Survey of activated FLT3 signaling in leukemia.

Activating mutations of FMS-like tyrosine kinase-3 (FLT3) are found in approximately 30% of patients with acute myeloid leukemia (AML). FLT3 is therefore an attractive drug target. However, the molecular mechanisms by which FLT3 mutations lead to cell transformation in AML remain unclear. To develop a better understanding of FLT3 signaling as well as its downstream effectors, we performed detailed phosphoproteomic analysis of FLT3 signaling in human leukemia cells. We identified over 1000 tyrosine phosphorylation sites from about 750 proteins in both AML (wild type and mutant FLT3) and B cell acute lymphoblastic leukemia (normal and amplification of FLT3) cell lines. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC), we were able to quantified over 400 phosphorylation sites (pTyr, pSer, and pThr) that were responsive to FLT3 inhibition in FLT3 driven human leukemia cell lines. We also extended this phosphoproteomic analysis on bone marrow from primary AML patient samples, and identify over 200 tyrosine and 800 serine/threonine phosphorylation sites in vivo. This study showed that oncogenic FLT3 regulates proteins involving diverse cellular processes and affects multiple signaling pathways in human leukemia that we previously appreciated, such as Fc epsilon RI-mediated signaling, BCR, and CD40 signaling pathways. It provides a valuable resource for investigation of oncogenic FLT3 signaling in human leukemia.

Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma.

Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23) of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.

Akt-RSK-S6 kinase signaling networks activated by oncogenic receptor tyrosine kinases.

Receptor tyrosine kinases (RTKs) activate pathways mediated by serine-threonine kinases, such as the PI3K (phosphatidylinositol 3-kinase)-Akt pathway, the Ras-MAPK (mitogen-activated protein kinase)-RSK (ribosomal S6 kinase) pathway, and the mTOR (mammalian target of rapamycin)-p70 S6 pathway, that control important aspects of cell growth, proliferation, and survival. The Akt, RSK, and p70 S6 family of protein kinases transmits signals by phosphorylating substrates on an RxRxxS/T motif (R, arginine; S, serine; T, threonine; and x, any amino acid). We developed a large-scale proteomic approach to identify more than 300 substrates of this kinase family in cancer cell lines driven by the c-Met, epidermal growth factor receptor (EGFR), or platelet-derived growth factor receptor alpha (PDGFRalpha) RTKs. We identified a subset of proteins with RxRxxS/T sites for which phosphorylation was decreased by RTK inhibitors (RTKIs), as well as by inhibitors of the PI3K, mTOR, and MAPK pathways, and we determined the effects of small interfering RNA directed against these substrates on cell viability. Phosphorylation of the protein chaperone SGTA (small glutamine-rich tetratricopeptide repeat-containing protein alpha) at serine-305 was essential for PDGFRalpha stabilization and cell survival in PDGFRalpha-dependent cancer cells. Our approach provides a new view of RTK and Akt-RSK-S6 kinase signaling, revealing previously unidentified Akt-RSK-S6 kinase substrates that merit further consideration as targets for combination therapy with RTKIs.

Signaling networks assembled by oncogenic EGFR and c-Met.

A major question regarding the sensitivity of solid tumors to targeted kinase inhibitors is why some tumors respond and others do not. The observation that many tumors express EGF receptor (EGFR), yet only a small subset with EGFR-activating mutations respond clinically to EGFR inhibitors (EGFRIs), suggests that responsive tumors uniquely depend on EGFR signaling for their survival. The nature of this dependence is not understood. Here, we investigate dependence on EGFR signaling by comparing non-small-cell lung cancer cell lines driven by EGFR-activating mutations and genomic amplifications using a global proteomic analysis of phospho-tyrosine signaling. We identify an extensive receptor tyrosine kinase signaling network established in cells expressing mutated and activated EGFR or expressing amplified c-Met. We show that in drug sensitive cells the targeted tyrosine kinase drives other RTKs and an extensive network of downstream signaling that collapse with drug treatment. Comparison of the signaling networks in EGFR and c-Met-dependent cells identify a "core network" of approximately 50 proteins that participate in pathways mediating drug response.

Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer.

