Transcription Of Target Genes Research Articles

Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind.

Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation.

Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind

Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation.

Transcriptional Landscape and Dynamics Involved in Sugar and Acid Accumulation during Apple Fruit Development.

In fleshy fruit, sugars and acids are central components of fruit flavor and quality. To date, the mechanisms underlying transcriptional regulation of sugar and acid during fruit development remain largely unknown. Here, we combined ATAC-seq with RNA-seq to investigate the genome-wide chromatin accessibility and to identify putative transcription factors related to sugar and acid accumulation during apple (Malus domestica) fruit development. By integrating the differentially accessible regions (DARs) and differentially expressed genes (DEGs), we generated a global dataset of promoter-accessibility- and expression-increased genes (PEIGs). Using this strategy, we constructed a transcriptional regulatory network enabling screening for key transcription factors and target genes involved in sugar and acid accumulation. Among these transcription factors, five fruit-specific Dof (DNA binding with one finger) genes were selected to confirm their regulatory effects, and our results showed that they could affect sugar or acid concentration by regulating the expression of sugar or acid metabolism-related genes in apple fruits. Our transcriptional regulatory network provides a suitable platform to identify candidate genes that control sugar and acid accumulation. Meanwhile, our dataset will aid in analyzing other characteristics of apple fruit that have not been illuminated previously. Overall, these findings support a better understanding of the regulatory dynamics during apple fruit development and lay a foundation for quality improvement of apple.

Persulfidation and phosphorylation of transcription factor SlWRKY6 differentially regulate tomato fruit ripening.

Cysteine desulfhydrase (LCD) catalyzes the generation of the signaling molecule hydrogen sulfide (H2S) in plants. In this study, we found that H2S can inhibit tomato (Solanum lycopersicum) fruit ripening and SlWRKY6 undergoes differential protein persulfidation in SlLCD1-overexpressing leaves. Then, further study indicated that SlWRKY6 could be persulfidated by H2S at Cys396. By construction of slwrky6 mutants and SlWRKY6-OE lines, we found that SlWRKY6 positively regulates leaf senescence and fruit ripening by activating the transcription of ripening-related genes STAYGREEN 1 (SlSGR1) and Senescence-Associated Gene 12 (SlSAG12). In addition, SlWRKY6 interacted with kinase SlMAPK4 and was phosphorylated at Ser33. Dual luciferase transient expression assays and electrophoretic mobility shift assays indicated that SlWRKY6 persulfidation attenuated its transcriptional regulation of target genes SlSGR1 and SlSAG12, whereas SlWRKY6 phosphorylation by SlMAPK4 activated the transcription of target genes to promote fruit ripening. Moreover, we provided evidence that SlWRKY6 persulfidation attenuated its SlMAPK4-mediated phosphorylation to inhibit tomato fruit ripening. By transient expression of SlWRKY6, SlWRKY6C396A, SlWRKY6S33A and SlWRKY6S33D in slwrky6 fruits, we found that SlWRKY6 persulfidation attenuated the expression of SlSGR1 and SlSAG12 thereby delaying tomato fruit ripening, while SlWRKY6 phosphorylation increased the expression of target genes. As tomato fruits ripened, endogenous H2S production decreased, while SlMAPK4 expression increased. Therefore, our findings reveal a model in which SlWRKY6 persulfidation due to higher endogenous H2S levels in un-ripened fruit inhibits its ability to activate SlSGR1 and SlSAG12 expression, while SlWRKY6 phosphorylation by SlMAPK4 activates its transcriptional activity, thereby promoting tomato fruit ripening.

TFTG: A comprehensive database for human transcription factors and their targets

Transcription factors (TFs) are major contributors to gene transcription, especially in controlling cell-specific gene expression and disease occurrence and development. Uncovering the relationship between TFs and their target genes is critical to understanding the mechanism of action of TFs. With the development of high-throughput sequencing techniques, a large amount of TF-related data has accumulated, which can be used to identify their target genes. In this study, we developed TFTG (Transcription Factor and Target Genes) database (http://tf.liclab.net/TFTG), which aimed to provide a large number of available human TF-target gene resources by multiple strategies, besides performing a comprehensive functional and epigenetic annotations and regulatory analyses of TFs. We identified extensive available TF-target genes by collecting and processing TF-associated ChIP-seq datasets, perturbation RNA-seq datasets and motifs. We also obtained experimentally confirmed relationships between TF and target genes from available resources. Overall, the target genes of TFs were obtained through integrating the relevant data of various TFs as well as fourteen identification strategies. Meanwhile, TFTG was embedded with user-friendly search, analysis, browsing, downloading and visualization functions. TFTG is designed to be a convenient resource for exploring human TF-target gene regulations, which will be useful for most users in the TF and gene expression regulation research.

