Dpep is a novel cell-penetrating peptide that selectively promotes the death of multiple tumor cell types in vitro and in vivo, and that is a potential therapeutic option for cancer. To better understand how it kills cancer cells, we compared gene expression levels in each of six diverse cancer cell lines before and after peptide treatment. An analysis of the data suggested a mechanism in which Dpep initiates a cascade of perturbations in gene expression that are particular to each cancer cell type, but that represent shared pathways that regulate cell growth and survival. These findings provide insight as to how drugs such as Dpep are able to impact the survival of a wide variety of tumor cell types, and identify how it might be combined with other cancer drugs for optimal efficacy.Dpep is a cell-penetrating peptide targeting transcription factors ATF5, CEBPB, and CEBPD, and that selectively promotes the apoptotic death of multiple tumor cell types in vitro and in vivo. As such, it is a potential therapeutic. To better understand its mechanism of action, we used PLATE-seq to compare the transcriptomes of six cancer cell lines of diverse origins before and after Dpep exposure. This revealed a context-dependent pattern of regulated genes that was unique to each line, but that exhibited a number of elements that were shared with other lines. This included the upregulation of pro-apoptotic genes and tumor suppressors as well as the enrichment of genes associated with responses to hypoxia and interferons. Downregulated transcripts included oncogenes and dependency genes, as well as enriched genes associated with different phases of the cell cycle and with DNA repair. In each case, such changes have the potential to lie upstream of apoptotic cell death. We also detected the regulation of unique as well as shared sets of transcription factors in each line, suggesting that Dpep may initiate a cascade of transcriptional responses that culminate in cancer cell death. Such death thus appears to reflect context-dependent, yet shared, disruption of multiple cellular pathways as well as of individual survival-relevant genes. The transcription factors ATF5 (activating transcription factor 5), CEBPB (CCAAT enhancer binding protein beta), and CEBPD (CCAAT enhancer binding protein delta) play key roles in the formation, growth, survival, metastasis, and treatment resistance of a wide variety of solid tumors . To bindBIRC5 gene) and elevation of the death gene BMF (BCL2-modifying factor) [BCL2 is reported to be a direct transcriptional target of ATF5 [The mechanisms by which the peptides cause the death of malignant cells are only partially understood. A variety of approaches have shown such death to be apoptotic ,9. Criti factor) . In some of ATF5 .However, beyond these findings, there is little evidence as to what may lie between target engagement by the peptides and activation of distal apoptotic pathways. Because the peptides target transcription factors, we hypothesized that their mechanism of action would include perturbation of the transcriptional landscape, which, in turn, would ultimately trigger elements of the apoptotic machinery. To approach this issue, we investigated and compared the transcriptomes of six different tumor cell lines of diverse origins before and after Dpep treatment. The findings presented herein indicate that the peptide initiates multiple context-dependent transcriptional responses. While the patterns of the responses are cell-specific, they include many alterations of individual gene expression and of functional pathways that are shared among different types of cancer cells and that lie upstream of apoptotic signaling. The data also suggest that such events are triggered by cascades of transcription factor activity.2. All lines were tested and found to be negative for mycoplasma .T98G, MDA-MB-231, MCF7, A549, HCT116, and A375 cells were obtained from and authenticated by the ATCC . Cells were cultured prior to the experiments in DMEM supplemented with 10% fetal bovine serum. All cultures were maintained at 37 °C in a humidified incubator in an atmosphere containing 5% CODpep was purchased as an acetate salt from Alan Scientific  and dissolved in 10% glycerol in PBS, pH 7.2, then stored as 2 mM aliquots at −80 °C prior to final dilution for the experiments. The Dpep sequence has been described previously . All cel6 and averaged 2.3 ± 0.1 × 106.For the PLATE-seq analysis , cells wDistribution testing of the expression data using quantro analysis showed significant differences in the shapes of the gene expression distributions of the samples, hence indicating the use of smooth quantile normalization. Multidimensional scaling, principal component analysis, and hierarchical clustering confirmed that the expression data for the different cell lines were segregated from one another, and that in each case, there was also segregation between Dpep-treated and untreated cells. For each line, differential gene expression for the untreated and Dpep-treated cells was determined using edgeR and included quasi-negative binomial error estimation, as well as robust estimation of both the dispersion and the model fit to reduce the effect of outliers. Significance was set at a cutoff of a Benjamini–Hochberg false discovery rate (FDR) of <0.05. All raw and processed data associated with this study are available at the Gene Expression Omnibus under accession number GSE244579.https://www.webgestalt.org/, accessed June to September 2023) using WebGestalt (WEB-based GEne SeT AnaLysis Toolkit) 2019 [Over-representation analysis (ORA) and gene set enrichment analysis (GSEA) were carried out online (it) 2019 , and we it) 2019 ,15,16,17https://depmap.org/portal/, accessed July 2023) and the Public 23Q2 database (accessed July 2023).Cell-line-specific dependency genes and common essential genes  were idehttps://maayanlab.cloud/chea3/, accessed July to September 2023) using ChEA3 , an enrichment tool based on “orthogonal omics integration” [Transcription factor enrichment analysis (TFEA) was performed online  of multiple samples ), on sixCEBPD was considerably more highly expressed in MDA-MB-231, T98G, and A549 cells compared with the other three lines  varied between lines. A549 (3350 DEGs) and MDA-MB-231 cells (3194 DEGs) showed the greatest number of changes; T98G cells (1621 DEGs) an intermediate number; and MCF7 (711 DEGs), HCT116 (638 DEGs), and A375 (517 DEGs) the fewest. A comparison of the individual DEGs elicited by Dpep in each line revealed distinct, line-dependent patterns of changes in both upregulated and downregulated DEGs D. This l2FC ≥ 1 or ≤−1 yielded a single gene, IGFBP3, that was upregulated in 5/6 lines. Although IGFBP3 is expressed by T98G cells, it was not regulated there by Dpep. IGFBP3 has been described as having tumor-suppressive activity in a variety of neoplasias via pleiotropic actions [While each line showed different patterns of regulated genes, about half of the DEGs for each line were shared with at least one other line . We furt actions ,21. Of p actions ,23, MDA- actions , and A54 actions ,26 cells actions , includi actions ,29.2FC ≥ 0.6 or ≤−0.6, a second gene, SPRY1, emerged as upregulated in five lines (but not in HCT116 cells). SPRY1 is described to have context-dependent tumor-suppressing or tumor-promoting activity and is reported to suppress the growth of A549 [When the cutoff was set to Log of A549 , A375 [3 of A549 , and MCF of A549  cells. I of A549  has indi of A549  indicate2FC ≥ 1 or ≤−1, five upregulated DEGs were common to four lines: CA9, MIR210HG, ANGPTL4, C3, and TXNIP. Of these, TXNIP (thioredoxin-interacting protein) upregulation in particular has been associated with cancer cell death [2FC ≥ 0.6 or ≤−0.6, the total number of upregulated DEGs shared by at least four of six lines reached 28, and the number of shared downregulated DEGS was 21   separately for these upregulated and downregulated DEGS (Logr ≤−0.6) B. A gener ≤−0.6) B. A pathWith regard to downregulated DEGs shared by four lines, gene ontology  and pathway (KEGG and Reactome) analyses identified significant enrichment of multiple categories related to the cell cycle and mitosis B. This cBIRC5 gene) and with upregulation of pro-apoptotic BMF (BCL2-modifying factor). The BCL2 protein was also downregulated to a variable degree, depending on the cell line [APOL6, CASP3, DAPK2, IGFBP3); MDA-MB-231 ; T98G ; A375 ; A549 ; and MCF7 . Interestingly, these upregulated DEGs represent both the intrinsic and extrinsic apoptotic mechanisms. Although each line displayed a distinct set of upregulated pro-apoptotic genes, several were upregulated by greater than twofold in three different lines: DAPK2  and XAF1 , and, in one case (IGFBP3), in five of the six lines.Prior studies have shown that Dpep promotes the apoptotic death of transformed cells . In all BCL2 was downregulated by about fourfold in MDA-MB-231 cells, a line that showed strong downregulation of the BCL2 protein by Dpep [BIRC5 (encoding SURVIVIN) was also downregulated by >twofold in MDA-MB-231 and A549 cells. A past study showed that diminished stability also contributes to a loss of the SURVIVIN protein in peptide-treated tumor cells [BCL2L12 was downregulated by >twofold in A549 cells. Taken together, these observations show that Dpep regulates multiple apoptotic genes and does so in a context-dependent, yet partially overlapping, manner.Regarding the downregulated pro-survival genes, there were relatively few, with none regulated by >twofold for HCT116, T98G, A375, or MCF7 cells C. It is or cells . Finally2FC was ≤−0.9 and for which the DepMap gene effect was ≤−0.5 and the Z score was ≤−1.6 according to either siRNA or CRISPR screens. As observed with other comparisons of DEGs described herein, the specific dependency genes regulated by Dpep in each line were distinct  encode proteins required for successful cell cycle completion. For the lines with <4 dependency DEGs, we also asked whether there were additional “common essential” genes identified in DepMap that were downregulated in response to Dpep. As listed in To identify single downregulated genes that could account for the inhibitory effects of Dpep on tumor cell growth and survival, we queried DepMap (Public 23Q2) to uncover candidates specific to each cell line studied herein. In particular, we searched for DEGs in each line in which the logdistinct D. The nuCDCA3, CDCA7, ERCC6L, and TICRR. For each of these genes, knockdown was reported to suppress proliferation and promote apoptosis in malignant lines, including lung and breast cancers [Apoptotic cell death is regulated by a variety of both transcriptional and non-transcriptional mechanisms. We, therefore, looked for upstream transcriptional changes that might affect the balance between pro- and anti-apoptotic components. Numerous studies have established a link between apoptosis and disruption of the activity/expression of cell cycle components ,37,38. T cancers ,40,41,42FAM83D (down by 2.4-fold) and RGCC/RGC32 (down by 4-fold), knockdown of which was reported to promote tumor cell apoptosis [MAD2L1 in T98G cells was also reported to cause cancer cell apoptosis when knocked down [HCT116 and T98G cells also exhibited numerous DEGs associated with the cell cycle, representing 12% and 7% of total DEGs in each line, respectively. In these lines, also, ORA and GSEA analyses identified significant enrichment of regulated genes associated with various phases of the cell cycle . Interespoptosis ,44. Downked down .CDK8 was downregulated by 30-fold in response to Dpep, and that CDK8 inhibition or knockdown promotes cancer cell apoptosis [Thus, in four