quantitative-mass-spectrometric-profiling-cancer-cell-proteomes

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Max Planck Institute for Biophysical Chemistry

In this study, multilocus sequence typing (MLST) was combined with the genetic detection of six genetic markers, ansB, dmsA, ggt, cj1585c, cjj81176-1367/71 (cj1365c), and the two-gene marker tlp7 (cj0951c plus cj0952c), to assess if their presence correlated with different C. jejuni clonal groups. Using a collection of 266 C. jejuni isolates from (in decreasing order of sample size) humans, chickens, cattle, and turkeys, it was further investigated whether the resulting genotypes correlated with the isolation source. We found combinations of the six marker genes to be mutually exclusive, and their patterns of presence or absence correlated to some degree with animal source. Together with MLST results, the obtained genotypes could be segregated into six groups. An association was identified for ansB, dmsA, and ggt with the MLST-clonal complexes (MLST-CC) 22, 42, 45, and 283, which formed the most prominent group, in which chickens were the most prevalent animal source. Two other groups, characterized by the presence of cj1585c, cjj81176-1367/71, and the two-gene marker tlp7, associated with either MLST-CC 21 or 61, were overrepresented in isolates of bovine origin. Mutually exclusive marker gene combinations were observed for ansB, dmsA, and ggt, typically found in CC 45 and the related CC 22, 42, and 283, whereas the other three marker genes were found mostly in CC 21, 48, and 206. The presence of the two-gene marker tlp7, which is typical for MLST 21 and 53 as well as for MLST-CC 61, strongly correlates with a bovine host; this is interpreted as an example of host adaptation. In cases of C. jejuni outbreaks, these genetic markers could be helpful for more effective source tracking.

Epidemiological association of different Campylobacter jejuni groups with metabolism-associated genetic markers.

Spleen tyrosine kinase (Syk) induces cell survival and proliferation in a high proportion of acute myeloid leukemia (AML) blasts, but the underlying molecular events of Syk signaling have not been investigated. Proteomic techniques have allowed us to identify the multiprotein complex that is nucleated by constitutively active Syk in AML cells. This complex differs from the B-lymphoid Syk interactome with respect to several proteins, especially the integrin receptor Mac-1, the Fc-γ receptor I (FcγRI), and the transcription factors STAT3 and STAT5. We show in several AML cell line models that tonic signals derived from the Fc-γ chain lead to Syk-dependent activation of STAT3 and STAT5, which in turn induces cell survival and proliferation. Moreover, stimulation of Mac-1 or FcγRI intensifies the constitutive Syk-mediated STAT3/5 activation in AML cells, a scenario likely to take place in the bone marrow niche. In accordance with these findings, we observed that β2 integrins, including Mac-1, trigger proliferation of AML cells in an AML cell/stroma coculture model. Taken together, we identified an oncogenic integrin/Syk/STAT3/5 signaling axis that might serve as a therapeutic target of AML in the future.

β2 integrin-derived signals induce cell survival and proliferation of AML blasts by activating a Syk/STAT signaling axis.

T cell antigen receptor (TCR) signaling depends on three interacting adaptor proteins: SLP-76, Gads, and LAT. Their mechanisms of signaling have been extensively explored, with the aid of fortuitously isolated LAT- and SLP-76-deficient T cell lines, but no such tools were available for Gads, a Grb2-family adaptor that bridges the TCR-inducible interaction between SLP-76 and LAT. TALEN-directed genome editing was applied to disrupt the first coding exon of human Gads in the Jurkat T cell line. Gads was dispensable for TCR-induced phosphorylation of SLP-76, but was a dose-dependent amplifier of TCR-induced CD69 expression. Gads conferred responsiveness to weak TCR stimuli, leading to PLC-γ1 phosphorylation and calcium flux. TALEN-derived, Gads-deficient T cell lines provide a uniquely tractable genetic platform for exploring its regulatory features, such as Gads phosphorylation at T262, which we observed by mass spectrometry. Upon mutation of this site, TCR responsiveness and sensitivity to weak TCR stimuli were increased. This study demonstrates the feasibility of TALEN-based reverse genetics in Jurkat T cells, while enriching our understanding of Gads as a regulated modulator of TCR sensitivity.

Modulation of TCR responsiveness by the Grb2-family adaptor, Gads.

Acute myeloid leukemia (AML) is driven by niche-derived and cell-autonomous stimuli. While many cell-autonomous disease drivers are known, niche-dependent signaling in the context of the genetic disease heterogeneity has been difficult to investigate. Here, we analyzed the role of Bruton's tyrosine kinase (BTK) in AML. BTK was frequently expressed and its inhibition strongly impaired the proliferation and survival of AML cells - also in the presence of bone marrow stroma. By interactome analysis, (phospho)proteomics and transcriptome sequencing, we characterized BTK signaling networks. We show that BTK-dependent signaling is highly context-dependent. In FLT3-ITD-positive AML, BTK mediates FLT3-ITD-dependent Myc- and STAT5 activation and combined targeting of FLT3-ITD and BTK showed additive effects. In FLT3-ITD-negative AML, BTK couples Toll-like receptor 9 (TLR9) activation to NFκB and STAT5. Both BTK-dependent transcriptional programs were relevant for cell cycle progression and apoptosis regulation. Thus, we identify context-dependent oncogenic driver events that may guide subtype-specific treatment strategies and point for the first time to a role of TLR9 in AML. Clinical evaluation of BTK inhibitors in AML seems warranted.

Article	Year
Epidemiological association of different Campylobacter jejuni groups with metabolism-associated genetic markers. Applied and environmental microbiology\| PubMed ID: 21278270	2011
β2 integrin-derived signals induce cell survival and proliferation of AML blasts by activating a Syk/STAT signaling axis. Blood\| PubMed ID: 23509157	2013
Modulation of TCR responsiveness by the Grb2-family adaptor, Gads. Cellular signalling\| PubMed ID: 25452106	2015
FLT3-ITD and TLR9 employ Bruton's tyrosine kinase to activate distinct transcriptional programs mediating AML cell survival and proliferation. Blood\| PubMed ID: 25605370	2015

Jasmin Corso

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