Zipper Domain Research Articles

CEBPA mutation in acute myeloid leukemia: prognostic impact of bZIP domain mutation

Abstract One of the most important prognostic genes for acute myeloid leukemia (AML) is CEPBA, and its mutation (CEBPAmu) is present in nearly 10%–15% of de novo AML cases. CEBPAdm is associated with a favorable prognosis; however, CEBPAsm does not appear to have a better prognosis than CEBPAdm. We reviewed CEBPAmu-bZIP in 8694 cases across studies for prognostic impact in patients with de novo AML. It was observed that CEBPAmu in the basic leucine zipper domain (bZIP) was strongly associated with a favorable prognosis, but CEBPAmu out of the bZIP domain was not. CEBPA-bZIP mutations were discovered in 562 (6.46%) of 8694 cases, with 366 (65.1%) harboring a second CEBPA mutation (CEBPA-double-mutated [CEBPAdm]) and 196 (34.9%) having a single CEBPA-bZIP only mutation. Multivariate analysis of three studies consisting of 1028, 4708, and 2958 patients showed that CEBPAmu in bZIP was the most potent predictor of overall survival (OS) with overall survivability of 53%, 62%, and 89%, respectively, independently. However, complete remission of disease with bZIP mutation in the same studies was found to be 80%, 62%, and 78.6%, respectively. These findings indicate that CEBPAmu in bZIP is a potent marker for AML prognosis.

A High-Throughput Screen for Antiproliferative Peptides in Mammalian Cells Identifies Key Transcription Factor Families.

Transcription factors (TFs) are a promising therapeutic target for a multitude of diseases. TFs perform their cellular roles by participating in multiple specific protein-protein interactions. For example, homo- or heterodimerization of some TFs controls DNA binding, while interactions between TFs and components of basal transcriptional machinery or chromatin modifiers can also be critical. While, in theory, small molecules could be used to disrupt specific protein-protein interfaces required for TF function, in practice, it is difficult to identify small molecules with the necessary specificity and efficacy, likely due to the extensive protein-protein interfaces that often underlie TF function. However, in contrast to small molecules, peptides have the potential to provide both the specificity and efficacy required to disrupt such interfaces. Here, we identified ∼15 peptides that inhibit the proliferation of leukemia cells using a high-throughput pooled screen of a library of 80-mer protein regions (peptides) derived from human nuclear-localized proteins. The antiproliferative peptides were enriched for regions known to be involved in specific TF dimerization, including the basic leucine zipper (bZIP) domain family. One of these bZIP domains, JDP2;bZIP_1, from the TF JDP2, was the top antiproliferative peptide, reducing the proliferation of K562 cells by 2-fold. JDP2;bZIP_1 inhibited AP-1 transcriptional activity and phenocopied JDP2 overexpression, suggesting that the peptide affected proliferation through a native JDP2 mechanism. Unexpectedly, given the strong conservation of the bZIP domain, residues outside of the annotated dimerization domain were critical for the peptide's antiproliferative potency. The peptide-mediated antiproliferative effect initiated erythrocyte differentiation in K562 cells and increased G0/G1 cells across multiple cell line models. We also found that many of the antiproliferative peptides identified in this study, including JDP2;bZIP_1, did not require a nuclear localization signal to function, a potential benefit for delivering these peptides in therapeutic applications.

The Aspergillus flavus hacA Gene in the Unfolded Protein Response Pathway Is a Candidate Target for Host-Induced Gene Silencing.

