Pax6 Binding Research Articles

Functional variant rs9344 at 11q13.3 regulates CCND1 expression in multiple myeloma with t(11;14).

Multiple myeloma (MM) is a plasma cell (PC) malignancy characterized by cytogenetic abnormalities, such as t(11;14)(q13;q32), resulting in CCND1 overexpression. The rs9344 G allele within CCND1 is the most significant susceptibility allele for t(11;14). Sequencing data from 2 independent cohorts, CoMMpass (n = 698) and Mayo Clinic (n = 661), confirm the positive association between the G allele and t(11;14). Among 80% of individuals heterozygous for rs9344 with t(11;14), the t(11;14) event occurs on the G allele, demonstrating a biological preference for the G allele in t(11;14). Within t(11;14), the G allele is associated with higher CCND1 expression and elevated H3K27ac and H3K4me3. CRISPR/Cas9 mediated A to G conversion resulted in increased H3K27ac over CCND1 and elevated CCND1 expression. ENCODE ChIP-seq data supported a PAX5 binding site within the enhancer region covering rs9344, showing preferential binding to the G allele. Overexpression of PAX5 resulted in increased CCND1 expression. These results support the importance of rs9344 G enhancer in increasing CCND1 expression in MM.

Rare germline genetic variation in PAX8 transcription factor binding sites and susceptibility to epithelial ovarian cancer.

Common genetic variation throughout the genome together with rare coding variants identified to date explain about a half of the inherited genetic component of epithelial ovarian cancer risk. It is likely that rare variation in the non-coding genome will explain some of the unexplained heritability, but identifying such variants is challenging. The primary problem is lack of statistical power to identifying individual risk variants by association as power is a function of sample size, effect size and allele frequency. Power can be increased by using burden tests which test for association of carriers of any variant in a specified genomic region. This has the effect of increasing the putative effect allele frequency. PAX8 is a transcription factor that plays a critical role in tumour progression, migration and invasion. Furthermore, regulatory elements proximal to target genes of PAX8 are enriched for common ovarian cancer risk variants. We hypothesised that rare variation in PAX8 binding sites are also associated with ovarian cancer risk, but unlikely to be associated with risk of breast, colorectal or endometrial cancer. We have used publicly available, whole-genome sequencing data from the UK 100,000 Genomes Project to evaluate the burden of rare variation in PAX8 binding sites across the genome. Data were available for 522 ovarian cancers, 2,984 breast cancers, 2,696 colorectal cancers, 836 endometrial cancers and 2253 non-cancer controls. Active binding sites were defined using data from multiple PAX8 and H3K27 ChIPseq experiments. We found no association between the burden of rare variation in PAX8 binding sites (defined in several ways) and risk of ovarian, breast or endometrial cancer. An apparent association with colorectal cancer was likely to be a technical artefact as a similar association was also detected for rare variation in random regions of the genome. Despite the null result this study provides a proof-of -principle for using burden testing to identify rare, non-coding germline genetic variation associated with disease. Larger sample sizes available from large-scale sequencing projects together with improved understanding of the function of the non-coding genome will increase the potential of similar studies in the future.

Abstract 1685: Investigating PAX8 interactions in ovarian cancer

Abstract Ovarian cancer is the most lethal gynecological cancer and there is a critical unmet need to develop novel therapies. The transcription factor, PAX8, is upregulated in ~80% of renal, ovarian, endometrial and thyroid tumors and its overexpression correlates with higher risk of death and recurrence. PAX8 represents a promising target because of its high specificity and low expected toxicity, but how it promotes tumorigenesis remains elusive. Using proximity labeling followed by mass spectrometry, we identified a list of PAX8 binding partners and have further confirmed these interactions at the genomic level through ChIP-seq and ATAC-seq. Our findings reveal novel PAX8 interactions and a potential therapeutic approach for ovarian cancer treatment. Citation Format: Kostianna Sereti, Anna Russo, Ryan Raisner, Karen Gascoigne. Investigating PAX8 interactions 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 1685.

Abstract 3589: Understanding the role of Pax5 in development of taxane-resistant NE-like prostate cancer

