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Lung cancer is the most common cause of death from cancer worldwide, and lung adenocarcinoma (LUAD) is the most common subtype of lung cancer. NKX2-1 (also known as TTF-1, TITF-1) is a lineage defining transcription factor throughout normal lung development. In LUAD, NKX2-1 is a highly specific and sensitive marker for 85-90% of both primary and metastatic LUADs. We and others identified NKX2-1 as the most significantly amplified gene in LUAD. NKX2-1 is amplified as the earliest stages of LUAD development, and NKX2-1 amplification is a truncal event in multi-region LUAD evolution. Despite strong evidence for an oncogenic role for NKX2-1, little is known about the mechanisms of NKX2-1 activation, or how its oncogenic regulation drives LUAD. Here, we identify recurrent focal amplification targeting a super-enhancer (SE) of NKX2-1 as a driving event in LUAD. Using epigenomic data from LUAD cell lines and primary samples, we identify this region as a lineage super-enhancer that is specifically active in NKX2-1(+) cell lines and patients. Notably, this region is co-amplified with NKX2-1 in 95.9% (143/149) of NKX2-1-amplified samples, suggesting this region is a hallmark of NKX2-1 activation, through focal or co-amplification with NKX2-1. Using endogenous ChIP-seq and exogenous luciferase assays, we show the enhancer activity of the NKX2-1 SE is comprised of three constituent enhancers, and map the transcriptional activity of the strongest enhancer to individual binding motifs for AP-1 and ETS. Using CRISPR inhibition (CRISPRi) and CRISPR activation (CRISPRa) in LUAD cell lines expressing high or low levels of NKX2-1, we show that activity of the NKX2-1 SE defines endogenous NKX2-1 expression. Using genome-wide shRNA and CRISPR screens, we identify a NKX2-1 dependency in NKX2-1 positive LUAD cell lines, and validate this dependency in clonogenic and proliferation assays. Using RNA sequencing (RNA-seq), we find that NKX2-1 knockdown activates an epithelial-mesenchymal transition (EMT) gene signature and downregulates an alveolar differentiation signature, including critical markers of LUAD differentiation, such as NAPSA, LMO3, and SLC34A2. Using a global clustering approach to CCLE RNA-seq of LUAD cell lines, we find that NKX2-1(+) LUAD cell lines cluster apart from NKX2-1(–), and that NKX2-1 knockdown regulates the majority (57/97) of genes that distinguish NKX2-1(+) LUADs. Similarly, we find that global clustering of H3K27ac ChIP-seq in LUAD cell lines separates NKX2-1(+) cells, and identify enhancers that are uniquely active NKX2-1(+) LUAD cell lines. We find these enhancers are bound and activated by NKX2-1, regulating genes we identified above such as NAPSA and LMO3. Our data demonstrates that enhancer amplification is a hallmark of oncogenic NKX2-1 activation in LUAD, through which NKX2-1 drives a lineage addicted state and oncogenic cell proliferation. This suggests that NKX2-1 is a critical defining oncogene for LUAD, and that targeting of NKX2-1 or its enhancer may suppress LUAD. Citation Format: John L. Pulice, Matthew Meyerson. Enhancer amplification defines lineage addiction in human lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A003.

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, "hyper-pluripotent" state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically, DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity, P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer, heterochromatin, and DNA methylation landscapes of undifferentiated cell types. Collectively, our data establish a link between P-body homeostasis, chromatin organization, and stem cell potency.

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.

Synovial sarcoma (SS) is defined by the hallmark SS18-SSX fusion oncoprotein, which renders BAF complexes aberrant in two manners: gain of SSX to the SS18 subunit and concomitant loss of BAF47 subunit assembly. Here we demonstrate that SS18-SSX globally hijacks BAF complexes on chromatin to activate an SS transcriptional signature that we define using primary tumors and cell lines. Specifically, SS18-SSX retargets BAF complexes from enhancers to broad polycomb domains to oppose PRC2-mediated repression and activate bivalent genes. Upon suppression of SS18-SSX, reassembly of BAF47 restores enhancer activation, but is not required for proliferative arrest. These results establish a global hijacking mechanism for SS18-SSX on chromatin, and define the distinct contributions of two concurrent BAF complex perturbations.

Mammalian SWI/SNF (BAF) chromatin remodeling complexes play critical roles in maintaining chromatin architecture and gene expression. Genomic sequencing efforts over the past several years have unveiled a major role for these complexes in the development of human cancer as well as neurologic disease, prompting the need to interrogate underlying mechanisms and to develop new methods to comprehensively understand mSWI/SNF complex function. Here we discuss the emerging insights from genetic, biochemical, and functional genomic studies in the field and suggest approaches toward further basic investigations, as well as therapeutic targeting of chromatin remodeling machinery.

