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.

Purpose Predictive biomarkers are required to identify patients who may benefit from the use of BH3 mimetics such as ABT-263. This study investigated the efficacy of ABT-263 against a panel of patient-derived pediatric acute lymphoblastic leukemia (ALL) xenografts and utilized cell and molecular approaches to identify biomarkers that predict in vivo ABT-263 sensitivity. Experimental Design The in vivo efficacy of ABT-263 was tested against a panel of 31 patient-derived ALL xenografts comprised of MLL-, BCP- and T-ALL subtypes. Basal gene expression profiles of ALL xenografts were analyzed and confirmed by quantitative RT-PCR, protein expression and BH3 profiling. An in vitro co-culture assay with immortalized human mesenchymal cells was utilized to build a predictive model of in vivo ABT-263 sensitivity. Results ABT-263 demonstrated impressive activity against pediatric ALL xenografts, with 19 of 31 achieving objective responses. Among BCL2 family members, in vivo ABT-263 sensitivity correlated best with low MCL1 mRNA expression levels. BH3 profiling revealed that resistance to ABT-263 correlated with mitochondrial priming by NOXA peptide, suggesting a functional role for MCL1 protein. Using an in vitro co-culture assay, a predictive model of in vivo ABT-263 sensitivity was built. Testing this model against 11 xenografts predicted in vivo ABT-263 responses with high sensitivity (50%) and specificity (100%). Conclusion These results highlight the in vivo efficacy of ABT-263 against a broad range of pediatric ALL subtypes and shows that a combination of in vitro functional assays can be used to predict its in vivo efficacy.

Purpose While the overall cure rate for pediatric acute lymphoblastic leukemia (ALL) approaches 90%, infants with ALL harboring translocations in the mixed-lineage leukemia (MLL) oncogene (infant MLL-ALL) experience shorter remission duration and lower survival rates (∼50%). Mutations in the p53 tumor suppressor gene are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be beneficial. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenografts. Experimental Design Eight MLL-ALL patient-derived xenografts were established in immune-deficient mice, and their molecular features compared with B-lineage ALL and T-ALL xenografts. The sensitivity of MLL-ALL xenografts to RG7112 was assessed in vitro and in vivo, and the ability of RG7112 to induce p53, cell cycle arrest and apoptosis in vivo was evaluated. Results Gene expression analysis revealed that MLL-ALL, B-lineage ALL and T-ALL xenografts clustered according to subtype. Moreover, genes previously reported to be over-expressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA family, were significantly up-regulated in MLL-ALL xenografts, confirming their ability to recapitulate the clinical disease. Exposure of MLL-ALL xenografts to RG7112 in vivo caused p53 up-regulation, cell cycle arrest and apoptosis. RG7112 as a single agent induced significant regressions in infant MLL-ALL xenografts. Therapeutic enhancement was observed when RG7112 was assessed using combination treatment with an induction-type regimen (vincristine/dexamethasone/L-asparaginase) against an MLL-ALL xenograft. Conclusion The utility of targeting the p53-MDM2 axis in combination with established drugs for the management of infant MLL-ALL warrants further investigation.

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Introduction While the overall cure rate for the most common pediatric cancer, acute lymphoblastic leukemia (ALL) now approaches 90%, infants (<12 months) diagnosed with ALL harboring translocations in the mixed-lineage leukemia oncogene (infant MLL-ALL) experience shorter remission duration and a significantly reduced likelihood of survival (∼50%). Therefore, new treatments that can be incorporated into conventional chemotherapy regimens to extend patient remission and improve survival are urgently required. Mutations in the p53 tumor suppressor are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be of therapeutic benefit. Nutlin cis-imidazole molecules selectively inhibit p53-MDM2 binding, resulting in activation of the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenograft models. Methods In vitro cytotoxicity was assessed by mitochondrial metabolic activity assay (Alamar blue) following 48h drug exposures. P53 protein levels and subcellular distribution were assessed by immunoblotting. Patient-derived xenografts were established from infant MLL-ALL, B-cell precursor (BCP)-ALL, or T-lineage ALL (T-ALL) bone marrow or peripheral blood (PB) biopsies in immune-deficient (NOD/SCID or NSG) mice, and their gene expression profiles generated using Illumina Human Ref-12 Expression BeadChips. Engraftment and drug responses were assessed by enumeration of the proportion of human versus mouse CD45+ cells in the PB. Mice with established disease received vehicle, RG7112 (100 mg/kg daily x 5 p.o.), a combination of vincristine (0.15 mg/kg once i.p.) dexamethasone (5 mg/kg daily x 5 i.p.) and L-asparaginase (1,000 IU/kg daily x 5 i.p.) (VXL), or RG7112 plus VXL. Anti-leukemic efficacy was assessed using an objective response measure modeled after the clinical setting, as well as the median event-free survival (EFS) of treated or control groups from treatment initiation. Therapeutic enhancement was considered to occur when the RG7112/VXL combination significantly extended mouse EFS compared with that of both of the RG7112 and VXL treated groups. Results Unsupervised hierarchical clustering of gene expression profiles revealed that the MLL-ALL (n=9), BCP-ALL (n=7) and T-ALL (n=13) xenografts clustered according to leukemia subtype. Moreover, genes previously reported to be overexpressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA gene family, were significantly upregulated in MLL-ALL xenografts. The specificity of RG7112 was validated by cytotoxicity assays against leukemia cell lines of known p53 status; p53 wild-type cell lines (RS4;11, IC50 1.4 µM; NALM-6, IC50 3.0 µM) were markedly more sensitive than those with mutant p53 (CEM, IC50 >10 µM; JURKAT, IC50 >10 µM). The in vitro sensitivity of BCP-ALL (n=3) and infant MLL-ALL (n=4) xenografts was consistent with wild-type p53 status, with IC50s of 0.11 - 2.2 µM. Exposure of ALL xenograft cells to nutlin-3 (10 µM, 6h) caused marked p53 up-regulation and nuclear translocation. Since we had previously shown that RG7112 administered as a single agent for 14 days induced significant regressions [Complete Responses (CRs) or greater] in 7/7 infant MLL-ALL xenografts in vivo, we assessed its efficacy in a 5-day combination treatment with an induction-type regimen (VXL) against two infant MLL-ALL xenografts (MLL-5 and MLL-14). The RG7112/VXL combination caused a Partial Response in MLL-5 compared with Progressive Disease for both RG7112 and VXL. The efficacy of RG7112/VXL was even more pronounced against MLL-14, causing a Maintained CR compared with CRs for both RG7112 and VXL, which met the criteria for Therapeutic Enhancement (the median EFS of RG7112/VXL-treated mice, 65.0 days, was significantly greater, P< 0.0001, than that of RG7112, 22.2 days, and VXL, 28.5 days). Conclusions RG7112 induces significant regressions in a high proportion of infant MLL-ALL xenografts and enhances the efficacy of an induction-type regimen. The utility of targeting the p53-MDM2 axis in combination with established drugs for the clinical management of infant MLL-ALL warrants further investigation. This study was supported by NCI NO1CM42216. The authors thank Roche Pharmaceuticals, Inc., for providing RG7112. Disclosures: No relevant conflicts of interest to declare.