Abstract Background: Resistance to the existing Androgen Receptor Signaling Inhibitor (ARSI) therapies led to higher incidences of aggressive therapy-induced neuroendocrine-like prostate cancer (t-NEPC) with predominant small cell-like characteristics, dismal overall survival, and an ultimate resistance to taxane chemotherapies. t-NEPCs are mostly treated with platinum-based drugs with etoposide and/or taxane but have less selectivity and high systemic toxicity, which often limits their clinical and therapeutic potential. During t-NEPC transformation, upon chromatin reprogramming associated with epigenetic alterations and altered transcriptional activity, adenocarcinoma loses its luminal features to adopt neurobasal characteristics. The following study aims to unravel the mechanism of developing t-NEPC taxane resistance upon acquiring such adaptive neuronal characteristics. Objective: The primary objective is to delineate how chromatin modifications along with epigenetic regulation facilitates the adaptive neuronal characteristics and dictates the development of t-NEPC taxane resistance. Methods: We generated various t-NEPC cell lines either by genetic modification (DKD) or therapeutic pressure (C4-2BER). We performed RNA-Seq, ATAC-Seq and acetylated Histone (H3K18 and H3K27) ChIP-Seq in our developed t-NEPC models and compared with adenocarcinoma. We next compared ATAC Seq and Acetylated histone ChIP Seq with RNA Seq in C4-2B and C4-2BER and performed functional assays. Results: Our ATAC Seq and acetylated histone footprints (Ac H3K18 and Ac H3K27 ChIP Seq) analyses revealed an enhanced chromatin accessibility during t-NEPC transformation. Overlapping RNA seq suggested newly transcribed neuronal genes with higher extent of promoter accessibility. Transcription Factor mapping among newly accessible gene promoters revealed preferential Pax5 binding, whose expression occurs selectively in t-NEPC unlike adenocarcinoma. Pathway analysis of differentially expressed neuronal genes in t-NEPC suggested an involvement of Pax5 in axonal guidance, neurotransmitter regulation, and neuronal adhesion, which are essential for strong cellular communication. Furthermore, Pax5 depletion disrupts cellular interaction in t-NEPC, thereby, sensitizing them to taxane therapies. Conclusions: Our study concludes that continuous AR ablation with ARSIs, leads to chromatin alterations with epigenetic changes, thereby allowing Pax5 transcriptional activation in t-NEPC. This altered transcriptional activity promotes gene expression necessary to govern taxane-resistant t-NEPC development. Citation Format: Sreyashi Bhattacharya, Ridwan Islam, Sanika Bodas, Juhi Mishra, Dipanwita Das, Kaustubh Datta, Samikshan Dutta. Understanding the role of Pax5 in development of taxane-resistant NE-like prostate 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 3589.

Multi ‐ omics studies of eye and lens development

Early eye development involves formation of a symmetric pair of optic vesicles from the anterior neuroectoderm and parallel formation of closely apposed lens placodes. Their reciprocal invaginations generate the 3D‐foundation of the optic cups. Embryonic lens development is initiated within the border between the anterior neuroectoderm and naïve ectoderm, called the anterior pre‐placodal region. The earliest stages of lens development require coordinated actions of DNA‐binding transcription factor Pax6, signalling growth factor BMP4 and a pair of chromatin remodelling enzymes p300 and CBP. Studies in model vertebrates postulated a common Pax6+ progenitor cell population to generate both the lens and olfactory placodes. Recent progress in technologies to probe chromatin structure, landscape of transcription factor binding, DNA methylation, coding (mRNA) and noncoding (e.g. eRNAs and ncRNAs) transcriptomes and chromatin conformation have been shown to generate unprecedented mechanistic insights into the earliest cell fate decisions and tissue organization. To examine lens development at E8.5, E9.5 and E10.5 mouse embryos, single cell RNA‐seq (scRNA‐seq) experiments using the 10XGenomics Chromium platform were conducted. E9.5 Pax6 null embryos and controls were also included. Whole genome bisulfite sequencing (WGBS) was performed in E14.5 and P0.5 microdissected mouse lenses to map DNA methylation and integrate the data with corresponding ATAC‐ and RNA‐seq data sets. Open chromatin and DNA methylation changes were defined through identification of differentially accessible regions (DARs), unmethylated/low methylated regions (UMRs/LMRs) coupled with differential gene expression during lens differentiation. To probe 3D‐organization of lens chromatin and to identify temporally and spatially regulated lens‐specific distal enhancers, Hi‐C was used in conjunction with ChIP‐seq analysis of transcription factor CTCF in microdissected newborn lenses. Previous ChIP‐seq data using whole newborn lenses include H3K27ac peaks to identify super‐enhancers. Pax6 proteins were found both in open and closed chromatin domains, including both closed and methylated regions. In vitro binding of Pax6 proteins was not inhibited by 5‐methylcytosines within one or two CpG dinucleotides present in the Pax6 binding sites. In summary, cell fate decisions are governed by complex changes in chromatin landscape controlled by sequence‐specific DNA‐binding transcription factors and their recruitment of chromatin remodelling complexes. A combination of different –omics is a powerful approach to examine molecular mechanisms governing eye development. These studies also reveal additional levels of genetic code complexity and stimulate follow up mechanistic studies of the individual cellular and molecular processes of lens morphogenesis and their underlying GRNs and identification of cataract‐causing mutation in non‐coding regulatory sequences.

