BAF Complex Research Articles

Abstract 3230: Discovery of selective BRM (SMARCA2) ATPase inhibitors for the treatment of BRG1 (SMARCA4) mutant cancers

Abstract BRM (SMARCA2) and BRG1 (SMARCA4) are highly homologous ATPases that are members of the BAF (also known as the mSWI/SNF) chromatin remodeling complex. BRM and BRG1 are mutually exclusive enzymatic subunits of BAF complexes, and functional genomic screens have shown a synthetic lethal relationship between the two genes. BRG1 is frequently mutated in cancer, including in approximately 10% of non-small cell lung carcinomas. Selective inhibition of BRM is a mechanism by which BRG1-mutated cancer cell growth would be affected by losing BRM function, while normal tissues should be spared as they still express functional BRG1. Given that the ATPase domains of BRM and BRG1 are 92% identical, the identification of selective enzymatic inhibitors of BRM has been challenging. Here, we report the discovery of novel, highly potent compounds that selectively inhibit BRM over BRG1 as seen in cell-based transcription and proliferation assays, that also possess excellent oral pharmacokinetics. Our lead compounds inhibit BRM in a cell-based reporter assay with an unbound IC50 of 1 nM and are greater than 20-fold selective over BRG1. They are also selective against other helicases and do not show any significant activity in off-target screening panels. Dosing of mice carrying BRG1-mutant xenografts causes target gene modulation that is correlated with compound exposure. Treatment of A549 and RERF-LC-AI xenografts with lead compounds led to tumor growth inhibition of 87% and 96%, respectively, at well-tolerated doses, and significant tumor growth inhibition was also achieved in multiple other BRG1-mutant models. The results suggest that our compounds have first-in-class potential as potent and selective BRM ATPase inhibitors for the treatment of BRG1-mutated cancers, and we are planning to file an IND in 2024. Citation Format: Janice Y. Lee, Nathan Brooks, Brandon Antonakos, Bryan Perria, Candace Langan, Bonita D. Jones, Robert Stephen Flack, Zhifang Li, David Terry, Ross Wallace, Robert Bondi, Gereint Sis, Ronee Baracani, Maralee McVean, Gabrielle Kolakowski, Dayo Osimboni, Kevin Wilson. Discovery of selective BRM (SMARCA2) ATPase inhibitors for the treatment of BRG1 (SMARCA4) mutant cancers [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 3230.

Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system

Aberrant stem cell-like activity and impaired differentiation are central to the development of colorectal cancer (CRC). To identify functional mediators of these key cellular programs, we engineer a dual endogenous reporter system by genome-editing the SOX9 and KRT20 loci of human CRC cell lines to express fluorescent reporters, broadcasting aberrant stem cell-like and differentiation activity, respectively. By applying a CRISPR screen targeting 78 epigenetic regulators with 542 sgRNAs to this platform, we identify factors that contribute to stem cell-like activity and differentiation in CRC. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominate SMARCB1 of the BAF complex (also known as SWI/SNF) as a negative regulator of differentiation across an array of neoplastic colon models. SMARCB1 is a dependency and required for in vivo growth of human CRC models. These studies highlight the utility of biologically designed endogenous reporter platforms to uncover regulators with therapeutic potential.

Ripks and Neuroinflammation.

Neuroinflammation is an immune response in the central nervous system and poses a significant threat to human health. Studies have shown that the receptor serine/threonine protein kinase family (RIPK) family, a popular research target in inflammation, has been shown to play an essential role in neuroinflammation. It is significant to note that the previous reviews have only examined the link between RIPK1 and neuroinflammation. However, it has yet to systematically analyze the relationship between the RIPK family and neuroinflammation. Activation of RIPK1 promotes neuroinflammation. RIPK1 and RIPK3 are responsible for the control of cell death, including apoptosis, necrosis, and inflammation. RIPK1 and RIPK3 regulate inflammatory responses through the release of damage in necroptosis. RIPK1 and RIPK3 regulate inflammatory responses by releasing damage-associated molecular patterns (DAMPs) during necrosis. In addition, activated RIPK1 nuclear translocation and its interaction with the BAF complex leads to upregulation of chromatin modification and inflammatory gene expression, thereby triggering inflammation. Although RIPK2 is not directly involved in regulating cell death, it is considered an essential target for treating neurological inflammation. When the peptidoglycan receptor detects peptidoglycan IE-DAP or MDP in bacteria, it prompts NOD1 and NOD2 to recruit RIPK2 and activate the XIAP E3 ligase. This leads to the K63 ubiquitination of RIPK2. This is followed by LUBAC-mediated linear ubiquitination, which activates NF-KB and MAPK pathways to produce cytokines and chemokines. In conclusion, there are seven known members of the RIPK family, but RIPK4, RIPK5, RIPK6, and RIPK7 have not been linked to neuroinflammation. This article seeks to explore the potential of RIPK1, RIPK2, and RIPK3 kinases as therapeutic interventions for neuroinflammation, which is associated with Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), ischemic stroke, Parkinson's disease (PD), multiple sclerosis (MS), and traumatic brain injury (TBI).

