The depth of our knowledge about the molecular genetics of glioblastoma (GBM) stands in stark contrast with our ability to treat it successfully. This work reports research aimed at developing combination therapies to effectively inhibit cell growth in GBM tumorspheres. In our first study, our goal was to identify synergistic pairs of drugs across three tumorsphere lines bearing genomic alterations representative of the established signaling subclasses of GBM tumors – the NF1 deleted (represented by the tumorsphere line TS565), PGDFRA amplified (represented by the tumorsphere line TS543) and EGFR activated (represented by the tumorsphere line TS676) types. Using 12 targeted drug pairs, we identified drug combinations whose effects were cell-line specific and reduced cell viability com-pared to the single drugs. We quantified synergy using two measures, the well known Combination Index, as well as a measure we defined as the Efficacy Index that is able to detect synergies in instances where the Combination Index defined at 50% is unable to capture synergy. Predominant among the synergistic drug combinations we report are the combination of MEK and AKT1/2 inhibition in the line TS543, that of the drugs gefitinib (EGFRi) and AG538 (IGFRi) in line TS565 and of gefitinib and stattic (STAT3i) in line TS676. In a second study, we sought to extend our findings from combination therapies to a clinically distinct, frequently observed subset of treatment resistant EGFR-driven GBM tumors. To improve the efficacy of EGFR inhibition, we rationally selected drugs that that may synergize with lapatinib based on the action of their respective targets on key oncogenic pathways, and explored the optimal sequence and timing of administration. In TS676 tumorspheres, which have an EGFR amplification, express the EGFRvIII mutantation and have low PTEN ex-pression, the combination of lapatinib and obatoclax was synergistic when obatoclax was applied before lapatinib. The observed synergy correlated positively with time delays from 3h to 24h. We then studied this combination in two other tumorsphere lines TS600, with an EGFR gain, and GBM39, which is EGFR amplified with the vIII mutation but PTEN intact. Sequential administration was only mildly beneficial in TS600 and not beneficial in GBM39. A time-course protein array experiment designed to illuminate the network aspects of the effects of lapatinib and obatoclax in TS676 and TS600 revealed that the most effective sequential combination in TS676 was obatoclax preceding lapatinib by 12h. This was associated with higher cleaved caspase-3 activation than the less effective co-treatment with lapatinib and obatoclax. We applied network based modeling methodologies to help test hypotheses that may explain the increased vulnerability of TS676 to lapatinib upon pretreatment with obatoclax. This study presents encouraging results demonstrating the role of drug combination timing and order on the observed effect and synergy of therapies aimed at treating EGFR driven GBM tumorspheres. We show that BCL2 inhibition by obatoclax offers a potent and promising means of increasing cellular sensitivity to lapatinib. However, further work in other EGFR driven GBM models that have lost PTEN expression is a desirable next step towards revealing the relationship of sequential synergy to this well studied co-occurrence of genetic alterations
