Purpose: BCR-ABL + B-ALL leukemic cells are highly dependent on the expression of endogenous anti-apoptotic MCL-1 to promote viability and are resistant to BH3-mimetic agents such as navitoclax (ABT-263) that targets BCL-2, BCL-XL, and BCL-W. However, the survival of most normal blood cells and other cell types are also dependent on Mcl-1. Despite the requirement for MCL-1 in these cell types, initial reports of MCL-1-specific BH3-mimetics have not described any overt toxicities associated with

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... single-agent use, but these agents are still early in clinical development. Therefore, we sought to identify FDAapproved drugs that could sensitize leukemic cells to ABT-263. Experimental Design: A screen identified dihydroartemisinin (DHA), a water-soluble metabolite of the anti-malarial artemisinin. Using mouse and human leukemic cell lines, and primary patient-derived xenografts, the effect of DHA on survival was tested and mechanistic studies were carried out to discover how DHA functions. We further tested in vitro and in vivo whether combining DHA with ABT-263 could enhance the response of leukemic cells to combination therapy. Results : DHA causes the down-modulation of MCL-1 expression by triggering a cellular stress response that represses translation. The repression of MCL-1 renders leukemic cells highly sensitive to synergistic cell death induced by ABT-263 in a mouse model of BCR-ABL + B-ALL both in vitro and in vivo. Furthermore, DHA synergizes with ABT-263 in human Ph + ALL cell lines, and primary patient derived xenografts of Ph + ALL in culture. Conclusions: Our findings suggest that combining DHA with ABT-263 can improve therapeutic response in BCR-ABL + B-ALL. The BH3-mimetic agents venetoclax (ABT-199) and ABT-263 are promising drug candidates, particularly for hematological malignancies that are dependent on the BCL-2 and BCL-XL survival genes respectively. Despite their promise, experimental evidence has indicated that elevated expression of other pro-survival molecules, such as MCL-1, represents a common resistance mechanism to these BH3-mimetics. Furthermore, in MCL-1-dependent malignancies, ABT-199 and ABT-263 resistance remains a significant barrier. We sought to identify agents that could render ABT-263-resistant BCR-ABL + B-ALL cells lines sensitive to ABT-263. Our efforts identified dihydroartemisinin, an antimalarial drug, as a repressor of MCL-1 protein expression. We demonstrate in mouse and human BCR-ABL + B-lineage leukemia that combining DHA-treatment with ABT-263 induces significant synergistic responses in cell culture, patient-derived xenografts, and in animal models. These findings suggest that combinatorial therapy of DHA with BH3mimetic agents can significantly improve leukemic response. Cell Death Experiments. Cells (6x10 4 ) were seeded in 96-well plates and DHA and ABT-263 (solubilized in DMSO or DMSO vehicle controls) added at indicated concentrations. After 24 hours, apoptotic cells were determined by staining with Annexin-V-APC and propidium iodide (BD Biosciences) and measured by flow cytometry. Real-time PCR. RNA extracted using Ambion RNA Extraction Kit (Life Technologies, CA) was reverse transcribed with SuperScript III (Life Technologies). Real-time PCR was performed using primers and Fast SYBR-green (Thermo Fisher, MA). Data were analyzed by the ΔΔCt method in a Quantstudio7 Flex real-time PCR machine (Thermo Fisher) with housekeeping gene (Ubiquitin) and compared to unstimulated cells. Primer sequences are available by request. Response Surface Modeling. Response surface modeling, implemented in Matlab version R2016a (Mathworks, MA), was used to determine changes in the response on viable cells Research. on October 24, 2017.