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J. Biol. Chem., Vol. 283, Issue 17, 11752-11762, April 25, 2008

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Combinatorial Targeting of the Macropinocytotic Pathway in Leukemia and Lymphoma Cells^*

Shinpei Nishimura, Shunsuke Takahashi, Hiromi Kamikatahira, Yuko Kuroki, Diana E. Jaalouk, Susan O'Brien, Erkki Koivunen, Wadih Arap¹, Renata Pasqualini², Hitoshi Nakayama, and Akihiko Kuniyasu³

From the Department of Molecular Cell Function, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, 5-1 Ohe-honmachi, Kumamoto 862-0973, Japan and The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030

Ligand-directed delivery of agents to leukemia and lymphoma cells has the potential to yield new mechanistic disease insights and targeted therapies. Here we set out to target the macropinocytotic pathway with a combinatorial approach. From the screening of acute T-lymphoblastic leukemia Molt-4 cells with a random phage-display peptide library, we isolated a phage displaying the sequence CAYHRLRRC. This peptide contains a lymph node-homing motif (Cys-Ala-Tyr) and a cell-penetrating motif (Arg-Leu-Arg-Arg). Binding of this ligand-directed phage to a large panel of leukemia/lymphoma cells and to patient-derived samples was much higher than to non-leukemia control cells. CAYHRLRRC phage internalization into Molt-4 cells is both energy- and temperature-dependent. Flow cytometry with fluorescein-labeled peptide and endocytosis blocking with specific inhibitors revealed that CAYHRLRRC is indeed taken up through macropinocytosis in Molt-4 and K562 human leukemia cells. Unexpectedly, the cell surface receptor for the CAYHRLRRC peptide is not a heparan sulfate proteoglycan as it would be predicted for other cell-penetrating peptides. Confirming this interpretation, a CAYHRLRRC-directed peptidomimetic-induced cell death in all the leukemia and lymphoma cells was evaluated, whereas a control transactivator of transcription protein (tat)-directed proapoptotic peptidomimetic was non-selective. In summary, the targeting peptide CAYHRLRRC is selectively internalized through macropinocytosis in leukemia and lymphoma cells and has potential as a drug lead for ligand-directed anti-leukemia therapies.

Received for publication, October 26, 2007 , and in revised form, February 5, 2008.

^* This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science number 1194 (to A. K.), Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan Grant 19659024 (to H. N.), and The University of Texas M. D. Anderson Cancer Center Leukemia Specialized Program of Research Excellence of the National Cancer Institute (to W. A. and R. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed. E-mail: warap{at}mdanderson.org.

² To whom correspondence may be addressed. E-mail: rpasqual{at}mdanderson.org.

³ To whom correspondence may be addressed. E-mail: kuniyasu{at}gpo.kumamoto-u.ac.jp.

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