Role Of Chromatin Remodeling Research Articles

Abstract P1116: Histone Deacetylase Complexes Regulate Direct Cardiac Conversion From Human Fibroblasts

Direct cardiac reprogramming is a promising approach to generate functional cardiomyocytes from non-myocytes for regenerative medicine, drug screening, and disease modeling. This method involves the transformation of specialized cells into functional cardiomyocytes using specific transcription factors or molecular signals. Epigenetic modulators, such as histone deacetylase (HDAC) complexes, may play a critical role in chromatin remodeling underlying cardiac reprogramming. To investigate the role of HDAC complexes in direct cardiac conversion, induced cardiomyocyte-like cells (iCMs) were derived from human fibroblasts through ectopic expression of reprogramming factors, and the changes of histone acetylation were evaluated by western blotting. The results showed decreased levels of H3K9ac, H3K14ac, and H3K27ac along with direct cardiac reprogramming, suggesting the significant chromatin remodeling and gene regulation. A loss-of-function screen of all the components associated with HDAC complex was performed to determine the responsible histone acetylation modifiers. The screen results showed that most of the HDAC components are necessary for direct cardiac reprogramming. Further investigation focused on a newly identified HDAC complex, mitotic deacetylase complex (MiDAC), consisting of mitotic deacetylase associated SANT domain protein MiDEAS (ELMSAN1), DNTTIP1, and HDAC1. Knocking down each of the MiDAC components led to similar reprogramming repression and cell death, indicating that MiDAC plays a critical role in cardiac reprogramming. In addition, the expression analysis of DNTTIP1 and HDAC1 showed a gradual decrease throughout the conversion, while ELMSAN1 diminished at the early stage of reprogramming, suggesting that ELMSAN1 is likely required for MiDAC stability. Furthermore, overexpression of ELMSAN1 significantly improved reprogramming efficiency and led to the increase of H3K9ac, H3K14ac, and H3K27ac levels globally, suggesting that ELMSAN1 and MiDAC might be responsible for the regulation of such histone modifications during the process. Overall, our findings provide novel insights into the epigenetic regulation of direct cardiac reprogramming and reveal the potential role of MiDAC in this process.

A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis.

The long interspersed element 1 (LINE-1 or L1) integration is affected by many cellular factors through various mechanisms. Some of these factors are required for L1 amplification, while others either suppress or enhance specific steps during L1 propagation. Previously, TRIM28 has been identified to suppress transposable elements, including L1 expression via its canonical role in chromatin remodeling. Here, we report that TRIM28 through its B box domain increases L1 retrotransposition and facilitates shorter cDNA and L1 insert generation in cultured cells. Consistent with the latter, we observe that tumor specific L1 inserts are shorter in endometrial, ovarian, and prostate tumors with higher TRIM28 mRNA expression than in those with lower TRIM28 expression. We determine that three amino acids in the B box domain that are involved in TRIM28 multimerization are critical for its effect on both L1 retrotransposition and cDNA synthesis. We provideevidence that B boxes from the other two members in the Class VI TRIM proteins, TRIM24 and TRIM33, also increase L1 retrotransposition. Our findings could lead to a better understanding of the host/L1 evolutionary arms race in the germline and their interplay during tumorigenesis.

Abstract 2440: HOTAIR functionality and regulation in ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the fifth leading cause of cancer-related death among American women. Persistence of OC stem cells (OCSCs) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse. We have previously shown that epigenetic changes in OCSCs play a role in post-therapy OCSC persistence and demonstrated the potential to target OCSC using epigenetic therapies. HOXC transcript antisense RNA (HOTAIR) has been shown to be associated with chemoresistance and overexpressed in high-grade serous OC (HGSOC). HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and plays a key role in chromatin remodeling. Because HOTAIR is a known epigenetic regulator of differentiation and developmental genes in OC, we hypothesized that HOTAIR regulates OCSCs and epigenetic targeting HOTAIR will prevent tumor relapse. To produce loss-of-function phenotypes of HOTAIR and investigate the function of this gene, we utilized the paired CRISPR guide RNA design to delete the functional sites of HOTAIR in a panel high grade serous OC cell lines (Kuramochi, OVCAR3) without affecting nearby protein-coding gene. Knockout of HOTAIR re-sensitized OC cells to platinum treatment and significantly decreased (P<0.001) the OCSC population and stemness-related phenotypes, including inhibiting stemness-associated gene expression ALDH1A1, Notch3, Sox9, and PROM1 and in vitro spheroid forming ability under low attachment conditions. Integrated analysis of RNA-seq and ATAC-seq on control and HOTAIR knockout cells revealed HOTAIR altered global chromatin dynamics to change downstream gene expression, particularly, the NF-kB pathway. In xenograft assays using HGSOC cells, combining a HOTAIR inhibitor with an EZH2 inhibitor and chemotherapy significantly decreased in vivo tumor formation and increased mouse survival. In summary, depletion of HOTAIR functionally decreased OCSC phenotypes and malignant potential and reprogrammed HGSOC to a less stem-like phenotype. There is an urgent need to develop new therapeutic strategies to target OCSCs and overcome chemoresistant OC, and the results of this study suggest that targeting HOTAIR in combination with epigenetic therapies may represent a novel therapeutic strategy to prevent tumor relapse in OC. Citation Format: Weini Wang, Yanchi Zhou, Ji Wang, Ali Ozes, Heather M. O’Hagan, Anirban Mitra, Kenneth P. Nephew. HOTAIR functionality and regulation in ovarian cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2440.

