Dataset Information

Phosphoproteomic Profiling of T-Cell Acute Lymphoblastic Leukemia Identifies Targetable Kinases and Combination Treatments

ABSTRACT: Protein kinase inhibitors are amongst the most successful cancer treatments, but targetable kinases activated by gene fusions are rare in T-ALL (e.g., NUP214-ABL1). Nevertheless, leukemic blasts rely on enhanced kinase signaling to sustain dysregulated proliferation. Protein kinases can be hyper-activated in the absence of defects in their genes. Thus, phospho-proteomics can provide information on pathway activation, signaling networks and aberrant kinases activities that offer important opportunities for targeted therapies. Here, we performed mass spectrometry-based global profiling of tyrosine, serine, and threonine phosphorylation in a panel of 11 T-ALL cell lines to identify potential targetable kinases. We report a comprehensive dataset consisting of 21,000 phosphosites on 4896 phosphoproteins, including 217 kinases. We identify several Src-family members (LCK, SRC, FYN, YES1) as well as the cyclin-dependent kinases CDK1 and CDK2 as broadly activated in our panel. We validate highly active kinases as putative target for therapy in vitro and propose potential novel combination treatment strategies, such as the inhibition of the insulin-like growth factor receptor (IGF-1R) signaling pathway to increase the sensitivity to dasatinib treatment in T-ALL.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Cell Culture

SUBMITTER: Sander Piersma

LAB HEAD: Connie Jimenez

PROVIDER: PXD024807 | Pride | 2022-02-21

REPOSITORIES: Pride

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Project description:Spermatogenesis is a complex cell differentiation process that includes marked genetic, cellular functional and structural changes. It requires tight regulation, since disturbances in any of the spermatogenic stages would lead to fertility deficiencies. In order to increase our knowledge of signal transduction during sperm development, we carried out a large-scale identification of the phosphorylation events that occur in the human gonad. Metal oxide affinity chromatography using TiOx combined with LC-MS/MS was conducted to profile the phosphoproteome of human testes with full spermatogenesis. A total of 8187 phosphopeptides derived from 2661 proteins were identified, resulting in the most complete report of human testicular phosphoproteins to date. Phosphorylation events were enriched in proteins functionally related to spermatogenesis, as well as to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle and apoptosis. Moreover, 174 phosphorylated kinases were identified. The most active and abundant human protein kinases in the testis were predicted both by the phosphorylation status of the kinase activation loop and the number of phosphopeptide spectra identified. The potential function of two of those kinases, cyclin-dependent kinase 12 (CDK12) and p21-activated kinase 4(PAK4), has been explored by protein-protein interaction analysis, immunodetection in human and mouse testicular tissue, and functional assay in a human embryonal carcinoma cell line. The co-localization of CDK12 with Golgi markers and probably pro-acrosomal vesicles suggests a potential crucial role of this protein kinase in sperm formation. PAK4 expression has been found limited to human spermatogonia, and a role in embryonal carcinoma cell response to apoptosis has been observed. Together, our data confirm that phosphoregulation by protein kinases is highly active in sperm differentiation, and open a window to detailed characterization and validation of potential targets for the development of drugs modulating male fertility, and tumor behavior.

Project description:Dendritic cells (DCs) are key initiators of the adaptive immunity, and upon recognition of pathogens are able to skew T cell differentiation to elicit appropriate responses. DCs possess this extraordinary capacity to discern external signals using receptors that recognize pathogen-associated molecular patterns. These can be glycan-binding receptors that recognize carbohydrate structures on pathogens or pathogen-associated patterns that additionally bind receptors, such as Toll-like receptors (TLRs). This study explores the early signaling events in DCs upon binding of α2-3 sialic acid (α2-3sia) that are recognized by Immune inhibitory Sialic acid binding immunoglobulin type lectins. α2-3sias are commonly found on bacteria, e.g. Group B Streptococcus, but can also be expressed by tumor cells. We investigated whether α2-3sia conjugated to a dendrimeric core alters DC signaling properties. Through phosphoproteomic analysis, we found differential signaling profiles in DCs after α2-3sia binding alone or in combination with LPS/TLR4 co-stimulation. α2-3sia was able to modulate the TLR4 signaling cascade, resulting in 109 altered phosphoproteins. These phosphoproteins were annotated to seven biological processes, including the regulation of the IL-12 cytokine pathway. Secretion of IL-10, the inhibitory regulator of IL-12 production, by DCs was found upregulated after overnight stimulation with the α2-3sia dendrimer. Analysis of kinase activity revealed altered signatures in the JAK-STAT signaling pathway. PhosphoSTAT3 (Ser727) and phosphoSTAT5A (Ser780), involved in the regulation of the IL-12 pathway, were both downregulated. Flow cytometric quantification indeed revealed de- phosphorylation over time upon stimulation with α2-3sia, but no α2-6sia. Inhibition of both STAT3 and -5A in moDCs resulted in a similar cytokine secretion profile as α-3sia triggered DCs. Conclusively, this study revealed a specific alteration of the JAK-STAT pathway in DCs upon simultaneous α2-3sia and LPS stimulation, altering the IL10:IL-12 cytokine secretion profile associated with reduction of inflammation. Targeted control of the STAT phosphorylation status is therefore an interesting lead for the abrogation of immune escape that bacteria or tumors impose on the host.

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Phosphoproteomic Profiling of T-Cell Acute Lymphoblastic Leukemia Identifies Targetable Kinases and Combination Treatments

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