Bendamustine(Treanda)DisplaysaDistinctPatternofCytotoxicity andUniqueMechanisticFeaturesComparedwith OtherAlkylatingAgents LorenzoM.Leoni, 1,2 Brandi Bailey, 1,3 JackReifert, 1,6 Heather H. Bendall, 1,4 Robert W.Zeller, 3 JacquesCorbeil, 7,9 GaryElliott, 1,5 and Christina C. Niemeyer 1,8 Abstract Purpose: Bendamustine has shown clinical activity in patients with disease refractory to conventional alkylator chemotherapy.The purpose of this study was to characterize the mechanisms ofactionof bendamustine and to compare it with structurally related compounds. Experimental Design: Bendamustine was profiled in the National Cancer Institute in vitro antitumor screen. Microarray-based gene expressionprofiling, real-time PCR, immunoblot, cell cycle, and functional DNA damage repair analyses were used to characterize response to bendamustine and compare it with chlorambuciland phosphoramide mustard. Results: Bendamustine displays a distinct pattern of activity unrelated to other DNA-alkylating agents. Its mechanisms of action include activation of DNA-damage stress response and apop- tosis, inhibition of mitotic checkpoints, and induction of mitotic catastrophe. In addition, unlike other alkylators, bendamustine activates a base excision DNA repair pathway rather than an alkyltransferaseDNArepairmechanism. Conclusion: These results suggest that bendamustine possesses mechanistic features that differentiate it from other alkylating agents and may contribute to its distinct clinical efficacy profile. Bendamustine, 4-{5-[bis(2-chloroethyl)amino]-1-methyl-2- benzimidazolyl} butyric acid hydrochloride, also known as Treanda (Cephalon, Inc.) and marketed in Germany as Ribo- mustin (Mundi Pharma Ltd.), is a purine analogue/alkylator hybrid cytotoxic with shown clinical activity against various human cancers including non–Hodgkin’s lymphoma (1, 2), chronic lymphocytic leukemia (3), multiple myeloma (4, 5), breast cancer (6), and small-cell lung cancer (7, 8). In addi- tion, both preclinical (9–11) and clinical (12) studies of ben- damustine have shown activity in cancer cells that are resistant to conventional alkylating agents. Bendamustine was originally designed to have both alkylat- ing and antimetabolite properties with acceptable toxicity (13). Structurally, bendamustine comprises three elements: a 2-chloroethylamine alkylating group, a benzimidazole ring, and a butyric acid side chain (Fig. 1). The 2-chloroethylamine alkylating group is shared with other members of the nitrogen mustard family of alkylators, which includes cyclophospha- mide, chlorambucil and melphalan, and the butyric acid side chain is shared with chlorambucil. The benzimidazole central ring system is unique to bendamustine; the intent of adding this structure to the nitrogen mustard was to include the antimetabolite properties shown for benzimidazole (14, 15). This heterocyclic ring structure may contribute to the unique antitumor activity of bendamustine and distinguish it from conventional 2-chloroethylamine alkylators (16). Although a large body of clinical data on bendamustine in a varietyoftumorshasbeenreported,studiesclearlydefiningthe mechanisms of action of bendamustine are lacking. Similar to other alkylators, bendamustine is a DNA cross-linking agent that causes DNA breaks. However, DNA single- and double- strand breaks caused by bendamustine are more extensive and significantly more durable than those caused by cyclophospha- mide, cisplatinum, or carmustine (bischloloroethyl nitro- sourea; ref. 9). The DNA damage mediated by alkylators has been associated with a regulated form of necrotic cell death (17). Bendamustine as a single agent (18–20) or in combina- tion with other anticancer agents (21) has also shown proapoptotic activity in several invitro tumor models. The objective of the current study was to describe potential mechanisms of action of bendamustine that distinguish it from other alkylators using large-scale screening technologies, including the National Cancer Institute (NCI) In vitro Cell Line Screening Project (IVCLSP) and gene microarrays. We report data from these analyses and confirm a unique anti- tumor activity profile for bendamustine compared with Cancer Therapy: Preclinical Authors’Affiliations: 1 Salmedix,acquiredbyCephalon,Inc.,Frazer,Pennsylvania; 2 Telormedix, Lugano, Switzerland; 3 Department of Biology, San Diego State University; 4 Neurocrine, Inc.; and 5 Galenic Strategies, San Diego, California; 6 University of California Santa Barbara, Santa Barbara, California; 7 Center forAIDS Research,CenterforMolecularMedicineandCancerCenter,UniversityofCalifornia San Diego and 8 Burnham Institute for Medical Research, La Jolla, California; and 9 QuebecCityGenomeCentre,LavalUniversity,Quebec,Canada Received5/3/07;revised9/25/07;accepted10/10/07. Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage charges.This article must therefore be hereby marked advertisement in accordance with18U.S.C.Section1734solely toindicatethisfact. Requests for reprints: Christina Niemeyer, Burnham Institute for Medical Research,10901NorthTorrey Pines Road, LaJolla, CA 92037. Phone: 858-646- 3100;Fax: 858-795-5221;E-mail:niemeyer@burnham.org. F 2008AmericanAssociationforCancerResearch. doi:10.1158/1078-0432.CCR-07-1061 www.aacrjournals.org ClinCancerRes2008;14(1)January1,2008 309