Expression data from thymic lymphomas of Lck-MyrAkt positive mice with WT or KD mTOR expression
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ABSTRACT: Lck-MyrAkt2 mice develop spontaneous thymic lymphomas at approximately 100-200 days of age, driven in part by a consitutatively-active AKT (due to myristoylation). mTOR Knock Down mice were crossed with Lck-MyrAkt postive mice to model the affects of decreasing mTOR activity on tumors with an activated PI3K/AKT/MTOR pathway. Lck-Akt/mTOR KD mice had prolonged survival compared to the Lck-Akt/mTOR WT mice. We used microarrays to compare the transcriptome in thymic lymphomas between Lck-Akt positive, mTOR WT and Lck-Akt positive, mTOR KD mice. Four thymic lymphomas from Lck-Akt/mTOR WT mice were compared to three thymic lymphomas from Lck-Akt/mTOR KD mice.
Project description:Purpose: The goals of this study is to uncover the cellular pathways that are essential for T-cell malignant transformation driven by ORP4L and HTLV-1 oncogene Tax. Methods: T-cell mRNA profiles of WT, ORP4L KI, Wild-type, LCK/R26Tax, ORP4Lcko;LCK/R26Tax mice were generated by deep sequencing, using Illumina NovaSeq 6000 sequencer for 318 cycles.Reads that passed the Illumina quality filters were kept for the subsequent analyses. Adapters were trimmed from the reads, and reads shorter than 17 nt were discarded. The reads were mapped to the Mouse mRNA reference database using FANSe3 algorithm on Chi-Cloud NGS Analysis Platform (Chi-Biotech Co. Ltd., Shenzhen, China). Results: We use edgeR to analysis ORP4kI-vs-WT, LCK/R26Tax-vs-Wild-type,ORP4Lcko;LCK/R26Tax-vs-Wild-type,with a |log2 (FoldChange)| > 1 and p value <0.01. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to promote T-cell malignant. Conclusions: Our study represents the first detailed analysis of transcriptomes that promote T-cell malignant transformation driven by HTVL-1 oncogene Tax.
Project description:Lck-Dlx5 induces T-cell lymphoma but the mechanism is unknown. We used microarrays to detail the global programme of gene expression underlying Dlx5-induced T-cell lymphoma T-cells were isolated from Lck-Dlx5 mice, Lck-MyrAkt2 mice and Wild type littermates for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Transcription profiling by array of spontaneous thymic lymphomas of Lck-MyrAkt positive mice with wild type or knocked-down mTOR expression
Project description:Lymphocyte-specific protein tyrosine kinase (Lck) is crucial for signaling from the T cell receptor (TCR) and is controlled through tyrosine phosphorylations. Phosphorylated Tyr505 (pTyr505) promotes a closed, inactive conformation of Lck, while pTyr394 is critical for kinase activity. Additionally, pTyr192 has been suggested to regulate Lck activity by changing the specificity of the Lck Src-homology 2 (SH2) domain and/or by affecting Lck association with CD45 thus drastically increasing pTyr505. However, little is known about how pTyr192 affects endogenously expressed Lck. Here we used CRISPR/Cas9 genome editing to generate Jurkat cell lines expressing Lck Glu192 mimicking Lck pTyr192, or Lck Phe192 mimicking unphosphorylated Lck Tyr192. We confirmed that Lck Glu192 is hyperphosphorylated on Tyr505, possibly explaining reduced association of Lck Glu192 with prototypic Lck-SH2 ligands. To isolate the effect of Lck Tyr192 mutations from the effect on Lck pTyr505, we subsequently generated Jurkat cells doubly mutated on Lck Tyr192 and Lck Tyr505. Both Lck Phe192/Phe505 and Lck Glu192/Phe505 mutants co-precipitated similar amounts of binding partners. Moreover, both mutants displayed hyperphosphorylation of Tyr394. Our results indicate that CD45 is the main phosphatase controlling Lck pTyr394 in steady-state T cells. Additionally, our data demonstrate that the prototypic specificity of the Lck SH2 domain (owned by the Lck Phe192 mutants) promotes transphosphorylation of Tyr394. These observations pinpoint the fundamental role of Tyr192 in regulation of Lck activity and simultaneously reveal the most potent Lck mutants so far described
Project description:Receptor-interacting protein kinase 3 (RIPK3) is the primary regulator of necroptotic cell death. RIPK3 expression is often silenced in various cancer cells, which suggests that it may have tumor suppressor properties. However, the exact mechanism by which RIPK3 negatively regulates cancer development and progression remains unclear. This report indicates that RIPK3 acts as a potent regulator of homeostatic proliferation of CD4+CD8+ double-positive (DP) thymocytes. Abnormal proliferation of RIPK3-deficient DP thymocytes occurs independently of the well-known role for RIPK3 in necroptosis (upstream of MLKL activation), and is associated with an incidental thymic mass, likely thymic hyperplasia. In addition, Ripk3-null mice developed increased thymic tumor formation accompanied by reduced host survival in the context of a N-ethyl-N-nitrosourea (ENU)-induced tumor model. Moreover, RIPK3 deficiency in p53-null mice promoted thymic lymphoma development via upregulated ERK signaling, which correlated with markedly reduced survival rates. Mechanistically, lymphocyte-specific protein tyrosine kinase (LCK) activates RIPK3, which in turn leads to increases in the phosphatase activity of protein phosphatase 2 (PP2A), thereby suppressing hyper-activation of ERK in DP thymocytes. Overall, these findings suggest that a RIPK3-PP2A-ERK signaling axis regulates DP thymocyte homeostasis and may provide a potential therapeutic target to improve thymic lymphoma therapies.
