Hox Expression Research Articles

Oil-soluble contrast medium bathing attenuated endometrial inflammation and improved endometrial receptivity in women with recurrent implantation failure: a descriptive study

BackgroundThe oil-soluble contrast medium used in hysterosalpingography has been shown to have a fertility-enhancing effect, but the underlying mechanism is unclear, especially regarding the role of window of implantation (WOI). This study aimed to assess the endometrial immunological impact of the WOI before and after bathing with the oil-soluble contrast medium in women with recurrent implantation failure (RIF).MethodsThis descriptive study involved two medical centers between December 18, 2019, and December 30, 2020. We included infertile women who underwent three or more transfer cycles, cumulative transplantation of at least four high-quality cleavage-stage embryos or three high-quality blastocysts without clinical pregnancy, and high-quality frozen embryos that were still available for implantation. Patients received 5 ml of ethiodized poppyseed oil bathing, endometrial biopsy around bathing, and frozen-thawed embryo transfer (FET) within four menstrual cycles after bathing. Patients were excluded if failure to complete anyone. Data on the baseline characteristics and clinical data of the FET cycles were collected, and endometrial biopsy specimens were collected in the luteal phase before and after bathing and subjected to immunohistochemistry. The number of CD56 and CD138 positive cells and H-score of expression of ανβ-3 and HOXA10 in endometrium were collected.ResultsThirty-four patients were initially enrolled in the study; ultimately, twelve patients with a median age of 32.5 years (range 27–40 years) completed the research. The median number of embryo transfer cycles was three (range 3–8). A total of 4 of 12 women (33.33%) were diagnosed with chronic endometritis before oil-soluble contrast bathing. After bathing, the median numbers of CD138-positive cells in endometrium decreased from 0.75 (range 0–13.5) to 0.65 (range 0–6), P = 0.035; additionally, the H-score of expression of ανβ-3 in endometrium increased from 148.50 ± 31.63 to 175.58 ± 31.83, P < 0.001. The thickness of the endometrium also significantly increased (8.90 ± 1.45 mm vs.10.11 ± 1.98 mm, P = 0.005). However, no consistent changes were found in the expression of CD56 and HOXA10 in the endometrium. Five patients experienced biochemical pregnancies (41.67%), four had clinical pregnancies (33.33%), and three achieved live births following oil-soluble contrast bathing (25%).ConclusionsThese results suggest that oil-soluble contrast medium bathing decreased CD138-positive cells and upregulated expression of ανβ-3 during WOI in patients with RIF. This histological impact of endometrium may result in enhanced fertility during FET cycles. Investigating the ability of intrauterine bathing with lower-dosage oil-soluble contrast to improve pregnancy in the RIF population is warranted.

Expression of Long Non-Coding RNA H19 in the Endometrium of Mice During Peri-Implantation and Its Regulation on Embryo Implantation

In this study, we utilized magnetic nanobeads for the extraction of nucleic acids from tissues to investigate the expression levels and correlation between lncRNA H19, miR-612, and their target gene HOXA10 in peri-implantation endometrium of mice. Furthermore, we conducted overexpression or gene knockout experiments on lncRNA H19 to observe its impact on the expression of miR-612 and HOXA10. The targeted binding relationship between lncRNA H19, miR-612, and HOXA10 was detected by dual luciferase reporter assay. The regulatory relationship between lncRNA H19, miR-612, and HOXA10 was verified through silencing or overexpression of these genes. Intrauterine transfection was used to modulate the expression of lncRNA H19 in endometria during pregnancy, followed by the detection of the expression levels of miR-612 and HOXA10 as well as ITGB3 and IGFBP-1 proteins. Compared with non-pregnant mice, we observed a significant upregulation of both lncRNA H19 and HOXA10 in the endometria of pregnant mice, while miR-612 was found to be downregulated (P &lt; 0.05). Further analysis revealed that the expression levels of lncRNA H19 and HOXA10 increased progressively with gestational days, peaking on Day 4 (P &lt; 0.05). Moreover, Through database analysis, we identified binding sites for lncRNA H19-miR-612 as well as HOXA10-miR-612 interactions. The dual-luciferase reporter assay further supported our conjecture that lncRNA H19 could specifically bind the miR-612, which in turn targets HOXA10 to regulate its expression (P &lt; 0.05). In conclusion, regulations of lncRNA H19 and HOXA10 expression contribute to enhancing endometrial receptivity and facilitating decidualization of endometrial stromal cells, ultimately promoting successful embryo implantation.

The Effect of Granulocyte Colony-Stimulating Factor on Endometrial Receptivity of Implantation Failure Mouse.