Association of ARID1A deficiency with invasive transformation in metastatic bladder cancer.

656 Background: ARID1A is an essential component of the BAF complex, a large SWI/SNF chromatin-remodeling complex which is implicated in bladder cancer (BC) pathogenesis. While ARID1A mutations are more highly represented in metastatic rather than localized tumors, the biological consequences of ARID1A loss in BC remain to be elucidated. We hypothesize that ARID1A deficiency primes an invasive transformation in BC, thus increasing the likelihood of disease progression and metastases. We used CRISPR/Cas9-mediated gene editing to knockout (KO) the ARID1A gene in several BC cell lines, thus providing a platform for cellular function assays. We show that ARIDA1 loss potentiates an invasive phenotype in these cell lines, giving evidence for its transformative role in the development of higher stage and metastatic BC. Methods: A CRISPR-Cas9 KO was performed with three cell lines with wild-type (WT) ARID1A (T24, SW1710, and 5637). This was accomplished by developing sgRNAs against ARID1A with a GFP marker and carrying out single-cell fluorescence-activated cell sorting (FACS) following nucleofection to identify ARID1A-deficient clones. Western blot analysis was performed on the resulting clones to confirm complete loss of ARID1A. Baseline proliferation studies were performed on the KO and WT cell lines. Finally, invasion studies were performed on the KO and WT cells with a scratch assay. Results: CRISPR-Cas9 directed KO of ARID1A yielded ARID1A-deficient clones for SW1710, T24, and 5637. The proliferation rates for each cell line were analyzed and compared which showed no significant differences. A scratch assay was performed with the cell lines in triplicate, with each ARID1A-deficient cell line showing a trend towards more rapid wound closure than its matched WT cell line (WT=31.5% vs. KO=53.5% for SW1710, p=0.15; WT=17.2% vs. KO=52.0% for T24, p=0.07; WT=10.4% vs. KO=13.9% for 5637, p=0.59). Conclusions: To explore the increased frequency of ARID1A alterations in metastatic BC compared to non-invasive tumors, we developed a model of ARID1A-deficient BC using CRISPR-Cas9. We compared the relative invasiveness of the lines using a scratch assay. All lines trended towards a more invasive phenotype following ARID1A KO, which was especially pronounced in T24. Taken together, these data suggest that ARID1A deficiency in BC may prime tumors for a more invasive phenotype, which may help to explain the higher frequency of ARID1A alterations in higher stage and metastatic samples.

The ncBAF Complex Regulates Transcription in AML Through H3K27ac Sensing by BRD9.

The bromodomain-containing protein BRD9 is essential for AML cell viability, but it is unclear whether this requirement is due to the protein's role as an epigenetic reader. We inhibited this activity and identified altered gene-distal chromatin regulation and transcription consistent with a more mature myeloid cell state.

Synovial sarcoma X breakpoint 1 protein uses a cryptic groove to selectively recognize H2AK119Ub nucleosomes.