Transcriptome-wide association analyses identify an association between ARL14EP and polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder, which is accompanied by a variety of comorbidities including metabolic, reproductive, and psychiatric disorders. Genome-wide association studies have identified several genetic variants that are associated with PCOS. However, these variants often occur outside of coding regions and require further investigation to understand their contribution to PCOS. A transcriptome-wide association study (TWAS) was performed to uncover heritable gene expression profiles that are associated with PCOS in two independent cohorts. Causal gene prioritization was subsequently performed and expression of genes prioritized through these analyses was examined in 49 PCOS patients and 30 controls. TWAS analyses revealed that increased expression of ARL14EP was significantly associated with PCOS risk in the discovery (P = 1.6 × 10-6) and replication cohorts (P = 2.0 × 10-13). Gene prioritization pipelines provided further evidence that ARL14EP is the most likely causal gene at this locus. ARL14EP gene expression was shown to be significantly different between PCOS cases and controls, after adjusting for body mass index, age and testosterone levels (P = 1.2 × 10-13). This study has provided evidence for the role of ARL14EP in PCOS. Given that ARL14EP has been reported to play an important role in chromatin remodeling, variants affecting the expression of ARL14EP may also affect the expression of other genes that contribute to PCOS pathogenesis.

Long Non-coding RNAs and CRISPR-Cas Edition in Tumorigenesis.

Long non-coding RNAs (lncRNAs) are one of the most abundant and heterogeneous transcripts with key roles in chromatin remodeling and gene regulation at the transcriptional and post-transcriptional levels. Due to their role in cell growth and differentiation, lncRNAs have emerged as an important biomarker in cancer diagnosis, prognosis, and targeted treatment. Recent studies have focused on elucidating lncRNA function during malignant transformation, tumor progression and drug resistance. The advent of the CRISPR system has made it possible to precisely edit complex genomic loci such as lncRNAs. Thus, we summarized the advances in CRISPR-Cas approaches for functional studies of lncRNAs including gene knockout, knockdown, overexpression and RNA targeting in tumorigenesis and drug resistance. Additionally, we highlighted the perspectives and potential applications of CRISPR approaches to treat cancer, as an emerging and promising target therapy.

Baz1b loss-of-function in zebrafish produces phenotypic alterations consistent with the domestication syndrome

BAZ1B is a ubiquitously expressed nuclear protein with roles in chromatin remodeling, DNA replication and repair, and transcription. Reduced BAZ1B expression disrupts neuronal and neural crest development. Variation in the activity of BAZ1B has been proposed to underly morphological and behavioral aspects of domestication through disruption of neural crest development. Knockdown ofbaz1b in Xenopus embryos and Baz1b loss-of-function (LoF) in mice leads to craniofacial defects consistent with this hypothesis. We generated baz1b LoF zebrafish using CRISPR/Cas9 gene editing to test the hypothesis that baz1b regulates behavioral phenotypes associated with domestication in addition to craniofacial features. Zebrafish with baz1b LoF show mild underdevelopment at larval stages and distinctive craniofacial features later in life. Mutant zebrafish show reduced anxiety-associated phenotypes and an altered ontogeny of social behaviors. Thus, in zebrafish, developmental deficits in baz1b recapitulate both morphological and behavioral phenotypes associated with the domestication syndrome in other species.

