Similar Gene Expression Profiles Research Articles

Abstract PS18-09: The DARC side of Breast Cancer - DARC, Duffy-null and African ancestry influence in the Triple Negative Breast Cancer tumor microenvironment

Abstract Background. Despite a recent convergence of breast cancer (BC) incidence rates between African American (AA) and European American (EA) women, AA women continue to have ~40% higher mortality rates. BC is a heterogeneous collection of diseases, where African ancestry women are disproportionately burdened with the most aggressive subtype of BC, triple negative BC (TNBC). We have previously reported that the African ancestry-specific Duffy-null allele (FY-) is a risk factor for TNBC diagnoses. FY- is a promoter variant that removes expression of the Duffy Antigen Receptor for Chemokines (DARC/ACKR1), from red blood cells (RBCs). DARC serves as a portal of entry for Plasmodium vivax malaria parasite, and FY- confers immunity, leading to fixation of FY- among Sub-Saharan African populations. DARC is an atypical chemokine receptor that functions to modulate chemokine levels in circulation and tissues to aid immune cell recruitment. DARC is also expressed on breast tumor epithelial cells, however the significance of DARC in the TNBC tumor microenvironment (TME) has not fully been explored. Leveraging our African ancestry enriched International Study for the Center of Breast Cancer Subtypes cohort and in vivo murine models, we employed multi-omics approaches to explore the function of FY- and DARC expression across cell types in the TNBC TME. Genomics. Using transcriptomic data from the TCGA BC cohort, we have previously reported that DARC expression is highly correlated with deconvoluted immune cell abundance using CIBERSORT. To build upon this finding among TNBC patients, we have analyzed RNAseq data from two cohorts of African ancestry women to explore correlation of DARC expression status and immune cell abundance. Among TNBC cases, we report that higher DARC expression was significantly associated with increased immune cell abundance, with increases in B cell, T cell and monocyte populations. Using whole genome sequencing data across 86 African ancestry cases, we are currently investigating germline FY- status with mutational signatures. Circulating biomarkers. Patient plasma was collected prior to surgery, where Luminex multiplex assays were performed to quantify levels of >40 circulating chemokines and cytokines. FY- status has been determined for ~550 patients using single-plex or multiplex PCR genotyping methods from germline saliva DNA. Our univariate analyses show several circulating chemokines, including CCL2, CXCL2, CXCL6 and CXCL11, have significant differences between FY- and non-FY- BC patients. Curation of clinicopathological variables for multivariate analysis is ongoing. Murine models. We crossed C3(1)Tag BC transgenic mice with a DARC knock-out (KO) line to generate mice lacking DARC expression that spontaneously develop tumors in the mammary fat pads. The C3(1)Tag model is reported to have similar gene expression profiles to basal-like TNBC tumors. Tumors were collected at pre-determined time points of 25 and 30 weeks, where DARC KO mice had increased tumor size and tumor burden at 30 weeks. Initial fluorescent staining revealed an increase in immune cell infiltration in the TME of DARC expressing mice compared to DARC KO mice. To further explore this, we employed multiplex staining across a representative cohort of DARC expressing and KO mice at 25 and 30 weeks to capture the specific immune cell populations infiltrating the TME. Conclusions. TNBC tumors have worse prognosis due to lack of targeted therapeutic options. Increased immunogenicity of TNBC tumors, especially among African ancestry women may be driven in part by DARC expression. Characterization of FY- status, in coordination with DARC among other cell types in the TNBC TME could present new opportunities for biomarker and/or therapeutic development to improve prognosis for these underserved populations. Citation Format: Rachel Martini, Stevens Patino, Emma Guyonnet, Brian Stonaker, Isra Elhussein, Julie Sahler, Avery August, Nancy Manley, Rick Kittles, Clayton Yates, Lisa Newman, Melissa Davis. The DARC side of Breast Cancer - DARC, Duffy-null and African ancestry influence in the Triple Negative Breast Cancer tumor microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS18-09.

A novel hypergraph model for identifying and prioritizing personalized drivers in cancer.

Cancer development is driven by an accumulation of a small number of driver genetic mutations that confer the selective growth advantage to the cell, while most passenger mutations do not contribute to tumor progression. The identification of these driver genes responsible for tumorigenesis is a crucial step in designing effective cancer treatments. Although many computational methods have been developed with this purpose, the majority of existing methods solely provided a single driver gene list for the entire cohort of patients, ignoring the high heterogeneity of driver events across patients. It remains challenging to identify the personalized driver genes. Here, we propose a novel method (PDRWH), which aims to prioritize the mutated genes of a single patient based on their impact on the abnormal expression of downstream genes across a group of patients who share the co-mutation genes and similar gene expression profiles. The wide experimental results on 16 cancer datasets from TCGA showed that PDRWH excels in identifying known general driver genes and tumor-specific drivers. In the comparative testing across five cancer types, PDRWH outperformed existing individual-level methods as well as cohort-level methods. Our results also demonstrated that PDRWH could identify both common and rare drivers. The personalized driver profiles could improve tumor stratification, providing new insights into understanding tumor heterogeneity and taking a further step toward personalized treatment. We also validated one of our predicted novel personalized driver genes on tumor cell proliferation by vitro cell-based assays, the promoting effect of the high expression of Low-density lipoprotein receptor-related protein 1 (LRP1) on tumor cell proliferation.