of six of the assessed lines, Dpep appeared to disrupt the expression of numerous genes involved in multiple steps of cell replication. Given the association between dysregulated cell cycle and apoptotic death, such effects are likely to contribute to Dpep’s inhibition of tumor cell growth and survival in these and, likely, additional cancer cell types. In contrast, A375 and MCF7 cells showed less robust regulation of cell-cycle genes, with only 1.5% and 2.7% of DEGs, respectively, assigned to this activity; neither ORA nor GSEA analysis revealed enrichment in this category. Nevertheless, it may be relevant that in MCF7 cells, the replication-related kinase gene poptosis ,47.BARD1, BRCA1, CHEK1, FANCB, FANCC, NEIL3, RAD51, TONSL, and XRCC3, were mainly associated with DNA repair. Such downregulation may tip the balance towards apoptosis, and supports the potential use of a combination of Dpep and DNA-damaging agents in certain cancers.In addition to cell-cycle-related DEGs, two lines, MDA-MB-231 and A549, exhibited conspicuous downregulation of numerous DEGs associated with DNA repair . ORA andIFI27, IFI44L, IFIT1, OAS1, and OAS2. Notably, each of these genes has been associated with the promotion of cancer cell apoptosis [An analysis of Dpep-responsive DEGs revealed the unanticipated upregulation of interferon-responsive genes in all six lines A. In parpoptosis ,50,51,52ANGPTL4, CA9, CXCL3, EGLN3, IGFBP3, MIR210HG, NDRG1, SOD2, TREM1, and TXNIP. An ORA gene ontology analysis of biological processes indicated significant enrichment in the categories “response to hypoxia” and “response to oxygen levels” in A375 cells, and in “response to oxygen levels” in MCF7 cells  and A549 cells ((log2FC = −2.08), while PLCE1, identified as an oncogene overexpressed in a variety of cancers [2FC = −1.98), MCF7 (log2FC = −4.31) and A549 cells (log2FC = −0.97). As is emblematic of many of the regulated oncogenes, knockdown of MERTK or PLCE1 has been reported to trigger cancer cell apoptosis [Additional scrutiny of the PLATE-seq data revealed cell-line-specific patterns of regulated DEGs encoding proteins described in multiple studies as either oncogenes or tumor suppressors . In each cancers ,54, was  cancers ,56, was poptosis ,54,55,56IGFBP3 (five lines); TXNIP and NDRG1 (four lines); and RAB37 and XAF1 (three lines). The upregulation of such genes by Dpep is anticipated to negatively influence growth and survival.Regarding DEGs encoding tumor suppressors, these were upregulated in all lines, with at least one in each line elevated by ≥2.7-fold. While each line had a distinct pattern of upregulated DEGs encoding tumor suppressors, several of such DEGs were upregulated in multiple lines, including the aforementioned HOXD10, ZNF528); MDA-MB-231 ; T98G ; A375 (BEND5); A549 ; and MCF7 . Four of the six lines also showed at least one transcription factor downregulated by at least eightfold: HCT116 (PAX9); MDA-MB-231 (LHX6); T98G (ATOH8); and A549 . While each line showed a distinct pattern of regulated transcription factors, there were several instances in which specific transcription factors were similarly responsive across multiple lines. These were IF27 ; ZNF467 ; FOXM1 ; ID2 ; ID3 ; MDX3 ; and MYBL1 . Taken together, these observations indicate that Dpep triggers dysregulated expression of multiple transcription factors in a context-dependent fashion. The presence of multiple regulated TFs suggests a cascade that is initiated by interference with ATF5, CEBPB, and CEBPD activities. This, in turn, may account in part for the large numbers of cell-type-specific DEGs elicited by Dpep treatment, and, ultimately, for the effects of the peptide on cancer cell growth and survival.Given the varied and context-dependent effects of Dpep on the transcriptome, we considered how it might affect the expression of transcription factors. Exploration of the PLATE-seq data revealed cell-specific patterns of multiple up- and downregulated DEGs encoding proteins that regulate transcription C. In eacTo further probe the regulatory networks of transcription factors that might contribute to the responses of cancer cells to Dpep, we carried out transcription factor enrichment analysis (TFEA) using ChEA3, an enrichment tool based on “orthogonal omics integration” . The anaAs is the case for other aspects discussed herein, the ChEA3 analyses of A375 and MCF7 DEGs differed from those in the other four lines, and the scores tended to be less robust. This underscores the context-dependent nature of the response to Dpep. One intriguing observation was the appearance of HIF1A  in the sets of lists for both cell lines, which may potentially be relevant to their enrichment for hypoxia-related DEGs, as discussed above.Another notable observation from the ChEA3 analysis was the inconsistent rankings of CEBPD, CEBPB, and ATF5 and their generally low scores. This suggests the interpretation that interference with their activities triggers a largely indirect cascade of downstream alterations in TF activities that ultimately accounts for the patterns of the observed DEGs. Consistent with this are the large numbers of Dpep-responsive DEGS that encode TFs, as described above.While the ChEA3 analysis did not provide consistent rankings for ATF5, CEBPB, or CEBPD when total DEGs were considered, we observed that such an analysis of only upregulated DEGs for each line returned enrichment scores in which at least one of the three factors was in the top 50 TFs in both the Mean and Top Rank libraries . In addiTo conceptualize potential networks through which Dpep might generate a transcriptional cascade by interfering with CEBPB/CEBPD activity, we used the TF co-regulatory tool in ChEA3 to analyze the top 25 and 43 enriched TFs in both the Mean and Top Rank libraries for upregulated DEGs in A549 cells D. Each aThe purpose of this study was to identify and compare the transcriptional responses of multiple tumor cell types to Dpep, a cell-penetrating peptide designed to interfere with ATF5, CEBPB, and CEBPD activity. We also inferred how such changes may lead to the apoptotic death of cancer cells. We achieved this goal using PLATE-seq, a low-cost, genome-wide mRNA profiling methodology which was initially applied for the purpose of screening drug effects across single cell lines . We achiThe overall finding was a context-dependent pattern of gene regulation that was unique to each of the six assessed cell lines. Within this general diversity, however, there were a number of shared regulated elements and pathways with the potential to negatively affect cell growth and survival . There wDAPK2 [XAF1 [TNFSF10 (TRAIL) and TNFSF14 (LIGHT), that participate in extrinsic apoptotic signaling. Of potential relevance, a past study showed that CP-dn-ATF5, a cell-penetrating peptide that targets CEBPB and CEBPD, sensitizes tumor cells to TRAIL-induced apoptosis [BCL2 in MDA-MB-231 cells. This correlates with a previously reported strong downregulation of the BCL2 protein by Dpep and Bpep in this line [A common endpoint thus far of all cancer lines treated with Dpep has been the onset of apoptotic cell death. At least two pro-apoptotic genes were upregulated by >twofold in each line assessed herein. While each line displayed a unique set of such genes, there was some overlap, with two pro-apoptotic genes, DAPK2  and XAF1K2 [XAF1 , upregulpoptosis . RegardiIGFBP3), as well as those unique to each line.Analysis of the PLATE-seq data uncovered a number of Dpep-regulated pathways and biological activity groups that can lie upstream of apoptotic activation. Four lines showed substantial downregulation of genes identified by DepMap as being “dependency” genes for those lines. Additionally, we found upregulation of multiple tumor suppressors and downregulation of multiple oncogenes in all lines. Again, while all lines were affected, the patterns of the regulated genes were context-dependent and included genes regulated in up to five of the six lines , were found by gene ontology analysis to show enrichment for hypoxia and interferon responses. These were also the two lines that showed the highest proportions of co-shared DEGS after normalization. As noted above, dysregulations of genes involved in the cell cycle or responsive to hypoxia, or interferons have been described as promoting the activation of apoptotic pathways.CDK8 in Dpep-treated MCF7 cells and the downregulation of “dependency” genes in susceptible cell lines. On the whole, the scenario emerges that in each cancer cell type, Dpep sets in motion multiple streams of altered gene expression that have the potential to culminate in apoptotic death.While our analysis has focused on pathways that show disruption by Dpep, it is important to consider that the dysregulation of only a single gene may interfere with cell growth and survival. The examples discussed above include depletion of replication-related transcripts for FOXM1, a well-studied oncogenic TF with a variety of roles in promoting cancers [How, then, might Dpep initiate such events? Interrogation of the transcriptome data revealed that in all six lines, Dpep regulated numerous transcription factors. While the patterns of the regulated TFs were context-dependent, it is also of interest that several were regulated in three different lines. Among them was downregulated  cancers ,60, inclGiven the design of Dpep, it is anticipated that the induction of such a cascade might start with altered expression of genes directly regulated by ATF5, CEBPB, and CEBPD. Notably, ChEA3 TF enrichment analysis based on total DEGs for each line did not highly rank the three factors, nor were their rankings consistent within or between cell lines. This is consistent with a mechanism in which interference with ATF5/CEBPB/CEBPD activity initiates cascading networks of downstream TFs that culminate in the production of the patterns of DEGs observed herein. The application of ChEA3 analysis to only upregulated DEGs consistently places CEBPB and/or CEBPD in the 50 top-ranked TFs. This enabled us to use the ChEA3 co-regulatory tool to produce a model of how a network of TFs might be interconnected with CEBPB and CEBPD to generate the observed pattern of DEGs in A549 cells.Regarding the inconsistent ranking of ATF5 in such analyses, this may reflect either its relative lack of involvement in generating DEGs or the present paucity of information regarding its transcriptional targets. For example, one report has indicated that ATF5  associates with a novel DNA binding site . In addiBesides directly repressing the transcriptional actions of ATF5, CEBPB, and CEBPD, there are several additional non-mutually-exclusive mechanisms by which Dpep might alter the transcriptomes of cancer cells. For example, the absence of available ATF5, CEBPB, and/or CEBPD may relieve competition for binding to DNA by other TFs, thus permitting an alternate pattern of gene regulation. It also cannot be ruled out that Dpep associates with and serves as an inhibitory decoy for TFs in addition to ATF5, CEBPB, and CEBPD . FinallyIn conclusion, the transcriptomic data described herein indicate that Dpep promotes the death of a wide range of tumor cells by setting in motion a cascade of TF-driven changes in multiple molecular pathways that converge on apoptosis. These events are context-dependent, yet fall into discrete sets of pathways shared by subsets of tumor cell types. The pathways include upregulation of tumor suppressors; pro-apoptotic genes; and genes responding to hypoxia, cytokines, and interferon. Downregulated pathways include oncogenes; dependency genes; and genes involved in cell cycle regulation, DNA repair, and cell survival.