Abstract IA021: Targeting epigenetic regulators of oncogenic transcription factors

Abstract The switch/sucrose non-fermentable (SWI/SNF) complex has a crucial role in chromatin remodeling and is altered in over 20% of cancers. We recently developed and evaluated a proteolysis-targeting chimera (PROTAC) degrader of the SWI/SNF ATPase subunits, SMARCA2 and SMARCA4, called AU-15330. Androgen receptor (AR)+ forkhead box A1 (FOXA1)+ prostate cancer cells are exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to normal and other cancer cell lines. SWI/SNF ATPase degradation rapidly compacts cis-regulatory elements bound by transcription factors that drive prostate cancer cell proliferation, namely AR, FOXA1, ERG and MYC, which dislodges them from chromatin, disables their core enhancer circuitry, and abolishes the downstream oncogenic gene programs. SWI/SNF ATPase degradation also disrupts super-enhancer and promoter looping interactions that wire supraphysiologic expression of the AR, FOXA1 and MYC oncogenes themselves. AU-15330 induces potent inhibition of tumor growth in xenograft models of prostate cancer and synergizes with the AR antagonist enzalutamide, even inducing disease remission in castration-resistant prostate cancer (CRPC) models without toxicity. Thus, impeding SWI/SNF-mediated enhancer accessibility represents a promising therapeutic approach for enhancer-addicted cancers. Citation Format: Arul M. Chinnaiyan. Targeting epigenetic regulators of oncogenic transcription factors. [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 IA021.

Potential of histone deacetylase inhibitors for the therapy of ovarian cancer.

Malignant ovarian tumors bear the highest mortality rate among all gynecological cancers. Both late tumor diagnosis and tolerance to available chemotherapy increase patient mortality. Accumulating evidence demonstrates that histone modifications play a key role in cancerization and progression. Histone deacetylases is associated with chromatin condensed structure and transcriptional repression and play a role in chromatin remodeling and epigenetics. Histone deacetylases are promising targets for therapeutic interventions intended to reverse aberrant epigenetic associated with cancer. Therefore, histone deacetylases inhibitors could be used as anti-cancer drugs. Preclinical studies have shown promising outcomes of histone deacetylases inhibitors in ovarian cancer while clinical trials have had mixed results and limited success as monotherapy. Therefore, combination therapy with different anticancer drugs for synergistic effects and newly selective histone deacetylases inhibitors development for lower toxicity are hot issues now. In this review, we summarize the latest studies on the classification and mechanisms of action of histone deacetylase and the clinical application of their inhibitors as monotherapy or combination therapy in ovarian cancer.

SMARCC2 mediates the regulation of DKK1 by the transcription factor EGR1 through chromatin remodeling to reduce the proliferative capacity of glioblastoma

Switch/sucrose-nonfermenting (SWI/SNF) complexes play a key role in chromatin remodeling. Recent studies have found that SMARCC2, as the core subunit of the fundamental module of the complex, plays a key role in its early assembly. In this study, we found a unique function of SMARCC2 in inhibiting the progression of glioblastoma by targeting the DKK1 signaling axis. Low expression of SMARCC2 is found in malignant glioblastoma (GBM) compared with low-grade gliomas. SMARCC2 knockout promoted the proliferation of glioblastoma cells, while its overexpression showed the opposite effect. Mechanistically, SMARCC2 negatively regulates transcription by dynamically regulating the chromatin structure and closing the promoter region of the target gene DKK1, which can be bound by the transcription factor EGR1. DKK1 knockdown significantly reduced the proliferation of glioblastoma cell lines by inhibiting the PI3K–AKT pathway. We also studied the functions of the SWIRM and SANT domains of SMARCC2 and found that the SWIRM domain plays a more important role in the complete chromatin remodeling function of SMARCC2. In addition, in vivo studies confirmed that overexpression of SMARCC2 could significantly inhibit the size of intracranial gliomas in situ in nude mice. Overall, this study shows that SMARCC2, as a tumor suppressor, inhibits the proliferation of glioblastoma by targeting the transcription of the oncogene DKK1 through chromatin remodeling, indicating that SMARCC2 is a potentially attractive therapeutic target in glioblastoma.

