KRAS-mediated upregulation of CIP2A promotes suppression of PP2A-B56α to initiate pancreatic cancer development.

Oncogenic mutations in KRAS are present in approximately 95% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) and are considered the initiating event of pancreatic intraepithelial neoplasia (PanIN) precursor lesions. While it is well established that KRAS mutations drive the activation of oncogenic kinase cascades during pancreatic oncogenesis, the effects of oncogenic KRAS signaling on regulation of phosphatases during this process is not fully appreciated. Protein Phosphatase 2A (PP2A) has been implicated in suppressing KRAS-driven cellular transformation. However, low PP2A activity is observed in PDAC cells compared to non-transformed cells, suggesting that suppression of PP2A activity is an important step in the overall development of PDAC. In the current study, we demonstrate that KRASG12D induces the expression of both an endogenous inhibitor of PP2A activity, Cancerous Inhibitor of PP2A (CIP2A), and the PP2A substrate, c-MYC. Consistent with these findings, KRASG12D sequestered the specific PP2A subunit responsible for c-MYC degradation, B56α, away from the active PP2A holoenzyme in a CIP2A-dependent manner. During PDAC initiation in vivo, knockout of B56α promoted KRASG12D tumorigenesis by accelerating acinar-to-ductal metaplasia (ADM) and the formation of PanIN lesions. The process of ADM was attenuated ex vivo in response to pharmacological re-activation of PP2A utilizing direct small molecule activators of PP2A (SMAPs). Together, our results suggest that suppression of PP2A-B56α through KRAS signaling can promote the MYC-driven initiation of pancreatic tumorigenesis.

Collaboration between distinct SWI/SNF chromatin remodeling complexes directs enhancer selection and activation of macrophage inflammatory genes.

Macrophages elicit immune responses to pathogens through induction of inflammatory genes. Here, we examined the role of three variants of the SWI/SNF nucleosome remodeling complex-cBAF, ncBAF, and PBAF-in the macrophage response to bacterial endotoxin (lipid A). All three SWI/SNF variants were prebound in macrophages and retargeted to genomic sites undergoing changes in chromatin accessibility following stimulation. Cooperative binding of all three variants associated with de novo chromatin opening and latent enhancer activation. Isolated binding of ncBAF and PBAF, in contrast, associated with activation and repression of active enhancers, respectively. Chemical and genetic perturbations of variant-specific subunits revealed pathway-specific regulation in the activation of lipid A response genes, corresponding to requirement for cBAF and ncBAF in inflammatory and interferon-stimulated gene (ISG) activation, respectively, consistent with differential engagement of SWI/SNF variants by signal-responsive transcription factors. Thus, functional diversity among SWI/SNF variants enables increased regulatory control of innate immune transcriptional programs, with potential for specific therapeutic targeting.

ARID1A suppresses R-loop-mediated STING-type I interferon pathway activation of anti-tumor immunity.

Clinical trials have identified ARID1A mutations as enriched among patients who respond favorably to immune checkpoint blockade (ICB) in several solid tumor types independent of microsatellite instability. We show that ARID1A loss in murine models is sufficient to induce anti-tumor immune phenotypes observed in ARID1A mutant human cancers, including increased CD8+ T cell infiltration and cytolytic activity. ARID1A-deficient cancers upregulated an interferon (IFN) gene expression signature, the ARID1A-IFN signature, associated with increased R-loops and cytosolic single-stranded DNA (ssDNA). Overexpression of the R-loop resolving enzyme, RNASEH2B, or cytosolic DNase, TREX1, in ARID1A-deficient cells prevented cytosolic ssDNA accumulation and ARID1A-IFN gene upregulation. Further, the ARID1A-IFN signature and anti-tumor immunity were driven by STING-dependent type I IFN signaling, which was required for improved responsiveness of ARID1A mutant tumors to ICB treatment. These findings define a molecular mechanism underlying anti-tumor immunity in ARID1A mutant cancers.

Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression.