The cancer-specific fusion oncoprotein SS18-SSX1 disturbs chromatin accessibility by hijacking the BAF complex from the promoters and enhancers to the Polycomb-repressed chromatin regions. This process relies on the selective recognition of H2AK119Ub nucleosomes by synovial sarcoma X breakpoint 1 (SSX1). However, the mechanism underlying the selective recognition of H2AK119Ub nucleosomes by SSX1 in the absence of ubiquitin (Ub)-binding capacity remains unknown. Here we report the cryo-EM structure of SSX1 bound to H2AK119Ub nucleosomes at 3.1-Å resolution. Combined in vitro biochemical and cellular assays revealed that the Ub recognition by SSX1 is unique and depends on a cryptic basic groove formed by H3 and the Ub motif on the H2AK119 site. Moreover, this unorthodox binding mode of SSX1 induces DNA unwrapping at the entry/exit sites. Together, our results describe a unique mode of site-specific ubiquitinated nucleosome recognition that underlies the specific hijacking of the BAF complex to Polycomb regions by SS18-SSX1 in synovial sarcoma.

BAF45D-binding to HOX genes was differentially targeted in H9-derived spinal cord neural stem cells

Chromatin accessibility has been used to define how cells adopt region-specific neural fates. BAF45D is one of the subunits of a specialised chromatin remodelling BAF complex. It has been reported that BAF45D is expressed in spinal cord neural stem cells (NSCs) and regulates their fate specification. Within the developing vertebrate spinal cord, HOX genes exhibit spatially restricted expression patterns. However, the chromatin accessibility of BAF45D binding HOX genes in spinal cord NSCs is unclear. In the present study, we found that in H9-derived spinal cord NSCs, BAF45D targets TBX6, a gene that regulates spinal cord neural mesodermal progenitors. Furthermore, BAF45D binding to the NES gene is much more enriched in H9-derived spinal cord NSCs chromatin compared to ESCs chromatin. In addition, BAF45D binding to anterior and trunk/central HOX genes, but not to lumbosacral HOX genes, was much more enriched in NSCs chromatin compared to ESCs chromatin. These results may shed new light on the role of BAF45D in regulating region-specific spinal cord NSCs by targeting HOX genes.

Abstract A056: Treatment with dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 ablates tumor-associated androgen response elements (AREs) in prostate cancer

Abstract The Androgen Receptor (AR) cistrome is critical in the development of prostate cell identity while its misregulation promotes prostate cancer development. The pioneer transcription factor Forkhead box A1 (FOXA1) has been shown to be essential for the recruitment of AR to androgen response elements (AREs) allowing for reprogramming of the AR cistrome resulting in prostate cell transformation. FHD-286 is a BRM/BRG1 dual ATPase inhibitor currently in clinical trials for AML. Here, we show that treatment of prostate cancer cell lines with FHD-286 results in ablation of FOXA1 mediated AREs at tumor-associated AR binding sites (T-ARBS). Dual ATPase treatment subsequently reduces the expression level of various oncogenic AR target genes, leading to a decrease in tumor cell viability. Both patient derived organoid and In vivo studies provide further validation by showing a decrease in tumor growth. Strikingly, inhibition of BAF complex activity bypasses AR resistance mechanisms commonly seen after castration and Enzalutamide treatment as cell lines containing AR-V7 splice variants and neuroendocrine organoids display sensitivity. Taken together, our data illustrates a novel mechanism of treating AR mediated prostate cancer though the inhibition tumor associated AREs by treatment with FHD-286. Citation Format: Gabriel J Sandoval, Richard C Centore, Sal Topal, Dave Lahr, GiNell Elliott, Heena Gandevia, Ammar Adam, Jessica Piel, Holly M Nguyen, Eva Corey, Yu Chen, Steven Bellon, Murphy Hentemann. Treatment with dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 ablates tumor-associated androgen response elements (AREs) in prostate cancer [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 A056.

SWI/SNF Chromatin Remodeling Complex Orchestrates Sequential Binding of Key Transcription Factors in B Cells and Restricts Aggressive Lymphoma