Fungal HacA/Hac1 transcription factors play a crucial role in regulating the unfolded protein response (UPR). The UPR helps cells to maintain endoplasmic reticulum (ER) protein homeostasis, which is critical for growth, development, and virulence. The Aspergillus flavus hacA gene encodes a domain rich in basic and acidic amino acids (Bsc) and a basic leucine zipper (bZip) domain, and features a non-conventional intron (Nt20). In this study, CRISPR/Cas9 was utilized to dissect the Bsc-coding, bZip-coding, and Nt20 sequences to elucidate the relationship between genotype and phenotype. In the Bsc and bZip experimental sets, all observed mutations in both coding sequences were in frame, suggesting that out-of-frame mutations are lethal. The survival rate of transformants in the Nt20 experiment set was low, at approximately 7%. Mutations in the intron primarily consisted of out-of-frame insertions and deletions. In addition to the wild-type-like conidial morphology, the mutants exhibited varied colony morphologies, including sclerotial, mixed (conidial and sclerotial), and mycelial morphologies. An ER stress test using dithiothreitol revealed that the sclerotial and mycelial mutants were much more sensitive than the conidial mutants. Additionally, the mycelial mutants were unable to produce aflatoxin but still produced aspergillic acid and kojic acid. RNAi experiments targeting the region encompassing Bsc and bZip indicated that transformant survival rates generally decreased, with a small number of transformants displaying phenotypic changes. Defects in the hacA gene at the DNA and transcript levels affected the survival, growth, and development of A. flavus. Thus, this gene may serve as a promising target for future host-induced gene-silencing strategies aimed at controlling infection and reducing aflatoxin contamination in crops.

BZW2 is a potential regulator of non-small cell lung cancer progression

BackgroundPersonalized targeted therapy has become an important strategy for cancer treatment owing to its remarkable therapeutic efficacy and safety. However, drug resistance remains the primary cause of treatment failure. Basic leucine zipper and W2 domain 2 (BZW2), which is aberrantly expressed in cancer, has been implicated in tumor progression and may serve as a new therapeutic target. Therefore, the role of BZW2 in non-small cell lung cancer (NSCLC) requires further investigation. MethodsThe expression and genetic alterations of BZW2 in pan-cancers were explored using The Cancer Genome Atlas (TCGA) PanCancer databases. The mRNA and protein levels of BZW2 in patients with NSCLC were verified in our cohort. Functional experiments including CCK8, colony formation, and transwell assays were performed to evaluate the impact of BZW2 on the proliferative, migratory, and invasive capacities of SK-MES-1 cells. Gene Set Enrichment Analysis was used to identify underlying biological processes and pathways. Single-cell RNA (scRNA) sequencing data were employed to investigate the tumor microenvironment of NSCLC and the co-expression of BZW2 and stemness-related genes. ResultsDysregulated BZW2 expression was observed in various malignant tumors. BZW2 expression was found to be significantly elevated in NSCLC. BZW2 depletion inhibited the growth, mobility, and invasive abilities of lung squamous cell carcinoma SK-MES-1 cells. BZW2 may be related to signaling pathways such as nucleotide excision repair, ubiquitin-mediated proteolysis, and the P53 signaling pathway. Biological processes, including translational initiation, tRNA processing, and RNA methylation, were observed to be enriched in the high-BZW2 group. Furthermore, there was a positive correlation between BZW2 and the m6A- and m5C-related genes. scRNA analysis revealed a co-expression relationship between BZW2 and stemness-related genes such as CD44, SOX9, and CD133. ConclusionsElevated BZW2 expression is associated with the proliferation, migration, and invasion of NSCLC, and BZW2 may be a potential therapeutic target for NSCLC.

DKK1 as a chemoresistant protein modulates oxaliplatin responses in colorectal cancer

Oxaliplatin is effective against colorectal cancer (CRC), but resistance hampers treatment. We found upregulated Dickkopf-1 (DKK1, a secreted protein) in oxaliplatin-resistant (OR) CRC cell lines and DKK1 levels increased by more than 2-fold in approximately 50% of oxaliplatin-resistant CRC tumors. DKK1 activates AKT via cytoskeleton-associated protein 4 (CKAP4, a DKK1 receptor), modulating oxaliplatin responses in vitro and in vivo. The leucine zipper (LZ) domain of CKAP4 and cysteine-rich domain 1 (CRD1) of secreted DKK1 are crucial for their interaction and AKT signaling. By utilizing the LZ protein, we disrupted DKK1 signaling, enhancing oxaliplatin sensitivity in OR CRC cells and xenograft tumors. This suggests that DKK1 as a chemoresistant factor in CRC via AKT activation. Targeting DKK1 with the LZ protein offers a promising therapeutic strategy for oxaliplatin-resistant CRC with high DKK1 levels. This study sheds light on oxaliplatin resistance mechanisms and proposes an innovative intervention for managing this challenge.