PURINE-DERIVED PHENYLHYDROXAMATE COMPOUNDS INDUCES CELL DEATH IN HEMATOLOGICAL NEOPLASM MODELS

The histone acetylation process is the best-studied type of post-translational modification, playing a key role in chromatin remodeling regulated by the activity of two enzymes: histone acetyltransferases and histone deacetylases (HDAC). High levels of HDAC have been observed in patients with hematological and solid cancers and are associated with poor prognosis. The present study aimed to evaluate the cellular effects of three novel potential HDAC inhibitors in hematological neoplasm models. A panel containing myeloid (OCI-AML3, Kasumi 1, HL60, THP1, MOLM13, MV4-11, U937, NB4, NB4-R2, K562, KU812, SET2, and HEL) and lymphoid (Jurkat, CEM, Namalwa, NALM6, Daudi, Raji, SUP-B15, REH, U266, MM1.S, MM1.R, and Karpas 422) neoplasm cells were used. Peripheral blood mononuclear cells (normal leukocytes) from healthy donors were used for toxicity evaluation (n = 4). Three novel purine-derived phenylhydroxamate with potential HDAC inhibitor activity (named here as 82, 83, and 84) were used. Vorinostat was used as a reference drug. Cell viability was assessed by MTT assay, apoptosis by annexin V/propidium iodide labeling and flow cytometry (FC), clonogenicity by autonomous colony formation, cell cycle by propidium iodide and FC, and protein expression by Western Blot. IC 50 values were obtained by non-linear regression. Statistical analysis was performed by ANOVA and Bonferroni post-test. A p < 0.05 was considered significant. All compounds tested reduced cell viability at submicromolar concentrations being compound 82 more potent than the others exhibiting less concentration necessary to reduce cell viability by 50% (IC 50 ranged from 0.09 to 2.04 μM). THP1, MOLM13, NB4-R2, and HEL leukemia cells were more sensitive to these compounds and were selected for the next experiments. In normal leukocytes, vorinostat and compound 82 did not reduce cell viability at the same extension as observed for leukemia cells, indicating a selective antineoplastic activity. All compounds induced time- and dose-dependent manner apoptosis in THP1, MOLM13, NB4-R2, and HEL cells (all p < 0.05). Vorinostat and compound 82 strongly reduced colony formation, being compound 82 more efficient, especially in MOLM13 and NB4-R2 cells (all p < 0.05). Low concentration of vorinostat and compound 82 lead to reduced cell cycle progression (all p < 0.05). THP1, MOLM13, NB4-R2, and HEL cells were exposed to vorinostat or compound 82 and the expression of proteins involved in apoptosis and HDAC activity was modulated. Our results indicate that the three purine-derived phenylhydroxamate tested presented antineoplastic effects being a future candidate to compose the list of HDAC inhibitors in the antineoplastic arsenal. CNPq, CAPES, and FAPESP.

Abstract B011: Two epigenetically distinct cellular states in osteosarcoma are regulated by a cluster-specific set of pioneer transcription factors

Abstract The Osteosarcoma (OS) genome is characterized by aneuploidy and dramatic structural rearrangements often leading to copy number gain and loss. There is significant heterogeneity between patients. Additionally, there is less known regarding the epigenetic heterogeneity within osteosarcoma and how this may impact critical phenotypes such as therapy response and metastasis. To characterize the osteosarcoma epigenome, we integrated ATAC-seq and RNA-seq across a unique set of patient samples, PDXs and PDX-derived cell lines obtained across the disease continuum and including biopsies, resections, and metastases. Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) analysis revealed the presence of two distinct epigenetic cell states. Differential chromatin accessibility between the clusters showed ~3000 unique open regions per cluster. Using GREAT to associate the differential genomic regions to genes, the enriched pathways of Cluster 1 (C1) are related to mesenchymal cell proliferation, skeletal system morphogenesis, osteoblast differentiation, and Wnt signaling pathway, whereas Cluster 2 (C2) enriched pathways are related to cell motility and migration, extracellular matrix organization and regulation of protein serine/threonine kinase activity. Accessible regions in the chromatin were enriched for a unique set of transcription factor binding sites. C1 accessible regions are enriched with transcription factor binding motifs (TFBM) related to development including RUNX2/3, MEOX2, HOXA2/5, VAX1/2, MEF, VSX and DLX; whereas members of AP1 complex including FOSL1/2, c-FOS, JUN are TFBM enriched in open regions in C2. This is correlated with high protein expression of these transcription factors in a cluster-specific manner and differential binding to chromatin. As expected, gene expression showed a positive correlation with accessible regions in promoters. RUNX2 plays a major role in chromatin remodeling in C1, modifying the chromatin accessibility of 5300 regions, most located in &amp;lt;1kb from the promoters. In conclusion, we uncovered two epigenetically cell states regulated by a cluster-specific set of transcription factors which are able to remodel the chromatin. Citation Format: Eunice Lopez Fuentes, Andrew Clugston, Leanne C. Sayles, E. Alejandro Sweet-Cordero. Two epigenetically distinct cellular states in osteosarcoma are regulated by a cluster-specific set of pioneer transcription factors [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B011.

