD-286 on pre-clinical models of non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A064.

Abstract A079: FHD286 is a BRG1/BRM ATPase inhibitor showing efficacy in NSCLC models and is applicable to NSCLC patients both as a single agent treatment and in combination with current standards of care

Abstract The BAF (BRG/BRM-associated factors) complex plays a critical role in shaping the chromatin landscape. The two mutually exclusive ATPases BRM/BRG1 (SMARCA2/4, respectively) drive chromatin remodeling activity in the BAF complex. Genetic alterations in the SMARCA4 gene, encoding BRG1, are found in approximately 10% of non-small cell lung cancer (NSCLC) patients, leaving this subset of BRG1-deficient tumors highly susceptible to inhibition of BRM (Hoffman, 2014; Oike, 2013). However, BRG1 can also have oncogenic activity in NSCLC (Li, 2019) and can serve as a predictive biomarker for chemotherapy outcome in patients (Bell, et al 2015), rationalizing the use of a dual BRG1/BRM ATPase inhibitor (FHD286) in NSCLC patients in combination with chemotherapy. Here, we demonstrate that a panel of in vitro and in vivo NSCLC models respond to BAF ATPase inhibition as a single agent treatment, irrespective of SMARCA4 mutation status. Analysis of gene expression and dependency data suggests that FHD286 sensitivity correlates with activity of genes related to DNA damage response and repair. We furthermore demonstrate an enhancing effect on tumor growth inhibition in vitro and in vivo driven by apoptosis and cell cycle arrest when combining dual FHD286 with current first and second line standards of care. Collectively, these results indicate a path forward for the development of FHD-286, a clinical stage BRM/BRG1 ATPase inhibitor, for the treatment of NSCLC patients both as a single agent treatment and in combination with current standards of care. Citation Format: Oliver Mikse, Ammar Adam, GiNell Elliott, Dave Lahr, Victoria Amaral, Priyadharshini Viswanathan, Liv Johannessen, Hafiz Ahmad, Jessica Wan, Steven Bellon, Murphy Hentemann. FHD286 is a BRG1/BRM ATPase inhibitor showing efficacy in NSCLC models and is applicable to NSCLC patients both as a single agent treatment and in combination with current standards of care [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A079.

Preliminary Results from a Phase 1 Dose Escalation Study of FHD-286, a Novel BRG1/BRM (SMARCA4/SMARCA2) Inhibitor, Administered As an Oral Monotherapy in Patients with Advanced Hematologic Malignancies

