DDDR-62. Dissecting cellular state alterations critical for the synergistic response and therapy resistance of the combined Abemaciclib, Temozolomide, and Radiation in DIPG PDOX models

Abstract Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain cancer. Here, we demonstrated that combining the CDK4/6 inhibitor abemaciclib with temozolomide and radiation (the triple therapy) yields synergistic anti-tumor effects in DIPG organoids and significantly extends survival times in patient-derived orthotopic (intra-brain stem) xenograft (PDOX) models. Median survival times increased from 42 days in the control group to 54 days (19% increase) in triple combination in IBs-9119DIPG (P < 0.001) and from 103 days to 140.5 days (30% increase) in IBs-A0317DIPG (P < 0.05). Mechanistically, we performed scRNA-seq and identified dynamic reprogramming of tumor cell states, including the expansion of NPC-like, and NPC-like cells displayed a consistent treatment-resistant profile across both DIPG models, with notable enrichment under triple therapy. In IBs-A0317DIPG, NPC-like cells rose from 39.7% in the control to 76.8% (1.9-fold) under the triple therapy. This trend was paralleled by increasing median potency scores, peaking at 0.378 with the triple combination, as well as decreasing pseudotime values across all treatment groups. In IBs-9119DIPG, although baseline NPC-like fractions were low (~1%), they also rose by ~1.5-fold (to 1.5%) following triple therapy. Concurrently, persistent MES-like and mitotic-like populations contributed significantly to therapeutic resistance, while OPC- and AC-like subpopulations were depleted, potentially enhancing therapy responsiveness. Pseudotime trajectory analysis further revealed therapy-induced cell fate transitions within NPC- and OPC-like compartments, including the enrichment of stem-like cells. More importantly, we discovered that an alternative trajectory—exit from stemness toward more differentiated states—may also contribute to therapeutic failure. Additionally, we identified a novel radiation-resistant subpopulation and found a new set of transcriptional targets indicative of therapeutic vulnerability. This study demonstrated the triple therapy as a promising DIPG regimen that can be rapidly translated into clinical trials and nominated new cellular and molecular targets to overcome residual disease.