Functional Characteristics of Diverse PAX6 Mutations Associated with Isolated Foveal Hypoplasia.

The human fovea is a specialized pit structure in the central retina. Foveal hypoplasia is a condition where the foveal pit does not fully develop, and it is associated with poor vision. Autosomal dominant isolated foveal hypoplasia (FVH1) is a rare condition of foveal hypoplasia (FH) that lacks any other ocular manifestations. FVH1 is associated with hypomorphic mutations in the PAX6 gene that encodes a sequence-specific DNA-binding transcription factor for morphogenesis and evolution of the eye. We report our findings in 17 patients with PAX6 mutations associated with FVH1 or FH with aniridia and corneal opacities. Patients with three mutations, p.V78E, p.V83F and p.R128H, in the C-terminal subdomain of the paired domain (CTS) consistently have severe FH. Luciferase assays for a single reporter containing a representative PAX6 binding site indicated that the transcriptional activities of these mutations were significantly reduced, comparable to that of the truncation mutation of p.G65Rfs*5. Patients with p.P20S in the N-terminal subdomain of the paired domain, and a patient with p.N365K in the proline-serine-threonine-rich domain (PSTD) had mild FH. A patient with p.Q255L in the homeodomain had severe FH. The P20S and Q255L mutants did not affect the transcriptional activity. Mutant N365K has a retained DNA-binding activity but a reduced transcriptional activity, due to a low PSTD transactivation. These findings demonstrated that mutations associated with FVH1 underlie a functional divergence between DNA-binding ability and transcriptional activity. We conclude that a wide range of mutations in the PAX6 gene is not limited to the CST region and are responsible for FVH1.

PAX5 and circ1857 affected DLBCL progression and B-cell proliferation through regulating GINS1.

PAX5, a member of the paired box gene family of transcription factors, is a B-cell-specific activator protein that plays important roles during B lymphopoiesis. Two putative PAX5 binding sites in the human GINS1 promoter region were identified. EMSA, ChIP and luciferase assay showed that PAX5 functions as a positive transcription factor for GINS1 expression. Furthermore, coordinated expression of PAX5 and GINS1 was observed in mice B cells under physiological conditions and LPS stimulation situations. A similar pattern was also observed in human DLBCL cell lines under differentiation-inducing conditions. In addition, both PAX5 and GINS1 were highly expressed and significantly correlated in DLBCL specimens and cell lines. These findings suggested that dysregulation of PAX5 played an extremely important role in controlling the universal phenomenon of tumor progression through increased expression of GINS1 in DLBCL. In addition, circ1857 that was generated using back splicing of PAX5 pre-mRNA could further stabilize GINS1 mRNA, modulate GINS1 expression and promote lymphoma progression. To the best of our knowledge, this report is the first to demonstrate the role of GINS1 in DLBCL progression, and the mechanism of GINS1 upregulation using both circ1857 and PAX5 in DLBCL was revealed. Our results suggested that GINS1 may be a possible therapeutic target for DLBCL.

PAX5 Epigenetically Orchestrates CD58 Transcription and Modulates Blinatumomab Response in Acute Lymphoblastic Leukemia