The BRG/Brahma-associated factors (BAF) family of chromatin remodeling complexes (also referred to as the mSWI/SNF complex) regulates the chromatin landscape of the genome. Through its adenosine triphosphate (ATP)-dependent chromatin remodeling activity, BAF complexes regulate the accessibility of gene-control elements, allowing for the binding of transcription factors. Thus, BAF is a major regulator of lineage- and disease-specific transcriptional programs. FHD-286 is a first-in-class, orally administered compound that potently and selectively inhibits the ATPase components of the BAF complexes, SMARCA4 and SMARCA2 (also called BRG1 and BRM, respectively). In preclinical models, acute myeloid leukemia (AML) cell lines were highly sensitive to BAF inhibition. FHD-286-C-002 (NCT04891757) is a multicenter, open-label, Phase 1 dose escalation study designed to determine the safety, tolerability, dose-limiting toxicities (DLTs), and maximum tolerated dose and/or the recommended phase 2 dose(s), as well as to evaluate the preliminary clinical activity of FHD-286, in patients with advanced hematologic malignancies. Patients with relapsed or refractory (R/R) AML or myelodysplastic syndrome (MDS) failing all available standard therapies were eligible to receive FHD-286 doses ranging from 2.5 mg once daily (QD) to 10 mg QD on a continuous dosing regimen. Patients had regularly scheduled assessments to evaluate safety, pharmacokinetics (PK), pharmacodynamics, preliminary clinical activity, and exploratory biomarkers. As of 02 Aug 2022, 40 patients with a median age of 65.5 years (range 25 to 84) with R/R AML (36 patients) or MDS (4 patients) had received at least 1 dose of FHD-286. 67.5% of patients had received ≥3 prior lines of therapy for AML/MDS/other antecedent hematologic disorder, with 22.5% having received ≥5 prior lines. 32.5% of patients had prior hematopoietic stem cell transplant. 65% of patients had adverse risk genetics by 2017 ELN recommendations. 85% of patients experienced a treatment-related adverse event (TRAE) of any grade; the most common (≥20% of patients) were dry mouth (27.5%), increased blood bilirubin (22.5%), and alanine aminotransferase (ALT) increased and rash (20% each). 50% of patients experienced a Grade ≥3 TRAE, the most common of which was increased blood bilirubin (12.5%); stomatitis, ALT increased, differentiation syndrome (DS), and hypocalcemia occurred in 7.5% (each). 2 DLTs were reported: Grade 3 hyperbilirubinemia in 1 patient receiving 5 mg QD and Grade 3 muscular weakness in 1 patient receiving 10 mg QD. Treatment-related DS of any grade was investigator-reported in 4 patients (10%). An independent adjudication committee determined that the rate of suspected DS was 15% (6 patients). 16 patients (15 AML; 1 MDS) had a best overall response of stable disease. Markers of myeloid differentiation with neutrophil recovery and reductions in bone marrow and/or peripheral blasts have been observed in a subset of patients in the study who had a broad range of cytogenetic backgrounds, including patients with enhancer-driven leukemias such as MECOM and KMT2A. Preliminary PK analysis indicates that FHD-286 appears to have a long half-life, at a minimum of >24 hours, and that plasma concentrations of FHD-286 increase with increasing dose. Exploratory flow cytometry assessment of markers of differentiation, hematopoietic stem cell identity, and apoptosis suggested dose-dependent target engagement. Additionally, exploratory sequencing analysis on bone marrow blasts revealed comprehensive impacts on AML-specific expression pathways and stemness genes in response to FHD-286. An exposure-response analysis using the percentage of peripheral blasts showed a trend toward lower blast count with higher FHD-286 area-under-the-curve. Enrollment into the single-agent dose escalation phase of the study is complete. Based on nonclinical, translational, and single-agent clinical data, FHD-286 in combination with decitabine or low-dose cytarabine is being evaluated in the combination dose escalation phase of the study.

Selective Globin Gene Regulation By the Non-Canonical Baf Chromatin Remodeling Complex

The regulation of the beta-type globin genes has served as a powerful model for developmental transcriptional control, with humans expressing embryonic (HBE), two fetal (HBG1/2) and two adult (HBB and HBD) versions at the corresponding developmental stages. Reactivation of HBG in adult erythroid cells is of benefit to patients with sickle cell disease and β-thalassemia. While past efforts focused on the identification of repressors of the HBG genes, including BCL11A, ZBTB7A, and NFI-A/X, to date no transcriptional activators have been identified with selectivity for any of the globin genes. Using a CRISPR-Cas9 screen in the HUDEP2 erythroid cell line, we recently identified BRG1/SMARCA4 as a potential activator of HBG production. BRG1 is a core ATPase component of the multi-subunit BAF (BRG1/BRM-associated factor) complex; different subtypes of this large chromatin remodeler family are specific to particular cell types or developmental stages. BRG1 was previously implicated in globin gene regulation but its molecular composition and whether it has selectivity for any of the genes in the cluster is unknown. Notably, we found that loss of BRG1 in both HUDEP2 and primary erythroid cells impairs transcription of HBG to a much greater extent than that of HBB. Here we functionally dissected BAF subunits to decipher composition of the relevant BAF complex and the mechanism for this unexpected HBG gene selectivity. We used CRISPR-Cas9 in HUDEP2 cells and human primary erythroid cultures to test the requirement for 14 different BAF subunits for selective HBG transcription. Among these, the BAF60A component emerged as critically required for the normal production of HBG but not HBB in HUDEP2 cells. Depletion of BAF60A in human primary erythroid cultures reduced HBG by up to 80% with no reduction in HBB as assessed by western blot, flow cytometry, HPLC, RT-PCR, and droplet digital RT-PCR. We also observed a reduction in HBD expression, suggesting that BAF60A functions in a gene selective manner but not necessarily favoring the fetal over the adult-type globin genes. α-type globin genes were unaffected by BAF60A loss, further demonstrating the gene selectivity of BAF60A action. RNA-seq of BAF60A-targeted cells showed that the most downregulated transcripts were from the two HBG genes and the BGLT3 long non-coding RNA, which typically parallels HBG in expression. These results revealed an unexpected gene selectivity of the BAF complex, conveyed at least in part by BAF60A. To assess global chromatin accessibility upon BAF60A loss in primary erythroid cells we utilized ATAC-seq. Among ~132,000 accessibility peaks, ~10,000 were reduced in BAF60A targeted cells. Interestingly, we saw a strong decrease in chromatin accessibility at the HBG and HBD genes with minimal to no effect on the HBB or α-globin genes, in line with gene expression data, again reflecting the gene specificity of BAF60A. BAF60A can be found in different classes of BAF complexes, but is absolutely required for the non-canonical BAF (ncBAF) subtype. ncBAF is a relatively recently described BAF complex that is distinguished from other classes of BAF complexes by the presence of the bromodomain protein BRD9. Therefore, we hypothesized that ncBAF is the relevant BAF complex required for selective HBG transcription. Genetic or pharmacologic disruption of BRD9 in HUDEP2 cells recapitulated the BAF60A phenotype, with decreased HBG and HBD expression but sparing of the HBB and α-globin genes. This is the first study of the role of the non-canonical BAF complex in hemoglobin gene regulation. Our results demonstrate remarkable selectivity of this subtype of a broadly active chromatin remodeling complex for specific hemoglobin genes in adult erythroid cells. Further work on the ncBAF complex will focus on rerouting the BAF complex to promote the transcription of HBG and HBD in hemoglobinopathies.

