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Chronic Lymphocytic Leukemia

Chemoproteomics-based kinome profiling and target deconvolution of clinical multi-kinase inhibitors in primary chronic lymphocytic leukemia cells

Leukemia volume 25, pages 89–100 (2011)Cite this article

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Abstract

The pharmacological induction of apoptosis in neoplastic B cells presents a promising therapeutic avenue for the treatment of chronic lymphocytic leukemia (CLL). We profiled a panel of clinical multi-kinase inhibitors for their ability to induce apoptosis in primary CLL cells. Whereas inhibitors targeting a large number of receptor and intracellular tyrosine kinases including c-KIT, FLT3, BTK and SYK were comparatively inactive, the CDK inhibitors BMS-387032 and flavopiridol showed marked efficacy similar to staurosporine. Using the kinobeads proteomics method, kinase expression profiles and binding profiles of the inhibitors to target protein complexes were quantitatively monitored in CLL cells. The targets most potently affected were CDK9, cyclin T1, AFF3/4 and MLLT1, which may represent four subunits of a deregulated positive transcriptional elongation factor (p-TEFb) complex. Albeit with lower potency, both drugs also bound the basal transcription factor BTF2/TFIIH containing CDK7. Staurosporine and geldanamycin do not affect these targets and thus seem to exhibit a different mechanism of action. The data support a critical role of p-TEFb inhibitors in CLL that supports their future clinical development.

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References

  1. Druker BJ . Translation of the Philadelphia chromosome into therapy for CML. Blood 2008; 112: 4808–4817.

    Article  CAS  PubMed  Google Scholar 

  2. Kumar C, Purandare AV, Lee FY, Lorenzi MV . Kinase drug discovery approaches in chronic myeloproliferative disorders. Oncogene 2009; 28: 2305–2313.

    Article  CAS  PubMed  Google Scholar 

  3. Zhang J, Yang PL, Gray NS . Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer 2009; 9: 28–39.

    Article  PubMed  Google Scholar 

  4. Veldurthy A, Patz M, Hagist S, Pallasch CP, Wendtner CM, Hallek M et al. The kinase inhibitor dasatinib induces apoptosis in chronic lymphocytic leukemia cells in vitro with preference for a subgroup of patients with unmutated IgVH genes. Blood 2008; 112: 1443–1452.

    Article  CAS  PubMed  Google Scholar 

  5. Wendtner CM, Eichhorst BF, Hallek MJ . Advances in chemotherapy for chronic lymphocytic leukemia. Semin Hematol 2004; 41: 224–233.

    Article  CAS  PubMed  Google Scholar 

  6. Phelps MA, Lin TS, Johnson AJ, Hurh E, Rozewski DM, Farley KL et al. Clinical response and pharmacokinetics from a phase 1 study of an active dosing schedule of flavopiridol in relapsed chronic lymphocytic leukemia. Blood 2009; 113: 2637–2645.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wiestner A, Rosenwald A, Barry TS, Wright G, Davis RE, Henrickson SE et al. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood 2003; 101: 4944–4951.

    Article  CAS  PubMed  Google Scholar 

  8. Gaiger A, Heintel D, Jager U . Novel molecular diagnostic and therapeutic targets in chronic lymphocytic leukaemia. Eur J Clin Invest 2004; 34 (Suppl 2): 25–30.

    Article  CAS  PubMed  Google Scholar 

  9. Bantscheff M, Eberhard D, Abraham Y, Bastuck S, Boesche M, Hobson S et al. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat Biotechnol 2007; 25: 1035–1044.

    Article  CAS  PubMed  Google Scholar 

  10. Nita-Lazar A, Saito-Benz H, White FM . Quantitative phosphoproteomics by mass spectrometry: past, present, and future. Proteomics 2008; 8: 4433–4443.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sharma K, Weber C, Bairlein M, Greff Z, Keri G, Cox J et al. Proteomics strategy for quantitative protein interaction profiling in cell extracts. Nat Methods 2009; 6: 741–744.