Project description:Circulating tumor cells (CTCs) play an important role in metastasis formation. Aberrant signaling of oncogenic pathways (e.g. PI3K/AKT/mTOR pathway) drives tumor progression. In this work, the susceptibility of the colon cancer CTC derived cell line, CTC-MCC-41, for AKT and mTOR inhibitors was evaluated. Additionally, the functional role of the expressed AKT isoforms was characterized in this cell line. The efficacy of AKT inhibitor MK2206, the mTOR inhibitor RAD001 and the combination was examined in CTC-MCC-41 cells in a murine intracardiac xenotransplantation model. Furthermore, stable isoform specific AKT1 or AKT2 knockdowns (KD) as well as AKT1/AKT2 double KD cells were generated. Differentially regulated proteins and phospho-peptides were identified using LC-MS. CTC-MCC-41 cells show a high susceptibility for dual targeting of AKT and mTOR in vivo indicating that selective eradication of CTCs may be considered as a new treatment option in cancer. KD of AKT1 or AKT2 significantly reduced the proliferation of CTC-MCC-41 cells. AKT KDs share commonly regulated proteins and phospho-proteins, but also regulate a large number uniquely. AKT1/AKT2 double KD cells show a strongly dysregulated replication machinery, as well as a decrease in cell cycle activity and stem cell associated processes underlining the non-redundant role of AKT isoforms.
Project description:MED23, a subunit of the Mediator coactivator complex, is important for the expression of a subset of MAPK/ERK pathway-dependent target genes; however, the genes in this subset varies between cell types. MAPK/ERK pathway-dependent processes are essential for T-cell development and function, but whether MED23 has a role in this context is unknown. We generated Med23 conditional knockout mice and induced Med23 deletion in early T cell development using the lineage specific Lck-Cre transgene. While the total cell number and distribution of cell populations in the thymuses of Med23flox/flox;Lck-Cre mice were essentially normal, MED23 null T-cells failed to efficiently populate the peripheral lymphoid organs. MED23 null thymocytes displayed decreased expression of the MAPK/ERK-responsive genes Egr1, Egr2, as well as of the membrane glycoprotein Cd52 (CAMPATH-1). MED23 null CD4 single-positive thymocytes also showed decreased expression of KLF2 (LKLF), a T cell master regulatory transcription factor. Indeed, similarities between the phenotypes of mice lacking MED23 or KLF2 in T-cells suggest that KLF2 deficiency in MED23 null T-cells is one of their key defects. Mechanistic experiments using MED23 null MEFs further suggest that MED23 is required for full activity of the MAPK-responsive transcription factor MEF2, which has previously been shown to mediate Klf2 expression. In summary, our data indicate that MED23 has critical roles in enabling T-cells to populate the peripheral lymphoid organs, possibly by potentiating MEF2-dependent expression of the T-cell transcription factor KLF2. 12 samples, 2 of each genotype (Lck-Cre, Med23flox/flox and Med23flox/flox;Lck-Cre) both with mock and anti-CD3 treatment
Project description:Inactivation of Pten occurs by multiple mechanisms including epigenetic silencing, point mutations, insertion, and deletion, which are tissue dependent. Although frequent loss of heterozygosity around Pten locus and plausible involvement of epigenetic silencing have been reported in radiation-induced thymic lymphomas, the frequency of Pten inactivation and the spectrum of causal aberrations have not yet been extensively characterized. Here, we assessed the principal mode of inactivation by comprehensively analyzing expression and alterations of Pten gene in 23 radiation-induced thymic lymphomas developed in B6C3F1 mice. We found no evidence for methylation-associated silencing of Pten gene. Instead, we found complex structural abnormalities in 8 lymphomas (35%) that included missense and nonsense mutations, 1- and 3-bp insertions, and focal deletions. Sequencing of deletion breakpoints suggested that illegitimate V(D)J recombination and microhomology-mediated rearrangement were responsible for the focal deletions. Seven out of these 8 lymphomas had biallelic alterations, and 4 of them did not express any Pten protein. These aberrations of Pten were well coincided with downstream Akt phosphorylation on Ser473. In conclusion, Pten inactivation, which is frequently biallelic and is caused by a variety of structural abnormalities but not by epigenetic silencing, is involved in radiation-induced lymphomagenesis. Three thymic lymphomas were analyzed by array-CGH method.
Project description:Lck-Dlx5 induces T-cell lymphoma but the mechanism is unknown. We used microarrays to detail the global programme of gene expression underlying Dlx5-induced T-cell lymphoma