Klotho, a gene found on chromosome 13q12, is involved in a variety of processes and signaling pathways in the human body related to vitamin D metabolism; cardiovascular, renal, musculoskeletal, and skin diseases; and cancer biology. However, more importantly, it has been linked to beneficial effects related to anti-aging. The levels of soluble Klotho in the blood have been found to decline with age, increasing the risk of age-related diseases. When the Klotho gene was silenced or defective, it caused a shorter lifespan. However, when the gene was overexpressed, it resulted in a longer lifespan. Klotho has positive benefits on the neurological system by causing a higher representation of useful longevity genes, preventing further neuronal damage, and offering neuroprotection. Thus, it has the potential to become a new treatment for many age-related diseases that cause dementia, including multiple sclerosis, Alzheimer’s disease, and Parkinson’s disease. In this review, we discuss the mechanisms of Klotho’s benefits and roles on various organ systems, specifically on nervous system disorders that lead to dementia. Klotho is a membrane-bound anti-aging protein that functions as an endocrine, autocrine, and paracrine hormone targeting different cells . There aThe Klotho gene is greatly exhibited in the kidneys, where it modulates renal function, parathyroid glands, and choroid plexus ,4. It plDementia is characterized by global cognitive impairment, consisting of an acquired decline in cognitive and emotional functioning, as well as behavioral and psychiatric disturbances severe enough to interfere with daily life. It is one of the most prevalent clinical syndromes seen in the old age group (age >65 years) . It has In this article, we aim to further explore the associations between the Klotho gene and dementia and try to help build a platform so that work can be done to investigate the therapeutic potential of Klotho for neurodegenerative conditions such as Alzheimer’s disease.Role, synthesis, and functions of Klotho in humansKlotho, a gene present on chromosome 13q12, has three protein forms, namely, a secretory, a cell membrane-associated, and an intracellular form. Membrane protease can digest the extracellular Klotho domain and can be seen in CSF, urine, or blood . SecretoThe circulating levels of soluble Klotho have been observed to decrease with age, which increases the risk of age-related illnesses. Torbus-Paluszczak et al. 2018) demonstrated accelerated aging and a shortened lifespan in mice when the Klotho gene was silenced or deficient . In cont18 demonsExpression of Klotho in the nervous systemIn the CNS, a secreted version of Klotho plays an important role in neuronal activity. It is a critical component of processes such as aging and neurodegeneration. In CSF, the expression of cytokine/chemokine is suppressed by Klotho, thereby supporting the activation of neuronal cells.Klotho is abundant in hippocampus neurons, choroid plexus ependymal cells, Purkinje EC cells, and the white matter of the brain . Its relRole of Klotho on oxidative stress/free radical injury and age-related changesKlotho was identified as a product of an anti-aging gene . AccordiIn contrast, KL-OE is expected to prolong the lifespan by reducing oxidative stress. It works like a hormone; thus, it attaches to the membrane receptor and prevents insulin-like growth factor-1 (IGF-1) from sending an intracellular signal . Its conMechanism of action of Klotho and signaling pathwaysMany biological activities of Klotho have been found; however, their molecular processes are unknown and must be explained. Research has shown that circulating Klotho has a role in intracellular signaling cascades by modulating the activity of multiple cell surface receptors, including TGF-β1 ,19, p53/Klotho’s Effect on the TGF-β1 Signaling PathwaySeveral growth factors and cytokines induce epithelial-to-mesenchymal transition (EMT). TGF-1 is the most efficient and ubiquitous growth factor. It works by suppressing the expression of genes with epithelial activities such as E-cadherin and by increasing the production of genes with mesenchymal functions such as vimentin, collagen-1, and N-cadherin. TGF-1 signaling is disrupted by secreted Klotho, which binds directly to the type II TGF receptor and inhibits TGF-1 binding. Secreted Klotho inhibits EMT in cancer cells and prevents the spread of cancer ,31.Klotho’s Effect on the p53/p21 Signaling PathwayCell cycle arrest by tumor suppressor p53, which activates the downstream p53-dependent cyclin-dependent kinase factors p-16 and p-21, results in growth arrest and cell death. Klotho reduces cellular senility in MRC5 primary human fibroblast and human umbilical vascular endothelial cells by inhibiting the p53/p21-dependent pathway. According to one theory, Akt/protein kinase B (PKB) decreases cell lifetime via the p53/21-dependent pathway. However, these hypotheses need to be validated .Klotho’s Effect on the c-AMP/PKA Signaling Pathwayc-AMP is generated from ATP by the enzyme adenylate cyclase (AC). c-AMP is required for protein kinase A (PKA). It stays catalytically inactive throughout the typical cell cycle. When c-AMP levels rise, an active form of PKA is released, phosphorylating and regulating several target proteins, including enzymes. By promoting superoxide generation, angiotensin-II (ANG-II) contributes to endothelial dysfunction. Membrane Klotho increases the angiotensin-converting enzyme (ACE) activity by stimulating the c-AMP/PKA pathway in human umbilical vein endothelial cells. Overexpressed Klotho suppresses the nicotinamide adenine dinucleotide phosphate oxidase 2 expression, thereby reducing ANG-II-induced oxidative damage, superoxide production, and apoptosis in smooth muscle cells. Hence, Klotho protects smooth muscle cells .Klotho’s Effect on the PKC Signaling PathwayPKC is an isoenzyme that forms a family related to serine/threonine kinases that regulate the target proteins. 25-OH vitamin D3 1-α-hydroxylase gene expression is regulated by both c-AMP and PKC. Klotho, on the other hand, inhibits the gene for 25-OH vitamin D3 1-α-hydroxylase in kidney cells. It is believed that Klotho suppresses the 1-alpha hydroxylase genes through a c-AMP or PKC-independent pathway. However, Klotho activates the c-AMP and PKC signaling pathways. PKC signaling pathway activation only occurs in the kidney and testes. In contrast, c-AMP activation occurs in each cell line .Klotho’s Effect on the Wnt Signaling PathwayWnt1, a proto-oncogene, is an important secreted factor that is responsible for cell proliferation and its maintenance. Its continuous exposure leads to accelerated cellular senescence. The biological activity of Wnt is inhibited when it interacts with Klotho. This tumor suppressor effect of Klotho is due to the inhibition of Wnt signaling and p53/21 and insulin/IGF-1. Klotho plays a key role in tumor genesis, its progression, and its prognosis. In tumor cells, Klotho is silenced by histone deacetylation and by hypermethylation of its promoter. Low levels of Klotho are accompanied by raised levels of Wnt/beta-catenin, which lead to fibrogenesis. Consequently, the role of Klotho in CKD is evident, as it regulates Wnt/beta-catenin activity .Regulation of FGF-23 Signaling Via KlothoFGFR23 is a protein that targets the membrane expressions of Klotho and FGFR. FGF-23 initiates downstream signaling effects, such as GRB 2-associated binding protein 1 (GAB1), ERK1/2-SGK1, phospholipase C (PLC), Shc, fibroblast growth factor receptor substrate 2 (FRS2), or signal transducer and activator of transcription 1 (STAT1), through this complex, providing a likely mechanism for different genes to be expressed. According to some experiments, FGF-23 inhibits the biosynthesis of phosphate and vitamin D reabsorption in the proximal renal tubules. At the same time, its homozygous missense mutation in humans can lead to soft-tissue calcification due to severe hyperphosphatemia. Klotho and FGF-23, through apoptosis, prevent vitamin D-induced cell death and, through cell proliferation, prevent tissue atrophy. This signaling inhibits vitamin D-mediated cell death by downregulating 1-alpha hydroxylase gene expression and suppressing the caspase activity through phosphoinositide-3 kinase .Klotho’s Effects on the Insulin/IGF-1 Signaling PathwaysThe intracellular IGF-1 signaling cascade, which is blocked by the aging suppressor Klotho protein, extends lifespan. With Klotho binding, the insulin/IGF-1 receptor’s tyrosine kinase activity is blocked, followed by insulin receptor substrate inhibition (IRS). IRS inhibits PKB and phosphoinositol 3-kinase (PI3K) serine phosphorylation. The capacity of the Klotho protein to block the insulin/IGF1 receptor or the direct suppression of IRS, P13K, and PKB/Akt still needs to be determined. Klotho inhibits IGF-1-mediated signaling pathways, which suppress various tumor types. It stimulates the Foxo forkhead transcription factors, which are downregulated by insulin/IGF-1 signaling. Hence, it increases the mitochondrial MnSOD expression, eliminating the reactive oxygen species and providing oxidative stress tolerance in animals at the organismal and cellular levels. The Klotho hormone is also involved in oxidative stress resistance, which may add to its anti-aging properties .Relation of Klotho deficiency and neurological abnormalitiesWith age, both the expression and circulating levels of klotho decline. In a study by Xiao et al., Klotho levels decreased with age in human blood serum samples . Klotho Neuroprotective effect of Klotho in different types of dementiaDementia is a neurological disorder categorized by impairment in memory in addition to one or more cognitive domains . This impairment constitutes a decline from the previous level of function and should be significant enough to result in impairment in daily life .It is estimated that about 47 million people suffer from dementia worldwide. As age is the most important risk factor leading to dementia, this number is set to increase in the coming years . Other rAlzheimer’s disease is characterized by extensive cortical atrophy caused by the deposition of hyperphosphorylated tau protein and amyloid plaques. Interleukin-6 (IL-6) is thought to activate microglia, which, in turn, leads to an increase in tau phosphorylation and the production of acute-phase reactions in neural tissue. A positive correlation was found between Alzheimer’s disease and elevated IL-6 levels . Lewy-boAmyloid plaques in Alzheimer’s disease consist of β-amyloid which is created through the processing of APP. It is postulated that the extracellular processing of APP promotes Klotho expression which protects against the harmful effects of β-amyloid on the neurons. β-amyloid is also thought to damage neurons through reactive oxygen species, and Klotho has been illustrated to protect against these oxidative insults . Higher Klotho contributes crucially to protecting hippocampus neurons from amyloid formation and plays a role against glutamate toxicity. Klotho is also considered key for oligodendrocytes maturation and myelin integration. The role of Klotho in the cellular life span regulation by p53 repression and downregulating p21 protein levels may be related to enhancing signaling through the insulin/IGF-1 pathway is already known . On a miMultiple SclerosisMultiple sclerosis is a demyelinating condition causing inflammation and myelin sheaths destruction in different parts of the CNS. A study showed that serum Klotho concentration is higher in multiple sclerosis patients with a longer disease duration when compared to the control group . ZeldichAlzheimer’s DiseaseThe disease process is centrally associated with the accumulation of amyloid plaques, neurofibrillary tangles, and neurodegeneration of various neuronal pathways of the brain. Multiple theories such as hyperphosphorylated tau proteins abnormalities and oxidative stress initiate the disease progression. A study done in Baltimore demonstrated that there are lower Klotho levels in CSF of Alzheimer’s disease compared to the control group, lower levels in older adults compared to younger adults, and higher levels in males than females . PretreaCognitionAll types of dementia are neurodegenerative, and protein toxicity is the principal feature. The leading causes are Alzheimer’s disease and Parkinson’s disease causing deterioration of brain functions. Klotho expression is reported in the adult hippocampus, and it enhances cognitive performance in mammals . As mentParkinson’s DiseaseA study conducted in Iran showed findings denoting the neuroprotective role of exogenous Klotho in the rat model of Parkinson’s disease and the protective effect against astrogliosis, programmed cell death, and oxidative insults by different signaling cascades . ZimmermRole of Klotho in other organ systemsReduced serum Klotho level has been linked to a greater density of epicardial fat, carotid artery intima-media, and reduced flow-mediated dilation of the brachial artery in the cardiovascular system; hence, reduced levels of serum Klotho must be addressed as a preliminary prognosticator of atherosclerosis . The redIn the gastrointestinal system, the role of abnormal Klotho levels was observed in colorectal carcinoma. DNA hypermethylation specifically in the promoter region leads to suppression and notable downregulation of Klotho in malignant colon cells which leads to their multiplication. Adequate Klotho levels can cause apoptosis of malignant colon cells . Klotho In the renal system, Klotho deficiency is greatly linked with ionic imbalance, calcification in vessels, inflammation and fibrosis in the renal parenchyma, and skeletal disorders, which represents CKD. Stage 2 CKD shows a premature reduction in Klotho protein levels in the blood, and stage 1 CKD shows even an earlier premature reduction in urinary Klotho levels. Hence, deficient Klotho levels are responsible for the growth and succession of CKD and other complications. It shows an analytical capacity that acts as a demonstrative biomarker of renal disorders such as CKD . Any treKlotho is also found in the skeletal system, specifically in osteoblasts and osteoclasts. Klotho protein plays an important role as a co-receptor for FGF-23. It has an effective activity in the development of the skeletal system. Further research is required to use this discovery to form therapy for bone disorders such as osteogenesis imperfecta, osteoporosis, osteodystrophy related to renal system disorders, and other bone-related ailments .Therapeutic role of Klotho on dementiaA randomized control trial on middle-aged healthy males conducted in Slovenia (2019) showed that a lower dose therapy of fluvastatin and valsartan causes a higher representation of useful longevity genes sirtuin-silent mating type information regulation 2 homolog, protein kinase AMP-activated catalytic subunit alpha, and Klotho, so it can be expressed as a favorable novel therapeutic perspective for senile disorders . Dubal eKlotho is one of the essential proteins that function as neuroprotective, anti-apoptotic, and suppresses oxidative stress. Since the detection of this protein, multiple studies have expanded our understanding of the significant role Klotho plays in the aging process. The levels of Klotho level decrease with age, which, in turn, increases the risk of developing dementia. Maintaining optimum levels of serum Klotho can slow this process. Furthermore, Klotho has been found to reduce neural damage and neurodegeneration, thus protecting against cognitive decline and contributing to life extension in humans. However, further studies are needed to explore its therapeutic role in humans.