Genetic Susceptibility, Mendelian randomization and Nomogram Model Construction of Gestational Diabetes Mellitus

Abstract Context Gestational diabetes mellitus (GDM) is a pregnancy complicated disease that poses a risk to maternal and infant health. However, the etiology of the disease has been not yet elucidated. Objective To detect the genetic susceptibility and construct a nomogram model with significantly associated polymorphisms and key clinical indicators for early prediction of gestational diabetes mellitus (GDM). Methods 11 functional single nucleotide polymorphisms (SNPs) screened by genome-wide association study (GWAS) were genotyped in 554 GDM cases and 641 healthy controls. Functional analysis of GDM positively associated SNPs, Multivariate mendelian randomization (MVMR) and a GDM early predictive nomogram model construction were performed. Result rs1965211, rs3760675 and rs7814359 were significantly associated with genetic susceptibility to GDM after adjusting age and pre-pregnancy BMI (pre-BMI). It seems that GDM associated SNPs have effects on regulating target gene transcription factor binding, post transcriptional splicing, and translation product structure. Besides, rs3760675 can be expression quantitative trait locis (eQTLs) and increase the XAB2 mRNA expression level (P = 0.047). The MVMR analysis showed that the increase of clinical variables of BMI, HbA1c and FPG had significant causal effects on GDM (BMI-ORMVMR = 1.52, HbA1c-ORMVMR = 1.32, FPG-ORMVMR = 1.78), P <0.05. A nomogram model constructed with pre-BMI, FPG, HbA1c, and genotypes of rs1965211, rs3760675 and rs7814359 showed an area under the ROC curve of 0.824. Conclusion Functional polymorphisms can change women's susceptibility to GDM and the predictive nomogram model based on genetic and environmental factors can effectively distinguish individuals with different GDM risks in early stages of pregnancy.

OTUD6A orchestrates complex modulation of TEAD4-mediated transcriptional programs.

TEAD transcription factors play a central role in the Hippo signaling pathway. In this study, we focused on transcriptional enhancer factor TEF-3 (TEAD4), exploring its regulation by the deubiquitinase OTU domain-containing protein 6A (OTUD6A). We identified OTUD6A as a TEAD4-interacting deubiquitinase, positively influencing TEAD-driven transcription without altering TEAD4 stability. Structural analyses revealed specific interaction domains: the N-terminal domain of OTUD6A and the YAP-binding domain of TEAD4. Functional assays demonstrated the positive impact of OTUD6A on the transcription of YAP-TEAD target genes. Despite no impact on TEAD4 nuclear localization, OTUD6A selectively modulated nuclear interactions, enhancing YAP-TEAD4 complex formation while suppressing VGLL4 (transcription cofactor vestigial-like protein 4)-TEAD4 interaction. Critically, OTUD6A facilitated YAP-TEAD4 complex binding to target gene promoters. Our study unveils the regulatory landscape of OTUD6A on TEAD4, providing insights into diseases regulated by YAP-TEAD complexes.

Phosphorylation of IDH1 Facilitates Progestin Resistance in Endometrial Cancer.

The progestin regimen is one of the main therapeutic strategies for women with endometrial cancer who undergo conservative management. Although many patients respond well to initial therapy, progestin-refractory disease inevitably emerges, and the molecular basis underlying progestin resistance has not been comprehensively elucidated. Herein, they demonstratedprogestin results in p38-dependent IDH1 Thr 77 phosphorylation (pT77-IDH1). pT77-IDH1 translocates into the nucleus and is recruited to chromatin through its interaction with OCT6. IDH1-produced α-ketoglutarate (αKG) then facilitates the activity of OCT6 to promote focal adhesion related target gene transcription to confer progestin resistance. Pharmacological inhibition of p38 or focal adhesion signaling sensitizes endometrial cancer cells to progestin in vivo. The study reveals p38-dependent pT77-IDH1 as a key mediator of progestin resistance and a promising target for improving the efficacy of progestin therapy.