Direct inhibition of tumor hypoxia response with synthetic transcriptional repressors.

Many oncogenic transcription factors (TFs) are considered to be undruggable because of their reliance on large protein-protein and protein-DNA interfaces. TFs such as hypoxia-inducible factors (HIFs) and X-box-binding protein 1 (XBP1) are induced by hypoxia and other stressors in solid tumors and bind to unfolded protein response element (UPRE) and hypoxia-induced response element (HRE) motifs to control oncogenic gene programs. Here, we report a strategy to create synthetic transcriptional repressors (STRs) that mimic the basic leucine zipper domain of XBP1 and recognize UPRE and HRE motifs. A lead molecule, STR22, binds UPRE and HRE DNA sequences with high fidelity and competes with both TFs in cells. Under hypoxia, STR22 globally suppresses HIF1α binding to HRE-containing promoters and enhancers, inhibits hypoxia-induced gene expression and blocks protumorigenic phenotypes in triple-negative breast cancer (TNBC) cells. In vivo, intratumoral and systemic STR22 treatment inhibited hypoxia-dependent gene expression, primary tumor growth and metastasis of TNBC tumors. These data validate a novel strategy to target the tumor hypoxia response through coordinated inhibition of TF-DNA binding.

The basic leucine zipper domain (bZIP) transcription factor BbYap1 promotes evasion of host humoral immunity and regulates lipid homeostasis contributing to fungal virulence in Beauveria bassiana.

Entomopathogenic fungi (EPF) offer an effective and eco-friendly alternative to curb insect populations in biocontrol strategy. When EPF enter the hemolymph of their host, they encounter a variety of stress reactions, such as immunological and oxidative stress. Basic leucine zipper domain transcription factors, of which yeast activator protein (Yap) is a significant class, have diverse biological functions related to metabolism, development, reproduction, conidiation, stress responses, and pathogenicity. This study demonstrates that BbYap1 of Beauveria bassiana regulates cellular enzyme lipid homeostasis and fungal virulence by eluding host humoral defense, which contributes to fungal chemical stress and vegetative development. These findings offer fresh perspectives for comprehending molecular roles of YAP in EPF.

Identification of differentially expressed genes controlling the expression of flowering in Bambara groundnut (Vigna subterranea [L.] Verdc.)

Bambara groundnut flowering is a crucial developmental stage in the vegetative to reproductive period. The earliness to lateness of flowering is regulated by various interconnected genetic pathways encoded by genes. Deoxyribonucleic acid (DNA) of the selected accessions was extracted through leaf samples at 3 weeks old, using Dellaporta Miniprep for Plant DNA Isolation procedure. The high-quality DNA was sequenced using Diversity Arrays Technology (DArT) markers and single nucleotide polymorphisms (SNP’s) associated with flowering was identified. There is need to investigate the genetic make-up of the cleistogamous flower of Bambara through associated genes for improvement. This research work identified four markers associated with the flowering of Vigna subterranea and the role of variant identified genes in flowering. The identified markers from the sequence and the selected amino acid sequence were used as a query to search the legume protein database in Vigna radiata. The four markers with adequate information associated with flowering in the sequence were 24385352|F|0–28:T > C-28:T > C; 27641816|F|0–17:C > T-17:C > T; 24384204|F|0–24:C > T-24:C > T and 24346601|F|0–67:T > C-67:T > C and significant at P < 1.68 × 10−4 at chromosomes 7, 11, 4, and 5. The identified genes including histones, Polyketide, cyclase/dehydrase, Transcription factor MYC/MYB N-terminal, Rhamnogalacturonate lyase, DHHC-type zinc finger protein, Putative S-adenosyl-L-methionine-dependent methyltransferase, Ribosomal protein L2, D-galactoside/L-rhamnose binding SUEL lectin domain, Lipase GDSL, Histone deacetylase superfamily, Basic-leucine zipper domain, TUP1-like enhancer of split, Zinc finger ZZ-type, Homeodomain-like, Phosphatidylethanolamine-binding protein PEBP, Leucine-rich repeat which are tools in controlling flowering in Bambara groundnut. This study revealed that Bambara groundnut flowering is controlled by the interplay of genes.

