Antiapoptotic Bcl-2 proteins are overexpressed in a number of cancers, including leukemias, and are frequently associated with resistance to conventional chemotherapeutic drugs. ABT-737, a Bcl-2 homology domain 3 mimetic (for structure, see Nature 435: [677][678][679][680][681] 2005) inhibits the prosurvival function of Bcl-2, Bcl-X L , and Bcl-w. We show that ABT-737 was effective as a single agent against a panel of pediatric acute lymphoblastic leukemia (ALL) xenografts, previously established, from patient biopsies, in immunodeficient mice. Although in vitro resistance of leukemia cell lines correlated with expression of the prosurvival protein Mcl-1, there was no relationship between Mcl-1 expression and in vivo xenograft response to ABT-737. However, expression of the proapoptotic protein Bim, and the extent of its association with Bcl-2, significantly correlated with in vivo ABT-737 sensitivity. ABT-737 potentiated the antileukemic effects of L-asparaginase, topotecan, vincristine, and etoposide against drug-resistant xenografts in vitro and in vivo. Finally, we show that the combination of L-asparaginase (by specifically down-regulating Mcl-1 protein levels), topotecan (by activating p53 via DNA damage), and ABT-737 (by inhibiting antiapoptotic Bcl-2 family members) caused profound synergistic antileukemic efficacy both in vitro and in vivo. Rational targeting of specific components of the apoptotic pathway may be a useful approach to improve the treatment of refractory or relapsed pediatric ALL. Overall, this study supports the inclusion of the clinical derivative of ABT-737, ABT-263 (for structure, see Cancer Res 68:3421-3428, 2008), into clinical trials against relapsed/refractory pediatric ALL.

IntroductionSignificant improvements in primary therapy for childhood acute lymphoblastic leukemia (ALL), including induction therapy using vincristine, L-asparaginase amidohydrase (L-ASP), doxorubicin, and a glucocorticoid, have led to an overall cure rate of approximately 80%. 1,2 For patients with ALL who have a slow response to initial therapy, modification of postinduction therapy based on early response to treatment has been shown to improve the survival of children with high-risk ALL. 3 Of the 20% of patients who relapse, most die, 4-6 and treatment outcome for early bone marrow relapse is especially poor. 7 Patients who experience a second relapse have a 3-year survival rate of only 8%, although third remissions are attainable. 5 Patients with longer duration of first remission are more likely to achieve remission again, and those who relapse at a site other than bone marrow are more likely to respond to reinduction chemotherapy. 6,[8][9][10] Several multiagent regimens, including the combination of vincristine, prednisone, and L-ASP, are reported to provide complete response (CR) rates of approximately 40% in multiple-relapse patients. 6 L-ASP, which depletes asparagine and glutamine in leukemic cells, 11 is a critical component of therapy for childhood ALL. As a single agent, L-ASP induced complete remissions in 40% to 60% of patients with ALL, and in combination with vincristine and prednisone is associated with an initial remission rate of 95%. 12,13 Both in vitro and in vivo resistance to L-ASP has been associated with poor long-term outcome. [14][15][16] In addition, relapsed patients with greater asparagine depletion on day 14 of reinduction were more likely to achieve a second remission in the context of 6-drug therapy. 11 However, expression of asparagine synthetase (AS), which may oppose the action of L-ASP by resynthesis of asparagine, has varied widely in clinical ALL samples, but a relationship of AS levels to drug resistance has not been reported. 15,17 ABT-737 is a small molecule that binds to and inhibits the Bcl-2 family antiapoptotic proteins Bcl-X L , Bcl-2, and Bcl-w. Similar to the BH3-only "sensitizing" protein Bad, ABT-737 does not directly activate Bax or Bak or induce cytochrome c release. Instead, ABT-737 binds to multidomain antiapoptotic Bcl-2 family proteins, preventing them from sequestering proapoptotic BH3-only proteins. 18,19 Overexpression of antiapoptotic Bcl-2 (Bcl-2 and Bcl-X L ) family proteins has been observed in acute myeloid leukemia (AML), [20][21][22]23,24 and other cancers. 24,25 Bcl-X L overexpression has been reported as an independent predictor of poor event-free survival (EFS) in pediatric ALL. 23 Effective pharmacologic inhibition of the Bcl-2 family of proteins could lower the apoptotic threshold in leukemia cells, resulting in synergy with other chemotherapeutic agents, including drugs commonly used for remission induction in primary and relapsed leukemia, such as vincristine, glucocorticoids, and L-ASP.Using both in vitro and in vivo models of ALL, we ...

Current regimens for induction therapy of pediatric acute lymphoblastic leukemia (ALL), or for re-induction post relapse, use a combination of vincristine (VCR), a glucocorticoid, and l -asparaginase (ASP) with or without an anthracycline. With cure rates now approximately 80%, robust pre-clinical models are necessary to prioritize active new drugs for clinical trials in relapsed/refractory patients, and the ability of these models to predict synergy/antagonism with established therapy is an essential attribute. In this study, we report optimization of an induction-type regimen by combining VCR, dexamethasone (DEX) and ASP (VXL) against ALL xenograft models established from patient biopsies in immune-deficient mice. We demonstrate that the VXL combination was synergistic in vitro against leukemia cell lines as well as in vivo against ALL xenografts. In vivo , VXL treatment caused delays in progression of individual xenografts ranging from 22 to >146 days. The median progression delay of xenografts derived from long-term surviving patients was 2-fold greater than that of xenografts derived from patients who died of their disease. Pharmacokinetic analysis revealed that systemic DEX exposure in mice increased 2-fold when administered in combination with VCR and ASP, consistent with clinical findings, which may contribute to the observed synergy between the 3 drugs. Finally, as proof-of-principle we tested the in vivo efficacy of combining VXL with either the Bcl-2/Bcl-xL/Bcl-w inhibitor, ABT-737, or arsenic trioxide to provide evidence of a robust in vivo platform to prioritize new drugs for clinical trials in children with relapsed/refractory ALL.