The polybromo, brahma-related gene 1-associated factors (PBAF) chromatin remodeling complex subunit polybromo-1 (PBRM1) contains six bromodomains that recognize and bind acetylated lysine residues on histone tails and other nuclear proteins. PBRM1 bromodomains thus provide a link between epigenetic posttranslational modifications and PBAF modulation of chromatin accessibility and transcription. As a putative tumor suppressor in several cancers, PBRM1 protein expression is often abrogated by truncations and deletions. However, ∼33% of PBRM1 mutations in cancer are missense and cluster within its bromodomains. Such mutations may generate full-length PBRM1 variant proteins with undetermined structural and functional characteristics. Here, we employed computational, biophysical, and cellular assays to interrogate the effects of PBRM1 bromodomain missense variants on bromodomain stability and function. Since mutations in the fourth bromodomain of PBRM1 (PBRM1-BD4) comprise nearly 20% of all cancer-associated PBRM1 missense mutations, we focused our analysis on PBRM1-BD4 missense protein variants. Selecting 16 potentially deleterious PBRM1-BD4 missense protein variants for further study based on high residue mutational frequency and/or conservation, we show that cancer-associated PBRM1-BD4 missense variants exhibit varied bromodomain stability and ability to bind acetylated histones. Our results demonstrate the effectiveness of identifying the unique impacts of individual PBRM1-BD4 missense variants on protein structure and function, based on affected residue location within the bromodomain. This knowledge provides a foundation for drawing correlations between specific cancer-associated PBRM1 missense variants and distinct alterations in PBRM1 function, informing future cancer personalized medicine approaches.

Identification of SWI/SNF Subcomplex GBAF Presence, Intra-Complex Interactions, and Transcriptional Dynamics during Early Porcine Development.

Understanding the complex interplay between genetics and environmental factors is vital for enhancing livestock production efficiency while safeguarding animal health. Despite extensive studies on production-specific genes in livestock, exploring how epigenetic mechanisms and heritable modifications govern animal growth and development remains an under-explored frontier with potential implications across all life stages. This study focuses on the GBAF chromatin remodeling complex and evaluates its presence during embryonic and fetal development in swine. Immunocytochemistry and co-immunoprecipitation techniques were employed to investigate the presence and interactions of GBAF subunits BRD9 and GLTSCR1 in porcine oocytes, preimplantation embryos, and cell lines, and transcriptional dynamics of GBAF subunits across these key developmental stages were analyzed using existing RNA-seq datasets. BRD9 and GLTSCR1 were identified across all represented stages, and an interaction between GLTSCR1 and BAF170 was shown in PTr2 and PFF cells. Our findings highlight the ubiquitous presence of GBAF in porcine early development and the potentially novel association between GLTSCR1 and BAF170 in swine. The transcriptional dynamics findings may suggest GBAF-specific contributions during key developmental events. This study contributes to the growing understanding of epigenetic regulators in both swine and mammalian development, emphasizing the implications of GBAF as a modulator of key developmental events.

Rational Design and Development of Selective BRD7 Bromodomain Inhibitors and Their Activity in Prostate Cancer.

Bromodomain-containing proteins are readers of acetylated lysine and play important roles in cancer. Bromodomain-containing protein 7 (BRD7) is implicated in multiple malignancies; however, there are no selective chemical probes to study its function in disease. Using crystal structures of BRD7 and BRD9 bromodomains (BDs) bound to BRD9-selective ligands, we identified a binding pocket exclusive to BRD7. We synthesized a series of ligands designed to occupy this binding region and identified two inhibitors with increased selectivity toward BRD7, 1-78 and 2-77, which bind with submicromolar affinity to the BRD7 BD. Our binding mode analyses indicate that these ligands occupy a uniquely accessible binding cleft in BRD7 and maintain key interactions with the asparagine and tyrosine residues critical for acetylated lysine binding. Finally, we validated the utility and selectivity of the compounds in cell-based models of prostate cancer.

PBRM1 bromodomains associate with RNA to facilitate chromatin association.