Introduction: The ligand-activated transcription factors, nuclear hormone receptors (NHRs), remain unexplored in hematological malignancies except for retinoic acid receptor alpha (RARA).Methods: Here we profiled the expression of various NHRs and their coregulators in Chronic myeloid leukemia (CML) cell lines and identified a significant differential expression pattern between inherently imatinib mesylate (IM)-sensitive and resistant cell lines.Results: Retinoid-X-receptor alpha (RXRA) was downregulated in CML cell lines inherently resistant to IM and in primary CML CD34+ cells. Pre-treatment with clinically relevant RXRA ligands improved sensitivity to IM in-vitro in both CML cell lines and primary CML cells. This combination effectively reduced the viability and colony-forming capacity of CML CD34+ cells in-vitro. In-vivo, this combination reduced leukemic burden and prolonged survival. Overexpression (OE) of RXRA inhibited proliferation and improved sensitivity to IM in-vitro. In-vivo, RXRA OE cells showed reduced engraftment of cells in the bone marrow, improved sensitivity to IM, and prolonged survival. Both RXRA OE and ligand treatment markedly reduced BCR::ABL1 downstream kinase activation, activating apoptotic cascades and improving sensitivity to IM. Importantly, RXRA OE also led to the disruption of the oxidative capacity of these cells.Conclusion: Combining IM with clinically available RXRA ligands could form an alternative treatment strategy in CML patients with suboptimal response to IM. Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by a reciprocal chromosomal translocation t that generates the BCR::ABL1 fusion protein, a constitutively active tyrosine kinase essential for the pathogenesis of the disease. Molecular targeted therapy with tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM) has dramatically improved the outcome of this disease. However, a proportion of patients fail to achieve or retain molecular response with TKIs leading to disease progression.The mechanism of TKI resistance in CML has been extensively studied and broadly classified as BCR::ABL1 dependent and independent. BCR::ABL1-dependent mechanisms include mutations in the BCR::ABL1 kinase domain and over expression of BCR::ABL1 or gene expression. BCR::ABL1 independent resistance mechansim include the persistance of leukemic stem cells, activation of multiple signaling pathways, and epigenetic events . MutatioRXRA) ligand bexarotene in combination with chemotherapy was more effective associated with better survival, and improved response in refractory cutaneous T-cell lymphoma (CTCL) (PPARG and RXRA were downregulated in chemoresistant acute myeloid leukemia (AML) cell lines  , metastaa (CTCL)  and non-a (CTCL) . The rolll lines , suggestRXRA, RXRB, and RXRG , vitamin-D-receptor (VDR), peroxisome proliferator-activated receptors (PPAR), liver-X-receptor (LXR), pregnane-X-receptor (PXR), and the constitutive androsterone receptor (CAR)  in SH-SY5Y cells  are members of the NHR superfamily . 9-cis rand RXRG . RXRs eior (CAR) . RARA ansyndrome . Severalll lines . AML pat, methyl thiazolyldiphenyl-tetrazolium bromide (MTT), 9-cis retinoic acid (9cRA), Bexarotene (Bexa), acitretin (ACI), SR11237, all trans retinoic acid (ATRA), testosterone, 17β-estradiol (17βE), pioglitazone (PIO), rosiglitazone (rosi), Triiodothyronine (T3) and GSK4716 were from Sigma-Aldrich . Rabbit monoclonal p-CRKL , p-STAT5 , p-AKT , caspase-3 , PARP , and RXRA  were from Cell Signaling Technologies . β-actin (A5441) was from Sigma-Aldrich , and horseradish peroxidase-conjugated goat anti-rabbit IgG , goat anti-mouse IgG  from Cell Signaling Technologies . Imatinib Mesylate used for mice model experiments is a gift from NATCO, India.2 at 37°C.CML cell lines JURL-MK1, KCL22, Lama84, KYO1, EM2, and KU812, were a kind gift from Dr. Markus Muschen, University of California San Diego (UCSD), with approval from DSMZ, Germany. Cell lines were cultured in RPMI medium with 10%-FBS and antibiotics (100 U/ml-penicillin and 100 μg/mL-streptomycin) in a humidified atmosphere with 5% CO+ cells, as reported previously . Primary CML cells from blood or bone marrow were processed and enriched for CML CD34eviously .Total RNA was extracted from CML cell lines  and treated with or without NHR ligands using Trizol reagent. RNA (1 μg) was converted to cDNA using random hexamer and reverse transcriptase enzyme .2 PCR array  (https://geneglobe.qiagen.com/product-groups/rt2-profiler-pcr-arrays). The expression of each NHR was normalized using five housekeeping genes , as recommended by the manufacturers. The data was analyzed using SA biosciences web-based analysis software .To identify the basal expression of NHRs and coregulators in CML cell lines, we used an NHR RTAHR (TaqMan assay ID: Hs00169233_m1), AR (Hs00171172_m1), ESR1 (Hs01046816_m1), ESRRG (Hs00976243_m1), PPARG (Hs01115513_m1), RXRA (Hs0107640_m1), RXRB (Hs 00232774_m1) and THRA (Hs00268470_m1) were evaluated by qRT-PCR. Imatinib influx transporter SLC22A1/hOCT1 (HS00427554-m1) expression was measured using TaqMan assay after treating the primary CML cells with NHR ligands. GAPDH was used as the housekeeping gene for these targets. Fold change in the expression of other NHRs such as RARA, PPARG, and VDR was measured by RQ-PCR using SYBR green chemistry (SYBR premix Ex TaqII (Tli RNAseH Plus), Takara, Bio, Shiga, Japan), and ACTB was used as the housekeeping gene to calculate the relative expression (∆∆Ct) of these targets.The expression of 5], were treated in a 96-well plate with increasing concentrations of imatinib (0–500 nM) for 48 h. Cell viability was measured at standard wavelength 570 nm and reference wavelength 630 nm using EL800 microplate reader with KC junior software .CML cell lines JURL-MK1, KCL22, Lama84, KYO1, EM2, and KU812 [1 × 10CML cell lines (KCL22 & Lama84) were pretreated with NHR agonists  of imatinib for 48 hrs.As suggested by previous reports , bulk pr+ cells (n = 39) and healthy donor CD34+ cells (n = 9) using total RNA purification plus kit . Expression of RXRA was assessed using nCounter, and data counts were normalized with housekeeping genes–ACTB, GAPDH & GUS using nSolver software .RNA was extracted from CML CD34+) or healthy donors  cells (2 × 105) were treated with and without RXRA ligands 9-cis retinoic acid, acitretin, or bexarotene in combination with imatinib for 24 h; 1 × 104 cells were seeded in methylcellulose medium . Colonies were visualized using a phase-contrast microscope (×10 objective) and enumerated after 14 days.Primary CML (bulk & CD34+ cells (2 × 105) were treated with RXR ligands 9-cis retinoic acid, acitretin, or bexarotene for 24 h, followed by imatinib for 48 h. Cells were washed with 1XPBS and stained with Annexin-V conjugated with allophycocyanin (APC) and 7-Aminoactinomycin D (7AAD) . BD-Accuri C6  with BD Accuri C6 software (Version 1.0.264.21) was used for flow cytometry analysis.Primary CML CD34CML cell lines  or primary CML cells were treated with RXRA ligands  for 24 h, followed by imatinib for 48 h. The whole-cell lysate was prepared using radioimmunoprecipitation assay (RIPA) buffer supplemented with protease-phosphatase inhibitor cocktail  and 2 mM phenyl methyl sulfonyl fluoride (Sigma Aldrich). Total protein concentration was measured using the Bradford assay. 50 ug of protein was resolved in a 10% SDS PAGE. Anti-p-CRKL, anti-p-AKT, anti-p-STAT5, anti-caspase3, and anti-PARP antibodies were used, and β-actin was used as the loading control.The RXRA plasmid containing the full-length cDNA, pLX304 (clone ID- HsCD00437134), was purchased from the DNASU plasmid repository . Lentivirus particles were prepared by transfecting 293T cells with the RXRA cDNA plasmid and packaging plasmids. Lentiviruses were collected after 48, 60, and 72 h, pooled together, filtered, and concentrated using a lenti-X concentrator , and stored at −80°C until use. Lama84 cells were transduced with lentiviruses in the presence of polybrene, followed by spinfection. RXRA OE virus transduced Lama84 cells were selected by blasticidin (1 mg/mL).6 cells) were then transplanted into sub-lethally irradiated (2.5Gy) 8–10 weeks old NSG mice  via tail-vein. Disease development was monitored by measuring luciferase expression by injecting D-luciferin substrate and imaging using an IVIS Spectrum in-vivo imaging system . One week after transplantation, the mice were treated orally with either imatinib mesylate (100 mg/kg dissolved in water)  or acitretin (10 mg/kg dissolved in corn oil) or in combination for 21 days. Engraftment of KCL22 cells in the mice was assessed by measuring luciferase expression every week for 6–7 weeks. All animal experiments were carried out per protocols approved by the Institutional Animal Care and Use Committee (IAEC No. 5/2019).293T cells were transfected with plasmid lentiX-Luc2 containing a luciferase reporter for lentivirus production. KCL22 cells were transduced with lentivirus containing the luciferase reporter. Luciferase transduced KCL22 (1*106) cells were transplanted into sub-lethally irradiated (2.5Gy) 8–10 weeks old NSG mice via tail-vein. The leukemic burden in peripheral blood was assessed using flow cytometry by measuring human CD45 expression . The percentage of survival of mice was evaluated by Kaplan-Meir survival analysis.RXRA/empty vector transduced Lama84 (2*105cells per well (4 replicates) were seeded in a 24-well XF24 plate coated with retronectin . Thirty minutes before analysis, the medium was replaced with Seahorse XF media , and the plate was incubated at 37°C. Three sequential measurements of OCR and ECAR were taken at basal, proton leak (oligomycin), maximal respiration (FCCP), and OXPHOS inhibition (rotenone and Antimycin-A) to assess bioenergetics.Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse-XFe24 Analyzer (Agilent Technologies). Briefly, Lama84 EV and RXRA OE 4 × 10t-test, non-parametric t-test, and ANOVA with Kruskal-Wallis’s correction were used where appropriate. All statistical analysis were done using GraphPad Prism-v5 software, and a p-value < 0.05 was considered statistically significant.Paired in-vitro cytotoxicity assay to identify CML cell lines that are sensitive or resistant to IM. KU812, EM2, and KYO1 cell lines were identified as sensitive, and JURL-MK-1, KCL22, and Lama84 cell lines as resistant to IM. The kill curves and IC-50 of the cell lines tested are listed  and ten coregulators  were upregulated and RORA & 4 coregulators  were downregulated in imatinib-sensitive cell lines. A Representative Heatmap illustrates the expression profile of NHRs and coregulators  were upregulated in at least one of the IM-sensitive cell lines  could improve IM sensitivity. The concentration of the ligands used for this treatment is listed . Treatmein-vitro in primary bulk cells obtained from imatinib naive CML patients  improved IM IC-50 patients . Other Npatients . Based oe status . Primarye status .