Blinatumomab is a bi-specific molecule that engages CD3+ T cells with CD19-expressing malignant B cells, thus inducing tumor lysis by T-cell immune response. While large frontline ALL trials of blinatumomab are lacking, existing data strongly indicate substantial inter-patient variability in response and toxicities. Complete remission rate ranges from 36 to 66%, depending on the regimen used and patient characteristics. Moreover, the duration of remission varies among trials, with a significant proportion of patients experiencing relapse or progressive disease following blinatumomab treatment, raising concerns about acquired drug resistance. In particular, mechanisms of resistance in CD19+ relapse remain largely unclear. Using genome-wide CRISPR screen in B-ALL cell line in vitro, we comprehensively identified leukemia intrinsic factors of blinatumomab sensitivity. In addition to CD19 deletion the loss of CD58 emerged as a top driver for resistance, plausibly compromising the CD2-CD58-mediated interaction between T cells and B-ALL. Genomic editing of the CD58 gene or blocking with anti-CD58 antibody completely abrogated blinatumomab sensitivity across different B-ALL cell lines. Screening 1,639 transcription factor genes, we then identified PAX5 as the key activator of CD58. Interestingly, primary ALL samples with PAX5 P80R mutation also showed complete loss of CD58 expression, based on RNA-seq of 1,988 B-ALL samples across 20 molecular subtypes. To further validate this, we developed isogenic clones of the B-ALL cell line 697, with different PAX5 diplotypes: wildtype/wildtype (WT/WT), wildtype/frameshift (WT/fs), and P80R/frameshift (P80R/fs). P80R/fs and WT/fs clones exhibited significantly lower CD58 expression as compared to the WT/WT clone, and both were markedly resistant to blinatumomab. PAX5 mutation did not influence CD19 expression in these B-ALL cells. By testing a panel of primary ALL samples in vitro, we found that blast harboring the PAX5 P80R mutation has the lowest CD58 expression and was the most resistant to blinatumomab, which was reversed by ectopic expression of CD58. To delineate the mechanism by which PAX5 mutation affects CD58 suppression in ALL, we comprehensively profiled epigenomic marks within the CD58 locus in 697 cells with WT/fs, P80R/fs, and WT/WT genotypes. Using ChIP-seq, we identified PAX5 binding sites within Intron 1 of the CD58 genes. By contrast, PAX5 binding at this locus was decreased in WT/fs cells and completely eliminated in the P80R/fs clones, suggesting a loss-of-function effect of the P80R mutation. This region was also characterized by H3K27ac and H3K4me3 marks, signifying its enhancer activity, which was also lost in P80R/fs cells. ATAC-seq profiling of these samples showed a similar pattern of PAX5-dependent open chromatin status. CRISPR interference showed that blocking the genomic sequence encompassing this enhancer resulted in significant downregulation of CD58 expression. PAX5 binding was also observed in the CD19 promoter region, but P80R mutation had only modest effects on histone modification and chromatin accessibility at this locus. Finally, we explored the effects of CD58 loss on T cell activation. T cell cocultured with CD58KO Nalm6 clone showed a substantial decrease in activation and proliferation, as compared to that cocultured with parental Nalm6. Defects of activation and proliferation were also observed in T cells cocultured with PAX5 P80R/fs 697 clone as compared to those with parental 697. Using RNA-seq, we identified 111 genes that were highly expressed in T cells cocultured with parental (relative to those with CD58KO Nalm6). Among these hits, there was significant enrichment of genes involved in oxygen metabolisms, immune response, and cytokine signaling pathways, indicating the loss of CD58 abolished blinatumomab-induced T cell activation. ATAC-seq of these samples further confirmed the findings of RNA-seq studies. In conclusion, our study identified the critical role of CD58-mediated interaction between B-ALL and T cells for blinatumomab sensitivity in this type of leukemia, pointing to potential strategies for genomics-guided blinatumomab treatment individualization.

Biphasic cell cycle defect causes impaired neurogenesis in down syndrome.

Impaired neurogenesis in Down syndrome (DS) is characterized by reduced neurons, increased glial cells, and delayed cortical lamination. However, the underlying cause for impaired neurogenesis in DS is not clear. Using both human and mouse iPSCs, we demonstrate that DS impaired neurogenesis is due to biphasic cell cycle dysregulation during the generation of neural progenitors from iPSCs named the “neurogenic stage” of neurogenesis. Upon neural induction, DS cells showed reduced proliferation during the early phase followed by increased proliferation in the late phase of the neurogenic stage compared to control cells. While reduced proliferation in the early phase causes reduced neural progenitor pool, increased proliferation in the late phase leads to delayed post mitotic neuron generation in DS. RNAseq analysis of late-phase DS progenitor cells revealed upregulation of S phase-promoting regulators, Notch, Wnt, Interferon pathways, and REST, and downregulation of several genes of the BAF chromatin remodeling complex. NFIB and POU3F4, neurogenic genes activated by the interaction of PAX6 and the BAF complex, were downregulated in DS cells. ChIPseq analysis of late-phase neural progenitors revealed aberrant PAX6 binding with reduced promoter occupancy in DS cells. Together, these data indicate that impaired neurogenesis in DS is due to biphasic cell cycle dysregulation during the neurogenic stage of neurogenesis.

Co-expression and Interaction of Pax6 with Genes and Proteins of Immunological Surveillance in the Brain of Mice.

The Pax6 binds to promoter sequence elements of genes involved in immunological surveillance and interacts with Iba1, p53, Ras-GAP, and Sparc in the brain of mice. The Pax6 also affects the expression pattern of genes involved in neurogenesis and neurodegeneration. However, the expression and association of Pax6 in the brain under immunologically challenged conditions are still elusive. Therefore, it has been intended to analyze the association of Pax6 in the immunity of the brain using the immune-challenged Dalton's lymphoma (DL) mice model. The expressions of Pax6, Iba1, and Tmem119 decreased, but expressions of Ifn-γ, Tnf-α, Bdnf, and Tgf-β increased in the brain of immune-challenged mice as compared to the control. The level of co-expression of Pax6 decreased in dual positive cells with Iba1, Tmem119, Sparc, p53, Bdnf, and Tgf-β in the brain of immune-challenged mice. Binding of Pax6 to multiple sites of the promoter sequences of Bdnf and Tgf-β indicates their Pax6-associated differential expression and association with immune responsive gene. The levels of binding of Pax6 to Tmem119, Iba1, Ifn-γ, and Tnf-α got altered during the immune-challenged state as compared to control. Results provide the first evidence of the association of Pax6 in brain-specific immunity.