PBRM1 is a subunit of the PBAF chromatin remodeling complex, which is mutated in 40-50% of clear cell renal cell carcinoma patients. It is thought to largely function as a chromatin binding subunit of the PBAF complex, but the molecular mechanism underlying this activity is not fully known. PBRM1 contains six tandem bromodomains which are known to cooperate in binding of nucleosomes acetylated at histone H3 lysine 14 (H3K14ac). Here, we demonstrate that the second and fourth bromodomains from PBRM1 also bind nucleic acids, selectively associating with double stranded RNA elements. Disruption of the RNA binding pocket is found to compromise PBRM1 chromatin binding and inhibit PBRM1-mediated cellular growth effects.

Activity-assembled nBAF complex mediates rapid immediate early gene transcription by regulating RNA Polymerase II productive elongation.

Signal-dependent RNA Polymerase II (Pol2) productive elongation is an integral component of gene transcription, including those of immediate early genes (IEGs) induced by neuronal activity. However, it remains unclear how productively elongating Pol2 overcome nucleosomal barriers. Using RNAi, three degraders, and several small molecule inhibitors, we show that the mammalian SWI/SNF complex of neurons (neuronal BAF, or nBAF) is required for activity-induced transcription of neuronal IEGs, including Arc . The nBAF complex facilitates promoter-proximal Pol2 pausing, signal-dependent Pol2 recruitment (loading), and importantly, mediates productive elongation in the gene body via interaction with the elongation complex and elongation-competent Pol2. Mechanistically, Pol2 elongation is mediated by activity-induced nBAF assembly (especially, ARID1A recruitment) and its ATPase activity. Together, our data demonstrate that the nBAF complex regulates several aspects of Pol2 transcription and reveal mechanisms underlying activity-induced Pol2 elongation. These findings may offer insights into human maladies etiologically associated with mutational interdiction of BAF functions.

Selective and Cell-Active PBRM1 Bromodomain Inhibitors Discovered through NMR Fragment Screening.

PBRM1 is a subunit of the PBAF chromatin remodeling complex that uniquely contains six bromodomains. PBRM1 can operate as a tumor suppressor or tumor promoter. PBRM1 is a tumor promoter in prostate cancer, contributing to migratory and immunosuppressive phenotypes. Selective chemical probes targeting PBRM1 bromodomains are desired to elucidate the association between aberrant PBRM1 chromatin binding and cancer pathogenesis and the contributions of PBRM1 to immunotherapy. Previous PBRM1 inhibitors unselectively bind SMARCA2 and SMARCA4 bromodomains with nanomolar potency. We used our protein-detected NMR screening pipeline to screen 1968 fragments against the second PBRM1 bromodomain, identifying 17 hits with Kd values from 45 µM to >2 mM. Structure-activity relationship studies on the tightest-binding hit resulted in nanomolar inhibitors with selectivity for PBRM1 over SMARCA2 and SMARCA4. These chemical probes inhibit the association of full-length PBRM1 to acetylated histone peptides and selectively inhibit growth of a PBRM1-dependent prostate cancer cell line.

Altered BAF occupancy and transcription factor dynamics in PBAF-deficient melanoma.

ARID2 is the most recurrently mutated SWI/SNF complex member in melanoma; however, its tumor-suppressive mechanisms in the context of the chromatin landscape remain to be elucidated. Here, we model ARID2 deficiency in melanoma cells, which results in defective PBAF complex assembly with a concomitant genomic redistribution of the BAF complex. Upon ARID2 depletion, a subset of PBAF and shared BAF-PBAF-occupied regions displays diminished chromatin accessibility and associated gene expression, while BAF-occupied enhancers gain chromatin accessibility and expression of genes linked to the process of invasion. As a function of altered accessibility, the genomic occupancy of melanoma-relevant transcription factors is affected and significantly correlates with the observed transcriptional changes. We further demonstrate that ARID2-deficient cells acquire the ability to colonize distal organs in multiple animal models. Taken together, our results reveal a role for ARID2 in mediating BAF and PBAF subcomplex chromatin dynamics with consequences for melanoma metastasis.