Abstract LB033: Identification of small molecule Pan-TEAD inhibitors disrupting YAP-TEAD protein-protein interaction and targeting gastric cancer cells

Abstract Because of the Hippo pathway’s unique ability to restrict TAZ/YAP-TEAD in promoting regeneration, any abnormality of its core components, may promote the migration, invasion, and malignancy of cancer cells. Aberrant overexpression of YAP/TAZ-TEAD promotes tumorigenesis and is therefore considered oncogenes in many solid tumors. Drug resistance is a major factor undermining the efficacy of cancer drugs. As YAP/TAZ-TEAD complex plays a role in intrinsic and acquired resistance to various chemotherapeutic and targeted therapy drugs, there is increasing interest in combining a TEAD inhibitor with various cancer therapies. Since elevated YAP/TAZ-TEAD activity has been implicated in multiple cancer types at various stages of cancer progression and cancer types, Drugging the TEAD hydrophobic pocket, which was discovered in 2015, remains as an attractive and proven strategy to modulate the activity. Despite the progresses, only 3 small molecule TEAD inhibitors are currently being tested in Phase I clinical trials which includes: VT3989b NCT04665206 from Vivacare, IK-930b NCT05228015 from Ikena Oncology and IAG-933, NCT0485737 from the Novartis Oncology. As such, there is a need to uncover alternative TEAD inhibitors for cancer therapeutics. We report novel small molecule inhibitors that form a covalent complex with the conserved cysteine of the TEAD palmitoylation site. Compounds inhibited TEAD transcriptional target genes in cancer cells. Cellular proliferation and colony forming assay data strongly displayed cellular sensitivity in Hippo signaling altered cancer cell lines. TEAD1 dependency of gastric cancer cell lines enhance cellular sensitivity in response to small molecule inhibitors. Transcriptional profiling and RNA seq data indicate that the covalent inhibitors specifically downregulate down-stream transcriptional target genes. With our exciting key data, we are exploring multiple other oncology targets to use our Pan-TEAD inhibitors either as a monotherapy or combination therapy. We expect to complete our Technology Disclosure on additional targets before the meeting and to add exciting data for the Cancer drug development community. Citation Format: Ramesh Kumar, Joel Toh, Joanne Thian, Noorul Farzana Mohideen, Jialin Sun, Sayan Chakraborty, Jayantha Gunaratne, Wanjin Hong. Identification of small molecule Pan-TEAD inhibitors disrupting YAP-TEAD protein-protein interaction and targeting gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB033.

Abstract ND02: Discovery of BMS-986365, a ligand-directed androgen receptor degrader (AR LDD) with a dual mechanism-of-action and best-in-class potential, for the treatment of advanced prostate cancer

Abstract The androgen receptor (AR) is a key driver of the cellular processes that contribute to the pathology of prostate cancers at most stages, and drugs that inhibit the activity of this receptor or interfere with androgen biosynthesis have become the cornerstone of treatments for prostate cancer. While both types of therapy have had a significant positive impact on disease progression and overall survival, de novo and acquired resistance remains an impediment to a durable clinical response in patients with metastatic castration-resistant prostate cancer (mCRPC). The mechanisms underlying drug resistance are complex and multifactorial, being attributable to AR overexpression, point mutations in the receptor that alter drug pharmacology, and to the upregulation of receptor-associated coregulators, which render AR less dependent on an activating ligand. Regardless, absolute inhibition of AR signaling remains the objective of contemporary approaches to treat prostate cancer. Recently, there has been specific interest in the development of approaches to eliminate AR proteins using drugs that enable targeted degradation of AR in cancer cells.This puts in context the significance of our discovery of BMS-986365 (also known as CC-94676), a heterobifunctional ligand-directed degrader (LDD) that enables the CRL4CRBN E3 ligase-dependent ubiquitination and degradation of AR (DC50 10 to 40 nM, Ymin 7 to 19%). BMS-986365 is a highly potent and selective AR degrader that induces rapid and deep degradation of both wildtype and mutant forms of the receptor residing in either the cytoplasmic or nuclear compartments of the cell. The drug is ~100-fold more potent than enzalutamide (ENZ) at inhibiting androgen-stimulated transcription of AR target genes, and 10 to 120-fold more potent than ENZ at inhibiting AR-dependent proliferation of multiple prostate cancer cell lines in vitro. In animal models of advanced prostate cancer, BMS-986365 demonstrates on-target activity, degrading AR, suppressing AR signaling, and inhibiting tumor growth. Indeed, tumor volume reductions of 63-92% were achieved by BMS-986365 in validated models of advanced CRPC and therapy resistant patient-derived xenografts, including those with acquired resistance to ENZ. Collectively our preclinical data suggest that the AR degrader BMS-986365 is superior to standard-of-care AR antagonists, such as ENZ, in both preclinical and disease-relevant animal models, and support its clinical development for treatment of prostate cancer. BMS-986365 has advanced into clinical studies where it has demonstrated encouraging clinical activity in patients with mCRPC. Citation Format: Shuichan Xu, Surendra Nayak, John D. Norris, Massimo Ammirante, Emyly Rychak, Suzanne E. Wardell, Toshiya Tsuji, Ken Liu, Joseph Meiring, Joseph R. Piccotti, Deepak Dalvie, Debbie Liao, Raju Kandimalla, Nadia Guerrero, Lisa Sapinoso, Jennifer G. Baker, Yeeun Bae, Joshua Baughman, Brandon Toyama, Celia Fontanillo Fontanillo, Stephen Norris, Evan J. Horn, Veronique Plantevin-Krenitsky, Deborah Mortensen, Brian Cathers, Marie Hong Nguyen, Joshua D. Hensen, Lawrence G. Hamann, Donald P. McDonnell, Rama Krishna Narla, Mark Rolfe. Discovery of BMS-986365, a ligand-directed androgen receptor degrader (AR LDD) with a dual mechanism-of-action and best-in-class potential, for the treatment of advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND02.

