Therapy-associated lineage plasticity in DIPG following combined CDK4/6 inhibitor, temozolomide, and radiation.

Diffuse intrinsic pontine glioma (DIPG) is a highly lethal and therapeutically refractory pediatric brain tumor, and the effects of distinct disease backgrounds on treatment response and residual cell-state plasticity remain poorly understood. We evaluated abemaciclib, an FDA-approved CDK4/6 inhibitor, combined with temozolomide (TMZ) and radiation (XRT) in two patient-derived orthotopic xenograft (PDOX) models established from a treatment-naïve biopsy (IBs-9119DIPG) and a previously treated autopsy tumor (IBs-A0317DIPG). Treatment activity was assessed in PDOX-derived 3D tumor organoids and in randomized DIPG PDOX studies, followed by survival analysis, immunohistochemistry, and endpoint single-cell RNA sequencing (scRNA-seq). The triple therapy generated synergistic antitumor effects in PDOX-derived organoids and significantly prolonged survival in both PDOX models (P < 0.05) despite their distinct baseline molecular and cell-state differences. Endpoint scRNA-seq revealed reduced oligodendrocyte-progenitor-like (OPC-like) cells in both models and decreased astrocyte-like cells in the IBs-A0317DIPG model as cell-state changes associated with treatment response. In contrast, neural progenitor-like (NPC-like) cells expanded in IBs-A0317DIPG, whereas mesenchymal-like and Mitotic populations persisted in IBs-9119DIPG as candidate therapy-tolerant states. Pseudotime trajectory analysis uncovered a resistance-associated trajectory characterized by an exit from stemness toward differentiation in OPC-like cells in treatment-naïve IBs-9119DIPG, in contrast to the enrichment of stem-like OPC-like and NPC-like cells in therapy-resistant IBs-A0317DIPG. A transcriptionally defined radiation-resistance-associated subpopulation with candidate radiosensitization target genes (NPAS3, TBC1D15, and INPP4B) was also identified. Overall, the triple therapy improved survival in clinically distinct DIPG PDOX models and revealed therapy-associated residual cell-state changes that may inform future strategies to improve durable DIPG tumor control.