Abstract LB037: Chromatin modifications guide Pax5 dependent gene expression in NE like prostate cancer

Abstract Background: Prolonged ablation of androgen receptor pathway in prostate cancer leads to the emergence of therapy resistant neuroendocrine-like (NE-like) aggressive lineage. This NE development is associated with chromatin reprogramming underlying epigenetic alterations and changes in transcriptional network. Clinically, NE development follows a poorer progression with multifaceted therapeutic challenges. Current lack of effective understanding of this NE trans-differentiation process limits the stratification of therapeutic window with further worsened patient management. The following study aims to identify the sequential molecular players responsible for therapy resistant NE like development. Objective: The primary objective of this study is to understand how chromatin modifications along with epigenetic regulation dictates the establishment of NE trans-differentiation process in Prostate Cancer in response to Androgen Receptor blockade therapy. Methods: We generated various NE-like cell lines either by genetic modification or therapeutic selection. We performed RNA Seq, ATAC Seq and acetylated Histone (H3K18 and H3K27) ChIP Seq in our developed NE and adenocarcinoma cell lines. We next compared ATAC Seq and Acetylated histone ChIP Seq with RNA Seq in C4-2B and C4-2BER. Results: Our ATAC Seq and acetylated histone footprints (Ac H3K18 and Ac H3K27 ChIP Seq) analyses revealed an enhanced chromatin accessibility during adenocarcinoma to NE transformation. Overlapping RNA seq results further suggested newly transcribed neuronal genes with higher degrees of promoter accessibility. Transcription Factor scanning analysis among newly active gene promoters revealed preferential binding of Pax5, whose expression has been validated to be selectively occurring in NE lineage unlike adenocarcinoma. Further analysis of Pax5 promoter site suggested that changes in 5-hydroxymethylation pattern during adenocarcinoma to NE transformation guided the recruitment of PBX1 at Pax5 promoter. Validating NE patient expression in silico supported that PBX1/Pax5 expressions are selective in NE lineage development. Conclusions: Our study concludes that specific chromatin alterations guide the recruitment of PBX1 to upregulate Pax5 to maintain a therapy resistant neuronal surrounding in NE-like prostate cancer development. Citation Format: Sreyashi Bhattacharya, Ridwan Islam, Sanika Bodas, Juhi Mishra, Dipanwita Das, Kaustubh Datta, Samikshan Dutta. Chromatin modifications guide Pax5 dependent gene expression in NE like 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 LB037.

Association of colorectal cancer with genetic and epigenetic variation in PEAR1-A population-based cohort study.

Platelet Endothelial Aggregation Receptor 1 (PEAR1) modulates angiogenesis and platelet contact-induced activation, which play a role in the pathogenesis of colorectal cancer. We therefore tested the association of incident colorectal cancer and genetic and epigenetic variability in PEAR1 among 2532 randomly recruited participants enrolled in the family-based Flemish Study on Environment, Genes and Health Outcomes (51.2% women; mean age 44.8 years). All underwent genotyping of rs12566888 located in intron 1 of the PEAR1 gene; in 926 participants, methylation at 16 CpG sites in the PEAR1 promoter was also assessed. Over 18.1 years (median), 49 colorectal cancers occurred, all in different pedigrees. While accounting for clustering of risk factors within families and adjusting for sex, age, body mass index, the total-to-HDL cholesterol ratio, serum creatinine, plasma glucose, smoking and drinking, use of antiplatelet and nonsteroidal anti-inflammatory drug, the hazard ratio of colorectal cancer contrasting minor-allele (T) carriers vs. major-allele (GG) homozygotes was 2.17 (95% confidence interval, 1.18–3.99; P = 0.013). Bootstrapped analyses, from which we randomly excluded from two to nine cancer cases, provided confirmatory results. In participants with methylation data, we applied partial least square discriminant analysis (PLS-DA) and identified two methylation sites associated with higher colorectal cancer risk and two with lower risk. In-silico analysis suggested that methylation of the PEAR1 promoter at these four sites might affect binding of transcription factors p53, PAX5, and E2F-1, thereby modulating gene expression. In conclusion, our findings suggest that genetic and epigenetic variation in PEAR1 modulates the risk of colorectal cancer in white Flemish. To what extent, environmental factors as exemplified by our methylation data, interact with genetic predisposition and modulate penetrance of colorectal cancer risk is unknown.