Meningioma achieves malignancy and erastin-induced ferroptosis resistance through FOXM1-AURKA-NRF2 axis

The oncogene Aurora kinase A (AURKA) has been implicated in various tumor, yet its role in meningioma remains unexplored. Recent studies have suggested a potential link between AURKA and ferroptosis, although the underlying mechanisms are unclear. This study presented evidence of AURKA upregulation in high grade meningioma and its ability to enhance malignant characteristics. We identified AURKA as a suppressor of erastin-induced ferroptosis in meningioma. Mechanistically, AURKA directly interacted with and phosphorylated kelch-like ECH-associated protein 1 (KEAP1), thereby activating nuclear factor erythroid 2 related factor 2 (NFE2L2/NRF2) and target genes transcription. Additionally, forkhead box protein M1 (FOXM1) facilitated the transcription of AURKA. Suppression of AURKA, in conjunction with erastin, yields significant enhancements in the prognosis of a murine model of meningioma. Our study elucidates an unidentified mechanism by which AURKA governs ferroptosis, and strongly suggests that the combination of AURKA inhibition and ferroptosis-inducing agents could potentially provide therapeutic benefits for meningioma treatment.

Type IV Collagen Promotes Adipogenic Differentiation of Adipose Stem Cells.

Type IV collagen is a major component of the extracellular matrix in adipose tissue. It is secreted during the lipogenic differentiation of mesenchymal stem cells, but its direct impact and mechanism on the differentiation of adipose-derived stem cells (ASCs) into lipids are unclear. In this study, ASCs were obtained from human liposuction samples and cultured. Lipogenic induction of ASCs was achieved using lipogenic induction medium. Immunofluorescence analysis revealed differential expression of type IV collagen during the early and late stages of adipogenic induction, displaying a distinct morphological encapsulation of ASCs. Silencing of type IV collagen using siRNA resulted in a significant decrease in adipogenic capacity, as indicated by reduced lipid droplet formation and downregulation of adipogenic-related gene transcription. Conversely, supplementation of the culture medium with synthetic type IV collagen demonstrated enhanced adipogenic induction efficiency, accompanied by upregulation of YAP/TAZ protein expression and its downstream target gene transcription. Furthermore, inhibition of the YAP/TAZ pathway using the inhibitor Blebbistatin attenuated the functionality of type IV collagen, leading to decreased lipid droplet formation and downregulation of adipocyte maturation-related gene expression. These findings highlight the crucial role of type IV collagen in promoting adipogenic differentiation of ASCs and suggest its involvement in the YAP/TAZ-mediated Hippo pathway.No Level Assigned This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Abstract 1093: Small molecule inhibitors of the PAX3::FOXO1 fusion protein in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. It is commonly divided into subtypes based on histopathological appearance. The oncogenic fusion proteins PAX3::FOXO1 or PAX7::FOXO1 are present in the alveolar subtype of RMS (ARMS). These chimeric proteins are formed by reciprocal chromosomal translocations and consist of an N-terminal portion of the PAX3 or PAX7 transcription factor fused to a C-terminal portion of the FOXO1 transcription factor. These fusion proteins have been shown to drive malignant transformation in ARMS, and patients with fusion-positive ARMS have a worse prognosis than patients with fusion-negative ARMS and embryonal RMS. We recently reported that piperacetazine, a phenothiazine derivative and first-generation antipsychotic, is capable of binding to and inhibiting the transcriptional activity of PAX3::FOXO1, reducing the expression of PAX3::FOXO1 target genes, and inhibiting the ability of fusion-positive RMS cells to grow in soft agar. We hypothesize that other phenothiazine derivatives may have stronger binding affinity for PAX3::FOXO1 and greater ability to reduce tumor growth in vivo. We employed a structure-activity relationship campaign to develop new phenothiazine derivatives that may be more potent inhibitors of PAX3::FOXO1 than piperacetazine. We synthesized and tested 10 new compounds, in addition to 9 existing FDA-approved phenothiazine derivatives, for their ability to bind to PAX3::FOXO1 and inhibit its transcriptional activity and target gene expression as well as inhibiting the growth of fusion-positive RMS cells in 3D culture. Seven derivatives inhibited PAX3::FOXO1 reporter activity better than piperacetazine, nine of them performed worse, and three of them showed comparable activity to piperacetazine. We demonstrated that piperacetazine does not alter PAX3::FOXO1 levels, subcellular localization, binding to a PAX3::FOXO1 target DNA sequence, or phosphorylation at Ser256 in the FOXO1 portion of the fusion protein. We therefore hypothesize that piperacetazine may be disrupting interactions between PAX3::FOXO1 and its partner proteins. We are employing a proximity-based biotinylation method to identify proteins that are competed away from PAX3::FOXO1 by piperacetazine. Our data suggest that it is possible to target PAX3::FOXO1 protein using small molecules that can directly bind to it and inhibit its activity in fusion-positive RMS cells. Citation Format: Taryn Shaw, Kay Nakazawa, Purushottam Tiwari, Eryn Nelson, Jeffrey Formen, Christian Wolf, Jeffrey Toretsky, Aykut Üren. Small molecule inhibitors of the PAX3::FOXO1 fusion protein in rhabdomyosarcoma [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 1093.