The opposition between Polycomb repressive complexes (PRCs) and BAF (mSWI/SNF) complexes has a critical role in both development and disease. Mutations in the genes encoding BAF subunits contribute to more than 20% of human malignancies, yet the underlying mechanisms remain unclear, owing largely to a lack of assays to assess BAF function in living cells. To address this, we have developed a widely applicable recruitment assay system through which we find that BAF opposes PRC by rapid, ATP-dependent eviction, leading to the formation of accessible chromatin. The reversal of this process results in reassembly of facultative heterochromatin. Surprisingly, BAF-mediated PRC eviction occurs in the absence of RNA polymerase II (Pol II) occupancy, transcription, and replication. Further, we find that tumor-suppressor and oncogenic mutant BAF complexes have different effects on PRC eviction. The results of these studies define a mechanistic sequence underlying the resolution and formation of facultative heterochromatin, and they demonstrate that BAF opposes PRC on a minute-by-minute basis to provide epigenetic plasticity.

Perturbations to mammalian SWI/SNF (mSWI/SNF or BAF) complexes contribute to more than 20% of human cancers, with driving roles first identified in malignant rhabdoid tumor, an aggressive pediatric cancer characterized by biallelic inactivation of the core BAF complex subunit SMARCB1 (BAF47). However, the mechanism by which this alteration contributes to tumorigenesis remains poorly understood. We find that BAF47 loss destabilizes BAF complexes on chromatin, absent significant changes in complex assembly or integrity. Rescue of BAF47 in BAF47-deficient sarcoma cell lines results in increased genome-wide BAF complex occupancy, facilitating widespread enhancer activation and opposition of Polycomb-mediated repression at bivalent promoters. We demonstrate differential regulation by two distinct mSWI/SNF assemblies, BAF and PBAF complexes, enhancers and promoters, respectively, suggesting that each complex has distinct functions that are perturbed upon BAF47 loss. Our results demonstrate collaborative mechanisms of mSWI/SNF-mediated gene activation, identifying functions that are co-opted or abated to drive human cancers and developmental disorders.

Synovial sarcoma (SS) is a soft-tissue malignancy driven by a recurrent chromosomal translocation (t(X;18)) that uniformly produces the SS18-SSX oncogenic fusion protein. SS18 is a core subunit of the mammalian SWI/SNF (BAF) complexes, which remodel nucleosomes in an ATP-dependent manner and antagonistically oppose gene-silencing activity of polycomb-repressive complexes to maintain transcriptional control throughout development and differentiation. We previously discovered that in SS, incorporation of the oncogenic SS18-SSX fusion into BAF complexes leads to eviction of the tumor-suppressor BAF47 (INI1/SMARCB1) subunit, and aberrant activation of polycomb target genes by displacement of H3K27me3-mediated repression. However, uncoupling the oncogenic consequences of two co-occurrent BAF complex perturbations, gain of 78- amino acids of SSX to SS18 and loss of BAF47, has remained a challenge for the field. To identify effective targeted therapeutics for this patient population, it is critical that we understand the contribution of the gain- versus loss-of-function properties of these molecular events in this malignancy. Here we demonstrate that the SSX 78aa tail engages mononucleosomes and targeted, quantitative mass spectrometry proteomics reveals preferential engagement to nucleosomes decorated with histone modifications associated with transcriptional repression. Using biochemical affinity assays, we find that SSX dramatically increases the affinity of SS18-SSX-containing BAF complexes for chromatin, thereby decreasing the dynamic mobility of BAF complexes. Furthermore, we show that SS18-SSX-containing BAF complexes possess a broader genomic footprint and exhibit distinct chromatin localization in that expression of SS18-SSX drives a near complete retargeting of BAF complexes genome-wide. SS18-SSX directs BAF complexes to polycomb-repressed sites to activate embryonic development and neuronal gene pathways hallmark to SS primary tumors. This targeting by SSX results in a transcriptional signature markedly distinct from sarcomas such as malignant rhabdoid tumors, which are driven solely by biallelic loss of BAF47. Moreover, using CRISPR/Cas9-mediated KO of BAF47 in SS cell lines, we show that the proliferative arrest of SS cell lines upon suppression of SS18-SSX is independent of BAF47 reassembly into BAF complexes, thereby demonstrating that SSX targeting of BAF complexes drives oncogenesis in a manner distinct from BAF47 loss. Taken together, these studies uncover a novel functionality of the SSX tail that is required for SS oncogenesis, and inform the selection of appropriate targeted therapeutic agents for this gain-of-function BAF complex-driven cancer. This abstract is also being presented as Poster B25. Citation Format: Matthew J. McBride, John L. Pulice, Robert T. Nakayama, Nazar Mashtalir, Davis R. Ingram, Jacob D. Jaffe, Jack F. Shern, Javed Khan, Jason L. Hornick, Alexander J. Lazar, Cigall Kadoch. SSX-mediated chromatin engagement and targeting of BAF complexes activates oncogenic transcription in synovial sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR11.

