Detection of a rare BCR–ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS)

Abstract

Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.targeted therapies | personalized medicine | protein-protein interactions P roteomics studies over the last decade have provided significant advancements in our understanding of the functional landscape of proteins, their posttranslational modifications and the protein-protein interactions (PPI) (1-3). This is especially important for diseases such as cancer because proteins and their transient posttranslational modifications (PTMs) reflect the changes cells undergo during pathogenic development. Modern high-resolution mass spectrometers are well suited for these analyses and have led the efforts in this field of proteomics, allowing researchers to analyze dynamic changes in cell lines or tissue and to distinguish between normal and unhealthy states (4, 5). PPI networks and phosphoproteomics are important for understanding the function and regulation of pathways leading to cell growth and proliferation in diseases such as cancer. The advantage 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. This can be achieved by performing an immunoprecipitation (IP) with a bait protein and analyzing the prey proteins using microcapillary-tandem mass spectrometry (LC-MS/MS) (6-1...

Reply to MacLeod et al.: Multiple myeloma plasma cells have chameleon characteristics

“…It appears that MacLeod et al performed only cytogenetic tests (i.e., FISH and RT-PCR) to make a determination of contamination, and we agree that our cells possessed the e14a2 form of BCR-ABL fusion, as we reported using similar genetic tests (1,2). Together with e13a2 fusion form, e14a2 and e13a2 represent the most common forms of BCR-ABL.…”

“…To summarize, our lot of H929 MM cells showed positive markers for plasma cells such as CD138 but were negative for standard markers of CML (K562) cells, including CD34, CD68, and myeloperoxidase. Additionally, inspection of the cells with Wright-Giemsa staining showed an abundant blue cytoplasm more deeply stained toward the periphery, prominent Golgi complex, eccentrically located nuclei, and a large and prominent central nucleolus, all features characteristic of plasma cells but not of CML cells (2).…”

“…Because our H929 cells lacked interferon regulatory factor 4 expression, this may have contributed to the expression of BCR-ABL (6). Our lot of H929 cells may have been a special case because we tested other lots of H929 cells, and because they contained significantly lower levels of BCR-ABL by Western blotting, we did not follow up with cytogenetic tests (2). It is possible that a BCR-ABL fusion occurred somewhere in the cultured history of our H929 plasma cells that was similar to the fusion found in K562 cells, and our IP-LC-MS/MS technology showed that one could detect such cell abnormalities and use it to our advantage when testing patient cancers.…”

Transcriptional repression by the HDAC4–RelB–p52 complex regulates multiple myeloma survival and growth

“…On the basis of FISH and reverse transcription (RT)-PCR analysis, MacLeod et al 62 have reported the H929 cell line on the International Cell Line Authentication Committee (ICLAC) list due to a suspected cross-contamination with K562 cell line in a study by Brietkopf et al 63 However, Brietkopf et al, in a subsequent and more detailed study 64 , have shown that their lot of H929 cell line was clearly of plasma cell origin and lacked most of the standard markers for the K562 cell line. Nevertheless, we obtained our MMCLs including the H929 from Dr Kuehl (NIH) and Dr Bergsagel (Mayo Clinic) and hence our study is not affected by the MacLeod's report.…”

Peptide–Drug Conjugate GnRH–Sunitinib Targets Angiogenesis Selectively at the Site of Action to Inhibit Tumor Growth

“…Images were analyzed with ImageJ software (v1.28) by loading the image as a grayscale picture. Each kinase array dot was manually selected, normalized, and an average intensity for each kinase was calculated as described previously (33).…”