Polycomb group proteins in cancer: multifaceted functions and strategies for modulation.

Polycomb repressive complexes (PRCs) are a heterogenous collection of dozens, if not hundreds, of protein complexes composed of various combinations of subunits. PRCs are transcriptional repressors important for cell-type specificity during development, and as such, are commonly mis-regulated in cancer. PRCs are broadly characterized as PRC1 with histone ubiquitin ligase activity, or PRC2 with histone methyltransferase activity; however, the mechanism by which individual PRCs, particularly the highly diverse set of PRC1s, alter gene expression has not always been clear. Here we review the current understanding of how PRCs act, both individually and together, to establish and maintain gene repression, the biochemical contribution of individual PRC subunits, the mis-regulation of PRC function in different cancers, and the current strategies for modulating PRC activity. Increased mechanistic understanding of PRC function, as well as cancer-specific roles for individual PRC subunits, will uncover better targets and strategies for cancer therapies.

MicroRNA-directed pathway discovery elucidates an miR-221/222-mediated regulatory circuit in class switch recombination.

MicroRNAs (miRNAs, miRs) regulate cell fate decisions by post-transcriptionally tuning networks of mRNA targets. We used miRNA-directed pathway discovery to reveal a regulatory circuit that influences Ig class switch recombination (CSR). We developed a system to deplete mature, activated B cells of miRNAs, and performed a rescue screen that identified the miR-221/222 family as a positive regulator of CSR. Endogenous miR-221/222 regulated B cell CSR to IgE and IgG1 in vitro, and miR-221/222-deficient mice exhibited defective IgE production in allergic airway challenge and polyclonal B cell activation models in vivo. We combined comparative Ago2-HITS-CLIP and gene expression analyses to identify mRNAs bound and regulated by miR-221/222 in primary B cells. Interrogation of these putative direct targets uncovered functionally relevant downstream genes. Genetic depletion or pharmacological inhibition of Foxp1 and Arid1a confirmed their roles as key modulators of CSR to IgE and IgG1.

Polycomb Paralog Chromodomain Inhibitors Active against Both CBX6 and CBX8*.

Methyllysine reader proteins bind to methylated lysine residues and alter gene transcription by changing either the compaction state of chromatin or by the recruitment of other multiprotein complexes. The polycomb paralog family of methyllysine readers bind to trimethylated lysine on the tail of histone 3 (H3) via a highly conserved aromatic cage located in their chromodomains. Each of the polycomb paralogs are implicated in several disease states. CBX6 and CBX8 are members of the polycomb paralog family with two structurally similar chromodomains. By exploring the structure-activity relationships of a previously reported CBX6 inhibitor we have discovered more potent and cell permeable analogs. Our current report includes potent, dual-selective inhibitors of CBX6 and CBX8. We have shown that the -2 position in our scaffold is an important residue for selectivity amongst the polycomb paralogs. Preliminary cell-based studies show that the new inhibitors impact cell proliferation in a rhabdoid tumor cell line.

A Potent, Selective CBX2 Chromodomain Ligand and Its Cellular Activity During Prostate Cancer Neuroendocrine Differentiation.

Polycomb group (PcG) proteins are epigenetic regulators that facilitate both embryonic development and cancer progression. PcG proteins form Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). PRC2 trimethylates histone H3 lysine 27 (H3K27me3), a histone mark recognized by the N-terminal chromodomain (ChD) of the CBX subunit of canonical PRC1. There are five PcG CBX paralogs in humans. CBX2 in particular is upregulated in a variety of cancers, particularly in advanced prostate cancers. Using CBX2 inhibitors to understand and target CBX2 in prostate cancer is highly desirable; however, high structural similarity among the CBX ChDs has been challenging for developing selective CBX ChD inhibitors. Here, we utilize selections of focused DNA encoded libraries (DELs) for the discovery of a selective CBX2 chromodomain probe, SW2_152F. SW2_152F binds to CBX2 ChD with a Kd of 80 nM and displays 24-1000-fold selectivity for CBX2 ChD over other CBX paralogs in vitro. SW2_152F is cell permeable, selectively inhibits CBX2 chromatin binding in cells, and blocks neuroendocrine differentiation of prostate cancer cell lines in response to androgen deprivation.