Mutations in the SWI/SNF chromatin remodeling complex BAF are a recurring feature in many cancers, with a high prevalence in follicular lymphoma (FL; >19%) and diffuse large B-cell lymphoma (DLBCL; >34%). Despite this, the mechanism that links mutations in ARID1A, most frequently mutated BAF subunit, to the development of lymphoma is still not understood. To this aim, we crossed Arid1a-floxed mice with Cγ1-Cre mice to yield offspring with a conditional (cKO) Arid1a deletion in germinal center (GC) B cells, FL and DLBCL cell-of-origin. We immunized WT/WT ( Arid1a+/+; Cγ1Cre/+) and cKO/WT ( Arid1a+/-; Cγ1Cre/+) mice with sheep red blood cells and analyzed 10 days later at the peak of the GC reaction. Upon Arid1a deletion, we found a decrease in the total number of GC B cells relative to total B cells (WT/WT vs. cKO/WT; p<0.001). However, we observed skewing of GC polarity manifesting as increased proportions of proliferating centroblasts (CB) vs. centrocytes (CC) (WT/WT vs. cKO/WT; p=0.004). These observations imply a substantial dysfunction at the CC stage, which could account for the overall reduced number of GC B-cells. To investigate this, we performed a detailed molecular characterization of these cells. We performed the ATAC-seq assay on sorted CB and CC from our mouse model and observed an extensive loss of chromatin accessibility. Changes in chromatin accessibility are thought to be dependent on the recruitment of the BAF complex by specific transcription factors. We discovered strong enrichment for DNA motifs of PU.1 and NF-kB factors in closing chromatin (p-value <0.001), indicating their dependence on the BAF complex. Furthermore, we noted that this chromatin closing occurred in proximity to canonical GC exit programs, including genes induced by the CD40, NF-kB signaling, IRF4, STAT3, IL2, IL4, IL6 and Notch pathways (hypergeometric p-value <0.001), indicating GC-exit perturbation. Expanding upon our findings, we leveraged a combined single-cell RNA and ATAC assay (Multiome) to scrutinize subtle shifts within GC populations. Determining the chromatin accessibility within the pseudo-time trajectory of GC transitions, we found that PU.1 and NF-kB factors are chronologically dependent on ARID1A with PU.1 accessibility initially compromised, followed by a subsequent decrease at binding sites for NF-kB factors. This sequence reveals ARID1A's unrecognized function as a regulator of temporal dynamics of these key transcription factors. Strikingly, our Multiome data revealed expansion of the pre-memory B cells accompanied by a decrease in the proportion of pre-plasma cells upon Arid1a deletion (p-value <0.001), suggesting that Arid1a loss favors GC exit towards the memory cell fate. To validate our findings, we performed immunophenotyping and observed an increase in memory B cells in cKO/WT mice (p=0.019), and a decrease in long-lived plasma cells (p=0.012) upon NP-KLH immunization. Furthermore, we observed that the absence of ARID1A tilts GC cell-fate towards immature IgM+CD80-PDL2- memory B cells (p=0.0016), known for their potential to re-enter new GCs. Linking this critical role of ARID1A in chromatin regulation to lymphomagenesis, we observed a reduction in overall survival for mice carrying VavP-Bcl2; Arid1a+/- allele (compared to VavP-Bcl2; Arid1a+/+; p=0.0087). Remarkably, we further show that FL patients with ARID1A-inactivating mutations display an immature memory B-cell-like state with increased transformation risk to aggressive disease (p=0.0391). These observations offer mechanistic understanding into the emergence of both indolent and aggressive lymphomas in ARID1A-mutant patients through formation of immature memory-like clonal precursors.

Notable Efficacy of Co-Treatment with FHD-286, a Dual BRG1/BRM ATP-Ase Inhibitor, and Menin or BET Inhibitor, Decitabine or Venetoclax Against AML with MLL-r or Mutant NPM1