Abstract 5217: Genome instability in renal tumors

Abstract Genome instability (GI), a recognized cancer hallmark, encompasses CNV burden, tumor mutational burden (TMB), and microsatellite instability and is variably associated with several clinical/prognostic features in solid tumors. GI and intratumor mutational heterogeneity in clear cell renal cell carcinoma (ccRCC) were associated with poor prognosis in a large multi-site sequencing study. As 10-15% of renal tumors exhibit copy number burden associated GI, the development of proteogenomic biomarkers to identify this subset of tumors will be of high clinical value. In our in-depth analysis of CPTAC renal cancer cohorts namely, ccRCC discovery and rare RCC, we first estimated absolute copy number using CNVEX TPO analysis pipeline. Copy number burden-based genome instability was estimated by calculating whole genome instability index (wGII) from DNA sequencing data, in addition to the estimation of genome ploidy and purity of the tumors. Genome instability was associated with survival outcomes in both ccRCC and rare RCC (Ref. 1 and 2). Differential proteogenomic analysis identified IGF2BP3, IGF2BP1, PYCR1 RNA and protein, to be upregulated among high wGII tumors. High wGII tumors showed upregulation of cell proliferation and other cell cycle/proliferation concepts in gene set enrichment analysis. Interestingly, transcriptional module analysis revealed transcription factors and target genes specifically upregulated in GI cases and this pattern showed certain degree of overlap to 9p loss ccRCC tumors (another molecular ccRCC subset that is also associated with poor prognosis). Differential expression analysis also identified, kinases, phosphosites and co-regulated kinase-substrates that are dysregulated in genome instable renal tumors. IGF2BP3 immunohistochemistry staining showed specific overexpression in high wGII tumors and the high positivity was observed exclusivity in the tumor cells. Prognostic biomarkers IGF2BP3, IGF2BP1, PYCR1, and UCHL1 show great promise to be developed as clinical prognostic assays and warrants additional characterization. Citation Format: Hanbyul Cho, Ginny X. Li, Rahul Mannan, Noshad Hosseini, Yi Hsiao, Yuping Zhang, Aniket Dagar, Chandan Kumar-Sinha, Marcin P. Cieslik, Saravana M. Dhanasekaran, Alexey I. Nesvizhskii, Arul M. Chinnaiyan, Clinical Proteomic Tumor Analysis Consortium. Genome instability in renal tumors [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 5217.

Abstract 7355: Integration analysis of lncRNAs and R-loops in 3D genome organization and gene regulation in the cancer genome

Abstract Existing evidence shows that some long non-coding RNAs (lncRNAs) can recruit chromatin regulators and form R-loops to mediate CCTCC-binding factor (CTCF) binding at topologically associated domain (TAD) boundaries to activate transcription of target genes. However, it remains unclear to what extent such lncRNA-dependent chromatin organization events occur genome-wide in normal and cancer cells. By curated public data of lncRNA, CTCF, RAD21 profiles and integrative analysis of multi-omics data using innovative computational approaches, we investigated global associations of lncRNAs and R-loops with activities of transcription factors and chromatin regulators in gene expression regulations across multiple human cell types. We developed a comprehensive computational pipeline for cross-cell-type analysis of lncRNA and protein-coding gene expression profiles and transcription factor binding profiles. We identified a total of 542 candidate genomic regions where lncRNAs play a pivotal role in regulating target genes by facilitating CTCF/cohesin binding. R-loops are enriched at CTCF/cohesin binding sites, suggesting their potential contribution to CTCF binding. Additionally, our findings underscore the significant cell-type specificity of CTCF binding patterns across the genome. This work adds knowledge to cancer genome organization and gene regulation, and builds foundations for further functional and mechanistic studies of lncRNAs and R-loops in the human genome. Citation Format: Shengyuan Wang, Zhenjia Wang, Yuh-Hwa Wang, Hui Li, Peter Todd Stukenburg, Chongzhi Zang. Integration analysis of lncRNAs and R-loops in 3D genome organization and gene regulation in the cancer genome [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 7355.