Cancer therapeutics strategy using nano-carrier mediated natural drugs

Nucleolin is a multifactorial protein, having a significant role in chromatin remodelling, mRNA stability, ribosome biogenesis, stemness, angiogenesis, etc., thus, it is potential therapeutic target in cancer. The purpose of this paper is to study porous silicon (pSi) nanocarrier-based natural drug delivery system targeting dysregulated nucleolin expression for cancer therapeutics. Quercetin was loaded in pre-synthesized and characterized pSi nanoparticles, and release kinetics was studied. The study compared the inhibitory concentration (IC50) of quercetin, synthetic drug doxorubicin, and quercetin-loaded pSi nanoparticles. Further, mRNA expression of a target gene, nucleolin, was tested with a quercetin treated breast cancer cell line (MCF-7). Quercetin-loaded pSi nanoparticles followed first-order release kinetics. IC50 was determined at concentrations of 312 nM, 160 µM, and 50 µM against doxorubicin, quercetin, and quercetin-loaded pSi nanoparticles, respectively. The results further indicated 16-fold downregulation of nucleolin mRNA expression after 48h of quercetin treatment of exponentially growing MCF-7 cells. Whether pSi nanoparticle loaded quercetin can significantly downregulate nucleolin protein expression and its impact on apoptosis, cell proliferation, and angiogenic pathways need further investigation. The practical application of the proposed nanocarrier-based drug delivery system potentially lays out a path for developing targeted therapy against nucleolin-dysregulated cancer using natural products to minimize the side effects of conventional chemotherapeutic drugs. Inhibition of nucleolin and nucleolin regulated pathways using natural compounds and its targeted delivery with nanocarrier is not yet done.

Potential of histone deacetylase inhibitors in the control and regulation of prostate, breast and ovarian cancer.

Histone deacetylases (HDACs) are enzymes that play a role in chromatin remodeling and epigenetics. They belong to a specific category of enzymes that eliminate the acetyl part of the histones’ -N-acetyl lysine, causing the histones to be wrapped compactly around DNA. Numerous biological processes rely on HDACs, including cell proliferation and differentiation, angiogenesis, metastasis, gene regulation, and transcription. Epigenetic changes, specifically increased expression and activity of HDACs, are commonly detected in cancer. As a result, HDACi could be used to develop anticancer drugs. Although preclinical outcomes with HDACs as monotherapy have been promising clinical trials have had mixed results and limited success. In both preclinical and clinical trials, however, combination therapy with different anticancer medicines has proved to have synergistic effects. Furthermore, these combinations improved efficacy, decreased tumor resistance to therapy, and decreased toxicity. In the present review, the detailed modes of action, classification of HDACs, and their correlation with different cancers like prostate, breast, and ovarian cancer were discussed. Further, the different cell signaling pathways and the structure-activity relationship and pharmaco-toxicological properties of the HDACi, and their synergistic effects with other anticancer drugs observed in recent preclinical and clinical studies used in combination therapy were discussed for prostate, breast, and ovarian cancer treatment.

Multiple Sclerosis: Enzymatic Cross Site-Specific Recognition and Hydrolysis of H2A Histone by IgGs against H2A, H1, H2B, H3 Histones, Myelin Basic Protein, and DNA.

Histones have a paramount role in chromatin remodeling and gene transcription. Free histones are damage-associated molecules in the blood; administration of histones to animals drives systemic inflammatory and toxic effects. Myelin basic protein (MBP) is the most crucial component of the axon myelin-proteolipid sheath. Antibodies-abzymes with different enzymatic activities are very toxic and an essential feature of some autoimmune diseases. Electrophoretically homogeneous IgGs against H1, H2A, H2B, H3, H4, MBP, and DNA were derived from sera of multiple sclerosis (MS) patients by several affinity chromatographies. Using MALDI-TOFF mass spectrometry, it was shown that IgGs against H2A split H2A at 12 sites; the number of H2A hydrolysis sites by antibodies against other antigens is different: H1 (19), H2B (11), H3 (15), H4 (9), MBP (10), and DNA (23), and they only partly match. Thus, the complex formation polyreactivity and the enzymatic cross-activity of pernicious humans IgGs against five histones, MBP, and DNA have been shown for the first time. The data obtained indicate that the formation of such polyspecific-polyreactive abzymes, whose single active center can recognize and hydrolyze different substrates, can occur due to the formation of antibodies against hybrid antigenic determinants consisting of several histone protein sequences. IgGs with high affinity for DNA with DNase and protease activities may be antibodies against DNA-histone complex antigenic determinants, including protein and DNA sequences. Polyreactive IgGs-abzymes against MBP, five histones, and DNA with extended cytotoxicity can play a very negative role in the pathogenesis of multiple sclerosis and probably other different diseases.