BRG1 (SMARCA4) and BRM (SMARCA2) are the core ATPase within the multi-protein, ATP-dependent, chromatin remodeling BAF complexes that regulate gene transcription. FHD-286 is a potent, selective, orally administered, inhibitor of BRG1/BRM in early clinical development as a therapy for AML. In present studies, we determined that exposure to FHD-286 (10 to 100 nM) for 4 to 7 days induced CD11b and morphologic features of differentiation and loss of viability in AML cell lines (MOLM13, MV4-11 and OCI-AML3) and patient-derived (PD) AML cells with MLL1r or mtNPM1. Notably, FHD-286 also induced significant lethality in Menin inhibitor (MI) resistant AML cell lines and PD AML cells lacking Menin mutations. ChIP-Seq analysis showed that exposure to FHD-286 significantly reduced the occupancy of BRG1 and H3K27Ac on the chromatin and reduced the core transcriptional regulatory circuitry in MOLM13 cells. FHD-286 treatment concordantly reduced genome-wide ATAC-Seq and RNA-Seq peaks, with negative enrichment at the gene-sets of MYC and E2F targets, mTORC1 signaling, translation initiation/elongation and DNA replication. scRNA-Seq analysis in PD AML bone marrow cells with MLL1r or mtNPM1 showed significant decline in the myeloid/erythroid progenitor (MEP) cells. Mass spectrometry conducted on FHD-286-treated vs control cells revealed a significant decline in the protein levels of c-Myc and its targets, as well as of PLK1, BCL2, PU.1 and CDK4, but exhibited increased levels of HEXIM1, CDKN1A/1B, CEBPα and CD11b. Moreover, CyTOF analysis of PD AML cells with MLL1r or mtNPM1 showed significant decline in the protein levels of c-Myc, PU.1, BRG1, MEF2C, PBX3, BCL2 and MCL1, but exhibited increased levels of cleaved PARP and Caspase-3 in the AML stem/progenitor cells defined by high expression of CLEC12A, CD123, CD99 and CD33 but low expression of CD11b. A domain-specific CRISPR screen targeting epigenetic regulators revealed significant co-enrichments with FHD-286 treatment of the gRNAs against BRD4 and LSD1, suggesting them as the epigenetic mechanisms of FHD-286 resistance. Consistent with findings noted above, co-treatment with FHD-286 and venetoclax, BET inhibitor (OTX015), decitabine or MI (SNDX-50469) exerted synergistic lethality in AML cell lines (MOLM13, MV4-11 and OCI-AML3) and PD AML cells with MLL1r or mtNPM1 (delta synergy scores between 10 to 33 by the ZIP method). In a PD xenograft (PDX) model of MLL1r AML with FLT3 mutation, monotherapy with FHD-286 (oral gavage) for 4 to 6-weeks was significantly effective in reducing AML burden and improving overall survival. Separately, in vivo FHD-286 treatment vs vehicle control, also significantly inhibited the AML-initiating potential, by reducing the AML burden and improving survival of the re-engrafted mice with the FHD-286-treated PDX cells. Moreover, co-treatment with FHD-286 and venetoclax or decitabine or OTX015, vs each drug alone, or vehicle control, significantly reduced the AML burden and improved survival, without significant toxicity in the PDX model. Finally, co-treatment with FHD-286 and OTX015 or SNDX-5613 (oral gavage) was significantly more effective than each drug alone in reducing the AML burden and overall survival of mice engrafted with a separate PDX model of AML cells with mtNPM1 and FLT3-ITD, without significant toxicity. These findings demonstrate the pre-clinical efficacy of FHD-286-based rational combinations and underscore their promise against AML with MLL1r or mtNPM1.

The BAF chromatin remodeling complex licenses planarian stem cells access to ectodermal and mesodermal cell fates

BackgroundThe flatworm planarian, Schmidtea mediterranea, has a large population of adult stem cells (ASCs) that replace any cell type during tissue turnover or regeneration. How planarian ASCs (called neoblasts) manage self-renewal with the ability to produce daughter cells of different cell lineages (multipotency) is not well understood. Chromatin remodeling complexes ultimately control access to DNA regions of chromosomes and together with specific transcription factors determine whether a gene is transcribed in a given cell type. Previous work in planarians determined that RNAi of core components of the BAF chromatin remodeling complex, brg1 and smarcc2, caused increased ASCs and failed regeneration, but how these cellular defects arise at the level of gene regulation in neoblasts is unknown.ResultsHere, we perform ATAC and RNA sequencing on purified neoblasts, deficient for the BAF complex subunits brg-1 and smarcc2. The data demonstrate that the BAF complex promotes chromatin accessibility and facilitates transcription at target loci, as in other systems. Interestingly, we find that the BAF complex enables access to genes known to be required for the generation of mesoderm- and ectoderm-derived lineages, including muscle, parenchymal cathepsin, neural, and epithelial lineages. BAF complex knockdowns result in disrupted differentiation into these cell lineages and functional consequences on planarian regeneration and tissue turnover. Notably, we did not detect a role for the BAF complex in neoblasts making endodermal lineages.ConclusionsOur study provides functional insights into how the BAF complex contributes to cell fate decisions in planarian ASCs in vivo.