The purpose of this study was to investigate the effect of G-CSF on the endometrial receptivity of implantation failure mice.Sixty female mice were treated mifepristone to establish an implant failure model. The treatment groups received different doses of G-CSF. Endometrial tissue and serum were collected on day 5 after mating. The abundance of pinopodes on the endometrium was observed by scanning electron microscopy. The expressions of LPAR3, COX2, and HOXA10 were detected by RT-qPCR and Western blotting. Serum levels of E2, P, VEGF, LIF, TNF-α and IL-10 were measured by ELISA. The expressions of VEGF, CD34, CD57, TNF-α, and IL-10 were assessed by immunohistochemistry. Immunofluorescence analysis was performed to determine the number of CD57, Treg, and Th17 cells.G-CSF increased implantation and pregnancy rates of mifepristone-induced implantation failure mice, with the most significant effect seen at the intermediate dose. G-CSF increased the serum levels of E2 and P, the abundance of endometrial pinopodes, and the level of LIF in the endometrium. It also promoted the expression of VEGF, HOXA10, LPAR3, and COX2. Moreover, G-CSF reduced the level of CD57 cells and the ratio of Th17/Treg cells in endometrium. G-CSF reduced the inflammatory factor TNF-α, but IL-10 did not change significantly.G-CSF can enhance embryo implantation rate and pregnancy rate and improve endometrial receptivity by attenuating degeneration of pinopodes, upregulating estrogen and progesterone, facilitating angiogenesis, maintaining immune cell homeostasis, and reducing the production of inflammatory cytokines in implantation failure mouse.

Myocardial Expression of Pluripotency, Longevity, and Proinflammatory Genes in the Context of Hypercholesterolemia and Statin Treatment.

Background: This study sought to assess the effect of statin therapy on myocardial inflammation in a White New Zealand rabbit model of atherogenesis. Methods: The mRNA expression levels of pro-inflammatory, pluripotency, and aging-related markers were quantified following a controlled feeding protocol and statin treatments. Results: Following high-cholesterol diet induction, we observed significant upregulation in the myocardial mRNA levels of MYD88, NF-κB, chemokines (CCL4, CCL20, and CCR2), IFN-γ, interleukins (IL-1β, IL-2, IL-4, IL-8, IL-10, and IL-18), and novel markers (klotho, KFL4, NANOG, and HIF1α). In contrast, HOXA5 expression was diminished following a hyperlipidemic diet. Both statin treatments significantly influenced the markers studied. Nevertheless, rosuvastatin administration resulted in a greater reduction in MYD88, NF-kB, chemokines (CCL4, CCL20, and CCR2), and interleukins IL-1β, IL-8, KLF4, NANOG, and HIF1α than fluvastatin. Fluvastatin, on the other hand, led to a stronger decrease in IL-4. Downregulation of IL-2 and IL-18 and upregulation of IFNβ and HOXA5 were comparable between the two statins. Notably, rosuvastatin had a stronger effect on the upregulation of klotho and IL-10. Conclusion: Overall, statin therapy significantly attenuated inflammatory, pluripotency, and klotho expression in myocardial tissue under atherogenic conditions. Our findings also highlight the differential efficacy of rosuvastatin over fluvastatin in curtailing proatherogenic inflammation, which could have profound implications for the clinical management of cardiovascular disease.

Abstract 5599: Role of HOX gene expression in differentiation of colon cancer stem cells

Abstract HOX genes encode a highly conserved set of transcription factors critical for stem cell (SC) function and embryonic development. Several HOX genes have also been implicated in a myriad of cancers, including colorectal cancer (CRC). However, how HOX genes play a role in SC regulation and contributes to cancer development is unclear. HOX expression is regulated by WNT, FGF, and retinoic acid (RA) signaling - pathways which also become dysregulated in cancer. Goal: To determine how regulation of HOX genes specifies differentiation of colonic SCs into specialized cell lineages and how dysregulation of HOX genes contributes to CRC development. Hypotheses: i) Each of the different cell types within human colonic epithelium has a distinct HOX gene expression signature; ii) All-trans retinoic acid (ATRA) induced differentiation of CRC cells changes HOX expression that correlates with specific HOX expression signatures in normal colon and CRC cells. Results: Immunostaining shows that specific HOX proteins (e.g., HOXA9 &amp; HOXC9) are selectively expressed in ALDH-positive SCs and expression of these HOX genes is increased in CRC tissues compared to normal epithelium. Bioinformatics analysis predicted that these HOX genes (plus HOXA4, HOXA10, HOXC8) are regulated by RA signaling. Accordingly, we studied how ATRA influences HOX expression in CRC cells (HT29 &amp; SW480). We found ATRA treatment: 1) Decreases proliferation and increases neuroendocrine cell (NEC) differentiation; and 2). Leads to specific changes in HOX gene expression patterns. Using NanoString profiling, we also discovered that LGR5+ SCs, ALDH+ SCs and GLP2R+ NECs have unique HOX signatures. By defining how these HOX gene expression changes in response to ATRA treatment in comparison to the HOX gene signatures of different colonic cell lineages will help us understand the role that HOX genes play in SC differentiation and how dysregulation of HOX gene expression contributes to CRC development. Citation Format: Brian T. Osmond, Caroline O. Facey, Lynn O. Opdenaker, Chi Zhang, Bruce M. Boman. Role of HOX gene expression in differentiation of colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5599.

