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1.
Front Oncol ; 10: 448, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32309216

RESUMO

The interaction of lymphoma cells with their microenvironment has an important role in disease pathogenesis and is being actively pursued therapeutically using immunomodulatory drugs, including immune checkpoint inhibitors. Diffuse large B-cell lymphoma (DLBCL) is an aggressive high-grade disease that remains incurable in ~40% of patients treated with R-CHOP immunochemotherapy. The FOXP1 transcription factor is abundantly expressed in such high-risk DLBCL and we recently identified its regulation of immune response signatures, in particular, its suppression of the cell surface expression of major histocompatibility class II (MHC-II), which has a critical role in antigen presentation to T cells. Using CRISPR/Cas9 genome editing we have depleted Foxp1 expression in the aggressive murine A20 lymphoma cell line. When grown in BALB/c mice, this cell line provides a high-fidelity immunocompetent disseminated lymphoma model that displays many characteristics of human DLBCL. Transient Foxp1-depletion using siRNA, and stable depletion using CRISPR (generated by independently targeting Foxp1 exon six or seven) upregulated cell surface I-Ab (MHC-II) expression without impairing cell viability in vitro. RNA sequencing of Foxp1-depleted A20 clones identified commonly deregulated genes, such as the B-cell marker Cd19, and hallmark DLBCL signatures such as MYC-targets and oxidative phosphorylation. Immunocompetent animals bearing Foxp1-depleted A20 lymphomas showed significantly-improved survival, and 20% did not develop tumors; consistent with modulating immune surveillance, this was not observed in immunodeficient NOD SCIDγ mice. The A20 Foxp1 CRISPR model will help to further characterize the contribution of Foxp1 to lymphoma immune evasion and the potential for Foxp1 targeting to synergize with other immunotherapies.

2.
Wellcome Open Res ; 5: 42, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-36865034

RESUMO

Background: The leukaemia-derived Jurkat E6.1 cell line has been used as a model T cell in the study of many aspects of T cell biology, most notably activation in response to T cell receptor (TCR) engagement. Methods: We present whole-transcriptome RNA-Sequencing data for Jurkat E6.1 cells in the resting state and two hours post-activation via TCR and CD28. We compare early transcriptional responses in the presence and absence of the chemokines CXCL12 and CCL19, and perform a basic comparison between observed transcriptional responses in Jurkat E6.1 cells and those in primary human T cells using publicly deposited data. Results: Jurkat E6.1 cells have many of the hallmarks of standard T cell transcriptional responses to activation, but lack most of the depth of responses in primary cells. Conclusions: These data indicate that Jurkat E6.1 cells hence represent only a highly simplified model of early T cell transcriptional responses.

3.
SLAS Discov ; 24(2): 133-141, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30359161

RESUMO

Eleven-nineteen leukemia (ENL) contains an epigenetic reader domain (YEATS domain) that recognizes lysine acylation on histone 3 and facilitates transcription initiation and elongation through its interactions with the super elongation complex (SEC) and the histone methyl transferase DOT1L. Although it has been known for its role as a fusion protein in mixed lineage leukemia (MLL), overexpression of native ENL, and thus dysregulation of downstream genes in acute myeloid leukemia (AML), has recently been implicated as a driver of disease that is reliant on the epigenetic reader activity of the YEATS domain. We developed a peptide displacement assay (histone 3 tail with acylated lysine) and screened a small-molecule library totaling more than 24,000 compounds for their propensity to disrupt the YEATS domain-histone peptide binding. Among these, we identified a first-in-class dual inhibitor of ENL ( Kd = 745 ± 45 nM) and its paralog AF9 ( Kd = 523 ± 53 nM) and performed "SAR by catalog" with the aim of starting the development of a chemical probe for ENL.


Assuntos
Descoberta de Drogas , Fatores de Elongação da Transcrição/antagonistas & inibidores , Fatores de Elongação da Transcrição/química , Fenômenos Biofísicos , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Histonas/metabolismo , Humanos , Concentração Inibidora 50 , Peptídeos/metabolismo , Domínios Proteicos , Relação Estrutura-Atividade
4.
Angew Chem Int Ed Engl ; 57(50): 16302-16307, 2018 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-30288907

RESUMO

YEATS domain (YD) containing proteins are an emerging class of epigenetic targets in drug discovery. Dysregulation of these modified lysine-binding proteins has been linked to the onset and progression of cancers. We herein report the discovery and characterisation of the first small-molecule chemical probe, SGC-iMLLT, for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). SGC-iMLLT is a potent and selective inhibitor of MLLT1/3-histone interactions. Excellent selectivity over other human YD proteins (YEATS2/4) and bromodomains was observed. Furthermore, our probe displays cellular target engagement of MLLT1 and MLLT3. The first small-molecule X-ray co-crystal structures with the MLLT1 YD are also reported. This first-in-class probe molecule can be used to understand MLLT1/3-associated biology and the therapeutic potential of small-molecule YD inhibitors.


Assuntos
Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Bibliotecas de Moléculas Pequenas/química , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Cristalografia por Raios X , Histonas/metabolismo , Humanos , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Domínios Proteicos , Mapas de Interação de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/metabolismo
5.
Haematologica ; 101(7): 861-71, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27056922

RESUMO

Strong FOXP1 protein expression is a poor risk factor in diffuse large B-cell lymphoma and has been linked to an activated B-cell-like subtype, which preferentially expresses short FOXP1 (FOXP1S) proteins. However, both short isoform generation and function are incompletely understood. Here we prove by mass spectrometry and N-terminal antibody staining that FOXP1S proteins in activated B-cell-like diffuse large B-cell lymphoma are N-terminally truncated. Furthermore, a rare strongly FOXP1-expressing population of normal germinal center B cells lacking the N-terminus of the regular long protein (FOXP1L) was identified. Exon-targeted silencing and transcript analyses identified three alternate 5' non-coding exons [FOXP1-Ex6b(s), FOXP1-Ex7b and FOXP1-Ex7c], downstream of at least two predicted promoters, giving rise to FOXP1S proteins. These were differentially controlled by B-cell activation and methylation, conserved in murine lymphoma cells, and significantly correlated with FOXP1S protein expression in primary diffuse large B-cell lymphoma samples. Alternatively spliced isoforms lacking exon 9 (e.g. isoform 3) did not encode FOXP1S, and an alternate long human FOXP1 protein (FOXP1AL) likely generated from a FOXP1-Ex6b(L) transcript was detected. The ratio of FOXP1L:FOXP1S isoforms correlated with differential expression of plasmacytic differentiation markers in U-2932 subpopulations, and altering this ratio was sufficient to modulate CD19 expression in diffuse large B-cell lymphoma cell lines. Thus, the activity of multiple alternate FOXP1 promoters to produce multiple protein isoforms is likely to regulate B-cell maturation.


Assuntos
Linfócitos B/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Processamento Alternativo , Animais , Antígenos CD19/genética , Antígenos CD19/metabolismo , Linhagem Celular Tumoral , Éxons , Fatores de Transcrição Forkhead/química , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/química
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