Useful targeted therapeutics for high risk subgroups of acute leukemia remain an unmet medical need. Here we report the results of a large-scale screening strategy that tested thousands of compounds targeting many signalling pathways for their ability to inhibit the survival and growth in vitro of human leukemic cells from multiple sources; i.e., patients’ samples, de novo generated human leukemia models, and established human leukemic cell lines. The responses of the cells from the de novo models exhibited similar responses to those of the patients’ samples both of which showed similar differences with the cell-line responses. Analysis of differences in subtype-specific therapeutic vulnerabilities made possible by the scale of this screen enabled the identification of a new specific modulators of apoptosis, shikonin, exemplifying its power to identify a relevant mechanism of action of small molecules with a complex polypharmacology. In summary, the results create a new platform for uncovering new therapeutic options for high-risk human leukemias, in addition to reinforcing the importance of the test sample choice for effective drug discovery.