Abstract 1227: Novel combination therapies to overcome non-genetic/adaptive menin inhibitor resistance in AML with MLL1r or mtNPM1

Abstract In AML with MLL1 rearrangement or MLL1 fusion proteins (FP), the N-terminus of MLL1 becomes fused to over 80 partner proteins resulting in an aberrant leukemic transcriptional program including dysregulated expression of HOXA9, MEIS1, PBX3, FLT3, MEF2C and CDK6. On the other hand, in mutant NPM1 AML cells, wild type MLL1 is the main regulator of HOXA9, MEIS1 and FLT3, promoting self-renewal of myeloid progenitor cells. Menin inhibitors have been developed that disrupt the binding of Menin to MLL1 leading to reduced activity of HOXA9 and MEIS1 and repression of MLL1 or MLL1-FP target genes. Clinical trials have demonstrated that Menin inhibitors (MI) are well-tolerated and clinical remissions have been observed in patients with relapsed/refractory AML harboring MLLr. Unfortunately, most patients either fail to respond or eventually relapse. MI-resistance in AML with MLL1r or mtNPM1 is either due to recently described mutations in the MI-binding domain of Menin or is non-genetic/adaptive, due to dysregulated epigenome/transcriptome/proteome. In the present studies, through repeated shocks with LD90 dosing of MI, followed by drug washout and recovery, we have generated MLL1r (MV4-11-MITR) and mtNPM1 (OCI-AML3-MITR) AML cell lines that are tolerant/resistant (TR) to MI with LD50 values greater than 1 μM. Whole exome sequencing confirmed that these cells were lacking in new driver mutations. We determined that resistance in the MITR cells was not due to presence of hotspot mutations in Menin. ChIP-Seq analysis of H3K27Ac showed that the activities of super enhancers and core transcriptional regulatory circuitry was altered in both MITR cell types. Non-genetic/adaptive resistance to MI also concordantly reduced genome wide ATAC-Seq and RNA-Seq peaks in the OCI-AML3-MITR cells with significant depletion of MEIS1, IGF2BP2, BCL2, and PBX3, with concomitant induction of leukemia stem cell associated CLEC12A and CD244 expressions. A domain-specific CRISPR screen in MV4-11-MITR cells identified several druggable co-dependencies (e.g. EP300 and MOZ) and co-enrichments (SMARCA4, CREBBP and BRD4) with MI, suggesting them as potential mechanisms of non-genetic/adaptive MI resistance. Consistent with these findings, co-treatment with SMARCA2/SMARCA4 (chromatin remodeler ATPases) inhibitor FHD-286 and the BETi OTX015 or the MI SNDX-50469 exerted synergistic lethality in MV4-11-MITR and OCI-AML3-MITR cells as well as in PD MITR AML cells with MLL1r or mtNPM1. Co-treatment with CBP/p300 inhibitor GNE049 or GNE781 was also synergistic with MI in these cells. In vivo treatment with FHD-286 and OTX015 or SNDX-5613 significantly reduced the AML burden in mice bearing OCI-AML3-MITR xenografts. These findings underscore preclinical activity of epigenetically-targeted agent-based combinations and highlight their promise in overcoming MI resistance in AML with MLL1r or mtNPM1. Citation Format: Warren C. Fiskus, Christopher P. Mill, Jessica Piel, Murphy Hentemann, Christine E. Birdwell, Kaberi Das, John A. Davis, Hanxi Hou, Tapan M. Kadia, Naval Daver, Sanam Loghavi, Branko Cuglievan, Courtney D. DiNArdo, Kapil N. Bhalla. Novel combination therapies to overcome non-genetic/adaptive menin inhibitor resistance in AML with MLL1r or mtNPM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1227.

Hypermethylation leads to the loss of HOXA5, resulting in JAG1 expression and NOTCH signaling contributing to kidney fibrosis

Epigenetic regulations, including DNA methylation, are critical to the development and progression of kidney fibrosis, but the underlying mechanisms remain elusive. Here, we show that fibrosis of the mouse kidney was associated with the induction of DNA methyltransferases and increases in global DNA methylation and was alleviated by the DNA methyltransferase inhibitor 5-Aza-2’-deoxycytidine (5-Aza). Genome wide analysis demonstrated the hypermethylation of 94 genes in mouse unilateral ureteral obstruction kidneys, which was markedly reduced by 5-Aza. Among these genes, Hoxa5 was hypermethylated at its gene promoter and this hypermethylation was associated with reduced HOXA5 expression in fibrotic mouse kidneys after ureteral obstruction or unilateral ischemia-reperfusion injury. 5-Aza prevented Hoxa5 hypermethylation, restored HOXA5 expression, and suppressed kidney fibrosis. Down-regulation of HOXA5 was verified in human kidney biopsies from patients with chronic kidney disease and correlated with the increased kidney fibrosis and DNA methylation. Kidney fibrosis was aggravated by conditional knockout of Hoxa5 and alleviated by conditional knockin of Hoxa5 in kidney proximal tubules of mice. Mechanistically, we found that HOXA5 repressed Jag1 transcription by directly binding to its gene promoter, resulting in the suppression of JAG1-NOTCH signaling during kidney fibrosis. Thus, our results indicate that loss of HOXA5 via DNA methylation contributes to fibrogenesis in kidney diseases by inducing JAG1 and consequent activation of the NOTCH signaling pathway.

MiR-135a-5p regulates window of implantation by suppressing pinopodes development and decidualization of endometrial stromal cells.