The BAF chromatin complex component SMARCC1 does not mediate GLI transcriptional repression of Hedgehog target genes in limb buds

Transcriptional responses to the Hedgehog (HH) signaling pathway are primarily modulated by GLI repression in the mouse limb. Previous studies suggested a role for the BAF chromatin remodeling complex in mediating GLI repression. Consistent with this possibility, the core BAF complex protein SMARCC1 is present at most active limb enhancers including the majority of GLI enhancers. However, in contrast to GLI repression which reduces chromatin accessibility, SMARCC1 maintains chromatin accessibility at most enhancers, including those bound by GLI. Moreover, SMARCC1 binding at GLI-regulated enhancers occurs independently of GLI3. Consistent with previous studies, some individual GLI target genes are mis-regulated in Smarcc1 conditional knockouts, though most GLI target genes are unaffected. Moreover, SMARCC1 is not necessary for mediating constitutive GLI repression in HH mutant limb buds. We conclude that SMARCC1 does not mediate GLI3 repression, which we propose utilizes alternative chromatin remodeling complexes.

Essential role of the Pax5 C-terminal domain in controlling B cell commitment and development.

The B cell regulator Pax5 consists of multiple domains whose function we analyzed in vivo by deletion in Pax5. While B lymphopoiesis was minimally affected in mice with homozygous deletion of the octapeptide or partial homeodomain, both sequences were required for optimal B cell development. Deletion of the C-terminal regulatory domain 1 (CRD1) interfered with B cell development, while elimination of CRD2 modestly affected B-lymphopoiesis. Deletion of CRD1 and CRD2 arrested B cell development at an uncommitted pro-B cell stage. Most Pax5-regulated genes required CRD1 or both CRD1 and CRD2 for their activation or repression as these domains induced or eliminated open chromatin at Pax5-activated or Pax5-repressed genes, respectively. Co-immunoprecipitation experiments demonstrated that the activating function of CRD1 is mediated through interaction with the chromatin-remodeling BAF, H3K4-methylating Set1A-COMPASS, and H4K16-acetylating NSL complexes, while its repressing function depends on recruitment of the Sin3-HDAC and MiDAC complexes. These data provide novel molecular insight into how different Pax5 domains regulate gene expression to promote B cell commitment and development.

GNE-235: A Lead Compound Selective for the Second Bromodomain of PBRM1.

Bromodomains are acetyl-lysine binding modules that are found in different classes of chromatin-interacting proteins. Among these are large chromatin remodeling complexes such as BAF and PBAF (variants of human SWI/SNF). Previous work has identified chemical probes targeting a subset of the bromodomains present in the BAF and PBAF complexes. Selective inhibitors of the individual bromodomains have proven challenging to discover, as the domains are highly similar. Here, elaboration of an aminopyridazine scaffold used previously to develop probes for the bromodomains of SMARCA2, SMARCA4, and the fifth bromodomain of PBRM1 yielded compounds with both potency and unusual selectivity for the second bromodomain of PBRM1. One of these, GNE-235, and its enantiomer control GNE-234 are suggested for initial cellular investigations of the function of the second bromodomain of PBRM1.

Comprehensive synergy mapping links a BAF- and NSL-containing “supercomplex” to the transcriptional silencing of HIV-1

Host repressors mediate HIV latency, but how they interactively silence the virus remains unclear. Here, we develop "reiterative enrichment and authentication of CRISPRi targets for synergies (REACTS)" to probe the genome for synergies between HIV transcription repressors. Using eight known host repressors as queries, we identify 32 synergies involving eleven repressors, including BCL7C, KANSL2, and SIRT2. Overexpression of these three proteins reduces HIV reactivation in Jurkat Tcells and in CD4 Tcells from people living with HIV on antiretroviral therapy (ART). We show that the BCL7C-containing BAF complex and the KANSL2-containing NSL complex form a "supercomplex" that increases inhibitory histone acetylation of the HIV long-terminalrepeat (LTR) and its occupancy by the short variant of the acetyl-lysine reader Brd4. Collectively, we provide a validated platform for defining gene synergies genome wide, and the BAF-NSL "supercomplex" represents a potential target for overcoming HIV rebound after ART cessation.

