We identified a synthetic lethality between PLK1 silencing and the expression of an oncogenic Epidermal Growth Factor Receptor, EGFRvIII. PLK1 promoted homologous recombination (HR), mitigating EGFRvIII induced oncogenic stress resulting from DNA damage accumulation. Accordingly, PLK1 inhibition enhanced the cytotoxic effects of the DNA damaging agent, temozolomide (TMZ). This effect was significantly more pronounced in an Ink4a/Arf(-/-) EGFRvIII glioblastoma model relative to an Ink4a/Arf(-/-) PDGF-β model. The tumoricidal and TMZ-sensitizing effects of BI2536 were uniformly observed across Ink4a/Arf(-/-) EGFRvIII glioblastoma clones that acquired independent resistance mechanisms to EGFR inhibitors, suggesting these resistant clones retain oncogenic stress that required PLK1 compensation. Although BI2536 significantly augmented the anti-neoplastic effect of EGFR inhibitors in the Ink4a/Arf(-/-) EGFRvIII model, durable response was not achieved until TMZ was added. Our results suggest that optimal therapeutic effect against glioblastomas requires a "multi-orthogonal" combination tailored to the molecular physiology associated with the target cancer genome.