Ravi S. Narayan

Supplementary Figure S2: Overview of averaged cell viability (n = 3) of all concentration-titrated combination therapies per cell line (continued)

Supplementary Figure S7: HRAS over expression in NF1 wildtype or NF1 mutant GB cell lines did not result in stronger synergy in dual mTOR/MEK inhibition.

Supplementary Figure S1: Overview of averaged cell viability (n = 3) of all concentration-titrated combination therapies per cell line.

Supplementary Figure S5: Gene signature analysis of transcriptomic data on primary cell lines.

Glioblastoma (GB) is the most common and aggressive brain-derived tumor. It often shows genetic alterations in kinase signaling pathways, such as the Pi3K/mTOR and RAS/MAPK pathways, which frequently converge onto oncogenic processes. However, it is unknown to what extend co-vulnerabilities exist within this network and which kinase drug targets are promising for GB treatment. We investigated the drug sensitivity of GB cell line models to monotherapy and synergy effects in dual combination therapy to targeting components of Pi3K/mTOR and RAS/MAPK pathways. In addition, we examined cell line drug sensitivities in relation to their individual genetic tumor-driving lesions [i.e., neurofibromin 1 (NF1) alterations as well as transcriptomic defined GB subtypes]. Synergy levels were correlated to in-lab generated phosphoproteomic data. Lastly, serial or simultaneous addition of MEK and mTOR inhibitors was investigated in longitudinal experiments. Dual inhibition of MEK and mTOR resulted in synergistic effects, which were associated with NF1 deficiency. Strong synergy effects were also associated with the mesenchymal subtype. Dual inhibition of MEK and mTOR led to prolonged growth inhibition in GB spheroids. In addition, sequential drug treatment resulted in similar growth inhibitory effects compared with simultaneous combination therapies. Our findings highlight the potential of dual inhibition strategies targeting multiple kinases for the treatment of GB, particularly in NF1-deficient and mesenchymal tumors, the most lethal subtype of GB.

Supplementary Table S8 shows Two-way ANOVA Tukey's multiple comparisons test U87 spheroid normalised drug effect

Supplementary Table S6 shows correlations between synergy and phosphorylation level

Supplementary Figure S8: Heatmap displaying the correlation between duplicate phosphoproteomic data.

Supplementary Table S7 shows two-way ANOVA Tukey's multiple comparisons test U87 spheroid size

Supplementary Figure S6: Titration analysis showing the synergy effect of drug combination therapy on GB subtypes and the two most outstanding cell lines (U251 and A172, respectively).