Combinatorial targeting of a chromatin complex comprising Dot1L, menin and the tyrosine kinase BAZ1B reveals a new therapeutic vulnerability of endocrine therapy-resistant breast cancer

BackgroundTargeting vulnerabilities of cancer cells by inhibiting key regulators of cell proliferation or survival represents a promising way to overcome resistance to current therapies. In breast cancer (BC), resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor alpha (ERα) signaling to the genome. Targeting components of the ERα pathway in these tumors represents, therefore, a rational way toward effective new treatments. Interaction proteomics identified several proteins associated with ERα in BC cells, including epigenetic complexes controlling gene transcription comprising the scaffold protein menin and the histone methyltransferase Dot1L.MethodsWe combined chromatin immunoprecipitation, transcriptome sequencing, siRNA-mediated gene knockdown (kd), pharmacological inhibition coupled to cellular and functional assays and interaction proteomics in antiestrogen (AE)-sensitive and AE-resistant human BC cell models to: map menin and Dot1L chromatin localization, search for their common and specific target genes, measure the effects of single or combinatorial knockdown or pharmacological inhibition of these proteins on cell proliferation and survival, and characterize their nuclear interactomes.ResultsDot1L and menin associate in MCF-7 cells chromatin, where they co-localize in a significant fraction of sites, resulting in co-regulation of genes involved, among others, in estrogen, p53, HIF1α and death receptor signaling, regulation of cell cycle and epithelial-to-mesenchymal transition. Specific inhibitors of the two factors synergize with each other for inhibition of cell proliferation of AE (tamoxifen or fulvestrant)-sensitive and AE-resistant BC cells. Menin and Dot1L interactomes share a sizeable fraction of their nuclear partners, the majority being known BC fitness genes. Interestingly, these include B-WICH and WINAC complexes that share BAZ1B, a bromodomain protein comprising a tyrosine–protein kinase domain playing a central role in chromatin remodeling and transcriptional regulation. BAZ1B kd caused significant inhibition of ERα expression, proliferation and transcriptome changes resulting in inhibition of estrogen, myc, mTOR, PI3K and AKT signaling and metabolic pathways in AE-sensitive and AE-resistant BC cells.ConclusionsIdentification of a functional interplay between ERα, Dot1L, menin and BAZ1B and the significant effects of their co-inhibition on cell proliferation and survival in cell models of endocrine therapy-resistant BC reveal a new therapeutic vulnerability of these aggressive diseases.

Abstract 3592: Targeting SWI/SNF ATPases in enhancer-addicted human cancers

Abstract In mammalian cells, DNA is wrapped around histone octamers (collectively referred to as nucleosomes) which form a physical barrier to all DNA-based processes. The switch/sucrose non-fermentable (SWI/SNF) is a multi-subunit chromatin remodeling complex that uses energy from ATP hydrolysis to reposition or eject nucleosomes at non-coding regulatory elements, thereby enabling access to the underlying DNA for transcriptional activation. Notably, the SWI/SNF complex plays a crucial role in chromatin remodeling and is recurrently altered in over 20% of human cancers, with the revised complex in cancer cells enabling central oncogenic gene programs. Yet, no studies have assessed the therapeutic efficacy of complete SWI/SNF inactivation across human cancers. Here, we developed a proteolysis targeting chimera (PROTAC) degrader of ATPase subunits of the SWI/SNF complex, SMARCA2 and SMARCA4. In a panel with over 90 normal and cancer cell lines from 18 different lineages, we found MYC-driven multiple myeloma and androgen receptor (AR)/forkhead box A1 (FOXA1)-positive prostate and breast cancers to be preferentially sensitive to dual SMARCA2 and SMARCA4 degradation relative to benign prostate as well as other cancer cell lines, including cancer cell lines with inactivating SMARCA4 mutations. We found complete SWI/SNF ATPase degradation to instantaneously compact the cis-regulatory elements that are bound and activated by transcription factors that drive cancer proliferation, namely MYC, IRF4, TCF3, AR, FOXA1, and ERG. This ensued in parallel untethering of these oncogenic drivers from the chromatin, with subsequent chemical decommissioning of their core enhancer circuitry and attenuation of downstream gene programs. Furthermore, using chromatin conformation assays we found SWI/SNF inactivation to disrupt super-enhancer and promoter DNA looping interactions that wire supra-physiologic expression of the MYC, AR, ERG, IRF4, and TCF3 oncogenes themselves, thereby tempering their expression in cancer cells. Treatment with the SMARCA2/4 degrader alone induced potent inhibition of tumor growth in cell line-derived xenograft models of multiple myeloma, as well as prostate cancer, and synergized with AR antagonists, inducing disease remission in several drug-resistant disease models. Notably, no major toxicities were seen in mice upon prolonged treatment with the SMARCA2/4 degrader, including no indications of thrombocytopenia, gastrointestinal goblet cell depletion, or germ cell degeneration—all being major toxicities associated with the BRD4-targeting therapeutics. To our knowledge, this study is the first preclinical proof of concept that targeted obstruction of chromatin accessibility at non-coding regulatory elements can be a potent therapeutic strategy in enhancer-addicted tumors, warranting the safety and efficacy assessments of SWI/SNF inhibitors and degraders in human clinical trials. Citation Format: Abhijit Parolia, Lanbo Xiao, Yuanyuan Qiao, Pushpinder Bawa, Sanjana Eyunni, Eleanor Young, Rahul Mannan, Sandra E. Carson, Yu Chang, Yuping Zhang, James George, Mustapha Jaber, Fengyun Su, Rui Wang, Sanjita Sasmal, Leena Khare, Subhendu Mukerjee, Chandrasekhar AbbinenI, Kiran Aithal, Xuhong Cao, Yuzhuo Wang, Susanta Samajdar, Murali Ramachandra, Arul M. Chinnaiyan. Targeting SWI/SNF ATPases in enhancer-addicted human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3592.