Abstract 2113: HMPL-506, a novel, highly potent and differentiated menin-MLL inhibitor for the treatment of MLL-rearrangedand NPM1mutant acute leukemia in preclinical models

Abstract Background: Mixed-lineage leukemia (MLL, or KMT2A) gene rearrangements (MLL-r) occur in 5%-10% acute leukemias and are associated with poor prognosis. Nucleophosmin 1 mutations (NPM1m) are the most common genetic alterations in acute myeloid leukemia (AML). Multiple studies have illuminated that those leukemogenesis are dependent on the interaction of menin-MLL, which controls downstream gene expressions associated with cell proliferation and differentiation, e.g. HOXA9 and CD11b. Herein, we introduce HMPL-506, a novel and highly differentiated small molecule compound targeting menin-MLL interaction. Methods and Results: Biochemical assay revealed that HMPL-506 potently blocked menin-MLL binding with IC50 of 1.0 nM. In cell lines carrying MLL-r or NPM1m, HMPL-506 substantially down-regulated menin-MLL target genes transcription of MEIS1 and HOXA9 and upregulated the differentiation marker of CD11b by RT-PCR assay, consequently, HMPL-506 attenuated tumor cell growth, with GI50 ranging from 3.0 to 12.1 nM in MLL-r cell lines (MV-4-11, MOML-13 and RS4;11) and 22.4 nM in NPM1m cell line (OCI-AML-3) in CellTiter-Glo cell viability assay. Notably, compared with the other 5 menin inhibitors in clinical stage, HMPL-506 showed the strongest inhibitory potency in MLL-r and NPM1m cell line models. In vivo studies demonstrated the dose- and exposure- dependent target regulation of MEIS1 and anti-tumor efficacy following HMPL-506 treatment in multiple tumor xenograft models. For instance, in MV-4-11 subcutaneous model, once daily oral administration of HMPL-506 at 5, 10 and 25 mg/kg for 4 days in BALB/c nude mice induced dose-dependent inhibition of MEIS1 gene expression, positively correlating with compound exposures in tumor tissues; continuous dosing of HMPL-506 at 5 mg/kg for 28 days reduced the tumor growth by 86%, which was comparable to the efficacy induced by SNDX-5613 at 50 mg/kg. Treatment of HMPL-506 at 10 mg/kg and 25 mg/kg resulted in tumor shrinkage in all treated animals, with tumor regression rates of 72% and 100%, respectively. Furthermore, HMPL-506 synergistically improved anti-tumor effect of azacytidine, venetoclax and gilteritinib against MLL-r leukemias both in vitro and in vivo. HMPL-506 displayed favorable PK profiles and high selectivity among multiple kinases, methyltransferases and safety related targets. More importantly, the IC50 of HMPL-506 on hERG patch clamp was over 60 µM, indicating low risk of QTc prolongation in human. Conclusion: HMPL-506 is a novel and highly differentiated menin-MLL inhibitor with robust anti-tumor activities, favorable ADME properties and low risk of cardiac toxicity, warranting it further clinical development for treatment of MLL-r and NPM1m acute leukemias. HMPL-506 IND is expected to be cleared in H1 2024. Citation Format: Min Cheng, Liang Ge, Zhihu Gao, Zeyu Zhong, An Jiang, Wei Zhang, Jia Hu, Shuwen Jiang, Na Li, Na Yang, Jian Wang, Yang Sai, Weiguo Qing, Yongxin Ren, Weiguo Su. HMPL-506, a novel, highly potent and differentiated menin-MLL inhibitor for the treatment of MLL-rearrangedand NPM1mutant acute leukemia in preclinical models [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 2113.

