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Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1707-1707
Author(s):  
Wesley Wilson ◽  
Fei Miao ◽  
Alfred L Garfall ◽  
Dexiu Bu ◽  
Elena J Orlando ◽  
...  

Abstract Background: Autologous chimeric antigen receptor T (CAR-T) cell therapies show significant clinical activity against hematologic malignancies including multiple myeloma (MM). Numerous factors influence the effectiveness of this therapeutic approach. The fitness of a CAR-T cell product is an important factor affecting T cell engraftment and persistence, a pre-requisite for effective CAR-T cell therapy. In an effort to deconstruct the cellular heterogeneity of CAR-T cell products and explore relationships between T cell states within the cellular product, gene expression and CAR-T cell engraftment following adoptive transfer, we performed single cell RNA-sequencing (scRNA-seq) on CAR-T cell products from a phase I trial of BCMA-specific CAR-T cells in relapsed/refractory MM patients that included cohorts receiving a similar CAR-T cell dose with and without preconditioning with cyclophosphamide (NCT02546167, Cohen et al, J Clin Invest 2019). Method: scRNA-seq was performed on 25 unique products with an average of 8823 cells captured per product at an average depth of 40779 reads per cell. Batch effects were controlled by integration using established metrics for quality control. The linked inference of genomic experimental relationships (LIGER) method exhibited the most effective integration of 228,752 single cells across the 25 products. Annotation was completed by comparing transcriptomes of cells to the Blueprint/ENCODE references of pure immune cells. Differential gene expression (DGE) analysis was conducted by both Wilcoxon rank sum test testing and negative binomial distribution. Gene enrichment analysis was performed by comparing DGE with the Molecular Signatures Database v7.4. Cell to cell communication utilized the KEGG signaling pathway maps and curated lists of interactions from the literature. The intercellular communication probability was estimated on the DE genes before statistically significant intercellular communications were calculated by a permutation test, with dominant senders, receivers, mediators, and influencers identified using graph theory. Patients were grouped based upon "good" or "poor" engraftment using a peak blood vector copies per cell cutoff of 10,000. Association of transcriptional variation with neurotoxicity was explored for all patients. Results: The CD4:CD8 ratio of 2.51 single cells in the data was comparable to the observed average CD4:CD8 ratio of total T cells in the product based upon flow cytometric analysis of the products at harvest. Cell annotation showed significant heterogeneity of CD4+ and CD8+ T cells with cells exhibiting a CD4 Tcm transcriptional profile comprising the largest subset of T cells within the product. DGE analysis found 205 genes that were up or down regulated between the "good" vs "poor" engraftment phenotypes with 75 genes being detected using both mathematical approaches. CAR-BCMA expression was increased in "good" engraftment groups which has not previously been shown in CAR sorted cells. Within both CD4+ and CD8+ T cell subsets, transcriptional pathways associated with the RXF5/RFXAP/RFXANK transcriptional activator complex and the IL-2/STAT5 signaling were identified as upregulated in the "good" engrafted products. Cell to cell communication analysis for both secreted signaling and cell to cell contact revealed similar ligand-receptor interaction differences between the engraftment groups. Within the neurotoxicity groups CAR-BCMA expression was not associated with either neurotoxicity or with high and low neurotoxicity effects in the product. High vs low neurotoxicity showed a shift with interferon gamma and cytokine-cytokine interactions. We also detected IL-6/JAK/STAT3 signaling activation increases in this group. Conclusion: The RFX5/RFXAP/RFXANK pathway associated with MHC class II expression and IL-2/STAT5 signaling show a significant association with engraftment of BCMA-specific CAR-T cells. Although IL-2 signaling is well known to be critical to T cell survival and a potential key driver for long-term persistence, the role of the RFX5 transcriptional activator complex in T cells, outside of its important role in regulating MHC expression, is largely unknown. Both pathways deserve further investigation. Cell to cell communication between engraftment groups suggests CD4/CD8 interactions that might be beneficial to engraftment at the product manufacturing stage. Figure 1 Figure 1. Disclosures Garfall: Amgen: Honoraria; CRISPR Therapeutics: Research Funding; GlaxoSmithKline: Honoraria; Janssen: Honoraria, Research Funding; Novartis: Research Funding; Tmunity: Research Funding. Bu: Novartis: Current Employment, Patents & Royalties: Co-inventor on patent applications. Orlando: Novartis: Current Employment. Brogdon: Novartis Institutes for Biomedical Research: Current Employment. Pruteanu-Malinici: Novartis: Current Employment. Cohen: Janssen: Consultancy; Oncopeptides: Consultancy; Genentech/Roche: Consultancy; BMS/Celgene: Consultancy; AstraZeneca: Consultancy; Novartis: Research Funding; Takeda: Consultancy; GlaxoSmithKline: Consultancy, Research Funding.