Abstract 6099: Identification of intrinsic precursors of Barrett's and gastric intestinal metaplasia

Abstract The origin of Barrett's esophagus (BE) and gastric intestinal metaplasia (GIM), obligate precursors of esophageal adenocarcinoma (EAC) and intestinal gastric cancer (iGC), has intrigued investigators for decades and would likely guide preemptive strategies. From endoscopic biopsies of clinically confirmed BE and GIM, we have cloned stem cells committed to intestinal metaplasia in vitro. Remarkably, the gene expression profiles of BE and GIM stem cells are highly related down to broad arrays of transcription factors compared with stem cells of the normal gastric mucosa. Using cell surface markers in common between BE and GIM stem cells, we have identified clusters of cells at the squamocolumnar junction and the distal stomach in mice and have used Fluorescence-activated cell sorting (FACS) to clone these cells from both sites. These murine clones can be differentiated in air-liquid interface cultures, and both are committed to intestinal metaplasia marked by goblet cells and Alcian blue staining. To ask whether similar cells exist in humans, we generated stem cell libraries from endoscopic biopsies taken from the gastroesophageal junction (GEJ) and the antrum-body junction (ABJ) in patients without BE or GIM. FACS sorting of these stem cell libraries using the same cell surface markers common to BE and GIM stem cells and the murine precursors, we have identified stem cells from the GEJ and ABJ that show similar gene expression profiles and share a commitment to intestinal metaplasia upon in vitro differentiation. Using identical transformation protocols involving retrovirally transduced protooncogenes, we find that these transformed intrinsic cells give rise to tumors with expression profiles similar to EAC and iGC in TCGA datasets, whereas transformed gastric mucosal stem cells yield tumors marked by signet ring cells typical of diffuse gastric cancer. Lastly, we find that drugs we have developed that target the stem cells of BE and GIM also eliminate the putative precursor cells of BE and GIM, suggesting the potential of therapeutics that might eliminate the emergence of intestinal metaplasia and the cancers derived from them. Citation Format: Wa Xian, Frank McKeon, Yusuke Yamamoto, Melika Khorrami, Melina Khorrami, Zaal Mory, Jeremy Siegelman, Amber Su, Raul Caballero Montes, Ashley Hoffman, Jaffer Ajani, Christopher Crum, William Bachovchin, Shan Wang, Matthew Vincent, Crystal Nguyen. Identification of intrinsic precursors of Barrett's and gastric intestinal metaplasia [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 6099.

Abstract P35: High-grade B-cell lymphomas with 11q aberrations show a dark zone expression profile similar to Burkitt lymphoma but with additional extension towards intermediate germinal center B-cells

Abstract P35: High-grade B-cell lymphomas with 11q aberrations show a dark zone expression profile similar to Burkitt lymphoma but with additional extension towards intermediate germinal center B-cells

Tyrosine kinase inhibitor response of ABL-class acute lymphoblastic leukemia: the role of kinase type and SH3 domain

AbstractAcute lymphoblastic leukemia (ALL) with fusion of ABL-class tyrosine kinase genes other than BCR::ABL1 occurs in ∼3% of children with ALL. The tyrosine kinase genes involved in this BCR::ABL1-like (Ph-like) subtype include ABL1, PDGFRB, ABL2, and CSF1R, each of which has up to 10 described partner genes. ABL-class ALL resembles BCR::ABL1-positive ALL with a similar gene expression profile, poor response to chemotherapy, and sensitivity to tyrosine kinase inhibitors (TKIs). There is a lack of comprehensive data regarding TKI sensitivity in heterogeneous group of ABL-class ALL. We observed variability in TKI sensitivity within and among each ABL-class tyrosine kinase gene subgroup. We showed that ALL samples with fusions for any of the 4 tyrosine kinase genes were relatively sensitive to imatinib. In contrast, the PDGFRB-fused ALL samples were less sensitive to dasatinib and bosutinib. Variation in ex vivo TKI response within the subset of samples with the same ABL-class tyrosine kinase gene was not associated with the ALL immunophenotype, 5′ fusion partner, presence or absence of Src-homology-2/3 domains, or deletions of IKZF1, PAX5, or CDKN2A/B. In conclusion, the tyrosine kinase gene involved in ABL-class ALL is the main determinant of TKI sensitivity and relevant for specific TKI selection.

Transcriptomic analysis of lipoteichoic acid‑treated undifferentiated and neutrophil‑like differentiated HL‑60 cells.

