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BH3 mimetics are a new class of proapoptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the prosurvival function of one or more Bcl-2-related proteins. Agents that inhibit Bcl-x L induce rapid platelet death that leads to thrombocytopenia; however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time-and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ib␣ and glycoprotein VI, and functional downregulation of integrin ␣ IIb ␤ 3 . Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a timedependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time.Overall, these studies demonstrate that Bcl-x L -inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets. (Blood. 2011;118(6):1663-1674) IntroductionApoptosis is an evolutionarily conserved process important for mammalian development, tissue homeostasis, and immune tolerance. Apoptosis also acts as an important barrier against malignant transformation. Resistance to apoptosis is a hallmark feature of cancer, and several pathways regulating apoptosis are commonly altered in human malignancies. 1,2 For example, Ͼ 50% of all human cancers contain mutations in the p53 tumor suppressor gene, with nearly all of these mutations preventing p53 from triggering apoptosis. 3 Furthermore, the overexpression or enhanced function of certain prosurvival proteins, such as Bcl-2, 2 Bcl-x L , 2 PI3K/Akt/mTOR, 4 and nuclear factor-B, 5 is found in many types of human cancers. As a consequence, strategies targeting specific prosurvival pathways have gained prominence as novel anticancer therapies.Progress unraveling the complex pathways underlying apoptosis and cell survival, and elucidation of their roles in human cancers, has led to the development of several potential anticancer drugs, such as inhibitors of the Bcl-2 protein family, 6 inhibitors of apoptosis (IAPs), 7 MDM2, 8 PI3K/Akt/mTOR, 9 as well as TNFrelated apoptosis-inducing ligand. 10 A particularly promising approach is the ability to inhibit tumor cell survival through the use of agents that mimic the proapoptotic Bcl-2 homology 3 (BH3) domain proteins. BH3 proteins play an important role in inducing apoptosis by antagonizing the function of prosurvival Bcl-2 family proteins. Preclinical studies with the BH3 mimetic ABT-737, which selectively antagonizes Bcl-2, Bcl-x L , and Bcl-w, have demonstrated potent cytotoxic activity a...

Rationale : Platelets are increasingly recognized as mediators of tumor growth and metastasis. Hypothesizing that activated platelets in the tumor microenvironment provide a targeting epitope for tumor-directed chemotherapy, we developed an antibody-drug conjugate (ADC), comprised of a single-chain antibody (scFv) against the platelet integrin GPIIb/IIIa (scFv GPIIb/IIIa ) linked to the potent chemotherapeutic microtubule inhibitor, monomethyl auristatin E (MMAE). Methods : We developed an ADC comprised of three components: 1) A scFv which specifically binds to the high affinity, activated integrin GPIIb/IIIa on activated platelets. 2) A highly potent microtubule inhibitor, monomethyl auristatin E. 3) A drug activation/release mechanism using a linker cleavable by cathepsin B, which we demonstrate to be abundant in the tumor microenvironment. The scFv GPIIb/IIIa -MMAE was first conjugated with Cyanine7 for in vivo imaging. The therapeutic efficacy of the scFv GPIIb/IIIa -MMAE was then tested in a mouse metastasis model of triple negative breast cancer. Results : In vitro studies confirmed that this ADC specifically binds to activated GPIIb/IIIa, and cathepsin B-mediated drug release/activation resulted in tumor cytotoxicity. In vivo fluorescence imaging demonstrated that the newly generated ADC localized to primary tumors and metastases in a mouse xenograft model of triple negative breast cancer, a difficult to treat tumor for which a selective tumor-targeting therapy remains to be clinically established. Importantly, we demonstrated that the scFv GPIIb/IIIa -MMAE displays marked efficacy as an anti-cancer agent, reducing tumor growth and preventing metastatic disease, without any discernible toxic effects. Conclusion : Here, we demonstrate the utility of a novel ADC that targets a potent cytotoxic drug to activated platelets and specifically releases the cytotoxic agent within the confines of the tumor. This unique targeting mechanism, specific to the tumor microenvironment, holds promise as a novel therapeutic approach for the treatment of a broad range of primary tumors and metastatic disease, particularly for tumors that lack specific molecular epitopes for drug targeting.