At the Crossroad of Gene Regulation and Genome Organization: Potential Roles for ATP-Dependent Chromatin Remodelers in the Regulation of CTCF-Mediated 3D Architecture.

In higher order organisms, the genome is assembled into a protein-dense structure called chromatin. Chromatin is spatially organized in the nucleus through hierarchical folding, which is tightly regulated both in cycling cells and quiescent cells. Assembly and folding are not one-time events in a cell's lifetime; rather, they are subject to dynamic shifts to allow changes in transcription, DNA replication, or DNA damage repair. Chromatin is regulated at many levels, and recent tools have permitted the elucidation of specific factors involved in the maintenance and regulation of the three-dimensional (3D) genome organization. In this review/perspective, we aim to cover the potential, but relatively unelucidated, crosstalk between 3D genome architecture and the ATP-dependent chromatin remodelers with a specific focus on how the architectural proteins CTCF and cohesin are regulated by chromatin remodeling.

BRD9 Is a Critical Regulator of Androgen Receptor Signaling and Prostate Cancer Progression.

Switch/sucrose-nonfermentable (SWI/SNF) chromatin-remodeling complexes are critical regulators of chromatin dynamics during transcription, DNA replication, and DNA repair. A recently identified SWI/SNF subcomplex termed GLTSCR1/1L-BAF (GBAF; or "noncanonical BAF", ncBAF) uniquely contains bromodomain-containing protein BRD9 and glioma tumor suppressor candidate region 1 (GLTSCR1) or its paralog GLTSCR1-like (GLTSCR1L). Recent studies have identified a unique dependency on GBAF (ncBAF) complexes in synovial sarcoma and malignant rhabdoid tumors, both of which possess aberrations in canonical BAF (cBAF) and Polybromo-BAF (PBAF) complexes. Dependencies on GBAF in malignancies without SWI/SNF aberrations, however, are less defined. Here, we show that GBAF, particularly its BRD9 subunit, is required for the viability of prostate cancer cell lines in vitro and for optimal xenograft tumor growth in vivo. BRD9 interacts with androgen receptor (AR) and CCCTC-binding factor (CTCF), and modulates AR-dependent gene expression. The GBAF complex exhibits overlapping genome localization and transcriptional targets as bromodomain and extraterminal domain-containing (BET) proteins, which are established AR coregulators. Our results demonstrate that GBAF is critical for coordinating SWI/SNF-BET cooperation and uncover a new druggable target for AR-positive prostate cancers, including those resistant to androgen deprivation or antiandrogen therapies. SIGNIFICANCE: Advanced prostate cancers resistant to androgen receptor antagonists are still susceptible to nontoxic BRD9 inhibitors, making them a promising alternative for halting AR signaling in progressed disease.

Chemical Inhibitors of a Selective SWI/SNF Function Synergize with ATR Inhibition in Cancer Cell Killing.

SWI/SNF (BAF) complexes are a diverse family of ATP-dependent chromatin remodelers produced by combinatorial assembly that are mutated in and thought to contribute to 20% of human cancers and a large number of neurologic diseases. The gene-activating functions of BAF complexes are essential for viability of many cell types, limiting the development of small molecule inhibitors. To circumvent the potential toxicity of SWI/SNF inhibition, we identified small molecules that inhibit the specific repressive function of these complexes but are relatively nontoxic and importantly synergize with ATR inhibitors in killing cancer cells. Our studies suggest an avenue for therapeutic enhancement of ATR/ATM inhibition and provide evidence for chemical synthetic lethality of BAF complexes as a therapeutic strategy in cancer.