Toll-like receptor 2 (TLR2) is an important sensor for innate immune cells, including neutrophils, for the recognition of pathogen infection. Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is a TLR2 ligand. LTA-induced TLR2 signaling pathways are well established in neutrophils. However, experimental studies regarding transcriptional regulation and the molecular mechanisms in primary human neutrophils are limited due to their short lifespan. The promyelocytic leukemia cell line, HL-60, can differentiate into a neutrophil-like phenotype following treatment with dimethyl sulfoxide. The aim of the present study was to investigate whether differentiated HL-60 (dHL-60) cells induced a similar gene expression profile upon LTA treatment as that previously determined for primary human neutrophils. After 4 or 24 h of Staphylococcus aureus LTA treatment, undifferentiated HL-60 (uHL-60) and dHL-60 cells were collected for RNA sequencing. The results demonstrated that hundreds of identical differentially expressed genes (DEGs) were observed in 1 and 10 µg/ml LTA-treated dHL-60 cells following 4 and 24 h of incubation, while almost no DEGs between LTA-treated HL-60 and dHL-60 cells were observed. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG), it was noted that the pathways of shared DEGs between the 1 and 10 µg/ml LTA-treated dHL-60 cells at both time points were significantly enriched in immune and inflammatory response-related pathways, such as cellular response to tumor necrosis factor, interleukin-1, interferon γ, neutrophil chemotaxis, the NF-κB signaling pathway and the Toll-like receptor signaling pathway. In addition, when comparing the effect of 1 and 10 µg/ml LTA treatment on dHL60 cells, it was found that all enriched GO and KEGG pathways were associated with the TLR signaling pathways of neutrophils. The results of the present study provided important information for the implementation of mRNA profiling in LTA-treated dHL-60 cells and may indicate the feasibility of using dHL-60 cells as a research model for TLR2 signaling in human neutrophils.

Comparative transcriptomic analysis of Illumina and MGI next-generation sequencing platforms using RUNX3- and ZBTB46-instructed embryonic stem cells.

Introduction: We have previously observed phenotypic and developmental changes upon the ectopic expression of the RUNX3 or the ZBTB46 transcription factors in mouse embryonic stem cell (ESC) derived progenitors. In this study, we evaluated the gene expression profiles of the RUNX3- and the ZBTB46-instructed murine ESCs with RNA-seq testing two next-generation sequencing technologies. Methods: We compared the DNA nanoball-based DNBSEQ G400 sequencer (MGI) with the bridge-PCR-based NextSeq 500 instrument (Illumina) for RNA sequencing. Moreover, we also compared two types of MGI sequencing reagents (Standard versus Hot-massive parallel sequencing (MPS)) with the DNBSEQ G400. Results: We observed that both sequencing platforms showed comparable levels of quality, sequencing uniformity, and gene expression profiles. For example, highly overlapping RUNX3- and ZBTB46-regulated gene lists were obtained from both sequencing datasets. Moreover, we observed that the Standard and the Hot-MPS-derived RUNX3- and ZBTB46-regulated gene lists were also considerably overlapped. This transcriptome analysis also helped us to identify differently expressed genes in the presence of the transgenic RUNX3 or ZBTB46. For example, we found that Gzmb, Gzmd, Gzme, Gdf6, and Ccr7 genes were robustly upregulated upon the forced expression of Runx3; on the other hand, Gpx2, Tdpoz4, and Arg2 were induced alongside the ectopic expression of Zbtb46. Discussion: Similar gene expression profile and greatly overlapping RUNX3- and ZBTB46-regulated gene sets were detected with both DNA sequencing platforms. Our analyses demonstrate that both sequencing technologies are suitable for transcriptome profiling and target gene selection. These findings suggest that DNBSEQ G400 represents a cost-effective alternative sequencing platform for gene expression monitoring. Moreover, this analysis provides a resource for exploration of the RUNX3- and ZBTB46-dependent gene regulatory networks.

The gene expression profile and cell of origin of canine peripheral T-cell lymphoma

BackgroundPeripheral T-cell lymphoma (PTCL) refers to a heterogenous group of T-cell neoplasms with poor treatment responses and survival times. Canine PTCL clinically and immunophenotypically resembles the most common human subtype, PTCL-not otherwise specified (PTCL-NOS), leading to interest in this canine disease as a naturally occurring model for human PTCL. Gene expression profiling in human PTCL-NOS has helped characterize this ambiguous diagnosis into distinct subtypes, but similar gene expression profiling in canine PTCL is lacking.MethodsBulk RNA-sequencing was performed on tumor samples from 33 dogs with either CD4+ (26/33), CD8+ (4/33), or CD4-CD8- (3/33) PTCL as diagnosed by flow cytometry, and sorted CD4+ and CD8+ lymphocytes from healthy control dogs. Following normalization of RNA-seq data, we performed differential gene expression and unsupervised clustering methods. Gene set enrichment analysis was performed to determine the enrichment of canine CD4+ PTCL for human PTCL-NOS, oncogenic pathways, and various stages of T-cell development gene signatures. We utilized gene set variation analysis to evaluate individual canine CD4+ PTCLs for various human and murine T-cell and thymocyte gene signatures. Cultured canine PTCL cells were treated with a pan-PI3K inhibitor, and cell survival and proliferation were compared to DMSO-treated controls. Expression of GATA3 and phosphorylated AKT was validated by immunohistochemistry.ResultsWhile the canine CD4+ PTCL phenotype exhibited a consistent gene expression profile, the expression profiles of CD8+ and CD4-CD8- canine PTCLs were more heterogeneous. Canine CD4+ PTCL had increased expression of GATA3, upregulation of its target genes, enrichment for PI3K/AKT/mTOR signaling, and downregulation of PTEN, features consistent with the more aggressive GATA3-PTCL subtype of human PTCL-NOS. In vitro assays validated the reliance of canine CD4+ PTCL cells on PI3K/AKT/mTOR signaling for survival and proliferation. Canine CD4+ PTCL was enriched for thymic precursor gene signatures, exhibited increased expression of markers of immaturity (CD34, KIT, DNTT, and CCR9), and downregulated genes associated with the T-cell receptor, MHC class II associated genes (DLA-DQA1, DLA-DRA, HLA-DQB1, and HLA-DQB2), and CD25.ConclusionsCanine CD4+ PTCL most closely resembled the GATA3-PTCL subtype of PTCL-NOS and may originate from an earlier stage of T-cell development than the more conventionally posited mature T-helper cell origin.

