Detection of a rare BCR–ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS) Susanne B. Breitkopfa,b, Min Yuana, German A. Pihanc, and John M. Asaraa,b,1 aDivision of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115; bDepartment of Medicine, Harvard Medical School, Boston, MA 02115; and cDepartment of Hematopathology, Beth Israel Deaconess Medical Center, Boston, MA 02115 Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved August 23, 2012 (received for review July 26, 2012) Hypothesis directed proteomics offers higher throughput over Here, we focused on a hypothesis-directed approach to identify global analyses. We show that immunoprecipitation (IP)–tandem the active signaling pathways that drive cancers. To this end, we mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) immunoprecipitated proteins that have clinical significance in cell cancer cells led to the discovery of a rare and unexpected BCR– proliferation such as the central nodes in the AKT and ERK sig- ABL fusion, informing a therapeutic intervention using imatinib naling pathways. The p85 regulatory subunit of phosphoinositide- (Gleevec). BCR–ABL is the driving mutation in chronic myeloid leu- 3-kinase (PI3K) binds pYXXM motif-containing proteins to the kemia (CML) and is uncommon to other cancers. Three different IP– SRC homology 2 (SH2) domains of p85, thus recruiting the p110 MS experiments central to cell signaling pathways were sufficient to catalytic subunit to the plasma membrane for activation (5, 17). Activated p110 phosphorylates its lipid substrate phosphatidyli- discover a BCR–ABL fusion in H929 cells: phosphotyrosine (pY) pep- nositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-tri- tide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) phosphate (PIP3) and binds to the pleckstrin homology (PH) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase domain of the serine/threonine kinase AKT, which is frequently activity, p85 IP informs the activating adaptors and receptor tyro- elevated in cancers (18). We recently showed that we can immu- fi sine kinases (RTKs) involved in AKT activation and GRB2 IP identi es nopurify p85 and quantify the activating PI3K adaptors and RTKs RTKs and adaptors leading to ERK activation. Integration of the by targeted LC-MS/MS (5). In addition, we show here that we can CELL BIOLOGY bait–prey data from the three separate experiments identified the IP the growth-factor-receptor-bound protein 2 (GRB2) adaptor to BCR–ABL protein complex, which was confirmed by biochemistry, identify associated activators of the extracellular signal-regulated cytogenetic methods, and DNA sequencing revealed the e14a2 fu- kinase (ERK), which is also frequently elevated in cancers (19). sion transcript. The tyrosine phosphatase SHP2 and the GAB2 adap- GRB2 is an important SH2/SH3 adaptor protein linking receptor tor protein, important for MAPK signaling, were common to all tyrosine kinases (RTK) and downstream targets and has implica- three IP–MS experiments. The comparative treatment of tyrosine tions in malignancies (20). The SH2 domain of GRB2 binds to the kinase inhibitor (TKI) drugs revealed only imatinib, the standard of phosphorylated tyrosine residue of an adaptor protein or RTK and care in CML, was inhibitory to BCR–ABL leading to down-regulation the SH3 domain binds proteins such as SOS leading to RAS/RAF of pERK and pS6K and inhibiting cell proliferation. These data sug- binding and ERK activation (20, 21). Tyrosine phosphorylation gest a model for directed proteomics from patient tumor samples (pY) is one of the most important and most studied post- fi for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. translational modi cation (PTM) of proteins because it regulates The data also suggest that MM patients, in addition to CML patients, signaling pathway activity and important cellular functions in- may benefit from BCR–ABL diagnostic screening. cluding cell proliferation, growth, survival, etc. (22). Abnormalities in tyrosine kinases play a role in the pathogenesis of numerous inherited or acquired human diseases from cancer to immune targeted therapies | personalized medicine | protein–protein interactions deficiencies (23, 24). Enrichment of low-abundance tyrosine phosphorylated proteins is necessary for a successful compre- roteomics studies over the last decade have provided signifi- hensive LC-MS/MS analysis. One common method for gaining Pcant advancements in our understanding of the functional insight into a broad range of active tyrosine signaling pathways in landscape of proteins, their posttranslational modifications and a cell can be achieved by an IP–MS analysis of phosphotyrosine