Trisomy 12 chronic lymphocytic leukemia expresses a unique set of activated and targetable pathways.

Although trisomy 12 (+12) chronic lymphocytic leukemia (CLL) comprises about 20% of cases, relatively little is known about its pathophysiology. These cases often demonstrate atypical morphological and immunophenotypic features, high proliferative rates, unmutated immunoglobulin heavy chain variable region genes, and a high frequency of NOTCH1 mutation. Patients with +12 CLL have an intermediate prognosis, and show higher incidences of thrombocytopenia, Richter transformation, and other secondary cancers. Despite these important differences, relatively few transcriptional profiling studies have focused on identifying dysregulated pathways that characterize +12 CLL, and most have used a hierarchical cytogenetic classification in which cases with more than one recurrent abnormality are categorized according to the abnormality with the poorest prognosis. In this study, we sought to identify protein-coding genes whose expression contributes to the unique pathophysiology of +12 CLL. To exclude the likely confounding effects of multiple cytogenetic abnormalities on gene expression, our +12 patient cohort had +12 as the sole abnormality. We profiled samples obtained from 147 treatment-naïve patients. We compared cases with +12 as the only cytogenetic abnormality to cases with only del(13q), del(11q), or diploid cytogenetics using independent discovery (n=97) and validation (n=50) sets. We demonstrate that CLL cases with +12 as the sole abnormality express a unique set of activated pathways compared to other cytogenetic subtypes. Among these pathways, we identify the NFAT signaling pathway and the immune checkpoint molecule, NT5E (CD73), which may represent new therapeutic targets.

Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics.

The cellular proto-oncogene c-Src is a nonreceptor tyrosine kinase involved in cell growth and cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise functions are not fully understood. Identification of the substrates of c-Src remains a major challenge, because there is no simple way to directly stimulate its activity. Here we combine the chemical rescue of mutant c-Src and global quantitative phosphoproteomics to obtain the first high resolution snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 29 potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the identification of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable isotope labeling of amino acids in cell culture-based quantitation allowed the detection of 97 nonredundant tyrosine phosphopeptides whose level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-Src putative protein targets and phosphorylation sites are presented here, a majority of which play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin signaling and focal adhesion kinase signaling pathway are two of the most altered pathways upon c-Src activation through chemical rescue. In this context, our study revealed the temporal connection between c-Src activation and the GTPase Rap1, known to stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the spatiotemporal mechanism of activation of the Rap1 guanine exchange factor, C3G, one of the identified potential c-Src substrates that plays a role in focal adhesion signaling. In addition to unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of the functions of nonreceptor tyrosine kinases with high specificity and kinetic resolution.