Dimorphic Regulation of the MafB Gene by Sex Steroids in Hamsters, Mesocricetus auratus.

MafB is a transcription factor that regulates macrophage differentiation. Macrophages are a traditional feature of the hamster Harderian gland (HG); however, studies pertaining to MafB expression in the HG are scant. Here, the full-length cDNA of the MafB gene in hamsters was cloned and sequenced. Molecular characterization revealed that MafB encodes a protein containing 323 amino acids with a DNA-binding domain, a transactivation domain, and a leucine zipper domain. qPCR assays indicated that MafB was expressed in different tissues of both sexes. The highest relative expression levels in endocrine tissues were identified in the pancreas. Gonadectomy in male hamsters was associated with significantly higher mRNA levels in the HG; replacement with dihydrotestosterone restored mRNA expression. The HG in male hamsters contained twofold more MafB mRNA than the HG of female hamsters. Adrenals revealed similar mRNA relative expression levels during the estrous cycle. The estrous phase was associated with higher mRNA levels in the ovary. A significantly up-regulated expression and sexual dimorphism of MafB was found in the pancreas. Therefore, MafB in the HG may play an active role in the macrophage differentiation required for phagocytosis activity and intraocular repair. Additionally, sex steroids appear to strongly influence the MafB expression in the HG and pancreas. These studies highlight the probable biological importance of MafB in immunological defense and pancreatic β cell regulation.

M6A demethylation of FOSL1 mRNA protects hepatoma cells against necrosis under glucose deprivation.

Stress-adaptive mechanisms enabling cancer cells to survive under glucose deprivation remain elusive. N6-methyladenosine (m6A) modification plays important roles in determining cancer cell fate and cellular stress response to nutrient deficiency. However, whether m6A modification functions in the regulation of cancer cell survival under glucose deprivation is unknown. Here, we found that glucose deprivation reduced m6A modification levels. Increasing m6A modification resulted in increased hepatoma cell necrosis under glucose deprivation, whereas decreasing m6A modification had an opposite effect. Integrated m6A-seq and RNA-seq revealed potential targets of m6A modification under glucose deprivation, including the transcription factor FOSL1; further, glucose deprivation upregulated FOSL1 by inhibiting FOSL1 mRNA decay in an m6A-YTHDF2-dependent manner through reducing m6A modification in its exon1 and 5'-UTR regions. Functionally, FOSL1 protected hepatoma cells against glucose deprivation-induced necrosis in vitro and in vivo. Mechanistically, FOSL1 transcriptionally repressed ATF3 by binding to its promoter. Meanwhile, ATF3 and MAFF interacted via their leucine zipper domains to form a heterodimer, which competed with NRF2 for binding to antioxidant response elements in the promoters of NRF2 target genes, thereby inhibiting their transcription. Consequently, FOSL1 reduced the formation of the ATF3-MAFF heterodimer, thereby enhancing NRF2 transcriptional activity and the antioxidant capacity of glucose-deprived-hepatoma cells. Thus, FOSL1 alleviated the necrosis-inducing effect of glucose deprivation-induced reactive oxygen species accumulation. Collectively, our study uncovers the protective role of m6A-FOSL1-ATF3 axis in hepatoma cell necrosis under glucose deprivation, and may provide new targets for cancer therapy.

Unraveling the interplay between the leucine zipper and forkhead domains of FOXP2: Implications for DNA binding, stability and dynamics.