Prediction of Cancer Driver Genes Using a Deep Convolutional Network

The paper describes a method for predicting genes associated with the development of cancer. The method applies the convolutional neural network for the purpose of predicting disease driver genes. Distinctive features of the method are the use of gene expression data to determine the topological structure of the network, the efficiency of prediction with limited information about genes associated with the disease, and the possibility of jointly including information on mutations and similarity of gene expression profiles to improve the accuracy of prediction.

Acute myeloid leukemia with t(16;21)(q24;q22), CBFA2T3::RUNX1, in post-treatment setting, a case report

Abstract Introduction/Objective Acute myeloid leukemia (AML) with t(16;21)(q24;q22), CBFA2T3::RUNX1 is an uncommon entity that has been reported in both pediatric and adult patients as de novo AML or secondary to prior chemo/radiation therapy. CBFA2T3::RUNX1 AMLs share similar clinico-pathological features and gene expression profile with RUNX1::RUNX1T1 AML. Methods/Case Report We report a case of AML with CBFA2T3::RUNX1 in a 52 year old female who had history of small cell cervical cancer treated with chemotherapy (4 cycles of cisplatin and etoposide) and pelvic radiation therapy. About 17 months after completion of therapy, she presented with anemia and thrombocytopenia, normal WBC/neutrophil count, eosinophilia and increased blasts (up to 10%) in blood. Bone marrow biopsy shows AML with maturation (FAB-M2), erythropoiesis is present, whereas megakaryocytes are decreased. Flow cytometry analysis shows myeloblasts with aberrant CD19 and CD56 expression; in addition, increased CD117/tryptase positive cells are noted, mostly interstitial and more concentrated around bone trabeculae. By immunohistochemical stains, these cells are at least partially positive for CD25, negative CD2, CD30. Karyotype studies demonstrate t(16;21)(q24;q22) and trisomy 8 in all metaphases. FoundationOne heme NGS studies confirmed CBFA2T3::RUNX1 fusion, and no additional mutations were identified. Patient received induction chemotherapy with Idarubicin/Cytarabine plus GO (Gemtuzumab ozogamicin); day 14 bone marrow showed chemotherapeutic effect and she is now in morphologic remission 5 months after diagnosis. Results (if a Case Study enter NA) NA Conclusion Our case adds to the limited knowledge of CBFA2T3::RUNX1 AMLs. Our case shows morphological and immunophenotypic features similar to AML with RUNX1::RUNX1T1, with unusual presentation of peripheral eosinophilia, and increased CD117/tryptase positive cells in bone marrow, the latter has been reported occasionally in core binding factor AMLs. We emphasize the importance of comprehensive genetic studies in AML patients, lack of adverse genetic abnormalities in our patient indicates favorable risk category regardless of the post-treatment clinical setting.

The Upregulation of the FBLN1 and HS3ST4 Genes May Represent a Potential Mechanism Driving the Fibrotic Progression in Primary Myelofibrosis and Leading to Adverse Prognosis

