Cancer is a complex heterogeneous disease for which there is no single one-size-fits-all treatment. Precision medicine is becoming a powerful tool for medical practice, as focus is put on inter-patient variability and understanding of the molecular signaling perturbations induced by malignant transformation. Conversely, deciphering the molecular mechanism of drugs in tumor cells can reveal insights into disease biology. We set out to research therapeutics using systems-level approaches. First we developed a refined methodology to characterize the cellular target profiles of small molecules based on chemical proteomics. We have implemented a new strategy that evidently enhanced cognate target elution efficiency and proved to be effective and generically applicable, since the enhancement was evident in either in chemical immobilization of compounds on an inert matrix or biotinylated compounds on avidin-functionalized resins. This knowledge may lead to exploitation of the full potential of drug candidates, while revealing off-target effects that often lead to toxicity. Using chemical proteomics, we discovered that MTH1 (NUDT1), a nucleotide pool sanitizing enzyme, has a global role in tumorigenesis. We showed that loss-of-function of MTH1 impairs the growth of KRAS mutant tumor cells and that MTH1 inhibitors cause DNA damage in cancer cells. Moreover, we found that the (S)-enantiomer of the kinase inhibitor crizotinib is a nanomolar inhibitor of MTH1 catalytic activity while(R)-crizotinib was inactive. All in all, our results suggest nucleotide pool homeostasis as an interesting intervention point for cancer therapy. Combinations of inhibitors are proposed as a method to overcome the resistance caused by compensatory pathways and to lessen the toxic side effects through reduced dosing, which is especially appealing in pediatric tumors. ^Using a parallel phenotypic combinatorial screening approach, we identified disease specific interactions of targeted agents. We observed a highly potent synergy in neuroblastoma, between the kinase inhibitor lapatinib and anticancer compound YM155. We found that the inhibition of ABCB1 efflux transporter by lapatinib led to considerable increase in intracellular concentration of YM155; this allowed the prolonged and elevated cytotoxicity specific for resistant neuroblastoma cells expressing high levels of ABCB1. Next, we retrieved combinations specific for Ewing sarcoma; e.g. concomitant treatment with the clinically evaluated multikinase inhibitor PKC412 and IGF1R/INSR inhibitors proved to be strongly synergistic. We profiled PKC412 by chemical proteomics and found that the compound exerts its cytotoxic effect by inhibiting crucial Ewing sarcoma signaling routes. We showed that a particular drug combination-induced alteration of phosphorylation events was responsible for the synergistic effect since a large portion of signaling events were unique for the combinatorial treatment. Finally, we focused on the phenomenon of drug resistance and drug action. Although YM155 was developed as a survivin inhibitor, the precise molecular mechanism was unknown. We used a haploid genetic screen to reveal an absolute interdependency between YM155 action and SLC35F2, a member of the solute carrier protein family that is overexpressed in a number of malignancies. We further showed that YM155 conferred its cytotoxicity via DNA intercalation in cells expressing SLC35F2, leading to a DNA damage response and apoptotic cell death. This gene-drug interaction might offer a specific targeting strategy of DNA damage to tumor cells with elevated levels of SLC35F2 expression. We used different techniques to profile drugs and drug combinations and characterize cancer vulnerabilities. The results demonstrate that a holistic, integrated systems pharmacology approach can contribute towards better understanding of cancer biology and identify new alternative therapeutic regimens.