Abstract 141: A human induced pluripotent stem cell model of KMT2A rearranged leukemia

Abstract Acute lymphoblastic leukemia in infants (iALL) is a high-risk subtype of childhood leukemia with poor survival outcomes despite intensive therapies. Rearrangement of KMT2A (KMT2A-r) occurs in 70% of cases and is associated with refractoriness to therapy, early relapse, and rapid leukemia progression. KMT2A-r generates a driver fusion oncogene, most commonly KMT2A::AFF1 in iALL, which leads to epigenetic dysregulation of target gene transcription. iALL with KMT2A::AFF1 is an unusual cancer in that other somatic mutations are uncommon, there are no known genetic risk factors, and phenotypic switch from lymphoid to myeloid, mixed, or undifferentiated phenotype is common during treatment and particularly resistant to therapies. The cell of origin is thought to be a very early hematopoietic precursor, with transcriptomic studies of iALL blasts showing similarities to hematopoietic stem and progenitor cells (HSPCs), multipotent progenitors and early lymphoid progenitors (ELPs). Unfortunately, research into this rare but devastating disease has been hindered by a lack of appropriate, representative models. Interestingly, KMT2A-r originates in HSPCs in utero and the age of onset of iALL is a strong prognostic factor, with age younger than six months associated with the highest risk of relapse. However, little is known regarding how KMT2A-r subverts early hematopoiesis or drives the severe disease phenotype. In an effort to understand the role of the developmental state of the cell of origin in iALL, we have created a highly controlled induced pluripotent stem (iPS) cell model system of KMT2A::AFF1 leukemia. Specifically, we engineered human iPS cell lines with doxycycline regulatable expression of KMT2A::AFF1 fusion and we are employing CRISPR gene editing technology to introduce clinically identified variants of interest. Utilizing directed differentiation, we have produced functional HSPCs from iPS cells. Notably, this model recapitulates hematopoietic ontogeny, with the ability to control iALL induction at specific developmental stages. Following doxycycline treatment, we demonstrate that the cells express KMT2A::AFF1 fusion transcripts. We are utilizing single cell genomics to investigate transcriptomic changes during hematopoietic differentiation of our KMT2A::AFF1 iPS cells. We will compare these results to published data sets of single cell RNA sequencing data from healthy fetal HSPCs and to our existing iALL and pediatric KMT2A-r ALL single cell sequencing data sets. Our iPS cell based iALL model system provides the opportunity to investigate a range of critical and outstanding questions of iALL disease initiation, progression, and treatment. Citation Format: Erin Guest, Meagan Vacek, Jacqelyn Nemechek, Irina Pushel, Bradley T. Thornton, Molly Leyda, Midhat S. Farooqi, John M. Perry, Jay L. Vivian. A human induced pluripotent stem cell model of KMT2A rearranged leukemia [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 141.

Abstract 6441: Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer

Abstract Background. HSF1 helps cancer cells cope with multiple stresses caused by oncogene activation. Methods. See details below. Results. We discovered the bisamide NXP800 as an inhibitor of HSF1-mediated transcription through phenotypic screening and med chem optimization. Gene expression microarray analysis of human cancer cell lines and tumor xenografts treated with bisamide inhibitors indicated activation of the ATF4 axis of the ISR as a key mechanism of action. In SK-OV-3 ovarian cancer xenografts, NXP800 showed clear PK/PD relationships with increased expression of ATF4 transcriptional target genes alongside decreased expression of HSF1 transcriptional targets. NXP800 also caused tumor regressions in this model. Expanding in additional human ovarian cancer xenograft models, we observed efficacy in five and a complete lack of response in three others. The sensitive models all had homozygous deleterious mutations in the ARID1A gene, whereas the non-responding xenografts were all wild type (WT). ARID1A is a component of the SWI/SNF chromatin remodelling complex involved in repression and activation of target genes. Subsequent screening of the large Sanger human cancer cell line panel confirmed ARID1A as the most significant common disease-related alteration predicting sensitivity to NXP800 in ovarian cancer cell lines. This predictive relationship was confirmed in an ARID1A isogenic HCT-116 cell line pair; sensitivity was greater in the homozygous ARID1A mutant cells compared to WT, resulting in PARP cleavage in the mutant cells only. In vivo there was no effect in WT HCT-116 xenografts whereas growth inhibition was observed in the homozygous mutant cells, resulting in significantly smaller tumors. In addition, the induction of ATF4 target genes was stronger and more prolonged in the mutant cells. It is known that whereas short term ATF4 activation is adaptive, persistent activation can promote the induction of apoptosis. CHIP-seq analysis confirmed clear relationships between ARID1A status, ATF4 and HSF1 promoter occupancy, and the distribution of BRG1 and RNA pol II at target sites, although these were often gene-specific and complex. A relatively simple example is the regulation of the INHBE gene (expression of which is a PD biomarker). In untreated samples there is no binding of ATF4, BRG1 and RNA pol II at the INHBE promoter. NXP800 treatment results in the recruitment of BRG1, ATF4 and RNA pol II in ARID1A mutant TOV-21G cells but not in the RMGI WT cells, leading to increased expression in the mutant cells only. Conclusions. We propose that ARID1A loss alters the binding and recruitment of ATF4 and HSF1 leading to the altered and prolonged expression of ATF4 target genes and increased sensitivity to NXP800. NXP800 is currently in phase Ib for the treatment of ARID1A mutant platinum resistant ovarian cancer (NCT05226507). Citation Format: Robert H. te Poele, Marissa Powers, Swee Sharp, Emmanuel de Billy, Maria Taskinen, Loredana Pellegrino, Sharon Gowan, Asadh Miah, Angela Hayes, Matthew Cheeseman, Keith Jones, Suzanne Eccles, Florence Raynaud, Paul Clarke, Paul Workman. Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer [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 6441.