Despite the success of tyrosine kinase-based cancer therapeutics, for most solid tumors the tyrosine kinases that drive disease remain unknown, limiting our ability to identify drug targets and predict response. Here we present the first large-scale survey of tyrosine kinase activity in lung cancer. Using a phosphoproteomic approach, we characterize tyrosine kinase signaling across 41 non-small cell lung cancer (NSCLC) cell lines and over 150 NSCLC tumors. Profiles of phosphotyrosine signaling are generated and analyzed to identify known oncogenic kinases such as EGFR and c-Met as well as novel ALK and ROS fusion proteins. Other activated tyrosine kinases such as PDGFRalpha and DDR1 not previously implicated in the genesis of NSCLC are also identified. By focusing on activated cell circuitry, the approach outlined here provides insight into cancer biology not available at the chromosomal and transcriptional levels and can be applied broadly across all human cancers.

Profiling of UV-induced ATM/ATR signaling pathways.

To ensure survival in the face of genomic insult, cells have evolved complex mechanisms to respond to DNA damage, termed the DNA damage checkpoint. The serine/threonine kinases ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) activate checkpoint signaling by phosphorylating substrate proteins at SQ/TQ motifs. Although some ATM/ATR substrates (Chk1, p53) have been identified, the lack of a more complete list of substrates limits current understanding of checkpoint pathways. Here, we use immunoaffinity phosphopeptide isolation coupled with mass spectrometry to identify 570 sites phosphorylated in UV-damaged cells, 498 of which are previously undescribed. Semiquantitative analysis yielded 24 known and 192 previously uncharacterized sites differentially phosphorylated upon UV damage, some of which were confirmed by SILAC, Western blotting, and immunoprecipitation/Western blotting. ATR-specific phosphorylation was investigated by using a Seckel syndrome (ATR mutant) cell line. Together, these results provide a rich resource for further deciphering ATM/ATR signaling and the pathways mediating the DNA damage response.

Phosphotyrosine profiling identifies the KG-1 cell line as a model for the study of FGFR1 fusions in acute myeloid leukemia.

The 8p11 myeloproliferative syndrome (EMS) is associated with translocations that disrupt the FGFR1 gene. To date, 8 fusion partners of FGFR1 have been identified. However, no primary leukemia cell lines were identified that contain any of these fusions. Here, we screened more than 40 acute myeloid leukemia cell lines for constitutive phosphorylation of STAT5 and applied an immunoaffinity profiling strategy to identify tyrosine-phosphorylated proteins in the KG-1 cell line. Mass spectrometry analysis of KG-1 cells revealed aberrant tyrosine phosphorylation of FGFR1. Subsequent analysis led to the identification of a fusion of the FGFR1OP2 gene to the FGFR1 gene. Small interfering RNA (siRNA) against FGFR1 specifically inhibited the growth and induced apoptosis of KG-1 cells. Thus, the KG-1 cell line provides an in vitro model for the study of FGFR1 fusions associated with leukemia and for the analysis of small molecule inhibitors against FGFR1 fusions.

A common phosphotyrosine signature for the Bcr-Abl kinase.

The Bcr-Abl fusion kinase drives oncogenesis in chronic myeloid leukemia (CML). CML patients are currently treated with the Abl tyrosine kinase inhibitor imatinib, which is effective in early stages of the disease. However, resistance to imatinib arises in later disease stages primarily because of a Bcr-Abl mutation. To gain deeper insight into Bcr-Abl signaling pathways, we generated phosphotyrosine profiles for 6 cell lines that represent 3 Bcr-Abl fusion types by using immunoaffinity purification of tyrosine phosphopeptides followed by tandem mass spectrometry. We identified 188 nonredundant tyrosine-phosphorylated sites, 77 of which are novel. By comparing the profiles, we found a number of phosphotyrosine sites common to the 6 cell lines regardless of cellular background and fusion type, several of which are decreased by imatinib treatment. Comparison of this Bcr-Abl signature with the profile of cells expressing an alternative imatinib-sensitive fusion kinase, FIP1L1-PDGFRalpha, revealed that these kinases signal through different pathways. This phosphoproteomic study of the Bcr-Abl fusion kinase highlights novel disease markers and potential drug-responsive biomarkers and adds novel insight into the oncogenic signals driven by the Bcr-Abl kinase.