FOXP2 is a transcription factor associated with speech and language. Like other FOX transcription factors, it has a DNA binding region called the forkhead domain (FHD). This domain can exist as a monomer or a domain swapped dimer. In addition to the FHD, the leucine zipper region (LZ) of FOXP2 is also believed to be associated with both DNA binding and oligomerization. To better understand the relationship between DNA binding and oligomerization of FOXP2, we investigated its structure, stability and dynamics, focusing specifically on the FHD and the LZ. We did this by using two constructs: one containing the isolated FHD and one containing both the LZ and the FHD (LZ-END). We demonstrate in this work, that while the FHD maintains a monomeric form that is capable of binding DNA, the LZ-END undergoes a dynamic transition between oligomeric states in the presence of DNA. Our findings suggest that FOXP2's LZ domain influences DNA binding affinity through a change in oligomeric state. We show through hydrogen exchange mass spectroscopy that certain parts of the FHD and interlinking region become less dynamic when in the presence of DNA, confirming DNA binding and oligomerization in these regions. Moreover, the detection of a stable equilibrium intermediate state during LZ-END unfolding supports the idea of cooperation between these two domains. Overall, our study sheds light on the interplay between two FOXP2 domains, providing insight into the protein's ability to respond dynamically to DNA, and enriching our understanding of FOXP2's role in gene regulation.

CEBPA mutations in acute myeloid leukemia: implications in risk stratification and treatment.

Mutations in CCAAT enhancer binding protein α (CEBPA) occur in approximately 10% of patients with de novo acute myeloid leukemia (AML). Emerging evidence supports that in-frame mutations in the basic leucine zipper domain of CEBPA (CEBPAbZIP-inf) confer a survival benefit, and CEBPAbZIP-inf replaced CEBPA double mutations (CEBPAdm) as a unique entity in the 2022 World Health Organization (WHO-2022) classification and International Consensus Classification (ICC). However, challenges remain in daily clinical practice since more than 30% patients with CEBPAbZIP-inf die of AML despite intensive treatment. This review aims to provide a comprehensive summary of the heterogeneities observed in AML with CEBPAdm and CEBPAbZIP-inf, and will discuss the prognostic implications of concurrent mutations and novel mechanistic targets that may inform future drug development. The ultimate goal is to optimize clinical management and to provide precision medicine for this category of patients.

Self-assembling lipolytic hydrogels

Background: Enzymatically active biomaterials, which are used in a variety of applications, such as tissue engineering and biosensors, have been the subject of research. Protein engineers created self-assembling enzymes as a result of improvements in the manufacturing and design of peptide-based biomaterials. In this regard, enzymes with cross-linking domains make it possible to create biomaterials that have a number of benefits, such as simple low-cost production, homogenous dispersion of activity in the hydrogel, and the ability to co-localize enzymes, which can all work together to increase the protein’s stability. Method: To test this theory, we combined lipases with leucine zipper domains to produce enzyme building blocks that self-assembled into lipolytic hydrogels. In this work, a chimeric protein with self-assembling capabilities was produced by introducing leucine zippers through a pET plasmid into the hydrogel-forming triblock polypeptide and the gene of interest. The chimaera was shown to have improved solubility and stability as well as the ability to form soluble aggregates. Additionally, the hydrogel’s catalytic capabilities for several substrates were investigated. Results: It could be concluded that the triblock peptide increased the solubility of protein by reducing the inclusion body formation. Thereby, increasing the purified protein concentration. In order to create bifunctional lipolytic hydrogels, the study combines the self-assembly function of leucine zippers with the catalytic activity of lipase. The self-assembling enzymatic gels offer an attractive method for in vivo enzyme entrapment with wide applications.

Molecular characterization of transcription factor CREB3L2 and CREB3L3 and their role in melanogenesis in Pacific oysters (Crassostrea gigas)

Colorful shells in mollusks are commonly attributable to the presence of biological pigments. In Pacific oysters, the inheritance patterns of several shell colors have been investigated, but little is known about the molecular mechanisms of melanogenesis and pigmentation. cAMP-response element binding proteins (CREB) are important transcription factors in the cAMP-mediated melanogenesis pathway. In this study, we characterized two CREB genes (CREB3L2 and CREB3L3) from Pacific oysters. Both of them contained a conserved DNA-binding and dimerization domain (a basic-leucine zipper domain). CREB3L2 and CREB3L3 were expressed highly in the mantle tissues and exhibited higher expression levels in the black-shell oyster than in the white. Masson-Fontana melanin staining and immunofluorescence analysis showed that the location of CREB3L2 protein was generally consistent with the distribution of melanin in oyster edge mantle. Dual-luciferase reporter assays revealed that CREB3L2 and CREB3L3 could activate the microphthalmia-associated transcription factor (MITF) promoter and this process was regulated by the level of cAMP. Additionally, we found that cAMP regulated melanogenic gene expression through the CREB-MITF-TYR axis. These results implied that CREB3L2 and CREB3L3 play important roles in melanin synthesis and pigmentation in Pacific oysters.