+ cells in-vitro, and the cell viability and colony-forming capacity were assessed. RXRA ligands, either alone or combined with IM, significantly decreased cell viability , accelerated phase (AP), and blast crisis (BC) patient samples from the GSE4170 dataset and found significantly reduced expression of RXRA in BC-CML samples compared to CP and AP  in CML cell lines and primary cells. RXRA ligands decreased p-CRKL, p-AKT, and p-STAT5 at the protein level in combination with IM  in CML cnd generation TKIs, dasatinib and nilotinib  of RXRA in the Lama84 cells with deficient basal protein expression , showed a differential effect, sensitizing the KCL22 cell line but no impact on the Lama84 cell line in combination with IM. Similarly, THRA agonist T3 treatment sensitized Lama84 and did not affect KCl22. Although 17BE and stanozolol treatment improved viability in both cell lines, these ligands did not improve sensitivity to IM in bulk CML cells PPARG agonist pioglitazone. When we assessed the BCR-ABL downstream signaling pathways, we observed that RXRA agonist in combination with IM resulted in decreased p-CRKL, p-STAT5, and p-AKT along with activation of the apoptotic cascade in CML cell lines and primary cells.RXRA in imatinib-resistant Lama84 cell line showed decreased proliferation, decreased BCR-ABL downstream signaling, and improved imatinib sensitivity in-vitro. RXRA expression in CML CD34+ cells compared to HD CD34+ and a reduction in the expression of RXRA in blast crisis CML patients compared to chronic phase, accelerated phase CML from the GSE4170 dataset indicating a potential role of RXRA in hindering leukemogenic capacity in CML.Lentiviral-mediated overexpression of + cells have a high oxidative capacity, which could be inhibited using tigecycline , a more robust in-vivo model to evaluate the therapeutic efficacy of this combination and anti-leukemogenic potential RXRA has on CML LSCs is needed. We are currently exploring the mechanism by which RXRA activation by both molecular/pharmacological means could orchestrate the downstream kinase activities.While our findings suggest the potential use of RXRA agonists in combination with IM in eliminating CML CD34in-vitro and in-vivo results suggest that using RXRA ligands could be an effective combination therapy to improve the imatinib response in patients with CML.We demonstrated differential NHR expression between imatinib-resistant and sensitive cell lines. The ligand-dependent activation of RXRA could be an effective CML target, affecting BCR-ABL1 downstream signaling and apoptotic pathway. Our


Graft versus host disease (GVHD) represents the major complication after allogeneic hematopoietic stem cell transplantation (Allo-SCT). GVHD-prone patients rely on GVHD prophylaxis (e.g. methotrexate) and generalized anti-GVHD medical regimen (glucocorticoids). New anti-GVHD therapy strategies are being constantly explored, however there is an urgent need to improve current treatment, since GVHD-related mortality reaches 22% within 5 years in patients with chronic GVHD. This review is an attempt to describe a very well-known receptor in lipoprotein studies – the low-density lipoprotein receptor related protein 1 (LRP1) - in a new light, as a potential therapeutic target for GVHD prevention and treatment. Our preliminary studies demonstrated that LRP1 deletion in donor murine T cells results in significantly lower GVHD-related mortality in recipient mice with MHC (major histocompatibility complex) -mismatched HSCT. Given the importance of T cells in the development of GVHD, there is a significant gap in scientific literature regarding LRP1’s role in T cell biology. Furthermore, there is limited research interest and publications on this classical receptor molecule in other immune cell types. Herein, we endeavor to summarize existing knowledge about LRP1’s role in various immune cells to demonstrate the possibility of this receptor to serve as a novel target for anti-GVHD treatment. Allogeneic hematopoietic stem cell transplantation (AHSCT) is a treatment for hematological malignancies by transferring stem cells from a healthy person (the donor) to the patient (the recipient) after high dose chemotherapy or radiation. Graft versus host disease (GVHD) is one of the major causes of morbidity and mortality in patients undergoing AHSCT . During Studies of the pathophysiological mechanisms of GVHD, as well as deeper understanding of T cell activation, have laid a foundation for novel therapeutic approaches in GVHD prophylaxis and management . Due to The low-density lipoprotein receptor-related protein (LRP1) is a member of the LDL receptor family. LRP1 plays diverse roles in multiple biological processes including lipoprotein metabolism, endocytosis, cell growth, cell migration, inflammation, and apoptosis. It is also involved in the regulation of platelet derived growth factor receptor, vascular tone, and blood brain barrier (BBB) permeability. Additionally, LRP1 is implicated in conditions such as neurodegenerative diseases, atherosclerosis, and cancer –7.via direct interaction with LRP1 on T cells  was first described through homology cloning with the LDL (low-density lipoprotein) receptor by Herz in 1988 . Based oLRP1 is now readily identified as a member of the LDL receptor family. This family of proteins contains structurally homologous receptors that are composed of uniformed structures. This receptor family currently consists of seven family members that are closely related, which includes the LDL receptor, very-low-density lipoprotein (VLDL) receptor, apoE receptor 2 or LRP8, multiple epidermal growth factor-like domains 7 (MEGF7), megalin or LRP2, LRP1, and LRP1b . NeverthLRP1 gene encodes a large 4525 amino acid protein, composed of a 515 kDa N-terminal extracellular subunit and an 85 kDa C-terminal transmembrane subunit, which are noncovalently linked with each other . LRP1 islrp1 gene deletion as it terminates mouse embryo development around the implantation stage  and T cells reveal interesting findings about the function of this protein, that are going to be discussed further.Murine and human LRP1 gene have more than 98% gene sequence homology when analyzed  biology . LRP1 kn biology . HoweverBeing ubiquitously expressed, especially with higher levels in the liver, brain and lung, LRP1 is also found on the surface of the immune cells in both mice and humans .Innate immunity is the first line of defense against pathogens. Innate immune cells such as neutrophils, NK cells, monocytes and macrophages initiate the first quick powerful, although not specific, immune response. The innate immune cells mediate nonspecific protection through the production of pre-synthesized effector molecules or acting as direct killers of the invading cells.A subset of innate immune cells serves as antigen presenting cells (APCs). The expression of LRP1 on APCs has been shown to enhance T- and B- cell responses by facilitating antigen uptake , 48. LRPLRP1 is expressed on the surface of human peripheral blood monocytes, as well as monocytic cancer cell lines, such as THP1 , 53, 54.+CD16−, whereas intermediate are CD14+CD16+ and non-classical are CD14dimCD16+  and inhibit the production of inflammatory proteins.−/− macrophages demonstrate a decreased ability to engulf ACs, when compared with wild-type cells (The phagocytosis of apoptotic cells (ACs) - efferocytosis, is critical for self-tolerance and host defense. It is one of the many functions of phagocytic macrophages, where it is driven by LRP1. It was demonstrated that LRP1 together with its coreceptor calreticulin, interact with complement factor C1q to engulf apoptotic cells . To supppe cells . Macrophpe cells . Additiope cells .Mouse model of atherosclerosis is a perfect representation for LRP1-driven efferocytosis of macrophages. Under normal conditions, macrophages enter the developing plaques at the early stage of the disease. To remove the excess of the cholesterol by the liver, the cholesterol is needed to be picked up by the macrophages first. In an unfavorable condition, such as when LRP1 levels are low on the macrophages, cholesterol-overloaded macrophages can become stuck and start breaking down in the arteries. Macrophage LRP1 exports excess cholesterol out of the cell, thus lowering the risk of atherosclerosis . Interesvia the process of regulated intramembrane proteolysis (RIP), where intracellular LRP1 beta chain is released into the cytoplasm and, under certain conditions, translocates into the nucleus to regulate gene transcription. In the case of atherosclerosis, lipopolysaccharide and other inflammatory mediators specifically promote LRP1 shedding. The cytoplasmic fragment can then relocate to the nucleus, where it binds to and promotes the export of interferon regulatory factor-3 from the nucleus leading to suppression of expression of pro-inflammatory target genes, thus inhibiting inflammation  and the plasmacytoid dendritic cells (pDC). Dendritic cells play an important role in initiating T cell responses. LRP1 expressed on DCs is associated with activation of DCs and cross-priming of T cells , 83. Not+ DCs with low levels of MHC Class II expression, exhibit LRP1 at higher levels compared to CD11c+ DCs with high MHC Class II -induced eosinophilic airway inflammation. Furthermore, it is proposed that LRP1can be a modulator of type 2 asthma, since circulating myeloid DCs of asthmatic patients demonstrate a reduced LRP1 expression .Clearance of apoptotic cells through efferocytosis is imperative to impede the possibility of necrosis and further inflammation caused by the release of intracellular contents . DendritIn addition to the innate mechanisms of host defense there is the adaptive immune system that has specificity for its target antigens. Responses of the adaptive immune system require the interaction of antigen-specific receptors expressed on the surfaces of T- and B- lymphocytes and their cognate antigens. After recognizing and binding specific antigens, these cells can then directly or indirectly initiate the killing and/or removal of the cell expressing the antigen of interest .It is known that expression of LRP1 on B cells is very low, which makes exploration of its role in B cell biology difficult . It is lvia TSP1 and CD47  can bind to LRP1 on tumor-infiltrating CD8mulation .+ human peripheral blood mononuclear cells (PBMCs) with proteinase-3 (P3)-expressing polymorphonuclear neutrophils (PMNs) resulted in inhibition of T cell replication. Interestingly, physical cell to cell contact is imperative for this inhibition, since using a trans-well system prevented this effect. The anti-LRP1 blocking antibody significantly reversed the inhibition and restored T cell proliferation  bound to LRP1 can be internalized to be trafficked to MHCI for representation by APCs. These HSPs-loaded MHCI complexed can be important in further stimulation of CD8 T cells . As far Free unattached LRP1 has been shown to affect T cell function as well. It has been shown that the i.p. administration of LRP1-derived peptide into abortion-prone pregnant mice significantly decreased the abortion rate by increasing immunomodulating Tregs. Increased LRP1-derived peptide-Treg expansion coupled with upregulated IL2 and IL10 in mouse serum, as well as decreased expression of co-stimulatory molecules of APCs resulted in successful pregnancies in mice .We have been investigating the role of leukemia specific antigen Proteinase 3 (P3) in modulating anti-tumor immune response and identified LRP1 on T cells as the direct mediator of T cell proliferation . Membranfx/fxLckCre+) mice to investigate the capacity of LRP1 in regulating T cell proliferation in vivo (fx/fx). Using the MHC-mismatched mouse model of GVHD, we demonstrate that LRP1 regulates alloimmune responses in GVHD. Recipient mice with LRP1-deificient T cell donor have significantly lower GVHD-related mortality and morbidity compared to WT control. The numbers of donor-derived cells and CD8 T cell subset were reduced in the spleen of recipient mice; while the percentage of naïve T cells (CD62L+CD44+) including both CD4 and CD8 subsets, and Tregs were increased. In addition, proinflammatory cytokines such as TNFα, IL17, IFNγ and IL2 were decreased in the serum of these recipient mice  cell and macrophage in feration . Our preferation . OverallJM, OS, LS, and QM substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; provide approval for publication of the content, agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors contributed to the article and approved the submitted version.