Whole exome sequencing identifies common mutational landscape of cervix and endometrium small cell neuroendocrine carcinoma.

Primary small cell neuroendocrine carcinomas of the cervix and endometrium are rare gynecological malignancies with limited treatment options. This study aimed to improve the understanding of the carcinogenesis process and identify potential therapeutic targets for these two tumor types by constructing the mutational landscape at the whole exome level. Primary tumor tissues and their matched blood samples were obtained from 10 patients with small cell cervical neuroendocrine carcinoma (NECC) and five patients with small cell endometrial neuroendocrine carcinoma (NECE). Whole exome sequencing was performed to construct the somatic mutation profiles. Mutational signature and recurrent mutated gene analysis were used to identify tumor subtypes and common carcinogenesis processes. Based on the burden of different mutational signatures, the NECCs in this work can be divided into two subtypes, including the mismatch repair deficiency like (dMMR-like) type (4/10) and the high spontaneous deamination type (6/10). Components of the PI3K/AKT signaling and RAS signaling were exclusively mutated in these two subtypes, respectively. The integration of human papillomavirus made a limited contribution to tumorigenesis in NECC (20%). The dysfunction of the mismatch repair system and microsatellite instability are the major features of NECE. PI3K/AKT, JAK/STAT signaling, and chromatin remodeling activity were the common mutated pathways in NECE. PIK3CA, WNK2, and KMT2B underwent mutations in both the dMMR-like subtype of NECC (50% - 75%) and in NECE (60% - 80%) specimens, while exhibiting infrequent mutational occurrences in publicly available data pertaining to neuroendocrine carcinomas of the lung or bladder (< 10%). We identified the two subtypes of NECC with distinct mutated pathways and potential therapy targets. The dMMR-like type NECC and NECE may share a similar carcinogenesis process that include dysfunction of PI3K/AKT signaling, cell cycle, antiapoptotic processes, and chromatin remodeling activity.

OR01-02 Epigenomic Dysregulation Of PPP1R17 Drives The Cell Cycle In Adenomas Causing Cushing’s Disease