Myelofibrosis is a group of diseases characterized by increased proliferation of bone marrow fibrous connective tissue and a decrease in hematopoietic cells, leading to impaired bone marrow hematopoietic function. Myelofibrosis includes primary myelofibrosis（PMF）, myelofibrosis secondary to polycythemia vera（post-PV MF）, and myelofibrosis secondary to essential thrombocythemia（post-ET MF）. Studies suggest that certain genes may be responsible for driving the development of myelofibrosis and causing adverse prognosis. By conducting RNA-Seq analysis, we investigated the differentially expressed genes and their impact on prognosis in primary myelofibrosis and secondary myelofibrosis. Furthermore, we analyzed the potential mechanisms driving the progression of fibrosis. Additionally, we performed a comparative analysis of the clinical features among various types of myelofibrosis. In this study, we performed transcriptome sequencing to analyze gene expression differences in 14 overt-PMF samples, 5 post-ET MF samples, and 3 post-PV MF samples.Functional and pathway analyses were conducted on these differentially expressed genes, and cluster heatmaps were used to visualize their expression patterns.Simultaneously, we performed a comparative analysis of clinical characteristics among 370 overt-PMF patients, 96 post-ET MF patients, and 52 post-PV MF patients.The study revealed that compared to post-PV MF, overt-PMF showed 81 upregulated and 2 downregulated differentially expressed genes, while compared to post-ET MF, overt-PMF displayed 19 upregulated and 3 downregulated differentially expressed genes (Figure a) (Figure b). Based on the similarity of gene expression profiles among the samples, a clustering analysis was performed (Figure c).The results demonstrated that in overt-PMF, the FBLN1 gene was significantly upregulated compared to post-PV MF and was enriched in pathways related to extracellular matrix formation and cell adhesion among the differentially expressed genes (Figure d). Additionally, the HS3ST4 gene was significantly upregulated in overt-PMF compared to post-ET MF and was enriched in pathways related to interleukin-8 generation and regulation of cytokine production involved in inflammatory responses.In the analysis of clinical characteristics among different types of myelofibrosis patients, it was observed that overt-PMF was more prone to transform into acute leukemia compared to post-PV MF, had a higher tendency to develop transfusion dependency (Figure e), and exhibited lower hemoglobin levels and hematocrit at the time of diagnosis (Figure f). Moreover, compared to post-ET MF, overt-PMF had a higher likelihood of transforming into acute leukemia and lower platelet counts at the time of diagnosis. The research findings indicate that the expressions of FBLN1 and HS3ST4 genes are significantly upregulated in overt-PMF, and patients with overt-PMF have a worse prognosis compared to those with post-PV MF and post-ET MF. Previous studies have shown that FBLN1 is involved in extracellular matrix assembly, promoting the formation and stabilization of collagen fibers. It can also interact with integrin receptors and other cell adhesion molecules, participating in cell-extracellular matrix signaling processes. On the other hand, HS3ST4 is involved in processes such as cell adhesion, cell migration, and cell signaling. Based on these findings, it is speculated that the upregulation of FBLN1 and HS3ST4 gene expressions may potentially drive the progression of fibrosis and lead to an adverse prognosis in patients with myelofibrosis. Acknowledgement: This research was funded by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; Zhejiang Medical Association Clinical Medical Research special fund project, No. 2022ZYC-D09. Correspondence to: Dr Jian Huang, Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine. No. 79 Qingchun Road. Hangzhou, Zhejiang, PR China. househuang@zju.edu.cn

Heterogeneous Gene Fusion Transcripts Found in t(7;12)(q36;p13) Acute Myeloid Leukemia but with Similar Gene Expression Profile

Cytogenetic aberrations are often involved in acute myeloid leukemia (AML) and can serve as diagnostic markers, prognosis predictors and impact the choice of therapy. A translocation t(7;12)(q36;p13) is reported in up to 20-30% of AML patients typically diagnosed before 24 months of age (Beverloo, Panagopoulos et al. 2001, Cancer Res 61(14): 5374-5377). However, in a retrospective survey of the Nordic Organization of Pediatric Hematology and Oncology (NOPHO) registry, only 4% of reported AML diagnosed before 24 months of age had t(7;12) (Espersen, Noren-Nystrom et al. 2018, Genes Chromosomes Cancer 57(7): 359-365). The t(7;12) is difficult to identify with conventional karyotyping, why the true frequency of t(7;12) may be underestimated. Similarly, the reported prognosis for t(7;12) patients has varied ranging from good (Espersen, Noren-Nystrom et al. 2018) to dismal (von Bergh, van Drunen et al. 2006, Genes Chromosomes Cancer 45(8): 731-739). The translocation has been reported to give rise to an in-frame fusion transcript, MNX1::ETV6. However, detection of this fusion is reported only in 50% of cases in contrast to high expression of MNX1 in 100% (von Bergh, van Drunen et al. 2006). The aim of this study was to determine the frequency of the t(7;12) in pediatric AML patients in the NOPHO AML-2004/2012 protocols, their event-free and overall survival and to investigate the presence of additional genetic alterations. Patients in Sweden, Denmark, or Iceland between 2004 to 2020 diagnosed with AML before 2 years of age that had not been reported with a recurrent genetic aberration plus all patients diagnosed with t(7;12) AML were identified. Bone marrow, or peripheral blood samples from patients were retrieved from the NOPHO biobank. This cohort constituted 86% (31 out of 36) of all patients that fulfilled these criteria. In total 89 AML patients were diagnosed before 2 years of age. The t(7;12) AML cases were identified by screening for high expression of MNX1 and presence of fusion transcripts using whole transcriptome sequencing (WTS). Whole genome sequencing (WGS) was done to identify chromosomal rearrangements, and translocation breakpoints. At diagnosis, four patients were diagnosed with t(7;12). A central karyotype review of all patients reported two additional cases. No further cases were identified using WTS. Thus, the frequency of t(7;12) AML was 7% (6 out of 89) and for patients diagnosed before 12 months of age 11% (5 out of 47). In total 12 cases with t(7;12) were identified in the complete NOPHO registry but only 11 patients received treatment according to NOPHO protocol. The relapse rate was 54% (6 out of 11). All relapse cases underwent allogeneic hematopoietic stem cell (HSC) transplantation in complete remission two (CR2), and their overall survival (OS) was 83% (5 out of 6). Hence OS for all t(7;12) patients treated according to the NOPHO2004 and NOPHO2012 protocols was 91% (10 out of 11). Analysis of WTS data showed similar gene expression profile between our t(7;12) cases and t(7;12) cases retrieved from the TARGET database. Genes that were typically overexpressed in all t(7;12) AML were, MNX1, MNX1-AS1, MNX1-AS2, LIN28B, BAMBI, MAF, CRISP3, EDIL3, CTTNBP2, KRT72, and AGR2, where MNX1 seems to be uniquely expressed in this type of leukemia (Nilsson et al. 2022, Int J Cancer 151(5): 770-782). Analysis of fusion transcripts showed presence of MNX1::ETV6 in just one case. The remaining cases instead showed presence of other fusion transcripts involving ETV6, NOM1::ETV6, ETV6::NOM1, ETV6::LMBR1 and ETV6::BMAL2. WGS verified a fusion between NOM1 and ETV6 in 3 cases. The patient with ETV6::BMAL2 showed that 16 Mb of chromosome 12 was inserted into chromosome 7 placing a large part of ETV6 near MNX1. The fusion transcript ETV6::BMAL2 is the product of what remains on chromosome 12. In conclusion, frequency of t(7;12) AML in the NOPHO treatment cohort was approximately 7%, with 54% relapse rate, but most patients were salvaged by HSCT in CR2. Heterogeneous gene fusion transcripts were identified in this subgroup of AML where ETV6::NOM1 was most frequent. However, all cases showed similar gene expression signatures which underlines that the leukemia driving event is ectopic expression of MNX1 (Waraky, A., 2023, Haematologica, DOI: 10.3324/haematol.2022.282255) and should therefore be the defining classifying criteria of this type of AML.