    Article  CAS  PubMed  Google Scholar 

  12. Apsel B, Blair JA, Gonzalez B, Nazif TM, Feldman ME, Aizenstein B et al. Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases. Nat Chem Biol 2008; 4: 691–699.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Peters EC, Gray NS . Chemical proteomics identifies unanticipated targets of clinical kinase inhibitors. ACS Chem Biol 2007; 2: 661–664.

    Article  CAS  PubMed  Google Scholar 

  14. Hopkins AL, Mason JS, Overington JP . Can we rationally design promiscuous drugs? Curr Opin Struct Biol 2006; 16: 127–136.

    Article  CAS  PubMed  Google Scholar 

  15. Pallasch CP, Schulz A, Kutsch N, Schwamb J, Hagist S, Kashkar H et al. Overexpression of TOSO in CLL is triggered by B-cell receptor signaling and associated with progressive disease. Blood 2008; 112: 4213–4219.

    Article  CAS  PubMed  Google Scholar 

  16. Bantscheff M, Boesche M, Eberhard D, Matthieson T, Sweetman G, Kuster B . Robust and sensitive iTRAQ quantification on an LTQ Orbitrap mass spectrometer. Mol Cell Proteomics 2008; 7: 1702–1713.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 2004; 3: 1154–1169.

    Article  CAS  PubMed  Google Scholar 

  18. Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT et al. A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 2008; 26: 127–132.

    Article  CAS  PubMed  Google Scholar 

  19. Rawstron AC, Bennett F, Hillmen P . The biological and clinical relationship between CD5+23+ monoclonal B-cell lymphocytosis and chronic lymphocytic leukaemia. Br J Haematol 2007; 139: 724–729.

    Article  PubMed  Google Scholar 

  20. Kitada S, Zapata JM, Andreeff M, Reed JC . Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia. Blood 2000; 96: 393–397.

    CAS  PubMed  Google Scholar 

  21. Gill AL, Verdonk M, Boyle RG, Taylor R . A comparison of physicochemical property profiles of marketed oral drugs and orally bioavailable anti-cancer protein kinase inhibitors in clinical development. Curr Top Med Chem 2007; 7: 1408–1422.

    Article  CAS  PubMed  Google Scholar 

  22. Brehmer D, Godl K, Zech B, Wissing J, Daub H . Proteome-wide identification of cellular targets affected by bisindolylmaleimide-type protein kinase C inhibitors. Mol Cell Proteomics 2004; 3: 490–500.

    Article  CAS  PubMed  Google Scholar 

  23. Wisniewski D, Lambek CL, Liu C, Strife A, Veach DR, Nagar B et al. Characterization of potent inhibitors of the Bcr-Abl and the c-kit receptor tyrosine kinases. Cancer Res 2002; 62: 4244–4255.

    CAS  PubMed  Google Scholar 

  24. Knight ZA, Shokat KM . Features of selective kinase inhibitors. Chem Biol 2005; 12: 621–637.

    Article  CAS  PubMed  Google Scholar 

  25. Misra RN, Xiao HY, Kim KS, Lu S, Han WC, Barbosa SA et al. N-(cycloalkylamino)acyl-2-aminothiazole inhibitors of cyclin-dependent kinase 2. N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4- piperidinecarboxamide (BMS-387032), a highly efficacious and selective antitumor agent. J Med Chem 2004; 47: 1719–1728.

    Article  CAS  PubMed  Google Scholar 

  26. Chen R, Keating MJ, Gandhi V, Plunkett W . Transcription inhibition by flavopiridol: mechanism of chronic lymphocytic leukemia cell death. Blood 2005; 106: 2513–2519.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Loisel S, Ster KL, Quintin-Roue I, Pers JO, Bordron A, Youinou P et al. Establishment of a novel human B-CLL-like xenograft model in nude mouse. Leuk Res 2005; 29: 1347–1352.