Tubuloside B, a major constituent of Cistanche deserticola, inhibits migration of hepatocellular carcinoma by inhibiting Hippo-YAP pathway

BackgroundStudies have shown that phenylethanoid glycosides (PhGs) have multiple pharmacological effects such as anti-inflammatory, hepatoprotective or neuroprotective functions, whereas their anti-tumor effects are rarely studied. Tubuloside B (Tub B) is a PhG isolated from Cistanche deserticola, a traditional Chinese medicine. To date, there is a lack of comprehensive research regarding the biological activity of Tub B. PurposeThe subject of the current study was to investigate the anti-hepatocellular carcinoma (HCC) cell activity and the underlying mechanism of Tub B. MethodsWe evaluated the in vitro anti-migratory effect of Tub B by scratch and transwell assays. RNA-seq was employed to identify the differential genes by Tub B. Besides, the functional mechanism of Tub B was investigated by distinct molecular biology techniques including immunofluorescent staining, quantitative PCR, as well as western blot analysis. Subsequently, we utilized Hep3B cells for in vivo metastasis assays through spleen injection and evaluated the anti-migratory effect of Tub B in hepatocellular carcinoma (HCC). ResultsTub B exhibited in vitro and in vivo inhibition of HCC cell migration. Tub B decreased the expression of transcriptional target genes downstream of the Hippo pathway, including CTGF, CYR61, and N-cadherin as determined by RNA-seq. Furthermore, mechanistic studies confirmed that Tub B increased phosphorylation of YAP at S127, which contributes to YAP cytoplasmic localization. Additionally, overexpression of YAP abrogated Tub B-induced inhibition of HCC migration and the mRNA levels of CTGF, CYR61, and N-cadherin. ConclusionsTaken together, these results illustrated that Tub B demonstrated great potential in inhibiting migration of HCC, and a portion of its impact can be attributed to the modulation of the Hippo-YAP pathway.

Utility of LEF1 to differentiate desmoid fibromatosis from its histologic mimics.

Diagnosis of desmoid-type fibromatosis (DF) may be challenging on biopsy due to morphologic overlap with reactive fibrosis (scar) and other uniform spindle cell neoplasms. Evaluation of nuclear β-catenin, a surrogate of Wnt pathway activation, is often difficult in DF due to weak nuclear expression and high background membranous/cytoplasmic staining. Lymphoid enhancer-factor 1 (LEF1) is a recently characterized effector partner of β-catenin which activates the transcription of target genes. We investigated the performance of LEF1 and β-catenin immunohistochemistry in a retrospective series of 156 soft tissue tumors, including 35 DF, 3 superficial fibromatosis, and 121 histologic mimics (19 soft tissue perineurioma, 8 colorectal perineurioma, 4 intraneural perineurioma, 26 scars, 23 nodular fasciitis, 6 low-grade fibromyxoid sarcomas, 6 angioleiomyomas, 5 neurofibromas, 5 dermatofibrosarcoma protuberans, 3 low-grade myofibroblastic sarcomas, 3 synovial sarcomas, 3 inflammatory myofibroblastic tumors, 2 schwannomas, and 1 each of Gardner-associated fibroma, radiation-associated spindle cell sarcoma, sclerotic fibroma, dermatofibroma, and glomus tumor). LEF1 expression was not only seen in 33/35 (94%) of DF but also observed in 19/23 (82%) nodular fasciitis, 7/19 (37%) soft tissue perineurioma, 2/3 (66%) synovial sarcoma, and 6/26 (23%) scar, as well as in 1 radiation-associated spindle cell sarcoma. The sensitivity and specificity of LEF1 IHC for diagnosis of DF were 94% and 70%, respectively. By comparison, β-catenin offered similar sensitivity, 94%, but 88% specificity. Positivity for LEF1 and β-catenin in combination showed sensitivity of 89%, lower than the sensitivity of β-catenin alone (94%); however, the combination of both LEF1 and β-catenin improved specificity (96%) compared to the specificity of β-catenin alone (88%). Although LEF1 has imperfect specificity in isolation, this stain has diagnostic utility when used in combination with β-catenin.