The window of implantation (WOI) is a brief period during which the endometrium is receptive to embryo implantation. This study investigated the relationship between miR-135a-5p and endometrial receptivity. Peripheral blood was collected on the day of ovulation and the 5th day after ovulation for high-throughput sequencing from women who achieved clinical pregnancy through natural cycle frozen embryo transfer. RT-qPCR assessed miR-135a-5p expression in the endometrium tissue or cells during the mouse implantation window or decidualization. Scanning electron microscopy was utilized to observe pinopode morphology and quantity in mice overexpressing miR-135a-5p during the WOI. Human endometrial stromal cells (HESC) and artificial induction of mouse uterine decidualization were used to explore whether miR-135a-5p overexpression inhibits decidualization by regulating HOXA10 and BMPR2. Furthermore, the impact of miR-135a-5p on HESC proliferation and HTR8/SVneo invasion was explored. A total of 54 women were enrolled in the study. bioinformatics analysis and animal models demonstrated that miR-135a-5p was significantly downregulated during the WOI, and its high expression can lead to abnormal pregnancy outcomes. Overexpression of miR-135a-5p resulted in the absence of pinopode in mouse endometrial tissue during the WOI. High miR-135a-5p levels were found to potentially inhibit endometrial tissue decidualization by downregulating HOXA10 and BMPR2 expression. Finally, CEBPD was identified as a potential regulator of miR-135a-5p, which would explain the decreased miR-135a-5p expression during the WOI. MiR-135a-5p expression is significantly downregulated during the WOI. High miR-135a-5p levels suppress pinopode development and endometrial tissue decidualization through HOXA10 and BMPR2, contributing to inadequate endometrial receptivity.

Uterine histopathology and steroid metabolism in a polycystic ovary syndrome rat model

The aim of this study was to investigate uterine lesions, uterine endocrine status and expression of genes involved in uterine differentiation in a rat model of polycystic ovary syndrome (PCOS). The possible involvement of the androgen receptor (AR) was also investigated. PCOS rats showed an increased incidence of uterine epithelial and glandular lesions and elevated serum testosterone level, which was not detected in uterine tissue. Uterine 17β-estradiol, estrone and progesterone were detected in 100%, 75% and 50% of the animals, respectively. This was associated with a decrease in Star and an increase in Hsd17b2, Srd5a1 and Cyp19a1, suggesting that uterine steroids are not synthesized de novo in PCOS and that alterations in these enzymes may explain the absence of testosterone and low progesterone. In addition, ESR2 decreased and AR increased, suggesting possible steroid receptor crosstalk. Genes associated with uterine differentiation, PTEN and WNT5a, also showed reduced expression. PCOS rats treated with flutamide, an AR antagonist, were similar to PCOS rats in terms of uterine lesions, serum steroid levels, ESR2, PTEN and WNT5a expression. However, testosterone, AR and aromatase levels were similar to control rats, with decreased expression of ESR1 and HOXA10, suggesting that these expressions are AR dependent. Our results suggest that the primary cause of the observed uterine lesions in the PCOS rat model is the altered endocrine status and consequently changes in genes related to uterine differentiation.

Evaluation of endometrial receptivity in recurrent pregnancy loss and recurrent implantation failure.

The cause of implantation defects in patients with recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) has not been clearly established. We aimed to evaluate the immunohistochemical changes in HOXA-11, β1 integrin, focal adhesion kinase (FAK), cluster of differentiation 44 (CD44), and extracellular matrix protein 1 (ECM1) molecules during the receptive endometrial period in patients with RIF and RPL. This study was retrospectively conducted at a university hospital. After the exclusion of cases with pathology that may cause a change in the level of receptors in the endometrium, biopsies performed during the receptive period were selected, and the patients were categorized into RPL (n=15), RIF (n=16), control (n=16) groups. All preparations were immunohistochemically stained for HOXA-11, β1 integrin, FAK, CD44, and ECM1. HOXA-11 and β1 Integrin expression changes were similar between the RIF and control groups. However, FAK expression was significantly increased in the RIF group (p<0.01). Additionally, ECM1 and CD44 expressions were significantly decreased in the RIF group compared with the control group (p<0.01). There was no significant difference in the endometrial staining of HOXA-11, FAK, and ECM1 in patients with a history of RPL. However, β1 Integrin and CD44 levels were significantly decreased in the RPL group compared with the control group (p<0.05). Implantation is a complex process, and altered adhesion mechanisms involved in endometrial receptivity may be related to defective implantation in patients with RIF and RPL. Among the adhesion molecules, the expression of CD44, β1 integrin, FAK, and ECM1 molecules varies in inappropriate implantation compared with the normal population.

Transcriptomic analysis of bovine endometrial epithelial cells in response to interferon tau and hormone stimulation.