Elucidating the clinical and molecular spectrum of SMARCC2-associated NDD in a cohort of 65 affected individuals

PurposeCoffin-Siris and Nicolaides-Baraitser syndromes are recognizable neurodevelopmental disorders caused by germline variants in BAF complex subunits. The SMARCC2 BAFopathy was recently reported. Herein, we present clinical and molecular data on a large cohort. MethodsClinical symptoms for 41 novel and 24 previously published affected individuals were analyzed using the Human Phenotype Ontology. For genotype-phenotype correlations, molecular data were standardized and grouped into non-truncating and likely gene-disrupting (LGD) variants. Missense variant protein expression and BAF-subunit interactions were examined using 3D protein modeling, co-immunoprecipitation, and proximity-ligation assays. ResultsNeurodevelopmental delay with intellectual disability, muscular hypotonia, and behavioral disorders were the major manifestations. Clinical hallmarks of BAFopathies were rare. Clinical presentation differed significantly, with LGD variants being predominantly inherited and associated with mildly reduced or normal cognitive development, whereas non-truncating variants were mostly de novo and presented with severe developmental delay. These distinct manifestations and non-truncating variant clustering in functional domains suggest different pathomechanisms. In vitro testing showed decreased protein expression for N-terminal missense variants similar to LGD. ConclusionThis study improved SMARCC2 variant classification and identified discernible SMARCC2-associated phenotypes for LGD and non-truncating variants, which were distinct from other BAFopathies. The pathomechanism of most non-truncating variants has yet to be investigated.

Different SWI/SNF complexes coordinately promote R-loop- and RAD52-dependent transcription-coupled homologous recombination.

The SWI/SNF family of ATP-dependent chromatin remodeling complexes is implicated in multiple DNA damage response mechanisms and frequently mutated in cancer. The BAF, PBAF and ncBAF complexes are three major types of SWI/SNF complexes that are functionally distinguished by their exclusive subunits. Accumulating evidence suggests that double-strand breaks (DSBs) in transcriptionally active DNA are preferentially repaired by a dedicated homologous recombination pathway. We show that different BAF, PBAF and ncBAF subunits promote homologous recombination and are rapidly recruited to DSBs in a transcription-dependent manner. The PBAF and ncBAF complexes promote RNA polymerase II eviction near DNA damage to rapidly initiate transcriptional silencing, while the BAF complex helps to maintain this transcriptional silencing. Furthermore, ARID1A-containing BAF complexes promote RNaseH1 and RAD52 recruitment to facilitate R-loop resolution and DNA repair. Our results highlight how multiple SWI/SNF complexes perform different functions to enable DNA repair in the context of actively transcribed genes.

DIPG-19. BAF COMPLEX PERTURBATION AS A NOVEL THERAPEUTIC OPPORTUNITY IN H3K27M PEDIATRIC GLIOMA

Abstract Epigenetic dysregulation resulting in stalled development plays a crucial role in pediatric cancer tumorigenesis. Diffuse midline gliomas (DMG) are universally fatal pediatric brain cancers refractory to standard of care treatment modalities. These malignancies are driven by heterozygous mutations in genes encoding histone 3 (H3K27M) which create an aberrant epigenetic landscape that keeps glioma cells in an undifferentiated stem-like state. Consequently, targeting epigenetic regulators to restore the epigenome and force glioma cells to exit this stem-like cell state represents a promising new therapeutic strategy for H3K27M-DMG. To interrogate for epigenetic dependencies, we performed a CRISPR/Cas9 inactivation screen in patient-derived H3K27M-DMG neurospheres using an epigenetically focused sgRNA library and identified several core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex as genetic vulnerabilities. Validation assays revealed that knockout of the BAF catalytic subunit BRG1 results in decreased glioma cell proliferation and tumor growth in orthotopic mouse models. Mechanistically, genome wide localization and DNA accessibility studies combined with regulatory network analysis demonstrated that BRG1 controls the transcription factor and enhancer landscapes that maintain H3K27M-DMG cells in a cycling, oligodendrocyte precursor cell-like state. Single cell transcriptome analysis in vitro and immunofluorescence studies in vivo confirmed that genetic perturbation of this chromatin remodeler promotes progression of differentiation along the astrocytic lineage. Similarly, pharmacological suppression of BRG1 activity, using both catalytic inhibitors as well as recently developed degraders, opposes tumor cell proliferation, stimulates cell state transition, and improves overall survival of patient-derived xenograft models. Interestingly, these effects seem to be restricted to H3K27M mutant glioma, as H3 wildtype glioma cells were less sensitive to BRG1 inhibition both in vitro and in vivo. In summary, we demonstrate that the BAF complex contributes to the maintenance of glioma cells in a proliferative stem-like state and that its therapeutic inhibition has translational potential for children bearing H3K27M-DMG.