Differential expression of PPP1R12A transcripts, including those harbouring alternatively spliced micro-exons, in placentae from complicated pregnancies

IntroductionPlacenta-associated pregnancy complications, including pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are conditions postulated to originate from initial failure of placentation, leading to clinical sequelae indicative of endothelial dysfunction. Vascular smooth muscle aberrations have also been implicated in the pathogenesis of both disorders via smooth muscle contractility and relaxation mediated by Myosin Light Chain Phosphatase (MLCP) and the oppositional contractile action of Myosin Light Chain Kinase. PPP1R12A is a constituent part of the MLCP complex responsible for dephosphorylation of myosin fibrils. We hypothesize that alternative splicing of micro-exons result in isoforms lacking the functional leucine zipper (LZ) domain which may give those cells expressing these alternative transcripts a tendency towards contraction and vasoconstriction. MethodsExpression was determined by qRT-PCR. Epigenetic profiling consisted of bisulphite-based DNA methylation analysis and ChIP for underlying histone modifications. ResultsWe identified several novel transcripts with alternative micro-exon inclusion that would produce LZ- PPP1R12A protein. qRT-PCR revealed some isoforms, including the PPP1R12A canonical transcript, are differentially expressed in placenta biopsies from PE and IUGR samples compared to uncomplicated pregnancies. DiscussionWe propose that upregulation of PPP1R12A expression in complicated pregnancies may be due to enhanced promoter activity leading to increased transcription as a response to physiological stress in the placenta, which we show is independent of promoter DNA methylation.

Meta-QTL analysis reveals the important genomics regions for biotic stresses, nutritional quality and yield related traits in pearl millet

AbstractPearl millet (Cenchrus americanus) is the sixth most significant cereal crop cultivated on 30 million ha and a staple diet for 90 million poor people across the globe. Besides abiotic stresses several biotic stresses have been limiting production of pearl millet in the semi-arid and arid regions. Although, the Quantitative Trait Loci (QTLs) associated with key diseases like blast, rust and downy mildew resistance and nutritional content has been reported, the use of these QTLs is limited in breeding programs. To identify highly stable consensus genomic regions, we conducted Meta-QTL analysis using 191 QTLs reported in 12 independent studies over the last two decades. As a result, we report 34 Meta-QTLs regions on a consensus genetic map comprising of 692 markers and spanning 2070.7 cM. The confidence interval of Meta-QTLs was reduced by 3.63 folds (0.18–7.49 cM), in contrast to projected QTLs interval of 1.11–60.63 cM. Further, a total of 1198 genes were identified in 34 Meta-QTL regions. Among 34 Meta-QTL regions, Meta-QTL1.1 is found to be region of significant importance as it harbours genes for enhanced biotic stress tolerance, plant growth and development as well as genes related with enhanced seed development. Meta-QTL2.4 has highest number of genes with a significant role in disease resistance which contains basic leucine zipper domain, zinc family, leucine rich repeat regions. Meta-QTL3.1 has ABC transporter like activity coupled with the ATPase activity which has a role in Fe and Zn uptake in leaves and root tissues. These Meta-QTL regions can be used in genomics-assisted breeding for enhancing the blast, rust downy mildew resistance as well as yield and nutritional traits.

Abscisic acid activates transcription factor module MdABI5-MdMYBS1 during carotenoid-derived apple fruit coloration.