Chromatin topology defines estradiol-primed progesterone receptor and PAX2 binding in endometrial cancer cells.

Estrogen (E2) and Progesterone (Pg), via their specific receptors (ERalpha and PR), are major determinants in the development and progression of endometrial carcinomas, However, their precise mechanism of action and the role of other transcription factors involved are not entirely clear. Using Ishikawa endometrial cancer cells, we report that E2 treatment exposes a set of progestin-dependent PR binding sites which include both E2 and progestin target genes. ChIP-seq results from hormone-treated cells revealed a non-random distribution of PAX2 binding in the vicinity of these estrogen-promoted PR sites. Altered expression of hormone regulated genes in PAX2 knockdown cells suggests a role for PAX2 in fine-tuning ERalpha and PR interplay in transcriptional regulation. Analysis of long-range interactions by Hi-C coupled with ATAC-seq data showed that these regions, that we call 'progestin control regions' (PgCRs), exhibited an open chromatin state even before hormone exposure and were non-randomly associated with regulated genes. Nearly 20% of genes potentially influenced by PgCRs were found to be altered during progression of endometrial cancer. Our findings suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with PAX2 to accessible chromatin regions. What maintains these regions open remains to be studied.

Epigenetic activation of the elongator complex sensitizes gallbladder cancer to gemcitabine therapy

BackgroundGallbladder cancer (GBC) is known for its high malignancy and multidrug resistance. Previously, we uncovered that impaired integrity and stability of the elongator complex leads to GBC chemotherapy resistance, but whether its restoration can be an efficient therapeutic strategy for GBC remains unknown.MethodsRT-qPCR, MS-qPCR and ChIP-qPCR were used to evaluate the direct association between ELP5 transcription and DNA methylation in tumour and non-tumour tissues of GBC. EMSA, chromatin accessibility assays, and luciferase assays were utilized to analysis the DNA methylation in interfering PAX5-DNA interactions. The functional experiments in vitro and in vivo were performed to investigate the effects of DNA demethylating agent decitabine (DAC) on the transcription activation of elongator complex and the enhanced sensitivity of gemcitabine in GBC cells. Tissue microarray contains GBC tumour tissues was used to evaluate the association between the expression of ELP5, DNMT3A and PAX5.ResultsWe demonstrated that transcriptional repression of ELP5 in GBC was highly correlated with hypermethylation of the promoter. Mechanistically, epigenetic analysis revealed that DNA methyltransferase DNMT3A-catalysed hypermethylation blocked transcription factor PAX5 activation of ELP5 by disrupting PAX5-DNA interaction, resulting in repressed ELP5 transcription. Pharmacologically, the DNA demethylating agent DAC eliminated the hypermethylated CpG dinucleotides in the ELP5 promoter and then facilitated PAX5 binding and reactivated ELP5 transcription, leading to the enhanced function of the elongator complex. To target this mechanism, we employed a sequential combination therapy of DAC and gemcitabine to sensitize GBC cells to gemcitabine-therapy through epigenetic activation of the elongator complex.ConclusionsOur findings suggest that ELP5 expression in GBC is controlled by DNA methylation-sensitive induction of PAX5. The sequential combination therapy of DAC and gemcitabine could be an efficient therapeutic strategy to overcome chemotherapy resistance in GBC.

Abstract 1316: PAX8-directed nanotherapeutics for high-grade serous ovarian cancer

