Acute lymphoblastic leukemia (ALL) is one of the most common cancers among children in the world, with almost 90% complete remission rate after the primary treatment. However, some patients are resistant to treatment and some relapse later. Moreover, existing treatments are toxic and induce life-long adverse effects. Potentially, it can be avoided if a more specific drug is used. Therefore, there is a need for new therapeutic targets and reliable validation of the developed drugs to avoid potential off-target effects. The family of serine-threonine polo-like kinases (Plk) is important for cell cycle. Among other family members, Polo-like kinase 1 is a crucial regulator of mitosis, and it is particularly important for the proliferation of cancer cells This thesis aimed to improve understanding of Plk1 as a treatment target in pediatric leukemia. In Paper I we aimed to identify the potential off-target proteins of Plk1 smallmolecule inhibitors such as BI2536, volasertib and NMS-1286937. The drugs are already involved in the late-stage clinical trials and result in severe side-effects that may affect the survival of the patients. We used cellular thermal shift assay and thermal proteome profiling to identify the proteins that have a change in the thermal stability after treatment. We found that the BI2536 and volasertib bind to Prostaglandin reductase 2 (PTGR2), an enzyme that regulates prostaglandin E2 metabolism and contributes to immune response. Also, we found that volasertib and NMS-1286937 affect the stability of a number of transcriptional coactivators, RNA splicing regulators, and proteins involved in the intracellular transport. In Papers II and III we investigated the potential of Plk1 as a treatment target in primary cells from pediatric T-cell and B-cell ALL. We showed that Plk1 is highly expressed both in patient cells and T/B-cell ALL cell lines, compared to peripheral blood mononuclear cells (PBMCs) from healthy donors. Further we targeted primary patient cells with RNAi prodrugs – short interfering ...