The embryonic loss during early stage of gestation is one of the major causes of infertility for domestic ruminants, causing huge economic losses to pasture. Maternal recognition of pregnancy and implantation are the crucial process for determining the successful establishment and development of pregnancy in cattle. The research on molecular mechanisms of pregnancy recognition will facilitate illustrating the complex process of pregnancy establishment and help to improve pregnancy outcomes. In this study, we performed transcriptomic analysis of primary bovine endometrial epithelial cells (BEND) with or without IFNT and hormones intervention through RNA sequencing. We eventually identified 608 differentially expressed genes (DEGs) including 409 up-regulated genes and 199 down-regulated genes in IFNT and hormones-treated group compared with control group. Gene Ontology (GO) enrichment analysis demonstrated that the majority of DEGs were implicated in immune system process, response to external stimulus, response to cytokine, regulation of response to stress. Results from KEGG analysis showed a significant enrichment of NOD-like receptor signaling pathway, antigen processing and presentation, necroptosis, oxidative phosphorylation, RIG-I-like receptor signaling pathway. Additionally, a set of promising candidate genes, including (USP18, STAT1, PSMB8, IFIH1, MX2, IFI44, DHX58, CASP8, DRAM1, CXCR4), were characterized by constructing an integrated interaction network. Specifically, the mRNA expression of HOXA11, PTGS1 and PTGS2 were remarkably suppressed by silencing DRAM1 under IFNT and hormone administration, thus speculating that DRAM1 might play a crucial role in early pregnancy by regulating endometrial function. The results of this study depicted a relatively comprehensive transcriptional profiles of BEND in response to IFNT and hormones, which contributes to a better understanding of gene interaction network and underlying regulatory mechanisms in endometrium of ruminants during early pregnancy.

Effect of Knocking Out HOXA5 Gene by CRISPR-Cas9-Mediated Gene Editing Technique on Proliferation of Acute Myeloid Leukemia Cells

To construct a acute myeloid leukemia (AML) cell line in which HOXA5 gene is stably knocked out by CRISPR-Cas9-mediated gene editing technique, so as to clarify the effect of HOXA5 gene knockout on the proliferation of AML cells, and preliminarily explore the role of HOXA5 gene in the pathogenesis of AML. The expression of HOXA5 in bone marrow mononuclear cells (BMMC) of non-tumor hematological patients and newly diagnosed AML patients was detected by quantitative real-time PCR (qRT-PCR) and Western blot, respectively. The AML cell line KO-HOXA5-THP-1 was constructed in which HOXA5 gene was knocked out by CRISPR-Cas9-Mediated gene editing technique, and the knockout of HOXA5 gene was verified by qRT-PCR and Western blot, and the cell proliferation was detected by CCK-8 assay. Compared with non-tumor hematological patients, the levels of HOXA5 gene and protein in BMMC of newly diagnosed AML patients were significantly increased (P <0.05). The stable HOXA5 knockout cell line can be obtained by CRISPR-Cas9-Mediated gene editing technique, and the proliferation ability of THP-1 cells with HOXA5 gene knockout was significantly decreased (P <0.05). HOXA5 is highly expressed in AML cells, and knocking out HOXA5 can significantly affect the proliferation ability of AML cells, which provides a new potential therapeutic target for the precise treatment of AML.

RNA-binding protein RBM5 plays an essential role in acute myeloid leukemia by activating the oncogenic protein HOXA9

BackgroundThe oncogenic protein HOXA9 plays a critical role in leukemia transformation and maintenance, and its aberrant expression is a hallmark of most aggressive acute leukemia. Although inhibiting the upstream regulators of HOXA9 has been proven as a significant therapeutic intervention, the comprehensive regulation network controlling HOXA9 expression in leukemia has not been systematically investigated.ResultsHere, we perform genome-wide CRISPR/Cas9 screening in the HOXA9-driven reporter acute leukemia cells. We identify a poorly characterized RNA-binding protein, RBM5, as the top candidate gene required to maintain leukemia cell fitness. RBM5 is highly overexpressed in acute myeloid leukemia (AML) patients compared to healthy individuals. RBM5 loss triggered by CRISPR knockout and shRNA knockdown significantly impairs leukemia maintenance in vitro and in vivo. Through domain CRISPR screening, we reveal that RBM5 functions through a noncanonical transcriptional regulation circuitry rather than RNA splicing, such an effect depending on DNA-binding domains. By integrative analysis and functional assays, we identify HOXA9 as the downstream target of RBM5. Ectopic expression of HOXA9 rescues impaired leukemia cell proliferation upon RBM5 loss. Importantly, acute protein degradation of RBM5 through auxin-inducible degron system immediately reduces HOXA9 transcription.ConclusionsWe identify RBM5 as a new upstream regulator of HOXA9 and reveal its essential role in controlling the survival of AML. These functional and molecular mechanisms further support RBM5 as a promising therapeutic target for myeloid leukemia treatment.

A new genomic framework to categorize pediatric acute myeloid leukemia.

Recent studies on pediatric acute myeloid leukemia (pAML) have revealed pediatric-specific driver alterations, many of which are underrepresented in the current classification schemas. To comprehensively define the genomic landscape of pAML, we systematically categorized 887 pAML into 23 mutually distinct molecular categories, including new major entities such as UBTF or BCL11B, covering 91.4% of the cohort. These molecular categories were associated with unique expression profiles and mutational patterns. For instance, molecular categories characterized by specific HOXA or HOXB expression signatures showed distinct mutation patterns of RAS pathway genes, FLT3 or WT1, suggesting shared biological mechanisms. We show that molecular categories were strongly associated with clinical outcomes using two independent cohorts, leading to the establishment of a new prognostic framework for pAML based on these updated molecular categories and minimal residual disease. Together, this comprehensive diagnostic and prognostic framework forms the basis for future classification of pAML and treatment strategies.

Expression of HOXA10 and HOXA11 in the endometrium of infertile patients with chronic endometritis.