Abstract 5469: Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer

Abstract The switch/sucrose non-fermentable (SWI/SNF) complex plays a crucial role in chromatin remodeling and is recurrently altered in over 20% of human cancers. Here, we developed a proteolysis targeting chimera (PROTAC) degrader of ATPase subunits of the SWI/SNF complex, SMARCA2 and SMARCA4. Intriguingly, we found androgen receptor (AR)/forkhead box A1 (FOXA1)-positive prostate cancer to be exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to benign prostate as well as other cancer cell lines, including those with inactivating SMARCA4 mutations. Mechanistically, SWI/SNF inhibition rapidly compacts the cis-regulatory elements that are bound and activated by transcription factors that drive cancer proliferation, namely AR, FOXA1, ERG, and MYC. This ensues in chromatin untethering of these oncogenic drivers, chemical decommissioning of their core enhancer circuitry, and attenuation of downstream gene programs. Furthermore, we found SWI/SNF inhibition to disrupt super-enhancer and promoter DNA looping interactions that wire supra-physiologic expression of the AR, FOXA1, and MYC oncogenes, thereby tempering their expression in cancer cells. Monotherapy with the SMARCA2/4 degrader induced potent inhibition of tumor growth in cell line-derived xenograft models of prostate cancer and remarkably synergized with AR antagonists, inducing disease remission in models of castration-resistant prostate cancer. We also found the combinatorial treatment to significantly inhibit the growth of enzalutamide resistant disease using in vitro as well as patient-derived xenograft models. Notably, no major toxicities were seen in mice upon prolonged treatment with the SMARCA2/4 degrader, including no indications of thrombocytopenia, gastrointestinal goblet cell depletion, or germ cell degeneration. Taken together, these results suggest that impeding enhancer accessibility through SWI/SNF ATPase inactivation represents a novel therapeutic approach in enhancer addicted human cancers. Citation Format: Lanbo Xiao, Abhijit Parolia, Yuanyuan Qiao, Pushpinder Bawa Pushpinder, Sanjana Eyunni, Rahul Mannan, Sandra E. Carson, Yu Chang, Xiaoju Wang, Yuping Zhang, Josh Vo, Steven Kregel, Stephanie A. Simko, Andrew D. Delekta, Mustapha Jaber, Heng Zheng, Ingrid Apel, Lisa McMurry, Fengyun Su, Rui Wang, Sylvia Wang, Sanjita Sasmal, Leena K. Satyam, Subhendu Mukherjee, Chandrasekhar AbbinenI, Kiran Aithal, Mital S. Bhakta, Jay Ghurye, Xuhong Cao, Nora M. Navone, Alexey Nesvizhskii, Rohit Mehra, Ulka Vaishampayan, Marco Blanchette, Yuzhuo Wang, Susanta Samajdar, Murali Ramachandra, Arul M. Chinnaiyan. Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5469.