DNAH5 and NME5, respectively, recapitulate the respective diseased phenotype on a molecular, structural and functional level.Primary ciliary dyskinesia (PCD) is a rare heterogenic genetic disorder associated with perturbed biogenesis or function of motile cilia. Motile cilia dysfunction results in diminished mucociliary clearance (MCC) of pathogens in the respiratory tract and chronic airway inflammation and infections successively causing progressive lung damage. Current approaches to treat PCD are symptomatic, only, indicating an urgent need for curative therapeutic options. Here, we developed an in vitro model for PCD based on human induced pluripotent stem cell (hiPSC)-derived airway epithelium in Air-Liquid-Interface cultures. Applying transmission electron microscopy, immunofluorescence staining, ciliary beat frequency, and mucociliary transport measurements, we could demonstrate that ciliated respiratory epithelia cells derived from two PCD patient-specific hiPSC lines carrying mutations in  DNAH5-mutated hiPSC-derived epithelial cells do not express the DNAH5 protein and consequently carry non-motile cilia with a reduced number of outer dynein arms, Sone et al. recently described the modeling of ciliary function using hiPSC-derived epithelial cells in more detail  = RTotal − RBlankR(2)2] = RTissue [Ω] × surface area [cm2]TEER [Ω × cmALI cultures were washed with PBS as described above and subsequently fixed and stored in 150 mM HEPES buffer containing 1.5% PFA and 1.5% glutaraldehyde. Membranes were cut off the ALI inserts and fixation and embedding were carried out as described in For the assessment of ultrastructural dynein arm defects, PCD Detect Software was used, following the developers’ workflow and using the same alignment settings . From eak (the fraction of the FOV containing MCC) of each FOV k using customized code in Matlab (Mathworks), as reported previously [k and Ck for all k = 1…n FOVs, respectively) per condition were compared with the non-parametric Kruskal–Wallis test, followed by Tukey-Kramer multi-comparison test, to test for different median values.Only cultures at day 35 of ALI or older were used for MCC measurements to ensure substantial maturity of MCC. To remove accumulated mucus and debris, ALI cultures were submerged with PBS for 10 min, then the PBS was carefully suctioned off. To visualize MCC, fluorescent microspheres  were diluted 1:1000 in PBS, and 20 µL of this suspension was added to the surface of each ALI culture. Particle movement was recorded using a Zeiss Axioscope fluorescence microscope equipped with an Orca Flash 4.0 camera  and a temperature-controlled chamber that was preheated to 37 °C. Apart from a few initial test recordings, standardized acquisition settings using a 10× objective were 1024 × 1024 pixels with 1.3 µm/pixel resolution at a framerate of 20 frames per second for a total duration of 10 s. For each condition, at least 3 experiments (different differentiations or clones (hiPSCs), or different isolations (pALI)) were performed. In each experiment, 5 fields of view (FOVs) with visible particle movement (sometimes only Brownian motion) were recorded from 2 insert cultures each. The particle trajectories were extracted from the raw movies using ImageJ/Fiji  with theeviously . For stamut and NME5mut cultures, as the movement of cilia could not be resolved at the tissue level. For tissue-level CBF measurements, ALI cultures were washed as above. High-speed movies of CBF (ca. 140 frames per second for a duration of at least 1.5 s) were recorded using a 40× long-distance phase contrast objective at a spatial resolution of 0.3 µm/pixel in a 512 × 512 pixel frame. For each condition, at least 3 experiments (different differentiations (hiPSCs) or different isolations (pALI)) were performed. From the cultures of each experiment, 6 FOVs with visible CBF were recorded. We measured cilia beat frequencies by applying Fourier spectral analysis, as previously described [Only cultures at day 35 of ALI or older were used for CBF measurements. CBF was recorded using a Zeiss Axioscope microscope, allowing for oblique phase contrast recordings, equipped with an Orca Flash 4.0 camera  and a temperature-controlled chamber that was preheated to 37 °C. Koehler illumination was set up for all recordings. Two different procedures were performed to measure CBF. In non-PCD pALI and non-PCD iALI cultures, CBF was measured at the tissue level, and single-cell CBF was measured in DNAH5escribed . The meaTM in a conical tube. DNase I at a concentration of 200 µg/mL was added to remove any free DNA resulting from the dissociation process. After 20 min warm culture medium was added at a ratio of 3:1 and the cells were centrifuged at 170 to 240× g for 5 min. The resulting pellet was resuspended in 1 mL culture medium to obtain a high cell density and 10 µL of this suspension was placed onto a glass bottom imaging dish . To confirm the viability of the cells, the same amount of life-dead stain  was added to the cell suspension droplet, and the solution was covered with a round glass coverslip in order to prevent evaporation. The measurements were performed using a 40× oil phase contrast objective (NA 1.4) under standardized acquisition settings, which were 800 frames per second for a duration of 2 s (NME5mut) and up to 10 s (DNAH5mut) of recording time and a spatial resolution of 0.16 µm/pixel in a 265 × 265 pixel frame. For each condition, at least 3 experiments (different differentiation (hiPSCs) or different isolations (pALI)) were performed, and in each experiment, at least 9 FOVs, i.e., 9 ciliated cells, were recorded. After each CBF movie recording a brightfield image and fluorescent images of the life-dead staining were recorded to confirm viability. The recordings were analyzed using the kymograph function in ImageJ. Kymographs of individual or groups of cilia were generated and the frequency was calculated by counting the amount of pixel intensity changes over time in the kymograph. Whenever a wave pattern was observed, the number of peaks were counted, else the dark lines were counted and divided by two to obtain a full beat cycle count. In the case of non-harmonic oscillation observed in DNAH5mut-derived ciliated cells, stationary vibrations were not included in the count. A median was calculated from all counted frequencies per ciliated cell. For statistical analysis, the pooled tissue-level medians and single-cell medians per condition were compared with the non-parametric Kruskal–Wallis test, followed by Tukey-Kramer multi-comparison test, to test for different median values.For single-cell CBF measurements, the cell layer was dissociated by incubating the ALI cultures for 20 min in prewarmed Accutasemut Clone 22 and Clone 24) and NME5 , respectively, were previously generated [For the establishment of a PCD in vitro disease model, two hiPSC lines from patients carrying homozygous mutations in DNAH5 , anterior foregut endoderm (AFE), and lung progenitor cells A. The prmut Cl. 22: 13.1 ± 4.1%; DNAH5mut Cl. 24: 14.0 ± 7.3% and NME5mut: 11.6 ± 3.1%) of the cells at day 14 of differentiation. In order to enrich lung progenitor cultures, magnetically activated cell sorting (MACS) using the cell surface maker carboxypeptidase M (CPM) was performed [mut Cl. 22: 50.2 ± 22.4%; DNAH5mut Cl. 24: 50.8 ± 14.7%; NME5mut: 57.4 ± 6.4%)  C.mut-derived cultures generated thicker cell layers and fewer AcTub, MUC5AC, and CCSP positive cells as compared to DNAH5mut and non-PCD diseased counterparts. Primary bronchial epithelial cell (PBEC)-derived ALI cultures served as control (non-PCD pALI). Additionally, airway epithelial cell-specific marker expression was confirmed by RT-qPCR  measurements were performed after 28 days of ALI culture and showed TEER values of 302–476 Ω × cm2, indicating intact and comparable barrier function in cultures derived from all cell lines , goblet cells (MUC5AC), club cells (CCSP) and basal cells (P63 and CK5)) in all cell lines D. Remark RT-qPCR E. Expresll lines F.In order to visualize ciliary protein expression, ALI cultures at day 28 post airlift were dissociated into single cells and stained for DNAH5 and NME5 protein, respectively. Immunofluorescence staining against AcTub was used to identify cilia and confirmed the presence of cilia with a normal appearance in all cell lines. In ciliated cells generated from non-PCD diseased primary cell-derived ALI cultures  of generated iALI cultures was performed. Analysis of cilia of non-PCD diseased hiPSC-derived cells showed a normal 9 + 2 ultrastructure pattern in more than 95% of analyzed cilia axonemes A,B as exFor investigating outer dynein arm (ODA) defects in DNAH5-mutated cilia, TEM cross-sections were analyzed using the PCD Detect software . This soTo evaluate the maturation and functional consequences of the DNAH5 and NME5 mutations in PCD, we measured ciliary beat frequencies (CBF) using high-speed microscopy on day 35 after airlift or later. In non-PCD cells (pALI and iALI) ciliary beat was visible directly in ALI cultures, enabling us to use tissue-level recordings and automated analysis of CBF A. In PCDSince functional MCC depends on many factors, including ciliary beat coordination and orientation ,40, CBF To date, mainly primary cells or non-airway immortalized cell lines  have been used for PCD in vitro modeling or drug screening ,47,48. HCurrent