The study aimed to evaluate the impact of CE on the expression of HOXA10 and HOXA11 during the late proliferative phase in the endometrium of infertile women. A prospective, translational cohort study was conducted in partnership with the Hospital Universitário Antônio Pedro in Niterói and the Clínica Ginendo in Rio de Janeiro after approval by the Ethics Committee. The patients were selected to participate in the study after showing an indication for hysteroscopy. All participants were divided into three groups: infertile women with endometritis (n=10), infertile women without endometritis (n=17) and fertile women without endometritis (n=10). At hysteroscopy, two endometrial samples were obtaneid, with one sent for histopathological examination per the gynecologist's request and the other used for immunohistochemistry procedures to evaluate the expression of CD138, HOXA10 and HOXA11. CD138 was used to confirm the diagnosis of CE. The analysis of HOXA10 and HOXA11 was performed using the HScoring method for immunohistochemistry with polyclonal antibodies. Women with and without endometritis had lower HOXA10 and HOXA11 expression values than women in the control group (fertile women without endometritis). The expression of HOXA10 and HOXA11 during the proliferative phase is not significantly different between infertile women with endometritis and infertile women without endometritis. Translational studies with a larger number of patients should be performed.

Exploration of Diagnostic Markers Associated with Inflammation in Chronic Kidney Disease Based on WGCNA and Machine Learning.

Chronic kidney disease (CKD) is a common disorder related to inflammatory pathways; its effective management remains limited. This study aimed to use bioinformatics analysis to find diagnostic markers that might be therapeutic targets for CKD. CKD microarray datasets were screened from the GEO database and the differentially expressed genes (DEGs) in CKD dataset GSE98603 were analyzed. Gene set variation analysis (GSVA) was used to explore the activity scores of the inflammatory pathways and samples. Algorithms such as weighted gene co-expression network analysis (WGCNA) and Lasso were used to screen CKD diagnostic markers related to inflammation. Then functional enrichment analysis of inflammation-related DEGs was performed. ROC curves were conducted to examine the diagnostic value of inflammation-related hub-genes. Lastly, quantitative real-time PCR further verified the prediction of bioinformatics. A total of 71 inflammation-related DEGs were obtained, of which 5 were hub genes. Enrichment analysis showed that these genes were significantly enriched in inflammation-related pathways (NF-κB, JAK-STAT, and MAPK signaling pathways). ROC curves showed that the 5 CKD diagnostic markers (TIGD7, ACTA2, ACTG2, MAP4K4, and HOXA11) also exhibited good diagnostic value. In addition, TIGD7, ACTA2, ACTG2, and HOXA11 expression was downregulated while MAP4K4 expression was upregulated in LPS-induced HK-2 cells. The present study identified TIGD7, ACTA2, ACTG2, MAP4K4, and HOXA11 as reliable CKD diagnostic markers, thereby providing a basis for further understanding of CKD in clinical treatments.

HOXA9 Regulome and Pharmacological Interventions in Leukemia.

HOXA9, an important transcription factor (TF) in hematopoiesis, is aberrantly expressed in numerous cases of both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and is a strong indicator of poor prognosis in patients. HOXA9 is a proto-oncogene which is both sufficient and necessary for leukemia transformation. HOXA9 expression in leukemia correlates with patient survival outcomes and response to therapy. Chromosomal transformations (such as NUP98-HOXA9), mutations, epigenetic dysregulation (e.g., MLL- MENIN -LEDGF complex or DOT1L/KMT4), transcription factors (such as USF1/USF2), and noncoding RNA (such as HOTTIP and HOTAIR) regulate HOXA9 mRNA and protein during leukemia. HOXA9 regulates survival, self-renewal, and progenitor cell cycle through several of its downstream target TFs including LMO2, antiapoptotic BCL2, SOX4, and receptor tyrosine kinase FLT3 and STAT5. This dynamic and multilayered HOXA9 regulome provides new therapeutic opportunities, including inhibitors targeting DOT1L/KMT4, MENIN, NPM1, and ENL proteins. Recent findings also suggest that HOXA9 maintains leukemia by actively repressing myeloid differentiation genes. This chapter summarizes the recent advances understanding biochemical mechanisms underlying HOXA9-mediated leukemogenesis, the clinical significance of its abnormal expression, and pharmacological approaches to treat HOXA9-driven leukemia.