Carotenoids are major pigments contributing to fruit coloration. We previously reported that the apple (Malus domestica Borkh.) mutant fruits of "Beni Shogun" and "Yanfu 3" show a marked difference in fruit coloration. However, the regulatory mechanism underlying this phenomenon remains unclear. In this study, we determined that carotenoid is the main factor influencing fruit flesh color. We identified an R1-type MYB transcription factor (TF), MdMYBS1, which was found to be highly associated with carotenoids and abscisic acid (ABA) contents of apple fruits. Overexpression of MdMYBS1 promoted, and silencing of MdMYBS1 repressed, β-branch carotenoids synthesis and ABA accumulation. MdMYBS1 regulates carotenoid biosynthesis by directly activating the major carotenoid biosynthetic genes encoding phytoene synthase (MdPSY2-1) and lycopene β-cyclase (MdLCYb). 9-cis-epoxycarotenoid dioxygenase 1 (MdNCED1) contributes to ABA biosynthesis, and MdMYBS1 enhances endogenous ABA accumulation by activating the MdNCED1 promoter. In addition, the basic leucine zipper domain TF ABSCISIC ACID-INSENSITIVE5 (MdABI5) was identified as an upstream activator of MdMYBS1, which promotes carotenoid and ABA accumulation. Furthermore, ABA promotes carotenoid biosynthesis and enhances MdMYBS1 and MdABI5 promoter activities. Our findings demonstrate that the MdABI5-MdMYBS1 cascade activated by ABA regulates carotenoid-derived fruit coloration and ABA accumulation in apple, providing avenues in breeding and planting for improvement of fruit coloration and quality.

Abstract 3051: JunAP, a peptide antagonist against the activator protein 1 transcription factor complex, demonstrates cancer cell cytotoxicity and reduced invasion in vitro and tumor regression in vivo in TNBC models

Abstract The activator protein 1 (AP-1) transcription factor is a dimeric complex consisting of proteins from the JUN, FOS, MAF, and ATF families. AP-1 complexes have been identified as novel therapeutic targets in cancer due to their role in tumor growth, invasion, metastasis, angiogenesis and chemoresistance. Dimerization of components of the AP-1 complex via leucine zipper domain interactions is required for DNA binding and subsequent transcriptional activity. We designed a peptide (Jun antagonizing peptide, JunAP) targeting the basic leucine zipper motif of AP-1 to antagonize AP-1 complex formation and prevent associated activity. Fluorescence polarization and DNA ELISA assays demonstrate target binding and selectivity of JunAP towards AP-1 family members, co-immunoprecipitation experiments reveal that JunAP blocks protein-protein interactions between cJun and its key binding partners, and reporter assays confirm inhibition of AP-1 transcriptional activity in vitro. Pathway analysis of RNA sequencing data confirmed by qPCR analysis shows that JunAP disrupts key pathways required for cell survival and migration. Corresponding functional in vitro assays reveal that JunAP exhibits an inhibitory effect on invasion in Boyden chamber assays and demonstrates potent cytotoxicity in Jun-dependent cells. Further, in a BT549 triple negative breast cancer subcutaneous xenograft model, administration of JunAP results in tumor regression, demonstrating the anti-cancer potential of targeting the AP-1 complex. In summary, these data support JunAP as a potent peptide antagonist of the AP-1 transcription factor family that warrants further development as a potential therapeutic option for AP-1 driven tumors. Citation Format: Karen Mendelson, Zachary F. Mattes, Siok Leong, Ricardo Ramirez, Mark Koester, Claudio Scuoppo, Julia Diehl, Erin Gallagher, Jerel Gonzales, Franco Abbate, Lila Ghamsari, Gene Merutka, Barry J. Kappel, Abi Vainstein-Haras, Jim A. Rotolo. JunAP, a peptide antagonist against the activator protein 1 transcription factor complex, demonstrates cancer cell cytotoxicity and reduced invasion in vitro and tumor regression in vivo in TNBC 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 3051.