    Article  CAS  PubMed  Google Scholar 

  28. Essakali S, Carney D, Westerman D, Gambell P, Seymour JF, Dobrovic A . Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail. BMC Biotechnol 2008; 8: 6.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Hillmen P . Advancing therapy for chronic lymphocytic leukemia--the role of rituximab. Semin Oncol 2004; 31 (Suppl 2): 22–26.

    Article  CAS  PubMed  Google Scholar 

  30. Olaharski AJ, Gonzaludo N, Bitter H, Goldstein D, Kirchner S, Uppal H et al. Identification of a kinase profile that predicts chromosome damage induced by small molecule kinase inhibitors. PLoS Comput Biol 2009; 5: e1000446.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Bres V, Yoh SM, Jones KA . The multi-tasking P-TEFb complex. Curr Opin Cell Biol 2008; 20: 334–340.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Bitoun E, Oliver PL, Davies KE . The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling. Hum Mol Genet 2007; 16: 92–106.

    Article  CAS  PubMed  Google Scholar 

  33. Tkachuk DC, Kohler S, Cleary ML . Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell 1992; 71: 691–700.

    Article  CAS  PubMed  Google Scholar 

  34. Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S et al. Large-scale mapping of human protein-protein interactions by mass spectrometry. Mol Syst Biol 2007; 3: 89.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Smit LA, Hallaert DY, Spijker R, de GB, Jaspers A, Kater AP et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocytic leukemia cells correlates with survival capacity. Blood 2007; 109: 1660–1668.

    Article  CAS  PubMed  Google Scholar 

  36. Contri A, Brunati AM, Trentin L, Cabrelle A, Miorin M, Cesaro L et al. Chronic lymphocytic leukemia B cells contain anomalous Lyn tyrosine kinase, a putative contribution to defective apoptosis. J Clin Invest 2005; 115: 369–378.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hantschel O, Rix U, Schmidt U, Burckstummer T, Kneidinger M, Schutze G et al. The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib. Proc Natl Acad Sci USA 2007; 104: 13283–13288.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Braselmann S, Taylor V, Zhao H, Wang S, Sylvain C, Baluom M et al. R406, an orally available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation. J Pharmacol Exp Ther 2006; 319: 998–1008.

    Article  CAS  PubMed  Google Scholar 

  39. Quiroga MP, Balakrishnan K, Kurtova AV, Sivina M, Keating MJ, Wierda WG et al. B cell antigen receptor signaling enhances chronic lymphocytic leukemia cell migration and survival: specific targeting with a novel Syk inhibitor, R406. Blood 2009; 114: 1029–1037.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Conroy A, Stockett DE, Walker D, Arkin MR, Hoch U, Fox JA et al. SNS-032 is a potent and selective CDK 2, 7 and 9 inhibitor that drives target modulation in patient samples. Cancer Chemother Pharmacol 2009; 64: 723–732.

    Article  CAS  PubMed  Google Scholar 

  41. Chen R, Wierda WG, Chubb S, Hawtin RE, Fox JA, Keating MJ et al. Mechanism of action of SNS-032, a novel cyclin-dependent kinase inhibitor, in chronic lymphocytic leukemia. Blood 2009; 113: 4637–4645.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Wang S, Fischer PM . Cyclin-dependent kinase 9: a key transcriptional regulator and potential drug target in oncology, virology and cardiology. Trends Pharmacol Sci 2008; 29: 302–313.

    Article  PubMed  Google Scholar 

  43. Estable MC, Naghavi MH, Kato H, Xiao H, Qin J, Vahlne A et al. MCEF, the newest member of the AF4 family of transcription factors involved in leukemia, is a positive transcription elongation factor-b-associated protein. J Biomed Sci 2002; 9: 234–245.