Targeting Oncogenic Enhancers in ASXL1-Mutant Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm with limited therapeutic options and dismal outcomes. Truncating mutations in the epigenetic modifier ASXL1 are common and lead to a proliferative disease phenotype, resistance to epigenetic therapies, and higher rates of AML transformation. ASXL1-mutant CMML is characterized by widespread epigenetic remodeling and up-regulation of key leukemogenic driver genes (e.g. HOXA9 and its co-factor MEIS1). There is a genotype-specific activation of enhancers in ASXL1-mutant CMML that is associated with up-regulation of these leukemogenic driver genes. Here, we test the hypothesis that disrupting the oncogenic interactions at these genotype-specific enhancers can be exploited for therapeutic benefit. Methods: We identified activated enhancers specific to ASXL1-mutant CMML as previously described ( Binder et al. Nat Commun 2022;13(1):1434). To disrupt the E2542605 enhancer, we created unique mapped single guide RNAs (confirmed by BLAT). We generated a ribonucleoprotein complex using the Alt-R CRISPR-Cas9 system (IDT Technologies, Coralville IA). We combined the RNP along with Electroporation Enhancer to 3x10^5 cells of the monocytic cell line U937 in 20uL strip-wells provided by the SF Cell Line 4D-Nucleofector X Kit (Lonza, Basel, Switzerland). To deliver the RNP, U937 cells were electroporated via listed cell line specific protocol by the Amaxa 4D-Nucleofector (Lonza). Forty-eight hours post-electroporation, cells were split and sequenced for E2542605 genomic structure disruption. In parallel, RNA was harvested, converted to cDNA and qPCR for HOXA9 and MEIS1 expression was performed using PrimeTime qPCR FAM labeled probe assays (IDT). Gene expression was determined using delta-delta Ct method. For assessment of selective sensitivity of ASXL1 / enhancer interactions, we performed progenitor colony forming assays using primary bone marrow mononuclear cells from CMML patients with or without ASXL1 mutations and healthy normal controls (n=2 in each category), exposed to increasing concentrations of the dual p300/CBP and BET bromodomain inhibitor EP31670. Briefly, we plated cells in methocellulose fortified with recombinant human cytokines supporting hematopoietic growth in duplicate, in a range of EP31670 concentrations. After 10 days, visible hematopoietic colonies were quantified and characterized. IC 50 values were generated using non-linear regression log(EP31670) vs. normalized response. Results: We computationally identified candidate transcriptional regulators enriched at the activated enhancers specific to ASXL1-mutant CMML, including BRD4 (FDR&amp;lt;2.57x10 -370), CBP (FDR=2.56x10 -217), and p300 (FDR=6.36x10 -93). We selected the ASXL1-mutant-specific enhancer E2542605 (predicted to drive HOXA9 expression), given the up-regulation of HOXA9 in ASXL1-mutant CMML (log 2-fold change +2.05, FDR=0.006, n=14). We disrupted E2542605 using two different CRISPR guides, leading to a PCR trace detection of a 14bp deletion and a 26bp indel in the E2542605 enhancer region, respectively. Both enhancer disruptions led to a down-regulation of its putative target gene HOXA9: Mean relative fold-change 0.36 (p=0.001, n=3) and 0.65 (p=0.046, n=3), respectively ( Fig. 1a). We then used a pharmacologic approach (EP31670) to target the candidate transcriptional regulators involved in the oncogenic interaction at these ASXL1-mutant-specific enhancers. We observed selective sensitivity to growth of CMML colonies (particularly ASXL1-mutant colonies) compared to ASXL1-wild type cases and normal bone marrow controls ( Fig. 1b). Conclusions: In CMML patients, truncating ASXL1 mutations are associated with the activation of genotype-specific enhancers and the recruitment of an extended repertoire of transcriptional regulators including BRD4, CBP, and p300. In a model system, the disruption of an enhancer predicted to govern the expression of the leukemogenic driver HOXA9, led to a significant down-regulation of HOXA9 expression. Pharmacologically disrupting the oncogenic interaction at such enhancers with EP31670 in primary patient samples led to significant therapeutic effects at nanomolar drug concentrations in a genotype-specific manner. Oncogenic interactions at activated enhancers are viable therapeutic targets in ASXL1-mutant CMML and warrant further clinical investigation.

Investigating the Role of the SETDB1 Target TANGO6 (TMCO7) in Leukemia

Despite advancements in AML therapies over the last 2 decades, 1 out of 3 patients diagnosed with AML succumb to their disease. The mainstay of treatment remains systemic chemotherapy, however better understanding of the genetic drivers in AML has generated new therapeutic opportunities. Targeted therapies have emerged to more precisely tailor leukemic treatment based on genetic and epigenetic drivers. We previously demonstrated that SETDB1, a H3K9 methyltransferase involved in heterochromatin regulation, negatively regulates the expression of proleukemic genes Hoxa9 and Meis1. Further, overexpression of SETDB1 correlates with improved overall AML patient survival and extends leukemic disease latency in mouse models (Ropa et al. Haematologica 2020). Here we describe a novel SETDB1 target, TANGO6, and aim to explore its role in leukemogenesis. We mined our genome wide expression and epigenetic datasets generated upon overexpression of SETDB1 to identify targets that may be important for leukemic progression (Ropa et al. Haematologica 2020). TANGO6 was found to be transcriptionally and epigenetically regulated by SETDB1, with little known about its role in leukemia. We performed a targeted CRISPR-Cas9 mediated knock out of TANGO6 to investigate its importance in several human leukemic cell lines: MV4;11, K562 and KOPN8. We established the above cell lines with Cas9 constitutively expressed and introduced sgRNAs targeting TANGO6 by lentivirus. We then performed a competitive growth assay using flow cytometry to assess cell fitness on days 3, 14, 10, 21. We observed that loss of TANGO6 resulted in decreased fitness across all three leukemic cell lines compared to the negative control. This phenotype was more striking in the K562 and KOPN8 cell lines. In lieu of having a reliable antibody to visualize changes in protein expression, we assessed mutational changes in the DNA sequence to evaluate efficiency of our CRISPR/Cas9 system. Genomic DNA was prepared from sg TANGO6 transduced K562 cells on days 5 and 10. The targeted region of TANGO6 was PCR amplified and TOPO cloned for direct Sanger sequencing. 92% (12 out of 13 samples) showed a mutation involving the target sequence. Of these mutations, ~67% resulted in frameshift mutations leading to early truncation of the protein. The remaining mutations involved deletions ranging from 1 to 28 amino acids, with one sample resulting in two single point mutations conferring a change of leucine to serine and lysine to arginine. These mutations may or may not affect the folding of the protein. This data demonstrates we are successfully targeting TANGO6, which produces the biological phenotype in our competitive growth assay. To further understand the potential importance of TANGO6 in human leukemia, we performed qPCR on 5 leukemic cell lines (2 MLL-rearranged AML cell lines, 2 B-lymphoblastic cell lines, and 1 CML cell line). Our data showed that TANGO6 was expressed at higher levels across all leukemic cell lines compared to normal CD34+ hematopoietic stem/progenitor cells, consistent with a role in leukemogenesis. Our preliminary data suggests that TANGO6 plays an important role in leukemogenesis. Ongoing work focuses on validating this hypothesis using in vivo leukemic mouse models and protein localization studies. Given the lack of available information on human TANGO6, characterizing its location and mechanism is essential to understanding its role in disease. Our work has established the need for further exploration of TANGO6 as a potential therapeutic target for AML.