Oncogene ◽  
2021 ◽  
Author(s):  
Daniel J. García-Domínguez ◽  
Nabil Hajji ◽  
Sara Sánchez-Molina ◽  
Elisabet Figuerola-Bou ◽  
Rocío M. de Pablos ◽  
...  

AbstractEwing sarcoma (EWS) is an aggressive bone and soft tissue tumor of children and young adults in which the principal driver is a fusion gene, EWSR1-FLI1. Although the essential role of EWSR1-FLI1 protein in the regulation of oncogenesis, survival, and tumor progression processes has been described in-depth, little is known about the regulation of chimeric fusion-gene expression. Here, we demonstrate that the active nuclear HDAC6 in EWS modulates the acetylation status of specificity protein 1 (SP1), consequently regulating the SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of the activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. High expression of HDAC6 in primary EWS tumor samples from patients correlates with a poor prognosis in two independent series accounting 279 patients. Notably, a combination treatment of a selective HDAC6 and doxorubicin (a DNA damage agent used as a standard therapy of EWS patients) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for patients with EWS.


2021 ◽  
Author(s):  
Dominik A. Herbst ◽  
Meagan N. Esbin ◽  
Robert K. Louder ◽  
Claire Dugast-Darzacq ◽  
Gina M. Dailey ◽  
...  

AbstractHuman SAGA is an essential co-activator complex that regulates gene expression by interacting with enhancer-bound activators, recruiting transcriptional machinery, and modifying chromatin near promoters. Subunit variations and the metazoan-specific requirement of SAGA in development hinted at unique structural features of the human complex. Our 2.9 Å structure of human SAGA reveals intertwined functional modules flexibly connected to a core that distinctively integrates mammalian paralogs, incorporates U2 splicing subunits, and features a unique interface between the core and the activator-binding TRRAP. Our structure sheds light on unique roles and regulation of human coactivators with implications for transcription and splicing that have relevance in genetic diseases and cancer.


2021 ◽  
Author(s):  
Jun Miao ◽  
Chengqi Wang ◽  
Amuza Lucky ◽  
Xiaoying Liang ◽  
Hui Min ◽  
...  