Stochastic analysis of a complex gene-expression model

Gene expression has been extensively studied in terms of Markov processes, but its stochastic mechanisms including how noisy sources contribute to expression levels still remain not fully understood. To reveal more reasonable mechanisms, it is needed to consider more molecular events occurring in gene-expression processes. Here we introduce and analyze a general gene-expression model described by a chemical master equation. This model simultaneously considers negative feedback, bursting and promoter cyclic structure, thus extending most gene-expression models in the existing literature. First, we uncover the hierarchy of this complex transcription process by deriving the probability-generating function expressed by an Euler integral formula. Second, we further derive analytical mRNA distribution, which includes previous results as its special cases and provides new insights into the roles of chromatin remodeling and feedback in fine-tuning the mRNA noise. Third, we show that the multi-OFF mechanism always reduces the mRNA noise, but the effect of feedback on the mRNA noise is complex, depending on the detail of the multi-OFF mechanism. The overall analysis provides a paradigm for investigation into expression noise and gene-expression mechanisms in more complex cases.

453-P: Role of JARID2 in Regulating Inflammatory Genes via Epigenetic Mechanisms in Monocyte/Macrophages in Type 2 Diabetes

The activation of monocytes and macrophages plays a central role in chronic inflammation associated with type 2 diabetes mellitus (T2D) and cardiovascular disease. However, the precise epigenetic mechanisms involved are unclear. From RNA-seq analysis we found dysregulated expression of several genes, including downregulation of JARID2 in human CD14+ monocytes obtained from T2D patients versus controls. JARID2 protein functions as a transcription repressor by facilitating the recruitment of polycomb repressor complex 2 and associated histone H3K27-trimethylation to target genes. RT-qPCR and RNA-seq analysis showed that high glucose +TNF-α (HT) , key pathological factors in diabetes, downregulated JARID2 in human monocytes along with genes involved in inflammation and cytokine related signaling networks. Western blotting also showed HT reduces JARID2 protein levels in human monocytes. Furthermore, JARID2 knockdown with siRNAs increased proliferation and enhanced LPS and HT induced inflammatory genes in human monocytes. ATAC-seq revealed that HT increased chromatin-accessibility at promoters of inflammatory genes like IL1B that were upregulated by JARID2 knockdown. Interestingly, microRNA-seq analysis revealed that CD14+ monocytes from T2D patients showed increased expression of several microRNAs with complementarity to the JARID2 3’-UTR, indicating cross-talk between epigenetic factors and microRNA-dependent mechanisms. Mouse orthologous Jarid2 was downregulated in macrophages from diabetic db/db mice and in macrophages from normal C57BL/6 mice treated with HT, similar to human JARID2. These results demonstrate that JARID2 plays a key role in chromatin remodeling and restraining inflammatory genes in monocytes, and its downregulation in T2D can augment inflammation. Further, understanding of epigenetic regulators like JARID2 can lead to novel therapies for chronic inflammatory diabetic complications. Disclosure M.A.Reddy: None. V.S.Tanwar: None. L.Zhang: None. L.L.Lanting: None. M.Wang: None. P.Pandey: None. Z.Chen: None. X.Wu: None. R.Natarajan: None. Funding National Institutes of Health (DK065073) to RN AR-DMRI Endowment Pilot grant to MAR

Characterization of Chromatin Remodeling Genes Involved in Thermal Tolerance of Biologically Invasive Bemisia tabaci.

As an invasive species, Bemisia tabaci Mediterranean (MED) has notable potential to adapt to a wide range of environmental temperatures, which enables it to successfully spread after invasion and occupy habitats over a wide latitude range. It has been postulated that chromatin remodeling mechanisms are related to the rapid acquisition of adaptive traits and thermal resistance in invasive species; however, relevant experimental evidence is scarce. To identify the molecular characteristics and assess the role of chromatin remodelers in thermal stress within invasive MED and native Asia II 1 of the B. tabaci species complex, we identified 13 switching defective/sucrose non-fermenting (SWI/SNF) and 10 imitation switch (ISWI) family members in the B. tabaci genome, analyzed their molecular characteristics and structures, and identified key mutation sites between MED and Asia II 1, then cloned the catalytic subunits, and revealed the difference in thermal tolerance function. The results showed that the expression levels of Bt-BRM-1 and Bt-BRM-2 were significantly higher in MED than in Asia II 1 during heat stress, and Bt-BRM-2 expression was significantly higher during cold stress. In addition, RNA interference results indicated that the two target genes had similar temperature tolerance function in the both two cryptic species. This study is the first to identify and analyze the molecular characteristics of SWI/SNF and ISWI family members and reveal their potential key roles in temperature tolerance in poikilothermic ectotherms. The results will assist in understanding the underlying temperature adaptation mechanism of invasive insects and will enrich stress adaptation research systems from an epigenetic perspective.