Acute Myeloid Leukemia (AML) from Older HSCs Is Resistant to Chemotherapy and Has an Altered DNA Damage Response

Acute myeloid leukemia (AML) is an aggressive malignancy that leads to the accumulation of immature myeloid precursors, resulting in progressive marrow failure and death. AML arises from the hematopoietic stem cell (HSC), either directly or through an early myeloid precursor. HSCs must survive for the entire human lifespan to provide the 200+ billion blood cells that are produced daily. They are subjected to the entirety of the aging process. Further, while AML is diagnosed in every age range, it disproportionately affects older patients, with a median age at diagnosis of 68. Older age, typically defined as 60 years of age or older, is a powerful, poor prognostic factor. Clinical trials show an estimated 5-year survival of less than 10% in these patients. Ten-year survival is even worse with one study estimating 2.4% of patients over 60 years of age alive at ten years when treated with chemotherapy. Importantly, this poor outcome is the result of disease that is significantly more resistant to therapy. Even though this clinical data is well known the underlying mechanisms of therapy resistance in older AML are unknown. To address this, we generated genetically identical AML cells from fetal, 12-week-old (Young) or 24-month-old (Old) murine HSCs. Retroviral vectors were used to express the oncogenic MLL-ENL fusion protein and NRas G12D. AML was confirmed by morphology, immunophenotype and all AML cells were lethal when injected into syngeneic recipients. Old AML cells demonstrated reduced autophagy, decreased NAD levels and impaired mitochondria consistent with a significant effect of biological aging. When treated with DNA damaging chemotherapy, Old AML cells were significantly more resistant than either Young or Fetal cells to both cytarabine and the anthracycline doxorubicin. To examine the DNA damage response AML cells were treated with doxorubicin and stained for the early DNA damage marker gH2AX. Old AML cells generated significantly more gH2AX than either Fetal or Young AML cells (Figure 1). We next examined the induction of p53 in response to treatment and in contrast to the gH2AX results Old AML cells induced the least amount of p53 in response to doxorubicin treatment (Figure 2). This data demonstrates that Old AML cells were more adept at recognizing DNA damage but least likely to induce p53 in response to it suggesting a difference in DNA repair. To better understand these differences RNA sequencing was performed and differentially expressed genes (DEGs) identified. When Fetal and Young AML cells were compared, no DEGs reached the false discovery threshold for significance, demonstrating that Fetal and Young AMLs have highly similar gene expression profiles. In contrast, when Old AML cells were compared to either Young or Fetal cells, hundreds of highly significant DEGs were identified, and KEGG pathway enrichment analysis revealed that several DNA repair pathways were significantly upregulated in Old AML. These include base excision repair (FDR p= 8.60E-06), mismatch repair (FDR p=3.26E-05), and nucleotide excision repair (FDR p= 0.00043). To further validate these pathways, the Beat AML TCGA dataset was assessed for the prognostic effect of increased expression of key genes in these pathways. Patients with increased expression of these genes had a trend toward worse survival (p=0.07) consistent with these pathways being clinically relevant. These data suggest that AML arising in older patients may overexpress DNA damage repair pathways leading to chemotherapy resistance and poor outcomes. Additional work on ways to increase sensitivity to Old AML cells is in progress.

Complex dynamics of coral gene expression responses to low pH across species.