    Article  CAS  PubMed  Google Scholar 

  44. Mueller D, Bach C, Zeisig D, Garcia-Cuellar MP, Monroe S, Sreekumar A et al. A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification. Blood 2007; 110: 4445–4454.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Krivtsov AV, Armstrong SA . MLL translocations, histone modifications and leukaemia stem-cell development. Nat Rev Cancer 2007; 7: 823–833.

    Article  CAS  PubMed  Google Scholar 

  46. Lin C, Smith ER, Takahashi H, Lai KC, Martin-Brown S, Florens L et al. AFF4, a component of the ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia. Mol Cell 2010; 37: 429–437.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Yokoyama A, Lin M, Naresh A, Kitabayashi I, Cleary ML . A higher-order complex containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and physiologic MLL-dependent transcription. Cancer Cell 2010; 17: 198–212.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Tong WG, Chen R, Plunkett W, Siegel D, Sinha R, Harvey RD et al. Phase I and pharmacologic study of SNS-032, a potent and selective Cdk2, 7, and 9 inhibitor, in patients with advanced chronic lymphocytic leukemia and multiple myeloma. J Clin Oncol 2010; 28: 3015–3022.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Jones DT, Addison E, North JM, Lowdell MW, Hoffbrand AV, Mehta AB et al. Geldanamycin and herbimycin A induce apoptotic killing of B chronic lymphocytic leukemia cells and augment the cells’ sensitivity to cytotoxic drugs. Blood 2004; 103: 1855–1861.

    Article  CAS  PubMed  Google Scholar 

  50. Malek SN, Dordai DI, Reim J, Dintzis H, Desiderio S . Malignant transformation of early lymphoid progenitors in mice expressing an activated Blk tyrosine kinase. Proc Natl Acad Sci USA 1998; 95: 7351–7356.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are thankful to Reinhild Brinker, Anja Podszuweit and Jessica Perrin for expert technical assistance, to Valérie Reader for compound synthesis, to Judith Schlegl, Christine Gmünd and Vincent Wolowski for software and database development, and to Yann Abraham for help with data analysis. We also thank Frank Weisbrodt for help with the figures. We would like to thank Tim Edwards, Jason Fisherman, Carsten Hopf, Gitte Neubauer and David Simmons for suggestions and support. This work was supported by a grant from the German Bundesministerium für Bildung und Forschung (Spitzencluster BioRN, Verbundprojekt Inkubator/Teilprojekt BioRN-IND-TP02) to Cellzome AG. CPP and CMW were supported by the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Köln Fortune, the CLL Global Research Foundation, and the Marga and Walter Boll-Stiftung.

Author contributions

UK, CPP and MB designed and performed research, analyzed data and wrote the paper; DE, LF, SKM, SG and TW performed research; CMW provided CLL patient samples and wrote the paper; GD conceptualized the project, analyzed data and wrote the paper.

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  1. U Kruse, C P Pallasch and M Bantscheff: These authors contributed equally to this study.

Authors and Affiliations

  1. Cellzome AG, Heidelberg, Germany

    U Kruse, M Bantscheff, D Eberhard, S Ghidelli, S K Maier, T Werner & G Drewes

  2. Department I of Internal Medicine, Laboratory of Molecular Biology and Immunology of CLL, Center of Integrated Oncology and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany

    C P Pallasch, L Frenzel & C M Wendtner

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Correspondence to C M Wendtner or G Drewes.

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UK, MB, DE, SG, TW and GD are employees of Cellzome AG. CPP and CMW received research funding from Cellzome AG. All the authors declare no other conflict of interest.

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Kruse, U., Pallasch, C., Bantscheff, M. et al. Chemoproteomics-based kinome profiling and target deconvolution of clinical multi-kinase inhibitors in primary chronic lymphocytic leukemia cells. Leukemia 25, 89–100 (2011). https://doi.org/10.1038/leu.2010.233

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