Abstract Disclosure: D. Asuzu: None. D. Mullaney: None. S. Stoica: None. D. Mandal: None. D. Maric: None. A. Elkahloun: None. B. Sisay: None. C. Tatsi: None. L.K. Nieman: None. P. Chittiboina: None. Introduction: Pituitary adenomas causing Cushing’s disease are largely (60%) mutationally bland. Previously, with syngeneic, pairwise transcriptomic analysis, we found a robust upregulation of PPP1R17 in canonically mutated (USP8, USP48 and BRAF) and ‘wildtype’ CD adenomas. PPP1R17 is an endogenous inhibitor of tumor suppressor phosphatase PP2A. Here, we investigated the epigenomic dysregulation of PPP1R17, its interaction with PP2A, and downstream effects on cell cycle regulation. Methods: We performed single nucleus assays for transposase accessible chromatin (snATACseq, 10X Genomics) in adenomas from 4 CD patients, and two syngeneic adenoma-normal pairs along with parallel single nucleus RNAseq (snRNAseq). Transcription factors bound to open chromatin were identified by reverse chromatin immunoprecipitation (rChIP) using CRISPR dCas9-3XFLAG-Biotin (Millipore Sigma) and promoter-specific synthetic guide RNAs (Invitrogen TrueGuide). Bound transcription factors were quantified using mass spectrometry (SimulTOF 300). Fixed human CD adenomas (n=5) were sectioned and stained using multiplexed immunohistochemistry (mIHC). Association between R17 and protein phosphatase 2A (PP2A) was assessed using proximity ligation assays (PLA, Sigma). We overexpressed R17 or GFP controls in murine corticotroph (mCort) cell lines and assessed for cell-cycle changes (Click-IT EdU) and cell proliferation (OneGlo). Results: We mapped the chromatin accessibility landscape of CD adenomas compared to normal corticotrophs. We identified coordinated chromatin accessibility (snATACseq) and transcriptional upregulation (snRNAseq) at the R17 locus exclusively within adenomatous corticotrophs. Using rChIP, we identified enrichment at the R17 promoter of the RNA polymerase II transcriptional coactivator p15 (SUB1) and the histone variant H1.4, indicating active chromatin remodeling. PPP1R17 protein and its target PP2A were overexpressed in the adenoma compartment but not in the adjacent normal gland. PLA confirmed robust interaction between R17 and PP2Ac within the CD adenoma compartment. Mechanistically, mCortR17 cells showed inhibition of PP2A with phosphorylation its known targets - EGFR and ERK. mCortR17 cells showed accelerated cell cycle progression compared to mCortGFP (S-phase 16.4% versus 24.1%, P &amp;lt; 0.001) with increased proliferation (two-way ANOVA P &amp;lt; 0.001). PP2A agonists Fingolimod, DT061 and ABL127 led to proliferation arrest in mCortR17 cells. Conclusions: We identified increased R17 expression as an underlying mechanism of tumor hyperproliferation in CD adenomas. We found chromatin accessibility underlying PPP1R17 overexpression. PP2A agonists reversed the effects of PPP1R17 overexpression in-vitro. Our findings uncover a novel therapeutic phosphatase target for patients with CD. Presentation: Thursday, June 15, 2023

Nucleosome repositioning in chronic lymphocytic leukemia.

The location of nucleosomes in the human genome determines the primary chromatin structure and regulates access to regulatory regions. However, genome-wide information on deregulated nucleosome occupancy and its implications in primary cancer cells is scarce. Here, we conducted a genome-wide comparison of high-resolution nucleosome maps in peripheral blood B cells from patients with chronic lymphocytic leukemia (CLL) and healthy individuals at single-base-pair resolution. Our investigation uncovered significant changes of nucleosome positioning in CLL. Globally, the spacing between nucleosomes-the nucleosome repeat length (NRL)-is shortened in CLL. This effect is stronger in the more aggressive IGHV-unmutated CLL subtype than in the IGHV-mutated CLL subtype. Changes in nucleosome occupancy at specific sites are linked to active chromatin remodeling and reduced DNA methylation. Nucleosomes lost or gained in CLL marks differential binding of 3D chromatin organizers such as CTCF as well as immune response-related transcription factors and delineated mechanisms of epigenetic deregulation. The principal component analysis of nucleosome occupancy in cancer-specific regions allowed the classification of samples between cancer subtypes and normal controls. Furthermore, patients could be better assigned to CLL subtypes according to differential nucleosome occupancy than based on DNA methylation or gene expression. Thus, nucleosome positioning constitutes a novel readout to dissect molecular mechanisms of disease progression and to stratify patients. Furthermore, we anticipate that the global nucleosome repositioning detected in our study, such as changes in the NRL, can be exploited for liquid biopsy applications based on cell-free DNA to stratify patients and monitor disease progression.