ARID1A Regulates Transcription and the Epigenetic Landscape via POLE and DMAP1 while ARID1A Deficiency or Pharmacological Inhibition Sensitizes Germ Cell Tumor Cells to ATR Inhibition.

Germ cell tumors (GCTs) are the most common solid malignancies found in young men. Although they generally have high cure rates, metastases, resistance to cisplatin-based therapy, and late toxicities still represent a lethal threat, arguing for the need of new therapeutic options. In a previous study, we identified downregulation of the chromatin-remodeling SWI/SNF complex member ARID1A as a key event in the mode of action of the histone deacetylase inhibitor romidepsin. Additionally, the loss-of-function mutations re-sensitize different tumor types to various drugs, like EZH2-, PARP-, HDAC-, HSP90- or ATR-inhibitors. Thus, ARID1A presents as a promising target for synthetic lethality and combination therapy. In this study, we deciphered the molecular function of ARID1A and screened for the potential of two pharmacological ARID1A inhibitors as a new therapeutic strategy to treat GCTs. By CRISPR/Cas9, we generated ARID1A-deficient GCT cells and demonstrate by mass spectrometry that ARID1A is putatively involved in regulating transcription, DNA repair and the epigenetic landscape via DNA Polymerase POLE and the DNA methyltransferase 1-associated protein DMAP1. Additionally, ARID1A/ARID1A deficiency or pharmacological inhibition increased the efficacy of romidepsin and considerably sensitized GCT cells, including cisplatin-resistant subclones, towards ATR inhibition. Thus, targeting ARID1A in combination with romidepsin and ATR inhibitors presents as a new putative option to treat GCTs.

Optimization of Ligands Using Focused DNA-Encoded Libraries To Develop a Selective, Cell-Permeable CBX8 Chromodomain Inhibitor.

Polycomb repressive complex 1 (PRC1) is critical for mediating gene expression during development. Five chromobox (CBX) homolog proteins, CBX2, CBX4, CBX6, CBX7, and CBX8, are incorporated into PRC1 complexes, where they mediate targeting to trimethylated lysine 27 of histone H3 (H3K27me3) via the N-terminal chromodomain (ChD). Individual CBX paralogs have been implicated as drug targets in cancer; however, high similarities in sequence and structure among the CBX ChDs provide a major obstacle in developing selective CBX ChD inhibitors. Here we report the selection of small, focused, DNA-encoded libraries (DELs) against multiple homologous ChDs to identify modifications to a parental ligand that confer both selectivity and potency for the ChD of CBX8. This on-DNA, medicinal chemistry approach enabled the development of SW2_110A, a selective, cell-permeable inhibitor of the CBX8 ChD. SW2_110A binds CBX8 ChD with a Kd of 800 nM, with minimal 5-fold selectivity for CBX8 ChD over all other CBX paralogs in vitro. SW2_110A specifically inhibits the association of CBX8 with chromatin in cells and inhibits the proliferation of THP1 leukemia cells driven by the MLL-AF9 translocation. In THP1 cells, SW2_110A treatment results in a significant decrease in the expression of MLL-AF9 target genes, including HOXA9, validating the previously established role for CBX8 in MLL-AF9 transcriptional activation, and defining the ChD as necessary for this function. The success of SW2_110A provides great promise for the development of highly selective and cell-permeable probes for the full CBX family. In addition, the approach taken provides a proof-of-principle demonstration of how DELs can be used iteratively for optimization of both ligand potency and selectivity.

Selection of DNA-Encoded Libraries to Protein Targets within and on Living Cells.

We report the selection of DNA-encoded small molecule libraries against protein targets within the cytosol and on the surface of live cells. The approach relies on generation of a covalent linkage of the DNA to protein targets by affinity labeling. This cross-linking event enables subsequent copurification by a tag on the recombinant protein. To access targets within cells, a cyclic cell-penetrating peptide is appended to DNA-encoded libraries for delivery across the cell membrane. As this approach assesses binding of DELs to targets in live cells, it provides a strategy for selection of DELs against challenging targets that cannot be expressed and purified as active.