Synovial sarcoma (SS) is a soft-tissue malignancy driven by a recurrent chromosomal translocation (t(X;18)) that uniformly produces the SS18-SSX oncogenic fusion protein. SS18 is a core subunit of the mammalian SWI/SNF (BAF) complexes, which remodel nucleosomes in an ATP-dependent manner and antagonistically oppose gene-silencing activity of polycomb complexes to maintain transcriptional control throughout development and differentiation. We previously discovered that in SS, incorporation of the oncogenic SS18-SSX fusion into BAF complexes leads to eviction of the tumor-suppressor BAF47 (INI1/SMARCB1) subunit, and aberrant activation of polycomb target genes by displacement of H3K27me3-mediated repression. However, uncoupling the oncogenic consequences of two co-occurrent BAF complex perturbations, gain of 78- amino acids to SS18 and loss of BAF47 has remained a challenge for the field; understanding gain- versus loss-of-function properties of these molecular events is critical to the identification of effective targeted therapeutics for this patient population. Here we show that synovial sarcoma primary tumors and cell lines harbor a transcriptional signature markedly distinct from sarcomas such as malignant rhabdoid tumors, which are driven solely by biallelic loss of BAF47. Indeed, we show that SS18-SSX-containing BAF complexes exhibit distinct chromatin localization in that suppression of SS18-SSX results in a near complete genome-wide retargeting of BAF complexes. We find that SS18-SSX directs BAF complexes to polycomb-repressed sites to activate embryonic development and neuronal gene pathways hallmark to SS primary tumors. Strikingly, using biochemical affinity assays, we demonstrate that the SSX 78aa tail dramatically increases the affinity of BAF complexes for chromatin as well as their genomic footprint sizes upon ChIP-seq analyses. This is in stark contrast to the demonstrated decrease in chromatin affinity and genomic occupancy resulting from the loss of function of BAF47 in MRT. Moreover, using CRISPR/Cas9-mediated KO of BAF47 in SS cell lines, we show that the proliferative arrest of SS cell lines upon suppression of SS18-SSX is independent of the BAF47 reassembly into BAF complexes, thereby demonstrating a unique SSX-driven oncogenic mechanism distinct from BAF47 loss. Taken together, these studies uncover a novel functionality of the SSX tail that is required for SS oncogenesis, and inform the selection of appropriate targeted therapeutic agents for this gain-of-function BAF complex-driven cancer. Citation Format: Matthew J. McBride, John L. Pulice, Robert T. Nakayama, Nazar Mashtalir, Davis R. Ingram, Jack F. Shern, Javed Khan, Jason L. Hornick, Alexander J. Lazar, Cigall Kadoch. SSX drives gain-of-function BAF complex chromatin affinity and genomic targeting in synovial sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3875. doi:10.1158/1538-7445.AM2017-3875

Prostate cancer remains one of the leading causes of cancer-related death in men. Chromosomal rearrangements resulting in the fusion of the androgen regulated gene TMPRSS2 and the ETS-family transcription factor ERG occur in over 50% of all prostate cancer cases. Recent studies enabled by genome-wide methodologies have implicated altered epigenomic landscapes and changes in DNA accessibility as major contributors to ERG-driven oncogenesis, however the precise mechanism underlying the ERG transcriptional signature has to date remained unclear. Here we performed the first endogenous purification and SILAC-mass spectrometric analysis of ERG in TMPRSS2-ERG prostate cancer cells. Remarkably, we demonstrate that ERG directly interacts with the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which was recently shown to be mutated in >20% of human malignancies. ERG co-localizes with BAF complexes genome-wide, resulting in specific ERG-dependent BAF complex targeting to sites enriched in known ERG, FOXA1, and HOXB13 motifs; additionally, loss of ERG in TMPRSS2-ERG driven cell lines results in dramatic retargeting of BAF complexes away from ERG-dependent sites, to sites enriched in known AR and CTCF motifs. Importantly, ERG-driven BAF complex retargeting contributes to activation of TMPRSS2-ERG prostate cancer gene expression signatures. We map the ERG-BAF interaction to a specific region within the ERG amino acid sequence and find that this region is required to bind BAF complexes. These studies reveal a novel, unexpected mechanism of action of ERG-driven oncogenesis and offers new strategies for therapeutic intervention. Citation Format: Gabriel J. Sandoval, John L. Pulice, David Y. Takeda, Monica A. Schenone, Marius Pop, Gaylor Boulay, Miguel N. Rivera, Lucienne Ronco, William C. Hahn, Cigall Kadoch. TMPRSS2-ERG drives global mistargeting of mammalian SWI/SNF (BAF) complexes in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 882.