AbstractThe histone acetyltransferase GCN5-associated SAGA complex is evolutionarily conserved from yeast to human and functions as a general transcription co-activator in global gene regulation. In this study, we identified a divergent GCN5 complex in Plasmodium falciparum, which contains two plant homeodomain (PHD) proteins (PfPHD1 and PfPHD2) and a plant apetela2 (AP2)-domain transcription factor (PfAP2-LT). To dissect the functions of the PfGCN5 complex, we generated parasites with the bromodomain deletion in PfGCN5 and the PHD domain deletion in PfPHD1. The two deletion mutants closely phenocopied each other, exhibiting significantly reduced merozoite invasion of erythrocytes and elevated sexual conversion. These domain deletions caused dramatic decreases not only in histone H3K9 acetylation but also in H3K4 trimethylation, indicating synergistic crosstalk between the two euchromatin marks. Domain deletion in either PfGCN5 or PfPHD1 profoundly disturbed the global transcription pattern, causing altered expression of more than 60% of the genes. At the schizont stage, these domain deletions were linked to specific downregulation of merozoite genes involved in erythrocyte invasion, many of which harbor the DNA-binding motifs for AP2-LT and/or AP2-I, suggesting targeted recruitment of the PfGCN5 complex to the invasion genes by these specific transcription factors. Conversely, at the ring stage, PfGCN5 or PfPHD1 domain deletions disrupted the mutually exclusive expression pattern of the entire var gene family, which encodes the virulent factor PfEMP1. Correlation analysis between the chromatin state and alteration of gene expression demonstrated that up- and down-regulated genes in these mutants are highly correlated with the silenct and active chromatin states in the wild-type parasite, respectively. Collectively, the PfGCN5 complex represents a novel HAT complex with a unique subunit composition including the AP2 transcription factor, which signifies a new paradigm for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.Author SummaryEpigenetic regulation of gene expression plays essential roles in orchestrating the general and parasite-specific cellular pathways in the malaria parasite Plasmodium falciparum. Using tandem affinity purification and proteomic characterization, we identified a divergent transcription co-activator – the histone acetyltransferase GCN5-associated complex in P. falciparum, which contains nine core components, including two PHD domain proteins (PfPHD1 and PfPHD2) and a plant apetela2-domain transcription factor. To understand the functions of the PfGCN5 complex, we performed gene disruption in two subunits of this complex, PfGCN5 and PfPHD1. We found that the two deletion mutants displayed very similar growth phenotypes, including significantly reduced merozoite invasion rates and elevated sexual conversion. These two mutants were associated with dramatic decreases in histone H3K9 acetylation and H3K4 trimethylation, which led to global changes in chromatin states and gene expression. Genes significantly affected by the PfGCN5 and PfPHD1 gene disruption include those participating in parasite-specific pathways such as invasion, virulence, and sexual development. In conclusion, this study presents a new model of the PfGCN5 complex for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.


2021 ◽  
Author(s):  
Daniel J. García-Domínguez ◽  
Nabil Hajji ◽  
Sara Sánchez-Molina ◽  
Elisabet Figuerola-Bou ◽  
Rocío M. de Pablos ◽  
...  

ABSTRACTEwing sarcoma (EWS) is an aggressive developmental sarcoma driven by a fusion gene, EWSR1-FLI1. However, little is known about the regulation of EWSR1-FLI1 chimeric fusion gene expression. Here, we demonstrate that active nuclear HDAC6 in EWS modulates acetylation status of specificity protein 1 (SP1), consequently regulating SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. Overexpression of HDAC6 in primary EWS tumor clinical samples correlates with a poor prognosis. Notably, a combination treatment of a selective HDAC6 inhibitor and doxorubicin (standard of care in EWS) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for EWS patients.


2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Marianna Holczer ◽  
Bence Hajdú ◽  
Tamás Lőrincz ◽  
András Szarka ◽  
Gábor Bánhegyi ◽  
...  

Abstract Autophagy is an intracellular digestive process, which has a crucial role in maintaining cellular homeostasis by self-eating the unnecessary and/or damaged components of the cell at various stress events. ULK1, one of the key elements of autophagy activator complex, together with the two sensors of nutrient and energy conditions, called mTORC1 and AMPK kinases, guarantee the precise function of cell response mechanism. We claim that the feedback loops of AMPK–mTORC1–ULK1 regulatory triangle determine an accurate dynamical characteristic of autophagic process upon cellular stress. By using both molecular and theoretical biological techniques, here we reveal that a delayed negative feedback loop between active AMPK and ULK1 is essential to manage a proper cellular answer after prolonged starvation or rapamycin addition. AMPK kinase quickly gets induced followed by AMPK-P-dependent ULK1 activation, whereas active ULK1 has a rapid negative effect on AMPK-P resulting in a delayed inhibition of ULK1. The AMPK-P → ULK1 ˧ AMPK-P negative feedback loop results in a periodic repeat of their activation and inactivation and an oscillatory activation of autophagy, as well. We demonstrate that the periodic induction of self-cannibalism is necessary for the proper dynamical behaviour of the control network when mTORC1 is inhibited with respect to various stress events. By computational simulations we also suggest various scenario to introduce “delay” on AMPK-P-dependent ULK1 activation (i.e. extra regulatory element in the wiring diagram or multi-phosphorylation of ULK1).