Coral capacity to tolerate low pH affects coral community composition and, ultimately, reef ecosystem function. Low pH submarine discharges ('Ojo'; Yucatán, México) represent a natural laboratory to study plasticity and acclimatization to low pH in relation to ocean acidification. A previous >2-year coral transplant experiment to ambient and low pH common garden sites revealed differential survivorship across species and sites, providing a framework to compare mechanistic responses to differential pH exposures. Here, we examined gene expression responses of transplants of three species of reef-building corals (Porites astreoides, Porites porites and Siderastrea siderea) and their algal endosymbiont communities (Symbiodiniaceae) originating from low pH (Ojo) and ambient pH native origins (Lagoon or Reef). Transplant pH environment had the greatest effect on gene expression of Porites astreoides hosts and symbionts and P. porites hosts. Host P. astreoides Ojo natives transplanted to ambient pH showed a similar gene expression profile to Lagoon natives remaining in ambient pH, providing evidence of plasticity in response to ambient pH conditions. Although origin had a larger effect on host S. siderea gene expression due to differences in symbiont genera within Reef and Lagoon/Ojo natives, subtle effects of low pH on all origins demonstrated acclimatization potential. All corals responded to low pH by differentially expressing genes related to pH regulation, ion transport, calcification, cell adhesion and stress/immune response. This study demonstrates that the magnitude of coral gene expression responses to pH varies considerably among populations, species and holobionts, which could differentially affect acclimatization to and impacts of ocean acidification.

Dietary Amino Acid Source Elicits Sex-Specific Metabolic Response to Diet-Induced NAFLD in Mice.

Non-alcoholic fatty liver disease (NAFLD) is a sexually dimorphic disease influenced by dietary factors. Here, the metabolic and hepatic effects of dietary amino acid (AA) source is assessed in Western diet (WD)-induced NAFLD in male and female mice. The AA source is either casein or a free AA mixture mimicking the composition of casein. As expected, males fed a casein-based WD display glucose intolerance, fasting hyperglycemia, and insulin-resistance and develop NAFLD associated with changes in hepatic gene expression and microbiota dysbiosis. In contrast, males fed the AA-based WD show no steatosis, a similar gene expression profile as males fed a control diet, and a distinct microbiota composition compared to males fed a casein-based WD. Females are protected against WD-induced liver damage, hepatic gene expression, and gut microbiota changes regardless of the AA source. Free dietary AA intake prevents the unhealthy metabolic outcomes of a WD preferentially in male mice.

ScribbleDom: Using Scribble-Annotated Histology Images to Identify Domains in Spatial Transcriptomics Data.

Spatial domain identification is a very important problem in the field of Spatial Transcriptomics (ST). The state-of-the-art solutions to this problem focus on unsupervised methods, as there is lack of data for a supervised learning formulation. The results obtained from these methods highlight significant opportunities for improvement. In this paper, we propose a potential avenue for enhancement through the development of a semi-supervised convolutional neural network (CNN) based approach. Named ScribbleDom, our method leverages human expert's input as a form of semi-supervision, thereby seamlessly combines the cognitive abilities of human experts with the computational power of machines. ScribbleDom incorporates a loss function that integrates two crucial components: similarity in gene expression profiles and adherence to the valuable input of a human annotator through scribbles on histology images, providing prior knowledge about spot labels. The spatial continuity of the tissue domains is taken into account by extracting information on the spot micro-environment through convolution filters of varying sizes, in the form of Inception blocks. By leveraging this semi-supervised approach, ScribbleDom significantly improves the quality of spatial domains, yielding superior results both quantitatively and qualitatively. Our experiments on several benchmark datasets demonstrate the clear edge of ScribbleDom over state-of-the-art methods-between 1.82% to 169.38% improvements in Adjusted Rand Index (ARI) for 9 of the 12 Human DLPFC samples, and 15.54% improvement in the Melanoma cancer dataset. Notably, when the expert input is absent, ScribbleDom can still operate, in a fully unsupervised manner like the state-of-the-art methods, and produces results that remain competitive. Source code is available at Github (https://github.com/1alnoman/ScribbleDom) and Zenodo (https://zenodo.org/badge/latestdoi/681572669). Supplementary data are available at Bioinformatics online.

Prenatal Sildenafil and Fetal-placental Programming in Human Pregnancies Complicated by Fetal Growth Restriction: A Retrospective Gene Expression Analysis

Objective: Fetal growth restricted (FGR) offspring are more susceptible to develop cardiovascular and renal disease. The potential therapeutic value of sildenafil to improve fetal growth has recently been evaluated in several randomized clinical trials. Here we investigate whether administration of sildenafil during pregnancies complicated by FGR influences fetal-placental programming profiles, especially related to cardiorenal development and disease. Methods: We collected human umbilical vein endothelial cells (HUVECs) and placental tissue within the Dutch STRIDER trial, in which sildenafil versus placebo treatment were randomly assigned to pregnancies complicated by severe early-onset FGR. Differential expression of genes of these samples were studied by whole genome RNA-sequencing. In addition, we performed gene set enrichment analysis focused on cardiovascular and renal gene sets to examine differentially expressed gene sets related to cardiorenal development and health. Results: Our study showed similar gene expression profiles between treatment groups in HUVECs (n=12 sildenafil; n=8 placebo) and placentas (n=13 per group). Prenatal sildenafil exposure did not change cardiovascular or renal programming in pregnancies complicated by FGR. In placental tissue, prenatal sildenafil altered a few gene sets involved with the nitric oxide pathway potentially reflecting the mechanism of action of sildenafil. Prenatal sildenafil also upregulated gene sets related to immune pathways in placental tissue. Conclusions: Overall, our study showed that sildenafil has the potential to alter placental (but not fetal) expression of gene sets related to immune pathways and did not support (in)direct reprogramming of cardiovascular or renal health in human pregnancies complicated by FGR.

