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Supplementary Figures S6-S9. Correlations between BCL2 family gene expression and in vivo ABT-263 sensitivity in the MLL-ALL xenograft panel; S7. Correlations between BCL2 family gene expression and in vivo ABT-263 sensitivity in the BCP-ALL xenograft panel; S8. Correlations between BCL2 family gene expression and in vivo ABT-263 sensitivity in the T-ALL xenograft panel; S9. Representative immunoblot of MCL1 protein expression in ALL xenografts.

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 composed 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 coculture 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 coculture 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. Clin Cancer Res; 20(17); 4520–31. ©2014 AACR.

Introduction: Manipulation of the mitochondrial apoptosis pathway is an appealing strategy for cancer treatment using small molecule BH3 mimetics such as ABT-737, ABT-263 and ABT-199. Defining a reliable cell or molecular signature that indicates a particular cancer's susceptibility to these drugs is a high priority to optimize their use for effective cancer chemotherapy. The goal of this study was to identify cell or molecular biomarkers predictive of the in vivo responses of pediatric acute lymphoblastic leukemia (ALL) xenografts established in immune-deficient (NOD/SCID) mice to single-agent ABT-263 treatment. Methods: The in vivo efficacy of ABT-263 (100 mg/kg x 21 days) was assessed against 31 patient-derived ALL xenografts derived from infant ALL with rearrangement of the Mixed Lineage Leukemia (MLL) oncogene (infant MLL-ALL, n=9), B-cell precursor (BCP)-ALL (n=7) and T-cell ALL (T-ALL, n=15) patients. Engraftment and drug responses were assessed by enumeration of the proportion of human versus mouse CD45+ cells in the peripheral blood. 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 post-treatment. Three methods were applied to identify correlates of in vivo ABT-263 responses: (1) analysis of differentially expressed genes by gene expression profiling (GEP, Illumina HT12 BeadChips); (2) functional assessment of the mitochondrial priming status (BH3 profiling); (3) in vitro responses to single-agent ABT263 using a co-culture system with immortalized (hTERT) human mesenchymal stromal cells. Results: ABT-263 showed impressive single-agent efficacy across all three ALL subtypes, with 29 of 31 xenografts exhibiting significant progressive delays (range: 3.1 – 78 days), and 19 achieving Objective Responses (3 Maintained Complete Responses, 11 Complete Responses, and 5 Partial Responses). GEP showed that MCL1 exhibited the strongest correlation with in vivo ABT-263 sensitivity amongst BCL2 family members across the xenograft panels. Moreover, MCL1 protein function, assessed via mitochondrial depolarization mediated by Noxa peptide, also significantly correlated with in vivo ABT-263 resistance. Using an in vitro co-culture assay and cell survival assessed by flow cytometry after ABT-263 exposure (0.001-10 µM, 72 h), a training set of 17 xenografts was used to build a prediction model for in vivo xenograft responses. Testing this model against 11 other xenografts for which the in vivo results were blinded demonstrated that the fraction of cell survival at 10 nM ABT-263 predicted the in vivo responses with high sensitivity (67%) and specificity (100%). Conclusions: ABT-263 exhibits broad in vivo efficacy against pediatric ALL xenografts with no apparent subtype specificity. Cell and molecular analysis revealed MCL1 to be an important determinant of in vivo ABT-263 sensitivity, while an in vitro co-culture cytotoxicity assay was able to predict in vivo sensitivity. These in-principle approaches could be used to predict the individual responses of pediatric ALL patients to second-generation BH3 mimetics such as ABT-199. Supported by NCI NO1CM42216. Citation Format: Santi Suryani, Hernan Carol, Viktoras Frimantas, Beat Bornhauser, Chintanu Sarmah, Triona Chonghaile, Kathryn Evans, Elise Tonna, Luke Jones, Anthony Letai, Jean-Pierre Bourquin, Peter J. Houghton, Malcolm A. Smith, Richard B. Lock. Cell and molecular determinants of in vivo efficacy of the BH3 mimetic ABT-263 against pediatric acute lymphoblastic leukemia xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B26.