The XPO1-FOXC1-HOX Functional Axis Opens New Therapeutic Avenues to Treat DEK-NUP214 AML Patients

Introduction: t(6;9)(p23;q34.1)/DEK-NUP214 patients represent a discrete group of younger AML patients recognised as a separate disease entity in the World Health Organization classification of myeloid neoplasms. They typically display a dismal prognosis, higher relapse rate and a striking co-occurrence with FLT3-ITD mutations in &amp;gt; 70% of cases. DEK is a nuclear factor that has been attributed multifunctional roles, including gene regulation, while its fusion partner, NUP214, plays a pivotal role in nuclear export by interacting with transport receptors such as XPO1. However, the precise mechanism by which DEK-NUP214 drives leukaemia remains unclear. Aims: Application of a multi-omics profiling to understand the oncogenic mechanisms underlying DEK-NUP214 fusion protein and identify potential therapeutical vulnerabilities. Methods: A comprehensive multi-omics screening (whole genome sequencing, targeted sequencing, transcriptomics, (phosphor)proteomics, mass cytometry (CyTOF) and in vitro drug screening) was conducted on 57 untreated primary AML samples from cytogenetically poor-risk AML, including four cases with t(6;9) translocation (complex karyotype (n=20), -7/del(7) (n=15), KMT2A rearrangements (not including t(9;11)) (n=12), t(6;9) (n=4) and other poor-risk karyotypes (inv(3), -5/del(5), t(3;12)/+8 or -17/del(17)) (n=6)). The t(6;9)/DEK-NUP214 cell line FKH-1, was utilised as a model for in vitro experiments and compared with other AML cell lines (OCI-AML3, KASUMI-1, THP-1, P31-FUJ, MV4-11 and K562). In vitro drug treatments were conducted in the cell lines and patient samples using Cell-Titer&amp;Glo, and transcriptional changes were evaluated using qPCR and RNA-seq. To examine the function of selected target genes of DEK-NUP214, we used lentiviral shRNA system followed by cell cycle, apoptosis, differentiation, and cell proliferation assays. Results: An integrative RNA-seq analysis of our cohort and a separate series of 691 AML cases that included three additional t(6;9) cases (Leucegene, https://leucegene.ca/) revealed 128 genes significantly upregulated and 74 downregulated in t(6;9) patients compared to other AML samples. The overexpressed genes included some known leukaemia mediators (FOXC1, HOXA and HOXB genes), the genes previously reported in t(6;9) patients (EYA3, SESN1 and PRDM2) and novel genes with roles in haematopoiesis or cancer cell survival that showed the most significant differences (NFIX and GGT5). The significant overexpression of these genes was also confirmed in the t(6;9) FKH-1 cell line. In vitro drug screening of 527 licenced or investigational drugs allowed us to identify the compounds that were most effective and selective for t(6;9) patients in comparison with other AML cytogenetic groups. The two XPO1 inhibitors (Selinexor and Eltanexor) included in our drug panel were within the four top-ranked compounds. Of note, qPCR and RNA-seq analyses in the cell lines and primary patient samples showed that inhibition of XPO1 resulted in significant downregulation of the expression of FOXC1, NFIX, EYA3, HOXA and HOXB genes, highlighting a functional axis linking these target genes, XPO1 and the fusion. In line with this finding, silencing of FOXC1 in the FKH-1 cells led to an increase in apoptosis, lower cell proliferation and reduced clonogenic capacity, accompanied by a concomitant downregulation of HOXA and HOXB genes. Collectively, these findings support a key role of FOXC1 in DEK-NUP214-driven AML. Conclusion: This study offers valuable insights into the molecular and pathological mechanisms underlying t(6;9)-AML, revealing a functional interaction between DEK-NUP214, XPO1 and FOXC1, and providing evidence to consider XPO1 inhibition as a potential new avenue to treat these patients.