Abstract 7166: Pharmacokinetic analysis of omomyc therapeutic protein in preclinical and human tumor tissue using targeted mass spectrometry

Abstract MYC is a pleiotropic transcription factor consisting in three paralogs which coordinate gene expression and regulates cell proliferation, cell differentiation, cell cycle, metabolism and apoptosis. The 3 paralogs (c-, N-, and L-MYC) have similar biological functions but show distinct tissular and timing expression patterns. To function, MYC must heterodimerize with its partner MAX and bind DNA via its basic, helix-loop-helix, leucine zipper domain (B-HLH-LZ). It has been estimated that MYC is aberrantly expressed in up to 70% of human cancers and hence MYC has been termed a “most wanted target for cancer treatment”. However, due to its nuclear localization, its intrinsic disorder and lack of an enzymatically active site, development of clinical grade potent &amp; selective small molecules targeting MYC remains an obstacle. Omomyc is a 91-amino-acids-long polypeptide that derives from the B-HLH-LZ domain of human MYC, containing four amino acid substitutions. Omomyc is capable of forming homodimers and heterodimers with MYC and with MAX and such dimers blunt MYC activity by preventing its access to target DNA sequences (E-boxes) and replacing it by inactive complexes. Previous studies showed the cell-penetrating properties of recombinant Omomyc and its subsequent nuclear localization, where it displaces MYC from its genomic locations, causing a specific shutdown of MYC transcriptional signature and effectively stopping cell cycle progression. Further in vivo preclinical experiments showed that Omomyc blocks MYC-driven transcription and proliferation, induces apoptosis and recruitment of intratumoral immune infiltrates, overall significantly abrogating tumor progression and reducing tumor grading. A targeted LC-MS/MS assay for the absolute quantification of 4 Omomyc proteotypic peptides was developed to assess the structural integrity of Omomyc and quantify functional Omomyc in tumor tissue. LLOQ and linear range of the panel was determined by spiking in dilution series of recombinant Omomyc protein into preclinical tumor tissue lysates. Finally, in the context of Omomyc clinical trial (NCT04808362), we used the panel to for quantification of functional Omomyc in FFPE tumor biopsies from 18 patients who received i.v. injection of Omomyc therapeutic. Data show that 2 h after i.v. administration, higher concentrations of the drug are reached in the tumors compared with serum and persist there with at least one order of magnitude higher concentration than in serum 72 h after administration in preclinical models. Functional Omomyc protein could be quantified in all of the 18 clinical tumor biopsies from treated patients showing that characterized LC-MS/MS proteomic assay can be used in the normal routine of a clinical trial for accurate quantification of therapeutic miniproteins. Citation Format: Marie-Eve Beaulieu, Sandra Martinez-Martin, Maik Mueller, Yuehan Feng, Jakob Vowinckel, Antoine Amaury Lachaud, Laura Soucek. Pharmacokinetic analysis of omomyc therapeutic protein in preclinical and human tumor tissue using targeted mass spectrometry [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 7166.

The Prognostic and Immune Significance of BZW2 in Pan-Cancer and its Relationship with Proliferation and Apoptosis of Cervical Cancer.

Basic leucine zipper and W2 domains 2 (BZW2), a member of the basic domain leucine zipper superfamily of transcription factors, has been implicated in the development and progression of various cancers. However, the precise biological role of BZW2 in pan-cancer datasets remains to be explored. This study aimed to assess the prognostic significance of BZW2 and its immune-related signatures in various tumors. Our study investigated the expression, epigenetic modifications, and clinical prognostic relevance of BZW2 using multi-omics data in different cancer types. Additionally, the immunological characteristics, tumor stemness, drug sensitivity, and correlation of BZW2 with immunotherapy response were explored. Finally, in vitro experiments were conducted to assess the impact of BZW2 knockdown on Hela cells, a cell line derived from cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). BZW2 exhibited elevated expression levels in various tumor tissues and significantly impacted the prognosis of different cancer types. BZW2 emerged as an independent prognostic factor in CESC. We found that copy number amplification and methylation levels of BZW2 were associated with its mRNA expression. Immunological analyses revealed that BZW2 shapes a non-inflamed immunosuppressive tumor microenvironment across multiple cancers. Furthermore, our cell experiments demonstrated that BZW2 knockdown reduced proliferation, migration, and apoptosis activities in CESC cells. BZW2 promotes cancer progression by shaping a non-inflamed immunosuppressive tumor microenvironment. Additionally, BZW2 was shown to significantly influence the proliferation, migration, and apoptosis of CESC cells.