differentiation protocols for the generation of airway epithelium from hiPSCs often include an intermediate 3D culture step, in which lung progenitor cells are embedded in Matrigel droplets, prior to differentiation to airway epithelium ,26. HencDNAH11, HEATR2, and PIH1D3 using a complex airway-on-a-chip technology [Sone et al. previously reported a hiPSC-based disease model of PCD-causing mutations in chnology . By applchnology ,50, are chnology ,52. Morechnology  does notIn contrast, transwell ALI cultures are compatible with standard readouts, easier to establish due to the commercial availability and handling simplicity, and available in various formats including 96-well plates , allowing higher throughput screening of pharmacological compounds. Altogether, the airway-on-a-chip system is a valuable tool to investigate specific research questions under dynamic flow conditions; however, the transwell ALI culture system remains indispensable to be used as a complementary system, in particular for screening purposes.DNAH5 [NME5 [DNAH5 results in loss of DNAH5 protein, altered ODA ultrastructure, and reduced CBF, consistent with previously published iPSC-based models [DNAH5-mutated cultures. Moreover, we demonstrate the application of the newly developed PCD Detect software [DNAH5 can also result in less prominent ODA defects such as dynein arm truncation [DNAH5-mutated cultures. Altogether, the detailed characterization of our DNAH5-mutated iALI cultures serves as a proof-of-concept to demonstrate that our in vitro model recapitulates the mutDNAH5-specific phenotype and can be captured in detail by utilizing improved readout methods.In the present study, we describe the establishment of a hiPSC-based PCD in vitro disease model using patient lines containing mutations in the most frequently mutated and well-described PCD-associated gene DNAH5 ,42, and H5 [NME5 , respectd models . Additiosoftware  that allsoftware , but mutuncation . Hence, uncation  to identNME5, which was recently identified as PCD-associated. NME5 is part of the radial spoke neck; however, so far, only little is known about the function of NME5 and the pathomechanism of its mutation [NME5 in more detail. Our TEM results are consistent with the previously reported loss of central pair (9 + 0) in NME5-mutated cilia, and moreover, show additional ultrastructural abnormalities, such as outer doublet transposition  and presence of additional microtubules (9 + 4), which have not been described for this mutation before. Importantly, similar ultrastructural abnormalities were reported in other radial spoke protein defects [Next, we aimed at modeling the impact of mutations in the gene mutation ,53. Solemutation . Here, w defects ,54,55,56NME5 on single-cell and tissue levels, we performed high-speed video microscopy (HSVM). Single-cell functional analysis revealed that NME5-mutant ciliated cells beat in an uncoordinated way and exhibited partially circular cilia movements that resembled 9 + 0 nodal cilia beating. These nodal-like beat kinematics have been previously reported to be a consequence of central pair and transposition defects [NME5-mutated ciliated cells showed an increased variance of CBFs, compared to controls. By assessing the mucociliary transport capacity, we could additionally demonstrate that these alterations lead to a significantly diminished mucociliary clearance capacity (coverage and speed) compared to non-PCD controls (iALI and pALI). Altogether, these findings indicate that mutated NME5 not only impairs the beating synchrony and pattern of a given cell but also potentially prevents tissue-level metachronal wave formation due to divergent beat frequencies, which breaks down mucus clearance [To further investigate the effect of mutated  defects ,43,44,45learance .mutNME5 cells). Further underlying causes for the observed differences between pALI and iALI cultures may include an insufficient maturation of the iALIs. Despite analyzing the cultures earliest at day 35 after the airlift, maturation of ciliary beating  might be not fully completed. So far, the maturation of cilia beating during differentiation is not well understood, and investigation of bead transport over the course of differentiation and analysis of cilia length remains to be carried out for further understanding. In addition, the higher heterogeneity of hiPSC-derived cultures (ciliated cells appearing in clusters and/or uneven epithelium surface) compared to pALIs might reduce MCC coverage and average trajectory speed, since bead transport slows down in non-ciliated areas.Notably, also non-PCD iALIs showed reduced MCC compared to primary cell controls (non-PCD pALI), despite comparable CBF parameters (median and variance) and synchronous cilia beating. This heterogeneity may have been neglected in other studies that used smaller fields of view for measuring bead transport; e.g., Sone et al. used a 100 µm × 100 µm fields of view (FOV)  comparedTaking together, here we present a hiPSC-based PCD model platform utilizing a simple air-liquid interface culture system that recapitulates mutation-specific phenotypes by detailed characterization on a molecular  and functional  level. In addition, our system allows down-scaling of the culture format and up-scaling of cell yield, which are essential prerequisites for upcoming higher throughput screening applications.


ZDHHC1, a gene previously implicated in immunity and STING activation, as the top upregulated AA-specific gene under hypoxia. Together, these data reveal a differential chromatin landscape in TNBC associated with donor ancestry. The open chromatin structure of AA TNBC may contribute to a more lethal disease.African American (AA) women have an excessive risk of developing triple-negative breast cancer (TNBC). We employed Assay for Transposase-Accessible Chromatin using sequencing to characterize differences in chromatin accessibility between nine commonly used TNBC cell lines derived from patients of European and African ancestry. Principal component and chromosome mapping analyses of accessibility peaks with the most variance revealed separation of chromatin profiles by patient group. Motif enrichment and footprinting analyses of disparate open chromatin regions revealed differences in transcription factor activity, identifying 79 with ancestry-associated binding patterns (FDR < 0.01). AA TNBC cell lines exhibited increased accessibility for 62 transcription factors associated with epithelial-to-mesenchymal transition, cancer stemness/chemotherapeutic resistance, proliferation, and aberrant p53 regulation, as well as KAISO, which has been previously linked to aggressive tumor characteristics in AA patients with cancer. Differential Assay for Transposase-Accessible Chromatin signal analysis identified 1,596 genes located within promoters of differentially open chromatin regions in AA-derived TNBC, identifying DNA methyltransferase 1 as the top upregulated gene associated with African ancestry. Pathway analyses with these genes revealed enrichment in several pathways, including hypoxia. Culturing cells under hypoxia showed ancestry-specific stress responses that led to the identification of a core set of AA-associated transcription factors, which included members of the Kruppel-like factor and Sp subfamilies, as well as KAISO, and identified in vitro studies to advance health equity at all levels of scientific research.We identify an ancestry-associated open chromatin landscape and related transcription factors that may contribute to aggressive TNBC in AA women. Furthermore, this study advocates for the inclusion of diversely sourced cell lines in experimental  Under the proper growth conditions, cancer cell lines can retain many of the genetic properties of the parental tumor from which they were derived  patients  cell lines derived from women of two different ancestral backgrounds—EA and African American (AA). We chose to focus our investigation on TNBC due to the well-documented racial disparities in tumor biology and breast cancer survival . AA womeMycoplasma testing was not performed. The biological sex of all cell lines was female. To perform the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis, following regular cell culture procedure, one million cells  were suspended in 1.5 mL freezing medium . The above cryopreserved cells were sent to the Sequencing Facility at Frederick National Laboratory for Cancer Research, Frederick, MD, where sample processing and sequencing was done.All human breast cancer lines were obtained from ATCC. MDA-MB-157 , MDA-MB-231 , MDA-MB-436 , and HCC1806  cells were grown in DMEM (Gibco Laboratory) with 2 mmol/L glutamine (Millipore Sigma) and 10% FBS (HyClone Laboratories). MDA-MB-468 , Hs578T , HCC70 , BT549 , and HCC2157  cells were grown in RPMI media (Gibco Laboratory) supplemented with 10% FBS. HCC2157 cells grow in suspension whereas all other cell lines are adherent. We obtained authentication of these cell lines through ATCC services using a short tandem repeat analysis; as cell lines were obtained directly from ATCC, 2) conditions, cells  were cultured at 37°C in a 5% CO2 humidified environment as an adherent monolayer. Hypoxia experiments were performed with the above eight adherent cell lines in a hypoxia chamber (BioSpherix) at 1% O2 at 37°C in a 5% CO2-humidified environment. Briefly, breast cancer cells were plated in T75 flask (1 × 106 cells/flask). Media was changed after 24 hours, and cells were exposed to normoxia (21% O2) and hypoxia (1% O2) for 48 hours. Experiments were conducted in duplicates. After 48 hours, the cells were harvested from the flasks by 0.25% trypsin (Gibco Laboratory) and 1 million cells were suspended in 1.5 mL of freezing medium . The above cryopreserved cells were sent to the Sequencing Facility at Frederick National Laboratory for Cancer Research, Frederick, MD, where sample processing and sequencing were done.For normoxic (21% O2 in water). Cells were centrifuged at 500 rcf for 5 minutes in a prechilled (4°C) fixed-angle centrifuge. After centrifugation, 900 µL of supernatant was aspirated, which left 100 µL of supernatant. This remaining 100 µL of supernatant was carefully aspirated by pipetting with a P200 pipette tip to avoid the cell pellet. Cell pellets were then resuspended in 50 µL of ATAC-seq RSB containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin by pipetting up and down three times. This cell lysis reaction was incubated on ice for 3 minutes. After lysis, 1 mL of ATAC-seq RSB containing 0.1% Tween-20 (without NP40 or digitonin) was added, and the tubes were inverted to mix. Nuclei were then centrifuged for 10 minutes at 500 rcf in a prechilled (4°C) fixed-angle centrifuge. Supernatant was removed with two pipetting steps, as described before, and nuclei were resuspended in 50 µL of transposition mix by pipetting up and down six times. Transposition reactions were incubated at 37°C for 30 minutes in a thermomixer with shaking at 