Systematic design of combination therapy by targeting master regulators of coexisting diffuse midline glioma cell states

Intratumor heterogeneity fundamentally challenges cancer treatment, as coexisting, molecularly distinct cell states with non-overlapping drug sensitivities can drive therapeutic resistance. We establish and validate a generalizable, network-based framework to systematically identify combination therapies targeting complementary tumor cell states. Applied to diffuse midline glioma (DMG)—a universally fatal pediatric malignancy—this approach identified master regulator protein dependencies in seven coexisting cell states, confirmed by pooled CRISPR–Cas9 assays. Perturbational transcriptional profiles for 372 clinically relevant drugs prioritized candidates predicted to invert state-specific master regulator activity. State-selective drug sensitivity was validated for eight out of nine (89%) drugs in vivo, including avapritinib, ruxolitinib and larotrectinib. Compared with monotherapy, co-administering drugs targeting complementary states significantly prolonged survival across virtually all combinations, with avapritinib plus ruxolitinib extending median survival nearly threefold versus vehicle and 1.5-fold versus avapritinib alone. These findings establish clinically actionable DMG combinations and a tumor-agnostic and mutation-agnostic framework for rational combination therapy design. This paper identifies the ‘master regulators’ that maintain cell transcriptional states in diffuse midline gliomas and shows that targeting them in combination could represent a promising therapeutic avenue for the disease.