Abstract Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer subtype. Targeted therapy for HGSOC is challenging as the disease is characterized by copy number variation and not somatic mutations. HGSOC displays well-conserved features such as expression of transcription factor PAX8. We propose employing therapies that capitalize on interactions between PAX8 and specific DNA-binding sites to develop therapeutic approaches to target HGSOC. Our hypothesis is to use a biomimetic approach wherein PAX8 is induced to transcribe an ectopically transfected CRISPR-base editor (BE)-guide RNA (gRNA) plasmid containing a PAX8 binding site in its promoter. The gRNA will impart a dominant-negative allele of ataxia telangiectasia and Rad3-related protein (ATR), which has been shown to be synthetically lethal with mutant p53 in cancer cells. In the presence of PAX8, the plasmid will be transcribed, while in the absence of PAX8, the plasmid is degraded, with minimal toxicity observed in healthy cells. Methods: PAX8-expressing ovarian cancer cell lines including COV362, Kuramochi, OVCAR8 and OVSAHO or PAX8-null cell lines including MCF-7 and HeLa were transfected with polyplexes bearing the plasmid of interest. Polyplex diameter, polydispersity indices and surface zeta potentials were measured on a Malvern Zetasizer. Polyplexes were imaged for uptake and functionality using fluorescence microscopy and Amnis ImageStream. Polyplex toxicity was assessed in vitro using cell viability assays. Preliminary in vivo experiments to demonstrate luciferase plasmid-bearing polyplexes have been performed in NSG mice. To monitor in vivo transfection efficiencies, we will deliver fluorescently labeled plasmids or luciferase-encoding plasmids to test bio-distribution and pharmacokinetics. Results: We have synthesized sub-200nm (diameter) polyplexes using the polyamine PPLG-g-azidopropylamine (PPLG) and BE or gRNA plasmids. Using -/- ATR gRNA, we observed that the BE and the gRNA can be efficiently packaged into polymeric nanoplexes and delivered to ovarian cancer cells or control cells to impart a dominant-negative mutant of ATR. Ongoing work includes cell cycle analysis using flow cytometry to monitor checkpoint activation, quantifying percent of total sequencing reads with target base pair conversions and testing gRNAs against other targets for efficacy in ovarian cancer targeting. Conclusion: Combining nanotechnology and gene editing can improve specificity toward ovarian cancer cells as well as incorporate precision therapy aimed at the underlying genetic architecture of this disease. Due to the synthetic lethality nature of ATR and p53, addition of a combinatorial PARP inhibitor to the proposed therapy will reduce tumor burden. While resistance can develop to ATR inhibitors, ovarian cancer cells are unable to downregulate PAX8, thus ensuring continued response to the PAX8-directed therapy. Citation Format: Akshaya Chandrasekaran, Mariam Ahmed, Nicole Holub, Wade Wang, Paula T. Hammond, Kevin M. Elias. PAX8-directed nanotherapeutics for high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1316.

Analysis of the FBXO7 promoter reveals overlapping Pax5 and c-Myb binding sites functioning in B cells

Fbxo7 is a key player in the differentiation and function of numerous blood cell types, and in neurons, oligodendrocytes and spermatocytes. In an effort to gain insight into the physiological and pathological settings where Fbxo7 is likely to play a key role, we sought to define the transcription factors which direct FBXO7 expression. Using sequence alignments across 28 species, we defined the human FBXO7 promoter and found that it contains two conserved regions enriched for multiple transcription factor binding sites. Many of these have roles in either neuronal or haematopoietic development. Using various FBXO7 promoter reporters, we found ELF4, Pax5 and c-Myb have functional binding sites that activate transcription. We find endogenous Pax5 is bound to the FBXO7 promoter in pre-B cells, and that the exogenous expression of Pax5 represses Fbxo7 transcription in early pro-B cells.

Paxbp1 controls a key checkpoint for cell growth and survival during early activation of quiescent muscle satellite cells

Adult mouse muscle satellite cells (MuSCs) are quiescent in uninjured muscles. Upon muscle injury, MuSCs exit quiescence, reenter the cell cycle to proliferate and self-renew, and then differentiate and fuse to drive muscle regeneration. However, it remains poorly understood how MuSCs transition from quiescence to the cycling state. Here, we report that Pax3 and Pax7 binding protein 1 (Paxbp1) controls a key checkpoint during this critical transition. Deletion of Paxbp1 in adult MuSCs prevented them from reentering the cell cycle upon injury, resulting in a total regeneration failure. Mechanistically, we found an abnormal elevation of reactive oxygen species (ROS) in Paxbp1-null MuSCs, which induced p53 activation and impaired mTORC1 signaling, leading to defective cell growth, apoptosis, and failure in S-phase reentry. Deliberate ROS reduction partially rescued the cell-cycle reentry defect in mutant MuSCs. Our study reveals that Paxbp1 regulates a late cell-growth checkpoint essential for quiescent MuSCs to reenter the cell cycle upon activation.

EBF1 and PAX5 control pro-B cell expansion via opposing regulation of the Myc gene

Genes encoding B lineage–restricted transcription factors are frequently mutated in B-lymphoid leukemias, suggesting a close link between normal and malignant B-cell development. One of these transcription factors is early B-cell factor 1 (EBF1), a protein of critical importance for lineage specification and survival of B-lymphoid progenitors. Here, we report that impaired EBF1 function in mouse B-cell progenitors results in reduced expression of Myc. Ectopic expression of MYC partially rescued B-cell expansion in the absence of EBF1 both in vivo and in vitro. Using chromosome conformation analysis in combination with ATAC-sequencing, chromatin immunoprecipitation–sequencing, and reporter gene assays, six EBF1-responsive enhancer elements were identified within the Myc locus. CRISPR-Cas9–mediated targeting of EBF1-binding sites identified one element of key importance for Myc expression and pro-B cell expansion. These data provide evidence that Myc is a direct target of EBF1. Furthermore, chromatin immunoprecipitation–sequencing analysis revealed that several regulatory elements in the Myc locus are targets of PAX5. However, ectopic expression of PAX5 in EBF1-deficient cells inhibits the cell cycle and reduces Myc expression, suggesting that EBF1 and PAX5 act in an opposing manner to regulate Myc levels. This hypothesis is further substantiated by the finding that Pax5 inactivation reduces requirements for EBF1 in pro–B-cell expansion. The binding of EBF1 and PAX5 to regulatory elements in the human MYC gene in a B-cell acute lymphoblastic leukemia cell line indicates that the EBF1:PAX5:MYC regulatory loop is conserved and may control both normal and malignant B-cell development.