1,000 rpm. Reactions were cleaned up with Zymo DNA Clean and Concentrator 5 columns. The remainder of the ATAC-seq library preparation was performed as described previously . Sequencing was performed on an Illumina NextSeq 2000 machine at the Sequencing Facility, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research using standard protocols. Briefly, 16 mRNA sequencing samples were pooled and sequenced on NextSeq 2000 P2 using Illumina Stranded mRNA Ligation Kit and paired-end sequencing. The samples had 56 to 69 million pass filter reads with more than 95% of bases above the quality score of Q30. The RNA sequencing (RNA-seq) data for the human breast cancer cell lines were deposited in the NCBI's GEO database under accession number GSE223181.After verifying that the pair-end reads are of acceptable sequencing quality, sequencing adapters and primers were trimmed away using Cutadapt . TrimmedThe dimensionality of the Tn5-nicking site counts matrix was reduced by calculating the principal components and plotting the first two components which explain a significant proportion of the overall variation.Loading the count matrix into DESeq2 v.1.45.5 , were then imported into the Enrichr tool  to identify groups of TF proteins that have known and/or predicted relationships . Using Sq value (FDR) < 0.05, the absolute value of fold change > 2. We then performed gene set enrichment analysis (GSEA) using the gene transcription regulation reference (Gene Transcription Regulation Database: GTRD) from the Molecular Signatures Database: http://www.gsea-msigdb.org/gsea/msigdb/human/collections.jsp).The Illumina FASTQ files were assessed for quality control using FastQC v.0.11.6. Reads of the samples were trimmed for adapters and low-quality bases using Cutadapt before alignment with the reference genome (hg38) and the annotated transcripts using STAR v.2.7.5b. The gene expression quantification was performed using RSEM v1.3.2. The average mapping rate of all samples was 97% and unique alignment was above 88%. The mapping statistics were calculated using Picard software. Percent coding bases were between 51% and 61%. Percent untranslated region bases were 32%–43%, and mRNA bases were between 89% and 94% for all the samples. Library complexity was measured in terms of unique fragments in the mapped reads using Picard's MarkDuplicate utility. The samples had 58%–71% nonduplicate reads. Differential expression analysis was done using DESeq2 package in R. Differential expression genes (DEG) were filtered by a https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/Software.cgi), which is a fast distance–based method to infer ancestry based on references from multiple genotype datasets, including those of populations of European, African, and Asian descent. The method assigned a predominant African ancestry to MDA-MB-468, MDA-MB-157, HCC1806, and HCC70 cells and a predominant European ancestry to the MDA-MB-231, MDA-MB-436, Hs578T, and BT549 cells, consistent with donor ethnicities reported by ATCC for these cell lines. Ancestry estimates of our cell lines aligned closely with publicly available data from the Estimated Cell Line Ancestry (ECLA) database from the Moffitt Cancer Center (http://ecla.moffitt.org).Germline variants were called using GATK's HaplotypeCaller v.4.2.6.1 from available RNA-seq data for eight cell lines cultured under normoxic conditions, MDA-MB-468, MDA-MB-157, HCC1806, HCC70, MDA-MB-231, MDA-MB-436, Hs578T, and BT549. For admixture analysis, only SNPs that were biallelic were retained in the analysis. We used GRAF-pop v.1.0 (https://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/BRCA/20160128/) covering 112 TNBC patient tumors  to infer differences in TF activity by genetic ancestry, which was assigned using previously reported quantified genetic ancestry estimates  breast cancer data were downloaded from the Broad Institute's GDAC  and AA (n = 5) donors of nine commonly used TNBC cell lines (http://ecla.moffitt.org) indicates an average European ancestry of 93% for the four EA TNBC cell lines and 79.5% West African ancestry for the five AA TNBC cell lines (2FC| > 2) in EA compared with AA TNBC cells confirmed distinctive patterning of open chromatin across a number of chromosomes  and BCL6B (Pnom = 0.26). In AA, 11 TFs overlapped, including SNAI1 (Pnom = 0.19), NR1H4 (Pnom < 0.0001), MSX2 (Pnom = 0.03), and KLF14 (Pnom = 0.08). Taken together, these data suggest that TF activities show significant differences by donor ancestry, with African ancestry displaying an ancestry-specific TF activity profile associated with increased aggressiveness in breast cancer.Next, we performed a digital footprinting analysis of differentially open chromatin regions using TOBIAS. We uncovered significant differences in TF binding by ancestry  and AA (n = 34) patients within TCGA to perform an upstream regulator analysis to infer critical transcriptional regulators based on ancestry-related differences in downstream gene expression , JUNB , BATF , and FOSL1 . In TNBC tumors from AA patients, validated TFs totaled 25 and notably included TP53 , TP63 , TCF3  and TCF4 , TFAP2A , and various members of the FOX TF family. These patient data were concordant with some of the top TFs identified in our cell line data.We next sought to extend these findings from human TNBC cell lines to TNBC patient tumors to investigate if ancestry-specific TF activity showed similar patterning by patient ancestry group. ATAC-seq data were not available within TCGA for validation of TF binding and motif enrichment analyses; however, we leveraged RNA-seq data from triple-negative breast tumor tissues in EA , a long noncoding RNA suspected to function as a tumor suppressor, followed by LRIG1, which interestingly also has an anti-invasive role in basal-like breast cancer cells through encouraging mesenchymal-to-epithelial transition , a DNA methyltransferase  upregulated in promoter regions. In EA TNBC, enriched Hallmark-annotated cancer pathways (FDR < 0.2) included epithelial-to-mesenchymal transition (Padjusted = 2.3 × 10−4), downregulation of UV response (Padjusted = 0.04), complement (Padjusted = 0.1), and hedgehog signaling  are negative regulators of this process , apical junction (Padjusted = 2.9 × 10−3), and hypoxia (Padjusted = 0.1), among others  and hypoxic (1% O2) conditions and performed ATAC-seq. Genome-wide analysis of differentially accessible chromatin sites (FDR < 0.01 and |log2FC| > 2) in EA compared with AA TNBC under hypoxia revealed distinctive patterning of open chromatin across a number of chromosomes , with a high degree of TF coexpression and shared functions  among the small network ; as expected, DETFs from AP-1 family, namely the JUN and FOS TF subfamilies, showed highly robust interactions, likely reflecting the homodimeric and heterodimeric functional relationships between these proteins. We performed a pathway enrichment analysis using REACTOME based on the TF network analysis to identify shared biologic function of significant TF clusters (P < 1.0 × 10−16), which was more expansive and extensive than that of the EA PPI network . TP53 is one of the most prominent nodes featured in this network, with high degree of connectivity and central to several other key nodes, such as SP-1, EGR-1, and KLF-4. In pathway enrichment analyses using REACTOME, 33 pathways were identified with an FDR < 0.01, 15 of which are shown in To better understand how differentially bound TFs involved in the ancestry-driven hypoxia response might act in concert to dictate downstream gene expression, we constructed a protein-protein interaction (PPI) network using the STRING database , allowinclusters ; the topMAGI1, a junctional scaffold protein that acts as a tumor suppressor in the context breast cancer through inhibition of the p38 stress pathway . For 111/112 TCGA samples used in our study, genetic ancestry consensus was concordant with SRR. Genetic ancestry consensus classification of African or admixed African ancestry captured 100% of TCGA participants who self-identified as “Black or African American”. Genetic ancestry consensus classification of EUR ancestry captured 98.7% of TCGA participants who self-identified as “White”; in one case, a participant that self-identified as White demonstrated a higher proportion of admixed American genetic ancestry. Among TCGA participants who self-identified as Black or African American, the average percentage of African ancestry was 83.2%, which is comparable to the quantified genetic ancestry estimates for the human cell lines used in this study that were classified as African American-derived (average African ancestry of 79.5%). Among TCGA participants who self-identified as White, the average percentage of European ancestry was 96.7%, which is comparable to the quantified genetic ancestry estimates for the human cell lines used in this study that were classified as European American-derived (average European ancestry of 93.0%).Click here for additional data file.Supplementary Figure 2Supplemental Figure 2. Expression of genes by donor ancestry in TNBC cell lines. Top-ranked genes identified to be located in open chromatin regions with an ancestry relationship were individually confirmed to be differentially expressed at the transcript level in matched RNA-seq data from the same human TNBC cell lines. Significance testing with two-sided t-test.Click here for additional data file.Supplementary Table 1Supplementary table 1 displaying data output from motif enrichment analysis performed on ATAC-seq data from TNBC cell lines.Click here for additional data file.Supplementary Table 2Supplementary table 2 displaying data output from digital footprinting analysis performed on ATAC-seq data from TNBC cell lines.Click here for additional data file.Supplementary Table 3Supplementary table 3 displaying data output from gene set enrichment analysis for transcriptional regulators performed on RNA-seq data from TNBC cell lines.Click here for additional data file.Supplementary Table 4Supplementary table 4 displaying data output from upstream regulator analysis performed on RNA-seq data from TNBC patients in TCGA.Click here for additional data file.Supplementary Table 5Supplementary table 5 displaying data output from differential ATAC signal performed on ATAC-seq data from TNBC cell lines.Click here for additional data file.Supplementary Table 6Supplementary table 6 displaying data output from differential gene expression analysis performed on RNA-seq data from TNBC cell lines.Click here for additional data file.Supplementary Table 7Supplementary table 7 displaying data output from digital footprinting analysis performed on ATAC-seq data from TNBC cell lines cultured under hypoxia.Click here for additional data file.Supplementary Table 8Supplementary table 8 displaying data output from differential ATAC signal analysis performed on ATAC-seq data from TNBC cell lines cultured under hypoxia.Click here for additional data file.