2020 ◽  
Vol 133 (18) ◽  
pp. jcs241174
Author(s):  
Priya Crosby ◽  
Carrie L. Partch

ABSTRACTMammalian circadian rhythms drive ∼24 h periodicity in a wide range of cellular processes, temporally coordinating physiology and behaviour within an organism, and synchronising this with the external day–night cycle. The canonical model for this timekeeping consists of a delayed negative-feedback loop, containing transcriptional activator complex CLOCK–BMAL1 (BMAL1 is also known as ARNTL) and repressors period 1, 2 and 3 (PER1, PER2 and PER3) and cryptochrome 1 and 2 (CRY1 and CRY2), along with a number of accessory factors. Although the broad strokes of this system are defined, the exact molecular mechanisms by which these proteins generate a self-sustained rhythm with such periodicity and fidelity remains a topic of much research. Recent studies have identified prominent roles for a number of crucial post-transcriptional, translational and, particularly, post-translational events within the mammalian circadian oscillator, providing an increasingly complex understanding of the activities and interactions of the core clock proteins. In this Review, we highlight such contemporary work on non-transcriptional events and set it within our current understanding of cellular circadian timekeeping.


2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Yueqing Li ◽  
Xiaotong Shan ◽  
Ruifang Gao ◽  
Taotao Han ◽  
Jia Zhang ◽  
...  

AbstractFloral anthocyanin has multiple ecological and economic values, its biosynthesis largely depends on the conserved MYB-bHLH-WD40 (MBW) activation complex and MYB repressors hierarchically with the MBW complex. In contrast to eudicots, the MBW regulatory network model has not been addressed in monocots because of the lack of a suitable system, as grass plants exhibit monotonous floral pigmentation patterns. Presently, the MBW regulatory network was investigated in a non-grass monocot plant, Freesia hybrida. FhMYB27 and FhMYBx with different functional manners were confirmed to be anthocyanin related R2R3 and R3 MYB repressors, respectively. Particularly, FhMYBx could obstruct the formation of positive MBW complex by titrating bHLH proteins, whereas FhMYB27 mainly defected the activator complex into suppressor via its repression domains in C-terminus. Furthermore, the hierarchical and feedback regulatory loop was verified, indicating the synergistic and sophisticated regulatory network underlying Freesia anthocyanin biosynthesis was quite similar to that reported in eudicot plants.


Author(s):  
Wen Luo ◽  
Irene Garcia-Gonzalez ◽  
Macarena Fernandez-Chacon ◽  
Veronica Casquero-Garcia ◽  
Rui Benedito

AbstractArteries are thought to be formed by the induction of a highly conserved arterial genetic program in a subset of vessels experiencing an increase in pulsatile and oxygenated blood flow. Both VEGF and Notch signalling have been shown to be essential for the initial steps of arterial specification. Here, we combined inducible genetic mosaics and transcriptomics to modulate and understand the function of these signalling pathways on cell proliferation, arterial-venous differentiation and mobilization. We observed that endothelial cells with high VEGF or Notch signalling are not genetically pre-determined and can form both arteries and veins. Importantly, cells completely lacking the Notch-Rbpj transcriptional activator complex can form arteries when the Myc-dependent metabolic and cell-cycle activity is suppressed. Thus, arterial development does not require the induction of a Notch-dependent arterial differentiation program, but rather the timely suppression of the endothelial metabolism and cell-cycle, a process preceding arterial mobilization and complete differentiation.


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