Comprehensive single-cell analysis reveals novel anergic antigen-presenting cell subtypes in human sepsis.

Sepsis is a life-threatening condition with high mortality. A few studies have emerged utilizing single-cell RNA sequencing (scRNA-seq) to analyze gene expression at the single-cell resolution in sepsis, but a comprehensive high-resolution analysis of blood antigen-presenting cells has not been conducted. All published human scRNA-seq data were downloaded from the single cell portal database. After manually curating the dataset, we extracted all antigen-presenting cells, including dendritic cells (DCs) and monocytes, for identification of cell subpopulations and their gene profiling and intercellular interactions between septic patients and healthy controls. Finally, we further validated the findings by performing deconvolution analysis on bulk RNA sequencing (RNA-seq) data and flow cytometry. Within the traditional DC populations, we discovered novel anergic DC subtypes characterized by low major histocompatibility complex class II expression. Notably, these anergic DC subtypes showed a significant increase in septic patients. Additionally, we found that a previously reported immunosuppressive monocyte subtype, Mono1, exhibited a similar gene expression profile to these anergic DCs. The consistency of our findings was confirmed through validation using bulk RNA-seq and flow cytometry, ensuring accurate identification of cell subtypes and gene expression patterns. This study represents the first comprehensive single-cell analysis of antigen-presenting cells in human sepsis, revealing novel disease-associated anergic DC subtypes. These findings provide new insights into the cellular mechanisms of immune dysregulation in bacterial sepsis.

Ehrlichia Wnt SLiM ligand mimic deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

Ehrlichia chaffeensis TRP120 effector has evolved short linear motif (SLiM) ligand mimicry to repurpose multiple evolutionarily conserved cellular signaling pathways, including Wnt, Notch, and Hedgehog. In this investigation, we demonstrate that E. chaffeensis and recombinant TRP120 deactivate Hippo signaling, resulting in the activation of Hippo transcription coactivator Yes-associated protein (Yap). Moreover, a homologous 6 amino acid (QDVASH) SLiM shared by TRP120 and Wnt3a/5a ligands phenocopied Yap and β-catenin activation induced by E. chaffeensis, rTRP120, and Wnt5a. Similar Hippo gene expression profiles were also stimulated by E. chaffeensis, rTRP120, SLiM, and Wnt5a. Single siRNA knockdown of Hippo transcription co-activator/factors, Yap, and transcriptional enhanced associate domain (TEAD) significantly decreased E. chaffeensis infection. Yap activation was abolished in THP-1 Wnt Frizzled-5 (Fzd5) receptor knockout cells (KO), demonstrating Fzd5 receptor dependence. In addition, the TRP120-Wnt-SLiM antibody blocked Hippo deactivation (Yap activation). Expression of anti-apoptotic Hippo target gene SLC2A1 (encodes glucose transporter 1; GLUT1) was upregulated by E. chaffeensis and corresponded to increased levels of GLUT1. Conversely, siRNA knockdown of SLC2A1 significantly inhibited infection. Higher GLUT1 levels correlated with increased B cell lymphoma-extra large (BCL-xL) and decreased BCL2-associated X, apoptosis regulator (Bax) levels. Moreover, blocking Yap activation with the inhibitor Verteporfin induced apoptosis that corresponded to significant reductions in GLUT1 and BCL-xL levels and activation of Bax and Caspase-3 and -9. This study identifies a novel shared Wnt/Hippo SLiM ligand mimic and demonstrates that E. chaffeensis deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

Acetylation-Mimic Mutation of TRIM28-Lys304 to Gln Attenuates the Interaction with KRAB-Zinc-Finger Proteins and Affects Gene Expression in Leukemic K562 Cells.

TRIM28/KAP1/TIF1β is a crucial epigenetic modifier. Genetic ablation of trim28 is embryonic lethal, although RNAi-mediated knockdown in somatic cells yields viable cells. Reduction in TRIM28 abundance at the cellular or organismal level results in polyphenism. Posttranslational modifications such as phosphorylation and sumoylation have been shown to regulate TRIM28 activity. Moreover, several lysine residues of TRIM28 are subject to acetylation, but how acetylation of TRIM28 affects its functions remains poorly understood. Here, we report that, compared with wild-type TRIM28, the acetylation-mimic mutant TRIM28-K304Q has an altered interaction with Krüppel-associated box zinc-finger proteins (KRAB-ZNFs). The TRIM28-K304Q knock-in cells were created in K562 erythroleukemia cells by CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein nuclease 9) gene editing method. Transcriptome analysis revealed that TRIM28-K304Q and TRIM28 knockout K562 cells had similar global gene expression profiles, yet the profiles differed considerably from wild-type K562 cells. The expression levels of embryonic-related globin gene and a platelet cell marker integrin-beta 3 were increased in TRIM28-K304Q mutant cells, indicating the induction of differentiation. In addition to the differentiation-related genes, many zinc-finger-proteins genes and imprinting genes were activated in TRIM28-K304Q cells; they were inhibited by wild-type TRIM28 via binding with KRAB-ZNFs. These results suggest that acetylation/deacetylation of K304 in TRIM28 constitutes a switch for regulating its interaction with KRAB-ZNFs and alters the gene regulation as demonstrated by the acetylation mimic TRIM28-K304Q.