Selected Articles from This Issue

Highlights| June 01 2023 Selected Articles from This Issue Author & Article Information Online ISSN: 1557-3125 Print ISSN: 1541-7786 ©2023 American Association for Cancer Research2023American Association for Cancer Research Mol Cancer Res (2023) 21 (6): 495. https://doi.org/10.1158/1541-7786.MCR-21-6-HI Related Content A commentary has been published: Interdependence of SS18-SSX–driven YAP1 and β-Catenin Activation in Synovial Sarcoma A commentary has been published: USP10 Regulates ZEB1 Ubiquitination and Protein Stability to Inhibit ZEB1-Mediated Colorectal Cancer Metastasis A commentary has been published: UBE2C-mediated Autophagy Inhibition via Ubiquitination of SIRT1 Contributes to Endometrial Cancer Progression View more A commentary has been published: Targeting Unc51-like Autophagy Activating Kinase 1 (ULK1) Overcomes Adaptive Drug Resistance in Acute Myelogenous Leukemia View less Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record June 1 2023 Citation Selected Articles from This Issue. Mol Cancer Res 1 June 2023; 21 (6): 495. https://doi.org/10.1158/1541-7786.MCR-21-6-HI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Synovial sarcoma arises from a reciprocal t(X;18)(p11;q11) translocation and resulting SS18-SSX fusion protein, which integrates into the BAF chromatin remodeling complex and dysregulates global transcription. While YAP and β-catenin are known SS18-SSX targets, whether YAP/β-catenin crosstalk occurs is not known. Using YAP and β-catenin genetic depletion and pharmacologic inhibition alongside reporters for each transcriptional coactivator in synovial sarcoma cell lines, Isfort and colleagues found that YAP and β-catenin mutually enhance each other's activity. Furthermore, the authors demonstrated that YAP/β-catenin coactivation occurs exclusively in SS18-SSX–expressing synovial sarcoma cell lines, and that abrogating SS18-SSX expression using siRNA eliminates YAP/β-catenin crosstalk. siRNA-mediated BAF complex component ablation also inhibits YAP/β-catenin coactivation, demonstrating that the SS18-SSX–containing BAF complex is required for YAP/β-catenin crosstalk. In sum, this study presents YAP/β-catenin coactivation as a SS18-SSX–driven transcriptional phenomenon that may represent a unique therapeutic vulnerability in synovial sarcoma. While targeted therapies provide efficacy against acute myelogenous leukemia (AML),... You do not currently have access to this content.

Canonical BAF complex activity shapes the enhancer landscape that licenses CD8+ T cell effector and memory fates

CD8+ Tcells provide host protection against pathogens by differentiating into distinct effector and memory cell subsets, but how chromatin is site-specifically remodeled during their differentiation is unclear. Due to its critical role in regulating chromatin and enhancer accessibility through its nucleosome remodeling activities, we investigated the role of the canonical BAF (cBAF) chromatin remodeling complex in antiviral CD8+ Tcells during infection. ARID1A, a subunit of cBAF, was recruited early after activation and established de novo open chromatin regions (OCRs) at enhancers. Arid1a deficiency impaired the opening of thousands of activation-induced enhancers, leading to loss of TF binding, dysregulated proliferation and gene expression, and failure to undergo terminal effector differentiation. Although Arid1a was dispensable for circulating memory cell formation, tissue-resident memory (Trm) formation was strongly impaired. Thus, cBAF governs the enhancer landscape of activated CD8+ Tcells that orchestrates TF recruitment and activity and the acquisition of specific effector and memory differentiation states.