Muscle progenitor specification and myogenic differentiation are associated with changes in chromatin topology

Using Hi-C, promoter-capture Hi-C (pCHi-C), and other genome-wide approaches in skeletal muscle progenitors that inducibly express a master transcription factor, Pax7, we systematically characterize at high-resolution the spatio-temporal re-organization of compartments and promoter-anchored interactions as a consequence of myogenic commitment and differentiation. We identify key promoter-enhancer interaction motifs, namely, cliques and networks, and interactions that are dependent on Pax7 binding. Remarkably, Pax7 binds to a majority of super-enhancers, and together with a cadre of interacting transcription factors, assembles feed-forward regulatory loops. During differentiation, epigenetic memory and persistent looping are maintained at a subset of Pax7 enhancers in the absence of Pax7. We also identify and functionally validate a previously uncharacterized Pax7-bound enhancer hub that regulates the essential myosin heavy chain cluster during skeletal muscle cell differentiation. Our studies lay the groundwork for understanding the role of Pax7 in orchestrating changes in the three-dimensional chromatin conformation in muscle progenitors.

Abstract 6241: PAX8-directed nanotherapeutics for high-grade serous ovarian carcinoma

Abstract Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer subtype. Targeted therapy for HGSOC is challenging because the disease is characterized by copy number variation rather than recurrent somatic mutations. HGSOC displays well-conserved epigenetic features such as expression of transcription factor PAX8. We propose employing epigenetic therapies that capitalize on interactions between PAX8 and specific DNA-binding sites to develop potential therapeutic approaches to target HGSOC. Our hypothesis is to use a biomimetic approach wherein PAX8 is induced to transcribe an ectopically transfected CRISPR-base editor (BE)-guide RNA (gRNA) plasmid containing a PAX8 binding site in its promoter. The gRNA will impart a dominant-negative allele of Polo Like Kinase 1 (PLK1) which has been shown to be synergistically lethal with deficient tp53 in cancer cells. In the presence of PAX8, the plasmid will be transcribed, while in the absence of PAX8, the plasmid is degraded, with minimal toxicity observed in healthy cells. Methods: PAX8-expressing ovarian cancer cell lines including COV362, Kuramochi, OVCAR8 and OVSAHO or PAX8-negative control cell lines including JEG3, MCF-7, HeLa and HEK293T were transfected with polyplexes bearing the plasmid of interest. Polyplex diameter, polydispersity indices, and surface zeta potentials were measured on a Malvern Zetasizer. Polyplexes were imaged for uptake and functionality using fluorescence microscopy and Amnis ImageStream. Polyplex toxicity was assessed in vitro using cell viability assays. Preliminary in vivo experiments to demonstrate luciferase plasmid-bearing polyplexes have been performed in NOD-SCID mice. To monitor in vivo transfection efficiencies, we will deliver fluorescently labeled plasmids or luciferase-encoding plasmids to test bio-distribution and pharmacokinetics. Results: We have synthesized sub-200nm (diameter) polyplexes using the polyamine PPLG-g-azidopropylamine (PPLG) and BE or gRNA plasmids. Using -/-PLK1 gRNA, we observed that the BE and the gRNA can be efficiently packaged into two distinct polymeric nanoplexes and co-delivered to ovarian cancer cells or control cells to impart a dominant-negative mutant of PLK1, which is also synthetically lethal with mutated tp53, as observed from cancer cell death compared to controls. Ongoing work includes repetitive polyplex injections in vitro and in vivo, quantifying percentage of total sequencing reads with target base pair conversions after treatment, determination of plasmid loading efficiency, and testing gRNAs against other targets for efficacy in ovarian cancer targeting. Conclusion: Well-defined lineage transcription factors have the potential to serve as novel targets for gene-editing therapies in ovarian cancer and other malignancies. Combining nanotechnology and gene editing can improve specificity toward ovarian cancer cells as well as incorporate precision therapy aimed at the underlying genetic architecture of this disease. Citation Format: Akshaya Chandrasekaran, Mariam Ahmed, Wade Wang, Paula T. Hammond, Kevin M. Elias. PAX8-directed nanotherapeutics for high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6241.