the protein–protein interactions (PPI) (1–3). This is especially containing peptides from tryptic digested cell lysates (25, 26). important for diseases such as cancer because proteins and their Here we show a clinically relevant example using our three- transient posttranslational modifications (PTMs) reflect the pronged IP–MS technology (p85 IP, GRB2 IP, and pY IP) in fi changes cells undergo during pathogenic development. Modern multiple myeloma (MM) cells and how we identi ed a rare high-resolution mass spectrometers are well suited for these breakpoint cluster region-Abl tyrosine kinase (BCR-ABL) fusion analyses and have led the efforts in this field of proteomics, protein by converging protein complexes and activated tyrosine kinases. BCR–ABL is unexpected in MM because, unlike the allowing researchers to analyze dynamic changes in cell lines or leukemias and lymphomas, MM typically has no specific chro- tissue and to distinguish between normal and unhealthy states (4, mosomal abnormalities and is characterized by a genomic in- 5). PPI networks and phosphoproteomics are important for un- stability that involves both ploidy and structural translocation (27). derstanding the function and regulation of pathways leading to cell There have only been two reports of MM patients with a Phila- growth and proliferation in diseases such as cancer. The advantage delphia chromosome t(9;22)(q34;q11), a 64-y-old woman of analyzing purified protein complexes is the ability to identify specific interacting proteins and posttranslational modifications that may otherwise go undetected in large-scale global analyses. Author contributions: J.M.A. designed research; S.B.B., M.Y., G.A.P., and J.M.A. performed This can be achieved by performing an immunoprecipitation (IP) research; G.A.P. contributed new reagents/analytic tools; S.B.B., M.Y., G.A.P., and J.M.A. with a bait protein and analyzing the prey proteins using micro- analyzed data; and S.B.B. and J.M.A. wrote the paper. capillary–tandem mass spectrometry (LC-MS/MS) (6–13). These The authors declare no conflict of interest. experiments have included various types of affinity tags and have This article is a PNAS Direct Submission. been overlaid with RNAi genetic screens (14, 15). Undoubtedly, 1To whom correspondence should be addressed. E-mail: [email protected]. PPI networks provide a valuable framework for a better un- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. derstanding of the functional organization of the proteome (16). 1073/pnas.1212759109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1212759109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 presenting a P190 BCR–ABL hybrid transcript by PCR analysis ments and the protein products from identified phosphopeptides (28) and a 61-y-old man showing BCR–ABL-positive bone mar- from the pY IP are then correlated in a simple network to clarify row after autologous bone marrow transplantation (29). which upstream RTK and adaptor proteins may be involved in The mass spectrometry results were verified using biochemistry promoting cell proliferation in cancers. and histological analyses as well as DNA sequencing and com- pared with an array of MM cell lines and the K562 chronic my- Case Study Using H929 MM Cells. H929 cells as well as other MM eloid leukemia (CML) cell line known to contain a BCR–ABL cells were investigated to explore which kinase signaling pathways fusion. We show that imatinib (trade name Gleevec) selectively may be responsible for driving proliferation and to explore whether inhibits signaling in H929 cells and halts cell proliferation. This any recently developed TKI drugs would be useful in inhibiting these report shows the molecular investigation of BCR–ABL fusion in cell lines. H929 cells are known to have a rearrangement of the MM, and the results should serve as a template for the use of c-Myc proto-oncogene and express c-Myc RNA and an activated N hypothesis directed proteomics on human tumor samples or cell -RAS allele. They are positive for plasma cell antigen 1 (PCA-1), lines derived from tumors for aiding the appropriate therapeutic transferrin receptor and CD38 (T10), but are negative for HLA selection of single or combinations of TKI drugs in patients. DR, CALLA, and markers of early B-cell development (31, 32). p85 IP–MS PPI landscape. The PPI landscape of p85 (PI3K) was in- Results vestigated as the first phase in identifying drivers of cell pro- liferation in MM cells. H929 cells were lysed, and a polyclonal Analysis of Activating Signaling Pathways Using a Multifaceted antibody recognizing the p85 regulatory subunit of PI3K was used Hypothesis-Directed Proteomics Approach.